Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols (Methods in Molecular Biology, 2546) 1071625640, 9781071625644

Table of contents :
Dedication
Preface
Contents
Contributors
Chapter 1: Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical Laboratory
1 Introduction
2 Fundamentals of Mass Spectrometry
2.1 Gas Chromatography Mass Spectrometry (GC-MS)
2.2 Liquid Chromatography Mass Spectrometry (LC-MS)
2.3 Electrospray Ionization (ESI)
2.4 Time-of-Flight (TOF) MS
3 Clinical Applications
3.1 Therapeutic Drug Monitoring and Toxicology
3.2 Endocrinology
3.3 Inborn Errors of Metabolism
3.4 Clinical Proteomics
3.5 Other Emerging Applications
4 Introducing Mass Spectrometry in the Clinical Laboratory
5 Conclusion
References
Chapter 2: System Performance Monitoring in Clinical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
1 Introduction
2 Calibration Monitoring
3 Retention Time Monitoring
4 Signal Intensity Monitoring
5 Ion Ratio Monitoring
6 Challenges and Limitations
7 Conclusions
References
Chapter 3: Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidu...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Quality Controls
2.4 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 4: Quantification of Plasma S-adenosylmethionine and S-adenosylhomocysteine Using Liquid Chromatography-Electrospray-T...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Sample Preparation
3.2 Data Analysis
4 Notes
References
Chapter 5: Quantitation of Aldosterone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
1 Introduction
2 Materials
2.1 Patient Preparation and Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Analysis
4 Notes
References
Chapter 6: Comprehensive Determination of Amino Acids for Diagnosis of Inborn Errors of Metabolism
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Calibration Standards
2.4 Working Internal Standard
2.5 Instruments and Columns
3 Methods
3.1 External Calibration
3.2 Sample Extraction and Butylation
3.3 LC-MS/MS Operation and Data Acquisition
3.4 Data Analysis
4 Notes
References
Chapter 7: Quantification of Branched-Chain Amino Acids in Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectr...
1 Introduction
2 Materials
2.1 Plasma Samples
2.2 Solvents and Reagent
2.3 Standards and Calibrators
2.4 Internal Standards and Quality Controls
2.5 Supplies
2.6 Equipment
3 Methods
3.1 Stepwise Procedure
3.2 Data Analysis
4 Notes
References
Chapter 8: Quantitation of Butyrylcarnitine, Isobutyrylcarnitine, and Glutarylcarnitine in Urine Using Ultra-Performance Liqui...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Reference Standards and Calibrators
2.4 Internal Standards, Urine Controls, and Quality Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 9: Quantification of Free and Total Carnitine in Serum Using Liquid Chromatography Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standard
2.4 Calibrators
2.5 Quality Controls
2.6 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
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Chapter 10: Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Standards and Calibrators
2.4 Quality Control Samples
2.5 LC Reagents
2.6 Equipment
2.7 Supplies
3 Methods
3.1 Procedure
3.1.1 Sample Collection: Correct Protocol with the Salivette Device
3.1.2 Stepwise Sample Preparation Procedure
3.1.3 Extraction: Part 1
3.1.4 Dilution with Formate and Vial Preparation: Part 2
3.1.5 LC Method
3.1.6 MS Method
3.2 Data Analysis
4 Notes
References
Chapter 11: Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Standards, Calibrators, and Quality Controls
2.4 Equipment
2.5 Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Chromatographic Conditions
3.3 Mass Spectrometer Conditions
3.4 Data Analysis
4 Notes
References
Chapter 12: Laboratory Diagnosis of Cerebral Creatine Deficiency Syndromes by Determining Creatine and Guanidinoacetate in Pla...
1 Introduction
2 Materials
2.1 Specimen Requirements
2.2 Reagents
2.3 Standards and Calibrators
2.4 Internal Standard and Quality Controls
2.5 Equipment
3 Methods
3.1 Preparation of Plasma Samples
3.2 Preparation of Urine Samples
3.3 Sample Preparation for UPLC/MS/MS Analysis
3.4 Setup for HPLC and Tandem Mass Spectrometer (See Note 4)
3.5 Data Analysis
3.6 Result Interpretation
4 Notes
References
Chapter 13: Quantitation of Estradiol and Testosterone in Serum Using LC-MS/MS
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
2.6 Instrument Operating Conditions
3 Methods
3.1 Stepwise Procedure
3.2 Analysis
4 Notes
References
Chapter 14: Quantitation of Fatty Acids in Serum/Plasma and Red Blood Cells by Gas Chromatography-Negative Chemical Ionization...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Standards and Calibrators
2.4 Internal Standards and Quality Controls
2.5 Analytical Equipment and Supplies
2.6 Instrumentation and Software
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Conditions
3.3 Data Analysis
3.4 Dilution
4 Notes
References
Chapter 15: Quantitation of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Sp...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Sample Preparation (Free GABA)
3.2 Sample Preparation (Total GABA)
3.3 Data Analysis
4 Notes
References
Chapter 16: A Simple, Fast, and Reliable LC-MS/MS Method for the Measurement of Homovanillic Acid and Vanillylmandelic Acid in...
1 Introduction
2 Materials
2.1 Specimens
2.2 Reagents
2.3 Calibration Standards
2.4 Internal Standards
2.5 Quality Control Materials
2.6 Equipment
2.7 Supplies
3 Method
3.1 Extraction
3.2 LC-MS/MS Analysis
3.3 Data Analysis
3.4 Result Interpretation
4 Notes
References
Chapter 17: Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chro...
1 Introduction
2 Materials
2.1 Sample
2.2 Reagents
2.3 Standards and Calibrators
2.4 Internal Standard and Quality Control Samples
2.5 Supplies
2.6 Equipment
3 Methods
3.1 Stepwise Sample Preparation Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 18: Quantification of 5-Hydroxyindoleacetic Acid in Urine by Ultra-performance Liquid Chromatography Tandem Mass Spect...
1 Introduction
2 Materials
2.1 Specimens
2.2 Reagents and Buffers
2.3 Standards and Calibrators
2.4 Quality Control (QC) Materials
2.5 Equipment
2.6 Supplies
3 Methods
3.1 Stepwise Procedure
3.2 LC-MS/MS Analysis
3.3 Data Analysis
4 Notes
References
Chapter 19: Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Sample Preparation Procedure
3.2 Analysis
4 Notes
References
Chapter 20: Quantification of Insulin Analogs by Liquid Chromatography-High-Resolution Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Calibrators, Internal Standards, and Quality Control (See Note 1)
2.4 Supplies and Equipment
3 Methods
3.1 Sample Preparation
3.2 Analysis
4 Notes
References
Chapter 21: Differentiation of Common IGF-1 Variants Using HRMS COM Determination with Follow-Up MS/MS Verification
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Calibrators, Internal Standard, and Quality Control Samples
2.4 Supplies and Equipment
3 Methods
3.1 Sample Preparation
3.2 HPLC Conditions
3.3 MS Conditions
3.4 Center of Mass Calculations
3.5 MS/MS Conditions
4 Notes
References
Chapter 22: Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography-High-Resolution Mass Spe...
1 Introduction
2 Materials
2.1 Specimens
2.2 Reagents, Calibrators, and QCs
2.3 Equipment, Supplies, and Software
3 Methods
3.1 IGF-1 Variants List
3.2 TraceFinder Setup
3.3 Sample Preparation for IGF-1 Quantitation and Variant Detection
3.4 Liquid Chromatography-High-Resolution Mass Spectrometry
3.5 Novel Variant Detection and Identification (NVDI) Workflow
3.6 DNA Sequencing
4 Notes
References
Chapter 23: Quantitation of Lactate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Sample Preparation
3.2 Data Analysis
4 Notes
References
Chapter 24: Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples and Controls
2.2 Reagents and Buffers
2.3 Equipment and Supplies
3 Methods
3.1 First Day: Incubation and Enzymatic Reactions
3.2 Second Day (Sample Clean-Up)
3.3 MS/MS and Analysis
4 Notes
References
Chapter 25: Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Standards and Calibrators
2.4 Internal Standards
2.5 Quality Controls
2.6 Supplies
2.7 Equipment
3 Methods
3.1 Lyso-GL1 and Psychosine
3.2 Lyso-Gb3 and Lyso-SPM
3.3 Data Analysis
4 Notes
References
Chapter 26: Detection of 13C-Mannitol and Other Saccharides Using Tandem Mass Spectrometry for Evaluation of Intestinal Permea...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Standards
2.3 Solutions
2.4 Calibrators, Internal Standards, and Quality Control Samples
2.5 Supplies and Equipment
3 Methods
3.1 Sample Preparation
3.2 Chromatography
3.3 Mass Spectrometry
4 Notes
References
Chapter 27: LC-MS/MS Method for High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine: Method for Analyzi...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Standards and Calibrators
2.4 Quality Control (QC) Samples
2.5 Equipment
2.6 Supplies
3 Methods
3.1 Procedure
3.2 LC-MS/MS
3.3 Data Analysis
4 Notes
References
Chapter 28: Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Ma...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Sample Preparation
3.2 Data Analysis
4 Notes
References
Chapter 29: Screening of Organic Acidurias by Gas Chromatography-Mass Spectrometry (GC-MS)
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Chemicals
2.3 Standards and Calibrators
2.4 Quality Controls
2.5 Internal Standards
2.6 Supplies
2.7 Equipment
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 30: Identification of Urine Organic Acids for the Detection of Inborn Errors of Metabolism Using Urease and Gas Chroma...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Solutions
2.3 Standards and Calibrators
2.4 Internal Standards
2.5 Quality Control
2.6 Supplies
2.7 Measuring Equipment
2.8 Equipment
3 Methods
3.1 Sample Preparation
3.2 Creatinine Determination
3.3 GC/MS Operating Conditions
3.4 Data Analysis
4 Notes
References
Chapter 31: Quantitative Organic Acids in Urine by Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry (GCxGC-...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Standards and Calibrators
2.4 Quality Controls
2.5 Internal Standards
2.6 Supplies
2.7 Equipment
3 Methods
3.1 Stepwise Procedure
3.2 Data Analysis
4 Notes
References
Chapter 32: Quantification of Parathyroid Hormone and its Fragments in Serum by Liquid Chromatography-High-Resolution Mass Spe...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Calibrators, Internal Standard, and Quality Control Samples
2.4 Supplies and Equipment
3 Methods
3.1 Sample Preparation
3.2 HPLC Conditions
3.3 Mass Spectrometry and Analysis (See Note 5)
4 Notes
References
Chapter 33: High Sensitivity Measurement of Parathyroid Hormone-Related Protein (PTHrP) in Plasma by LC-MS/MS
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Standards and Calibrators
2.4 Quality Controls (QCs)
2.5 Equipment
2.6 Supplies
3 Methods
3.1 Procedure
3.2 LC-MS/MS
3.3 Data Analysis
4 Notes
References
Chapter 34: Quantitation of Phenylalanine in Dried Blood Spot Using Liquid Chromatography Tandem Mass Spectrometry for Monitor...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Solvents
2.3 Internal Standard
2.4 Calibrators
2.5 Quality Controls
2.6 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 35: An Optimized Procedure for Proteomic Analysis of Extracellular Vesicles Using In-Stage Tip Digestion and DIA LC-MS...
1 Introduction
2 Materials
3 Methods
3.1 In-Stage Tip Digestion for Enrichment and Concentration of ECVs
3.2 DIA LC-MS/MS Method and Downstream Analysis
4 Notes
References
Chapter 36: High-Throughput Plasma Proteomic Profiling
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Equipments
3 Methods
3.1 Abundant Protein Depletion
3.2 Tryptic Digestion of Depleted Plasma
3.3 Sample Clean-up Using 96-Well C18 MacroSpin Plates
3.4 Basic pH Reversed-Phase (BPRP) Chromatography
3.5 Mass Spectrometry Parameters for Spectral Library Generation and diaPASEF Analysis
3.6 Data Analysis and Protein Quantification
4 Notes
References
Chapter 37: Quantitation of Purine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Standards and Calibrators
2.4 Quality Controls and Internal Standards
2.5 Supplies
2.6 Equipment
3 Methods
3.1 Stepwise Procedure
3.2 Data Analysis
4 Notes
References
Chapter 38: Quantitation of Pyrimidine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Standards and Calibrators
2.4 Quality Controls and Internal Standards
2.5 Supplies and Equipment
3 Methods
3.1 Stepwise Procedure
3.2 Data Analysis
4 Notes
References
Chapter 39: Quantitation of Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Step-Wise Procedure
3.2 Analysis
4 Notes
References
Chapter 40: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Steroids Androstenedion...
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 41: A User-Friendly Sample Preparation Alternative for Manual and Automated LC-MS/MS Quantification of Testosterone
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Calibrators
2.4 Internal Standards and Quality Control Samples
2.5 Chromatography and Extraction Supplies
2.6 LC-MS/MS Equipment and Supplies
3 Methods
3.1 Stepwise Manual Extraction Procedure
3.2 Stepwise Automated Liquid Handler (Tecan Freedom EVO) Extraction Procedure
3.3 LC-MS/MS Conditions and Data Analysis
3.4 Development Notes
3.4.1 LC Optimization
3.4.2 Extraction Optimization
3.4.3 Liquid Handling Optimization
3.4.4 Plate Handling
3.4.5 Matrix Effect
3.4.6 Collection Container Validation
3.4.7 Calibration Design
3.4.8 Trueness and Patient Correlation Studies
4 Notes
References
Chapter 42: Quantitation of Thyroglobulin in Serum Using SISCAPA and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
1 Introduction
2 Materials
2.1 Samples
2.2 Solvents and Reagents
2.3 FSP Antibody
2.4 Internal Standards
2.5 Calibrators and Controls
2.6 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise procedure
3.1.1 Preparation of Antibody Bound to Protein G Magnetic Beads
3.1.2 Sample Denature and Alkylation Procedure
3.1.3 Sample Digestion
3.1.4 Antibody Peptide Capture
3.1.5 Magnetic Bead Washing and Elution of FSP Peptide
3.2 Analysis
4 Notes
References
Chapter 43: Quantitation of Free Thyroxine by Equilibrium Dialysis and Liquid Chromatography-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Specimens
2.2 Reagents
2.3 Dialysis Buffer
2.4 Calibrator Preparation
2.5 Internal Standard Solution Preparation
2.6 Quality Control
2.7 Equipment
3 Methods
3.1 Equilibrium Dialysis
3.2 Chromatographic Conditions (Table 2)
3.3 Mass Spectrometer Conditions
3.4 Data Analysis
4 Notes
References
Chapter 44: Quantification of Tryptophan, Indole, and Indoxyl Sulfate in Urine Using Liquid Chromatography-Tandem Mass Spectro...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Solvents
2.3 Internal Standard
2.4 Calibrators
2.5 Quality Controls
2.6 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 45: Very-Long-Chain Fatty Acids Quantification by Gas-Chromatography Mass Spectrometry
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Buffers
2.3 Equipment
3 Methods
3.1 Sample Preparation
3.2 Mass Spectrum Analysis
3.3 Data Analysis
4 Notes
References
Chapter 46: Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass Sp...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents
2.3 Internal Standards and Standards
2.4 Calibrators and Quality Controls
2.5 Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions
3.3 Data Analysis
4 Notes
References
Chapter 47: High-Throughput Analysis of 25-OH-Vitamin D2 and D3 Using Multichannel Liquid Chromatography-Tandem Mass Spectrome...
1 Introduction
2 Materials
2.1 Samples
2.2 Reagents and Consumables (see Note 2)
2.3 Stock Solutions and Diluents
2.4 Standards and Calibrators (see Notes 1, 2, and 3)
2.5 Quality Control Samples (see Notes 2 and 3)
2.6 LC Reagents
2.7 Equipment
2.8 Supplies and Other TECAN Accessories (see Note 4)
3 Methods
3.1 Procedure for Sample Preparation (see Notes 1 and 4)
3.2 High-Throughput HPLC Operating Conditions
3.3 TSQ-Endura Operating Conditions
3.4 Data Analysis
3.5 Linear Calibration Curves
3.6 Data Analysis Procedure
4 Notes
References
Chapter 48: Quantification of 25-Hydroxyvitamin D2 and D3 Using Liquid Chromatography-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 Patient Samples
2.2 Chemicals and Reagents
2.3 Standards and Calibrators
2.4 Quality Controls
2.5 Analytical Equipment and Supplies
3 Methods
3.1 Stepwise Procedure
3.2 Instrument Operating Conditions and Ion Pairs (MRMs)
3.3 Data Analysis
4 Notes
References
Correction to: Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass
Index

Citation preview

Methods in Molecular Biology 2546

Uttam Garg Editor

Clinical Applications of Mass Spectrometry in Biomolecular Analysis Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Clinical Applications of Mass Spectrometry in Biomolecular Analysis Methods and Protocols Second Edition

Edited by

Uttam Garg Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA

Editor Uttam Garg Department of Pathology and Laboratory Medicine Children’s Mercy Hospital Kansas City, MO, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-2564-4 ISBN 978-1-0716-2565-1 (eBook) https://doi.org/10.1007/978-1-0716-2565-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022, Corrected Publication 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Dedication This book is dedicated to my wife, Jyotsna, who is the epitome of strength and inspiration, daughters Megha and Mohini, and son-in-law Yafeez.

v

Preface Once only considered for specialized laboratories, mass spectrometry is increasingly being used by routine clinical laboratories. This has been made possible by the compact bench-top, relatively inexpensive, and user-friendly systems. With these benefits, applications of mass spectrometry have grown in all laboratory fields including endocrinology, biochemical genetics, drug analysis, proteomics, and pathogen identification. Mass spectrometry has key advantages including excellent specificity, multi-component analysis, and limited need for specialized reagents. Similar to the first edition, the second edition of Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols provides stepwise instructions for the analysis of a number of analytes of clinical importance. This edition includes mostly new chapters, and a few chapters from the previous editions have been updated. I am indebted to the authors and my colleagues for their excellent contributions to the contents of this book. Also, I would like to thank Dr. John Walker, the series editor, and Anna Rakovsky and David C. Casey, Springer Protocols editors. Kansas City, MO, USA

Uttam Garg

vii

Contents Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Y. Victoria Zhang and Uttam Garg 2 System Performance Monitoring in Clinical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Shannon Haymond 3 Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidurias and Fatty Acid Oxidation Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 David Scott, C. Clinton Frazee III, Bryce Heese, and Uttam Garg 4 Quantification of Plasma S-adenosylmethionine and S-adenosylhomocysteine Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry . . . . . 35 Erland Arning, Brandi Wasek, and Teodoro Bottiglieri 5 Quantitation of Aldosterone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) . . . . . . . . . . . . . . . . 45 J. Grace van der Gugten and Daniel T. Holmes 6 Comprehensive Determination of Amino Acids for Diagnosis of Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Stephen M. Roper, Annette L. Weindel, and Dennis J. Dietzen 7 Quantification of Branched-Chain Amino Acids in Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Hamed Piri-Moghadam, Alan Miller, Debra Pronger, Faye Vicente, Joel Charrow, Shannon Haymond, and David C. Lin 8 Quantitation of Butyrylcarnitine, Isobutyrylcarnitine, and Glutarylcarnitine in Urine Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Judith A. Hobert, Stephen A. Brose, and Marzia Pasquali 9 Quantification of Free and Total Carnitine in Serum Using Liquid Chromatography Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Serena Baird, C. Clinton Frazee III, and Uttam Garg 10 Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Wayne B. Anderson, Putuma P. Gqamana, and Y. Victoria Zhang 11 Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS . . . . . . . . 119 Julie A. Ray, Erik Kish-Trier, and Lisa M. Johnson

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Laboratory Diagnosis of Cerebral Creatine Deficiency Syndromes by Determining Creatine and Guanidinoacetate in Plasma and Urine . . . . . . . . . Ning Liu and Qin Sun Quantitation of Estradiol and Testosterone in Serum Using LC-MS/MS . . . . . . Ryan C. Schofield, Daniel Kirchoff, and Dean C. Carlow Quantitation of Fatty Acids in Serum/Plasma and Red Blood Cells by Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erik Kish-Trier and Tatiana Yuzyuk Quantitation of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erland Arning, Brandi Wasek, and Teodoro Bottiglieri A Simple, Fast, and Reliable LC-MS/MS Method for the Measurement of Homovanillic Acid and Vanillylmandelic Acid in Urine Specimens . . . . . . . . . . Vrajesh Pandya and Elizabeth L. Frank Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography–Mass Spectrometry (GC/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Melissa Beals, Bheemraj Ramoo, C. Clinton Frazee III, and Uttam Garg Quantification of 5-Hydroxyindoleacetic Acid in Urine by Ultra-performance Liquid Chromatography Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . Heather A. Nelson and Elizabeth L. Frank Quantitation of IgG Subclasses in Serum Using Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS) . . . . . . . . . . . . . . . . J. Grace van der Gugten, Daniel T. Holmes, and Andre Mattman Quantification of Insulin Analogs by Liquid Chromatography–HighResolution Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erica M. Fatica, Nicholas E. Larkey, and Ravinder J. Singh Differentiation of Common IGF-1 Variants Using HRMS COM Determination with Follow-Up MS/MS Verification . . . . . . . . . . . . . . . . . . . . . . . . Nicholas E. Larkey, Erica M. Fatica, and Ravinder J. Singh Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography–High-Resolution Mass Spectrometry in a Clinical Laboratory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ievgen Motorykin, Allison Li, and Zengru Wu Quantitation of Lactate in Cerebrospinal Fluid Using Liquid Chromatography–Electrospray-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . Brandi Wasek and Erland Arning Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hsuan-Chieh (Joyce) Liao and Hsiao-Jan Chen Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brandon B. Stauffer and Chunli Yu

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Detection of 13C-Mannitol and Other Saccharides Using Tandem Mass Spectrometry for Evaluation of Intestinal Permeability or Leaky Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicholas E. Larkey, Erica M. Fatica, and Ravinder J. Singh LC–MS/MS Method for High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine: Method for Analyzing Isomers Without Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark M. Kushnir, Gordon J. Nelson, Elizabeth L. Frank, and Alan L. Rockwood Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erland Arning, Brandi Wasek, and Teodoro Bottiglieri Screening of Organic Acidurias by Gas Chromatography–Mass Spectrometry (GC–MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David Scott, C. Clinton Frazee III, and Uttam Garg Identification of Urine Organic Acids for the Detection of Inborn Errors of Metabolism Using Urease and Gas Chromatography–Mass Spectrometry (GC/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stanley F. Lo, Keely Pierzchalski, Velta Young, and William J. Rhead Quantitative Organic Acids in Urine by Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry (GCxGC-TOFMS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erland Arning, Paula Ashcraft, Jeanna Bennett-Firmin, and Lawrence Sweetman Quantification of Parathyroid Hormone and its Fragments in Serum by Liquid Chromatography–High-Resolution Mass Spectrometry . . . . . . . . . . . . Erica M. Fatica, Nicholas E. Larkey, and Ravinder J. Singh High Sensitivity Measurement of Parathyroid Hormone–Related Protein (PTHrP) in Plasma by LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark M. Kushnir and Alan L. Rockwood Quantitation of Phenylalanine in Dried Blood Spot Using Liquid Chromatography Tandem Mass Spectrometry for Monitoring of Patients with Phenylketonuria (PKU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serena Baird, C. Clinton Frazee III, and Uttam Garg An Optimized Procedure for Proteomic Analysis of Extracellular Vesicles Using In-Stage Tip Digestion and DIA LC-MS/MS: Application to Liquid Biopsy in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rajesh Kumar Soni, Lyssa Dimapanat, Manpreet S. Katari, and Alex J. Rai High-Throughput Plasma Proteomic Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rajesh Kumar Soni Quantitation of Purine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Qin Sun

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Quantitation of Pyrimidine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ning Liu and Qin Sun 39 Quantitation of Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) . . . . . . . . . . . . . . . . J. Grace van der Gugten and Daniel T. Holmes 40 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Steroids Androstenedione, Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ada Munar, C. Clinton Frazee III, and Uttam Garg 41 A User-Friendly Sample Preparation Alternative for Manual and Automated LC-MS/MS Quantification of Testosterone . . . . . . . . . . . . . . . . . Judy A. Stone, Dawn Francisco, Heather Tone, Joshua Akin, and Robert L. Fitzgerald 42 Quantitation of Thyroglobulin in Serum Using SISCAPA and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) . . . . . . . . . . . . . . . . J. Grace van der Gugten, Morty Razavi, and Daniel T. Holmes 43 Quantitation of Free Thyroxine by Equilibrium Dialysis and Liquid Chromatography-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Weijia (William) Wu, Michael J. McPhaul, and Zengru Wu 44 Quantification of Tryptophan, Indole, and Indoxyl Sulfate in Urine Using Liquid Chromatography-Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . Serena Baird, C. Clinton Frazee III, and Uttam Garg 45 Very-Long-Chain Fatty Acids Quantification by Gas-Chromatography Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anna I. Scott 46 Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass Spectrometry . . . . . . . . . . . Irene De Biase and Marzia Pasquali 47 High-Throughput Analysis of 25-OH-Vitamin D2 and D3 Using Multichannel Liquid Chromatography-Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Putuma P. Gqamana and Y. Victoria Zhang 48 Quantification of 25-Hydroxyvitamin D2 and D3 Using Liquid Chromatography-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ada Munar, C. Clinton Frazee III, and Uttam Garg Correction to: Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass Spectrometry . . . . . Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors JOSHUA AKIN • UC San Diego Health, Center for Advanced Laboratory Medicine, San Diego, CA, USA WAYNE B. ANDERSON • Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA ERLAND ARNING • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA PAULA ASHCRAFT • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA SERENA BAIRD • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA MELISSA BEALS • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA JEANNA BENNETT-FIRMIN • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA TEODORO BOTTIGLIERI • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA STEPHEN A. BROSE • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA DEAN C. CARLOW • Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA JOEL CHARROW • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA; Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA HSIAO-JAN CHEN • The Chinese Foundation of Health, Newborn screening Center, Taipei, Taiwan C. CLINTON FRAZEE III • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA; University of Missouri School of Medicine, Kansas City, MO, USA IRENE DE BIASE • Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA DENNIS J. DIETZEN • Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA; Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA; St. Louis Children’s Hospital, St. Louis, MO, USA LYSSA DIMAPANAT • Department of Pathology & Cell Biology, New York, NY, USA ERICA M. FATICA • Mayo Clinic, Rochester, MN, USA; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA ROBERT L. FITZGERALD • UC San Diego Health, Center for Advanced Laboratory Medicine, San Diego, CA, USA DAWN FRANCISCO • UC San Diego Health, Center for Advanced Laboratory Medicine, San Diego, CA, USA ELIZABETH L. FRANK • Department of Pathology, University of Utah Health, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT, USA

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UTTAM GARG • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA; University of Missouri School of Medicine, Kansas City, MO, USA PUTUMA P. GQAMANA • Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA SHANNON HAYMOND • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA; Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA BRYCE HEESE • Department of Pediatrics, Children’s Mercy Hospital, Kansas City, MO, USA JUDITH A. HOBERT • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA DANIEL T. HOLMES • Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, BC, Canada LISA M. JOHNSON • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA MANPREET S. KATARI • Department of Biology, New York University, New York, NY, USA DANIEL KIRCHOFF • Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA ERIK KISH-TRIER • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA MARK M. KUSHNIR • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA; Department of Pathology, University of Utah Health School of Medicine, Salt Lake City, UT, USA NICHOLAS E. LARKEY • Mayo Clinic, Rochester, MN, USA; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA ALLISON LI • Quest Diagnostics, San Juan Capistrano, CA, USA HSUAN-CHIEH (JOYCE) LIAO • Departments of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA DAVID C. LIN • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA; Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA NING LIU • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Baylor Genetics, Houston, TX, USA STANLEY F. LO • Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Pathology, Children’s Wisconsin, Milwaukee, WI, USA ANDRE MATTMAN • Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, BC, Canada MICHAEL J. MCPHAUL • Quest Diagnostics, San Juan Capistrano, CA, USA ALAN MILLER • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA IEVGEN MOTORYKIN • Quest Diagnostics, San Juan Capistrano, CA, USA ADA MUNAR • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA; University of Missouri School of Medicine, Kansas City, MO, USA GORDON J. NELSON • ARUP Laboratories, Salt Lake City, UT, USA

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HEATHER A. NELSON • Department of Pathology, University of Utah Health, Salt Lake City, UT, USA VRAJESH PANDYA • Department of Pathology, University of Utah Health, Salt Lake City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA MARZIA PASQUALI • Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; Department of Pediatrics, University of Utah, Salt Lake City, UT, USA KEELY PIERZCHALSKI • Department of Pathology, Children’s Wisconsin, Milwaukee, WI, USA HAMED PIRI-MOGHADAM • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA DEBRA PRONGER • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA ALEX J. RAI • Herbert Irving Comprehensive Cancer Center, New York, NY, USA; Department of Pathology & Cell Biology, New York, NY, USA; Special Chemistry Laboratories, New York, NY, USA BHEEMRAJ RAMOO • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA JULIE A. RAY • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA MORTY RAZAVI • SISCAPA Technologies Inc, Washington, DC, USA WILLIAM J. RHEAD • Department of Pediatrics, Medical College of Wisconsin and Children’s Wisconsin, Milwaukee, WI, USA ALAN L. ROCKWOOD • Department of Pathology, University of Utah Health School of Medicine, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA STEPHEN M. ROPER • Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA; Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA; St. Louis Children’s Hospital, St. Louis, MO, USA RYAN C. SCHOFIELD • Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA ANNA I. SCOTT • Department of Laboratories, Seattle Children’s Hospital, Seattle, WA, USA; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA DAVID SCOTT • Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA RAVINDER J. SINGH • Mayo Clinic, Rochester, MN, USA; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA RAJESH KUMAR SONI • Herbert Irving Comprehensive Cancer Center, New York, NY, USA; Proteomics and Macromolecular Crystallography Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, NY, USA BRANDON B. STAUFFER • Sema4, Stamford, CT, USA JUDY A. STONE • University of California-San Francisco, San Francisco, CA, USA QIN SUN • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Baylor Genetics, Houston, TX, USA LAWRENCE SWEETMAN • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA HEATHER TONE • UC San Diego Health, Center for Advanced Laboratory Medicine, San Diego, CA, USA

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J. GRACE VAN DER GUGTEN • Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, BC, Canada FAYE VICENTE • Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA BRANDI WASEK • Institute of Metabolic Disease, Baylor Scott & White Research Institute, Dallas, TX, USA ANNETTE L. WEINDEL • St. Louis Children’s Hospital, St. Louis, MO, USA WEIJIA (WILLIAM) WU • Quest Diagnostics, San Juan Capistrano, CA, USA ZENGRU WU • Quest Diagnostics, San Juan Capistrano, CA, USA VELTA YOUNG • Department of Pathology, Children’s Wisconsin, Milwaukee, WI, USA CHUNLI YU • Sema4, Stamford, CT, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA TATIANA YUZYUK • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA Y. VICTORIA ZHANG • Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA; Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA

Chapter 1 Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical Laboratory Y. Victoria Zhang and Uttam Garg Abstract Mass spectrometry is a technique that identifies analytes based on mass-to-charge (m/z) ratio and structural fragments. Although this technique has been used in research and specialized clinical laboratories for decades, only in recent years has mass spectrometry become popular in routine clinical laboratories. Mass spectrometry, especially when coupled with gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of mass spectrometry include simultaneous analysis of multiple analytes (>100) and generally limited requirement for specialized reagents. Commonly measured analytes by mass spectrometry include metabolites, drugs, hormones, and proteins. Key words Clinical laboratory, Mass spectrometry, Liquid chromatography, Gas chromatography, Tandem mass spectrometry, Endocrinology, Newborn screening, Hormones and proteins

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Introduction The use of mass spectrometry (MS) technology in clinical laboratories has been growing rapidly in the last few decades. Once considered too specialized and costly for routine use, mass spectrometry has made its way in many routine clinical laboratories [1, 2]. This has been made possible with the advent of benchtop, inexpensive, and user-friendly mass spectrometry systems. The major advantages of mass spectrometry include excellent specificity, multiplexing capability, limited need for specialized reagents, and flexibility for laboratories with laboratory-developed tests to support patient needs. Mass spectrometry is a preferred technology for the measurement of many laboratory analytes particularly drugs and hormones. Other well-established clinical applications of mass spectrometry are in the field biochemical genetics such as newborn screening and diagnosis of inherited metabolic disorders.

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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In recent years, clinical applications in the areas of fast pathogen identification and proteomics have emerged [3, 4].

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Fundamentals of Mass Spectrometry It is beyond the scope of this chapter to describe the details of mass spectrometry technology. We here only provide a brief summary of the fundamentals of mass spectrometry mostly related to the assays and techniques described in this book. A mass spectrometer can be viewed as a device to weigh positively or negatively charged individual molecules or fragments of molecules in the gas phase. Biological samples such as serum, plasma, and urine cannot be analyzed by MS directly. They require certain levels of sample preparation and then most often chromatographic separation of analytes of interest before subjected to MS analysis. Figure 1 shows a general workflow for analysis of biological samples by MS platform. Mass spectrometers are typically considered consisting of three closely coupled elements—the ion source, the mass analyzer, and the detector (Fig. 1). The ion source is an interface to the sample introduction system and where the ions are formed. Commonly used ion sources are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted laser desorption ionization (MALDI). The mass analyzer, in most instruments, separate ions from one another based on their mass-to-charge (m/z) ratio, and the detector converts the separated masses into a signal that is fed into the instrument data processing system. There can be one or several mass analyzers, and the latter is referred as tandem mass spectrometry (MS/MS or MS/MS/MS). Some examples of tandem mass spectrometry are triple quadrupole (QQQ) and quadrupole time of flight (Q-TOF). The data processing system (computer) controls the entire instrument. Below we briefly describe a few key concepts in sample analysis using mass spectrometry.

Fig. 1 Typical workflow of sample analysis using mass spectrometry platform

Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical. . .

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2.1 Gas Chromatography Mass Spectrometry (GC-MS)

GC-MS has been used in clinical laboratories for several decades. A typical GC-MS uses helium gas, sample injection port, capillary column, mass spectrophotometer, vacuum system, detector, and a data analysis system. Typical analysis by GC-MS involves analyte extraction from a sample, concentration of the extract, sample derivatization if the compound of interest is not volatile or is heat labile, and injection of the extract in GC-MS. Once analyte ions are fragmented, either selected ions can be analyzed (selected ion monitoring) or all fragments can be analyzed (total ion monitoring) to produce a mass spectrum. Selected ion monitoring is more sensitive than total ion monitoring and is generally used in quantitative analysis. Total ion monitoring is very useful in the identification of unknown compounds. It is like a fingerprint since a specific mass spectrum are produced from the signature fragments of a specific analyte. GC-MS is most suitable for analysis of small molecules that are volatile, nonpolar, and thermally stable.

2.2 Liquid Chromatography Mass Spectrometry (LC-MS)

In recent years, LC-MS, particularly LC-MS/MS, has gained popularity and has become the method of choice, particularly for the analysis of metabolites, hormones, and proteins. Wide array of analytes can be measured by LC-MS/MS as compared to GC-MS. Analytes that are heat labile and difficult to derivative are more suited for LC-MS analysis. Furthermore, sample preparation is generally less involved as compared to GC-MS. Disadvantages of LC-MS/MS are less reproducible mass spectra, with higher maintenance and instrument cost as compared to GC-MS. In clinical laboratories, commonly used LC-MS techniques are LC-MS/MS involving electrospray ionization and time-of-flight (TOF) MS.

2.3 Electrospray Ionization (ESI)

Electrospray ionization (ESI) is by far the most commonly used ionization method in clinical applications [5]. Although not completely understood yet, the most acceptable explanation for ESI mechanism is that electrospray ionization process starts with a solution containing preformed ions being nebulized. The droplets shrink, as solvent molecules evaporate. This process lasts until columbic forces cause the droplets to fragment, desolvate, and eventually form charged species. These charged species are introduced into the mass spectrometer through a small orifice. The nebulization and evaporation processes can be assisted by coaxial and additional gas flows. Electrospray often produces singly charged ions for small molecules and can produce multiply charged ions for large molecules. The latter increases the effective mass range of the mass spectrometer which made analysis of proteins possible.

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2.4 Time-of-Flight (TOF) MS

3

In TOF-MS, an ion’s mass-to-charge ratio is determined by calculating the time required by the ion to travel a fixed distance into a flight tube. Under a fixed electrical field, lighter ions travel faster than the heavier ions. The major advantages of TOF-MS analyzers are high mass resolution and exact mass measurements. For example, at m/z of 100 and resolution of 2 ppm, mass error is less than m/z 0.0002. This provides adequate information to assign initial molecular formulae to a compound for further identification and confirmation. High-resolution TOF-MS is commonly being used in drug and metabolite screening and identification. MALDI-TOF MS is being increasingly used in the analysis of proteins and pathogen identification [6].

Clinical Applications MS is increasingly being used in clinical laboratory for the analysis of wide array of analytes. Most common applications of MS are in the fields of therapeutic drug monitoring, toxicology, endocrinology, and inborn error of metabolism. In recent years, emerging application include pathogen identification, proteomics, and genomics [7–9].

3.1 Therapeutic Drug Monitoring and Toxicology

Immunoassays are the commonly used methods for therapeutic drug monitoring and screening for drugs of abuse. Since immunoassays are available only for a limited number of drugs and they are nonspecific, MS has been used for the analysis of these drugs for therapeutic functions and for confirmation of drug of abuse samples with positive immunoassay results. Although GC-MS is still widely used in therapeutic drug monitoring and toxicology, LC-MS/MS has been increasingly used due to its ease of operations and less involved sample preparations. In addition, using GC-MS or LC-MS/MS, tens or hundreds of drugs and toxins can be identified and/or quantified at the same time.

3.2

Although immunoassays remain mainstream for the analysis of hormones, due to its specificity, MS has gained popularity in this field [10–12]. Immunoassays have inherent limitations on specificity and commutability among different assays particularly for steroid hormones and catecholamines and their metabolites [2, 12–14]. For example, steroid hormone immunoassays generally overestimate real concentrations, and there is a significant interlaboratory variability among different immunoassay vendors and platforms [15]. This makes difficult to follow up a patient over a long period. Although both GC-MS and LC-MS/MS are used for the determination of hormones, the latter is becoming a preferred technique due to specificity and sometime sensitivity. Table 1 lists commonly assayed hormones using mass spectrometry.

Endocrinology

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Table 1 Hormones commonly measured by mass spectrometry Aldosterone Androstenedione Catecholamines Coenzyme Q Cortisol Corticosterone Cortisone Deoxycortisol Deoxycortisone Dehydroepiandrosterone Estrone Estradiol Insulin Insulin-like growth factor-1 (IGF-1) Metanephrines Pancreatic polypeptide Parathyroid hormone (PTH) Parathyroid hormone (PTH)-related protein Pregnenolone (17-hydroxy) Progesterone (17-hydroxy) Testosterone Thyroxine (T4, total or free) Thyroglobulin Vitamin D (1,25-dihydroxy) Vitamin D (25-hydroxy)

3.3 Inborn Errors of Metabolism

Mass spectrometry has shown great promise in the screening and confirmation of inborn errors of metabolism. LC-MS/MS is widely used in newborn screening to detect a wide array of metabolic disorders including disorders of amino acid, organic acids and fatty acid metabolism [16–18]. GC-MS has been used for several decades and still remains the most commonly used technique for the analysis of urine organic acids. HPLC coupled with spectrophotometry had been the mainstream method for analysis of amino acids. This method is very time-consuming and is being replaced by

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Table 2 Metabolic disorders screened by tandem mass spectrometry through newborn screening Organic acidurias Isovaleric acidemia Glutaric aciduria 1 and 2 3-Hydroxy-3-methylglutaric aciduria Multiple carboxylase deficiency Methylmalonic acidemia due to mutase deficiency 3-Methylcrotonyl-CoA carboxylase deficiency Methylmalonic acidemia due to cobalamin A and B defects Propionic acidemia 3-Ketothiolase Fatty acids oxidation defects Medium chain acyl-CoA dehydrogenase Very-long-chain acyl-CoA dehydrogenase Long-chain 3-hydroxy-acyl-CoA dehydrogenase Trifunctional protein deficiency Carnitine uptake defect Short-chain acyl-CoA dehydrogenase Amino acids/urea cycle Phenylketonuria Maple syrup urine disease Homocystinuria Citrullinemia Argininosuccinic aciduria Tyrosinemia

LC-MS/MS. Other commonly measured analytes for the detection of metabolic disorders include acylcarnitines, bile acids, purines, and pyrimidines. Tables 2 and 3 list the disorders screened by tandem mass spectrometry in newborn screening and other metabolic disorders and metabolites diagnosed/assayed by mass spectrometry. 3.4 Clinical Proteomics

Analysis of large proteins and peptides such as thyroglobulin, insulin, and growth hormone has been introduced to the clinical laboratories on LC-MS/MS platform recently [19, 20]. Different from small molecules, analysis of large molecules will require new

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Table 3 Other metabolic disorders/metabolites commonly diagnosed/assayed by mass spectrometry Confirmation of disorders in Table 2 Many other disorders of organic, amino, and fatty acids not listed above under screening Acylcarnitines S-adenosylhomocysteine S-adenosylmethionine γ-Aminobutyric acid Bile acid synthesis Cholesterol synthesis Disorders of glycosylation Glycosphingolipids Neurotransmitters Lysosomal disorders Pterins Purine and pyrimidine disorders Succinylacetone

Fig. 2 Schematic workflow comparison for small and large molecules using mass spectrometry analysis

workflows and approaches on mass spectrometry (Fig. 2). Analysis of small molecules needs to remove proteins via approaches such as protein crash, while analysis of proteins or peptides requires protein capture, digestion, peptide enrichment, and final cleanup before LC-MS/MS analysis (Fig. 2).

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3.5 Other Emerging Applications

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Until recently, most of the clinical applications of MS have been in the analysis of small molecules. However, in recent years, applications of MS are expanding in the analysis of large molecules such as proteins, lipids, polysaccharides, and tissue imaging [21]. For example, MALDI mass spectrometry is now commonly used for rapid pathogen identification [7]. Protein profiling is showing great promise in the area of cancer diagnosis [22–24].

Introducing Mass Spectrometry in the Clinical Laboratory Before brining mass spectrometry into the laboratory, both patient needs and financial considerations should be assessed. Mass spectrometers need specific infrastructure to accommodate special needs such as consistent electric supply, high purity gases, and ventilation. A dedicated electricity supply and an uninterrupted power supply (UPS) are recommended for the best performance of the instrument. Other major challenge is acquiring competent staff who covers the day-to-day operational and troubleshooting functions for mass spectrometry in clinical laboratories. Implementation of mass spectrometry depends on specific needs. Sometimes the primary driving force for the adoption of mass spectrometry is substantial cost-savings over other methods such as immunoassays or send-out costs to a reference laboratory. Other times, the driving force is to provide analytically superior results (e.g., steroid hormone analysis or confirmation of immunoassay-positive drugs of abuse results) or develop mass spectrometry methods due to lack of other methods. Given many choices of instrument availability and need for current and future test selection, implementing mass spectrometry in a clinical laboratory could be challenging and needs careful financial and human resource evaluation [25–30]. Major considerations in implementing mass spectrometry are listed in Table 4. While modern mass spectrometry companies provide highquality products, consulting with colleagues and site visits can help narrow down the choices of instruments for further investigation. Analyzing small set of test samples on different platforms can provide better insight into the capabilities of different instruments. Before the final decision, the level of service capabilities from individual manufactures such as service coverage and response time should be considered seriously. More often than not, the lab can find peer-reviewed publications on the methods or similar methods as reference for assays to be brought in the laboratory. However, substantial efforts are still required to implement and validate the method in one’s own lab. Therefore, it is recommended to have dedicated individuals for method development and validation for clinical mass spectrometry laboratories.

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Table 4 Major steps in implementing mass spectrometry in a clinical laboratory Clinical needs Is the primary consideration Reduce turn-around time Control over sample handling process Instrument selection Based on intended analyses and economics Site visit and communication with colleagues and vendors Service types and response time for service requests Assay selection Based on the type of instrumentation, analytes, and clinical needs Literature search and communication with colleagues Consider lab staff experience and training Financial justification Key is to have an institutionally acceptable return on investment (ROI) Benefits include bringing test in-house and reduce send-out costs Major huddle is capital or leasing cost for instrument itself Other financial considerations should include the following: Service contract costs Infrastructure renovation expenses Costs for interfacing to the LIS if desirable Ongoing operating cost (e.g., high-grade reagents, gas) Professional personnel expenses (such as a dedicated person for assay development) Infrastructure planning Space for instrumentation and HPLC Nitrogen gas dewars or nitrogen generator Ventilation and noise blocking Lab space rearrangements (e.g., fixed vs. movable bench) Dedicated electric system and uninterrupted power supply IT support and data backup Staff and personnel training Essential for a successful implementation Is an ongoing process (continued)

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Table 4 (continued) Onsite training with manufacturers Online training courses Conferences workshops, symposia, and short courses Method development and validation Meets CLIA requirements for high complex testing Use highest-grade reagents available (MS grade or at least HPLC grade) Choose proper internal standards Validation shall include the following: Precision Accuracy Analytical sensitivity (LoQ) Reportable range Specificity and interference

Mass spectrometric assays are considered high-complexity assays under CLIA and laboratory-developed tests (LDTs) by FDA. Laboratory is responsible for developing and evaluating performance characteristics of the assays. A number of CLSI guidelines are available as a reference for mass spectrometry and for evaluation of other components of method development such as limit of detection (LoD), limit of quantitation (LoQ), accuracy, precision, analytical measurement range, and reference intervals. Sample preparation and use of isotope-labeled compounds as internal standards are unique features to mass spectrometry assays. Isotopiclabeled compounds behave very similar to the analytes and thus reduce the variability in sample extraction and analysis. A mass difference between the analyte of interest and the internal standard of at least 3 mass units is desirable, although a difference of at least 5 is preferred to eliminate cross-talk. Once the mass spectrometry is introduced, instruments, methods, and users need ongoing support and upgrades.

5

Conclusion In conclusion, in recent years, mass spectrometry has emerged as an important analytical platform in the clinical laboratory to provide essential services to patient needs. Its current major clinical applications are in therapeutic drug monitoring, metabolic screening, and endocrinology. Its applications are increasing in the areas of protein profiling, microorganism identification, and biomarker discovery.

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References 1. Strathmann FG, Hoofnagle AN (2011) Current and future applications of mass spectrometry to the clinical laboratory. Am J Clin Pathol 136(4):609–616 2. Holmes DT (2019) A brief update on mass spectrometry applications to routine clinical endocrinology. Clin Mass Spectrom 13:18–20 3. Grenga L, Pible O, Armengaud J (2019) Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns. Clin Mass Spectrom 14(Pt A):9–17 4. Fung AWS, Sugumar V, Ren AH, Kulasingam V (2020) Emerging role of clinical mass spectrometry in pathology. J Clin Pathol 73(2): 61–69 5. Kebarle P (2000) A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J Mass Spectrom 35(7):804–817. https://doi.org/10.1002/ 1 0 9 6 - 9 8 8 8 ( 2 0 0 0 0 7 ) 3 5 : 7 3.3.Co;2-H 6. Brais CJ, Ibanez JO, Schwartz AJ, Ray SJ (2021) Recent advances in instrumental approaches to time-of-flight mass spectrometry. Mass Spectrom Rev 40(5):647–669 7. Lagace-Wiens P (2015) Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS)-based identification of pathogens from positive blood culture bottles. Methods Mol Biol 1237:47–55 8. Kriegsmann J, Kriegsmann M, Casadonte R (2015) MALDI TOF imaging mass spectrometry in clinical pathology: a valuable tool for cancer diagnostics (review). Int J Oncol 46(3):893–906 9. Hou TY, Chiang-Ni C, Teng SH (2019) Current status of MALDI-TOF mass spectrometry in clinical microbiology. J Food Drug Anal 27(2):404–414 10. Pagotto U, Fanelli F, Pasquali R (2013) Insights into tandem mass spectrometry for the laboratory endocrinology. Rev Endocr Metab Disord 14(2):141 11. Vogeser M, Parhofer KG (2007) Liquid chromatography tandem-mass spectrometry (LC-MS/MS)–technique and applications in endocrinology. Exp Clin Endocrinol Diabetes 115(9):559–570 12. Ketha SS, Singh RJ, Ketha H (2017) Role of mass spectrometry in clinical endocrinology. Endocrinol Metab Clin N Am 46(3):593–613 13. Soldin SJ, Soldin OP (2009) Steroid hormone analysis by tandem mass spectrometry. Clin Chem 55(6):1061–1066

14. Wudy SA, Schuler G, Sanchez-Guijo A, Hartmann MF (2018) The art of measuring steroids: principles and practice of current hormonal steroid analysis. J Steroid Biochem Mol Biol 179:88–103 15. Albrecht L, Styne D (2007) Laboratory testing of gonadal steroids in children. Pediatr Endocrinol Rev 5(Suppl 1):599–607 16. Garg U, Dasouki M (2006) Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: clinical and laboratory aspects. Clin Biochem 39(4):315–332 17. Jones PM, Bennett MJ (2002) The changing face of newborn screening: diagnosis of inborn errors of metabolism by tandem mass spectrometry. Clin Chim Acta 324(1–2):121–128 18. El-Hattab AW, Almannai M, Sutton VR (2018) Newborn screening: history, current status, and future directions. Pediatr Clin N Am 65(2):389–405 19. Macklin A, Khan S, Kislinger T (2020) Recent advances in mass spectrometry based clinical proteomics: applications to cancer research. Clin Proteomics 17:17 20. Lehmann S, Brede C, Lescuyer P, Cocho JA, Vialaret J, Bros P, Delatour V, Hirtz C (2017) Clinical mass spectrometry proteomics (cMSP) for medical laboratory: what does the future hold? Clin Chim Acta 467:51–58 21. Banerjee S (2020) Empowering clinical diagnostics with mass spectrometry. ACS Omega 5(5):2041–2048 22. Jimenez CR, Verheul HM (2014) Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications. Am Soc Clin Oncol Educ Book 2014:e504–e510 23. Kriegsmann J, Kriegsmann M, Casadonte R (2014) MALDI TOF imaging mass spectrometry in clinical pathology: a valuable tool for cancer diagnostics (review). Int J Oncol 46(3):893–906 24. Li Y, Song X, Zhao X, Zou L, Xu G (2014) Serum metabolic profiling study of lung cancer using ultra high performance liquid chromatography/quadrupole time-of-flight mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 966:147–153 25. Clarke W, Rhea JM, Molinaro R (2013) Challenges in implementing clinical liquid chromatography-tandem mass spectrometry methods–the light at the end of the tunnel. J Mass Spectrom 48(7):755–767

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26. Roux A, Lison D, Junot C, Heilier JF (2011) Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: a review. Clin Biochem 44(1):119–135. https://doi. org/10.1016/j.clinbiochem.2010.08.016 27. Wu AH, French D (2013) Implementation of liquid chromatography/mass spectrometry into the clinical laboratory. Clin Chim Acta 420:4–10. https://doi.org/10.1016/j.cca. 2012.10.026 28. Vogeser M, Kirchhoff F (2011) Progress in automation of LC-MS in laboratory medicine.

Clin Biochem 44(1):4–13. https://doi.org/ 10.1016/j.clinbiochem.2010.06.005 29. Carvalho VM (2012) The coming of age of liquid chromatography coupled to tandem mass spectrometry in the endocrinology laboratory. J Chromatogr B Analyt Technol Biomed Life Sci 883–884:50–58. https://doi. org/10.1016/j.jchromb.2011.08.027 30. Himmelsbach M (2012) 10 years of MS instrumental developments--impact on LC-MS/MS in clinical chemistry. J Chromatogr B Analyt Technol Biomed Life Sci 883-884:3–17. https://doi.org/10.1016/j.jchromb.2011. 11.038

Chapter 2 System Performance Monitoring in Clinical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Shannon Haymond Abstract Quality assurance (QA) activities enable continuous improvement through ongoing post-implementation monitoring to identify, evaluate, and correct problems. QA for clinical liquid chromatography tandem mass spectrometry (LC-MS/MS) assays should include specific components that address the unique aspects of these methods. This chapter briefly describes approaches for clinical LC-MS/MS system performance monitoring using batch and peak review metrics, largely following CLSI-C62A guidance. Though routine checks ensure the quality of results reported for each run, there is also a need to evaluate metrics between runs over time. Post-implementation performance monitoring of LC-MS/MS methods is typically focused on calibration curves, retention times, peak intensities, and ion ratios. Key words LC-MS/MS, Quality assurance, Data analytics, Mass spectrometry

1

Introduction Clinical mass spectrometry methods are subject to regulations specified in the Clinical Laboratory Improvement Amendments of 1988 (CLIA or CLIA 1988). Among the regulatory standards described by CLIA is the need for a quality management system (QMS). A QMS comprises two primary components—quality control (QC) and quality assurance (QA)—and includes all policies and procedures needed for a laboratory to achieve its quality goals across the total testing process. QA activities enable continuous improvement through ongoing post-implementation monitoring to identify, evaluate, and correct problems. QA for clinical liquid chromatography tandem mass spectrometry (LC-MS/MS) assays should include specific components that address the unique aspects of these methods [1–4]. The CLSI-C62A guideline was designed for use by clinical laboratories and draws from several of these preceding resources, including such recommendations. CLSIC62A calls out four areas for QA in clinical mass spectrometry

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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assays: (1) system suitability, (2) batch and peak review, (3) reagent and system changes, and (4) long-term accuracy [2]. The error tolerances for these activities are set by each laboratory, typically during method validation, and may differ based on the individual assay performance and clinical needs. Guidance documents provide acceptable limits for some cases. The results of a recent survey suggest there is heterogeneity in clinical laboratory QA practices, even for a single type of LC-MS/MS assay, 25-OH vitamin D, indicating additional detail may be needed [5]. This chapter briefly describes approaches for clinical LC-MS/MS system performance monitoring using batch and peak review metrics. Following a successful system suitability test, an analytical run is started and typically contains a sequence of samples comprised of blanks, knowns (multiple levels of quality controls and calibrators), and unknowns (patients). For quantitative methods, every sample includes an internal standard (IS) for each analyte at known concentration. It is best practice to analyze at least two transitions, referred to as the quantifier and qualifier peaks, for each analyte and internal standard. These various features provide a rich source of metadata that can be leveraged to monitor each peak, analyte, sample, and batch, yielding information about the real-time and long-term performance of a particular assay and instrument. Deviations, shifts, or trends may indicate a problem with a sample or assay system that can negatively impact patient results. Review of an LC-MS/MS run typically begins at the batch level, checking for carryover, calibrator and QC sample acceptance, and overall IS recovery. This is followed by individual peak review for compliance with peak shape, signal intensity, retention time, and ion ratio acceptance criteria. Though these checks ensure the quality of results reported for each run, there is also a need to evaluate metrics within a run and between runs and analytes. Postimplementation performance monitoring of LC-MS/MS methods is typically focused on calibration curves, retention times, peak intensities, and ion ratios.

2

Calibration Monitoring Calibrator accuracy and calibration slope acceptance are paramount for obtaining accurate results. CLSI-C62A recommends that calibration curve slope r2 and individual calibrator concentration bias be monitored for deviation from their respective allowable ranges [2]. The limits should be determined in advance, at the discretion of the laboratory director, based on method verification studies. Calibration strategies vary widely among laboratories and are often based on practical considerations related to instrument use for a particular assay and desire to gain efficiencies through alternative calibration schemes. Though strides have been made to

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demonstrate that performing calibration curve analysis with each sample batch is unnecessary and there is no regulatory requirement to do so [6–8], it remains common practice in many laboratories for a variety of reasons. Longitudinal monitoring of calibration parameters makes the most sense if the calibration curve is constructed with the same target values and weighting scheme over time. Calibration curve data taken from all method verification studies can be analyzed to yield estimates for the expected variation in calibration concentrations and calibrator slope values. Guidance documents indicate that the calibration curve r2 should be greater than or equal to 0.995 and the allowable bias should be within 15% for all calibrators greater than the lower limit of the measurement interval (LLMI) and 20% for those at the LLMI [2, 4]. CLSI-C62A notes that for certain analytes, more stringent criteria may be appropriate and derived from biological variation, clinical guidelines, or regulatory requirements [2]. To monitor performance over time, laboratories may plot calibration r2 values (Fig. 1a) of each run for each analyte with indication of the acceptable threshold. There may be utility in trending calibration slope (Fig. 1b) and intercept values as well or instead, as outliers may be more easily detected with such plots. For example, Fig. 1b shows a single batch with a spurious slope value outlier that is not obvious when calibration r2 data from the same batches are plotted (Fig. 1a). When alternative calibration strategies are used, laboratories should monitor metrics that indicate calibration stability, which will vary by the approach but may include response factors [7, 8]. Calibration standard percent bias may be plotted for each calibrator by analyte over time with the appropriate thresholds (Fig. 1c). Time series plots of calibration curve parameters can be helpful to identify calibrator drift or degradation (in individual levels or lots of calibrator) and to spot outliers attributable to specific runs or calibrators. These deviations from expected values may indicate problems such as contamination, loss of detector sensitivity, or issues with sample preparation.

3

Retention Time Monitoring Chromatographic retention time is a key parameter in LC-MS/MS methods for ensuring appropriate specificity in analyte detection. It is best practice to select an IS that has identical physiochemical and analytical behaviors as the analyte of interest, leading to similar chromatographic retention times for the analyte and IS. Retention time changes may also indicate LC system issues that need to be addressed. For these reasons, retention times should be consistent for analytes and IS in unknown and standard (i.e., calibrator) samples within a run and be maintained from

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Fig. 1 (a) Calibration r2 over time for a single analyte on a single instrument. The dashed red line represents the acceptable limit. (b) Calibration slope over time for a single analyte on a single instrument. The solid blue

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run-to-run. CLSI-C50 and CLSI-C62A similarly recommend laboratories to monitor relative retention time (analyte to IS) for consistency over time and between unknowns and standards [1, 2]. This relative retention time for unknown samples should be within 2.5% of the mean value calculated for the calibrator samples within and across runs. The absolute retention times may also be monitored for each run and across runs to not exceed a specified acceptable range, determined from method verification studies and within the 2.5% tolerance. Though not prescribed by any guidance documents, some examples for monitoring retention time may include (a) violin or box plots with jittered strip plots of relative or absolute retention times for each analyte by run over time, illustrating both the mean or median and the distribution relative to an established threshold (Fig. 2a); (b) time series jittered strip plots for each analyte showing the percent difference of relative retention time for unknowns and QCs from the mean value for the calibrators, compared to the 2.5% threshold (Fig. 2b); or (c) time series plots for samples in a batch depicting run order within the batch (Fig. 2c). Each of these plots provides a different type of information. The plots showing retention times in aggregate over time can alert users to shifts or drifts and outliers in specific runs. The deviation plot provides more granular insight into the behavior by sample type. A shift due to an LC-related issue that affects all samples similarly would not be detected with the plot that only assesses deviations between sample types. Similarly, the distribution-focused plots make it more difficult to determine how the different types of samples are affected. When used in conjunction, these types of plots could be informative about potential issues affecting an assay or a particular analyte in a multiplex assay. Retention time shifts can be sudden or can manifest as more gradual deviations that are observed over time. Changes in retention time are likely due to LC-related issues, including column conditions, improper equilibration, temperature changes, mobile phase composition, and problems affecting the flow rate.

4

Signal Intensity Monitoring Mass spectrometry methods have a very useful internal quality tool in the IS. In addition to serving as a marker for expected retention time and peak shape, a well-chosen IS can correct for variability in

ä Fig. 1 (continued) line represents the target value, and the dashed red lines represent 2SD of the target value. (c) Calibrator percent bias over time for a single analyte on a single instrument tiled by calibrator (values are expected concentrations). The dashed blue lines represent 10% deviation from expected, and the dashed red lines represent 20% deviation from expected

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Fig. 2 (a) Boxplots with overlaid jitter plots showing relative (analyte/IS) retention time by batch for a single analyte on a single instrument. Points are colored by sample type. The solid blue line represents the target

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signal intensity due to differences in sample preparation, injection, and ionization steps [9]. Fluctuations in IS peak intensity are common given the normal changes encountered with an LC-MS/ MS assay system and its various components that impact signal. In addition, clinical sample matrices may introduce additional, unexpected issues like ion suppression. Therefore, results from method validation studies should be used to estimate the expected value and allowable limits for IS peak area. Tolerance levels for peak area may be represented as a minimum threshold or an acceptable range of values, as either counts or precision. Within a run and across runs, IS peak area should be consistent for calibrators and QCs. Multiplexed LC methods should demonstrate consistent signal intensity across channels. CLSI-C62A recommends that the thresholds for within run % CV (or relative SD) and minimal acceptable limit for the IS peak area in calibrators and controls be specified from method validation studies [2]. More information on considerations and general experimental design for determining the appropriate IS concentration can be found in this excellent review on method development [10]. Laboratories may choose to monitor the within run %CV (or relative SD) calculated for IS peak areas of calibrators and controls (Fig. 3a) or the mean IS peak area for calibrators and controls (Fig. 3b) to evaluate whether IS peak area is consistent within and across runs. Figure 3a and b demonstrates different utility of these alternatives. The same raw data was used to construct the graphs. The spurious outlier in Fig. 3b suggests that IS solution was erroneously added twice to all samples. In a multiplex assay, this could be further investigated by looking for a similar trend in all analytes that share the same IS solution. Because the error was consistent across all samples, it is not reflected in the graph that shows %CV over time (Fig. 3a). This should be identified during batch review. The effects are also manifested in the calibration slope time series plot (Fig. 1b). In addition to IS peak area, CLSI-C62A notes recommendations for calibrator signal intensity. It recommends that signal-tonoise (S/N) for any reportable peak, and particularly at the LLMI, must be >10 [2]. Assuming there is a calibrator at or near the LLMI, trending its S/N over time and setting a threshold of ä Fig. 2 (continued) value, calculated as the historical mean relative retention time of calibrators. The dashed red lines represent thresholds for 2.5% deviation from target value. (b) Boxplots with overlaid jitter plots showing relative (analyte/IS) retention time percent bias by batch for a single analyte on a single instrument tiled by sample type. Bias was calculated compared to the historical mean relative retention time of calibrators (the same target value in Fig. 2a and represented here with a solid blue line). The dashed red lines represent thresholds for 2.5% deviation from target value. (c) Retention time by run index over time for a single analyte on a single instrument. The solid blue line represents the target retention time determined from historical mean of calibrators. The dashed red lines represent thresholds for 2.5% deviation from the target value

Fig. 3 (a) Within batch %CV of IS area for QC and calibrator samples over time for a single analyte on a single instrument. (b) Within batch mean of IS area for QC and calibrator samples over time for a single analyte on a single instrument. The dashed red line represents the minimum acceptable limit for IS area. (c) Calibrator peak area:IS peak area over time for a single analyte on a single instrument, colored by calibrator level (expected concentration)

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10 would be one way to monitor this aspect of signal intensity. If employing a batch-based calibration scheme, calibrator peak areas and concentrations should be consistent across runs. CLSI-C62A indicates that calibrator/IS peak area for each calibrator should fall within defined limits with a CV 250.1), SAH (m/z 385.1 > 136.1), SAM-D3 (m/z 402.1 > 250.1), and SAH-D4 (m/z 389.1 > 138.1). Concentration of SAM and SAH is 95 nM and 41 nM, respectively

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Table 1 HPLC-ESI-MS/MS operating conditions A. HPLC (SAM and SAH)a Column temp.

40  C

Flow rate

0.20 mL/min

Gradient

Time (min)

Mobile Phase A (%)

0.0

75

7.0

0

7.1

75

10.0

Stop

B. MS/MS Tune Settings

b

Entrance Potential (V)

10

Curtain gas (psi)

20

CAD gas

Medium

Ion Spray (V)

5000

Temp ( C)

700

GS1 (psi)

40

GS2 (psi)

50

Resolution Q1 and Q3

Unit

Optimized for Shimadzu Nexera liquid chromatography system equipped with Phenomenex EZfaast, 3 μm, 250
2 mm analytical column; mobile phase A: 4 mM of ammonium acetate, 0.1% formic acid and 0.1% heptafluorobutyric acid in water; mobile phase B: 0.1% formic acid in methanol b Optimized for Sciex 5500QTRAP®. Tune settings may vary slightly between instruments a

3.2

Data Analysis

1. Instrumental operating parameters are given in Tables 1 and 2. 2. Data are analyzed using Analyst software 1.6.2 (Sciex). 3. Standard curves are generated based on linear regression of the analyte/IS peak-area ratio (y) versus analyte concentration (x) using the primary ions indicated in Table 2. 4. Acceptability of each run is confirmed if the quality controls are within previously defined limits using Westgard rules. Inter-day precision was evaluated by repeated analysis of bi-level quality control material analyzed in duplicate over a period of 20 different days. 5. Liquid chromatography retention time window limits for SAM and SAM-D3 are set at 5.8 (0.2) min—SAH and SAH-D4 5.5 (0.2) min. 6. The assay has a lower limit of quantitation of 1 nM for both SAM and SAH, with precisions of 141.2 (C4) 2.08e6

4

5

8 6 7 1: MRM of 5 channels ES+ 291.3 > 85.1 (C4-d3) 9.21e6

4

5

6 7 8 2: MRM of 3 channels ES+ 388.4 > 85.1 (C5DC) 2.68e7

2.41 C4

2

IsoC4-d3 2.27

1: MRM of 5 Channels ES+ 288.3 > 85.1 (C4) 1.29e7

2.41 C4

3

2.41 C4-d3

2

3

C5DC 4.79

%

100 0

E

1

2

3

4

5

C5DC 4.79

%

100 0

F

1

2

3

4

5

C5DC-d3 4.78

%

100 0

1

2

3

4

Time (minutes)

5

6

7

8

2: MRM of 3 channels ES+ 388.4 > 199.2 (C5DC) 3.07e6 6 7 8 2: MRM of 3 channels ES+ 391.4 > 85.1 (C5DC-d3) 7.57e6 6

7

8

Fig. 1 Representative UPLC-MS/MS ion chromatograms of isobutyrylcarnitine, butyrylcarnitine (A, B, C), glutarylcarnitine (D, E, F) and corresponding d3-internal standards in calibrator level 4. Chromatograms A and D show quantifying ions, B and E qualifying ions, and C and F internal standard transitions

5. Controls (a) Urine controls: Negative control, mid-positive control, and high-positive control are prepared and analyzed with each batch. Post-analysis, analyte concentrations are normalized to creatinine and evaluated as mmol/mol creatinine. Acceptable ranges for negative, mid-positive, and high-positive controls are determined based upon calculated mean, standard deviation, and %CV from 30 data points gathered over four to five separate batches. (b) Quality controls: Quality control values are accepted when they fall within 15% of their mean when qualified as described above. 6. Samples with measured concentrations above the highest calibrator may be diluted tenfold in plate with sample suspension solvent and re-injected.

Quantitation of Butyrylcarnitine, Isobutyrylcarnitine, and Glutarylcarnitine

4

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Notes 1. Recommend purchasing a metered vial of glutarylcarnitine reference standard. 2. Exact concentration of each stock is calculated as follows: concentration (mM) ¼ ((weight (mg)/formula weight)/ 10 mL)  1000  purity. 3. The exact volume of each individual stock required is based on the exact concentration and is calculated using the equation: volume (μL) ¼ (5 mL  20 μM)/concentration of stock (mM). 4. Remember to account for creatinine normalization when spiking urine controls to achieve the desired target concentrations post normalization. To simplify this process, the creatinine concentration of pooled urine may be adjusted by dilution with Nanopure water or concentrated by addition of concentrated urine to achieve a specific creatinine concentration (such as 5 or 10 mg/dL). 5. Use positive displacement pipettes for transferring the calibrators and quality controls to the plate. 6. Preparing samples in a plate is highly recommended over preparation in microfuge tubes. We found microfuge tubes cumbersome to use; they also increase the likelihood of contamination. 7. When using the SPE Dry 96 Evaporator, be sure to keep probe tips above samples to prevent contamination. 8. Remove plate immediately after samples are dry; overdrying can negatively impact this assay. 9. The temperature of the metal block can affect recovery of acylcarnitine species. 10. Dried, derivatized samples in a sealed sample collection plate are stable at least for 24 h at room temperature. 11. Confirm the instrument is ready for analysis before reconstituting the dried, derivatized samples. 12. Derivatized samples should be analyzed without a delay after sample preparation; reconstituted samples are stable for at least 13 h (enough time for the analysis of a full plate of samples) when kept in UPLC sample manager compartment at 10
C.

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References 1. Pasquali M, Longo N (2019) Newborn screening and inborn errors of metabolism. In: Rifai N, Horvath AR, Wittwer C (eds) Tietz textbook of clinical chemistry and molecular diagnostics, 8th ed. Elsevier, St. Louis, pp 882–897 2. Rinaldo P, Cowan TM, Matern D (2008) Acylcarnitine profile analysis. Genet Med 10: 151–1563 3. Miller M, Cusmano-Ozog K, Oglesbee D, Young S (2021) Laboratory analysis of

acylcarnitines, 2020 update: a technical standard of the American College of Medical Genetics and Genomics. Genet Med 23:249–258 4. Tortorelli S, Hahn SH, Cowan TM, Brewster TG, Rinaldo P, Matern D (2005) The urinary excretion of glutarylcarnitine is an informative tool in the biochemical diagnosis of glutaric acidemia type I. Mol Genet Metab 84:137–143

Chapter 9 Quantification of Free and Total Carnitine in Serum Using Liquid Chromatography Tandem Mass Spectrometry Serena Baird, C. Clinton Frazee III, and Uttam Garg Abstract L-carnitine is a crucial component for transporting long-chained fatty acids from the cytosol into the mitochondrial matrix for fatty acid oxidation. During this process, carnitine forms numerous acylcarnitines before being recycled into the cytosol. Abnormal levels of free carnitine, total carnitine, and acylcarnitines in serum can be indicative of a metabolic disorder before symptoms are present. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is described for the determination of free and total carnitine in serum. To measure total carnitine, samples are spiked with deuterated carnitine (internal standard) and hydrolyzed with potassium hydroxide to convert acylcarnitines to carnitine. The reaction is quenched by the addition of hydrochloric acid. Carnitine is extracted via a methanolic protein precipitation. The solution is then injected on LC-MS/MS for analysis to determine the carnitine concentration using multiple-reaction monitoring. Key words Carnitine, Acylcarnitine, Fatty acid oxidation, Tandem mass spectrometry

1

Introduction L-carnitine is an essential nutrient made from the amino acids lysine and methionine [1]. Daily carnitine requirements are primarily met through a person’s diet via consumption of meat and dairy products, but it is also produced endogenously in the human body. In certain organic acidurias and fatty acid oxidation defects, carnitine supplements can also be taken to increase overall levels. Carnitine is essential for fatty acid oxidation as it is required for the transportation of long-chain fatty acids into the mitochondria. Carnitine palmitoyltransferase 1 (CPT1), an enzyme in the outer mitochondrial membrane, activates the reaction between carnitine and long-chain acyl-CoA species to form corresponding long-chain acyl-carnitines to be transported into the mitochondria by carnitine-acylcarnitine translocase (CACT). Once inside the mitochondrial matrix, carnitine palmitoyl transferase 2 (CPT-2)

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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converts acylcarnitine to carnitine and acyl-CoA. Carnitine is recycled back to the cytoplasm for the cycle to continue [2]. Primary carnitine deficiency, CPT-1 deficiency, CACT deficiency, and CPT-2 deficiency are autosomal recessive disorders that interrupt this transportation process, leading to fatty acid oxidation defects [3]. Early detection of these disorders can be diagnosed through free and total carnitine levels before symptoms are present. Also, levels help in monitoring the patients who are supplemented with carnitine. Quantification of free and total carnitine is commonly achieved using liquid chromatography tandem mass spectrometry (LC-MS/ MS) [4–8]. By measuring free and total carnitine, total acylcarnitine can be calculated. Abnormal levels of free carnitine, total carnitine, calculated acylcarnitine, and the ratio between free carnitine and acylcarnitine are used to help diagnose several metabolic disorders. Here we describe a simple LC-MS/MS method to determine free and total carnitines in serum without the need for derivatization or extended hydrolysis time in a heated environment.

2 2.1

Materials Samples

2.2 Solvents and Reagents

Collect a minimum of 0.5 mL of blood in a red-top tube. Gel tubes are acceptable. Sample should be centrifuged at 2000 g for 7 min. Separate the serum and refrigerate at 4
C. Stable for up to 2 months. 1. 88% formic acid, mass spec grade. 2. 7.5-M ammonium acetate solution (Sigma Chemicals). 3. L-carnitine hydrochloride (Sigma Chemicals). 4. L-carnitine-D3 chloride (Toronto Research Chemicals). 5. Mass spect gold human serum (Golden West Diagnostic). 6. C2 Acetylcarnitine (Cambridge Isotope Laboratories). 7. C3 Propionylcarnitine (Cambridge Isotope Laboratories). 8. C4 Butyrylcarnitine (Cambridge Isotope Laboratories). 9. C5 Isovalerylcarnitine (Cambridge Isotope Laboratories). 10. C8 Octanoylcarnitine (Cambridge Isotope Laboratories). 11. C14 (Myristoyl) tetradecanoylcarnitine (Cambridge Isotope Laboratories). 12. C16 (Palmitoyl) hexadeanoylcarnitine (Cambridge Isotope Laboratories). 13. Normal human serum (UTAK). 14. 0.9% Sodium chloride. 15. Concentrated hydrochloric acid. 16. Potassium hydroxide pellets.

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17. Hydrolysis solution (1 M potassium hydroxide): Add approximately 100 mL of deionized water to a 250 mL volumetric flask. Add 14.025 g of KOH to the volumetric flask and stir until dissolved. Fill to the mark with deionized water. Stable for 1 year at room temperature. 18. 1-M HCl Solution: Add approximately 100 mL of deionized water to a 250-mL volumetric flask. Slowly add 20.5 mL of concentrated hydrochloric acid to the volumetric flask. Adjust the final volume to 250 mL with deionized water. Stable for 1 year at room temperature. 19. Mobile phase A (40.5 mM of ammonium acetate and 0.05% formic acid in water): Add 5.4 mL of 7.5 M of ammonium acetate and 570 μL of 88% formic acid to 1 L of deionized water. Mix and degas. Stable for 1 month when stored at room temperature. 20. Mobile phase B (40.5 mM of ammonium acetate and 0.05% formic acid in methanol): Add 5.4 mL of 7.5 M of ammonium acetate and 570 μL of 88% formic acid to 1 L of optima-grade methanol. Mix and degas. Stable for 1 month when stored at room temperature. 2.3

Internal Standard

1. Primary (1
) internal standard (10-mM L-carnitine-D3): Weigh approximately 5.0 mg of L-carnitine-D3 chloride into a glass vial, and add water (~2.5 mL) to make a final concentration of 10 mM. Calculate the amount of water needed using the following equation: Volume of water ðmLÞ ¼

mg of L‐carnitine D3 powder 2:0068

2. Secondary (2
) internal standard (1-mM L-carnitine-D3): Add 500 μL of L-carnitine-D3 primary internal standard to a 5 mL volumetric flask and fill to the line with deionized water. Store at 20
C. 3. Working internal standard (25-μM L-carnitine-D3): Add 1.25 mL of L-carnitine-D3 secondary internal standard to a 50 mL volumetric flask and fill to the line with deionized water. Store at 20
C. 2.4

Calibrators

1. L-carnitine primary standard stock solution (10 mM): Weigh approximately 19.77 mg of L-carnitine into a glass vial, and add water (~10 mL) to make a final concentration of 10 mM. Calculate the amount of water needed using the following equation: Volume of water ðmLÞ ¼

mg of L‐carnitine powder 1:9766

2. L-carnitine secondary standard (1 mM): Add 500 μL of L-carnitine primary standard stock solution to a 5 mL volumetric flask and fill to the line with mass spect gold. Store at 20
C.

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Table 1 Preparation of calibrators in mass spect gold Control

Concentration (μM)

μL of 1.00 mM

μL of 10 μM

Mass spect gold (μL)

Cal 1

1

–

100

900

Cal 2

2.5

–

250

750

Cal 3

10

–

1000 (3
std.)

–

Cal 4

25

25

975

Cal 5

100

100

900

Cal 6

500

500

500

Cal 7

1000

1000 (2
std)

–

3. L-carnitine tertiary standard (10 μM): Add 50 μL of L-carnitine secondary standard stock solution to a 5 mL volumetric flask and fill to the line with mass spect gold. Store at 20
C. 4. Prepare L-carnitine calibrators 1 and 2 from the tertiary standard and calibrators 4, 5, and 6 from the secondary standard using Table 1. 2.5

Quality Controls

All quality controls were prepared in-house. 1. QC 1 (low/free): Add 1000 μL of normal human serum to a 5 mL volumetric flask and fill to the line with 0.9% sodium chloride solution. Treat as a free carnitine specimen. Target mean established in-house. Stable for 1 year when stored at 20
C. 2. QC 2 (middle/free): Spiked mass spect gold with a target value of 250 μM. Treat as a free carnitine specimen. Prepare as follows (see Note 1): (a) L-carnitine control standard stock solution (10 mM): Weigh approximately 19.77 mg of L-carnitine into a glass vial and add water (~10 mL) to make a final concentration of 10 mM. Calculate the amount of water needed using the following equation: Volume of water ðmLÞ ¼

mg of L‐carnitine powder 1:9766

(b) Add 125 μL of the standard stock solution to a 5 mL volumetric flask and fill to the line with mass spect gold. Stable for 1 year when stored at 20
C. 3. QC 3 (middle/hydrolysis): Undiluted normal human serum. Treat as a total carnitine specimen. Target mean established in-house. Stable for 1 year when stored at 20
C. 4. QC 4 (high/hydrolysis): A mixture of acylcarnitines without free carnitine to verify hydrolysis reached completion. Target mean established in-house.

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Table 2 Preparation of 10 μM primary stock acylcarnitine solutions Acylcarnitine

M.W. (g/mol)

Amount in vial (μg)

Methanol added to vial (mL)*

C2

240

5000

2.08

C3

254

5000

1.97

C4

268

5000

1.87

C5

282

5000

1.77

C8

324

5000

1.54

C14

408

5000

1.23

C16

436

5000

1.15

*The formula for the table is as follows: Volume (mL) ¼ mass (μg)/(MW  10)

Table 3 Preparation of quality control 4 Acylcarnitine

Primary stock solution (μL)

Concentration (μM)

C2

200

400

C3

10

20

C4

10

20

C5

10

20

C8

10

20

C14

10

20

C16

10

20

Total

–

520

(a) Primary stock solutions (10 mM): Reconstitute the vials of acylcarnitine stock compounds according to Table 2. Stable for 2 years at 20
C. (b) Add the primary solutions to a 5 mL volumetric flask according to Table 3 and fill to the line with mass spect gold. Upon hydrolysis, all acylcarnitines should be converted to carnitine for a total concentration of approximately 520 μM. Stable for 1 year when stored at 20
C. 2.6 Analytical Equipment and Supplies

1. LC-MS/MS: Waters MS/MS Xevo TQ-Smicro with Acquity UPLC and autosampler. 2. Analytical column: Supelcosil LC-18; 5 cm  4.6 mm, 3 μm particle size (Phenomenex).

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3. Guard column: Pinnacle II C18 5um (Restek). 4. Autosampler vials with glass inserts and screw caps. 5. Microcentrifuge tubes, 0.6 mL graduated tube with flat cap. 6. Centrifuge.

3

Methods

3.1 Stepwise Procedure

1. Pipette 20 μL of well-mixed calibrator, patient serum, blank, or control into an appropriately labeled microcentrifuge tube. Aliquot each patient sample in duplicate, one tube to measure free carnitine and one to measure total carnitine. 2. Add 20 μL of working internal standard to all tubes (see Note 2). 3. To the free carnitine tubes (including calibrators, blank, QC 1, QC 2, and one set of patient samples): (a) Add 40 μL of D.I. water to each tube. (b) Vortex the tubes and proceed to step 5. 4. To the total carnitine tubes (including QC 3, QC 4, and the second set of patient samples): (a) Add 20 μL of 1-M KOH to the tubes and then immediately cap and vortex (see Note 3). (b) Allow tubes to stand at room temperature for 20 min. (c) Add 20 μL of 1 M HCl to each tube. (d) Vortex the tubes and proceed to step 5. 5. Add 400 μL of methanol to all tubes and vortex. 6. Centrifuge the tubes for 5 min at 3500 RPM and ambient temperature. 7. Add 100 μL of methanol to each labeled autosampler vial (see Note 4). 8. Transfer 50 μL of the supernatant from each microcentrifuge tube to the corresponding autosampler vial. 9. Mix the contents of each autosampler vial thoroughly using the pipette. 10. Inject 3 μL into LC-MS/MS for analysis.

3.2 Instrument Operating Conditions

3.3

Data Analysis

The UPLC autosampler temperature was 10
C and the column temperature was 40
C. Samples were injected in partial loop mode with a loop volume of 10 μL. Further instrument’s operating conditions are given in Tables 4, 5, and 6. 1. Data is processed and analyzed using MassLynx Software (Waters Corp, Milford, MA).

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Table 4 UPLC gradient programming and operating conditions Event

Time

Flow (mL/min)

% MPA

% MPB

Curve

1

Initial

0.500

98.0

2.0

Initial

2

0.50

0.500

98.0

2.0

6

3

2.50

0.500

60.0

40.0

6

4

3.0

0.500

2.0

98.0

6

5

3.5

0.500

2.0

98.0

6

6

3.6

0.500

98.0

2.0

6

7

4.5

0.500

98.0

2.0

6

Table 5 MS source parameters Ionization mode

ES+

Scan mode

MRM

Retention window

0.75–2.50 min

Capillary voltage

0.5 kV

Desolvation temp

600
C

Desolvation gas

950 L/h

Cone gas

20 L/h

Source temp

150
C

Table 6 MS acquisition parameters Analyte

Parent (m/z)

Daughter (m/z)

Dwell (s)

Cone (V)

CE (V)

Comment

L-carnitine

161.90

102.65

0.025

42.00

14.00

Quant

L-carnitine

161.90

84.80

0.025

42.00

18.00

Qualifier

L-carnitine—D3

165.03

102.86

0.025

42.00

14.00

Quant

L-carnitine—D3

165.03

84.89

0.025

42.00

18.00

Qualifier

Acetylcarnitine

204.03

84.89

0.025

42.00

16.00

Hydrolysis

2. Standard curves are generated per run based on inversely weighted linear regression analysis of the analyte/IS peak area ratio ( y) versus analyte concentration (x) using the quant ions for carnitine (102.65 m/z) and L-carnitine-D3 (102.86 m/z).

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3. The assay is linear from 1 to 1000 μM. 4. Calibration curve is acceptable if correlation coefficient (R2) is 0.99. 5. QC values are acceptable if they fall within 2 standard deviation of the established mean. 6. Retention times are acceptable within 0.1 min of the calibrators. 7. Ion suppression is monitored using a trend plot of internal standard areas. 8. Acylcarnitine is calculated using the following equation: Acylcarnitine concentration ðμMÞ ¼ Total carnitine ðμMÞ  free carnitine ðμMÞ 9. Carnitine ratio is calculated using the following equation: Calculated carnitine ratio ¼

Acylcarnitine concentration ðμMÞ Free carnitine concentration ðμMÞ

10. A representative chromatograph is given in Fig. 1.

Carnitine RT: 1.46 min

Carnitine D3 RT: 1.45 min

Fig. 1 Typical extracted ion chromatogram of carnitine and carnitine D3 for a patient sample

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A) Unhydrolyzed Specimen

B) Hydrolyzed Specimen

Fig. 2 Extracted ion chromatogram of acetyl carnitine (m/z 204.03 ! 84.89). Acetyl carnitine is hydrolyzed to carnitine with the addition of KOH. Therefore, a peak should be present in samples where hydrolysis has not taken place (while measuring free carnitine), and a peak should not be present when hydrolysis has taken place (while measuring total carnitine). A chromatogram of the same specimen when it is unhydrolyzed (a) and hydrolyzed (b) is shown

11. For total carnitine specimens, completion of hydrolysis should be verified by an absence of an acetyl carnitine peak (see Fig. 2).

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Notes 1. Calibrators and controls should be prepared from different lots of carnitine stock solution. 2. Free carnitine samples can be prepped while the total carnitine samples are hydrolyzing, allowing all samples to proceed to step 5 at the same time. 3. Verify the liquid is in the bottom of the tube with no bubbles, so that the KOH can hydrolyze the sample. 4. Diluting the sample with methanol was used to ensure the detector was not overloaded. Sample dilution is instrument specific and should be a part of method validation.

References 1. Rebouche CJ (1992) Carnitine function and requirements during the life cycle. FASEB J 6(15):3379–3386 2. Jones PM, Bennett MJ (2012) Disorders of the carnitine cycle and mitochondrial fatty acid oxidation. In: Garg U, Smith LD, Heese BA (eds) Laboratory diagnosis of inherited metabolic diseases. AACC Press, Washington D.C., pp 65–91 3. Longo N, Amat di San Filippo C, Pasquali M (2006) Disorders of carnitine transport and the carnitine cycle. Am J Med Genet C Semin Med Genet 142C(2):77–85 4. Ho CS, Cheng BS, Lam CW (2003) Rapid liquid chromatography-electrospray tandem mass spectrometry method for serum free and total carnitine. Clin Chem 49(7):1189–1191 5. Minkler PE, Stoll MS, Ingalls ST, Kerner J, Hoppel CL (2015) Validated method for the

quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples. Anal Chem 87(17):8994–9001 6. Morand R, Donzelli M, Haschke M, Krahenbuhl S (2013) Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction. Anal Bioanal Chem 405(27):8829–8836 7. Scott D, Heese B, Garg U (2016) Quantification of free carnitine and acylcarnitines in plasma or serum using HPLC/MS/MS. Methods Mol Biol 1378:11–19 8. Sowell J, Fuqua M, Wood T (2011) Quantification of total and free carnitine in human plasma by hydrophilic interaction liquid chromatography tandem mass spectrometry. J Chromatogr Sci 49(6):463–468

Chapter 10 Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode Wayne B. Anderson, Putuma P. Gqamana, and Y. Victoria Zhang Abstract Cortisol is one of the most important glucocorticoids involved in the regulation of human metabolism and physiological stress. Monitoring of levels of cortisol is of immense clinical benefit. In particular, salivary cortisol levels have been shown to correlate well with diurnal changes in cortisol levels in serum and have been used widely for monitoring of cortisol levels for diagnosis and prognosis purposes. We present a sensitive, fast, and robust quantitative liquid chromatography and tandem mass spectrometry (LC-MS/ MS) assay for salivary cortisol in negative mode. This assay employs protein precipitation followed by reversed-phase liquid chromatographic separation, negative-mode electrospray ionization (ESI), and MS/MS detection. This assay has a total run time of 5.8 minutes and a limit of quantification of 0.5 ng/ mL with a linear range up to 100 ng/mL. No carryover was observed at 10 μg/mL. This assay also incorporates the routine monitoring of prednisolone, a potential interferent to salivary cortisol. Key words Salivary cortisol, Cushing’s syndrome, LC-MS/MS, Prednisolone, Interferents, Quality control

1

Introduction Cushing’s syndrome is one of the most common diseases caused by overproduction of cortisol [1–4]. Physicians have relied on a number of primary tests for the diagnosis of Cushing’s syndrome in different matrices and biofluids such as urine, plasma, serum, and saliva [5–9]. Assays developed and validated in saliva have since gained prominence in recent years due to several unique advantages that include noninvasive sampling, general specimen stability, and the good correlation to free cortisol in serum, which is not affected by globulin binding or saliva flow rate [10–12]. In addition, literature shows that the salivary cortisol tests have demonstrated high accuracy diagnosis of Cushing’s syndrome, with a specificity of 93–100% and a sensitivity of 92–100% [10, 11, 13–17]. As a result, late-night salivary cortisol was recommended as one of the first-line

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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diagnostic tests for Cushing’s syndrome by the Endocrine Society [18]. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) assays have delivered the required specificity and sensitivity performance for the analysis of salivary cortisol in comparison to traditional immunoassays or HPLC assays [6–8, 19–23], so much the technology is currently considered as the most popular and reliable reference for measuring salivary cortisol [24–27]. We present a rapid, robust, and quantitative LC-MS/MS assay for salivary cortisol. The key attributes of the method include protein precipitation with acetonitrile, direct dilution of the supernatant with formate, reversed-phase chromatography, and subsequent analysis of the formate-analyte adducts in negative ion electrospray MS/MS mode. The assay has a short run time (5.8 min) and an analytical range of 0.5–100 ng/mL set by six levels of calibrators normalized against 20 ng/mL cortisol-d4 internal standard. The robustness is derived from the use of tandem mass spectrometry analysis in the negative ion mode, as well as the novel quality control monitoring of prednisolone. Here, a 3.5-ng/ mL prednisolone standard in comparable matrix is injected once per batch run and used as a reference quality control to set a threshold for the reporting of cortisol levels in samples likely adulterated with high prednisolone interferent.

2 2.1

Materials Samples

1. Saliva samples are collected using a Salivette® collection device (Sarstedt, Nu¨mbrecht, Germany) with a minimum sample volume of 0.5 mL (see Note 1). 2. Samples are stable at room temperature for 7 days, or refrigerated for 2 weeks, or frozen for 12 months.

2.2

Reagents

1. 1 mg/mL of cortisol reference standards in methanol (Cerilliant). 2. 100 μg/mL: 1 mL of cortisol reference standards in methanol (Lipomed). 3. 100 μg/mL: 1 mL of prednisolone reference standard in acetonitrile (Cerilliant, Millipore Sigma). 4. Cortisol-9,11,12,12-d4, >98% purity (CDN Isotopes). 5. Ammonium formate, LCMS Grade. 6. Methanol and acetonitrile, LCMS grade. 7. 99.5% Molecular Biology Grade NaCl, KCl, Na2HPO4, and KH2PO4. 8. ACS-grade NaOH and concentrated HCl.

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9. Phosphate-buffered saline (PBS)—diluent: Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of ultrapure deionized water. Mix and adjust pH to 7.4 using 0.05 M HCl or 0.5 M NaOH. Stable at 2–8  C for up to 2 months. See Note 2. 2.3 Standards and Calibrators

1. 100-μg/mL cortisol stock solution: Measure 1000 μL of the 1 mg/mL cortisol stock with a Class A volumetric pipette to a 10 mL Class A volumetric flask. Bring to volume with methanol. Mix and transfer to a labeled screw-top tube. Be sure to include vendor on label (cap should have a PTFE liner). Stable at 10  C to 30  C for 1 year. 2. 10-μg/mL cortisol stock solution: Measure 100 μL of the 1-mg/mL cortisol stock with a Class A volumetric pipette to a 10-mL Class A volumetric flask. Bring to volume with methanol. Stable at 20  C for 1 year. 3. 100-ng/mL cortisol (level 7) stock solution in PBS (see Note 2): Measure 1.0 mL of the 10 μg/mL cortisol stock for calibrators into a 100 mL Class A measuring flask. Bring to volume with PBS. Mix carefully and let stand 20 minutes before further dilution. The final concentration is 100 ng/mL which is used as the level 7 stock for the preparation of calibrators. Stable at 10  C to 30  C for 1 year. 4. A set of six calibrators of concentration (ng/mL) 0.50, 1.00, 10.00, 25.00, 50.00, and 75.00 in PBS (see Note 2) can be prepared from the level 7 stock (100 ng/mL) accordingly. 5. 1.0 mg/mL cortisol-d4 intermediate IS stock solution in methanol: Weigh out approximately 1 mg of CDN isotope cortisold4 into a labeled screw-cap tube. Add sufficient methanol to obtain a 1-mg/mL solution, e.g., ~1 L. Cap and Mix (cap should have a PTFE liner). Stable at 10  C to 30  C for 3 years. 6. Cortisol- d4 100-μg/mL intermediate IS stock solution in methanol: Measure 1.0 mL of the 1.0-mg/mL stock to a 10 mL Class A volumetric flask. Bring to volume with LCMS-grade methanol. Mix and transfer to labeled screw-cap tube (cap should have a PTFE liner). Stable at 10  C to 30  C for 2 years. 7. Internal standard working solution (cortisol-d4 500 ng/mL in methanol): Dilute 50 μL of cortisol-d4 stock solution to a final volume of 10.0 mL in methanol. Stable at 20  C for 1 year. 8. Precipitation reagent solution (20-ng/mL IS in acetonitrile): Dilute 10 μL of cortisol-d4 500-ng/mL working solution to 2.5 mL in acetonitrile. Stable at 20  C for 1 year. (See Note 5).

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2.4 Quality Control Samples

1. 10-μg/mL cortisol (Lipomed) stock Solution in Methanol: Measure 100 μL of the 1 mg/mL (Lipomed) stock with a Class A volumetric pipette to a 10 mL Class A volumetric flask. Bring to volume with methanol. Mix and transfer to a labeled screw-top tube. Be sure to include Vendor on label. (Cap should have a PTFE liner.) Stable at 10  C to 30  C for 1 year. 2. Level 4 working stock (80-ng/mL cortisol in PBS): Measure 400 μL of the 10 μg/mL cortisol (Lipomed) stock solution with a Class A volumetric pipette to a 50 mL Class A volumetric flask. Bring to volume with PBS (see Note 2). Mix carefully and let stand for 20 minutes before further dilution (see Note 4). Stable at 20  C for 1 year after preparation. 3. Quality control standards, viz., QC 1, QC 2, and QC 3, of target concentrations of 1.50 ng/mL, 8 ng/mL, and 40 ng/ mL, respectively, are prepared by spiking PBS with the level 4 working stock to 25 mL volumes. Stable at 10  C to 30  C for 1 year. 4. URTS (unextracted retention time standard): Cortisol and cortisol-d4, each at 5.0 ng/mL in 50:50 water:methanol (see Note 3). Stable at 20  C for 1 year after preparation (see Note 4). 5. Prednisolone stock solution (10 μg/mL): Measure 1 mL of the 100-μg/mL Cerilliant stock with a Class A volumetric pipette to a 10-mL Class A volumetric flask. Bring to volume with acetonitrile. Mix and transfer to a labeled screw-top tube (Cap should have a PTFE liner). Stable at 10  C to 30  C for 1 year (see Notes 7, 8, and 9). 6. Prednisolone intermediate stock solution (351 ng/mL in acetonitrile): Measure 351.4 μL of the 10 μg/mL prednisolone stock to a 10-mL Class A volumetric flask. Bring to volume with acetonitrile. Mix and transfer to a labeled screw-top tube (cap should have a PTFE liner). Stable at 10  C to 30  C for 1 year (see Notes 7, 8, and 9). 7. PLN interference quality control (3.5 ng/mL in PBS, see Note 2): Measure 250 μL of prednisolone intermediate stock to 20 mL PBS in a 25-mL Class A volumetric flask. Bring to volume with PBS. Mix and transfer to five labeled 16x100 screw-top tube (cap should have a PTFE liner). Stable at 10  C to 30  C for 1 year (see Notes 7, 8, and 9).

2.5

LC Reagents

1. 1 M aqueous ammonium formate solution: Weigh out 6.306 g of ammonium formate, and dissolve in Millipore water to a 100 mL Class A volumetric flask. Bring to volume with water. Mix and Transfer to labeled screw-top bottle. Stable at 2  C– 8  C for 2 years.

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2. 1 mM ammonium formate working solution: Measure 1 mL of the 1 M ammonium formate stock with a Class A volumetric pipette, and add to a 1 L bottle. Bring to volume with water. Mix and transfer to a labeled screw-top bottle. Stable at 2  C– 8  C for 2 years. 3. Mobile phase A: 1 mM of ammonium formate in 5% acetonitrile: Add 50 mL of acetonitrile to a 1 L bottle. Add 1 ml of 1 M ammonium formate. Bring to 1 L volume with water. Mix and label. Stable at room temperature for 1 month. 4. Mobile phase B: 1 mM of ammonium formate in 95% acetonitrile:5% water. Add 50 mL of water to a 1 L bottle. Add 1 ml of 1 M ammonium formate. Bring to 1 L volume with acetonitrile. Mix and label. Stable at room temperature for 6 months. 5. Wash reagent: 100% methanol poured into a labeled 1-L bottle. Stable at room temperature for 1 year. 2.6

Equipment

1. Triple quadrupole mass spectrometer API-5000 equipped with Turbo V™ ion source, with QJet™ ion guide technology and pulse-counting CEM detector. 2. Shimadzu Prominence HPLC system, consisting of autosampler (SIL-20A HT), solvent delivery modules (x2) (LC 20-AD), degasser (DGU-20A3), and system controller (CBM-20A lite, installed in Pump A) Valco ten-port two-way switching valve. Operating software: Analyst 1.5 Software (AB Sciex, Framingham, MA). 3. Vortex with adaptor for microcentrifuge tubes. 4. Ultracentrifuge for 1.5 mL tubes. 5. Centrifuge with buckets for 96-well plates.

2.7

Supplies

1. HPLC column: Bonshell Phenyl-Hexyl column; 50 mm by 4.6 mm (90 A˚, 2.7 μm) (Agela Biosciences—Phenomenex, Torrance, CA). 2. Guard cartridge: Onyx C8, 4 mm x 3 mm (Phenomenex, Torrance, CA). 3. 1.5 mL centrifuge tubes with cap. 4. 500 μL Eppendorf tips. 5. Sarstedt Salivette® cotton swab for saliva collection 100/pk. 6. 2.0 mL screw-top glass vials (see Note 3). 7. 1.5 mL snap glass vials with caps (see Note 3).

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Methods Procedure

3.1.1 Sample Collection: Correct Protocol with the Salivette® Device

(a) Remove the top cap of the tube to expose the swab. (b) Place swab directly into the mouth by tipping the tube, so the swab falls into the mouth. Do NOT touch the swab with your fingers. (c) Roll the swab in your mouth and chew lightly to stimulate saliva flow. Keep the swab in your mouth for approximately 2 minutes. (d) Spit the swab back into the tube. Do NOT touch the swab with your fingers. (e) Replace the cap. Make sure cap is pushed on tightly. (f) Label the tube with your name, second ID, and collection date and time. (g) Minimum sample volume (recovered from Salivette after centrifugation) is 250 μL. (h) Salivary cortisol specimens have been shown to be stable at room temperature for 14 days, refrigerated at 2 –8 for 14 days, and frozen at 10  C to 30  C for 3 months. (i) Specimens may be transported at room temperature/refrigerated/or frozen. (j) Storage: Sample will be stored at 10 to 30 after analysis is completed for 2 months and then discarded. (k) The sample must be properly labeled with two identifiers and collection information. (l) Sample will be rejected if the previously stated criteria are not met.

3.1.2 Stepwise Sample Preparation Procedure

(a) Use standard laboratory safety practices for handling of samples with only one calibrator, control, or patient sample opened and aliquoted at a time, to avoid cross contamination and/or mix-up. (b) Bring the calibrators and controls to room temperature. Mix prior to using. (c) Spin patient Salivette® collection device in the centrifuge for 3 minutes at 3000 rpm. (d) Afterward remove cap, discard insert, and replace cap onto tube.

3.1.3

Extraction: Part 1

(a) Label polypropylene microcentrifuge tubes for each of the following samples: Control Blank (CB), Zero, PrednisoloneQC (PLNQC), Calibrators (six levels, Cal 1 to Cal 6), and QC (four levels, QC 1 to QC 4).

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(b) Add 200 μL of sample and 300 μL of precipitation reagent. For CB and Zero, use 200 μL of PBS diluent (see Note 2) as a dummy sample. In addition, the CB uses 300 μL of acetonitrile as a dummy precipitation reagent. (c) QCs and patient specimens must be set up at the same time. (d) After reagent addition, vortex the sample mixtures for 10 seconds, and centrifuge afterward for 3 minutes at 12,700 rpm. 3.1.4 Dilution with Formate and Vial Preparation: Part 2

(a) Label glass autosampler vials for CB, Zero, Calibrators, QC, and patient specimens. (b) Using a repeater pipette: Add 800 μL of 1 mM ammonium formate working solution to each labeled glass vial. (c) Transfer 400 μL supernatant from each microcentrifuge tube to the labeled glass vial. (d) Cap and vortex for 10 seconds. (e) Place on the autosampler, and inject into the LC-MS instrument according to a batch injection sequence of choice.

3.1.5

LC Method

(a) The analyte mixture is injected and separated by RPLC using the Bonshell Phenyl-Hexyl column; 50 mm by 4.6 mm (90 A˚, 2.7 μm). (b) Briefly, a dual mobile phase comprises 1 mM ammonium formate in 5% acetonitrile:95% water and 1-mM ammonium formate in 95% acetonitrile:5% water flowing initially at 0.6 mL/min with 80:20 composition maintained for 4.3 minutes. (c) The composition is switched to 5:95 for 0.6 mins with a flow rate of 1 mL/min for rapid column reconditioning and thereafter returned to the initial state at ~5 for 0.8 mins of re-equilibration time. (d) The MS acquisition of the LC eluate is delayed for the first 2.8 minutes after sample injection, whereby the column eluate is diverted to waste (valve A). (e) Acquisition starts at 2.8 minutes until 4.4 minutes of run time, after which the LC flow again returns to waste (valve A) for 1.4 minutes.

3.1.6

MS Method

(a) The MS acquisition of the LC eluate is based on the multiplereaction monitoring (MRM) analysis of the formate-analyte adducts (M + HCOO ), i.e., M represents cortisol, prednisolone, and IS, in negative ion mode electrospray MS. (b) The MS/MS source and operating parameters such as gas, temperature, voltage settings, and collision-induced

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Table 1 Operating parameters: ESI source and MS analyzer Curtain gas (CUR)

30 au

Ion source gas (GS1)

60 au

Ion source gas (GS2)

50 au

Ion spray voltage (IS)

3500 V

Temperature (TEM)

650 C

Collision gas (CAD)

5 au

Declustering potential (DP)

90 V

Entrance potential (EP)

10 V

Scan in period/cycles

200

MRM pause

5 ms

Dwell

65 ms

Table 2 MRM parameters Analyte

Q1 m/z

Q3 m/z

CE (V)

CXP (V)

Cortisol 1

407.2

297.1

44

25

Cortisol 2

407.2

282.1

51

25

PLN 1

405.2

295.1

44

25

PLN-2

405.2

280.1

51

23

Cortisol-d4

411.2

286.1

52

23

dissociation (CID) are summarized in Table 1, whereas the specific MRM transitions are shown in Table 2 (see Note 6). (c) Briefly, the M + HCOO precursors are selected in the first set of quadrupoles (Q1). They are detected as m/z values of 407.2, 405.2, and 411.2 for cortisol, prednisolone, and cortisol-d4, respectively. (d) Ions are fragmented in the collision chamber, and specific fragments, corresponding to the target and confirming fragment ions, are selected for detection in the third set of quadrupoles (Q3). These correspond to m/z values of 297/282 and 295/280 for cortisol and prednisolone, respectively. For cortisol-d4, the IS, the confirmatory fragment, is not a requirement, and as a result only the 411/286 SRM is useful. Quantitation is based on the integrated peak area of the

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Fig. 1 Extracted ion chromatograms (XIC) of the 0.5-ng/mL calibrator illustrates the sensitivity of the method and the product ion ratio, i.e., transition 2/transition 1

analyte fragment ions normalized to the IS target fragment intensity, e.g., 297/286 SRM. (e) This does not apply to prednisolone, since the 405.2/295.1 and 405.2/280.1 MRMs are only utilized as interference thresholds to the analyte (see above). (f) An example of extracted ion chromatogram (XIC), collected from the analysis of the LOQ sample of 0.5 ng/mL, is shown in Fig. 1. Here the XIC shows both transitions 1 and 2 overlaid. 3.2

Data Analysis

1. The LC-MS/MS data is analyzed using the quantitation functions in Analyst Software (ABSciex). 2. The measured cortisol peak areas corresponding to the quantifying ions and internal standards are used to construct calibration curves, by plotting the ratio of cortisol/IS areas against cortisol concentration. From this curve, the concentration of unknown and controls can be determined. A least squares regression curve with 1/x weighting is employed. 3. Ion ratio agreement: The quantifier ion in the sample is considered acceptable if the ratios of qualifier ions to quantifying ion are within +/ 20% of the average ion ratios for the calibrators. 4. Procedure for prednisolone interference quality control: see Notes 7, 8, and 9. (a) If there are peaks present at the expected prednisolone RT (refer to PLNQC if needed), integrate if needed.

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Fig. 2 An illustration of extracted ion chromatograms obtained from a PLN-negative patient saliva specimen spiked with methanol (Panels a, b) and from PLNQC sample (3.5 ng/mL; Panels c, d)

(b) Once integrated follow the prednisolone EXCEL sheet to determine if prednisolone is present and at an amount to interfere with the patients’ cortisol results, i.e., > 3.5 ng/ mL, PLNQC cutoff. (c) If interference is determined enter calculated cortisol results with the comment: Interpret with caution: potential interference present. (d) An illustration of extracted ion chromatograms obtained from a PLN-negative patient saliva specimen spiked with methanol (Panels A, B) and from PLNQC sample (3.5 ng/mL; Panels C, D) is shown in Fig. 2. Panels A and C show the SRM response of cortisol (407/297), and Panels B and D show the SRM response of prednisolone (405/295). The signal in Panel D is equivalent to the signal observed in PBS spiked with prednisolone (~3.5 ng/mL), which is an equivalent of 10% of the LOQ, and therefore below the 0.5 ng/mL cutoff. However, patient samples which are adulterated with significantly higher prednisolone levels than PLNQC will significantly interfere with the cortisol MRM track.

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Notes 1. The following collection instructions for the salivary cortisol sample are provided to the patient, along with the Salivette® device(s): (a) Do not brush teeth before collecting sample. (b) Do not eat or drink for 15 min prior to sample collection. (c) Collect a sample between 11 p.m. and midnight or a different collection time instructed by the doctor such as 7–9 a.m. or 3–5 p.m. 2. We used PBS as dilution buffer. Mobile phase A can also be used as the dilution buffer to replace PBS. The comparison from our results did not show significant difference between those two types of diluent buffer. 3. Though polypropylene centrifugation tubes are usable during sample extraction, they are not recommended in long-term storage of reference standards, calibrators, and QC samples. Storage in glass vials is preferred and has markedly higher recovery. 4. No carryover was observed up to a spiked cortisolCortisol level of 10 μg/mL. 5. Micropipetting errors associated with the use of 10 μL volumes are a challenge best overcome by bulk preparation of specialized reagents and solvents. 6. Similar assay parameters will also work in positive ion mode ESI mass spectrometryMass spectrometry (MS). However, negative mode ESI enabled very clean mass spectra and minimized manual integration of the peaks. 7. Detected cortisol can be falsely elevated in the presence of prednisolone. Although the cortisol precursor m/z differs from the analogous prednisolone precursor m/z by 2 units, the prednisolone isotopologue at M + 2 can be enhanced enough in samples containing high levels to interfere with the cortisol MRMs. Since this interference is reflected in the calculated peak ratio values, significant prednisolone interference will cause a rejection of the analytical data (because the ratios are out of range) rather than the reporting of an incorrect value. The PLNQC approach mitigates against high rejection rates. Long-term continuous monitoring of this strategy will inform clinical benefit and utility. 8. Simultaneous quantitative analysis of both cortisol and prednisolone is a potential alternative to the PLNQC approach, preferably in higher-resolution MRM or MS1 settings, or even in higher-resolution triple-quadrupole MS instruments,

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whereby an FWHM setting is significantly smaller than 0.7 m/ z units, for example. However, both suggestions point toward more expensive MS instrumentation. 9. When it is no longer necessary, this assay can be easily modified to remove the monitoring of prednisolone and to only perform the quantitative analysis of cortisol. References 1. van Eck M, Berkhof H, Nicolson N, Sulon J (1996) The effects of perceived stress, traits, mood states, and stressful daily events on salivary cortisol. Psychosom Med 58(5):447–458 2. Jacobson L, Sapolsky R (1991) The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis*. Endocr Rev 12(2):118–134. https://doi.org/ 10.1210/edrv-12-2-118 3. Giubilei F, Patacchioli FR, Antonini G, Monti MS, Tisei P, Bastianello S, Monnazzi P, Angelucci L (2001) Altered circadian cortisol secretion in Alzheimer’s disease: clinical and neuroradiological aspects. J Neurosci Res 66(2):262–265. https://doi.org/10.1002/ jnr.1219 4. Cleare AJ, Bearn J, Allain T, McGregor A, Wessely S, Murray RM, O’Keane V (1995) Contrasting neuroendocrine responses in depression and chronic fatigue syndrome. J Affect Disord 34(4):283–289. https://doi. org/10.1016/0165-0327(95)00026-j 5. Taylor RL, Machacek D, Singh RJ (2002) Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 48(9):1511–1519 6. Me´száros K, Karvaly G, Márta Z, Magda B, ˝ke J, Szu¨cs N, To´th M, Rácz K, Pato´cs A To (2018) Diagnostic performance of a newly developed salivary cortisol and cortisone measurement using an LC–MS/MS method with simple and rapid sample preparation. J Endocrinol Investig 41(3):315–323. https://doi. org/10.1007/s40618-017-0743-6 7. Antonelli G, Ceccato F, Artusi C, Marinova M, Plebani M (2015) Salivary cortisol and cortisone by LC–MS/MS: validation, reference intervals and diagnostic accuracy in Cushing’s syndrome. Clin Chim Acta 451:247–251. https://doi.org/10.1016/j.cca.2015.10.004 8. Mezzullo M, Fanelli F, Fazzini A, Gambineri A, Vicennati V, Di Dalmazi G, Pelusi C, Mazza R, Pagotto U, Pasquali R (2016) Validation of an LC-MS/MS salivary assay for glucocorticoid status assessment:

evaluation of the diurnal fluctuation of cortisol and cortisone and of their association within and between serum and saliva. J Steroid Biochem Mol Biol 163:103–112. https://doi. org/10.1016/j.jsbmb.2016.04.012 9. El-Farhan N, Rees DA, Evans C (2017) Measuring cortisol in serum, urine and saliva – are our assays good enough? Ann Clin Biochem 54(3):308–322. https://doi.org/10.1177/ 0004563216687335 10. Woolston JL, Gianfredi S, Gertner JM, Paugus JA, Mason JW (1983) Salivary cortisol: a nontraumatic sampling technique for assaying cortisol dynamics. J Am Acad Child Psychiatry 22(5):474–476. https://doi.org/10.1016/ S0002-7138(09)61512-0 11. Vining RF, Mcginley RA, Maksvytis JJ, Ho KY (1983) Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem 20(6):329–335. https:// doi.org/10.1177/000456328302000601 ˚ M, Garde AH, Persson R (2008) 12. Hansen A Sources of biological and methodological variation in salivary cortisol and their impact on measurement among healthy adults: a review. Scand J Clin Lab Invest 68(6):448–458. h t t p s : // d o i . o r g / 1 0 . 1 0 8 0 / 00365510701819127 13. Laudat MH, Cerdas S, Fournier C, Guiban D, Guilhaume B, Luton JP (1988) Salivary cortisol measurement: a practical approach to assess pituitary-adrenal function. J Clin Endocrinol Metabol 66(2):343–348. https://doi.org/10. 1210/jcem-66-2-343 14. Martinelli CE, Sader SL, Oliveira EB, Daneluzzi JC, Moreira AC (1999) Salivary cortisol for screening of Cushing’s syndrome in children. Clin Endocrinol 51(1):67–71. https:// doi.org/10.1046/j.1365-2265.1999. 00749.x 15. Putignano P, Toja P, Dubini A, Giraldi FP, Corsello SM, Cavagnini F (2003) Midnight salivary cortisol versus urinary free and midnight serum cortisol as screening tests for Cushing’s syndrome. J Clin Endocrinol

Analysis of Salivary Cortisol by LC-MS/MS Metabol 88(9):4153–4157. https://doi.org/ 10.1210/jc.2003-030312 16. Raff H, Raff JL, Findling JW (1998) Latenight salivary cortisol as a screening test for Cushing’s syndrome1. J Clin Endocrinol Metabol 83(8):2681–2686. https://doi.org/ 10.1210/jcem.83.8.4936 17. Yaneva M, Mosnier-Pudar H, Dugue´ M-A, Grabar S, Fulla Y, Bertagna X (2004) Midnight salivary cortisol for the initial diagnosis of Cushing’s syndrome of various causes. J Clin Endocrinol Metabol 89(7):3345–3351. https://doi.org/10.1210/jc.2003-031790 18. Nieman LK, Biller BMK, Findling JW, NewellPrice J, Savage MO, Stewart PM, Montori VM (2008) The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice guideline. J Clin Endocrinol Metabol 93(5): 1526–1540. https://doi.org/10.1210/jc. 2008-0125 19. Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, De Palo EF (2009) Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clin Biochem 42(12): 1205–1217. https://doi.org/10.1016/j. clinbiochem.2009.04.011 20. Miller R, Plessow F, Rauh M, Gro¨schl M, Kirschbaum C (2013) Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry. Psychoneuroendocrinology 38(1):50–57. https://doi. org/10.1016/j.psyneuen.2012.04.019 21. Kataoka H, Matsuura E, Mitani K (2007) Determination of cortisol in human saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry. J Pharm Biomed Anal 44(1): 160–165. https://doi.org/10.1016/j.jpba. 2007.01.023

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22. De Palo EF, Antonelli G, Benetazzo A, Prearo M, Gatti R (2009) Human saliva cortisone and cortisol simultaneous analysis using reverse phase HPLC technique. Clin Chim Acta 405(1–2):60–65. https://doi.org/10. 1016/j.cca.2009.04.006 23. Perogamvros I, Owen LJ, Newell-Price J, Ray DW, Trainer PJ, Keevil BG (2009) Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography– tandem mass spectrometry: application in basal and stimulated conditions. J Chromatogr B 877(29):3771–3775. https://doi.org/10. 1016/j.jchromb.2009.09.014 24. Rauh M (2009) Steroid measurement with LC–MS/MS in pediatric endocrinology. Mol Cell Endocrinol 301(1–2):272–281. https:// doi.org/10.1016/j.mce.2008.10.007 25. Shackleton C (2010) Clinical steroid mass spectrometry: a 45-year history culminating in HPLC–MS/MS becoming an essential tool for patient diagnosis. J Steroid Biochem Mol Biol 121(3–5):481–490. https://doi.org/10. 1016/j.jsbmb.2010.02.017 26. Rege J, Nakamura Y, Satoh F, Morimoto R, Kennedy MR, Layman LC, Honma S, Sasano H, Rainey WE (2013) Liquid chromatography–Tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation. J Clin Endocrinol Metabol 98(3):1182–1188. https://doi.org/10.1210/jc.2012-2912 27. Jones RL, Owen LJ, Adaway JE, Keevil BG (2012) Simultaneous analysis of cortisol and cortisone in saliva using XLC–MS/MS for fully automated online solid phase extraction. J Chromatogr B 881-882:42–48. https://doi. org/10.1016/j.jchromb.2011.11.036

Chapter 11 Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS Julie A. Ray, Erik Kish-Trier, and Lisa M. Johnson Abstract Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing’s syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement. We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotopelabeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection. Key words Urinary free cortisol, Cortisone, Cushing’s syndrome, APCI, LC-MS/MS

1

Introduction Cortisol is a glucocorticoid produced in the adrenal glands. It is the primary stress hormone, which enables the brain to control mood, motivation, and fear. It plays an important role in lowering inflammation [1] and in regulating metabolism of carbohydrates (e.g., glucose), fats, and protein [2]. Cortisol also helps control the sleep/wake cycle and is an energy booster. While high levels of this glucocorticoid are expected in “flight-or-fight” situations, concentrations of cortisol are expected to normalize after the danger or stress has passed. Persistently high concentrations of cortisol may be indicative of Cushing’s syndrome, a disorder where either a pituitary or adrenal tumor is causing the adrenal glands to synthesize and secrete too much cortisol [3]. Symptoms of Cushing’s syndrome include central obesity, tiredness, moon faces, and purple striae due to thinning of the skin. High concentrations of

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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glucocorticoids may also be caused by using corticosteroid medications, such as prednisone or prednisolone [4]. This potential for interference necessitates accurate and specific measurement of cortisol. Screening for Cushing’s syndrome consists of an algorithm [5] beginning with testing for urinary free cortisol (UFC), midnight salivary cortisol, or serum cortisol after a dexamethasone suppression test. While most of the cortisol is protein bound (inactive), a small percentage of circulating cortisol is free, or biologically active, in healthy individuals. During certain disease states, the concentration of free cortisol increases due to excess secretion of cortisol from the adrenal glands. The unbound cortisol is subsequently filtered through the kidneys and can be measured in the urine. While serum cortisol measures the analyte at a given point in time, measurement of 24-h UFC excretion provides a combined index of cortisol production over a 24-h period. It is also an easy and noninvasive method for measuring free cortisol during symptoms of hypercortisolism, provided the patient follows the instructions in collecting all urine samples throughout the 24-h period. For these reasons, UFC is one of the main screening tests for Cushing’s syndrome. Follow-up testing may include imaging and additional suppression or stimulation tests in conjunction with serum adrenocorticotropic hormone and cortisol measurements to determine the cause of Cushing’s syndrome for the patient. Although cortisol can be measured by immunoassays [3, 6, 7], there are potential interferences from synthetic corticosteroids or metabolites [8]. Measuring the cortisol concentrations by LC-MS/ MS provides more accurate results [6, 9–14]. In the present work, we have improved our current urine free cortisol LC-MS/MS assay [10] to a more specific assay which is free from interference from the cholesterol-controlling drug fenofibrate [15] and monitors known interferences such as prednisone and prednisolone.

2

Materials All solvents should be LCMS grade when possible.

2.1

Samples

2.2 Reagents and Buffers

Refrigerated 24-h urine or random urine collected in transport tubes. 1. 1 mg/mL of cortisol (Cerilliant, Round Rock, TX). 2. 1 mg/mL of cortisone (Cerilliant, Round Rock, TX). 3. 100 μg/mL of cortisol-[13C3] (IsoSciences, Ambler, PA). 4. 100 μg/mL of cortisone-[13C3] (IsoSciences, Ambler, PA).

Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS

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5. 100 mg of estriol (Sigma-Aldrich, St. Louis, MO). 6. Ethyl acetate. 7. Hexanes. 8. 10 mM of formic acid in water. 9. 10 mM of formic acid in acetonitrile. 10. Synthetic urine: Water (4.0 L), potassium diphosphate (14.0 g), sodium phosphate (2.75 g), sodium chloride (35.0 g), creatinine (5.0 g), and urea (100 g) are mixed until the chemicals are completely dissolved. 11. Needle wash solution: 20% water, 80% methanol, and 0.1% trifluoroacetic acid. 12. Reconstitution solution: Mixture of water and methanol (3:1) spiked with 10,000 ng/mL estriol. 2.3 Standards, Calibrators, and Quality Controls

1. Stock standards of cortisol and cortisone (1 mg/mL) are stable for 3.5 years at 10 C, and cortisol-[13C3] and cortisone[13C3] (100 μg/mL) are stable for 6 months at 70 C per manufacture. 2. Working standard mix of cortisol and cortisone (1000 ng/mL) is prepared in water and methanol (1:1) and fortified with 10,000 ng/mL estriol according to Table 1a. 3. Calibrators 1–5 (1, 5, 50, 100, 200 ng/mL) are prepared by separately aliquoting the volume of intermediate standard specified in Table 1b to 1 L of synthetic urine. 4. Working internal standard mix of cortisol-[13C3] and cortisone-[13C3] (200 ng/mL) is prepared in water and methanol (1:1) according to Table 2. 5. Quality control samples are prepared from a stock mix of standards of cortisol, cortisone, and estriol in 1:1 water:methanol as specified in Table 3a. Levels 1–4 (1, 50, 100, 200 ng/ mL) are prepared by spiking appropriate volumes of the stock mix into synthetic urine along with 1500 μL of stock estriol (1 mg/mL) to all the levels as shown in Table 3b. These controls are stable for 3 months at 70  C. Synthetic urine is used as the negative control, and it is stable for 1 year at 2–8  C.

2.4

Equipment

1. Triple quadrupole mass spectrometer: API 5500 (AB SCIEX, Foster City, CA). 2. Ion source: Turbo V ion source, APCI (AB SCIEX, Foster City, CA). 3. Software: Analyst® 1.6.3 (AB SCIEX, Foster City, CA). 4. Autosampler: Shimadzu Columbia, MD).

LC

30

(Shimadzu

America,

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Table 1 Preparation of calibration standards (a) Preparation of working standard mix Analyte

Standard (ng/mL)

Cortisol

1,000,000

50

1000

100,000

500

1000

1,000,000

500

10,000

Cortisone Estriol Total volume (mL)

Final concentration Volume of standard added (μL) (ng/mL)

50

(b) Dilutions for preparation of calibration standards Volume of working Calibrator standard mix added (μL)

Volume of estriol solution added (μL)

Total volume (mL)

1

10

500

10

1

2

50

500

10

5

3

500

500

10

50

4

1000

500

10

100

5

2000

500

10

200

Concentration cortisol/ cortisone (ng/mL)

Table 2 Cortisol-[13C3] and cortisone-[13C3] working internal standard mix

Internal standard

Standard (ng/mL)

Final concentration Volume of standard added (μL) (ng/mL)

Cortisol-[13C3]

100,000

100

200

100,000

100

200

13

Cortisone-[ C3] Total volume (mL)

50

5. HPLC pumps: Shimadzu LC 20 (Shimadzu America, Columbia, MD). 6. HPLC column: Ultra Aromax 100 mm  3.0 mm  3 μm (Restek, Bellefonte, PA). 7. Security guard cartridge: C18 4  2.0 mm (Phenomenex, Torrance, CA). 8. Liquid handler: Hudson, NH).

ViaFlow

096

(Integra

Biosciences,

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Table 3 Preparation of quality controls (a) Preparation of stock mix Analyte

Standard (ng/mL)

Cortisol

1,000,000

100

10,000

100,000

1000

10,000

1,000,000

100

10,000

Cortisone Estriol Total volume (mL)

Final concentration Volume of standard added (μL) (ng/mL)

10

(b) Dilutions for preparation of quality controls Volume of stock mix QC added (μL)

Volume of estriol solution Total volume Concentration cortisol/ added (μL) (mL) cortisone (ng/mL)

1

15

1500

150

1

2

225

1500

150

15

3

900

1500

150

60

4

2250

1500

150

150

2.5

Supplies

1. Glassware: assorted sizes of beakers, graduated cylinders, and class A volumetric flasks, 2. Pipettes: assorted sizes of volumetric pipettes and syringes. 3. Stir plate with multiple sizes of magnetic stir bars. 4. Analytical balance with weigh paper and reagent spatulas. 5. Specimen rockers. 6. Multi-tube vortex. 7. ViaFlow 096 tips (Integra Biosciences, Hudson, NH). 8. High-speed centrifuge. 9. Collection plate: 96-well, 2-mL, square/round conical (Phenomenex, Torrance, CA). 10. Sealing mats: pre-slit, 96-square-well silicone (Phenomenex, Torrance, CA). 11. Repeater pipette with assorted tips. 12. 2 mL bottle-top dispenser. 13. 96-well plate evaporator. 14. Positive displacement pipette and tips (1–10 μL and 10–100 μL).

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Methods

3.1 Stepwise Procedure

1. 250 μL of calibrators, quality controls, and patient samples are aliquoted into a 96-well plate. 2. 25 μL of working internal standard mix is added to each sample. 3. The plate is covered and vortexed for 10 min at speed setting of 500 rpm on a microplate shaker. 4. The plate is transferred to the ViaFlow 096 and 1.1 mL of the extraction solvent (3:1 ethyl acetate:hexanes) is added to each sample, mixed for 50 cycles, and allowed to sit for 5 min. 5. 800 μL of the top organic layer is transferred by the ViaFlow 096 into a 96-well collection plate. 6. The collection plate is dried under N2 at 45  C for 15 min or until completely dry. 7. 100 μL of reconstitution solvent is added to each well. 8. The plate is sealed and vortexed gently for 2 min. 9. The plate is centrifuged at approximately 1300 RCF for 2 min and submitted for analysis. 10. The plate is stored at 4  C if not immediately analyzed.

3.2 Chromatographic Conditions

1. Chromatographic gradient used in the method is listed in Table 4. 2. Mobile phase A: water with 10 mM of formic acid. 3. Mobile phase B: acetonitrile with 10 mM formic acid. 4. Injection volume: 5 μL.

3.3 Mass Spectrometer Conditions

1. Mass transitions for cortisol, cortisone, and their internal standards are listed in Table 5. 2. Optimized mass spectrometric voltages and gas flow rates are as follows: (a) Ion source: APCI (b) Curtain gas: 20 (c) Collison gas (CAD): 9 (d) Nebulizer current: 2 (e) Temperature: 400 (f) Gas 1: 40 Mass analyzer Q1 and Q3 are tuned for unit resolution.

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Table 4 Chromatographic gradient Step

Time (min)

Flow (mL/min)

%A

%B

1

0.01

0.520

65

35

2

0.10

0.520

65

35

3

3.80

0.520

60

40

4

3.81

0.520

5

95

5

4.80

0.520

5

95

6

4.81

0.520

65

35

7

6.49

0.520

65

35

8

6.50

0.520

Controller stop

Table 5 Mass transitions and voltages for analytes, interferences, and internal standards

Q1 Q3 Dwell (m/z) (m/z) (msec)

Declustering potential (DP)

Exit potential (EP)

Collision energy (CE)

Collison exit potential (CXP)

Transition ID

363.1 121.1 60

80

9

37

14

Cortisol quant

363.1 267.1 60

80

9

25

14

Cortisol qual

366.2 124.1 60

90

9

31

8

Cortisol[13C3] quant

366.2 270.1 60

80

8

25

13

Cortisol-[13C3] qual

361.1 163.1 60

85

6

32

15

Cortisone quant

361.1 121.1 60

85

6

39

14

Cortisone qual

364.2 166.1 60

111

6

30

14

Cortisone[13C3] quant

364.2 124.1 60

111

6

35

9

361.1 147.1 60

80

9

37

14

Prednisone

359.1 147.1 60

85

6

32

15

Prednisolone

Cortisone[13C3] qual

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4.0e5

XIC of +MRM (10 pairs): 361.100/163.100 Da ID: CN quant 163 from Sample 1.wiff... 3.57

3.6e5

Max. 2.1e4 cps.

Cortisone-[13C3]

3.2e5

Intensity, cps

2.8e5

2.4e5 3.06

Cortisol-[13C3]

2.0e5

1.6e5

1.2e5

8.0e4

Cortisol

4.0e4

0.0 0.0

0.5

1.0

1.5

2.0

Cortisone 2.5

3.0 3.5 Time, min

4.0

4.5

5.0

5.5

6.0

6.5

Fig. 1 Chromatogram of QC1 with 1.02 ng/mL of cortisol and 1.09 ng/mL of cortisone 3.4

Data Analysis

1. Data analysis is performed on Analysis 1.6.3 (Applied Biosystems SCIEX, Foster City, CA). 2. A calibration curve is prepared with every batch of samples in synthetic urine. The regression is linear with 1/x weighting; ignore origin. 3. Concentrations are determined using the response of the quantitative transitions of cortisol and cortisone and normalized by internal standard response (see Note 1). 4. Specificity of the analysis is evaluated using ratios of concentrations determined from the quantifier and the qualifier mass transitions. If the ratio of concentrations determined from the quantifier (363.1/121.1) and the qualifier mass transitions (363.1/267.1) for cortisol and the quantifier (361.1/163.1) and the qualifier mass transitions (361.1/121.1) for cortisone are outside of the acceptability range (e.g., 30% of calibrators), interference may be present, and the sample should be repeated. 5. Each chromatogram should be evaluated for acceptable peak quality (Fig. 1; see Note 2).

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Notes 1. If a run contains a sample with concentration of free cortisol or cortisone greater than 8000 or 3000 ng/mL, respectively, the following sample should be evaluated for carryover. 2. Although corticosteroids prednisolone and prednisone are baseline separated from cortisol and cortisone, respectively, the method suffers interferences from high concentrations of these analytes. When cortisol concentrations are low in a patient sample and the prednisolone concentration is very high (>1000 ng/mL), there could be significant interference from the tail of the prednisolone peak resulting in apparent elevated results for cortisol. The MRM transitions monitored in the method, 361.1/147.1 for prednisolone and 359.1/ 147.1 for prednisone, are more sensitive for the two interferences than for cortisol and cortisone by mass, respectively. Peaks at the retention times of prednisolone and prednisone in MRM channels for cortisol (363.1/121.1) and cortisone (361.1/163.1) in addition to large peaks for 361.1/147.1 and 359.1/147.1 indicate the presence of these isomeric interferences.

Acknowledgments We thank the ARUP Institute for Clinical and Experimental Pathology® for supporting this project. References 1. Yeager MP, Pioli PA, Guyre PM (2011) Cortisol exerts bi-phasic regulation of inflammation in humans. Dose Response 9(3):332–347 2. Christiansen JJ, Djurhuus CB, Gravholt CH et al (2007) Effects of cortisol on carbohydrate, lipid, and protein metabolism: studies of acute cortisol withdrawal in adrenocortical failure. J Clin Endocrinol Metab 92(9): 3553–3559 3. Perrin P, Plotton I, Berthiller J et al (2020) Urinary free cortisol: an automated immunoassay without extraction for diagnosis of Cushing’s syndrome and follow-up of patients treated by anticortisolic drugs. Clin Endocrinol 93(1):76–78 4. Lin CL, Wu TJ, Machacek DA et al (1997) Urinary free cortisol and cortisone determined by high performance liquid chromatography in the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 82(1):151–155

5. Gilbert R, Lim EM (2008) The diagnosis of Cushing’s syndrome: an endocrine society clinical practice guideline. Clin Biochem Rev 29 (3):103–106 6. Aranda G, Careaga M, Hanzu FA et al (2016) Accuracy of immunoassay and mass spectrometry urinary free cortisol in the diagnosis of Cushing’s syndrome. Pituitary 19:496–502 7. Horie H, Kidowaki T, Koyama Y et al (2007) Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations. Clin Chim Acta 378(2):66–70 8. Berthod C, Rey F (1988) Enormous crossreactivity of hydrocortisone hemisuccinate in the “RIANEN” RIA kit for cortisol determination. Clin Chem 34:1358 9. Wear JE, Owen LJ, Duxbury K et al (2007) A simplified method for the measurement of urinary free cortisol using LC-MS/MS. J

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Chromatogr B Analyt Technol Biomed Life Sci 858:27–31 10. Kushnir MM, Rockwood AL, Nelson GJ et al (2003) Liquid chromatography-tandem mass spectrometry analysis of urinary free cortisol. Clin Chem 49:965–967 11. Ceccato F, Antonelli G, Barbot M et al (2014) The diagnostic performance of urinary free cortisol is better than the cortisol:cortisone ratio in detecting de novo Cushing’s syndrome: the use of a LC-MS/MS method in routine clinical practice. Eur J Endocrinol 171(1):1–7 12. Cuzzola A, Mazzini F, Petri A (2014) A comprehensive study for the validation of a LC-MS/MS method for the determination of free and total forms of urinary cortisol and its metabolites. J Pharm Biomed Anal 94: 203–209

13. Ceccato F, Barbot M, Zilio M et al (2015) Screening tests for Cushing’s syndrome: urinary free cortisol role measured by LC-MS/ MS. J Clin Endocrinol Metab 100(10): 3856–3861 14. Taylor RL, Machacek D, Singh RJ (2002) Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 48(9):1511–1519 15. Meikle AW, Findling J, Kushnir MM et al (2003) Pseudo-Cushing syndrome caused by fenofibrate interference with urinary cortisol assayed by high-performance liquid chromatography. J Clin Endocrinol Metab 88(8): 3521–3524

Chapter 12 Laboratory Diagnosis of Cerebral Creatine Deficiency Syndromes by Determining Creatine and Guanidinoacetate in Plasma and Urine Ning Liu and Qin Sun Abstract Cerebral creatine deficiency syndromes are caused by the dysfunctional creatine biosynthesis or transport and comprise three hereditary neurodevelopmental defects including arginine-glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and creatine transporter deficiencies. All conditions are characterized by seizures, intellectual disability, and behavioral abnormalities. Laboratory diagnosis of these disorders relies on the determination of creatine and guanidinoacetate concentrations in both plasma and urine. Here we describe a rapid quantitative UPLC/MS/MS method for the simultaneous determination of these analytes using a normal-phase HILIC column after analyte derivatization. The approach is suitable for neonatal screening follow-ups and monitoring of the treatment for creatine deficiency syndromes. Key words Creatine, Guanidinoacetate, Cerebral creatine deficiency, Arginine-glycine amidinotransferase, AGAT, Guanidinoacetate methyltransferase, GAMT, Creatine transporter, Laboratory diagnosis, Mass spectrometry

1

Introduction Creatine (CRT) acts as a temporal and spatial buffer for ATP and is critical for intracellular energy transmission and storage mainly in tissues with high energy demand like the brain, skeletal muscle, and heart [1–4]. For humans, the dietary consumption of meat and fish supplies approximately half of the creatine daily requirement with the remainder originating from endogenous synthesis. The creatine biosynthetic pathway contains two steps of reactions. The first and rate-limiting step, catalyzed by arginine:glycine amidinotransferase (AGAT) mainly in the kidney, occurs with the synthesis of ornithine and guanidinoacetate (GAA) from glycine and arginine. The second step, catalyzed by guanidinoacetate methyltransferase (GAMT) in the liver, occurs via methylation of GAA to yield creatine, concomitant with conversion of the methyl donor

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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S-adenosylmethionine (SAM) into S-adenosylhomocysteine. Subsequently, the target tissues take up creatine from the blood stream into the cells via a sodium-/chlorine-dependent membrane creatine transporter (CRTR) [5, 6]. Cerebral creatine deficiency syndromes (CCDS) have been described involving both creatine synthesis disorders, AGAT and GAMT deficiencies, and CRTR deficiency [7–12]. All are rare with only fewer than 20 cases of AGAT deficiency, fewer than 120 cases of GAMT deficiency, and fewer than 200 cases of CRTR deficiency described to date, but it is likely that more will be identified with increased awareness [13, 14]. AGAT and GAMT deficiencies are inherited in an autosomal recessive manner, while CRTR deficiency is an X-linked disorder estimated to be the most prevalent CCDS condition. All three CCDS share common clinical features including development delay, seizures, intellectual disability, speech delay, and autistic behaviors. However, unique biochemical phenotypes are characterized for each CCDS, facilitating differential diagnosis [15]. High GAA concentrations in plasma are considered pathognomonic for GAMT deficiency, and creatine is low to within normal limits in infancy but declines by adolescence if the proband is on strict vegetarian diet. The toxicity of overproduction of GAA and the depletion of creatine are thought to be the pathogenesis of GAMT deficiency. In AGAT deficiency, biochemical testing reveals extremely decreased concentration of creatine and creatinine, as well as low GAA in body fluids. Plasma is the sample of choice for diagnosing AGAT and GAMT deficiency as unaffected individuals may present low concentrations of GAA and creatine in urine, and GAMT deficiency patient may have normal GAA-to-creatinine ratio in the newborn period [16, 17]. Magnetic resonance spectroscopy (MRS) in the brain reveals creatine depletion in both disorders and guanidinoacetate phosphate accumulation in GAMT deficiency [18]. Prenatal diagnosis is possible by measuring guanidinoacetate in amniotic fluids [19]. The creatine transporter, encoded by an X chromosome gene (SLC6A8), is important in providing adequate energy to the brain, skeletal, and cardiac muscle. The transporter is also critical for renal reabsorption of creatine and responsible for cerebral distribution and uptake of GAA [20, 21]. Mutations of this gene are also found in patients with X-linked mental retardation [8, 9, 22–24]. Biochemical diagnosis is revealed by an elevated urinary creatine-tocreatinine ratio, and urine is the appropriate sample type for investigating this condition as plasma creatine levels are typically normal in this disorder. Interestingly, changes in guanidinoacetate and/or creatine excretion are also reported in disorders in which onecarbon methyl group metabolism is disturbed, for example, in S-adenosylhomocysteine hydrolase deficiency and cobalamin deficiency [25, 26].

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Early detection and diagnosis of CCDS are imperative because the treatment with oral creatine monohydrate in the neonatal or early infantile period in patients with creatine biosynthesis disorders is reported to be effective [27–29]. The investigation of suspected CCDS may be accomplished by measuring GAA and creatine in the blood and/or urine, and several analytical methods have been reported using techniques such as HPLC, GC/MS, and HPLC/ tandem MS with or without derivatization [30–35]. While high guanidinoacetate and creatine concentrations in urine were quantified without difficulty using these methods, the methods were not sufficiently sensitive to accurately measure the low plasma guanidinoacetate essential for diagnosing AGAT deficiency. We describe an improved tandem MS protocol utilizing derivatized analytes and a hydrophilic interaction column (HILIC) which is a variation of normal-phase chromatography designed for separation of polar analytes. This approach is reliable and most importantly sufficiently sensitive to permit the measurement of low concentrations of guanidinoacetate, allowing simultaneous analysis of both urine and plasma samples.

2

Materials

2.1 Specimen Requirements

1. Plasma samples (sodium heparin): Immediately centrifuge whole blood and transfer the plasma (0.5–1.0 mL) to a labeled tube. Store frozen at 16  C or colder. Frozen samples are stable up to 6 months. Samples should be transported between the collection site and laboratory on dry ice (see Note 1). 2. Urine samples: Freeze 2–5 mL of urine. Store frozen at 16  C or colder. Frozen samples are stable up to 6 months. Samples should be transported between the collection site and laboratory on dry ice (see Note 1).

2.2

Reagents

1. 1% Ammonium formic acid: Add 10 mL of formic acid to 800 mL of HPLC grade H2O, adjust pH to 4.0 with concentrated ammonium hydroxide, and bring total volume to 1 liter. Store at room temperature. Stable for up to 2 years. 2. HPLC running buffer: 95% acetonitrile containing 0.05% ammonium formate. Add 50 mL of the 1% ammonium formate solution to 950 mL of acetonitrile. Store at room temperature. Stable for up to 2 years. 3. Derivatizing reagent, 3 N HCl in n-butanol (Regis Technologies, Inc., Morton Grove, IL).

2.3 Standards and Calibrators

1. 6 mM Creatine primary stock standard: Add 44.75 mg of creatine (Sigma, C3630) into 50 mL of acetonitrile:H2O (50:50). Store frozen ( 16  C or colder) for up to 2 years.

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Table 1 Standard solutions for GAA and CRT

GAMT A μL

GAMT B μL

Internal standard μL

CRT final concentration (μM)

GAA final concentration (μM)

G1

100

–

40

1000

83.3

G2

45

–

40

450

37.5

G3

10

–

40

100

8.33

G4

–

30

40

30

2.5

G5

–

15

40

15

1.25

G6

–

10

40

5

0.4

2. 600 μM Creatine secondary stock solution: Prepare by diluting 1 mL of the primary stock solution with 9 mL acetonitrile:H2O (50:50) for effective concentration of 600 μM. Store frozen ( 16  C or colder) for up to 1 year. 3. Creatine quality control solution (7.6 mM): Add 50 mg of creatine into 50 mL of acetonitrile:H2O (50:50). 4. 5 mM Guanidinoacetate primary stock standard: Add 29.3 mg of guanidinoacetate (Sigma) into 50 mL of acetonitrile:H2O (50:50). Store the stock solution frozen ( 16  C or colder) for up to 2 years. 5. 50 μM Guanidinoacetate secondary stock standard: Dilute the primary stock solution by mixing 100 μL with 9.9 mL of acetonitrile:H2O (50:50). Store in freezer ( 16  C or colder) for up to 1 year. 6. 850 μM Guanidinoacetate quality control solution: Add 25 mg of GAA into 250 mL of acetonitrile:H2O (50:50). 7. Prepare working creatine and guanidinoacetate standards as follows (see Note 1): (a) GAMT A standard solution, 300 μM creatine and 25 μM GAA: Mix equal volumes of 600 μM creatine and 50 μM guanidinoacetate secondary stock standards. (b) GAMT B standard solution, 30 μM creatine and 2.5 μM GAA: Dilute GAMT A standard solution 1:10 with 95% acetonitrile. 8. Prepare six calibrators according to Table 1. Mix all calibrators thoroughly and dry using SpeedVac. Drying time is approximately 30 min. 2.4 Internal Standard and Quality Controls

1. Stock internal standard (D3-creatine): Prepare by adding 10 mg of D3-creatine (C/D/N isotopes) to 20 mL of

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133

acetonitrile:H2O (50:50) to give a solution of approximately 3.3 mM. The stock solution is stable when stored frozen at 16  C or colder for up to 5 years. 2. Stock internal standard (D2-guanidinoacetate): Prepare by adding 20 mg of D2-guanidinoacetate (C/D/N isotopes) to 45 mL of acetonitrile:H2O (50:50) to give a solution of approximately 3.75 mM. The stock solution is stable when stored frozen at 16  C or colder for up to 5 years. 3. IS working solution: Mix the above internal standard stock solutions to yield a 50 μM working solution by adding 227.3 μL of 3.3 mM D3-creatine and 199.5 μL of 3.75 mM D2-guanidinoacetate into 14.573 mL of acetonitrile:H20 (50:50). The stock solution is stable when stored frozen at 16  C or colder for up to 1 year. 4. Urine controls: Pooled urine from volunteers or drug-free urine may be used. Dilute pool 1:10 using water to prepare a “normal” urine control. Use undiluted urine as a “high” control. Aliquot control materials and store frozen at 16  C or colder for up to 18 months. 5. Plasma controls: Fresh frozen plasma or similar material is used to prepare controls. A portion of the pool should be aliquoted to prepare a “normal” control. Prepare a “high” control by adding 100 μL of the 7.6-mM creatine and 100 μL of the 850μM guanidinoacetate quality control standard solutions to 4 mL of fresh frozen plasma. Aliquot control materials and store frozen at 16  C or colder for up to 18 months. 2.5

Equipment

1. Acquity UPLC with TQD tandem mass spectrometer (Waters, MA). 2. Atlantis HILIC (Hydrophilic Interaction Chromatography) HPLC column, 2.1  100 mm, 5 μm (Waters, MA). 3. MassLynx software (Waters, MA). 4. SPD1010 SpeedVac (Thermo Fisher Scientific, MA).

3

Methods

3.1 Preparation of Plasma Samples

1. Thaw the sample and mix thoroughly. Sample may be maintained at room temperature while processing. 2. Transfer 30 μL of the plasma sample and add 40 μL of the internal standard working solution to an appropriately labeled 1.5 mL microfuge tube. 3. Add 300 μL of acetonitrile into the above mixture and vortex to precipitate the protein. 4. Centrifuge for 10 min at 10,500 g in a desktop microfuge.

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5. Transfer the supernatant to a new, labeled tube and dry liquid completely under nitrogen in heating block at temperature of 45  C for 45 min. 3.2 Preparation of Urine Samples

1. Determine the creatinine concentration of urine samples. Creatinine values are used to determine the appropriate urine dilution in subsequent steps. The assay is not valid for samples with creatinine less than 0.1 mg/mL. See Note 2. 2. Dilute samples 1:10 with deionized water if creatinine is 0.1– 0.4 mg/mL and 1:75 if creatinine is above 0.4 mg/mL. See Note 3. 3. Dilute normal and high urine controls 1:10 times with deionized water. 4. Transfer 30 μL of the diluted urine sample and control into a labeled 1.5 mL microfuge tube. Add 40 μL of the internal standard working solution and vortex to mix. 5. Dry liquid completely using a SpeedVac with heating temperature of 45  C for 45 min. Trace amount of liquid does not affect final results.

3.3 Sample Preparation for UPLC/ MS/MS Analysis

1. Add 70 μL of the 3 N HCl in n-butanol to all dried tubes, including standard solutions, controls, and the samples, and mix thoroughly. 2. Place the tubes in the 65  C heating block for 20 min. Vortex at least once during the time period. 3. Remove the tubes and dry the contents under nitrogen with heating temperature of 45  C. Drying time is approximately 30 min. Trace amount of liquid does not affect final results. 4. Add 1 mL of 95% acetonitrile to each tube and mix thoroughly. 5. Centrifuge the tubes at 10,500 g for 6 min to remove any particulate matter. 6. Transfer 150 μL of supernatant to 150 μL sample vials for chromatographic analysis.

3.4 Setup for HPLC and Tandem Mass Spectrometer (See Note 4)

1. The chromatographic separation of hydrophilic creatine and guanidinoacetate is performed at room temperature using an Atlantis HILIC (Hydrophilic Interaction Chromatography) HPLC column, 2.1  100 mm, 5 μm. The mobile phase is maintained at a flow rate of 0.2 mL/min. 2. The settings for the mass spectrometer are as follows: positive ion mode of electrospray (ESI+), capillary voltage 3.5 kV, source cone voltage 25 V, extract voltage 4 V, source temperature 130  C, desolvation temperature 350  C, and analyzer collision 15. Nitrogen gas flows at 90 and 600 l/hr for nebulizer and desolvation, respectively.

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Fig. 1 Chromatogram of LC-MS/MS analyses of a sample. The individual traces of MRM transitions are as follows: (a) D3-creatine (m/z 191.05>93); (b) creatine (m/z 188.05>90), (c) C2-GAAand (m/z 176.1>102.9), and (d) GAA (m/z 174.1>101)

3. Multiple-reaction monitoring (MRM) transitions are utilized: guanidinoacetate 174.1>101, D2-guanidinoacetate 176.1>102.9, creatine 188.05>90, and D3-creatine 191.05>93. A typical chromatogram is shown in Fig. 1. 3.5

Data Analysis

1. Concentrations of unknowns and quality control samples are calculated from standard curves using MassLynx software. The concentration values in Table 2 take into account the sample volume being used in the assay. Urine dilution factors need to be entered to allow MassLynx to calculate the value. No further calculations are necessary as long as the sample volume does not change. 2. The coefficient of correlation (r2) for the standard curve should be above 0.97. 3. For results to be acceptable, the control values should be within 2 SD of the established control means. Examples of quality control ranges are shown in Tables 3 and 4. 4. If the values of clinical samples are above the upper limit of analytical reportable ranges (AMR), the sample must be diluted

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Table 2 Dilution factor calculation for urine samples Creatinine mg/mL

Dilution in H2O

Urine sample volume analyzed (μL)

Final dilution factor

0.1–0.4

10

30

10X

0.4 and above

75

30

75X

Table 3 Example of urine quality control ranges Compound

High tolerance limits (μM)

Normal tolerance limits (μM)

Creatine

660–1260

50–165

Guanidinoacetate

160–330

10–40

Table 4 Example of plasma quality control ranges Compound

High tolerance limits (μM)

Normal tolerance limits (μM)

Creatine

75–240

10–55

Guanidinoacetate

8–45

0.5–2.5

and re-analyzed. AMR should be verified every 6 months and may cover wider ranges in comparison with calibration curves used in daily operation. 3.6 Result Interpretation

1. Reference ranges for both plasma and urine are shown in Table 5. These ranges should only serve as a guide and should be established by each laboratory. The concentration of guanidinoacetate was reported to decline with age in urine but increases with age in plasma [30]. Others have similarly reported age-related changes in these reference values [36]. However, this trend is not reflected in our reference range (Table 5). This could be due to either differences in age groups or sample population (our “normal” ranges are established using hospital patient samples received in the laboratory). 2. Abnormalities in creatine deficiencies: The following general guidelines are used to interpret patient results. 3. Diet and supplements may have a major impact on creatine concentrations. Adults may obtain up to 50% of creatine from diet, mostly from meat and fish. Thus, patients on Western diet rarely have low creatine concentrations. In addition, creatine is

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137

Table 5 Reference ranges for CRT and GAA Plasma (μM)

Urine (mmoles/mole creatinine)

0–3 years

Above 3 years

1–7 years

Above 7 years

CRT

28–102

20–110

20–900

12–500

GAA

0.3–1.6

0.3–2.1

22–123

24–112

Table 6 Interpretation of results Plasma (μM)

Urine (mmoles/mole creatinine)

GAA

CRT

GAA

CRT

AGAT deficiency

Low

Low

Low

Normal/low

GAMT deficiency

High

Low

Normal/high

Normal/low

CRT transporter defect

Normal

Normal/low

Normal

High

the most common nutritional supplement used by athletes. Creatine supplementation is known to increase plasma or urine creatine concentrations [37]. A patient’s diet and any history of supplement usage are important information for interpreting guanidinoacetate and creatine results. Since patients who have AGAT or GAMT deficiency are often found to have creatine concentrations within or only slightly above the reference range, plasma guanidinoacetate concentrations (Table 6) are significant and should be sufficient for diagnoses. It is interesting that plasma guanidinoacetate is suggested to increase in combined methylmalonic aciduria and homocystinuria due to cobalamin deficiencies [25]. Creatinuria is also found following trauma, likely due to muscle damage [38]. Such patients should be retested at a later date.

4

Notes 1. GAA and creatine are unstable. Plasma and urine samples should be stored frozen at 16  C or colder. Use dry ice when shipping from collection site to laboratory. Samples should be analyzed within 2 weeks of collection. Plasma samples that are hemolyzed are unacceptable due to contamination from erythrocyte creatine. Stock solutions are stable up to 1 year when kept frozen. Working solutions are stable for 6 weeks when stored at 4  C.

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2. The urinary excretion of creatine and guanidinoacetate varies considerably even in normal populations. To minimize the numerous issues associated with a random urine collection, it is critical that these results are normalized to the urinary creatinine concentration before interpretation. Extremely low creatinine levels are problematic and tend to falsely elevate creatinine-normalized creatine and guanidinoacetate levels. It is recommended to cancel urine samples with very low creatinine (0.4 mg/mL, respectively. These dilution factors are entered in running sequence list in MassLynx software for later calculation. See Table 2 for dilution factor calculation for urine samples. 4. The sensitivity of this tandem mass spectrometry method is improved compared to previous methods [35]. Shifting retention times often indicate deterioration of the column. Changing the guard column and/or washing HILIC column with running buffer are usually sufficient to solve the problem. For persistent column-related problems, it may be useful to perform the following: wash the column for 30 min with 5% acetonitrile (95% water), continue for an additional 30 min with 95% acetonitrile, and conclude with 30 min of 5% acetonitrile (95% water) again. The time for each step could vary from 30 min to a few hours depending on different situations. It is also recommended that the column be washed with 5–10 column volumes of running buffer before each routine run to assure equilibration and improve consistency of retention times. 5. Calibration curves may be prepared in stripped plasma or artificial urine to monitor matrix effects. References 1. Joncquel-Chevalier Curt M, Voicu PM, Fontaine M et al (2015) Creatine biosynthesis and transport in health and disease. Biochimie 119:146–165 2. Monge C, Beraud N, Kuznetsov AV et al (2008) Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and

mitochondrial creatine kinase. Mol Cell Biochem 318:147–165 3. Saks V, Kaambre T, Guzun R et al (2007) The creatine kinase phosphotransfer network: thermodynamic and kinetic considerations, the impact of the mitochondrial outer membrane and modelling approaches. Subcell Biochem 46:27–65

Determining Creatine and Guanidinoacetate in Plasma and Urine 4. Saks V, Kuznetsov A, Andrienko T et al (2003) Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells. Biophys J 84:3436–3456 5. Braissant O, Bachmann C, Henry H (2007) Expression and function of AGAT, GAMT and CT1 in the mammalian brain. Subcell Biochem 46:67–81 6. Fons C, Campistol J (2016) Creatine defects and central nervous system. Semin Pediatr Neurol 23:285–289 7. Ganesan V, Johnson A, Connelly A et al (1997) Guanidinoacetate methyltransferase deficiency: new clinical features. Pediatr Neurol 17:155–157 8. Item CB, Stockler-Ipsiroglu S, Stromberger C et al (2001) Arginine:glycine amidinotransferase deficiency: the third inborn error of creatine metabolism in humans. Am J Hum Genet 69:1127–1133 9. Salomons GS, Van Dooren SJ, Verhoeven NM et al (2001) X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome. Am J Hum Genet 68:1497–1500 10. Stockler S, Hanefeld F, Frahm J (1996) Creatine replacement therapy in guanidinoacetate methyltransferase deficiency, a novel inborn error of metabolism. Lancet 348:789–790 11. Stockler S, Marescau B, De Deyn PP et al (1997) Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis. Metabolism 46:1189–1193 12. Van Der Knaap MS, Verhoeven NM, Maaswinkel-Mooij P et al (2000) Mental retardation and behavioral problems as presenting signs of a creatine synthesis defect. Ann Neurol 47:540–543 13. Bahl S, Cordeiro D, Macneil L et al (2020) Urine creatine metabolite panel as a screening test in neurodevelopmental disorders. Orphanet J Rare Dis 15:339 14. Clark JF, Cecil KM (2015) Diagnostic methods and recommendations for the cerebral creatine deficiency syndromes. Pediatr Res 77:398–405 15. Mercimek-Andrews S, Salomons GS (1993) Creatine deficiency syndromes. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A (eds) GeneReviews((R)), Seattle 16. Bodamer OA, Iqbal F, Mu¨hl A et al (2009) Low creatinine: the diagnostic clue for a treatable neurologic disorder. Neurology 72:854–855 17. Pasquali M, Schwarz E, Jensen M et al (2014) Feasibility of newborn screening for

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guanidinoacetate methyltransferase (GAMT) deficiency. J Inherit Metab Dis 37:231–236 18. Schulze A, Hess T, Wevers R et al (1997) Creatine deficiency syndrome caused by guanidinoacetate methyltransferase deficiency: diagnostic tools for a new inborn error of metabolism. J Pediatr 131:626–631 19. Cheillan D, Salomons GS, Acquaviva C et al (2006) Prenatal diagnosis of guanidinoacetate methyltransferase deficiency: increased guanidinoacetate concentrations in amniotic fluid. Clin Chem 52:775–777 20. Braissant O, Henry H (2008) AGAT, GAMT and SLC6A8 distribution in the central nervous system, in relation to creatine deficiency syndromes: a review. J Inherit Metab Dis 31 (2):230–239 21. Guimbal C, Kilimann MW (1993) A Na(+)dependent creatine transporter in rabbit brain, muscle, heart, and kidney. cDNA cloning and functional expression. J Biol Chem 268:8418–8421 22. Clark AJ, Rosenberg EH, Almeida LS et al (2006) X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology. Hum Genet 119:604–610 23. Rosenberg EH, Almeida LS, Kleefstra T et al (2004) High prevalence of SLC6A8 deficiency in X-linked mental retardation. Am J Hum Genet 75:97–105 24. Salomons GS, Van Dooren SJ, Verhoeven NM et al (2003) X-linked creatine transporter defect: an overview. J Inherit Metab Dis 26:309–318 25. Bodamer OA, Sahoo T, Beaudet AL et al (2005) Creatine metabolism in combined methylmalonic aciduria and homocystinuria. Ann Neurol 57:557–560 26. Buist NR, Glenn B, Vugrek O et al (2006) Sadenosylhomocysteine hydrolase deficiency in a 26-year-old man. J Inherit Metab Dis 29:538–545 27. El-Gharbawy AH, Goldstein JL, Millington DS et al (2013) Elevation of guanidinoacetate in newborn dried blood spots and impact of early treatment in GAMT deficiency. Mol Genet Metab 109:215–217 28. Stockler-Ipsiroglu S, Apatean D, Battini R et al (2015) Arginine:glycine amidinotransferase (AGAT) deficiency: clinical features and long term outcomes in 16 patients diagnosed worldwide. Mol Genet Metab 116:252–259 29. Stockler-Ipsiroglu S, Van Karnebeek C, Longo N et al (2014) Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for

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diagnosis, treatment and monitoring. Mol Genet Metab 111:16–25 30. Arias A, Ormazabal A, Moreno J et al (2006) Methods for the diagnosis of creatine deficiency syndromes: a comparative study. J Neurosci Methods 156:305–309 31. Benoit R, Samir M, Boutin J et al (2019) LCMS/MS measurements of urinary guanidinoacetic acid and creatine: method optimization by deleting derivatization step. Clin Chim Acta 493:148–155 32. Bodamer OA, Bloesch SM, Gregg AR et al (2001) Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry. Clin Chim Acta 308:173–178 33. Sharer JD, Bodamer O, Longo N et al (2017) Laboratory diagnosis of creatine deficiency syndromes: a technical standard and guideline of the American College of Medical Genetics and Genomics. Genet Med 19:256–263 34. Struys EA, Jansen EE, Ten Brink HJ et al (1998) An accurate stable isotope dilution gas

chromatographic-mass spectrometric approach to the diagnosis of guanidinoacetate methyltransferase deficiency. J Pharm Biomed Anal 18:659–665 35. Young S, Struys E, Wood T (2007) Quantification of creatine and guanidinoacetate using GC-MS and LC-MS/MS for the detection of cerebral creatine deficiency syndromes. Curr Protoc Hum Genet Chapter 17:Unit 17 13 36. Valongo C, Cardoso ML, Domingues P et al (2004) Age related reference values for urine creatine and guanidinoacetic acid concentration in children and adolescents by gas chromatography-mass spectrometry. Clin Chim Acta 348:155–161 37. Derave W, Marescau B, Vanden Eede E et al (2004) Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans. J Appl Physiol 97:852–857 38. Threlfall CJ, Maxwell AR, Stoner HB (1984) Post-traumatic creatinuria. J Trauma 24:516–523

Chapter 13 Quantitation of Estradiol and Testosterone in Serum Using LC-MS/MS Ryan C. Schofield, Daniel Kirchoff, and Dean C. Carlow Abstract Adult and pediatric endocrinology and oncology often requires measuring serum estrogens and testosterone at very low concentrations. Conventional immunoassay methods often lack the required performance to meet this analytical need, and mass spectrometry techniques must be employed. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum, utilizing commercially available calibrators and without the need for chemical derivatization. Serum samples, after the addition of an internal standard, are combined with a hexane:ethyl acetate extraction solution. The samples are vortexed, and the organic layer is decanted into a clean sample tube and evaporated to dryness under a stream of nitrogen. The samples are reconstituted in a water:methanol solution and separated chromatographically using a reversed-phase HPLC column. Subsequent mass spectrometry is performed using both positive ion mode for Te and negative ion mode for E2. Key words Estradiol, Testosterone, Mass spectrometry, Liquid chromatography, Commercial calibrators, Serum

1

Introduction Sensitive measurements of serum estradiol (E2) and testosterone (Te) are important in both adult and pediatric endocrinology and oncology. Very low measurements of E2, typically less than 20 pg/ mL, are required for the determination of menopausal status, diagnosing E2 deficiency, performing E2 measurements in men, and monitoring E2 levels in cancer patients treated with antiestrogen medications [1]. Very low-level Te measurements, less than 50 ng/ dL, are needed for adult women, prepubescent children, and men undergoing anti-androgen treatment [2]. Conventional immunoassay methods can meet the needs for routine steroid analysis; however, they cannot meet the sensitivity and specificity requirements needed to monitor Te and E2 levels at these very low concentrations [3–6]. In addition, many immunoassays are not standardized against internationally recognized standards, and few

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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are currently certified by the Centers for Disease Control and Prevention (CDC) Hormone Standardization (HoSt) Program at the very lowest concentrations required [7, 8]. To meet this need, a number of sensitive and specific assays have been developed to measure E2 and Te by mass spectrometry. Sensitive assays by mass spectrometry for Te typically do not require chemical modification; however, the same is not true for E2. To increase the sensitivity of E2 assays by mass spectrometry, many previously published reports have resorted to chemical derivatization which is an additional step that increases complexity and may introduce variability [1, 9–11]. In this protocol we describe a method for a very sensitive HPLC-MS/MS assay for simultaneous measurement of serum E2 and Te using commercially available calibrators and quality controls and without the need for chemical derivatization or extended extraction protocols. Furthermore, the accuracy has been ensured by comparison with internationally accepted reference standards and direct comparison with a reference assay using LC/MS-MS ([11–13]; see Note 1).

2

Materials Samples

Serum samples are required. All samples should be processed and analyzed within 7 days of collection or refrigerated up to 14 days after collection or frozen at 20  C for 6 months.

2.2 Solvents and Reagents

1. Mobile phase A, 0.2 mM of ammonium fluoride in water: Use an analytical balance weigh of 30 mg of ammonium fluoride and place in a new 4 L water (LC/MS grade) bottle. Cap and invert the bottle ten times followed by sonication for 5 min. The mobile phase is stable at room temperature, 18–24  C, for 1 month.

2.1

2. Mobile phase B, acetonitrile (LC/MS grade): Use a new unopened bottle. The mobile phase is stable at room temperature, 18–24  C, for 6 months. 3. Mobile phase C, acetonitrile/2-propanol/acetone (LC/MS grade; 6:3:1 v/v): In a 1000 mL graduated cylinder, add 600 mL of acetonitrile, 300 mL of 2-propanol, and 100 mL of acetone. Two full cylinders are poured and degassed into a 2-liter HPLC solvent bottle. Degas for 5 min by sonication. The mobile phase is stable at room temperature, 18–24  C, for 6 months. 4. Autosampler aqueous wash, water/acetic acid/acetonitrile (LC/MS grade; 9.7:0.1:0.2 v/v): In a 1000-mL graduated cylinder, add 970 mL of water, 10 mL of acetic acid, and 20 mL of acetonitrile. Two full cylinders are poured into a

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2-liter HPLC solvent bottle and degassed for 5 min by sonication. The mobile phase is stable at room temperature, 18–24  C, for 1 month. 5. Autosampler organic wash, acetonitrile/2-propanol/acetone (6:3:1 v/v): In a 1000-mL graduated cylinder, add 600 mL of acetonitrile, 300 mL of 2-propanol, and 100 mL of acetone. Two full cylinders are poured into a 2-liter HPLC solvent bottle and degassed for 5 min by sonication. The mobile phase is stable at room temperature, 18–24  C, for 6 months. 6. Reconstitution solution, water:methanol (1:1 v/v): Use a 100 mL graduated cylinder, add 50 mL of water and 50 mL of methanol into the cylinder, and then decant into a 250 mL solvent bottle. Degas the solution for 5 min by sonication. The reconstitution solution is stable at room temperature, 18–24  C, for 1 month. 7. Extraction solution, hexanes:ethyl acetate (LC/MS grade; 9: 1 v/v): Using a 1000 mL graduated cylinder, add 900 mL of hexanes and 100 mL of ethyl acetate into the cylinder, and decant into a 1 liter HPLC solvent bottle. Degas the solution for 5 min by sonication. The extraction solution is stable at room temperature, 18–24  C, for 6 months. 2.3 Internal Standards and Standards

1. 100-μg/mL primary internal standards (IS): estradiol-d5 and testosterone-d3, 100 μg/mL (Cerilliant) or equivalent. 2. Standard stock solutions. (a) 10-μg/mL estradiol-d5: Add 100 μL of the primary standard to 900 μL of methanol in an amber glass vial, and mix well. Both stock solutions are stable for 6 months at 20  C. (b) 10-μg/mL testosterone-d3: Add 100 μL of the primary standard to 900 μL of methanol in an amber glass vial and mix well. Both stock solutions are stable for 6 months at 20  C. 3. Working internal standard solution (3750 pg/mL of estradiold5 and 1125 ng/dL of testosterone-d3 in methanol): Add approximately 150 mL of methanol into a 200 mL volumetric flask. Add 75 μL of estradiol-d5 stock (10 μg/mL) and 225 μL of testosterone-d3 stock (10 μg/mL) to the same volumetric flask. Bring to volume with methanol and mix well. The internal standard solution is stable for 6 months when at 20  C.

2.4 Calibrators and Controls

1. Calibrators: E2 and Te calibrators were purchased from Chromsystems (Grafelfing, Germany) utilizing their 6PLUS1® Multilevel Serum Calibrator Set (MassChrom® Steroid Panel 2) kit.

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2. Controls: E2 and Te controls were purchased from Chromsystems utilizing their MassCheck® Steroid Panel 2 (serum control levels I, II, and III) kit. 3. Check the new lot of standards by verifying five unknown patient sample concentrations with the current lot of calibrators. The agreement between the two calculated concentrations must be within 10%. 4. Establish a range for the new lot of controls by collecting data points over 20 consecutive runs and establish the mean and standard deviation. 2.5 Analytical Equipment and Supplies

1. Thermo Scientific (San Jose, CA) Transcend™ TLX-2 coupled to a SCIEX Triple Quad™ (Framingham, MA) 6500 mass spectrometer running Aria™ software 1.6.3 (Thermo Scientific) and MultiQuant™ 3.0.2 (SCIEX). 2. Analytical column: Accucore™ C18 (3.0  50 mm, 2.6 μm particle size); (Thermo Scientific). 3. Column heater (Thermo Scientific, Hot Pocket) or equivalent. 4. Multi-tube equivalent.

vortex

(Fisherbrand;

Ottawa,

Canada)

or

5. Solvent evaporator (Biotage, TurboVap® LV; Salem, NH) or equivalent. 6. Borosilicate test tubes (13  100 mm), caps, and assorted pipette tips. Additionally, 2-mL glass autosampler vials with inserts and pre-slit caps are needed. 2.6 Instrument Operating Conditions

1. High-pressure liquid chromatography (HPLC) (Table 1): Chromatographic separations were performed using a Thermo Scientific Transcend™ TLX-2 which was comprised of a autosampler, a low-pressure mixing quaternary pump (loading pump), a high-pressure binary pump (eluting pump), and a six-valve switching module with six-port valves. The system was controlled via Aria™ software, version 1.6.3. The analytical HPLC column was an Accucore™ C18 (3.0  50 mm, 2.6 μm particle size). The temperature of the analytical column was maintained at 70  C using a column heater. The analytes were loaded on the analytical column in 85% mobile phase A and 15% mobile phase B. The retention times of the HPLCESI-MS/MS ion chromatograms of the analytes can be seen in Fig. 1. 2. Tandem mass spectrometry: Mass spectrometric detection was performed using a SCIEX Triple Quad™ 6500 mass spectrometer equipped with an electrospray ionization (ESI) source operating in alternating positive/negative ion mode. Additional mass spectrometer parameters are referenced in

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Table 1 HPLC operating parameters HPLC parameters Step

Time (s)

Flow (mL/min)

Gradient

%A

%B

1

30

0.5

Step

85

15

2

60

0.5

Ramp

67.5

32.5

3a

135

0.5

Ramp

50

50

4

60

0.5

Step

0

100

5

60

0.5

Step

85

15

a

Data acquisition window: 1.75–3.25 min

Fig. 1 HPLC-ESI-MS/MS ion chromatograms of estradiol, testosterone, estradiol-d5, and testosterone-d3 product ions

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Table 2 Analyte precursor and product ions

Analyte

Precursor ion (m/z)

Product ion (m/z)

CE (V)

DP (V)

EP (V)

CXP (V)

Estradiol

271.1

143.1

75

20

10

12

Testosterone

289.2

97.1

30

95

10

10

Estradiol-d5

279.1

145.1

75

20

10

12

Testosterone-d3

292.3

97.2

30

95

10

10

a

Optimized m/z may change based on instrument and tuning parameters

[12, 13]. Nitrogen (99.995% purity) was used as the desolvation gas and collision gas. The mass transitions from the protonated molecular ion [M+H]+ to the most abundant product ions were used as the quantifying ions for each analyte (Table 2). The MRM acquisition method was run in unit resolution with a scan rate of 50 ms.

3

Methods

3.1 Stepwise Procedure

1. Aliquot 500 μL of standards, controls, or specimens into appropriately labeled 13  100 mm glass test tubes. 2. Aliquot 50 μL of the internal standard solution into each test tube. 3. Add 2.5 mL of the extraction solution to each tube. 4. Place the samples on the multi-tube vortex and vortex at 2000 RPM for 2 min. 5. Place the samples in the 15 min.

80  C explosion proof freezer for

6. Label a second set of glass test tubes (13  100 mm). Remove the samples from the freezer, and decant the organic layer (unfrozen portion) into the new labeled tubes (see Note 2). 7. Place the samples in the solvent evaporator at 35  C for 15 min, and evaporate to dryness under a stream of nitrogen at 15 PSI (see Note 3). 8. Appropriately label autosampler vials and place glass inserts in each vial. 9. Reconstitute the samples with 150 μL of the reconstitution solution, and place the solution in the autosampler vials. 10. Place all samples in the HPLC autosampler, and inject the sample (40 μL) into the HPLC-ESI-MS/MS. Ion chromatograms for all analytes are shown in Fig. 1.

Estradiol and Testosterone by LC-MS/MS

3.2

Analysis

147

1. Instrumental operating parameters are described in [12]. 2. The data are analyzed using MultiQuant™ 3.0.2 software (SCIEX). 3. Standard curves are based on linear regression analysis for estradiol and testosterone. Weighted linear regression models with weights inversely proportional to the X values were used. The analysis compared IS peak area to sample peak area (y-axis) versus analyte concentration (x-axis) using the quantifying ions indicated in Table 2. 4. Typical coefficients of correlation of the standard curve are >0.995.

4

Notes 1. While this assay is very accurate at low steroid levels, it should be noted that this method is intended for a low to moderate throughput laboratory. Due to the lengthy manual sample preparation protocol, it is recommended for less than 100 specimens per day. Our laboratory reflexes Te samples 0.990, % accuracy at the lower limit of quantitation (LLOQ) within 30%, and % accuracy for all other calibrators within 15%. 4. QCs are acceptable if the concentrations fall within two standard deviations of the target mean values established in at least 20 runs. 3.4

Dilution

1. Results above the highest calibrator for a given analyte require dilution. 2. Bring the stored extracts to room temperature, vortex for ~2 min at 1200 rpm, and then centrifuge tubes at 1200  g for 4 min. 3. For each sample to be diluted, prepare a new autosampler vial with 100 μL of hexane. 4. Transfer 50 μL of the organic phase to the appropriate autosampler vials. 5. Inject samples (see Note 4).

4

Notes 1. Pre-wet the pipette tip twice before pipetting organic solutions. Rinse tip once after transferring solutions containing fatty acids (standards, calibrators, or internal standards). 2. Soak glassware in CONTRAD 70 liquid overnight. Alternatively, immerse glassware in CONTRAD 70 liquid with a clean container, and apply sonication for 30 min. Remove and save the CONTRAD 70 liquid for the next use (CONTRAD 70 liquid can be reused for five to ten times, if it is not diluted). Rinse the glassware with DI water. It may take 5–7 good washes (well emptied each time) to rinse off the very basic solution. Use a pH strip to test the wash solution residual from the glassware. It must be less than or equal to pH 7. Rinse the glassware with methanol twice to remove residual DI water from glassware. Place the glassware upside down in racks or clean containers until dry.

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3. Fatty acid contamination is ubiquitous in plastic consumables. Pre-rinse all plastic surfaces coming in contact with the sample or extract with methanol or hexane. Ensure plastic consumable is completely dry prior to use. 4. If patient samples are reinjected within 24 h of calibrator injections, it is not necessary to reinject the calibrators; process samples with the existing batch. If it has been greater than 24 h, calibrators and QCs must be re-injected. References 1. Van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9(2): 112–124 2. Calder PC (2015) Functional roles of fatty acids and their effects on human health. JPEN J Parenter Enteral Nutr 39(1 Suppl):18S–32S 3. Burke PA, Ling PR, Forse RA et al (1999) Conditionally essential fatty acid deficiencies in end-stage liver disease. Nutrition 15(4): 302–304 4. Williard DE, Nwankwo JO, Kaduce TL et al (2001) Identification of a fatty acid delta6desaturase deficiency in human skin fibroblasts. J Lipid Res 42(4):501–508 5. Harris WS, Baack ML (2015) Beyond building better brains: bridging the docosahexaenoic acid (DHA) gap of prematurity. J Perinatol 35(1):1–7 6. Hadley KB, Ryan AS, Forsyth S et al (2016) The essentiality of arachidonic acid in infant development. Nutrients 8(4):216 7. Holman RT (1998) The slow discovery of the importance of omega 3 essential fatty acids in human health. J Nutr 128(2 Suppl):427S–433S 8. Lagerstedt SA, Hinrichs DR, Batt SM et al (2001) Quantitative determination of plasma c8–c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol Genet Metab 73(1):38–45

9. Ren J, Mozurkewich EL, Sen A et al (2013) Total serum fatty acid analysis by GC–MS: assay validation and serum sample stability. Curr Pharm Anal 9(4):331–339 10. Kish-Trier E, Schwarz EL, Pasquali M et al (2016) Quantitation of total fatty acids in plasma and serum by GC-NCI-MS. Clin Mass Spectrom 2:11–17 11. Harris WS, Thomas RM (2010) Biological variability of blood omega-3 biomarkers. Clin Biochem 43:338–340 12. Yuzyuk T, Lozier B, Schwarz EL et al (2018) Intra-individual variability of long-chain fatty acids (C12-C24) in plasma and red blood cells. Prostaglandins Leukot Essent Fatty Acids 135:30–38 13. Harris WS, Sands SA, Windsor SL et al (2004) Omega-3 fatty acids in cardiac biopsies from heart transplantation patients: correlation with erythrocytes and response to supplementation. Circulation 110(12):1645–1649 14. Franco RS (2012) Measurement of red cell lifespan and aging. Transfus Med Hemother 39(5):302–307 15. Van der Vusse GJ (2009) Albumin as fatty acid transporter. Drug Metab Pharmacokinet 24(4):300–307 16. Holman RT (1964) The essential fatty acid requirement of infants and the assessment of their dietary intake of linoleate by serum fatty acid analysis. Am J Clin Nutr 14:70–75

Chapter 15 Quantitation of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry Erland Arning, Brandi Wasek, and Teodoro Bottiglieri Abstract We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of CSF GABA has clinical utility in diagnosing inborn errors of GABA metabolism, specifically for deficiencies of GABA-transaminase and succinic semialdehyde dehydrogenase. Quantitation of CSF GABA is performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Quantitation of total GABA in CSF requires additional sample preparation in order to hydrolyze all the conjugated GABA in the sample to free GABA. Complete hydrolysis is performed incubating sample at >100  C in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90% acetonitrile/0.1% formic acid solution and injected into the HPLCESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 to 1000 nM and 0.63 to 80 μM for free and total GABA, respectively. Key words GABA, Cerebrospinal fluid, Seizures, Mass spectrometry

1

Introduction γ-Aminobutyric acid (GABA), a primary inhibitory neurotransmitter in the brain, is synthesized from glutamate by the pyridoxinedependent enzyme glutamic acid decarboxylase (GAD). The first step in the catabolism of GABA involves degradation to succinic semialdehyde in a reaction catalyzed by GABA-transaminase (GABA-T). Succinic semialdehyde is then converted to succinic acid by the enzyme succinic semialdehyde dehydrogenase (SSADH). GABA is also present in non-neuronal tissues and may exist in peripheral and central tissues in the form of a dipeptide with

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_15, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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histidine, as a compound known as homocarnosine [1]. Several inherited disorders of GABA metabolism have been identified; these are SSADH deficiency, GABA-T deficiency, and homocarnosinosis [2]. The clinical presentation of these cases may vary considerably and include psychomotor retardation, convulsions, ataxia, hypotonia, hyperreflexia, and delayed speech development. In addition, an autosomal recessive disorder has been characterized in a group of patients with pyridoxine-dependent seizures [3]. In these cases, the presumed abnormality is due to reduced binding of pyridoxal-5-phosphate to GAD. Elevation of CSF free and total GABA are characteristic of GABA-T and SSADH deficiency, whereas low CSF GABA has been reported in pyridoxal-5-phosphate dependent seizures. Lastly, patients treated with antiepileptic drugs, including valproic acid and vigabatrin, have been shown to have significantly increased CSF GABA compared to untreated epileptic patients [4, 5]. A variety of methods for determining CSF GABA have been published, which include HPLC-fluorescence [6], HPLCelectrochemical detection [7], GC-MS [8], CE-MS [9], and LC-MS [10]. Many of these methods employ lengthy and laborintensive sample preparation, which may require derivatization prior to analysis. We have developed a method which involves a minimal sample preparation for both free and total GABA and does not require prior sample cleanup or derivatization. Analysis of both free and total GABA requires two individual sample preparations and analyses. Approximately 98% of GABA in CSF is present in the conjugated form as homocarnosine, homoanserine, GABA-lysine, GABA-cystathionine, and possibly other unknown GABA conjugates [11]. Extra care must be taken to ensure accurate determination of free GABA in CSF caused by delayed freezing and/or repeated freeze/thaw cycles, which will result in an artifactual increase in free GABA resulting from the breakdown of conjugated GABA. Free GABA is performed by simple stable isotope dilution (1:2) followed by analysis by HPLC-ESI-MS/MS. Analysis of total GABA requires boiling the CSF sample in the presence of 6 N hydrochloric acid (HCl) for 4 h to ensure complete hydrolysis of all conjugated GABA. Following boiling step, sample is diluted 1: 10 with a 90% acetonitrile/0.1% formic acid solution and analyzed by HPLC-ESI-MS/MS.

2 2.1

Materials Samples

Human lumbar CSF specimen drawn any time during the day will be acceptable. No patient preparation is required. Optimal volume of 1.0 mL (minimum 0.25 mL) CSF should be collected in tube 5 (or 1, 2, or 4) of the collection kit provided by the testing laboratory or in a regular sterile CSF collection tube. If the CSF is

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clear, the sample should be immediately frozen at the bedside on dry ice. If blood contaminated, the sample should be placed on wet ice, centrifuged within 5 min and the clear CSF transferred to another vial and frozen on dry ice. CSF is stored at 80  C until time of testing (see Note 1 for more information regarding sample stability). 2.2 Solvents and Reagents

1. All solvents and reagents should be ACS or LC/MS grade. 2. Mobile phase A (0.5% formic acid/0.25% heptafluorobutyric acid in water): In a hood add 5 mL of formic acid and 2.5 mL of heptafluorobutyric acid to a 1 L glass cylinder, bring to volume with water, and mix to combine. Stable at room temperature, 18–24  C, up to 3 months. 3. Mobile phase B (0.5% formic acid/0.25% heptafluorobutyric acid in acetonitrile): In a hood add 5 mL of formic acid and 2.5 mL of heptafluorobutyric acid to a 1 L glass cylinder, bring to volume with acetonitrile, and mix to combine. Stable at room temperature, 18–24  C, up to 3 months. 4. Autosampler wash solution (10% methanol): In a hood add 100 mL of methanol to a 1 L glass cylinder, and bring volume up to 1 L with water, and mix. Stable at room temperature, 18–24  C, up to 1 year. 5. Deproteinizing solution (90% acetonitrile/0.1% formic acid in water): Add 49.5 mL of acetonitrile and 50 μL of formic acid to a 50-mL glass bottle, and mix to combine. Stable at room temperature, 18–24  C, up to 8 months. 6. 2X artificial CSF (aCSF): (a) Solution A: Weigh the following and combine in a 500-mL volumetric flask containing a magnetic stir bar and 250 mL of HPLC-grade water: 8.66 g NaCl, 0.224 g KCl, 0.206 g CaCl2 2H2O, and 0.163 g MgCl2 6H2O. Mix on a magnetic stirrer until dissolved. Bring volume to 500 mL with water and mix to combine. Stable for 1 year in the refrigerator, 2–8  C. (b) Solution B: Weigh the following and combine in a 500-mL volumetric flask containing a magnetic stir bar and 250 mL of HPLC-grade water: 0.214 g Na2HPO4 2H2O and 0.027 NaH2PO2 H2O. Mix on a magnetic stirrer until dissolved. Bring volume to 500 mL with water and mix to combine. Stable for 1 year in the refrigerator, 2–8  C. 7. 1X aCSF: Mix equal parts of 2X aCSF solution A and 2X aCSF solution B, and mix to combine. Stable for 8 h in the refrigerator, 2–8  C.

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2.3 Internal Standards and Standards

1. Primary standard: GABA (γ-aminobutyric acid). 2. Primary internal standard (2H2-γ-aminobutyric acid).

(I.S.):

GABA-D2

3. 1 mM GABA standard stock solution: In a 100 mL volumetric flask, dissolve 10.3 mg of GABA in 90 mL of 0.1 M HCL, and adjust volume to 100 mL with 0.1 M HCL. Store in 200 μL of aliquots at 80  C for up to 4 years (see Note 2). 4. 1 mM GABA-D2 IS stock solution: In a 100-mL volumetric flask, dissolve 10.5 mg of GABA-D2 in 90 mL of 0.1 M HCL, and adjust volume to 100 mL with 0.1 M HCL. Store in 200 μL of aliquots at 80  C for up to 4 years (see Note 2). 5. 2 μM Free GABA-D2 IS working solution: Add 2 μL of 1 mM GABA-D2 to 998 μL of water and mix well by vortex. Stable at 2–8  C for up to 1 week. 6. 40 μM Total GABA-D2 IS working solution: Add 40 μL of 1 mM GABA-D2 to 960 μL of water and mix well by vortex. Stable at 2–8  C for up to 1 week. 2.4 Calibrators and Controls

1. Calibrators: Free GABA working standard curve: Dilute 1-mM GABA standard stock solution as follows: (a) Dilution A (100 μM): Add 100 μL of 1 mM GABA stock solution to 900 μL of 1X aCSF, and mix well by vortex. (b) Dilution B (10 μM): Add 100 μL of Dilution A to 900 μL of 1X aCSF, and mix well by vortex. (c) Dilution C (1 μM): Add 100 μL of Dilution B to 900 μL of 1X aCSF, and mix well by vortex. (d) Free GABA working standard curve (25–400 nM): Add 400 μl of Dilution C to 600 μL of 1X aCSF, and mix well by vortex. Perform four additional serial dilutions by adding 500 μL of previous standard to 500 μL of 1X aCSF. This will provide a calibration curve of 400, 200, 100, 50, and 25 nM. Working standard curve may be stored in the refrigerator, 2–8  C, for up to 8 h (see Note 3). 2. Calibrators: Total GABA working standard curve: Dilute 1 mM GABA standard stock solution as follows: Total GABA working standard curve (2.5–40 μM): Add 40 μL of 1 mM GABA stock solution to 960 μL of 1X aCSF, and mix well by vortex. Perform four additional serial dilutions by adding 500 μL of previous standard to 500 μL of 1X aCSF, and mix well by vortex. This will provide a calibration curve of 40, 20, 10, 5, and 2.5 μM. Working standard curve may be stored in the refrigerator, 2–8  C, for up to 8 h (see Note 3).

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3. Low control: Free GABA target value: 60–100 nM. (a) Prepare 10 mL of pooled CSF. (b) Assay pooled CSF to quantitate the native concentration of free GABA. (c) Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 60–100 nM free GABA. Store in 80 μL aliquots at 80  C for up to 4 years (see Notes 1 and 3). 4. High control: Free GABA target value: 300–400 nM. (a) Prepare 10 mL of pooled CSF. (b) Assay pooled CSF to quantitate the native concentration of free GABA. (c) Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 300–400 nM free GABA. Store in 80 μL aliquots at 80  C for up to 4 years (see Notes 1 and 3). 5. Low control: Total GABA target value: 4–8 μM. (a) Prepare 10 mL of pooled CSF. (b) Assay pooled CSF to quantitate the native concentration of total GABA. (c) Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 4–8 μM total GABA. Store in 80 μL aliquots at 80  C for up to 4 years (see Note 2). 6. High control: Total GABA target value: 30–40 μM. (a) Prepare 10 mL of pooled CSF. (b) Assay pooled CSF to quantitate the native concentration of total GABA. (c) Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 30–40 μM total GABA. Store in 80 μL aliquots at 80  C for up to 4 years (see Note 2). 2.5 Analytical Equipment and Supplies

1. Shimadzu Prominence liquid chromatograph system with Sciex 4000QTRAP® with Analyst software version 1.6.2 2. Analytical column: Phenomenex EZfaast, 3 μm, 250  2 mm. 3. Guard column: Phenomenex Security Guard, 5 μm, 4  3 mm. 4. Boiling water bath. 5. Metal 1.5 mL screw-top rack. 6. 1.5 mL Microcentrifuge tubes. 7. 96-well microtiter plate. 8. 96-well silicone mat.

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Methods

3.1 Sample Preparation (Free GABA)

1. To labeled 1.5 mL microcentrifuge tubes, pipette 50 μL of CSF (calibrators, controls, patient CSF, or 1 aCSF). 2. Add 50 μL of free GABA IS working solution. 3. Cap and vortex mix tubes at maximum speed for 3 s. 4. Transfer 90 μL of prepared sample into corresponding work list position in 96-well microtiter plate, and cover with silicone cover. 5. Place completed 96-well microtiter plate onto refrigerated autosampler (4  C). 6. Inject 12 μL of sample onto HPLC-ESI-MS/MS. Representative HPLC-ESI-MS/MS ion chromatograms for free GABA and IS are shown in Fig. 1 (see Notes 4 and 5).

3.2 Sample Preparation (Total GABA)

1. To labeled 1.5 mL microcentrifuge tubes, pipette 50 μL of CSF (calibrators, controls, patient CSF, or 1X aCSF). 2. Add 50 μL of total GABA IS working solution.

Fig. 1 HPLC-ESI-MS/MS ion chromatogram of free GABA 1 (m/z 104.1 > 87.1), GABA 2 (m/z 104.1 > 69.1), and GABA-D2 (m/z 106.0 > 89.1). Concentration of GABA shown is 169 nM

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3. Add 200 μL of 6 N HCl. 4. Cap and vortex mix tubes at maximum speed for 3 s. 5. Place tubes in a metal rack with screw-top rack. 6. Place metal rack with tubes in boiling water bath. 7. Boil samples for 4 h. 8. After boiling, remove samples and allow to reach room temperature, 18–24  C. 9. To new labeled 1.5-mL microcentrifuge tubes, add 180 μL of deproteinizing solution. 10. Transfer 20 μL of hydrolyzed (boiled) sample to 1.5-mL microcentrifuge tube containing deproteinizing solution, and mix well by vortex. 11. Transfer 160 μL of prepared sample into corresponding work list position in 96-well microtiter plate, and cover with silicone cover. 12. Place completed 96-well microtiter plate onto refrigerated autosampler (4  C). 13. Inject 10 μL of sample onto HPLC-ESI-MS/MS. Representative HPLC-ESI-MS/MS ion chromatograms for total GABA and IS are shown in Fig. 2 (see Notes 4 and 5).

Fig. 2 HPLC-ESI-MS/MS ion chromatogram of total GABA 1 (m/z 104.1 > 87.1), GABA 2 (m/z 104.1 > 69.1), and GABA-D2 (m/z 106.0 > 89.1). Concentration of GABA shown is 8.8 μM

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Table 1 HPLC-ESI-MS/MS operating conditions A. HPLC (Free GABA)a Column temp.

40°C

Flow rate

0.230 mL/min

Gradient

Time (min) 0.0 5.0 5.1 6.0 6.1 10.0

Mobile Phase A (%) 90 25 0 0 90 Stop

B. HPLC (Total GABA) a Column temp.

40°C

Flow rate

0.230 mL/min

Gradient

Time (min) 0.0 4.0 4.1 8.0

Mobile Phase A (%) 90 40 90 Stop

C. MS/MS Tune Settings b Entrance Potential (V) Curtain gas (psi) CAD gas Ion Spray (V) Temp (°C) GS1 (psi) GS2 (psi) Resolution Q1 and Q3

10 10 Medium 5500 600 40 40 Unit

a Optimized for Shimadzu Prominence liquid chromatography system equipped with Phenomenex EZfaast, 3 μm, 250  2 mm analytical column; Mobile phase A: 0.5% formic acid-0.25% heptafluorobutyric acid in water; Mobile phase B: 0.5% formic acid-0.25% heptafluorobutyric acid in methanol b Optimized for AB Sciex 4000QTRAP®. Tune settings may vary slightly between instruments

3.3

Data Analysis

1. Instrumental operating parameters are given in Table 1a, b, and c. 2. Data are analyzed using Analyst software version 1.6.2. 3. Standard curves are generated based on linear regression of the analyte/IS peak-area ratio ( y) versus analyte concentration (x) using the primary ions indicated in Table 2. The curve is weighted 1/x. 4. Acceptability of each run is confirmed if quality controls are within previously defined limits using Westgard rules. Inter-day

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Table 2 HPLC-ESI-MS/MS operating conditions Compound

Q1 (m/z)

Q3 (m/z)

Dwell time DP (msec) (V)

CE (V)

CXP (V)

GABA 1

104.1a

87.1a,b

300

36

15

6

GABA 2

a

104.1

a,c

69.1

300

36

23

4

GABA-D2

106a

89.1a,b

300

26

15

6

a

Optimized m/z may change based on tuning parameters and instrument used Primary ion for GABA quantification c Secondary ion used for MRM ratio b

Table 3 Age-specific reference range for CSF GABA (total and free) Age < 2 years > 2 years

Free GABA (nM) 17 - 67 32 - 170

Total GABA (µM) 4.2 - 13.4 3.3 - 12.2

See reference [6]

precision was evaluated by repeated analysis of bi-level quality control material analyzed in duplicate over a period of 20 different days. 5. Confirmatory analysis based on the ratio of two MRM transitions per assay: free GABA 1 or total GABA 1 (primary)/free GABA 2 or total GABA 2 (secondary) should be 20% of the established value for the assay based upon the calibration curve. 6. The assays have a lower limit of quantitation of 6 nM and 0.6 μM for free and total GABA, respectively, with precisions of 2 h) due to equipment failure or other delays. 5. The controls are analyzed at the beginning and end of analysis batch for assay verification. 6. Ion suppression effects were evaluated by sample infusion method. No significant interferences or ion suppression were identified. References 1. Perry TL, Kish SJ, Sjaastad O, Gjessing LR, Nesbakken R, Schrader H, Loken AC (1979) Homocarnosinosis: increased content of homocarnosine and deficiency of homocarnosinase in brain. J Neurochem 32(6):1637–1640 2. Pearl PL, Jakobs C, Gibson KM (2019) Acknowledgments: disorders of β- and γamino acids in free and peptide-linked forms. Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA(eds) The Online Metabolic and Molecular Bases of Inherited Disease. McGraw Hill. https://ommbid.mhmedical. com/content.aspx?bookid=2709§ionid=2250 85392 3. Goto T, Matsuo N, Takahashi T (2001) CSF glutamate/GABA concentrations in pyridoxine-dependent seizures: etiology of pyridoxine-dependent seizures and the mechanisms of pyridoxine action in seizure control. Brain Dev 23:24–29 4. Loscher W, Siemes H (1985) Cerebrospinal fluid gamma-aminobutryic acid levels in children with different types of epilepsy: effect of anticonvulsant therapy. Epilepsia 26(4):314–319 5. Cortes-Saladelafont E, Molero-Luis M, Cuadras D, Casado M, Armstrong-Moron J, Yubero D, Montoya J, Artuch R, GarciaCazorla A, Institut De Recerca Sant Joan De Deu Working Group (2018) Gammaaminobutyric acid levels in cerebrospinal fluid in neuropaediatric disorders. Dev Med Child Neurol 60(8):780–792

6. Goldsmith RF, Earl JW, Cunningham AM (1987) Determination of γ-aminobutyric acid and other amino acids in cerebrospinal fluid by reversed-phase liquid chromatography. Clin Chem 33(10):1736–1740 7. Naini AB, Vontzalidou E, Cote LJ (1993) Isocratic HPLC assay with electrochemical detection of free γ-aminobutyric acid in cerebrospinal fluid. Clin Chem 39(2):247–250 8. Struys EA, Gue´rand WS, ten Brink HJ, Jakobs C (1999) Combined method for the determination of gamma-aminobutyric and betaalanine in cerebrospinal fluid by stable isotope dilution mass spectrometry. J Chromatogr B 732:245–249 9. Song Y, Shenwu M, Dhossche DM, Liu Y (2005) A capillary liquid chromatographic/ tandem mass spectrometric method for the quantification of γ-aminobutyric acid in human plasma and cerebrospinal fluid. J Chromatogr B 814:295–302 10. Eckstein JA, Ammerman GM, Reveles JM, Ackermann BL (2008) Analysis of glutamine, pyroglutamate, and GABA in cerebrospinal fluid using ion pairing HPLC with positive electrospray LC/MS/MS. J Neurosci Methods 171:190–196 11. Schechter PJ, Sjoerdsma A (1990) Clinical relevance of measuring GABA concentrations in cerebrospinal fluid. Neurochem Res 15(4): 419–423

Chapter 16 A Simple, Fast, and Reliable LC-MS/MS Method for the Measurement of Homovanillic Acid and Vanillylmandelic Acid in Urine Specimens Vrajesh Pandya and Elizabeth L. Frank Abstract Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are catecholamine metabolites used in the diagnostic workup of neuroendocrine tumors. Here we describe a simple dilute-and-shoot method for simultaneously quantitating HVA and VMA in human urine specimens. The method employs analyte separation on a reverse-phase liquid chromatography column followed by detection using electrospray ionization triple quadrupole mass spectrometry (ESI-MS/MS), wherein qualifier and quantifier ion transitions are monitored. This is a simple and fast analytical method with an injection-to-injection time of 4 min. Key words Homovanillic acid, Vanillylmandelic acid, Neuroblastoma, Pheochromocytoma, Adrenal medulla

1

Introduction Catecholamines are structurally related compounds released in response to stress and mediate the fight-or-flight response [1, 2]. Chromaffin cells of the adrenal medulla are a type of neurosecretory cells that secret catecholamines including dopamine, epinephrine, and norepinephrine [1, 2]. Tumors originating from chromaffin cells (e.g., pheochromocytoma) or neural crest (e.g., neuroblastoma) may secret excessive catecholamines [1]. These catecholamines as well as their metabolites can aid in the diagnosis of these tumors [3, 4]. Homovanillic acid (HVA) is the end product of dopamine metabolism, whereas vanillylmandelic acid (VMA) is the principal end product of epinephrine and norepinephrine metabolism [1, 2]. HVA and VMA are used as tumor markers in suspected cases of neuroblastoma and pheochromocytoma [1, 2]. Since approximately 90% of the neuroblastoma tumors produce HVA and VMA, their elevated concentrations in the

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_16, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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urine support the diagnosis of neuroblastoma [5] but may also aid in diagnosing pheochromocytomas [1]. Traditional assays for HVA and VMA involved highperformance liquid chromatography (HPLC) followed by electrochemical detection [6]. However, these assays were timeconsuming (~21 min) and susceptible to interferences [6]. Relatively recently, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/ MS)-based assays have been developed that offer the advantages of speed and improved specificity [6–8]. The analytical method described here is highly specific and can measure both HVA and VMA in a single run in approximately 5 min [2].

2

Materials

2.1

Specimens

2.2

Reagents

To reduce potential interferences, patients may need to abstain from medications for 72 h prior to specimen collection. Timed (24-h) or random urine collections are acceptable specimens (see also Notes 1 and 2). A 24-h timed collection should be refrigerated during collection. Refrigerated specimens are stable for 1 week, and frozen samples are stable for 2 weeks. 1. Deionized water, reagent grade. 2. Formic acid: 98%, reagent grade. 3. Methanol: HPLC grade. 4. Synthetic urine: 16-mM dipotassium phosphate, 4-mM monosodium phosphate, 120-mM sodium chloride, 7-mM creatinine, and 330-mM urea (ARUP Laboratories, UT, USA). 5. HVA: 4-hydroxy-3-methoxyphenyl Aldrich, USA).

acetic

6. VMA: 4-hydroxy-3-methoxymandelic Aldrich, USA).

acid acid

(Sigma(Sigma-

7. Mobile phase A (0.05% formic acid in deionized water). 8. Mobile phase B (0.05% formic acid in HPLC grade methanol). 2.3 Calibration Standards

1. HVA and VMA (1000 mg/L): Dissolve 25 mg each of HVA and VMA in deionized water, and bring the final volume to 25 mL (see Note 3). 2. Working calibration standards (0.5, 2, 10, 50, and 100 mg/L): Take appropriate volumes of the stock solution, and dilute in synthetic urine to a final volume of 50 mL. Aliquots of the working calibration standards may be prepared and stored at 70  C for up to 1 year.

LC-MS/MS Method to Measure HVA and VMA in Urine Specimens

2.4 Internal Standards

177

1. Internal standard stock solution (1000 mg/L): Dissolve 10 mg of ring-13C6, 4-hydroxy-18O (HVA-13C618O; Cambridge Isotope Laboratories, Inc., MA, USA) and 10 mg of 4-hydroxy-3-methoxy-d3-mandelic acid (VMA-d3; C/D/N Isotopes Inc., QC, Canada) in 10 mL of deionized water (see Note 3). 2. To prepare working solutions, add 50 μL of the IS stock solution into 10-mL water, which will result in a 5 mg/L concentration. Keep protected from light and use within 1 day.

2.5 Quality Control Materials

Two concentrations of quality control (QC) solution (i.e., low and high) may be used to determine the accuracy and precision of the method. Take the following steps to prepare the QC materials (see also Note 4). 1. Dissolve 25 mg each of HVA and VMA in deionized water, and bring the final volume to 25 mL to produce a QC stock solution (1000 mg/L) (see Note 5). 2. To prepare the low-concentration QC (0.8 mg/L), add 160 μL of QC stock solution to deionized water, and make up the volume to 200 mL using deionized water. 3. To prepare the high-concentration QC (8 mg/L), add 16 mL of QC stock solution to deionized water, and make up the volume to 200 mL using deionized water. 4. Prepare 1-mL aliquots, and store at 70  C protected from light for up to 2 years. Validate the controls per the laboratory’s requirements.

2.6

Equipment

1. Analytical HPLC column: Phenomenex Kinetex XB-C18, 2.1  50 mm, 100 A˚, 1.7 μm (00B-4498-AN, Phenomenex, USA). 2. Waters® Xevo™ TQ MS ACQUITY UPLC® instrument equipped with Waters MassLynx™ software (v4.1 or higher): This setup consists of an ACQUITY UPLC® system consisting of a binary solvent manager, sample manager, sample organizer, and column manager. The instrument control and data analysis are performed using the Waters MassLynx™ software. 3. Refrigerated centrifuge with 96-well plate adapters and a minimal centrifugal force of 3500  g.

2.7

Supplies

1. 96-well 2 mL/well collection plate (Phenomenex, USA). 2. 96-well plate polypropylene mat lid (VWR, USA). 3. Amber and/or colored microcentrifuge tubes, 1.5 mL (Eppendorf, USA). 4. Screw neck glass maximum recovery vials (Waters, USA). 5. Amber solvent storage bottles of appropriate volumes.

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6. Pipette and repeater pipette tips. 7. Glassware: volumetric flasks, 50 mL, 1000 mL; beakers. 8. Graduated cylinders: 100 and 1000 mL. 9. Vial and tube racks.

3 3.1

Method Extraction

1. Allow urine specimens, calibration standards, IS, and QC solutions to reach room temperature. Start processing within 1 h. 2. Mix thawed specimens and reagents thoroughly. 3. Take two 96-well plates and label them as (1) mix plate and (2) transfer plate. Mix plate will be used throughout the extraction, whereas the transfer plate will be used at the end of extraction to transfer samples for LC-MS/MS analysis. 4. Transfer 50 μL of blank (synthetic urine), calibrators, QC, and patient specimens into individual wells (see Note 6). 5. Add 50 μL of the internal standard followed by 400 μL of mobile phase A to each well containing the blank, calibrators, QC, and patient specimens. 6. Seal the plate with a plate mat and centrifuge at 3500  g for 5 min at room temperature. 7. Carefully remove the plate mat, and transfer 250 μL of solution from each well in the mix plate to the corresponding well of the transfer plate. 8. Apply a fresh plate mat to the transfer plate, and transfer to the LC-MS/MS system for analysis.

3.2 LC-MS/MS Analysis

The instrument setup consists of an ultra-high-performance liquid chromatography (UPLC) system coupled to an MS/MS instrument set in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). Optimized LC-MS/MS parameters were as follows: 1. Specimen manager temperature: 10  C. 2. Injection volume: 5 μL. 3. Injection type: no load ahead. 4. Fill mode: full loop injection. 5. Full loop overfill factor: four times. 6. Weak wash volume: 600 μL (use mobile phase A). 7. Strong wash volume: 500 μL (use mobile phase B). 8. Flow rate: 0.400 mL/min. 9. Approximate elution time: VMA, 0.86 min; HVA, 2.32 min.

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Table 1 LC gradient program Time (min.)

% Mobile phase B

Flow rate (mL/min)

Curve setting

0.00

5.0

0.400

Initial

2.30

30.0

0.400

6 (linear)

2.31

95.0

0.400

6 (linear)

2.60

95.0

0.400

6 (linear)

2.61

5.0

0.400

6 (linear)

3.00

5.0

0.400

6 (linear)

Specific LC gradient parameters, MS instrument settings, and MRM transitions are listed in Tables 1, 2, and 3, respectively. 3.3

Data Analysis

3.4 Result Interpretation

Data analysis is performed using Waters MassLynx™ software (v4.1 or higher). TargetLynx module is used to determine analyte concentrations using peak response and a calibration curve generated for each run. Chromatographic quality and qualitative ion ratios (QIR) are used to assess the run quality and to determine the identity of the analytes. Chromatographic quality is assessed by visual inspection and using the retention time acceptability criteria outlined in Subheading 3.4. QIR is determined by dividing the peak area for the quantifying MRM transition by the peak area for the qualifying MRM transition. The following criteria should be fulfilled for a run to be deemed successful. 1. Retention times of HVA and VMA should be 2.32 min (3%) and 0.86 min (3%), respectively. 2. Relative retention times for HVA and VMA should be within 0.005 min of their respective isotope-labeled IS. 3. Peaks for HVA and VMA should be symmetrical in shape with minor tailing as the column ages. 4. A blank should not show the presence of HVA or VMA but should contain an appropriate signal for isotope-labeled IS. 5. Internal standard peak area should be between 50% and 175% of the combined median IS peak area of all injections in the batch. Lower or higher IS recovery may be indicative of issues with extraction. 6. The QIR averages should be approximately 6.6 (HVA), 6.2 (HVA-13C618O), 2.0 (VMA), and 1.8 (VMA-d3). 7. The calibration curve, which is generated using five calibrators, should be quadratic in shape, and the coefficient of determination (r2) should be >0.99.

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Table 2 MS parameters Source parameters Capillary

3.00 kV

Extractor

3.00 V

Source temperature

150  C

Desolvation temperature

600  C

Desolvation gas flow

1000 L/h

Cone gas flow

25 L/h

Collision gas flow

0.20 mL/min

Analyzer parameters Mass resolution of Q1 and Q2

Unit mass (0.7 Da at half-height)

LM1 resolution

2.80

HM1 resolution

14.78

Ion energy 1

0.6

Aperture

0.10

Entrance

0.4

Exit

0.4

LM2 resolution

2.88

HM2 resolution

14.77

Ion energy 2

0.8

Scan speed of Q1 and Q2

10 Da/s

CEM

1800.0

Inter-scan delay

0.005 s

Interchannel delay

0.005 s

8. The low- and high-concentration quality controls should be within 15% of the assigned values. 9. Chromatograms from a typical HVA:VMA run are shown in Fig. 1. 10. Reference intervals for 24-h excretion in adults 18 years of age and older are HVA, 0–15.0 mg/d, and VMA, 0–7.0 mg/d. Reference values for HVA and VMA in random urine specimens are reported as a ratio to creatinine and vary with age.

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Table 3 MRM transitions (m/z) Analyte

Transition (m/z)

Dwell time

Conea (V)

Collision (V)

HVA (quant)

183.1 > 122.1

0.032

12

25

HVA (qual)

183.1 > 137.1

0.032

12

12

HVA-13C18O (quant)

191.1 > 130.1

0.032

12

27

HVA- C O (qual)

191.1 > 145.1

0.032

12

12

VMA (quant)

181.1 > 149.1

0.032

22

14

VMA (qual)

181.1 > 153.1

0.032

22

14

VMA-d3 (quant)

184.1 > 149.1

0.032

22

14

VMA-d3 (qual)

181.1 > 156.1

0.032

22

13

13

18

a

Cone and collision voltages are for example only and may vary slightly with the instrument

4

Notes 1. Typically, a 24-h urine specimen is tested using this method for patients 18 years of age and older. 2. Urine creatinine measurements should be performed for urine specimens analyzed for HVA and VMA. The results may be reported as HVA and VMA ratio to creatinine (mg/g of creatinine). The ratio to creatinine concentration can be particularly useful for pediatric patients where collection of a 24-h urine might be challenging. 3. While preparing calibration and internal standards, ensure thorough mixing, and visually inspect to ensure complete dissolution. Protect from light. Use immediately for working standard preparation or alternately, and store at 70  C for up to 1 year. 4. The preparation of QC material should be done independently of the calibration standards. 5. Ensure thorough mixing of QC solutions, and visually inspect for complete dissolution. Protect from light. Use immediately to prepare working QC solutions. Alternately, store at 70  C for up to 2 years. 6. Synthetic urine is used as a negative control, whereas synthetic urine spiked with internal standard (see Subheading 3.1 Step 4) is used as a blank to determine extraction efficiency.

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Fig. 1 LC-MS/MS chromatograms of (a) 2 mg/L calibration standard, (b) healthy patient urine, and (c) abnormal patient urine: vanillylmandelic acid (VMA), vanillylmandelic acid-d3 (VMA-d3), homovanillic acid (HVA), and homovanillic acid-13C618O (HVA-13C618O). (Reproduced with permission from ELSEVIER with the following acknowledgment: “This article was published in “Clinica Chimica Acta; Volume: 468; Authors: Zlatuse D. Clark, Jeaneah M. Cutler, Igor Y. Pavlov, Frederick G. Strathmann, Elizabeth L. Frank; Title: Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography-tandem mass spectrometry; Pages: 201–208; Copyright Elsevier (2017)”)

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Acknowledgments This work was supported by the ARUP Institute of Clinical and Experimental Pathology®. References 1. Alter DN (2020) Chapter 41 – disorders of the adrenal cortex and medulla. In: Clarke W, Marzinke MA (eds) Contemporary practice in clinical chemistry, 4th edn. Academic Press, pp 729–746. https://doi.org/10.1016/B978-012-815499-1.00041-7 2. Eisenhofer G, Grebe S, Cheung NKV (2018) Chapter 63 - Monoamine producing tumors. In: Horvath AR, Rifai N, Wittwer C (eds) Tietz textbook of clinical chemistry and molecular diagnostics, Sixth Edition. Elsevier Inc, pp 1421. e75. 3. Irwin MS, Park JR (2015) Neuroblastoma: paradigm for precision medicine. Pediatr Clin N Am 62(1):225–256. https://doi.org/10. 1016/j.pcl.2014.09.015 4. Maris JM, Hogarty MD, Bagatell R, Cohn SL (2007) Neuroblastoma Lancet 369(9579): 2106–2120. https://doi.org/10.1016/s01406736(07)60983-0 5. Øra I, Eggert A (2011) Progress in treatment and risk stratification of neuroblastoma: impact

on future clinical and basic research. Semin Cancer Biol 21(4):217–228. https://doi.org/10. 1016/j.semcancer.2011.07.002 6. Clark ZD, Cutler JM, Pavlov IY, Strathmann FG, Frank EL (2017) Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography tandem mass spectrometry. Clin Chim Acta 468:201–208. https://doi.org/10.1016/j.cca. 2017.03.004 7. de Jong WH, de Vries EG, Kema IP (2011) Current status and future developments of LC-MS/MS in clinical chemistry for quantification of biogenic amines. Clin Biochem 44(1): 9 5 – 1 0 3 . h t t p s : // d o i . o r g / 1 0 . 1 0 1 6 / j . clinbiochem.2010.07.006 8. Magera MJ, Thompson AL, Matern D, Rinaldo P (2003) Liquid chromatography-tandem mass spectrometry method for the determination of vanillylmandelic acid in urine. Clin Chem 49(5): 825–826. https://doi.org/10.1373/49.5.825

Chapter 17 Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography–Mass Spectrometry (GC/MS) Melissa Beals, Bheemraj Ramoo, C. Clinton Frazee III, and Uttam Garg Abstract Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2 mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis (trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography–mass spectrometry (GC/MS). Key words Homovanillic acid, Vanillylmandelic acid, Neuroblastoma, Catecholamines, Epinephrine, Norepinephrine, Dopamine, Gas chromatography–mass spectrometry

1

Introduction Neuroblastoma, ganglioneuroblastoma, and ganglioneuroma are tumors of neural crest origin that are characterized by abnormalities in the production and excretion of catecholamines and their metabolites [1–3]. Neuroblastomas are the most common neuroblastic tumors in infants and account for nearly 15% of pediatric cancer fatalities [1]. More than 90% of neuroblastoma tumors are associated with excessive excretion of one or another of the catecholamines and/or their metabolites [1, 2]. Homovanillic acid (HVA) is the major metabolite of dopamine, and vanillylmandelic acid (VMA) is the primary metabolite of epinephrine and norepinephrine. The pattern and ratio of specific catecholamine production can vary based on tumor differentiation and presentation, thereby making the measurement of HVA and VMA an important

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_17, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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diagnostic tool [1, 4]. Methods for analysis of HVA and VMA include spectrophotometric techniques and liquid or gas chromatography [5–10]. Due to poor specificity, most spectrophotometric testing has been replaced by chromatographic methods. Liquid and gas chromatography–mass spectrometry are the most frequently used methods for the determination of HVA and VMA [5–9]. In this chapter, we discuss a robust GC/MS method involving an acidic extraction and simultaneous measurement of HVA and VMA.

2

Materials

2.1

Sample

2.2

Reagents

Randomly collected urine is an acceptable sample for this procedure. Samples are valid for testing within 14 days of receipt if kept frozen (<
5  C) or refrigerated ( 0.990.

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4. The linearity/limit of quantitation of the method is 0.1 to 5.0 mg/dL. Samples in which the analyte concentrations exceed the upper limit of quantitation should be prepared at a dilution with DI water and reanalyzed. Dilutions should not exceed a factor of 50 (see Note 8). 5. Quality control: The run is considered acceptable if calculated concentrations of HVA/VMA in the controls are within 20% of target values. For all samples, ratios of qualifier ions to the quantifying ion must be within 20% of the ion ratios for the calibrators. 6. Typical intra- and inter-assay imprecision is 71.0), and lactate-D3 (m/z 92.1 > 73.1). Concentration of lactate shown is 20.0 mM standard

5. Confirmatory analysis based on the ratio of two MRM transitions lactate 1 (primary)/lactate 2 (secondary) should be 20% of the established value for the assay based upon the calibration curve. 6. The assay has a lower limit of quantitation of 0.3 mM for lactate, with imprecision of 43.1), lactate 2 (m/z 89.0 > 71.0), and lactate-D3 (m/z 92.1 > 73.1). Concentration of lactate shown is abnormal 7.2 mM

2. Individual sets of lactate standard and internal standard stock solutions and controls can be pre-aliquoted and frozen until use in each analytical run. For each set pipette specified volume of stock standard/control solution into 1.5-mL microcentrifuge tubes, and freeze at
80  C until use. Thaw at room temperature 19–27  C, completely before use; do not use heat or water to accelerate the process. Controls are stable for 2 years and stock standards/internal standards for 4 years. 3. A new standard curve should be prepared with each analytical run to optimize method performance. 4. The controls are analyzed at the beginning and end of the assay as analysis verification. 5. Ion suppression effects were evaluated by sample infusion method. No significant interferences or ion suppression was identified.

Quantitation of Lactate in Cerebrospinal Fluid

259

Table 1 HPLC–ESI–MS/MS operating conditions A. HPLCa Column temp.

40  C

Isocratic flow rate

0.600 mL/min

B. MS/MS tune settingsb Entrance potential (V)


10

Curtain gas (psi)

20

CAD gas

High

Ion spray (V)


4500



Temp. ( C)

300

GS1 (psi)

20

GS2 (psi)

40

Resolution Q1 and Q3

Unit

Optimized for Shimadzu Prominence liquid chromatography system equipped with Phenomenex C18, 5 μm, 150  3 mm analytical column; mobile phase: 0.1% formic acid/75% acetonitrile b Optimized for Sciex 4000QTRAP. Tune settings may vary slightly between instruments a

Table 2 HPLC–ESI–MS/MS operating conditions MRM transition Compound

Q1 (m/z)

Q3 (m/z)

Dwell time (ms)

DP (V)

CE (V)

CXP (V)

Lactate 1

89.0a

43.1a,b

150


50


17


5

Lactate 2

89.0

a

71.0

a,c

150


40


15


10

92.1

a

73.1

a,b

150


70


19


4

Lactate-D3 a

Optimized m/z may change based on tuning parameters and instrument used Primary ions for lactate quantification c Secondary ion used for MRM ratio confirmation b

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Fig. 3 CSF lactate proficiency testing References 1. Chow SL, Rooney ZJ, Cleary MA, Clayton PT, Leonard JV (2005) The significance of elevated CSF lactate. Arch Dis Child 90(11):1188–1189 2. Baheerathan A, Pitceathly RDS, Curtis C, Davies NWS (2020) CSF lactate. Pract Neurol 20(4):320–323 3. Gordon N, Newton RW (2003) Glucose transporter type1 (GLUT-1) deficiency. (2003). Brain and Development 25(7):477–480 4. Klepper J, Leiendecker B (2007) GLUT1 deficiency syndrome – 2007 update. Develop Med Child Neurol 49(9):707–716 5. de Almeida SM, Faria FL, de goes fontes K, Buczenko GM, Berto DB, Raboni SM, Vidal

LR and Nogueira MB. (2009) Quantitation of cerebrospinal fluid lactic acid in infectious and non-infectious neurological diseases. Clin Chem Lab Med 47(6):755–761 6. Abassi M, Bangdiwala AS, Nuwagira E, Kandole Tadeo K, Okirwoth M, Williams DA, Mpoza E, Tugume L, Ssebambulidde K, Huppler Hullsiek K, Musubire AK, Muzoora C, Rhein J, Meya DB, Boulware DR (2021) Cerebrospinal fluid lactate as a prognostic marker for disease severity and mortality in cryptococcal meningitis. Clin Infect Dis 73(9):e3077–e3082

Chapter 24 Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry Hsuan-Chieh (Joyce) Liao and Hsiao-Jan Chen Abstract Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography–mass spectrometry (LC–MS/MS) system. Key words Dried blood spot, Lysosomal storage disorders, Newborn screening, Enzyme activity assay, Liquid chromatography–mass spectrometry

1

Introduction Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders that result from deficiency of specific enzymes responsible for the degradation of substances present in lysosomes. Over 70 LSDs are known with a collective incidence of approximately one in 5000 live births [1]. The deficient enzyme causes an accumulation of normally degraded substrates within lysosomes, and the influence is usually systemic and causes multiple organ damage, such as cardiomyopathy, neuropathy, and renal disorders [2, 3]. The main treatments for LSD include enzyme replacement therapy (ERT), bone marrow transplantation, substrate reduction therapy, and gene therapy [4]. Timely initiation of treatments can prolong survival, delay disease progression, and improve long-term outcomes [5–7]. With the availability of existing and new therapies, there is increasing interest in newborn screening (NBS) and

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_24, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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high-risk group screening for LSDs with the goal of reducing disease-related morbidity and mortality through early detection. Screening for LSDs was initially carried out by Chamoles et al., who developed 4-methylumbelliferone-conjugated substrates to detect enzyme activities from dried blood spots (DBS) samples [8, 9]. The fluorescent method has been successfully proven as a useful tool allowing for large-scale newborn screenings [10] and was further translated to digital microfluidics fluorimetry platform in recent years [11]. The other methodology, tandem mass spectrometry (MS/MS), has been well established and widely implemented in newborn screening fields since 1980 for inborn error metabolic disorders. The MS/MS method for LSD screening was first described by Gelb et al. [12], who used synthesized substrates and internal standards to detect multiple enzymatic products for Fabry, Gaucher, Krabbe, mucopolysaccharidosis-I (MPS-I), Niemann-Pick-A/B, and Pompe diseases [13, 14]. The reagents and cocktails for six-plex assays were distributed by the CDC along with DBS quality control materials then later finalized and commercialized by PerkinElmer. A key advantage of the multiplexed MS/MS assay is based on the fact that multiple enzymes could be analyzed from a single DBS punch in a single buffer [15, 16]. The assay now is authorized test approved by FDA and implemented to NBS laboratories worldwide [17, 18]. Subsequently, 18-plex UPLC– MS/MS assays [19] were established, and they nicely highlight the multiplexing capacity of MS/MS. Those screening methods and these efforts provide critical, detailed data to help guide objective decisions and facilitate optimal treatment outcomes [20]. This chapter focuses on the use of tandem mass spectrometry (MS/MS) to measure the activity of four lysosomal enzymes, acid alpha-glucosidase (GAA), alpha-galactosidase (GLA), β-glucocerebrosidase (GBA), and alpha-iduronidase (IDUA). Two enzymes, acid sphingomyelinase (ASM) and galactocerebrosidase (GALC), can also be included into the screening panel. These enzymes are deficient in Pompe disease, Fabry disease, Gaucher disease, mucopolysaccharidosis type I, Niemann-Pick type A/B, and Krabbe disease, respectively. The enzymes are extracted from dried blood spots and incubated with multiplexed substrate and internal standard. The substrates are the structural analogues to the natural substrates, and the internal standard are deuterated structural analog to the enzymatic product (Fig. 1). The enzyme cocktail is prepared directly from the commercially available mixtures of six substrates and internal standards at the predetermined and optimized concentrations. After the enzymatic reactions are quenched, the products and internal standards are prepared using liquid/liquid extraction and quantified simultaneously using selected ion monitoring on a LC–MS/MS system.

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263

Fig. 1 NeoLSD substrates and internal standards

2

Materials

2.1 Samples and Controls

1. Patient samples: The newborn’s blood is collected from the heel and spots in five circles on the qualified filter paper (Whatman 903 filter paper). The DBS are collected within 3 days of birth and shipped at ambient temperature (see Note 1). DBSs are submitted to analyze on the day of arrival in the newborn screening laboratory. Afterward, they were stored at 4
C for 2–3 weeks before being transferred to 20
C storage for 2–3 years. 2. DBS controls (C1, C2, C3: low, medium, high controls; NeoLSD Kit, PerkinElmer): The unopened controls are stable at 20
C until the expiry date. Once opened, the kit controls are stable for up to 8 weeks. The values for the kit controls measured by the manufacturer are given on the lot-specific quality control certificate included in the kit (see Note 2). 3. External quality controls: DBS with low, medium, and high enzyme activities are provided by CDC Foundation (Atlanta, GA).

2.2 Reagents and Buffers

1. NeoLSD substrates and internal standards (S/IS) (Fig. 1): The unopened S/IS powders are stable at 20
C until the expiry date. Once reconstituted with NeoLSD Assay Buffer, the assay cocktails are stable for up to 2 weeks in the dark at +2 to +8
C or 1 month at 20
C (see Note 3).

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2. NeoLSD Assay Buffer (1 bottle, 35 mL): The unopened buffer is stable at +2 to +8
C until the expiry date. The assay buffer can also be homemade: adding 10.5-g succinic acid to 950-mL water until it is completely dissolved. Add 20-g NaOH, adjust pH to 4.71, and then add 2.15-mg acarbose, 11.18-g N-acetylgalactosamine, 8.73-mg saccharic acid lactone, 14.8-g sodium taurocholate, and 81.97-mg ZnCl2. Mix and adjust pH to 4.7 and add water to 1000 mL. Once opened or prepared, the Assay Buffer can be used for up to 8 weeks (see Notes 3 and 4). 3. Quench solution (1 bottle, 4000 mL): Contains 2000 mL of ethyl acetate and 2000 mL of methanol. The solution is stable for up to 4 weeks. Store in the dark (see Note 4). 4. Flow solvent (1 bottle, 800 mL): 80% acetonitrile and 20% water with 0.2% of formic acid. 5. Weak wash buffer: Water with 0.1% of formic acid; strong wash buffer: 80% acetonitrile and 20% isopropanol with 0.1% of formic acid (see Note 4). 2.3 Equipment and Supplies

1. UPLC system. 2. CTC Autosampler. 3. TSQ Vantage. 4. Plate centrifuge. 5. Temperature-controlled incubator. 6. Aluminum plate heat sealer. 7. Dried blood spot puncher (BSD 700 puncher, BSD Technologies). 8. Liquid handler (Tecan Freedom EVO). 9. Sample concentrator. 10. Deep 96-well polypropylene plates and PS-V plates (Greiner). 11. Aluminum film.

3

Methods

3.1 First Day: Incubation and Enzymatic Reactions

1. Preparation cocktail: Bring NeoLSD Assay Buffer to room temperature before use. Add 6.6 mL of NeoLSD Assay Buffer to one vial of NeoLSD substrates and internal standards. Sonicate for 10 minutes at ambient temperature in a bath sonicator. Rock the vial for an additional 20 minutes on a laboratory rocker (see Note 3). 2. Punch out one 3.2-mm DBS from blank, controls, and patient samples in polypropylene 96-well PS-V plate by using a DSB puncher. In the beginning, each plate should contain two blank

Lysosomal Enzyme Activities on Dried Blood Spots

265

wells that have filter paper only, following the controls C1, C2, and C3 after the blank samples. Also, place DBS controls and blanks at the end of the plate after the patient samples. Blanks are carried out exactly as above except the 3.2-mm punches of filter papers without blood. 3. Transfer DBS from PS-V plate to 96 deep well plate. Pipette 30 μL of incubation cocktail into each well by a 12-channel pipet. Cover the plate tightly with an aluminum foil microplate, and seal the aluminum film by heat sealer. 4. Centrifuge the plate at 2000 rpm (805  g) for 2 minutes. Incubate the plate in 37
C incubators for 18 hours while shaking at 360 rpm. Put the worksheet in the LC–MS (Thermo Xcalibur Sequence Setup). 3.2 Second Day (Sample Clean-Up)

1. Remove the plate(s) from the incubator, centrifuge the plate at 2000 rpm (805  g) for 2 minutes, and remove the aluminum foil cover carefully avoiding spillover. 2. Quench the reaction by adding 100 μL of Quench solution into each well by Tecan Liquid handler. Mix the solution by patting the plates gently. 3. Add 200 μL of water, then 400 μL of ethyl acetate to each deep well, and mix the contents of each well by pipetting up and down 50 times by liquid handler (see Note 5). Cover the plate with a plastic blue mat, and centrifuge at 3500 rpm (2465  g) for 20 mins. 4. The contents of the plate wells will be separated into an upper organic layer and a lower aqueous layer. Transfer 200 μL of the organic top layer into the corresponding wells of a U-bottom microplate by the liquid handler. Be careful not to touch the lower aqueous layer while transferring the upper organic layer into the U-bottom microplate. 5. Put the plates on 96-well dry baths at +40
C. Dry the contents of the plate(s) on the microplate evaporator using streams of clean, dry air or nitrogen, about 10–20 minutes until all wells are completely dry. 6. Add 150 μL of flow solvent to each well. Cover each plate with an aluminum plate cover, and shake at ambient temperature at 500 rpm for 5 minutes. Remove the plate(s) from the incubator shaker unit, keeping the adhesive cover(s) in plate(s), and load them onto the MS/MS screening system.

3.3 MS/MS and Analysis

1. MS/MS analysis is performed on a TSQ Vantage quadrupole mass (see Note 6). The instrument is operated in positive ion mode. All analytes are monitored by selected reaction monitoring (SRM). The typical experimental setup on TSQ Vantage is listed in Tables 1 and 2.

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Table 1 Typical parameters used on the TSQ Vantage Tune parameters

MS method function

Spray voltage

3800

Type

SRM

Vaporizer T

180
C

MS acquire time (min)

1

Sheath gas pressure

10

Chrom filter peak width (s)

2

Aux gas pressure

103

Collision gas pressure (m Torr)

1.5




Capillary T

180 C

Scan width (m/z)

0.01

Tube lens offset

56

Scan time (s)

0.05

Skimmer offset

10

Q1 peak width

0.7

Collision pressure

1.1

Q3 peak width

0.7

Collision energy

10

Ionization mode

ES+

Table 2 Exact MW of the internal standards (IS) and enzymatic products (P) measured with the NeoLSD MSMS Kit Name

Parent mass

Product mass

Collision energy

ABG_P

384.3

264.3

19

ABG_IS

391.4

271.3

19

ASM_P

398.4

264.3

15

ASM_IS

405.4

264.3

15

GALC_P

412.4

264.3

17

GALC_IS

417.4

264.3

17

IDUA_P

426.2

317.2

10

IDUA_IS

431.3

322.2

10

GLA_P

484.3

384.2

11

GLA_IS

489.3

389.3

11

GAA_P

498.3

398.2

11

GAA_IS

503.3

403.3

11

2. Twenty microliters of sample solution is injected by an autosampler and delivered at the flow rate of 150 μL/minute with the UHPLC system. The total run time on the LC–MS system is 3 minutes per sample (including injection and equilibration of the column).

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Table 3 Linearity, within-lab variation, and reference ranges determined using the TSQ Vantage (enzyme activity μmol/L/h) Linearity μmol/hr/L

Precision (%)

GAA

0.04–29.0

GLA

Reference range μmol/hr/L

% of mean

5.0–9.0

>1.00 15 < 6*

0.06–33.6

4.0–5.0

>1.75

>25

ABG

0.05–85.2

4.0–7.0

>1.00

>12

IDUA

0.04–29.0

6.0–8.0

>0.75

>15

3. Enzyme activity is calculated by dividing the measured enzymatic product intensities by those of the internal standards in the final sample analyzed according to the following equation: Enzyme activity ¼

Product intensity IS intensity 

ðIS concentration  IS volumeÞ  RRF ðblood volume  incubation timeÞ

Product and IS intensities (or integrated area) are in MS/ MS instrument (LC Quan); IS concentration is in μM; IS volume is 30 ul, and blood volumes are 3.1 μL; and incubation time is 18 hours. The constants in the equation are IS volume (30 μL) and blood volume (3.1 μL). A relative response factor (RRF) is set up as one that aligns with MS/MS instruments. Make sure that the IS concentrations (concentrations of internal standards) correspond to those given on the lot-specific quality control certificate. 4. The blank activity is subtracted from the measured activity to obtain the final activity of each sample. The blank activity is the average activity of blank filter paper samples on the same plate with samples. 5. The cutoff values mentioned in this chapter should only be used as a reference only, and each laboratory shall establish its own reference range and cutoff values. Do not use a cutoff value that is based on data collected at another site. 6. Cases with extremely low GAA activity should be referred and confirmed immediately (see Table 3). Treatment for patients with infantile Pompe disease should be initiated as early as possible before irreversible damage occurs [21].

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Notes 1. The transfusion cases should wait at least 1 week to collect the blood samples. 2. Internal standards will be the same concentrations in every lot. C1 (control level 1) will serve as positive patients, and the enzyme activities should be below the established cutoff values. The enzyme activities from C2 and C3 should fall into the linear ranges. 3. Do not shake or vortex the assay buffer or assay cocktail. If an assay cocktail looks hazy or particulates are visible, heat it briefly in warm tap water, and/or sonicate it at room temperature for 5 minutes. 4. All solutions and buffers should be prepared with HPLC grade water. Sterile filter buffers to remove particulates and debris. Buffers do not need to be handled as sterile solutions. Discard any solutions showing evidence of contamination. 5. Make sure the organic and aqueous phases are mixed homogeneously. This is a critical step for extraction efficiency. 6. The assay has been performed on other mass spectrometers, including Sciex 3200, Waters Quattro Micro, and TQD. Comparable results have been obtained on all instruments.

References 1. Meikle PJ, Hopwood JJ, Clague AE, Carey WF (1999) Prevalence of lysosomal storage disorders. JAMA 281(3):249–254. https://doi. org/10.1001/jama.281.3.249 2. Zarate YA, Hopkin RJ (2008) Fabry’s disease. Lancet 372(9647):1427–1435. https://doi. org/10.1016/S0140-6736(08)61589-5 3. van der Ploeg AT, Reuser AJ (2008) Pompe’s disease. Lancet 372(9646):1342–1353. https://doi.org/10.1016/S0140-6736(08) 61555-X 4. Tuttolomondo A (2020) Treatment of Lysosomal Storage Disorders (LSDs). Curr Pharm Des 26(40):5087–5088. https://doi.org/10. 2174/138161282640201111114453 5. Escolar ML, Poe MD, Provenzale JM, Richards KC, Allison J, Wood S, Wenger DA, Pietryga D, Wall D, Champagne M, Morse R, Krivit W, Kurtzberg J (2005) Transplantation of umbilical-cord blood in babies with infantile Krabbe’s disease. N Engl J Med 352(20): 2069–2081. https://doi.org/10.1056/ NEJMoa042604 6. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, Caplan L, Linthorst GE,

Desnick RJ, Group ICFDS (2001) Safety and efficacy of recombinant human alphagalactosidase A replacement therapy in Fabry’s disease. N Engl J Med 345(1):9–16. https:// doi.org/10.1056/NEJM200107053450102 7. Chien YH, Lee NC, Thurberg BL, Chiang SC, Zhang XK, Keutzer J, Huang AC, Wu MH, Huang PH, Tsai FJ, Chen YT, Hwu WL (2009) Pompe disease in infants: improving the prognosis by newborn screening and early treatment. Pediatrics 124(6):e1116–e1125. https://doi.org/10.1542/peds.2008-3667 8. Chamoles NA, Blanco M, Gaggioli D (2001) Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin Chim Acta 308(1–2):195–196. https://doi.org/10. 1016/s0009-8981(01)00478-8 9. Chamoles NA, Niizawa G, Blanco M, Gaggioli D, Casentini C (2004) Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper. Clin Chim Acta 347(1–2):97–102. https://doi.org/10. 1016/j.cccn.2004.04.009 10. Chien YH, Chiang SC, Zhang XK, Keutzer J, Lee NC, Huang AC, Chen CA, Wu MH,

Lysosomal Enzyme Activities on Dried Blood Spots Huang PH, Tsai FJ, Chen YT, Hwu WL (2008) Early detection of Pompe disease by newborn screening is feasible: results from the Taiwan screening program. Pediatrics 122(1): e39–e45. https://doi.org/10.1542/peds. 2007-2222 11. Hopkins PV, Campbell C, Klug T, Rogers S, Raburn-Miller J, Kiesling J (2015) Lysosomal storage disorder screening implementation: findings from the first six months of full population pilot testing in Missouri. J Pediatr 166(1):172–177. https://doi.org/10.1016/j. jpeds.2014.09.023 12. Li Y, Scott CR, Chamoles NA, Ghavami A, Pinto BM, Turecek F, Gelb MH (2004) Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 50(10):1785–1796. https://doi.org/10. 1373/clinchem.2004.035907 13. Li Y, Brockmann K, Turecek F, Scott CR, Gelb MH (2004) Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for Krabbe disease. Clin Chem 50(3):638–640. https:// doi.org/10.1373/clinchem.2003.028381 14. Blanchard S, Sadilek M, Scott CR, Turecek F, Gelb MH (2008) Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis I. Clin Chem 54(12):2067–2070. https://doi.org/ 10.1373/clinchem.2008.115410 15. Spacil Z, Tatipaka H, Barcenas M, Scott CR, Turecek F, Gelb MH (2013) High-throughput assay of 9 lysosomal enzymes for newborn screening. Clin Chem 59(3):502–511. https://doi.org/10.1373/clinchem.2012. 189936

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16. Gelb MH, Scott CR, Turecek F (2015) Newborn screening for lysosomal storage diseases. Clin Chem 61(2):335–346. https://doi.org/ 10.1373/clinchem.2014.225771 17. Liao HC, Chiang CC, Niu DM, Wang CH, Kao SM, Tsai FJ, Huang YH, Liu HC, Huang CK, Gao HJ, Yang CF, Chan MJ, Lin WD, Chen YJ (2014) Detecting multiple lysosomal storage diseases by tandem mass spectrometry–a national newborn screening program in Taiwan. Clin Chim Acta 431:80–86. https:// doi.org/10.1016/j.cca.2014.01.030 18. Schielen PCJI, Kemper EA, Gelb MH (2017) Newborn screening for lysosomal storage diseases: a concise review of the literature on screening methods, therapeutic possibilities and regional programs. Int J Neonatal Screen 3(2). https://doi.org/10.3390/ijns3020006 19. Hong X, Sadilek M, Gelb MH (2020) A highly multiplexed biochemical assay for analytes in dried blood spots: application to newborn screening and diagnosis of lysosomal storage disorders and other inborn errors of metabolism. Genet Med 22(7):1262–1268. https:// doi.org/10.1038/s41436-020-0790-9 20. Wasserstein MP, Orsini JJ, Goldenberg A, Caggana M, Levy PA, Breilyn M, Gelb MH (2021) The future of newborn screening for lysosomal disorders. Neurosci Lett 760: 136080. https://doi.org/10.1016/j.neulet. 2021.136080 21. Yang CF, Yang CC, Liao HC, Huang LY, Chiang CC, Ho HC, Lai CJ, Chu TH, Yang TF, Hsu TR, Soong WJ, Niu DM (2016) Very early treatment for infantile-onset Pompe disease contributes to better outcomes. J Pediatr 169:174–180.e171. https://doi.org/10. 1016/j.jpeds.2015.10.078

Chapter 25 Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry Brandon B. Stauffer and Chunli Yu Abstract Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann–Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC–MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann–Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC–MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lysoGb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann–Pick and successfully distinguished different subtypes reflecting the disease severity. Key words Glucosylsphingosine (lyso-GL1), Galactosylsphingosine (psychosine), Globotriaosylsphingosine (lyso-Gb3), Sphingosylphosphorylcholine (lyso-SPM), Liquid chromatography, Mass spectrometry, Gaucher disease, Niemann–Pick disease, Krabbe disease, Fabry disease

1

Introduction Sphingolipids make up a significant fraction of cell membrane lipid mass and are ubiquitous among mammalian cell types. They are characterized by the presence of a long-chain sphingoid base, with the prototypical example being sphingosine. During synthesis, the amine group in the second position of the sphingosine base is conjugated to a long-chain fatty acid in the endoplasmic reticulum to form ceramide. Upon reaching the Golgi apparatus, hydrophilic

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_25, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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groups are added to the hydroxyl group located at position 1 of ceramide. Sphingolipids are classified into sphingomyelins and glycosphingolipids depending on the chemical structures of the modification on 1-OH position. Sphingomyelin consists of phosphocholine (or phosphoethanolamine) and ceramide. It is a major phosphosphingolipid species in the outer leaflet of cell membranes and is abundant in the membranes that constitute the myelin sheath of the nervous system. Glycosphingolipids are derived from the addition of glycan moieties onto the ceramide backbone. Glycans can be either mono sugar (cerebrosides) or two to four sugar moieties (globosides). Other glycosphingolipid species include sulfatides (sulfated galactosylcerebroside) and gangliosides which include at least one sialic acid linked to the galactose residue on globosides. Deficiencies of the lysosomal enzymes that are required for the degradation and recycling of these sphingolipids result in sphingolipidoses. Excess accumulation of primary disease substrates is subject to deacylation by acid ceramidase leading to the formation of lysosphingolipids. These lysosphingolipid species are normally present at very low levels in human plasma but have been shown to be highly elevated in patients with sphingolipidoses and contribute to the pathophysiology in these diseases. Examples of lysosphingolipids include glucosylsphingosine (lyso-GL1) in Gaucher disease, galactosylsphingosine (psychosine) in Krabbe disease, globotriaosylsphingosine (lyso-Gb3) in Fabry disease, and sphingosylphosphorylcholine (lyso-SPM) in Niemann–Pick disease [1, 2]. The chemical structures of these lyso- species are illustrated in Fig. 1. Loss of glucocerebrosidase activity in Gaucher disease leads to the accumulation of glucosylceramide (GL1) and lyso-GL1 in monocytes and macrophages. The lipid-laden “Gaucher cells” have a characteristic “wrinkled-tissue-paper” or “crumpled-silk” appearance in cytoplasm. Their accumulation in the liver and spleen of Gaucher patients leads to the characteristic hepatosplenomegaly, while progressive infiltration of Gaucher cells into bone tissue results in the skeletal osteopenia, osteosclerosis, and osteonecrosis seen in these patients. Significant amounts of GL1 and lyso-GL1 are also present in the CNS in neuropathic Gaucher disease; however, plasma GL1 is only slightly elevated because most is bound to lipoproteins. Conversely, plasma lyso-GL1 has been found to be highly elevated (>200-fold) in clinically manifesting type I Gaucher patients [3–5]. The concentration tends to decrease with enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and their combination [5]. The decrease of plasma lyso-GL1 is reportedly associated with improvement of clinical parameters in multiple clinical trial studies [6, 7]. The value of plasma lyso-GL1 correlates with chitotriosidase, a well-established biomarker of macrophage activation and lipid storage that is commonly used for monitoring the disease state and therapeutical efficacy in

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273

Fig. 1 Structures of glucosylsphingosine (lyso-GL1), galactosylsphingosine (psychosine), globotriaosylsphingosine (lyso-Gb3), and sphingosylphosphorylcholine (lyso-SPM)

Gaucher patients [6]. Additionally, there is evidence supporting lyso-GL1 has value as a prognostic biomarker that is useful for the prediction of disease severity [5, 7]. In Krabbe disease, galactosylceramide elevation is less than its deacylated species psychosine. Psychosine is a neurotoxin and is absent in unaffected brain tissue but was shown to be elevated in brain tissue of early-onset Krabbe disease patients [8]. Recent studies have shown psychosine is also elevated in DBS samples taken from both early-onset and late-onset Krabbe patients. Elevations were more dramatic in patients with the infantile-onset form and tended to decrease following hematopoietic stem cell transplantation [9–11]. In Fabry disease, loss of α-galactosidase A activity leads to the accumulation of globotriaosylceramide (Gb3) in endothelial, epithelial, and smooth muscle cells of the vascular system diminishing function of the kidney, heart, eyes, and the autonomic nervous system. In this x-linked disease, female heterozygotes can be affected due to skewed random X-inactivation. As seen in Gaucher disease, the primary disease substrate Gb3 is only mildly elevated compared to the ~250-fold elevation of its deacylated derivative lyso-Gb3 [12]. Males with the attenuated form of the disease tend to have lesser elevations of lysoGb3 as compared to those carrying classic mutations [13]. Interestingly, plasma lyso-Gb3 levels are also increased in heterozygous females carrying classic mutations even in the context of normal

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enzyme activity suggesting lyso-Gb3 is a more sensitive biomarker for the diagnosis of female Fabry patients [14]. Finally, lyso-Gb3 levels tend to be reduced by ERT, particularly in males with classic disease [15] . In vitro studies have shown that lyso-Gb3 can inhibit α-galactosidase A activity and exposure of smooth muscle cells to lyso-Gb3 causes marked proliferation. These findings suggest important roles of lyso-Gb3 in disease pathophysiology of Fabry [16, 17]. In Niemann–Pick disease, deficiency of acid sphingomyelinase leads to the accumulation of sphingomyelin and secondary increases of cholesterol in monocytes and macrophages which ultimately affects multiple organ systems including the liver, spleen, nervous system, bone marrow, kidney, and lungs. Plasma lyso-SPM was shown to be elevated in the tissues and blood of Niemann–Pick patients [18]. Levels were reduced with ERT and tended to be higher in type A vs type B patients, though there was some overlap in the groups [19, 20]. Taken together, these lysosphingolipids show potential as valuable biomarkers of disease severity, progression, and/or treatment for these sphingolipidoses. In this chapter we describe two liquid chromatography tandem mass spectrometry (LC–MS/MS) methods to measure plasma concentrations of lyso-GL1, psychosine, lyso-Gb3, and lyso-SPM. Total preparation time is approximately 1 hour, and the LC–MS/ MS programs take 6 and 5 minutes per sample for lyso-GL1+psychosine and lyso-SPM+lyso-Gb3, respectively. Briefly, plasma is deproteinated by adding methanol containing isotope-labeled internal standards. After thorough mixing via vortex, precipitates are removed by centrifugation. Supernatant is then transferred to a 96-well plate and dried under nitrogen before being resuspended in initial mobile phase for analysis. Chromatographic separations are obtained by using either a BEH amide column (lyso-GL1+psychosine) or a reverse phase C18 column (lyso-SPM+lyso-Gb3). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. See Fig. 2 for distributions of lyso-GL1 in type I and III Gaucher patients, lyso-Gb3 in hemizygote Fabry patients and heterozygous females with classis GLA mutations, and lyso-SPM in Niemann–Pick, types A, A/B, and B, compared to normal controls.

2 2.1

Materials Samples

These assays were optimized for measurement of lysosphingolipids from plasma samples. Plasma can be collected from either heparinized or EDTA-treated whole blood and should be separated and frozen as soon as possible (see Note 1). Plasma should be kept frozen until testing is performed. Sample conditions (lipemic, icteric, or hemolysis) should be recorded; some analyte values may be impacted (see Note 2).

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275

Fig. 2 Distributions of lyso-GL1 in type I and III Gaucher patients, lyso-Gb3 in hemizygous (Hemi) and heterozygous (Het) Fabry patients carrying classic GLA mutations, and lyso-SPM in types A, A/B, and B Niemann–Pick patients compared to normal controls (NR)

2.2 Reagents and Buffers

1. Lyso-GL1 and psychosine. (a) Mobile phase A (10-mM ammonium acetate in 10% acetonitrile): Dissolve 0.77-g ammonium acetate in 900-mL LC–MS grade water, add 100-mL LC–MS grade acetonitrile, and mix. (b) Mobile phase B (10-mM ammonium acetate in 90% acetonitrile): Dissolve 0.77-g ammonium acetate in 100-mL LC–MS grade water, add 900-mL LC–MS grade acetonitrile, and mix. The mobile phase solutions should be made fresh and kept less than 2 weeks (see Note 3). When making mobile phase, ammonium acetate should be dissolved in water first then be mixed with acetonitrile (see Note 4). 2. Lyso-SPM and lyso-Gb3. (a) Mobile phase A (0.1% formic acid in water): Add 1 mL formic acid to 1 L of LC–MS grade water, and mix. (b) Mobile phase B (0.1% formic acid in methanol): Add 1-mL formic acid to 1 L of LC–MS grade methanol.

2.3 Standards and Calibrators

1. Lyso-GL1 and psychosine (Table 1). (a) Lyso-GL1 stock solution (1 mg/mL): Dissolve 1.0 mg lyso-GL1 in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (b) Lyso-GL1 intermediate solution (100 μg/mL): 100 μL of 1-mg/lyso-GL1 is diluted to 1 mL with methanol. (c) Lyso-GL1 working solution (1 μg/mL in plasma): 10 μL of 100 μg/mL lyso-GL1 intermediate solution is added to 990 μL of plasma. (d) Psychosine stock solution (1 mg/mL): Dissolve 1 mg psychosine in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years.

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Table 1 Lyso-GL1 and psychosine calibrator preparation

Calibrator

Concentration (ng/mL)

Additive

Plasma (mL)

Total volume (mL)

8

100

500 μL of each working solution

4

5

7

50

2.5 mL cal 8

2.5

5

6

25

2.5 mL cal 7

2.5

5

5

10

2.0 mL cal 6

3

5

4

1

0.5 mL cal 5

4.5

5

3

0.5

2.5 mL cal 4

2.5

5

2

0.2

2.0 mL cal 3

3

5

1

0.1

2.5 mL cal 2

2.5

5

Table 2 Lyso-Gb3 and lyso-SPM calibrator preparation

Calibrator

Lyso-Gb3/Lyso-SPM concentration (ng/mL)

8

100/500

7

Additive

Plasma (mL)

Total volume (mL)

1.2 mL of each working solution

9.6

12.0

50/250

5.25 mL cal 8

5.25

10.5

6

25/125

4.5 mL cal 7

4.5

9.0

5

10/50

3.0 mL cal 6

4.5

7.5

4

1/5

1.1 mL cal 5

9.9

11.0

3

0.5/2.5

5.0 mL cal 4

5.0

10.0

2

0.2/1.0

3.6 mL cal 3

5.4

9.0

1

0.1/0.5

3.0 mL cal 2

3.0

6.0

(e) Psychosine intermediate solution in methanol (100 μg/ mL): 100 μL of 1 mg/mL lyso-GL1 diluted to 1 mL with methanol. (f) Psychosine working solution in plasma (1 μg/mL): 10 μL of 100 μg/mL lyso-GL1 intermediate solution is added to 990 μL of plasma. 2. Lyso-Gb3 and lyso-SPM (Table 2). (a) Lyso-Gb3 stock solution (1 mg/mL): Dissolve 1 mg lysoGb3 in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years.

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(b) Lyso-Gb3 intermediate solution (100 μg/mL): 0.1 mL of 1 mg/mL lyso-Gb3 is diluted to 1 mL with methanol. Store at 20  C for up to 1 year. (c) Lyso-Gb3 working solution (1 μg/mL in plasma): 15 μL of 100 μg/mL lyso-Gb3 intermediate solution is diluted to 1.5 mL with plasma. Store at 20  C for up to 1 year. (d) Lyso-SPM stock solution (1 mg/mL): Dissolve 1 mg lyso-SPM in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (e) Lyso-SPM intermediate solution (500 μg/mL): 0.5 mL of 1 mg/mL lyso-SPM is diluted to 1 mL in methanol. Store at 20  C for up to 1 year. (f) Lyso-SPM working solution (5 μg/mL in plasma): 15 μL of 500 μg/mL lyso-SPM is diluted to 1.5 mL with plasma. Store at 20  C for up to 1 year. 2.4 Internal Standards

1. Lyso-GL1 and psychosine. (a) d5-Lyso-GL1 individual stock solution (1 mg/mL): Dissolve 1 mg d5-lyso-GL1 in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (b) d5-psychosine individual stock solution (1 mg/mL): Dissolve 1 mg d5-PSY in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (c) d5-Lyso-GL1 and d5-psychosine mixture stock solution (1 μg/mL): Dilute 0.01 mL each of 1 mg/mL d5-LysoGL1 and d5-psychosine to final volume of 10 mL with LC–MS grade methanol. Store at 20  C for up to 2 years. (d) d5-Lyso-GL1 and d5-psychosine mixture (1 ng/mL): Dilute 0.05 mL of 1 μg/mL d5-Lyso-GL1 and d5-psychosine mixture stock solutions to a final volume of 50 mL with LC–MS grade methanol. Store at 20  C for up to 3 months. 2. Lyso-Gb3 and lyso-SPM. (a) d7-Lyso-Gb3 individual stock solution (1 mg/mL): Dissolve 1 mg d7-lyso-Gb3 in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (b) d7-lyso-SPM individual stock solution (1 mg/mL): Dissolve 1 mg d7-lyso-SPM in 1 mL LC–MS grade methanol. Store at 20  C for up to 2 years. (c) d7-Lyso-Gb3 intermediate stock solution (1 μg/mL): Dilute 0.01 mL of 1 mg/mL d7-Lyso-Gb3 to final volume of 10 mL with LC–MS grade methanol. Store at 20  C for up to 2 years.

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(d) d7-lyso-SPM intermediate stock solution (1 μg/mL): Dilute 0.01 mL each of 1 mg/mL d7-lyso-SPM and psychosine to final volume of 10 mL with LC–MS grade methanol. Store at 20  C for up to 2 years. (e) 1 ng/mL d7-Lyso-Gb3 and 5 ng/mL d7-lyso-SPM working solution: Dilute 0.05 mL of 1 μg/mL d7-LysoGb3 and 0.25 mL of 1 μg/mL d7-lyso-SPM intermediate stock solutions to a final volume of 50 mL with LC–MS grade methanol. Store at 20  C for up to 6 months. 2.5

Quality Controls

1. Lyso-GL1 and psychosine. (a) QCH: Spike 1.0 mL of lyso-GL1 and 1.0 mL of psychosine working solutions (1 μg/mL) to 8.0 mL of plasma, and mix well. Make 60 μL aliquots and store at 80  C. The final concentration of each analyte is 100 ng/mL. (b) QCL: Dilute 100 μL of QCH with 9.90 mL plasma and mix well. Make 60 μL aliquots and store at 80  C. The final concentration of each analyte is 1 ng/mL. 2. Lyso-Gb3 and lyso-SPM. (a) QCH: Spike 500 μL of lyso-Gb3 working solution (1 μg/ mL) and 500 μL of lyso-SPM working (5 μg/mL) to 9.0 mL of plasma, and mix well. The final concentration of lyso-Gb3 is 50 ng/mL, whereas lyso-SPM is 250 ng/ mL. (b) QCL: Dilute 100 μL of QCH with 9.9 mL plasma and mix well. The final concentration of lyso-Gb3 is 0.5 ng/ mL, whereas lyso-SPM is 2.5 ng/mL.

2.6

Supplies

1. Microtube vortexer. 2. Microtube centrifuge. 3. Microcentrifuge tubes. 4. Nitrogen gas. 5. Micropipette. 6. 96-well microplates (300 μL volume, round bottom). 7. 96-well microplate closing mats. 8. Waters UPLC BEH Amide column, 2.1 mm  100 mm, 1.7 μm particle size (lyso-GL1 and psychosine). 9. Waters UPLC CSH C18 column, 2.1 mm  50 mm, 1.7 μm particle size (lyso-Gb3 and lyso-SPM).

2.7

Equipment

10. Agilent 6470B triple quadrupole mass spectrometer coupled to an HPLC and autosampler. 11. Microplate evaporator.

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3

279

Methods Mass spectrometer and chromatography setting are shown for lysoGL1 and psychosine in Table 3a–c and for lyso-Gb3 and lyso-SPM in Table 4a–c. Examples of extracted ion chromatograms for lysoGL1 and psychosine and lyso-GB3 and lyso-SPM are shown in Fig. 3a, b, respectively.

3.1 Lyso-GL1 and Psychosine

1. Remove patient samples, calibration aliquots, and quality control aliquots from 80  C to thaw at room temperature. 2. Generate a worksheet which includes blank, eight levels of calibrators, two levels of QCs, and patient samples. 3. Label 1.5 mL Eppendorf tube with sample sticker according to the worksheet. 4. Pipette 50 μL plasma samples into its respective test tubes; 50 μL water is used for blank sample. 5. Add 250 μL of internal standard working solution to each tube. 6. Vortex for 10 seconds. 7. Centrifuge at 20,000 rcf for 5 min. 8. Transfer 200 μL supernatant to 96-well plate, and dry under nitrogen at 40  C for 20 min. 9. Reconstitute with 50 μL mobile phase B and mix well. 10. Cover the plate tightly with a closing mat, and place in autosampler for analysis.

3.2 Lyso-Gb3 and Lyso-SPM

1. Remove patient samples, calibration aliquots, and quality control aliquots from 20  C to thaw at room temperature. 2. Generate a worksheet which includes blank, six levels of calibrators, two levels of QCs, and patient samples. 3. Label 1.5 mL Eppendorf tube with sample sticker according to the worksheet. 4. Add 50 μL of plasma samples to each labeled tube. 5. Add 250 μL of internal standard working solution to each tube. 6. Vortex for 10 seconds and wait for 5 min. 7. Centrifuge at 20,000 rcf for 5 min. 8. Transfer 200 μL supernatant to 96-well plate, and dry under nitrogen at 40  C for 20 min. 9. Reconstitute with 50 μL methanol. 10. Cover the plate tightly with a closing mat, and place in the autosampler for analysis.

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Table 3 Instrument settings and parameters for lyso-GL1 and psychosine A. LC–MS/MS instrument operating parameters for lyso-GL1 and psychosine LC settings Injection volume (μL)

1

Flow rate (mL/min)

0.4 

Column temperature ( C)

40

Mass spectrometer source settings Gas temperature ( C)

300

Gas flow (L/min)

10

Nebulizer (psi)

45 

Sheath gas temp ( C)

300

Sheath gas flow (L/min.)

10

Capillary voltage

4000

Nozzle voltage

500

B. Chromatography program for lyso-GL1 and psychosine Time (min.)

% Mobile phase B

Flow (ml/min.)

0

100

0.4

1.2

100

0.4

4.0

80

0.4

4.2

100

0.6

5.8

100

0.6

6.0

100

0.4

C. SRM parameters for lyso-GL1 and psychosine Compound

Precursor (m/z)

Product (m/z)

Fragmentor (V)

Collision energy

Polarity

Lyso-GL1

462.3

282.2

115

20

Positive

d5-lyso-GL1

467.3

287.2

115

20

Positive

Psychosine

462.3

282.2

135

20

Positive

d5-psychosine

467.3

287.3

135

25

Positive

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Table 4 Instrument settings and parameters for lyso-Gb3 and lyso-SPM A. LC–MS/MS instrument operating parameters for lyso-Gb3 and lyso-SPM LC settings Injection volume (μL)

1

Flow rate (mL/min)

0.4 

Column temperature ( C)

40

Mass spectrometer source settings Gas temperature ( C)

340

Gas flow (L/min)

10

Nebulizer (psi)

20 

Sheath gas temp ( C)

350

Sheath gas flow (L/min.)

10

Capillary voltage (V)

5000

Nozzle voltage (V)

500

B. Chromatography program for lyso-Gb3 and lyso-SPM Time (min.)

% Mobile phase B

Flow (ml/min.)

0

70

0.4

2.0

70

0.4

2.5

100

0.4

3.5

100

0.4

4.0

70

0.4

5.0

70

0.4

C. SRM parameters for lyso-Gb3 and lyso-SPM Compound

Precursor (m/z)

Product (m/z)

Fragmentor (V)

Collision energy

Polarity

Lyso-Gb3

786.2

282.2

180

35

Positive

d7-lyso-Gb3

793.5

289.3

180

35

Positive

Lyso-SPM

465.3

184.0

180

20

Positive

d7-lyso-SPM

472.3

184.1

180

20

Positive

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Fig. 3 Extracted ion chromatogram (EIC) of lysosphingolipids in the high-concentration quality control specimen (QCH). (a) 100 ng/mL lyso-GL1 and psychosine, along with their isotope-labeled internal standards. (b) 50 ng/mL lyso-Gb3, 250 ng/mL lyso-SPM, and their isotope-labeled internal standards

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Data Analysis

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1. Extracted ion chromatograms for lyso-GL1 and psychosine in QC material are shown in Fig. 3a. Separation of the isobaric cerebrosides is achieved with this chromatography procedure. Fig. 3b shows a similar panel of results for the lyso-Gb3 and lyso-SPM transitions. 2. Agilent MassHunter software is used for quantification. A linear equation is fit to the relative responses of analyte versus known concentration across the calibration samples and is used to calculate the final concentration in each sample. 3. The calibration curve is considered acceptable if r2 > 0.95 and calculated concentrations for each level of calibration is 85–115% of the expected concentration. 4. Intra- and inter-assay variability for these assays is 2.5 min), consider a new column. 2. If chromatography is poor (such as peaks that are excessively wide, have excessive tailing, or have low intensity), replace guard and analytical columns. 3. If poor chromatography persists with new columns, replace PEEK tubing. 4. If chromatography at analyte retention time makes it difficult to evaluate peak concentration, compare resulting concentration to the alternate transition for mannitol and lactulose. Currently, only one transition exists for rhamnose and sucralose. 5. The sucralose I.S. and analyte retention times consistently differ by 0.16–0.20 min. The analyte has the longer retention time.

References 1. Camilleri M (2019) Leaky gut mechanisms, measurement and clinical implications in humans. Gut 68(8):1516–1526. https://doi. org/10.1136/gutjnl-2019-318427 2. Lostia AM, Lionetto L, Principessa L, Evangelisti M, Gamba A, Villa MP, Simmaco M (2008) A liquid chromatography/mass spectrometry method for the evaluation of intestinal permeability. Clin Biochem 41(10–11):887–892. https://doi.org/10. 1016/j.clinbiochem.2008.03.016

3. Rao AS, Camilleri M, Eckert DJ, Busciglio I, Burton DD, Ryks M, Wong BS, Lamsam J, Singh R, Zinsmeister AR (2011) Urine sugars for in vivo gut permeability: validation and comparisons in irritable bowel syndromediarrhea and controls. Am J Physiol Gastrointest Liver Physiol 301(5):G919–G928. https:// doi.org/10.1152/ajpgi.00168.2011 4. Miki K, Butler R, Moore D, Davidson G (1996) Rapid and simultaneous quantification of rhamnose mannitol, and lactulose in urine

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by HPLC for estimating intestinal permeability in pediatric practice. Clin Chem 42(1):71–75 5. Barboza MS Jr, Silva TMJ, Guerrant RL, Lima AAM (1999) Measurement of intestinal permeability using mannitol and lactulose in children with diarrheal diseases. Braz J Med Biol Res 32:1499–1504. https://doi.org/10. 1590/s0100-879x1999001200008 6. Vilela EG, Torres HO, Ferrari ML, Lima AS, Cunha AS (2008) Gut permeability to lactulose and mannitol differs in treated Crohn’s disease and celiac disease patients and healthy subjects. Braz J Med Biol Res 41(12): 1105–1109. https://doi.org/10.1590/ s0100-879x2008001200010 7. Grover M, Camilleri M, Hines J, Burton D, Ryks M, Wadhwa A, Sundt W, Dyer R, Singh RJ (2016) 13C-mannitol as a novel biomarker for measurement of intestinal permeability. Neurogastroenterol Motil 28:1114–1119 8. Van Nieuwenhoven MA, Geerling BJ, Deutz NEP, Brouns F, Brummer R-JM (1999) The sensitivity of the lactulose/rhamnose gut permeability test. Eur J Clin Investig 29:160–165. https://doi.org/10.1046/j.1365-2362.1999. 00421.x 9. Fujii T, Seki T, Maruoka M, Tanaka J, Kawashima Y, Watanabe T, Sawamura T, Inoue K (2001) Lactulose-L-rhamnose intestinal permeability test in patients with liver

cirrhosis. Hepatol Res 19(2):158–169. https://doi.org/10.1016/S1386-6346(00) 00099-1 10. Sanderson IR, Boulton P, Menzies I, WalkerSmith JA (1987) Improvement of abnormal lactulose/rhamnose permeability in active Crohn’s disease of the small bowel by an elemental diet. Gut 28(9):1073–1076. https:// doi.org/10.1136/gut.28.9.1073 11. Faubion WA, Camilleri M, Murray JA, Kelly P, Amadi B, Kosek MN, Enders F, Larson J, Boe G, Dyer R, Singh R (2016) Improving the detection of environmental enteric dysfunction: a lactulose, rhamnose assay of intestinal permeability in children aged under 5 years exposed to poor sanitation and hygiene. BMJ Glob Health 1:e000066 12. Khoshbin K, Khanna L, Maselli D, Atieh J, Breen-Lyles M, Arndt K, Rhoten D, Dyer RB, Singh RJ, Nayar S, Bjerkness S, Harmsen WS, Busciglio I, Camilleri M (2021) Development and validation of test for "Leaky Gut" small intestinal and colonic permeability using sugars in healthy adults. Gastroenterology S0016-5085(21):00642–00649. https://doi. org/10.1053/j.gastro.2021.04.020. Epub ahead of print 13. CLSI (ed) (2009) Urinalysis, CLSI guideline GP16-A3, 3rd edn. Clinical and Laboratory Standards Institute, Wayne

Chapter 27 LC–MS/MS Method for High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine: Method for Analyzing Isomers Without Chromatographic Separation Mark M. Kushnir, Gordon J. Nelson, Elizabeth L. Frank, and Alan L. Rockwood Abstract Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-toinjection cycle time of approximately 1 minute. Key words Methylmalonic acid, Succinic acid, Isomers, Derivatization, Liquid chromatography, Tandem mass spectrometry, Data analysis, Deconvolution

1

Introduction Vitamin B12 is a water-soluble vitamin that plays an essential role in proper functioning of the nervous system, production of red blood cells, and DNA. The human body does not make vitamin B12; the only sources of vitamin B12 are animal-derived foods and supplements. Vitamin B12 is also an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_27, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of methylmalonic acid (MMA) [1]. Longterm vitamin B12 deficiency causes demyelination of nerves that lead to serious and often irreversible neurological and cognitive disorders (dementia, psychiatric illness, megaloblastic anemia, suppressed immune system, etc.) [1–4]. Therefore, measurement of MMA plays an important role in diagnosing vitamin B12 deficiency [1]. Common causes of vitamin B12 deficiency include gastrointestinal diseases (e.g., gastritis, Crohn’s disease, celiac disease), excessive use of alcohol, long-term use of certain medications, malnutrition, malabsorption, and stress. Some of the classes of medications known to cause vitamin B12 deficiency are macrolide antibiotics, oral contraceptives, proton pump inhibitors, antihypertensives, and anticonvulsants. Higher prevalence of vitamin B12 deficiency among patients with type 2 diabetes, who were treated with metformin, has been reported in a recent study [4]. A moderately increased MMA concentration (greater than 0.4 μM in serum or plasma and greater than 3.6 mmol/mol creatinine in urine) is an early indicator of acquired vitamin B12 deficiency; a massive elevation of MMA in serum, plasma, or urine (100- to 1000-fold above the concentrations characteristic for vitamin B12 deficiency) is indicative of methylmalonic acidemia, an inborn metabolic disorder [2]. An accurate prevalence of vitamin B12 deficiency is difficult to estimate because published reports are based on diverse inclusion criteria and methods. Prevalence of vitamin B12 deficiency reported for elderly individuals in the Framingham Heart Study was 12% [5]. Both serum MMA and serum cyanocobalamin measurements can be used to detect B12 deficiency; however, multiple studies have demonstrated that MMA is a better biomarker of vitamin B12 deficiency than serum cyanocobalamin [1–3]. Advantages of measuring MMA instead of cobalamin include (i) MMA concentrations are indicative of vitamin B12 status in tissues, while concentration of vitamin B12 in serum or plasma may not adequately reflect tissue cobalamin status; (ii) concentration of MMA in serum is up to 1000 fold greater than serum cyanocobalamin concentration; (iii) increased (rather than decreased) MMA concentration is found in vitamin B12 deficiency, and this places less demand on the analytical sensitivity of the method; (iv) MMA is more stable than cyanocobalamin; (v) elevated MMA represents a functional deficiency, while concentration of cyanocobalamin in blood represents B12 status at the time of specimen collection; and (vi) concentrations of cyanocobalamin may not be indicative of vitamin B12 status in patients whose blood is tested soon after a vitamin B12 injection. The major obstacle for MMA analysis in biological fluids is the potential interference from other low molecular weight organic acids, especially from the naturally occurring structurally related

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isomer, SA, which is present in the blood and urine at a significantly higher concentration than MMA. SA interference with MMA in mass spectrometry-based methods is difficult to overcome, because the chromatographic retention characteristics and mass spectra of SA are almost identical to those of MMA. Chromatographic separation of peaks of MMA and SA requires a lengthy analysis time; injection-to-injection time in reported methods is 4–7 min per sample [6–11]. In past, the method of choice for measuring MMA was gas chromatography–mass spectrometry (GC-MS) [6, 7]. The main disadvantage of GC-MS methods for MMA measurement is relatively low instrument throughput. Using liquid chromatography tandem mass spectrometry (LC–MS/MS), organic acids are typically analyzed in negative ion mode. In methods using negative ion mode detection, all organic acids are ionized in the ion source and may be detected in the mass spectrometer [8–11]. The majority of endogenous organic acids are chromatographically retained longer than MMA; therefore, such methods require extensive column conditioning and re-equilibration after every injection. The described method utilizes extraction that is specific to acidic compounds, while derivatization and positive ion mode ionization make only polycarboxylic acids (MMA and SA are dicarboxylic acids) ionizable and detectable using tandem mass spectrometry [12–14]. In addition, this method takes advantage of distinctive fragmentation of the dibutyl MMA and dibutyl SA derivatives (Fig. 1), which in combination with mathematical deconvolution [15, 16] allows high-throughput quantitative analysis of MMA without the need for chromatographic separation of the MMA and SA peaks. Compared to other published methods, the present LC–MS/MS method allows increased throughput of analysis by five- to tenfold, which is a significant practical advantage for high-throughput clinical laboratories.

2 2.1

Materials Samples

1. Serum or plasma (sodium heparin or EDTA anticoagulant): Samples are stable for 4 days if refrigerated or 6 months frozen at or below
20  C. 2. Timed (24-hour) or random urine: Samples are stable for 4 days refrigerated or 6 months frozen at or below
20  C (see Note 1).

2.2 Reagents and Buffers

1. MMA and SA (Sigma-Aldrich). 2. Methyl-d3-malonic acid (d3-MMA) (Cambridge Isotope). 3. 3 M Hydrochloric acid in 1-butanol (Regis Technologies) (see Note 2).

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Fig. 1 Product ion mass spectra of protonated molecular ion of the n-dibutyl esters of (a) MMA, (b) SA, and (c) mixture of MMA and SA. Ratio of the MMA/SA concentrations 1:50 (this is comparable to physiologically observed concentrations of MMA and SA)

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4. Extraction solvent (3% phosphoric acid in MTBE): To a 4-L bottle of MTBE, add 84 mL of concentrated phosphoric acid (85%, specific gravity 1.685 g/mL). Cap and mix thoroughly. Stable for 2 weeks at ambient temperature. 5. Dialyzed plasma (MMA and SA free): Stable for 1 year at or below
20  C (see Note 3). 6. Synthetic urine: In a 1-L glass beaker containing 1 L water, dissolve 2.8 g of K2HPO4, 0.55 g of NaH2PO4, 7 g of NaCl, 20 g of urea, and 1 g of creatinine. Mix solution until completely dissolved. Stable for 2 years at or below
20  C. 7. Mobile phase (5 mM ammonium formate in 15% water/85% methanol): In a 1-L bottle, add 0.315 g ammonium formate and 150 mL water; mix until dissolved. Using separate graduated cylinders, measure 850 mL of methanol, transfer into the bottle, mix, and filter through a 0.5-μm filter. Stable for 3 days at ambient temperature. 8. Needle wash solution [methanol: 2-propanol: water, 60:20:20 (v/v)]. Using separate graduated cylinders, measure 600 mL methanol, 200 mL 2-propanol, and 200 mL water; transfer into a 1-L glass bottle, mix, and cap. Stable for 10 days at ambient temperature. 2.3 Standards and Calibrators

1. 10 mM MMA stock calibration standard: Weigh 0.0118 g methylmalonic acid (if needed, account for the purity of the standard), transfer to a 10-mL volumetric flask, and fill to volume with methanol. Mix by inversion until the solid is dissolved. Stable for 1 year at or below
20  C (see Note 3). 2. 10 μM MMA working calibration standard: Add 100 μL of 10 mM MMA stock calibration standard into a 100-mL volumetric flask containing approximately 50 mL water. Fill to volume with nanopure water. Aliquot 400 μL in microcentrifuge tubes. Stable for 1 year at or below
20  C (see Note 3). 3. 10 mM d3-MMA stock internal standard: Weigh 0.0121 g (12.1 mg) of MMA d3 to a 10-mL volumetric flask. Fill to volume with methanol. Stable for 2 years at or below
20  C (see Note 3). 4. 15 μM d3-MMA working internal standard: To a 500-mL volumetric flask, add 400 mL nanopure water and 750 μL d3-MMA stock internal standard. Fill to volume with nanopure water and mix. Aliquot 5 mL in polypropylene tubes. Stable for 1 year at or below
20  C (see Note 3). 5. 10 mM SA stock standard: Weigh 0.0118 g succinic acid in a 10-mL volumetric flask. Fill to volume with methanol. Stable for 2 years at or below
20  C (see Note 3).

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6. 200 μM SA-dibutyl derivative: Transfer 100 μL of SA stock standard into a 10-mL glass tube and evaporate the solvent at room temperature. Add 40 μL of the derivatizing reagent and incubate at 70  C for 10 minutes. Evaporate the derivatizing reagent; reconstitute the residue with 5.0-mL water/methanol (15:85). Stable for 3 months refrigerated. 2.4 Quality Control (QC) Samples

1. Dialyzed plasma spiked with 6 μM succinic acid (used as a matrix for preparation of quality controls): Add 120 μL succinic acid stock standard to 200 mL dialyzed plasma. Mix for 30 minutes. Stable for 6 months at or below
20  C (see Note 4). 2. 10 mM MMA stock standard for preparation of QC: Weigh 0.0118 g methylmalonic acid, add to a 10 mL volumetric flask, and fill to volume with methanol. Mix by inversion until completely dissolved. Stable for 1 year at or below
20  C (see Note 3). 3. 1 mM MMA working standard for preparation of QC: In a 10-mL volumetric flask, add 1000 μL MMA stock standard, add nanopure water to volume, and mix. Stable for 1 week refrigerated. 4. 0.4 μM MMA and 6-μM SA (control I, serum/plasma): Add 500 mL dialyzed plasma into a 1-L beaker with a stir bar, and begin mixing. Add 200 μL of MMA working standard for preparing QC (1 mM), and stir for 15 minutes. Add 300 μL succinic acid stock standard (10 mM), cover, and mix for additional 30 minutes Aliquot in 1.5 mL microcentrifuge tubes. Stable for 1 year at or below
20  C. 5. 1 μM MMA and 6 μM SA (control II, serum/plasma): Add 500 mL dialyzed plasma in a beaker with a stir bar, add 50 μL MMA stock for preparation of QC, and mix for 15 minutes. Add 300 μL succinic acid stock standard, cover, and mix for additional 30 minutes. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below
20  C (see Note 3). 6. Negative control. Dialyzed plasma (free of MMA). 7. 10 μM MMA and 6-μM SA (control III, urine): Into a 500-mL beaker, pour 250 mL of synthetic urine, add 250 μL of MMA stock standard, and mix for 15 minutes. Add 150 μL succinic acid stock standard, fill to volume with synthetic urine, cover, and mix for additional 30 minutes. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below
20  C (see Note 3). 8. 20 μM MMA and 6-μM SA (control IV, urine): Into a 500-mL beaker, pour 250 mL of synthetic urine, add 500 μL of MMA stock standard for preparation of QC, and mix for 15 minutes. Add 150 μL succinic acid stock standard, fill to volume with

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synthetic urine, cover, and mix for additional 30 minutes. Aliquot 1.5 mL into microcentrifuge tubes. Stable for 1 year at or below
20  C (see Note 3). 2.5

Equipment

1. Triple quadrupole mass spectrometer AB3200 with TurboV ion source (AB Sciex, Foster City, CA) with built-in switching valve. 2. Binary HPLC pump series 1260 (Agilent Technologies, Santa Clara, CA), vacuum degasser, and autosampler CTC PAL (Carrboro, NC) equipped with fast-wash station. 3. Vortex with adaptor for microcentrifuge tubes. 4. Evaporator for 96-well plates. 5. Centrifuge for microcentrifuge tubes. 6. Centrifuge with buckets for 96-well plates. 7. Shaker for 96-well plates.

2.6

Supplies

1. Microcentrifuge tubes, 2 mL (Eppendorf, Westbury, NY). 2. Deep 96-well plates (2 mL well volume) and sealing mats for the plates (Phenomenex, Torrance, CA). 3. Transfer pipettes. 4. HPLC column: Luna C18 30 mm  3 mm, 5 μm particles; SecurityGuard cartridge holder and C18 cartridges (Phenomenex, CA).

3 3.1

Methods Procedure

1. Label a set of 2 mL microcentrifuge tubes. 2. Prepare calibrators and negative control by adding working calibration standard and dialyzed plasma to the corresponding labeled tubes: (a) Aliquot in the tubes 500 μL of dialyzed plasma. (b) Add in the tubes working calibration standard in amounts according to Table 1. 3. Aliquot patient samples and controls into labeled tubes: (a) Serum/plasma samples: 500 μL of patient sample or control. (b) Urine: 50 μL of patient sample or control and 450 μL of nanopure water. 4. Add to each tube 50 μL of working internal standard. 5. Add to each tube 1 mL extraction solvent (MTBE/3% phosphoric acid), and cap tubes.

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Table 1 Preparation of calibration standards Concentration of standard, μM

Working calibration standard, μL

Dialyzed plasma, μL

0.20

10

500

0.40

20

500

0.80

40

500

1.00

50

500

1.50

75

500

2.00

100

500

6. Set rack with tubes on platform of vortex mixer and shake for 5 minutes. 7. Centrifuge the tubes at 14,000 rpm for 3 minutes. 8. Transfer the top organic layer from the tubes into the corresponding wells of 96-well plate. 9. Set 96-well plate on evaporator and evaporate organic phase (50  C) until completely dry. 10. Add into each well of the plate 40 μL of derivatizing reagent (3 M HCl in 1-butanol) and cover the plate with a sealing mat. 11. Incubate the plate at 70  C for 10 minutes. 12. Remove the plate from the incubator and take off the mat. 13. Set 96-well plate on evaporator and evaporate organic solvent (50  C). 14. Add in each well 200 μL of 5 mM ammonium formate in methanol/water (1:1); cover plate with the sealing mat. 15. Set plate on shaker for 96-well plates and vortex on medium setting for 3 min. 16. Centrifuge plate for 1 min at 4000 g. 17. Inject the samples. 3.2

LC–MS/MS

1. Mobile phase flow rate 750 μL/min; flow diverted to waste before 0.3 min and after 1 min. 2. Mobile phase gradient is shown in Table 2 (see Note 4). 3. Injection volume 20 μL. 4. Autosampler syringe wash solution: 60% methanol, 20% isopropanol, 20% water. 5. Instrumental analysis was performed in positive ion mode using electrospray ion source on a triple quadruple mass spectrometer 3200 (AB Sciex, Framingham, MA). Mass transitions

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Table 2 Mass transitions Compound

Primary mass transition, m/z

Secondary mass transition, m/z

MMA

231.2 to 119.1

231.2 to 175.1

d3-MMA

234.2 to 122.1

234.2 to 178.1

are listed in Table 2; dwell time for each mass transition is 150 ms. 6. Voltages and gas flow rates for the mass spectrometer were optimized for maximum sensitivity and were as follows: (a) Ionspray voltage: 5000 V. (b) Ion source temperature: 450  C. (c) Nebulizer gas: 60, heating gas: 60. (d) Collision gas: 6 (arbitrary units). (e) Declustering potential: 50 V. (f) Entrance potential: 10 V. (g) Collision energy: 15 V. (h) Collision cell exit potential: 6 V. (i) Mass analyzers Q1 and Q3 are set for unit resolution (0.7 Da width at 50% height). 3.3

Data Analysis

Data analysis is performed using Analyst™ 1.7 software (AB Sciex) and a spreadsheet for deconvolution of signal corresponding to the peak area of MMA from the total peak area of MMA/SA. Calculations are performed using peak areas of the two mass transitions of MMA/SA and d3-MMA. The algorithm and equations used for deconvolution of the signal corresponding to MMA are described in Notes 5 and 6. 1. Export from Analyst to Microsoft Excel worksheet (Fig. 2) table with peak areas of mass transitions m/z 231.2 to 119.1 and m/z 231.2 to 175.1 (MMA/SA) and m/z 234.2 to 122.1and m/z 234.2 to 178.1 (d3-MMA). 2. Coefficients of regression equation and correlation coefficient are displayed in cells O13–O15 (Fig. 2). 3. The calculated deconvoluted peak area corresponding to the signal of MMA is shown in columns “I” (m/z 231.2 to 119.1) and “J” (m/z 231.2 to 175.1). 4. Calculated MMA concentration is shown in column “M.” 5. The test results are considered acceptable if the correlation coefficient (r) for the calibration curve is greater than 0.995. The calculated ratio of the peak areas of two mass transitions

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Fig. 2 Example of an Excel worksheet for calculating concentrations of isomers from unresolved chromatographic peaks

d3-MMA should be within 30% of the mean value of the ratio observed in the calibration standards of the batch [16, 17]. Concentration of MMA in the negative control must be below the limit of quantitation of the method, concentration of MMA in the controls should be within the limits of the QC rules established by the laboratory. 6. Method performance characteristics: total CV of the method is below 10%; limit of quantitation 0.1 μM; upper limit of linearity 150 μM. 7. Reference intervals: serum/plasma 313.3), 5-MTHF 2 (m/z 460.3 > 194.5), and 5-MTHF-13C5 (m/z 465.3 > 313.3)]

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Fig. 2 HPLC–ESI–MS/MS ion chromatogram of 5-MTHF abnormal QC (30 nM) [5-MTHF 1 (m/z 460.3 > 313.3), 5-MTHF 2 (m/z 460.3 > 194.5), and 5-MTHF-13C5 (m/z 465.3 > 313.3)] 3.2

Data Analysis

1. Instrumental operating parameters are given in Table 1a, b. 2. Data are analyzed using Analyst software version 1.6.2. 3. Standard curves are generated based on linear regression of the analyte/I.S. peak-area ratio (y) versus analyte concentration (x) using the primary ions indicated in Table 2. The curve is weighted 1/x. 4. Acceptability of each run is confirmed if quality controls are within previously defined limits using Westgard rules. Intra-day and inter-day precision was evaluated by analysis of bi-level quality control material with CV < 4% and CV < 6%, respectively. 5. Confirmatory analysis based on the ratio of two MRM transitions 5-MTHF 1 (primary)/5-MTHF 2 (secondary) should be 20% of the established value for the assay based upon the calibration curve.

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Table 1 HPLC–ESI–MS/MS operating conditions A. HPLC (SAM and SAH)a Column temp.

40°C

Flow rate

0.375 mL/min

Gradient

Time (min) 0.0 1.5 2.0 2.1

Mobile Phase A (%) 100 0 0 100

B. MS/MS Tune Settings b Entrance Potential (V) Curtain gas (psi) CAD gas Ion Spray (V) Temp (°C) GS1 (psi) GS2 (psi) Resolution Q1 and Q3

10 20 Medium 5500 500 50 50 Unit

a

Optimized for Shimadzu Prominence liquid chromatography system equipped with Phenomenex SynergiHydro, 4 μm, 150  3 mm analytical column; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol. b Optimized for Sciex 40000QTRAP®. Tune settings may vary slightly between instruments.

Table 2 HPLC–ESI–MS/MS operating conditions

Compound

MRM transition Q1 Q3 (m/z) (m/z)

Dwell time DP (msec) (V)

CE (V)

CXP (V)

5-MTHF 1

460.3a

313.3a,b

150

96

29

18

5-MTHF 2

a

460.3

194.5a,c

150

96

48

8

13

a

a,b

150

96

29

18

5-MTHF- C5

465.3

313.3

a

Optimized m/z may change based on tuning parameters and instrument used Primary ion for 5-MTHF quantitation c Secondary ion used for MRM ratio confirmation b

6. The assay has a lower limit of quantitation of 3 nM for 5-MTHF, with imprecision of 10 years

5-MTHF (nmol/L) 40 - 240 40 - 187 40 - 150 40 - 128 40 - 120

Notes 1. Individual sets of 5-MTHF standard and internal standard stock solutions and controls can be pre-aliquoted and frozen until use in each analytical run. For each set, pipette specified volume of stock standards/control solution into 1.5-mL microfuge tubes, and freeze at 80  C until use. Thaw at room temperature 18–24  C completely before us; do not use heat or water to accelerate the process. Stable for 4 years at 80  C. 2. A new standard curve should be prepared with each analytical run to optimize method performance. 3. The controls are analyzed at the beginning and end of the assay as analysis verification. 4. Ion suppression effects were evaluated by sample infusion method. No significant interferences or ion suppression was identified.

References 1. Antony A (1992) The biological chemistry of folate receptors. Blood 79(11):2807–2820 2. Westerhof G, Rijnboutt S, Schornagel J, Pinedo H, Peters G, Jansen G (1995) Functional activity of the reduced folate carrier in KB, MA104, and IGROV-I cells expressing folate-binding protein. Cancer Res 55(17): 3795–3802 3. Serrano M, Perez-Duenas B, Montoya J, Ormazabal A, Artuch R (2012) Genetic causes of cerebral folate deficiency: clinical, biochemical and therapeutic aspects. Drug Discov Today 17(23–24):1299–1306 4. Pope S, Artuch R, Heales S, Rahman S (2019) Cerebral folate deficiency: analytical tests and

differential diagnosis. J Inherit Metab Dis 42: 655–672 5. Sequeira JM, Ramaekers VT, Quadros EV (2013) The diagnostic utility of folate receptor autoantibodies in blood. Clin Chem Lab Med. 51(3):545–554 6. Ramaekers VT, Blau N (2004) Cerebral folate deficiency. Dev Med Child Neurol 46(12): 843–851 7. Ramaekers VT, Hausler M, Opladen T, Heimann G, Blau N (2002) Psychomotor retardation, spastic paraplegia, cerebellar ataxia and dyskinesia associated with low 5-methyltetrahydrofolate in cerebrospinal fluid: a novel neurometabolic condition

Quantitation of 5-Methyltetrahydrofolate in CSF responding to folinic acid substitution. Neuropediatrics 33(6):301–308 8. Ormazabal A, Garcia-Cazorla A, PerezDuenas B, Gonzalez V, Fernandez-Alvarez E, Pineda M, Campistol J, Artuch R (2006) Determination of 5-methyltetrahydrofolate in cerebrospinal fluid of paediatric patients: reference values for a paediatric population. Clin Chim Acta 371:159–162 9. Al-Baradie R, Chudary M (2014) Diagnosis and management of cerebral folate deficiency:

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a form of folinic acid-responsive seizures. Neurosciences 19(4):312 10. Willemse E, Vermeiren Y, Garcia-Ayllon M, Bridel C, De Deyn P, Engelborghs S, van der Flier W, Jansen E, Lopez-Font I, Mendes V, Manadas B, de Roeck N, Saez-Valero J, Struys E, Vanmechelen E, Andreasson U, Teunissen C (2019) Pre-analytical stability of novel cerebrospinal fluid biomarkers. Clin Chim Acta 497:201–211

Chapter 29 Screening of Organic Acidurias by Gas Chromatography–Mass Spectrometry (GC–MS) David Scott, C. Clinton Frazee III, and Uttam Garg Abstract Organic acidurias or acidemias are a group of diverse disorders caused by decreased or diminished activity of specific enzyme or transporter involved in the metabolism of amino acids, carbohydrates, fatty acids, and nucleic acids. Organic acidurias are generally inherited but may be acquired due to deficiency of certain cofactors or vitamins. As clinical symptoms are of nonspecific nature, definitive diagnosis of organic aciduria requires measurement of organic acids in urine or blood and sometimes enzyme activity in the cells. Gas chromatography–mass spectrometry (GC–MS) is a commonly used method for screening of organic acidurias. GC–MS procedure described here involves the use of urine volume that contains 1 μmole (113 μg) of creatinine. Internal standards (tropic and 2-ketocaproic acids) are added to the samples, followed by treatment with hydroxylamine to form oxime derivatives of the ketoacids. The mixture is then acidified, and organic acids are extracted in ethyl acetate. The organic extract is concentrated to dryness, and the residue is treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS)/pyridine to form the trimethylsilyl (TMS) derivatives of the organic acids. The derivatized extract is then directly injected onto GC–MS for analysis. Key words Organic acidurias, Organic acidemias, Trimethylsilyl derivatization, Gas chromatography–mass spectrometry

1

Introduction Organic acidurias, commonly also referred as organic acidemias, are a group of diverse disorders caused by deficiency of specific enzyme or transporter involved in the metabolism of carbohydrates, amino acids, fatty acids, and nucleic acids [1–4]. Most of the organic acidurias are inherited but may be acquired due to deficiency of certain cofactors or vitamins. As clinical symptoms of organic acidurias are nonspecific, definitive diagnosis requires measurement of organic acids in urine or blood. Further workup may involve genetic testing or measurement of enzyme activity in fibroblasts, leucocytes, and other tissue cells [1, 3, 5]. Once the diagnosis is

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_29, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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made, organic acids may be measured for follow-up of clinical management of diagnosed patients. Gas chromatography–mass spectrometry (GC–MS) is a commonly used method for the measurement of organic acids in urine or blood [2, 6–9]. Urine is a preferred sample as organic acids are excreted and concentrated in urine. In this chapter, we describe a GC–MS method for the estimation of urine organic acids. Urine volume containing a specific amount of creatinine (e.g., 1 μmole, 113 μg) is used for the organic acid analysis. Internal standards (2-ketocaproic acid and tropic acid) are added to the samples to account for differences in extraction efficiency, any interferences, and other factors. This is followed by treatment with hydroxylamine to form stable oxime derivatives of the ketoacids. The mixture is then acidified, and organic acids are extracted in ethyl acetate. The organic extract is concentrated to dryness, and the residue is treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS)/pyridine to form the trimethylsilyl (TMS) derivatives of the organic acids. The derivatized extracts are then directly injected onto GC–MS for analysis. Organic acids are identified by their retention time and mass spectra.

2 2.1

Materials Samples

2.2 Reagents and Chemicals

5-mL random urine. Freeze sample at

20  C as soon as possible.

1. Ethyl acetate, analytical grade. 2. 6.0-N HCL: Add 50-mL deionized water to a 100-mL volumetric flask and qs with concentrated HCL (stable for 1 yr. at room temperature). 3. 0.1-N HCL: Add ~50-mL deionized water to a 100-mL volumetric flask. Add 0.83-mL concentrated HCL to the flask and qs with deionized water (stable 1 yr. at room temperature). 4. 6.0-N NaOH: Add 30-mL deionized water to a 100-mL volumetric flask. Add 60 mL of 10-N NaOH and qs to 100 mL with deionized water (stable 6 mos.). 5. 250-mg/mL Hydroxylamine-HCl: Add 500 mg of hydroxylamine-HCl to a 16  100 mm tube. Add 2-mL deionized water and mix (see Note 1). 6. Derivatization reagent: BSTFA with 1% TMCS: pyridine (2:1). 7. Lyphochek-2 urine control (Bio-Rad). 8. ERNDIM, MCA control (Winterswijk, Netherlands). 9. Organic acids, see Table 1.

Screening of Organic Acidurias by GC-MS

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Table 1 Organic acid standards for calibration 2-Ketoadipic

4-OH-Phenylacetic

Malic

2-Ketoglutaric

4-OH-Phenyllactic

Malonic

2-Ketoisocaproic

Aconitic

Methylmalonic

2-Ketoisovaleric

Adipic

Methylsuccinic

2-OH-3-Methylvaleric

Benzoic

N-Acetylaspartate

2-OH-butyric

Citric

N-Acetyltyrosine

2-OH-Glutaric

Ethylmalonic

Orotic

2-OH-Isocaproic

Fumaric

Phenylacetic

2-OH-Isovaleric

Glutaric

Phenyllactic

3-Methyladipic

Glyceric

Pimelic

3-Methylglutaric

Glycolic

Pyruvic

3-OH-butyric

Hippuric

Succinic

3-OH-Isovaleric

HVA

Succinylacetone

3-OH-Octanoic

Lactic

VMA

4-OH-butyric

2.3 Standards and Calibrators

1. For each available organic acid (see Table 1), four calibrators at the concentrations of 50, 100, 400, and 800 nmoles/tube are prepared. These concentrations are equivalent to patients’ results of 50, 100, 400, and 800 moles/mole of creatinine, when 1 μmole of creatinine is used for each sample. 2. For unavailable organic acids and organic acids with stability and/or solubility problems, quantitation is performed by comparing the counts of total ion chromatogram of tropic acid (50 nmoles/tube) with the counts of total ion chromatogram of the analyte. This is further corrected by accounting for the difference in contribution of the quant ion to the total ion chromatogram (TIC) area, estimated or observed extraction efficiency of the compound, difference in the molecular weight of the derivatized tropic acid, and the derivatized compound (see Note 2).

2.4

Quality Controls

1. Bio-Rad Lyphochek-2 urine control: Reconstitute with 10-mL deionized water, and add 80-μL 6-N HCl. Make 1-mL aliquots and store at 20  C (stable 1 year). 2. Abnormal urine control (or spiked urine): Previously diagnosed patients’ samples with known organic acidurias are pooled to make this control.

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3. ERNDIM, MCA Control (Winterswijk, Netherlands): Reconstitute with 5-mL deionized water, and add 40-μL 6-N HCl. Make 0.5-mL aliquots and store at 20  C (stable 1 year). 4. Negative control: Deionized water. 2.5 Internal Standards

4. Internal standard (0.5-mM tropic acid and 1.0-mM 2-ketocaproic acid: Weight 83 mg of tropic acid, and dissolve in 500 mL of 0.1 N HCL (this will yield a 1.0-mM solution). Weigh out 152 mg of 2-ketocaproic acid, and dissolve in 500 mL of 0.1 N HCL (this will yield a 2.0-mM solution). Mix equal portions of the tropic acid solution with the 2-ketocaproic acid solution, and store at 20  C (stable 1 year).

2.6

1. 16  100 mm tubes with snap caps.

Supplies

2. 13  100 mm tubes with screw caps. 3. Glass transfer pipets. 4. Autosampler vials (12  32 mm), crimp caps, and 0.3-mL tapered bottom glass inserts (P.J. Cobert Associates, Inc., St. Louis, MO). 5. GC column: Zebron ZB-1MS with dimensions of 30 m  0.25 mm  0.25 μm (Phenomenex, Torrance, CA). 6. Nitrogen: Tank or laboratory supply from liquid nitrogen. 2.7

Equipment

1. TurboVap® LV Evaporator (Biotage, Uppsala, Sweden). 2. Lab-Line Multi-Blok® Heater 2052 (Lab-Line Instruments, Inc., Melrose Park, IL). 3. A gas chromatograph/mass spectrometer (GC–MS), model 7890A/5975, operated in electron impact mode (Agilent Technologies, Santa Clara, CA).

3

Methods

3.1 Stepwise Procedure

1. Centrifuge urine to remove any debris or sediment. Analyze urine for creatinine using standard chemistry analyzer. 2. Pipet a volume of patient urine containing 1-μmole (113 μg) creatinine into a 13  100 mm tube (see Note 3). 3. Add deionized water to bring volume to 2 mL. 4. Add 100 μL of internal standard (0.5-mM tropic acid and 1.0mM 2-ketocaproic acid). This contains 50 nmoles of tropic acid and 100 nmoles of 2-ketocaproic acid. 5. Add 100 μL of 0.25-g/mL hydroxylamine-HCl in DI water (see Note 1).

Screening of Organic Acidurias by GC-MS

325

6. Add 200 μL of 6.0-M NaOH to each tube. 7. Cap the tubes (Teflon-lined preferred), and place in a heating block at 60  C for 30 min. 8. Cool the tubes to room temperature. 9. Add 500 μL of 6-N HCl. 10. Add 2 mL of ethyl acetate and rock the tubes for 5 min. 11. Centrifuge the tubes for 5 minutes, and transfer the upper ethyl acetate layer to a clean concentration tube. 12. Repeat extraction step with another 2-mL ethyl acetate, and add to the previous extract. 13. Dry the extracts at 30  C under nitrogen (see Note 4). 14. Add 150 μL of derivatizing reagent (BSTFA/TMCS/pyridine) to each tube. 15. Cap and heat in a dry block at 65  C for 30 min. 16. Cool the tubes, and transfer the derivatized extracts to injection vials for GC–MS analysis. 17. Inject 1 μL on to GC–MS. 3.2 Instrument Operating Conditions 3.3

Data Analysis

The instrument’s operating conditions are given in Table 2.

1. Analyze data using ChemStation (Agilent Technologies) and Target software (Thermo) or similar linear regression software. Ions used for identification and quantification are listed in Table 3. 2. The quantifying ions, m/z 1, (Table 3) of available organic acids (Table 1) are used to construct standard calibration curves of the calibrator/internal standard peak area ratios vs concentration. These curves are then used to determine the concentrations of the controls and the patient samples. 3. Typical calibrator curves have a correlation coefficient (r2) > 0.990. 4. Quality control: The run is considered acceptable if calculated concentrations of >80% of organic acids in controls are within +/ 50% of target values. For all samples, ratios of qualifier ions to the quantifying ion must be within +/ 40% of the ion ratios for the calibrators (see Note 5). 5. Patient results are calculated as mmol/mole creatinine (see Table 4 for approximate reference ranges). 6. Representative chromatogram of ERNDIM-MCA control is shown in Fig. 1.

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Table 2 GC–MS operating conditions Column pressure

5 psi

Injector temp.

250  C

Purge time on

0.5 min.

MDS transfer line

280  C

Initial oven temp.

60  C

Initial time

1.0 min.

Temperature ramp

7  C/min.

Final oven temp.

280  C

Final time

3 min.

Total run time

35.4 min.

Solvent delay

7.75 min.

MS source temp

230  C

MS mode

Electron impact at 70 eV, Scan mode: 40–550 m/z

MS tune

Standard tune

GC–MS software

MSD ChemStation Agilent Technologies, Inc.

Table 3 Organic acids monitored on patient samples

Compound name

Retention time (min)

Relative retention time

m/z 1 Quant

m/z 2 Qual

m/z 3 Qual

Lactic DTMS

8.9

0.46

219

190

147

Hexanoic (caproic) MTMS

9.0

0.47

173

117

131

Glycolic DTMS

9.2

0.48

177

205

161

Oxalic DTMS

10.1

0.52

219

190

133

Glyoxylic MTMS

10.2

0.53

218

233

188

2-OH-butyric DTMS

10.4

0.54

233

205

190

3-OH-propionic DTMS

10.6

0.55

177

219

133

Pyruvic DTMS

10.7

0.55

232

204

130

3-OH-butyric DTMS

11.0

0.57

191

117

204

3-OH-Isobutyric DTMS

11.0

0.57

177

103

218

2-OH-Isovaleric DTMS

11.2

0.58

145

219

133

Malonic DTMS

11.7

0.61

233

133

99 (continued)

Table 3 (continued)

Compound name

Retention time (min)

Relative retention time

m/z 1 Quant

m/z 2 Qual

m/z 3 Qual

2-Methyl-3-OH-butyric-DTMS

11.8

0.61

117

147

218

Methylmalonic DTMS

12.0

0.62

218

247

133

3-OH-Isovaleric DTMS

12.1

0.63

205

131

115

2-Ketoisovaleric MTMS

12.1

0.63

275

186

232

4-OH-butyric DTMS

12.4

0.64

233

133

117

Benzoic MTMS

12.5

0.65

179

105

135

2-Ethyl-3-OH-propionic DTMS

12.5

0.65

177

217

233

Acetoacetic-1 DTMS

12.5

0.65

261

246

156

3-OH-Valeric DTMS

12.6

0.65

205

131

189

2-OH-Isocaproic DTMS

12.7

0.66

103

177

261

2-OH-3-Methylvaleric DTMS

12.8

0.66

159

261

103

Octanoic (caprylic) MTMS

13.0

0.67

201

117

129

Acetoacetic-2 DTMS

13.0

0.67

246

261

82

4-OH-Isovaleric DTMS

13.2

0.68

204

247

157

2-Keto-3-methylvaleric

13.3

0.69

274

200

289

2-Methyl-3-OH-valeric D

13.4

0.69

203

115

247

Ethylmalonic MTMS

13.4

0.69

217

261

232

Phenylacetic MTMS

13.4

0.69

193

164

91

2-Ketoisocaproic DTMS

13.7

0.71

110

184

129

Succinic DTMS

14.0

0.73

247

172

129

Methylsuccinic DTMS

14.3

0.74

261

217

186

2-methyl-acetoacetic MTMS

14.3

0.74

171

127

113

2-Ketocaproic acid DTMS (internal Std)

14.5

0.75

274

200

172

Uracil DTMS

14.5

0.75

241

256

99

Mevalonolactone MTMS

14.6

0.76

115

187

101

Propionylglycine MTMS

14.6

0.76

188

159

144

Glyceric TTMS

14.7

0.76

189

292

307

Fumaric DTMS

14.8

0.77

245

143

115

5-OH-Hexanoic DTMS

15.0

0.78

204

261

171

Isobutyrylglycine MTMS

15.1

0.78

202

158

173

Glutaric DTMS

15.8

0.82

261

158

186

Thymine DTMS

15.8

0.82

255

270

113

Isobutyrylglycine DTMS

16.2

0.84

274

289

199

3-Methylglutaric DTMS

16.2

0.84

204

275

185 (continued)

Table 3 (continued)

Compound name

Retention time (min)

Relative retention time

m/z 1 Quant

m/z 2 Qual

m/z 3 Qual

Propionyglycine DTMS

16.2

0.84

260

232

158

Succinylacetone-2pk MTM

16.2

0.84

212

227

110

Butyrylglycine MTMS

16.2

0.84

158

145

202

3-Methylglutaconic DTMS

16.4

0.85

198

183

170

Glutaconic DTMS

16.5

0.85

259

217

230

2-Methylbutyrlglycine M

16.8

0.87

216

203

172

Decanoic (capric) MTMS

16.9

0.88

129

229

201

Isovalerylglycine MTMS

17.0

0.88

189

216

172

Butyrylglycine DTMS

17.3

0.90

172

102

246

3-OH-Octanoic DTMS

17.4

0.90

233

289

217

2-Methylbutyrylglycine

17.6

0.91

288

186

303

Malic-TTMS

17.7

0.92

335

265

307

Adipic DTMS

17.7

0.92

111

275

159

5-Oxoproline DTMS

17.9

0.93

156

258

230

Isovalerylglycine DTMS

17.9

0.93

261

186

288

3-Methyladipic DTMS

18.3

0.95

125

289

155

Tiglylglycine MTMS

18.5

0.96

139

214

83

3-Methylcrotonylglycine MTMS

18.5

0.96

139

214

83

Tiglylglycine DTMS

18.7

0.97

286

184

168

2-OH-Phenylacetic DTMS

18.8

0.97

253

296

281

Mevalonic TTMS

18.8

0.97

233

247

259

3-Methylcronylglycine DTMS

18.9

0.98

286

211

301

2-OH-Glutaric TTMS

19.2

0.99

203

247

129

3-OH-Glutaric TTMS

19.2

0.99

185

246

259

4-OH-Cyclohexylacetic-DTMS-Pk2

19.2

0.99

197

212

287

Phenyllactic DTMS

19.3

1.00

193

267

220

Tropic acid-DTMS (internal Std)

19.3

1.00

280

118

267

Pimelic DTMS

19.4

1.01

289

173

217

3-OH-3-methylglutaric TTMS

19.7

1.02

273

363

199

Hexanoylglycine MTMS

19.7

1.02

230

158

189

4-OH-Cyclohexylacetic-DTMS-Pk1

19.8

1.03

287

212

195

4-OH-Phenylacetic DTMS

20.0

1.04

252

179

281

2-Ketoglutaric-TTMS

20.1

1.04

362

377

260 (continued)

Table 3 (continued)

Compound name

Retention time (min)

Relative retention time

m/z 1 Quant

m/z 2 Qual

m/z 3 Qual

Phenylpyruvic-DTMS

20.1

1.04

323

308

280

N-Acetylaspartic DTMS

20.2

1.05

158

202

304

Hexanoylglycine DTMS

20.3

1.05

302

200

176

N-Acetylaspartic TTMS

20.8

1.08

274

230

230

2-OH-Adipic TTMS

20.8

1.08

261

363

203

3-OH-Adipic TTMS

21.0

1.09

247

363

233

Suberic DTMS

21.0

1.09

303

169

187

2-Ketoadipic-TTMS

21.4

1.11

376

258

302

Orotic TTMS

22.0

1.14

254

357

372

Aconitic TTMS

22.0

1.14

285

375

229

Glutaconic TTMS (Peak2)

22.1

1.15

331

156

346

HVA DTMS

22.1

1.15

326

209

296

Azelaic DTMS

22.7

1.18

317

217

201

Hippuric MTMS

22.9

1.19

105

236

206

Isocitric TetraTMS

23.4

1.21

245

319

285

Homogenetisic TTMS

23.5

1.22

384

341

252

Citric TetraTMS

23.5

1.22

183

257

231

Methylcitric 2S,3R-TetraTMS

23.8

1.23

287

361

389

VMA TriTMS

24.0

1.24

297

371

399

Sebacic DTMS

24.2

1.25

331

215

287

4-OH-phenyl-lactic TTMS

24.5

1.27

308

179

281

4-OH-Phenylpyruvic TTMS

25.0

1.30

277

179

396

Phenylpropionylglycine MTMS

25.4

1.32

279

264

189

Phenylpropionylglycine DTMS

25.5

1.32

351

336

261

3-OH-Decanedioic TTMS

26.8

1.39

233

419

303

N-Acetyltyrosine TTMS

27.2

1.41

260

218

NA

N-Acetyltyrosine DTMS

27.3

1.41

352

208

249

Suberylglycine DTMS

28.9

1.50

360

189

244

Suberylglycine TTMS

28.9

1.50

330

432

274

Relative retention times are retention time of organic acid/retention time of tropic acid

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David Scott et al.

Table 4 Approximate reference ranges for various organic acids (mmole/mole of creatinine) 0–30 days

1–6 months

6–24 months

2–10 years

>10 years

Lactic

275

208

141

139

139

Hexanoic (caproic)

2

2

2

1

1

Glycolic

142

142

154

228

265

2-OH-butyric

17

11

9

10

23

3-OH-propionic

54

54

34

28

28

Pyruvic

131

85

76

39

44

3-OH-butyric

31

29

42

7

8

3-OH-Isobutyric

126

87

83

55

49

2-OH-Isovaleric

7

2

3

2

2

Malonic

15

15

15

12

7

2-Methyl-3-OH-butyric

11

11

20

14

13

Methylmalonic

13

13

11

9

7

3-OH-Isovaleric

39

39

40

37

39

2-Ketoisovaleric

33

23

10

2

2

4-OH-butyric

8

8

8

8

8

Benzoic

71

28

24

30

12

2-Ethyl-3-OH-propionic

25

25

22

26

14

3-OH-Valeric

1

1

1

0

0

2-OH-Isocaproic

2

2

1

0

1

2-OH-3-Methylvaleric

2

2

1

0

1

Octanoic (caprylic)

6

6

3

2

1

Acetoacetic

63

52

26

10

14

4-OH-Isovaleric

0

0

0

0

0

2-Keto-3-methylvaleric

16

10

10

5

2

2-Methyl-3-OH-valeric

0

0

0

0

2

Ethylmalonic

28

28

23

10

7

Phenylacetic

2

2

4

3

2

2-Ketoisocaproic

44

44

19

4

4

Succinic

154

154

171

74

39

Methylsuccinic

9

9

10

8

4

2-methyl-acetoacetic

0

0

0

0

0

Mevalonolactone

0

0

0

0

0

Glyceric TTMS

152

152

131

68

44 (continued)

Screening of Organic Acidurias by GC-MS

331

Table 4 (continued) 0–30 days

1–6 months

6–24 months

2–10 years

>10 years

Fumaric

28

28

16

7

3

5-OH-Hexanoic

24

24

15

10

6

Glutaric

14

14

20

9

7

Isobutyrylglycine

0

0

0

0

0

3-Methylglutaric

3

3

3

3

2

Propionyglycine

0

0

0

0

0

Succinylacetone

0

0

0

0

0

3-Methylglutaconic

12

12

17

10

10

Glutaconic

0

0

0

0

0

Decanoic (capric)

3

3

2

1

1

Butyrylglycine

1

1

0

0

0

3-OH-Octanoic

4

4

2

2

1

2-Methylbutyrylglycine

0

0

0

0

1

Malic

94

73

49

25

12

Adipic

72

72

53

24

12

5-Oxoproline

331

331

207

228

131

Isovalerylglycine

1

1

4

4

2

3-Methyladipic

7

6

6

6

4

Tiglylglycine

0

0

0

0

0

2-OH-Phenylacetic

13

13

11

7

4

Mevalonic

0

0

0

0

0

3-Methylcronylglycine

0

0

0

0

0

2-OH-Glutaric

105

87

89

51

30

3-OH-Glutaric

16

9

10

7

4

Phenyllactic

3

2

3

3

3

Pimelic

9

9

8

8

4

3-OH-3-Methylglutaric

65

32

24

15

6

4-OH-Cyclohexylacetic

1

1

5

5

2

4-OH-Phenylacetic

160

144

134

62

60

2-Ketoglutaric

526

484

471

135

74

Phenylpyruvic

9

6

8

1

2

N-Acetylaspartic

54

54

47

21

18

Hexanoylglycine

1

1

1

1

0 (continued)

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David Scott et al.

Table 4 (continued) 0–30 days

1–6 months

6–24 months

2–10 years

>10 years

2-OH-Adipic

3

3

3

2

1

3-OH-Adipic

15

15

19

11

4

Suberic

30

30

26

13

4

2-Ketoadipic

15

14

17

5

2

Orotic

10

8

7

6

4

Aconitic

170

170

177

154

76

Azelaic

35

35

37

15

7

Hippuric

974

974

867

816

544

Isocitric

168

131

149

133

68

Homogenetisic

2

2

1

1

1

Citric tetra

1227

918

994

659

528

Methylcitric 2S,3R

8

8

7

5

4

Sebacic

15

15

10

5

1

4-OH-phenyl-lactic

33

12

15

4

5

4-OH-Phenylpyruvic

87

43

22

4

12

Phenylpropionylglycine

1

0

0

1

0

3-OH-Decanedioic

74

74

85

24

7

N-Acetyltyrosine

6

6

2

1

2

Suberylglycine

1

1

0

0

0

Fig. 1 GC–MS total ion chromatogram of ERNDIM-MCA control showing various organic acids

Screening of Organic Acidurias by GC-MS

4

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Notes 1. This solution must be made fresh with each run. 2. This approach provides only semi-quantitation of the unavailable or unstable organic acids. Clinically this approach works as exact quantitation of organic acids is not needed for the diagnosis and follow-up of organic aciduria. 3. Volume of sample containing of 1-μmol (133 mg) creatinine can be calculated as follows: 11,300/creatinine (mg/dL). 4. Do not overdry the extract. This will result in low recovery. 5. These acceptability limits are broader than a typical quantitative method. However, they are acceptable for semiquantitative method as exact quantitation of organic acids is not needed for the diagnosis and follow-up of organic aciduria.

References 1. Goodman SI, Markey SP (1981) Diagnosis of organic acidemias by gas chromatography–mass spectrometry. Lab Res Methods Biol Med 6: 1–158 2. Lo SF, Young V, Rhead WJ (2010) Identification of urine organic acids for the detection of inborn errors of metabolism using urease and gas chromatography-mass spectrometry (GC-MS). Methods Mol Biol 603:433–443 3. Sweetman L (1991) Organic acid analysis. In: Hommes FA (ed) Techniques in diagnostic human biochemical genetics: a laboratory manual. Wiley-Liss, New York, pp 143–176 4. Villani GR, Gallo G, Scolamiero E, Salvatore F, Ruoppolo M (2017) “Classical organic acidurias”: diagnosis and pathogenesis. Clin Exp Med 17(3):305–323 5. Dimitrov B, Molema F, Williams M, Schmiesing J, Muhlhausen C, Baumgartner MR, Schumann A, Kolker S (2021) Organic acidurias: major gaps, new challenges, and a yet unfulfilled promise. J Inherit Metab Dis 44(1): 9–21

6. Gallagher RC, Pollard L, Scott AI, Huguenin S, Goodman S, Sun Q (2018) Laboratory analysis of organic acids, 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med 20(7): 683–691 7. Kumps A, Duez P, Genin J, Mardens Y (1999) Gas chromatography-mass spectrometry analysis of organic acids: altered quantitative response for aqueous calibrators and dilute urine specimens. Clin Chem 45(8 Pt 1):1297–1300 8. Matsumoto I, Kuhara T (1996) A new chemical diagnostic method for inborn errors of metabolism by mass spectrometry-rapid, practical, and simultaneous urinary metabolites analysis. Mass Spectrom Rev 15(1):43–57 9. Mouskeftara T, Virgiliou C, Theodoridis G, Gika H (2021) Analysis of urinary organic acids by gas chromatography tandem mass spectrometry method for metabolic profiling applications. J Chromatogr A 1658:462590

Chapter 30 Identification of Urine Organic Acids for the Detection of Inborn Errors of Metabolism Using Urease and Gas Chromatography–Mass Spectrometry (GC/MS) Stanley F. Lo, Keely Pierzchalski, Velta Young, and William J. Rhead Abstract A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography–mass spectrometry (GC/MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules is available for separation and quantification by GC/MS. The urease method is a useful adjunct to newborn screening follow-up, and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. This method describes the urease treatment, derivatization, and the organic acids and other biochemical metabolites that can be identified. Key words Urease, Organic acids, Inborn errors of metabolism, Gas chromatography–mass spectrometry

1

Introduction The identification of urinary organic acids is a key component in the diagnosis and treatment of inborn errors of metabolism. The addition of expanded newborn screening using tandem mass spectrometry has significantly increased the number of infants identified with fatty acid oxidation and organic acid disorders, thus increasing the value in determining the presence of urinary organic acids for both confirmation and treatment of disease. The Shoemaker and Elliott urease method [1] differs from the classical organic acid methods [2–4] by taking advantage of urease to remove urea in urine. The elimination of extracting from the acidic fraction of urine, as done in the classical method, is replaced by the enzymatic removal of urea

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_30, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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and enables specimens to have an abundance of biochemical metabolites to derivatize and detect using GC/MS. While the method described here is limited to organic acids, it can easily be used to detect amino acids, carbohydrates, acyglycines, neurotransmitters, purines and pyrimidines, vitamin and peroxisomal metabolites, and dipeptides. The method can also be adapted to measure metabolites in plasma, cerebral spinal fluid, and amniotic fluid.

2 2.1

Materials Samples

2.2 Reagents and Solutions

1. Random urine: Preferred specimen volume is 3–10 mL. Minimum volume is 1.5 mL. Store in
20  C freezer. Samples that will not be analyzed within 2 weeks should be stored at
70  C. Retained specimen samples are stored in the
70  C freezer for up to 2 years and as space permits. 1. Dichloromethane (Biotech grade 99.9%). 2. Triethylammonium trifluoroacetic acid (TEA/TFA): Purchase from Santa Cruz Biotechnology or prepare as follows. 3. TEA/TFA (1:1): in-house preparation method (see Note 1): (a) Triethylamine (TEA): Volume to mole to determine TFA volume (8-mL TEA * 0.73 g/mL) / 101.2 g/ mol ¼ 0.058-mol TEA. (b) Trifluoroacetic acid (TFA): Mole to volume to determine TFA volume (0.058-mol TFA * 114 g/mol) / 1.49 g/ mL ¼ 4.4-mL TFA. (c) Dichloromethane volume: (8-mL TEA +4.4-mL TFA) * 50 ¼ 620 mL (maximum reaction volume in 1-L round bottom flask (RBF) is 700 mL). (d) To dissolve TEA and TFA into dichloromethane, position 1-L RBF in an ice bucket, on a stir plate, held up with clamp apparatus. (e) Add 620-mL dichloromethane to 1-L RBF with stir bar. Stir and chill for 10 minutes. (f) Slowly add 8-mL TEA with volumetric pipet to chilled dichloromethane while continuously stirring. Chill for 5 minutes while stirring after full-volume TEA has been added. (g) Measure 4.4-ml TFA using volumetric pipets, and add to a glass test tube. Using a Pasteur pipet, slowly add TFA to stirring dichloromethane/TEA in a dropwise fashion. Allow solution to cool between drops to limit generation of smoke/heat. Once completely added, chill for 10 minutes while continuously stirring.

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(h) To remove dichloromethane, set up Rotovap apparatus (Buchi) with water bath set to 35  C for dichloromethane evaporation (dichloromethane boiling point is 40  C). (i) Lower 1-L RBF with TEA/TFA/dichloromethane into water bath so solution is mostly submerged, and begin rotation. (j) Start vacuum (with second cold trap to prevent dichloromethane from getting into vacuum lines) which will pull the evaporating dichloromethane into the condenser. Collect dichloromethane into round bottom receiving flask until no more drops are being formed on the condenser. (k) To measure TEA/TFA yield, measure volumes of TEA/TFA left in 1-L RBF and dichloromethane in receiving flask. TEA/TFA: (mL recovered / 12.4 mL)  100 ¼ % recovered. Dichloromethane: (mL recovered / 620 mL)  100 ¼ % recovered. 4. Urease (from Jackbeans), (Calzyme Laboratories). Store stock bottle at -20  C. 5. Urease aqueous solution preparation: Allow aliquot to warm to room temperature for 10 minutes. Reconstitute with clinical laboratory reagent water (CLRW), and vortex to mix being sure to dissolve completely. Let sit for 5 minutes and vortex to mix just before use. 6. Aliquot preparation from stock. Store aliquots at
20  C. (a) Desired activity: 150 U / 50 uL. (b) μL needed / sample: 50 μL  two additions ¼ 100 μL. (c) μL needed / set: 100 μL * eight samples +100 μL ¼ 900 μL (volume needed for reconstitution of aliquot). (d) U needed / set: 150 U / 50 μL ¼ 3 U / μL * 900 μL ¼ 2700 U. (e) Urease activity: U / mg protein. (f) mg urease needed per set: 2700 U / (U / mg urease) ¼ mg urease (amount needed per aliquot). Example: Urease activity ¼ 300 U / mg 2700 U / (300 U / mg) ¼ 9 mg (amount needed per aliquot). 7. Acetonitrile (ACS grade, 99.9%). 8. Acetone (HPLC grade, 99.9%): Chill in ice bath or freezer blocks at time of use. 9. Methanol (HPLC grade, 99.9%). 10. N-methyl-N-trifluoroacetamide Technologies). 11. Water (HPLC grade).

(MSTFA)

(Regis

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2.3 Standards and Calibrators

2.4 Internal Standards

Calibration is performed by separating the analytes of interest into groups (Table 1). Stock solutions for each analyte are prepared and then combined with other analytes to create group master mix solutions. Master mix solutions are prepared by adding each stock solution to a class A 10-mL volumetric flask and filling to volume with the appropriate diluent. A five-point standard curve is prepared by adding 10, 100, 250, 500, and 1000 μL to ReactiVials with internal standard which are processed for analysis (see Note 2). 1. Internal standard stock solution: 100 nmol each d3-lactate (C/D/N Isotopes), 13C3-pyruvate (Isotec Sigma), d3-methylmalonic acid (C/D/N Isotopes), d3-serine (Cambridge Isotopes), d5-phenylalanine (C/D/N Isotopes), 15N2orotic acid (Cambridge Isotopes), d4-sebacic acid (C/D/N Isotopes), 13C6-glucose (Isotec Sigma), d6-inositol (C/D/N Isotopes), d5-tryptophan (C/D/N Isotopes), and 500 nmol d3-creatine (C/D/N Isotopes) are used as internal standards (IS). From purchased chemicals, 0.01-M stock aliquots are prepared to make IS working solutions from 0.01 M stock solutions for each IS and are stable for 2 years at
70  C (Table 2). 2. Internal standard working solution: Place 1000 μL of each 0.01-M stock solution in a class A 25-mL volumetric flask as described in Table 2 to create the IS working solution. 250 μL of the IS working solution is added to each Reacti-Vial. Each sample Reacti-Vial contains 100 nmol each of ten internals standards and 500 nmol d3-creatine. The prepared set of IS Reacti-Vials is kept frozen at
70  C until ready for use and is stable for 1 year.

2.5

Quality Control

1. Quality control matrix: Urine free from inborn errors of metabolism are pooled (10–20 collections, >10 mL each) and filtered (Nalgene bottle top filter connected to vacuum). This pooled, filtered urine is diluted with CLRW or HPLC grade water to reach specific gravity of ~1.012. 50-mL aliquots are prepared and kept frozen at
70  C for up to 2 years. From the 50-mL aliquots, 1-mL aliquots are prepared and kept frozen at
70  C for up to 1 year. At time of use, the 1-mL aliquots are thawed and diluted (0.9-mL urine +2.1-mL CLRW) to make diluted pooled urine for normal and abnormal control samples. The diluted pooled urine should have a specific gravity of ~1.002–1.004 and a target creatinine of ~20 mg/dL. Adjust pooled and filtered matrix stock with filtered urine or CLRW as needed to achieve this target. Reference ranges are determined for each new batch of quality control matrix, with the normal control (n ¼ 20+) +/
3SD and the abnormal control (n ¼ 40+) +/
2SD.

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339

Table 1 Calibration groups Group 1 Organic acids Prepared in 10-mL HPLC water

RT (minute)

Qualifying Standard stock ion conc. (M)

Volume (mL)

Glutaric acid (sigma)

14.90

261

0.01

0.2

3-Hydroxy-3-methylglutaric acid (sigma)

19.80

247

0.01

0.2

3-Hydoxypropionic acid (TRC)

7.35

219

0.01

0.2

Suberic acid (sigma)

21.55

187

0.01

0.2

2-Hydroxybutyric acid (sigma)

7.00

131

0.01

0.1

2-Hydroxyglutaric acid (TRC)

19.25

129, 349

0.01

0.2

2-Methylcitric acid (TRC)

24.65

287

0.01

0.2

3-Hydroxybutyric acid (sigma)

7.80

191

0.01

0.1

3-Hydroxyisovaric acid (sigma)

9.17

131

0.01

0.2

3-Methylglutaric acid (sigma)

15.46

275, 159

0.01

0.1

4-Hydroxybutyric acid (sigma)

9.90

233

0.000793

1.27

4-Hydroxyphenylacetic acid (sigma)

20.36

179

0.01

0.3

Adipic acid (sigma)

17.49

111

0.01

0.5

Glyceric acid (sigma)

13.03

189

0.01

0.5

Glycolic acid (sigma)

5.64

205

0.01

0.4

Hippuric acid (sigma)

23.64

206, 105

0.01

0.25

Lactic acid (sigma)

5.33

117

0.2

0.5

Maleic acid (sigma)

11.95

245

0.01

0.1

Malic acid (sigma)

17.33

233, 245

0.01

0.2

Group 2 organic acids Prepared in 10-mL HPLC water

RT Qualifying Standard stock (minute) ions conc. (M)

Volume (mL)

2-Hydroxyisovaleric acid (sigma)

7.96

219

0.01

0.1

2-Hydroxysebacic acid (TRC)

27.40

317

0.01

0.1

3-Hydroxyglutaric acid (sigma)

19.26

185, 349

0.01

0.1

3-Hydroxyisobutyric acid (sigma)

7.82

177, 218

0.01

0.5

3-Methylglutaconic acid (TRC)

15.89

229, 273

0.01

0.4

4-Hydroxyphenyllactic acid (sigma)

25.37

179, 308

0.01

0.2

Cis-Aconitic acid (sigma)

22.67

229

0.01

1.0

Citric acid (sigma)

24.15

375

0.02

0.5

Glycerol (sigma)

11.30

218

0.01

0.5

Hexanoic acid (sigma)

5.49

173, 117

0.01

0.2 (continued)

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Table 1 (continued) Ketoisoleucine (sigma)

12.13

259

0.01

0.2

Ketoleucine (sigma)

10.38

259

0.01

0.2

Ketovaline (sigma)

10.60

245

0.01

0.2

Methylsuccinic acid (sigma)

12.61

261

0.01

0.2

Mevalonic lactone (sigma)

13.65

187, 249

0.01

0.2

Octanoic acid (sigma)

10.61

201

0.01

0.2

Pantothenic acid (sigma)

26.73

291, 103

0.01

0.2

Phenylpyruvic acid (sodium) (sigma)

21.78

293, 308

0.01

0.2

Pyroglutamic acid (sigma

17.87

230, 156

0.02

0.25

Sebacic acid (sigma)

25.09

331, 185

0.02

0.25

Succinic acid (sigma)

12.23

247, 72

0.01

0.5

Succinylacetone (sigma)

21.77

257, 359

0.01

0.2

Group 3 organic acids Prepared in 10-mL dilute pooled urine

RT Qualifying Standard stock (minute) ions conc. (M)

Volume (mL)

2-Ketoglutaric acid (sigma)

19.92

347

0.04

1.0

5-Aminolevulinic acid (sigma)

22.06

174, 260

0.01

0.4

5-Hydroxyhexanoic acid (TRC)

13.59

171

0.01

0.4

Ethylmalonic acid (sigma)

11.32

261, 248

0.01

0.4

Guanidine acetate (sigma)

16.26

187, 146

0.01

0.2

Malonic acid (sigma)

9.02

233

0.01

0.2

Methylmalonic acid (sigma)

9.35

247, 218

0.02

0.5

Orotic acid (sigma)

22.64

254, 357

0.01

0.2

Pyruvic acid (sodium) (sigma)

5.92

217

0.02

0.1

Group 4 organic acids Prepared in 10-mL methanol (100%)

RT Qualifying Standard stock (minute) ions conc. (M)

Volume (mL)

Fumaric acid (sigma)

13.21

245

0.01

0.2

Tetradecanedioic acid (sigma)

29.48

387

0.01

0.1

Group 5 Acylglycines Prepared in 10-mL HPLC water

RT Qualifying Standard stock (minute) ions conc. (M)

Volume (mL)

2-Methylbutyrylglycine (TRC)

17.27

288

0.01

0.1

3-Methylcrotonylglycine (TRC)

19.05

286, 184

0.01

0.1

Butyrylglycine (TRC)

17.09

274, 172

0.01

0.1

Hexanoylglycine (TRC)

20.66

200, 261

0.01

0.1

Isobutyrylglycine (TRC)

15.55

274, 172

0.01

0.1 (continued)

Urine Organic Acids by GC/MS

341

Table 1 (continued) Isovalerylglycine (TRC)

17.74

288

0.01

0.2

Phenylpropionylglycine (TRC)

26.55

351

0.01

0.1

Propionylglycine (sigma)

15.53

158

0.01

0.1

Suberylglycine (TRC)

29.14

261, 330

0.01

0.1

Tiglylglycine (TRC)

18.74

286, 212

0.01

0.1

Group 6 amino acids, neurotransmitters, and pyrines and pyrimidines Prepared in 10-mL HPLC water

RT Qualifying Standard stock (minute) ions conc. (M)

Volume (mL)

Acetyl-aspartic acid (AA) (sigma)

21.29

274, 376

0.01

0.2

Acetyl-tyrosine (AA) (sigma)

28.00

260

0.01

0.1

Aminoadipic (AA) (sigma)

22.07

362, 260

0.01

0.2

Beta-alanine (AA) (sigma)

8.58

176

0.01

0.1

Cystathionine (AA) (sigma)

29.11

128, 278

0.01

0.2

Homocystine (AA) (sigma)

31.48

128, 278

0.01

0.2

Pipecolic acid (AA) (sigma)

13.73

156

0.01

0.1

Sarcosine (AA) (sigma)

7.14

116

0.01

0.2

Tyrosine (AA) (sigma)

26.00

218

0.01

0.5

5-Hydroxyindole-3-acetic acid (NT) (sigma)

29.01

290

0.01

0.4

Gamma-aminobutyric acid (NT) (sigma)

18.10

174, 304

0.01

0.4

Homovanillic acid (NT) (sigma)

22.98

209

0.01

0.4

Metanephrine (NT) (TRC)

25.70

116, 297

0.01

0.1

4-Hydroxy-3-methoxyphenyl glycol (NT) (TRC)

24.29

297, 267

0.01

0.4

Vanillylmandelic acid (NT) (sigma)

25.09

297

0.01

0.4

Hypoxanthine (PP) (sigma)

23.59

265, 280

0.01

1.0

Thymine (PP) (sigma)

14.89

255

0.01

0.1

Uracil (PP) (sigma)

13.02

241, 255

0.01

0.4

Uridine (PP) (sigma)

30.95

258, 517

0.01

0.2

Xanthine (PP) (sigma)

27.05

353

0.01

0.4

. Groups are designed to avoid overlapping/interfering peaks and prepared in diluents most appropriate to for calibration. All reagents are from Millipore/Sigma/Aldrich (Sigma) or Toronto Research Chemical (TRC). Standard stocks are prepared ahead of time and frozen at
70  C. Standard stock aliquots expire 1 year from preparation date. Standard stock aliquot concentrations (M) are listed and the volumes (mL) used to make the master mix (MM). MM should be used fresh

342

Stanley F. Lo et al.

Table 2 Internal standard (IS) preparations IS stock preparation

F.W.

Purity Standard stock (%) conc. (M)

Volume Amount (L) needed (g)

Prep notes

d3-lactic acid (CDN) 115.08 97.6

0.01

0.01

0.01179

HPLC water

13

C3-pyruvic acid (sigma)

113.02 99.0

0.01

0.01

0.01142

HPLC water

d3-Methylmalonic acid (CDN)

121.11 98.0

0.01

0.01

0.01236

HPLC water

d3-serine (CAM)

108.11 100

0.01

0.01

0.01081

HPLC water

d5-phenylalanine (CDN)

170.22 98.6

0.01

0.01

0.01726

Warm HPLC water

15

158.08 99.0

0.01

0.01

0.01597

NH3 gas

d4-Sebacic acid (CDN)

206.27 99.4

0.01

0.01

0.02075

50% methanol

13

186.11 99.0

0.01

0.01

0.01880

HPLC water

d6-Insitol (CDN)

186.19 97.1

0.01

0.01

0.01918

HPLC water

d5-tryptophan (CDN)

209.26 98.7

0.01

0.01

0.02120

NH3 gas/HPLC warm water

d3-Creatine (CDN)

152.17 99.0

0.05

0.01

0.07685

Warm HPLC water

N2-Orotic acid (CAM)

C6-glucose (sigma)

Deuterated internal standards are purchased from C/D/N Isotopes (CDN), Millipore/Sigma/Aldrich (Sigma), or Cambridge Isotope Laboratories (CAM). IS standard stock aliquots are prepared and frozen at
70  C. To calculate amount to weigh: (FW  M  L)  (%/100) ¼ (g/mol)  (mol/L)  (L)  (%/100) ¼ g. The working solution is prepared by adding 1.0 mL each IS into a 25-mL volumetric flask and diluted with water. A 25-mL working solution will prepare ~96 Reacti-Vials with 250 μL/vial

2. Abnormal control spike solution: 0.05-M stock solution of each standard, glutaric acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxypropionic acid, and suberic acid are prepared in class A 10-mL volumetric flasks. A master mix solution is made of these four standard solutions by combining 8 mL each of the 0.05-M standard solutions plus 8-mL HPLC grade water using volumetric pipets for a total of 40 mL into a beaker; cover, and mix with a stir bar. The final concentration of each standard is 0.01 M. 200-μL aliquots are prepared in 0.5-mL Eppendorf tubes and stored at
70  C for 1 year. 3. Normal control: Add 1-mL diluted pooled urine to Reacti-Vial with IS. 4. Abnormal control: Add 1-mL diluted pooled urine +30-μL abnormal control spike solution to Reacti-Vial with IS. 5. Negative control: Add 1-mL CLRW to Reacti-Vial with IS.

Urine Organic Acids by GC/MS

2.6

Supplies

343

1. Reacti-Vials 2 mL and 3 mL (Supelco). 2. Red rubber plug septa (Wheaton). 3. Teflon septa for 7- and 22-mL vials (Supelco). 4. Disposable 3-mL glycerol-free syringe. 5. 0.2-μm SFCA 25-mm syringe filters (Nalgene). 6. 0.2-μm SFCA sterile bottle top filter (500 mL) (Nalgene). 7. Borosilicate Pasteur pipets, 5.7500 . 8. Disposable 12  75-mm glass test tube. 9. Falcon 5-mL polystyrene round bottom tube with cap. 10. 2.0-mL microcentrifuge tubes. 11. 21-gauge disposable needles (Becton Dickinson). 12. MS autosampler vials 1.8 mL (Thermo). 13. Aluminum seals for autosampler vials (Thermo). 14. Aluminum seal crimper. 15. Vial inserts (Supelco). 16. Vacutainer® blood collection tube, used to hold ammonium hydroxide to generate ammonia gas to aid dissolution of standards in water. 17. 21-gauge (butterfly) (Becton Dickinson) used to transfer ammonia gas from the collection tube to the volumetric flask holding a standard in water. 18. Cryovials 1.2 mL (Nalgene). 19. Carbon dioxide gas (CO2).

2.7 Measuring Equipment

1. Single-channel adjustable pipettors for 1–10, 10–100, 20–200, and 100–1000 μL. 2. 1-mL disposable syringes (Becton Dickinson). 3. 25-gauge disposable needles (Becton Dickinson).

2.8

Equipment

1. A gas chromatography–mass spectrometry system (GC/MS; Agilent 7890B/5977A) with autosampler and operated in electron impact mode and GC column (Agilent) fused silica capillary column, DB-5, with dimensions of 25 m  0.32 mm  0.5-μm film thickness. 2. Refractometer, TS Meter (Leica). 3. Water bath, 37  C, filled with CLRW and treated with water conditioner to prevent growth of any kind. 4. Vortex. 5. Microcentrifuge for 2.0-mL tubes, refrigerated (4 12,000 rpm, or 13,900  g.



C),

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6. Reacti-Vap Evaporator III (Thermo) with nitrogen source (set up in chemical fume hood). 7. Reacti-Therm III Heating Module (Thermo) (set up in chemical fume hood). 8. Reacti-Blocks (Thermo), T-1 (2-mL Reacti-Vials), and B-1 (3-mL Reacti-Vials).

3

Methods

3.1 Sample Preparation

1. Prepare materials needed: (a) Label tubes and vials appropriately. (b) Pull urease aliquot(s) to warm. (c) Set urine specimen into water bath to thaw. (d) Set up Reacti-Therm III heating module to 70  C, and verify nitrogen tank supply is sufficient. (e) Perform instrument maintenance and set-up for the day. (f) Record reagent documentation.

lot#/expiration

dates

and

QC

2. Prepare control samples in 2.0-mL Reacti-Vials with internal standards. (a) Prepare diluted pooled urine: 0.9-mL pooled urine +2.1mL CLRW in 5-mL Falcon tube. Vortex to mix. (b) Normal control: 1.0-mL diluted pooled urine. Vortex to mix. (c) Abnormal control: 1.0-mL diluted pooled urine +30-μL abnormal spike solution. Vortex to mix. (d) Negative control: 1.0-mL CLRW only. 3. Determine the specific gravity and dilution factor of each urine sample to be analyzed. (a) Specific gravity 1.010, no dilution, use 1.0 mL. (b) Specific gravity 1.011–1.015, (1.0 mL + 1.0 mL CLRW).

2

dilution

(c) Specific gravity 1.016–1.020, (0.5 mL + 1.5 mL CLRW).

4

dilution

(d) Specific gravity 1.021–1.025, (0.4 mL + 1.6 mL CLRW).

5

dilution

(e) Specific gravity >1.026, (0.2 mL + 1.8 mL CLRW).

10

dilution

4. Using a 3-mL glycerol-free syringe, filter urine through a 0.2-micron aqueous syringe filter into a clean glass test tube. 5. Make dilution with filtered urine in appropriately labeled 5-mL Falcon tube with cap, and vortex to mix.

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6. Transfer 1 mL of filtered and diluted urine to the appropriately labeled 2.0-mL Reacti-Vial containing 250 μL of the working internal standard mixture. Cap vial with a rubber septum plug and vortex to mix. 7. Proceed with sample preparation with all QC and sample vials processed together. 8. Add CO2 into the vial with 21-gauge needle connected to a CO2 line through the septum with the cap loose, and after 10 seconds, tighten the cap, and quickly remove the needle, wiping needle with clean Kimwipe between samples. 9. Add the 50 μL of the urease solution to each sample. Vortex to mix. Reintroduce CO2 blanket and reseal. 10. Incubate sample at 37  C for 15 minutes. 11. Add additional CO2 without loosening the cap to maintain pressure. 12. Incubate sample at 37  C for 15 minutes. 13. Add 50 μL of urease solution. Vortex to mix. Reintroduce CO2 blanket and reseal. 14. Incubate at 37  C for an additional 15 minutes. 15. Chill samples in an ice bath or in freezer blocks (Reacti-Blocks stored at -20  C) for 5 minutes. 16. Change existing septum to a Teflon-lined rubber septum. 17. Add 1.4-mL chilled acetone to each vial and vortex to mix. Chill in ice bath or cold block for 15 minutes. Wet pipet tip before use to prevent excessive acetone dripping. Change tips between samples. 18. Transfer sample using a glass Pasteur pipet to a 2.0-mL microcentrifuge tube, and spin for 5 min at 13,900  g in refrigerated centrifuge. 19. Pour supernatant over into a clean 3-mL Reacti-Vial. 20. In chemical fume hood for the, add 20-μL TEA/TFA and vortex to mix. 21. Add acetonitrile using a glass Pasteur pipet to the 3-mL line on the Reacti-Vial and vortex to mix. 22. In a 70  C heating block, evaporate the sample under nitrogen stream for ~10 minutes or when evaporated to ~1.0-mL mark for up to 8 samples. More samples may need longer evaporation times. Use this evaporation time for the subsequent acetonitrile additions. 23. Add 0.5 mL-acetonitrile, vortex briefly, and evaporate under nitrogen. Continue adding acetonitrile until volume is 5 mmol/ mol creatinine will be used for the normal control. Take 400 mL aliquot of the pooled urine to spike as the abnormal control. The following organic acids and their desired concentrations are used to spike Normal Urine (the organic acid standards should be of the highest grade, purity, and quality available): lactic 200 nmoles/ umole creatinine, methylmalonic 200 nmoles/umole creatinine, 2-hydroxyisocaproic 100 nmoles/umole creatinine, ethylmalonic 90 nmoles/umole creatinine, glutaric 100 nmoles/umole creatinine, 2-hydroxyglutaric 90 nmoles/umole creatinine, 4-hydroxyphenylacetic 160 nmoles/umole creatinine, orotic

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357

50 nmoles/umole creatinine, 4-hydroxyphenyllactic 100 nmoles/umole creatinine, N-acetylaspartic 80 nmoles/ umole creatinine, 3-hydroxybutyric 200–300 nmoles/umole creatinine (NOTE: 3-hydroxybutyric is spiked when the normal pooled urine contains less than 100 mmol/mol creatinine). 2.5 Internal Standards

1. 5 mM methylsuccince-d6 and 5 mM 2-oxocaproic (1:1 mixture of the two in 20 mM HCL). Intermediate: 10 mM 2-Methylsuccinic-d6 (Cambridge, F.W.138.16) in 20 mM HCl. (used for all other analytes not covered by the other two Internal Standards). 2. Intermediate: 10 mM 2-Oxocaproic (2-ketohexanoic) (Sigma, F.W.152.16) in 20 mM HCl. (used for oxo-acids). 3. Working Internal Standard: Combine equal volumes of the 10 mM 2-methylsuccinic-d6 and 10 mM 2-oxocaproic to make a final concentration of 5 mM each in a 20 mM HCl solution. Aliquot and store at 20  C, stable for 6 months. 4. Working Internal Standard: 5 mM Heptanoic-d5 (CDN Isotopes, F.W. 135.18) in Methanol. Aliquot and store at 20  C, stable for 6 months (used for octanoic and decanoic).

2.6

Supplies

1. 1.5 ml microtubes (extraction). 2. 1.5 mL tubes with screw caps (urease). 3. Wheaton Scintillation Vials with screw caps (PFBO and calibration mixes). 4. Glass transfer pipets. 5. VWR Precleaned Trace Clean Clear Borosilicate 20 mL vials with Teflon-lined closure (Stock Standards). 6. Agilent certified vials (used for internal standards and reaction vials). 7. Xpertek 0.1 mL autosampler vials, plastic with glass inner cone. 8. Xpertek 11 mm Teflon faced silicon/rubber seals (for autosampler and reaction vials). 9. SGE Syringe 10F-CTC-0.63 10uL. 10. Columns-Primary column, Restek 30 m  0.25 mm ID – BPX50 0.25 um, secondary column, Restek 25 m  0.32 mm ID – BPX5 0.025 um (2 m used in the secondary oven). 11. Liquid nitrogen, very dry nitrogen gas and air, ultrahigh purity helium gas.

2.7

Equipment

1. Pegasus4D GCxGC-TOFMS System (Leco, St. Joe, MI) with autosampler (Gerstal). 2. Microcentrifuge.

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3. Heat block. 4. Concentrator. 5. Analytical balance. 6. Mini vortex. 7. Adjustable and fixed volume pipettes. 8. Oven. 9. Crimper and decapper.

3

Methods

3.1 Stepwise Procedure

1. Analyze urine samples for creatinine (LC-MS-MS method is used). 2. Pull an Artificial Urine Control, a Normal Urine Control, an Abnormal Urine Control, urease, urine samples, and internal standards out and allow them to come to room temperature. 3. Obtain 1.5 mL microtubes for sample setup; verify that the tubes are clean and without any particulate matter in it before using. Label tubes. 4. Prepare PFBO solution (10 mg/mL). Each sample requires 40 uL of PFBO. PFBO must be made fresh daily and should not be made up more than 1 hour prior to setting up samples. Vortex the PFBO solution and verify that all of the PFBO is dissolved prior to use. 5. Add 10 μL of combined working internal standard 5 mM Methylsuccinic-d6 and 5 mM 2-oxocaproic acid to each tube. Add 10 μL of 5 mM Heptanoic-d5 internal standard to each tube. 6. Vortex the urine sample and pipette the urine into appropriately labeled 1.5 mL microtube. Pipette an aliquot of urine (plus deionized water if indicated) based on creatinine result. (a) 0.2–0.99 mmoles take 0.200 mL of urine. (b) 1.0–4.0 mmoles take 0.100 mL of urine. (c) 4.0–13.0 mmoles take 0.050 mL of urine. (d) >13.0 mmoles take 0.025 mL of urine +0.025 mL deionized H2O). Any sample with creatinine less than 0.2 mmoles will need to be tested for proper sample type; if deemed urine, the sample will need to be concentrated before it can be tested. Concentration procedure: take a large aliquot of urine (at least 1500 uL) place in a scintillation vial and dry down over night in a vacuum concentrator. Reconstitute with deionized water at a concentration 10X less (150 μL of water).

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7. Add 40 μL of working urease to each tube, cap, vortex, and place in a 37  C heat block for 10 minutes. 8. Add 40 μL of PFBO to each sample, cap, vortex, and incubate at room temperature for 30 minutes. 9. Add 900 μL of 200 proof ethyl alcohol to each tube, cap, and vortex. Centrifuge tubes at 5000 rpms for 10 minutes. 10. Transfer solution to a prelabeled Agilent certified reaction vial, Transfer as much of the liquid as possible without disturbing the pellet at the bottom of the vial, place on a concentrator, and evaporate under nitrogen at 50  C until completely dry (~30 minutes). 11. Pipette 300 μL of BSTFA:Tri-Sil Derivatization mixture into each vial. Cap and vortex. Place in an oven at 60–65  C for 30 minutes. 12. Remove the samples from the oven and allow the samples to cool to room temperature prior to transferring to the GC vials. 13. Pipette 100 μL of the derivatized sample to a labeled autosampler vial. Cap with a GC autosampler cap immediately. Place on instrument to run. 14. One microliter of sample is injected using spitless mode. The inlet temperature is at 280  C with helium as the carrier gas. A flow rate of 1 mL/minute for the entire run is used using the corrected constant flow via pressure ramps. The initial oven temperature is 75  C, and 5 minutes after the injection, the oven temperature is raised to 280  C at a rate of 4  C/minute and held at 280  C for 5 minutes. The secondary oven temperature offset is 25  C with the modulator offset at 30  C. Modulation timing is set for 6-second periods, with 0.6-second hot pulse and 2.40-second cool time. Transfer line and source temperatures are set at 250  C and 230  C, respectively. Mass range is 45–750 with an acquisition rate of 200 spectra/second. The acquisition voltage setting is 1500 with the electron energy of 70. 3.2

Data Analysis

1. The identification criteria for organic acids (and internal standards), which are quantified by areas of extracted ion chromatograms, include a combination of the following: a quantitation ion and a confirming ion, which are unique to that organic acid peak, retention times, ratios of areas of extracted ion chromatograms, and total spectra. Additionally, for accurate identification, the retention time of the quantitation ion extracted ion chromatogram must be near the center (expected retention time of the acid) of the 7 seconds time range of the primary chromatogram and 0.05 seconds of the secondary. The quantitation ion and confirming ion of each organic acid were chosen to be as uniquely characteristic as

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possible within this narrow time window. Therefore, the retention times for the extracted ion chromatograms must be very close and the ratios of areas for the confirming ion as percent of the quantitation ion should be within 45% (relative) of the expected values in the calibration table; most compounds will be within 20%. In the identification of each organic acid, the full ion spectrum is compared to the authentic spectra in the mass spectral library and calibration reference and a match of greater than 700 is considered to be acceptable for identification (see Note 4). 2. However, because there are many coeluting compounds in urine, (see Note 5), which can lower the library match, the definitive criteria for identification from ion ratios and the total spectra are at the discretion of carefully trained, highly experienced technicians who can recognize characteristic masses and % intensities to identify the different compounds in mixed spectra. A representative GCxGC-TOFMS chromatogram and a 2D-chromatogram (contour plot) of the TMS derivatives of all compounds seen is shown in Fig. 1. GCxGC-TOFMS selected ion chromatogram is shown in Fig. 2 (see Note 3). 3. Analyze data using ChromaTOF Software (Leco, St. Joseph, MI) with a data-processing method, which includes the calibration curves for the 76 analytes previously performed. The quantifying ions previously established are used to construct standard curves of the peak area ratios (calibrator/internal standard pair) versus concentration. These curves are then used to determine the concentrations of the controls and unknown samples. All results are reported in mmol/mole of creatinine; a calculation factor is created using the creatinine value and volume of sample taken to calculate the final result. This factor is multiplied by the result from the calibration curve to give the final result in mmol/mole of creatinine. 4. The linearity/limit of quantitation of the method is 0.25–500 mmol. Samples in which the analyte concentrations exceed the upper limit of quantitation should be reanalyzed using a smaller aliquot of urine. 5. A typical calibration curve has correlation coefficient (r2) of >0.99. 6. Typical intra- and interassay imprecision is 136.2

0.011

26

15

Guanosine

+

284 > 152.2

0.011

20

18

Hypoxanthine

+

136.9 > 110.2 0.011

49

20

Inosine




266.9 > 134.9 0.011

42

22

Succinyladenosine

+

384 > 252

0.011

40

20

Xanthine




151 > 108

0.011

48

17

13

C5 adenosine

+

273 > 136.2

0.011

28

18

15

N5 deoxyadenosine +

257 > 141.2

0.011

26

15

15

N5 guanosine

+

289 > 157.25

0.011

21

18

13

C5 hypoxanthine

+

142 > 114

0.011

49

20

15

N4 inosine




270.9 > 138.9 0.011

42

22




153 > 109

48

17

1,3-

3.2

15

N2 xanthine

Data Analysis

0.011

1. A sample of MRM chromatograms of purine metabolites is shown in Fig. 1. MRM transitions used to quantify these analytes are listed in Table 4. 2. Quantlynx software (Waters, MA) was used to quantitate data. Linear standard curves based on calibrator/internal standard responses versus target concentration are established to quantitate unknown patient samples. Acceptable calibration curves have correlation coefficient (R2) >0.96 but preferable >0.99. 3. Normal reference ranges are listed in Table 5.

4

Notes 1. Urine purine values are normalized with creatine level of specimen. Diluted urine may cause artificial elevations of analyte concentrations. Extremely low urine samples with creating levels 0.96 but preferable >0.99.

4

Notes 1. Urine pyrimidine values are normalized with creatine level of specimen. Diluted urine may cause artificial elevations of analyte concentrations. Extremely low urine samples with creating levels 70

0.25

24

14

15

N2-uracil

114.8 > 70.7

0.25

24

15

Dihydrouracil

114.85 > 72.9

0.25

25

11

13

121.0 > 76.7

0.25

26

10

Thymine

127 > 110.2

0.25

26

13

D4-thymine

131 > 114.1

0.25

35

16

Dihydrothymine

129 > 69

0.25

22

14

D9-Dihydrothymine

135 > 74

0.25

28

15

Thymidine

243.2 > 127.2

0.25

10

12

D4-thymidine

247.2 > 131.2

0.25

15

10

15

C4, N-Dihydrouracil

Fig. 1 LCMS chromatograms of pyrimidine metabolites. From the top, thymidine, dihydrothymine, thymine, dihydrouracil, and uracil. Note that thymine elutes at 5.52 minute. A contamination with the same 127 > 110.2 MRM is eluted later at 8.99 minute

sample is required for measurement. Infantile urine samples frequently have low creatinine values and are cancelled. To

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Table 2 Normal reference rangesa Analyte

0–2 years old

>2 years old

Uracil

0–40

0–17

Dihydrouracil

0–158

3–58

Thymine

0–1.5

0–0.9

Dihydrothymine

0–4

0–2.4

Thymidine

0–0.2

0–0.2

aAll values are expressed as mmol/mole creatinine

Table 3 Diseases associated with elevated analyte concentrations Disease

Metabolites elevated

Dihydropyrimidine dehydrogenase deficiency

Uracil, thymine

Dihydropyrimidinase deficiency

Dihydrouracil, dihydrothymine, uracil, thymine

MNGIE

Thymidine, deoxyuridine

improve the chances of sample acceptability, 24-hour randomly collected urine is recommended. In such cases, individual urine samples may be pooled and frozen immediately after collection. 2. Occasionally, urine samples may give low responses for some pyrimidine analytes. Signals of corresponding internal standards are usually low as well. This signal-quenching phenomenon is most likely due to interferences by unknown medications or compounds in the urine sample. A quick solution is to dilute the urine specimen. This can help in many cases without further modification of LC settings. In rare cases in which dilution does not improve the signal intensity, a replacement specimen is required for accurate measurement. 3. It was found that an unidentified compound was eluting closely with dihydrouracil based on our experiences. This peak is only present in some urine samples and yields the same 114.85 > 72.9 MRM transition. In order to accurately measure dihydrouracil, it is important to ensure the LC gradient is optimized and able to clearly separate this contamination peak from real dihydrouracil peak (using isotopic internal standard as guidance).

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References 1. Aherne GW, Hardcastle A, Raynaud F et al (1996) Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells. Biochem Pharmacol 51:1293–1301 2. Andre Bp Van Kuilenburg AVC, Nico Ggm Aberling (ed) (2008) Screening for disorders of purine and pyrimidine metabolism using HPLC-electrospray tandem mass spectrometry. Springer-Verlag, Berlin Heidelberg 3. Balasubramaniam S, Duley JA, Christodoulou J (2014) Inborn errors of pyrimidine metabolism: clinical update and therapy. J Inherit Metab Dis 37:687–698 4. Burton K (ed) (1974) Spectral data and pK values for purines, pyrimidines, nucleosides, and nucleotides. Oxford University Press, London 5. Georges Van Den Berghe M-FV, Marie S (eds) (2012) Disorders of purine and pyrimidine metabolism. Springer-Verlag, Berlin Heidelberg 6. Jordheim LP, Durantel D, Zoulim F et al (2013) Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat Rev Drug Discov 12: 447–464 7. Jurecka A (2009) Inborn errors of purine and pyrimidine metabolism. J Inherit Metab Dis 32:247–263

8. Kelley RE, Andersson HC (2014) Disorders of purines and pyrimidines. Handb Clin Neurol 120:827–838 9. Mollick T, Laı´n S (2020) Modulating pyrimidine ribonucleotide levels for the treatment of cancer. Cancer Metab 8:12 10. Nyhan WL (2005) Disorders of purine and pyrimidine metabolism. Mol Genet Metab 86: 25–33 11. Ohyama T, Matsubara C, Takamura K (1996) Highly sensitive densitometry for Sugarphosphoeters and nucleotides using enzymatic hydrolysis on TLC plate. Chem Pharm Bull 44:1252–1254 12. Schaefer GB, Mendelsohn NJ (2013) Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions. Genet Med 15:399–407 13. Van Gennip Ah BH, WI N (eds) (2006) Inborn error of purine and pyrimidine metabolism. Springer-Verlag, Berlin, Heidelberg 14. Van Kuilenburg AB, Dobritzsch D, Meijer J et al (2010) Dihydropyrimidinase deficiency: phenotype, genotype and structural consequences in 17 patients. Biochim Biophys Acta 1802:639–648 15. Van Kuilenburg AB, Meijer J, Dobritzsch D et al (2007) Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency. Mol Genet Metab 91: 157–164

Chapter 39 Quantitation of Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) J. Grace van der Gugten and Daniel T. Holmes Abstract Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison’s disease, congenital adrenal hyperplasia, Bartters and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semiautomated and simple method for the accurate and precise measurement of PRA from 500 μL of plasma (250 μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37
C in buffering conditions at pH 6, the reaction is quenched with 10% formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/ MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34–30.0 ng/mL, which corresponds to PRA values of 0.11–10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8–5.0% for BioRad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal-to-noise ratio is 50:1, indicating that the limit of quantitation is well below the level required for clinical use. Key words Primary aldosteronism, Mineralocorticoid hypertension, Secondary hypertension, Hypokalemia, Angiotensin I, Plasma renin activity, Mass spectrometry

1

Introduction Renin (3.4.23.15) is an enzyme released into circulation by the juxtoglomerular apparatus of the nephron. Its action is to cleave the decapeptide angiotensin I (AngI, DRVYIHPFHL) from angiotensinogen, an α2-globulin secreted by the liver. Under the action of the angiotensin-converting enzyme (ACE), which is primarily expressed in pulmonary vascular endothelium, AngI is converted to the octapeptide angiotensin II (AngII), a potent vasoconstrictor

Uttam Garg (ed.), Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2546, https://doi.org/10.1007/978-1-0716-2565-1_39, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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that also upregulates the production of aldosterone at the level of the adrenal cortex. The principal action of aldosterone, as mediated by the mineralocorticoid receptor, is to cause the retention of sodium ions in exchange for potassium ions through expression of basolateral K+/Na+ channels in the renal collecting duct. This leads to the expansion of the plasma volume, the maintenance of blood pressure, and urinary wasting of K+. In pathological states, the adrenal production of aldosterone can become relatively autonomous due to bilateral adrenal hyperplasia, aldosterone-producing adenoma, and a number of other less frequent causes [1, 2]. The unregulated production of aldosterone leads to physiologically appropriate suppression of plasma renin activity (PRA) but pathological Na+ retention and hypertension, which may be accompanied by hypokalemia and metabolic alkalosis [1]. The accepted means of screening for PA is the determination of simultaneously collected plasma aldosterone and PRA and the calculation of the aldosterone-to-renin ratio (ARR). Ratios over a specific threshold, usually between 20 and 40 ng/dL:ng/mL/ h depending on method-specific biases, represent a positive screen for PA in a clinically appropriate setting [2]. PRA determination is also useful in the diagnosis of hypertension caused by secondary (hyperreninemic) aldosteronism, which is seen in renal artery stenosis (whether attributable to atherosclerosis or fibromuscular dysplasia) or, rarely, renin-producing tumors of the kidney. Other conditions where PRA determination may be diagnostically useful include primary adrenal insufficiency (Addison’s Disease), Bartter and Gitelman syndromes, and neonatal congenital or acquired neonatal pseudohypoaldosteronism. PRA is determined by measuring the amount of AngI generated after incubation of plasma in appropriately buffered conditions (usually pH ¼ 6) for a fixed time period, which is at least 1 h, typically 3 h and occasionally 18 h if additional analytical sensitivity is desired [3]. In traditional assays, a “blank” specimen is also prepared by incubating an aliquot of plasma in identical buffering conditions but cooled on an ice-water bath so that renin is enzymatically inactive. The AngI concentration in the blank sample can then be used to correct for the presence of low-level AngI in the plasma at the time of (or inadvertently generated after) the collection. PRA is then calculated as follows: PRA ¼

½AngI37
C  ½AngIblank , Δt

ð1Þ

where [AngI]37
C represents the concentration of AngI after of after incubation at 37
C, [AngI]blank represents the concentration of AngI after incubation on ice-water bath, and Δt is the duration of the incubation.

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441

In this method, the AngI is generated using a 3 h incubation at pH ¼ 6 where renin is approximately twofold more enzymatically active than at physiological pH [4]. Generated AngI is extracted from the plasma samples using solid phase extraction (SPE) and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The implicit assumption is that the endogenous angiotensinogen in the specimen will remain at a sufficiently high concentration in the 3 h incubation so as not to be rate limiting. Proteolytic degradation of AngI by ACE and nonspecific peptidases is inhibited during AngI generation by both ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonylfluoride (PMSF), which are present in the generation buffer. Analytically, AngI was traditionally determined by radioimmunoassay (RIA), but with the advent of LC-MS/MS instruments into routine clinical laboratories, several authors have published methods suitable for high-throughput environments [5–8]. In addition to the benefit of avoiding radiotracers used in RIA, LC-MS/MS has a linear relationship between analyte concentration and instrument response. This affords a very wide analytical range, thereby permitting the direct analysis of specimens from neonates (and from other high-renin states) without predilution, which causes unpredictable and patient-specific effects on recovery [4]. LC-MS/MS also permits the use of ion ratios to verify the absence of analytical interferents and, by virtue of its analytical sensitivity and specificity, likely obviates the need for blank subtraction, provided that the generation step is adequately long [7].

2

Materials Samples

Plasma collected in lavender-top K2EDTA or K3EDTA tubes is the only acceptable sample type for this assay [9]. Specimens should be collected into EDTA plasma tubes and centrifuged within 30 min (preferably within 10 min) and rapidly frozen until analysis [3] (see Note 1).

2.2 Solvents and Reagents

1. Bovine Serum Albumin, Fraction V, Omnipur® (EMD Millipore. Billerica, MA).

2.1

2. Phenylmethanesulfonylfluoride (PMSF), >98.5% (SigmaAldrich St. Louis, MO). 3. 100 mM PMSF in methanol: Dissolve 0.174 g of PMSF into 10 mL of methanol. Store at 2–8
C. Expected stability: >6 months. 4. Buffer A (0.1 M Tris Base pH 6): Dissolve 12.11 g of Tris Base into 1000 mL of deionized water. Adjust to pH 6 with glacial acetic acid. Store at 2–8
C. Expected stability: >6 months.

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5. Working Buffer B (1% BSA (w/v) in Stock Buffer A): Dissolve 0.1 g of BSA into 10 mL of 0.1 M Tris Buffer pH 6. Prepare fresh for each batch. 6. Generation Buffer: Weigh 121.1 g of Tris Base +74 g EDTA into a 1000 mL volumetric flask. Add DI water to about 900 mL. Sonicate for 30 min or until the Tris Base and EDTA are fully dissolved. Add DI water to the volume mark and mix well. Transfer to a labeled polypropylene storage container. Adjust to a pH range of 5.45–5.50 with glacial acetic acid. Store at 2–8
C. Expected stability: >6 months. 7. On the day of analysis, add 100 μL of the 100 mM PMSF solution to 10 mL of generation buffer. 8. Mobile Phase A (0.2% formic acid in water): Add 2 mL of formic acid to 1 L of DI water. Mix well. 9. Mobile Phase B: 100% LCMS grade methanol. 10. 10% Formic Acid in water: Add 50 mL of formic acid to 450 mL of DI water. Mix well. Store at room temperature. Expected stability: >3 months. 11. 5% formic acid/20% methanol in water: Add 25 mL of formic acid and 100 mL of methanol to 375 mL of DI water. Mix well. Store at room temperature. Expected stability: >3 months. 12. Lyphochek™ Hypertension Markers (Bio-Rad, Montreal QC, Canada). 2.3 Internal Standards and Standards

Control,

Trilevel

1. Primary standard: AngI: 3  10 μg (Proteochem, Loves Park IL). 2. Stable isotopically labelled internal standard (SIS): AngI (DRVYIHPFHL) with isotopically labelled arginine residue (13C,15N) was synthesized by the University of Victoria Genome BC Proteomics Centre (see Note 2). 3. AngI stock solution (5000 ng/mL AngI in Working Buffer B): contents of 10 μg vials of AngI are dissolved in a total of exactly 2 mL of Working Buffer B to make a solution of 5000 ng/mL. Note that the dissolution process must be performed in 0.5 mL aliquots as the Proteochem vials are small in volume. Aliquot 500 μL of the 5000 ng/mL stock solution to microvials with lids, seal with parafilm, and store at 70
C. 4. AngI SIS Solutions: (a) Stock Solution, 1 mg/mL: dissolve 1 mg of AngI-SIS in DI water. Mix well to dissolve. (b) Intermediate Working Solution, 10 μg/mL: dilute the 1 mg/mL Stock Solution 100-fold to a resulting concentration of 10 μg/mL: add 100 μL of the 1 mg/mL stock solution to 9.90 mL of DI water. Mix well. Aliquot 10  1 mL to labelled cryovials and store at 70
C.

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(c) Working Solution, 10 ng/mL in 10% formic acid: Remove a vial of the 10 μg/mL Ang1-SIS Intermediate Working Solution and allow it to thaw. Mix well. Aliquot 0.5 mL into 500 mL of 10% formic acid. The resulting concentration is 10 ng/mL. Store at 2–8
C. 2.4 Calibrators and Controls

1. Controls used are BioRad Hypertension controls Levels 1, 2, and 3. The target mean and standard deviations are set based on results of 20 analyses over 10 runs. Controls are run in duplicate with each analytical run. 2. An in-house patient pool is prepared approximately once yearly by pooling discarded anonymized previously analyzed patient plasma samples. The patient pool target value is set as per the BioRad QC. The patient pool is run in duplicate with each analytical run. 3. Calibrators are prepared in-house using the AngI stock solution in 1% BSA in Buffer A (Working Buffer B) according to details provided in Table 1.

2.5 Analytical Equipment and Supplies

1. Strata-X 33u Polymeric Reversed Phase 96-Well Plate, 60 mg/ well (Phenomenex, Torrance, CA). 2. Vacuum manifold or positive pressure manifold, installed in robotic liquid handler or manually controlled. 3. 2 mL 96 deep square well, V-bottom plates (Corning, Corning NY). 4. Silicone cap mats with PTFE barrier for square well plates (Microliter Analytical Supplies, Suwanee GA).

Table 1 Volumes required to prepare final calibrant solutions. Calibrators are prepared by placing approximately 15 mL of Buffer B into a 20 mL class A volumetric flask. Spiking solution is added and 20 mL volume is filled with Buffer B followed by thorough mixing. Calibrators are aliquoted 0.5 mL to labeled 12  75 mm polystyrene tubes, capped, and stored at 70 ˚C. Stability 1 year Calibrator Ang 1 spiking level solution (ng/mL)

Volume of spiking solution (μL)

Final volume Buffer B (mL)

Final concentration (ng/mL)

Std1

100

67

20

0.335

Std2

100

120

20

0.6

Std3

1000

27

20

1.35

Std4

1000

54

20

2.7

Std5

1000

180

20

9

Std6

5000

120

20

30

Std7

5000

400

20

100

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5. 2 mL Nunc® 96 DeepWell™ round-bottom well plates, (Thermo Scientific, Waltham, MA). 6. Nunc® cap mats for round bottom plates (Thremo Scientific, Waltham, MA). 7. API5000 or 5500 QTRAP® triple quadrupole mass spectrometer (SCIEX, Concord, ON) or other mass spectrometer capable of reaching the required limit of detection, equipped with appropriate software (e.g., Analyst®). 8. Shimadzu 20 AC LC System with pumps, column oven, degasser, autosampler, or equivalent system (Kyoto, Japan). 9. Analytical column: 4u Proteo 90 Å, 50  2.0 mm (Phenomenex, Torrance, CA). 10. Guard column, Torrance, CA).

3

C12,

4



2.0

mm

(Phenomenex,

Methods

3.1 Step-Wise Procedure

1. Calibrators are thawed at room temperature. 2. Prepare Working Buffer B. 3. Prepare calibration standards by hand or on robotic liquid handler (see Note 3). The calibration standards (see Note 4) are serial dilutions of S7 as follows: (a) S6: 420 μL of S7 is mixed with 980 μL of Working Buffer B––resulting concentration is 30.00 ng/mL. (b) S5: 420 μL of S6 is mixed with 980 μL of Working Buffer B––resulting concentration is 9.000 ng/mL. (c) S4: 420 μL of S5 is mixed with 980 μL of Working Buffer B––resulting concentration is 2.700 ng/mL. (d) S3: 700 μL of S4 is mixed with 700 μL of Working Buffer B––resulting concentration is 1.350 ng/mL. (e) S2: 700 μL of S3 is mixed with 700 μL of Working Buffer B––resulting concentration is 0.6750 ng/mL. (f) S1: 700 μL of S2 is mixed with 700 μL of Working Buffer B––resulting concentration is 0.3375 ng/mL. (g) Blank: Working Buffer B with no AngI added. 4. Prepare Generation Buffer as described above. 5. Samples and QCs are thawed in room temperature water bath for 5 min. 6. Samples and QCs are transferred to an ice bath to complete thawing. 7. Samples and QCs mixed and centrifuged for 5 min at 2100 g at 0.99. 4. Compound-specific parameters for each analyte are given in Table 4. 5. Quality control samples are evaluated with each run. The run is considered acceptable if calculated concentrations of controls are within the 20% of target values. 6. Samples with results greater than upper limit of linearity should be diluted with blank serum. 7. A typical ion chromatogram for various steroids is shown in Fig. 1.

4

Notes 1. Validate serum before use for calibrators and quality controls to ensure the absence of each analyte. 2. When possible, calibrators and controls should be prepared from different lots of stock solution on separate days.

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Table 4 Compound-specific parameter Q1 mass (amu)

Q3 mass (amu)

287.2

Androstenedione- C3

Qualifier ion

Declustering potential (DP)

Collision energy (CE)

Cell exit potential (CXP)

97.0

109.0

70

32

10

290.3

100.1

None

81

31

16

17-OHP

331.3

97.1

109.2

80

38

17

17-OHP D8

339.2

100.0

None

80

40

17

DHEA

271.2

213.1

253.2

56

32

10

DHEA-D5

276.2

258.3

None

61

21

16

11-deoxycortisol

347.1

97.0

109.2

71

37

6

11-deoxycortisol-D5

352.2

100.0

None

66

41

6

Testosterone

288.9

97.1

109.1

36

35

16

Testosterone-D3

292.4

97.1

None

61

35

16

Analyte Androstenedione 13

Fig. 1 A typical ion chromatogram for different steroids

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References 1. Shaw AM (2010) 21-hydroxylase deficiency congenital adrenal hyperplasia. Neonatal Netw 29(3):191–196 2. White PC, Bachega TA (2012) Congenital adrenal hyperplasia due to 21 hydroxylase deficiency: from birth to adulthood. Semin Reprod Med 30(5):400–409 3. Heather NL, Seneviratne SN, Webster D, Derraik JG, Jefferies C, Carll J, Jiang Y, Cutfield WS, Hofman PL (2015) Newborn screening for congenital adrenal hyperplasia in New Zealand, 1994-2013. J Clin Endocrinol Metab 100(3):1002–1008 4. White PC (2013) Optimizing newborn screening for congenital adrenal hyperplasia. J Pediatr 163(1):10–12 5. Vats P, Dabas A, Jain V, Seth A, Yadav S, Kabra M, Gupta N, Singh P, Sharma R, Kumar R, Polipalli SK, Batra P, Thelma BK, Kapoor S (2020) Newborn screening and diagnosis of infants with congenital adrenal hyperplasia. Indian Pediatr 57(1):49–55 6. Taieb J, Benattar C, Birr AS, Lindenbaum A (2002) Limitations of steroid determination by direct immunoassay. Clin Chem 48(3): 583–585 7. Taylor AE, Keevil B, Huhtaniemi I (2015) Mass spectrometry and immunoassay; how to measure steroid hormones today and tomorrow. Eur J Endocrinol 173(2):D1–D12 8. Abdel-Khalik J, Bjorklund E, Hansen M (2013) Simultaneous determination of endogenous steroid hormones in human and animal plasma and serum by liquid or gas chromatography coupled to tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 928:58–77

9. Bulska E, Gorczyca D, Zalewska I, Pokrywka A, Kwiatkowska D (2015) Analytical approach for the determination of steroid profile of humans by gas chromatography isotope ratio mass spectrometry aimed at distinguishing between endogenous and exogenous steroids. J Pharm Biomed Anal 106:159–166 10. McDonald JG, Matthew S, Auchus RJ (2011) Steroid profiling by gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry for adrenal diseases. Horm Cancer 2(6):324–332 11. Fanelli F, Belluomo I, Di Lallo VD, Cuomo G, De Iasio R, Baccini M, Casadio E, Casetta B, Vicennati V, Gambineri A, Grossi G, Pasquali R, Pagotto U (2011) Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults. Steroids 76(3):244–253 12. Kulle AE, Welzel M, Holterhus PM, Riepe FG (2011) Principles and clinical applications of liquid chromatography – tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones. J Endocrinol Investig 34(9):702–708 13. Stanczyk FZ, Clarke NJ (2010) Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Biol 121(3–5):491–495 14. Munar A, Frazee C, Garg U (2016) Quantification of Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone by Liquid ChromatographyTandem Mass Spectrometry (LC/MS/MS). Methods Mol Biol 1378:273–279

Chapter 41 A User-Friendly Sample Preparation Alternative for Manual and Automated LC-MS/MS Quantification of Testosterone Judy A. Stone, Dawn Francisco, Heather Tone, Joshua Akin, and Robert L. Fitzgerald Abstract We describe an LC-MS/MS method for serum testosterone using a novel extraction media, AC Extraction Plate™ (AC Plate,Tecan Schweiz). The AC Plate principle is essentially that of a liquid-liquid extraction (LLE) but employs a stationary nonpolar phase coated on the wells of 96-well plates instead of a nonmiscible organic solvent for partitioning testosterone out of serum, leaving interfering substances behind. This low complexity sample preparation protocol has been validated for and used in production in our laboratory with both manual and automated liquid handling. The primary advantage of this method is the highly reproducible nature of an extraction method that does not require LC-MS/MS expertise or specialized extraction equipment. We modified the existing vendor application and validated the method for matrix effect, recovery, precision, trueness [accuracy relative to certified reference material (CRM)], specificity, reportable range, sample stability, various sample containers, and correlation with other methods. Method performance is excellent, with a reportable range of 4–750 ng/dL, between-day quality control coefficient of variation (CV) over 12 months of 109.2 divided by the peak area of the m/z 289.3 > 97.1. The average qualifier ion ratio is calculated as

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Fig. 1 LC gradient for testosterone Table 1 LC gradient table Time (min)

Flow rate (mL/min)

%A

%B

Curve

1.

Initial

0.4

90

10

Initial

2.

0.01

0.4

90

10

6

3.

2.80

0.4

60

40

6

4.

3.80

0.6

10

90

6

5.

4.18

0.6

10

90

6

6.

4.19

0.6

5

95

6

7.

4.93

0.6

5

95

6

8.

4.94

0.4

90

10

6

9.

5.44

0.4

90

10

6

the mean of the standards and +/
20% is used as the acceptance criteria. 5. This method is linear from 4 ng/dL to 750 ng/dL of testosterone. 6. The accuracy and precision for this LC-MS/MS method is shown in Table 3. 3.4 Development Notes 3.4.1

LC Optimization

The choices for LC column and mobile phases for this method were limited. A requirement was to use the existing LC-MS/MS instrument configuration that was designed for unattended, overnight, sequential analysis of multiple batches using six other methods. Therefore, the only options for investigation were four different

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Table 2 MS conditions Capillary (kV)

0.60

Cone (V)

40

Source offset (V)

50

Source temperature ( C)

150 

Desolvation temperature ( C)

650

Cone gas flow (L/Hr)

150

Desolvation gas flow (L/Hr)

1000

Collision gas flow (mL/min)

0.18

Nebulizer gas flow (Bar)

7.0

Fig. 2 MRM chromatogram for testosterone obtained from a female specimen that contained 8 ng/dL testosterone

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Table 3 Assessment of accuracy and precision for testosterone LC-MS/MS assay Sample

Target

Cal A

4

Cal B

Mean

%Bias

SD

%CV

n

3.8


5

0

6

10

52.5

55.6

6

3

6

10

Cal C

750

747.1

0

3

0

10

SRM 971 F

27.7

28.3

2

1

4

5

SRM 971 M

643.5

620.7


4

41

7

6

SRM 971a F

32.3

31.1


4

1

2

3

SRM 971a M

580.8

572.1


2

19

3

3

LC columns and two organic, two aqueous mobile phases that were already in use. If additional signal, better resolution, and/or shorter run times are necessary—additional column and mobile phase optimization may be worthwhile. 3.4.2 Extraction Optimization

A limitation of the AC Plate is that the total volume of liquid in the plate well (e.g., sample + extraction reagent) cannot exceed 350 μL to avoid cross-well contamination during orbital shaking [15]. This constraint is chiefly of concern when optimizing the volumes of sample and extraction reagent needed to achieve optimal extraction recovery and the desired limit of quantitation. We interrogated and adapted a testosterone application note from the AC Plate vendor (Tecan Schweiz) by evaluating the following extraction conditions for optimal extraction recovery, matrix effect, and LC signal [5, 15]: (a) Selection of organic solvent type (methanol, acetonitrile) and ratio to water for the working internal standard. (b) Comparison of two agents in the extraction buffer for separating testosterone from binding proteins (ZnSO4, LiCl). (c) Adjustment of pH (10 with ammonium hydroxide, no pH adjustment) for the extraction buffer and wash reagent. (d) Selection of organic solvent type (methanol, acetonitrile) and ratio to aqueous components in the extraction buffer. (e) Selection of organic solvent type (methanol, acetonitrile) and ratio to water in the elution/injection reagent. (f) Sample and extraction reagent volumes.

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3.4.3 Liquid Handling Optimization

For the manual protocol, air displacement pipets were used for samples, a positive displacement repeater pipet was used for the internal standard and reagents, and an 8-channel multi-pipet was used for removing waste. For the automated liquid handler (ALH) protocol, the big challenges were a) optimizing precision for the internal standard dispense and b) preventing dripping from tips during the extraction/wash reagent removal. Because the ALH AC Plate extraction itself is quite reproducible, we discovered that total precision of the method was highly dependent on the precision of the internal standard (IS) pipetting. We optimized the IS liquid class (based on guidance from Tecan and experimentation). Best precision was obtained by prewettting single-use tips via three aspiration/dispense cycles of the working IS prior to the final aspiration/dispense. Disposal of the extraction waste also required single-use tips to avoid cross-well contamination, but reuse of tips for reagent dispense steps and disposal of wash waste is feasible. Best performance for waste disposal was obtained by optimizing the size of the trailing air gap, the speed of waste aspiration, and the travel pattern from extraction plate to the waste trough.

3.4.4

Plate Handling

We did not use all 96 wells of an AC Plate for each batch during method development or once the method was moved to production. We wrote the locations (e.g., C5-E8) of wells used in each batch on the side of uniquely barcoded plates and in an Excel tracking spreadsheet. Partially used, sealed AC Plates were stored at room temperature. We found no difference in extraction recovery between wells of freshly unsealed, never used AC Plates and wells of plates that had been partially used and stored in this manner for up to 6 months. We observed large differences in recovery of 13C-testosterone internal standard using polypropylene injection plates (extraction eluent was transferred from the AC Plate to an injection plate). We switched to glass inserts (see Methods) in the injection plate and saw much more reproducible IS peak areas between wells. There was no difference in testosterone recovery between deactivated and untreated glass inserts. Plates are sealed and centrifuged after the transfer of eluents from the extraction plate to glass inserts in the injection plate to eliminate air bubbles. Sample stability in the injection plates was validated for up to 120 hours at 2–8  C.

3.4.5

Matrix Effect

Our optimization of extraction and LC conditions was focused more on reducing and standardizing matrix effect between samples than on increasing recovery. We performed qualitative [postcolumn infusion] [16] and quantitative [17] matrix effect experiments and monitored phospholipid MRMs to select the best extraction and LC conditions that met this objective. Recovery was quite low (mean 26%) but very reproducible (CV for IS peak areas was

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10%). The signal-to-noise ratio at a 4 ng/dL limit of quantitation was good: it was 11–63 during validation. Removal of phospholipids and ion suppression was very successful, with a mean decrease in signal from matrix of only 5% and only a 5% CV for IS normalized matrix effect across all 10 patient samples tested. 3.4.6 Collection Container Validation

A major advantage of this method is the option to use either serum or heparinized plasma SST tubes for sample collection. No interference from or loss of analyte to the SST gel was detected, and testosterone was stable in serum/plasma stored on the SST gel for up to 7 days (2–8  C). Primary barcoded SST tubes or aliquot tubes can be loaded on the ALH for sampling.

3.4.7

Calibration Design

We demonstrated excellent and similar between-day precision and accuracy using either an intensive (10 calibrators) versus sparse (3 calibrators) calibration design and for in-house prepared (primary testosterone standard added to double charcoal-stripped female serum from Golden West Biological) versus commercially available, traceable to NIST CRM, purchased calibrators (see Methods). Between-run imprecision for QC material during validation met the analytical CV “desirable” goal of 5.3% based on biological variability. Between-day CVs in production are typically 339.4

0.08

38

23

3.50

d3-Phy

401.3 > 342.4

0.08

38

23

3.49

C20:0

398.3 > 339.4

0.08

38

23

4.01

d3-C20:0

401.3 > 342.4

0.08

38

23

4.00

C22:0

426.3 > 367.4

0.115

40

24

4.58

d4-C22:0

430.3 > 371.4

0.115

40

24

4.57

C24:0

454.3 > 395.4

0.04

40

26

5.00

d4-C24:0

458.3 > 399.4

0.04

40

26

5.00

C26:0

482.3 > 423.45

0.55

43

27

5.41

d4-C26:0

486.3 > 427.45

0.55

43

27

5.40

Table 7 Recommended maximum value for the calibration curve y-intercept Compound

y-intercept maximum value

Pri