Drug Monitoring by Hplc : Recent Developments [1 ed.] 9781613244722, 9781608761838

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

PHARMACOLOGY – RESEARCH, SAFETY TESTING AND REGULATION SERIES

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DRUG MONITORING BY HPLC: RECENT DEVELOPMENTS

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

PHARMACOLOGY – RESEARCH, SAFETY TESTING AND REGULATION SERIES

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

DRUG MONITORING BY HPLC: RECENT DEVELOPMENTS

VICTORIA SAMANIDOU AND

EFTICHIA KARAGEORGOU

Nova Science Publishers, Inc. New York

Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

PHARMACOLOGY – RESEARCH, SAFETY TESTING AND REGULATION SERIES Antibiotic Resistance: Causes and Risk Factors, Mechanisms and Alternatives Adriel R. Bonilla and Kaden P. Muniz (Editors) 2009. ISBN: 978-1-60741-623-4 Antibiotic Resistance: Causes and Risk Factors, Mechanisms and Alternatives Adriel R. Bonilla and Kaden P. Muniz (Editors) 2009. ISBN: 978-1-61668-162-3

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Poisons: Physiologically Active Substances S. B. Zotov and O. I. Tuzhikov 2009. ISBN: 978-1-60741-973-0 Drug Monitoring by HPLC: Recent Developments Victoria Samanidou and Eftichia Karageorgou 2010. ISBN: 978-1-60876-183-8

Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Samanidou, Victoria. Drug monitoring by HPLC : recent developments / Victoria Samanidou and Eftichia Karageorgou. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61324-472-2 (eBook) 1. High performance liquid chromatography. 2. Drugs--Analysis. I. Karageorgou, Eftichia. II. Title. [DNLM: 1. Drug Monitoring--methods. 2. Chromatography, High Pressure Liquid. WB 330 S187d 2009] RS189.5.H54S26 2009 615'.1901--dc22 2009034548

Published by Nova Science Publishers, Inc.  New York

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CONTENTS

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Preface

vii

Chapter 1

Introduction

1

Chapter 2

Brief Introduction to HPLC

5

Chapter 3

Sample Preparation

11

Chapter 4

Anti-Cancer Drugs

21

Chapter 5

Bronchodilators

29

Chapter 6

Cardiovascular Drugs

33

Chapter 7

Antipsychotics

41

Chapter 8

Antibiotics

49

Chapter 9

Antiepileptic Drugs

53

Chapter 10

Antidepressants

83

Chapter 11

Immunosuppressants

105

Chapter 12

Conclusions

111

Index

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115

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PREFACE Drug monitoring in clinical chemistry refers to the measurement of drug concentration in blood serum/plasma so that an optimal concentration is obtained to benefit patient with minimal toxic adverse effects. Drug monitoring addresses drugs with narrow effective range or a narrow therapeutic/toxic index. It is required to individualise and optimise drug therapy. Moreover it supports pharmacokinetic and drug metabolism studies. Drugs that usually require monitoring include: cardioactive medications, antiepileptic drugs, antibiotics (e.g. aminoglycosides), anti-cancer drugs, immunosuppressants, antidepressants (e.g. tricyclic antidepressants), bronchodilators (Theophylline), antipsychotics etc. Monitoring of medication is also important to detect poisoning with above drugs in forensics. In pharmacology, many drugs are used without monitoring of blood levels, as their dosage can generally be varied according to the clinical response of the patient. In some drugs insufficient levels will lead to undertreatment or resistance, and excessive levels can lead to toxicity and tissue damage. The available analytical methods for monitoring drug levels in patient specimens in human blood or serum/plasma include: immunoassays, such as microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT), fluorescent polarization immunoassay (FPIA), radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) and most frequently fluorescence polarisation immunoassay (FPIA) and liquid chromatography-based methods. Chromatography-based methods include high performance liquid chromatography (HPLC) with ultraviolet detection, HPLCmass spectrometry (HPLC-MS), and HPLC-tandem mass spectrometry (HPLCMS/MS). In clinical practice, immunochemical assays are applied to screen the drugs in biological fluids. These assays while they are sensitive, they are not

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Victoria Samanidou and Eftichia Karageorgou

sufficiently selective and can only be used to eliminate negative samples. Confirmation of the initial screening should always be implemented by a separation assay usually a chromatographic one, which should have sufficient sensitivity and selectivity to confidently identify the analyte down to the cut-off level. Currently HPLC-MS/MS has gained increasing popularity in clinical laboratories due to the advantages of the technology over other methods, while the capital cost for instruments has been decreased. HPLC-MS/MS provides high specificity and sensitivity for the simultaneous measurement of several drugs and/or their major metabolites in one single analytical run. In this book chromatographic methods published in the last decade are reviewed regarding their applicability to drug monitoring.

VICTORIA SAMANIDOU

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Assistant Professor of Analytical Chemistry, Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, R54124 Thessaloniki, Greece,Tel. +302310-997698, Fax. +302310-997719, e-mail: [email protected], http://users.auth.gr/samanidu.

EFTICHIA KARAGEORGOU MSc in Analytical Chemistry, Laboratory of Analytical Chemistry Department of Chemistry Aristotle University of Thessaloniki GR-54124 Thessaloniki,Greece.

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Chapter 1

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INTRODUCTION Incessant discovery of new drugs is a worldwide demand. New drugs may be derived from natural products or be produced synthetically. New compounds have to be comparatively tested with their predecessors. Toxicity, biological half-life, ease of administration are important factors to be checked when a new drug is under development. Research on absorption, distribution, metabolism and excretion of the new drugs must proceed at the same time with development process. Therapeutic Drug Monitoring (TDM) is the measurement of specific drugs at different time intervals in order to maintain a relatively constant concentration of the medication in the bloodstream. Drugs that require monitoring tend to have a narrow ―therapeutic range‖, which means that the necessary quantity to be effective is not far from the quantity that causes significant side effects and/or signs of toxicity. Maintaining this steady state is mandatory and not as simple as compared with a standard dose of medication. Personalization of dosage is a significant issue, since absorption, metabolism, utilization, and elimination of drugs will be different in each person, based upon their age, general state of health, genetic makeup, and the interference of other medications that they are taking. This rate may change over time and vary from day to day. [1] Luckily not all medications require therapeutic monitoring. Most drugs have a wide therapeutic range and can be prescribed based upon pre-established dosing schedules. The effectiveness of these treatments is evaluated, while it is not usually necessary to determine the concentration of the drug in the bloodstream. Many of the drugs that are monitored therapeutically are taken for a lifetime. They must be maintained at steady concentrations year after year, while the patient ages and life events such as pregnancies, temporary illnesses, infections, emotional and physical stress, accidents, and surgeries may occur. Over time other

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Victoria Samanidou and Eftichia Karageorgou

chronic conditions such as cardiovascular disease, kidney disease, thyroid disease, liver disease, and HIV/AIDS may arise that also require lifetime medication and that may affect the processing of their monitored drugs. Therapeutic drug monitoring put up with these changes. Patient non-compliance is assessed by identifying the effect of drug interactions caused by drug higher or lower concentrations. This helps to individualize dosages to fit the current needs of the specific patient helping tolp identify decreases in the efficiency of and dysfunctions in the body in metabolizing and eliminating therapeutic drugs. [1] It has become clear by now that therapeutic drug monitoring is of great value for specific classes of drugs. The following criteria should be fulfilled so that TDM measurements are clinically worthwhile:

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Relationship between plasma drug concentration and therapeutic response and/or toxicity. Relationship between plasma concentration and drug dosage. Clinical indication for no response to treatment; suspected noncompliance; signs of toxicity. [2] In vivo–factors affecting therapeutic drug levels include: Patient compliance, Bioavailability (access to circulation, interaction with related receptors; ionized and free, or bound to a carrier molecule e.g. albumin), and Pharmacokinetics. Drug concentrations are affected by: Interaction with diet or other medication. Absorption on large molecule size components, e.g. proteins. Lipid solubility, which affects the volume of distribution as highly lipidsoluble substances have high affinity for adipose tissue and a low tendency to remain in the vascular compartment. Biotransformation, by hepatic metabolism, in which polar groups are introduced into relatively insoluble molecules by oxidation, reduction or hydrolysis. Physiological factors like age affect drug levels. Lower doses are necessary in both infants and the elderly, in the former because the metabolism mechanism is not fully operational. In elderly people the organism may suffer from decrease in cardiac and/or renal function, enzyme activity etc. Enzyme induction may reduce the drug's activity,

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Introduction

3

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Enzyme inhibition may result in the increase of drug activity, prolonging the action of various drugs e.g. metronidazole. Genetic factors. Associated diseases may also affect drug distribution or metabolism, e.g. renal disease with decrease in clearance and increase of drug levels, or hepatic disease, in which there is a decrease in albumin production and enzyme activity resulting in a functional increase in drug levels. [3] As it has been already mentioned not all medications require therapeutic monitoring. For example many anti-infective agents (e.g. aminoglycosides antibiotics) have a wide therapeutic range, which means that standard doses can be used. However, with some antibiotics, toxicity is associated with persistently high concentrations whereas with others, treatment failure can result from persistently low concentrations. Antibiotics are ―time-dependent‖. The aim when this medication is prescribed is to maintain concentrations above the Minimum Inhibitory Concentration (MIC) for most of the dosage interval or ―concentrationdependent‖. In the latter situation the aim is to achieve high peak concentrations, while allowing the concentration to fall to low levels between doses. A field most commonly requiring TDM is psychiatry. The routine analysis of drugs used in psychiatry is popular in many European countries. Monitoring concentrations of antiepileptic drugs became popular during the 1980‘s, when it was recognized that it could help to reduce variability in response and toxicity. Most of the older anticonvulsants are eliminated by hepatic metabolism, leading to a wide range of dose requirements and a high incidence of drug interactions. Target ranges have been identified for a number of antidepressants and antipsychotics, many of which are metabolized by cytochrome P450 2D6. Significant differences in dosage requirements have been demonstrated between poor or extensive metabolisers. Although anticancer drugs have narrow therapeutic ranges, concentrations are not routinely monitored because of a lack of data on concentration-effect relationships. One exception, however, is methotrexate, where folic acid rescue therapy is based on monitoring the methotrexate concentration 24-48 hours after high dose therapy and continuing until concentrations are below 0.05 μmol/L. For other anticancer drugs, monitoring the enzyme responsible for their metabolism has been used to help adjust doses and reduce toxicity. Bronchodilators belong also to drugs requiring TDM. Although theophylline has largely been replaced by other drugs with less potential for adverse effects, it is still used in some patients. It is principally cleared by hepatic metabolism, thus dosage requirements vary widely and drug interactions remain a major concern.

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Victoria Samanidou and Eftichia Karageorgou

Bronchodilator effects have been demonstrated within the normal target range of 10-20 mg/L, but lower concentrations are associated with anti-inflammatory and steroid sparing effects. Consequently, there is some support for reducing the target range to 5-15 mg/L, which might reduce the incidence of toxic effects. [4] It can be concluded by now that the appropriate use of therapeutic drug monitoring requires measuring the concentration of a drug in the patient‘s blood. In the following paragraphs drugs requiring TDM are classified according to their activity and target and methods are summarized to provide information on their application. Figure 1 illustrates the chemical structures of most common analytes requiring drug monitoring.

REFERENCES

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[1] [2] [3] [4]

www.labtestonline.org (Accessed January 2009) www.toxlab.co.uk (Accessed January 2009) www.thefreedictionary.com (Accessed January 2009) www.pjonline.com (The Pharmaceutical Journal Vol.273 2004) (Accessed January 2009)

SUGGESTED FURTHER READING [1] [2]

[3] [4] [5]

Dasgupta, A. Handbook of Drug Monitoring Methods, Therapeutics and Drugs of Abuse.p.446, 44 2008. Hempel, G. Drug Monitoring and Clinical Chemistry, 5. Institut fur Pharmazeutische und Medizinische Chemie Westfalische WilhelmsUniversitat Munster. Hardbound, 2004. Dasgupta, A. Advances in Chromatographic Techniques for Therapeutic Drug Monitoring CRC Press, 2009. Evans, W.E.; Schentag, J.J.; Jusko, W.J. Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring. Lippincott Williams & Wilkins Bauer, L.A. Applied Clinical Pharmacokinetics. McGraw-Hill, 2001.

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Chapter 2

BRIEF INTRODUCTION TO HPLC

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2.1. Introduction Chromatography is a science that counts more than one century, since its introduction, while in the past twenty years has expanded exponentially because of the availability to be applied for the analysis of various analytes in a very wide variety of matrices. In principle, chromatographic techniques belong to separation science, where separation is achieved by regulating the magnitude of the distribution coefficient between two distinguished phases: one stationary and one mobile. Mixture components are separated while they migrate with different rates in the stationary phase by means of the mobile phase. Depending on the type of these phases there are several chromatographic techniques. When the mobile phase is a liquid, the chromatography is called liquid chromatography. Liquid chromatography in its various forms, where HPLC is the most important and dominant, is of major importance in all areas related to chemistry. The most sophisticated type of liquid chromatography is HPLC where the mobile phase runs through the stationary by means of a pump at elevated pressures. HPLC has been used in an extremely wide range of analytical methods and it is impossible to give a comprehensive set of examples that would illustrate its wide applicability in a variety of matrices. HPLC is probably the most universal type of analytical procedure; it has achieved this position as a result of the constant evolution of the equipment used to provide higher efficiencies at faster analysis time with a constant incorporation of new column packings.

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Victoria Samanidou and Eftichia Karageorgou Five different mechanisms are responsible for separation in HPLC:

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1. Adsorption of the analytes on the sorbent used as a stationary phase. 2. Partitioning of the solutes between two liquid phases. These two mechanisms can be applied in normal or reversed phase mode, where analytes are retained due to their polarity. In normal phase, most nonpolar compounds elute first and the most polar compounds elute last, because the mobile phase is less polar than the stationary phase. The opposite stands for reversed phase, which covers the vast majority of applications. In this mode the mobile phase is more polar than the stationary phase, so that more polar compounds elute earlier. 3. Ion exchange, where separation is based upon electrostatic interactions between electrically charged ions or ionizable compounds. 4. Size exclusion, where biomolecules can be separated based on their size, rather than on their charge or polarity, by passing, or filtering, them through a controlled-porosity packing material. 5. Affinity, where molecules are separated due to a highly specific interaction between targeted analytes and the packing material e.g. the isolation of antibodies in a serum sample specific for a particular antigenic determinant.

2.2. HPLC Instrumentation The main components of an HPLC system are shown in Figure 2.1. These include: 1. Mobile phase reservoir: Solvent Reservoirs are used to store MobilePhase. 2. Pump: The HPLC pump is used to deliver the mobile phase at constant flow so that the separation of the components of the mixture occurs in a reasonable time. There are two types of pumping systems Isocratic and Gradient.

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Brief Introduction to HPLC

Pump

Mobile Phase Reservoir

Chromatographic Column

7

Detector

Injector

Chromatogram

Waste

A typical HPLC apparatus

Figure 2.1. Main components of an HPLC system.

Figure 2.1. Main components of an HPLC system.

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Isocratic: In this mode the mobile phase composition is kept constant throughout the chromatographic run. Gradient: In this mode the mobile phase composition can be changed during chromatographic run to achieve a better or/and faster separation. There are two types of gradient systems: Low-pressure mixing and High-pressure mixing. In the second mode two pumps are required. 1. Injection Port: Sample introduction unit. Usually this is a six port valve injection with a loop which can be partially or fully filled. 2. Analytical column: This contains the stationary phase for separating the components of the sample. It is regarded as the ―heart‖ of the chromatographic system responsible for the efficiency of chromatographic separation. 3. Detector: The device used to detect the separated compounds. There are several detectors available. However UV-VIS Detector, PhotoDiode Array Detector, fluorescence Detector, are more commonly used. The Photo Diode Array Detector has the advantage of identification by means of UVvis spectrum comparison. It can give a three dimensional view of chromatogram (Intensity Vs Time) and Spectra (Intensity Vs Wavelength) simultaneously. It also gives information on peak purity. The new ELSD detector is proving to be an important detector especially in polymer analysis; while the MS as detector is outstanding. Figure 2.2 cites the most common detection techniques used in HPLC.

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8

DETECTION TECHNIQUES Identification

Electrochemical

•Conductivity •Potentiometry •Amperometry •Coulometry

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Figure 2.2. Most common detection techniques in HPLC.

FTIR NMR MS

Spectroscopic

Molecular Spectroscopic techniques •UV/vis •Photodiode array (PDA) •Fluorescence

Atomic Spectroscopic techniques •Atomic Absorption Spectrometry •Atomic Emission Spectrometry

•Refractive index

6. Data Acquisition System: to process the detector output and integrate it to form the chromatogram. Personal computers with sophisticated software are used to process the chromatogram for quantitative analysis, identification, peak purity test etc. The same software may also be used to control various parameters of the system. Beside these main parts there may also be optional units such as 1. 2. 3. 4.

Precolumn or guard column to protect the analytical column. Autosampler. Column thermostat to maintain the column at an elevated temperature. Fraction collector to obtain the separated analytes for preparative chromatography.

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Brief Introduction to HPLC

9

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2.3. Liquid Chromatography-Mass Spectrometry Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an analytical technique that combines the separation capabilities of HPLC with the mass detection capabilities of mass spectrometry. Ion Sources: used for the ionization of sample molecules include Electrospray Ionisation (ESI), EI (Electron Ionisation), Matrix-Assisted Laser desorption/Ionisation (MALDI), Chemical Ionisation (CI) Fast Atom Bombardment (FAB), Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photoionization (APPI), Thermospray Ionization. Mass analysers: used to Sort Ions by Mass (m/z) include: Quadrupole, Ion Trap, Magnetic Sector Field, Electric Sector Field, Time-Of-Flight (TOF). Detectors: Detection of ions is achieved by a Microchannel Plate, an Electron Multiplier, a Faraday cup, a photomultiplier etc. Tandem mass spectrometry, known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation during different stages of mass analysis. This hyphenated to HPLC is a powerful technique which can also provide structural information. It is beyond the scope of this book to give more details upon this well known technique, since it has been comprehensively covered by textbooks dedicated to the analytical technique theory, instrumentation and applications. The aim of this chapter is to give supportive fundamental information to the clinical chemist.

SUGGESTED FURTHER READING-REFERENCES [1] [2] [3] [4] [5] [6]

www.chromatography-online.org/HPLC.html Papadoyannis, I.N. ΗPLC in Clinical Chemistry. Editor J. Cazes, Marcel Dekker Inc. New York & Basel, 1990. Snyder, L.R.; Glajch, J.L.; Kirkland, J.J. Practical HPLC Method Development. Wiley John & Sons, 1997. Neue, U.D. HPLC Columns: Theory, Technology, and Practice. WileyVCH Inc. Milford, MA USA, 1997. Dong, M.W. Modern HPLC for Practicing Scientists. John Wiley & Sons Inc. New Jersey, 2006. Kazakevich, Y.V. & LoBrutto, R. HPLC for Pharmaceutical Scientists. Wiley-Interscience, New Jersey USA, 2007.

Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

10 [7]

Snyder, L.R. & Dolan, J.W. High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. John Wiley & Sons Inc. New Jersey, 2007. Samanidou, V.F. & Papadoyannis, I.N. HPLC: the dominant separation technique with a wide range of applications, edited collection: "Chromatography: Types, Techniques and Methods." Nova Publishers, 2009.

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[8]

Victoria Samanidou and Eftichia Karageorgou

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Chapter 3

SAMPLE PREPARATION 3.1. Introduction

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Sample preparation is widely accepted to be the most important part of any analytical practice. It is usually time consuming and since it requires many steps it arises to be the most error-prone phase in any sample processing. The objectives of any sample preparation technique include: 1) Modification or elimination of sample matrix to prepare it for introduction (injection) on to the chromatographic column. 2) Impurities removal and sample purification. 3) Analytes‘ enrichment by pre-concentration when being in not detectable amounts. The latter should be done with precautions because impurities and matrix interferences if present are inevitably concentrated as well. [1,2]. Therapeutic drug monitoring (TDM) may be used as a tool for the improvement of drug therapy. The ideal sample preparation technique for biological samples prior to HPLC or LC/MS analysis should be ―no preparation‖. A similar approach is to avoid the preparation step by using a technique known as "dilute and shoot". This technique could only be efficient when levels of targeted analytes are relatively high, and the matrix components do not co-elute. When HPLC is the analytical technique of choice then sample pre-treartment is especially necessary: to prolong instrument‘s and above all column life time. to improve method detectability providing lower LOD and LOQ values

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12

to change the sample solvent and make it suitable for introduction to the chromatographic system. Sample pretreatment may include various steps after sampling such as: 1. 2. 3. 4.

Preservation during storage. Dilution, freeze drying, filtration, centrifugation, pH adjustment etc. Extraction-Isolation of analytes of interest. Derivatization prior to analysis to improve either chromatographic separation or detection capability of the method.

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There is no ideal or universal sample pretreatment technique. The analytical chemist should take into consideration all requirements in order to choose the most efficient sample preparation protocol, which should: be simple and fast provide high extraction efficiency be accurate, precise and reproducible be specific for the targeted analytes provide sample free from interference and impurities be easily automated in order to be readily applied in routine analysis provide high sample throughput involve the minimum manipulation to reduce analytes‘ loss be solvent-free or use as less solvent amount as possible, so as to be friendly to the environment be of low cost. Final decision upon the optimal sample preparation technique depends also on sample volume and required quantities for measurement. [3-6]

3.2. Sample Preparation in Drug Monitoring In drug monitoring sample preparation aims to eliminate endogenous inteference from biological samples. Biological fluids may need dilution, protein precipitation and an extraction step for the isolation of analytes of interest. Sample dilution can only be applied in a few cases if the concentrations of the analytes are sufficiently high that they can still be detectable.

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Sample Preparation

13

Blood serum and plasma are routinely used for drug monitoring. Plasma samples contain a significant amount of salt and proteins that can be precipitated or adsorbed on reversed phase packings. The adsorbed protein can easily foul the column resulting in changes in the separation and ultimately clogging the column. Serum is the straw-colored liquid that separates from the clot that forms in whole blood. Plasma is prepared from whole blood that has treated with an anti-clotting substance such as heparin. It is the supernatant that results when the cellular components of blood are removed by centrifugation. A method developed for plasma can normally be applied without modification or slightly modified to serum as well. Urine is one of the most commonly studied for drug analysis, particularly because of its relative ease of collection, and because it is nearly universal means of excretion of parent drug compounds metabolites or both. As a matrix, it has moderate complexity, and typically contains both organic and inorganic constituents, as well as a relatively high variability. A protein - free sample can be obtained by protein precipitation, for example by addition of acetonitrile or an acidic solution such as perchloric acid, trichloroacetic acid to the sample. After centrifugation the supernatant can be directly injected into the chromatographic system, if it is free from endogenous compounds. However, in most cases matrix components still interfere. Therefore biological samples prior to HPLC analysis should be further purified in an organic solvent. This is accomplished mainly by liquid-liquid extraction (LLE) and solid-phase extraction (SPE). In the extraction method the compound of interest is removed from the biological matrix (plasma, serum, urine, etc.) under suitable conditions that selectively isolate (extract) the desired components and leave behind unwanted materials. A typical sample preparation protocol is shown in Figure 3.1. [7-10] Nearly all analytical problems can be solved by LLE and SPE. Therefore, these methods can be characterized as universal from a scientific and technical view. Liquid-liquid extraction (LLE) is the traditional extraction technique by which analytes of interest are extracted from the sample matrix with an organic solvent with a large affinity for the analyte. This technique has some drwbacks such as: 1. The use of large volumes of extraction solvent, 2. The formation of emulsions during the mixing procedure. 3. Time-consuming evaporation procedures. 4. Co-extraction of proteins and other matrix components. 5. It is not suitable for very polar analytes. Since many samples also have very polar metabolites this method has limitations.

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14

Sample SamplePreparation Preparation Generic GenericProtocol Protocol Blood plasma/serum

Steps Steps

Protein precipitation

1. Deproteinization

e.g. Acetonitrile, TFA etc

Centrifugation

2. Cleanup-isolation Solid Phase Extraction (e.g. RP-18, HLB, mixed mode) 1. Wash to remove interference 2. Elute anaytes of interest

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3. Analysis

HPLC, HPLC-MS/MS

Figure 3.1 Typical sample preparation protocol for blood plasma/serum samples in drug monitoring.

So called modern extraction techniqes are preferred nowadays such as solid pahse extraction (SPE) or solid phase microextarction (SPME). Comparing solid-phase extraction with classical liquid- liquid extraction, it displays the following advantages: -

An almost quantitative recovery can be obtained, provided that the optimal conditions regarding sorbent, wash and elution solvents have been chosen.

-

The extracts obtained with solid-phase extraction are cleaner than those obtained with liquid-liquid extraction due to the better selectivity of solid-phase extraction.

-

Lower volumes of both samples and solvents are required.

-

Problems such as the formation of emulsions are absent in solid-phase extraction.

-

Solid-phase extraction methods are not time consuming and have proven to be applicable with large numbers of samples. Since 10, 12, 24 or even more samples can be processed simultaneously, solid-phase extraction is considerably faster than liquid-liquid extraction.

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Sample Preparation

15

Solid - phase extraction (SPE) is an extraction process that comprises a solid and a liquid phase. In SPE the sorbent is packed between two fritted disks in a polypropylene cartridge. The basic principle of SPE is that intercepts the components of interest, mainly organic molecules, by a special sorbent placed in a disposable extraction minicolumn. Sorbents used in SPE involve: Reversed phase, high - hydrophobic octadecyl (C18), octadecyl (C18), octyl (C8), ethyl (C2), cyclohexyl, phenyl. Wide-pore reversed phase, butyl (C4 ). Normal phase, silica modified by cyano (-CN), amino (-NH2), diols (-COHCOH). Adsorption, silica gel (-SiOH), florisil (Mg2SiO3), alumina (Al2O3). Ion-exchangers, amino (-NH2), quaternary amine (N+), carboxylic acid (-COOH), aromatic sulfonic acid (ArSO2OH). Wide-pore ion exchangers, carboxylic acid (-COOH), polyethyleneimine [-(CH2CH2NH)n - ]. The particle size allows the use of small pressure to force the sample and wash solutions through the column. Separation mechanisms involved in SPE are similar to those encontered in liquid chromatography: adsorption (silica), bonded-phase partition (normalphase= sample solvent less polar than the adsorbent, reversed phase with or without ion-painting= sample solvent more polar than the adsorbent) ionexchange, size exclusion.

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SPE consists of four steps as shown in Figure 3.2: 1. Sorbent conditioning which is the preparation of the sorbent. This is also called solvation (activation of functional groups). It aims to facilitate interactions between analytes of interest and sorbent. After conditioning removal of the excess of solvation solvent then washing with a solvent suitable for analyte retention of the sorbent is required; however drying of sorbent should be prevented. 2. Sample Loading: The sample solution is forced through the sorbent of the cartridge. Analyte are retained in this step. Some undesired compounds may also be adsorbed by the sorbent. 3. Washing with an appropriate solvent for undesired matrix components removal. 4. Elution: Selective desorbing the compounds of interest from the sorbent with a suitable elution solvent, and collecting the cartridge effluent. The extract is directly injected or evaporated and reconstituted in the mobile phase or after addition of internal standard.

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16

SOLID PHASE EXTRACTION STEPS

interferences

Analyte of interest

1. Conditioning

2. Sample loading

3. Washing

4. Elution

Figure 3.2. Steps in Solid Phase Extraction

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Figure 3.2. Steps in Solid Phase Extraction.

Optimization assays are required in each of these steps to reassure high recoveries and purification efficiency. The suitable sorbent type and mass as well as washing and elution solvents are critical for high recovery rates without loss in recovery. Some polymeric sorbents such as Hydrophilic-lipophilic polymers used in OASIS by WATERS and Nexus Abselut by Varian are designed to extract extensive spectrum of analytes: lipophilic, hypophilic, acidic, basic and neutral, where no conditioning is required. These sorbents significantly simplify extraction protocols. [11] Two different approaches can be chosen for SPE: either the analyte is retained on the sorbent whilst components pass through the waste. The analyte will be eluted later from the sorbent with a suitable solvent to be analysed. Or the matrix components are adsorbed, whilst the analyte is evacuated. The first approach is generally prefered as less sorbent is required and isolate preconcentration is possible. Advantages of this principle should be greater than those of other extraction methods with only a very low quantity (micro) of the extraction agent, for example, SPE with disc technology. In contrast to conventional SPE with packedbed columns, micro or non micro columns, this arrangement allows the combination of all steps of sample preparation in one step as described above. [12, 13]

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Sample Preparation

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SPE cartridges can be used either with centrifugation, positive pressure or under vacuum manually or in a manifold as shown in Figure 3.3.

Vacuum Manifold

Centrifugation Positive Pressure (Manually)

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Figure 3.3. Solid Phase Extraction modes (From Biotage Sweden AB, with Permission).

Some promising approaches in SPE are based on special packings such as restricted access materials (RAMs), and(From molecular imprinting Figure 3.3. Solid Phase Extraction modes Biotage Sweden AB, with materials Permission) (MIPs). On column sample preparation with column-switching techniques and on-line SPE in LC using RAMs, LC-GC coupling and membrane-based sample preparation dialysis, electrodialysis, ultrafiltration are also introduced as a useful tool for sample pretreatment and sampling for complicated matrix samples. On line dialysis has been used in on-line bioprocess monitoring. It has the advantages of easy operation rapid isolation of components of interest from complicate and dirty matrix and free or less use of organic solvents. Serum, plasma, muscle tissue can be analyzed after sampling by means of on line dialysis. [14-17] Although these methods have their own merits, most of them are only found in isolate applications, oftentimes, they do not achieve sensitivity and selectivity of LLE and SPE and, finally, some methods need expensive equipment. Other problems are fouling of membranes in membrane-based sample preparation and irreversible in on-line SPE. Solid Phase Microextraction SPME is a modern solvent-free adsorption/desorption technique used to analyze volatile and nonvolatile compounds in both liquid and gaseous samples. An SPME unit consists of a fused silica fiber coated with a cross linked polymeric organic liquid such as polydimehylsiloxane phase and bonded to a stainless steel plunger and a holder. The fiber assembly consists of an outer protective septum piercing needle and an inner fiber attachment needle to which the fiber is epoxied. Organic compounds can be directly introduced into any gas chromatograph or GC/MS system, as well as in an HPLC system with the proper interface consisting

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Victoria Samanidou and Eftichia Karageorgou

of a six port injection valve and a desorption chamber that replaces the injection loop in the HPLC system. Analytes can be removed in a moving stream of mobile phase (dynamic desorption) or when analytes are more strongly adsorbed to the fiber the fiber can be soaked in mobile phase or another stronger solvent for a specific period of time (e.g. 1 minute) before the material is injected onto the column (static desorption). SPME is an alternative to the most common sample preparation techniques used in any laboratory such as liquid-liquid extraction or SPE for semivolatile compounds. As no solvent is used, SPME saves preparation time and cost and often improves the limits of detection in an analysis. However SPME can display these advantages only in some areas of biomedical analysis, i.e., the matrix and the volatility of target analyte have to be taken into account. First of all, the combination of low volatility of analyte and a complex matrix with polymer components, e.g. proteins in plasma or cell cultures, considerably limits the application of SPME. The extraction is very slow in contrast to LLE and SPE with packed bed columns. The advantages of SPME can be used for both the assay of low volatile and highly volatile analytes in urine or other body fluids, with no or only a low concentration of polymer biomolecules. The problems of sensitivity and delayed time are considerably decreased in comparison to plasma. A number of methods with good precision, accuracy, sensitivity and selectivity were demonstrated which were also simple and fast. SPME is an encouraging development for sample preparation in biomedical analysis. The most striking attribute of SPME is a low recovery as reported for many methods. This is not unexpected because SPME is an equilibrium extraction but not an exhaustive extraction. A wider application of SPME in TDM is expected for the near future. However, more studies are necessary, for example, the potential of SPME to analyze directly the free concentration of drugs in plasma. [18-22]

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REFERENCES [1]

[2]

[3] [4] [5]

[6] [7]

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[8] [9]

[10] [11] [12] [13]

[14] [15]

Papadoyannis, I.N. & Samanidou, V.F. Sample Preparation Prior to HPLC. Encyclopedia of Chromatography. p. 1499 – 1515. Marcel Dekker. Jack Cazes Ed. Second Edition, 2003 Hassan, E.Y.; Enein. A.; Papadoyannis, I.N.; Samanidou,V. F. Sample Pretreatment in Clinical Chemistry. Chapter 1: Separation Techniques in Clinical Chemistry. Marcel Dekker. NewYork,USA; 2003. Henion, J.; Brewer, E.G. LC-MS Sample Preparation. Today’s Chemist at Work p.36-42 February 1999. Majors, R. E. New Approaches to Sample Preparation. LC-GC International 1995 8, 128-133. Pearce, J.C.; McDowall, R. D.; El Sayed, A.; Pichon, B. Automated analysis of drugs in plasma using liquid-solid extraction and HPLC. International Laboratory November/December 34-40, 1990. Rossi, D. T. ; Zhang, N. Automating solid-phase extraction: current aspects and future prospects. Journal of Chromatography A 2000 885, 97-113. Wells, M. Principles of extraction and the extraction of semivolatile organics from liquids in Sample Preparation Techniques in Analytical Chemistry. John Wiley & Sons, Inc.; 2003. Snow, N.H. Solid phase extraction of drugs from biological matrices. Journal of Chromatography A 2000 885, 445-455. Hennion, M.C. Solid-phase extraction: method development, sorbent, and coupling with liquid chromatography. Journal of Chromatography A 1996 85, 63-54. Majors, R.E. Liquid Extraction Techniques for Sample Preparation. LC-GC International 1997 10, 93-101. Huck, W.; Bonn, G.K. Recent developments in polymer-based sorbents for solid-phase extraction. Journal of Chromatography A 2000 885, 51-72. Fritz, J.S.; Masso, J.J. Miniaturized solid-phase extraction with resin disks. Journal of Chromatography A 2000 909, 79-85. Bert Ooms, J.A.; Van Gils, G.J.M.; Duinkerken, A.R.; Halmingh, O. Development and validation of protocols for solid-phase extraction coupled to LC and LC-MS. International Laboratory 2000 11, 18-23. Johnsson, J.A.; Mathiasson, L. Membrane based techniques for sample enrichment. Journal of Chromatography A 2000 902, 205-225. Majors, R. E. The use of membranes in sample preparation. LC-GC International 1995 13, 364-373.

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[16] Delaunay-Bertoncini, N.; Pichon, V.; Hennion, M.-C. Immunoextraction: A highly selective method for sample preparation. LC/GC Europe 2001 14, 164-172. [17] Boos, K.S.; Rudolphi, A. The Use of restricted-Access media in HPLC, Part I- Classification and Review. LC-GC International 1998 11, 84-95. [18] Queiroz, M.E.C.; Lanças, F.M. Practical Tips on Preparing Plasma Samples for Drug Analysis Using SPME. LC-GC North America 2004 22, 970-980. [19] Lord, H.; Pawliszyn, J. Microextraction of drugs. Journal of Chromatography A 2000 902, 17-63. [20] Lord, H. ; Pawliszyn, J. Review Evolution of solid-phase microextraction technology. Journal of Chromatography A 2000 885, 153-193. [21] Lord, H.L.; Pawliszyn, J.; Recent Advances in Solid-Phase Microextraction. LC-GC International 1998 11, 776-785. [22] Ulrich, S. Solid-phase microextraction in biomedical analysis. Journal of Chromatography A 2000 902, 167-194.

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Chapter 4

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ANTI-CANCER DRUGS The available anticancer drugs have discrete mechanisms of action, which may vary in their effects on different types of normal and cancer cells which demonstrate very few biochemical differences. There is no single cure for cancer, since there is not a single type of cancer. On the contrary there are more than a hundred different types of cancer. The effectiveness of many anticancer drugs is limited by their toxicity to normal rapidly growing cells in the intestinal and bone marrow areas. A final problem is that cancerous cells, which are initially suppressed by a specific drug, may develop a resistance to that drug. For this reason cancer chemotherapy may consist of using several drugs in combination for varying lengths of time. Besides their beneficial effect, chemotherapy drugs may sometimes cause toxic effects. The common approach in chemotherapy is to decrease the growth rate (cell division) of the cancer cells. Side effects may be seen in systems that naturally have a rapid turnover of cells including skin, hair, gastrointestinal, and bone marrow. These healthy, normal cells also end up damaged by the chemotherapy program. The most commonly-used anticancer agents can be divided into three main categories based on their mechanism of action. a. Those which stop the synthesis of pre DNA molecule building blocks: These agents work by blocking some step in the formation of nucleotides or deoxyribonucleotides (necessary for making DNA).

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b. Those which directly damage the DNA in the nucleus of the cell: These agents chemically damage DNA and RNA. They disrupt replication of the DNA and either totally prevent replication or cause the manufacture of false DNA or RNA. c. Those which affect the synthesis or breakdown of the mitotic spindles: These drugs disrupt the formation of mitotic spindles and therefore interrupt cell division. [1] Resveratrol is a phytoalexin produced naturally by several plants, when under attack by pathogens such as bacteria or fungi. In mouse and rat experiments, anticancer, anti-inflammatory, blood-sugar-lowering, chelating and other beneficial cardiovascular effects of resveratrol have been reported. Since 1997 when Jang reported that topical resveratrol applications prevented the skin cancer development in mice treated with a carcinogen, dozens of studies of the anti-cancer activity of resveratrol in animal models have been reported. In vitro resveratrol interacts with multiple molecular targets, and has positive effects on the cells of breast, skin, gastric, colon, esophageal, prostate, and pancreatic cancer, and leukemia. The study of pharmacokinetics of resveratrol in humans concluded that even high doses of resveratrol might be insufficient to achieve resveratrol concentrations required for the systemic prevention of cancer due to its poor systemic bioavailability. [2, 3] Table 1 summarises the chromatographic methods for therapeutic drug monitoring of anti-cancer drugs. In the following paragraphs an overview of published methods on the analysis of anti-cancer drugs is provided.

4. 1. Analytical Methods Monitoring of drug substances and metabolism products is routinely accomplished using HPLC. (E)-3,5,4Ά-trimethoxystilbene (BTM-0512) (trans-3,5,4Ά-trihydroxystilbene) is a resveratrol analog with a variety of pharmacological action, including anticancer properties, anti-allergic activity, estrogenic activity, antiangiogenic activity, and vascular-targeting activity against microtubule-destabilization. Pharmacokinetic data and suitable methods for determination of the compound in plasma are mandatory. A rapid and sensitive liquid chromatographic–mass spectrometric method for determination of (E)-3,5,4Ά-trimethoxystilbene in rat plasma, using, has been developed and validated. Plasma samples were treated with acetonitrile to precipitate proteins. Samples were then analyzed by HPLC on

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a 250mm x 4.6 mm, 5-μm, C18 column with methanol–water, 80:20 (v/v), containing 10 mM ammonium acetate and 0.2% formic acid (pH 3.0), as mobile phase, delivered at 0.85 mL/min. Carbamazepine was used as internal standard A single-quadrupole mass spectrometer with an electrospray interface operated in selected-ion monitoring mode was used to detect [M + H]+ ions at m/z 271.3 for (E)-3,5,4Ά- trimethoxystilbene and m/z 237.5 for the internal standard. Linearity was observed at concentrations ranging from 0.01 to 5.0 μg/mL. Intra-day and inter-day precision provided RSD values lower than 12.9%, while accuracy was in the range 94.8–104.7%. The limit of detection in plasma was 0.005 μg/mL. The method was successfully applied to determine the concentration of (E)-3,5,4Άtrimethoxystilbene after oral administration of 86 mg/kg of the drug to rats and can be used to investigate the pharmacokinetics of the compound. [4] In vitro and in vivo biotransformation studies play an important role during the drug development process. A major goal of studying the biotransformation of a drug in both in vitro and in vivo models is to ensure toxicology studies expose animals to the same therapeutic agent and metabolites as those observed in humans. Wabnitz et al in their study, investigated the metabolism of CI-1040 using in vitro and in vivo models compared with metabolism observed in a human bile sample. Samples sample were obtained from a cancer patient receiving a MAP-Erk Kinase (MEK) inhibitor CI-1040 during an efficacy study against tumor growth. MAP-Erk Kinase inhibitors are atypical in selectively inhibiting various signals in the mitogenic cascade. MEK is involved in the transmission of oncogenic and proto-oncogenic signals. A substance that blocks these keysignaling molecules that are involved in tumor growth, progression and metastasis, would be of significance in the treatment of cancer, and inflammatory diseases. CI-1040 (2-(2-chloro-4-iodo-phenylamino)-Ncyclopropylmethoxy-3, 4difluoro-benzamide, is a potent and highly selective inhibitor of MEK. It directly inhibits purified MEK with a 50% inhibitory concentration (IC50) of 17 nM (23). CI-1040 reduces gene over-expression, when an abnormality is present in the MEK pathway leading to increased cell growth and tumor production. Pre-clinical data from various animal models has indicated significant tumor growth inhibition. CI-1040 recently underwent phase II clinical trials. Human bile and plasma samples were obtained immediately after administration of CI-1040 to a patient with advanced colon cancer. A combination of HPLC-radiochromatography (HPLC-RAM), LC/MS and LC/MS/MS experiments were used to analyze all resulting metabolites.

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Table 4.1. Overview of HPLC methods for therapeutic drug monitoring of anti-cancer drugs. Analytes Resveratrol (trans-3,5,4Άtrihydroxystilbene)

Sample type Rat plasma

Sample Preparation Deproteinization with ACN

Dup 941 and its impurities

Standard solutions of drug

6-thioguanine (6-TGN) and methyl 6mercaptopurine nucleotides (Me6-MPNs)

Human red blood cell

Deproteinization by HClO4 with dithiothreitol by heating the sample for 45 min at 100 °C.

6-Thioguanine, 6mercaptopurine, and 6methylmercaptopurine Dithiothreitol 6-MP, 6-MMP, dithiothreitol, allopurinol, S-adenosyl-L-methionine

Human red blood cell

Acid hydrolysis

Blood samples

Incubation

Methotrexate (MTX),

Human urine

Oxidation using 10-3M KMnO4 (pH 5.0) and 35 min of oxidation time

5-fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP)

Εnvironmen tal samples of large general hospital

Chromatography C18 250mm x4.6 mm, 5 μm, MP: MeOH–water, 80:20 (v/v), 10 mM ammonium acetate and 0.2% formic acid (pH 3.0), FR: 0.85 mL/min). IS: carbamazepine

Recovery % 94.8–104.7% Linearity: 0.01 to 5.0 μg/mL

Zorbax SB-C8 4.6 mm x 15 cm, MP: ACN– water–TFA (10:90:0.1, v:v:v) gradient to ACN– water–TFA (40:60:0.1, v:v:v).

Purospher RP 18-e, 5 μm. Gradient. MP: 0.02 mol/L potassium phosphate (pH 3.5) :MeOH (40:60 v/v). FR: 1.2 mL/min. C18 (25 x 0.46 cm)

73.1% and 84.0% for 6-TGN and Me6MPN derivatives, respectively

C18 5 μm (125x4 mm) LiChrocart. MP: acetic acid 0.1% and ACN 100% in linear gradient. FR: 1 mL /min. MP: Tris–NaCl buffer solution with 15 mM Tris and 1mM NaCl , pH 6.8 with HCl. FR: 1.0 mL/min Synergi 4u Max-RP capillary column (0.5 x 50 mm, 5 μm, 80 A° ). MP: 20 mM ammonium acetate, pH 4 with acetic acid and MeOH. Gradient. FR: 10 μL/min.

Recovery: 70-89.9% Linear range: 6-MP (0.15–8 mM) SAM (1–12 mM) 89-122%

Recovery: 92.4-99.9% Linearity: 1.1 and 3333.3 μg/L for MTX and CP and between 33.3 and 3333.3 for 5FU.

Detection Single-quadrupole mass spectrometer ESI in SIM mode [M + H]+ at m/z 271.3 for (E)3,5,4Άtrimethoxystilbene and m/z 237.5 for IS. UV diode array 210– 600 nm.

Reference 4

6-TG at 341 nm and Me6-MP at 304 nm

7

UV: 6-TG at 342 nm, Me6-MP at 303 nm

8

UV: 290 nm

9

Photometric and Fluorimetric detectors, in series, at 230 nm and 444 nm (λex=280 nm) MS/MS

10

5

11

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Unlabeled CI-1040 was administered (100 mg/day, QD) for 15 days to a patient suffering from colon cancer. Bile was collected by the insertion of a Ttube directly into the bile duct over a 14-h period. Metabolites were also monitored in the patient‘s plasma. [5] A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 · 108 red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6methylmercaptopurine metabolites, respectively. [6] 6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6MPNs) are the two major metabolites found in erythrocytes after administration of azathioprine. In order to understand the role of these metabolites in the pharmacologic and toxic activity of thiopurines, an HPLC method was developed for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes after deproteinization by perchloric acid with dithiothreitol by Dervieux and Boulieu. The nucleotides were hydrolyzed to their bases by heating the sample for 45 min at 100 °C. During acid hydrolysis Me6-MP was converted into a compound analyzed on a Merck Purospher RP 18-e column, 5-μm, protected by a Purospher RP 18-e guard column with a linear gradient elution mode with 0.02 mol/L potassium phosphate (pH 3.5) and 0.02 mol/L potassium phosphate (pH 3.5):methanol (40:60 v/v). The flow rate was 1.2 mL/min. Detection of 6-TG and Me6-MP derivative was performed at 341 nm and 304 nm respectively. With this procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN derivatives, respectively, were found. [7] Stefan et al in their paper presented in-depth methodological analysis and optimization of the two previously described HPLC procedures [10 and 11] to improve precision, turn-around time, ruggedness, and cost effectiveness. Reversed-phase chromatography with UV detection was performed on a Waters HPLC system. The two protocols were improved with regards to chromatographic conditions, as well as reagent preparation, storage, and use. 6-Thioguanine nucleotides (6-TGNs) were analyzed by optimized techniques in samples from patients on thiopurine therapy (n = 24) and the results were compared. 6Mercaptopurine (6-MP) was used as internal standard in both procedures. Isocratic elution with 5% acetonitrile (ACN) in 20 mmol/L phosphate buffer pH 2.5 allowed for minimal background interference in both protocols. 6Thioguanine, 6-mercaptopurine, and 6- methylmercaptopurine (6-MMP) eluted at

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around 4, 5, and 6 min, respectively. Dithiothreitol (DTT) was critical only during the acid hydrolysis step. With these optimized protocols recovery of 6-TGNs was on average 1.38-fold higher in the Dervieux–Boulieu method and no interfering peaks hindered analysis. [8] Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs. A genetic polymorphism is responsible for large inter-individual differences observed in TPMT activity. An HPLC method was reported avoiding an extraction step and the use of radioactive reagents, based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and between-run variations showed high precision. The formation of 6-MMP was linear with respect to the lysate concentration and time. The results of HPLC method correlated with those of the radiochemical method. Because of the absence of extraction step, this method of TPMT activity determination reduces analysis variation and is time-saving and suitable for routine monitoring of TPMT activity and for fundamental studies. [9] An HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established after oxidation process using 10-3 M KMnO4 (pH 5.0) and 35 min of oxidation time to transform the analytes in the highly fluorescent pteridinic rings. Chromatographic separation was achieved on a C18 Nova-Pack column (150 × 3.9mm, 5 μm, Waters, Millipore Iberica, Barcelona, Spain). The mobile phase consisted of Tris–NaCl buffer solution containing 15 mM Tris and 1 mM NaCl and adjusted to pH 6.8 with HCl delivered at a flow rate of 1.0 mL/min. MTX and the assayed pteridines were monitored by Fluorescence at λem=444 nm and λex=280 nm and creatinine was monitored by absorption measurements at 230 nm. All components were well resolved in approximately 7 min. Detection limits were 10 ng/mL for MTX, less than 1 ng/mL for all of the pteridines, and 4 ng/mL for CREA. [10] A high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric (HPLC/ESI-MS/MS) method was developed for the simultaneous quantification of 5-fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP) in environmental samples. Micro-HPLC analysis was performed at a flow-rate of 10 μL/min using 20 mM ammonium acetate, adjusted to pH 4 with acetic acid (solvent A), and methanol (solvent B). Separation was accomplished on a Synergi 4u Max-RP capillary column (0.5 × 50 mm, 5 μm, 80

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Anti-Cancer Drugs

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A° ), (Phenomenex, Torrance, CA, USA), by gradient elution as follows: after 1 min of isocratic elution, solvent B was increased from 15 to 50% in 0.5 min along a linear gradient curve, then increased to 65% in 15 min. Finally, solvent B was decreased from 65 to 15% in 0.5 min and the column equilibration was conducted isocratically for 8 min. The total run time was 25 min. Transitions of the protonated and deprotonated molecular ions ([M- H]- for 5FU, [M+ H]+ for the other analytes and IS were monitored for quantitative analysis in the multiple reaction monitoring (MRM) mode. Transitions m/z 129 42, 455  308, 261 140 and 323 154 were selected for 5FU, MTX, CP and TP (IS), respectively. The present method offers high sensitivity, with detection limits of 1.1 μg/L for MTX and CP and 33.3 μg/L for 5FU, avoiding any sample preconcentration procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and 99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the method suitable for the routine determination of these three antineoplastic drugs. [11]

REFERENCES

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[1] [2] [3] [4]

[5]

[6]

[7]

www.elmhurst.edu (February 2009) Farina, A.; Ferranti, C.; Marra, C. An improved synthesis of resveratrol. Nat. Prod. Res. 2006 20, 247–52. Elliott, P.J.; Jirousek, M. Sirtuins: Novel targets for metabolic disease. Current Opion in Investigational Drugs 2008 9, 1472–4472. Ma, N.; Liu, W.-Y.; Li, H.-D.; Jiang, X.-Y.; Zhang, B.-K.; Zhu, R.-H.; Wang, F.; Liu, W.; Liu, X.; Xiang, D.-X. Determination of (E)-3,5,4 ATrimethoxystilbene in Rat Plasma by LC with ESI-MS. Chromatographia 2007 66, 251–255. Wabnitz, P.A.; Mitchell, D.; Wabnitz, D.A.M. In Vitro and in Vivo Metabolism of the Anti-Cancer Agent CI-1040, a MEK Inhibitor, in Rat, Monkey, and Human. Pharmaceutical Research 2004 21, 9. Lennard. L.; Singleton. H. High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6 mercaptopurine: quantification of red blood cell 6 thioguanine nucleotide, 6 thioinosinic acid and methylmercaptopurine metabolites in a single sample. Journal of Chromatography 1992 58, 383–390. Dervieux, T.; Boulieu, R. Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC. Clinical Chemistry 1998 44, 551-555.

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Stefan, C.; Walsh, W.; Banka, T.; Adeli, K.; Verjee, Z.; Improved HPLC methodology for monitoring thiopurine metabolites in patients on thiopurine therapy. Clinical Biochemistry 2004 37, 764– 771. [9] Anglicheau , D.; Sanquer , S.; Loriot , M.-A.; Beaune , P.; Thervet, E. Thiopurine methyltransferase activity: new conditions for reversed-phase high-performance liquid chromatographic assay without extraction and genotypic–phenotypic correlation. Journal of Chromatography B 2002 773, 119–127. [10] Duran Meras, I.; Espinosa Mansilla, A.; Rodriguez Gomez, M. J. Determination of methotrexate, several pteridines, and creatinine in human urine, previous oxidation with potassium permanganate, using HPLC with photometric and Xuorimetric serial detection. Analytical Biochemistry 2005 346, 201–209. [11] Sabatini, L.; Barbieri, A.; Tosi, M.; Saverio Violante, F. A new highperformance liquid chromatographic/ electrospray ionization tandem mass spectrometric method for the simultaneous determination of cyclophosphamide, methotrexate and 5-fluorouracil as markers of surface contamination for occupational exposure monitoring. Journal of Mass Spectometry 2005 40, 669–674.

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[8]

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Chapter 5

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BRONCHODILATORS Bronchodilators are medicines that help open the bronchial airways of the lungs, so that more air can flow through them. People with asthma have problematic breathing, since their airways are inflamed and become narrowed. Air moves smoothly through the airways and into the lungs during inhaling. Exhaling happens automatically, when the person stops breathing in. In a person with asthma incoming air can slide around the blockage, because the act of breathing in makes the airways expand. The problem in people with asthma comes during exhaling. The air remains trapped in the lungs so that the patient can then only take shallow breaths. Bronchodilators work by relaxing the smooth muscles lining the airways. This results in opening of the airways wider and allows air to leave the lungs. These drugs also are used to relieve breathing problems associated with emphysema, chronic bronchitis, and other lung diseases. Some bronchodilators are inhaled, using a nebulizer or an inhalation aerosol. Others are taken as injections or orally. Dosage depends on the type of bronchodilator and may be different for different patients. [1] Theophylline, also known as 1,3 dimethylxanthine, is a methylxanthine drug used in therapy for respiratory diseases. Due to its numerous side-effects, is are now less administered for clinical use. The mechanism of action of theophylline involves relaxing of bronchial smooth muscle, increasing of heart muscle contractility and efficiency, increasing heart rate, increasing blood pressure, increasing renal blood flow, anti-inflammatory effects and central nervous system stimulatory effect. Its bioavailability is quantitative. Theophylline is distributed in the extracellular fluid, in the placenta, in the mother's milk and in the central nervous system. The protein binding is 40%. The volume of distribution is 0.5 L/kg and may increase in neonates and those suffering from cirrhosis or

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30

malnutrition, whereas the volume of distribution may decrease in those suffering from obesity. Theophylline is metabolized extensively (up to 70%) in the liver, by Ndemethylation via cytochrome P450 1A2. It is metabolized by parallel first order and Michaelis-Menten pathways. Metabolism may become saturated (non-linear), even within the therapeutic range. Small dose increases may result in large increases in serum concentration. Clearance of the drug is influenced by age, so that it is increased in children 1 to 12, teenagers 12 to 16, adult smokers, elderly smokers or decreased in elderly. Other dieseases such as cystic fibrosis, hyperthyroidism, acute congestive heart failure, cirrhosis, hypothyroidism and febrile viral illness may also affect theophylline‘s clearance positively or negatively. [2,3] Table 5.1. summarises the chromatographic methods for therapeutic drug monitoring of bronchodilators. Table 5.1. Overview of HPLC methods for therapeutic drug monitoring of bronchodilators.

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Analytes Theophylline Ciprofloxacin

Sample type Rat Blood samples

Theophylline

Human serum

Theophylline

Intestinal fluid

Sample Preparation

Chromatography

LLE with isopropyl alcohol. Vortex Mix. Centrif. Evap. organic Layer and reconstitution of residue with MP. LLE with chloroform/ isopropanol. Vortex Mix and Centrif. Organic phase dried under N2. Extract dissolved with MP. LLE with chloroform– isopropyl alcohol 1:1. Dilution in KH2PO4. Extraction vortex and centrif. The organic layer evap. to dryness. The residue dissolved in MP.

Novapak C18 (3.9x150 mm) MP: ACN, MeOH, TCA pH=3 with 1M NaOH. IS: isopropyl analog of ciprofloxacin. Waters RP C18, 39x150 mm.MP:ACN: phosphate buffer (94:6 v:v). FR: 1.6 mL /min. IS: (b-OH ethyltheophylline 40 mg /L) Waters Novapak C-18 (4 μm). MP: 10mM KH2PO4–ACN– MeOH (900:25:90 v/v). FR: 2mL/min. IS: diprophylline

Detection

Reference 4

UV: 267 nm

5

UV: 271nm

6

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Bronchodilators

31

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5.1. Analytical Methods Theophylline requires frequent control because of its narrow therapeutic index. Theophylline and ciprofloxacin were determined in blood samples of cadmium-exposed and control rats. Cadmium has been associated with a number of adverse health effects but the impact of those effects on the pharmacokinetics of different drugs has not been investigated. The pharmacokinetics of theophylline and ciprofloxacin were studied in cadmium-exposed and control rats (72 rats) following i.p. (6.5 mg/kg) and p.o. (10 mg/kg) administration, respectively. The third-generation off springs of rats exposed to 100 mg/mL of cadmium chloride in drinking water were used in this study. Following eight weeks of exposure, animals received the drugs as a single dose. Blood samples were withdrawn at different time-points and the plasma concentrations of both drugs were analyzed by HPLC after LLE extraction with 2mL of chloroform. The pharmacokinetic parameters of theophylline and ciprofloxacin were altered significantly in the cadmium exposed animals. HPLC analysis was performed within 8 min using a Novapak C18 column (3.9x150 mm, 5 μm). A significant effect of chronic exposure to cadmium on the pharmacokinetics of the two selected drugs was demonstrated in this study. Theophylline Cl/F was found to be reduced by 41% due to the effect of Cd exposure on the liver, which could be hazardous to humans, since theophylline has a narrow therapeutic window. The pharmacokinetics of ciprofloxacin on exposure to cadmium was not altered as drastically as that of theophylline. [4] Theophyline was determined in human serum. Samples were extracted by chloroform and isopropanol mixture. After centrifugation, the organic phase was dried under nitrogen. Finally the extract was dissolved in 100 mL of mobile phase. HPLC–UV measurements were performed using a Waters RP C18, 39 × 150 mm column. The mobile phase consisted of acetonitrile: phosphate buffer (94:6 v:v) delivered at a flow rate of 1.6 mL/min. The detector wavelength was set at 267 nm and the temperature of the column maintained at 50°C. [5] Theophylline was determined in intestinal fluid after LLE with 2 mL chloroform–isopropyl alcohol 1:1, vortex mixing for 30 s and centrifugation, the water layer was discarded and the organic layer evaporated to dryness under a gentle stream of air. The residue was dissolved in 200 μL mobile phase, of which 100 μL was injected into the HPLC system. The column used was a Waters Novapak C-18 column (4 μm) and the mobile phase was 10 mM KH2PO4– acetonitrile– methanol (900:25:90 v/v). The flow was maintained at 2 mL/min.

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Victoria Samanidou and Eftichia Karageorgou

Spectrophotometric detection at 271 nm was applied. Diprophylline theophylline was used as internal standard. Less than 20% of the total dose was dissolved from the slow-release pellets after 120min, while dissolution from the immediaterelease formulation was found to be complete after 45–90 min in the different media. [6]

REFERENCES [1] [2] [3] [4]

[5]

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[6]

www.healthatoz.com (Accessed February 2009) www.theophyllineatpriory.com (Accessed February 2009) www.rxlist.com (Accessed February 2009) Zaghloul, I. Y.; Radwan, M.A.; Aly, Z.H. The effect of chronic cadmium exposure on the pharmacokinetics of theophylline and ciprofloxacin in rats. Journal of Trace Elements in Medicine and Biology 2007 21, 132–137. Jourquin, G.; Kauffmann, J.-M. Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water/in oil microemulsion. Journal of Pharmaceutical and Biomedical Analysis 1998 18, 585–596. Brouwers, J.; Ingels, F.; Tack, J.; Augustijns, P. Determination of intraluminal theophylline concentrations after oral intake of an immediate and a slow-release dosage form. Journal of Pharmacy and Pharmacology 2005 57, 987–995

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Chapter 6

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CARDIOVASCULAR DRUGS The angiotensin converting enzyme (ACE) inhibitor captopril, which was developed around 1975 was the first drug designed to block a particular target protein, and has subsequently become the preferred therapy for hypertension and congestive heart failure. [1] The statins (or HMG-CoA reductase inhibitors) are a class of drugs that lower cholesterol levels in people with or at risk of cardiovascular disease. They lower cholesterol by inhibiting the enzyme HMG-CoA reductase, which is the ratelimiting enzyme of the mevalonate pathway of cholesterol synthesis. Inhibition of this enzyme in the liver stimulates low-density lipoprotein (LDL) receptors, resulting in an increased clearance of LDL from the bloodstream and a decrease in blood cholesterol levels. The first results can be seen after one week of use and the effect is maximal after four to six weeks. Based on clinical trials and the increasing focus on aggressively lowering LDL-cholesterol, the statins continue playing an important role in the primary as well as in secondary prevention of coronary heart disease, myocardial infarction, stroke and peripheral artery disease. Statins also appear to have a favorable effect on inflammation, cancer etc. [2,3] Table 6.1 summarises the chromatographic methods for therapeutic drug monitoring of cardiovascular drugs. In the following paragraphs an overview of published methods on the analysis of these drugs is provided.

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Table 6.1. Overview of HPLC methods for therapeutic drug monitoring of cardiovascular drugs. Table 6.1. Overview of HPLC methods for therapeutic drug monitoring of cardiovascular drugs. Analytes

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Eprosartan

Sample type Plasma Samples

Sample Preparation SPE

XUESETONG injection: saponins made from Panax notoginseng

XUESETO NG injection

Filtration through a 0.45 μm membrane.

Pholedrine (40hydroxymethamphet amine)

Blood plasma and urine

SPE (SPEC C18)

Homocysteine

Total plasma

Captopril and its disulfide metabolites (total captopril)

Human plasma

Derivatization with ammonium-7fluorobenzo-2-oxa-1,3diazole-4-sulphonate after reduction with tri-nbutylphosphine LLE

Chromatography

Recovery %

Waters Atlantis dC18, 100Χ3.9mm, 3μm, 100 °A, 35±0.2 ◦C, MP: 0.031% TFA and ACN 0.026% TFA, gradient. FR=1.25 mL/min. IS=irbesartan A ZORBAXSB-C18 (2.1x150mm, 5μm) Gradient. MP: ammonium acetate and ACN, FR: 0.4 ml/min, at 25 ◦C.

93.4-102.8%. Linearity: 150–4000 ng/mL

UV: 232 nm

5

95.5 - 100.0 % Lineariry: 0.00021.0900 (μg/μl) 67% linearity: 1– 100 ng/mL

DAD detector: 203 nm ESI-MS/MS:

6

MS/MS positive ionization, (MRM) mode

8

95%

Fluorimetric λ (Ex): 385 and λ(Em): 515 nm

9

Linearity: 5 ng/mL (LOQ) and 300 ng/mL

Fluorescence detection at ex 235 nm and em 440 nm

11

LC- MS/MS: RP-18 gradient 50 to 70% of B MeOH/ACN 3/1 (v/v), 0.02% acetic acid) in A (5 mM ammonium acetate/ACN 95/5 (v/v), 0.02% acetic acid. IS: D11methamphetamine, D5methylenedioxymethampheta mine Discovery C18, MP: ACN– potassium dihydrogenphosphate buffer 0.1 M (8:92, v:v) (pH=2.1). C-18. MP: MeOH-ACNphosphate buffer (0.02 molΒ·L-1, pH 6.4) (30:30:135, v/v), FR: 1 mL/min

Detection

Reference

Cardiovascular Drugs

35

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6. 1. Analytical Methods The 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, more commonly known as ‗statins‘, are a novel class of drugs widely used for the treatment of hypercholesterolaemia in patients with established cardiovascular disease as well as those at high risk of developing atherosclerosis. High performance liquid chromatography (HPLC) in combination with tandem mass spectrometry (MS/MS) is the dominant analytical technique for the quantification of statins in biological samples. An excellent and comprehensive review has be written on the most of the methods used for quantification of statins are in plasma and they are suitable for therapeutic drug monitoring of these drugs. [4] A solid phase extraction-reversed phase high performance liquid chromatographic (SPE-RP-HPLC) method with photometric detection for monitoring the antihypertensive drug eprosartan has been validated in order to assure good quantitation of eprosartan in plasma samples obtained from patients under cardiovascular treatment. No interferences were observed from endogenous compounds of plasma and other drugs which are commonly co-administered in elderly patients. The recoveries of eprosartan from plasma samples, measured at three levels of the linear concentration range (150–4000 ng/mL) were found to be between 93.4 and 102.8%. The intra-day and inter-day precision and accuracy were always lower than 13% with regards to RSD and 4% with regards to RE. The analytical column was a Waters Atlantis dC18, 100 × 3.9mm, 3 μm, thermostated at 35±0.2 ◦C, protected with a guard column Waters Bondapak C18 10 μm. The mobile phase consisted of a mixture of water 0.031% TFA and acetonitrile 0.026% TFA, low pressure mixed, and delivered in gradient mode at a flow rate of 1.25 mL/min. Irbesartan was used as internal standard. Photometric detection was performed at 232 nm. The use of this method can save effort when monitoring patients who take several medications, especially when polar drugs are mixed. The validity, LOQ and the linearity range of the method make it acceptable for eprosartan monitoring during 24 h after dose intake which is necessary to assure that antihypertensive drug plasma levels are included in the therapeutic range during all the interdose range to decrease the incidence of cardiovascular events. [5] An interested paper has been reported on the development of an HPLC–ESIMS/MS method for the qualitative and quantitative determination of gensinosides and notoginsenosides, which is helpful to improve the quality control of Panax notoginseng and its pharmaceutical preparations such as Xuesetong injection. The latter is one of the most widely used proprietary medicines in traditional

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Victoria Samanidou and Eftichia Karageorgou

Chinese medicine, which consists of total saponins made from Panax notoginseng, used to treat cardiovascular diseases. Analysis was performed within 25 min, on a ZORBAXSB-C18 column (2.1 × 150 mm, 5μm) and a ZORBAX ODS C18 guard column (4.6 × 12.5mm, 5 μm). Gradient elution was applied with mobile phase consisted of aqueous ammonium acetate and acetonitrile, delivered at a flow-rate of 0.4 mL/min and the system operated at 25 ◦C. Full scan and time programmed selected reaction monitoring (SRM) were used for qualitative and quantitative analysis of saponins, respectively in negative mode. Twenty-seven saponins were identified and nine of them were quantified. Ten XUESETONG injections were analyzed and compared. The results showed that there is a great variation among different samples. The developed method is rapid, accurate and sensitive for qualitative and quantitative analysis of saponins in Xuesetong injection. Moreover, it also can be used for the quality control of Panax notoginseng raw material and its preparations. The method can be modified to include the analysis of biofluids. [6] An interesting study investigated the interaction of exercise training and chronic nitroglycerin treatment on blood pressure and alterations in nitric oxide (NO), glutathione (GSH), antioxidant enzyme activities and lipid peroxidation in rats. Fisher rats were divided into four groups: (1) sedentary control, (2) exercise training for 8 weeks, (3) nitroglycerin (15 mg/kg, s.c. for 8 weeks) and (4) training + nitroglycerin for 8 weeks. Blood pressure, heart rate and respiratory exchange ratio were monitored weekly for 8 weeks using tail-cuff method and oxygen/carbon dioxide analyzer, respectively. The animals were sacrificed 24 h after last treatment and plasma was isolated and analyzed using HPLC, ELISA and UV-VIS spectrophotometric techniques. Biochemical changes were accompanied by a significant increase in respiratory exchange ratio (p < 0.001) without a significant change in blood pressure and heart rate. Chronic nitroglycerin administration significantly increased nitric oxide levels (p < 0.05), glutathione levels (p < 0.001), superoxide dismutase activity (p < 0.05), Glutathione S-transferase activity (p < 0.05), and decreased malon-dialdehyde levels (p < 0.05). These biochemical changes were accompanied by a significant decrease in blood pressure (p < 0.05) and without any significant changes in heart rate and respiratory exchange ratio. Interaction of exercise training and chronic nitroglycerin treatment resulted in normalization of plasma nitric oxide, malon- dialdehyde, lactate levels, and catalase activity. The combination of exercise and nitroglycerin significantly enhanced glutathione levels (p < 0.05), and the up-regulation of superoxide dismutase (p < 0.001), glutathione peroxidase (p < 0.05), glutathione reductase (p < 0.05) and Glutathione S-transferase (p < 0.001) activities. These biochemical changes were

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Recent Developments in Drug monitoring by HPLC

37

accompanied by normalization of blood pressure and a significant increased in respiratory exchange ratio (p < 0.001). The data suggest that the interaction of physical training and chronic nitroglycerin treatment resulted in the maintenance of blood pressure and the up-regulation of plasma antioxidant enzyme activities and glutathione levels in the rat. [7] Pholedrine (40-hydroxymethamphetamine) is a cardiovascular agent exerting hypertensive and adrenergic effects. High doses may cause a drop in the peripheral circulation blood flow and increase blood pressure, heart rate and body temperature up to a state of central respiratory paralysis. A 15-year-old girl who suffered from heavy agitation and hallucinations was admitted to the intensive care unit in a comatose state. The toxicological analysis using GC/MS revealed a considerable amount of pholedrine in blood and urine. A method for determining pholedrine in human body fluids utilizing high-performance liquid chromatography (HPLC)/tandem mass spectrometry (LC-MS/MS) with a turbo ion-spray source was developed, using D11-methamphetamine and D5methylenedioxymethamphetamine as internal standards. Samples were prepared by SPE extraction using SPEC-C18AR columns. Chromatographic separation was achieved on an RP-18 stationary phase applying gradient elution from 50 to 70% of B (methanol/acetonitrile 3/1 (v/v), 0.02% acetic acid) in A (5 mM ammonium acetate/acetonitrile 95/5 (v/v), 0.02% acetic acid, at pH 5). Supra-pure acetic acid was added to the post-column effluent with a flow rate of 0.2 mL/min to optimize ionization. Detection was carried out in the positive ionization, multiple reaction monitoring (MRM) mode. The chromatograms showed no interference from other substances. The limit of detection (LOD, S/N=3) of pholedrine was 0.8 ng/mL. The intra-day R.S.D. between 5 and 80 ng/mL were 3.8–8.7% and the inter-day R.S.D. between 5 and 100 ng/mL were 6.7–10.7%. The pholedrine concentrations in blood and urine collected when the girl was still alive were 16.1 mg/mL (R.S.D. 10.5%) and 1120 mg/mL (R.S.D. 8%), respectively. In post-mortem samples, they were 23.0 mg/mL (R.S.D. 5.1%) in heart blood and 27.3 mg/g (R.S.D. 6.6%) in the liver. Interactions with endogenous or exogenous substances, such as metabolites, were not observed. Calibration was linear in a range from 1 to 100 ng/mL, and hence was suited for determining therapeutic concentrations. Concentrations of toxicological relevance need dilution of the matrix and appropriate calibration. [8] Total plasma homocysteine (tHcy) in newborn children may be a useful biochemical marker for genetic risk of premature cardiovascular disease. A rapid, isocratic HPLC method able to process very small amount of newborn plasma samples was developed by Bartesaghi et al. Plasma sample from 1 to 10 mL was

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Victoria Samanidou and Eftichia Karageorgou

derivatized with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate after reduction with tri-n-butylphosphine and analyzed on Discovery C18 column, with a solution of acetonitrile–dihydrogenphosphate 0.1 M (8:92 v:v at pH=2.1). Fluorimetric detector with excitation (Ex) and emission (Em) wavelengths set at 385 and 515 nm respectively was used. The method is suitable for routine analysis of tHcy and other aminothiols (Cys, Cys-Gly, Glutathione) assessed for clinical and research purposes. The method was applied for monitoring tHcy levels in 1400 apparently healthy newborn babies. This HPLC method allows measurement of tHcy in newborn during the routinary capillary blood collection in the fourth living day without any other invasive procedure. [9] Metoprolol and captopril were determined in human plasma of volunteers. [10] Captopril and its disulfide metabolites (total captopril) human plasma liquidliquid extraction HPLC using a C-18 reversed phase column. The mobile phase consisted of a methanol-acetonitrile-phosphate buffer (0.02 molΒ·L-1, pH 6.4) mixture (30:30:135, v/v), and was set at a flow rate of 1 mL/min. Linearity was observed with 5 ng/mL (lower limit of quantitation) and 300 ng/mL for total captopril in plasma fluorescence detection at the excitation and emission wavelengths of 235 nm and 440 nm. [11]

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REFERENCES [1] [2] [3] [4]

[5]

[6]

[7]

www.bmb.leeds.ac.uk (Accessed January 2009) www.jlr.org (Accessed January 2009) www.bmj.com (Accessed January 2009) Nirogi, R.; Mudigonda, K.; Kandikere, V. Chromatography–mass spectrometry methods for the quantitation of statins in biological samples. Journal of Pharmaceutical and Biomedical Analysis 2007 44, 379–387. Ferreiros, N.; Iriarte, G.; Alonso, R.M.; Jimenez, R.M.; Ortız, E. Validation of a solid phase extraction-high performance liquid chromatographic method for the determination of eprosartan in human plasma. Journal of Chromatography A 2006 1119, 309–314. Lai, C.M.; Li, S.P.; Yu, H.; Wan, J.B.; Kan, K.W.; Wang, Y.T. A rapid HPLC–ESI-MS/MS for qualitative and quantitative analysis of saponins in ―XUESETONG‖ injection. Journal of Pharmaceutical and Biomedical Analysis 2006 40, 669–678. Husain, K.; Somani, S.M.; Boley T.M.; Hazelrigg, S.R. Interaction of physical training and chronic nitroglycerin treatment on blood pressure and

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39

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plasma oxidant/antioxidant systems in rats. Molecular and Cellular Biochemistry 2003 247, 37–44. [8] Romhild, W.; Krause, D.; Bartels, H.; Ghanem, A.; Schoning, R.; Wittig, H. LC-MS/MS analysis of pholedrine in a fatal intoxication case. Forensic Science International 2003 133, 101–106. [9] Bartesaghi, S.; Accinni, R.; De Leo, G.; Cursano, C.F.; Campolo, J.; Galluzzo, C.; Vegezzi, P.G.; Parodi, O. A new HPLC micromethod to measure total plasma homocysteine in newborn. Journal of Pharmaceutical and Biomedical Analysis 2001 24, 1137–1141. [10] Qu, F.-J.; Zhang, X.-J.; Song, L.; Xu, K.-J. Studies on the pharmacokinetic interaction between metoprolol and captopril in healthy volunteers. Chinese Pharmaceutical Journal 2001 36, 111-113. [11] Zhong, D.; Li, X.; Wang, A.; Chen, X. Determination of captopril plus its disulfide metabolites in human plasma. Yaoxue Xuebao 1998 33, 605-609.

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Chapter 7

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ANTIPSYCHOTICS Antipsychotics are a group of psychoactive drugs commonly used to treat psychosis typified by schizophrenia. A wide range of antipsychotics have been developed till now. The first generation of antipsychotics, known as typical antipsychotics, was discovered in the 1950s. The second generation is known as atypical antipsychotics. Both classes of medication act by blocking receptors in the brain's dopamine pathways. Excess release of dopamine in the mesolimbic pathway has been linked to psychotic experiences. Besides beneficial effects a number of side effects have been observed in relation to specific medications. The development of new antipsychotics, and the relative efficacy of different ones, is an important ongoing field of research. The decision upon the most appropriate drug for an individual patient requires careful consideration. Antipsychotics are used in the treatment of schizophrenia, mania, and delusional disorder. They might be also used to counter psychosis associated with a wide range of other diagnoses, such as psychotic depression, as well as to treat non-psychotic disorders e.g. Tourette syndrome, or Asperger's syndrome. Typical antipsychotics are not particularly selective and also block dopamine receptors in other pathways, leading to unwanted side effects. They are classified on a spectrum of low potency to high potency.Potency refers to the ability of the drug to bind to dopamine receptors. High-potency antipsychotics such as haloperidol, in general, have doses of a few milligrams and cause less sleepiness and calming effects than low-potency antipsychotics, such as chlorpromazine and thioridazine, which have dosages of several hundred milligrams. The latter have a greater degree of anticholinergic and antihistaminergic activity, which can counteract dopamine-related side effects. Atypical antipsychotic drugs have a similar blocking effect on D2 receptors. Some also block or partially block serotonin receptors (particularly 5HT2A, C and

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5HT1A receptors): ranging from risperidone, which acts on serotonin receptors, to amisulpride. The latter has no serotonergic activity. The additional effects on serotonin receptors may be why some of them can benefit the 'negative symptoms' of schizophrenia. [1,2] Aripiprazole has been approved by the Food and Drug Administration (FDA) in 2002 for the treatment of schizophrenia. More recently it received FDA approval for the treatment of acute manic and mixed episodes associated with bipolar disorder, and as an adjunct for the treatment of depression. Its mechanism of action is different from other atypical antipsychotics (e.g., clozapine or risperidone). Aripiprazole appears to be a D2 partial agonist and selective agonist instead of antagonizing the D2 receptor. It acts also as a partial agonist at the 5HT1A receptor, and like the other atypical antipsychotics displays an antagonist profile at the 5-HT2A receptor. It has moderate affinity for histamine and αadrenergic receptors and for the serotonin transporter, and no appreciable affinity for cholinergic muscarinic receptors. [3] Chlorpromazine, a phenothiazine antipsychotic, is the oldest of the antipsychotic drugs, synthesized in 1950. It is a typical antipsychotic, mainly used in the treatment of schizophrenia, though it has also been used to treat severe manic episodes in people with bipolar disorder. Its use has been characterised as the single biggest advance in psychiatric treatment. Currently its use has been largely supplanted by the newer atypical antipsychotics. Chlorpromazine works on a variety of receptors in the central nervous system including anticholinergic, antidopaminergic and antihistamine effects as well as some antagonism of adrenergic receptors. It has minimal effect on the serotonergic pathways. It also has anxiolytic (alleviation of anxiety) properties. [4,5] Clozapine is an antipsychotic medication used in the treatment of schizophrenia. The first of the atypical antipsychotics to be developed, it was first introduced in Europe in 1971. It was voluntarily withdrawn by the manufacturer in 1975, after it was shown to cause agranulocytosis that led to death in some clozapine-treated patients. In 1989, the FDA approved clozapine's use only for treatment-resistant schizophrenia. In 2002 it was approved by the FDA that clozapine for patients with schizophrenia, reduces the risk of suicidal behavior. Due to its potential to cause many severe side effects, it is relegated to third-line use. It is only used in patients when all other anti-psychotics have failed. Safer use of clozapine requires weekly blood monitoring for around five months followed by four weekly testing thereafter. [6, 7] .

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Antipsychotics

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Table 7.1. Overview of HPLC methods for therapeutic drug monitoring of antipsychotics. Analytes Chlorpromazine, haloperidol, Loxapine, clotiapine), (clozapine, quetiapine and risperidone) and their active metabolites (Ndesmethylclozapine, clozapine N-oxide and 9-hydroxyrisperidone 48 antidepressants and antipsychotics

Sample type Human plasma

Sample Preparation Plasma with EDTA as anticoagulant. Centrif. SPE with cyanopropyl cartridges.

Chromatography Chromsep C8 (150×4.6 mm, 5 μm) at RT. MP: ACN (30%, v/v) and a pH 3.0, 30 mM phosphate buffer with 0.5% triethylamine (70%, v/v). FR: 1.0 mL/min. IS: amitriptyline

Recovery % ≥93%

Detection UV: 238 nm

Reference 9

Human serum

Protein precipitation

92-111% Linearity: 0.5–2000 ng/mL

MS/MS in positive MRM mode

10

18 antidepressants, four atypical antipsychotics and active metabolites

Human serum

SPE

75-99%

UV: 230 nm

11

Aripiprazole and dehydroaripiprazole

Human plasma

LLE with heptane and isopropanol 98:2 (v/v)

98 to 113% linear :2–1000 ng/mL

DAD: 200 to 350 nm

12

Quetiapine

Human plasma

SPE on Oasis HLB. Elution with MeOH.

Chromolith Speed ROD C18, 50 mm x 4.6mm , 5 μm. MP:MeOH and 5mM acetic acid, pH 3.9 and ammonia solution, gradient. FR: 1.0 mL/min. Nucleosil (250x4.6 mm ) containing 100-5-Protect 1 (endcapped), MP: 25 mM KH2PO4 (pH 7.0)–ACN (60:40). FR: 1 mL/min. IS: melperone C18 X Bridge® C18 3.5-μm 100 mm x 4.6mm. MP: ACN: ammonium buffer (10mM; pH 8.35) (60:40, v/v) FR: 1mL/min. IS: chlorohaloperidol C8 (150x4.6 mm , 5 mm). MP: ACN, MeOH and pH 1.9 phosphate buffer. IS: triprolidine.

92% Linearity: 4/400 ng/ml

UV: 254 nm

13

44

Victoria Samanidou and Eftichia Karageorgou

Risperidone was approved by FDA in 1993 for the treatment of schizophrenia. In 2007 risperidone was approved as the only drug agent available for treatment of schizophrenia in ages from 13 to 18 years old; it was also approved for treatment of bipolar disorder in youth and children ages 10–18, joining lithium. In 2003 the FDA approved risperidone for the short-term treatment of the mixed and manic states associated with bipolar disorder. Like other atypical antipsychotics, risperidone has also been used off-label for the treatment of anxiety disorders, such as obsessive-compulsive disorder; severe, treatment-resistant depression with or without psychotic features; Tourette syndrome; disruptive behavior disorders in children; and eating disorders. [8] Table 7.1 summarises the chromatographic methods for therapeutic drug monitoring of antipsychotics. In the following paragraphs an overview of published methods on the analysis of antipsychotics is provided

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7.1. Analytical Methods Antipsychotics neuroleptics (chlorpromazine, haloperidol, loxapine and clotiapine), atypical antipsychotics (clozapine, quetiapine and risperidone) and their active metabolites (N-desmethylclozapine, clozapine N-oxide and 9hydroxyrisperidone were determined in human plasma. The blood samples were collected from patients subjected to therapy with two or more of the drugs of interest for at least 2 weeks at constant daily doses. Blood samples were usually drawn 12 h after the last drug administration. Blood was stored in glass tubes containing EDTA as the anticoagulant, and then centrifuged (within 2 h from collection) at 1,400×g for 15 min; the supernatant (plasma) was then transferred to polypropylene tubes and stored at −20 °C until HPLC analysis. Control plasma was obtained in the same way from blood drawn from healthy volunteers not subjected to any pharmacological treatment. The solid-phase extraction procedure was carried out on IST Isolute cyanopropyl (CN) cartridges (50 mg/1 mL). Separation was obtained using a C8 Chromsep column (150 × 4.6-mm, 5 μm) at room temperature. The mobile phase consisted of a mixture of acetonitrile (30%, v/v) and a pH 3.0, 30 mM phosphate buffer containing 0.5% triethylamine (70%, v/v), delivered at a flow rate of 1.0 mL/min. Amitriptyline was used as the internal standard. Column effluent was monitored at 238 nm. The limits of quantitation (LOQ) were lower than 2.6 ng/mL, while the limits of detection (LOD) were lower than 0.9 ng/mL, for all analytes. The method was applied successfully to plasma samples from

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schizophrenic patients undergoing polypharmacy with two or more different antipsychotics. [9] An interested HPLC method was developed for the simultaneous determination of 48 antidepressants and antipsychotics in human serum. This method describes the simultaneous determination of amisulpride, amitriptyline, aripiprazole, benperidol, chlorpromazine, chlorprothixene, citalopram, clomipramine, clozapine, desipramine, doxepin, fluoxetine, flupentixol, fluphenazine, fluvoxamine, haloperidol, hydroxyrisperidone, imipramine, levomepromazine, maprotiline, mianserine, mirtazapine, moclobemide, norclomipramine, nordoxepin, norfluoxetine, nortriptyline, Odesmethylvenlafaxine, olanzapine, opipramol, paroxetine, perazine, perphenazine, pimozide, pipamperone, quetiapine, reboxetine, risperidone, sertraline, sulpiride, thioridazine, trazodone, trimipramine, venlafaxine, viloxazine, ziprasidone, zotepine and zuclopenthixol with a single sample/ triple injection approach. Drugs were assigned to subgroups covering low, medium and high concentrations (overall range of therapeutic levels to be considered: 0.5–2000 ng/mL) by further dilution of the supernatant obtained after the first protein precipitation. Chromatographic separation was necessary for isobaric mass fragments and performed on a monolithic column Chromolith Speed ROD C18, 50 mm x 4.6 mm, 5 μm. The mobile phase was a mixture of methanol and 5mM acetic acid, pH 3.9 and ammonia solution delivered by a gradient elution program. The flow rate was 1.0 mL/min. The injection interval was 8 min. A set of three internal standards was used for quantification of drugs, which are characterised by widely different hydrophobicity. After electrospray ionization positive ion fragments were detected in the multiple reaction monitoring (MRM) mode. Regression parameters of calibration curves and limits of quantification showed good covering of therapeutic and subtherapeutic ranges, within the range 0.5–2000 ng/mL for all analytes. Average coefficients of variation were 6.1% for intra-assay and 7.4% for inter-assay comparisons, while average deviations from spiked concentrations were 4.8% for intra-assay and 4.2% for inter-assay comparisons, respectively. Recovery rates, measured as the percent recoveries of spiked serum samples against standard solutions without serum matrix, varied between 92 and 111%, with an average of 101%, except for olanzapine for which response was much higher (185%) in serum matrix than in matrix-free controls. The method allows a general view on the individual intake of psychoactive drugs and its accurate quantification as well. [10] Therapeutic drug monitoring of antidepressants necessitates efficient, fast and reliable analytical methods validated by external quality control. An isocratic

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reversed-phase HPLC method with ultraviolet detection was developed and optimised to quantify eighteen antidepressants, four atypical antipsychotics and active metabolites mirtazapine, reboxetine, moclobemide, venlafaxine, Odesmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column. The mobile phase consisted of 25 mM potassium dihydrogenphosphate (pH 7.0)–acetonitrile (60:40) delivered at a flow-rate of 1 mL/min. Melperone was used as internal standard 75-99%. UV detection was applied at 230 nm. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r=0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9–10.2% and 0.9–9.7%, respectively. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics, since it allows an efficient and rapid analysis of serum concentrations within 24 h with a single system. [11] A high-performance liquid chromatographic method with diode array detection (HPLC-DAD) was developed for quantification of aripiprazole and dehydro-aripiprazole, in human plasma after LLE with heptane and isopropanol in a ratio of 98:2 (v/v) as extracting solvents. The HPLC chromatographic separation of compounds was carried out on a C18 column X Bridge® 3.5-μm, 100mm × 4.6mm (Waters). Compounds were eluted isocratically using a mobile phase consisting of acetonitrile: ammonium buffer (10mM; pH 8.35) (60:40, v/v) with a flow rate of 1 mL/min. Column effluent was monitored using a diode array detector in the range 200-350 nm. Chlorohaloperidol was used as internal standard. Recovery obtained ranged from 98 to 113%. The total run time was 7 min at a flow-rate of 1.0 mL/min. The precision values were less than 12% and the accuracy values were ranging from 98 to 113% and the lower limit of quantification was 2 ng/mL for both compounds. Calibration curves were linear over a range of 2–1000 ng/mL. The mean plasma concentrations in patients treated with aripiprazole were 157 and 29 ng/mL for aripiprazole and dehydroaripiprazole, respectively. This method is a rapid, reproducible and accurate method to quantify both aripiprazole and dehydroaripiprazole. Due to the fact that

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is is easy and less expensive than those based on mass spectrometry detection, which is not available in many laboratories, it represents therefore an alternative procedure for routine therapeutic drug monitoring of patients treated with aripiprazole. [12] A precise and feasible high-performance liquid chromatographic (HPLC) method for the analysis of the novel antipsychotic drug quetiapine in plasma has been developed. The analysis was carried out on a C8 (150 × 4.6 mm, 5 μm) column, using a mixture of acetonitrile, methanol and pH 1.9 phosphate buffer as the mobile phase. Triprolidine was used as the internal standard. Mean recovery was 92%. Linearity ranged from 4 to 400 ng/mL. UV detection was performed at 254 nm. Careful pretreatment of the biological samples was implemented by means of solid-phase extraction (SPE) Oasis HLB (Hydrophilic/Lipophilic Balance) cartridges (30 mg/1 mL). The application to some plasma samples of patients treated with quetiapine containing tablets gave satisfactory results. The accuracy was good (quetiapine mean recovery 92%), as well as the precision (mean RSD 3.3%). The method seems to be suitable for the clinical monitoring of patients treated with quetiapine. The proposed method for the determination of quetiapine in human plasma based on the use of liquid chromatography with spectrophotometric detection resulted to be simple, accurate and precise, fast and feasible. Furthermore, it has good selectivity; in fact, no interference was found upon examining 21 different CNS drugs. The method is suitable for the analysis of quetiapine in human plasma, applicable for the TDM of patients undergoing chronic treatment with quetiapine. [13]

REFERENCES [1] [2]

[3]

[4] [5] [6]

Swainston, H.T.; Perry, C.M. Aripiprazole: a review of its use in schizophrenia and schizoaffective disorder. Drugs 2004 64, 1715–1736. Zuardi, A.W; Crippa, J.A.S.; Hallak, J.E.C.; Moreira, F.A.; Guimarães, F.S. Cannabidiol as an antipsychotic drug. Brazilian Journal of Medical and Biological Research 2006 39, 421–429. Lawler, C.P. et al. Interactions of the novel antipsychotic aripiprazole (OPC-14597) with dopamine and serotonin receptor subtypes. Neuropsychopharmacology 1999 20, 612–27. www.curedisease.com (Accessed January 2009) www.cochrane.org (Accessed January 2009) Benkert, H. Kompendium der Psychiatrischen Pharmakotherapie (German), Springer Verlag, 4th. ed.

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48 [7] [8] [9]

[10]

[11]

[12]

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[13]

Victoria Samanidou and Eftichia Karageorgou Bandelow, B.; Bleich, S.; Kropp, S. Handbuch Psychopharmaka (German), Hogrefe, 2nd. ed. www.fda.gov (Accessed January 2009) Mercolini, L.; Grillo, M.; Bartoletti, C.; Boncompagni, G.; Raggi, M.A. Simultaneous analysis of classical neuroleptics, atypical antipsychotics and their metabolites in human plasma. Anaytical Bioanaytical Chemistry 2007 388, 235–243. Kirchherr, H.; Kuhn-Velten, W.N. Quantitative determination of forty-eight antidepressants and antipsychotics in human serum by HPLC tandem mass spectrometry: A multi-level, single-sample approach. Journal of Chromatography B 2006 843, 100–113. Frahnert, C.; Rao, M.L.; Grasmader, K. A nalysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring. Journal of Chromatography B 2003 794, 35–47. Lancelin, F.; Djebrani, K.; Tabaouti, K.; Kraoul, L.; Brovedani, S.; Paubel, P.; Piketty, M.-L.; Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma. Journal of Chromatography B 2008 867, 15–19. Mandrioli, R.; Fanali, S.; Ferranti, A.; Raggi, M.A. HPLC analysis of the novel antipsychotic drug quetiapine in human plasma. Journal of Pharmaceutical and Biomedical Analysis 2002 30, 969-977.

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Chapter 8

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ANTIBIOTICS Antibiotics are chemical substances produced by microorganisms that either destroy (bactericidal) or inhibit the growth of other microorganisms (bacteriostatic). Antibiotics can be either broad spectrum, which means that they are active against a wide range of microorganisms. Narrow spectrum drugs target a specific group of microorganisms and are able to interfere with a metabolic process specific to those organisms. In general antibiotics work by: (i) preventing the synthesis of bacterial cell wall components (e.g. penicillins); (ii) damaging the bacterial cytoplasmic membrane; (iii) interfering with protein or nucleic acid synthesis. Not all antibiotic classes require therapeutic drug monitroring. Aminoglycosides are a group of antibiotics that are used to treat certain bacterial infections. They can be used against certain Gram-positive bacteria, but are not typically employed because other antibiotics are more effective and have fewer side effects. They are primarily used to combat infections due to aerobic, Gramnegative bacteria: Pseudomonas, Acinetobacter, and Enterobacter species, among others. Aminoglycosides are also effective against mycobacteria, the bacteria responsible for tuberculosis. They are not effective against anaerobic bacteria (bacteria that cannot grow in the presence of oxygen), viruses, and fungi, only one aminoglycoside, paromomycin, is used against parasitic infection. This group of antibiotics includes at least eight drugs: amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin. All of these drugs have the same basic chemical structure. [1] Streptomycin was the first of aminoglycosides to be discovered, and was the first antibiotic used to treat tuberculosis. It is is a bactericidal antibiotic derived from the actinobacterium Streptomyces griseus. It kills sensitive microbes by inhibiting protein synthesis; it binds to the 16S rRNA of the bacterial ribosome,

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interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This prevents initiation of protein synthesis thus leading to the death of microbial cells. Humans have structurally different ribosomes from bacteria, thereby allowing the selectivity of this antibiotic for bacteria. Streptomycin must be administered by regular intramuscular injection. [2] Spiramycin is a 16-membered ring macrolide (antibiotic). It was discovered in 1952 as a product of Streptomyces ambofaciens, used as preparations for oral administration, since 1955, while in 1987 its parenteral form was introduced into practice. The antibacterial action involves inhibition of protein synthesis in the bacterial cell during translocation. Resistance to spiramycin can develop by several mechanisms and its prevalence is to a considerable extent proportional to the frequency of prescription in a given area. The antibacterial spectrum comprises Gram-positive cocci and rods, Gram-negative cocci and also Legionellae, mycoplasmas, chlamydiae, some types of spirochetes, Toxoplasma gondii and Cryptosporidium sp., Enterobacteria, pseudomonads and pathogenic moulds are resistant. Its action is mainly bacteriostatic, on highly sensitive strains it exerts a bactericide action, with the significant advantage its great gastrointestinal tolerance. [3] Metronidazole is a nitroimidazole anti-infective medication used mainly in the treatment of infections caused by susceptible organisms, mainly anaerobic bacteria and protozoa. Metronidazole is also used in the treatment of the dermatological conditions. It is a pro-drug, converted in anaerobic organisms by the redox enzyme pyruvate-ferredoxin oxidoreductase. The nitro group of metronidazole is chemically reduced by ferredoxin (or a ferredoxin-linked metabolic process) and the products are responsible for disrupting the DNA helical structure, thus inhibiting nucleic acid synthesis. Metronidazole is selectively taken up by anaerobic bacteria and sensitive protozoal organisms, because of the ability of these organisms to reduce metronidazole to its active form intracellularly. [4,5] Table 8.1. summarises the chromatographic methods for therapeutic drug monitoring of antibiotics. In the following paragraphs an overview of published methods on the analysis of antibiotics is provided.

8. 1. Analytical Methods Metronidazole and spiramycin I were determined in human plasma, saliva and gingival crevicular fluid. For plasma, the samples were thawed at room temperature and vortexed, then centrifuged for 5 min at 3500 rpm at

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51

approximately +4 ◦C. To 500 μL of plasma, 50 μL of 0.1 M sodium hydroxide solution, and 0.1 M 1mL of pH 9 buffer solution were added, followed by 6 mL of ethyl acetate after vortexing for a few seconds. A similar extraction procedure was applied for saliva. Table 8.1 Overview of HPLC methods for therapeutic drug monitoring of antibiotics.

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Analytes

Sample type

Sample Preparation

Metronidazole and spiramycin I

Human plasma, saliva and gingival crevicular fluid

LLE with ethyl acetate.

Evernimicin

Human plasma

Ultrafiltration

Chromatography Kromasil C18, 5μm (150mm×4.6mm,), Gradient. MP: ACN, water and formic acid. FR: 0.9 ml/min. IS: Ornidazole

PRP-1 5-mm at 40 oC MP: 57% 0.2 M ammonium acetate (pH 8.75, with triethylamine), 43% ACN, FR: 1.0 mL/min.

Recovery % Human plasma: 77, 76 and 92% for metronidazole and spiramycin I. Human saliva: 67, 82 and 76%,. Human GCF approximately 100% for both. 99.4-103% Linearity: 25 to 2500 ng/mL

Detection

Reference

MS/MS

6

UV: 302 nm

7

For the human gingival crevicular fluid, the samples were thawed at room temperature. Twenty microlitres internal standard at 0.4 ng/ μL was added, followed by 50 μL of acetonitrile to precipitate proteins. Separation was achieved by LC–MS/MS on a 5 μm Kromasil C18 column (150 mm×4.6 mm, 5 μm), with a gradient using acetonitrile, water and formic acid at a flow rate of 0.9 mL/min. Ornidazole was used as an internal standard. [6] Evernimicin was determined in human plasma after ultrafiltration. An aliquot of the ultrafiltrate mixed with 40 μL of acetonitrile was transferred into the HPLC system. The analytical column was a 5-mm polymeric reversed-phase PRP-1 column maintained at 40oC. The mobile phase, consisting of 57% 0.2 M ammonium acetate (pH 8.75, adjusted with triethylamine) and 43% acetonitrile, was delivered at 1.0 mL/min. Linearity was observed in the range 25 to 2500 ng/mL. UV detection was used at 302 nm. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies. The assay was successfully used to support single- and multiple-rising dose safety and tolerance studies in the clinic as well as to assess the effect of liver dysfunction or renal insufficiency on the binding of evernimicin to human plasma proteins. [7]

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REFERENCES [1] [2]

[3] [4] [5] [6]

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[7]

www.healthatoz.com (Accessed January 2009) Kingston, W.; Waksman, S. Streptomycin and the Balance of Credit for Discovery. Journal of the History of Medicine and Allied Sciences 2004 59, 441-462. www.drugs.com (Accessed January 2009) http://ntp.niehs.nih.gov (Accessed January 2009) http://cmr.asm.org (Accessed January 2009) Sagan, C.; Salvador, A.; Dubreuil, D.; Poulet, P.; Duffaut, D.; Brumpt, I. Simultaneous determination of metronidazole and spiramycin I in human plasma, saliva and gingival crevicular fluid by LC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis 2005 38, 298–306. Zhong, R.; Hernandez, A.; Alton, K.B.; Kishnani, N.S.; Patrick, J.E. Highperformance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate. Journal of Chromatography B 2002 772, 191–195.

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Chapter 9

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ANTIEPILEPTIC DRUGS Modern treatment of seizures started in 1850 with the introduction of bromides. In 1910, phenobarbital, which then was used to induce sleep, was found to have antiseizure activity and became the drug of choice for many years. A number of medications similar to phenobarbital were developed, including primidone. In 1940, phenytoin (PHT) was found to be an effective drug for the treatment of epilepsy, and since then it has become a major first-line antiepileptic drug (AED) in the treatment of partial and secondarily generalized seizures. In 1968, carbamazepine (CBZ) was approved, for the treatment of trigeminal neuralgia; and in 1974, for partial seizures. Ethosuximide has been used since 1958 as a first-choice drug for the treatment of absence seizures without generalized tonic-clonic seizures. Valproate was licensed in Europe in 1960 and in the United States in 1978, and today is worldwide used for generalized epilepsies. Novel antiepileptic drug with good efficacy, fewer toxic effects, better tolerability, and no need for blood level monitoring were developed after 1990, approved in the United States as add-on therapy only, with the exception of topiramate and oxcarbazepine; lamotrigine is approved for conversion to monotherapy. Understanding the mechanism of action and pharmacokinetics of antiepileptic drugs is important in clinical practice, so that they can be used effectively, especially in multi-drug treatment. Many structures and processes are involved in the development of a seizure, including neurons, ion channels, receptors, glia, and inhibitory and excitatory synapses. The antiepileptic drugs are designed to modify these processes to favor inhibition over excitation in order to stop or prevent seizure activity. The antiepileptic drugs can be grouped according to their main mechanism of action, although some of them have several actions and others have unknown mechanisms of action. The main groups include sodium channel blockers, calcium

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current inhibitors, gamma-aminobutyric acid (GABA) enhancers, glutamate blockers, carbonic anhydrase inhibitors, hormones, and drugs with unknown mechanisms of action. [1] Carbamazepine was discovered in 1953. It was initially introduced as a drug to treat trigeminal neuralgia in 1962. In 1971, Drs. Takezaki and Hanaoka first used carbamazepine to control mania in patients refractory to antipsychotics. Dr. Okuma, working independently, did the same successfully. Carbamazepine would be studied for bipolar disorder throughout the 1970s. The mechanism of action of carbamazepine is stabilization of the inactivated state of sodium channels, meaning that fewer of these channels are available to open, making brain cells less excitable. Common side effects include drowsiness, headaches and migraines, motor coordination impairment and/or upset stomach. With normal use, small reductions in white cell count and serum sodium are common, however, in rare cases, the loss of platelets may become life-threatening. Therefore frequent blood tests during the first few months of use are mandatory, followed by three to four tests per year for established patients. [2] Oxcarbazepine is an anticonvulsant and mood stabilizing drug, used primarily in the treatment of epilepsy and bipolar disorder. It is a derivative of carbamazepine, adding an extra oxygen atom on the dibenzazepine ring. This difference helps reducing the impact on the liver of metabolizing the drug, and also prevents the serious forms of anemia or agranulocytosis occasionally associated with carbamazepine. It has a similar mechanism as carbamazepine sodium channel inhibition and is generally used to treat the same conditions. Oxcarbazepine has been recently found associated with a greater enhancement in mood and reduction in anxiety symptoms than other drugs employed to treat epilepsy. [3] Lamotrigine is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder. For epilepsy it is used to treat partial seizures, primary and secondary tonic-clonic seizures, and seizures associated with Lennox-Gastaut syndrome. The exact way lamotrigine works is unknown. One proposed mechanism of action for lamotrigine involves an effect on sodium channels, although this remains to be established in humans. In vitro pharmacological studies suggest that lamotrigine inhibits voltage-sensitive sodium channels, thereby stabilizing neuronal membranes and consequently modulating presynaptic transmitter release of excitatory amino acids (e.g. glutamate and aspartate). Lamotrigine shares very few side-effects with other, unrelated anticonvulsants known to inhibit sodium channels, (e.g. Oxcarbazepine), which

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may suggest that lamotrigine has a different mechanism of action. It is inactivated by hepatic glucuronidation. Lamotrigine has recently been reported to be a useful treatment for some people with post-traumatic stress disorder and borderline personality disorder. A recent study reported beneficial effects on individuals with schizoaffective disorder, bipolar subtype with depression. The pharmacokinetics of lamotrigine are quite complicated, with highly varying half-life and blood plasma levels. Lamotrigine has fewer drug interactions than many anticonvulsant drugs, although a pharmacokinetic interaction with sodium valproate in particular is an indication for blood monitoring. [4, 5] Phenobarbital is the most used anticonvulsant worldwide and the oldest still commonly used. It has sedative and hypnotic properties but it has been replaced by the benzodiazepines for these indications. In most countries it is no longer recommended as a first-line medication, rather it is considered as an alternate when a patient fails to respond to treatment with more modern anti-epilepticdrugs. It is still commonly used around the world to treat neonatal seizures. Phenobarbital is indicated in the treatment of all types of seizures except absence seizures. Phenobarbital is no less effective at seizure control than more modern drugs such as phenytoin and carbamazepine. It is, however, significantly less well tolerated. For treatment of status epilepticus benzodiazepines such as diazepam or lorazepam are the first choice. Phenobarbital has an oral bioavailability of approximately 90%. Peak plasma concentrations are reached 8 to 12 hours after oral administration. It is one of the longest-acting barbiturates available – it remains in the body for a very long time (half-life of 2 to 7 days) and has very low protein binding (20 to 45%). Phenobarbital is metabolized by the liver, mainly through hydroxylation and glucuronidation, and induces many isozymes of the cytochrome P450 system. It is excreted primarily by the kidneys. [6] Phenytoin sodium is a commonly used antiepileptic. Phenytoin acts to decrease the unwanted, runaway brain activity seen in seizure by reducing electrical conductance among brain cells by stabilizing the inactive state of voltage gated sodium channels. Aside from seizures, it is an option in the treatment of trigeminal neuralgia as well as certain cardiac arrhythmias. [7] Primidone is an anticonvulsant of the pyrimidinedione class whose active metabolites, phenobarbital (major) and phenylethylmalonamide (minor), are also anticonvulsants. It is used mainly to treat complex partial, simple partials, generalized tonic-clonic seizures, myoclonic, akinetic seizures. Unlike other anticonvulsants such as carbamazepine and valproic acid, primidone is rarely used in the treatment of bipolar disorder or any other psychiatric problem.

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Primidone was once a mainstay anticonvulsant in the treatment of partial and generalized seizures and was the treatment of choice for secondarily generalized seizures originating in the temporal lobes, especially when combined with phenytoin. Primidone works via interactions with voltage-gated sodium channels, which inhibit high-frequency repetitive firing of action potentials. It is much less toxic in overdose than phenobarbital. Along with carbamazepine, phenobarbital, and phenytoin, primidone is an inducer of metabolic enzymes in the liver, as it accelerates the metabolism of many other pharmaceuticals. [8] Table 9.1 summarises the chromatographic methods for therapeutic drug monitoring of antiepileptics. In the following paragraphs an overview of methods on the analysis of antiepileptics is provided.

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9. 1. Analytical Methods The therapeutic efficacy of anti-epileptic drugs depends on the achievement of well-defined plasma concentrations. A validated analytical method is essential to yield results that satisfactorily allow the monitoring of patients during therapy. In a routine laboratory, where a large number of samples have to be analyzed every day, requirements such as short analysis times, simple instrumentation, and chromatographic conditions, as well as an easily applicable technique, without affecting the accuracy and reproducibility of the analytical methods are mandatory. A sensitive and reproducible stir bar-sorptive extraction and highperformance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10, 11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Chromatographic separation was achieved at room temperature on a LiChrospher 100 RP-18 column (125 mm × 4 mm, 5 μm, Merck, Damstadt, Germany). The mobile phase consisted of water: acetonitrile (78:22, v/v) delivered at a flow-rate of 1 mL/min. The ultraviolet detector was set at 220 nm.

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Table 9.1. Overview of HPLC methods for therapeutic drug monitoring of antiepileptics.

Analytes

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Carbamazepine, carbamazepine10,11-epoxide, phenytoin phenobarbital

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Stir bar-sorptive extraction vs LLE

SBSE/HPLC-UV vs LLE/HPLC-UV: Lichrospher 100 RP-18 (125 mm × 4 mm, 5μm). RT. MP: water: ACN (78:22, v/v). FR:1.0 mL/min. (IS): tolylbarbituric Acid

LLE with diethylether

Glass column (3×150 mm) with stationary Phase Separon SGX C18, 5 μm. MP: water/ACN/MeOH 72:23:5 (v/v/v) with TEA. pH: 3.5–7.0. FR: 1 mL/min. IS: 5-Ethyl-5-ptolylbarbituric acid

72 to 86%, except for phenytoin (62%) Linearity: 0.08– 40.0 μgmL−1 for carbamazepine, carbamazepine10,11-epoxide and phenobarbital and 0.125–40.0 μgmL−1 for phenytoin Linearity: 0.5–25 mg/Lfor PEMA and LAM; 1.25–25 mg/Lfor PD and CMZ; 0.625 –12.5 mg/Lfor EPO; 1.5–60 mg/Lfor PB; and 1.25–50 mg/Lfor DPH

and

Lamotrigine (LAM), primidone (PD), phenobarbital (PB), phenytoin (DPH), carbamazepine (CMZ), and two active metabolites 2-phenyl2-ethyl-malonamide (PEMA) and 10,11dihydro-10,11epoxycarbamazepine (EPO)

Human serum

Detection

Reference

UV: 220 nm

9

UV: 220 nm

10

Table 9.1. Continued.

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Analytes Primidone, phenobarbital, Phenytoin, carbamazepine with its two major metabolites carbamazepine10,11-epoxide and carbamazepine-10,11(trans)-dihydrodiol lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide Lamotrigine, oxcarbazepine and its metabolite 10monohydroxycarbazepine (MHD)

Sample type Human serum

Human serum

Sample Preparation

Chromatography

Recovery %

Detection

Reference

SPE. Elution with ACN/MeOH (7/3, v/v)

Alltima 3 C18 (15 cm × 0.46 cm) at 45◦C. MP: MeOH (14.5 vol.%), ACN (19.5 vol.%) and 25 mm Phosphate buffer with 12.5mm of sodium chloride, pH 6.2 (66 vol.%). FR: 0.9 mL/min. IS:5-ethyl-5-para-tolyl barbituric acid

98-103%. Linearity (mg/L): PRM 0–21.8 ZNS 0–21.8 CBZD 0–9.28 LTG 0–21.6 HCB 0–38.8 PHB 0–50.4 CBZE 0–6.26 PHT 0–31.0 CBZ 0–14.8

DAD: 215 and 275 nm

11

Samples were cleaned from interfering proteins and lipids by transfer onto a precolumn, using a Perfect bond C-8 material, With 8% ACN in water as a pre-column eluent

Betasil C-6, 250 mm × 4.6 mm, 5μm. 25◦C. At 0–5 min

95–107% for lamotrigine, 101– 103% for MHD and 97 to 120% for oxcarbazepine. Linearity: 30–5000 ng/mL for lamotrigine, 60–10000 ng/mL for 10-monohydroxycarbazepine (MHD) and 90–1000 ng/mL For oxcarbazepine

UV: 215 nm

12

Table 9.1. Continued.

Analytes

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Drug vigabatrin and R-(−)and S-(+)-enantiomers

Zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine (CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD) and carbamazepine 10,11-epoxide (CBZE)

Sample type Human plasma

Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

Reference

Deproteinize with ACN and the supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA). The Trinitrobenzene derivatives were then concentrated on High Performance Extraction Disk Cartridges Plasma samples Vortex-mixed For 30 s then centrifuged at 12,000g for 10 min at 4 oC.

Chiralcel-ODR (250 mm × 4.6 mm, 10 μm) at 25 ◦C. MP: potassium hexafluorophosphate/AC N/ethanol. Gradient. FR: 0.9 mL/min. IS: 1aminomethyl-cycloheptylacetic acid

96±7% for S-(+)vigabatrin and 94±3% For R-(−)-vigabatrin. Linearity:0.5–40 μg/mL

UV: 340 nm

13

4.6 mm · 250 mm, 5 μm, Zorbax RX-C8 MP: MeOH–ACN– 0.1% TFA, 235: 120: 645 (v/v), FR: 1.5 mL/min. 40oC

94.57 ± 6.33to 103.49 ± 5.78%. Linearity (μg/mL): Carbamazepine1–25, Carbamazepine 10,11-epoxide 1–10, Lamotrigine 1–25, Monohydroxycarbam azepine 1–50, Oxcarbazepine 0.5– 25, Phenobarbital 5– 100, Phenytoin 1–50, Primidone 5–50, Zonisamide 1–80.

UV: 215 nm for PRI, LTG, MHD, PB, PHT, and CBZE, and at 235 nm for ZNS, OXC, and CBZ.

14

Table 9.1. Continued.

Analytes

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

Reference

Deproteinization by ACN

15

Human serum

Deproteinized with TCA and precolumn derivatization with ophtaldialdehyde (OPA) and 3-Mercaptopropionic acid

100-104% Linearity: 1–20 μg/mL for lamotrigine, 2–40 μg/mL for monohydroxycarbam azepine and 10–120 μg/mL for felbamate Linearity: 63 mg/l For PGB, 40 mg/Lfor GBP and 62 mg/Lfor VGB

UV: 210 nm

Pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB)

Fluorescence detector: λex =330 nm and λem = 450 nm.

16

Piracetam(2-oxo-1pyrrolidineacetamide, VPANa (2-propylpentanoic acid, sodium salt, PRM (2desoxyphenobarbital And CBZ (5H-dibenz[b, f]azepine-5-carboxamide

Standard solutions

Synergi 4 μm Hydro-RP, 150 mm × 4 mm, MP: potassium dihydrogen phosphate buffer (50 mm, ph 4.5) and ACN/MeOH (3/1) (65:35, v/v) FR: 1.0 mL/min. I.S:4-methylprimidone. Home-made Column (15 cm × 0.46 cm) packed with Alltima 3 C18 kept at 30 ◦C. MP: MeOH (8.0 vol. %), ACN (17.5 vol.%) and 20 mm phosphate buffer ph 7.0 (74.5 vol.%). FR: 0.8 mL/min. pH 7.50 IS: Norvaline Hibar RT 250-4, Lichrosorb RP-8. 250 mm × 4 mm, 5μm, 25 ◦C. FR: 0.5 mL/min. Gradient. MP: (A) ammonium acetate (B) ethanol and (C) isopropyl alcohol.

ELSD

17

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Lamotrigine (LTG), oxcarbazepine‘s (OXC) main active metabolite monohydroxycarbamazepine and felbamate

Table 9.1. Continued.

Analytes

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Oxcarbazepine and metabolites

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

Reference

SPE (Elution MeOH)

Microsorb MV Rainin (C18 , 150 × 4.6 mm, 5 mm) MP: (FR, 1 mL min ) 15 mM phosphate buffer–MeOH–ACN– triethylamine (62.25:20.0:17.5:0.25, v/v/v/v) pH= 3.5 with 1 M HCl. IS: 10,11-dihydro10,11epoxycarbamazepine Symmetry C18, 3.5 μm 2.1mm × 100 mm, (40 ◦C). MP: ACN 40% with 0.02% formic acid. FR: 350 μL/min. IS: Cyheptamide (CYE)

>94% Linearity: 100–4000 ng mL for OXCBZ, 1.0–40.0 mgmL for CBZ-10OH and 0.3– 12.0 mg mL for CBZ-dioh; 2.0 mg mL (constant) for the I.S.

UV: 237 nm

18

60±4% for OXC, 88±1% for MHD and 62±15% for DHD (mean±SD) Linearity: 0.78–50 mg/L for MHD and 0.078–5.0 mg/Lfor OXC and DHD Linearity: 1.0–15.0 and 0.5–12.0 μg/mL of CBZ and CBZE respectively

MS/MS

19

UV: 190–350 nm

20

Oxcarbazepine (OXC), 10hydroxycarbazepine (MHD) and Trans-diol-carbazepine (DHD)

Human serum

Deproteinization with acetone and SPE on C8 cartridge.

Carbamazepine (CBZ), carbamazepine-10,11Epoxide (CBZE)

Human plasma

LLE with dichloromethane

Table 9.1. Continued.

Analytes

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Eslicarbazepine acetate, oxcarbazepine, Slicarbazepine and Rlicarbazepine

Oxcarbazepine (OXC) and its active metabolite, 10,11dihydro-10hydroxycarbamazepine (MHD)

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

Reference

SPE on Oasis cartridges, conditioned with MeOH, ACN and water–ACN (95:5, v/v). Elution with ethyl acetate

Lichrocart 250-4 Chiradex (β-cyclodextrin, (5 μm) 30°C.isocratic MP: water– MeOH (88:12, v/v), FR: 0.7 mL/min

UV: 225 nm

21

Human plasma

LLE with diethyl ether– diclhoromethane (60:40 v/v) using deuterade carbamazepine (d10carbamazepine) as IS.

Phenomenex® Luna C18 5 μm (150 mm × 4.6 mm) isocratic MP: (ACN/water (50:50 v/v) + 20mm acetic acid). FR: 1.0 mL/min. IS: d10-carbamazepine

94.00-102.23% Linearity: 0.4–8 μg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4 – 80 μg/ mL for each licarbazepine enantiomer. 95.8% for OXC and 86.7% for MHD Linearity: 20–5250 ng/mL for OXC and 40–10,500 ng/mL for MHD

MRM positive Electrospray ionization (ESI+). m/z 253 > 208 for OXC, m/z 255 > 194 for MHD and m/z 247 > 204 for IS.

22

Table 9.1. Continued.

Analytes

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Lamotrigine, carbamazepine and zonisamide

Oxcarbazepine and its main metabolites 10-hydroxy-10, 11- Dihydrocarbamazepine and 10,11-dihydroxy-trans10,11-dihydrocarbamazepine

Sample type Human plasma and serum

Human plasma and cerebros pinal fluid

Sample Preparation

Chromatography

Recovery %

Detection

Reference

LLE with ethylacetate

Μbondapak C18 22°C, MP: aqueous 30 mm potassium phosphate buffer (pH 3.7 with 5% phosphoric acid) and ACN (65:35). FR: 1.2 mL/min. IS: chloramphenicol X-TERRA C18 (150mm x 4.6 mm, 5μm at 40 ◦C. MP: 20mm KH2PO4, ACN, And n-octylamine (76:24:0,05, v/v/v). Isocratic. FR: 0.7 mL/min IS: bromazepam

94%-98% Linearity:1–30 μg/mL for lamotrigine, 2– 20 μg/mL for carbamazepine, and 1–40 μg/mL for zonisamide 80% for oxcarbazepine and 55% for metabolites. Linearity: 25–1000 ng/mL, 1000–25 000 ng/mL, and 100– 4000 ng/mL for Oxcarbazepine, 10hydroxy-10, 11-dihydrocarbamazepinea nd 10,11-di-hydroxytrans-10,11-dihydrocarbamazepine respectively.

UV:270 nm

23

UV: 237 nm

26

LLE with methyl-tertbutyl ether.

Table 9.1. Continued.

Analytes

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Fluvoxamine

Sample type Rat plasma

Levomepromazine, Oxcarbazepine (OXCBZ) and its two metabolites, 10hydroxycarbazepine (CBZ10OH) and trans-diolcarbazepine (CBZ-dioh)

Blood and hair samples

Carbamazepine And Carbamazepine epoxide

Human serum

Sample Preparation

Chromatography

Recovery %

Detection

Reference

LLE with n-hexane. Pre-column derivatization with 4fluoro-7- nitro-2,1,3benzoxadiazole (NBDF). SPE

C18 150 × 4.6 mm, 5 μm. MP: ACN- TFA (0.1% v/v) 60:40 v/v. 25°C. FR: 1.0, mL/min.IS: Propafenone Hydrochloride Lichrocart 125 mm × 3 mm., 5μm, filled with Purospher RP 18. Gradient MP= 0.1% formic acid and 95% ACN + 5% of the phase [A]. FR: 4 mL/min. IS: CBZ epoxy and levomepromazine.

97.3–104.7% Linearity: 0.015–1.5 μg/mL

Fluorometric λex= 470 nm and λem =540 nm.

27

62.0–94.4% and 39.0–77.1% Linearity: 0.05 to 20.00 μg/mL for OXCBZ and its metabolites and levomepromazine (blood) and 0.50– 100.00 ng/mg for OXCBZ and its metabolites (hair). Average recovery: 96.56% Linearity: 014.0 μg/mL for CBZ and 0-4.2 μg/mL for CBZ-EP

MS ion trap equipped with an APCI

28

UV: 280–350 nm

29

LLE

Table 9.1. Continued.

Analytes

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Lamotrigine

Sample type Human plasma and Serum

Sample Preparation

Chromatography

Recovery %

Detection

Reference

LLE with dichloromethane with 5% 3-methyl-2butanol (isoamyl alcohol).

C18. Aqueous phosphoric buffer, ACN and diethylamine. FR: 0.9 mL/minIS:tyramine cloride

86% Linearity: 1.95–39.0 mm

UV: 210 or 285 nm

30

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66

Victoria Samanidou and Eftichia Karageorgou

The commercial stir bar Twister for sorptive extraction was obtained from Gerstel (Gerstel GmbH, Mulheim an der Ruhr, Germany), consisting of a 10 mm long glass-encapsulated magnetic stir bar, externally coated with 22 μg of PDMS. This layer is 0.5 mm thick, corresponding to a volume of 24 μL of PDMS. Prior to the first use, the stir bars were conditioned for 24 h with an acetonitrile: methanol solution (80:20, v/v). Among the successive extractions, the used stir bars were cleaned in methanol for 30 min at 50 ◦C, under magnetic stirring rate of 1200 rpm, followed by a drying step using a lint-free tissue. For the desorption, the stir bars were rinsed lightly with Milli-Q water (1.0 mL), dried with lint-free tissue, and placed in a glass vial containing 1.0 mL of solvent, ensuring total immersion. Desorption was performed by ultrasonic treatment for 15 min at room temperature (25 ◦C) or by magnetic agitation for the same period at the same temperature. After the desorption process, the stir bars were removed by means of a magnetic rod and the solvent was evaporated until dryness. The dry residues were re-dissolved in 200 μL of the mobile phase, and 100 μL of this extract were injected into the HPLC-UV system. LLE was performed using 1 mL of sodium acetate buffer 0.75 M (pH 5.0) and 5mL dichloromethane to 1mL of plasma. The SBSE/HPLC-UV method was linear over a working range of 0.08–40.0 μg/mL for carbamazepine, carbamazepine-10, 11-epoxide and phenobarbital and 0.125–40.0 μg/mL for phenytoin. The intra-assay and inter-assay precision and accuracy expressed as coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08 μg/mL for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125 μg/mL for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. The SBSE/HPLC-UV methodology developed presents high sensitivity and enough reproducibility to permit the quantification of carbamazepine, carbamazepine-10, 11-epoxide, phenytoin and phenobarbital in human plasma. The method has been successfully applied to analysis of real samples demonstrating that it works equally as well as the routine extraction method for therapeutic drug monitoring of antiepileptic drugs. [9] An HPLC procedure for the determination of lamotrigine (LAM) simultaneously with other antiepileptic drugs, primidone (PD), phenobarbital (PB), phenytoin (DPH), carbamazepine (CMZ), and two active metabolites 2phenyl-2-ethyl-malonamide (PEMA) and 10,11-dihydro-10,11epoxycarbamazepine (EPO) was developed and validated for their determination in human serum for routine TDM in patients. The method involves an ordinary RP system and a liquid –liquid extraction with diethylether. Α glass column (36

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Antiepileptic Drugs

mm) with stationary phase Separon SGX C18, 5 μm was used. Mobile phase consisted of ternary mixtures of water/ACN/methanol (pH = 7) in ratio 72:23:5 (v/v/v) with addition of TEA as a modifier. UV detection was carried out at a wavelength of 220 nm and the whole analysis took 15 min. The method was linear in the range of 0.5–25 mg/L for PEMA and LAM; 1.25–25 mg/L for PD and CMZ; 0.625 – 12.5 mg/L for EPO; 1.5–60 mg/L for PB; and 1.25–50 mg/L for DPH, respectively. Within-day CV% and between-day CV% were within 10%. The mixture consisting of water/ACN /methanol/TEA with was selected as the universal mobile phase for both basic drugs and acid drugs, which were analyzed. Preparation of patient samples is rapid, simple, accurate, and reproducible. A low volume of a sample, only 50 μL of blood serum which is needed for the whole analysis, is highly convenient namely for TDM in children. The developed HPLC method allows simultaneous analysis of five AEDs with their two active metabolites and now is used for the routine therapeutic drug monitoring of epileptic patients both in children and adults. [10] A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by HPLC-diode array detector (DAD) at 215-275 nm. After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 ◦C. The method was validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly coadministered antiepileptic drugs did not interfere with the method. Intra-day precision (RSD < 1.9%), linearity, lower limit of quantitation (LOQ < 0.065 mg/L) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies. Prior to SPE 0.1 mL of serum is diluted with 0.5 mL of the internal standard working solution. After conditioning the extraction column with 1mL methanol followed by 1mL water the sample mixture was poured into the column reservoir. The sample was then drawn through the column with a maximum flow-rate of 1 mL/min. Consequently the column was washed with 2mL of water at maximum speed. The drugs were then eluted with three times 0.1 mL of a mixture of acetonitrile/methanol (7/3, v/v). After diluting the eluate with 1 mL of water 50 μL was injected onto the HPLC system. ×150

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The method is in daily use by the authors for routine therapeutic drug monitoring for a considerable time without any problems. The experimental results with respect to linearity, accuracy, precision, specificity and sensitivity demonstrate the reliability of the procedure for its intended application. [11] Using isocratic column-switching HPLC a group method for automated quantitative analysis of the antiepileptic drugs lamotrigine, oxcarbazepine and its metabolite 10-monohydroxycarbazepine (MHD) that are also used in psychiatry as mood stabilizers was developed in human serum. Samples were cleaned from interfering proteins and lipids by transfer onto a pre-column, using a PerfectBond® C-8 material, with 8% acetonitrile in water as a pre-column eluent. Separation was performed by isocratic elution onto the analytical column (Betasil® C6 5 μm, 250 mm × 4.6 mm) at a flow rate of 1.0 mL/min with potassium dihydrogenphosphate buffer (20 mmol/L, pH3.0)/acetonitrile (70/30; v/v) as analytical eluent. UV- detector was set to 215 nm for all three compounds. The process of switching from the pre-column to the analytical column was executed by an electric 10-port valve incorporated in a thermostatted column compartment, set to 25 ◦C. At 0–5 min, serum samples were delivered to the precolumn by pre-column eluent (8% acetonitrile in water). At the same time the analytical eluent flushed the analytical column for preparing separation of the drug mixture. At 5–10 min, the switching valve was set to the analytical position and analytical eluent delivered the matrix free drug mixture to the analytical column in backflush mode. The valve was set back to the starting position from 10 on to 18 min to prepare the pre-column for the next sample injection. The analytical run was finished within 18 min. Detection limit was 30 ng/mL for lamotrigine, 35 ng/mL for oxcarbazepine and 25 ng/mL for 10-monohydroxycarbazepine. The method was found to be suitable for automated analysis of serum samples of patients treated with lamotrigine and oxcarbazepine. The HPLC method described for the determination of lamotrigine, oxcarbazepine and its active metabolite 10- monohydroxycarbazepine turned out to be rapid, precise, accurate and specific over the entire therapeutic range, using the advantages of an on-line sample preparation by column-switching procedure. For separation of lamotrigine, oxcarbazepine and MHD in human serum, injection of 100 μL serum turned out to be sufficient. Intraday variations within the concentration levels specified were always below 2% for all analytes, indicating high precision of the method. An imprecision of up to 2% for lamotrigine and MHD and below 8% for oxcarbazepine among interday measurement experiments is considered acceptable. The application of this HPLC method offers the possibility of a simultaneous detection of various antidepressants and neuroleptics

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within the same run that might be prescribed to patients suffering from bipolar disorders. [12] A rapid and simple HPLC method for the determination of the R-(−)- and S(+)-enantiomers of the antiepileptic drug vigabatrin in human plasma is described by Franco et al. After adding the internal standard (1-aminomethyl-cycloheptylacetic acid), plasma samples (200 μL) are deproteinized with acetonitrile and the supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA). Separation is achieved on a reversed-phase cellulose-based chiral column (Chiralcel-ODR, 250 mm × 4.6 mm) using 0.05 M potassium hexafluorophosphate (pH 4.5)/acetonitrile/ethanol (50:40:10 v/v/v) as mobile phase at a flow-rate of 0.9 mL/min. Chromatographic selectivity is improved by concentrating the derivatives on High Performance Extraction Disk Cartridges prior to injection. Detection was performed at 340 nm. Calibration curves are linear over the range of 0.5–40 μg/mL for each enantiomer, with a limit of quantification of 0.5 μg/mL for both analytes. The assay is suitable for therapeutic drug monitoring and for single-dose pharmacokinetic studies in man. For sample preparation 200 μL aliquot of plasma was mixed with 200 μL of internal standard working solution (12.5 μg/mL) and with 200 μL of acetonitrile. After vortexing for 15 s and centrifuging for 10 min at 1400g, 200 μL of supernatant were transferred into glass tubes. To each tube were added 70 μL of saturated sodium borate solution and 5 μL of the derivatizing agent (5% TNBSA), resulting in an amber-orange colored solution. The tubes were then tightly capped, incubated at 50 ◦C for 10 min, and vortexed for 10 s. The caps were then removed and the derivatization was stopped by adding 250 μL of 0.25 M acetic acid, which resulted in a color change from amber-orange to yellow. The trinitrobenzene derivatives were then concentrated on High Performance Extraction Disk Cartridges and interfering substances were washed away. The method described offers significant advantages in terms of simplicity and ease of use, and it has sufficient sensitivity to allow quantification of the concentrations of each enantiomer which are observed after administration of single oral doses of the racemate in infants and in adults. Therefore, this method can provide a useful tool not only for therapeutic drug monitoring purposes, but also for the conduction of pharmacokinetic studies in a variety of clinical settings. Τhe assay is currently being applied to the investigation of R-(−)- and S-(+)- vigabatrin pharmacokinetics during pregnancy in women with epilepsy. [13] A simple reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of the antiepileptic drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine

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(CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD) and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL) was pretreated by deproteinization with 300 μL methanol containing 20 μg/mL propranolol hydrochloride as internal standard. HPLC was performed on a C8 column (4.6 mm × 250 mm; 5 μm) with methanol–acetonitrile–0.1% trifluoroacetic acid, 235:120:645 (v/v), as mobile phase at a flow rate of 1.5 mL/min. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm, and PRI, LTG, MHD, PB, PHT, and CBZE by UV detection at 215 nm. Relationships between response and concentration were linear over the concentration ranges 1– 80 μg/mL for ZNS, 5–50 μg/mL for PRI, 1–25 μg/mL for LTG, 1–50 μg/mL for MHD, 5–100 μg/mL for PB, 1–10 μg/mL for CBZE, 0.5–25 μg/mL for OXC, 1– 50 μg/mL for PHT, and 1–25 μg/mL for CBZ. Intra-day and inter-day reproducibility were adequate (coefficients of variation were < 11.6%) and absolute recovery ranged from 95.2 ± 6.13 to 107.7 ± 7.76% for all the analytes; for the IS recovery was 98.69 ± 1.12%. The main advantages of the assay are the rapid, single-step, protein precipitation procedure, isocratic elution, and short analysis time, which enabled the method to be cost-effective, and suitable for analysis of a large number of samples in routine work for therapeutic monitoring of the nine analytes. [14] A very simple and fast method has been developed and validated for the simultaneous determination of the antiepileptic drugs: lamotrigine (LTG), oxcarbazepine‘s (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using HPLC with spectrophotometric detection. Plasma sample (500 μL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 μm Hydro-RP, 150 mm × 4 mm column, using a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. UV detection was performed at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1– 20 μg/mL for lamotrigine, 2–40 μg/mL for monohydroxycarbamazepine and 10– 120 μg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples. Venous blood samples (5 mL) were drawn from patients at 8 a.m., before their first morning dose of AEDs. Τhe proposed method proved to possess adequate specificity, sensitivity, accuracy and precision for a reliable

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71

simultaneous determination of LTG, MHD and FBM in patients with epilepsy. By minimizing plasma preparation steps and grouping different new AEDs in the same assay the method allows a large series of patient samples to be processed in a single analytical session, a task which can be very advantageous in a TDM setting. [15] A rapid, simple and robust method is developed for the simultaneous determination of the γ-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by HPLC. Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant are precolumn derivatized with o-phtaldialdehyde (OPA) and 3mercaptopropionic acid. Separation is achieved on an Alltima 3 C18 column using isocratic elution; the drugs are monitored using fluorescence detection (excitation wavelength 330 nm and emission wavelength 450 nm). Norvaline was used as an internal standard. The method was linear up to at least 63 mg/L for PGB, 40 mg/L for GBP and 62 mg/L for VGB. Lower limits of quantitation (LOQ) are 0.13 mg/L for PGB, 0.53 mg/L for GBP and 0.06 mg/L for VGB. No interferences were found from commonly coadministered antiepileptic drugs and from endogenous amino acids. The method is suitable for routine therapeutic drug monitoring and for pharmacokinetic studies. The experimental results with respect to linearity, accuracy, precision, specificity and sensitivity demonstrate the reliability of the procedure for its intended application. [16] A novel, rapid, accurate, sensitive, reproducible and robust HPLC–ELSD method for the simultaneous separation and quantitation of four antiepileptic drugs: Piracetam(2-oxo-1-pyrrolidineacetamide, VPA-Na (2-propylpentanoic acid, sodium salt, PRM (2-desoxyphenobarbital and CBZ (5H-dibenz[b, f]azepine-5-carboxamide has been developed and validated in terms of precision, accuracy, linearity of detector response and robustness. Chromatographic separation was performed on a C8 Hibar pre-packed column 250 mm × 4 mm, 5 μm, Lichrosorb RP-8 column maintained at 25 ◦C during analysis. Mobile phase consisted of ammonium acetate, ethanol and isopropyl alcohol delivered under gradient elution at a flow rate of 0.5 mL/min. Detection was performed using evaporative light scattering detector (ELSD). Optimal instrumental conditions were obtained by assessing the effect of various critical experimental parameters such as evaporator tube temperature, carrier gas flow rate, photomultiplier gain on separation efficiency, accuracy, reproducibility and sensitivity of measurement on all four AEDs. This study illustrates the potential for use of HPLC–ELSD in drug level monitoring of patients undergoing mono- or polytherapy for epilepsy. [17]

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A method based on high-performance liquid chromatography with UV detection in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of the antiepileptic drug oxcarbazepine and its main metabolites in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective solid-phase extraction procedure using hydrophilic–lipophilic balance cartridges. The separation was obtained on a Varian Microsorb MV Rainin reversed-phase column (C18, 150 × 34.6 mm, 5 μm) with a Varian C18 precolumn (30 × 34.6 mm, 5 μm). The mobile phase was a 15 mM phosphate buffer–methanol–acetonitrile– triethylamine (62.25:20.0:17.5:0.25, v/v/v/v) mixture, at pH= 3.5 with 1 M HCl delivered at a flow-rate of 1 mL/min. UV detection was performed at 237 nm. Under these chromatographic conditions, oxcarbazepine and its metabolites 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and the internal standard are baseline separated in less than 9 min. The extraction yield values were 94% for all analytes. The LOQs reached (15 ng/mL) are below the concentrations expected in patients. The SPE procedure appropriately adopted for sample pre-treatment enabled avoidance of chromatographic interference when analysing samples from patients treated with multiple drugs. The method was applied to plasma samples from patients undergoing chronic treatment with oxcarbazepine, both in monotherapy and in polytherapy. Based on the analytical parameters precision, accuracy, limit of quantitation and analysis time the method is suitable for routine application in therapeutic drug monitoring. [18] A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diolcarbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) after sample pretreatment with acetone to precipitate the proteins and separated in less than 3 min on column (Symmetry C18 3.5 μm 2.1 mm × 100mm, Waters, USA) at 40◦C. The mobile phase consisted of acetonitrile 40% containing 0.02% formic acid delivered isocratically. Analytical flow rate of 350 μL/min. A split was included so that only approximately 1/3 of the column eluent entered into ESI probe. The temperature of the autosampler was maintained at 8 ◦C and 10 μL were automatically injected into the HPLC. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78–50 mg/L for MHD and 0.078–5.0 mg/L for OXC and DHD). Within-day and between-days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard.

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Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r = 0.88) in comparison to the routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies. [19] Carbamazepine (CBZ) undergoes enzyme biotransformation through epoxidation with the formation of its metabolite, carbamazepine-10, 11- epoxide (CBZE). Carbamazepine (CBZ) and carbamazepine-10, 11-epoxide (CBZE) were determined in human plasma using a simple chemometrics-assisted spectrophotometric method. A liquid extraction procedure using dichloromethane was operated to separate the analytes from plasma, and the UV absorbance spectra of the resultant solutions were subjected to partial least squares (PLS) regression. An HPLC method was also employed for comparison. The respective mean recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 % and 103.00 % for PLS and 99.40 % and 102.20 %. Analysis of the CBZ and CBZE in the real patients‘ plasma by the two methods indicated excellent agreement between the results obtained by both methods. Thus, both can be used to monitor the levels of CBZ and CBZE in plasma for pharmacokinetic studies. [20] Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis® HLB cartridges. Chromatographic separation was achieved by isocratic elution with water–methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (β-cyclodextrin, 5 μm) column at 30°C. BIA 2-265 used as internal standard. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4–8 μg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4 – 80 μg/ mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy was less than 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 μg/mL for all compounds. The method seems to be a useful tool to individually assess the pharmacologically active metabolites licarbazepine enantiomers and their prodrugs ESL and OXC. It can be applied to clinical trials but also in the routine therapeutic drug monitoring

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assays or in bioequivalence studies that involve the new antiepileptic drug ESL and the already marketed OXC. [21] A fast and sensitive method to quantify oxcarbazepine (OXC) and its active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (MHD) in human plasma using HPLC–MS/MS has been developed. The method involved liquid–liquid extraction (LLE), with diethyl ether–dichloromethane (60:40 v/v) using deuterade carbamazepine (d10-carbamazepine) as internal standard (IS). The analytes and IS were separated using an isocratic mobile phase (acetonitrile/water (50:50 v/v) + 20mM acetic acid) on the analytical column Phenomenex® Luna C18, 5 μm, (150mm × 4.6 mm) at room temperature. Detection was performed by a Micromass Quatro LC mass spectrometer in the reaction monitoring mode using positive electrospray ionization (ESI+). The MS–MS ion transition monitored were m/z 253 > 208 for OXC, m/z 255 > 194 for MHD and m/z 247 > 204 for IS. Linearity was observed over the range 20–5250 ng/mL for OXC and 40–10,500 ng/mL for MHD. The lower limits of quantification obtained as a result of the LLE procedure was 20 ng/mL for OXC and 40 ng/mL for MHD. The suitability of the assay for pharmacokinetics studies was determined by measuring OXC and MHD concentration after administration of a single 10 mL of OXC oral suspension (6%) in plasma human of healthy volunteers. The method has proved to be fast and reliable, with each sample requiring less than 4 min of analysis time. Finally, the suitability of LC–MS/MS method to identify and quantify OXC and MHD in human plasma has been successfully demonstrated. [22] An HPLC assay with ultraviolet detection was developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid–liquid extraction under alkaline conditions using ethylacetate. The method was linear in the range 1–30 μg/mL for lamotrigine, 2– 20 μg/mL for carbamazepine, and 1–40 μg/mL for zonisamide. Within- and between-run precision studies demonstrated coefficient of variation 91% recovery for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy.

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The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine. It has been successfully applied to the analysis of plasma samples from patients subjected to treatment with LMP and CLZ. [25] An isocratic reversed-phase HPLC-UV procedure for the determination of oxcarbazepine and its main metabolites 10-hydroxy-10,11- dihydrocarbamazepine and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine in human plasma and cerebrospinal fluid has been developed and validated. After addition of bromazepam as internal standard, the analytes were isolated from plasma and cerebrospinal fluid by liquid–liquid extraction with 0.5 mL of 0.1M NaOH and 5mL of methyl-tert-butyl ether. Separation was achieved on a X-TERRA C18 column using a mobile phase composed of 20 mM KH2PO4, acetonitrile, and noctylamine (76:24:0.05, v/v/v) delivered isocratically at a flow rate of 0.7 mL/min, at 40 ◦C and detected at 237 nm. Bromazepam was used as internal standard. The described assay was validated in terms of linearity, accuracy, precision, recovery and lower limit of quantification according to the FDA validation guidelines. Linearity was observed within the range: 25–1000 ng/mL, 1000–25 000 ng/mL, and 100–4000 ng/mL for oxcarbazepine, 10-hydroxy-10,11dihydrocarbamazepine, and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine, respectively Accuracy ranged from 92.3% to 106.0% and precision was between 2.3% and 8.2%. The method was simple, precise, accurate, selective and sufficiently sensitive and seems suitable for the quantitative determination of oxcarbazepine, 10hydroxy-10,11-dihydrocarbamazepine and 10,11-dihydroxytrans10,11dihydrocarbamazepine in plasma and cerebrospinal fluid samples, obtained in the conduct of clinical pharmacokinetic studies, after oral administration of oxcarbazepine, both in monotherapy and polytherapy. [26] A sensitive, simple and reliable method using HPLC was reported for the determination of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after LLE with n-hexane and pre-column derivatization with 4-fluoro-7- nitro-2,1,3-benzoxadiazole (NBD-F). Extracted plasma samples were mixed with NBD-F at 60°C for 5 min and injected into HPLC. The HPLC C18 column was 150 × 4.6 mm, 5μm. The mobile phase consisted of acetonitrile and trifluoroacetic acid (0.1% v/v) in water, at a volume ratio 60:40. The samples were eluted from the column at 25°C at a flow rate of 1.0 mL/min. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. Linearity was observed in the range 0.015– 1.5 μg/mL. The lower limits of detection and quantification of FLU were 0.008 and 0.015 μg/mL, respectively. The coefficients of variation for intra-day and

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inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Propafenone hydrochloride solution in water was added as an internal standard. Fluorometer detector was operating at an excitation wavelength of 470 nm and an emission wavelength of 540 nm. No interference was observed by other SSRIs and centrally acting drugs. Results indicate that the method is useful to determine the FLU levels in rat plasma of volumes as small as 100 μL and can be applied to pharmacokinetic studies and therapeutic drug monitoring of FLU in patients. [27] Levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) were determined in blood and hair samples after SPE. A typical use of hair analysis in forensic toxicology is the documentation of previous drug administration. This is illustrated in a suicidal death of a 58-yearold epileptic patient who was treated with oxcarbazepine and probably with levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS included hair (6 cm length) besides postmortem blood. The method was validated for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in various biological matrices. The analysis revealed differences between the concentration levels of oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and hair, suggesting different mechanisms of penetration of metabolites and their precursors into this matrix. Samples were extracted by means of solid phase extraction (SPE). The method provided good relative and absolute recoveries of 62.0–94.4% and 39.0–77.1%, respectively, for all analytes across the linear dynamic range. The chromatographic separation was performed with a LiChroCART column 125 mm×3mm, 5μm, filled with Purospher RP 18 and a LiChroCART precolumn 4 mm×4 mm, 5 μm, filled with LiChrospher 60 RP – select. The mobile phase consisted of a gradient mixture of A: 0.1% formic acid in demineralised water and B: 95% acetonitrile + 5% of A delivered at a flow rate of 0.4 mL/min. Linearity was obsreved in the range: 0.05 to 20.00 μg/mL for OXCBZ and its metabolites and levomepromazine (blood) and 0.50–100.00 ng/mg for OXCBZ and its metabolites in hair. [28] Carbamazepine and carbamazepine epoxide were determined in human serum of real patients by a procedure assisted by chemometric tools. First, a response surface methodology based on a mixture design was applied in order to select the best conditions for the extraction step. Finally, partial least squares multivariate calibration (PLS-1) was applied to second-derivative UV spectra, eliminating a shift baseline effect that originated in the extraction procedure. The performance

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assessment included: (a) a three-level precision study, (b) a recovery study analyzing spiked samples, and (c) a method comparison with high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) applied on real patient samples. The obtained results show the potentiality of the presently studied methodology for the monitoring of patients treated with this anticonvulsant. The combination of UV spectrophotometry coupled to both optimized-analyte extraction and multivariate calibration (PLS-1) leads to a powerful tool to be applied to drug monitoring. The results obtained by applying the developed method on real patient serum samples and by comparing it with reference methods show the enormous potentiality of this analytical strategy. Carbamazepine was determined with high accuracy and precision by using a very simple, quick and inexpensive method. Average recovery was 96.56%. Linearity ranged from 0 to14.0 μg/mL for CBZ and 0-4.2 μg/mL for CBZ-EP. [29] Lamotrigine was determined in human plasma and serum. Aliquots of sample (patient, calibrators and controls) were mixed with internal standard solution, NaOH and dichloromethane containing 5% 3-methyl-2-butanol (isoamyl alcohol). After centrifugation, the aqueous (upper) phase was discarded and the organic phase was evaporated to dryness and reconstituted in 200 mL methanol. Separation was performed on a reversed-phase C18 column. The mobile phase was prepared by mixing aqueous phosphoric buffer, acetonitrile and diethylamine. The flow-rate was 0.9 mL/min. Tyramine chloride was used as internal standard. Linearity was observed in the range 1.95–39.0 mM. UV detection was performed at 210 or 285 nm. [30] Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. Two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiplecomponent LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. High flow LC was achieved by the use of 4 mL/min flow rate on a 1 × 50 mm Waters Oasis column. A 2 × 100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. The on-line extraction HFCS LC/MS/MS method has been successfully used not only in the plasma assays of N-in-1 PK studies but also in urine and synovial

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fluid assays where the biological fluids contain dirty matrices. In fact, good sensitivity and separation efficiency were achieved from all biological fluids including directly injected plasma, serum, whole blood, urine, and bile samples. The resulting dynamic range, lower limit of quantification, QC accuracy and precision were within the acceptable range for discovery research and development. [31]

REFERENCES

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[1] [2] [3]

emidicine.medscape.com (Accessed January 2009) www.blackwell-synergy.com (Accessed January 2009) Mazza, M.; Della Marca, G.; Di Nicola, M. Oxcarbazepine improves mood in patients with epilepsy. Epilepsy Behaviour 2007 10, 397–401. [4] www.drug.co.il (Accessed January 2009) [5] www.tevapharm.com (Accessed January 2009) [6] www3.interscience.wiley.com (Accessed January 2009) [7] www.ncbi.nlm.nih.gov (Accessed January 2009) [8] www.pubmedcentral.nih.gov (Accessed January 2009) [9] Costa Queiroz, R.H.; Bertucci, C.; Malfara, W.R.; Carvalho Dreossi, S.A.; Rodrigues Chaves, A.; Rodrigues Valerio, D.A.; Costa Queiroz, M.E. Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis 2008 48, 428–434. [10] Budakova, L.; Brozmanova, H.; Grundmann, M.; Fischer, J. Simultaneous determination of antiepileptic drugs and their two active metabolites by HPLC. Journal of Separation Science 2008 31, 1–8. [11] Vermeij, T.A.C.; Edelbroek, P.M. Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction. Journal of Chromatography B 2007 857, 40–46. [12] Greiner, C.; Haen, E. Development of a simple column-switching highperformance liquid chromatography (HPLC) method for rapid and simultaneous routine serum monitoring of lamotrigine, oxcarbazepine and 10-monohydroxycarbazepine (MHD). Journal of Chromatography B 2007 854, 338–344.

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[13] Franco, V.; Mazzucchelli, I.; Fattore, C.; Marchiselli, R.; Gatti, G.; Perucca, E. Stereoselective determination of vigabatrin enantiomers inhuman plasma by high performance liquid chromatography using UV detection. Journal of Chromatography B 2007 854, 63–67. [14] Ma, C.-L.; Jiao, Z.; Jie, Y.; Shi, X.-J. Isocratic Reversed-Phase HPLC for Simultaneous Separation and Determination of Seven Antiepileptic Drugs and Two of their Active Metabolites in Human Plasma. Chromatographia 2007 65, 267–275. [15] Contin, M.; Balboni, M.; Callegati, E.; Candela, C.; Albani, F.; Riva, R.; Baruzzi, A. Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy. Journal of Chromatography B 2005 828, 113–117. [16] Vermeij, T.A.C.; Edelbroek, P.M. Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection. Journal of Chromatography B 2004 810, 297–303. [17] Manoj Babu, M.K. Simultaneous separation and quantitation of four antiepileptic drugs—a study with potential for use in patient drug level monitoring. Journal of Pharmaceutical and Biomedical Analysis 2004 34, 315–324. [18] Mandrioli, R.; Ghedini, N.; Albani, F.; Kenndler, E.; Raggi, M.A. Liquid chromatographic determination of oxcarbazepine and its metabolites in plasma of epileptic patients after solid-phase extraction. Journal of Chromatography B 2003 783, 253–263. [19] Paglia, G.; D‘Apolito, O.; Garofalo, D.; Scarano, C.; Corso, G. Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum. Journal of Chromatography B 2007 860, 153–159. [20] Hemmateenejad, B.; Rezaei, Z.; Khabnadideh, S.; Saffari, M. A PLS-based extractive spectrophotometric method for simultaneous determination of carbamazepine and carbamazepine-10,11-epoxide in plasma and comparison with HPLC. Spectrochimica Acta Part A 2007 68, 718–724. [21] Alves, G.; Figueiredo, I.; Castel-Branco, M.; Loureiro, A.; Fortuna, A.; Falcao, A.; Caramona, M. Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma. Biomedical Chromatography 2007 21, 1127–1134. [22] de Sousa Maia, M.B.; do Nascimento, D. F.; Martins, I.L.; Nunes Cunha, A.; Goncalves de Lima, F. E.; Frota Bezerra, F. A.; de Moraes, M.O.; de

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Antiepileptic Drugs

[23]

[24]

[25]

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[26]

[27]

[28]

[29]

[30]

81

Moraes, M.E.A. Simultaneous quantitative analysis of oxcarbazepine and 10,11-dihydro-10 hydroxycarbamazepine in human plasma by liquid chromatography–electrospray tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2007 45, 304–311. Greiner-Sosanko, E.; Lower, D.R.; Virji, M.A.; Krasowski, M.D. Simultaneous determination of lamotrigine, zonisamide, and carbamazepine in human plasma by high-performance liquid chromatography. Biomedical Chromatography 2007 21, 225–228. Ma, C.-L.; Jiao, Z.; Jie, Y.; Shi, X.-J. Isocratic Reversed-Phase HPLC for Simultaneous Separation and Determination of Seven Antiepileptic Drugs and Two of their Active Metabolites in Human Plasma. Chromatographia 2007 65, 267–275. Mercolini, L.; Bugamelli, F.; Kenndler, E.; Boncompagni, G.; Franchini, L.; Raggi, M.A.; Simultaneous determination of the antipsychotic drugs levomepromazine and clozapine and their main metabolites in human plasma by a HPLC-UV method with solid-phase extraction. Journal of Chromatography B 2007 846, 273–280. Kimiskidis, V.; Spanakis, M.; Niopas, I.; Kazis, D.; Gabrieli, C.; Kanaze, F.I.; Divanoglou, D. Development and validation of a high performance liquid chromatographic method for the determination of oxcarbazepine and its main metabolites in human plasma and cerebrospinal fluid and its application to pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 2007 43, 763–768. Higashi, Y.; Matsumura, H.; Fujii, Y. Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole. Biomedical Chromatography 2005 19, 771–776. Kłys, M.; Rojek, S.; Bolechała, F. Determination of oxcarbazepine and its metabolites in postmortem blood and hair by means of liquid chromatography with mass detection (HPLC/APCI/MS). Journal of Chromatography B 2005 825, 38–46. Camara, M. S.; Mastandrea, C.; Goicoechea, H.C. Chemometrics-assisted simple UV-spectroscopic determination of carbamazepine in human serum and comparison with reference methods. Journal of Biochemical and Biophysical Methods 2005 64, 153–166. Theurillat, R.; Kuhn, M.; Thormann, W. Therapeutic drug monitoring of lamotrigine using capillary electrophoresis. Evaluation of assay performance and quality assurance over a 4-year period in the routine arena. Journal of Chromatography A 2002 979, 353–368.

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[31] Mandrioli, R.; Fanali, S.; Ferranti, A.; Raggi, M.A. HPLC analysis of the novel antipsychotic drug quetiapine in human plasma. Journal of Pharmaceutical and Biomedical Analysis 2002 30, 969-977.

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Chapter 10

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ANTIDEPRESSANTS An antidepressant is a psychiatric medication used for treeatment of major depression or dysthymia. Drug groups most commonly prescribed by psychiatrists include monoamine oxidase inhibitors (MAOIs), tricyclics, and second-generation antidepressants such as Selective serotonin reuptake inhibitors (SSRIs), and Serotonin norepinephrine reuptake inhibitors (SNRI's). The effectiveness and adverse effects are the subject of many studies. Most antidepressants have a delayed onset of action and are usually administered over a long-term period of weeks, months, or sometimes years. Antidepressants are often used in the treatment of other conditions, including anxiety disorders, bipolar disorder, obsessive compulsive disorder, eating disorders, chronic pain, mood disorders, and dysmenorrheal and other hormonemediated conditions. Selective serotonin reuptake inhibitors (SSRIs) are a family of antidepressants acting by preventing the reuptake of serotonin (also known as 5hydroxytryptamine, or 5-HT) by the presynaptic neuron, thus maintaining higher levels of 5-HT in the synapse. It is thought that one cause of depression may be an inadequate amount of serotonin, a chemical used in the brain to transmit signals between neurons. Members of this family include fluoxetine, paroxetine, escitalopram, citalopram, sertraline, and fluvoxamine. These antidepressants typically have fewer adverse events and side effects than the tricyclics or the MAOIs. Some side effects may decrease as a person adjusts to the drug, but other side effects may be persistent. They are considered to be safer than the first generation antidepressants. Serotonin-norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine, milnacipram and duloxetine are a newer form of antidepressant that work on both

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norepinephrine and 5-HT. They typically have similar side effects to the SSRIs, although there may be a withdrawal syndrome on discontinuation that may necessitate dosage control. Noradrenergic and specific serotonergic antidepressants (NASSAs) are a newer class of antidepressants which increase norepinephrine (noradrenaline) and serotonin neurotransmission by blocking presynaptic alpha-2 adrenergic receptors while at the same time minimizing serotonin related side-effects by blocking certain serotonin receptors. Mirtazapine is the only member of this class in clinical use. Mianserin has also similar mechanism of action. Norepinephrine (noradrenaline) reuptake inhibitors (NRIs) act via norepinephrine (known also as noradrenaline). NRIs are thought to have a positive effect on concentration and motivation. Norepinephrine-dopamine reuptake inhibits the neuronal reuptake of dopamine and norepinephrine (noradrenaline). Tricyclic antidepressants (TCAs) are the oldest class of antidepressant drugs. Representative members are amitriptyline and desipramine. They act by blocking the reuptake of certain neurotransmitters such as norepinephrine (noradrenaline) and serotonin. They are used less commonly now due to the development of more selective and safer drugs. In overdoses they can be lethal, as they may cause a fatal arrhythmia. However, tricyclic antidepressants are still used because of their effectiveness, especially in severe cases of major depression. Monoamine oxidase inhibitor (MAOIs) may be used if other antidepressant medications are ineffective. Because there are potentially fatal interactions between this class of medication and certain foods (particularly those containing Tyramine), red wine, as well as certain drugs, classic MAOIs are currently rarely prescribed. MAOIs work by blocking the enzyme monoamine oxidase which breaks down the neurotransmitters dopamine, serotonin, and norepinephrine (noradrenaline). MAOIs can be as effective as tricyclic antidepressants, although they can have a higher incidence of dangerous side effects (as a result of inhibition of cytochrome P450 in the liver). Duloxetine is a serotonin-norepinephrine reuptake inhibitor (SNRI) used for major depressive disorder, generalized anxiety disorder, pain related to diabetic neuropathy and fibromyalgia. Preclinical studies demonstrate that duloxetine potently inhibits neuronal serotonin and norepinephrine reuptake, and this inhibition is balanced throughout the dosing range. It is also considered a less potent inhibitor of dopamine reuptake. Since it has no significant affinity for dopaminergic, adrenergic, cholinergic, histaminergic, opioid, glutamate, and GABA receptors it can be considered as a selective reuptake inhibitor at the 5-HT and NA receptors. Duloxetine undergoes extensive metabolism, but the major

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circulating metabolites do not contribute significantly to the pharmacologic activity. Its pharmacokinetic is dose proportional over the therapeutic range. After 3 days a steady-state is usually achieved. [1] Mianserin is a tetracyclic antidepressant that has antihistaminic and hypnosedative, but almost no anticholinergic, effect. Mianserin is a weak inhibitor of norepinephrine reuptake and strongly stimulates the release of norepinephrine. Interactions with serotonin receptors in the central nervous system have been also found. Mianserin blocks inhibitory α2-autoreceptors on central noradrenergic nerve endings, and so may increase the amount of noradrenaline in the synaptic cleft. It may also cause agranulocytosis and aplastic anaemia. [2,3] Fluoxetine hydrochloride is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. Fluoxetine is approved for the treatment of major depression, obsessive-compulsive disorder, bulimia nervosa, anorexia nervosa, panic disorder and premenstrual dysphoric disorder. Despite the availability of newer agents, it remains extremely popular. The bioavailability of fluoxetine is relatively high (72%), and peak plasma concentrations are reached in 6 to 8 hours. It is highly bound to plasma proteins, mostly albumin. Fluoxetine is metabolized in the liver by isoenzymes of the cytochrome P450 system, including CYP2D6. Only one metabolite of fluoxetine, norfluoxetine (demethylated fluoxetine), is biologically active. The concentration of the drug and its active metabolite in the blood increases through the first few weeks of treatment, and their steady concentration in the blood is achieved only after four weeks. Complete excretion of the drug may take several weeks. [4] Venlafaxine is an antidepressant of the serotonin-norepinephrine reuptake inhibitor (SNRI). It is prescribed for the treatment of major depression and anxiety disorders. Due to the side effects and suspicions that venlafaxine may significantly increase the risk of suicide, it is not recommended as a first line treatment of depression. However, it is often effective for depression not responding to SSRIs.

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Table 10.1. Overview of HPLC methods for therapeutic drug monitoring of antidepressants. Analytes

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

SPE: C8 cartridges (50 mg, 1 mL). Elution with MeOH.

>91% Linearity: TRZ 10–2000 ng/mL m-CPP: 10–1000 ng/mL

UV: 255nm

Mianserin hydrochloride

Human plasma

LLE with N-hexane.

81.3–84.1% Linearity: 1.0– 200.0 ng/mL

ESI–MS: (SIM) at m/z 265 [M+H]+ for mianserin and m/z 369 [M+H]+ for cinnarizine.

8

Trazodone, fluoxetine, Mianserine Desipramine imipramine nortryptiline amitryptiline trimipramine clomipramine

Urine samples

SPE: laboratory made 30 mg MWCNTs cartridges

Genesis C8 (15 mm × 4.6 mm, 5 μm) RT. MP: ACN (30%, v/v) and pH 3.5, 50mM phosphate buffer with 0.3% (v/v) triethylamine (70%, v/v). FR: 1.2mL/min. (IS): Loxapine Hypersil-Hypurity C18 (150 mm × 2.1mm, 5μm) at 45 ◦C. MP: 10mM ammonium acetate (pH 3.4)– MeOH–ACN (35:50:15, v/v/v). FR: 0.22 mL/min (I.S.) cinnarizine Eclipse X-DB-C8 (4.6 × 150 mm). MP: NaH2PO4 buffer (pH 3.0)–ACN– MeOH 70:25:5 (v/v). FR: 1.2 mL min−1

72.4-97%

UV: 254 nm.

9

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Trazodone and its main active metabolite 3-(1Clorophenyl)piperazine (m-cpp)

Referenc e 7

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Table 10.1. Continued. Analytes Amitriptyline, Imipramine and sertraline

Sample type Urine, plasma and tap water

Amitriptyline doxepin Clomipramine Imipramine

Pharmaceutical formulations and biological Fluids

Duloxetine

Human plasma

Sample Preparation (HF-LPME): Extraction from 11.0 mL of aqueous solution with pH 12.0 into ndodecane impregnated in a hollow fiber and back extracted into 24 μL of aqueous solution located inside the lumen of the hollow fiber and adj. pH 2.1 using 0.1M of H3PO4 Human plasma samples were pretreated to remove proteins. Urine samples were analysed both directly after twofold dilution and filtration and after SPE. SPE: Waters Oasis mixed-mode reversed phase—strong cation exchange (MCX) cartridges (30 mg/ 1 mL). Elution with ammonia/ water/MeOH (5/15/80, w/w/v).

Chromatography Zorbax Extend C18 (100 mm × 2.1 mm). MP: 0.02 M acetic acid (pH 4.0) and MeOH (54:46) FR: 0.25mL/min. IS: chloropromazine.

Recovery % Linearity: 5–500 μg/L

Detection UV: 215 nm

Reference 10

Kromasil C8 (250 × 4 mm), 5 μm, RT. MP: CH3COONH4 0.05 M– CH3CN 45:55 v/v, FR: 1.5 mL/min. IS: bromazepam.

91.0 -114.0% Linearity: 1–5 μg/mL

UV: 238 nm

11

C8. MP: 60% aqueous phosphate buffer with triethylamine, pH 3.0 and 40% CAN. FR: 1 mL/min. IS: Loxapine.

>90% Linearity: 2–200 ng/mL

UV: 230 nm

12

Table 10.1. Continued. Analytes

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Imipramine, amitriptyline, clomipramine, fluoxetine, sertraline, paroxetine, citalopram, mirtazapine, moclobemide and duloxetine

Levomepromazine, clozapine and their main Metabolites: n-desmethyllevomepromazine, levomepromazine sulphoxide, odesmethyl-levomepromazine, ndesmethylclozapine and clozapine N-oxide.

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

LLE

72%-86%, except for moclobemide (59%). Linearity: 2.5–2000 ngmL−1

UV: 230 nm

Human plasma

SPE: BondElut C1 cartridges (100 mg, 1 mL). Elution with MeOH.

LiChrospher 60 RPselect B (250 mm × 4 mm, 5μm). MP: 35% ACN: MeOH (92:8, v/v) and 65% of sodium acetate buffer, pH 4.5. FR: 1.0mLmin−1. IS: etidocaine. C8 (150 mm × 4.6 mm, 5 μm; Phenomenex. MP: ACN and a phosphate buffer (34 mM, pH 2.0) with 0.3% triethylamine (29:71, v/v). FR: 1.0 mL/min. IS: Loxapine.

Referenc e 13

>91% Linearity: LMP 9–200 ng/mL; NDLM 10–150 ng/mL; LMSO 5–500 ng/mL; ODLM 7– 150 ng/mL; CLZ 20–2500 ng/mL; DMC 15–1000 ng/mL; NOX 10– 200 ng/mL; IS 200 ng/mL

UV: 254 nm

14

Table 10.1. Continued.

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Analytes Venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine, Melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and trazodone) together with eight of their metabolites (odesmethylvenlafaxine, norfluoxetine, desmethylmianserine,desmethy lmirtazapine,desmethylcitalopr am,didesmethylcitalopram,des methylsertraline and m chlorophenylpiperazine) Amitriptyline and nortriptyline

Three intermediates in the synthesis of S-duloxetine, the antidepressant drug, viz., 2acetyl thiophene (AT), N,Ndimethyl-3-keto(2-thienyl)-propanamine (DKTP) and (S)-N,N-dimethyl3-hydroxy-(2-thienyl)propanamine (DHTP)

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

Reference

SPE apolar, polymeric, ion-exchange, mixed modes combining ionexchange properties Elution with MeOH and 3mL of buffer (pH 6.5 or 2.5).

HPLC-DAD GC-MS

70 and 109% for all compounds, except for trazodone (39%)

DAD: 210-380 nm MS: the spectra obtained were measured in the SIM mode

15

Serum

Dilution (1:10) in 0.15 M SDS-6% pentanol at pH 7, and filtration through 0.45 μm nylon membranes. DKTP as a hydrochloride salt was added. Incubation at 30 ◦C in an orbital shaker 200 rpm. LLE with ethyl acetate.

Kromasil C18 with 5 μm, 250 mm × 4.6 mm. MP: a 0.15 M SDS-6% (v/v) pentanol at pH 7. FR: 1.5 mL/min. 250mm × 4.6 mm, 5 μM LichroCART RP-18

98.5-101.6%

UV: 240 nm Electrochemical detection at 650 mV.

16

99.7- 101.5% Linearity: AT 5– 500 (μg/mL) DKTP &DHTP 20–500 (μg/mL)

UV: 241 nm

17

Standard solutions

Table 10.1. Continued. Analytes

Sample type Human plasma

Sample Preparation

Chromatography

Recovery %

Detection

SPE on Waters Oasis. Elution with % HAcMeOH.

> 73.2% Linearity: 5.0–1000.0 ng/mL

(ESI-MS) in the selected ion recording (SIR) mode

Eighteen antidepressants, four atypical antipsychotics And active metabolites

Human serum

SPE

75-99%

UV: 230 nm

19

Amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI)

Plasma and Serum

LLE using hexane at pH 11

92-105% Linearity: 20 and 400 ng /mL

UV: 242 nm

20

Fluoxetine, norfluoxetine

Human plasma

SPE on C8 cartridge. Elution with MeOH.

C18 (250 mm × 4.6 mm, 5 μm), MP: (formic acid 0.6‰, ammonium acetate: 30 mmol/L)– ACN (35:65, v/v), FR: 0.85 mL/min. IS: fluvoxamine Nucleosil 100-5- (250 ×4.6 mm), MP: 25 mM potassium dihydrogen phosphate (pH 7.0)– ACN (60:40). FR: 1 mL/min. IS: melperone Nova-Pak C18, (4.6 × 150 mm). MP: KH2PO4 buffer, ACN and diethylamine, pH= 8 with H3PO4. FR: 0.9 mL/ min. RT. IS: econazole. C8 (150 × 4.6 mm). MP: ACN and water with HClO4 and tetramethylammonium perchlorate FR: 1 mL/min.

Linearity: 8–200 ng /mL

Fluorescence: λem =290 Nm, λexc = 230 nm.

21

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Fluoxetine, citalopram, paroxetine and venlafaxine

Referenc e 18

Table 10.1. Continued. Analytes

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Modafinil (provigil)

Sample type Human plasma and urine

Sample Preparation

Chromatography

Recovery %

Detection

LLE with ethyl acetate and ethyl acetate– acetic acid (100:1, v/v).

4.6 mm × 250mm Symmetry C18, MP: MeOH–water– acetic acid 500:500:1, v/v) FR: 1.0 mL/min. IS: Phenylthioacetic acid

80.0- 98.9%. Linearity: 0.1–20.0 μg/mL

UV:220-233 nm

Referenc e 22

Victoria Samanidou and Eftichia Karageorgou

92

Venlafaxine is a bicyclic antidepressant, and is usually categorized as a serotonin-norepinephrine reuptake inhibitor (SNRI), but it has been referred to as a serotonin-norepinephrine-dopamine reuptake inhibitor. It acts by blocking the transporter "reuptake" proteins for key neurotransmitters affecting mood, thereby leaving more active neurotransmitters in the synapse. In high doses it weakly inhibits the reuptake of dopamine. Venlafaxine is well absorbed with at least 92% of an oral dose being absorbed into systemic circulation. It is extensively metabolized in the liver via the CYP2D6 isoenzyme to O-desmethylvenlafaxine, which is just as potent a serotonin-norepinephrine reuptake inhibitor as the parent compound, meaning that the differences in metabolism between extensive and poor metabolizers are not clinically important in terms of efficacy. Steady-state concentrations of venlafaxine and its metabolite are attained in the blood within 3 days. Therapeutic effects are usually achieved within 3 to 4 weeks. [5,6] Table 10.1 summarises the chromatographic methods for therapeutic drug monitoring of antidepressants. In the following paragraphs an overview of published methods on the analysis of antidepressants is provided. Figure 10.2 illustrates an example of a chromatographic separation of SNRI‘s and SSRI‘s in blood plasma.

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AU 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 0,5

2,5

4,5

6,5 t (min)

Figure 10. 2. HPLC chromatogram of SNRIs and SSRIs determination in human plasma in the presence of BAM as the internal standard. Experimental data from author‘s laboratory. VEN: venlafaxine, BAM: bamifylline (IS), PAR: paroxetine, DUL: duloxetine and FLU: fluoxetine.

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10.1. Analytical Methods Trazodone and its main active metabolite 3-(1-clorophenyl) piperazine (mCPP) were determined in human plasma 12 h after the last drug administration. Trazodone is a second-generation antidepressant with serotonin antagonist activity. The metabolite is considered to be involved in some side effects of trazodone therapy, and for this reason its determination is very important during therapeutic drug monitoring. Analysis was performed with a Jones Chromatography Genesis C8 column (150mm × 4.6mm, 5 μm) operated at room temperature using a mobile phase composed of aqueous phosphate buffer (70%), containing triethylamine, at pH 3.5 and acetonitrile (30%). UV detection at 255 nm was applied. Loxapine was used as the internal standard. C8 reversed phase cartridges IST (Mid Glamorgan, UK) Isolute (50 mg/1 mL) were applied for sample preparation with extraction yields values better than 90%. The method was successfully applied to plasma samples from depressed patients undergoing therapy with trazodone; accuracy results were satisfactory (recovery >91%). The method is very selective since no endogenous compounds or any of the tested Central Nervous System drugs caused any interference in the analysis of TRZ and m-CPP in depressed patients‘ plasma. The proposed method has high accuracy and a wide response function range, which allows the determination of the analytes not only at therapeutic doses of TRZ, but also in overdose cases and whentakenatsub-therapeuticdoses.[7] A rapid, convenient and selective method using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC– ESI/MS) has been developed and validated to determine mianserin in human plasma. Mianserin and the internal standard (I.S.), cinnarizine were extracted from plasma by N-hexane:dimethylcarbinol (98:2, v/v) after addition of sodium hydroxide. LC separation was performed on a Thermo Hypersil-Hypurity C18 (5 μm, 150mm × 2.1mm) with the mobile phase consisting of 10 mM ammonium acetate (pH 3.4)–methanol–acetonitrile (35:50:15, v/v/v) at 0.22 mL/min. The retention time of mianserin and cinnarizine was 3.4 and 2.1 min, respectively. Quadrupole MS detection and quantitation was performed by monitoring at m/z 265 [M+H]+ for mianserin and m/z 369 [M+H]+ for cinnarizine (IS). The method was validated over the concentration ranges of 1.0–200.0 ng/mL for mianserin. The recovery was 81.3–84.1%. Limit of quantitation was 1.0 ng/mL for mianserin. This method proved to be suitable for the bioequivalence study of mianserin hydrochloride tablets. [8] As it has already been obvious antidepressants are widely used for the treatment of psychiatric disorders and therefore their monitoring in biological

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fluids is quite important taking into account that they can produce dangerous biochemical imbalances in toxic doses. A method for the determination of antidepressants: Trazodone, fluoxetine, Mianserine, Desipramine, Imipramine, Nortryptiline, Amitryptiline Trimipramine and Clomipramine in urine samples is presented using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Home-made cartridges containing 30 mg multiwall carbon nanotubes are employed for isolation of the analytes from the sample, allowing also the preconcentration of the analytes prior to the HPLC analysis. Chromatographic separation was carried out on an Eclipse X-DB-C8 column (4.6 × 150 mm) using as mobile phase sodium dihydrogen phosphate buffer (pH 3.0)–acetonitrile– methanol 70:25:5 (v/v). The ionic liquid 1-butyl-3-methylimidazolium trifluoromethanesulfonate was added at a concentration of 20 mmol/L in order to suppress the effect of the silanol groups on the chromatographic separation. The flow rate of the isocratic separation was 1.2 mL/min and the injection volume was 20 μL. Analytes were detected at 254 nm. Limits of detection were 12.3 ng/mL for trazodone and 90.1 ng/mL for fluoxetine. The method, validated for sensitivity and precision, has been successfully applied to the determination of antidepressants in urine samples from hospital patients after a course of treatment with antidepressants and it proved suitable for the therapeutic monitoring of antidepressants in urine samples. [9] The applicability of hollow fiber-based liquid phase microextraction (HFLPME) was evaluated for the extraction and preconcentration of three antidepressant drugs (amitriptyline, imipramine and sertraline) prior to their determination by HPLC-UV in urine and plasma. The extraction was performed due to pH gradient between the inside and outside of the hollow fiber membrane. In order to obtain high extraction efficiency, the parameters affecting the HFLPME were studied and optimized. All the extractions were carried out using an Accurel Q3/2 polypropylene hollow fiber membrane (Wuppertal, Germany) with a 0.2 μm pore size, 600 μm internal diameter and 200 μm wall thickness. Separations were carried out on a Zorbax Extend C18 column (100 mm × 2.1 mm, with 3.5 μL particle size). A mixture of 0.02 M acetic acid solution (pH 4.0) and methanol (54:46) at a flow rate of 0.25 mL/min was used as a mobile phase in isocratic elution mode. Linearity was observed in the range 5–500 μg/L. UV detection was applied at 215 nm. Chloropromazine was used as internal standard. The limits of detection (LODs) ranged between 0.5 and 0.7 μg/L (based on S/N = 3). Finally, the applicability of the proposed method was evaluated by extraction and determination of the drugs in urine, plasma and tap water samples. The results indicated that hollow fiber microextraction method has excellent clean-up and

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high-preconcentration factor and can be served as a simple and sensitive method for monitoring of antidepressant drugs in the biological samples. [10] Α simple, rapid and sensitive HPLC method was developed and validated for the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin, clomipramine (CLO) and imipramine, in pharmaceutical formulations and biological fluids. A Kromasil C8 analytical column (250 × 4 mm, 5 μm) was used for the separation within 6 min, using a mobile phase consisting of 0.05 M CH3COONH4 and CH3CN (45:55 v/v) delivered at 1.5 mL/min isocratically. Quantification was performed at 238 nm, with bromazepam (1.5 ng/μL) as internal standard. Linearity was observed within the range: 1–5 μg/mL The determination of TCAs in blood plasma was performed after protein precipitation with ACN. Urine analysis was performed by means of SPE using Lichrolut RP-18 Merck cartridges providing high absolute recoveries (higher than 94%). Direct analysis of urine was also performed after two-fold dilution. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5) revealed RSD lower than 13%. Recoveries from biological samples ranged from 91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1– 0.6 ng in blood plasma and 0.2–0.5 ng in extracted urine or 0.4–0.7 in diluted urine. The method was applied to real samples of plasma from a patient under CLO treatment. The method is suitable for the pharmacokinetics and bioavailability studies of TCAs. It is also a useful tool in human medicine for estimating and personalising the effective drug dose in patients. [11] Duloxetine is the most recent serotonin and norepinephrine reuptake inhibitor (SNRI) drug introduced for the therapy of depression. Thus, it is evident that there is a need for having on hand new reliable analytical methods for the determination of duloxetine plasma levels in depressed patients. A method dealing with the development of a rapid and sensitive high-performance liquid chromatographic method for duloxetine analysis in human plasma has been presented. The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 60% aqueous phosphate buffer containing triethylamine at pH 3.0 and 40% acetonitrile. The UV detector was set at 230 nm and loxapine was used as the internal standard. Pre-treatment of plasma samples was developed, based on solidphase extraction (SPE) Waters (Milford, MA, USA) Oasis mixed-mode reversed phase—strong cation exchange (MCX) cartridges (30 mg/1 mL). The extraction yields values were higher than 90%. Linearity was found in the 2–200 ng/mL range; the limit of quantitation was 2.0 ng/mL. The method was applied to plasma samples from depressed patients undergoing therapy with duloxetine. Precision

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data and accuracy results were satisfactory and no interference from other drugs was found. The HPLC method presented here for the analysis of DLX is feasible and rapid: a chromatographic run lasts less than five minutes. The SPE procedure implemented for the sample pre-treatment, based on MCX cartridges, gives good extraction yields (>90%) and satisfactory precision (RSD% < 5.1%). Thus, the method seems to be suitable for the therapeutic drug monitoring of duloxetine in depressed patients‘ plasma. [12] A high-performance liquid chromatography method is presented for the determination of 10 frequently prescribed tricyclic and nontricyclic antidepressants: imipramine, amitriptyline, clomipramine, fluoxetine, sertraline, paroxetine, citalopram, mirtazapine, moclobemide and duloxetine in human plasma. The simple and accurate sample preparation step, consisted of liquid:liquid extraction with recoveries ranging between 72% and 86%, except for moclobemide (59%). The extraction consisted of the addition of 25 μL of etidocaine (IS), 200 mg NaCl, 50μL of sodium hydroxide 1.5 M, and 5 mL hexane-isoamyl alcohol (99:1, v/v) to 1mL of plasma. Chromatographic separation was achieved isocratically, at room temperature, on a LiChrospher 60 RP-select B column (250 mm × 4 mm, 5 μm). The mobile phase consisted of 35% a mixture of acetonitrile: methanol (92:8, v/v) and 65% of sodium acetate buffer, pH 4.5 delivered at a flow-rate of 1.0mL/min under isocratic conditions with UV detection at 230 nm. Linearity was observed over a working range of 2.5–1000 ng/mL for moclobemide, 5–2000 ng/mL for citalopram, duloxetine, fluoxetine, 10–2000 ng/mL for sertraline, imipramine, paroxetine, mirtazapine and clomipramine. The intra-assay coefficients of variation (CVs) studied at three concentrations (50, 200, and 500 ng/mL) for all compounds were less than 8.8%, and all inter-CVs were less than 10%. Limits of quantification were 2.5 ng/mL for moclobemide, 5 ng/mL for citalopram, duloxetine and amitriptyline, and 10 ng/mL for mirtazapine, paroxetine, imipramine, fluoxetine, sertraline, and clomipramine. No interference of the drugs normally associated with antidepressants was observed. The method has been successfully applied to the analysis of real samples, for monitoring of ten frequently prescribed tricyclic and non-tricyclic antidepressant drugs, so it can be readily applied to the routine therapeutic drug monitoring. [13] An HPLC method with UV detection has been developed for the simultaneous determination of levomepromazine, clozapine and their main metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, Odesmethyl-levomepromazine, N-desmethylclozapine and clozapine

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N-oxide in human plasma. The analytes were separated on a C8 (150 mm × 4.6 mm, 5 μm; Phenomenex) column supplemented with a C8 cartridge precolumn, using a mobile phase composed of acetonitrile and a pH2.0, 34 mM phosphate buffer containing 0.3% triethylamine (29:71, v/v). UV detection was performed at 254 nm. Loxapine was used as the internal standard. Baseline separation of the 7 analytes (and the IS) was achieved in less than 20 min. A reliable biological sample pre-treatment procedure by means of solid-phase extraction on BondElut C1 cartridges was implemented, which allows to obtain good extraction yields (>91%) for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy. Linearity was observed in the ranges: LMP 9–200 ng/mL; NDLM 10–150 ng/mL; LMSO 5–500 ng/mL; ODLM 7–150 ng/mL; CLZ 20–2500 ng/mL; DMC 15–1000 ng/mL; NOX 10–200 ng/mL. These assays gave RSD% values for precision always lower than 4.9% and mean accuracy values higher than 92%. The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine. [14] A solid phase extraction procedure (SPE) for 13 ‗new‘ antidepressants (venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine, melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and trazodone) together with eight of their metabolites (O desmethylvenlafaxine, norfluoxetine, desmethylmianserine, desmethylmirtazapine, desmethylcitalopram, didesmethylcitalopram, desmethylsertraline and m-chlorophenylpiperazine) from plasma was optimized using HPLC-DAD at 210-380 nm as monitoring system. A total number of 10 sorbents (apolar, polymeric, ion-exchange and mixed mode combining ion-exchange properties with, respectively, C8 or a styrene– divinylbenzene polymer) was evaluated. Based on recovery, reproducibility and absence of interfering substances the strong cation exchanger gave the best results. Recoveries were determined at low and high therapeutic and toxic levels and ranged between 70 and 109% for all compounds, except for trazodone (39%). [15] Amitriptyline and nortriptyline are tricyclic antidepressants which act by enhancing the actions of norepinephrine and serotonin caused by blocking the reuptake of various neurotransmitters at the neuronal membrane. A micellar liquid chromatographic procedure was developed to determine these drugs in serum samples for use in clinical monitoring. The chromatographic determination was carried out using a 0.15 M SDS-6% (v/v) pentanol buffered at pH 7, in a Kromasil 5 C18 column, 5 μm, 250 mm × 4.6 mm from Scharlab (Barcelona, Spain). The mobile phase was 0.15 M SDS-6% (v/v) pentanol at pH 7 delivered at a flow-rate

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of 1.5 mL/min. Detection was performed at 240 nm and 650 mV. The analysis time was 14 min. The limits of detection (ng/mL) in serum were 0.25 and 0.31 for amitriptyline and nortriptyline, respectively. Repeatability and intermediate precision were evaluated at three different concentrations in serum samples. Untreated serum samples were injected directly into the HPLC system after filtration, leading to be a simple procedure that can be applied in routine analyses for Therapeutic Drug Monitoring. No interference was observed from endogenous compounds and other drugs. Recoveries obtained were from 95 to 102%. The applicability of the procedure developed to determine amitriptyline and nortriptyline was verified by analysing them in spiked and real serum samples. [16] A simple reversed-phase high-performance liquid chromatographic method employing C18 column has been developed for simultaneous analysis of three intermediates in the synthesis of S-duloxetine, the antidepressant drug, viz., 2acetyl thiophene (AT), N,N-dimethyl-3-keto- (2-thienyl)-propanamine (DKTP) and (S)-N,N-dimethyl-3-hydroxy-(2-thienyl)-propanamine (DHTP). Good separations were achieved on a 250 mm × 4.6 mm, 5 μM particle LichroCART RP-18 column (Merck, Germany) by employing an isocratic system using acetonitrile and 0.05M phosphate buffer (pH 7.0) containing 0.02% diethylamine. The detection was carried out at 241 nm. High recovery rates were obtained 99.7101.5%. The method was validated in terms of linearity, range, accuracy and precision. Linearity was demonstrated within the range: AT 5–500 (μg/mL), DKTP & DHTP 20–500 (μg/mL). The developed HPLC method is simple, accurate and reproducible and can be used as a routine analytical tool during the synthesis of S-duloxetine. [17] The four most commonly prescribed non-tricyclic antidepressants: Fluoxetine, citalopram, paroxetine and venlafaxine were simultaneously determined by high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–MS/ESI) in human plasma of patients undergoing antidepressant treatment. The analytes in plasma were extracted by solid-phase-extraction column on (Waters Oasis) after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C18 (250mm × 4.6 mm, 5 μm, Germany) column, using water (formic acid 0.6‰, ammonium acetate: 30 mmol/L)–acetonitrile (35:65, v/v) as mobile phase, with a flow-rate of 0.85 mL/min. Fluvoxamine was used as internal standard. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode.

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The calibration curves were linear in the 5.0–1000.0 ng/mL range for all compounds. The average extraction recoveries for all the four analytes were above 73.2%. The limits of detection (LODs) were 0.5, 0.3, 0.3 and 0.1 ng/mL for fluoxetine, citalopram, paroxetine and venlafaxine, respectively. The intra- and inter-day variation coefficients were less than 15.0%. The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) as well as toxicologic screening, and for the study of the pharmacokinetics and metabolism of the four drugs. [18] An isocratic reversed-phase HPLC method with ultraviolet detection was developed and optimised to quantify eighteen antidepressants and four atypical antipsychotics commonly prescribed psychotropic drugs including seven metabolites. Among these 29 compounds are new psychotropic drugs for which so far only single-component assays are described in the literature: mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OHrisperidone in human serum. After solid-phase extraction on 3M-Empore highperformance extraction disk cartridges (Varian, Darmstadt, Germany) and a Baker spe-12G vacuum instrument of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-5-Protect 1 (endcapped) column with acetonitrile–potassium dihydrogenphosphate buffer as mobile phase. Melperone (3000 ng/mL) was used as internal standard. UV detector was used at 230 nm. The method was validated for therapeutic and toxic serum ranges. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9–10.2% and 0.9–9.7%, respectively. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics, within 24 h with a single system, thus reducing the time for apparatus preparation and system in stabilities linked to this process. [19] A reversed-phase high performance liquid chromatography (HPLC) method for the determination of plasma and serum levels of amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI) is described, based upon single step liquid:liquid extraction with hexane of these compounds at pH 11 (recovery between 92 and 105%). A a Nova-Pack C-18 HPLC cartridge column was used for separation while the mobile phase composed of a phosphate buffer with 50%

Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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100

Victoria Samanidou and Eftichia Karageorgou

(v:v) acetonitrile and about 0.2% (v:v) diethylamine (final pH: 8) and solute detection at 242 nm. Using 1 mL of plasma or serum and econazole as internal standard, drug levels between 20 and 400 ng/mL were found to provide linear calibration graphs. For drug concentrations in the range of 70–120 ng/mL, intraday and interday imprecisions (n=5) were determined to be