Ambient mass spectroscopy techniques in food and environment 9781138505568, 1138505560

Table of contents :
Content: Section I: Ambient mass spectroscopy techniques and principles. 1. Introduction to Ambient mass spectrometry techniques / Raquel Sero, Maria Teresa Galceran, and Encarnacion Moyano --
Section II: Spray or jet ionization. 2. Desorption electrospray ionization / Alexandros G. Asimakopoulos and Susana V. Gonzalez
3. Desorption atmospheric pressure photoionization / Tiina Kauppila
4. Easy ambient sonic-spray ionization mass spectrometry in food analysis and foodomics / Rosana M. Alberici, Gab riel D. Fernandes, and Daniel Barrera-Arellano --
Section III: Electric discharge ambient ionization. 5. Direct analysis in real time mass spectrometry / Semih Otles and Vasfiye Hazal Ozyurt
6. Low-temperature plasma ionization / Sandra Marínez-Jarquín and Robert Winkler
7. Plasma-assisted desorption ionizaton / Kirsty McKay --
Section IV: Ambient gas, heat, or laser-assisted desorption/ionization. 8. Extractive electrospray ionization / Sheetal Mital
9. Electrospray laser desorption ionization / Leo M. L. Nollet
10. Sorptive tape-like extraction coupled with laser desorption ionization / Leo M. L. Nollet --
Section V: Other techniques. 11. Rapid evaporative ionization mass spectrometry / Leo M. L. Nollet
12. Paper spray mass spectrometry and related techniques applied to food and environmental analysis / Ildefonso Binatti, Hebert Vinicius Pereira, Victoria Silva Amador, Marina Jurisch, Camila Cristina Almeida de Paula, Evandro Piccin, and Rodinei Augusti.

Citation preview

Ambient Mass Spectroscopy Techniques in Food and the Environment

Food Analysis and Properties Leo M.L. Nollet University College Ghent, Belgium This CRC series Food Analysis and Properties is designed to provide a state-of-art coverage on topics to the understanding of physical, chemical, and functional properties of foods including (1) recent analysis techniques of a choice of food components, (2) developments and evolutions in analysis techniques related to food, and (3) recent trends in a­ nalysis techniques of specific food components and/or a group of related food components.

Flow Injection Analysis of Food Additives Edited by Claudia Ruiz-Capillas and Leo M.L. Nollet

Marine Microorganisms: Extraction and Analysis of Bioactive Compounds Edited by Leo M.L. Nollet

Multiresidue Methods for the Analysis of Pesticide Residues in Food Edited by Horacio Heinzen, Leo M.L. Nollet, and Amadeo R. Fernandez-Alba

Spectroscopic Methods in Food Analysis Edited by Adriana S. Franca and Leo M.L. Nollet

Phenolic Compounds in Food: Characterization and Analysis Edited by Leo M.L. Nollet and Janet Alejandra Gutierrez-Uribe

Testing and Analysis of GMO-containing Foods and Feed Edited by Salah E.O. Mahgoub and Leo M.L. Nollet

Fingerprinting Techniques in Food Authenticity and Traceability Edited by K.S. Siddiqi and Leo M.L. Nollet

Hyperspectral Imaging Analysis and Applications for Food Quality Edited by Nrusingha Charan Basantia, Leo M.L. Nollet, and Mohammed Kamruzzaman

Ambient Mass Spectroscopy Techniques in Food and the Environment Edited by Leo M.L. Nollet and Basil K. Munjanja For more information, please visit the Series Page: https://www.crcpress.com/ Food-Analysis--Properties/book-series/CRCFOODANPRO

Ambient Mass Spectroscopy Techniques in Food and the Environment

Edited by

Leo M.L. Nollet Basil K. Munjanja

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2019 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-138-50556-8 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents Series Preface vii Preface ix Acknowledgments

xi

Editors xiii Contributors xv

Section I  AMBIENT MASS SPECTROSCOPY TECHNIQUES AND PRINCIPLES Chapter 1

Introduction to Ambient Mass Spectrometry Techniques 3 Raquel Sero, Maria Teresa Galceran, and Encarnacion Moyano

Section II  SPRAY OR JET IONIZATION Chapter 2

Desorption Electrospray Ionization 39 Alexandros G. Asimakopoulos and Susana V. Gonzalez

Chapter 3

Desorption Atmospheric Pressure Photoionization 57 Tiina Kauppila

Chapter 4

Easy Ambient Sonic-Spray Ionization Mass Spectrometry in Food Analysis and Foodomics 75 Rosana M. Alberici, Gabriel D. Fernandes, and Daniel Barrera-Arellano

Section III  ELECTRIC DISCHARGE AMBIENT IONIZATION Chapter 5

Direct Analysis in Real Time Mass Spectrometry 95 Semih Otles and Vasfiye Hazal Ozyurt

Chapter 6

Low-Temperature Plasma Ionization 105 Sandra Martínez-Jarquín and Robert Winkler

Chapter 7

Plasma-Assisted Desorption Ionization 125 Kirsty McKay

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Contents

Section IV  AMBIENT GAS, HEAT, OR LASERASSISTED DESORPTION/IONIZATION Chapter 8

Extractive Electrospray Ionization 145 Sheetal Mital

Chapter 9

Electrospray Laser Desorption Ionization 171 Leo M.L. Nollet

Chapter 10

Sorptive Tape-Like Extraction Coupled with Laser Desorption Ionization 175 Leo M.L. Nollet

Section V OTHER TECHNIQUES Chapter 11

Rapid Evaporative Ionization Mass Spectrometry 181 Leo M.L. Nollet

Chapter 12

Paper Spray Mass Spectrometry and Related Techniques Applied to Food and Environmental Analysis 187 Ildefonso Binatti, Hebert Vinicius Pereira, Victoria Silva Amador, Marina Jurisch, Camila Cristina Almeida de Paula, Evandro Piccin, and Rodinei Augusti

Index 221

Series Preface There will always be a need for analyzing methods of food compounds and properties. Current trends in analyzing methods include automation, increasing the speed of analyses, and miniaturization. The unit of detection has evolved over the years from micrograms to pictograms. A classical pathway of analysis is sampling, sample preparation, cleanup, derivatization, separation, and detection. At every step, researchers are working and developing new methodologies. A large number of papers are published every year on all facets of analysis. So, there is a need for books that gather information on one kind of analysis technique or on analysis methods of a specific group of food components. The scope of the CRC series on Food Analysis and Properties aims to present a range of books edited by distinguished scientists and researchers who have significant experience in scientific pursuits and critical analysis. This series is designed to provide state-of the-art coverage on topics such as 1. Recent analysis techniques on a range of food components 2. Developments and evolution in analysis techniques related to food 3. Recent trends in analysis techniques of specific food components and/or a group of related food components 4. The understanding of physical, chemical, and functional properties of foods. The book Ambient Mass Spectroscopy Techniques in Food and the Environment is the ninth volume of this series. I am happy to be a series editor of these books for the following reasons: • I am able to pass on my experience in editing high-quality books related to food. • I get to know colleagues from all over the world more personally. • I continue to learn about interesting developments in food analysis. I dedicate this series to • My wife, for her patience with me (and all the time I spend on my computer) • All patients suffering from prostate cancer; knowing what this means, I am hoping they will have some relief

vii

Preface Mass spectrometry has been used as a great tool in food and environmental analysis for a very long time. However, a disadvantage of the technique is that the run times may be long, and complex sample preparation may be required. However, a significant breakthrough in this technique was the shift from the ionization process from the vacuum to the atmospheric environment. This forms the basis of ambient mass spectrometry. Since then, ambient mass spectrometry has been widely used in food and environmental analysis. In addition, it provides results that are comparable to those that would have been obtained with other conventional techniques. To date, several publications have reported on the application of the techniques in food safety, authenticity, and traceability screening. In addition, mass spectrometry has also been utilized in the monitoring of environmental pollutants such as pharmaceuticals, pesticides, and drugs of abuse. Thus, in-depth discussion of the applications of the various techniques, advances in instrumentation, and future perspectives in this field are needed. For this reason, this book, Ambient Mass Spectrometry Techniques in Food and the Environment aims to furnish readers with the theoretics of mass spectrometry, benefits and pitfalls of ambient mass spectrometry in the analysis of food and environmental parameters, and the latest developments of the techniques in the analysis of food and environmental parameters. The specialized view presented will give an insight to starters, as well as professionals practicing in food and the environment.

ix

Acknowledgments A lot of work is involved in the preparation of a book. I have been assisted and supported by a number of people, all of whom I would like to thank. I would especially like to thank the team at CRC Press/Taylor & Francis, with a special word of thanks to Steve Zollo, Senior Editor. Many, many thanks to all the editors and authors of this volume and future volumes. I very much appreciate all their effort, time, and willingness to do a great job.

xi

Editors Leo M.L. Nollet, PhD, earned an MS (1973) and PhD (1978) in biology from the Katholieke Universiteit Leuven, Belgium. He is an editor and associate editor of numerous books. He edited for M. Dekker, New York—now CRC Press of Taylor & Francis Publishing Group—the first, second, and third editions of Food Analysis by HPLC and Handbook of Food Analysis. The last edition is a two-volume book. Dr. Nollet also edited the Handbook of Water Analysis (first, second, and third editions) and Chromatographic Analysis of the Environment (third and fourth editions; CRC Press). With F. Toldrá, he coedited two books published in 2006, 2007, and 2017: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing—now Wiley). With M. Poschl, he coedited the book Radionuclide Concentrations in Food and the Environment, also published in 2006 (CRC Press). Dr. Nollet has also coedited with Y. H. Hui and other colleagues on several books: Handbook of Food Product Manufacturing (Wiley, 2007), Handbook of Food Science, Technology, and Engineering (CRC Press, 2005), Food Biochemistry and Food Processing (first and second editions; Blackwell Publishing—now Wiley—2006 and 2012), and the Handbook of Fruits and Vegetable Flavors (Wiley, 2010). In addition, he edited the Handbook of Meat, Poultry, and Seafood Quality (first and second editions; Blackwell Publishing—now Wiley—2007 and 2012). From 2008 to 2011, he published five volumes on animal product-related books along with F. Toldrá: Handbook of Muscle Foods Analysis, Handbook of Processed Meats and Poultry Analysis, Handbook of Seafood and Seafood Products Analysis, Handbook of Dairy Foods Analysis, and Handbook of Analysis of Edible Animal By-Products. Also in 2011, with F. Toldrá, he coedited two volumes for CRC Press: Safety Analysis of Foods of Animal Origin and Sensory Analysis of Foods of Animal Origin. In 2012, F. Toldrá and L.M.L. Nollet published the Handbook of Analysis of Active Compounds in Functional Foods. In a coedition with Hamir Rathore, Handbook of Pesticides: Methods of Pesticides Residues Analysis was marketed in 2009; Pesticides: Evaluation of Environmental Pollution in 2012; Biopesticides Handbook in 2015; and Green Pesticides Handbook: Essential Oils for Pest Control in 2017. Other finished book projects include Food Allergens: Analysis, Instrumentation, and Methods (with A. van Hengel; CRC Press, 2011) and Analysis of Endocrine Compounds in Food (Wiley-Blackwell, 2011). Dr. Nollet’s recent projects include Proteomics in Foods with F. Toldrá (Springer, 2013)

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Editors

and Transformation Products of Emerging Contaminants in the Environment: Analysis, Processes, Occurrence, Effects, and Risks with D. Lambropoulou (Wiley, 2014). In the series, Food Analysis and Properties, he edited (with C. Ruiz-Capillas) Flow Injection Analysis of Food Additives (CRC Press, 2015) and Marine Microorganisms: Extraction and Analysis of Bioactive Compounds (CRC Press, 2016). With A.S. Franca, he edited Spectroscopic Methods in Food Analysis (CRC Press, 2017), and with Horacio Heinzen and Amadeo R. Fernandez-Alba, he edited Multiresidue Methods for the Analysis of Pesticide Residues in Food (CRC Press, 2017). Basil K. Munjanja  received his BSc (Hons) in applied chemistry (2013) from the National University of Science and Technology, Bulawayo, Zimbabwe. Since 2013, he has authored 13 book chapters on various topics such as biopesticides, spectroscopy, and mass spectrometry for CRC Press, Taylor & Francis, in books, namely, Handbook of Food Analysis (3rd edition), Biopesticides Handbook, Flow Injection Analysis of Food Additives, and Chromatographic Analysis of the Environment: Mass Spectrometry Based Approaches (4th edition). All these books were under the editorship of Dr. Leo M.L. Nollet. He is an associate member of the South African Chemical Institute (SACI) and the Society of Environmental Toxicology and Chemistry (SETAC) Africa.

Contributors Rosana M. Alberici Fats and Oils Laboratory, Faculty of Food Engineering UNICAMP São Paulo, Brazil

Gabriel D. Fernandes Fats and Oils Laboratory, Faculty of Food Engineering UNICAMP São Paulo, Brazil

Camila Cristina Almeida de Paula Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil

Maria Teresa Galceran Department of Chemical Engineering and Analytical Chemistry University of Barcelona Barcelona, Spain

Victoria Silva Amador Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil Alexandros G. Asimakopoulos Department of Chemistry Norwegian University of Science and Technology Trondheim, Norway Rodinei Augusti Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil Daniel Barrera-Arellano Fats and Oils Laboratory, Faculty of Food Engineering UNICAMP São Paulo, Brazil Ildefonso Binatti Centro Federal de Educação Tecnológica de Minas Gerais Belo Horizonte, Brazil

Susana V. Gonzalez Department of Chemistry Norwegian University of Science and Technology Trondheim, Norway Marina Jurisch Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil Tiina Kauppila Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, Drug Research Program University of Helsinki Helsinki, Finland Sandra Martínez-Jarquín CINVESTAV Unidad Irapuato Irapuato, Mexico

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Contributors

Kirsty McKay Department of Electrical Engineering and Electronics University of Liverpool Liverpool, United Kingdom

Vasfiye Hazal Ozyurt Faculty of Engineering Department of Food Engineering Near East University Nicosia, Turkey

Sheetal Mital Applied Sciences Krishna Institute of Engineering & Technology Ghaziabad, Uttar Pradesh, India

Hebert Vinicius Pereira Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil

Encarnacion Moyano Department of Chemical Engineering and Analytical Chemistry University of Barcelona Barcelona, Spain Leo M.L. Nollet University College Ghent Ghent, Belgium Semih Otles Department of Food Engineering Ege University Izmir, Turkey

Evandro Piccin Departamento de Química Universidade Federal de Minas Gerais Belo Horizonte, Brazil Raquel Sero Department of Chemical Engineering and Analytical Chemistry University of Barcelona Barcelona, Spain Robert Winkler CINVESTAV Unidad Irapuato Irapuato, Mexico

Section

I

Ambient Mass Spectroscopy Techniques and Principles

Chapter

1

Introduction to Ambient Mass Spectrometry Techniques Raquel Sero, Maria Teresa Galceran, and Encarnacion Moyano University of Barcelona

CONTENTS Introduction 3 Ambient MS Techniques 8 One-Step Ambient MS Techniques 9 Solid–Liquid Extraction 9 Thermal and Chemical Desorption 11 Two-Step Ambient MS Techniques 13 Liquid–Solid Extraction 13 Laser Desorption/Ablation 14 Three-Step Ambient MS Techniques 14 Other Ambient MS Techniques 15 Rapid Evaporative Ionization Mass Spectrometry 15 Physical Designs and Experimental Approaches 15 Probe Assembly Configurations 15 Solvents, Gases, and Sample Devices 18 Ambient MS Imaging 20 Sample Handling 21 Applicability and Method Performance 23 Concluding Remarks 25 Acknowledgments 26 References 26

INTRODUCTION Cooks and coworkers introduced the term “ambient ionization” in mass spectrometry (MS) in 2004 (Takáts et al. 2004). This term grouped those emerging desorption/­ ionization techniques that operated at atmospheric pressure and were able to subject an object for its immediate mass spectral analysis by handling it in front of the interface without the need of sample preparation or sample pretreatment. However, nowadays, it would be more accurate to say that these methods frequently do not require other sample preparation than the sample processing that takes place during the analysis (Venter et al.

3

4

Mass Spectroscopy Techniques in Food and the Environment

2014; Monge et al. 2013). To classify an ionization source in this new group of ambient MS techniques, it should meet the following requirements: • The ability to perform ionization of compounds in the open air on objects of unusual shape or size, not typically amenable to direct MS analysis. • The capability to perform direct surface analysis, avoiding time-consuming sample preparation steps typically required in MS-based chemical analysis of solid samples. • The easiness to be swappable in any mass spectrometer equipped with an atmospheric pressure interface. • The capacity to generate ions without significant in-source fragmentation, which would simplify the mass spectral data interpretation and the compound identification when analyzing complex samples. The two first introduced ambient mass spectrometry (ambient MS) techniques were desorption electrospray ionization (DESI) (Takáts et al. 2004) and direct analysis in real time (DART) (Cody, Laramee, and Durst 2005), which boosted the development of a growing number of new ambient MS methods and a large number of acronyms. Table 1.1 summarizes the ambient MS techniques mostly used in food and environmental analysis. A number of excellent books and book chapters (Domin and Cody 2015; Gross 2017; Moyano and Galceran 2015; Kauppila and Vaikkinen 2014; Sero, Nunez, and Moyano  2016), review articles (Ding and Duan 2015; Monge et al. 2013; Espy et al. 2014; Cooks and Mueller 2013; Badu-Tawiah et al. 2013; Cody 2013; Javanshad and Venter 2017; Snyder et al. 2016; Klampfl and Himmelsbach 2015; Kauppila and Kostiainen 2017; Venter et al. 2014), and tutorials (Huang et al. 2011; Bodzon-Kulakowska et al. 2014), which have already been published under the topic of “ambient ionization,” can be recommended for a further in-depth study of ambient MS technique fundamentals and state of the art. The ambient MS techniques developed until now combine different desorption methods with different ionization techniques, thus making their classification very challenging. Some authors have classified ambient MS techniques based only on the ionization mechanism (spray or jet ionization, electric discharge ionization, and gas-, heat- or laser-assisted desorption/ionization), although more thorough classifications have been made based on the intrinsic extraction/desorption/ionization mechanisms involved in these techniques (Black, Chevallier, and Elliott 2016). However, these divisions are sometimes debatable, especially when several of these mechanisms concur in the same technique. Ambient MS techniques can offer advantageous characteristics to analytical laboratories working in the field of food and environmental analysis (Kauppila and Vaikkinen 2014; Chen et al. 2017; Nielen et al. 2011; Black, Chevallier, and Elliott 2016; Luo et al. 2017; Porcari et al. 2016). For instance, real-time and in situ analysis, low sample requirements with little sample invasion, fast and high-throughput analysis, minimal or no sample prior preparation, small or no use of organic solvents, and relatively low matrix effects are some of the ambient MS characteristics that can be attractive for food and environmental applications. These features allow facing some requirements such as workload, turnaround time, and cost per sample frequently demanded by modern analytical laboratories. Table 1.2 summarizes some of the most recent ambient MS applications in food and environmental analysis. As can be seen, most of these applications used DESI and DART, probably because they were the first techniques developed, and they have been more widely studied than others introduced later. Furthermore, the availability of commercial devices for both DESI and DART, which can be coupled to many mass

Ambient Mass Spectrometry Techniques

5

TABLE 1.1  Acronyms and Primary References for Ambient MS Techniques Used in Environmental and Food Analysis Abbreviation DAPCI

Name

DART

Desorption atmospheric pressure chemical ionization Desorption atmospheric pressure photoionization Direct analysis in real time

DBDI DESI DICE EASI

Dielectric barrier discharge ionization Desorption electrospray ionization Desorption ionization by charge exchange Easy ambient sonic-spray ionization

EESI ELDI

Extractive electrospray Electrospray-assisted laser desorption ionization Flowing atmospheric pressure afterglow Laser ablation electrospray ionization Leaf spray Liquid extraction surface analysis Liquid microjunction surface sampling probe Low-temperature plasma

DAPPI

FAPA LAESI Leaf spray LESA LMJ-SSP LTP MALDESI Nano-DESI

Matrix-assisted laser desorption electrospray ionization Nano-desorption electrospray ionization

ND-EESI

Neutral desorption extractive electrospray

PADI PESI PS REIMS

Plasma-assisted desorption ionization Probe electrospray ionization Paper spray Rapid evaporative ionization mass spectrometry

First Reference Song and Cooks (2006) Haapala et al. (2007) Cody, Laramee, and Durst (2005) Na, Zhao, et al. (2007) Takáts et al. (2004) Chan et al. (2010) Haddad, Sparrapan, and Eberlin (2006) Chen, Venter, and Cooks (2006) Shiea et al. (2005) Andrade et al. (2008) Nemes and Vertes (2007) Liu et al. (2011) Kertesz and Van Berkel (2010) Berkel, Sanchez, and Quirke (2002) Cotte-Rodríguez and Cooks (2006) Sampson, Hawkridge, and Muddiman (2006) Roach, Laskin, and Laskin (2010b) Chen, Wortmann, and Zenobi (2007) Ratcliffe et al. (2007) Hiraoka et al. (2008) Wang et al. (2010) Schäfer et al. (2009)

Bold italics acronyms: Commercially available Ambient MS techniques.

spectrometer brands, may also affect the increase of their use. The simplicity of ambient MS methods allows the easy modification of the already existing ones to adapt them to both the sample type and the analytical problem. For this reason, many prototypes, home-made new approaches, are continuingly being introduced, and most of them have been explored for their application to target analysis of organic contaminants in food and environmental samples, food fraud, food authentication, and screening of emerging pollutants, among others. Although most of the characteristic aspects of ambient MS techniques can be very attractive and “revolutionary,” one should be aware of intrinsic limitations. For instance, the detection of a compound largely depends on the ionization

Ambient MS Technique

Mass Analyzer

Sample

Scope of the Analysis

6

TABLE 1.2  Applications of Ambient MS Methods in Environmental and Food Analysis

References

Food Analysis IT, Q-LIT, QqQ, miniature MS

Fruits, vegetables, cereals, olive oil, fish, meat, wine, and coffee

Analysis of pesticides and lipids, food authentication, and food profiling

EASI PS

Q, Q-TOF, FT-ICR Q, IT, QqQ

Leaf spray LESA

Q-Orbitrap IT

Meat and vegetable oils Fruits, vegetables, coffee, meat, milk, olive oil, and sport drinks Plants Meat

DART

QqQ, TOF, Orbitrap, QTOF, LIT-Orbitrap

Fruits, vegetables, cereals, sweets, fish, meat, and beverages

Analysis of lipids, authentication, and lipid profiling Analysis of pesticides, drugs, and foodstuffs contaminants; authentication; metabolomics; and food profiling Discrimination analysis Proteomics and peptidomic analysis Analysis of pesticides, xenobiotics, mycotoxins, phytohormones, melamine, and caffeine; authentication; metabolomics; screening; and characterization

DAPPI

QTOF, IT

Fruits

DAPCI

IT

Fruits

Analysis of pesticides and screening of contaminants Screening and discrimination analysis

Cajka, Riddellova, Zomer, et al. (2011), Gerbig and Takáts (2010), Garcia-Reyes, Jackson, et al. (2009), Schurek et al. (2008), Mainero-Rocca et al. (2017), Berchtold et al. (2013), Hartmanova et al. (2010), Joyce et al. (2013), Montowska et al. (2014), Zhou et al. (2008), B. Li et al. (2011) Porcari et al. (2016) Evard et al. (2015), Garrett, Rezende, and Ifa (2013), Guo et al. (2017), Zhang and Lee (2013)

Pereira et al. (2017) Montowska et al. (2015) Avula et al. (2015), Kalachova et al. (2011), Crawford and Musselman (2012), Farré, Picó, and Barceló (2013), Novotná et al. (2012), Hrbek et al. (2014), Yang et al. (2015), Danhelova et al. (2012), Lojza et al. (2012), Rajchl et al. (2013), Luo et al. (2017) Räsänen et al. (2014), Suni et al. (2012), Luosujärvi et al. (2010) Yang et al. (2009) (Continued)

Mass Spectroscopy Techniques in Food and the Environment

DESI

TABLE 1.2 (Continued )  Applications of Ambient MS Methods in Environmental and Food Analysis Ambient MS Technique

Mass Analyzer IT, miniature MS

EESI

Q-LIT, Q-TOF

REIMS ELDI

Q-TOF IT

Fruits, vegetables, wine, coffee, milk, and fish Fruits, vegetables, olive oil, beverages, and cheese Meat Fungus

Scope of the Analysis Analysis of pesticides, melamine, and volatile compounds, and authentication analysis Analysis of lead complexes, screening, and authentication

References Garcia-Reyes, Mazzoti, et al. (2009), Gerbig et al. (2017) Zhang et al. (2017), Bai et al. (2012), Sun et al. (2015)

Authentication/classification Determination of chemical surface composition

Verplanken et al. (2017) Huang et al. (2012)

Analysis of pesticides, phthalates, toxins, and PAHs

Ewing et al. (2015), Gerbig et al. (2015), Cain et al. (2014), Boone et al. (2015), Mattarozzi et al. (2016)

Analysis of amines, malachite green, crystal violet, and their metabolites Analysis of domoic acid

Jjunju et al. (2016), Fang et al. (2016)

Analysis of pesticides and PAHs

X. Wang et al. (2014), S. Zhou, Forbes, and Abbatt (2015) N. N. Wang et al. (2014) Mirabelli, Wolf, and Zenobi (2016)

Environmental Analysis DESI, nanoDESI

IT, Orbitrap, LIT-Orbitrap

PS

IT, QqQ, Orbitrap

LAESI

IT, Q-Orbitrap TOF

DART DAPCI DBDI EESI

IT IT, Q-LIT, LIT-Orbitrap IT

Consumer goods, clams, air, plants, soil, aerosols, and water particles Water

Mussels Lake water and aerosols Soil Spiked water Water

Analysis of plasticizers Analysis of pesticides and drugs

Beach et al. (2016)

Tian et al. (2014), Fang et al. (2016) 7

Analysis of malachite green and tetrabromobisphenol A

Ambient Mass Spectrometry Techniques

LTP

Sample

8

Mass Spectroscopy Techniques in Food and the Environment

mechanism as well as on the nature of the analyte to yield ions under the concrete ionization mechanism. Therefore, not a single ionization method, especially when is used under one set of conditions, can deliver ions of all constituents in a complex sample. However, all ionizable compounds on the sample surface will contribute to the generation of complex mass spectral data. On the other hand, ambient ionization is generally a soft ionization that yields ions with low internal energy, which suffer no or little fragmentation in the atmospheric pressure region. Mono-charged and multiple-charged ions, as well as adduct ions, can be expected from those ambient MS methods shearing electrospraylike ionization mechanisms, while plasma-based techniques only will generate monocharged ions and radical molecular ions. Under this scenario, the quality of the results would depend on the degree of information that the mass spectrometer can provide. Ambient MS techniques have been coupled to low-resolution mass analyzers (quadrupole and ion traps) mainly for the analysis of target compounds. However, the complexity of mass spectral data and the application of ambient MS techniques for the screening of complex samples require the use of MS instruments capable of acquiring data at highresolution and/or performing tandem MS experiments. High-resolution MS instruments are required to overcome interference problems, avoid overlapping isotope clusters, and provide high-quality mass spectral information to identify both the chemical formula and the chemical structure. Today, highly sensitive and selective instruments, such as linear ion trap (LIT), time of flight (TOF), Orbitrap and hybrid instruments such as triple quadrupole (QqQ), quadrupole-TOF (Q-TOF), quadrupole-Orbitrap or LIT-Orbitrap, have demonstrated to provide the necessary performance to design reliable methods with minimal sample preparation, thus facilitating high throughput analysis. This chapter aims to provide a general picture of ambient MS techniques used for the analysis of organic compounds in environmental and food samples. Classification and a brief description of the ambient MS methods used in these fields as well as the most important and critical features of the desorption/ionization techniques are included.

AMBIENT MS TECHNIQUES What really differentiates ambient MS from other direct MS techniques is that the sample processing takes place in real time and proximal to ionization. Among the ambient MS techniques, the difference relies on the way of how sample processing is combined with well-known ionization mechanisms. For instance, electrospray ionization (ESI), sonic-spray ionization (SSI), gas-phase ion–molecule reactions, and photochemical ionization have been coupled in real time with sample processing steps such as liquid extraction, thermal desorption, and laser ablation, among others. The classification of ambient MS techniques in subclasses is not easy and differs depending on the reviews published until now, with a certain degree of overlap. In addition, it is sometimes difficult to distinguish between a new ambient MS technique and the simple rebranding of an already reported ambient ionization source. Despite the difficulties to categorize the ambient MS ­techniques, the approximations of Venter et al. (2014) and Monge et al. (2013) seem to provide the most logical arrangement since both the sample processing and the desorption/ionization mechanisms are taken into account. Venter et al. (2014) classified ambient MS techniques based on the predominant sample processing method: liquid extraction (spray desorption, liquid junction, and substrate spray), thermal desorption, and laser ablation. On the other hand, the classification of Monge et al. (2013) is based on the intrinsic desorption/ionization mechanism of surface ambient MS techniques. They are classified into one-step and two-step

Ambient Mass Spectrometry Techniques

9

techniques: one-step technique where desorption works in parallel with the ionization and two-step technique where desorption/ablation is followed by a secondary independent ionization step. It must be mentioned that these authors do not include paper spray (PS) and extractive electrospray ionization (EESI) in their classifications. In this work, we propose a classification (Table 1.3) based on the number of steps necessary to desorb/ionize the analytes from the sample (Monge et al. 2013), but also considering the sample processing that takes place in each technique (Venter et al. 2014). Thus, this classification allows including PS and EESI, which are classically considered as ambient MS techniques. One-Step Ambient MS Techniques As mentioned earlier, in this group of techniques, sample processing occurs in parallel with the ionization mechanism. Liquid–solid extraction, thermal desorption, or chemical sputtering occurs in the same space and time than the ionization of compounds desorbed from the sample surface. Solid–Liquid Extraction Those one-step ambient MS techniques having liquid–solid extraction and liquid–liquid extraction (LLE) as the most important sample processing steps can be further divided into two subgroups depending on how the extraction product is presented to the ionization event: spray desorption or liquid microjunction. DESI (Takáts et al. 2004) is the most well-known spray desorption ambient MS technique (Figure 1.1), in which the sample processing starts by generating a charged spray plume through pneumatically assisted electrospray of a solvent. This charged spray plume is directed onto the sample surface to create TABLE 1.3  Classification of Ambient MS Techniques Used in Environmental and Food Analysis Techniques One step

Sample Processing Liquid–solid extraction

Spray desorption

Liquid microjunction Thermal/chemical desorption

Two step

Liquid–solid extraction

Laser desorption/ ablation Three step Others

Spray desorption Substrate spray Ultrasonic nebulization Laser ablation Matrix-assisted laser desorption Substrate spray

Solid–liquid extraction Thermal ablation/chemical ionization

Ambient MS Techniques

Ionization ESI Sonic spray ESI Plasma based

DESI, EESI EASI LMJ-SSP, LESA Nano-DESI DART, DAPCI, FAPA, LTP, DBDI, PADI,

Photoionization

DAPPI

ESI ESI APPI

ND-EESI PESI EAPPI

ESI ESI

LAESI (ELDI) MALDESI

ESI

PS REIMS

10

Mass Spectroscopy Techniques in Food and the Environment

FIGURE 1.1  (See color insert after page 124.) Schematic illustrations of some commer-

cially available ambient MS techniques: DESI (Reprinted with permission from reference (Sero, Nunez, and Moyano 2016), Copyright (2016) Elsevier.), DART and LAESI (Reprinted with permission from reference (Stopka et al. 2014), Copyright (2014) Royal Society of Chemistry.), and PS (Reprinted with permission from reference (Wang et al. 2010), Copyright (2010) Wiley-VCH.). a microlocalized liquid layer where compounds are dissolved/extracted. Finally, after the impact of subsequent arriving primary charged droplets on the surface, the liberation of secondary droplets containing analytes is produced, thus generating analyte ions through traditional ESI mechanisms. Contrary to DESI, easy ambient sonic-spray ionization (EASI) (Haddad, Sparrapan, and Eberlin 2006) operates free of high voltage, in spite of sharing the same practical setup (nebulizing gas flow and polar solvent systems). The nebulizer gas flows at sonic speed (2–5 times higher than DESI) coaxially to a solvent flow, which generates charged droplets through a statistical imbalance of charge. Although DESI is commercially available for its installation in several mass spectrometers, EASI is still a homemade device. However, DESI commercial ionization source can be adapted to perform EASI analysis with little custom readjustments. Regarding EESI (Chen, Venter, and Cooks 2006), the main difference when comparing with DESI and EASI is the use of two orthogonal sprays: the first spray contains charged droplets generated by the electrospray of a solvent, and the second spray can be either a neutral aerosol produced from a liquid sample or a gas stream containing volatile compounds (Figure 1.2). In this technique, collisions between neutral (containing analytes) and solvent charged droplets produce the extraction of analytes and subsequently the ionization takes place through ESI mechanisms. Regarding liquid microjunction-based techniques, a solvent is delivered through a capillary to form a semistatic liquid junction where analytes are dissolved/extracted from the sample surface. The extracting liquid is transferred to a position where analytes can be ionized by well-known ionization mechanisms, typically by electrospray. Techniques such as liquid microjunction surface sampling probe (LMJ-SSP) (Berkel, Sanchez, and Quirke 2002), nano-DESI (Roach, Laskin, and Laskin 2010b), and liquid extraction surface analysis (LESA) (Kertesz and Van Berkel 2010) belong to this group of ambient MS methods (Figure 1.2). The LMJ-SSP, already commercialized as flowprobeTM , uses two concentric tubes to supply the extraction solvent and to suction out the extracting liquid

Ambient Mass Spectrometry Techniques

11

FIGURE 1.2  (See color insert after page 124.) Schematic views of some one-step spraybased setup techniques: EESI (Reprinted with permission from reference (X. Li et al. 2011), Copyright (2011) Nature America, Inc.), LMJ-SSP (Reprinted and adapted with permission from reference (Berkel, Sanchez, and Quirke 2002), Copyright (2002) American Chemical Society.), LESA (Reproduced with permission from reference (Montowska et al. 2014), Copyright (2014) American Chemical Society.), and ­nano-DESI (Reprinted with permission from reference (Roach, Laskin, and Laskin 2010a), Copyright (2010) American Chemical Society.).

phase with analytes, which are further ionized by ESI or atmospheric pressure chemical ionization (APCI). Nano-DESI operates more similar to LMJ-SSP than to DESI, since the lack of nebulizing gas removes the momentum transfer of incoming droplets to the liquid film. Instead, a solvent delivered by a fused silica capillary dissolves the compounds on the sample surface and forms a solvent bridge with a self-aspirating nanospray emitter. Regarding LESA, which is fully automated, microliquid extraction from a solid surface is combined with a nano-electrospray, thus being considered an adaptation of the infusion nano-ESI automated device (Nanomate). Thermal and Chemical Desorption The thermal-assisted desorption is the most common way to remove analytes from the sample surface before gas-phase ionization through plasma-base techniques and ­atmospheric pressure photoionization (APPI). However, other mechanisms such as sputtering, where the sample surface is bombarded with high-energy species generated in atmospheric pressure plasmas, can also contribute to remove compounds with very low or no vapour pressure. The plasma-based techniques falling into this group rely on an electrical discharge involving metastable and reactive charged species, which interact, directly or indirectly,

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Mass Spectroscopy Techniques in Food and the Environment

through proton- and charge-transfer reactions with thermally/chemically desorbed analytes. Despite the different type of atmospheric pressure plasma used, such as coronas and dielectric barrier discharges (DBDs) and direct current (dc) and radio frequency (rf) glows, the design of the plasma source and how the plasma interacts with the sample allow classifying these techniques. In DART (Cody, Laramee, and Durst 2005), flowing atmospheric pressure afterglow (FAPA) (Andrade et al. 2008), and desorption atmospheric pressure chemical ionization (DAPCI) (Song and Cooks 2006), the plasma is generated in a physically and/or electrically isolated region from the sample introduction zone. Regarding DART, the plasma species generated by a dc corona-to-glow discharge are electrically filtered and heated before their interaction with the sample (Figure 1.1). Therefore, metastable species, formed in the discharge supporting gas (He or N2), interact with gas-phase water molecules to generate protonated water clusters by Penning ionization, which participate in proton-transfer reactions with the thermal desorbed analytes. In the FAPA source, the dc atmospheric pressure glow discharge is generated in a sealed discharge cell, and plasma species are transported into the open air producing an afterglow discharge (Figure 1.3). Although FAPA source seems to be similar to DART, it behaves quite differently in practice, which is due to the differences in the source design and the operating conditions that will be discussed later. In DAPCI, a dc corona discharge ionizes gas-phase solvent vapours, as in APCI, producing reagent species in a heated chamber, and the flowing gas transfers the reagent ions onto the sample surface inducing “chemical sputtering” of adsorbed analytes (Figure 1.4). By contrast, in low-temperature plasma (LTP) (Cotte-Rodríguez and Cooks 2006) and dielectric barrier discharge ionization (DBDI) (Na et al. 2007), the plasma is generated in the sample region, and all reactive species are used for ionization of compounds (Figure 1.3). In these techniques, a “cold” (~30°C) nonequilibrium plasma is generated by

FIGURE 1.3  (See color insert after page 124.) Schematics of some one-step plasma-based

techniques: FAPA (Reprinted with permission from reference (Shelley, Wiley, and Hieftje 2011), Copyright (2011) American Chemical Society.), DBDI (Reprinted with permission from reference (Na, Zhang, et al. 2007), Copyright (2007) John Wiley & Sons, Ltd.), and LTP (Reprinted with permission from reference (Benassi et al. 2013), Copyright (2013) John Wiley & Sons, Ltd.).

Ambient Mass Spectrometry Techniques

13

FIGURE 1.4  (See color insert after page 124.) Schematic illustrations of DAPCI (Reprinted with permission from reference (Song and Cooks 2006), Copyright (2006) John Wiley & Sons, Ltd.) and DAPPI (Reprinted with permission from reference (Haapala et al. 2007), Copyright (2007) American Chemical Society.).

a DBD, sustained by an inert gas, so no damage to the surface due to heating is expected. Moreover, plasma-assisted desorption ionization (PADI) (Ratcliffe et al. 2007) uses an rf glow discharge plasma in direct contact with the sample to desorb/ionize target analytes. As in LTP, the plasma plume in PADI operates at room temperature, which favors the use of both techniques for the analysis of thermally labile compounds. Finally, DAPPI (Haapala et al. 2007) (Figure 1.4) is the only non-plasma-based technique included in this group, in which analytes thermally/chemically desorbed are ionized by APPI mechanisms. Thus, a heated mix containing the carrying gas and solvent vapours (dopant) is directed onto the sample surface. The analytes are desorbed by the hot vapour, after which the ionization is produced by ultraviolet (UV) radiation involving photoionization, charge-transfer reactions, and ion–molecule reactions with solvent/dopant species. Two-Step Ambient MS Techniques In the ambient MS techniques included in this group, the sample is nebulized, and/or analytes are desorbed/extracted through thermal desorption, mechanical ablation, or laser desorption/ablation in a first step that is followed by an independent secondary ionization step. Liquid–Solid Extraction The extraction atmospheric pressure photoionization (EAPPI) (Liu et al. 2016) is a twostep technique that uses an ultrasonic nebulizer for sample processing (extraction, nebulization, and vaporization). The generated aerosol is transported by a carrier gas to the photoionization region (vacuum UV lamp), while mixing with a gaseous dopant. By contrast, in probe electrospray ionization (PESI) (Hiraoka et al. 2008) (Figure 1.5), which involves a solid sampling electrospray probe, a needle is inserted into a sample to pick up or to coat the needle surface with sample material before positioning it close to the mass spectrometer inlet. Finally, the ionization step is performed by applying a high voltage to the needle probe to induce an electrospray.

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Mass Spectroscopy Techniques in Food and the Environment

FIGURE 1.5  Schematic setup of PESI.

Laser Desorption/Ablation This group of techniques uses an infrared (IR) or UV laser to produce the desorption or ablation of analytes from a surface with and without a matrix. The sample surface is broken into small pieces by the sudden delivery of energy. The fine clusters or particles are dispersed by the expanding plume, which is subsequently merged with an electrospray plume or a plasma stream depending on the ionization source used in the second step. One advantage of not being the sample in direct contact with the ionizing plume is that desorption and ionization can be optimized independently. The first technique that coupled laser sampling to an ESI source was electrosprayassisted laser desorption ionization (ELDI) (Shiea et al. 2005). In ELDI, a pulsed nitrogen laser is used, while in laser ablation electrospray ionization (LAESI) (Nemes and Vertes 2007) (Figure 1.1), which is commercially available, the desorption/ablation takes place applying a mid-IR laser. By contrast, matrix-assisted laser desorption electrospray ionization (MALDESI) (Sampson, Hawkridge, and Muddiman 2006) uses an IR or UV laser to excite an exogenous matrix that cocrystallizes with the analyte, while applying a voltage to the stainless-steel target plate. Three-Step Ambient MS Techniques In this classification, a three-step ambient MS group has been proposed to include PS as an ambient MS technique, since it incorporates two sampling/processing steps and the ionization as a third step. In PS (Wang et al. 2010; Liu et al. 2015), a prepared

Ambient Mass Spectrometry Techniques

15

liquid sample is spotted onto a paper triangle and a solid-phase extraction (SPE) and/or chromatographic separation occurs when delivering a solvent and applying a high voltage to the paper substrate (Figure 1.1). The extracting solvent transports the extracted compounds to the apex of the triangle paper, whereas the different interaction of sample components with the paper substrate could produce their spatial/temporal separation. Electrospray is the driven force that pulls the solvent to the apex of the paper triangle, and also it is responsible for compound ionization through ESI mechanisms. Leaf (Liu et al. 2011, 2015) is an ambient MS method sharing similar principles with PS. However, in this technique, the plant material serves both as a substrate and as a sample. For instance, plant materials having a natural sharp shape (e.g., bean sprout) can be analyzed directly by leaf spray, while a small nick cut with a sharp tip has to be made to different samples. As in PS, the spray solvent is supplied on the pointy plant material to extract the endogenous compounds and to transport them to the tip. Finally, by applying a high voltage directly on the plant material, a spray of charged droplets is generated at the tip through ESI mechanisms. Other Ambient MS Techniques Rapid Evaporative Ionization Mass Spectrometry Rapid evaporative ionization mass spectrometry (REIMS) is an ambient MS technique based on other principles for desorption or ionization that cannot be classified in any of the previous categories. Originally, this technique was developed for the real-time identification of tissues during surgical interventions (Schäfer et al. 2009), although it has recently been proposed as a new analytical approach for direct analysis of meat samples (Balog et al. 2016; Verplanken et al. 2017). In REIMS, surgical blades are used to ablate tissues in order to produce aerosols and charged species by the heat dissipated during the electrosurgical process, being subsequently transported to the MS inlet. The ionization mechanism proposed for REIMS is quite similar to that taking place in both APCI and thermospray ionization in filament-off mode (Schäfer et al. 2009). The desorption of neutral molecules is followed by gas-phase ionization via proton transfer reactions with the ionized water molecules as in APCI.

PHYSICAL DESIGNS AND EXPERIMENTAL APPROACHES As mentioned earlier, in ambient MS analysis, sample processing and ionization usually take place in a single platform near the inlet of the mass spectrometer. In this context, geometrical parameters, solvent and/or gas characteristics and flow rate, plasma formation approaches, surface properties, solubility, and temperature are important variables that critically affect the performance of the techniques. Probe Assembly Configurations In one-step spray desorption-based techniques (Table 1.3) such as DESI, EASI, and EESI, the geometrical configuration and operational parameters are important aspects to be considered (Weston 2010). For instance, when tuning DESI source, the response depends on the incident and the collection angles, the sample spot-to-MS inlet distance, as well as on

16

Mass Spectroscopy Techniques in Food and the Environment

both the tip-to-surface and the MS orifice-to-surface heights (Figure 1.1). Recommended parameters for DESI typically are within the range of 45°−60° and 5°−30° for incident and collection angles, respectively, 2−3 mm for the sample spot-to-MS inlet distance, and 1−3 mm and ~1 mm for the tip-to-surface and the MS orifice-to-surface heights, respectively (Monge et al. 2013). The liquid sample in EESI (Figure  1.2) is sprayed into an electrospray plume positioned orthogonally to the sample spray, being the distance and the angle between both sprays critical to guarantee an efficient overlapping. The geometrical configuration of liquid microjunction-based techniques is quite different, and the sampling probe rests on the surface of the sample for longer time than in spraybased ones. The differences among these techniques are due to the way of ­delivering and removing the extracting solvent from the sample surface. As can be seen in Figure 1.2, in LMJ-SSP, the coaxial capillaries are positioned perpendicularly (100−300 µm) above the sample. The outer capillary supplies the solvent to form the liquid junction and extracts compounds from the sample surface, whereas the inner ­capillary pulls the solution that contains the analytes to the ionization source. In contrast to the vertical arrangement of LMJ-SSP, in nano-DESI, the two fused silica capillaries that form the liquid microjunction are positioned at an angle above the sample surface (Figure 1.2). The primary capillary continuously supplies the solvent to create and maintain the liquid bridge, whereas the secondary capillary, an electrospray emitter, aspirates and ionizes the desorbed analytes. Regarding LESA (Figure 1.2), a robotic arm picks up a conductive disposable pipette tip and moves it above to a well containing the extracting solvent, which is aspirated by immersing the pipette tip into it. Afterwards, the pipette tip is positioned above the surface spot to form the liquid microjunction by dispensing a specific volume of solvent on the surface to extract the compounds. The latter liquid phase is aspirated back into the tip and positioned in one of the emitters of a chip-based nano-ESI for the ionization. The main advantages of LESA are the fully automatization and the use of disposable tips, which prevent contamination and carryover. As mentioned before, method performance of plasma-based techniques is more affected by the physical design of the plasma source and the kind of plasma–sample interaction than by geometrical configurations (Ding and Duan 2015). Thus, in LTP and PADI, the plasma is generated in the sampling zone, whereas in DART, FAPA, and DAPCI, the plasma is isolated from the sample ionization region. The DART source (Figure 1.1) consists of two chambers through which the DART gas flows. In the first region, a dc corona-to-glow discharge (1−5 kV) generates the plasma, which is filtered by one or several perforated plate electrodes and heated before passing through a final grid electrode to interact with compounds thermally desorbed from the sample surface. This design allows the selective removal of ionic species from the heated plasma gas (typically He), thus only electronically excited metastable atoms arrive to the outside region. In DART, the sample is placed near the source outlet with minimum disturb of the gas flow to the MS inlet, being geometries for direct desorption and for transmission commercially available. By contrast, in DAPCI, the plasma species are transferred to the sample region without any filtration (Figure 1.4). This ambient source is built by simple modifications of the conventional APCI source, where gaseous reagent ions, generated by atmospheric corona discharge, are sprayed through a pipette tip and impacted on the solid sample, thus producing the desorption and subsequent ionization of analytes. In FAPA, LTP, and DBDI, a two-electrode configuration with different power supplies, electrode shape, and discharge cells are used. For instance, FAPA operates in a current controlled glow discharge regime (~25 mA) applying a direct voltage (typically a few hundred volts) to a pin-to-plate or pin-to-capillary electrodes in a sealed discharge

Ambient Mass Spectrometry Techniques

17

cell that physically separates the source from the sample (Figure 1.3). Unlike DART, FAPA does not use any electrode to filter plasma species before interacting with the sample. Moreover, no external heating is needed in FAPA, since the temperature of the plasma stream is high enough (>200°C) for an efficient desorption of the analytes. Regarding ambient MS methods based on DBD, at least one dielectric layer has to be placed in between the electrodes, being necessary a high ac voltage to transport the current through the discharge gap. Typically, amplitudes of 1−100 kV and frequencies of a few Hz to MHz are commonly used. The difference between DBDI and LTP lays on the geometrical configuration. In DBDI source, a glass slide is inserted between a plate electrode and a needle electrode to function as dielectric material and sample plate (Figure 1.3). When ac high voltage (5−10 MHz) is applied between the electrodes, an LTP consisting of numerous transient micro-discharges is ignited, and analytes placed in the surface plate are desorbed and ionized. On the other hand, LTP uses a glass tube (typically, ~4 mm inner diameter and ~6 mm outer diameter) as dielectric material to physically separate the ring outer electrode from the metal pin internal grounded electrode, which is directly in contact with the flowing gas (Figure 1.3). The generated plasma interacts with the sample, thus desorbing and ionizing analyte molecules. Since thermal desorption processes clearly play a major role in plasma-based techniques, temperature becomes a critical parameter to improve sensitivity. As commented before, FAPA does not use additional heating since the heat generated in the source is enough to desorb analytes from the sample surface. By contrast, DART uses an external heating to desorb semi-volatile and low-volatile compounds. In fact, a selective detection of thermal-dependent compounds can be performed in DART by ramping the temperature of the discharge gas to produce the thermal fractionation of the compounds in the sample surface. PADI is another “proximal” plasma-based technique, where high-energy plasma species interact directly with the sample. Hence, an rf glow discharge is set at the end of a pin electrode, which comes in direct contact with the sample, that works as ground electrode. Some thermal desorption techniques are not plasma-based. Among them, DAPPI has increased its popularity in recent years because it can be applied for the analysis of low-polarity compounds (Kauppila and Kostiainen 2017). In this technique, a heated microchip is used to deliver both a narrow jet of vaporized solvent (dopant) and a nebulizer gas onto the sample surface, thus achieving surface temperatures up to 350°C for the desorption (Figure 1.4). Ionization is initiated by photons emitted by a vacuum UV lamp, typically a krypton discharge lamp that emits 10 eV photons, although dopants such as toluene, acetone, or anisole (typically added at 10 µL/min) are required to ionize those compounds with ionization energies below that of the photons. For the two-step laser-based desorption/ablation techniques, the key parameters affecting their figures of merit are the characteristics of the lasers used for ablation or desorption of the analytes from the sample surface, the ionization method, the use of matrix, and the geometrical configuration (Javanshad and Venter 2017). Both UV and IR lasers are used to generate the plume of particles, clusters, and free molecules, which are subsequently ionized in a separated step. For instance, UV nitrogen lasers, mainly at 337 nm operating at 10 MHz with a pulse length of 4 ns and a pulse energy of 20 µJ, are used in ELDI. Although, in most of the techniques as for instance in LAESI, IR lasers typically tuned at 2,940 nm with pulses of 5 ns duration at 2−20 Hz and pulse energy between 100 and 52 mJ are used (Figure 1.1). As regards the ionization step, the most popular is electrospray ionization, which is used in techniques such as LESI, LAESI, and in MALDESI where an exogenous matrix is used to improve ionization. Although both

18

Mass Spectroscopy Techniques in Food and the Environment

reflection and transmission approaches are used in laser-based techniques, most of the geometrical implementations use reflective geometries with incident laser beams hitting perpendicularly the sample. However, other incident angles such as 45° with respect to the sample have also been used in some cases. In the less-used inverted geometry (transmission mode), the laser beam from the backside illuminates the sample, and the plume of desorbed material exits from the front side in the opposite direction of laser application. Solvents, Gases, and Sample Devices In liquid extraction-based methods, a solvent is always used, and its selection is a key aspect to be considered. In spray desorption techniques such as DESI, a spray of solvent charged droplets is used to perform the desorption/extraction of compounds and to ionize the analytes, whereas in EESI and in liquid microjunction techniques, the solvent extraction takes place in line with an electrospray emitter. Solvent composition affects both the initial spray droplet size that depends on both the surface tension and the dielectric constant of the solvent, as well as on the capacity of solubilizing sample component (Javanshad and Venter 2017). In general, organic/aqueous binary mixtures containing 50%−80% of methanol or acetonitrile are used to dissolve analytes. Moreover, the solvent system has to be electrically conductive and typically, when working in positive ion mode, acetic or formic acid (0.1%−0.5%) is used to increase ionization efficiency. DESI is generally indicated for polar compounds that can be easily ionized by controlling the pH of the solvent mixture. Even so, low polar and hydrophobic compounds have also been analyzed using nonpolar solvents such as toluene (Green et al. 2010). In this case, the solvent is ionized by electrochemical oxidation at the metal spray needle interface with the subsequent ionization of analytes by charge-transfer processes. As regards flow velocities, both sheath gas and liquid solvent flow rates, which are interdependent, affect the performance of the technique. A threshold velocity of gas flow is required for an efficient production of small secondary droplets, but values above the optimum generate a spray focused in a small spot, reducing the amount of desorbed material. This effect can be compensated by increasing the solvent flow rate. Typical gas pressure values (N2) ranging from 120 to 150 psi and solvent flow rates from 2 to 10 µL/min are used to maximize the ion signal (Bodzon-Kulakowska et al. 2014). In nano-DESI, no sheath gas is needed since the flow rate for the liquid junction is governed by electrospray-induced flow generated by the nano-spray tip and additionally assisted by the vacuum in the MS inlet. In LMJ-SSP, typically suction flows of 10 µL/min are used by controlling inner and outer capillary diameters and gas flow. This technique can work in two operating modes: stepping and scanning. In stepping mode, a single spot of the sample is analyzed at a time, whereas in scanning mode, the liquid microjunction is dragged across the surface obtaining one- and two-dimensional images. In this last case, a thorough control of flow rate is needed since low flow rates worse the spatial resolution, while high flow rates and scanning speed decrease the signal intensity. Today, the reduction of gas consumption in miniaturized plasma-based ion sources is an attractive feature for their implementation in mass spectrometer instruments for fieldwork. The discharge gases most frequently used are helium, nitrogen, argon, or air at flow rates that depend on the technique, ranging from EA (O2) if EA(M) > 0 eV if ΔacidG(M) < ΔacidG(HO2•)

(1) (2) (3) (4) (5) (6) (7) (8)

SCHEME 3.1  Ionization reactions in positive (Reactions 1−3) and negative ion DAPPI

(Reactions 1, 4−8) (Luosujärvi et al. 2008). S, solvent/dopant; M, analyte; PA, proton affinity; EA, electron affinity; ΔacidG, gas-phase acidity. efficient for small molecules, but it prevents the analysis of large, low volatility, and/or thermolabile compounds. Thus so far, the above described DAPPI setup has been applied to the analysis of pharmaceuticals in tablets (Haapala et al. 2007), drugs of abuse in powder, tablet, resin, plant and paper form (Kauppila et al. 2008; Luosujärvi et al. 2009; Kauppila et al. 2011, 2013), drug metabolites and endogenic steroids from urine (Suni et al. 2011; Vaikkinen et al. 2015b), pesticides in plant material (Luosujärvi et al. 2010; Vaikkinen et al. 2015c), PAHs in soil (Luosujärvi et al. 2010), lipids from capsules, butter, milk and food oils (Suni et al. 2012; Vaikkinen et al. 2015a; Rejšek et al. 2016), explosives (Kauppila et al. 2016), insect defense compounds from insect skin (Rejšek et al. 2015), and mouse tissue (Pól et al. 2009). DAPPI has also been used for the characterization of pyrogenic black carbon in a setup, where the sample was held in front of the heated nebulizer of a commercial APPI source (Podgorski et al. 2012). Below, DAPPI applications in the field of food and environmental analysis are discussed more closely.

Desorption Atmospheric Pressure Photoionization

59

DAPPI IN FOOD ANALYSIS Luosujärvi et al. showed the feasibility of desorption atmospheric pressure ­photoionization-mass spectroscopy (DAPPI-MS) in the analysis of pesticides from fruit peel (Luosujärvi et al. 2010). First, standard samples of nine pesticides, aldicarb, carbofuran, ditalimfos, imazalil, methiocarb, methonyl, oxamyl, pirimicarb, and thiabendazole, were analyzed from PMMA (polymethylmethacrylate) surface by positive ion DAPPI. All the pesticides were successfully detected. With acetone as the DAPPI solvent, the pesticides formed protonated molecules, whereas with toluene as the DAPPI solvent, both protonated molecules and molecular ions were observed. In addition, some of the compounds showed fragments and substitution products in their DAPPI mass spectra. Limits of detection for the pesticides ranged from 30 to 300 pg. Finally, the peels of organic and conventionally produced oranges were analyzed directly with DAPPI-MS (Figure 3.2). An intense ion at m/z 297, with a distinctive 37Cl isotopic peak at m/z 299, was observed in the peel of the conventionally produced orange. The peaks at m/z 297 and 299 were identified as the [M+H]+ ions of imazalil, and this was further confirmed by MS/MS analysis and comparison with the MS2 spectrum of imazalil standard. The peel of the organically produced orange showed no signs of pesticides. DAPPI-MS was thus thought to be well suited to the fast screening of pesticides from fruits or other foodstuff. Räsänen et al. combined DAPPI with travelling wave ion mobility (IM) spectroscopymass spectrometry (TWIMS-MS) in order to improve the spectrum interpretability and to increase the signal-to-noise (S/N) ratio in the analysis of complicated samples, while retaining the speed provided by ambient MS (Räsänen et al. 2014). In addition, the sensitivities of DAPPI and direct analysis in real time (DART) for a variety of standards and compounds from food and pharmaceutical products were compared. DAPPI was found approximately 10–100 times more sensitive than DART for the tested standard compounds, bisphenol A (BPA), chloroquine, benzo[a]pyrene, ranitidine, cortisol, and α-tocopherol. DAPPI-TWIM-MS was used for the direct analysis of almond surface, where the M+• ion of α-tocopherol was successfully detected. The IM separation prior to MS analysis greatly reduced the chemical noise in the acquired mass spectrum. DAPPITWIM-MS was also used for the analysis of multivitamin products containing several vitamins, minerals, and excipients. Seven out of thirteen vitamins reported by the product manufacturer were detected. The main peaks observed from the multivitamin tablet (Figure 3.3) were due to nicotinamide (amide of niacin, vitamin B3), pyridoxine, (vitamin B6), and α-tocopherol. In addition, caffeine showed an intense peak. Less intense peaks were due to biotin (vitamin B7), thiamine (vitamin B1), and cholecalciferol (vitamin D3). The TWIM separation helped to distinguish the low-intensity signal of cholecalciferol from the other compounds, as the S/N was increased sevenfold. Compounds that were reported by the product manufacturer, but were not be detected with DAPPI-TWIM-MS in positive ion mode, were ascorbic and folic acids, retinol, β-carotene, cyanocobalamin, and riboflavin. Suni et al. compared DAPPI and DESI for the analysis of several lipid classes (fatty acids (FA), fat-soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids) and showed that both techniques are well suited to detect lipids from pharmaceutical and food products (Suni et al. 2012). Fish oil capsules containing FA eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) were analyzed with negative ion DAPPI and DESI. Oil from the capsules was taken with a syringe needle and injected onto an office paper attached on a poly(tetrafluoroethylene) surface. Both techniques showed the deprotonated molecules clearly in the mass spectra. α-Tocopherol capsules were analyzed

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Mass Spectroscopy Techniques in Food and the Environment

FIGURE 3.2  DAPPI mass spectra obtained from (a) the piece of an orange peel and

(b) the piece of an organically produced orange peel in positive ion mode with acetone (10 mL/min) as the spray solvent. In (a), the [M+H]+ ion of imazalil is seen at m/z 297 and the 37Cl isotopic peak at m/z 299. (Reprinted with permission from “Environmental and Food Analysis by Desorption Atmospheric Pressure Photoionization-Mass Spectrometry” by Laura Luosujärvi, Sanna Kanerva, Ville Saarela, Sami Franssila, Risto Kostiainen, Tapio Kotiaho, and Tiina J. Kauppila, Rapid Communications in Mass Spectrometry 24 (2010): 1343–50. Copyright (2010) John Wiley and Sons Ltd.) similarly to the fish oil capsules, but in positive ion mode. DAPPI showed an intense molecular ion of α-tocopherol without any background disturbances, while DESI showed a weak protonated molecule of α-tocopherol together with a high background. Butter was spread on a piece of office paper and analyzed by DAPPI-MS/MS. Cholesterol could be identified from butter by its characteristic product ions, as shown in Figure 3.4. Transmission mode (TM) DAPPI was developed for easy sampling of liquid samples and enrichment of dilute solutions prior to analysis (Vaikkinen et al. 2015a). In TM-DAPPI, the liquid sample was applied on a metal or polymer mesh, which was

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61

FIGURE 3.3  DAPPI-TWIM-MS analysis of the scratched surface of a multivitamin tablet:

(a) total IM spectrum; (b) combined mass spectrum from the total IM data, where the ion at m/z 123 is [M+H]+ of nicotinamide, 170 is [M+H]+ of pyridoxine, 195 is [M+H] of caffeine, 430 is M+• of α-tocopherol, and 472 is [M+H]+ of α-tocopheryl acetate; (c) single IM spectrum for biotin [M+H]+ at m/z 245; (d) combined mass spectrum from the IM data in (c); (e) single IM spectrum for the thiamine M+ ion at m/z 265 (and for thiamine fragment ion at m/z 144 with dashed line); (f) combined mass spectrum from the IM data in (e); (g) single IM spectrum for the cholecalciferol M+• at m/z 384; (h) combined mass spectrum from IM data in (g) (insert shows enlargement of the spectrum at mass range m/z 370–400). (Reprinted with permission from “Desorption Atmospheric Pressure Photoionization and Direct Analysis in Real Time Coupled with Travelling Wave Ion Mobility Mass Spectrometry” by RiikkaMarjaana Räsänen, Prabha Dwivedi, Facundo M. Fernández, and Tiina J. Kauppila, Rapid Communications in Mass Spectrometry 28 (2014): 2325–36. Copyright John Wiley and Sons.) placed perpendicularly to the MS inlet, and hot steam from the heated nebulizer microchip was directed to the backside of the mesh. The sample surface was faced by the VUV lamp photons on the top. The sample could be applied on the mesh as a droplet, or the mesh could be dipped in the sample solution. The meshes were used to extract

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FIGURE 3.4  The product ion mass spectrum of m/z 369 (a) from butter and (b) from cholesterol standard (10 mM, corresponding to 10 pmol) with positive ion DAPPI using toluene as the spray solvent. (Reprinted with permission from “Analysis of Lipids with Desorption Atmospheric Pressure Photoionization-Mass Spectrometry (DAPPI-MS) and Desorption Electrospray Ionization-Mass Spectrometry (DESI-MS)” by Niina M. Suni, Henni Aalto, Tiina J Kauppila, Tapio Kotiaho, and Risto Kostiainen, Journal of Mass Spectrometry 47 (2012): 611–19. Copyright (2012) John Wiley and Sons.)

and concentrate compounds from aqueous solutions. TM-DAPPI was shown to be feasible in the analysis of milk lipids. A piece of PEEK mesh was dipped in full-fat cow milk diluted with water (1:10) and analyzed by TM-DAPPI (Figure 3.5). The spectrum showed intense ion signals at 28 units apart, mainly in the range of m/z 300–600. The ions were suggested to be the [MH-FA]+ fragments of triglycerides, which are the main constituents in cow milk, and typically contain an even number of carbons in the FA chain (Jensen 2002). This interpretation was also supported by the product ion spectra of the main ions. Rejsek et al. used DAPPI for the direct detection of lipids from high-performance thin-layer chromatography (TLC) plates (Rejšek et al. 2016). The sample components were first separated by TLC, after which the sample zones were detected by DAPPI. DAPPI could be used to efficiently ionize several lipid groups, such as cholesterol, triacylglycerols, 1,2-diol diesters, wax esters, cholesteryl esters, and hydrocarbons from TLC and high-performance thin-layer chromatography (HPTLC) plates. TLC-DAPPI-MS was applied to the analysis of plant oils including caraway, parsley, safflower, and jojoba oils. Apart from jojoba oils, the plant oils showed strong signals for the [M+NH4]+ adducts of triacylglycerols and fragments. The mass spectrum of jojoba oil affirmed that it differs entirely from other plant oils by its chemical composition, being a mixture of wax esters,

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FIGURE 3.5  TM-DAPPI spectrum from a PEEK mesh dipped in full-fat milk/water (1:10). Microchip heating power was 3.0 W, nebulizer gas flow rate was 125 mL/min, and toluene flow rate was 0.5 mL/min. (Reprinted with permission from “Transmission Mode Desorption Atmospheric Pressure Photoionization” by Anu Vaikkinen, Juha Hannula, Iiro Kiiski, Risto Kostiainen, and Tiina J. Kauppila. 2015. Rapid Communications in Mass Spectrometry 29 (2012): 585–92. Copyright (2015) John Wiley and Sons Ltd.)

FIGURE 3.6  (See color insert after page 124.) Extracted ion chromatograms acquired by DAPPI-MS in MS mode. Caraway oil from (a) the normal-phase HPTLC (NP-HPTLC) and (b) the reversed-phase HPTLC (RP-HPTLC) plate. Jojoba oil from (c) the NP-HPTLC and (d) the RP-HPTLC plate. TLC mobile phases for the NP-HPTLC plate, hexane/ diethyl ether/acetic acid (93:7:1; v/v/v), and the RP-HPTLC plate, acetone/acetonitrile (90:10; v/v). The green line: TG 54:6 [M+NH4]+. The red line: TG 52:4 [M+NH4]+. The black line: TG 54:3 [M+NH4]+. The orange line: WE 38:2 [M+H]+. The blue line: WE 40:2 [M+H]+. The violet line: WE 42:2 [M+H]+. The brown line: WE 44:2 [M+H]+. (Reprinted with permission from Rejšek, Jan, Vladimír Vrkoslav, Anu Vaikkinen, Markus Haapala, Tiina J. Kauppila, Risto Kostiainen, and Josef Cvačka, Analytical Chemistry, 88 (2016): 12279–86. Copyright (2016) American Chemical Society.)

which were detected as [M+H]+ ions in DAPPI-MS. Figure 3.6 shows the separation of triacylglycerols and wax esters of the studied plant oils by TLC, and the detection of the lipid components by DAPPI-MS. The DAPPI-MS food applications are summarized in Table 3.1.

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TABLE 3.1  DAPPI in Food Applications MS Method

Analytes

Matrix

Sample Preparation

Imazalil

Orange peel

None

DAPPI-IT

Aldicarb, carbofuran, ditalimfos, imazalil, methiocarb, methonyl, oxamyl, pirimicarb, thiabendazole [MH-FA]+ fragments of triglycerides

Standards

None

Full-fat cow milk

1:10 dilution with water

DAPPI-IT, DESI-IT DHA and EPA

Fish oil capsules

DAPPI-IT, DESI-IT α-Tocopherol

α-Tocopherol capsules

DAPPI-MS/MS

Butter

Oil removed from the capsules by a syringe needle and applied on office paper; analysis from the paper Oil removed from the capsules by a syringe needle and applied on office paper; analysis from the paper Butter spread on office paper; analysis from the paper

TM-DAPPI-IT

Cholesterol

Imazalil detected from conventionally produced, but not from organically produced oranges

Reference Luosujärvi et al. (2010) Luosujärvi et al. (2010)

Deprotonated molecules detected in negative ion mode

Vaikkinen et al. (2015a) Suni et al. (2012)

Molecular ion detected with DAPPI, with very low background

Suni et al. (2012)

Cholesterol identified by its characteristic product ions

Suni et al. (2012) (Continued)

Mass Spectroscopy Techniques in Food and the Environment

DAPPI-IT

Results/Notes

MS Method

Analytes

TLC-DAPPIHRMS

Cholesterol, triacylglycerols, 1,2-diol diesters, wax esters, cholesteryl esters and hydrocarbons α-Tocopherol

DAPPI-TWIM-MS

DAPPI-TWIM-MS

Nicotinamide, pyridoxine, α-tocopherol, caffeine, biotin, thiamine, cholecalciferol

Matrix

Sample Preparation

Results/Notes

Reference

plant oils including Dissolution in chloroform caraway, parsley, safflower, and jojoba oils

Triacylglycerols and wax esters were separated by TLC, and analyzed directly from the TLC plates by DAPPI

Rejšek et al. (2016)

Almond surface

None

Chemical noise was significantly reduced by the IM separation

Multivitamin products

None

The TWIM separation helped to distinguish the low-intensity signals of, for example, cholecalciferol Ascorbic and folic acids, retinol and β-carotene, cyanocobalamin and riboflavin could not be detected

Räsänen et al. (2014) Räsänen et al. (2014)

Abbreviations: IT, ion trap; MS/MS, tandem mass spectrometry.

Desorption Atmospheric Pressure Photoionization

TABLE 3.1 (Continued )  DAPPI in Food Applications

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DAPPI IN ENVIRONMENTAL ANALYSIS Luosujärvi et al. showed the suitability of DAPPI in the analysis of several polycyclic aromatic hydrocarbons (PAHs) and one N-PAH (Luosujärvi et al. 2010). First, standards of naphthalene, phenanthrene, acridine, chrysene, benzo[a]pyrene, and benzo[k] fluoranthene were analyzed from PMMA surface. Toluene and acetone were both tested as DAPPI solvents. With toluene as the DAPPI solvent, the PAHs formed mainly molecular ions with some minor formation of [M+H]+ for the PAHs with highest proton affinities. Naphthalene was an exception, as it could not be ionized with either solvents, which was thought to be due to its IE, which is lower than the IEs of the larger PAHs. Acridine, which is an N-PAH, showed solely [M+H]+ ions due to its considerably higher proton affinity. With acetone as the DAPPI solvent, none of the PAHs was ionized, since the formation of molecular ions through charge exchange is not possible with acetone. Acridine, however, which ionizes through proton transfer, could be ionized also with acetone. The limits of detection for the PAHs were between 100 and 1,000 pg. DAPPI-MS was finally used for the direct analysis of a soil pellet spiked with phenanthrene, chrysene, and benzo[k]fluoranthene (10 µg/g of each in dry soil), and a blank soil pellet. All three compounds were clearly observed in the spectrum of the spiked soil pellet, as shown in Figure 3.7. The analysis of soil with high organic content shows the applicability of the method in the screening of contaminated soils, since PAHs (with three rings or more) tend to accumulate in nature into the humified organic part of soil. It should be noted, however, that the identification of individual PAHs by DAPPI is not possible, since the ions of compounds with the same molecular mass will overlap in the mass spectra, and the PAH compounds do not fragment readily enough to give clear MS/MS spectra. The feasibility of DAPPI-MS in the analysis of polybrominated flame retardants was shown by using tetrabromobisphenol A (TBBPA) as the test compound (Luosujärvi et al. 2010). TBBPA was analyzed in both positive and negative polarities. In positive ion DAPPI, TBBPA showed a molecular ion, whereas in negative ion DAPPI [M-H]− was observed. Due to the bromine substituents, a wide isotopic pattern of peaks was observed in both cases. The background noise in the analyte ion mass range was observed to be slightly lower in negative ion mode than that in positive ion mode. In addition, the MS2 spectrum of the [M-H]− ions was said to be more informative than those of the M+• ions, and therefore, the identification of TBBPA in negative ion mode was thought to be more reliable. Finally, DAPPI-MS in negative ion mode was used to analyze the surface of a printed circuit board. The mass spectrum showed an intense group of ions at m/z 539–547 with a typical isotopic pattern of a molecule containing four bromine atoms (Figure  3.8). The masses of the ions in the full-scan mass spectrum as well as those observed in the MS/MS spectrum matched those of the TBBPA standard. Neonicotinoids are widely used insecticides, which are suspected to play a part in the unexplainable large-scale loss of honeybee colonies (Jeschke et al. 2011). In a study by Vaikkinen et al., DAPPI and DESI were tested in the analysis of the five most used neonicotinoid compounds: thiacloprid, acetamiprid, clothianidin, imidacloprid, and thiamethoxam (Vaikkinen et al. 2015c). DAPPI was found to be more sensitive than DESI, with 2–11 times better S/N ratio, and limits of detection at 0.4–5.0 fmol. DAPPI was also used to analyze fresh rose leaves treated with commercially available thiacloprid insecticide (Figure 3.9). Thiacloprid could be detected from the rose

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FIGURE 3.7  DAPPI mass spectra obtained from (a) spiked soil pellet and (b) blank

soil pellet in positive ion mode with toluene (10 mL/min) as the spray solvent. In (a)  ions originating from benzo[k]fluoranthene, chrysene, and phenanthrene are seen at m/z 252 (M+•), 228 (M+•), and 178 (M+•), respectively. The amount of each PAH ­compound was 10 mg/g of soil. (Reproduced with permission from Reprinted with permission from “Environmental and Food Analysis by Desorption Atmospheric Pressure Photoionization-Mass Spectrometry” by Laura Luosujärvi, Sanna Kanerva, Ville Saarela, Sami Franssila, Risto Kostiainen, Tapio Kotiaho, and Tiina J. Kauppila, Rapid Communications in Mass Spectrometry, 24 (2010): 1343–50. Copyright (2010) John Wiley and Sons.) leaves even 2.5 months after the treatment. DAPPI could also detect thiacloprid from dried powdered turnip rape flowers, which had been collected from a field treated with ­thiacloprid-containing insecticide. The analysis of plant material by DAPPI did not require extraction or other sample preparation. DAPPI was thought to show promise as a fast tool for screening for forbidden insecticides, or studying the distribution of insecticides in nature.

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FIGURE 3.8  (a) DAPPI mass spectrum obtained from the circuit board in negative ion

mode with anisole (10 mL/min) as the spray solvent. The isotopic pattern of the [M−H]− ion of TBBPA is seen at m/z 539–547. (b) Theoretical isotopic pattern of TBBPA. (Reprinted with permission from “Environmental and Food Analysis by Desorption Atmospheric Pressure Photoionization-Mass Spectrometry” by Laura Luosujärvi, Sanna Kanerva, Ville Saarela, Sami Franssila, Risto Kostiainen, Tapio Kotiaho, and Tiina J. Kauppila, Rapid Communications in Mass Spectrometry, 24 (2010): 1343–50. Copyright (2010) John Wiley and Sons.) Parshintsev et al. used DAPPI-MS with high-resolution mass spectroscopy (HRMS) as a complementary technique for the fast analysis of atmospheric aerosols from city air (Parshintsev et al. 2015). The aerosols had been collected on a filter, from which they were directly analyzed by DAPPI-HRMS. The results were compared to those obtained by LC-MS after extraction. The DAPPI results agreed well with the measured aerosol particle number. DAPPI-HRMS showed several important oxidation products of terpenes, and numerous compounds were tentatively identified. Besides the targeted compounds, LC-MS and DAPPI-HRMS were found to detect different compounds and would therefore provide complementary information about the aerosol samples (Table 3.2). TM-DAPPI with metal and polymer meshes as sampling support (described in ­section “DAPPI in Food Analysis”) could be used for the enrichment of PAHs from aqueous solutions (Vaikkinen et al. 2015a). Pieces of stainless steel and PEEK meshes were soaked into aqueous sample containing analytes of different sizes and polarities,

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FIGURE 3.9  DAPPI-MS analysis of (a) control rose leaf, (b) thiacloprid-treated rose leaf

immediately after treatment with the insecticide Calypso, and (c) rose leaf 2.5 months after treatment with the insecticide. The inserts in (a–c) show the product ion spectra of m/z 253. The dopant was acetone at 10 μL/min. (Reprinted with permission from “Analysis of Neonicotinoids from Plant Material by Desorption Atmospheric Pressure Photoionization-Mass Spectrometry” by Anu Vaikkinen, Henning S. Schmidt, Iiro Kiiski, Sari Rämö, Kati Hakala, Markus Haapala, Risto Kostiainen, and Tiina J. Kauppila, Rapid Communications in Mass Spectrometry, 29 (5): 424–30. Copyright (2015) John Wiley and Sons.)

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TABLE 3.2  DAPPI in Environmental Applications MS Method DAPPI-IT

DAPPI-IT

DAPPI-IT

DAPPIHRMS, LC-MS

Analytes

Matrix

Sample Preparation

Standards None naphthalene, phenanthrene, acridine, chrysene, benzo[a]pyrene and benzo[k] fluoranthene A spiked soil None phenanthrene, sample chrysene, and benzo[k] fluoranthene TBBPA Standard/ circuit board Fresh rose thiacloprid, leaves and acetamiprid, dried clothianidin, powdered imidacloprid, and turnip rape thiamethoxam flowers neonicotinoids Atmospheric acids Atmospheric and terpene aerosols oxidation products collected on a filter

TM-DAPPI

Benzo[a]pyrene

Standard in aqueous solution

DAPPI-MS, DART-MS

BPA, benzo[a]pyrene Standards

Results/Notes

Reference Luosujärvi et al. (2010)

Luosujärvi et al. (2010)

Luosujärvi et al. (2010) Vaikkinen None Thiacloprid was detected from the et al. (2015c) rose leaves even 2.5 months after thiacloprid treatment Parshintsev None; direct DAPPI-HRMS et al. analysis of provided (2015) the filters complementary information to LC-MS Vaikkinen Collection to Efficient et al. enrichment of porous (2015a) B[a]P from metal and aqueous solutions polymer to the metal and mesh polymer meshes Räsänen None DAPPI-MS was et al. found 10–100 (2014) times more sensitive than DART-MS

Abbreviations: DART, direct analysis in real time; HRMS, high-resolution mass spectrometry; IT, ion trap; LC-MS, liquid chromatography-mass spectrometry; TM, transmission mode.

for 0–60 min. After evaporation of the solvent, the meshes were analyzed with TM-DAPPI. For all the test compounds, significantly higher signals were achieved after the extraction. The effect was most pronounced for benzo[a]pyrene (B[a]P), which showed a tenfold increase already after 1-min extraction time. With extraction times over 1 min, the signal for B[a]P remained on the same level, but the signal of the background was observed to decrease, possibly due to competitive adsorption on the mesh material. This resulted in significantly improved S/N for B[a]P, as shown in Figure 3.10.

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FIGURE 3.10  TM-DAPPI spectra from a PEEK mesh after (a) 1-min and (b) 40-min extraction times in 10 mL solution containing 100 nM benzo[a]pyrene, and 1 μM testosterone and verapamil. Microchip heating power was 3.0 W, nebulizer gas flow rate was 125 mL/min, and toluene flow rate was 0.5 μL/min (Vaikkinen et al. 2015a). (Reprinted with permission from “Transmission Mode Desorption Atmospheric Pressure Photoionization” by Anu Vaikkinen, Juha Hannula, Iiro Kiiski, Risto Kostiainen, and Tiina J. Kauppila. 2015. Rapid Communications in Mass Spectrometry 29 (2012): 585–92. Copyright (2015) John Wiley and Sons Ltd.)

REFERENCES Haapala, Markus, Jaroslav Pol, Ville Saarela, Ville Arvola, Tapio Kotiaho, Raimo A. Ketola, Sami Franssila, Tiina J. Kauppila, and Risto Kostiainen. 2007. Desorption atmospheric pressure photoionization. Analytical Chemistry 79(20): 7867–72. doi: 10.1021/ac071152g. Jensen, Robert G. 2002. The composition of bovine milk lipids: January 1995 to December 2000. Journal of Dairy Science 85(2): 295–350. doi: 10.3168/jds. S0022-0302(02)74079-4.

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Jeschke, Peter, Ralf Nauen, Michael Schindler, and Alfred Elbert. 2011. Overview of the status and global strategy for neonicotinoids. Journal of Agricultural and Food Chemistry 59(7): 2897–2908. doi: 10.1021/jf101303g. Kauppila, Tiina J., Ville Arvola, Markus Haapala, Jaroslav Pól, Laura Aalberg, Ville Saarela, Sami Franssila, Tapio Kotiaho, and Risto Kostiainen. 2008. Direct analysis of illicit drugs by desorption atmospheric pressure photoionization. Rapid Communications in Mass Spectrometry 22(7): 979–85. doi: 10.1002/rcm.3461. Kauppila, Tiina J., Anu Flink, Markus Haapala, Ulla-Maija Laakkonen, Laura Aalberg, Raimo A. Ketola, and Risto Kostiainen. 2011. Desorption atmospheric pressure ­photoionization–mass spectrometry in routine analysis of confiscated drugs. Forensic Science International 210(1–3): 206–12. doi: 10.1016/j.forsciint.2011.03.018. Kauppila, Tiina J., Anu Flink, Ulla-Maija Laakkonen, Laura Aalberg, and Raimo A. Ketola. 2013. Direct analysis of cannabis samples by desorption atmospheric pressure photoionization-mass spectrometry. Drug Testing and Analysis 5(3): 186–190. doi: 10.1002/dta.1412. Kauppila, Tiina J., Anu Flink, Jari Pukkila, and Raimo A. Ketola. 2016. Analysis of nitrogen-based explosives with desorption atmospheric pressure photoionization mass spectrometry. Rapid Communications in Mass Spectrometry 30(4): 467–75. doi: 10.1002/rcm.7469. Luosujärvi, Laura, Ville Arvola, Markus Haapala, Jaroslav Pól, Ville Saarela, Sami Franssila, Tapio Kotiaho, Risto Kostiainen, and Tiina J. Kauppila. 2008. Desorption and ionization mechanisms in desorption atmospheric pressure photoionization. Analytical Chemistry 80(19): 7460–66. doi: 10.1021/ac801186x. Luosujärvi, Laura, Sanna Kanerva, Ville Saarela, Sami Franssila, Risto Kostiainen, Tapio Kotiaho, and Tiina J. Kauppila. 2010. Environmental and food analysis by desorption atmospheric pressure photoionization-mass spectrometry. Rapid Communications in Mass Spectrometry 24(9): 1343–50. doi: 10.1002/rcm.4524. Luosujärvi, Laura, Ulla-Maija Laakkonen, Risto Kostiainen, Tapio Kotiaho, and Tiina J. Kauppila. 2009. Analysis of street market confiscated drugs by desorption atmospheric pressure photoionization and desorption electrospray ionization coupled with mass spectrometry. Rapid Communications in Mass Spectrometry 23(9): 1401–4. doi: 10.1002/rcm.4005. Parshintsev, Jevgeni, Anu Vaikkinen, Katriina Lipponen, Vladimir Vrkoslav, Josef Cvačka, Risto Kostiainen, Tapio Kotiaho, Kari Hartonen, Marja-Liisa Riekkola, and Tiina J. Kauppila. 2015. Desorption atmospheric pressure photoionization high-resolution mass spectrometry: A complementary approach for the chemical analysis of atmospheric aerosols. Rapid Communications in Mass Spectrometry 29(13): 1233–41. doi: 10.1002/rcm.7219. Podgorski, David C, Rasha Hamdan, Amy M. McKenna, Leonard Nyadong, Ryan P. Rodgers, Alan G. Marshall, and William T. Cooper. 2012. Characterization of pyrogenic black carbon by desorption atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry 84(3): 1281–87. doi: 10.1021/ac202166x. Pól, Jaroslav, Vidová Veronika, Kruppa Gary, Kobliha Václav, Novák Petr, Lemr Karel, Kotiaho Tapio, Kostiainen Risto, Havlíček Vladimír, and Volný Michael. 2009. Automated ambient desorption−ionization platform for surface imaging integrated with a commercial Fourier transform ion cyclotron resonance mass spectrometer. Analytical Chemistry 81(20): 8479–87. doi: 10.1021/ac901368q.

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Räsänen, Riikka-Marjaana, Prabha Dwivedi, Facundo M. Fernández, and Tiina J. Kauppila. 2014. Desorption atmospheric pressure photoionization and direct analysis in real time coupled with travelling wave ion mobility mass spectrometry. Rapid Communications in Mass Spectrometry 28(21): 2325–36. doi: 10.1002/rcm.7028. Rejšek, Jan, Vladimír Vrkoslav, Robert Hanus, Anu Vaikkinen, Markus Haapala, Tiina J. Kauppila, Risto Kostiainen, and Josef Cvačka. 2015. The detection and mapping of the spatial distribution of insect defense compounds by desorption atmospheric pressure photoionization orbitrap mass spectrometry. Analytica Chimica Acta 886(July): 91–7. doi: 10.1016/j.aca.2015.06.007. Rejšek, Jan, Vladimír Vrkoslav, Anu Vaikkinen, Markus Haapala, Tiina J. Kauppila, Risto Kostiainen, and Josef Cvačka. 2016. Thin-layer chromatography/desorption atmospheric pressure photoionization orbitrap mass spectrometry of lipids. Analytical Chemistry 88(24): 12279–86. doi: 10.1021/acs.analchem.6b03465. Suni, Niina M, Henni Aalto, Tiina J. Kauppila, Tapio Kotiaho, and Risto Kostiainen. 2012. Analysis of lipids with desorption atmospheric pressure photoionizationmass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS). Journal of Mass Spectrometry 47(5): 611–19. doi: 10.1002/ jms.2992. Suni, Niina M., Pia Lindfors, Olli Laine, Pekka Östman, Ilkka Ojanperä, Tapio Kotiaho, Tiina J. Kauppila, and Risto Kostiainen. 2011. Matrix effect in the analysis of drugs of abuse from urine with desorption atmospheric pressure photoionizationmass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS). Analytica Chimica Acta 699(1): 73–80. doi: 10.1016/j. aca.2011.05.004. Vaikkinen, Anu, Juha Hannula, Iiro Kiiski, Risto Kostiainen, and Tiina J. Kauppila. 2015a. Transmission mode desorption atmospheric pressure photoionization. Rapid Communications in Mass Spectrometry 29(7): 585–92. doi: 10.1002/rcm.7139. Vaikkinen, Anu, Jan Rejšek, Vladimír Vrkoslav, Tiina J. Kauppila, Josef Cvačka, and Risto Kostiainen. 2015b. Feasibility of desorption atmospheric pressure photoionization and desorption electrospray ionization mass spectrometry to monitor ­urinary steroid metabolites during pregnancy. Analytica Chimica Acta 880(June): 84–92. doi: 10.1016/j.aca.2015.03.029. Vaikkinen, Anu, Henning S. Schmidt, Iiro Kiiski, Sari Rämö, Kati Hakala, Markus Haapala, Risto Kostiainen, and Tiina J. Kauppila. 2015c. Analysis of neonicotinoids from plant material by desorption atmospheric pressure photoionization-mass spectrometry. Rapid Communications in Mass Spectrometry 29(5): 424–30. doi: 10.1002/rcm.7123.

Chapter

4

Easy Ambient Sonic-Spray Ionization Mass Spectrometry in Food Analysis and Foodomics Rosana M. Alberici, Gabriel D. Fernandes, and Daniel Barrera-Arellano Fats and Oils Laboratory, Faculty of Food Engineering, UNICAMP

CONTENTS Introduction 75 Background and Concepts 76 EASI Sister Techniques 78 Venture-EASI 78 Spartan-EASI 79 Strategies for Minimum Sample Preparation 79 Applications 80 Vegetable Oils 80 Food Emulsifiers 82 Meat Products 84 Fish and Seafood 85 Propolis 86 Coffee Beans 87 Quantitative Analysis 88 Conclusions and Perspectives 88 References 89

INTRODUCTION The term “foodomics,” introduced by Cifuentes in 2009 [1], is defined as “the discipline that studies the food and nutrition domains through the application and integration of advanced omics technologies to improve consumer’s well-being, health, and confidence” [2]. In recent years, foodomics has attracted increasing attention reflected in a growing number of published research within this new field of the omics family [3] in which mass spectrometry (MS) techniques play the most important role [4]. MS techniques can be used in combination with separation techniques (e.g., with high-pressure liquid chromatography (HPLC), gas chromatography (GC), capillary zone electrophoresis) or in a way that, after a few steps of pretreatment, sample is directly

75

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Mass Spectroscopy Techniques in Food and the Environment

TABLE 4.1  Applications of EASI-MS in Foodomics Studies Application

Reference

Characterization of vegetable oils by triacylglycerol profiles Quality control and certification of geographical origin of vegetable oils Typification and quality control of Amazon oils (andiroba oil, Brazilian nut oil, and buriti oil) via triacylglycerol profiles Differentiation of unusual plant seed oil of the Brazilian Cerrado Characterization of antioxidant potential of Chilean chia seeds and oil (Salvia hispanica L) Monitoring of triacylglycerol oxidation in edible oils and fats Lipid profiles of muscle tissue (beef, fish, caviar, dry cured hams, and Nile tilapia) Characterization of food emulsifiers Fingerprinting of propolis Differentiating coffee postharvest methods by analysis of intact coffee beans

[18–20] [21,22] [23–26] [27] [28] [29,30] [31–34] [35] [36] [37]

submitted to MS analysis (direct MS). The latter approach is advantageous not only if short analysis times are required, as in the case of the high-throughput analysis, but also in cases where more extensive sample pretreatment procedures can affect the analytical result, so that the sample is analyzed in its original form. Ambient mass spectrometry (AMS), a subgroup within direct MS techniques, provides an even simpler approach, allowing direct access to mass spectra directly from the samples with no (or minimal) sample preparation [5–8]. This fact makes the AMS a useful tool, particularly when a large number of samples have to be analyzed, which is common in the field of omics. Since the beginning of AMS, the analysis of food and food-related samples has always been one of its major fields of application [9,10]. However, when browsing through the relevant publications, one will notice that in most cases, an AMS technique is employed for the detection of a single (or a limited number) of predefined analytes within a food matrix. So, the large majority of these publications, although of importance for food chemists, do not follow a typical omics approach. Nevertheless, within the multitude of food-related AMS applications, some go beyond the scope of “single component” analysis, heading for a more comprehensive picture of the investigated sample and therefore may qualify to be discussed in the context of foodomics [11]. Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) technique [12] along with other AMS techniques, such as desorption electrospray ionization (DESI), direct analysis in real time (DART), and paper spray (PS), has been the most widely used in foodomics studies [13–15]. EASI-MS has also been used in the assessment of food quality and authenticity [16,17]. In Table 4.1, a number of applications of EASI-MS are listed that may be categorized according to these requirements.

BACKGROUND AND CONCEPTS EASI-MS technique, developed by Eberlin and coworkers in 2006 [12], is certainly the simplest AMS ionization technique since no temperature or voltage is required to promote

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the ionization of the molecules. Additionally, an EASI source can be easily built in a mass spectrometry laboratory without any special requirement, and therefore, it is low-cost. Due to its high versatility, EASI source can be adapted to any mass spectrometer, from a single quadrupole to a high-resolution ion cyclotron resonance (ICR) or Orbitrap. Structurally, an EASI source is constructed from a “T”-capillary ­connection (Figure  4.1c), in which the solvent is pumped through one end and the gas flows through the second; both, solvent and air, go out through the third end in a high velocity, enabling desorption and ionization of the sample. Since EASI source can be easily hand-constructed, the optimization of some parameters of the source is very important to reach the best performance of the sonic-spray [12,16]. In general, a polar solvent, such as methanol, is pumped at 20 µL/min and the gas, commonly N2 , but even air, flows at 3 L/min. Regarding the ionization mechanism, it is based on the sonic-spray ionization process (SSI), described as the first voltage, radiation, and temperature-free ionization technique by Hirabayashi and coworkers in 1994 [38]. The high-velocity flow of gas and solvent generates high shearing force, which promotes the formation of very tiny droplets with an unbalanced distribution of cations and anions. Thus, these minute droplets consist of a low-density bipolar stream, enough for molecules ionization. The ionization itself occurs when the solvent evaporates, the droplet collapses, and the charges are transferred to the molecules, making available the ionized molecules into the gas phase. A desorption step occurs before the ionization process, which the bipolar stream droplets strike the analyzed sample, on a surface, desorbing the molecules from the sample support by a droplet pickup process (Figure 4.1a,b). After easy ambient sonic-spray ionization, analytes can be analyzed by both positive and negative MS, EASI(+) and EASI(−), respectively. The most common EASI(+) ionization is cationization, by means of the formation of cation adducts, mainly with sodium, [M + Na]+, potassium, [M + K]+, and ammonium, [M + NH4]+, although the common

FIGURE 4.1  (a) Schematic diagram of EASI(±); (b) the ions that take place in the

­ionization/desorption process; and (c) detailed EASI source.

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protonation, [M + H]+, occurs as well. The deprotonation ionization [M – H]− is the most common process in the EASI(−); however, anionization can also happen. The molecular ion formed depends directly on the molecule structure and deserves to be studied in each case. In 2014, Fernandes and coworkers [39] described the ionization efficiencies of lipids. The authors compared, by experimental analysis and metadynamic calculations, the preferences of triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG), as well as phosphatidylcholine on the formation of Na and K adducts. Unsaturated lipids have a stronger interaction with Na and K than the saturated ones, due to the dipole–dipole interactions in the double bonds; in other words, unsaturated lipids have a more efficient ionization. When compared TAG to phosphatidylcholine, they have an equal behavior, the same ionization efficiencies, on the contrary to MALDI (matrix-assisted laser desorption/ionization), in which, using DNB (3,5-dinitrobenzoic acid) matrix, phospholipids suppress TAG. One of the most important features of the EASI-MS technique is its ability to analyze intact molecules, i.e., with no fragmentation, due to soft ionization provided by SSI. This fact significantly simplifies the mass spectra in which each ion corresponds to a molecule. The bipolar stream positively and negatively charged droplets also enables the analysis of both EASI(+) and EASI(−) in the same experiment, excluding the necessity of acidified or basified solvents [17]. All these features are advantages that make possible the analysis of complex mixtures with no, or reduced sample preparations [16,17]. The sample preparation, as well as the applications of EASI-MS technique to complex mixtures, will be further discussed.

EASI SISTER TECHNIQUES Based on the same ionization mechanism, other ionization techniques have been developed, called EASI sister techniques. These techniques were developed to facilitate, even more, the EASI application, reducing solvent consumptions and mainly becoming reality the application of EASI into mass spectrometer devices. The first sister technique was Venturi-EASI (V-EASI) [40], and the second was Spartan-EASI (S-EASI) [41]. During the next two topics, we will describe both techniques in details, and the schematics of both techniques is shown in Figure 4.2. Venture-EASI V-EASI is by concept a self-pumping ionization technique in which the Venturi effect is used to replace the solvent syringe pump. The Venturi effect is originated when a high-velocity fluid flows through a constricted section of a pipe, thus causing a reduction in fluid pressure and the self-suction effect. In this way, the solvent pump is withdrawn, and the free end of the capillary is dipped into a solvent (or analyte solution), which is “sucked” into the EASI system and self-pumped to the spray end. Thus, V-EASI can be used in two modes: in the first mode, the free end of the capillary is placed into a solvent that is bombarded against the sample to the desorption ionization of analytes from the surface; in the second mode, the solvent is replaced by the analyte solution, and in this case, the system sprays and ionizes the molecules to the mass spectrometer [40].

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FIGURE 4.2  (See color insert after page 124.) Schematic and actual pictures of the

V-EASI (above) and S-EASI (below) systems. Spartan-EASI The named S-EASI is not the most powerful ionization technique; however, it is the simplest and less expensive ionization source already described. S-EASI is able to work with no electricity supply or cylinders of compressed gas, pressure regulators, and even the T-capillary connection. A compressed air can replaces the gas cylinder and the regulators, while a two-way catheter replaces the T-capillary connection. As in V-EASI, the solvent pump is replaced by the Venturi effect. This device configuration enables the application of EASI in portable and compact mass spectrometers. It is very useful to perform in situ analysis in forensic and food authentication activities [41].

STRATEGIES FOR MINIMUM SAMPLE PREPARATION EASI-MS can be carried out directly on the sample surface; however, for liquid and tissues samples, such as meat products, it is not possible. In this way, some strategies were developed to reach high-throughput minimum sample preparations. Many surfaces have been tested as sample support, such as glass, Teflon, brown envelope paper, and silica TLC plates [16]. For liquid samples, such as vegetable oils, brown envelope paper was, until now, the best surface [18]. TLC plate coated with silica brings a different approach that can be used as previous step for sample separation/purification and direct EASI application [36]. In order to access specific analytes from complex food matrices without an extra extraction step, for instance, lipids from meat products, thermal imprinting (TI) protocols have been developed [31]. TI-EASI has been specifically applied to replace solvent protocols for lipid extraction, such as Folch and Bligh & Dyer. Under the application of TI-EASI, the lipid fraction can be achieved in a few minutes and just before the analysis, as a sample pretreatment. The lipid content is imprinted on a piece of paper through the application of few solvents and a heater. The heater device is made of a 150 W halogen bulb, and the solvent is based on the Bligh & Dyer mixture (MeOH–CHCl3,

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FIGURE 4.3  (See color insert after page 124.) Schematic of TI-EASI procedure for the

MS analysis of meats, fats, and related products such as caviar and ham. 2:1 v/v). For TI-EASI application, a small piece of the sample (ca. 1 cm 2 , 1 mm thick) is placed over a paper surface, four droplets of the solvent mixtures are dropped over the sample, and after that, the heater device is applied during 20–90 s. During this process, a mass transference of lipids occurs from the sample to the paper and it can be traditionally analyzed by EASI-MS. The schematic of TI-EASI-MS protocol is shown in Figure 4.3. The other technique named VALDI [37] was also developed to enhance the penetration into the sample surface, enabling more efficient biomarkers desorption from intact samples. The sample is treated with a laser source in the free entrance of the Venturi effect, and the EASI source is coupled to a commercially available API source to complete the ionization process. Until now, VALDI was applied in only one study to analyze coffee beans, the reason why we still consider it an underdevelopment technique. These strategies are very important to make the application of EASI-MS possible in different and complex food matrices; thus, new strategies have been constantly developed.

APPLICATIONS EASI-MS and its sister techniques have been applied with success to the analysis of different analytes and matrices in the food analysis and foodomics, as shown in Table 4.1. Its applications have been recently reviewed [16,17], and some examples are described below.

Vegetable Oils Vegetable oils are undoubtedly one of the matrices most studied by EASI-MS, perhaps due to the unique ability of this technique to provide simple, fast but comprehensive origin certification, quality control, and adulteration detection [21,22]. Not only most common vegetable oils such as soybean, palm, olive, hazelnut, canola, and grape seed oils but also exotic oils, for instance, from the Amazon jungle have been well directly characterized (no derivatization or extractions) by rich profiles of TAG, DAG, MAG, and free fatty acids (FFA) from a single oil droplet [18]. Figure 4.4a shows an unique profile of soybean oil dominated by [TAG+Na]+ ion attributed to PPL (C50:2, m/z 853), PPO (C50:1, m/z 855), PLL (C52:4, m/z 877), PLO (C52:3, m/z 879), POO (C52:2, m/z 881), LLLn or OLnLn (C54:7, m/z 899), LLL or OLLn (C54:6, m/z 901), OLL or OOLn

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FIGURE 4.4  Representative EASI(+)-MS of vegetable oils: (a) soybean oil and (d) ­oxidized soybean oil; (b) olive oil and (e) oxidized olive oil; (c) andiroba oil and (f) oxidized ­andiroba oil.

(C54:5, m/z 903), OOL (C54:4, m/z 905), and OOO (C54:3, m/z 907) (Figure 4.4b), where P = palmitic acid, O = oleic acid, L = linoleic acid, and Ln = linolenic acid. These profiles of TAG obtained by EASI-MS have also been found to closely reflect the profiles and relative concentrations of TAG in the original samples as revealed by classical GC-MS or LC-MS runs and to match known compositions of FFA. The TAG profiles of such oils are therefore a fast way to access their most abundant composition of FA. EASI-MS fingerprinting has also been demonstrated as a valuable tool for the rapid classification and quality control of less common oils such as those from the Amazon jungle [23–26] as exemplified for the andiroba, açaí, urucum, buriti, and Brazil nuts oils. The EASI(+)-MS profile for andiroba oils (Figure 4.4c) was found to be dominated by TAG containing mainly palmitic, as shown by the abundance of the ion of m/z 855 (PPO), oleic, and linoleic acids. Brazil nut oil – an important natural product from the Amazon – with several health benefits and applications in food and cosmetic industries, has also been characterized by EASI(+)-MS. Several samples of Brazil nut oil including authentic oils from different geographical origins, commercial oils, and some adulterated with soybean oil were analyzed [24]. The relative abundance of the ion of m/z 899, which

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corresponds to LLLn and/or OLnLn, [C54:7], was used to monitor adulteration through the addition of soybean oil. For this goal, Brazil nut oil was spiked with soybean oil (2%, 5%, 10%, 30%, and 50%), and the samples were analyzed by EASI(+)-MS. Indeed, the marker ion of m/z 899 had its relative abundance steadily increased as a function of blend composition. Consequently, by using this marker ion, an adulteration level as low as 5% of soybean oil in Brazil nut oil could be easily detected by EASI-MS. Plant seed oils of the Brazilian Cerrado [27] have also been characterized by the EASI(±)-MS profiles of TAG and FFA. The species Jatropha curcas, Bombacopsis glabra, Capparis flexuosa, Siparuna guianensis, Moringa oleifera, Hibiscus tiliaceus, Virola bicuhyba, Pouteria caimito, and Syagrus coronata were found to exhibit a predominance of TAG composed of the most common FA, that is, palmitic, oleic, stearic, and linoleic acids. Two exceptions were clearly observed: V. bicuhyba and S. coronata seed oils. These unique oils were found to be composed mainly of TAG containing lauric acid. These short-chain TAG make these two oils potential ingredients for spreads and toppings. EASI-MS has also been used to examine the maturation of Jatropha curcas L. seeds by monitoring changes in the profile of TAG of their oil [20]. The profiles of TAG were observed to substantially change during the maturation and drying processes. The profiles remained however nearly unaltered during storage monitoring. Another interesting application of EASI-MS is for the monitoring of the oxidation mechanism of edible oils [28]. Oxidation of such oils is the major problem during storage, since it may compromise its quality and nutritional value, resulting for vegetable oils in a product with a typical undesirable rancid aroma. The oxidation of TAG involves a complex set of autocatalytic reactions yielding TAG hydroperoxides as primary products. EASI(+)-MS has successfully been used to monitor such processes, and the results obtained through this methodology were compared to those obtained via the classical Rancimat test for oil oxidation. The oxidation levels of oils were continuously determined during the accelerated oxidation process, as well as the final overall oxidation. The detection of TAG mono-, bis-, and tris-hydroperoxide products was used for this purpose (Figure 4.4d,f). The relatively high degree of unsaturation of soybean oil makes this oil quite susceptible to oxidation. Thermally oxidized soybean oil and used frying oil were monitored by EASI(+)-MS, and the characteristic profiles of cations related to hydroperoxide oxidation products as well as DAG ions were observed. Food Emulsifiers Food emulsifiers are a very important group of additives for the food industry. They are applied in almost all formulated and/or processed food since they act between water and oil interface, enabling emulsion formations. Besides that, emulsifiers have a great influence on the sensory characteristics, such as texture and mouthfeel, as well as shelf life stability. Many groups of compounds have been developed and proposed to be used as food emulsifiers, such as MAG, DAG, sorbitan esters, and phospholipids. Phospholipids are commonly and commercially known as lecithins [42]. Vegetable lecithins were first developed as a by-product of the oil industry. Phospholipids are withdrawn from vegetable oils during the degumming process. Nowadays, soybean lecithin is the most applied lecithin in the food industry, mainly due to its low cost and high availability. Many types of lecithins are now available to food applications with different percentage of phospholipids as well as chemical modifications. In general, lecithins are chemically composed of a mixture of different phospholipids

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such as phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidylinositols, phosphatidylglycerols, phosphatidic acid, glycophospholipids (GPL), and their lysoforms [43]. Lecithins chemical characterization demands very laborious procedures such as incineration and/or acetone solubilization. Taking it account, Fernandes and coworkers [35] applied EASI-MS technique to direct characterization of the main lipid components from lecithins. Samples of commercial lecithins as well as standard, refined, deoiled, and modified soy lecithin were analyzed, and they showed characteristic profiles of PC and TAG by EASI(+)-MS. TAG molecules, corresponding mainly to TAG from soybean oil, were observed between 850 and 1,000 m/z range. PC molecules were observed between 750 and 850 m/z range being that the major compounds detected were PC 18:2/18:2 and/or 18:1/18:3 (m/z 820), PC 18:1/18:2 and/or 18:0/18:3 (m/z 822), PC 18:0/18:2 and/ or 18:1/18:1 (m/z 824), PC 16:0/18:2 (m/z 796), PC 16:0/18:1 (m/z 798), and LPC (PC lysoform) between 500 and 600 m/z range being LPC 18:2 (m/z 558) observed in an enzymatically hydrolyzed sample. EASI(−)-MS complemented the chemical information by providing profiles of PE, GPL and FFA. As expected, the FFA profile was very usual for soybean oil. Regarding phospholipids PE, lysophosphatidylethanolamine (LPE), acetylated phosphatidylethanolamine (PEAcet), and GPL were also detected (Figure 4.5).

FIGURE 4.5  (See color insert after page 124.) EASI-MS for commercial lecithin samples. Positive ion mode: (a) acetylated lecithin (SOLEC AA); (b) hydroxylated lecithin (SOLEC AE IP); (c) deoiled lecithin (SOLEC F). Negative ion mode: (d) acetylated lecithin (SOLEC AA); (e) hydroxylated lecithin (SOLEC AE IP); (f) deoiled lecithin (SOLEC F). Main positive ions assignment: m/z 558 (LPC 18:2); m/z 796 (PC 16:0/18:2); m/z 820 (PC 18:2/18:2 and/or 18:1/18:3); m/z 822 (PC 18:1/18:2 and/or 18:0/18:3); m/z 893 (PLL); m/z 919 (OLL/ OOLn). Main negative ions assignment: m/z 279 (linoleic acid); m/z 476 (LPE 18:2); m/z 518 (LPEAcet 18:2); m/z 714 (PE 16:0/18:2); m/z 738 (PE 18:2/18:2 and/or 18:1/18:3); m/z 756 (PEAcet 16:0/18:2); m/z 780 (PEAcet 18:2/18:2 and/or 18:1/18:3); m/z 833 (GPL).

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EASI-MS can be successfully applied to carry out a complete analysis of the lipidic components of lecithins and their products. Besides that, it is possible to extend this lipidomic approach to perform quality control analysis and/or process analysis since the modifications applied to commercial lecithins such as acetylation and enzymatic hydrolysis were clearly detected by a direct EASI evaluation. Meat Products Porcari and coworkers [31] have developed the previously described TI-EASI-MS, which enable to imprint the lipid content on a paper surface to later analyze by EASI-MS. This special procedure is very simple and takes less than 2 min, in a simplified way, using a few droplets of solvents. It is very revolutionary for the application of the foodomic concept in a complex matrix by AMS. Meat samples such as beef, bovine fat, chicken, pork, mutton, and fish (that will be further discussed) were analyzed by this approach, showing a quite diverse fingerprint. Except beef and bovine meat fat that have a very similar profile which POO TAG was the most abundant in both samples, however, detected as m/z 897 [POO + K]+ in the beef sample and as m/z 881 [POO + Na]+ in the bovine fat sample. The chicken and pork samples were marked by the presence of a significant amount of the ion of m/z 879 (PLO) in the spectra. However, when comparing both samples, the ion of m/z 879/881 (PLO/POO) ratio was very different. In the chicken samples, PLO was higher than POO while in the pork sample, the opposite was noticed (Figure 4.6a). The mutton profile was characterized by the presence of ion of m/z 883 (PSO) due to the high amount of stearic acid in the fatty acid profile of this tissue. During the development of this work, the authors have also demonstrated that it is possible to keep the lipid content on the paper with no loss of the chemical information. This is another advantage of this methodology since it is possible to perform a remote TI-EASI analysis. Fernandes and coworkers [33] have also applied TI-EASI(+)-MS to access the traceability and authenticity of dry-crude hams. The lipid content of five dry-crude ham samples was determined in order to establish a relationship between the lipid profile, the pig feed, and the ripening time. [FFA − H]− and [TAG or DAG + Na]+ profiles were detected by Parma, Serrano, Cebo, Recebo, and Bellota hams samples. Regarding the FFA, Bellota ham has presented higher amounts of oleic acid of m/z 281 and lower amounts of palmitic and linoleic acids, of m/z 255 and m/z 279, respectively. These results were directly related to pig feed since pigs directed to Bellota ham production are feed with a high oleic feed source. The same relationship was noticed in the TAG results, and Bellota sample displayed a higher relative abundance of triolein (OOO, of m/z 907) than the other samples. Besides that, other TAG were detected as PPL (m/z 853), PPO (m/z 855), PLL (m/z 877), POL (m/z 879), POO (m/z 881), OOL or LLS (m/z 905), OOO or OLS (m/z 907), and OOS or SSL (m/z 909) (Figure 4.6b). The TI-EASI-MS approach was also used to differentiate the samples according to the ripening time, showing a relationship between profile and the technological characteristics of the samples. Lipid metabolites of the ripening reactions that are very important to the development of ham flavors, especially DAG resulting from the TAG hydrolysis, were detected. The main [DAG + Na]+ detected were PO (m/z 617) and OO (m/z 643). The shorter ripening times samples, Parma and Serrano, were those with the low relative abundance of PO and OO. Cebo, Recebo, and Bellota samples had similar levels of DAG, and these samples were those with the largest ripening times. Bellota ham had a special fingerprint, and it was the only sample with a higher amount of OO than PO. This high

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FIGURE 4.6  TAG profile of diverse muscle products obtained by TI-EASI(+)-MS.

(a) beef, beef fat, chicken, and pork; (b) Parma, Cebo, Recebo, and Bellota hams. OO amounts in Bellota ham are also related to pig feed as it was previously noticed in TAG and FFA [33]. In conclusion, the application of TI-EASI-MS with a foodomic approach is a very promising technique, mainly for lipidomics evaluations. It is possible to correlate TI-EASI result to meet source and technological process, as well as applied it to quality control and development of new products. Fish and Seafood Fish TAG profiles from trout, salmon, and sardine were obtained by Porcari and coworkers [32] using TI sample preparation and further EASI-MS analysis. As well as for meats, TAG were the main molecule detected with very characteristic fingerprints, highlighting the presence of arachidonic (AA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) fatty acids. In the Trout sample, the most abundant ions were m/z 895 and 897, assigned as the POL and POO, respectively. For the salmon sample, the amounts of TAG with EPA and DHA were very characteristic, and in this way, O-L-EPA (m/z 925) and S-O-DHA (m/z 927) were the most abundant. In the Sardine sample, besides EPA and DHA, the presence of TAG with AA was also characteristic. As for the feeding characteristics, Bonafé and coworkers [34] showed that the effect of the diet on the incorporation of conjugated linoleic and α-linolenic fatty acids (CLA) into muscle tissue could be monitored by EASI-MS. The consumption of these

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supplemented fishes can contribute to best nutritional characteristics to consumers. In this study, Oreochromis niloticus (Nile tilapia) were fed with a diet supplemented with tung oil in comparison with a diet with sunflower oil. The incorporation of CLA into the tilapia muscle was observed after 10 days of supplementation, and besides that, changes in the metabolic pathways were also noticed as an improvement of EPA and DHA into the muscles. The profiles of FFA and chemometric analysis were used to group samples according to the type of diet employed for fish growth. TI-EASI-MS has also been used to monitor degradation as a function of the conservation protocol and compositional changes from different storage conditions (i.e., time and temperature) in caviar samples of Atlantic sturgeon (Acipenser sturio) [32]. The shelf life of freshly salted and commercially salted pasteurized caviar samples was monitored in the common conservation temperature, at 4°C, and under acceleration condition, at 25°C. Salted and pasteurized caviar showed different lipid profiles mainly related to the abundance of ions containing DHA (22:6) or EPA (20:5) such as POO (m/z 881), P-O-DHA or O-O-EPA (m/z 927), and P-P-DHA or P-O-EPA (m/z 901). Changes in the lipid profile from storage at room temperature and in the refrigerator could also be monitored as a function of the abundance of ions assigned as PC such as PC 16:0/18:1 (m/z 782), PC 16:0/20:5 or 18:2/18:3 (m/z 802), and PC 16:0/22:6 or 18:1/20:5 (m/z 828). During the shelf life experiment, a decrease in the PL ions was noticed, indicating a hydrolysis process of the PL in the samples, and DAG and MAG were generated as hydrolysis products (Figure 4.7). Propolis Propolis is a resin byproduct from honeybee production; bees produce and utilize propolis as an aseptic solution to keep the hive free of microorganism, and besides that, the application of propolis in the combs reinforce its structure. In this way, propolis has several functional applications such as antibacterial, antifungal, antioxidant, antiviral, and anticancer. However, these properties are closely related to the chemical composition which has a high variation depending on the group of plants from which the resin is obtained. Propolis characterization is therefore extremely important for its standardization, practical applications, and therapy. Sawaya and coworkers [36] evaluated the chemical fingerprint of ethanolic extracts of propolis by EASI(−)-MS, using TLC as sample surface. The authors have studied 49 samples from North and South America, Europe, Asia, and Oceania. Ions of interest were found only between 150 and 650 m/z range, and the results were submitted to principal component analysis (PCA) for the direct, fast, and reliable determination of the most important metabolites according to the origin, and determine the plant origins of their resins. Over half the samples analyzed had compounds derived from Populus resins as characteristic ions, thus indicating a clear preference for this plant genus by Apis mellifera bees. Propolis derived from Populus resins displayed ions related to dihydroxyflavone and other well-known flavonoids, such as chrysin, pinocembrin, apigenin/galangin, pinobanksin, caffeic acid phenethyl ester, and pinobanksin acetate. In Mexico and Brazil, stingless bees frequently use resins from different plant sources in relation to A. mellifera bees. Propolis resins originating from these bees showed therefore a different composition. The EASI-MS data allowed the grouping of samples for similar plants and geographical origins, enabling the identification of the plant sources of propolis resins. Besides that, EASI-MS procedure has several advantages such as simple (no) sample preparation, reproducibility, and the amount and quality of the results enabling even the identification of the metabolites and even the resin plant source [36].

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FIGURE 4.7  TAG profile of diverse muscle products obtained by TI-EASI(+)-MS. (a)

truta, (b) salmon, (c) sardine, and (d) caviar. Coffee Beans Coffee is one of the most commercialized food products and the most widely consumed beverage in the world. Coffee is also considered, a functional food because of its high content of antioxidants and other beneficial components. Coffee is an essential agricultural commodity, especially in Brazil, which is the largest producer. The analysis and especially those directed to the control of technological process are very laborious and time-­consuming procedures. Recently, Garret and coworkers [37] proposed to apply

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EASI-MS to characterize intact coffee beans, without any sample preparation, and use this high-throughput technique to differentiate technological procedures of postharvest. EASI-MS was found to function as fast and simple protocols to differentiate beans treated by the dry, semidry, and wet postharvest methods. Five coffee phytomarkers were monitored and three of them (βN-arachinoyl-5-hydroxytryptamide, βN-behenoyl5-hydroxytryptamide, and βN-lignoceroyl-5-hydroxytryptamide) were identified as components of the wax layer that covers the coffee bean, and these compounds are generally associated with stomach irritation in sensitive persons who consume coffee drinks. Using PCA, it was indeed possible to follow the differences among the coffee beans according to the postharvest treatments. EASI-MS could be applied to control and certification processes, mainly by using portable mass spectrometers that could be applied for field analysis.

QUANTITATIVE ANALYSIS Although EASI-MS has been widely applied in qualitative food analysis, it has also been shown to provide proper quantification. FFA can be quantified in crude vegetable oils by EASI(−)-MS, using internal standards, with reasonable linearity (r = 0.98) [18], allowing to access the acidity of vegetable oils. EASI(+)-MS seems to offer not only an appropriate qualitative tool for oil analysis but also a precise and detailed way to perform quantification as demonstrated by the determination of the composition of TAG of edible vegetable oils, hydrogenated vegetable fats, and cocoa butter [19]. Results were compared with those obtained by gas chromatography–flame ionization detection (GC-FID) and also by theoretical predictions of the composition of TAG performed by a software projection after detection. Acceptable correlation coefficients were observed between the three methods during the analysis of vegetable oils and hydrogenated vegetable fats. The reproducibility of the quantitative results was ca. 10%–15%. However, one should always consider that the main purpose when applying EASI-MS in food analysis is not to perform a comprehensive composition analysis or precise quantification but to mostly provide data on typification and quality control via characteristic chemical signatures.

CONCLUSIONS AND PERSPECTIVES EASI-MS and its sister techniques, because of its unique combination of figures of merits, including superior simplicity and voltage-free environment, as well as with no sampling preparation, seem to be quite attractive for food analysis. After a decade of investigations, several applications of EASI(±)-MS to food analysis have been demonstrated, and their main findings have been summarized in this chapter. The use of such a simple and robust technique, which can be most conveniently coupled to compact/portable mass spectrometers, may also facilitate on-site food screening. Such a monitoring could, for instance, be performed by control agencies directly on production sites or provide fast analysis in the laboratory in the cases of contamination, fraud, or authenticity check. The quite universal application of such techniques also allows their use in a myriad of different food products. A new, rapid, simple, quite comprehensive, and efficient tool is now available for food analysis at the molecular level.

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Section

III

Electric Discharge Ambient Ionization

Chapter

5

Direct Analysis in Real Time Mass Spectrometry Semih Otles and Vasfiye Hazal Ozyurt Ege University and Near East University

CONTENTS Introduction 95 Principle of the Technique and Instrumentation 96 Optimization of the Dart Technique 97 Application in Food Quality and Safety 98 Application in Enviromental Analysis 100 Conclusion and Future Outlook 100 References 100

INTRODUCTION Mass spectrometry (MS) is one of the most powerful techniques for food analysis because of its high resolution and/or fragment ions information. However, traditional MS such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) has some problems such as matrix effect, ionization efficiency, and tedious sample pretreatments. Moreover, both of them show the complexity and expertization of instrument operation and maintenance. LC-MS is a technique which is time/labor consuming and solvent wasting because of the requirements of hard sample preparation and various chemical reagents, whereas GC-MS also has complex sample preparation. To overcome these problems, new ionization techniques were used, and these techniques are collectively known as ambient MS. As a kind of ion source, direct analysis in real time (DART) possesses various merits. DART can be used in analyzing liquid, solid, and gas samples consisting of either polar or nonpolar compounds. Moreover, DART allows rapid introduction of analytes into MS with a minimal or even without sample preparation in most cases. Furthermore, DART ionization source works at atmosphere conditions instead of a vacuum (Guo et al., 2017). The working principle of DART is that a heated gas flow goes through the main chamber, and the plasma of ions, electrons, metastable atoms, and molecules can be produced in that chamber. Most of the charged particles are screened from the neutral gas molecules by the next grids to go to the orifice. This review has focused on summarizing the applications of food quality and safety assurance, and detection of trace compounds in food and environmental samples during production, processing, and storage and transportation by DART-MS.

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PRINCIPLE OF THE TECHNIQUE AND INSTRUMENTATION DART is a mass spectrometric atmospheric pressure ion source. In that ion source, solids, liquids, and gases ionize in open air under ambient conditions. DART has started to use as a commercial product since 2005 (Cody et al., 2005a; Laramee and Cody, 2006). DART can work the atmospheric pressure interactions of long-lived electronic excitedstate atoms or vibronic excited-state molecules with the sample and atmospheric gases (Cody et al., 2005b). The working principle of the DART ion source is that a gas flows through an enclosed chamber and generates ions, electrons, and excited-state neutral species (atoms and molecules). The gases such as helium, argon, and nitrogen can be used. The enclosed chamber has intermediate lenses or grids. Moreover, the excited-state species pass through, but neutral gas atoms/molecules remain in that chamber (Cody, 2009). The grid electrode which has positioned to that chamber helps to remove ions with opposite polarity to prevent signal loss by ion–ion interaction and ion–electron recombination (Cody et al., 2005b). The DART ion source can be formed in both positive and negative modes. These ions depend on the nature of the gas, ion polarity, and whether dopants are present (Cody et al., 2005a). In positive ion mode, molecular ions (M+.) are formed for low polarity or nonpolar molecular compounds when nitrogen is used, whereas protonated [M + H]+ cations are typically observed when helium is used. When an ammonia source is present nearby, the DART source adducts have also been observed [M + NH4]+. In negative ion mode, mass spectra are mainly dominated by deprotonated [M − H]− anions for most compounds while some negatively charged ions (M−.) are observed for specific compounds. Other adducts, such as [M + Cl]−, are observed when a suitable dopant is used (Cody et al., 2005a). Several ionization mechanisms in DART have been presented by Cody et al. (2005a). The first ionization mechanism is referred to as Penning ionization. Penning ionization can explain the transfer of energy from an excited gas M* to an analyte A with an ionization potential lower than the energy of M* (Equation 5.1) (Cody et al., 2005a):



A + M* → A +· + M + e− (5.1)

Second ionization mechanism can occur through the proton transfer mechanism and explain the interaction of helium metastables (He, 23S), and atmospheric water vapor generates water clusters, and this reaction can be followed by proton transfer reactions (Equation 5.2) (Cody et al., 2005b):

( )

( )

H 2O + He 23 S → He 11 S + H 2O+· + e− H 2O + H 2O+ → H3O+ + OH − H3O+ + nH 2O → ( H 2O) nH 

+

+



( H 2O) nH  + A → AH + + nH 2O

(5.2)

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The third mechanism is known as electron capture. In that mechanism, electrons (e−) that are produced by Penning ionization or surface Penning ionization are thermalized by collision with atmospheric pressure gas (Equation 5.3): M* + surface → M + surface + e− − e−fast + gas → eslow



− eslow + O2 → O2−

(5.3)

The fourth mechanism is the transient microenvironment mechanism. A transient microenvironment (TME) is generated the analytes from direct ionization by the DART gas stream. Then, the TME may be created through desorption and ionization of volatile matrix molecules (containing the analyte), and analytes are ionized by the matrix ion species through gas-phase ion/molecule reactions (Song et al., 2009). This reaction mechanism is a nine-stage reaction mechanism. He* + H 2O → He + H 2O+ + e− H 2O+· + ( H 2O)m → HO·+ ( H 2O)m + H 

+

He* + S → He + S+· + e− S+ + Sn → [ S − H ] + [ Sn + H ] −

+

+

( H 2O)m + H  + Sn → ( H 2O)m + [ Sn + H ]

+

[Sn + H ]+ + A → Sn + [ A + H ]+ + S+· + A → [ S − H ]· + [ A + H ]

S+· + A → S + A +· +



( H 2O)m + H  + A → ( H 2O)m + [ A + H ] (5.4) +

OPTIMIZATION OF THE DART TECHNIQUE Like other detection methods, the parameters including both DART and MS should be optimized to supply the required amount of analyte ions and higher sensitivity, detectability, and reproducibility (Hajslova et al., 2011). The temperature of an ionization gas is one of the most important factors which affect the experimental results. For that reason, the ionization gas temperature could be optimized. Moreover, the optimization of the ionization gas temperature should be supplied for the required intensity of analyte ions. After the optimization of the temperature of ionization gas, the position of the sample in the DART-ionization region is

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another important factor. To achieve the best efficiency, the sample should be placed in an upright position close to the DART-gun outlet, and slightly below the centerline of the ionization-gas plume (Harris et al., 2011). Another optimization parameter is the presence of dopant vapors in the sampling region, and the dopant vapors can help in the formation of ions from analytes, which are impossible to ionize with DART technology (Zhao et al., 2008). The impact of matrix effects is considerably an important factor for the optimization of DART ion source. Many researchers attempted to elucidate the nature of matrix effects in DART (Zhao et al., 2008; Yu et al., 2009; Song et al., 2009), and a matrix effect is defined as a physical or chemical process that inhibits or enhances detection or proper quantification of an analyte in the presence of one or more matrix species (Shelley and Hieftje, 2010). Matrix effects were evaluated during the analysis of mycotoxins in wheat or maize extracts using DART coupled to an ultrahigh-resolution mass spectrometer (Vaclavik et al., 2010b). Signal suppression was observed in crude acetonitrile–water extract, and these were removed by adding primary secondary amine sorbent. Moreover, even after the impurities were removed, the signals of the mycotoxins in the pure extracts were 12%–39% of the analyte intensity in solvent. The same authors found matrix effects to be pronounced in another study that they carried out to determine melamine and cyanuric acid in milk powder using DART-TOF-MS (Vaclavik et al., 2010a). Therefore, it is worth noting that the matrix effects limit the quantification capabilities of the technique (Gross, 2014). The “Application in Food Quality and Safety” section discusses the application of DART-MS in food quality and safety.

APPLICATION IN FOOD QUALITY AND SAFETY DART has been used for a multitude of applications because of its introduction as a readily available commercial product with the versatility in ionizing a wide range of chemicals without the need of extensive sample preparation. Vaclavik et al. (2009) developed a novel approach for the authentication of olive oil (OO) samples representing different quality grades and used DART, coupled to a high-resolution time-of-flight-mass spectrometer (TOF-MS). The comprehensive profiling of triacylglycerols (TAGs) and/or polar compounds extracted with a ­methanol–water mixture were detected. Direct analysis in real time-time-of-flightmass spectrometry (DART-TOF-MS) achieved differentiation among extra virgin olive oil (EVOO), olive pomace oil, and OO and supplied EVOO adulteration with commonly used adulterant. Cajka et al. (2008) described using DART-TOF-MS and obtaining negative and ­positive ion profiles of different soft drinks to determine the presence of various compounds, including antimicrobial preservatives, artificial sweeteners, acidulants, and ­saccharides, without any sample preparation and chromatographic separation. Lara et al. (2017) detected a number of highly polar pesticides in complex samples such as lettuce and celery using the Quick Polar Pesticides Extraction (QuPPe) method and DART. This method proved to be a fast tool with quantitative capabilities for at least seven compounds: amitrole, cyromazine, propamocarb, melamine, diethanolamine, triethanolamine, and 1,2,4-triazole. Fraser et al. (2013) studied the biochemical composition of oolong tea during the manufacturing and fermentation process using a nontargeted method utilizing ambient ionization with a DART ion source and MS.

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Alam et al. (2017) reported the characterization of TAGs and fatty acids in Leucaena (Leucaena leucephala) oil. DART ion sources were successfully applied and investigated to obtain the best detection limit with high-quality mass profile. Singh et al. (2012) characterized the diversity in terms of metabolite profiles of Cinnamomum tamala Nees and Eberm genotypes using DART ion sources. The result explained that the genotype of C. tamala could be differentiated using DART-MS data. Bajpai et al. (2010) used to detect phenols and their acetates such as chavicol, allylpyrocatechol, chavibetol, chavicol acetate, allylpyrocatechol acetate, chavibetol acetate, and allylpyrocatechol diacetate DART ion source in Piper betle, whereas Wang et al. (2014) detected saccharides such as glucose, mannose, galactose, rhamnose, arabinose, xylose, sucrose, trehalose, maltose, cellobiose, lactose, raffinose, and ginseng t­ etrasaccharide in Panax ginseng. Edison et al. (2011a) used DART-MS to analyze pesticides in apple, orange, and grape samples. About 132 pesticides were totally detected, and LODs were calculated as 2 ng/g (86%), 2 ng/g (86%), and 10 ng/g (86%) for apples, oranges, and grapes, respectively. Then Edison et al. (2011b) used DART-MS to detect pesticides in apple, kiwi, and peach samples, and LODs were calculated as 10, 10, and 10 ng/g, respectively, as well as 140, 60, and 132 kind of pesticides were analyzed respectively. Crawford and Musselman (2012) detected insecticides in cherry tomatoes, peaches, and oranges, and LODs were found as 20, 80, and 20 ppb, respectively. Martínez-Villalba et al. (2013) determined veterinary drug residues in milk and chicken feed and found 20 benzimidazoles and 4 coccidiostats, respectively. Kiguchi et al. (2014) detected organophosphorus insecticides in dumplings and grapefruits, whereas Wang et al. (2012) detected insecticides in agrochemicals. Kern et al. (2014) used to detect the pesticides in apples, oranges, and broccoli. Danhelova et al. (2012) used DART-MS to detect the amount of caffeine in coffee and coffee beverages. LODs were found as 0.1 ug/ml in extract. Bai et al. (2012) detected phytohormones in fruit juices, whereas Kim and Jang (2009) did curcuminoids in functional drink. In addition to those aims, DART-MS can be used to detect processing additives. Vail et al. (2007) detected melamine in pet food, while Haefliger and Jeckelmann (2007) were detected cooling agents in chewing gum. Moreover, Chernetsova and Morlock (2012) and Rajchl et al. (2013) were used to detect 5-HMF as processing by-product in honey and other processing by-products such as polycyclic aromatic hydrocarbon, polybrominated diphenyl ether, and polychlorinated biphenyl in fish and shrimps were detected by Kalachova et al. (2011). Busman et al. (2014) used to detect aflatoxin in corn. Bentayeb et al. (2013) found photoinitiators in food packing using DART-MS. Curtis et al. (2009) detected in drywall sulfur-containing materials using DART. Borges et al. (2009) studied to detect the organometallic compounds. Domin et al. (2010) screened insoluble polycyclic aromatic hydrocarbon contaminant. Ackerman et al. (2009) identified the food packaging additives including plasticizers, antioxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Vaclavik et al. (2010a,b) detected melamine and cyanuric acid contamination in powdered milk. Vaclavik et al. (2010a,b) and Zachariasova et al. (2010) used a DART ion source to detect mycotoxins in cereals and beer, respectively. Schrage et al. (2013) used to detect food adulteration in Chinese star anise. When Japanese star anise level was 1%, adulteration was determined using DART-MS. Cajka et al. (2013) detect chicken bone meal as an adulterant in chicken muscle. When adulterated level was 5%–8%, DART-MS was an excellent method.

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APPLICATION IN ENVIROMENTAL ANALYSIS DART‐MS can be used for the qualitative and quantitative analysis of pollutants in environmental contaminants derived from chemical industries, agricultural practices, or human personal care products. Zhou et al. (2015) applied DART-MS to the study of gas–surface heterogeneous reactions; moreover, the kinetics of a well-studied reaction of surface-bound polycyclic aromatic hydrocarbons with ozone were presented. Wang et al. (2012) applied to the rapid and direct analysis of any material (gases, liquids, and solids) with minimal or no sample preparation. A pilot study of rapid qualitative determination of hazardous pesticides was performed. DART-MS technology was able to qualitatively determine the existence of highly hazardous pesticides in commercial pesticide formulations. Bridoux et al. (2015) presented for the extraction of phosphoric acid alkyl esters [tri-(TnBP), di-(HDBP), and mono-butyl phosphate (H2MBP)] from aqueous samples using new SBSE/DART/Orbitrap-MS method. The application of this new SBSE/DART/ Orbitrap-MS method is valuable for on-site sampling/monitoring, limiting the transport of large volumes of water samples from the sampling site to the laboratory. DART-MS was also successfully used to determine seven organic UV filters in water. Stir bar sorptive extraction was used to preconcentrate the samples. However, it was reported that semiquantitative reports were obtained, and limits of detection were poorer than those obtained with thermal desorptive-gas chromatography-mass spectrometry (TD-GC-MS) (Haunschmidt et al., 2010). It is also worth noting that the technique has also been successfully used in the semiquantitative analysis of contaminants in soil (Grange, 2013), and analysis of aerosols and microsized particles (Nah et al., 2013).

CONCLUSION AND FUTURE OUTLOOK DART-MS has been used for the direct, rapid, and advantageous analysis of simple/ medium-complexity analytes in food samples or extracts with minimal or sometimes no sample preparation as well as has proved to be an effective and complementary alternative to prevalent chromatography-MSn strategies for food and environmental samples. Food safety-related indicators, veterinary drugs, pesticides, microbial metabolites, and enzymatic hydrolysates are investigated by DART-MS. Its versatility has been demonstrated by its ability to analyze compounds with different polarities, and molecular masses. However, a problem that still needs to be addressed is matrix effects. Moreover, emerging acute pathogenic analytes in food samples such as persistent organic pollutants like PCBs, and biomacromolecules such as some fatal bacterial/viral proteins, need to be investigated in detail. Furthermore, currently, the identification of genetically modified organisms in foods is of the most importance. Therefore, in the future, DART-MS could become the most chosen method for monitoring many of the aforementioned target analytes with/without the incorporation of matrix and desorption ionization methods.

REFERENCES Ackerman, L. K., Noonan, G. O., Begley, T. H. Assessing direct analysis in real-timemass spectrometry (DART-MS) for the rapid identification of additives in food packaging. Food Addit. Contam. 2009, 26, 1611–1618.

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Chapter

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Low-Temperature Plasma Ionization Sandra Martínez-Jarquín and Robert Winkler CINVESTAV Unidad Irapuato

CONTENTS Principles of Low-Temperature Plasma Ionization 105 Ionization and Desorption Mechanisms 106 LTP Probes and Coupling to Analyzers 108 Portability 111 Operational Costs and Environmental Footprint 111 Applications in Environmental Analyses 112 Organic Compounds in Air 113 Detection of Explosives 113 Food Analysis Applications 114 Direct Detection of Organic Compounds from Food Products 114 Plant Oil Analyses 114 Classification of Coffee Products 115 Plant Tissue Imaging 115 Detection of Agrochemicals 117 Melamine Contamination in Milk Products 118 Authenticity of Agave Spirits 119 Determination of Micronutrients 120 Concluding Remarks 120 Acknowledgments 120 Conflict of Interest 120 References 120

PRINCIPLES OF LOW-TEMPERATURE PLASMA IONIZATION The term “plasma” refers to gases with a significant proportion of particles in a non-­ neutral state (Langmuir, 1928). Plasma is the most common state of matter in the ­universe (Peratt, 1995) and can be observed on Earth (e.g., in lightning and “the polar fountain”). Artificially produced plasma is used in fluorescent lamps, TVs, and welding. Plasmas can be classified according to their observed temperature: “cold” or ­“low-temperature” plasmas (LTPs) have temperatures up to 104 K, whereas “hot” or “hightemperature” plasmas (HTPs) have temperatures of 107 K and above (Rutscher, 2008).

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The plasma is “nonthermal” if the plasma temperature is about ambient (below ~40°C) (Meichsner, 2005; Rutscher, 2008). In nonthermal plasmas (NTPs), the electron temperature is much higher than the ion and neutral gas temperature. As a consequence, the temperature of the plasma system is near the ambient temperature. In mass spectrometry (MS), NTPs are commonly called “LTP,” and this term will be used in the following text. Figure 6.1 shows an infrared (IR) image of an operating three-dimensional (3D)-printed LTP probe (Martínez-Jarquín, Moreno-Pedraza, Guillén-Alonso, & Winkler, 2016). The gas bulk temperature was determined to be about 28°C, which can be considered ambient. Whereas plasmas have been used for surface treatment since the 1970s (Chan, Ko, & Hiraoka, 1996; Collins, Lowe, & Nicholas, 1973) and for medical applications since the 2000s (Laroussi, Alexeff, & Kang, 2000), it was not until 2008 that its potential was recognized as an ion source for instrumental analysis, especially for MS (Harper et al., 2008). Many analytical applications have been reported since 2008, because LTP works for solid, liquid, and gaseous samples, is suitable for imaging, has low technical requirements, and is capable of ionizing a wide range of chemically diverse compounds (Harper et al., 2008; Liu et al., 2010; Martínez-Jarquín & Winkler, 2017). After presenting the fundamentals of LTP ionization and desorption processes as well as the state of the art of the technology, we present examples of the use of LTP-MS for environmental and food analyses. Ionization and Desorption Mechanisms For the analysis of molecules by LTP ionization coupled to MS under ambient conditions, four important aspects can be identified: (1) plasma generation and plasma processes,

FIGURE 6.1  (See color insert after page 124.) IR image of a “nonthermal,” or LTP probe. The apparent tip temperature of 28°C allows the direct sampling from heat-­sensitive surfaces (Martínez-Jarquín, Herrera-Ubaldo, Folter, & Winkler, 2018).

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FIGURE 6.2  Important mechanisms in LTP ionization coupled to MS (Martínez-Jarquín & Winkler, 2017).

(2) ionization and fragmentation of molecules, (3) desorption and surface reactions, and (4) ion transfer. These LTP processes are summarized in Figure 6.2. Plasma is a very reactive mixture consisting of multiple reactive species, such as free electrons, positively charged and metastable particles. The possible ionization m ­ echanisms include the ionization by electrons, Penning ionization, associative ionization, charge transfer reaction, photoionization, electron capture, and combinations with excitation processes IUPAC Gold Book Release 2.3.3b., 2017; Meichsner, 2005; Murray et al., 2013). Several reactions involve ambient molecules, such as N2 , H 2O, and O2 , and lead to the generation of reactive species, such as N2+ and H3O. Ozone is built in two steps, the second being a three-body reaction: (1) O2 + e− → 2 + e−; (2)  + O2 + M → O3 + M (Eliasson, Hirth, & Kogelschatz, 1987). Those ozone products were shown to form oxidation products with the terpene limonene (Nørgaard, Kofoed-Sørensen, Svensmark, Wolkoff, & Clausen, 2013). Frequently, clusters and adducts of the analyzed molecules (M) are found, such as [2M + H]+, [3M + H]+, and [M + H 2O]+, which can lead to very complex mass spectra. Compared to electrospray ionization (ESI) and atmospheric pressure chemical ­ionization, LTP is able to ionize more hydrophobic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and imines (see Figure 6.3) (Albert & Engelhard, 2012). Although the molecular weight range is limited to about 500 m/z, most biologically ­relevant classes of organic compounds can be ionized and detected by LTP-MS (GamboaBecerra et al., 2015). In addition to ionization, fragmentation of small molecules and peptides is possible. The mean kinetic energy of energy-carrying particles is a few eV (Meichsner, 2005), which results in less fragmentation compared to “harder” ionization sources, such as electron impact (EI) with 70 eV (Martínez-Jarquín & Winkler, 2017). Fragmentation spectra obtained by LTP ionization are similar to collision-induced dissociation data (Gamboa-Becerra et al., 2015; Xia, Ouyang, & Cooks, 2008). Desorption of nonvolatile molecules can occur either through thermal desorption or by momentum transfer (Venter, Nefliu, & Graham Cooks, 2008). Due to the relatively low temperature of the gas flow, the desorption of molecules from solid surfaces can be difficult to achieve by LTP alone. However, the detection of compounds with limited

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FIGURE 6.3  LTP is able to ionize compounds of relatively low molecular weight in a broad polarity range (Albert & Engelhard, 2012).

volatility can be improved by additional heating, such as heat plates (Albert, Kramer, Scheeren, & Engelhard, 2014; Jackson et al., 2010; Liu, Lin, Zhang, Yang, & Zhang, 2009) or lamps (Chen et al., 2013). Some examples will be presented below. The transfer of produced ions to the analyzer is another critical step that is not fully understood yet. In general, the fast separation of oppositely charged particles should avoid the neutralization of formed ions and improve the signal intensity. Charges can be lost by electron–ion, ion–ion, and electron–ion–neutral recombinations (Meichsner, 2005). For a more detailed review of plasma ion processes, please consult the reference by Martínez-Jarquín and Winkler (2017). LTP Probes and Coupling to Analyzers Currently, the commercial availability of LTP probes is just taking off. The still limited distribution of LTP devices is a major drawback for the broad application of this ambient ionization technique. However, interested MS users can construct an LTP probe without much difficulty. Multiple LTP probe designs have been reported that are often optimized for specific uses, such as for screening large surfaces, for MS imaging (MSI), or for gas sampling. We have reviewed different instrumental setups recently (Martínez-Jarquín & Winkler, 2017). All plasma probe designs are based on a dielectric barrier discharge principle. However, the individual constructions and setups are very diverse with respect to materials, discharge gas, electronic parameters, geometry, and dimensions. Therefore, to improve the reproducibility of LTP-MS studies, we proposed a set of parameters that should be documented (Figure 6.4 and Table 6.1) (Martínez-Jarquín & Winkler, 2017). A functional LTP probe can be assembled from 3D-printed and commercial parts (Figure 6.5) (Martínez-Jarquín et al., 2016). The use of this design is free for noncommercial use (Creative Commons Attribution-NonCommercial 4.0 International Public License http://creativecommons.org/licenses/by-nc/4.0/legalcode). However, intellectual property issues have to be considered for commercial applications (Martínez-Jarquín & Winkler, 2016). The company NovionX GmbH (www.novionx.de, Lindau, Germany) recently presented PlasmaChip that produces a radio frequency-driven plasma. Figure 6.6 illustrates the mounting of PlasmaChip in front of a mass spectrometer and a spectrum from the analysis of a cocoa preparation.

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FIGURE 6.4  Important parameters for LTP measurements. The variables used in this

drawing are listed in Table 6.1 (Martínez-Jarquín & Winkler, 2017). TABLE 6.1  Parameters for LTP Probe Setups Symbol V f Q ⌀C ⌀IE y α b d x

Parameter Voltage Frequency Gas flow Capillary diameter Inner electrode diameter Outer electrode length Probe angle Distance (outer electrode to plasma probe tip) Distance (inner to outer electrode) Distance (plasma probe to instrument inlet)

References and typical value ranges can be found in Martinez-Jarquin and Winkler (2017).

LTP probes have been coupled to various types of mass analyzers, such as linear ion trap (Harper et al., 2008), 3D ion trap (Martínez-Jarquín et al., 2016), quadrupole (Martínez-Jarquín & Winkler, 2013b), and Fourier transform ion cyclotron resonance spectrometers (Benassi et al., 2013). As demonstrated in Figures 6.5 and 6.6, the vacuum of the mass spectrometer can be used to suck the generated ions into the analyzers. For simple experiments, placing the sample and the LTP probe in front of the MS cone is sufficient. An extended ion-transfer tube as shown in Figure 6.5 facilitates advanced applications, such as MSI. Theoretically, there is no restriction of shape, size, or material for the analyzed samples (Liu et al., 2010). Usually, LTP probes are used for direct ambient analyses, for example, methods without upstream separation. Nevertheless, placing an LTP probe between a gas chromatograph and an EI mass analyzer delivered additional structural information for the identification of molecules (Nørgaard, Vibenholt, Benassi, Clausen, & Wolkoff, 2013). The coupling of LTP to ion mobility spectrometry (IMS) was demonstrated as well (Jafari, 2011; Kuklya et al., 2015). The analytical resolution of IMS is lower compared to MS, but IMS also has several advantages: (1) No vacuum is necessary for the analyzer, which drastically reduces the cost of the system and also facilitates the development of portable devices. (2) The technology of IMS devices is much simpler than the technology

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FIGURE 6.5  Direct analysis of a strawberry with a 3D-printed LTP probe (MartínezJarquín & Winkler, 2017).

FIGURE 6.6  Commercial LTP probe PlasmaChip with the mass spectrum of a raw cocoa

sample (contribution of Ralf Dumler, NovionX GmbH). of MS analyzers, which supports the rapid prototyping of economical and “fieldable” equipment. (3) IMS analyses provide the geometry of molecules as an additional analytical dimension, which helps to distinguish between isobaric species if the IMS resolution is sufficient. The sequential arrangement of IMS and MS analyzers also might be an option

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to quickly extract desired ions from an LTP ionization mixture and to optimize the ion transfer (Martínez-Jarquín & Winkler, 2017). Portability LTP sources do not require a solvent flow and are therefore pump-free. The necessary discharge gas can be provided by a small gas cylinder. Alternatively, atmospheric air can be used as a discharge gas instead of noble gases, eliminating the need of transporting a gas cylinder. However, the composition of air leads to spectra with more fragmentation signals (Harper et al., 2008). The power consumption of high-voltage circuits employed for LTP is usually a few watts (Martínez-Jarquín & Winkler, 2013b) easily provided by a battery. Hence, all components of a LTP source are suitable to develop portable systems. Figure 6.7 shows a small, portable, mass spectrometer with LTP ionization for the detection of pesticides in nanogram amounts. The probe can be operated with helium or air as a discharge gas (Wiley, Shelley, & Cooks, 2013). A complete LTP-MS system for the detection of illicit drugs, explosives, and chemical warfare agents (tested with analogs) was designed to fit into a backpack. The entire system weighed about 12 kg and consumed 65 W that allowed about 1.5 h of continuous use with the included battery (Hendricks et al., 2014). LTP probes are ideal for the development of mobile analysis devices. The miniaturization of mass analyzers is more difficult because of the necessity of a vacuum and the related weight and energy consumption. Operational Costs and Environmental Footprint The construction costs of LTP probes depend on the specific model and the requirements of the experiment. Simple probes can be built from basic commercial laboratory materials. As power supply, a car ignition coil or a plasma toy kit can be used (Harper et al., 2008; Martínez-Jarquín & Winkler, 2013b). In addition, a gas cylinder with volumetric flow control is required. A LTP probe can be fabricated for a few hundred U.S. dollars.

FIGURE 6.7  A handheld LTP ionization probe that can be coupled to different mass

analyzers (Wiley et al., 2013).

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Such a simple LTP probe works fine for casual use, such as rapid identification testing of a few samples. Technically more demanding experiments require components of higher quality. For example, MSI requires a small LTP jet diameter and a stable plasma for several hours. Whereas the regulation of the volumetric gas flow is relatively easy, optimizing the (electronic) parameters and the LTP probe design, as well as coupling to the MS analyzer, can be time-consuming and costly. To start, a commercial high-voltage alternating current voltage power supply and a 3D-printed LTP probe (Martínez-Jarquín et al., 2016) can be tested and optimized. If the jet diameter is not critical, the PlasmaChip (NovionX GmbH, Lindau, Germany) could be an option. The operational costs and the environmental impact of analyses are extremely low. The consumption of electric energy is only a few watts and therefore negligible. In most cases, no sample pretreatment is necessary, which saves solvents, chemical reagents, consumables, and time. Some ozone and reaction products with surrounding media are formed during the operation of the LTP source, but the concentration of those compounds should not be hazardous to the staff in well-ventilated laboratories. Moderate purity of the discharge gases is acceptable, since the plasma will interact with atmospheric molecules anyway. Therefore, the main cost factor is the initial setup of the LTP system. The running costs and the environmental footprint are insignificant.

APPLICATIONS IN ENVIRONMENTAL ANALYSES LTP probes enable the detection of a wide range of compounds that are relevant for ­environmental and food analyses. LTP ionization can be applied to solid, liquid, and gaseous samples, which is beneficial for environmental applications. Moreover, complex samples can be analyzed without preprocessing. Figure 6.8 shows the direct analysis of the stomach contents of a deceased dog. The LTP-MS analysis with N2 as a discharge gas clearly demonstrates the presence of ions corresponding to terbufos and terbufos sulfoxide, typical ingredients of terbufos-based pesticides (Harper et al., 2008).

FIGURE 6.8  LTP-MS analysis of the stomach content of a deceased dog indicates poisoning with a terbufos-based insecticide (Harper et al., 2008).

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In the next sections, examples for the use of LTP-MS in environmental monitoring will be given. Organic Compounds in Air Outdoor pollution is responsible for about 3.3 million cases of premature death, with carbon-containing particles being the most toxic (Lelieveld, Evans, Fnais, Giannadaki, & Pozzer, 2015). Surprisingly, ~72% of the indoor air pollution in residences of asthmatic children in New York City could be contributed to indoor sources, such as household activities (i.e., cooking, cleaning) and smoking (Habre et al., 2014). To reduce health hazards for humans, more studies regarding the toxicity of different types of particles and air quality monitoring in the most affected areas are necessary (Lelieveld et al., 2015). Using a simple LTP-MS setup with air as a discharge gas, the organic compounds formaldehyde, ethanol, acetone, and benzene could be detected in a mixture demonstrating its potential for indoor monitoring (Gong et al., 2011). The suitability of LTP-MS for real-time analysis of aerosols containing organic particles that originate from the pyrolysis of organic materials was also shown. Since those organic combustion products are among the most dangerous air contaminants, LTP-MS has a possible application in air quality and pollution studies (Spencer, Tyler, Tolocka, & Glish, 2015). Detection of Explosives Explosives are used for industrial uses (mining), but mainly for military activities. Since some of the commonly used substances, such as nitroacrylic compounds, degrade slowly, they contaminate the environment for long periods, accumulate in organisms, and may have toxic effects (Juhasz & Naidu, 2007; Lima, Bezerra, Neves, & Moreira, 2011). Since the very beginning of LTP-MS research, the monitoring of so-called homeland security compounds was investigated. The nitro group-containing explosives 2,4,6-­trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine were detected from a variety of surfaces with LTP-MS (Harper et al., 2008; Zhang et al., 2009). Related compounds (possible degradation products of explosives) could be identified from different matrices—PTFE (polytetrafluoroethylene), latex, fabric, glass, and paper—with limits of detection of picograms to nanograms (García-Reyes et al., 2011). The sensitivity for the detection of explosives by LTP-MS was enhanced by heating with a halogen lamp, which enables the measurement of compounds with low volatility. The method enabled the direct detection of 2,4-dinitrotoluene, 2,6-dinitrotoluene, and TNT in effluents of an explosives manufacturing plant (Chen et al., 2013). Addition of humid air to the discharge gas could further enhance the detection of explosives by LTP-MS, to reach sensitivities at low picogram levels (Chen, Hou, Hua, Xiong, & Li, 2014). A portable (backpack) LTP-MS system to detect chemical warfare agents was reported by Hendricks et al. (2014). The reported applications of the sensitive detection of pollutants from solid, ­liquid, and gaseous matter demonstrate the high potential of LTP-MS in environmental monitoring.

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FOOD ANALYSIS APPLICATIONS LTP-MS is ideal to obtain chemical fingerprints of complex food preparations in a short period of time (2–10 s per sample). The complete spectra can be used in a variety of ways, such as authenticity investigations, using specific quality markers, or detecting potentially hazardous substances above critical limits. However, ambient ionization MS methods in general has certain limitations concerning quantification. Typically, LTP-MS would be used for high-throughput screening applications followed by conventional analysis of suspicious samples by standard methods, such as gas chromatography (GC) or liquid chromatography (LC), coupled to MS (GC-MS and LC-MS). Various applications for food analyses will now be presented. Direct Detection of Organic Compounds from Food Products Due to the ionization characteristics of LTP, a broad range of organic compounds can be detected directly from food products, such as alcohols, aldehydes, alkaloids, alkanes, alkenes, arenes, capsaicinoids, esters, ketones, pyrazines, sulfur-containing substances, and terpenes. The mass range is limited to about 500 m/z (Gamboa-Becerra et al., 2015). The main limitation for the analysis of substances from organic materials is the desorption from the respective matrix, although ions are successfully generated. For coffee, it was shown that modulating the plasma temperature changed the mass spectra of the analytes. At a lower temperature, volatile compounds such as vanillin (important for the flavor) and guaiacol (“burnt” taste) were detected. Increasing the plasma temperature, less volatile substances and degradation products, such as caffeine and pyridine, were found (Martínez-Jarquín & Winkler, 2013a). The volatility of target molecules has to be kept in mind when optimizing the technical parameters of LTP-MS methods. Plant Oil Analyses Vegetable oils are frequently used in cooking. Besides a pleasant taste and the contribution of energy, plant oils are associated with nutraceutically beneficial effects, such as the prevention of cardiovascular diseases (Mozaffarian, 2013). Olive oil is very popular in Mediterranean regions and offered in different qualities and prices. Mixing expensive “extra virgin” olive oil with lower quality “virgin” and “lampante” oil is a fraudulent practice, which is difficult to detect since the chemical characterization of the vegetable oils is challenging and therefore costly. Direct analysis with LTP-MS led to the detection of typical oil constituents, such as free fatty acids (oleic, linoleic, and palmitic acids), phenolics (p-hydroxybenzoic acid, syringic acid, coumaric acid, ferulic acid, tyrosol, and hydroxytyrosol), and volatiles (hexanal, 2-hexen-1-ol, and 2-hexenal) (García-Reyes et al., 2009). Since signals above 500 m/z are weak with LTP ionization, information about the abundant di- and triacylglycerides is largely missing (Figure 6.9). To detect these compounds, paper spray (PS) ionization with the addition of Ag+ represents an alternative ambient ionization method (Lara-Ortega et al., 2018). MSI of cardamom (Amomum subulatum) seeds with an LTP microprobe revealed the presence of a likely fragment of 1,8-cineole, a main constituent of its essential oil (Bowfield et al., 2014), thus supporting the usability of LTP-MS for vegetable oil analyses.

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FIGURE 6.9  LTP-MS spectra (a–c) were generated in negative mode from hydroalcoholic extracts of extra virgin, virgin, and “lampante” oil, respectively (Lara-Ortega et al., 2018).

Classification of Coffee Products Coffee is one of the most popular beverages worldwide and is an important commercial product. Besides the plant-related parameters (i.e., genotype, geography, farming), the processing of the green beans is crucial for the “cup quality” and the commercial value of the final product. LTP-MS fingerprints demonstrate the distinct chemical composition in different stages of production (see Figure 6.10). Notably, LTP-MS permits the measurement of volatiles, such as vanillin, which are important for the sensorial quality of coffee. Combining the direct MS analyses with data mining strategies enabled the development of predictive classification models as well as the discovery of “important” variables for certain characteristics. Therefore, LTP-MS could be employed in process optimization and monitoring in coffee manufacturing (Gamboa-Becerra et al., 2017). Plant Tissue Imaging LTP ionization can be used for the nondestructive analysis of sensitive surfaces. Liu et al. (2010) investigated Chinese works of art by LTP-MS imaging to prove the authenticity

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FIGURE 6.10  (See color insert after page 124.) Classification of coffee products by hierarchical cluster analysis. Roasted (left) and lyophilized (right) are clearly separated by their LTP-MS data fingerprints (Gamboa-Becerra et al., 2017).

of the colors used (Liu et al., 2010). LTP-MS also allows for the spatiality-defined detection of compounds on surfaces or tissue sections for studying the metabolism of plants of agronomic interest and other uses. The diameter of the plasma beam is crucial for imaging applications, because it defines the maximum lateral resolution of the analyses. With a so-called microplasma probe, a jet diameter below 200 µm could be obtained (Bowfield et al., 2014). However, the signal intensity of mass analyses is reduced with the beam diameter. Further, higher spatial resolution increases the volume of MS data, making the data storage and processing more difficult without necessarily providing additional biological information. Therefore, an optimum beam diameter has to be found. Using a double dielectric barrier design of the probe and 3D-printed parts, the jet diameter can be adjusted to the needs of the application (Martínez-Jarquín & Winkler, 2013b, 2016; Martínez-Jarquín et al., 2016). The sample has to be displaced relatively to the LTP probe to create images. Moving the probe seems less advisable, because the orientation of the LTP probe and the ion transfer to the mass analyzer should be kept fixed to maintain stable signals.

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FIGURE 6.11  (See color insert after page 124.) Imaging of volatile and semivolatile compounds in a cross-cut section of chili (Capsicum annuum) pepper (Gamboa-Becerra et al., 2015).

With  simple  linear  server motors controlled by a USB (Universal Serial Bus) gadget, a resolution of 300 µm has been achieved making simple ambient MSI applications possible (Maldonado-Torres, López-Hernández, Jiménez-Sandoval, & Winkler, 2014). Figure 6.11 shows the LTP-MSI analysis of a cross-cut section of a chili pepper (Capsicum annuum). More than 100 compounds were localized in the different anatomic structures of the fresh tissue. Importantly, volatile and semivolatile metabolites could be detected that are lost in MSI techniques requiring vacuum conditions (Gamboa-Becerra et al., 2015). The chemical profile accessible by LTP-MSI is complementary to conventional methods, such as matrix-assisted laser desorption/ionization, but no sample pretreatment is necessary. Therefore, LTP-MSI is a highly attractive option for biological MS imaging. Detection of Agrochemicals Agrochemicals, such as pesticides, represent a serious hazard to food consumers. Therefore, most countries have established limits for allowed concentrations of ­pesticides in consumables. Due to the high toxicity of those compounds, the permitted maximum residual limits (MRLs) are low enough to assure safe accumulations ingested by

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the consumers. For example, the harmonized Regulation EC 396/2005 of the European Commission specifies a general default MRL of 0.01 mg/kg, if the specifications for a pesticide are not explicitly listed in the regulations (see https://ec.europa.eu/food/plant/ pesticides/max_residue_levels/eu_rules_en). The low concentrations and the chemical diversity of pesticides make the quantification of pesticides difficult. Usually, the compounds are extracted and enriched by the so-called Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) strategy (Anastassiades, Lehotay, Stajnbaher, & Schenck, 2003) and subsequently analyzed by analytical reference methods, such as GC or LC coupled to MS. A comparative study between direct LTP-MS and a conventional method (QuEChERS followed by LC-MS/MS) for 13 pesticides (ametryn, amitraz, atrazine, buprofezin, DEET (N,N-diethyl-3-methylbenzamide), diphenylamine, ethoxyquin, imazalil, isofenphosmethyl, isoproturon, malathion, parathion-ethyl, and terbuthylazine) demonstrated high detection sensitivity of LTP-MS for most of the compounds. Remarkably, detection of contamination levels below the MRLs was possible by the direct measurement of fruits and vegetables, and quantification over four orders of magnitude was shown for some substances. Although the analytical performance of the reference methods is not achieved, the fast LTP-MS strategy could serve as a screening tool in the food industries (Wiley et al., 2010). Meticulous evaluation of the analytical performances of LTP-MS and LC-ESI-MS methods for the quantification of acetamiprid, cyprodinil, fenhexamid, and fludioxonil in fruits confirmed the suitability of LTP-MS to reveal sub-MRL contaminations. Sample work-up using the QuEChERS method improved the limit of quantification for LTP-MS (Albert et al., 2014). Diphenylamine could be detected on apples by a direct LTP-MS technique with a portable handheld MS. Also, thiabendazole on oranges could be analyzed with this equipment using a simplified PS method (Soparawalla, Tadjimukhamedov, Wiley, Ouyang, & Cooks, 2011). The fungicides azoxystrobin, carbendazim, dimethomorph, fenhexamid, flusilazol, metalaxyl, penconazole, tebuconazole, imazalil, and thiabendazole could be quantified in sub-MRL concentrations in wine by LTP-MS after diluting the samples with acetonitrile (Beneito-Cambra, Pérez-Ortega, Molina-Díaz, & García-Reyes, 2015). The examples above demonstrate the usability of LTP-MS for the direct detection of multiclass agrochemicals from fresh or processed food. LTP-MS is limited for quantitative analyses; therefore, additional analyses with reference methods are necessary to confirm whether consumables exceed permitted MRLs. Nevertheless, the fast LTP-MSbased screening allows for more sampling along the food manufacture/distribution chain, thus improving the consumers’ safety. Melamine Contamination in Milk Products In 2008, the intentional admixture of melamine in baby formula poisoned more than 53,000 children, killing four. Melamine, an industrial product that is associated with the formation of kidney stones, was added to pretend a higher protein content in diluted milk products (Xin & Stone, 2008). LTP coupled to MS with fragmentation (MS/MS) demonstrated the possibility to detect and quantify melamine in trace amounts from the complex matrices (Huang, Ouyang, & Cooks, 2009).

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Coupling an LTP probe to a handheld miniature mass spectrometer enabled the rapid screening of melamine with critical concentrations. Nanogram levels of melamine could be detected in milk preparations, fish meat, and synthetic urine. The relative error was up to 23%, which is reasonable for a direct ambient method, especially considering the high throughput of about 120 samples per hour (Huang, Xu, Visbal-Onufrak, Ouyang,  & Cooks, 2010). Authenticity of Agave Spirits Tequila and mezcal are Mexican spirits that are produced by the distillation of cooked and fermented agave broths. 100% tequila is manufactured exclusively from blue Agave (Agave tequilana Weber var. azul). Tequila “mixto” may contain up to 49% sugars from other sources, usually from different agave species. In stark contrast, mezcal may be made from any agave species. Whereas tequila production is well defined (fermentation with defined yeasts, final product specifications, etc.), mezcal is often prepared under rustic conditions. Thus, the quality and safety of mezcal is extremely variable. The quality of tequila can also vary, because the fraudulent addition of cheaper spirits to tequila is a common practice. Two direct MS methods, direct liquid injection (DLI)-ESI and LTP ionization, were tested for their suitability to discriminate between tequila and mezcal. Classification is ­possible with both methods after applying a Random Forest algorithm. As illustrated in Figure 6.12, the important variables for differentiating between tequila and mezcal are minor signals. LTP-MS data were more variable than DLI-ESI-MS data, but the former analysis only took 20 s per sample, thus allowing for automated high-throughput measurements. Besides the possibility of quickly detecting counterfeit products by LTP-MS, this example demonstrates the power of data mining methods to extract relevant information from noisy MS data (Martínez-Jarquín, Moreno-Pedraza, Cázarez-García, & Winkler, 2017).

FIGURE 6.12  (See color insert after page 124.) Important mass signals for the differentiation between tequila and mezcal spirits, based on LTP-MS data. The most relevant variables are present in relatively low abundance (Martínez-Jarquín et al., 2017).

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Determination of Micronutrients The quantification of the essential micronutrients folic acid, iron, retinol, thyroxine, and zinc in biological samples was tested using LTP-MS and ESI-MS. All five compounds were detectable from a bovine serum albumin matrix using LTP-MS. Samples of “healthy” and “unhealthy” individuals were simulated by adding different physiological micronutrient concentrations to porcine blood plasma. Mixtures with different micronutrient content could be distinguished by principal component analysis, which indicates a potential use of LTP-MS in the quick evaluation of the nutritional state of animals and humans (Stein, Lo, Castner, & Ratner, 2012).

CONCLUDING REMARKS LTP ionization coupled to MS already provides a wide range of possible applications in environmental and food analyses. The lack of commercial probes on the market is currently the greatest obstacle for the development and adoption of LTP-MS methods. However, industry-grade products should be available on the market soon. The versatility of LTP-MS with respect to the range of detectable molecules and type of samples as well as the low technical requirements and high possible throughput supports the use of LTP-MS for analytical procedures in “green foodomics” (Gilbert-López, Mendiola, & Ibáñez, 2017). With cost-sensitive applications and improved analytical performance in combination with MS, the coupling of LTP to IMS has a high potential for future applications.

ACKNOWLEDGMENTS The work was supported by the CONACyT Fronteras Project 2015-2/814 and the bilateral grant CONACyT-DFG 2016/277850. SMJ acknowledges their CONACyT postgraduate scholarship. We also thank Carolyn Smith of Peace Corps Response for proofreading the manuscript.

CONFLICT OF INTEREST SMJ and RW hold a patent on the ionization probe (Nonthermal plasma jet device as source of spatial ionization for ambient mass spectrometry and method of application, US 9362100 B2, June 7, 2016). RW is an advisory board member of the company NovionX GmbH, Germany.

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Murray, K. K., Boyd, R. K., Eberlin, M. N., Langley, G. J., Li, L., & Naito, Y. (2013). Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013). Pure Appl. Chem. 85(7), 1515–1609. doi:10.1351/PAC-REC-06-04-06 Nørgaard, A. W., Kofoed-Sørensen, V., Svensmark, B., Wolkoff, P., & Clausen, P. A. (2013). Gas chromatography interfaced with atmospheric pressure ionization-­ quadrupole time-of-flight-mass spectrometry by low-temperature plasma ionization. Anal. Chem. 85(1), 28–32. doi:10.1021/ac301859r Nørgaard, A. W., Vibenholt, A., Benassi, M., Clausen, P. A., & Wolkoff, P. (2013). Study of ozone-initiated limonene reaction products by low temperature plasma ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 24(7), 1090–1096. doi:10.1007/ s13361-013-0648-3 Peratt, A. L. (1995). Plasma and the universe: Large scale dynamics, filamentation, and radiation. In A. L. Peratt (Ed.), Plasma Astrophysics and Cosmology (pp. 97–107). Springer: Dordrecht. Rutscher, A. (2008). Characteristics of low-temperature plasmas under nonthermal ­conditions – A short summary. In M. S. Hippler & H. Schmidt (Eds.), Low Temperature Plasmas: Fundamentals, Technologies and Techniques (2nd ed, pp.  1–14). John Wiley & Sons: Berlin, Heidelberg. Retrieved from http://media. johnwiley.com.au/product_data/excerpt/35/35274067/3527406735-2.pdf Soparawalla, S., Tadjimukhamedov, F. K., Wiley, J. S., Ouyang, Z., & Cooks, R. G. (2011). In situ analysis of agrochemical residues on fruit using ambient ionization on a handheld mass spectrometer. Analyst. doi:10.1039/c1an15493a Spencer, S. E., Tyler, C. A., Tolocka, M. P., & Glish, G. L. (2015). Low-temperature plasma ionization-mass spectrometry for the analysis of compounds in organic aerosol particles. Anal. Chem. doi:10.1021/ac5038889 Stein, M. J., Lo, E., Castner, D. G., & Ratner, B. D. (2012). Plasma pencil atmospheric mass spectrometry detection of positive ions from micronutrients emitted from surfaces. Anal. Chem. 84(3), 1572–1578. doi:10.1021/ac2028134 Venter, A., Nefliu, M., & Graham Cooks, R. (2008). Ambient desorption ionization mass spectrometry. TrAC—Trends Anal. Chem. 27(4), 284–290. doi:10.1016/j. trac.2008.01.010 Wiley, J. S., García-Reyes, J. F., Harper, J. D., Charipar, N. A., Ouyang, Z., & Cooks, R. G. (2010). Screening of agrochemicals in foodstuffs using low-temperature plasma (LTP) 611 ambient ionization mass spectrometry. Analyst 135(5), 971–979. doi:10.1039/b919493b Wiley, J. S., Shelley, J. T., & Cooks, R. G. (2013). Handheld low-temperature plasma probe for portable “point-and-shoot” ambient ionization mass spectrometry. Anal. Chem. 85(14), 6545–6552. doi:10.1021/ac4013286 Xia, Y., Ouyang, Z., & Cooks, R. G. (2008). Peptide fragmentation assisted by surfaces treated with a low-temperature plasma in NanoESI. Angew. Chem. 47(45), 8646–8649. doi:10.1002/anie.200803477 Xin, H., & Stone, R. (2008). Chinese probe unmasks high-tech adulteration with melamine. Science 322(5906), 1310–1311. doi:10.1126/science.322.5906.1310 Zhang, Y., Ma, X., Zhang, S., Yang, C., Ouyang, Z., & Zhang, X. (2009). Direct detection of explosives on solid surfaces by low temperature plasma desorption mass spectrometry. Analyst 134(1), 176–181. doi:10.1039/B816230A

FIGURE 1.1  Schematic illustrations of some commercially available ambient MS techniques: DESI (Reprinted with permission from reference (Sero, Nunez, and Moyano 2016), Copyright (2016) Elsevier.), DART and LAESI (Reprinted with permission from reference (Stopka et al. 2014), Copyright (2014) Royal Society of Chemistry.), and PS (Reprinted with permission from reference (Wang et al. 2010), Copyright (2010) Wiley-VCH.).

FIGURE 1.2  Schematic views of some one-step spray-based setup techniques: EESI

(Reprinted with permission from reference (X. Li et al. 2011), Copyright (2011) Nature America, Inc.), LMJ-SSP (Reprinted and adapted with permission from reference (Berkel, Sanchez, and Quirke 2002), Copyright (2002) American Chemical Society.), LESA (Reproduced with permission from reference (Montowska et al. 2014), Copyright (2014) American Chemical Society.), and nano-DESI (Reprinted with permission from reference (Roach, Laskin, and Laskin 2010a), Copyright (2010) American Chemical Society.).

FIGURE 1.3  Schematics of some one-step plasma-based techniques: FAPA (Reprinted with permission from reference (Shelley, Wiley, and Hieftje 2011), Copyright (2011) American Chemical Society.), DBDI (Reprinted with permission from reference (Na, Zhang, et al. 2007), Copyright (2007) John Wiley & Sons, Ltd.), and LTP (Reprinted with permission from reference (Benassi et al. 2013), Copyright (2013) John Wiley & Sons, Ltd.).

FIGURE 1.4  Schematic illustrations of DAPCI (Reprinted with permission from reference (Song and Cooks 2006), Copyright (2006) John Wiley & Sons, Ltd.) and DAPPI (Reprinted with permission from reference (Haapala et al. 2007), Copyright (2007) American Chemical Society.).

FIGURE 3.6  Extracted ion chromatograms acquired by DAPPI-MS in MS mode. Caraway

oil from (a) the normal-phase HPTLC (NP-HPTLC) and (b) the reversed-phase HPTLC (RP-HPTLC) plate. Jojoba oil from (c) the NP-HPTLC and (d) the RP-HPTLC plate. TLC mobile phases for the NP-HPTLC plate, hexane/diethyl ether/acetic acid (93:7:1; v/v/v), and the RP-HPTLC plate, acetone/acetonitrile (90:10; v/v). The green line: TG 54:6 [M+NH4]+. The red line: TG 52:4 [M+NH4]+. The black line: TG 54:3 [M+NH4]+. The orange line: WE 38:2 [M+H]+. The blue line: WE 40:2 [M+H]+. The violet line: WE 42:2 [M+H]+. The brown line: WE 44:2 [M+H]+. (Reprinted with permission from Rejšek, Jan, Vladimír Vrkoslav, Anu Vaikkinen, Markus Haapala, Tiina J. Kauppila, Risto Kostiainen, and Josef Cvačka, Analytical Chemistry, 88 (2016): 12279–12286.) Copyright (2016) American Chemical Society.

FIGURE 4.2  Schematic and actual pictures of the V-EASI (above) and S-EASI (bellow)

systems.

FIGURE 4.3  Schematic of TI-EASI procedure for the MS analysis of meats, fats, and

related products such as caviar and ham.

FIGURE 4.5  EASI-MS for commercial lecithin samples. Positive ion mode: (a) acety-

lated lecithin (SOLEC AA); (b) hydroxylated lecithin (SOLEC AE IP); (c) deoiled lecithin (SOLEC F). Negative ion mode: (d) acetylated lecithin (SOLEC AA); (e) hydroxylated lecithin (SOLEC AE IP); (f) deoiled lecithin (SOLEC F). Main positive ions assignment: m/z 558 (LPC 18:2); m/z 796 (PC 16:0/18:2); m/z 820 (PC 18:2/18:2 and/or 18:1/18:3); m/z 822 (PC 18:1/18:2 and/or 18:0/18:3); m/z 893 (PLL); m/z 919 (OLL/OOLn). Main negative ions assignment: m/z 279 (linoleic acid); m/z 476 (LPE 18:2); m/z 518 (LPEAcet 18:2); m/z 714 (PE 16:0/18:2); m/z 738 (PE 18:2/18:2 and/or 18:1/18:3); m/z 756 (PEAcet 16:0/18:2); m/z 780 (PEAcet 18:2/18:2 and/or 18:1/18:3); m/z 833 (GPL).

FIGURE 6.1  IR image of a “nonthermal,” or LTP probe. The apparent tip temperature of 28°C allows the direct sampling from heat-sensitive surfaces (Martínez-Jarquín, HerreraUbaldo, Folter, & Winkler, 2018).

FIGURE 6.10  Classification of coffee products by hierarchical cluster analysis. Roasted (left) and lyophilized (right) are clearly separated by their LTP-MS data fingerprints (GamboaBecerra et al., 2017).

FIGURE 6.11  Imaging of volatile and semivolatile compounds in a cross-cut section of chili (Capsicum annuum) pepper (Gamboa-Becerra et al., 2015).

FIGURE 6.12  Important mass signals for the differentiation between tequila and mezcal

spirits, based on LTP-MS data. The most relevant variables are present in relatively low abundance (Martínez-Jarquín et al., 2017).

FIGURE 8.1  Schematic diagram of atmospheric pressure ND-EESI-MS.

FIGURE 12.2  PS-MS ionization modes and size distributions of droplets resulted from the spray formation on the paper triangle tip. (Reproduced and adapted with permission from ©2012 Elsevier.)

FIGURE 12.3  Influence of key parameters to the performance of the PS-MS approach. (Reproduced and adapted with permission from ©2012 Elsevier.)

FIGURE 12.4  Graphic representation of related PS-MS techniques: (a) leaf spray mass spectrometry, (b) tissue spray mass spectrometry, (c) wooden-tip electrospray mass spectrometry, and (d) glass spray mass spectrometry. Reproduced and adapted with permission from ©2011 American Chemical Society (Figure 12.4a), ©2013 American Chemical Society (Figure 12.4b), ©2017 Elsevier (Figure 12.4c), and ©2015 American Chemical Society (Figure 12.4d).

FIGURE 12.6  Examples of chemical profiles (fingerprints) obtained via tissue spray

mass spectrometry of distinct species of fruits: (a) grape fruit; (b) tomato; (c) pepper. (Reproduced and adapted with permission from ©2011 American Chemical Society.)

FIGURE 12.8  Paper cone spray ionization mass spectrometry for analysis of ground beef, tea leaves, and infant formulas. (Reproduced and adapted with permission from ©2015 Royal Society of Chemistry.)

FIGURE 12.9  Schematic representation of the automatic device to perform rapid analysis by PS-MS with high reproducibility. (Reproduced and adapted with permission from ©2013 Elsevier.)

FIGURE 12.10  The nanosheet-modified N þ-nylon membrane used as the ionization substrate for PS-MS analysis of malachite green and leucomalachite green in fish meat. (Reproduced and adapted with permission from ©2018 Elsevier.)

FIGURE 12.11  Schematic representation of the slug-flow microextraction coupled with a PS-MS device. (Reproduced and adapted with permission from ©2016 Elsevier.)

Chapter

7

Plasma-Assisted Desorption Ionization Kirsty McKay University of Liverpool

CONTENTS Introduction 125 Low-Temperature Atmospheric Pressure Plasma 126 Background 126 Plasma Needle 127 Electrical Properties of the Plasma Needle 128 Plasma Needle Chemistry 129 Effect of Substrate/Sample Type on the Plasma Conditions 132 Plasma-Assisted Desorption Ionization 133 Desorption/Ionization Mechanisms 135 Micro-PADI 137 Comparison to Other Ambient Techniques 137 Food and Environmental Applications 139 References 140

INTRODUCTION Plasma-assisted desorption ionization (PADI) is an atmospheric pressure radio-frequency (RF) plasma-based technique that can be coupled with atmospheric pressure sampling mass spectrometry (MS). One of the main advantages of plasma-based desorption/­ ionization techniques is that they are able to yield mass spectral information from a range of analyte types without the need for surface/sample preparation. PADI was one of the original plasma-based sources developed for ambient MS and was first described for use as a desorption/ionization source for ambient MS by Ratcliffe et al.[1]; however, the PADI source was based upon the RF plasma needle developed by Stoffels et al.[2] and was originally designed for biomedical applications. PADI, however, has lost favor in recent years due to the number of other low-temperature plasma sources developed but still has great potential, especially in the areas of analyte detection for food and environmental applications. In this chapter, we will explore the properties of PADI sources which make them ideal for these types of applications. Understanding the basic physical, chemical, and electrical properties of plasma is fundamental to designing, building, and operating any

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plasma source; for this reason, we will start this chapter with a brief introduction into the plasmas and the key areas of understanding required for the RF plasma needle/PADI source that is of concern in this chapter. We will then discuss the development of the plasma needle and the characterization of its physical, chemical, and electrical properties. Finally, we will examine its use in surface analysis MS applications, the key desorption/ ionization mechanisms, and its applications to studying the chemicals which are relevant for food and environmental applications.

LOW-TEMPERATURE ATMOSPHERIC PRESSURE PLASMA Background Plasma makes up 99% of the visible matter in the universe and is often referred to as the fourth state of matter.[3] Common examples of naturally occurring plasmas include stars, nebula, the aurora borealis, and lightning. Plasma generally consists of a mixture of ions, electrons, metastable, excited atomic and molecular states, and neutral particles. To produce the plasma state, energy is added into gas until it begins to emit light (can be in the nonvisible spectrum) or glow. The energy can be electrical, chemical, or heat based and can be applied in a number of ways.[3–5] Plasmas can be divided into two categories: low and high temperature plasmas. High temperature plasmas are produced in the stars, in powerful explosions, and at the center of a fusion reactor. Low-temperature plasmas can then be further subdivided into thermal and nonthermal plasmas,[6] thermal plasmas have high ion and electron temperatures and are found, for example, in plasma welding torches. The plasmas of interest in this chapter are low-temperature nonthermal plasmas; these are often referred to as cold plasmas.[6] In cold plasmas, the electron temperature is typically on the order of 10 4 –105 K (1–10 eV), whereas the ion and neutral temperature (gas temperature) is approximately 300–400 K or room temperature.[6] Cold plasmas were developed due to their ability of causing chemical and physical reactions while keeping the gas at these low temperatures. The nonequilibrium nature of these plasmas results in a multitude of reaction pathways, which are inaccessible to conventional methods in chemistry. These properties mean that these plasmas are used in a variety of fields, such as microelectronic fabrication, surface coatings, biomedical applications, environmental applications, and MS.[6] The most common method of low-temperature plasma generation is using an external electric field to electrically ionize a gas.[7,8] To do this, a potential difference (electric field) is set up between two electrodes, for simplicity, let us consider a parallel plate system where one is grounded and the other has a voltage applied. As the voltage applied is increased, the available electrons (a small amount of free electrons are always present in gas) are accelerated by the applied electric field and gain kinetic energy. These electrons then collide with other particles in the background gas; if the electron energies are above the excitation and ionization thresholds of the gas, then they free an electron from the particle or excite the electron in the outer shell to a higher energy level. As more and more electrons are freed, a cascade occurs and continues until the gas breaks down and becomes plasma. The process continues until a certain degree of ionization is achieved (different degrees of ionization can be desired depending on the application the plasma is intended for).[9,10] The electrons and ions in the plasma are influenced by the applied electric field, the electrons move towards the positive electrode and the ions move towards the negative electrode. Electrons move more quickly than the ions due to their higher

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mobility and reach the electrode first resulting in a layer of positive charge forming; this is called a sheath.[4,11] The sheath acts to screen the electric field from the rest of the plasma, and the rest of the plasma is called the bulk and is quasi-neutral in nature (balance of positive and negative charge). Plasma sources come in many different shapes and sizes, with their dimensions and electrode configurations determined by their intended applications; they can operate at a range of pressures and can be operated using a variety of power sources. For example, plasmas can be operated using direct current or alternating current (AC) at low frequency (Hz–kHz), RF (MHz), or in the microwave regime (GHz). Different operating frequencies result in different physicochemical properties. In AC, the voltage discharged on the plates changes from positive to negative every half cycle, and thus, electrons and ions change direction every half cycle, the sheath is also controlled by this oscillation, and forms on opposite electrodes every half cycle.[4,12,13] At very high frequencies (such as RF), the time taken for the ions and electrons to move between the electrodes is larger than the half period of the applied electric field. This means that both species are retained between the electrodes, and this results in a lower voltage being required to initiate and sustain the AC discharge [e.g., for kilohertz (kHz) discharges, voltages of 6–12 kV are often required, whereas for RF discharges, only a few hundred volts are required]. The frequencies of interest for this chapter are RF. In an RF plasma, the frequency of the applied field is in the MHz range, and this means that only the electrons can oscillate with the electric field, whereas the lower mobility of the ion means that they do not respond significantly to the change in field.[4] This means that ions are diffusion controlled and are transported due to gas flow and random collisions rather than the electric field, and the electrons are drift controlled and their motion is heavily influenced by the electric field and only secondarily influenced by gas flow and random collisions. One of the advantage of RF plasmas is that they do not require a dielectric layer between the powered and ground electrode (but they can be included) to prevent glow-to-arc transitions; arc discharges can damage the plasma device and other electrical equipment in close proximity, and once an arc has occurred, it becomes the preferred state of the discharge. There are two main variants of RF discharges, capacitively coupled plasmas (CCPs) and inductively coupled plasmas (ICPs).[14,15] In CCPs, the plasma volume is confined by two electrodes that form a capacitor in the electric circuit of the system. One electrode is generally grounded and the other electrode is driven by the RF generator at a few hundred volts. The RF generator is normally connected by a matching unit (which helps ensure maximum power is delivered into the plasma). In ICPs, the electric field is coupled into the plasma by induction via an alternating magnetic field. In this case, a coil is connected to the RF generator and the alternating magnetic field induces an oscillating electric field within the plasma volume that drives an RF plasma current.[15] The plasma acts as the secondary coil of a transformer. The primary external coil can take the form of a planar antenna behind a quartz window or in the form of a few windings around a quartz tube confining the plasma. While ICPs are used in MS applications, PADI sources are CCP and they are the focus of this chapter. Plasma Needle The RF-driven “plasma needle” has been in use for over a decade and was originally pioneered by E. Stoffels and co-workers and was presented initially in a 2002 publication.[2]

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It was developed with the aim of producing a less aggressive plasma source, which could be applied directly on organic materials and living tissues. The properties desired in the source were that it was noncontact, was strictly local, and had a minimum penetration depth. While these properties make it desirable for medical treatment, they also make it ideal for ambient surface analysis MS.[2] The original “plasma needle” consisted of a 5 cm long, 1 mm diameter stainless steel wire, with a sharpened tip. The wire served as the powered electrode driven at 13.56 MHz, and it was positioned coaxially within a grounded metal cylinder with a 1 cm inner diameter. The cylinder was filled with flowing gas, the main feed gas used was typically helium at a flow rate of a few hundred mL/min, but other gases, such as argon, air, nitrogen, and hydrogen, had also been introduced as secondary gases to the main flow. The setup was closed with a quartz window to allow optical diagnostics to be performed without ambient air becoming a significant factor.[2] This original plasma needle was later modified by Kieft et al.[16] to allow for the treatment of large-sized objects. In this design, a metal-alloy pin (diameter 0.3 mm) is used as the central electrode housed in a Perspex tube, with the plasma generated at the tip. To prevent the discharge spreading along the length of the plasma needle, the pin was insulated with glass. This design was further modified by making the needle position adjustable with respect to the Perspex housing; this allowed the amount of ambient air in the discharge to be varied.[17] The final design is the one that is used widely for ambient MS surface analysis applications. Electrical Properties of the Plasma Needle Understanding the electrical properties of the plasma needle is important as it allows for a better understanding of the plasma formation and sustainment processes and how they can be tailored for specific applications. While experimental techniques can provide useful information about the discharge conditions, they cannot reveal the underlying physics behind the observed properties; to do this, computational modelling has been extensively used to better understand the physical and electrical properties of the RF plasma needle. One of the first simulation-based investigations was carried out by Brok et al.[18] The aim of this was to provide an initial numerical description of the plasma needle and provide estimates of the important plasma properties and insights into the physical processes that govern them. To do this, they used a versatile, two-dimensional time-dependent fluid model.[18] From this, the maximum electron density, for the conditions modeled, was found to be ~1011/cm3, and the electron energy near the needle electrode was 8 eV, but this was found to rapidly decrease below 1.5 eV at a distance of 0.5 mm away from the needle. They benchmarked the simulation against experimental data, and it was found that they were in good agreement. Sakiyama and Graves developed a finite element model (FEM) of the plasma needle which was used in several studies.[19] Their initial study in 2006 focused on the plasma structure as a function of power; as they varied the power applied to the plasma needle, they observed an apparent α-γ mode transition (from a diffuse glow to a constricted mode) at a critical discharge power[19]; in a later publication, they present evidence that this was not in fact a α-γ mode transition but two distinct modes—a corona glow at low power and a glow mode at a critical power.[20] When the discharge operated in corona mode, the plasma density and ionization were confined near the needle tip, whereas in glow mode, it spread back along the needle surface.[20] These modes have also been observed

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experimentally by Stoffels et al. and Kieft et al. where two different slopes were observed in the voltage–power curve and a significant increase in light emission was observed at a certain voltage, respectively.[16,21] Plasma Needle Chemistry As well as understanding the electrical properties of the plasma it is also important to understand the chemical properties, especially for ambient MS applications where the chemistry plays an important role in the ionization and desorption processes. Many factors, such as gas flow, power, gas mixture, and frequency influence the chemical species produced in the plasma. Low-temperature atmospheric-pressure plasmas are very chemical reactive and consist of positive and negative ions, electrons, excited and metastable species, and neutrals. Several studies have examined the chemical composition of the RF plasma needle under a variety of conditions. In these studies, the plasma chemistry has been determined using a variety of techniques, including two-photon absorption laserinduced fluorescence (TALIF),[22,23] optical emission spectroscopy (OES),[16,22,24] and molecular beam mass spectrometry (MBMS).[25–28] While these methods provide important information into the location and abundance of certain species in the plasma, they do not provide us with the underlying mechanisms for the production and destruction of the various species. For this, we must again rely on computational modeling and interpretation of theoretical and empirical data. MBMS has been used extensively to examine the plasma chemistry; it can identify positive and negative ions, neutral species, and also radical species by probing the threshold ionization energy of neutral species. The mass spectrometer which has been used by several different research groups to examine the plasma chemistry is the Hiden Analytical Ltd HPR-60, which is a quadrupole-based mass spectrometer (QMS).[29] It consists of three differential pumped vacuum stages which reduce the pressure from atmosphere to 10 −1, 10 −5, and 10 −7 Torr. The pressure gradient towards the sampling orifice causes the gas molecules to flow into the low-pressure region, allowing a molecular beam to form and be focused into the QMS. As the molecular beam enters the mass spectrometer, it transitions from atmospheric pressure to the reduced pressure region (P1) where it undergoes adiabatic expansion, which causes shock waves to form. The shock waves create regions of large pressure, temperature, and velocity gradients, which can affect the chemical species present within the mass spectrometer. To reduce the chances of sampling this modified chemistry, the HPR-60 is designed with conical skimmer cones that sample the molecular beam within a zone of silence, a region that has not been disturbed by the formation of shock waves and has an unmodified chemistry.[30,31] Consequently, we can be confident that the measured chemical composition is a true representation of that produced in the plasma needle and is not significantly impacted by the pressure reduction stages. The HPR-60 can detect species in the mass range of 0–1,000 amu. The HPR-60 can be operated in both time-averaged and time-resolved modes (time resolution ~2 µs), allowing for a more in-depth study of the plasma species and their behavior as a function of time.[28] Time-resolved MS also has the advantage of being able to identify transient effects in the chemistry that time-averaged data can miss, such as the periods of production and loss as a function of time. One drawback of MBMS is that it can only provide information on the species that make it to the mass spectrometer inlet, this means that we miss short-lived species or those produced not along the center line of the plasma (where the source is aligned with the inlet). However, in the case of surface

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analysis MS, this is not a major concern, as it is the species that interact with the surface which are of interest. Stoffels et al. examined the generation of nitric oxide (NO) by a He + O2 + N2 plasma needle discharge using MBMS,[25] NO is particularly important in plasma therapy applications. The authors found that NO can comprise up to 20% of the total plasma density, under certain conditions, and that the maximum NO density was found at a distance of ~2.5 mm from the tip of the needle for most power conditions. This was thought to be due to the reaction kinetics, which involved dissociation and three-body recombination, a process which can take several µs to complete. In addition, more oxygen (O) mixed into the discharge from the ambient air as the distance from the needle tip increased. In another study, Stoffels et al. also used MBMS to examine the excited species present in the discharge using threshold ionization MS.[26] The major radical species produced by the plasma needle were found to be NO, atomic nitrogen (N), atomic oxygen (O), and hydroxyl radicals (OH). Malovic et al. also used MBMS to examine the neutral and positive ion chemistry of a helium plasma needle operating with a gas running into open air.[27] As they increased the helium flow from 1 to 3 SLM (standard liter per minute) they saw a reduction in reactive species (N, O, NO, NO2 , and NO3), this is thought to be due to less mixing of ambient air at higher flow rates. The dominant positive ion species observed were O2+ , O+, H3O+, N 2+, N+, and NO+. McKay et al. used MBMS to examine the positive and negative ion chemistry of a helium into open-air RF plasma needle discharge, in both time-averaged and timeresolved mode.[28] In this case, the RF excitation was pulse modulated, this served to reduce the operating gas temperature of the plasma for a constant input power and allowed negative ions to be readily detected. From this it was found that, protonated water clusters (H+(H 2O)n) dominated the positive ion spectra and oxygenated water clusters O−n ( H 2O)n dominated the negative ion spectra. It was found that the relative yield of these clusters could be altered by changing the frequency of the modulation of the RF excitation. Time-resolved data showed that negative ions were predominately created or able to reach the MS in the off phase of the pulse, and positive water cluster ions were generated and increased in size during the on phase of the discharge. They also showed using a thermal camera that as the on time of the discharge increased, the temperature of plasma needle tip increased to over 100°C.[28] As can be seen from these MBMS studies, the interaction of the plasma plume with ambient air or the small amounts of other gasses added to the feed gas is a significant influencing factor in the chemistry detected. Several groups have made large-scale global and fluid models which examine the chemical reaction pathways in RF discharges[32–34]; however, modeling large chemistries is time consuming and computationally expensive, especially when considering one or more dimensions in the model, it is for this reason that most models only select a few important reactions in there simulations. From the large global and 1-D fluid models, the main reaction pathways for the production of the plasma species can be determined, for example, positive ions are thought to be generated through Penning ionization and step ionization with helium metastables and dimer metastables and direct electron ionization. Neutrals and excited species, such as atomic nitrogen, atomic oxygen, OH, and NO are thought to be formed through dissociation of molecules and three-body reactions. Negative ions are generally formed through attachment reactions; many of the negative ions formed in the plasma contain oxygen atoms or molecules due to its high electron affinity, which makes it particularly prone to attachment reaction processes. Water clusters also feature significantly in the plasma needle chemistry; these

(

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TABLE 7.1  Production Reaction Pathways Reaction Form He + e → He  + e He + e → He+ + 2e He* + M → M+ + He + e He+ + M → M+ + He He + N2/O2 + e → He + N+ + O + NO + 2e e + H2O → OH+ + H + 2e *

H 2O + H 2+ → H3O+ + H H 2O + H3O+ → H + ( H 2O)2 − 2

O2 + e → O

Reaction Type Electron impact excitation Direct electron impact ionization Penning ionization Charge exchange Three-body collision Dissociation of water Protonation Hydration reaction Attachment

are formed through dissociation and hydration reaction. Examples of these reactions are given below in Table 7.1; this list is far from exhaustive but provides a basic representation of the generation mechanisms at a work. Another common way to detect the chemical species present in the plasma is by examining the light emission from the plasma; this can be done using a technique called optical emission spectrometry (OES), the light collected is split into a spectra using a grating, from this spectra different emission lines can be associated with different atomic and molecular transitions. Kieft et al. used OES to detect the ultraviolet (UV) emission and determine the discharge composition of the plasma needle.[16] They were able to detect emission lines from He, N2 , N 2+, OH*, and O*. They also examined the effect of gas flow on the emission profiles. From this it was found that, as the helium flow was decreased, the total spectral intensity increased. This was due to an increase in N2 emission, as the strongest nitrogen lines were below 400 nm, the emission of UV light also increased. While this study was carried out to determine what effect these species might have on biological material, such as cells, it is also important to consider factors such as UV emission for the photoionization and energetic ejection of analyte material in desorption/ionization applications. While OES is a versatile technique which can give positional information, with resolution determined by the optical fiber size used and the distance from the plasma and gas temperature from line ratios, it cannot detect weakly emitting species.[22] A modification of the OES technique is phase-resolved OES (PROES); using PROES, the dynamics of the excitation processes within the discharge can be examined. To perform PROES, a high-repetition rate gateable intensified charge-coupled device (ICCD) camera is required; this is then coupled to a long-distance microscope. Benedikt et al. used PROES to examine the dynamics of excitation processes on axis for a coaxial plasma jet driven at 13.56 MHz.[30] TALIF spectroscopy is another experimental technique that is used to investigate ground state species. It works by the absorption of two photons followed by the emission of a third. To convert experimental data into absolute concentrations, kinetic models are often used.[22] Sakiyama et al. used TALIF spectroscopy and gas flow simulations to show that ground state oxygen forms in two different spatial patterns near a surface when different flow rates are used.[23] When the flow rate was low, the radial density peaked along the axis of the needle; however, at high flow rates, a ring-shaped density distribution appeared. The distribution of oxygen and potentially other oxygen-dependent species is

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important for surface analysis MS especially when used for imaging of surfaces, as different species may desorb surface material in unwanted areas, decreasing the achievable image resolution. Effect of Substrate/Sample Type on the Plasma Conditions For ambient MS applications the plasma source will have to interact with a variety of samples. Different samples (solid, liquid, gas) can have very different electrical properties (conducting, dielectric, insulating) all of which can alter the plasma physicochemical properties when the plasma is in direct or nondirect contact. It is important that we understand these effects so that we can tailor the plasma to maximize the signal intensity. Much of the research in this area has been carried on solid samples for the plasma needle, with a particular focus on the implications this has for biological materials; however, this is still applicable for PADI, especially food applications where the effect of plasma on biological material is also important. Hofmann et al. examined the effect that the conductive and dielectric substrates had on the electrical and optical properties of both continuous and pulsed RF plasmas.[35] They examine the plasma dissipated power and the gas temperature, when the plasma is in contact with different substrates (water, glass, and metal), using ICCD imaging and time-resolved plasma-dissipated power measurements combined with spatially resolved OES. They showed that, when in close proximity to a conductive substrate, the power dissipated in the plasma and the gas temperature increased. The substrate type has a visible and measureable effect on the plasma properties. Hofmann et al. determined that much of the changes in plasma properties were induced by a change in the electric field as it interacted with the surface. Simulations can also be used to provide us with a valuable insight into the effect of different substrates on the plasma needle properties. Sakiyama et al. used their FEM of the plasma needle to show that the plasma structure strongly depends on the electrical properties of the treated surface as well as the discharge mode.[19,36] For dielectric surface, in the low power mode, the particle fluxes were relatively low and followed a Gaussianlike radial profile. In the high-power mode, the particle fluxes to the surface were orders of magnitude higher and the spatial distribution of the particle fluxes became radially more uniform due to a uniform ionization layer just above the treated surface. When, however, a conductive plate replaced the dielectric surface, an intense ionization spot appeared near the surface closest to the needle tip, this resulted in the particle fluxes to the surface peaking near the symmetry axis. These results were confirmed experimentally by examining the light emission spatial profiles.[36] Sample properties not only affect the plasma properties but also affect the ability of different ambient techniques’ abilities to desorb and ionize the samples which can in turn effect detection. Salter et al.,[37] examined the effect of sample form and surface temperature on the signal intensities detected using PADI. They found that the sample form was very important, with powders of all volatilities being effectively analyzed; however, thin films with a low vapor pressure 7 W), surface erosion and charring of the tablet surface occurred. PADI has the ability to both positively and negatively ionize samples, which is advantageous for chemicals, such as pharmaceuticals, as many of the drugs contain both basic and acidic molecules with a range of polar and nonpolar properties. This is also beneficial for environmental and food applications as there are a range of chemistries present in both areas. After the introduction of PADI as a viable technique for ambient MS analysis, only a few research groups started to use and test its capabilities, with only 1% of publications on plasma-based sources focusing on PADI.[39] It is thought that this may be due to the range of other plasma-based techniques such as low-temperature probes (LTPs), which emerged shortly after PADI; these techniques do not have high operating temperatures and are generally run in the kHz regime, and kHz power supplies are normally cheaper than commercially available RF power supplies and matching networks. Much of the research into PADI sources for MS applications has been performed by Salter et al. and various associates.[37,38,40–42] The first publication by Salter et al. in

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2011[40] is a comparative study between PADI and DESI sources for the detection of personal care products (PCPs) on model skin surfaces. From this it was found that PADI was able to detect the molecular ion and other characteristic peaks (fragments and rearrangements of the molecule), from 12 of the PCP components, and detected unidentifiable ions from the thirteenth component tested. DESI on the other hand was only able to detect 9 out of 13 components. In this study, the authors also examine the optimal operating conditions of the PADI source for detection of two of the PCP components, hydroxycitronellal and triethanolamine.[40] For example, they examined how RF input power (not power dissipated in the plasma) effected the ion intensity; from this they found that, in general, the intensities increased with increased power; however, some fragment ions showed a decrease in intensity, and nearly all ion intensities decreased at the highest power tested. While they suggest that this may be due to reaction chemistries which depend on humidity, they do not consider the potential of a mode change with increasing power that had been reported by Sladek and Stoffels,[21] Sakiyama et al. and others for plasma needle discharges.[16,20] Another study by Salter et al. in 2013 looks again at optimization of the PADI source[38]; however, this time they also consider the geometry of the source, sample, and MS inlet. They found that the highest ion intensities were found when the plasma was 2 mm away from the surface, and the MS inlet was 7 mm away and the input RF power was set to 22 W, they also found that the maximum intensity was achieved when the PADI source was perpendicular to surface (not shown here).[38] In this study, they also used the PADI source to analyze polymer substrates; the ability to analyze polymers is important for industrial applications, as it is used in a variety of products and processes. It is also important for the food industry as a variety of plastics are used extensively in packaging materials. The four polymers examined were polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), polylactic acid (PLA), and polyethylene terephthalate (PET). All the polymers were detected in either positive or negative ion modes or both, and polymer fragment ions up to m/z 1,200 were detected for PTFE, which was higher than had previously been reported. While much of the work carried out by Salter et al. focused on optimizing the PADI source for analyte detection, they also examined the effect of sample form and surface temperature on the signal intensity achieved using PADI, these results have been discussed in “Effect of Substrate/Sample Type on the Plasma Conditions” and “Desorption/Ionisation Mechanisms” sections, respectively, so will not be discussed here again. McKay et al. performed a comparison of three plasma sources[42]: PADI, LTP, and a dielectric barrier discharge (DBD) plasma jet. In the case of the PADI source, the RF excitation was operated in continuous and pulse-modulated modes (duty cycles: 30%, 45%, 60%, and 90%). Unlike Salter et al.,[38] the source was positioned 4 mm away from the surface, this reduced sample surface damage significantly, and it was orientated at 45° to the surface, this gave good signal intensities. When the plasma needle was positioned further away from the sample, as in this case, gas turbulence and the lower vacuum pressure presented by the mass spectrometer may become a significant factor in species transport to the mass spectrometer from the surface. The sources were tested against a range of pharmaceuticals and polymer samples. It was found that, as the average power applied increased for the PADI source, the molecular ion intensity for ibuprofen, paracetamol, and caffeine increased, this was attributed in the increase in surface temperature.[42] It is also noted that the PADI source was able to extract larger polymer chains due to the increased surface temperature achieved. In addition to larger polymer chains that were observed, less fragmentation was also observed and this was thought to be due to the nondirect plasma treatment as the visible plasma is not in contact with the sample in this

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case. The authors also discuss in detail the plasma species transport mechanisms and the effect of surface temperature on the signal intensities detected, this will be expanded on further in “Desorption/Ionization Mechanisms” section. Desorption/Ionization Mechanisms The desorption/ionization mechanisms of PADI have been discussed in several publications, they are generally attributed to two main mechanisms, thermal desorption and plasma species interactions with the analyte surface and/or the analyte in the gas phase. In this section, we will describe and discuss these mechanisms in detail. Several studies have examined the thermal interaction of the plasma needle or PADI source with various substrates. In the original plasma needle study by Stoffels et al.,[2] it was established that increased power leads to increased gas heating, as does the addition of molecular species, such as nitrogen, into the feed gas. Increased heating was thought to be due to the increased electron temperature and more efficient energy transfer from electrons to heavy particles when molecular species were present.[2] The rotational ­temperature is close to the gas temperature in atmospheric pressure low-temperature plasmas. In a later publication, Stoffels et al. reviewed a number of studies where the gas and sample temperatures were examined. The variation in temperatures between the different methods was due to the location of the measurements. OES measurements were taken in the hottest part of the plasma, the active zone, which yields the highest emission intensity. MS measurements were taken downstream, recording the density of gas flowing into the mass spectrometer; correspondingly, the temperatures were lower in this region. The liquid crystal strips measured the temperature that the sample would experience, which was significantly lower than the measured gas temperature; this was dependent on the heat capacity and thermal conductivity of the sample. These reported results for the plasma needle are consistent with thermal camera measurements for the PADI source performed by Salter et al.[38] and McKay et al.,[42] suggesting that changes in source geometry make little difference to the gas heating mechanisms. Salter et al. measured the achieved sample temperature as a function of RF input power with a thermal camera. It can be seen that the temperature linearly increased with increased power; however, there was a discontinuity at 17 W.[38] Although not suggested by the authors, this discontinuity may suggest a mode change from corona

FIGURE 7.2  Micro-PADI source developed by Bowfield et al.[41]

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mode to glow mode, which has been widely reported for the plasma needle discharge. It should be noted that Salter et al. reports a higher input power compared to other studies, it is thought that there were significant losses in the matching unit and power cables and that the actual power dissipated in the plasma was much lower and more in line with other studies. This research established that PADI sources not only have a high gas temperature but also induce an elevated sample temperature, which will vary depending on thermal conductivity and heat capacity of the sample. However, from the data presented in a variety of publications, it is clear that increasing power increases sample temperature and increases signal intensity detected by the mass spectrometer. This is a good indication that thermal desorption plays an important role in desorption/ionization mechanisms for PADI. The second mechanism that is thought to be important for desorption/ionization, especially for ionization in the gas phase, is the interaction of the plasma-generated species with the analyte material. Many of the gas-phase interactions are similar to those suggested for the generation of plasma species shown in “Electrical Properties of the Plasma Needle and Plasma Needle Chemistry” sections. Many studies into PADI sources analyze the resultant adducts or fragmentation patterns in the detected analyte chemistry to suggest possible chemical desorption/­ ionization mechanisms.[1,39] However, care must be taken with this approach as the resultant analyte chemistry may be altered due to transport through the heat transfer tube into the MS and may undergo more than one process before being analyzed. In addition to this, the mass range of most analytical MS starts at ~50 Da; as a result, much of the plasma chemistry and any small analyte fragments are excluded. Ratcliffe et al. briefly commented on the desorption and ionization mechanisms of PADI.[1] For the ionization mechanisms they suggest that analyte ionization occurs through a combination of ionized water cluster formation and proton-transfer reactions,[1] they supported this by the observation of M+ and MH+ fragments in the analyte spectra. The role and importance of cluster ions in the ambient plasma ionization techniques is highlighted by Klee et al.[43] Negative ion formation was suggested to proceed via direct and dissociative electron attachment to oxygen species which then react with analyte molecules to produce predominantly [M − H]− groups.[1] The authors acknowledge that desorption mechanisms were less well understood but suggested that a combination of energy transfer from metastable helium, ion impact, and radical-surface interactions contribute to the desorption mechanisms.[1] While these could be potential desorption mechanisms and are key for other plasma-based techniques, thermal processes are thought to dominate for PADI.[42] Helium metastables are relatively short lived at atmospheric pressure, due to the collisions with the air, and it is only when the source is very close to the surface and the plasma is in contact with the surface that they reach the sample. Ion impact with the surface is also possible and is more likely when the electric field between the source and sample is strong, for example, when the sample is conductive (as mentioned in “Effect of Substrate/Sample Type on the Plasma Conditions” section). Radical interactions with the surface is also a valid suggestion; however, the timescales of radical interact processes are long in comparison with detection time so it is unlikely that this is a significant surface process unless there is a significant density of these radicals at the surface; however, gas phase oxidation is an important ionization mechanism (especially for negative ion formation) and this is evidenced by the appearance of oxygenated molecules in the mass spectra. More about these mechanisms and their importance is discussed in the study by McKay et al.[42]

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Micro-PADI Due to the “homemade” nature of PADI source, several modified versions exist; however, results have shown that small changes to the geometry have little effect on the operational capabilities of the source. One of the main drawbacks to PADI sources is that they can damage the surface due to excessive heating, one way of reducing this heating damage is to move the plasma further away from the surface; this results in an increase of the area that the plasma species interact and therefore reduces the achievable surface resolution, meaning that if the chemistry changes over a small distance, then resolving this change becomes very difficult. To circumvent this reduction in surface resolution, a micro-PADI source has been developed.[41,44] The micro-PADI source was designed to operate in ambient air without the need for independent noble gas flow.[41] This source consisted of a 200 µm diameter tungsten wire, which acted as the driven electrode; the wire protruded 5 mm out of a ceramic holder, 3 mm from the end of the holder a grounded brass ring was placed as shown in figure 7.2. The electrode was driven using a 19.2 kHz, 12.0 kV voltage pulse. The source was tested against a traditional PADI source,[41] for three different samples: paracetamol, ibuprofen, and PTFE. From this they found that the new micro-PADI source excelled as an ion source and provided a route to improving the spatial resolution of PADI for imaging without inducing thermal damage. One of the major differences between the micro and traditional PADI source was that the analyte fragmentation was higher for the traditional source than for the micro-PADI source. As the excitation mechanism and operating conditions for the two devices were significantly different, it is difficult to compare them directly in terms of their desorption and ionization mechanisms, and the authors do not discuss these differences in any detail. In another publication, Bowfield et al. presented another micro-PADI source,[45] this time the powered electrode (1.0 mm copper rod with sharpened tip) was encased in a tapered glass capillary (tapered diameter: 20–56 µm), with an aluminium ground electrode wrapped around the glass tube. The powered electrode was driven at 9.8 kV using a 14.25 kHz sinusoidal waveform. In this case, Helium was used as a feed gas. The plasma was generated inside the capillary and was allowed to exit through the tapered end where it could then interact with the surface. The author suggests that this device was similar to the LTP source with reversed electrodes; however, the breakdown mechanisms were probably more like the traditional PADI with a corona type discharge forming at the needle tip, the ionization channel then extends in the helium gas. As the excitation frequency is in the kHz range, it was suggested that discharge would extinguish every half cycle, which is similar to the LTP operation. A number of samples, including PTFE, a range of PCP components, amino acids, fluorenylmethyloxycarbonyl chloride-pentafluoro-L-phenylalanine, caffeine, phospholipid dipalmitoyl-phosphatidylcholine, and over the counter drugs, were used to test the viability of this source. To test the imaging capabilities of the source, PTFE and Cardamom seeds were examined. Using a capillary with a 30 µm diameter, a spatial resolution of 230 µm was achieved; by reducing the capillary to 20 µm, the spatial resolution was improved to 147 ± 28 µm. The achieved spatial resolution was better than any other reported plasma-based ion source. However, the small capillary size did not have a stable ion signal and performance varied with different sample types and substrates.[45] Comparison to Other Ambient Techniques Many plasma-based desorption/ionization sources exist, and they operate under a range of different conditions and have a range of different desorption and ionization

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mechanisms.[39] One study which examines these differences is by McKay et al.,[42] in this study, three plasma-based sources, PADI, dielectric barrier plasma jet, and a LTP were compared to determine their ability to detect a range of pharmaceuticals (paracetamol, ibuprofen, and caffeine) and polymers (PTFE, PMMA, and PLA). While all of the sources were able to desorb and ionize the majority of samples, it was found that the plasma needle could desorb and ionize the surface compounds without direct contact, this was thought to be due to its ability to induce thermal emission from the surfaces when operating at higher powers.[42] This was particularly advantageous when the sample was highly compacted, well adhered to the surface or had a high vapor pressure, such as paracetamol, which was only detected with the PADI source. Using MBMS it was found that the plasma positive ion chemistry for the plasma jet and the LTP were very similar, with a number of air species, such as N+, O+, N 2+, O2+ , dominating the spectrum. Helium ions were also detected in the plasma jet and LTP sources.[42] For the plasma needle, air species were also important; however, the spectrum was dominated by water clusters (H+(H 2O)n , n = 1–5) and oxygen species, such as O3+ . To compare the three sources abilities to desorb and ionize surface compounds, an Oribtrap MS was used in both positive and negative ion mode in m/z range 50–1,500 Da. For ibuprofen, the main ions detected in the different sources changed, for the PADI source the deprotonated and oxygenated ibuprofen molecule was detected, ([M − H]−, [M − H + O]−).[42] For the plasma jet and LTP, the main ions detected were deprotonated propylene glycol, clustered propylene glycol, and deprotonated ibuprofen ([C3H8O2 − H]−, [2(C3H8O2) − H]−, [M − H]−). The detected intensities were significantly higher for the PADI source compared to the other sources. To explain the differences in the analyte chemistry, the differences in electric field, propagation mechanisms, and the surface temperature of the samples achieved with the three sources were discussed. For the PADI source, it was noted that the main species transport mechanisms were diffusion and gas transport; this was due to the highly transient nature and orientation of the electric field, which acts to confine the plasma to a small region around the plasma needle tip. It was also found that at a distance of 4 mm from the source, the sample temperature was 87°C when the RF excitation was operated in continuous mode. Due to the noncontact nature of the PADI source with the sample, the plasma species interaction with the surface was reduced and it was said that thermal processes dominate the desorption and ionization mechanisms.[42] This is consistent with what has been reported in “Desorption/Ionization Mechanisms” section of this chapter. The transport mechanisms for the LTP and plasma jet were similar in nature, with ionization fronts or “plasma bullets” propagating from the tube towards the surface every half cycle of the excitation waveform. This resulted in the surfaces being exposed to strong self-induced electric fields and energetic particles that are associated with the streamer formed in this type of discharge. The surface temperature achieved for the plasma jet was ~30°C for all powers tested, for the LTP increased from 30°C to 40°C as the power applied increased. For the plasma jet thermal processes do not play a role in desorption and ionization mechanisms, but they may have some influence in the case of the LTP.[42] Salter et al. analyzed PCPs on model skin surfaces using DESI and PADI techniques.[40] They showed that PADI was able to identify all the pure PCP compounds presented  unlike DESI; however, they noted that the spectra obtained from PADI was more ­complex and harder to interpret, but this improved with optimization of the plasma operating parameters. This is discussed in further detail in the “PADI” section. PADI and DESI were also shown to be effective in obtaining useful information, such as siloxane content from PCPs and creams directly from skin-like surface. It was noted that PADI

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FIGURE 7.3  Comparison of technique sensitivity and analysis time (higher on the y-axis

less time taken). analysis time should be restricted to ≤5 seconds as it was shown to induce damage in fibroblast cells.[40] In this study, the plasma was in direct contact with the surface. Green et al. also conducted a review and comparison of DESI, direct analysis in real  time (DART), PADI, and extractive electrospray ionization, for forensic applications.[45] Figure 7.3 compares the sensitivity and analysis time for a variety of techniques used in forensic analysis. However, from their narrative, it was difficult to draw a clear conclusion as to the performance of PADI compared to the other techniques, but they do note that PADI was able to detect RDX (cyclotrimethylene trinitroamine),[46] which is an explosive, as well a range of pharmaceuticals, this matches results from other studies. They also indicate that PADI was able to detect molecules from urine and had been used for trace detection of an insecticide from dog stomach contents.[46] Smoluch et al. also compare a range of ambient plasma-based ionization sources, but in this case, for the broader field of bioanalytical sciences, however, again the main focus of this review is DART due to its domination in the field.[46]

FOOD AND ENVIRONMENTAL APPLICATIONS Food safety is an important area of research which impacts the whole of society. Factors which influence food safety include pesticide and chemical fertilizer residues in fruits and vegetables, excess and illegal food additives, inferior raw food, and heavy-metal contamination. Detection of these vectors prior to them entering the food chain is paramount to improving and ensuring food safety. In the environment, detection of pesticides and chemical fertilizers are also important for identifying contamination of the natural environment and water sources. Detection of airborne contaminates such as volatile organic compounds is also a very important area of research. While little work has been done to directly detect these vectors from the environment or from foodstuffs using PADI, some studies have included the analysis of chemicals

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which are typically found in these environments. For example, Ratcliffe et al.[1] used PADI to detect naturally occurring plant alkaloids. They examined tobacco and the vegetable alliums, garlic, and onion. Nicotine was detected in the tobacco, the predominate thiosulfate, Allicin, was detected in the freshly cut garlic, and propanethial-S-oxide was observed for freshly cut onion. Traditionally, the unstable volatile compound, which forms through the decomposition of sulfenic acids generated by enzymes released when the onion tissue is damaged, is difficult to detect using traditional MS techniques.[1] Another example of the detection of relevant chemistry is found in the study by Bowfield et al.[44] where they used the micro-PADI source for the imaging of cardamom seeds; while a chemical image was obtained, the ion chemistry detected was difficult to assign, and it was believed that a high level of fragmentation occurred. While PADI has not been extensively used for these applications, it has great potential in this area, while direct treatment of target samples must be carried out with care due to the higher operating temperature achieved, gas phase and liquid phase sample analysis should not require this consideration.

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34. T. Murakami, K. Niemi, T. Gans, D. O’Connell, and W. G. Graham, “Interacting kinetics of neutral and ionic species in an atmospheric-pressure helium–oxygen plasma with humid air impurities,” Plasma Sources Sci. Technol. 2013, 22, 4, 45010. 35. S. Hofmann, K. van Gils, S. van der Linden, S. Iseni, and P. Bruggeman, “Time and spatial resolved optical and electrical characteristics of continuous and time modulated RF plasmas in contact with conductive and dielectric substrates,” Eur. Phys. J. D 2014, 68, 3, 56. 36. Y. Sakiyama, D. B. Graves, and E. Stoffels, “Influence of electrical properties of treated surface on RF-excited plasma,” J. Phys. D: Appl. Phys. 2008, 41, 095204. 37. T. L. R. Salter, J. Bunch, and I. S. Gilmore, “The importance of sample form and surface temperature for analysis by ambient plasma mass spectrometry (PADI),” Anal. Chem. 2014, 86, 9264–9270. 38. T. L. Salter, I. S. Gilmore, A. Bowfield, O. T. Olabanji, and J. W. Bradley, “Ambient surface mass spectrometry using plasma-assisted desorption ionization: Effects and optimization of analytical parameters for signal intensities of molecules and polymers,” Anal. Chem. 2013, 85, 3, 1675–1682. 39. A. Albert, J. T. Shelley, and C. Engelhard, “Plasma-based ambient desorption/ionization mass spectrometry: State-of-the-art in qualitative and quantitative analysis,” Anal. Bioanal. Chem. 2014, 406, 6111–6127. 40. T. L. Salter, F. M. Green, N. Faruqui, and I. S. Gilmore, “Analysis of personal care products on model skin surfaces using DESI and PADI ambient mass spectrometry,” Analyst 2011, 44, 0, 3274–3280. 41. A. Bowfield, D. A. Barrett, M. R. Alexander, C. A. Ortori, F. M. Rutten, T. L. Salter, I. S. Gilmore, and J. W. Bradley, “Surface analysis using a new plasma assisted desorption/ionisation source for mass spectrometry in ambient air,” Rev. Sci. Instrum. 2012, 83, 063503. 42. K. McKay, T. L. Salter, A. Bowfield, J. L. Walsh, I. S. Gilmore, and J. W. Bradley, “Comparison of three plasma sources for ambient desorption/ionization mass spectrometry,” J. Am. Soc. Mass Spectrom. 2014, 25, 1528–1537. 43. S. Klee, V. Derpmann, W. Wißdorf, S. Klopotowski, H. Kersten, K. J. Brockmann, T. Benter, S. Albrecht, A. P. Bruins, F. Dousty, T. J. Kauppila, R. Kostiainen, R. O’Brien, D. B. Robb, and J. A. Syage, “Are clusters important in understanding the mechanisms in atmospheric pressure ionization? part 1: Reagent ion generation and chemical control of ion populations,” J. Am. Soc. Mass Spectrom. 2014, 25, 8, 1310–1321. 44. A. Bowfield, J. Bunch, T. L. Salter, R. T. Steven, I. S. Gilmore, D. A. Barrett, M. R. Alexander, K. McKay, and J. W. Bradley, “Characterisation of a micro-plasma for ambient mass spectrometry imaging,” Analyst 2014, 139, 5430–5438. 45. F. M. Green, T. L. Salter, P. Stokes, I. S. Gilmore, and G. O’Connor, “Ambient mass spectrometry: Advances and applications in forensics,” Surf. Interface Anal. 2010, 42, 5, 347–357. 46. M. Smoluch, P. Mielczarek, and J. Silberring, “Plasma-based ambient ionisation mass spectrometry in bioanalytical sciences,” Mass Spectrom. Rev. 2016, 35, 22–34.

Section

IV

Ambient Gas, Heat, or LaserAssisted Desorption/Ionization

Chapter

8

Extractive Electrospray Ionization Sheetal Mital KIET Group of Institutions

CONTENTS Introduction 145 Principle of the Technique 145 Mechanism 148 Applications in Food and Environment Matrices 151 Analysis in Food 155 Analysis in Biological Samples 156 In Vivo Skin Analysis 158 Analysis in Perfume 160 Analysis of Organic Aerosols 160 Merits and Demerits of Techniques 162 References 163

INTRODUCTION Extractive electrospray ionization (EESI)[1–2] mass spectroscopy (MS) is a dual-spray powerful and versatile analytical technique, which attracts much interest due to its advantages over conventional electrospray ionization (ESI).[3] It is a powerful ambient ionization technique[4–8] that can provide comprehensive MS information for many samples having complex mixtures, aerosols, complex liquids, or suspensions without any sample pretreatment and fast testing. It is widely used in many fields requiring highthroughput analysis, such as online detection of chemical reaction products and intermediates,[9] detection of native biomolecules,[10] environmental monitoring in the field,[11] and in vivo metabolomics.[12] It has also been employed for the rapid characterization of living objects,[13] native proteins,[14] and metabolic biomarkers,[15–17] proteomics (analysis of proteins), and petroleomics (analysis of petroleum).

PRINCIPLE OF THE TECHNIQUE EESI-MS, a technique derived from ESI, was first introduced by Chen et al.[1] in 2006 as a powerful and versatile analytical technique. In the traditional ESI process, the analytes are dissolved in solvent and delivered through a capillary. A very high voltage is applied at the tip of the capillary, which charges the molecules in the solvent. These highly charged molecules when exiting the capillary tip and pushing toward the evaporation chamber

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form the cone shape (Taylor cone) initially, which is further converted to a fine spray as droplets burst away due to the repulsion among the like charges. Thus, a plume of charged droplets are formed with the help of a high electric field and sometimes assisted with a sheath glass. The droplets in the spray go through a series of divisions because the solvent within these droplets gradually evaporates (with the assistance of nitrogen gas pumped into the chamber), forcing the charges in the molecules within these droplets (which, again, are identical) closer together. When these ions are pushed close enough together, they will repel each other (this behavior is known as the Coulomb force), causing the droplets to divide into two smaller droplets. This process repeats itself until the solvent is completely evaporated and the droplets have split up to the point that each is a single, charged molecule, hence producing gaseous ions after several desorption steps. One of the advantages of this ionization method is that the molecules remain intact and will not be broken apart, and this ionization is called soft.[18] The last step, the release of solvated ion, can be described either by the ion evaporation model[19] or by the charge residue model.[20] Chen et al.[21] reported neutral desorption (ND) sampling coupled with EESI-MS for a rapid differentiation of biosamples by metabolomic fingerprinting. Figure 8.1 represents a schematic diagram of atmospheric pressure ND-EESI-MS used for analyzing the compounds from the surface of biological samples such as frozen meat by desorbing without sample pretreatment by a room temperature nitrogen gas flow, which creates a neutral aerosol mixture containing molecular metabolites. The aerosol is transported to the EESI source through the desolvation gas inlet (I). The aerosol passes through the heated region (H), which is maintained at 80°C. The distance between the desorption gas flow tip and the sample surface is 2–10 mm; the desorption (α) and collecting (β) angles are both 60°. The aerosol transfer line is a flexible Teflon tube (i.d. 5 mm and 120 cm in length), thereby demonstrating the possibility for remote analysis. As ESI[22] charges neutral sample molecules infused along the spray solvent, it results in a relatively weak tolerance of matrices. It is also reported that ESI has relatively low tolerance to the presence of buffers, salts, and complex matrices, which can lead to serious ion suppression effects. Usually, a dilute sample is required for ESI; therefore, a process for sample cleanup is demanded before a practical biological sample or other complex matrices were analyzed.[22] Alternatively, ions of biomolecules can be generated by desorption/ionization techniques, for which the sample is usually placed on a solid substrate. In such a case, ions can be generated by a laser [e.g., protein–protein interactions, matrix-assisted laser

FIGURE 8.1  (See color insert after page 124.) Schematic diagram of atmospheric ­pressure ND-EESI-MS.

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desorption/ionization (MALDI)], charged particles [e.g., desorption ESI (DESI), secondaryion MS, desorption atmospheric pressure chemical ionization (DAPCI)], or atom bombardment [e.g., fast atom bombardment, direct analysis in real time (DART)]. In MALDI,[23–25] biological samples are deposited on a metal plate as a small spot and cocrystallized with suitable matrices. Generally, ion yield depends heavily on the sample molecules, the matrix, and how they are prepared.[26] Owing to the abundant matrix peaks, analytes of small molecular weight (≤1,000 Da) are extremely difficult to be characterized with MALDI.[27] Recently, progress in desorption/ionization on silicon has shown that some relatively small molecules (≤500 Da) can be detected without a matrix. An interesting development was made by Cooks and coworkers[5,28] in 2004 with the invention of DESI, with which highthroughput analyses of various ambient samples were demonstrated for the first time without sample pretreatment. In DESI, ions are generated via the reactive collisions occurring between the neutral sample molecules and the charged particles (e.g., protons, fine droplets) produced by the electrospray. Theoretically, ion suppression can be reduced by separating the analyte from the electrospray solvent[29,30]; thus, biological samples (e.g., urine,[31,32] animal tissue,[33] plant tissue,[34] Sudan dyes in foods[35]) could be directly analyzed by DESI without a tedious sample cleanup. Analogous to DESI, the primary ions generated from atmospheric pressure chemical ionization (APCI) can be directed to impact a sample surface for DAPCI.[36–38] Taking advantage of APCI for a high yield of primary ions, the sensitivity of DAPCI is higher than that of other desorption/ionization methods. In principle, all these new desorption/ionization techniques have in common the fact that the sample is present as a solid, although the physical state of the original sample can also be different.[32,39] Motivated by the need for a direct analysis of liquid samples with complex matrices, EESI[1] was introduced in 2006. EESI[1,2] is a MS that uses two colliding aerosols: one of which is generated by electrospray (an apparatus that employs high-voltage electricity to disperse a liquid or for the fine aerosol) and another sample spray. In standard EESI, syringe pumps provide the liquids for both an electrospray and a sample spray. In ND-EESI, the liquid for the sample aerosol is provided by a flow of nitrogen. In the EESI process, the liquid sample is atomized using a sheath glass in the absence of electric field and forms neutral droplets. These droplets are dispersed into a conventional ESI plume formed from pure solvent and collide with the charged ESI droplets in a Y-shaped intersection, where the analyte interacts with the charged ESI droplets. Finally, in the merged plume, the analyte molecules mixing with the charged solvent in the droplets become gaseous ions in a subsequent ESI-like process. In this neutral sample, in the form of a gas or aerosol flow, the analyte is ionized by directing it into a plume of charged droplets, generated by forcing a solution of pure solvent through a small heated capillary (at a flow rate of 1–10 μL/min) into an electric field. Because the analyte is dispersed over a large volume, EESI does not require any sample pretreatment, and also, ion suppression is drastically reduced. Suffering from no ion suppression, pure solvents such as methanol/water mixture maintain a constant ion yield in ESI for a long time, which ensures stable signals of analyte molecules in EESI so that matrices such as raw urine or milk can be directly infused to generate a constant signal for more than 7 h.[1,12,40] Chen et al. demonstrated with a homemade EESI source coupled to a linear ion trap mass spectrometer that EESI shows sensitivity similar to ESI.[1] Another merit of EESI is that a neutral sample (i.e., biological subject) is safely isolated from any high voltage or from the direct bombardment by charged particles, and therefore, biological samples can be analyzed in vivo, with neither sample pretreatment nor chemical contamination. Another advantage is that the solvents in the charged ESI spray can be tuned to selectively extract the analytes, which are needed for the MS analysis, in complex matrices.[41]

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These advantages render EESI an ideal secondary ionization method for the analysis of gaseous volatile, semivolatile, and even nonvolatile substances in various complex matrices[42] as human breath can be directly analyzed in vivo and that nonvolatile compounds up to relatively high molecular weight (≤1,000 Da) are detectable in breath,[17,42,43] for the detection of melamine in milk products,[44–45] and for the analysis of viscous liquids.[46–48] Therefore, metabolism dynamics can be followed easily, and metabolic changes can be measured in vivo by EESI-MS. Liquid samples were initially demonstrated in EESI by real-time monitoring of complex biological fluids; gaseous samples including breath aerosol were successfully analyzed without suffering from any notable ion suppression. Besides analytical applications, EESI has been used to reduce the charge state of biopolymers either by ion– molecule reactions[49] or by ion–ion reaction[50] in open air. Thus, a promising new area emerges, showing numerous potential applications of EESI in multiple disciplines.

MECHANISM An understanding of the EESI mechanism is critical for defining its range of applications, the advantages, and limitations and for improving its repeatability, sensitivity, and selectivity. However, few studies on EESI mechanisms have been conducted so far. Law et al.[41] have done fluorescence studies in the EESI plume using rhodamine 6G and H-acid sodium salt directly, which demonstrate that liquid–phase interactions occur between charged ESI droplets and neutral sample droplets. Moreover, the effect of the composition of the primary ESI spray and sample spray on signals of the analyte in EESI-MS was investigated systematically. The results show that the analyte signals strongly depend on its solubility in the solvents involved, indicating that selective extraction is the dominant mechanism involved in the EESI process and provides valuable insights for optimizing the performance of EESI in future applications. Zhu et al.[51] reported a method for simultaneous sampling of volatile and nonvolatile analytes in beer for fast fingerprinting by EESI-MS. In this method, a stream of inert gas was introduced into the liquid sample, which induces bubbling under the surface of the liquid. Due to the pressure difference, the bubbles eventually burst at the bulk liquid/air interface, generating varied size of aerosol droplets (sub-micrometer to many micrometers) via rupture of the bubble skin and the formation of a jet of liquid bubbles.[52,53] The aerosol droplets, which contain both volatile and nonvolatile analytes from the bulk solution, are then carried by the gas flow to the EESI ionization source for further analysis. The hypothesis for this mechanism is that neutral aerosol droplets first collide with the charged primary droplets in the ESI plume, and the analytes are then extracted into the charged ESI droplets. These undergo further desolvation steps, finally yielding gaseous ions in a normal ESI process. The successful detection of nonvolatile analytes, such as amino acids, lactic acid, and glucose, in beer samples is consistent with the proposed mechanism. However, the solvent cluster ions observed also support the notion that aerosol droplets are generated via a bubble-bursting process, although such solvent cluster ions were not observed in the mass spectra obtained when electrospraying pure ESI solvent. Furthermore, measurement of some of the beer samples using ultrasound-assisted nebulization for EESI[44] did not generate such cluster ions. Aerosol droplets formed by bubble bursting in beer samples may be so large[52,53] that solvent desolvation is incomplete, thus giving a high population of solvent clusters; in contrast, ultrasonic nebulization of beer is believed to be much more efficient in generating very small droplets or even free molecules.

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The presence of protonated clusters (m/z 223) composed of solvents both from the ESI spray (e.g., methanol) and from the beer sample (e.g., ethanol) experimentally confirms for the first time that droplet–droplet collisions must have occurred before ionization. It would be much less likely to form such solvent cluster ions via collisions of molecules in the gas phase. In other studies, multiply charged ions of peptides and proteins were clearly observed after these were desorbed in neutral form either with an ultrasonic transducer[44] or with an radio frequency (RF) source[54] and then postionized with a primary ESI plume. The formation of multiple charged macromolecular ions is known to follow the so-called charged residue model[55]; that is, it is unlikely to form these multiple charged macromolecular ions only from the interaction of gaseous peptide/protein molecules and gas-phase ions from the charging spray. Though, the extent of the ­droplet– droplet collisions is still unclear. Despite the wide scope of applications, there are still a lot of questions regarding the charging of analyte molecules.[41] Wang et al.[3] explained the interaction of the droplets from the ESI spray and sample spray with each other in the Y-shaped intersection and whether a total coalescence occurs between the droplets. When the droplets from the ESI spray and sample spray interact, four types of droplet–droplet collision are possible: bounce, coalescence, disruption, and fragmentation.[56,57]

1. Bounce: There is a thin intervening gas film between the surfaces of two droplets. If the collision kinetic energy (CKE) of the two droplets is not sufficient to penetrate this gas layer, then the droplets bounce off each other, meaning that there is no physical contact between two liquid droplets and they only flatten temporarily. 2. Total coalescence: When the CKE of two droplets is high enough so that the thickness of the gas film reaches a critical value, usually 10 nm,[58] two droplets will coalesce temporarily or permanently, depending on the CKE. If the CKE is not too high, the coalesced droplet will oscillate with an amplitude of a few nanometers and finally achieve a stable form. 3. Disruption: Two droplets temporarily coalesce/contact and afterward separate into two droplets. 4. Fragmentation: After temporary coalescence/contact, the droplet undergoes catastrophic breakup into numerous satellite droplets.[56] Understanding of dominant type of interaction between the droplets from the ESI spray and the sample spray, mechanism for the corresponding charging process of analytes helps for optimizing the performance of the EESI method in terms of sensitivity, universality, and reproducibility. Using laser-induced fluorescence, Law et al.[41] demonstrated that the charged ESI droplets and the neutral sample droplets do collide in liquid form before the final desolvation and gas-phase ion formation take place for nonvolatile compounds. In addition, a strong dependence of ion signals on the analyte solubility in both the ESI and sample spray solvents implies that a selective extraction occurs between the charged ESI droplets and the neutral sample droplets. This excludes total coalescence as the dominant type of droplet–droplet interaction between the ESI droplets and the neutral sample droplets with different solvents in the two sprays. To study whether the total coalescence of droplets happens in the EESI process when the solvents in the ESI spray and sample spray are identical, a detailed investigation was conducted based on a theoretical model to distinguish the type of droplet–droplet interaction. However, MS measurements cannot provide such information about the droplets in the EESI process[41]; thus, phase Doppler anemometry (PDA), a powerful tool, is used

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to directly measure the droplet size, velocity, and number density (essential for theoretical analysis) in dynamic processes such as electrospray.[59–61] The size, velocity, and density of droplets in the intersection where two sprays meet (referred to as the origin) and 2 mm downstream from the cross section were measured. The EESI setup consisted of two identical commercial electrospray sources (M955015DC6, Waters, Manchester, UK) that were constructed from two coaxial stainless capillaries of identical diameters for delivering the liquid and the sheath gas, respectively. In the PDA measurements, one of the sources was used to spray the neutral sample, with a sheath gas pressure of 2 bar (compressed air); the other one was used as the ESI source, with a sheath gas pressure of 2 bar and a fixed voltage of + 4 kV applied by a high-voltage supply (HCN140-12500, FuG, Rosenheim, Germany). In the EESI spray measurements, a water/ ethanol mixture (1:1 volume ratio) was used in both sprays. The solutions were delivered by two 500-μL syringes (Hamilton, Bonaduz, Switzerland) and a syringe pump (Harvard 22 syringe pump; Harvard Apparatus GmbH, March-Hugstetten, Germany) having a flow rate of 1–20 μL/min. The distance between the tips of the capillaries and the counter electrode was 10 cm. The measurements were taken with various geometries, at angles (α) of 30°, 80°, and 180° and distances between the tips of the two capillaries (d) of 3, 4, and 5 mm, respectively. A commercial-phase Doppler anemometer (TSI Inc., St. Paul, MN, USA) was used to measure droplet velocity, droplet size, and droplet density in the EESI spray and single-spray modes (i.e., one spray was turned off, and the other spray was kept on); the sheath gas was always on for both sprays. Two laser lines (488 and 514.5 nm) from a water-cooled 5-W argon ion laser (LA-70-5, Innova 70, Coherent, Santa Clara, CA, USA) were used in the PDA setup. The laser beams illuminated the EESI spray, and the scattered light from the droplets in the spray was collected by a fiber opticbased receiver, which was 45° off the axis of the incident beams. The ESI spray came from the negative X direction, whereas the sample spray came from the positive X direction; both flowed along the Z-axis after merging at the origin. The whole system was mounted on a three-dimensional (3D) translation stage (9450-XYZ500, Isel Germany AG, Dermbach, Germany) controlled by a stepping motor (C142-4.1, Isel Germany AG) to allow moving the measurement point and making automated profile measurements. The PDA measurement volume formed by the two intersecting laser beams had a half-axis of approximately 2.5 mm in length and 0.22 mm in width and height.[62] The measurements were done at 49 positions (7 × 7 grid) in the XY plane by ­moving the measurement point automatically. The measurement area was defined according to the size of the spray plume. The detection ranges of droplet size, velocity, and density were 1–100 μm, ≈0–640 m/s, and ≥20/cc, respectively.[63] Droplets with a size of 4 μm only take 10 μs to be accelerated to follow the gas flow,[64] a time short enough to consider the velocities of these droplets to be the same as the velocity of the sheath gas.[65] Thus, in this work, the gas velocity was derived from the velocities of droplets smaller than 4 μm from the raw PDA data. With both the ESI and sample sprays on, the size, velocity, and density of droplets were measured simultaneously in the EESI spray plume under various experimental conditions using PDA. The Sauter mean diameter (SMD, the total droplet volume divided by the total droplet surface area) is usually used to characterize the droplets in the spray. The volume density rather than the number density was used to avoid bias from the large number of small droplets. The volume density was normalized to the sum of all droplet volumes in the measurement area in order to avoid an influence of a varying volume density in different measurements. In the case of α = 80°, the SMD of

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the EESI spray was measured to be 7.85 μm and the average SMD was calculated to be 8.82 μm by averaging the results of the ESI spray and the sample spray in the singlespray mode, that is, quite similar. If permanent total coalescence happens between the droplets from the two sprays, the droplet size distribution of the EESI spray should shift to a significantly larger value and the density of small droplets should decrease compared with the averaged data from the ESI spray and the sample spray in the single-spray mode. However, no obvious shift of the distributions was observed; that is, there was no significant change in the droplet size or a dramatic decrease in the droplet density after two sprays met, implying that no permanent coalescence takes place at the origin of the EESI plume (z = 0) even when the solvents in the two sprays are identical. This is fully in line with the results where different solvents[41] were used for the two sprays. The droplet behavior 2 mm axially downstream from the origin was also measured. The droplet sizes for both sprays have decreased further compared with the ones at the origin due to the solvent evaporation; the decrease is stronger for the droplets in the sample spray. At 2 mm, the SMD of the EESI spray (6.65 μm) was similar to the average (6.00 μm), and the droplet size distributions of the EESI spray and the combination of the two single sprays were analogous, suggesting again that there is no total coalescence of droplets 2 mm downstream. The mechanism of EESI in the dual-spray configuration has been clarified based on the systematic PDA measurements, numeric simulations, and theoretical analysis. Measurements of droplet size, velocity, and density were taken in the EESI spray at the origin and downstream from the origin using various experimental parameters. The droplet behavior in the single-spray mode was also investigated to compare with the results in the EESI spray. The results show that the droplet size distribution of the EESI spray was very similar to the averaged results in single-spray mode. This suggests that there was no permanent coalescence at the origin as well as further downstream in the EESI plume, although the solvents in the two sprays were identical. Similar phenomena were observed with different geometries of the EESI source and various sample flow rates. The absence of coalescence probably has two reasons: (1) the overlap of the droplet distributions between the ESI spray and the sample spray was small, reducing the collision probability between the ESI droplets and the sample droplets. The interaction time between the ESI droplet and the sample droplet was estimated to be 1,000 Da) in biological samples. The mass spectral fingerprints yield comprehensive information about the molecular basis for the physiological states of biological samples and for the metabolic dynamics of microorganisms associated with the samples. The difference between samples in different physiological states can be further visualized with principal component analysis.[61] This study gives a starting point for more sophisticated applications in multiple disciplines, where analytes in complex matrices can be conveniently sampled in real time or in vivo. A new method for rapid on-line detection of metabolic markers in complex biological samples was developed by Chen et al.[21] to allow the interrogation of virtually any type of surface by a gentle stream of air or gas, followed by an efficient ionization of the neutral molecules released in an EESI step, such as frozen meat, spinach, and human skin. The samples were analyzed directly and in an on-line fashion by the gentle desorption of neutrals coupled with EESI-MS for rapid monitoring, without any chemical contamination or sample pretreatment. The mass spectral fingerprints display metabolites originating either from growing microorganisms or from the sample itself, and therefore, molecular signatures for a wide variety of biological samples are favorably obtained. This novel metabolomics-based strategy represents a “green” procedure for fast food quality assessment. The resulting mass spectral fingerprints are shown to be able to detect spoilage of meat even in the frozen (−20°C) state and the contamination of spinach by E. coli, and to identify metabolites and contaminants on human skin within seconds, in an on-line and high-throughput fashion. Typical molecular markers are identified using MS/MS data and by comparison with reference compounds. The detection limit achieved is 10 fg/cm 2 (S/N = 3) for histamine on the surface of frozen meat. It was validated by 15 meat samples from different origins and was

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successfully applied to fast screening of spinach samples contaminated by E. coli and to in vivo analysis of human skin. The physiological and/or pathological status of animals or plants can also be diagnosed in vivo on the basis of a molecular signature.[123] The technique shows potential for more advanced applications in multiple disciplines, including food regulation, homeland security, in vivo metabolomics, and clinical diagnosis. Peaks of interest such as m/z 122, 88, and 116 are tentatively assigned, from their MS/MS spectra to be –NH 2- and/or –SH-containing compounds. For example, the compound of the highest intensity (m/z 122) in the mass spectrum is interpreted as protonated CH3CH 2OC(O)NHSH (MW 121), which loses CH3 to yield m/z 107, CO to yield m/z 94, and CO2 to yield m/z 88 as major fragments. It could be cystein (MW 121); however, protonated cysteine loses water to generate a major fragment of m/z 104. But this fragment (m/z 104) was not observed. The second most abundant peak (m/z 88) is assigned to protonated C4H 9ON (MW 87), which loses water in the MS/MS spectrum to yield ions of m/z 70 or alternatively loses CH 2CO to give a fragment of m/z 46. Peak at m/z 116 was tentatively identified as protonated 4-amino-2-hydroxycyclopentanone (MW 115) since it loses NH3 to yield a small peak at m/z 99, whereas it loses CO to give a major peak at m/z 88, which further loses water to give an abundant peak at m/z 70. By comparing with the MS/MS data of authentic compounds (Table 8.2), many peaks detected are biogenic TABLE 8.2  MS/MS Data of Identified Molecular Markers Detected in Fish Meat at Different Stages of Spoilage Molecular Markers Trimethylamineb Dimethylamineb Dimethylacetylamineb N-Methylpyrrolidineb CH3CH2OC(O)NHSHc CH3NHCH2C(CH2)OHc N- Methylcyanamidec Putrescineb,d Cadaverineb,d Histamineb,d C2N2H8c Amino-2-hydroxyclyclopentanonec Tyramineb Spermidineb,d Tryptamineb Spermineb Pentanethiol C2H4N2c a b c

d

Molecular Weighta 59 45 73 117 121 87 56 88 102 111 60 115 137 145 160 202 104 57

Number of Days Samples Were Exposed to Room Temperature 0 0 0 0 0 0 1,2 1,2 1,2 0,1,2 0,1 1,2 2 2 2 2 2 1,2

All compounds are detected as protonated molecules in EESI-MS. Compounds identified with reference compounds using MS/MS. Compounds tentatively identified from EESI-MS and MS/MS data without confirmation by reference compounds. Compounds also commonly found in beef, lamb, and turkey meat samples, especially in samples at advanced stages of spoilage.

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amines, which are biomarkers known for microorganisms growing on meat[69–72] and can be used for detecting the spoilage of the food. For example, trimethylamine (MW 59) shows a high signal, whereas dimethylamine (MW 45) gives relatively low intensities in the fresh sample. All the alkylamines except trimethylamine are undetectable in fish samples after exposure to room temperature. Similar dynamic changes were also found for the peaks at m/z 122 and 88 owing to the metabolic dynamics of the microorganisms.[70,73,74] Since the fresh fish sample was not truly “fresh,”[75] the smell a typical “fresh fish odor” could not be smelled. Chemically, the change in smell was mainly due to variable levels of alkylamines and other predominant flavors such as the compounds detected at m/z 122 and 88. The peaks detected reflect the chemical dynamics of the changes in the smell. Interestingly, the peak at m/z 57, probably protonated C2H4N2 (MW 56), is quite strong in the fish sample exposed to room temperature for 2 days (3.27 × 106 counts) and 1 day (1.06 × 105 counts), whereas it was not detected in fresh fish. Meat spoilage results from the growth of microorganisms that produce characteristic metabolites such as biogenic amines.[69,70,72,73] For instance, histamine is a typical molecular marker for various spoiled foods[70–72,74] and is the main toxin involved in health problems such as histamine fish poisoning[76] and other biochemical disorders.[77] Histamine (m/z 112) and putrescine (m/z 89) were detected in fish exposed for 1 day. All the typical biogenic amines including putrescine, cadaverine, histamine, tyramine (MW 137), spermidine (MW 145), tryptamine (MW 160), and spermine (MW 202) were detected as protonated molecules with relatively increased intensities in fish exposed to room temperature for 2 days. The biogenic amines[69,70,72,73] and other strong peaks such as those at m/z 116 and 57 are proposed to account for the typical putrescent smell of spoiled fish at room temperature. The multiple peaks shown in the mass spectral fingerprints of spoiled fish suggest that there is actually more than one component (i.e., histamine) in the spoiled fish. If spoiled fish is consumed, many chemicals that are more toxic than pure histamine (including toxins generated from the metabolism of the microorganisms) are ingested, thus giving useful hints in understanding the finding that histamine consumed in spoiled fish is more toxic than pure histamine taken orally.[76] In Vivo Skin Analysis The metabolites in different areas of human skin[21] such as forehead, forearm, and abdominal can be fingerprinted in vivo by EESI-MS. The forehead skin of a male adult shows numerous peaks in the mass range from m/z 50 to 1,000 in the positive ion detection mode. Similarly, EESI mass spectral fingerprints are recorded from the abdominal skin, the forearm skin, and the foot skin of the same volunteer showing quite different spectral patterns of the different skin areas although some common peaks (e.g., m/z 282) are detected on the forehead, forearm, and abdominal skin, whereas the peak at m/z 538 is commonly detected on the skin of the forehead, abdomen, and the foot. The peak at m/z 181 detected in the forehead skin is identified, on the basis of a reference compound, to be protonated glucose, which losses HCHO and H 2O as major fragments in collision-induced dissociation (CID). Interestingly, the amount of glucose on the skin of the forehead is much higher than that of other samples in consideration, indicating that the difference in the metabolites (e.g., glucose) excreted from different skin areas might be due to the differential metabolic activities of the adjacent organs, which consumes glucose with differential speed due to different metabolic status.[124] Under the experimental conditions used, the foot skin presents a much simpler fingerprint than

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other skin samples, probably because the feet have harder skin and therefore fewer metabolites can be released to their surface. It is also observed that if a different desorption reagent (e.g., acetone instead of nitrogen) is used, different spectral patterns are obtained, which reveals rich molecular information. For example, when acetone vapor is added into the nitrogen gas beam for skin desorption sampling, it shows different mass spectral fingerprints from the same area of a hand skin before and after the addition of acetone in the gas beam. As a result, the intensity of peak at m/z 538 is dramatically enhanced so that it becomes the predominant peak and forms a proton bound cluster (m/z 598) with acetic acid. Potentially, this feature is very useful for chemical classification and metabolites profiling, especially when a specially designed reagent gradient is used for desorption sampling. It is also possible to detect cyclotrimethylene trinitroamine (RDX), a typical explosive, on skin after exposure to air containing traces of RDX. An RDX methanol/water (1: 1) solution (2 mL, 10 ppt) is sprayed into the open air above a bare hand, which is exposed to this plume for 2 min to receive part of the RDX sprayed into the air. The hand skin is directly analyzed using the ND-EESI-MS, and a mass spectrum shows the peak at m/z 223, which is identified as protonated RDX and yields a major fragment at m/z 177 by the loss of NO2 during CID. Successful rapid detection of RDX, a typical explosive, provides an example for fast screening of dangerous chemical or biological hazard reagents such as anthrax bacteria, explosives, and chemical warfare agents, or for rapid clinical diagnosis, showing promising applications in homeland security programs. Song et al.[78] have analyzed various meat samples by direct internal EESI-MS (iEESIMS) as hemoglobin (Hb) present in the blood and meat juice samples was selectively adsorbed by graphene oxide (GO) particles functionalized with amylopectin and was sensitively detected. Various samples including the whole blood samples of chicken, duck, sheep, mouse, pigeon, turtledove, and meat juice mixtures were successfully identified based on the difference in molecular composition of Hb reflected in MS. The adulteration of sheep blood with only 2% chicken blood could be detected, which demonstrated the high chemical specificity of the approach. The established method is featured by the high speed of analysis (4 min per sample, including the analyte extraction and sample loading), high sensitivity, minimal sample preparation, and low sample consumption (0.9 μL of whole blood or 300 mg of raw meat). In perspective, the reported method can be extended for the sensitive detection of trace analytes in complex matrices in broad molecular range using the selective enrichment on functionalized GO particles followed by iEESI-MS analysis. Meier et al.[79] investigated how binary mixtures of compounds influence each other’s signal intensity in ESI, EESI, and secondary ESI (SESI) experiments. The experiments were conducted using a series of homologous primary amines (from 1-butyl to 1-decylamine). In every experiment, two of the amines were present, and all 21 possible combinations were measured with EESI, ESI, and SESI as ionization sources. Except for the volatility, which decreases with increasing molecular weight, the physicochemical properties of the amines are very similar, so that the intensity ratio obtained in each experiment provides information about discrimination effects occurring during the ionization process. The results show that for the relatively volatile compounds investigated, the EESI ionization mechanism resembles the SESI-like gas-phase charge transfer more than ESI-like analyte ionization in solution. In addition, almost no discrimination effects were observed in the spectra obtained in EESI experiments. Quantitative EESI experiments with nonylamine as internal standard showed that EESI is capable of providing both more accurate and more precise results than SESI and ESI.

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Analysis in Perfume Diethyl phthalate (DEP) is found in abundance in perfume products for both men and women. Chingin et al.[16] had carried out EESI-MS/MS spectra of 18 perfume samples in same conditions to identify the DEP content as it is more toxic to males by causing reproductive failure[80–84]; thus, it is of greater concern for men to avoid using perfume products containing high amounts of DEP. It was detected in 13 of the 18 samples (detected in “Weekend,” “Beautiful,” “Hugo XY,” “le Male,” “ETH Zurich 150,” “Bright Crystal,” “Option,” “CK One,” “Miss Dior,” “Clinique Happy for Men,” “Opium,” “Opium Shanghai,” and “Opium Fleur Imperiale”; not detected in “Natural fragrance,” “White Musk,” “Midnight Poison,” “Be delicious,” and “Clinique Happy Heart”). For these samples, MS/MS spectral patterns of m/z 223 ions were identical to that of authentic DEP. The highest detected signal was from “le Male” by Jean Paul Gaultier. According to the Greenpeace survey, this fragrance holds about 1% DEP (w/w).[85] It was found that DEP formed ionic sodium adducts (m/z 245) rather than protonated molecules when a diluted sodium chloride solution (1 ppm) was electrosprayed in the EESI source, which helps in the specifying DEP detection from the complex perfume samples. When the sodium content decreased down to the low parts per billion range, both of the sodium adducts and the protonated molecules of DEP were simultaneously observed in the EESI-MS spectra. Cationization of DEP can provide a better sensitivity, probably due to higher affinity of DEP to sodium ions compared to protons. This is also supported by the CID experiments of DEP–sodium adducts, which gave no fragments with the possible highest energy for collisions in quadrupole time-of-flight (QTOF) instrument, showing the stability of the DEP–sodium adducts. Thus, a reliable identification without resorting to tandem MS can be done from a high-precision mass measurement experiments of the parent ions by Fourier-transform ion cyclotron resonance (FTICR)[86] or Orbitrap[87] analyzers, both of which feature very high mass accuracy. Also, it is well known that the CID fragment of DEP at m/z 149 is characteristic for other phthalates as well.[88] Therefore, surveying all parent ions of the fragment could be informative of other closely related phthalates present in a perfume sample. This can be easily achieved by neutral-loss scan available in a number of tandem mass spectrometers, for example, triple-stage quadrupoles.[89] Therefore, this method can be potentially extended to fast screening of any phthalic acid in perfume products. With the use of the method reported here, no sample pretreatment is required for perfume analysis, and a single sample analysis can be completed within a few seconds. The limit of determination for DEP in perfume was on the order of 100 ppb with tandem MS. This method provided a dynamic response range about 4 orders of magnitude, providing a rapid way to obtain semiquantitative information on DEP in bulk perfume analyses. For applications in which trace amounts of analytes need to be continuously monitored, the classic configuration of EESI using two spray beams[1,12,68] is probably preferable, because it enables online, real-time monitoring of complex samples with ease. However, when highthroughput analysis is required (e.g., identification of hazardous species in bulk commercial products on the market), the novel sample delivery method becomes particularly useful. Analysis of Organic Aerosols Real-time in situ MS analysis of airborne particles is important in several applications, including exposure studies in ambient air, industrial settings, and assessing impacts on

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visibility and climate. However, obtaining molecular and 3D structural information is more challenging, especially for heterogeneous solid or semisolid particles. Kumbhani et al.[90] reported a study of EESI-MS for the analysis of solid particles with an organic coating to elucidate the overall particle content that can be sampled, and to determine the sensitivity of the technique to the surface layers. It is shown that for NaNO3 particles coated with glutaric acid (GA), very little of the solid NaNO3 core is sampled compared to the GA coating, whereas for GA particles coated with malonic acid (MA), significant signals from both the MA coating and the GA core are observed. However, conventional ESI-MS of the same samples collected on a Teflon filter (and then extracted) detects much more core material compared to EESI-MS in both cases. The results show that EESI-MS does not sample the entire particle but, instead, is more sensitive to surface layers. Separate experiments on single-component particles of NaNO3, GA, or citric acid show that there must be a kinetics limitation to dissolution that is important in determining EESI-MS sensitivity. In conjunction with previous EESI-MS studies of organic particles, the results suggest that EESI does not necessarily sample the entire particle when solid and that not only solubility but also surface energies and the kinetics of dissolution play an important role. Gallimore et al.[91] have characterized and measured the detailed chemical composition of organic aerosol particles by EESI-MS, which was composed of soluble organic compounds. They were extracted into and ionized by a solvent electrospray, producing molecular ions from the aerosol with minimal fragmentation. It was demonstrated that the technique has a time resolution of seconds and is capable of taking stable measurements over several hours. The ion signal in the MS was linearly correlated with the mass of aerosol delivered to the EESI source over the range tested (3–600 μg/m3) and was independent of particle size and liquid water content, suggesting that the entire particle bulk is extracted for analysis. Tandem MS measurements enabled the detection of the ozonolysis of oleic acid aerosol (20 μg/m3), which revealed the formation of a variety of oxidation products. It demonstrates the technique’s potential for studying the productresolved kinetics of aerosol-phase chemistry at a molecular level with high sensitivity and time resolution. Jecklin et al.[92] compared the features and characteristics of standard ESI, chipbased nano-ESI, and ESSI mounted onto a hybrid QTOF mass spectrometer in their performance to determine the dissociation constant of the model system, hen egg white lysozyme (HEWL) binding to N,N,N-triacetylchitotriose (NAG3). The best K D value compared with solution data was found for ESSI, that is, 19.4 ± 3.6 μM. The K Ds of the two nucleotide-binding sites of adenylate kinase (AK) were also determined, thus showing K Ds of 2.2 ± 0.8 μM for the first and 19.5 ± 8.0 μM for the second binding site using ESSI. The current particle measurement techniques struggle to efficiently shift analytes from the condensed phase to the gas phase for molecular analysis by MS. Thermal desorption of aerosol components for gas-phase analysis is typical in most online aerosol mass spectrometers, but thermal decomposition during desorption can significantly alter the measured molecular composition. Even instruments that use soft ionization techniques still rely on some type of thermal desorption to evaporate material into the gas phase for analysis, leading to unpredictable changes in particle composition due to decomposition. Therefore, there remains a fundamental need for online, rapid-response chemical characterization of particles without artifacts from thermal decomposition. Lopez-Hilfiker et al.[93] present the design and the characterization of an online EESI source for online particle analysis and its application to atmospheric aerosol.

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Merits and Demerits of Techniques Many techniques including chromatography,[94–96] optical spectroscopy,[97,98] ion mobility spectrometry,[99] and MS[100–105] have been used for the detection of analytes in various samples. Because of the complexity of the real samples, a sample cleanup procedure was usually required for most techniques. Chromatographic techniques such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) are conventionally used to identify components as, for example, phthalate esters in cosmetic products,[106,107] but a single HPLC/GC run typically takes approximate 30 min. In addition to it, sample pretreatment may be required, especially for sensitive detection (e.g., extraction, preconcentration). However, when a large number of samples (e.g., in quality control laboratories) need to be processed, high throughput may become a major requirement. An appropriate analytical tool for practical sample analysis must meet the demanding requirements for high throughput, high sensitivity, and specificity since the concentration of analyte as well as the sample composition varies over a wide range. To facilitate high-throughput MS analysis, techniques such as DESI,[28,30,108] DAPCI,[36,38,109,110] DART,[36,108,111–113] atmospheric-pressure solids analysis probe (ASAP),[114,115] and thermal desorption APCI (TD-APCI),[116,117] have been used for fast detection of analytes on solid surfaces. Besides some common advantages of these ambient ionization techniques, each one has unique features for specific analytical applications. Liquid samples can be analyzed by these techniques but require sample pretreatment. For example, dilute urine dried on a paper surface[32] can be examined by DESI. Similar to DESI, other techniques such as DART, ASAP, and TD-APCI analyze liquid samples indirectly. Usually, deposition of the sample on a solid surface and solvent volatilization is employed as a sample preparation step. In DART, TD-APCI as well as ASAP, the solvent evaporation can be shortened to 2–3 min by heating the sample surface to a high temperature (e.g., 250°C–450°C). However, such a high temperature results in fast degradation of sensitive compounds, and thus, the mass spectrum of a sample can be significantly changed. This renders data interpretation difficult, especially for the analysis of heatsensitive samples such as perfumes. Liquids, gases, suspensions, and aerosol samples can be directly analyzed by EESI[1,12,15,17,21,66–68,108,118–122] and MS without any sample pretreatment. In this technique, very fragile biomolecules can be ionized, and even noncovalent complexes can be detected with no dissociation and large biomolecules (MW > 70,000) can be analyzed in a small m/z range ( 0.99), extensive linear ranges (5–15,000 ng/g), and high precision (relative standard deviations about 10%). The authors also observed that the agrochemicals were not homogeneously distributed throughout the different layers of the fruits/vegetables. They also reported that two or more agrochemicals were simultaneously detected in some samples. This is because producers usually apply mixtures of agrochemicals on the plantation, aiming at obtaining the best protection while respecting the maximum residue limits for each compound. Finally, the mass spectra of organic and nonorganic fruits and vegetables could be easily differentiated by the presence of characteristic ions arising from the main pesticides usually applied on the conventional cultures (Malaj, Ouyang, Sindona and Cooks 2012). Yang et al. made use of wooden tip electrospray mass spectrometry for the detection of pesticides in samples of apple juice and other foodstuffs. The sampling consisted of a simple immersion of wooden tips into the juices followed by the application of a polar solvent and a high voltage. For quantitative analysis, a calibration curve was built by spiking a blank sample with appropriate standards and using the selected reaction monitoring mode. The limit of detection of 30.0 ng/mL was in conformity with the Chinese legislation GB 2763-2012 that establishes the maximum residue limits of pesticides in food samples (Yang et al. 2015). Matrix effects caused by sample components have been extensively discussed by several authors that worked with the food analysis by ambient mass spectrometry methods. The presence of typical food components (e.g., sugars, lipids, peptides, and others) is the main cause of ionization suppression of target analytes. Therefore, some studies have been conducted aiming at verifying the appropriate methods for sample loading on the paper substrate. For instance, Evard and coworkers proposed the use of PS-MS as a fast-screening method to select samples to be later submitted to confirmative LC-MS analysis. They investigated the presence of the pesticides aldicarb, methomyl, methiocarb, imazalil, and thiabendazole in fruits and vegetables, such as oranges, grapefruits, lemons, limes, mandarins, tomatoes, apples, pears, strawberries, grapes, and sweet peppers. The authors evaluated two modes of sample loading onto the PS-MS substrate: (1) wiping the fruit surface with a wet paper; and (2) applying the homogenized sample directly on the paper. They could determine, by both methods, the presence of the pesticides at concentrations lower than the maximum residue limits stated by the legislation (e.g., 5 ug/kg for imazalil and thiabendazole). The authors observed a remarkable matrix effect in the homogenized samples, but they argue that additional experiments must be conducted to better understand this result. For the method evaluation, the authors acquired a total of 11 different fruits in a local market and analyzed them by the wiping method, which detected the presence of the pesticides thiabendazole and/or imazalil in four samples (Evard, Kruve, Lõhmus and Leito 2015). Besides the evaluation of the sample loading modes, the performance of PS-MS was also compared with some of its derived techniques. Pereira and coworkers evaluated the responses of two similar ionization modes, paper spray (PS) and leaf spray (LS), on the screening of agrochemicals. The pesticides atrazine, diuron, and methomyl were quantified by both methods in arugula, basil, cabbage, and lettuce. Atrazine was used as the internal standard for the diuron and methomyl analysis, whereas diuron was used as the internal standard for atrazine quantification. A calibration curve was built using the ratio of the intensities of the analytes and the internal standard ions. The results evidenced

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similar analytical responses for PS-MS and LS-MS, showing that both are excellent ionization techniques for agrochemical analysis (Pereira et al. 2017). Modifications in the paper surface have been performed to improve the detection and quantification of analytes in food samples. These modifications aim to reduce the voltage applied at the base of the triangular paper, change the nature of the interaction between matrix/analytes and the surface, and increase the selectivity/sensitivity of the analytical methodology (see further explanations on this matter previously in this chapter, “Modifications in the Paper Substrate” section). For instance, Narayanan and coworkers reported the use of a modified paper surface recovered with carbon nanotubes. The objective of these modifications was to minimize (or even eliminate) the occurrence of in-source fragmentation and formation of oxidized by-products, besides reducing the amount of sample required in the conventional PS-MS methodology. The authors reported that this modified PS-MS methodology was used for the detection of amino acids, drugs, and pesticides in orange. The modified paper was wiped on the peels of several fruits and three pesticides (carbofuran, methyl parathion, and parathion) were detected. The authors obtained high-quality MS spectra with the application of low potential (3 V) that could be easily obtained by using portable batteries (Narayanan, Sarkar, Cooks and Pradeep 2014). Wang and coworkers reported the use of a paper surface coated with silica for PS-MS analysis. This surface was used to analyze seven common pesticides (alachlor, acetochlor, pretilachlor, butachlor, metolachlor, napropamid, and benzeneacetamide) in milk samples. Silica was chosen due to its attractive physico-chemical properties, such as small particle size, high specific surface area, ease of application on solid surfaces, and the ability to change the paper surface porosity. All these aspects allowed for the attainment of mass spectra with an excellent signal-to-noise ratio. The authors used tandem MS for identification and quantification of these pesticides. The validation of the calibration curves showed that the use of the modified paper leads to a decrease in the limits of detection for all analytes (2–19 fold) when compared to the conventional PS-MS approach (Wang, Zheng, Zhang, Han, Wang and Zhang 2015). Recently, Pereira and coworkers proposed a new modification in the traditional PS-MS technique based on the application of molecularly imprinted polymers (MIPs) on the paper surface. This was done to improve the detection capability of the pesticides diuron and 2,4-di-chlorophenoxyacetic acid in apple, banana, and grape samples. The MIPs were used to selectively extract the analytes from the methanolic extracts of these ­samples thus minimizing ionization suppression effects. Two different types of MIPs were tested: monuron and 2,4,5-trichlorophenoxyacetic acid. These materials were chosen due to their routine use in the preparation of SPME and solid-phase extraction (SPE) devices. The results showed a significant increasing in the intensities of the pesticide ions in the mass spectra when compared to those obtained with nonmodified cellulose membrane. The MIP-modified paper was also used to quantify diuron and 2,4-di-chlorophenoxyacetic acid in simulated and real food samples (Pereira, Rodrigues, Chaves and Vaz 2018). PS-MS Chemical Profiles of Foodstuffs Foodomics is a topic that seeks for the understanding on how the food components interact with the metabolism and which are their effects on the human health. In order to conduct these studies, novel analytical methodologies have been introduced to access not only the identification/quantification of specific components but also to appraise the

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effects (beneficial or detrimental) caused by specific foods on human nutrition. Therefore, in foodomics, whose ultimate goal is to gain a whole perception of the feeding process, these two approaches are considered inseparable. As a consequence of the application of the foodomics concepts, several impacts have been noticed, such as (a) improvement in the human feed habits, (b) a better understanding on the effects caused by the use of genetically modified foods, and (c) enhancement in the production methods of foods with the consequent cost reduction (Capozzi and Bordoni 2013). However, the main limitation against the development of foodomics is associated to the inherent limitations of the conventional methods used for food analyses. Food is a complex matrix and, in most cases, analyses require laborious sample pretreatment steps to promote the isolation of specific target components. Therefore, the results from these experiments do not fully describe all possible antagonistic/synergistic interactions between a specific component and the metabolic pathways of a given organism. For this reason, the development of new methodologies able to simultaneously determine several classes of food components and metabolites is mandatory (Capozzi and Bordoni 2013). MS meets the main requirements of foodomics due to its high sensitivity and selectivity as well as its superior capacity to identify individual components present in complex matrices. Besides that, MS is able to simultaneously detect a wide range of compounds from several classes and, consequently, to provide the chemical profiles (fingerprints) of diverse types of samples. Recent developments in ambient ionization techniques for MS have expanded the possibilities of fast and direct analysis (screening) of foodstuffs. Among these methodologies, PS-MS (and correlated techniques) have attracted the foremost attention. For instance, tissue spray mass spectrometry was used by Liu and coworkers to analyze intact tissues from several plant species aiming at the attainment of their chemical profiles. Hence, the fingerprints of leaves (green tea, Brussels sprout, cauliflower, cabbage, tomato, green onion, and spinach), rhizomes (ginger), fruits (cranberry, tomato, grape fruit, pepper, cucumber, corn kernel, onion), seeds (peanut, mung bean, and ginkgo), tuber (potato), and sprout (mung bean) were obtained, and the results are displayed in Figure 12.6. The detection of several metabolites from distinct phytochemical classes, such as amino acids, sugars, alkaloids, flavonoids, organic acids, phospholipids, fatty acids, and lipids, is clearly noticeable in these samples (Liu, Wang, Cooks and Ouyang 2011). Chemical profiles of endogenous compounds were also successfully obtained using both bench top and portable mass spectrometry. In vivo analyses were performed, including on a plant species (bean) at different stages of development: seed, sprout, and leaf. The authors also demonstrated the usefulness of tissue spray mass Spectrometry in real time analyses by monitoring the metabolic changes experienced by spinach leaves submitted to a mechanical stress (chewing by an herbivore) (Liu, Wang, Cooks and Ouyang 2011). Zhang and coworkers described a methodology for direct analysis of steviol glycosides in fresh leafs of Stevia using leaf spray mass spectrometry. Steviol glycosides are of significant importance to the food industry. Because of their excellent sweetener properties and low-calorie content, these compounds have been extensively used as additives in beverages and foodstuffs. The authors obtained the MS fingerprints of Stevia leaves (both in the positive and negative modes) and compared them with those obtained by other ambient ionization methodologies. The results demonstrated the superior performance of leaf spray mass spectrometry in the detection of the steviol glycosides. These compounds were detected as adducts of Na+, K+, and Cl− and identified by their fragmentation profiles. Oxidation by-products formed during the ionization process were also characterized by using high-resolution mass spectrometry. The authors performed

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FIGURE 12.6  (See color insert after page 124.) Examples of chemical profiles (fingerprints) obtained via tissue spray mass spectrometry of distinct species of fruits: (a) grape fruit; (b) tomato; (c) pepper. (Reproduced and adapted with permission from ©2011 American Chemical Society.)

semiquantitative analysis by adding an isotopically labeled internal standard (rebaudioside D) on commercial stevia leaves. The results showed that the steviol glycosides were in the expected concentration range in such leaves. The authors also analyzed supplemental products and could confirm the presence of steviol glycosides and polyglucose in their formulation (Zhang, Li, Ouyang and Cooks 2012). Other technique related to PS-MS was proposed by Hu and coworkers to be applied to the analysis of several raw samples. The method, called by the authors as TLC-ESI-MS, was based on the use of a TLC device directly coupled to a MS instrument. The intention was to perform a partial elution of a sample to retain most of the interfering compounds (e.g., salts and detergents), thus reducing matrix effects. For this, a sorbent material was supported on the TLC substrate and, after sample elution, triangles were cut and positioned in front of the MS inlet to be analyzed. The authors applied the TLC-ESI-MS methodology to obtain chemical profiles of spinach leaves (Hu, Xin, So and Yao 2015). They also evaluated fruits that belong to two species of the genre Shisandrac: S. sphenanthera (FSS) and S. chinensis (FSC). Both fruits are used in traditional Chinese medicine for the treatment of the common cold and seasickness. Moreover, two different modes of sample analysis were evaluated: off-line and on-line. In the first, the TLC plate was initially used to promote the elution of the sample extracts, then different spots were individually analyzed. In the second mode, the extracts were analyzed with no previous elution step. The on-line method was used to analyze leaf spinach extracts, and some

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endogenous compounds were easily identified during the elution step. Compounds such as carotene, pheophytin A, and other pigment components were continuously visualized in the mass spectra during the analyses. As a result of the off-line method, several compounds (e.g., lignans) could be identified in each spot of the TLC plate after sample elution. This allowed for the differentiation among the fruit species not only by a visual TLC comparison but also by means of the MS identification of some diagnostic compounds (Hu, Xin, So and Yao 2015). A new sampling methodology based on the sequential generation of droplets was proposed by Liu and coworkers. This device, easily constructed and operated, was used to load samples onto the PS-MS substrate. The primary intention of the authors was to demonstrate that this apparatus could be used as a simple MS sensor for monitoring complex systems. The authors applied it to monitor chemical reactions in real time to detect dyes (rhodamine 6G) and amino acids (l-phenylalanine) in water and to obtain the chemical profiles of assorted types of fruits, such as apple, pear, and watermelon. Moreover, the method was applied to continuously generate charged micro-droplets from juices directly extracted from the fruit pulps. In the experimental setup, a capillary tube was inserted into the fruit pulp with the assistance of a plastic tip. The juice was then extracted from the pulp (by capillary action and assistance of an electric field) and directly transferred to the PS-MS substrate to generate MS fingerprints (Figure 12.7) (Liu, Mao, Wu and Lin 2013). Another PS-MS modification that provides new perspectives for the attainment of chemical profiles of food samples was described by Kim and Cha. The authors proposed a simple modification in the geometry of the PS-MS paper in order to turn possible direct analysis of raw solid samples, including foodstuffs. This modification consisted of using a paper cone working as a support chamber for samples (Figure 12.8) (Kim and Cha 2015). This method was called paper cone spray ionization. After sample loading and the application of an appropriate solvent, the analytes are extracted and transported to the cone tip. The application of electric potential results in the formation of a spray containing ionized analytes, which are then attracted into the mass spectrometer, and their mass-to-charge ratios were determined. In contrast with the conventional PS-MS, this technique showed better results when low wetting capacity papers were used as support. To demonstrate the performance of this technique, some solid samples were analyzed, e.g., green tea leaves, ground beef, and infant formulas. The authors concluded that this

FIGURE 12.7  Apparatus used to generate microdroplets directly from fruit pulps: (a)

extraction of juice from an apple pulp and (b) resulted chemical profile after performing PS-MS analysis. (Reproduced and adapted with permission from ©2013 Royal Society of Chemistry.)

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FIGURE 12.8  (See color insert after page 124.) Paper cone spray ionization mass s­ pectrometry for analysis of ground beef, tea leaves, and infant formulas. (Reproduced and adapted with permission from ©2015 Royal Society of Chemistry.)

technique can be used as a rapid method to achieve chemical profiles of raw solid samples and to verify the efficiency of solid–liquid extractions in real time (Kim and Cha 2015). Lara-Ortega and coworkers compared the performance of several atmospheric pressures, i.e., ESI and atmospheric pressure chemical ionization (APCI), and ambient ionization methods, i.e., low-temperature plasma (LTP) and PS-MS, to perform direct analysis of olive oils. The authors observed that the use of ESI and APCI, two of the most commonly used atmospheric pressure ionization methods, is difficult in consequence of strong carry over effects and the constant contamination of the instrument, even using diluted olive oil samples (1–1,000 fold). However, the ambient ionization methods, i.e., LTP and PS-MS, were easily used for the analysis of these samples furnishing mass spectra that better reflected their real chemical composition. The use of LTP allowed for the analysis of undiluted samples, whereas PS-MS demonstrated high sensitivity after spiking samples with AgNO3. The Ag+ binds with double bonds of the unsaturated compounds to form silver adducts that were detected with higher intensities. Finally, the authors suggest that this approach has the potential to be used to detect adulterations in olive oil (LaraOrtega, Beneito-Cambra, Robles-Molina, García-Reyes, Gilbert-López and Molina-Díaz 2018). Quality Control of Foodstuffs Modern mass spectrometry techniques have been largely applied by researchers, companies, and governmental agencies in quality control of foodstuffs. For instance, several teas and herbs, highly used in the traditional eastern medicine for containing beneficial compounds commonly used for the treatment of assorted diseases, have been frequently subjected to frauds. This is usually done by total or partial substitution of the authentic material by other less expensive and with similar morphological properties but with no (or deleterious) effects on human health. For instance, PS-MS and derived techniques have been applied to monitor and thus ensure an efficient and safe quality control of herbal medicines (Chan, Wong, Tang, Che and Ng 2011) This is the case of the work developed by Chan and coworkers who reported the use of tissue spray mass spectrometry for a direct analysis of the roots (cut as triangular slices) of American ginsengs (Panax quinquefolium), a valuable medicinal herb consumed as tea and used in alternative treatments for cancer and diabetes as

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well as to prevent aging effects. The authors detected the presence of important metabolites, such as amino acids, oligosaccharides, and ginsenosides, and noticed significant differences between the chemical profiles depending on the ginseng cultivate types. The authors also observed that the root slices could be analyzed morphologically, allowing the accomplishment of complementary microscopic assays for quality assurance (Chan, Wong, Tang, Che and Ng 2011). The first work that used conventional PS-MS in the direct analyses of tea leaves was performed by Deng and Yang in 2013, with no use of any sample pretreatment or derivatization procedures. The authors obtained MS fingerprints of Bansha herbal, a famous composed tea blend consumed in eastern countries for the treatment and prevention of flu, sore throat and fever. The mass spectra revealed the presence of several active compounds, such as organic acids, triterpenoids, saponins, amino acids, and sugars. The authors submitted the PS-MS data to exploratory analysis by principal component analysis (PCA), and the results revealed that the methodology can be used to (1) verify the tea authenticity, (2) differentiate locals of production, and (3) differentiate the quality of samples (appropriated or expired products) (Deng and Yang 2013). In 2015, Du and coauthors applied the wooden-tip electrospray mass spectrometry technique for the quality control of an herbal product known as Qingkailing. This is a formula constituted by eight medicinal materials and used in the treatment of diseases of the circulatory system, phlogistic illnesses, and viral infections. Sixteen batches of the Qingkailing solution and nine batches of Qingkailing granule were analyzed. The use of this technique associated with collision-induced dissociation (CID) allowed for the identification of 26 compounds. In addition, PCA applied to the MS data was successful to differentiate among the qualified and damaged samples (Du, Deng, Liu, Zhang, Yang and Chen 2015). Another traditional Chinese herb, famous for its healthy properties, is Aurantii Fructus Immaturus (aka Zhishi). This herb has been used to eliminate phlegm and disperse stagnation and contain several bioactive compounds such as flavonoids, alkaloids, and essential oils. Liu and coworkers obtained PS-MS profiles of this herbal product in both positive and negative ionization modes. Two exploratory methods, PCA and hierarchical cluster analysis (HCA), were successfully used to differentiate several batches of aka Zhishi and aka Qingpi, an herb used as adulterant. Moreover, the concentration of synephrine, an alkaloid commonly evaluated for the quality control of aka Zhishi, was determined using d2-isotopic-labeled synephrine as internal standard. To validate the PS-MS methodology, HPLC-UV was used as a comparative method. The authors described that no significant differences between the results from PS-MS and HPLC-UV methods were observed (Liu et al. 2017). Yuan Guo and coworkers used PS-MS as a rapid method to analyze Corni fructus, a crude herb with several medicinal properties. It has been widely used for the treatment of spontaneous sweating and associated to beneficial effects, such as antioxidant, antidiabetic, antineoplastic, anti-inflammatory, hepatoprotective, and neuroprotective. However, different types of herbs such as Crataegi fructus, Lycii fructus, and grape skin are illegally sold as Corni fructus at local markets. The authors developed a PS-MS method to distinguish among the chemical profiles of Corni fructus and Crataegi fructus. PCA was applied to evaluate the similarities between the chemical fingerprints of these samples, which resulted in the formation of two clearly distinguished clusters of each fruit. The authors also performed a semiquantitative analysis by PS-MS of loganin and morroniside in Corni fructus using genipin as internal standard and observed a good agreement between the results from PS-MS and HPLC-UV, used as a comparative method (Guo et al. 2017).

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Besides quality assurance, PS-MS and derivative techniques have been used to detect and quantify controlled substances or even contaminants in several types of food ­samples. Among these, beverages and meat have been largely studied. For instance, Li and coworkers reported the application of PS-MS for qualitative and quantitative analysis of  4-methylimizadole, a highly toxic compound with a maximum residue limit of 250  mg/kg, in soft drinks (lemonade and cola) and caramel with a minimum sample ­pretreatment procedure. Quantification was performed by constructing an internal calibration curve using isotopically labeled 4-methylimizadole as internal standard. The results from validation indicated that the developed method attends the requirements of the local legislations (Li, Wei, Hsu and Cooks 2013). Donna and coworkers applied PS-MS to quantify resveratrol in red wines. This compound is produced in grapes to self-protection and in human body it is associated with the prevention of cardiovascular/neurodegenerative diseases and presents antioxidant properties. The study was performed using the multiple reaction monitoring (MRM) mode and the isotopically labeled resveratrol as internal standard. The samples were submitted to a clean-up procedure by SPE, and the eluate was loaded on the PS-MS substrate for analysis in the positive ionization mode. The PS-MS method was validated by comparing its results to those arising from a traditional LC-MS procedure. Some figures of merit were calculated for both approaches, and excellent similarities were obtained (Di Donna, Taverna, Indelicato, Napoli, Sindona and Mazzotti 2017). Another substance in beverages that has been controlled is caffeine. Taverna and coworkers used the PS-MS technique to quantify caffeine in the following beverages: cola, espresso coffee, energy drink, and tea. The quantitative analyses were carried out in the MRM mode using 13C3 isotopically labeled caffeine as internal standard. In addition, the analyses were performed using LC-UV as a comparative method. The analytical parameters obtained by both methods revealed that PS-MS is a reliable technique that can be applied to a quantitative analysis of this analyte (Taverna, Di Donna, Bartella, Napoli, Sindona and Mazzotti 2016). In a similar study, Sneha and coworkers used PS-MS to analyze caffeine in energy drinks. The primary purpose of the experiment was to introduce ambient ionization mass spectrometry to first-year undergraduate students and its usefulness to qualitative and quantitative analyzes as well as in the identification of unknown molecules. The chemical profiles of some prototype drinks were obtained by direct PS-MS analyses. Tandem mass spectrometry (MS/MS) was also applied for the identification of other analytes, such as aspartame, fructose, and sucrose. In sequence, caffeine was quantified in the energy drink samples by using d9 isotopically labeled caffeine as internal standard. The proposed method showed accuracy higher than 97% (Sneha, Dulay and Zare 2017). Zhang and coworkers reported the application of PS-MS for qualitative and semiquantitative analyses of the following contaminants in several foodstuffs: beta-­antagonists in pork and beef (clenbuterol, terbutaline, salbutamol, and ractopamine), adulterant in milk and formula powder (melamine); plasticizers in sport juices (DEHP and DEHA). All compounds were analyzed using a chromatographic paper coated with silica, and the essays were performed using the MRM mode. All semiquantitative methods showed high linearity (R 2 > 0.99), low limits of detection (1–200 ng/g), and linear ranges with maximum limits between 1,000 and 10,000 ng/g. Because solvent has strong influence on the extraction efficiency, elution, and formation of spray droplets, the authors emphasize the importance of choosing the correct PS-MS solvent for each type of food (Zhang, Cooks and Ouyang 2012).

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Mazotti and coworkers reported the application of paper spray tandem mass spectrometry (PS-MS/MS) for the quantification of the nonsteroidal anti-inflammatory drugs (NSAIDs) in olive oils. These compounds are responsible for the anti-inflammatory properties of olive oils being easily characterized by the phenolic dialdehydes composition. These compounds are not directly ionized by conventional ambient mass spectrometry methods and must be submitted to derivatization methodologies (Mazzotti et al. 2013). The authors thus proposed the derivatization of the target analytes using methoxyamine directly fixed onto the paper substrate in order to produce alkyloxime (compounds easily detected by MS). The semiquantification of these analytes was performed by the precursor ion scan methodology. The authors were able to determine the concentration of four different NSAIDs with satisfactory relative standard deviations and acceptable recovery rates (between 92% and 107%) (Mazzotti et al. 2013). An instrumental development in the PS ionization was proposed by Shen and coworkers in 2013. The authors constructed an automatic device to perform rapid and highly reproducible PS-MS analyses. The method was applied to the quality control of meat, specifically in the quantitative determinations of erbutaline, clenbuterol, and ractopamine in bovine beef. The described device was able to perform PS-MS analysis in a rate of 7 seconds per sample (Figure 12.9). Besides the method quickness, low limits of detection (10 ng/g for terbutaline and 30 ng/g for clebuterol and ractopamine) were obtained, which were similar to those from previous PS-MS works (Shen, Zhang, Yang, Manicke and Ouyang 2013). Wei and coworkers applied graphene oxide on nylon paper (hybond-N+) membranes and utilized this device to analyze malachite green in water and fish meat by PS-MS. Malachite green is a type of dye commonly used by the aquaculture industry to prevent protozoal and fungal infections in fishes. In human body, malachite green has been reported to be carcinogenic and to provoke highly toxic effects. For this reason, the use of this substance is forbidden in most of the countries. However, this dye is still used in

FIGURE 12.9  (See color insert after page 124.) Schematic representation of the automatic device to perform rapid analysis by PS-MS with high reproducibility. (Reproduced and adapted with permission from ©2013 Elsevier.)

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aquaculture of several countries due to its highly disinfection efficiency and low costs. The authors used a nanosheet-modified functional graphene oxide membrane (GOM) for the extraction of the analyte in water samples and homogenized fish meat. Then, the GOM was used in PS-MS analysis as the ionization substrate, a method called GOMspray MS (Figure 12.10). Tandem mass spectrometry (MS/MS) was also applied to quantify the analyte. Moreover, in fish analysis, a malachite green metabolite (leucomalachite green) was quantified as well. As a result, GOM allowed the extraction of approximately 98% of the analyte. The proposed method could identify the dye in concentrations below the nanomolar levels (Wei et al. 2018). Another different modification was proposed in 2016 and consisted of the coupling of the slug-flow microextraction with paper spray mass spectrometry (SFME-PS-MS) (Figure 12.11). The extraction method was performed using a disposable capillary with a small amount of sample and an organic solvent. Then, the extract (sample plus the organic solvent) was dropped on the triangle paper for PS-MS analysis. The proposed method was applied to the analysis of three macrolide antibiotics: erythromycin, clarithromycin, and roxithromycin, in milk samples. The results showed that the SFME-PS-MS improved the sensitivity of the analysis of erythromycin by 45 ± 6 folds, clarithromycin 62 ± 9 folds, and roxithromycin 53 ± 7 folds when compared with the results arising from the application of the traditional PS-MS methodology (Deng et al. 2016). In the same context, Ma and coworkers used the SFME directly coupled to nanoESI in a miniature mass spectrometer to analyze milk and sport drinks (Figure 12.12). Organic and aqueous phases were sequentially injected into a borosilicate glass capillary,

FIGURE 12.10  (See color insert after page 124.) The nanosheet-modified N þ-nylon membrane used as the ionization substrate for PS-MS analysis of malachite green and leucomalachite green in fish meat. (Reproduced and adapted with permission from ©2018 Elsevier.)

FIGURE 12.11  (See color insert after page 124.) Schematic representation of the slug-flow

microextraction coupled with a PS-MS device. (Reproduced and adapted with ­permission from ©2016 Elsevier.)

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FIGURE 12.12  Slug-flow microextraction coupled to nano-ESI and to a miniature mass

spectrometer: (a) experimental apparatus and (b–e) MS and MS/MS spectra of bisphenol A (BPA) and bis-(2-ethylhexyl)-phthalate (DEHP) in samples of milk and sport drinks. (Reproduced and adapted with permission from ©2016 Elsevier.) where the analytes were extracted via liquid–liquid extraction procedure. Milk spiked with bisphenol A (BPA) and sport drink samples spiked with di-(2-ethylhexyl)-phthalate (DEHP) were analyzed. The MS profiles of each sample were acquired, and the MS/MS analyses were performed to confirm the presence of the analytes and to estimate the limits of detection. The results confirmed that the SFME coupled to a miniature mass spectrometer can be successfully used for the identification of illicit substances in food, even at low parts-per-billion levels (Ma et al. 2016). In the context of controlled or prohibited substances, the studies for detection and quantification of dyes in foodstuffs have been largely developed. Dyes are added on food products to provide a better visual aspect. Synthetic dyes (such as Sudan and Disperse dyes, Rhodamine B, and Malachite Green) are commonly used by the food industry due to their low cost and high coloring properties. However, the synthetic dyes have been associated with children hyperactivity and also with genotoxic and carcinogenic activities. So, the use of these substances is regulated in several countries. For instance, the

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Sudan azo-dyes are not allowed to be added to foodstuffs in European Union and other countries around the world. Other dyes, such as Fast Green, Orange B, and Citrus Red 2, are illegal in China and the European Union. However, contaminations of foodstuffs with these compounds have been frequently reported. As an example, Sudan azo-dyes were reported in powdered chilli pepper as an adulterant (Taverna, Di Donna, Mazzotti, Policicchio and Sindona 2013; Guo, Zhang, Yannell, Dong and Cooks 2017). The first work that used PS-MS for dye analysis was published in 2012 by Zhang and coworkers. The authors analyzed Sudan II, Sudan III, Sudan IV, and Sudan G in chilli powder. All analytes were analyzed using a chromatographic paper modified with silica coated, and the essays were executed using the MRM mode. Semiquantitative analysis showed very good correlation coefficients (R 2 > 0.99), low limits of detection (1–200 ng/g), and linear ranges with maximum limits between 1,000 and 10,000 ng/g (Zhang, Cooks and Ouyang 2012). In a similar work, Taverna and coworkers also applied PS-MS for the detection and quantification of Sudan I, II, III, IV, and para-red azo-dyes present in samples of powdered chili pepper. The authors simultaneously detected and quantified the dyes in a spiked powdered pepper by using deuterium-labeled compounds as internal standards. These standards were used to build the calibration curves applying the precursor ion scan mode for quantification purpose. The dyes could be detected in the chili pepper samples in concentrations up to 1 ppm (Taverna, Di Donna, Mazzotti, Policicchio and Sindona 2013). In the same context, Yang and coworkers evaluated the performance of the woodentip ESI mass spectrometry technique for qualitative and quantitative analysis of Sudan I in powdered pepper. For the quantitative analysis, the MRM mode was applied with no use of internal standards. The linearity of the analytical curve was evaluated by the correlation coefficient (R 2 = 0.97), with a linear range of 0.5–20 mg/kg and a limit of detection of 0.1 mg/kg. The results proved that the method is able to determine trace levels of dyes in food samples by such a direct and simple analytical methodology (Yang et al. 2015). Guo and coworkers, in 2017, reported a rapid methodology based on PS-MS and tandem MS for the detection and quantification of six prohibited azo-dyes (Sudan I, Sudan II, Malachite Green, Rhodamine B, crystal violet, and methylene blue) in soft drinks, teas, fruit juices, as well as alcoholic and energy beverages. Isotopically labeled internal standards and a blend of 21 noncolored soft drinks were used to build matrix-matched calibration curves. These conditions were used to improve the accuracy, precision, and robustness of the analysis (Guo, Zhang, Yannell, Dong and Cooks 2017). The optimized sample pretreatment consisted on the dilution of the beverage in methanol (1:10 dilution rate), addition of internal standards, and direct analysis of the resulting solution (30 L) using PS-MS. Upon validation, the calibration curves showed acceptable linear responses for all analytes, with limits of detection and quantification below 1.5 and 5 ng/mL, respectively. The accuracy of the method varied in the range of 80%–120% for all analytes. Lastly, the authors were able to analyze samples within 1 min and investigated the presence of illegal dyes in 20 different commercial colored samples. They did not detect the presence of any of the six dyes in these samples, which attested the conformity of the manufacturers to the US legislations (Guo, Zhang, Yannell, Dong and Cooks 2017). Forensic Studies and Pattern Recognition of Foodstuffs In the forensic context, PS-MS and related techniques have been used to verify the authenticity and the occurrence of adulterations and forgeries in foodstuffs. Mostly of these

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studies were carried out by the analysis of chemical fingerprints, which were used to distinguish among authentic and counterfeit samples based on the visual comparison of the MS profiles. The simplicity and fastness of some of these MS techniques, such as PS-MS, have allowed for the analysis of hundreds of samples in a short period of time. However, since the amount of data generated is quite huge, chemometric tools that encompass multivariate statistical and mathematical methods have been used to facilitate data interpretation. These methods used for pattern recognition and multivariate discrimination become obligatory in cases of samples with identical (or quite similar) MS fingerprints that cannot be distinguished by a simple visual inspection. The first application of PS-MS to the differentiation among food samples was reported by Garret and coworkers in 2013. The authors described the use of PS-MS for the analysis of coffee. In this work, PS-MS fingerprints were obtained to promote (1) the differentiation of two species of coffee, Coffea arabica and Coffea caneferra robusta, with remarkable distinct market value and sensorial properties, (2) to distinguish among samples grown in different regions of Brazil, and (3) to discriminate samples submitted to assorted postharvest treatment methods such as dry, semidry, and wet bean. PS-MS spectra were obtained by analyzing the methanolic/aqueous coffee extracts. The authors identified carbohydrates, alkaloids, and amino acids in the positive ion mode. Moreover, chemometric exploratory tools, PCA and HCA, were used to perform sample classification. Three models were able to differentiate samples accordingly to their origin and postharvest treatment method. The authors also described an adaptation of tissue spray mass spectrometry for the direct analysis of coffee bean. The authors called this method as coffee spray mass spectrometry. Hence, a slice of green coffee bean was analyzed in both modes of ionization, positive and negative, and stable spray and MS signals were obtained for more than 2 min. Several endogenous compounds responsible for the antioxidant activity and bitterness of coffee could be identified (Garrett, Rezende and Ifa 2013). In another manuscript, Pereira and coworkers applied PS-MS in association with chemometric tools, partial least square discriminant analysis (PLS-DA) and variable selection by ordered predictors selection (OPS), to identify counterfeit beers. In this practice, very common in Brazil, a low-cost beer is sold as a higher market price product. The fraud consisted in changing of the bottle caps and labels between beer brands. The authors analyzed three beer brands, leaders in the Brazilian market, and five brands with lower prices. The analyses were conducted in the positive ion mode, and the mass spectra revealed the predominant presence of malto-oligosaccharides for all beer brands. However, in some cases, significant differences in the mass spectra of the different brands could not be observed by visual inspection. Then, a PLS-DA model improved by variable selection with OPS was constructed. The model was validated and was able to discriminate among the higher price beers from those of lower price, with no prediction errors. These results indicated therefore that the proposed method has a potential to be used in routine investigations to prevent this type of illegal practice (Pereira, Amador, Sena, Augusti and Piccin 2016). In the same context of fraud in beverages, Teodoro and coworkers proposed a PS-MS method for the identification of counterfeit blended Scottish whiskies. The authors used PS-MS in the negative ion mode, which allowed for the identification of characteristic compounds, used as markers of authentic whiskeys, extracted from oak barrels during the aging process. The authors used chemometric tools (PCA and PLS-DA) to distinguish among the chemical profiles of authentic and counterfeit samples. In the PLS-DA model, all the authentic and counterfeit samples were correctly classified (Teodoro, Pereira, Sena, Piccin, Zacca and Augusti 2017).

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Another class of food largely subjected to forgeries is the natural products, such as medicinal herbs and essential oils. Taverna and coworkers proposed a PS-MS method for the analysis of the nonvolatile compounds of Bergamot essential oil. Bergamot essential oil has been used in several applications by the food and cosmetic industries due to its antioxidant and antimicrobial activities. To supply the market requirement, expensive oils are commonly reconstituted using cheaper ones. Despite the visual similarity, reconstituted oils do not have the same functionalities and quality than the authentic product. The authors analyzed bergamot, sweet orange, orange, cedar, grapefruit, and mandarin essential oils. PS-MS and MS/MS were used for the characterization of the chemical profiles and for the compound identification, respectively. Chemometric approaches, linear discriminant analysis (LDA) and soft independent modeling of class analogy (SIMCA), were applied to classify the different sample classes. Both recognition models, LDA and SIMCA, provided excellent results, distinguishing the authentic Bergamot essential oils from the other ones (Taverna et al. 2016). In another interesting forensic work, Yang and coworkers developed a wooden-tip ESI mass spectrometry method for qualitative and quantitative analysis of diazepam, an anxiety drug irregularly used as sedative in sexual crimes, in Coca-Cola. To simulate the crime scene, 350 mL of the drink was spiked with a piece of diazepam tablet. The ­sampling procedure consisted on immersing the wooden tip directly into the drinks. To confirm the presence of diazepam and to perform quantitative analysis, the selectedreaction monitoring mode was applied, avoiding the addition of an internal standard. The limit of detection and relative standard deviation obtained were 30 ng/mL (absolute amount: 30.0 pg) and 8.7 %, respectively (Yang et al. 2015).

APPLICATION OF PS-MS AND RELATED TECHNIQUES TO ENVIRONMENTAL ANALYSIS Up to the end of the 20th century, determinations of organic environmental pollutants at trace levels in environmental samples (for instance, water, soil, and air) were mostly conducted by using gas chromatography (GC-MS) and liquid chromatography (LC-MS) coupled to mass spectrometry. Both techniques demonstrate excellent analytical performance but require laborious sample preparation steps prior to the instrumental analysis. Extraction, separation, and derivatization procedures cause increase of costs and time of analyses (Kennedy et al. 2016; Maher et al. 2016; Fang et al. 2016). In this context, the PS-MS technique appears as a simple, efficient, and robust alternative for the analysis of complex environmental samples since it requires little or no sample preparation, providing almost instantaneous data acquisition (less than 1 min). Most of the environmental analyses are routinely performed using typical configuration of the PS source, as will be exposed following in this chapter. Moreover, internal standards are frequently applied to make possible performing quantitative essays and also to reduce errors caused by matrix effects (Kennedy et al. 2016; Maher et al. 2016; Fang et al. 2016; Reeber, Gadi, Huang and Glish 2015; Resende, Teodoro, Binatti, Gouveia, Oliveira and Augusti 2017). Despite these attractive possibilities, the application of PS-MS to environmental analysis has occurred in a much smaller number when compared to the food samples. For instance, qualitative and quantitative direct analyses of triazine (Atrazine/Propazine) and chloroacetanilide (Metolachlor) herbicides using spiked samples of water and crop extracts, at part-per-billion levels, were performed by Reber and coworkers. Triazines

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and Metolachlor are synthetic herbicides used to protect crops against grasses and weeds. When associated, these compounds act synergistically due to the different mechanisms of action. Quantifications of these herbicides were performed using isotopically labeled internal standards. The authors obtained good limits of detection (3.53 and 1.38 ppb) and quantification (9.78 and 1.79 ppb) for Atrazine and Metolachlor in surface water, respectively. However, these limits were higher than the maximum residue limits for these herbicides in drink water as established by the United States Environmental Protection Agency (USEPA) (Reeber, Gadi, Huang and Glish 2015). In another example, Maher and coworkers applied PS-MS to analyze metaldehyde in water samples. This compound is classified as a moderately hazardous pesticide (class II) by the Word Health Organization and widely used as a molluscicide in agricultural and horticultural crops. The direct detection of metaldehyde in water samples was possible by adding a sodium salt in the PS solvent to form a sodium adduct ion (of m/z 199) of this analyte. The sodiated adduct of metaldeyde was submitted to the CID for structure confirmation by loss of acetaldehyde units. By adding a solution of 0.1% formic acid to the PS solvent, an intense protonated ion of m/z 177 (protonated metaldehyde) and a major fragment ion of m/z 149, arising from the loss of ethylene from protonated metaldehyde, were observed in the resulting mass spectrum. A procedure for metaldehyde quantitation in water using an isotopically labeled internal standard was developed, and the following limits of detection were achieved: 2.69 ng/mL for [M + Na]+ and 0.05 ng/mL for [M + H]+. The limit of detection for the sodiated adduct was below the maximum residue limit permitted by the European Union legislation. The method was applied to a direct analysis of raw environmental water samples, without any dilution or sample preparation, and metaldehyde was found in some of them (Maher et al. 2016). PS-MS has also been used to monitor air quality as exemplified in a study on the chemical detection of organosulfate compounds directly from aerosol filter samples. Despite ample research into the atmospheric oxidation of α-pinene, an important precursor to biogenic secondary organic aerosol formation, the identification of its reaction products, specifically organic nitrates, which impact atmospheric NOx concentrations, is still incomplete. This negatively impacts our understanding of α-pinene oxidation chemistry and its relation to air quality. Photochemical chamber experiments were conducted and the oxidation products formed under these conditions were continuously collected on filters. These filters were directly analyzed by PS-MS and previously unobserved products, derived from the oxidation of α-pinene, were detected. According to the authors, these findings can help lower the uncertainty on α-pinene oxidation chemistry, whereas PS-MS can become a new platform to be used in the identification and quantification of important atmospheric compounds relevant to air quality (Rindelaub, Wiley, Cooper and Shepson 2016). The low levels of contaminants besides the frequent occurrence of matrix effects are generally the major issues in environmental analysis using ambient ionization techniques, such as PS-MS. For this reason, extraction and preconcentration methods have been used to improve the performance of mass spectrometry methods applied on these kind of complex matrices. In this context, liquid-phase microextraction (LPME) appears as a suitable methodology since it is able to effectively extract and concentrate target analytes for PS-MS analyses. For instance, LPME was used by Fang and coworkers to analyze malachite green, crystal violet, and their metabolites in water samples. PS-MS analysis was performed using a triple quadrupole mass spectrometer in the positive ion mode, and quantitative determinations were executed using isotopically labeled internal standards and the MRM acquisition mode. The results indicated that the method is suitable for the analysis of these target compounds in real water samples (Fang et al. 2016).

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Reactive paper spray ionization (R-PSI), a variant of traditional PS, consists of the addition of a reactive reagent into the PS solvent to react with a target molecule present in a complex mixture at the sampling spot. R PS aids sensitivity and selectivity to the traditional PS-MS analysis. Although no examples on the application of this methodology to environmental issues have been so far described, R-PS has an enormous potential to be used in the analyzes of this type of samples. To demonstrate this possibility, the manuscript by Zhou and coworkers is presented. Hence, these authors made use of reactive PS to detect prototype quinones, 1,4-benzo-quinone (BQ), 1,4-benzo-naphtho-quinone (NQ), and 1,4-anthra-quinone (AQ), in complex matrices, such as raw urine, raw serum, and cell culture. Quinones, formed from the degradation of polycyclic aromatic hydrocarbons (PAHs), are involved in the pathogenesis of respiratory diseases and are linked to mutations and cancer. Quinones has low proton affinity resulting in no significant MS response even using an acidic solution as the PS solvent. The reactive PS method was based on an in situ Michael reaction between cysteamine and the quinones to yield easily charged derivatives. The method can be potentially applied in fast screenings of raw urine, serum, and cell culture, as demonstrated by adding a fixed amount of NQ in this three different matrices and detecting the derivative (m/z 219) in the respective PS(+)-MS (Zhou, Pei and Huang 2015).

CONCLUSIONS This chapter presents some illustrative examples of applications of PS-MS, and its derived techniques, as effective alternatives for the analysis of complex food and environmental matrices. The auspicious examples exposed in this chapter demonstrate that PS-MS is quite simple (usually a minimal sample preparation is required), fast (each analysis takes usually few minutes), reliable (assorted analytes can be undoubtedly detected in real samples), sensitive (the presence of analytes at quite low levels can be easily confirmed), specific (different analytes in distinct matrices can be undoubtedly detected by using tandem mass spectrometry), and can provide quantitative information (once an adequate internal standard can be used). Besides that, a general information regarding the chemical profiles (fingerprints) of assorted types of samples can also be obtained. Moreover, PS-MS is an attractive option to be used instead of chromatographic techniques, thus saving time and resources, allowing for the execution of in situ and real-time experiments. Finally, PS-MS can be easily adapted to any laboratory where a mass spectrometer is available. Other PS-MS methods of potential interest for the food and environmental areas can, therefore, be developed and used as routine procedures.

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Index A Agave spirits, 119 Agrochemicals, 117–118, 196–199 Ambient ionization, 3 Ambient mass spectrometry (AMS), 8–9, 76 advantages, 4 application, 6–7, 23–24 imaging, 20–21 laser desorption/ablation, 14 liquid–solid extraction, 13–14 performance, 24–25 probe assembly configurations, 15–18 rapid evaporative ionization mass spectrometry, 15 requirements, 4 sample handling, 21–23 solid–liquid extraction, 9–11 solvents, gases, and sample devices, 18–20 thermal and chemical desorption, 11–13 three-step, 14–15 AMS, see Ambient mass spectrometry (AMS) APCI, see Atmospheric pressure chemical ionization (APCI) APPI, see Atmospheric pressure photoionization (APPI) Atmospheric pressure chemical ionization (APCI), 203 Atmospheric pressure photoionization (APPI), 57 Aurantii Fructus Immaturus (aka Zhishi), 204 B Bergamot essential oil, 211 Beverages caffeine, 99, 205 coffee, 115 coffee beans desorption electrospray ionization, 47–48 easy ambient sonic-spray ionization mass spectrometry, 86–88 low-temperature plasma-mass spectrometry, 115 Bronsted–Lowry acid–base reactions, 189

C Caffeine, 99, 205 Capacitively coupled plasmas (CCPs), 127 CCPs, see Capacitively coupled plasmas (CCPs) Chemical desorption, 12–13 Chlorpropham, 42–43 Chromatography papers, 191 CID, see Collision-induced dissociation (CID) Coffea arabica, 210 Coffea caneferra robusta, 210 Coffee beans desorption electrospray ionization, 47–48 easy ambient sonic-spray ionization mass spectrometry, 86–88 low-temperature plasma-mass spectrometry, 115 Cold plasmas, 126 Collision-induced dissociation (CID), 204 Corni fructus, 204 Cotoneaster horizontalis, 45 Crataegi fructus, 204 D DAPCI, see Desorption atmospheric pressure chemical ionization (DAPCI) DAPPI, see Desorption atmospheric pressure photoionization (DAPPI) DAPPI-MS, see Desorption atmospheric pressure photoionization-mass spectroscopy (DAPPI-MS) DART, see Direct analysis in real time (DART) DART-HRMS, see Direct analysis in real time ionization-high resolution mass spectrometry (DART-HRMS) screening method DART-MS, see Direct analysis in real timemass spectrometry (DART-MS) DBDI, see Dielectric barrier discharge ionization (DBDI) DDT, see Dichlorodiphenyltrichloroethane (DDT) DESI, see Desorption electrospray ionization (DESI)

221

222

Index

DESI-MSI, see Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) Desorption atmospheric pressure chemical ionization (DAPCI), 12, 16 Desorption atmospheric pressure photoionization (DAPPI), 13, 17, 57–58 environmental analysis, 66–71 in food analysis, 59–65 high-resolution mass spectroscopy, 68 neonicotinoids, 66–67 N-PAH, 66 polycyclic aromatic hydrocarbons, 66–68 tetrabromobisphenol A, 66 transmission mode, 60–63, 68, 70–71 travelling wave ion mobility spectroscopymass spectrometry, 59 Desorption atmospheric pressure photoionization-mass spectroscopy (DAPPI-MS), 59 Desorption electrospray ionization (DESI), 4, 9–10, 15–16, 18, 19, 21, 39–40 applications aging, foodstuff, 48 coffee beans, 47–48 marine toxins, 48–49 chlorpropham, 42–43 DESI-HRMS screening method, 43 DESI-MSI, 43–46 DESI-MS/MS method, 41–42 food fraud and adulteration, 46–47 herbicides, 42–43 imaging, 43–46 pesticides, 41–43 plasma-assisted desorption ionization and, 133–134 solvent, 42 source and performance, 41 Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI), 43–46 Desorption/ionization mechanisms, 135–136 Dichlorodiphenyltrichloroethane (DDT), 197 Dielectric barrier discharge ionization (DBDI), 12–13, 17 Diethyl phthalate (DEP), 160 Direct analysis in real time (DART), 4, 12, 16, 17, 19, 22, 59, 95 electron capture, 97 matrix effects in, 98 optimization, 97–98 Penning ionization, 96 principle, 96

proton transfer reactions, 96 transient microenvironment, 97 Direct analysis in real time ionization-high resolution mass spectrometry (DARTHRMS) screening method, 43 Direct analysis in real time-mass spectrometry (DART-MS), 24–25, 95 enviromental analysis, 100 food quality and safety, 98–99 Direct liquid injection (DLI)-ESI, 119 E EAPPI, see Extraction atmospheric pressure photoionization (EAPPI) EASI, see Easy ambient sonic spray ionization (EASI) EASI-MS, see Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) Easy ambient sonic spray ionization (EASI), 10 Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) ambient mass spectrometry, 76 applications coffee beans, 86–88 fish and seafood, 85–87 food emulsifiers, 82–83 meat products, 84–85 propolis, 86 vegetable oils, 80–82 in foodomics, 75–76 ionization process, 76–78 quantitative analysis, 88 Spartan-EASI, 78, 79 thermal imprinting, 79–80 VALDI, 80 Venture-EASI, 78, 79 EESI, see Extractive electrospray ionization (EESI) EESI-MS, see Extractive electrospray ionization-mass spectrometry (EESI-MS) ELDI, see Electrospray laser desorption ionization (ELDI) ELDI-MS, see Electrospray laser desorption ionization-mass spectrometry (ELDI-MS) Electron capture, 97 Electrospray ionization (ESI), 8, 10, 11, 15, 188, 203 Electrospray laser desorption ionization (ELDI), 14, 19, 21, 171–172 Electrospray laser desorption ionization-mass spectrometry (ELDI-MS), 171–172

Index

223

Environmental analysis, 5 ambient mass spectrometry, 6–7, 9, 23–25 desorption atmospheric pressure photoionization, 66–71 desorption electrospray ionization, 40 direct analysis in real time, 100 extractive electrospray ionization, 152–154 low-temperature plasma ionization, 112–113 paper spray-mass spectrometry, 211–213 plasma-assisted desorption ionization, 139–140 Escherichia coli, 155, 184 ESI, see Electrospray ionization (ESI) Explosives, 113 Extraction atmospheric pressure photoionization (EAPPI), 13 Extractive electrospray ionization (EESI), 9, 16, 18, 145 applications biological samples, 156–158 food, 155–156 in vivo skin analysis, 158–159 organic aerosols, 160–161 perfume, 160 sample matrix, 152–154 chromatographic techniques, 162 direct analysis in real time, 162 mechanism, 148–151 principle, 145–148 Extractive electrospray ionization-mass spectrometry (EESI-MS), 145–148 aerosol particles, 161 beer samples, 155–156 in vivo skin analysis, 158–159 perfume samples, 160 sample matrix, 152–154

and seafood, 85–87 fraud and adulteration, 46–47 olive oil, 114 plasma-assisted desorption ionization, 139–140 spinach, 155–157 Foodomics, 75–76, 199–200 Foodstuffs aging, 48 application agrochemicals, 196–199 chemical profiles, 199–203 forensic and pattern recognition, 209–211 quality control, 203–209 Fourier-transform ion cyclotron resonance (FTICR), 160 FTICR, see Fourier-transform ion cyclotron resonance (FTICR) Fusarium, 45

F FAPA, see Flowing atmospheric pressure afterglow (FAPA) FEM, see Finite element model (FEM) Finite element model (FEM), 128 Flowing atmospheric pressure afterglow (FAPA), 12, 16–17 Food analysis direct analysis in real time-mass spectrometry, 98–99 emulsifiers, 82–83 extractive electrospray ionization, 155–156 fish marine toxins in, 48–49 rapid evaporative ionization mass spectrometry, 184

I iKnife handheld sampling device, 182–183 Inductively coupled plasmas (ICPs), 127 in vivo skin analysis, 158–159 Ion mobility spectrometry (IMS), 109–111 Isopropyl N-(3-chlorophenyl)carbamate (chlorpropham), 42

G Gas chromatography-mass spectrometry (GC-MS), 95, 211 Glass spray mass spectrometry, 196 Graphene oxide membrane (GOM), 207 H Herbicides, 42–43, 211–213 Hiden Analytical Ltd HPR-60, 129–130 Hierarchical cluster analysis (HCA), 204 High-pressure liquid chromatographyultraviolet (HPLC-UV), 204 High-resolution mass spectroscopy (HRMS), 68 High-temperature plasmas (HTPs), 105–106, 126 Histamine, 158

K Kalanchoe blossfeldiana, 45 L LAESI, see Laser ablation electrospray ionization (LAESI) Laser ablation electrospray ionization (LAESI), 14, 19, 21

224

Index

Laser desorption/ablation, 14 LC-MS, see Liquid chromatography-mass spectrometry (LC-MS) LDA, see Linear discriminant analysis (LDA) Leaf spray mass spectrometry, 194–195 LESA, see Liquid extraction surface analysis (LESA) Limits of detection (LODs), 22–24, 43, 99 Linear discriminant analysis (LDA), 182, 183, 211 Liquid chromatography-mass spectrometry (LC-MS), 95, 211 Liquid extraction-based methods, 18 Liquid extraction surface analysis (LESA), 11, 16 Liquid–liquid extraction (LLE), 9 Liquid microjunction-based techniques, 10 Liquid microjunction surface sampling probe (LMJ-SSP), 10–11, 16, 18, 21 Liquid-phase microextraction (LPME), 212 Liquid–solid extraction, 9, 13–14 LMJ-SSP, see Liquid microjunction surface sampling probe (LMJ-SSP) LODs, see Limits of detection (LODs) Low temperature atmospheric pressure plasma, 126–127 plasma needle, 127–128 chemistry, 129–132 electrical properties of, 128–129 substrate/sample type, 132–133 Low-temperature plasma (LTP) ionization environmental analysis applications, 112–113 air quality and pollution, 113 detection of explosives, 113 food analysis applications agave spirits, 119 agrochemicals, 117–118 coffee products, 115 melamine contamination, 118–119 micronutrients, 120 organic compounds, 114 plant oil analyses, 114–115 plant tissue imaging, 115–117 to ion mobility spectrometry, 109–111 principles, 105–106 ionization and desorption mechanisms, 106–108 operational costs and environmental footprint, 111–112 portability, 111 probes and coupling to analyzers, 108–111 Low-temperature plasma-mass spectrometry (LTP-MS) system, 111, 118

coffee manufacturing, 115 explosives, 113 imaging, 115–117 with N2 gas, 112–113 organic compounds, 113 plant oil analyses, 114–115 Low-temperature plasmas (LTPs), 12–13, 16, 17, 105–106, 126, 138, 203 LPME, see Liquid-phase microextraction (LPME) LTP ionization, see Low-temperature plasma (LTP) ionization M Malachite green, 206 MALDESI, see Matrix-assisted laser desorption electrospray ionization (MALDESI) MALDI, see Matrix-assisted laser desorption ionization (MALDI) Marine toxins, 48–49 Mass spectrometry (MS), 95, 129, see also Easy ambient sonic-spray ionization mass spectrometry (EASI-MS); Paper spray-mass spectrometry (PS-MS) ambient mass spectrometry, 8–9, 76 advantages, 4 application, 6–7, 23–24 imaging, 20–21 laser desorption/ablation, 14 liquid–solid extraction, 13–14 performance, 24–25 probe assembly configurations, 15–18 rapid evaporative ionization mass spectrometry, 15 requirements, 4 sample handling, 21–23 solid–liquid extraction, 9–11 solvents, gases, and sample devices, 18–20 thermal and chemical desorption, 11–13 three-step, 14–15 desorption electrospray ionization-mass spectrometry imaging, 43–46 direct analysis in real time-mass spectrometry, 24–25, 95 enviromental analysis, 100 food quality and safety, 98–99 extractive electrospray ionization-mass spectrometry, 145–148 aerosol particles, 161 beer samples, 155–156 in vivo skin analysis perfume samples, 160

Index sample matrix, 152–154 ion mobility spectrometry, 109–111 liquid chromatography-mass spectrometry, 95, 211 low-temperature plasma-mass spectrometry system, 111, 118 coffee manufacturing, 115 explosives, 113 imaging, 115–117 with N2 gas, 112–113 organic compounds, 113 plant oil analyses, 114–115 molecular beam mass spectrometry, 129–130 one-step ambient mass spectrometry techniques solid–liquid extraction, 9–11 thermal and chemical desorption, 11–13 paper spray tandem mass spectrometry, 206 rapid evaporative ionization mass spectrometry, 15, 181–183 bacteria, 184 boar teint, 183–184 fish fraud detection, 184 meat, 183 sorptive tape-like extraction coupled with laser desorption ionization mass spectrometry, 175–176 tandem mass spectrometry, 8, 22, 161, 172, 199, 205, 207, 209 thermal desorptive-gas chromatographymass spectrometry, 100 thermal imprinting-easy ambient sonicspray ionization-mass spectrometry, 84, 86 two-step ambient mass spectrometry techniques laser desorption/ablation, 14 liquid–solid extraction, 13–14 wooden tip electrospray mass spectrometry, 195–196 Mass spectrometry imaging (MSI), 20 Matrix-assisted laser desorption electrospray ionization (MALDESI), 14 Matrix-assisted laser desorption ionization (MALDI), 172 Matrix effects, 98 Maximum residual limits (MRLs), 117–118 MBMS, see Molecular beam mass spectrometry (MBMS) Meat products, 84–85 Melamine contamination, 118–119 Metolachlor, 212

225

Mezcal, 119 Micronutrients, 120 Micro-plasma-assisted desorption ionization, 137 Minimal sample manipulation/treatment, 22 MIPs, see Molecularly imprinted polymers (MIPs) Molecular beam mass spectrometry (MBMS), 129–130 Molecularly imprinted polymers (MIPs), 199 MRLs, see Maximum residual limits (MRLs) MRM, see Multiple reaction monitoring (MRM) MS, see Mass spectrometry (MS) MS 4 fragmentation, 42–43 MSI, see Mass spectrometry imaging (MSI) Multiple reaction monitoring (MRM), 205, 209 N Nano-desorption electrospray ionization, 11 Neonicotinoids, 66–67 Nonthermal plasmas (NTPs), 106, 126 NTPs, see Nonthermal plasmas (NTPs) O OES, see Optical emission spectroscopy (OES) One-step ambient mass spectrometry techniques solid–liquid extraction, 9–11 thermal and chemical desorption, 11–13 OPS, see Ordered predictors selection (OPS) Optical emission spectroscopy (OES), 129, 131 Orbitrap, 160 Ordered predictors selection (OPS), 210 Oreochromis niloticus (Nile tilapia), 85 Organic aerosols, 160–161 Organic compounds, 113, 114 Orthogonal Projections to Latent StructuresDiscriminant Analysis (OPLS-DA) models, 184 P PADI, see Plasma-assisted desorption ionization (PADI) PAHs, see Polycyclic aromatic hydrocarbons (PAHs) Paper cone spray ionization, 202 Paper spray (PS), 9, 14–15, 20 Paper spray ionization (PSI), 190–191 Paper spray ionization mass spectrometry (PSI-MS), 188, 205 Paper spray-mass spectrometry (PS-MS), 188, 204

226

Index

Paper spray-mass spectrometry (PS-MS) (cont.) ambient ionization methods, 188 and derivative techniques, 205 electrospray ionization, 189 environmental application, 211–213 foodstuffs application agrochemicals, 196–199 chemical profiles, 199–203 forensic and pattern recognition, 209–211 quality control, 203–209 highly energetic sources, 188 mechanisms, 188 modifications glass spray mass spectrometry, 196 leaf spray mass spectrometry, 194–195 paper substrate, 193–194 tissue spray mass spectrometry, 194, 195 touch spray mass spectrometry, 195 wooden tip electrospray mass spectrometry, 195–196 performance, 191–192 procedure, 189–190 quantitative analysis, 192–193 soft ionization techniques, 188 Paper spray tandem mass spectrometry (PSMS/MS), 206 Paper substrate, 193–194 Paraffin, 194 Partial least square discriminant analysis (PLS-DA), 210 PCA, see Principal component analysis (PCA) Penning ionization, 12, 96 PESI, see Probe electrospray ionization (PESI) Pesticides, 24, 41–43, 45, 59, 98–100, 117– 118, 195, 197–199 Phase-resolved optical emission spectrometry (PROES), 131 Plant oil analyses, 114–115 Plant tissue imaging, 115–117 Plasma, 105–106, 126–127 Plasma-assisted desorption ionization (PADI), 13, 16, 17, 125–126, 133–135 desorption electrospray ionization and, 133–134, 138–139 desorption/ionization mechanisms, 135–136 food and environmental applications, 139–140 and low-temperature plasma, 138 micro-PADI, 137 Plasma-based techniques, 11–12 PlasmaChip, 108 Plasma needle, 127–128

chemistry, 129–132 electrical properties of, 128–129 nitric oxide, 130 substrate/sample type, 132–133 PLS-DA, see Partial least square discriminant analysis (PLS-DA) Polycyclic aromatic hydrocarbons (PAHs), 66–68 Polydimethylsiloxane (PDMS), 175 Principal component analysis (PCA), 47, 182, 183, 204 Probe assembly configurations, 15–18 Probe electrospray ionization (PESI), 13, 14, 20, 21 PROES, see Phase-resolved optical emission spectrometry (PROES) Propolis, 86 Proton affinity (PAs), 57 Proton transfer reactions, 96 PS, see Paper spray (PS) PSI, see Paper spray ionization (PSI) PSI-MS, see Paper spray ionization mass spectrometry (PSI-MS) PS-MS, see Paper spray-mass spectrometry (PS-MS) Q Qingkailing (herbal product), 204 Quadrupole-based mass spectrometer (QMS), 129 Quadrupole time-of-flight (QTOF) instrument, 160 Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) strategy, 118 Quick Polar Pesticides Extraction (QuPPe) method, 98 Quinones, 213 R Radio-frequency (RF) discharges, 127 excitation, 130, 134 generator, 127 plasma, 127 plasma needle, 127–128 chemistry, 129–132 electrical properties of, 128–129 nitric oxide, 130 substrate/sample type, 132–133 Rapid evaporative ionization mass spectrometry (REIMS), 15, 181–183 bacteria, 184 boar teint, 183–184 fish fraud detection, 184

Index meat authenticity, 183 and growth promoters, 183 Reactive desorption electrospray ionization, 19 Reactive paper spray ionization (R-PSI), 213 REIMS, see Rapid evaporative ionization mass spectrometry (REIMS) S Saxitoxins, 49 Sheath, 127 Shisandrac chinensis (FSC), 201–202 Shisandrac sphenanthera (FSS), 201–202 Silica plate imprinting laser desorption/ ionization mass spectrometry imaging (SPILDI-MSI), 176 Sister techniques Spartan-EASI (S-EASI), 78, 79 Venture-EASI (V-EASI), 78, 79 Slug-flow microextraction with paper spray mass spectrometry (SFME-PS-MS), 207 Soft independent modeling of class analogy (SIMCA), 211 Solid–liquid extraction, 9–11 Sorptive tape-like extraction coupled with laser desorption ionization mass spectrometry (STELDI-MS), 175–176 olive oil, 176 Spartan-EASI (S-EASI), 78, 79 Spray and ion formation, 190–191 T TALIF spectroscopy, see Two-photon absorption laserinduced fluorescence (TALIF) spectroscopy Tandem mass spectrometry, 8, 22, 161, 172, 199, 205, 207, 209 Tequila, 119 Terfenadine, 45 Tetrabromobisphenol A (TBBPA), 66 Thermal-assisted desorption, 11–13 Thermal desorptive-gas chromatography-mass spectrometry (TD-GC-MS), 100 Thermal imprinting-easy ambient sonic-spray ionization (TI-EASI), 79–80

227

Thermal imprinting-easy ambient sonicspray ionization-mass spectrometry (TI-EASI-MS), 84, 86 Thermal plasmas, 126 Thermo LTQ Orbitrap mass spectrometer, 47 Three-step ambient mass spectrometry techniques, 14–15 TI-EASI, see Thermal imprinting-easy ambient sonic-spray ionization (TI-EASI) TI-EASI-MS, see Thermal imprinting-easy ambient sonic-spray ionization-mass spectrometry (TI-EASI-MS) Tissue spray mass spectrometry, 194, 195, 200 TM DAPPI, see Transmission mode (TM) DAPPI TME, see Transient microenvironment (TME) Touch spray mass spectrometry, 195 Transient microenvironment (TME), 97 Transmission mode (TM) DAPPI, 60–63, 68, 70–71 Travelling wave ion mobility spectroscopymass spectrometry (TWIMS-MS), 59 Triazines, 211–212 TWIMS-MS, see Travelling wave ion mobility spectroscopy-mass spectrometry (TWIMS-MS) Two-photon absorption laserinduced fluorescence (TALIF) spectroscopy, 129, 131–132 Two-step ambient mass spectrometry techniques laser desorption/ablation, 14 liquid–solid extraction, 13–14 Two-step laser-based desorption/ablation techniques, 17–18 V Vacuum ultraviolet (VUV) krypton discharge lamp, 57 VALDI, 80 Vegetable oils, 80–82, 114 Venture-EASI (V-EASI), 78, 79 Venturi effect, 79 W Wooden tip electrospray mass spectrometry, 195–196

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