Food safety : innovative analytical tools for safety assessment 9781119160557, 1119160553

Table of contents :
Content: Preface xiii 1 Food Analysis: A Brief Overview 1 Giuseppe Cirillo, Donatella Restuccia, Manuela Curcio, Francesca Iemma and Umile Gianfranco Spizzirri 1.1 Introduction 1 1.2 Chromatographic Techniques in Food Analysis 2 1.3 Spectroscopic Methods 5 1.4 Biologically Based Methodologies in Food Analysis 7 References 8 2 Recent Analytical Methods for the Analysis of Sweeteners in Food: A Regulatory Perspective 13 Romina Shah and Lowri S. De Jager 2.1 Introduction 13 2.2 Sample Preparation 17 2.2.1 Internal Standards 20 2.3 Analytical Methods 21 2.3.1 Instrumental Analyses 21 2.3.1.1 HPLC-UV-VIS/DAD/ELSD Detection 21 2.3.1.2 HPLC-MS/Tandem MS Detection 24 2.3.1.3 Capillary Electrophoresis 28 2.4 Future Trends 28 References 29 3 Current Analytical Techniques for Food Lipids 33 Cynthia T. Srigley and Magdi M. Mossoba 3.1 Introduction 33 3.2 Official Methods for the Analysis of Fat in Foods 36 3.2.1 Importance of Official Methods of Analysis 36 3.2.2 Official Methods for the Gravimetric Determination of Total Fat 36 3.2.2.1 Solvent Extraction Procedures 37 3.2.2.2 Hydrolytic Procedures 40 3.2.3 Official Methods for the Determination of Total Fat by GC 42 3.2.3.1 Sample Preparation Procedures 42 3.2.3.2 Analysis of FAME by GC-FID 47 3.2.4 FTIR Spectroscopic Methods 51 3.2.5 Method Validation for Novel Sample Matrices 54 3.3 Conclusions 56 References 57 4 Detection of Allergenic Proteins in Food: Analytical Methods 65 Girdhari M. Sharma, Sefat E Khuda, Christine H. Parker, Anne C. Eischeid and Marion Pereira 4.1 Introduction 65 4.2 Immunochemical Methods 69 4.2.1 Lateral Flow Device (LFD)/Dipstick 69 4.2.2 ELISA 70 4.2.2.1 Milk 71 4.2.2.2 Egg 72 4.2.2.3 Fish 72 4.2.2.4 Crustacean Shellfish 73 4.2.2.5 Peanut 73 4.2.2.6 Tree Nuts 74 4.2.2.7 Wheat (Gluten) 75 4.2.2.8 Soy 76 4.3 Mass Spectrometry (MS) Methods 76 4.3.1 Milk 81 4.3.2 Egg 82 4.3.3 Fish and Crustacean Shellfish 82 4.3.4 Peanut 83 4.3.5 Tree Nuts 83 4.3.6 Wheat 84 4.3.7 Soy 84 4.4 DNA-Based Methods 85 4.4.1 Tree Nuts 89 4.4.2 Crustacean Shellfish 90 4.5 Method Validation 90 4.5.1 Specificity and Cross-Reactivity 97 4.5.2 Robustness and Ruggedness 97 4.5.3 Sensitivity, LOD and LOQ 97 4.5.4 Accuracy and Trueness 98 4.5.5 Precision 98 References 99 5 GMO Analysis Methods for Food: From Today to Tomorrow 123 Ozgur Cak r, Sinan Meric and ule Ar 5.1 Introduction 124 5.2 Methods for Detection, Identification and Quantification of GMOs in Food 135 5.2.1 Detection of GMOs by DNA-Based Methods 136 5.2.1.1 Polymerase Chain Reaction for GMO Detection 138 5.2.1.2 Real-Time PCR for GMO Quantification 140 5.2.2 Protein-Based Methods for GMO Detection and Quantification 141 5.2.2.1 ELISA (Enzyme-Linked Immunosorbent Assay) 142 5.2.2.2 Lateral Flow Strips 143 5.2.3 Phenotypic Detection of GMOs 144 5.2.4 Overall Assessment of Conventional Methods 145 5.2.5 New Detection Methods of GMOs 145 5.2.5.1 Amplification Based Detection Methods of GMOs 145 5.2.5.2 Biosensor-Based Detection Methods of GMOs 151 5.2.5.3 High-Throughput (HT) Techniques for GMO Detection 154 5.3 Conclusion 160 References 163 6 Determination of Antioxidant Compounds in Foodstuff 179 Amilcar L. Antonio, Eliana Pereira, Jose Pinela, Sandrina Heleno, Carla Pereira and Isabel C.F.R. Ferreira 6.1 Introduction 179 6.2 Common Antioxidants in Foodstuff 180 6.3 Antioxidants for Bioactive or Preservative Purposes 184 6.4 Analysis of Antioxidants in Foods 190 6.4.1 Extraction of Antioxidant Compounds 190 6.4.1.1 Conventional Methods 192 6.4.1.2 Nonconventional Methods 192 6.4.1.3 Extraction Solvents and Surfactants 196 6.4.2 Analytical Methodologies for Antioxidants 197 6.4.2.1 Detection of Antioxidant Compounds 197 6.4.2.2 Determination of Individual Antioxidant Molecules 198 6.5 Conclusion 202 References 203 7 Analytical Methods for Pesticide Detection in Foodstuffs 221 S. Hrouzkova 7.1 Introduction 221 7.1.1 Pesticide Residues in Foodstuffs 223 7.1.2 Analytical Methods for Pesticide Residue Analysis 224 7.2 Sample Preparation 225 7.2.1 Solvent-Based Extractions Liquid-Liquid Extraction (LLE) 227 7.2.1.1 QuEChERS Extraction 227 7.2.1.2 Accelerated Solvent Extraction 229 7.2.1.3 Microwave-Assisted Extraction (MAE) 230 7.2.1.4 Supercritical Fluid Extraction (SFE) 231 7.2.1.5 Liquid Phase Microextraction (LPME) 232 7.2.2 Sorption-Based Extractions 234 7.2.2.1 Solid-Phase Extraction (SPE) 234 7.2.2.2 Matrix Solid-Phase Dispersion (MSPD) 238 7.2.2.3 Microextraction by Packed Syringe (MEPS) 238 7.2.2.4 Solid-Phase Microextraction (SPME) 239 7.2.2.5 Stir-Bar SorptiveExtraction (SBSE) 240 7.3 Chromatographic Methods 241 7.3.1 Gas Chromatography 242 7.3.2 Fast Gas Chromatography 243 7.3.3 Liquid Chromatography 244 7.4 Detection of Pesticides 245 7.4.1 MS Detection 246 7.4.1.1 Ionization Techniques in GC-MS 246 7.4.1.2 Ionization Interfaces in LC-MS 247 7.4.1.3 MS Analyzers and Tandem MS 248 7.4.2 Ambient MS 250 7.5 Specific Problems of Pesticide Residue Analysis 252 7.6 Future Trends and Conclusions 254 Acknowledgment 254 References 255 8 Application of Chromatograpic Methods for Identification of Biogenic Amines in Foods of Animal Origin 271 Cesar Aquiles Lazaro De La Torre and Carlos Adam Conte-Junio 8.1 Biogenic Amines 272 8.1.1 Definition 272 8.1.2 Classification 272 8.1.3 Synthesis 272 8.2 Importance of Biogenic Amines in Food of Animal Origin 273 8.2.1 Toxicological Aspect 274 8.2.2 Quality Indicators 275 8.2.3 Control and Prevention 276 8.3 Procedures for Chromatographic Methods in Biogenic Amines 277 8.3.1 Sample Processing 278 8.3.2 Analytical Determination 286 8.4 Chromatography Applications in Food of Animal Origin 288 8.4.1 Milk and Dairy Products 289 8.4.2 Fish and Seafood Products 291 8.4.3 Meat, Meat Products and Edible Byproducts 292 8.4.4 Chicken Meat and Chicken Meat Products 293 8.4.5 Eggs and Egg Products 293 8.4.6 Honey 294 8.5 Conclusion 294 Acknowledgments 295 References 295 9 Advances in Food Allergen Analysis 305 Joana Costa, Telmo J.R. Fernandes, Caterina Villa, M. Beatriz P.P. Oliveira and Isabel Mafra 9.1 Introduction 305 9.2 Proteins versus DNA as Targets for Food Allergen Analysis 307 9.2.1 Protein-Based Methods 308 9.2.1.1 ELISA 308 9.2.1.2 Immunosensors 310 9.2.1.3 MS Platforms 321 9.2.2 DNA-Based Techniques 332 9.2.2.1 Real-Time PCR Coupled to HRM Analysis 332 9.2.2.2 Single-Tube Nested Real-Time PCR 333 9.2.2.3 Ligation-Dependent Probe Amplification 337 9.2.2.4 Genosensors 338 9.2.3 Aptasensors 343 9.3 Final Remarks 343 Acknowledgments 346 References 347 10 Food and Viral Contamination: Analytical Methods 361 Gloria Sanchez 10.1 Introduction 361 10.1.1 Virus Extraction from Food 364 10.1.2 Virus Extraction from Bilvalve Molluscs 364 10.1.3 Virus Extraction from Soft Fruits and Leafy Greens 367 10.1.4 Virus Extraction from Bottled Water 371 10.1.5 Virus Extraction from Other Food Products 373 10.2 Nucleic Acid Extraction and Purification 374 10.3 Virus Detection by Molecular Techniques 374 10.4 Assessment of Infectivity 376 10.5 Quality Controls 378 10.6 Conclusions 379 Acknowledgments 380 References 380 11 Application of Biosensors for Food Analysis 395 Viviana Scognamiglio, Amina Antonacci, Maya D. Lambreva, Fabiana Arduini, Giuseppe Palleschi, Simona C. Litescu, Udo Johanningmeier and Giuseppina Rea 11.1 The Agrifood Sector 396 11.2 Food Quality and Safety Concepts 397 11.3 Effect of Unsafe Food on Human Health 400 11.4 Revealing Methods for Food Components and Contaminants 402 11.5 Biosensors: Definition, Market and Application Fields 403 11.6 Biosensors and Bioassays for the Detection of Food Components and Contaminants 405 11.6.1 Biosensing Technologies for Glucose Detection 405 11.6.2 Biosensors and Bioassays to Reveal Glutamine 409 11.6.3 Biodetecting Methods for Gliadin 410 11.6.4 Enzyme Based-Biosensors for Phenols Detection 412 11.6.5 Biosensing Technology Trends for Pesticide Monitoring 414 11.6.6 Toxin Biodetection 419 11.6.7 Heavy Metal Monitoring by Biosensing Methodologies 420 11.7 Biosensors for Intelligent Food Packaging 422 11.8 Biosensor Technology to Sustain Precision Farming 423 11.9 Conclusions 424 Acknowledgments 426 References 426 12 Immunoassay Methods in Food Analysis 435 Pranav Tripathi, Satish Malik and Seema Nara 12.1 Introduction 436 12.2 Immunoassays 437 12.2.1 Principle and Significance of ELISA 438 12.2.2 Application of Immunoassays in Food Safety 439 12.3 Immunosensors 440 12.3.1 Electrochemical Transducers 441 12.3.1.1 Amperometric Transducers 441 12.3.1.2 Potentiometric Transducers 441 12.3.2 Piezoelectric Immunosensors 441 12.3.3 Optical Transducers 442 12.3.4 Application of Immunosensors in Food Safety 442 12.4 Lateral Flow Immunoassay (LFIA) 443 12.4.1 Applications of LFIA in Food Safety 444 12.5 Sample Processing in Food Analysis 445 12.6 Outlook 446 References 450

Citation preview

Food Safety: Innovative Analytical Tools for Safety Assessment

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Insights into Modern Food Science Th book series examines how modern society eff The ffects food science and it is intended to be an encyclopaedic knowledge base correlating the challenges of the XXI century to food science. The Th series will have five main themes: Food Production; Food Safety; Food and Health; Food Packaging; Food and the Law. Series Editor: Giuseppe Cirillo Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS), Italy E-mail: [email protected] People are encouraged to submit proposals to the series editor. Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Food Safety: Innovative Analytical Tools for Safety Assessment

Edited by

Umile Gianfranco Spizzirri and

Giuseppe Cirillo

Copyright © 2017 by Scrivener Publishing LLC. All rights reserved. Co-published by John Wiley & Sons, Inc. Hoboken, New Jersey, and Scrivener Publishing LLC, Beverly, Massachusetts. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts ff in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically fi disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profi fit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. For more information about Scrivener products please visit www.scrivenerpublishing.com. Cover design by Russell Richardson Library of Congress r Cataloging-in-Publication Data: ISBN 978-1-119-16055-7

Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents Preface 1 Food Analysis: A Brief Overview Giuseppe Cirillo, Donatella Restuccia, Manuela Curcio, Francesca Iemma and Umile Gianfranco Spizzirri 1.1 Introduction 1.2 Chromatographic Techniques in Food Analysis 1.3 Spectroscopic Methods 1.4 Biologically-Based Methodologies in Food Analysis References 2

3

Recent Analytical Methods for the Analysis of Sweeteners in Food: A Regulatory Perspective Romina Shah and Lowri S. de Jager 2.1 Introduction 2.2 Sample Preparation 2.2.1 Internal Standards 2.3 Analytical Methods 2.3.1 Instrumental Analyses 2.3.1.1 HPLC-UV-VIS/DAD/ELSD Detection 2.3.1.2 HPLC-MS/Tandem MS Detection 2.3.1.3 Capillary Electrophoresis 2.4 Future Trends References Current Analytical Techniques for Food Lipids Cynthia T. Srigley and Magdi M. Mossoba 3.1 Introduction 3.2 Offi fficial Methods for the Analysis of Fat in Foods 3.2.1 Importance of Offi fficial Methods of Analysis

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1 2 5 7 8

13 13 17 20 21 21 21 24 28 28 29 33 33 36 36 v

vi

Contents 3.2.2

Offi fficial Methods for the Gravimetric Determination of Total Fat 3.2.2.1 Solvent Extraction Procedures 3.2.2.2 Hydrolytic Procedures 3.2.3 Official ffi Methods for the Determination of Total Fat by GC 3.2.3.1 Sample Preparation Procedures 3.2.3.2 Analysis of FAME by GC-FID 3.2.4 FTIR Spectroscopic Methods 3.2.5 Method Validation for Novel Sample Matrices 3.3 Conclusions References 4

Detection of Allergen Markers in Food: Analytical Methods Girdhari M. Sharma, Sefat E Khuda, Christine H. Parker, Anne C. Eischeid and Marion Pereira 4.1 Introduction 4.2 Immunochemical Methods 4.2.1 Lateral Flow Device (LFD)/Dipstick 4.2.2 ELISA 4.2.2.1 Milk 4.2.2.2 Egg 4.2.2.3 Fish 4.2.2.4 Crustacean Shellfish fi 4.2.2.5 Peanut 4.2.2.6 Tree Nuts 4.2.2.7 Wheat (Gluten) 4.2.2.8 Soy 4.3 Mass Spectrometry (MS) Methods 4.3.1 Milk 4.3.2 Egg 4.3.3 Fish and Crustacean Shellfish fi 4.3.4 Peanut 4.3.5 Tree Nuts 4.3.6 Wheat 4.3.7 Soy 4.4 DNA-Based Methods 4.4.1 Crustacean Shellfish fi 4.4.2 Tree Nuts

36 37 40 42 42 47 51 54 56 57 65

65 69 69 70 71 72 72 73 73 74 75 76 76 81 82 82 83 83 84 84 85 89 90

Contents 4.5

Method Validation 4.5.1 Specifi ficity and Cross-Reactivity 4.5.2 Robustness and Ruggedness 4.5.3 Sensitivity, LOD and LOQ 4.5.4 Accuracy and Trueness 4.5.5 Precision References

vii 90 97 97 97 98 98 99

5 GMO Analysis Methods for Food: From Today to Tomorrow Özgür Çakır, Sinan Meriç and Şule Arı 5.1 Introduction 5.2 Methods for Detection, Identification fi and Quantification fi of GMOs in Food 5.2.1 Detection of GMOs by DNA-Based Methods 5.2.1.1 Polymerase Chain Reaction for GMO Detection 5.2.1.2 Real-Time PCR for GMO Quantification fi 5.2.2 Protein-Based Methods for GMO Detection and Quantification fi 5.2.2.1 ELISA (Enzyme-Linked Immunosorbent Assay) 5.2.2.2 Lateral Flow Strips 5.2.3 Phenotypic Detection of GMOs 5.2.4 Overall Assessment of Conventional Methods 5.2.5 New Detection Methods of GMOs 5.2.5.1 Amplification fi Based Detection Methods of GMOs 5.2.5.2 Biosensor-Based Detection Methods of GMOs 5.2.5.3 High-Th Throughput (HT) Techniques for GMO Detection 5.3 Conclusion References

123

6 Determination of Antioxidant Compounds in Foodstuff ff Amilcar L. Antonio, Eliana Pereira, José Pinela, Sandrina Heleno, Carla Pereira and Isabel C.F.R. Ferreira 6.1 Introduction 6.2 Common Antioxidants in Foodstuff ff 6.3 Antioxidants for Bioactive or Preservative Purposes

179

124 135 136 138 140 141 142 143 144 145 145 145 151 154 160 163

179 180 184

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Contents 6.4 Analysis of Antioxidants in Foods 6.4.1 Extraction of Antioxidant Compounds 6.4.1.1 Conventional Methods 6.4.1.2 Nonconventional Methods 6.4.1.3 Extraction Solvents and Surfactants 6.4.2 Analytical Methodologies for Antioxidants 6.4.2.1 Detection of Antioxidant Compounds 6.4.2.2 Determination of Individual Antioxidant Molecules 6.5 Conclusion References

7 Analytical Methods for Pesticide Detection in Foodstuffs ff S. Hrouzková 7.1 Introduction 7.1.1 Pesticide Residues in Foodstuff ffs 7.1.2 Analytical Methods for Pesticide Residue Analysis 7.2 Sample Preparation 7.2.1 Solvent-Based Extractions – Liquid-Liquid Extraction (LLE) 7.2.1.1 QuEChERS Extraction 7.2.1.2 Accelerated Solvent Extraction 7.2.1.3 Microwave-Assisted Extraction (MAE) 7.2.1.4 Supercritical Fluid Extraction (SFE) 7.2.1.5 Liquid Phase Microextraction (LPME) 7.2.2 Sorption-Based Extractions 7.2.2.1 Solid-Phase Extraction (SPE) 7.2.2.2 Matrix Solid-Phase Dispersion (MSPD) 7.2.2.3 Microextraction by Packed Syringe (MEPS) 7.2.2.4 Solid-Phase Microextraction (SPME) 7.2.2.5 Stir-Bar Sorptive Extraction (SBSE) 7.3 Chromatographic Methods 7.3.1 Gas Chromatography 7.3.2 Fast Gas Chromatography 7.3.3 Liquid Chromatography 7.4 Detection of Pesticides 7.4.1 MS Detection 7.4.1.1 Ionization Techniques in GC-MS

190 190 192 192 196 197 197 198 202 203 221 221 223 224 225 227 227 229 230 231 232 234 234 238 238 239 240 241 242 243 244 245 246 246

Contents 7.4.1.2 Ionization Interfaces in LC-MS 7.4.1.3 MS Analyzers and Tandem MS 7.4.2 Ambient MS 7.5 Specific fi Problems of Pesticide Residue Analysis 7.6 Future Trends and Conclusions Acknowledgment References 8 Application of Chromatograpic Methods for Identification fi of Biogenic Amines in Foods of Animal Origin César Aquiles Lázaro de La Torre and Carlos Adam Conte-Junio 8.1 Biogenic Amines 8.1.1 Defi finition 8.1.2 Classification fi 8.1.3 Synthesis 8.2 Importance of Biogenic Amines in Food of Animal Origin 8.2.1 Toxicological Aspect 8.2.2 Quality Indicators 8.2.3 Control and Prevention 8.3 Procedures for Chromatographic Methods in Biogenic Amines 8.3.1 Sample Processing 8.3.2 Analytical Determination 8.4 Chromatography Applications in Food of Animal Origin 8.4.1 Milk and Dairy Products 8.4.2 Fish and Seafood Products 8.4.3 Meat, Meat Products and Edible Byproducts 8.4.4 Chicken Meat and Chicken Meat Products 8.4.5 Eggs and Egg Products 8.4.6 Honey 8.5 Conclusion Acknowledgments References

ix 247 248 250 252 254 254 255

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272 272 272 272 273 274 275 276 277 278 286 288 289 291 292 293 293 294 294 295 295

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Contents

9 Advances in Food Allergen Analysis Joana Costa, Telmo J.R. Fernandes, Caterina Villa, M. Beatriz P.P. Oliveira and Isabel Mafra 9.1 Introduction 9.2 Proteins versus DNA as Targets for Food Allergen Analysis 9.2.1 Protein-Based Methods 9.2.1.1 ELISA 9.2.1.2 Immunosensors 9.2.1.3 MS Platforms 9.2.2 DNA-Based Techniques 9.2.2.1 Real-Time PCR Coupled to HRM Analysis 9.2.2.2 Single-Tube Nested Real-Time PCR 9.2.2.3 Ligation-Dependent Probe Amplification fi 9.2.2.4 Genosensors 9.2.3 Aptasensors 9.3 Final Remarks Acknowledgments References

305

10 Food and Viral Contamination: Analytical Methods Gloria Sánchez 10.1 Introduction 10.1.1 Virus Extraction from Food 10.1.2 Virus Extraction from Bilvalve Molluscs 10.1.3 Virus Extraction from Soft ft Fruits and Leafy Greens 10.1.4 Virus Extraction from Bottled Water 10.1.5 Virus Extraction from other Food Products 10.2 Nucleic Acid Extraction and Purifi fication 10.3 Virus Detection by Molecular Techniques 10.4 Assessment of Infectivity 10.5 Quality Controls 10.6 Conclusions Acknowledgments References

361

305 307 308 308 310 321 332 332 333 337 338 343 343 346 347

361 364 364 367 371 373 374 374 376 378 379 380 380

Contents

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11 Application of Biosensors for Food Analysis Viviana Scognamiglio, Amina Antonacci, Maya D. Lambreva, Fabiana Arduini, Giuseppe Palleschi, Simona C. Litescu, Udo Johanningmeier and Giuseppina Rea 11.1 Th The Agrifood Sector 11.2 Food Quality and Safety Concepts 11.3 Eff ffect of Unsafe Food on Human Health 11.4 Revealing Methods for Food Components and Contaminants 11.5 Biosensors: Defi finition, Market and Application Fields 11.6 Biosensors and Bioassays for the Detection of Food Components and Contaminants 11.6.1 Biosensing Technologies for Glucose Detection 11.6.2 Biosensors and Bioassays to Reveal Glutamine 11.6.3 Biodetecting Methods for Gliadin 11.6.4 Enzyme Based-Biosensors for Phenols Detection 11.6.5 Biosensing Technology Trends for Pesticide Monitoring 11.6.6 Toxin Biodetection 11.6.7 Heavy Metal Monitoring by Biosensing Methodologies 11.7 Biosensors for Intelligent Food Packaging 11.8 Biosensor Technology to Sustain Precision Farming 11.9 Conclusions Acknowledgments References

395

12 Immunoassay Methods in Food Analysis Pranav Tripathi, Satish Malik and Seema Nara 12.1 Introduction 12.2 Immunoassays 12.2.1 Principle and Significance fi of ELISA 12.2.2 Application of Immunoassays in Food Safety 12.3 Immunosensors 12.3.1 Electrochemical Transducers 12.3.1.1 Amperometric Transducers 12.3.1.2 Potentiometric Transducers

435

396 397 400 402 403 405 405 409 410 412 414 419 420 422 423 424 426 426

436 437 438 439 440 441 441 441

xii Contents 12.3.2 Piezoelectric Immunosensors 12.3.3 Optical Transducers 12.3.4 Application of Immunosensors in Food Safety 12.4 Lateral Flow Immunoassay (LFIA) 12.4.1 Applications of LFIA in Food Safety 12.5 Sample Processing in Food Analysis 12.6 Outlook References Index

441 442 442 443 444 445 446 450 455

Preface Food safety and quality are key objectives for food scientists and industries all over the world, both in developed and developing countries. Several diff fferent approaches have been proposed to characterize and eventually improve the nutritional value and the safety standards of food products, with innovative and fashionable techniques acting on food production, processing and analysis. With the aim to provide a detailed overview of recent developments in food science in a multidisciplinary context, from agriculture to chemistry and engineering, and from physics to biology and medicine, we planned a new book series highlighting how the knowledge and research in different ff fields can be applied to address quality and safety issues. fi In this first volume we show the recent developments in the analytical techniques (based on both destructive and nondestructive detection) proposed to fit the government regulations related to food quality. The development of effective ff analytical routes for the evaluation of food quality and safety is, indeed, a key objective for the food industry, for both safe and valuable production and storage. Th The distinctive aspect of this project is the evaluation of both the nutritional and contamination elements in foodstuff ffs, with specifi fic attention given to the effi fficiency and applicability in practical analyses. The volume is organized into two parts. The first part is related to the evaluation of the major food components (e.g., protein, polysaccharides, lipds, vitamins, etc.), with particular attention paid to recent developments in the fi field. In the second part, the risks associated with food consumption are evaluated by exploring recent advances in the detection of the key food contaminants (e.g., pollutants, pesticides, toxins, etc.). Thanks to the valuable contributions of scientists working in these speTh cialized fields, fi we present a detailed overview of the available techniques, with key advantages and limitations, highlighting the possibility to select

xiii

xiv Preface an innovative and eff ffective strategy to achieve the main goal of maximizing the nutritional values of foodstuff ffs while minimizing the risk of toxicity for consumers. Umile Gianfranco Spizzirri Giuseppe Cirillo Department of Pharmacy, Health and Nutritional Sciences University of Calabria September 2016

1 Food Analysis: A Brief Overview Giuseppe Cirillo, Donatella Restuccia, Manuela Curcio, Francesca Iemma and Umile Gianfranco Spizzirri* Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy

Abstract Food products are complex mixtures consisting of naturally occurring compounds with nutritional value and contaminating substances, generally originating from technological processes, agrochemical treatments, or packaging materials. Since the exact content of a food product must be assessed before it can be put on the market, routine and/or specialized analysis protocols have been pointed out for the characterization of all compounds present in food and beverages. The Th main approaches involve separation methods (e.g., chromatography), spectroscopic and biologically derived protocols. By developing effi fficient methodology with high reproducibility and low detection limits, high quality and safety standards can be achieved to fit fi the developed government regulations. Keywords: Food quality, food analysis, separation methods, molecular recognition

1.1 Introduction Th nutritional and health-related properties make food and beverages Their highly important products able to provide humans with different ff biologically active compounds [1, 2]. In the last few years, production, collection, storage, and distribution of food and beverages have been significantly fi infl fluenced by technological and scientifi fic developments with considerable advantages for both food quality and safety [3–5].

*Corresponding author: [email protected] Umile Gianfranco Spizzirri and Giuseppe Cirillo (eds.) Food Safety: Innovative Analytical Tools for Safety Assessment, (1–12) © 2017 Scrivener Publishing LLC

1

2 Food Safety: Innovative Analytical Tools Food products are complex mixtures consisting of naturally occurring compounds with nutritional value (e.g., lipids, carbohydrates, proteins, vitamins, phenolic compounds, organic acids and aromas) [6] and contaminating substances (e.g., pesticides, polycyclic aromatic hydrocarbons, chlorinated and brominated compounds, veterinary drugs, toxins, mutagenic compounds, metals, and inorganic compounds), generally originating from technological processes, agrochemical treatments, or packaging materials [7–9]. The exact composition (in terms of both natural components and conTh taminants) must be assessed before a foodstuff ff can be put on the market, and several limitations are imposed by national and international control agencies in order to assure safety and quality, and to avoid frauds [10–12]. For this reason, routine and/or specialized analysis protocols have been pointed out for the characterization of all compounds present in food and beverages [13–15]. Various types of methods, including microbial methods, sensory analysis, biochemical and physicochemical methods, are used in food analysis. Spectroscopic and chromatographic methods have become very popular for separation and identification fi of food components due to their high reproducibility and low detection limits [16]. Similarly, biologically based assays, including polymerase chain reaction (PCR) techniques, fi and immunological-based methods, are also used for detection of specific targets in food samples [17]. This chapter focuses on the principal instrumental techniques proposed in food and beverage analysis.

1.2 Chromatographic Techniques in Food Analysis Separation techniques, such as gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE), have largely been used for analysis of compounds in food samples [18]. The complexity of food matrices oft ften requires not only extensive sample preparation, but also online coupling techniques, which are used for their superior automation and high-throughput capabilities. Many detectors with different ff types of selectivity can be used in gas chromatography. A fi first classifi fication can be done in terms of detected compounds. Nonselective detectors are able to detect all compounds except the carrier gas, selective detectors respond to a range of compounds with a common physical or chemical property, while specific fi detectors are able to detect a single chemical compound. Detectors can also be grouped into concentration- and mass-flow-dependent fl detectors. The signal

Food Analysis: A Brief Overview 3 from a concentration-dependent detector is related to the concentration of solute in the detector, and does not usually destroy the sample, while mass-flow-dependent fl detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The coupling of separation techniques in tandem with mass spectrometry (MS) or high-resolution MS (time-of-fl flight) is a valuable tool for enhancing the selectivity and sensitivity of a detection system, giving precise information on the identity of compounds [19]. Nowadays, gas chromatography coupled to mass spectrometry (GCMS, GC-MS/MS) with electron impact ionization is a routine technique for analysis of nonpolar, semipolar, volatile and semivolatile food compounds such as polycyclic aromatic hydrocarbons, pesticides and dioxins [20, 21]. In contrast, for polar and nonvolatile substances, LC is the technique of choice, with increased application over the last few years [22]. Detection by liquid chromatography can be carried out in diff fferent ways, highly affecting ff its applicability to food analysis. Ultraviolet (UV) detection has been mostly used as well as mass spectrometers or refractive index (RI) detectors [23, 24]. Mass spectrometers are sensitive and universal detectors, but they are expensive; moreover, both UV and MS detectors suffer ff from non-uniform responses due to differences ff in absorptivity and ionization effi fficiencies as a function of chemical structure, respectively. Most types of MS can be used to analyze food components, including triple quadrupole, quadrupole time-of-fl flight, LTQ Orbitrap, ion trap, and magnetic sector mass spectrometers. Others, such as RI detectors, provide a more universal response but only for moderately high concentrations. Moreover, RI detectors are relatively insensitive and incompatible with gradient elution and diffi fficult to stabilize. In recent years, LC coupled to an evaporative light-scattering detector (ELSD) has represented a useful alternative [25]. The use of an ELSD approach for spectrophotometric derivatization (i.e., insertion of chromophoric groups) is feasible and therefore the drawbacks of derivatization (e.g., dependence on experimental parameters, incompleteness of derivatization reaction, use of salt-laden mobile phases, prolonged analysis time, additional cost for derivatization system and reagents) can be eliminated [26]. LC coupled to ELSD was successfully proposed to determine lipids and biogenic amines in different ff food matrices [27]. Within the past decade, the introduction of ultra-high pressure liquid chromatography (UHPLC) and rapid-scan and sensitive MS instruments has resulted in a seismic shift ft away from traditional chromatographic techniques towards multiclass, multiresidue methods with short injection cycle times and minimal sample preparation [28]. Comprehensive methods for some of the more important contaminant groups in residue analysis have

4 Food Safety: Innovative Analytical Tools been developed for UHPLC-MS/MS, including anthelmintics, β-agonists, steroids, quinolones and others. Expected future developments include the possibility to analyze larger numbers of classes of compounds; the use of ever higher temperatures and pressures to create more effective ff separation methods; and further reduction in sample preparation via online solidphase extraction and other techniques, to increase the speed of analysis beyond current standards. The use of supercritical fluids is another useful technology attracting Th increased interest from researchers in the food sector. Carbon dioxide is the most commonly used supercritical fl fluid, because it is nontoxic, nonexplosive, and the experimental conditions required are easily achievable, since the critical temperature and pressure are, respectively, 31 °C and 73 bar [29]. Supercritical fluid fl chromatography (SFC) was initially performed with pure CO2 as the mobile phase, but nowadays SFC is very often ft carried out under subcritical conditions because CO2 is modifi fied with an organic modifi fier or additive in order to increase the solubility of polar compounds [30]. In contrast to LC, SFC allows the use of higher flow fl rates with lower pressure falls through the column, leading to greater effi fficiency in short analysis times and reduced consumption of organic solvents. This Th implies sharper peaks, improved resolutions and faster methods due to the shorter times for column equilibration. Moreover, it offers ff the possibility of analyzing thermally labile and polar compounds which cannot be analyzed by GC without derivatization. Traditionally, SFC applications have been focused on lipid compounds, which could be due to the high solubility of these analytes in supercritical CO2, while more recent studies have shown their suitability for analyzing more polar compounds such as amino acids or carbohydrates [31]. Miniaturized separation techniques, such as electromigration methods (capillary electrophoresis, CE, and capillary electrochromatography, CEC), are alternative methodologies to GC and LC with a big potential regarding analysis time and costs, and off ffering diff fferent advantages, e.g., minor quantities of solvents, stationary phases and samples, easier coupling with MS, shorter analysis time, etc. [32]. Among these techniques, CEC has found a special place due to the combined advantages coming from CE and LC. On the one hand, the interaction between the analytes and the stationary phase inside the capillary column provides a high selectivity, while the presence of the electroosmotic fl flow reduces the solute dispersion in the column, highly increasing the effi fficiency. Diff fferent authors have reviewed the overall application of CEC [33, 34] for the analysis of certain compounds, such as nucleosides and nucleotides in food materials [35], proteins and peptides [36], natural/bioactive compounds [37] or phytochemicals [38].

Food Analysis: A Brief Overview 5

1.3 Spectroscopic Methods Th spectroscopic methods used for food analysis include ultravioletThe visible (UV-Vis) spectroscopy, fluorescence fl spectroscopy, Raman spectroscopy and infrared spectroscopy (IR), X-ray spectroscopy, and nuclear magnetic resonance (NMR), such as electron spin resonance. Th The underlying mechanism at the basis of their application in routine analysis is described below. UV/visible spectroscopy is a very simple physicochemical method with respect to experimental setup, developed in the middle of the 1900s. Recently it has been used for qualitative and quantitative analysis of food components such as carotenoids and related compounds [39], while a multivariate screening methodology based on UV-visible and multivariate classifi fication was proposed for testing adulteration in sauces with the banned Sudan I dye [40]. Fluorescence spectroscopy is a highly developed and noninvasive technique that enables the online measurement of substrate and product concentrations or the identifi fication of characteristic process states. The application of fluorescence spectroscopy, especially 2D fluoresence, is becoming more and more interesting for the analysis of fluorescent fl proteins and biological molecules (e.g., aminoacids, vitamins, and coenzymes) in foodstuffs ff [41]. Raman spectroscopy is another powerful technique for molecular analysis of foodstuffs ff since a fingerprint spectrum can be obtained for a target molecule. In this way, food components, additives, processes and changes during shelf life, adulterations and numerous contaminants, such as microorganisms, chemicals and toxins, can be determined [42]. Major advantages of this technique are its ability to provide information about concentration, structure, and interaction of biochemical molecules within intact cells and tissues nondestructively. In addition, it does not require homogenization, extraction, the use of dyes or any other labeling agent, or any pretreatment of samples [43]. Infrared spectroscopy embraces a number of techniques allowing the analysis of diff fferent types of samples (e.g., liquid, solids, pastes), determining specific fi applications such as attenuated total refl flection (ATR, ATR-MIR), Fourier transform (FT-MIR, FT-NIR), transmittance (T), transflectance fl or refl flectance (R), and diff ffuse refl flectance (DF) [44]. For a long time, infrared (IR) spectroscopy was not considered to be a method for fundamental research in food science [45]. More recently, the possibility to generate spectra containing hundreds of variables (absorbance intensities measured at each wavenumber or wavelength) resulting in the

6 Food Safety: Innovative Analytical Tools production of large data sets, allow the extension of this technique to food analysis, and methods and techniques based in IR spectroscopy are now used for the routine analysis of several foods for chemical properties such as moisture, fat and protein [46]. Vibrational spectroscopy in general and IR spectroscopy in particular presents an unique opportunity to interrogate or analyze the food matrix as a whole, on a chemical and biochemical level, as the fingerprints contain information from all components of the sample. X-ray, also called roentgen ray, is electromagnetic radiation with the wavelength range of 0.01–10 nm. Th The photon energy of an X-ray is in the range of 0.1–120 keV, which leads to strong penetrability. X-ray, similar to other electromagnetic waves, can show the following phenomena: refl flection, refraction, scattering, interference, diff ffraction, polarization and absorption. Usually, X-rays whose photon energy is up to about 10  keV (10–0.10 nm wavelength) are classifi fied as “soft ft” X-rays, and those of 10–120 keV (0.10–0.01 nm wavelength) are “hard” X-rays, due to their penetrating abilities. As hard X-rays pollute food, only the soft ft XRI technique is used in food inspection. X-ray has been employed for the evaluation of frozen products [47], in fruit-storage control [48], and fungal infection in wheat, namely Aspergillus niger, A. glaucus group, and Penicillium spp. [49]. Applications of X-ray in food manufacture were also reported [50], with the development of image-processing methods based on an X-ray instrument for the control of eye formation of cheese throughout the ripening period [51]. Foreign objects whose density is similar to that of water cannot be easily recognized by the X-ray technique [52]. The noninvasive, nondestructive nature of NMR relaxometry and magnetic resonance imaging (MRI) and the fact that both qualitative and quantitative data on physical and chemical properties of a wide range of samples can be gathered, have made them popular in food-related applications [53]. NMR techniques are applied in food science research and industrial processes to assess the product quality [54]. Changes in microcellular structure, diffusion ff of polymers and investigation of heat and mass transfer within the materials are performed by NMR/MRI measurements [55]. In addition, low-fi field NMR relaxometry and MRI have been used in the analysis of water content, mobility and distribution [56] as well as measurement of fat content and solid fat ratio and protein content [57]. Electron spin resonance (ESR) spectroscopy is a suitable tool useful in the detection of paramagnetic ion and free radicals with superior sensitivity limit and reduced acquisition time in respect to other analytical methods. ESR analysis has been approved as one of the standard reference methods for the detection of irradiated food containing bone, cellulose

Food Analysis: A Brief Overview 7 and crystalline sugar [58]. Furthermore, ESR technique was employed for the characterization of the antioxidant properties of sulfite fi and thiols in beer [59], determination of antocyanin in refined fi sugar [60], and sugar content in peony roots.

1.4

Biologically-Based Methodologies in Food Analysis

In food analyses, enzyme-linked immunosorbent assays (ELISAs), protein-based immunoassays lateral flow fl strip/protein strip tests, realtime polymerase chain reaction (RTPCR), and flow cytometry are widely explored techniques to assess food authenticity and detect biological contamination [17]. ELISA is an immunological technique involving the use of a selected enzyme to detect the presence of a specific fi antibody or antigen in a food sample in both a qualitative or quantitative format [61]. In food analysis, two variants of ELISAs have been widely explored, namely the indirect and sandwich ELISA. In the first fi protocol, two antibodies are employed, one of which is specific fi to the antigen to be detected, while the second is coupled to the enzyme and causes the production of a signal by a chromogenic or fluorogenic fl substrate [62]. In the sandwich ELISA protocol, the antigen is bound between two antibodies: one acting as capture and the other as detection antibody [63]. ELISA protocols are widely used for the detection of allergens, toxins, and antibiotic contamination with high specifi ficity [64, 65]. Protein immunoassays are based on the molecular recognition of antigens by antibodies to form a stable complex [66–68]. Although the high specificity of the underlying mechanism is the basis of molecular recognition, fi widely adopted for the detection of allergens [69], this technique cannot discriminate among phylogenetically related species and suffers ff from falsenegative results due to protein denaturation at high temperatures [70]. A direct upgrade of this technique is the lateral flow fl immunoassays, suitable for qualitative, semiquantitative and to some extent quantitative monitoring of pathogens, drugs, hormones, toxins and metabolites in foodstuffs ff [71]. Furthermore, the introduction of flow cytometric bead-based technology confers new opportunities for immunoassay protocols [72]. According to the literature, this technology allows (i) evaluation of multiple analytes in a single sample; (ii) utilization of minimal sample volumes; (iii) high reproducibility; (iv) direct comparison with already developed assays; and (v) a more rapid evaluation of multiple samples in a single platform.

8 Food Safety: Innovative Analytical Tools Another emerging method for food analysis is RTPCR, the method of choice for food analysis to detect and diff fferentiate between phylogenetically related species and to check the adulteration or the authenticity of food products [73], due to its rapid and highly sensitive identification fi capabilities [74]. Compared to conventional PCR, which is a cyclic process doubling the target sequences aft fter each cycle and involving denaturation, annealing, and extension steps, in RT-PCR no post-processing is required to analyze the amplification fi process, as it monitors the increasing copy number of amplicon in real time after ft each cycle [17]. In this technique, several fluorescent dyes, with emission ranges between 487 and 560 nm, have been developed for the quantitative estimation of PCR amplicons, as the total fluorescence intensity changes in direct proportion to the amount of DNA in the sample. RTPCR-based protocols have been developed for the detection of allergens, genetically modifi fied organisms, as well as bacterial and viral contamination [75].

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2 Recent Analytical Methods for the Analysis of Sweeteners in Food: A Regulatory Perspective Romina Shah* and Lowri S. de Jager U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, Maryland, USA

Abstract Non-nutritive or low calorie sweeteners are commonly used worldwide in the food industry, oft ften in combination in order to limit undesirable tastes. The list of allowable sweeteners varies among countries and it is important for regulatory agencies and food safety laboratories to monitor these highly consumed products to ensure compliance with worldwide regulations. Current analytical methods for confirmation fi and quantifi fication of sweeteners must allow for confi firmation of analyte identity in order to be compatible with today’s standards. Various methods for the determination of non-nutritive sweeteners have been reported in the literature. The most common multi-sweetener methods involve high performance liquid chromatography (HPLC) with different ff types of detection. The modern technique of HPLC-MS/MS is the current method of choice for the determination and confirmation of sweeteners in foods. In addition to multi-sweetener analyses there is also a need for single sweetener analytical methods in certain circumstances. Keywords: Non-nutritive sweeteners, foods, LC-MS/MS

2.1 Introduction Non-nutritive sweeteners are commonly used in foods as alternatives to sugar to provide a sweet taste with little or no calories [1]. They are an important class of food additives which are added to foods to cause *Corresponding author: [email protected] Umile Gianfranco Spizzirri and Giuseppe Cirillo (eds.) Food Safety: Innovative Analytical Tools for Safety Assessment, (13–32) © 2017 Scrivener Publishing LLC

13

14 Food Safety: Innovative Analytical Tools a technical effect ff such as sweetening [2]. Sweeteners are grouped into two main categories, bulk and intense sweeteners. Bulk sweeteners, such as sugar alcohols, provide texture and preservative effects ff to low calorie foods, with equivalent or less sweetening strength relative to sucrose. Sugar alcohols have been given quantum satis, meaning that they are harmless enough to have no specifi fic quantity restriction [3]. Intense sweeteners have sweetening capacities greater than sucrose with varying potencies. These compounds can be synthetic, semi-synthetic or natural. Th The majority are synthetic compounds, including aspartame (ASP), sucralose (SCL), saccharin (SAC), cyclamate (CYC), acesulfamepotassium (ACS-K), alitame (ALI), neotame (NEO) and dulcin (DUL). Neohesperidine dihydrochalcone (NHDC) is a semi-synthetic sweetener, while stevioside (STV) and rebaudioside (REB) A are natural sweeteners [4]. Th The list of allowable sweeteners varies among nations worldwide [5]. For example, CYC and NHDC are not approved for use as food additives by the US Food and Drug Administration (FDA) but are authorized in the European Union (EU) [6]. The oldest sweetener on the market, SAC is approved for use in nearly 90 countries. It has a sweetening strength about 450 times that of sucrose and exhibits high water solubility and storage stability [7]. In the 1980s, its consumption was linked with bladder cancer in rats and as such was prohibited in Canada [8]. Despite its bitter metallic aft ftertaste it is approved for use in many foods and beverages [9]. Unlike SAC, DUL does not have a bitter aftertaste ft and has a sweetening capacity about 250 times that of sucrose. However, DUL has not gained widespread use due to concerns over its toxicity [7]. It is not approved for use in the USA. Discovered in 1967, ACS-K exhibits good storage stability [9]. It is 200  times sweeter than sucrose and its use is associated with a slight bitter aft ftertaste at high concentrations [8]. ACS-K is widely used and approved in 90 countries with few health problems linked with its use [9]. It has very good water solubility and is stable at high cooking and baking temperatures [7]. In contrast, ASP is the most controversial artifi ficial sweetener regarding its health effects. ff There have been reports about adverse neurological eff ffects and cancer in rats. It is 180 times sweeter than sucrose and thus only small quantities are added to foods to achieve the desired sweetness. Since ASP is not heat-stable it degrades in liquids during prolonged storage [8]. Therefore, it cannot be used in baking or cooking and beverage products with ASP have expiry dates for acceptable consumption [9]. It has been approved for use by the US FDA and the EU. Phenylalanine is a metabolite of ASP, which cannot be metabolized by people with phenylketonuria, a

Recent Analytical Methods for the Analysis of Sweeteners 15 rare genetic disorder. Excessive intake of phenylalanine has been linked to brain damage [7]. As a result, all products containing ASP must be labeled to indicate the presence of a phenylalanine source [8]. A derivative of ASP, NEO is an odorless, white crystalline powder. It is safer for consumption by people with phenylketonuria because the 3,3-dimethyl group in its structure blocks the breakdown to phenylalanine [10]. NEO is 7000–13000 times sweeter than sucrose, with a taste very similar to sucrose. Its use is not associated with any bitter aftertaste ft and it has extensive shelf-life stability in dry conditions. It is also very stable in aqueous solutions in the neutral and acidic pH ranges [7]. In addition, NEO is heat stable and thus can be used in cooking and baking. It is approved for use in the USA, Australia, New Zealand and the EU. The dipeptide sweetener ALI has a sweetening capacity 2000 times greater than sucrose. Due to the presence of an amide moiety in its structure, ALI is relatively heat stable [7]. It has no aftertaste ft and is characterized by a clean, sweet fl flavor. It is approved for use as a sweetener in Australia and Mexico but not in the USA or EU [7]. Discovered in the 1960s, NHDC has a sweetening strength ~1500 times greater than sucrose. Industrially, it is produced by hydrogenation of a flafl vonoid (neohesperidin) found in citrus fruits. NHDC is known to have menthol-licorice-like aft ftertastes and antioxidant properties [8]. It exhibits good stability in aqueous solutions [7]. Sucralose is thermally stable and contains three chlorine atoms in its structure, making it an organochloride. It is about 600 times sweeter than sucrose and can be used during cooking and baking [9]. It is approved for use by the US FDA in a variety of foods and beverages. There Th is some concern about its safety due to the fact that other organochlorides such as dioxins and pesticides are linked with toxic and carcinogenic effects ff [8]. However, human and animal studies have shown SCL to be safe for human consumption [9]. Steviol glycosides are natural components in the extract of Stevia rebaudiana Bertoni, a plant native to Paraguay [11]. Stevia has been used for years in Japan, Korea, China, Brazil, and Paraguay as a food additive or as a household sweetener [12]. Steviol glycosides under certain conditions are considered Generally Recognized as Safe (GRAS) by the FDA and are approved in the EU. Stevia produces several diterpene glycosides, the most abundant being STV and REB A [13]. Five other steviol glycosides have been identifi fied as minor components of the stevia leaf, including Reb C, D, F, dulcoside A, and rubusoside. The steviol glycosides have similar structures: a steviol aglycone is connected at C-4 and C-13 to mono, di, or trisaccharides consisting of glucose and/or rhamnose residues [14, 15].

16 Food Safety: Innovative Analytical Tools Steviolbioside and Reb B are thought to be hydrolysis products of STV and Reb A formed during the extraction process of the glycosides from the plant [16]. The distribution of steviol glycosides in plant extracts can vary greatly depending on the extraction and purification fi process [17]. One issue preventing the wide use of stevia as an artificial fi sweetener is the presence of a bitter aft ftertaste in some extracts. REB A has been reported to have the least bitterness of the major steviol glycosides [18]. The Th sweetening power of the steviol glycosides also differ, ff with REB A being 400 times sweeter than sucrose while STV is about 300 times sweeter [16, 19]. Sweeteners are often ft used in combination to enhance sweetness and limit undesirable aft ftertastes [7]. A classic example is the blend of SAC-CYC formulated in a 1:10 ratio. The bitter aft ftertaste of SAC is masked by CYC and due to an additive effect ff the sweetening power of the mixture is greater. Food products containing sweeteners are heavily promoted as beneficial fi for the treatment of obesity and management of diabetes [7]. Sweeteners can be found in a large number of food products including the following: tabletop sweeteners, carbonated and non-carbonated beverages, baked goods, preserves and confectionery (icings, frostings, and syrups), alcoholic drinks, candies and dairy products such as yogurt and ice cream [20]. There is considerable controversy surrounding the adverse health eff Th ffects of non-nutritive sweeteners. Consumers worldwide have reported side eff ffects linked to sweetener consumption, including mood and behavioral changes, skin irritations, headaches, allergies, respiratory difficulties, ffi and cancer [7]. As such, it is important to monitor and control the concentration of sweeteners in foods to ensure compliance with diff fferent countryspecific fi regulations. The EU limits the amount of sweeteners added to food and sets a maximum usable dose (MUD) for specific fi food commodities [20]. In order to ensure that products are in compliance with regulations, it is necessary to have reliable, robust and quantitative methods for the simultaneous determination of several commonly used sweeteners in a single analysis. In addition to multi-sweetener analyses, there is also a need for single sweetener analytical methods such as in the case of CYC. The Th non-nutritive sweetener CYC was discovered in the 1930s [21]. It is 30–40 times sweeter than sucrose with its effi fficacy increasing when used in combination with other sweeteners [22]. It is widely used as a sweetening agent in a variety of low-calorie foods and beverages in many countries [21]. However, CYC is banned for commercial use as a food additive by the US FDA (Code of Federal Regulations 21, §189.135) because of research findings that linked its consumption with bladder cancer in rats [23]. Under the ban, CYC cannot be added to or be detectable in food. Since there is an increasing

Recent Analytical Methods for the Analysis of Sweeteners 17 number of foods sold in the USA that are imported from other countries, where CYC is approved for use as a food additive, it is important to have analytical methodology for the detection and confirmation fi of CYC in foods [22].

2.2 Sample Preparation Sample preparation/cleanup is the process of isolating target analytes from interferences in food matrices prior to instrumental analysis. This Th is oft ften the most time-consuming part of the analytical method and is essential to analyte determination. In order to be able to determine whether or not a sample contains sweeteners and authenticate the presence and concentrations of these analytes in various foods, simple to extensive sample cleanup is necessary. Sweeteners are widely used in drinks, candies and yogurts, which are commonly consumed products [24]. Foods are complex matrices due to the considerable diff fferences in their composition, which includes the presence of macromolecules, color additives and preservatives. Furthermore, sweeteners are present in food products at levels that require prepared samples to oft ften be signifi ficantly diluted in order to bring the analyte concentrations within the linear range of the method. There are many components in food matrices that have similar polarities Th to sweeteners, most of which are water soluble, with the exception of DUL and NHDC. Therefore, it is very diffi fficult to isolate sweeteners from food matrix. There are considerable diff fferences in the concentrations of sweeteners in drinks, possibly due to beverage manufacturing processes that may contribute to these variations. Differences ff are most likely due to the varying sweetening strengths of these compounds relative to sucrose. Therefore, Th diff ffering amounts of sweeteners are added to produce the desired sweetening effect ff [3]. Furthermore, there are signifi ficant diff fferences in chemical properties among sweeteners such as solubility and thermal stability [3]. As such, some sweeteners function better in certain food types while others are best suited for use in drinks. Generally, hard candies, drinks and tabletop sweeteners require minimal sample preparation prior to instrumental analysis. Normally, hard candies and tabletop sweeteners are weighed and dissolved in H2O by the process of shaking and/or vortexing. The Th samples are then diluted to obtain an analyte concentration within the linear range of the method. This procedure should produce complete dissolution of the candy or tabletop samples, resulting in transparent solutions with no visual insoluble

18 Food Safety: Innovative Analytical Tools material remaining aft fter shaking. Drink samples are simply diluted with H2O or mobile phase and filtered fi with sonication of carbonated beverages to remove dissolved gases [25]. Replicate analysis should be performed on all samples and if products are packaged in individual servings (candy, tabletop sweeteners), separate packages should be analyzed. Liquid-liquid extraction (LLE) is sometimes used as a simple, low-cost method to prepare samples prior to instrumental analyses [5]. LLE involves addition of an organic solvent to the food in liquid form. Sweeteners are then extracted from the liquid aqueous phase into the organic phase [6]. Solid-liquid extraction (SLE) is the process of partitioning target analytes from a solid state into a solvent prior to dilution and filtration. fi Solid samples can be homogenized, vortexed and centrifuged to separate the supernatant [5, 22, 26]. Yang and Chen [5] used LLE and SLE to extract sweeteners from a water/ methanol solution (50:50, v/v). Beverages were degassed when necessary and solid samples were homogenized and extracted. The Th method was applied to the determination of eight non-nutritive sweeteners in foods. Lim et al. modifi fied the LLE and SLE procedures developed by Yang and Chen to analyze nine artificial fi sweeteners in Korean foods. Samples analyzed included candies, beverages and yogurts. Sheridan and King [22] applied SLE with homogenization to the analysis of CYC in a wide range of foods, including dried prunes and beans, jarred mangos and peaches, grape tomatoes and strawberry cake. Since CYC is water-soluble the aqueous extract could be centrifuged, filtered and signifi ficantly diluted, which limits matrix interferences and MS signal suppression [22]. Scotter et al. also used LLE and SLE for the analysis of CYC in carbonated beverages, fruit juices, milk-based desserts, jams and spreads. Additionally, Carrez I and II solutions (reagents used to precipitate proteins and fats) a were prepared and added to the foods under slightly heated conditions for sample clarifi fication [7, 26]. This is followed by centrifugation to separate proteins and fatty material from the water-soluble supernatant in complex matrices such as ice-cream, chocolate syrup and coffee ff creamers [27]. The supernatant can then be fi filtered and diluted in preparation for instrumental analysis. Centrifugation without protein separation may be needed to separate solid particles present in some fruit juices [28]. Solvents that are commonly used for extraction are methanol (MeOH), acetonitrile (ACN) and water [28]. Another technique to prepare solid samples, such as dried fruits, uses a cryogenic grinder. Dried fruits are cut into small pieces and placed into a cryogenic blender. Liquid nitrogen is then poured over the pieces until they are immersed. Once the liquid nitrogen completely evaporates and

Recent Analytical Methods for the Analysis of Sweeteners 19 the pieces are frozen they are blended into a fine powder using an analytical mill. Solvent is then added to a weighed amount of the powder with subsequent vortex mixing, centrifugation, dilution and filtration [28]. This procedure results in a more homogeneous and uniform sample mixture than achieved with normal homogenization because the solid is broken down into very fine fi particles. One of the biggest challenges in food analysis is the effect ff of matrix composition on the performance of the analytical method. In order to determine method accuracy and selectivity, a representative from each food commodity containing no target analytes is fortified fi with known amounts of sweeteners. The sweeteners chosen for spiking experiments should encompass the range of polarities, including most polar, intermediate and nonpolar compounds. Food products are fortifi fied in triplicate at three diff fferent concentrations in accordance with agency guidelines and analyzed alongside an unfortified fi sample. Solid-phase extraction (SPE) is a reproducible technique that can be used to isolate sweeteners based on their affi ffinity to a stationary phase. The SPE sorbents are silica- or polymer-based beds that are modifi fied with polar or nonpolar functional groups [29]. There Th are many types of commercially available SPE cartridges that are packed with C8, C18 and ionexchange sorbent beds [29]. For the isolation of sweeteners from foods, the most successful SPE cartridges have been those with reversed-phase (RP) sorbents such as C8 or C18 [30]. Zygler et al. developed a method for the determination of nine nonnutritive sweeteners using Strata-X polymeric RP 3 mL cartridges packed with 200 mg sorbent bed for the cleanup of beverages, yogurts, and fish fi products [20]. These SPE cartridges were chosen because extensive testing of diff fferent SPE columns, including Chromabond C18ec, Strata-X RP, and Bakerbond Octadecyl, revealed optimal recoveries for all sweeteners were achieved [29]. Scheurer et al. [8] tested several diff fferent SPE cartridges and determined that Bakerbond Isolute SDB-1 achieved best recoveries for the extraction of ACS-K, SAC, ASP, CYC, NEO, SCL and NHDC in waste and surface waters. Yogurts represent a much more complex mixture of ingredients than beverages or hard candies, thus requiring a thorough sample cleanup prior to chromatographic analyses [31]. Th This ensures better long-term performance of the instrument and minimizes ion suppression effects ff when using mass spectrometric detection. fied and optimized a previous SPE method for the Shah et al. [32] modifi analysis of yogurts using Macherey-Nagel Chromabond C18ec 3 mL cartridges packed with 500 mg sorbent bed [29]. Several SPE parameters were

20 Food Safety: Innovative Analytical Tools tested, including sorbent phase type, cartridge size, sample load volume, and extraction buff ffer. As previously seen, the most critical factor aff ffecting analyte recoveries was the composition of the extraction buffer ff [29]. The use of formic acid and N,N- diisopropylethylamine (DIPEA) at pH 4.5 yielded the best recoveries for the sweeteners from yogurts. Compared to triethylamine (TEA), the ion pairing agent DIPEA allows for improved recoveries as it enables a stronger hydrophobic interaction between the sorbent bed and sweeteners [29]. As a result, this enables better retention of the sweeteners on the SPE cartridge, especially ACS-K and CYC. The Th authors reported that it is imperative to prevent the cartridge from drying out during the course of this SPE procedure. Yang and Chen [33] developed a SPE method using a Waters Oasis HLB cartridge for the isolation of NEO from beverages, preserved fruits and  cake. Dairy and fruit juice beverages were pretreated with MeOH, mixed, centrifuged and loaded on the SPE cartridge. Preserved fruits and cake were homogenized, vortexed, sonicated, centrifuged, and then loaded onto the SPE cartridge. Th The cartridge was conditioned prior to sample loading and then washed with water followed by MeOH to remove impurities. NEO was eluted with MeOH and concentrated to dryness by vacuum and reconstituted with MeOH prior to filtration fi into HPLC vials [33]. A dispersive SPE procedure was developed by Chen et al. for the determination of ACS-K, SAC, CYC, ASP, STV and NEO in red wine. The method allows for the quick magnetic separation of target analytes Th from matrix interferents using ethylenediamine-functionalized magnetic polymers (IEDA-MP) as the adsorbent. This technique allows for the easy clean-up of red wine using magnetic iron oxide particles to remove pigments, organic acids and sugars under a magnetic field. Recoveries ranged from 78.5% to 99.2% [34]. If available, a standard reference material containing certified fi values of sweeteners fortifi fied in a food matrix can be obtained from an institution, such as the National Institute of Standards and Technology (NIST) or the International Union of Pure and Applied Chemistry (IUPAC), and analyzed. This material is analyzed to confi firm that the method is valid and accurate for its designed purpose.

2.2.1 Internal Standards Generally, it is important to have internal standards for quantitation to account for possible ion suppression from matrix interferences in the complex composition of foods [5]. Although it is ideal to have isotopically labeled standards for MS detection methods for each compound

Recent Analytical Methods for the Analysis of Sweeteners 21 being analyzed, these are sometimes unavailable and cost prohibitive. Therefore, similar chemical and physical properties to the target anaTh lytes are the criteria used for internal standard choice. Shah et al. used saccharin-d4, sodium cyclamate-d11 and D-Sorbitol-1-13C as the three internal standards for the analysis of fourteen sweeteners in foods [32]. Cycloheptylamine was used as the internal standard for the determination of CYC in foods by RP HPLC-UV [26]. Huang et al. used tiopronin as the internal standard for the determination of CYC in foods using ionpair HPLC coupled to ESI-MS [21]. Sodium warfarin has been used as an internal standard in previous multi-sweetener methods for determination of several target analytes [5, 6]. Sucralose-d6 was used as the internal standard for the determination of SCL by ESI-LC/MS-MS in waste and surface waters [8].

2.3

Analytical Methods

2.3.1 Instrumental Analyses 2.3.1.1 HPLC-UV-VIS/DAD/ELSD Detection Non-nutritive sweeteners are a class of compounds that have significantly fi diff fferent physical and chemical properties. This makes it very challenging to develop a single method for their separation and isolation from matrix interferences. In the past, the most common technique for screening sweeteners was thin-layer chromatography (TLC). Th The FDA has used the AOAC Offi fficial Method #969.27, TLC method for the determination of some non-nutritive sweeteners in food samples [35]. This Th method lacks specificity fi and is limited to the qualitative determination of a select few sweeteners for routine regulatory analyses. In addition, this method lacks confi firmation criteria compatible with today’s standards. More recently, high performance liquid chromatography (HPLC) with reversed-phase (RP) ion-pair, ion and hydrophilic interaction chromatography (HILIC) have all been applied to the analysis of sweeteners. Gas chromatography is seldom used today for the analysis of sweeteners due to their low volatility and diffi fficulty to form volatile derivatives. Therefore, GC will not be further discussed here. The Th FDA has used ion chromatography (IC) coupled to suppressed conductivity detection for the determination of ASP, CYC, ACK-S and SAC [36]. However, IC has proved to lack selectivity in certain matrices such as those that contain citric acid. Th The authors report signifi ficant interference from a very large citric acid peak in this anion-exchange separation which can adversely impact target analyte

22 Food Safety: Innovative Analytical Tools determinations [36]. Furthermore, the scope of the method is narrow and does not incorporate all sweeteners of regulatory interest [37, 36]. Most sweeteners have poor chromophoric properties and determination by HPLC with an ultraviolet (UV) detector requires derivatization prior to analyses. Furthermore, HPLC-UV lacks specifi ficity especially in food matrices. Additionally, sweeteners encompass a wide range of polarities and molecular size with very different ff pKa values that makes chromatographic separation diffi fficult. For example, ERY is a very small highly polar compound compared to REB A, which is considerably larger and relatively more hydrophobic (Figure 2.1). Although several analytical methods for the determination of artifi ficial sweeteners have been published, many are not appropriate for routine regulatory analyses. Various detection techniques for the determination of non-nutritive sweeteners have been reported in the literature. The Th most common multisweetener methods involve HPLC with diff fferent types of detection [20].

OH

O

HO

OH

N H

OH

OH

O

N H

OH

HO

O

NH2

OH

O

OH

SAC MW = 183 g/mol

N H

O

ASP MW = 294 g/mol

HO HO

ALI MW = 331 g/mol

OH HO HO

H3C O O

O Me O HO HO OH

OH O OH OH O

OH

OH O

HO

O OH

H3C

CH3 O

O CH2

OH

REB A MW = 966 g/mol

NH

HO

N H

CH3

O NEO MW = 378 g/mol

CH3

HO HO

OMe HO

OH

O HO

OH O HO

HO NHDC MW = 612 g/mol

CI

O OH

SCL MW = 396 g/mol

OH

O

OH

HO O

HO O

O

H3C

OH

OH

H

HO

NH2 O

O

HO

MAL MW = 344 g/mol

O

O

H N

K+

H N

NH

DUL MW = 180 g/mol

O

–

O ACS-K MW = 152 +39 (201) g/mol

O

OH

O

O

S

OH

O

O

S

NH2

O

O

N

HO O

O

H3C

HO

HO

CYC MW = 179 g/mol

XYL MW = 152 g/mol

O

S

–

O S

OH

ERY MW = 122 g/mol

H3C

OH

OH

OH HO

OH CI

O

O

HO

CI O

HO

O OH OH

O

HO H

O

HO HO

O

H

STV MW = 804 g/mol

O

Figure 2.1 Chemical structures of the non-nutritive sweeteners of varying molecular sizes and polarities: ERY, erythritol; XYL, xylitol; CYC, cyclamate; DUL, dulcin; SAC, saccharin; ACS-K, acesulfame potassium; ASP, aspartame; ALI, alitame; MAL, maltitol; NEO, neotame; SCL, sucralose; NHDC, neohesperidine dihydrochalcone; STV, stevioside; REB A, rebaudioside A; and MW, molecular weight.

Recent Analytical Methods for the Analysis of Sweeteners 23 An HPLC-UV method is reported for the determination of CYC, SAC and ASP using a simple RP separation and detection at 196 nm. The Th method does not require derivatization of CYC or sample preparation prior to HPLC-UV. However, in order to achieve baseline resolution of CYC and SAC, the pH of the phosphate buffer ff mobile phase needs to be maintained at 2.5, which could severely compromise the integrity of a RP column [38]. Furthermore, many foods and beverages contain UV-active species which could interfere with the analysis if chromatographic separation was not achieved. This method was applied to the analysis of CYC, SAC, and ASP in beverages [38]. Scotter et al. developed a HPLC-UV method for the determination of CYC using peroxide oxidation of CYC to cyclohexylamine followed by derivatization with trinitrobenzene sulfonic acid. Analytes were separated by RP using a Spherisorb ODS2 C18 column (250 × 4.6 mm, 5 μm). The Th limit of detection (LOD) values ranged from 1–20 mg/kg in a variety of foods. Recoveries from spiking studies were in the range of 82% to 123%. The method was single-laboratory validated for the analysis of CYC in bevTh erages, fruit preserves, spreads and dairy desserts [26]. Serdar and Knezevic [39] reported two RP methods using diode array detection (DAD) for the determination of ASP, ACS-K, SAC, and CYC in beverages and nutritional products. The Th first method used a C18 column for the isocratic separation of ASP, ACS-K, and SAC with a mobile phase of phosphate buff ffer and ACN. The second method used a C18 column for the isocratic separation of CYC with a mobile phase of MeOH and water [39]. However, derivatization of CYC to cyclohexylsulfamic acid was required prior to instrumental analysis, which is unfavorable for routine laboratory use. A novel technique was reported by Cheng and Wu [40] for the determination of ASP and its hydrolysis products in Coca-Cola Zero. Th The authors described a two-dimensional HPLC-UV method using a C8 RP column for the fi first dimension and determination of ASP. The second dimension used a ligand-exchange column with online post-column derivation fluorescence detection for analysis of the hydrolysis products, L- and D-enantiomers of aspartic acid and phenylalanine. Electric or microwave heating was used to induce the formation of the hydrolysis products. The Th LOD and limit of quantitation (LOQ) for ASP were 1.3 and 4.3 μg/mL, respectively, with a linear range spanning 0–50 μg/mL. The Th LODs and LOQs for L- and D-aspartic acid were 0.16 and 0.17 μg/mL and for L- and D-phenylalanine were 0.52 and 0.55 μg/mL, respectively [40]. Determination of nine sweeteners by HPLC with evaporative light scattering detection (ELSD) has been published [2]. The Th method involves SPE

24 Food Safety: Innovative Analytical Tools cleanup of samples prior to HPLC-ELSD. Analyte recoveries ranged from 93–109% for spiked concentrations at MUD levels or LOQ concentrations. LOD and LOQ values were < 15 μg/g and < 30 μg/g, respectively, in three matrices, including an energy drink, canned fruit and yogurt. Precision was tested by fortification fi of sweeteners in three matrices at three different ff concentration levels on three diff fferent days. Intermediate precision was < 8% for all sweeteners with the exception of ASP in canned fruits, due to its degradation as a result of improper storage conditions [2]. This method is suitable for rapid screening of samples for sweeteners but Th may lack selectivity for target analytes, especially among interferences in the matrix. A rapid and sensitive method for the determination of steviol glycosides in Stevia rebaudiana and stevia products has been developed using ultra-high performance liquid chromatography (UHPLC) with UV and ELSD [41]. Five steviol glycosides are baseline separated on a Waters Acquity UPLC HSS T3 analytical column (100 × 2.1 mm, 1.8 μm) within 12 minutes. The LOD and LOQ values for the steviol glycosides were