Analysis of Endocrine Disrupting Compounds in Food

Analysis of Endocrine Disrupting Compounds in Food

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

Analysis of Endocrine Disrupting Compounds in Food Leo M.L. Nollet EDITOR

A John Wiley & Sons, Inc., Publication

Edition first published 2011 © 2011 Blackwell Publishing Ltd. Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Editorial Office 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services, and for information about how to apply for permission to reuse the copyright material in this book, please see our Website at www.wiley.com/ wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee code for users of the Transactional Reporting Service is ISBN-13: 978-0-8138-1816-0/2011. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Analysis of endocrine disrupting compounds in food / editor, Leo M.L. Nollet. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-1816-0 (hardback : alk. paper) 1. Food–Analysis. 2. Endocrine disrupting chemicals–Analysis. I. Nollet, Leo M. L., 1948– TX541.A75 2011 664'.07–dc22 2010016639 A catalog record for this book is available from the U.S. Library of Congress. Set in 10 on 12 pt Times by Toppan Best-set Premedia Limited Printed in Singapore Disclaimer The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation warranties of fitness for a particular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strategies contained herein may not be suitable for every situation. This work is sold with the understanding that the publisher is not engaged in rendering legal, accounting, or other professional services. If professional assistance is required, the services of a competent professional person should be sought. Neither the publisher nor the author shall be liable for damages arising herefrom. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. 1

2011

Table of Contents

Preface List of contributors

vii ix

Chapter 1

Endocrine Disrupting Chemicals. What? Where? Guang-Guo Ying

3

Chapter 2

Analysis of PCBs in Food Manuela Melis and Ettore Zuccato

19

Chapter 3

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food Luisa R. Bordajandi, Belén Gómara, and María José González

49

Chapter 4

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities M.J. Gómez, M.A. Martínez-Uroz, M.M. Gómez-Ramos, A. Agüera, and A.R. Fernández-Alba

75

Chapter 5

Pesticides: Herbicides and Fungicides Iván P. Román Falcó, Lorena Vidal, and Antonio Canals

127

Chapter 6

Pesticides: Organophosphates Juan F. García-Reyes, Bienvenida Gilbert-López, and Antonio Molina-Díaz

199

Chapter 7

Phytoestrogens Ashok K. Singh and Leo M.L. Nollet

219

Chapter 8

Mycoestrogens Jean-Denis Bailly

229

Chapter 9

Analysis of Hormones in Food John L. Zhou and Zulin Zhang

243

Chapter 10 Phthalates Jiping Zhu, Rong Wang, Yong-lai Feng, and Xu-liang Cao

255

Chapter 11 Organotin Compounds Analysis Maw-Rong Lee and Chung-Yu Chen

269

Chapter 12 Determination of Heavy Metals in Food by Atomic Spectroscopy Joseph Sneddon

289

v

vi

Contents

Chapter 13 Surfactants Bing Shao

305

Chapter 14 Polybrominated Biphenyls Antonia María Carro-Díaz and Rosa Antonia Lorenzo-Ferreira

325

Chapter 15 Bisphenol A Ana Ballesteros-Gómez and Soledad Rubio

349

Chapter 16 Perfluoroalkylated Substances Leo M.L. Nollet

367

Chapter 17 Flame Retardants D. Lambropoulou, E. Evgenidou, Ch. Christophoridis, E. Bizani, and K. Fytianos

377

Chapter 18 Personal Care Products Guang-Guo Ying

413

Chapter 19 Polycyclic Aromatic Hydrocarbons Peter Šimko

429

Chapter 20 Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene Leo M.L. Nollet

447

Index

471

Preface

Many chemical compounds used in the past, and others still used, may have hormonedisrupting properties. Such chemical compounds may interfere with the normal action of hormones in humans and animals. A great number of these endocrine-disrupting compounds are persistent organic pollutants (POPs). POPs may be found worldwide and in every compartment of the environment: water, air, and soil. Animals and humans may inhale or ingest residues of these chemical compounds. Other sources of endocrine disruptors are accidents and pollution. Analysis of Endocrine Disrupting Compounds in Food provides, first and above all, a unique and comprehensive professional reference source covering most of the recent analytical methodology used to study endocrine-disrupting compounds in food. A broad team of international authors addresses the most recent advances in analysis of endocrine-disrupting chemicals in food. The book further discusses the relationship between chemical compounds and hormone activity. What are the health impacts of different chemical compounds for humans and animals? How are these compounds entering into foodstuffs? While covering conventional (typically lab-based) methods of analysis, the book focuses on leading-edge technologies that have recently been introduced. The book looks at areas such as food quality assurance and safety. The topics of the presence of

persistent organic pollutants; monitoring pesticide and herbicide residues in food; determining heavy and other metals in food; discussing the impact of dioxins, PCBs, PCDFs, and many other suspected chemicals are covered. The book highlights the relevance and importance of sample preparation and cleanup. Applications of gas chromatography, high-pressure liquid chromatography, and related techniques, and the use of biosensors for evaluating the safety and quality of food and agricultural products are discussed. The reader will also find information on the principles and applications of immunodiagnostics and applications in food safety. A unique feature of the book is that the informational tables are structured the same way throughout the book; furthermore, most chapters are also structured similarly. For all their great efforts and their excellent work I thank all of the authors who contributed to this work. It is their efforts that give value to this book. A special thanks is directed to Mark Barrett and Susan Engelken of Wiley-Blackwell for their support. I dedicate this book to my fourth grandchild and first grandson, Naut. I hope he will become a respected and loved man in a green world, a world without endocrine disrupting compounds. Leo M.L. Nollet

Exert your talents, and distinguish yourself, and don’t think of retiring from the world, until the world will be sorry that you retire. (Samuel Johnson) vii

List of Contributors

A. Agüera Pesticide Residue Research Group University of Almería 04120 Almería, Spain Jean-Denis Bailly Mycotoxicology Research Unit National Veterinary School of Toulouse 23 Chemin des Capelles BP 87614 31076 Toulouse Cedex 03, France Ana Ballesteros-Gómez Department of Analytical Chemistry Campus of Rabanales Edificio Anexo Marie Curie University of Córdoba 14071 Córdoba, Spain E. Bizani Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece Luisa R. Bordajandi European Food Safety Authority (EFSA) Unit on Contaminants Largo N. Palli 5/A. 43100 Parma, Italy Antonio Canals Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante, Spain

Xu-liang Cao Food Research Division Health Canada 251 Sir Frederick Banting Driveway Tunney’s Pasture Ottawa Ontario K1A 0L2 Canada Antonia María Carro-Díaz Dpto. de Química Analítica Nutrición y Bromatología Facultad de Química Instituto de Investigacións e Análises Alimentarios Universidad de Santiago de Compostela Avda. de las Ciencias s/n 15782 Santiago de Compostela, Spain Chung-Yu Chen Department of Chemistry National Chung Hsing University Taichung Taiwan Ch. Christophoridis Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece E. Evgenidou Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece ix

x

Contributors

Iván P. Román Falcó Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante Spain Yong-lai Feng Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada A.R. Fernández-Alba IMDEA (Instituto Madrileño de Estudios Avanzados) Parque Científico Tecnológico de la Universidad de Alcalá 28805 Alcalá de Henares, Madrid Spain Pesticide Residue Research Group University of Almería 04120 Almería, Spain K. Fytianos Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece

Belén Gómara Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute, CSIC Juan de la Cierva 3, 28006 Madrid Spain

M.J. Gómez IMDEA (Instituto Madrileño de Estudios Avanzados) Parque Científico Tecnológico de la Universidad de Alcalá 28805 Alcalá de Henares, Madrid Spain Department of Chemical Engineering University of Alcala 28771 Alcala de Henares, Madrid Spain

M.M. Gómez-Ramos Pesticide Residue Research Group University of Almería 04120 Almería, Spain

María José González Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute, CSIC Juan de la Cierva 3, 28006 Madrid Spain

Juan F. García-Reyes Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

D. Lambropoulou Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece

Bienvenida Gilbert-López Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

Maw-Rong Lee Department of Chemistry National Chung Hsing University Taichung Taiwan

Contributors

Rosa Antonia Lorenzo-Ferreira Dpto. de Química Analítica Nutrición y Bromatología Facultad de Química Instituto de Investigacións e Análises Alimentarios Universidad de Santiago de Compostela Avda. de las Ciencias s/n 15782 Santiago de Compostela, Spain M.A. Martínez-Uroz Pesticide Residue Research Group University of Almería 04120 Almería, Spain Manuela Melis Laboratory of Food Toxicology Department of Environmental Health Sciences Mario Negri Institute for Pharmacological Research Via La Masa 19, 20156 Milan, Italy Antonio Molina-Díaz Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

xi

Bing Shao Institute of Nutrition and Food Hygiene Beijing Center for Disease Control and Prevention Beijing 100013, China Peter Šimko Food Research Institute Priemyselna 4 824 75 Bratislava Slovak Republic and Institute of Food Science and Biotechnology Faculty of Chemistry Brno University of Technology Purkynˇ ova 464/118, 612 00 Brno Czech Republic Ashok K. Singh Department of Veterinary Diagnostic Medicine College of Veterinary Medicine University of Minnesota, St Paul Campus 1333 Gortner Ave St Paul, MN 55108 USA Joseph Sneddon Department of Chemistry McNeese State University Lake Charles, LA 70609 USA

Leo M.L. Nollet University College Ghent Member of Ghent University Association Faculty of Applied Engineering Sciences Schoonmeersstraat 52 B9000 Gent Belgium

Lorena Vidal Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante, Spain

Soledad Rubio Department of Analytical Chemistry Campus of Rabanales Edificio Anexo Marie Curie University of Córdoba 14071 Córdoba, Spain

Rong Wang Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada

xii

Contributors

Guang-Guo Ying State Key Laboratory of Organic Geochemistry Guangzhou Institute of Geochemistry Chinese Academy of Sciences 511 Kehua Street, Tianhe District Guangzhou 510640, China

Jipin Zhu Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada

Zulin Zhang The Macaulay Institute Craigiebuckler Aberdeen, AB15 8QH, UK

Ettore Zuccato Laboratory of Food Toxicology Department of Environmental Health Sciences Mario Negri Institute for Pharmacological Research Via La Masa 19, 20156 Milan, Italy

John L. Zhou School of Life Sciences Science University of Sussex Falmer, Brighton BN1 9QG, UK

Chapter 1 Endocrine Disrupting Chemicals. What? Where? Guang-Guo Ying

Introduction There is a concern that some natural and synthetic chemicals can interfere with the normal functioning of endocrine systems, thus affecting reproduction and development in wildlife and humans. These chemicals are called endocrine disruptors or endocrinedisrupting chemicals (EDCs). Although endocrine disruption has been known since the 1930s (Dodds et al. 1938), this issue has regained attention and generated immense scientific and public interest since 1992 (Colborn and Clement 1992), and especially since the publication of the book Our Stolen Future (Colborn et al. 1996). The chemicals identified or suspected as being endocrine disruptors in the literature include pesticides (e.g., dichlorodiphenyltrichloroethane [DDT], dichlorodiphenyldichloroethylene [DDE], dieldrin, endosulfan), pharmaceuticals (e.g., diethylstilbestrol [DES]) and industrial chemicals or pollutants (e.g., polychlorinated biphenyls [PCBs], dioxins, bisphenol A) (Table 1.1). Since then, many studies have been carried out on endocrine disruption. Some reproductive problems in wildlife and humans have been linked to exposure to these chemicals. Wildlife and humans are exposed daily to these pervasive chemicals that have already caused numerous adverse effects in

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

wildlife and are most likely affecting humans as well. There is compelling evidence regarding the effects of exposure to EDCs on wildlife (Damstra et al. 2002). These include imposex of mollusks by organotin compounds (Alzieu 2000; Gibbs et al. 1990; Horiguchi et al. 1994); developmental abnormalities, demasculinization and feminization of alligators in Florida by organochlorines (Guillette et al. 1994, 2000); and feminization of fish by wastewater effluent from sewage treatment plants and paper mills (Table 1.2) (Jobling et al. 1998; Bortone et al. 1989). There is also evidence that human testicular and breast cancer rates have increased during the last four decades, especially in developed countries (Brown et al. 1986; Hakulinen et al. 1986; Adami et al. 1994; Feuer 1995; Moller 1993; Ries et al. 1991; Wolff et al. 1993). However, except in a few cases (e.g., DES), a causal relation between exposure to chemicals and adverse health effects in humans has not been firmly established. Owing to the scientific evidence and public concern about potential effects on humans and wildlife, the U.S. Congress made amendments to the Safe Drinking Water Act (SDWA) in 1996 and required the U.S. Environmental Protection Agency (U.S. EPA) to develop a screening program for endocrine disruptors (Fenner-Crisp et al. 2000). In April 2000, a meeting of the environment ministers of the G8 group of industrialized countries listed EDCs as one of the high priorities and called for a furtherance of knowledge acquisition 3

4

Analysis of Endocrine Disrupting Compounds in Food

Table 1.1. List of suspected/known EDCs. Classification

Endocrine-Disrupting Chemicals

Pesticides

2,4-D Atrazine Benomyl Carbaryl Chlordane (γ-HCH) Cypermethrin DDT and its metabolites Dicofol Dieldrin/Aldrin Endosulfan Endrin Heptachlor Hexachlorobenzene (HCB) Iprodione

Kepone (chlordecone) Lindane Malathion Mancozeb Methomyl Methoxychlor Mirex Parathion Pentachlorophenol Permethrin Simazine Toxaphene Trifluralin Vinclozolin

Organohalogens

Dioxins and furans PCBs

PBBs and PBDEs 2,4-Dichlorophenol

Alkylphenols

Nonylphenols Octylphenols Pentaphenols

Nonylphenol ethoxylates Octylphenol ethoxylates Butylphenols

Heavy metals

Cadmium Lead

Mercury Arsenic

Organotins

Tributyltin

Triphenyltin (TPhT)

Phthalates

Diethylhexyl phthalate Butyl benzyl phthalate Di-n-butyl phthalate Di-n-pentyl phthalate

Dihexyl phthalate Dipropyl phthalate Dicyclohexyl phthalate Diethyl phthalate

Natural hormones

17β-Estradiol Estrone

Estriol Testosterone

Pharmaceuticals

Ethinyl estradiol Mestranol

Tamoxifen Diethylstilbestrol (DES)

Phytoestrogens

Isoflavonoids Coumestans Lignans

Zearalenone β-sitosterol

Phenols

Bisphenol A

Bisphenol F

Aromatic hydrocarbons

Benzo(a)pyrene Benz(a)anthracene Benzo(b/h)fluoranthene 6-hydroxy-chrysene

Anthracene Pyrene Phenanthrene n-Butyl benzene

on EDCs through jointly planned and implemented projects and international information sharing (Loder 2000). Surveys of some new emerging endocrine disrupting chemicals (e.g., nonylphenol and steroids) in major rivers of some countries have been undertaken (e.g., Naylor et al. 1992; Blackburn et al. 1999; Ahel et al. 2000; Tabata et al. 2001; Kolpin et al. 2002). The U.S. EPA and the Organization of Economic and Cooperative

Development (OECD) have invested considerable resources to develop tiered procedures for the testing and assessment of EDCs (Fenner-Crisp et al. 2000; Huet 2000; Parrott et al. 2001). The U.S. EPA planned to screen 15,000 chemicals for their possible effects as endocrine disruptors in animals and humans (Macilwain 1998). This chapter will give some background information about the endocrine disruption

Endocrine Disrupting Chemicals. What? Where?

5

Table 1.2. Effects associated with exposure to EDCs in wildlife (representative examples). Organism Alligators (Lake Apopka)

Fish (roach, trout) (Rivers in UK) Fish (Rivers in Canada and Finland) Fish (salmon) (Great Lakes) Birds (gulls, bald eagles) (Great Lakes, and California coast)

Chemical(s) DDT and its metabolites (DDD and DDE), dicofol and other pesticides Hormone steroids and nonylphenols from STP effluents Chemicals from pulp and paper mill effluents PCBs, dioxins, and organochlorine pesticides PCBs, DDT, and DDE

Effect

References

Demasculinization, reproductive dysfunction

Guillette et al. (1994) Guillette et al. (1995) Guillette et al. (2000) Pickford et al. (2000)

Feminization, abnormal gonad and gonoduct development Delayed sexual maturity, smaller gonads, reduced fecundity and changed sex steroid levels Abnormal thyroid function

Purdom et al. (1994) Jobling and Sumpter (1993) Jobling et al. (1998) Aaltonen et al. (2000) Karels et al. (1999) McMaster et al. (1995) Munkittrick et al. (1997) Leatherland (1993) Bowerman et al. (2000) Fry et al. (1987) Moccia et al. (1986)

Invertebrates (snails, mussels, clams, oysters)

Tributyltin (TBT)

Feminization, abnormal thyroid function, supernormal clutches, and decreased hatchability Masculinization (imposex)

Sheep

Phytoestrogens

Decreased fertility

issue, EDCs in food, and potential effects associated with exposure to EDCs.

Endocrine-disruption chemicals Endocrine system An endocrine system is found in nearly all animals, including mammals, nonmammalian vertebrates (e.g., fish, amphibians, reptiles and birds) and invertebrates (e.g., snails, lobsters, insects, and other species). Along with the nervous system, the endocrine system is one of the two communication systems that regulate all responses and functions of the body. The endocrine system consists of glands and the hormones they produce that guide the development, growth, reproduction, and behavior of humans and animals. The major endocrine glands of the body include the pituitary, thyroid, parathyroids, adrenals, pancreas, pineal gland, and gonads (ovaries in females and testes in males).

Alzieu (1998, 2000) Gibbs and Bryan (1986) Gibbs et al. (1988) Jha et al. (2000a) Jha et al. (2000b) Morcillo and Porte (2000) Bennetts et al. (1946) Hughes (1988)

Hormones are biochemicals that are produced by endocrine glands in one part of the body, travel through the bloodstream, and cause responses in other parts of the body. They act as chemical messengers and interact with specific receptors in cells to trigger responses and prompt normal biological functions such as growth, reproduction, and development. Hormones generally fall into four main categories: (1) amino acid derivatives, (2) proteins, (3) steroids, and (4) eicosanoids (Lister and van der Kraak 2001). The unifying nature of hormone action is the presence of receptors on target cells, which bind a specific hormone with high affinity and stereospecificity. Steroid and thyroid hormones act by entering target cells and stimulating specific genes. All other hormones bind to receptors on the cell surface and activate second-messenger molecules within the target cells (Raven and Johnson 1999). The body has hundreds of different kinds of receptors; each one is

6

Analysis of Endocrine Disrupting Compounds in Food

Figure 1.1. Functioning of hormone system. VTG, vitellogenin; ER, estrogen receptor; Hsp90, heat shock protein 90 kDa; ERE, estrogen response element.

designed to receive a particular kind of chemical signal. The hormone and its receptor have a “lock-and-key” relationship (Figure 1.1). When a hormone encounters its receptor, they grab hold, engaging in a molecular embrace known as binding. Once joined, the hormone molecule and its receptor trigger the production of particular proteins that turn on the biological activity associated with the hormone. The actions of hormones have two types: organizational and activational (Lister and van der Kraak 2001). The first type of action occurs during critical periods of development and induces permanent effects such as the actions of sex steroids. The second type of action causes only transient changes in a myriad of cellular processes such as the effects of glucagon and insulin on glucose homeostasis. Organizational actions are more important in terms of effect with respect to environmental contaminants (Guillette et al. 1996). Timing of hormone release is often critical for normal function, especially during fetal development (Palanza et al. 1999).

Endocrine disruption The Society of Environmental Toxicology and Chemistry (SETAC) defined endocrine

disruption as follows: “Synthetic, and naturally occurring, chemical substances in the environment are disrupting the normal functions of the endocrine system and its hormones in humans and wildlife” (SETAC 2000). This hypothesis has received much attention in recent years because there is increasing evidence that some chemicals in our environment disrupt the endocrine systems in wildlife as well as humans. There are several ways that chemicals can interfere with the endocrine system (Sonnenschein and Soto 1998). They can mimic or block natural hormones and alter hormonal levels, thus affect the functions that these hormones control. Less direct disruption involves alteration of the body’s ability to produce hormones, interference with the ways hormones travel through the body, and changes in numbers of receptors. Regardless of the situation, having too much or too little of the hormones it needs may cause the endocrine system to function inappropriately. Very subtle disruptions of the endocrine system can result in changes in growth, development, or behavior that can affect the organism itself or the next generation (Guillette et al. 1996; vom Saal et al. 1997; Palanza et al. 1999).

Endocrine Disrupting Chemicals. What? Where?

Hormones play a crucial role in the proper development of the growing fetus. Embryos and fetuses are especially sensitive at particular times to low doses of endocrine disruptors (Guillette et al. 1996; vom Saal et al. 1997; Palanza et al. 1999). Substances that have no effect in an adult can become poisonous in the developing embryo. The timing of exposure may be more important than the dose of the substance. The ultimate effects of endocrine disruption might not be seen until later in life or even until the next generation (Colborn et al. 1996; U.S. EPA 1997).

Endocrine disruptors Endocrine disruptors have received growing attention in public media and the scientific community due to their potential impacts on humans and wildlife. There are several definitions used by scientists and policy makers. In the Organization of Economic and Cooperative Development (OECD), an endocrine-disrupting chemical has been defined as an exogenous substance or mixture that alters the function(s) of the endocrine systems and consequently causes adverse health effects in an intact organism or its progeny or (sub) populations (Lister and Van Der Kraak 2001). An environmental endocrine disruptor was also broadly defined by the U.S. EPA as “an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for maintenance of homeostasis and the regulation of developmental processes” (Kavlock et al. 1996). A potential endocrine disruptor can be simply defined as a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism. An extensive list of the chemicals (Table 1.1) (Colborn et al. 1996; Guillette et al. 1996; Sonnenschein and Soto 1998; U.S. EPA 1997; Depledge and Billninghurst 1999) that have been found to be or are suspected to be capable of disrupting the endocrine

7

systems include many pesticides that are designed to be bioactive (e.g., DDT, vinclozolin, tributyltin [TBT], atrazine), persistent organochlorines (e.g., PCBs, dioxins and furans), alkyl phenols (e.g. nonylphenol and octylphenol), heavy metals (e.g., cadmium, lead, mercury), phytoestrogens (e.g., isoflavoids, lignans, β-sitosterol), and synthetic and natural hormones (e.g., β-estradiol, ethinyl estradiol). Many of these compounds have little in common structurally or in terms of their chemical properties, but they evoke agonist or antagonist responses, possibly through comparable mechanisms of action. These chemicals are released from a wide variety of sources such as intensive agriculture, industrial wastes, mining activity, domestic sewage, and landfills. Suspected EDCs can be found in every division of our environment (air, water, soil, sediment, and biota), in industrial products and household items, and even in the food we eat. They are often found in mixtures, such as effluents from sewage treatment plants, paper mills, and textile factories. It is not clear whether the components in a mixture act additively, synergistically, or antagonistically. EDCs can be classified into the following categories: 1. 2. 3.

4.

5.

6.

Environmental estrogens, for example, methoxychlor, bisphenol A Environmental antiestrogens, for example, dioxin, endosulfan Environmental antiandrogens, for example, vinclozolin, DDE, kraft mill effluent Toxicants that reduce steroid hormone levels, for example, fenarimol and other fungicides, endosulfan Toxicants that affect reproduction primarily through effects on the central nervous system (CNS), for example, dithiocarbamate Toxicants that affect hormone status, for example, cadmium, benzidine-based dyes (Depledge and Billinghurst 1999)

8

Analysis of Endocrine Disrupting Compounds in Food

The chemistry of the potential endocrine disruptors varies greatly, as does potency, that is, the effectiveness in binding and turning on the response. Most endocrine disruptors have very low potency because their chemistry is significantly different from the hormones they mimic. In addition to potency, the potential for a hormonelike effect depends on dose. For most of the endocrine disruptors, the dose–response relationship has not yet been established, especially at the low-dose range, and this may differ from species to species. The risk of endocrine disruptors to humans and wildlife also depends on their behavior and fate in the environment. Chemicals behave differently in different media. For example, nonylphenol had a dissipation halflife of ≤1.2 days in the water column, 28– 104 days in sediment, and 8–13 days on macrophytes in an experimental littoral ecosystem (Liber et al. 1999). Some EDCs (e.g., DDT and PCBs) are ubiquitous and persistent in the environment (Atlas and Giam 1981). They accumulate in the fatty tissue of organisms and increase in concentration as they move up through the food web (biomagnification). Because of their persistence and mobility, they accumulate in organisms and harm

species far from their original source. In order to assess the risks, it is necessary to carry out monitoring of those chemicals possessing endocrine-disrupting characteristics in environmental media and foods we eat.

Human exposure of endocrinedisrupting chemicals Endocrine-disrupting chemicals need to enter an organism before they can disrupt its endocrine system. Humans can be exposed in a variety of ways (Figure 1.2): the food we eat, the air we breathe and the particles or vapors it contains, the pharmaceuticals we ingest for medical reasons, the water we drink, the soil we accidentally or intentionally eat, and in utero exposure from the mother ’s body burden (Crisp et al. 1998). For fat-soluble chemicals such as PCBs, for example, food is the major source for people. Dairy products, meat, and processed foods are all major contributors. Breast milk is also a contributor (Table 1.3). These fat-soluble chemicals remain in the body for a long time, and their accumulation early in life contributes significantly (approximately 15%) to the adult body burden (Patandin et al. 1999).

Dermal exposure Cosmetics Body creams Deodorants

Inhalation exposure PAHs, PBDEs Plasticizers Heavy metals

Accumulation of lipophilic chemicals (DDT/DDE, PCB, PBDE)

Oral exposure Food contaminants Plasticizers PAHs, organochlorines Pesticides or Fungicides Heavy metals

Transfer from mother to fetus or to amniotic fluid or both

Figure 1.2. Routes of human exposure to chemicals.

Transfer of lipophilic chemicals to offspring by breast feeding

9

1973 1995–1997 1972

New South Wales, Australia

Germany

Japan

2002 2000 1996 1997–1998

China

India

Canada

UK

0.006–0.27 (0.071)

α-HCH

1.6–10.67 (5.43) 0.042–0.969 (0.21)

0.004–0.25 (0.04)

0.001–4.4 (0.345)

β-HCH

(0.043)

(0.043)

(0.001)

(0.081)

0.0039–0.0228 (0.0136)

0.003–0.53 (0.08)

0.06–2.37 (0.468)

0.06–0.51 (0.24)

0.93–8.26 (2.22)

0.08–13.2 (2.04) 0.016–7.6 (0.411)

HCBa

ΣDDT

(0.47)

(0.47)

(0.43)

(2.1)

0.77–4.01 (2.224) 0.0813–1.119 (0.28)

0.027–1.54 (0.24)

0.45–5.31 (1.92)

0.48–3.24 (1.38)

3.26–21 (8.6)

0.6–23.2 (5.64) 0.156–4.86 (1.185)

(0.25)

(0.030)

(0.042)

0.076-0.385 (0.2)

0.118-1.81 (0.55)

0.039–1.571 (0.5)

PCBs

References

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Konishi et al. (2001)

Konishi et al. (2001)

Schade and Heinzow (1998)

Siyali (1973)

Stacey et al. (1985)

Miller and Fox (1973)

Quinsey et al. (1995)

Newton and Greene (1972)

Concentration range and mean in parentheses. HCH, hexachlorocyclohexane isomers (α, β-HCH); HCB, hexachlorobenze; ΣDDT, dichlorodiphenyltrichloroethane (DDT and its metabolite DDE); PCBs, polychlorinated biphenyls.

a

1979–1980

Western Australia

1998

1971–1972

1990

1970

Year

Queensland, Australia

Victoria, Australia

Location

Table 1.3. Comparison of levels of selected organochlorines (μg/g fat) in breast milk of women from Australia and other countries.

10

Analysis of Endocrine Disrupting Compounds in Food

Table 1.4. Concentrations of some EDCs in foods. Compounds

Concentrationsa

Food

References

0.1–19.4 μg/kg

Guenther et al. (2002)

13.4–56.3 (32) ng/ml 5.8–235.8 ng/g wet weight

Ademollo et al. (2008) Lu et al. (2007)

5–1220 (147) ng/g wet weight

Ferrara et al. (2008)

Canned fish

2–59 ng/g

Various food (fruit and vegetables, meat)

0.5–384 ng/g

Podlipna and Cichna-Markl (2007) Ballesteros-Gomez et al. (2009)

DEHP

Wine

12 h

Room temperature Room temperature

No

>14 h Room temperature

≅200

No No No No No No

>3 h 12 h 1h 2h Not indicated 2h Room temperature Room temperature 70°C (hydrolysis) Room temperature Room temperature Room temperature

650 ≅180 ≅70 ≅180 Not included 400

Yes Yes Yes No No

No Yes

No No No No No Yes No Yes No No

No

Including Fat Retainer

6h 6h 6h Not indicated Not indicated

Room temperature Room temperature Room temperature Room temperature Room temperature

18 h 24 h 18 h 24 h 15 min 30 min 30 min 20 min 30 min 20 min

Room temperature Room temperature Room temperature Room temperature 1500 psi 120°C; 1500 psi 120°C; (12 MPa) 100°C; FV 60% 100°C; 1500 psi 100–185°C; 1500 psi 100°C Room temperature 20 min 6h

24 h

Extraction Time

Room temperature

Extraction Conditions

≅400 ≅400 ≅400 Not included Not included

≅200 330

≅40 ≅150 ≅100 ≅100 ≅150 ≅200

≅300

Amount of Solvent (mL)

Chen et al. 2008

Papadopoulus et al. 2004

Baars et al. 2004

Knutzen et al. 2003 Tsutsumi et al. 2001 Otaka et al. 2004 Sasamoto et al. 2006 Fernandes et al. 2004 Chen et al. 2008

Bordajandi et al. 2004 Fernández et al. 2004 Gómara et al. 2005 Mayer 2001 Jacobs et al. 2002

Lund et al. 2008 Santillo et al. 2003

Abad et al. 2002 Kivirinanta et al. 2004 Papadoupoulus et al. 2004 Bocio and Domingo 2005 Focant et al. 2001 Bernsmann and Fürst 2004 El-Kady et al. 2007 Wiberg et al. 2007 Traag et al. 2008 Grümping et al. 2008

Wittsiepe et al. 2001

References

Table 3.3. Comparison of the experimental conditions of the most used extraction techniques for dioxin analysis in foodstuffs of animal origin.

56

Analysis of Endocrine Disrupting Compounds in Food

Substantial progress has been made toward developing enhanced extraction techniques such as microwave-assisted solvent extraction (MASE) (Camel 2000) and, especially, the widely used pressurized liquid extraction (PLE or ASE, accelerated solvent extraction, named after the PLE system marketed by Dionex). Saponification under alkaline and acidic conditions followed by extraction with organic solvents is often employed for the analysis of large amounts (up to 100 g) of fat. However, that method is known to lead to degradation of dioxins in proportion to their chlorine content, and in the case of PCDFs, to the production of artifacts such as lower chlorinated PCDFs and ethoxy-PCDFs (Ryan et al. 1989). Finally, it is worth noting that although the approach has not yet been studied extensively, some applications have already demonstrated the potential of sonication (ultrasound-assisted extraction, or USE) for food dioxin analysis (Lanbropoulou et al. 2006; Ahn et al. 2006).

Soxhlet extraction SOX is one of the most frequently used liquid– solid extraction methods, developed in the late nineteenth century and still routinely used for extraction of dioxins from food samples (Tables 3.2 and 3.3). However, the technique has a number of drawbacks, among them the large volume of solvent required (200 mL for 100 g of tissue), the long extraction time (more than 18 hours), the generation of dirty extracts that require extensive cleanup, and the impossibility of automation. In order to overcome these drawbacks, alternative extraction strategies have been developed, offering analysts a choice of newer techniques such as SPE, MSPD, and more recently, PLE (or ASE).

Solid-phase extraction and matrix solid-phase dispersion SPE is today a classic extraction system, thanks mainly to the popularization of SPE

cartridges, which have been successfully applied to biological human fluids (Chang et al. 1993) and liquid food samples (cow’s and breast milk samples) using C18-bound silica phase (Focant et al. 2003). However, in the case of solid samples, SPE is less popular and has almost never been used to extract dioxins from food and food products because of the large amount of sample needed. To the contrary, MSPD, using open conventional glass chromatography columns, is very often used in routine analysis of food samples (Tables 3.2 and 3.3). In MSPD, the sample is mixed or blended with an appropriate sorbent (e.g., C18, silica) until a homogeneous mixture is obtained. This mixture is then packed into a glass column from which the analytes of interest are eluted with a suitable organic solvent. The extraction and first cleanup steps are performed at the same time, and most of the artifacts are eliminated. Because a large amount of sample is needed, the method compares unfavorably with SOX in terms of the amount of solvent required (around 400 mL).

Supercritical fluid extraction SFE is another classic method for food dioxin analysis, but it is not as popular as SOX and MSPD. SFE has attracted intense interest during the past 20 years, mainly for extraction of solid samples, because it offers short extraction times and minimal use of organic solvents (Smith 1999; Ahmed 2003). Carbon dioxide (CO2) is mostly used as the extraction solvent because of its moderate critical temperature (31°C) and pressure (73 atm). In the 1990s, SFE instruments became commercially available, enabling larger sample sizes and rendering it more suitable for wider applications. For food matrices, fat retainers such as Florisil and silica are usually introduced in the extraction thimble to achieve a fat-free extract. Some applications for fatty food dioxin determinations were published several years ago (van Babel et al. 1996; van der

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

Velde et al. 1996), but currently the focus has been on environmental samples such as soils and sediments (Miyawaki et al. 2008). Although SFE extraction is automated and offers a short extraction time and minimal use of organic solvents with no additional cleanup step before GC-MS, it is not widely used because of the large number of parameters that have to be optimized, especially in the analyte collection chamber, and the high cost of the equipment. Like SPE, the use of SFE for dioxin analysis is not common due to the large amounts of sample required (5–10 g of lipid equivalents) to be able to reach the low levels at which dioxins are present in food samples.

Pressurized liquid extraction One of the most recent solid-sample extraction methods used as an alternative to classic extraction techniques such as SOX and MSPD is PLE or accelerated solvent extraction (ASE). Unlike SFE, an organic solvent replaces CO2, and increased pressures and temperatures are used to speed up the extraction procedure. PLE uses conventional liquid solvents at high pressures (1500 psi) and temperatures (100°–120°C) to extract solid samples quickly and with much less solvent than conventional techniques. Food samples are placed in extraction cells that are filled with an extraction solvent and then heated. The sample is statically extracted for 5–10 min, with the expanding solvent vented to a collection vial. Following this period, compressed nitrogen is used to purge the remaining solvent into the same vial. The entire procedure is completed in 15–30 min per sample and uses only 40–200 mL of solvent. The main disadvantage of PLE (or ASE) is that sample cleanup is still necessary. For this reason, applications dealing with selective extraction procedures, where integrated cleanup strategies are used to combine extraction and cleanup or fractionation to further simplify all the sample-preparation

57

steps, are of special interest. The first online cleanup attempt was performed for the extraction of PCBs from foodstuffs by using acidic alumina in the extraction cell (Dionex Application Note 322 1996). Other fat retainers, for example, basic alumina, neutral alumina, acidic alumina, Florisil, and sulfuric acid–impregnated silica, also were tested for PCB extraction (Björklund et al. 2001). Given the good results obtained, this approach was also applied to the extraction of dioxins. Bernsmann and Fürst (2004) first described the extraction of PCDD/PCDFs and PCBs from feeding stuffs using sulfuric acid– impregnated silica as a fat retainer with very good results. In this study, the integrated cleanup approach was found to be equivalent to SOX extraction. As part of the DIFFERENCE project, Wiberg and coworkers (2007) evaluated traditional extraction techniques versus alternative techniques such as PLE (or ASE) for PCDD/PCDF and dioxin-like PCB determinations in food and feed, including certified reference materials. They demonstrated that PLE is more of a quantitative extraction process than other conventional techniques. The PLE method, in combination with HRMS detection, meets the quality criteria for official control of dioxins in foodstuffs (Commission Regulation 1883/2006/EC). One of the last developments is the combination of PLE with integrated carbon fractionation (Nording et al. 2005), in which dioxins can be fractionated and obtained in backward elution, and only a small, miniaturized, multilayer silica column cleanup is required after PLE and before instrumental determination. Some attempts have also been made to combine PLE with automated cleanup systems, in particular the Power-Prep system (which is discussed below in the section on cleanup), to construct a fully automated system (extraction plus cleanup). However, the results have not been satisfactory; in this type of combination, PLE is used as a dynamic system and requires the incorporation of a

58

Analysis of Endocrine Disrupting Compounds in Food

concentration phase prior to Power-Prep, rendering automation virtually impossible and considerably increasing the analysis times (Focant et al. 2005a). At present, the PLE system is working in static mode, but the HPLC system (pump and valves) is located in an individual module separated from the low-pressure module and can operate at pressures up to 2500 psi (Focant et al. 2005a). More extensive information about this technique can be found in the literature, where there are some reviews dealing with PLE for POPs in foods (Björklund et al. 2006; Wiberg et al. 2007) and biological matrices (Focant et al. 2004). Tables 3.2 and 3.3 show some examples in which PLE has been used as a routine method in the analysis of dioxins in foods with and without the use of a fat retainer in the cell extraction.

Microwave-assisted solvent extraction MASE has been only recently introduced for the analysis of dioxins. To our knowledge, there are no published studies in which MASE was used for dioxin food extraction. In recent years, this technique has attracted growing interest because it allows rapid extraction of solutes from solid samples by employing microwave energy as a source of heat, with extraction efficiency comparable to that of classic techniques. The partitioning of the analytes from the sample matrix to the later extractant depends on the temperature and the nature of the extractant. Unlike conventional systems, microwaves heat the entire sample simultaneously without heating the vessel; thus, the solution reaches its boiling point very rapidly and the extraction time is very short (Camel 2000). In view of the good results of MASE in the extraction of PCBs and DDTs (Vetter et al. 1998; de Boer 1988) from biological tissues and dioxins from environmental samples (Eljarrat et al. 1998; Miyawaki et al. 2009), this technique is very attractive for dioxin analysis in food samples. Its main drawbacks are the loss of more vola-

tile solutes if the temperature rises rapidly and the need for the solution to be cooled to room temperature after extraction before the vessels can be opened, which increases the overall extraction time. In addition, it is not possible to automate the procedure to incorporate cleanup steps.

Comparison of extraction techniques Table 3.3 shows the experimental conditions and benefits of the extraction methods currently used in most literature. The data presented in Table 3.3 show that PLE (or ASE) with a fat retainer in the extraction chamber compares favorably with conventional classical extraction techniques for dioxin analysis in foodstuffs. The PLE extraction technique drastically reduces the amount of solvent used and the analysis time, mainly because extraction and primary cleanup could be automatically performed in one step within 90 min. Other advantages of this method are the possibility of working with six or more extractions in parallel and, in the future, the online automation including cleanup steps.

Cleanup methods Analytical procedures for the determination of PCDD/PCDFs in food samples involve sophisticated and tedious cleanup methods. Several steps are usually required to remove the bulk of coextractants (including lipids) in order to finish with an extract containing only PCDD/PCDFs in which the analytes can be detected at the ultratrace levels at which they occur in food samples. The choice of a particular sequence of steps will depend very much on the analytical system that is used. Sample extraction, cleanup, and GC method together form a delicately balanced combination, each part contributing to the ultimate specificity and selectivity. For the determination of dioxins, nearly all established schemes involve combinations of cleanup methods originally developed for the analysis of PCBs

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

59

and organochlorinated pesticides (OCPs) (solid–liquid adsorption chromatography using Florisil, silica and alumina, gelpermeation chromatography [GPC], and HPLC) in combination with an active carbon step to isolate the specific fraction containing the dioxins without chemical interference (Table 3.2). In many of the methods used today, the sample extraction and cleanup steps are combined online or at-line, and some are automated.

used for dioxin and other POP analysis in a large variety of fatty food samples such as oils and butters (Strandberg et al. 1998, Hess and Wells 2001; Grochowalski and Wojtalewicz 2005; Gómara et al. 2006). Although it is efficient, simple, versatile, and does not entail excessive solvent use, this procedure is not often used for dioxin analysis because it is time consuming (72 h). In addition, online coupling, either with extraction or with cleanup steps, is not possible.

Lipid removal

Isolation of uncommon chemical interferences

Lipid removal is the first step in the cleanup process, where usually other interference is also eliminated. Several different methods have been used to remove lipids and similar compounds in POP food analysis (Table 3.2), including destructive methods (e.g., sulfuric acid, silica modified with sulfuric acid, Florisil, and alumina) and nondestructive methods (e.g., GPC and dialysis). GPC, which is sometimes referred to as sizeexclusion chromatography (SEC), has been successfully applied to food samples for POP analysis using SX-3 Biobeds (200–400 mesh) in a range of column sizes and solvents. It can be fully automated and, unlike adsorption chromatography, it is also more suitable for the isolation of unknown contaminants on whose polarity or chemical functionality there is little information. The method can also handle a large mass of lipid in each sample (e.g., columns of about 500 × 25 mm ID can handle up to 500 mg of lipids) compared to adsorption columns that are limited to 50 mg of lipids per gram of adsorbent (Ahmed 2001; de Boer and Lau 2003; Hoh et al. 2008). Dialysis with semipermeable membranes (SPMs) in an organic solvent can separate other similar POPs from lipids. The method can eliminate more than 20 g of lipids in a single membrane with acceptable recoveries of internal standards, practically irrespective of the amount and type of lipid dialyzed. The method has been successfully

For dioxin analysis in a food matrix, an additional purification step is necessary to eliminate other interference (including any other lipids). A combination of adsorbents (neutral, basic and acid alumina, silica gel, modified silica gel with acids and basics, and AgNO3, C18, carbon, and Florisil, in multilayer or onelayer columns) and solvents with different polarities and dielectric constants are used to eliminate interference (Table 3.2). It is wellknown to experts that the application of the extract to a strongly basic adsorbent (potassium or cesium hydroxides) silica gel with a low-polarity solvent, for example, hexane, is very effective for removing trace residues of acidic compounds such as phenolic and carboxylic acids and sulfonamide compounds (Smith et al. 1984). On the other hand, sulfuric acid–impregnated silica gel (20–40% w/w) is very effective in removing numerous types of compounds by dehydration, acidcatalyzed condensation, and oxidation reactions (Lamparski et al. 1979). Alumina (basic, acid, and neutral), silica gel modified with AgNO3, and Florisil are used at different activation grades, mainly to eliminate all other lipids and other coextractants (Ramos et al. 1997; Liu et al. 2006). The literature gives no indication of preferences for any specific adsorbent or solvent, the choice of which depends more on the laboratory’s preferences than on performance. Cleaning up food

60

Analysis of Endocrine Disrupting Compounds in Food

samples for dioxin analysis is a laborious and tedious task, which has to be validated. The combination of adsorbents and solvents chosen to obtain a clean extract without any dioxin loss before the GC-MS analysis is up to each laboratory.

Fractionation/group separation Normally, a group separation is necessary before final analysis of dioxins by GC-HRMS. At this stage, the cleaned extract may contain other similar organohalogen compounds such as PCBs. With the exception of non-ortho PCBs, dioxins are present at substantially lower concentrations than the other POPs, and it is therefore necessary to separate dioxins from the bulk of POPs. The methods available for the isolation of POPs into separate fractions prior to GC analysis are based on the spatial planarity of dioxins, which are used to separate them as a distinct fraction. The available methods for fractionation have been extensively reviewed (Hess et al. 1995; Concejero et al. 2001). Open liquid chromatography columns of Florisil, alumina, active carbon, and graphitic carbon are among the most widely used methods (Table 3.2). HPLC with either porous graphitic carbon (PGC) (Creaser and Al-Haddad 1989; de Boer et al. 1993), active carbon (Lundgren et al. 2002), or 2-(1-pyrenyl)ethyldimethylsilylated silica gel (PYE) columns (Ramos et al. 1999; Hess et al. 1995; Díaz-Ferrero et al. 2005) do not have an extended use despite the inherent advantages of HPLC. Concejero and coworkers (2001) studied the feasibility of different adsorbents. Two active carbons (Amoco PX-21 and Carbosphere), two graphitic carbons (Carbopack B and C), and one HPLC stationary phase, PYE (typically used for PCB and PCDD/ PCDF fractionation), were studied for fractionation of PCBs and PCDD/PCDFs. Recoveries for fractionation of the target compounds with all the sorbents studied were generally good, and the reproducibility was

satisfactory. All were able to isolate PCDD/ PCDFs from PCBs, which could interfere in the final determination of the PCDD/PCDFs by GC-HRMS. As a result, Carbopack B (as SPE cartridges) and PYE (as HPLC column) were considered the most valuable alternatives for simultaneous fractionation of PCDD/ PCDFs and the different classes of PCBs typically investigated in environmental studies. An additional merit of this HPLC stationary phase is possible automation. Table 3.2 shows some examples in which different adsorbents were used in the fractionation step of cleanup methodology. It is worth noting that immunoaffinity chromatography (IAC) using monoclonal and polyclonal antibodies specifically developed to recognize 2,3,7,8-PCDD/PCDFs was considered a very attractive technique in the 1990s. Because of the good results achieved in the cleanup of aqueous samples (water and blood) for dioxin analysis (Concejero et al. 2004), it was initially thought to be very promising. However, the need for using fatfree extracts, the variability of the results, and the presence of cross-reactions rendered this alternative cleanup process without further applications.

Automation of extraction and cleanup In view of the extreme difficulty and tediousness of the extraction and cleanup process in food dioxin analysis, there have been many attempts at automation, but so far no one has come up with an automated procedure for simultaneous extraction and cleanup. Smith and coworkers (1984) made the first attempt at a semiautomated at-line extraction/ cleanup procedure. They developed a method for dioxin analysis of biological tissues (including fish) in two steps. In the first step, the extraction and a first cleanup step using active carbon were performed simultaneously. In the second step, the extract was applied to a second series of adsorbents

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

contained in two tandem columns. Based on this general scheme, in 1997 a semiautomated method for online extraction plus cleanup and fractionation of PCBs and PCDD/PCDFs was developed (Krokos et al. 1997). Up to 6 g of fat could be extracted with this method, which is very useful in the case of fatty food samples, as demonstrated by several studies (Jiménez et al. 1996; Serrano et al. 2000; Santillo et al. 2003). Nevertheless, despite its good performance, the method has not been widely used, as no apparatus is available commercially. The efficiency of the automated PowerPrep system in purifying sample extracts for dioxin analyses has already been demonstrated in recent years for different types of matrices, including food (Eljarrat 2001; Pirard et al. 2002). The multistep procedure is based on the use of disposable multilayer silica columns, basic alumina, and PX-21 carbon columns, which can be combined to suit the target analytes. This means that dioxins and non-ortho PCBs can be isolated in a fraction with good recoveries, and several samples can be analyzed in parallel, even those with high fat contents (Focant et al. 2001). Table 3.2 shows that the Power-Prep system is today used as a routine cleanup method for dioxin food analysis. This method has become increasingly popular over the years. As the only commercially available apparatus, it is gradually making its way into all laboratories that perform large numbers of analyses. Some efforts have been made to couple an online PLE (or ASE) extraction step, but for the moment, no satisfactory results have been achieved (Focant et al. 2004). Although laboratories can choose the method that best suits them for extraction plus cleanup of dioxins in foodstuffs, there is no doubt that PLE (or ASE) as an extraction method and the Power-Prep system for cleanup, both commercial products, are the only methods that, although not fully automated, permit large numbers of samples to be

61

analyzed in the shortest possible time. With this combination, it is possible to handle 10 samples at once in both the extraction and the purification steps and to deal with any food health emergency due to dioxin contamination of foodstuffs.

Instrumental determination As noted above, the choice of analytical procedure for extraction plus cleanup of the seventeen 2,3,7,8-PCDD/PCDF congeners is up to each laboratory, provided that the analyte recoveries are within the range laid down by the EU directive (Commission Regulation 1883/2006/EC). However, in the case of the instrumental determination, the EU directive requires the use of GC-HRMS, which was the only method able to reliably determine dioxins at levels appropriate for food analysis. Some other methods, such as DR CALUX bioassay and GC coupled to ion trap MS working in tandem mode, or GC-MS/MS(ITD), are accepted officially only for screening purposes. The most important specific requirements are recovery control by the addition of 13C12PCDD/PCDFs as standard. The recoveries of the individual internal standards should be between 50% and 130%. The GC separation of the isomers should be 0.998).

UV-Vis

Figures of Merit and Remarks LODs: 0.5–5 μg/kg Recoveries: 45–104% with RSDs: 0.905). Recoveries of the analytes ranged from 60.7 to 136.7%. Repeatability RSD: 3.0– 30.8%. LOQ: 0.0099 (for hexachlorobenzene) μg/mL and 2.45 μg /mL for propargite Linearity: r > 0.995. Recoveries: 83–92% RSDs: 4–8% LOQs: 0.8–2.2 μg/kg (NPD) LOQs: 0.1–0.7 μg/kg (MS)

References

Figures of Merit and Remarks LODs: 0.19 μg/L in red wine after SPE

138

Grape, pear, apple, lemon

QuEChERS

LLE-SPE: Oasis HLB SPE cartridge

Atrazine, chlorsulfuron, diuron, isoproturon, haloxyfop-methyl

Multiresidue (24-herbicides)

SLE

Chlorpropham

SLE

Atrazine, bromozynil, chlorotoluron, diuron, flufenacet, pendimethalin, simazine

Frozen and canned vegetables, canned fruit, and ready-toeat salads: tomato, peas, lettuce, apple puree Lemon and raisin

—

Alachlor, atrazine, dinoseb, isoproturon, metolachlor, metolcarb, simazine

Apple, cranberry, grape, and orange juices as well as fruit peel and salad leaves Strawberry

Extraction Technique SPE: HLB cartridges (200 mg) from Oasis

Compounds Multiresidue (24-herbicides)

Matrix Fruit-based soft drinks

LC: Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm I.D., 5 μm particle size)

TOF-MS: EI-positive ion mode MS-MS: hybrid triple quadrupole/ linear IT

MS-MS: orthogonal Z-Spray-ESI

Linearity: 10–500 μg/L. Recoveries: 71–108%. (except Haloxyfop-methyl in lemon 45–51%) RSDs: 0.9925 Absolute recoveries: 3.4–9.6%. LODs: 14–95 μg /L (FID). LODs: 10–90 μg /kg (onion and maize) LODs: 30–230 μg /kg (rice). Recoveries: 85–96.7%. RSDs: 5.56–10.8%.

(continued)

[112]

[111]

Recovery averaged: atrazine: 90.4% with an RSD of 3.3% Others >73.4%.

Linearity: r2 > 0.99 Recoveries: ECD: 73–101% with RSDs: 4–15%; TSD: 86–120% with RSDs: 3–20%.

[109]

Linearity: 1–1000 μg/kg (QqQ), 20–5000 μg/kg (QIT) and 70–7000 μg/kg (QqTOF). Repeatabilities (RSDs): QqQ (5–12%); QIT (6–15%); QqTOF (14–19%) Recoveries 88–92% (PLE), 64–77% (LLE) LODs: 1 μg /kg (QqQ), 20 μg / kg (QIT), 70 (QTOF). LOD: 3 μg/L Compared with ELISA

[110]

References

Figures of Merit and Remarks

140 SLE LLE-SPE

Atrazine, simazine, prometryn Atrazine, simazine, prometryn

Atrazine, diuron, simazine, terbuthylazine. Atrazine, diuron, simazine, trebuthylazine

Fresh and processed olives Olive oil

Olive oil and olives

Olive oil

LLE-MSPD

GPC

—

Diclofop-acid and diclofop-methyl

Herbicides

Extraction Technique SFE

Compounds Paraquat and diquat

Matrix Olive oil

LC and LCxLC: System A: C18-Luna column (150 × 4.60 mm, 5 μm). System B: ChiralAGP column (100 × 30 mm, 5 μm). GC: Trx-1701 column (30 m × 0.25 mm, df film thickness0.25 μm). GC-NPD: Zebron ZB-1 (30 m × 0.32 mm I.D., df film thickness 1 μm) GC-ECD: Zebron ZB-5 (30 m × 0.25 mm I.D., df film thickness 0.25 μm). GC-MS/MS: CP-SIL 5CB (30 m × 0.25 mm I.D., df film thickness 0.25 μm). LC-TOF-MS: Zorbax Eclipse XDB-C8 (150 mm × 4.6 mm, 5 μm particle size)

Separation Technique HPLC: μBondapak C18 (300 mm × 3.9 mm I.D., 10 μm particle size)

TOF-MS

IT-MS-MS: EI mode

NPD, ECD

NPD

UV-Vis

Detection Technique DAD: confirmation method (HPLC) Screening method: amperometric

Recoveries: 82–136, RSDs: 5–11%, LODs: 0.1–0.5 μg/ kg. LOQs: 0.2–5.0 μg/kg. Linearity: Matrix matched std. Range: 5–500 μg/kg (using extracted ion chromatograms ([XIC]) (r > 0.99). Matrix effects: 14–36% matrix induced suppression. LODs: 1–5 μg/ kg. RSD 80% in water samples. LODs ranged 20–70 ng/L (HPLC-UV) for tap water; 30–100 ng/L for surface water Linearity: r2 > 0.99; RSD