Methods of Analysis of Food Components and Additives Chemical and Functional Properties of Food Components Series SERIES EDITOR Zdzislaw E Sikorski Chemical and Functional Properties of Food Proteins Edited by Zdzislaw E Sikorski Chemical and Functional Properties of Food Components, Second Edition Edited by Zdzislaw E Sikorski Chemical and Functional Properties of Food Lipids Edited by Zdzislaw E Sikorski and Anna Kolakowska Chemical and Functional Properties of Food Saccharides Edited by Piotr Tomasik Toxins in Food Edited by Waldemar M Dabrowski and Zdzislaw E Sikorski Methods of Analysis of Food Components and Additives Edited by Semih Ötles, Methods of Analysis of Food Components and Additives EDITED BY Semih Ötles, Ege University Department of Food Engineering Izmir, Turkey Boca Raton London New York Singapore A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc Published in 2005 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2005 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S Government works Printed in the United States of America on acid-free paper 10 International Standard Book Number-10: 0-8493-1647-2 (Hardcover) International Standard Book Number-13: 978-0-8493-1647-0 (Hardcover) This book contains information obtained from authentic and highly regarded sources Reprinted material is quoted with permission, and sources are indicated A wide variety of references are listed Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including 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T&F Informa plc and the CRC Press Web site at http://www.crcpress.com Preface The ability to accurately separate, identify, and analyze nutrients, additives, and toxicological compounds found in food and food products has become critically important in recent decades, as knowledge of and interest in the relationships between diet and health have increased This requires training students and analysts in the proper application of the best methods, as well as improving, developing, or adapting existing methods to meet specific analytic needs This book aids the analyst by providing a valuable reference to both newly developed and established methods of analysis of food components and additives The book comprises 16 chapters, which take the reader through brief and accessible descriptions of methods of analysis of food components and additives Ranging from chemical analysis of food components and additives to infrared (IR), nuclear magnetic resonance (NMR), Fourier transform Raman (FTR), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), and more The book provides first-hand explanations of modern methods, contributed by 24 leading scientists, many of whom actually developed or refined the techniques, and presents new documented information on standard methods of analysis of food components and additives in a uniform format and in a style that can be understood by a reader who is not familiar with the analysis of each component Each chapter is structured to provide a description of the information about the component or additive that can be analyzed, a simple method explanation of how it works, examples of applications, and references for more detailed information This format also facilitates comparison of methods of analysis of each component The use of different authors to cover a broad spectrum of methods resulted in some differences of style, but overall the book achieves its goal The first chapter, “Selection of Techniques Used in Food Analysis,” covers topics relevant to all techniques, including sample preparation, quantitative measurements, and information management, and concentrates on what goals can be achieved by applying different techniques for various purposes in food analysis The second chapter, “Statistical Assessment of Results of Food Analysis,” provides an overview of the need for statistical assessment of the results of food analysis and the evaluation of most suitable methods for different situations at a level that is more complete than those found in most introductory analysis textbooks The remaining 14 chapters address the major areas of analysis of food components and additives: analysis of drinking waters, proteins, peptides, amino acids, carbohydrates, food lipids, metals and trace elements in foods, vitamins, carotenoids, chlorophylls, food polyphenols, aroma compounds, food volatiles, sensory analysis of foods and determination of food allergens, genetically modified components, pesticide residues, pollutants in foods, chemical preservatives in foods, radioactive contaminants in foods, and rapid analysis techniques in food microbiology In most chapters, many examples of applications of methods to analytical problems are provided The references provided in these chapters can be highly useful and valuable for those seeking additional information This comprehensive book should serve as a reference for scientists, analytical chemists, engineers, researchers, food manufacturers, personnel from government agencies, standards writing bodies, students majoring in various science disciplines (biology, biochemistry, chemistry, environmental science, engineering, and food chemistry, to name a few) interested in obtaining a stronger background in analysis, and all those involved in the analysis of both food components and food additives The Editor A native of Izmir, Turkey, Semih Ötles¸ obtained a B.Sc degree from the Department of Food Engineering (Ege University) in 1980 During his assistantship at Ege University in 1985, he received an M.S in food chemistry, and in 1989, after completing his thesis research on the instrumental analysis and chemistry of vitamins in foods, he earned a Ph.D in food chemistry from Ege University In 1991–92, he completed postdoctoral training, including an OECD postdoctoral fellowship, at the Research Center Melle at Ghent University, Belgium Afterward, Dr Ötles¸ joined the Department of Food Engineering at Ege University as a scientist of food chemistry, being promoted to associate professor in 1993 and to professor in 2000 During 1996–1998 he was deputy director at the Ege Vocational School of Higher Studies Since 2003 he has been vice dean of the engineering faculty, Ege University The research activities of Professor Ötles¸ have been focused on instrumental analysis of food compounds: he began a series of projects on the separation and analysis techniques of high-performance liquid chromatography (HPLC), first for analysis of vitamins in foods, then proteins and carbohydrates, and, most recently, carotenoids Other activities span the fields of GC, GC/MS analysis, soy chemistry, aromatics, medical and functional foods and nutraceutical chemistry; included are multiresidue analysis of various foods, and n-3 fatty acids in fish oils Professor Ötles¸ is the author or coauthor of more than 150 publications (technical papers, book chapters, and books) and a presenter of seminars He is a member of several scientific societies, associations, and organizations, including the Asian Pacific Organization for Cancer Prevention (APOCP) and the International Society of Food Physicists (ISFP) He is a member of the steering committee of APOCP’s local scientific bureau and is the Turkish representative of ISFP, and has organized international congresses on diet/cancer and food physics He is a member of editorial advisory boards for Asian Pacific Journal of Cancer Prevention; Food Science & Technology Abstracts of IFIS (International Food Information Service); Current Topics in Nutraceutical Research; Electronic Journals of Environmental, Agricultural and Food Chemistry; Newsline; Journal of Oil, Soap, Cosmetics; Trends World Food; Trends Food Science & Technology; Pakistani Journal of Nutrition; Journal of Food Technology; Academic Food; and Australian Journal of Science & Technology He is referee/reviewer for AOAC International, Journal of Experimental Marine Biology and Ecology, Journal of Medical Foods, die Nahrung, Journal of Alternative & Complementary Medicine, The Analyst, and Journal of Agricultural and Food Chemistry Acknowledgments Permission to reprint the following is gratefully acknowledged: Table 4.1: Kolakowski, E., Protein determination and analysis in food systems, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing, Lancaster/Basel, chap 4, pp 57–112, 2001 Figure 11.3: Orlandi, P.A et al., Analysis of flour and food samples for Cry9C from bioengineered corn, J Food Prot., 65, 426, 2002 Figure 11.4: Raybourne, R.B et al., Development and use of an ELISA test to detect IgE antibody to Cry9c following exposure to bioengineered corn, Int Arch Allergy Immunol., 132(4), 322, 2003 Contributors Aldert A Bergwerff Utrecht University Utrecht, The Netherlands Marek Biziuk Gdansk University of Technology Gdansk, Poland Richard Brereton University of Bristol Bristol, United Kingdom Stephen G Capar U.S Food and Drug Administration College Park, Maryland Francisco Diez-Gonzalez University of Minnesota St Paul, Minnesota Douglas G Hayward U.S Food and Drug Administration College Park, Maryland Yildiz Karaibrahimoglu U.S Department of Agriculture Wyndmoor, Pennsylvania Jae Hwan Lee Department of Food Science and Technology Seoul National University of Technology Seoul, Korea Steven J Lehotay U.S Department of Agriculture Wyndmoor, Pennsylvania Dan Levy U.S Food and Drug Administration Laurel, Maryland Kannapon Lopetcharat Unilever Corporation Edgewater, New Jersey Katerina Mastovska U.S Department of Agriculture Wyndmoor, Pennsylvania Mina McDaniel Oregon State University Corvallis, Oregon Edward Kolakowski Agricultural University of Szczecin Szczecin, Poland Malgorzata Michalska Institute of Maritime and Tropical Medicine Gdynia, Poland Keith A Lampel U.S Food and Drug Administration Laurel, Maryland Robert A Moreau U.S Department of Agriculture Wyndmoor, Pennsylvania 424 16.6.2 Methods of Analysis of Food Components and Additives MOLECULAR PROBES A molecular probe (MP) is a single-stranded oligonucleotide or small nucleic acid molecule that has been designed to be capable of specifically binding to a complementary DNA or RNA sequence and to trigger a detection signal as a result of this hybridization Typical MPs have between 15 and 30 base pairs, but in DNA hybridization, probes as large as 1.8 kb have been used.40 In 1983, Fitts et al.41 designed one of the first DNA hybridization methods to detect Salmonella in foods by using a radiolabeled probe obtained from a random chromosomal fragment with a sensitivity of approximately 106 cells per milliliter Radiolabeled probes were limited for widespread application, but the application of alternative labels and a variety of sensing systems allowed the active development and marketing of molecular probebased techniques In general, there are two major types of MP protocols: those that detect target nucleic acid bound to a membrane and those that use immobilized probes.11 The first group of molecular probe assays originated from the pioneering development of Southern blotting by E M Southern,42 who first employed membranes to immobilize DNA In this type of method, bacterial colonies or liquid culture are initially transferred to nylon or nitrocellulose membranes where they are lyzed and their DNA released The free DNA can be fixed onto the membranes by high temperature or UV light36 and then hybridized with specific probes that had been previously labeled with typically biotin, fluorescin, or digoxigenin The hybrid immobilized DNA can be visualized using avidin or antibody conjugates for the probe label that might be linked to enzymes, fluorescine, gold, or rhodamine The DNA hybridization assays that use immobilized probes are typically based on microtiter plates and also require the release of nucleic acids from a colony or a pure culture The microbial nucleic acid hybridizes with the capture probe, and these assays have frequently been designed to include a reporter probe that binds to the captured DNA and can be detected based on its specific label Among the many MP assays that have been reported in the literature, there are only two formats that have been successfully commercialized since the early 1990s The first of these systems is the GENE-TRAK® assay currently marketed by Neogen Corp (Lansing, MI) The first GENE-TRAK technique used random chromosomal radiolabeled DNA probes, and the second generation used a combination of three probes that captured rRNA fragments detected by an anti-fluorescine antibody–enzyme conjugate.39–41 The current GENE-TRAK format uses three types of DNA probes: a polydeoxythimidylic acid fragment attached to a dipstick or to a microtiter well, a probe containing a polydexydenylic acid at one extreme and a sequence specific for a region of rRNA, and another rRNA-specific probe covalently bound to fluorescin or directly to horseradish peroxidase.39,43 The detection of this “sandwich” complex can be achieved by light emission in a luminol-mediated reaction catalyzed by an antibody–horseradish peroxidase conjugate Specific GENE-TRAK kits exist for the analysis of E coli, Salmonella, Listeria, Campylobacter, and Staphylococcus aureus.3 The second commercially available nucleic acid hybridization kit is the AccuProbe® distributed by Gene-Probe, Inc (Salem, MA) Similar to GENE-TRAK, it Rapid Analysis Techniques in Food Microbiology 425 also targets rRNA sequences, but it does not involve immobilization In the AccuProbe assay, chemioluminescently labeled probes are subjected to differential hydrolysis after mixing with target DNA, and the protected double-stranded hybrids can be visualized by light emission.3 Accuprobe protocols have been developed for the detection of Listeria, Campylobacter, and Staphylococcus aureus.3,43 A number of researchers have compared the specificity and sensitivity of Accuprobe and GENE-TRAK assays for Campylobacter and Listeria, and for the most part the performance of both protocols is quite similar.44,45 Both assays have demonstrated very high sensitivity, but it is limited to approximately 106 CFU per assay.3,39 Because of this limitation, these commercial probe kits are mostly well used for a rapid identification of isolates, rather than for rapid detection Another nucleic acid method that is not normally classified among the molecular probe-based methods is ribotyping.33 Ribotyping is the first rapid automated DNAbased fingerprinting technique developed by Qualicon, Inc (Wilmington, DE) and commercialized as the RiboPrinter® microbial characterization system Ribotyping has been quite useful in the food industry to quickly identify isolates The RiboPrinter is capable of performing DNA restriction digestion, electrophoresis, and membrane hybridization automatically The core of the ribotyping is the use of specific probes designed against ribosomal RNA genes, which hybridizes to the DNA digestion fragments after these have been separated in a gel The DNA fragment bands can be visualized by means of a chemioluminescent agent Despite the automation capabilities, ribotyping has not been capable of replacing other more laborious DNA fingerprinting methods used in epidemiology, such as pulsed field gel electrophoresis (PFGE) because of their superior strain discriminatory power 16.6.3 16.6.3.1 POLYMERASE CHAIN REACTION (PCR) PCR Principles The polymerase chain reaction is an in vitro DNA amplification method that revolutionized every life sciences field and has become a routine procedure in most biology laboratories PCR was invented by Kary Mullins in 1983 by taking advantage of the unique properties of DNA replication catalyzed by DNA polymerase.46 The development of PCR was due to the ability of DNA probes to bind target DNA serving as primers for the enzymatic replication of DNA In contrast to the MP technology described above, PCR requires the use of pairs of primers specific for flanking regions of each of the two DNA strands in the target DNA The steps involved in PCR are the following: (1) denaturation, (2) primer hybridization (annealing), and (3) extension (replication).47 The separation of DNA strands (denaturation) is achieved by heating at temperatures greater than 94ºC for a few minutes, and the annealing of primers to complementary sequences in the target DNA occurs after cooling the reaction to less than 65ºC Once the primers bind, DNA replication catalyzed by a DNA polymerase proceeds in the 5′ to 3′ direction for a few minutes After the extension step, the cycle is repeated as many times as needed (typically from 15 to 40 cycles) Theoretically, each cycle duplicates the target DNA once, and the number of molecules increases exponentially with additional cycles 426 Methods of Analysis of Food Components and Additives The first PCR protocols utilized DNA polymerase that had been purified from E coli, but fresh enzyme had to be added to the reaction after each cycle because of its inactivation by denaturating temperatures.46 The discovery that allowed the development of current PCR formats was the use of thermostable polymerases The first of this type of enzymes was obtained from Thermus aquaticus bacteria and is known as Taq polymerase A wide variety of other thermostable polymerases have been successfully marketed The development of thermocycler devices also allowed the rapid application of PCR to almost any imaginable field.47 16.6.3.2 Traditional PCR PCR was first enthusiastically adopted for research and an extensive number of articles have reported the utilization of PCR for detection of food-borne pathogens.35,48 Once the reaction conditions are optimized, PCR protocols can easily be adapted as routine tests Traditional PCR is typically divided into three major stages: (1) DNA amplification stage, (2) separation of PCR products, and (3) detection of products The amplification stage involves all the reaction steps described above performed in a thermocycler Separation of produced DNA fragments is typically conducted by agarose gel electrophoresis, and the detection of PCR products is achieved by visualization of bands onto gels under UV light after staining with ethidium bromide Although traditional PCR has been a very powerful technique for research, its adoption for large-scale industrial analysis of foods was hampered by its relatively high sophistication and its labor intensiveness Suppliers of equipment and PCR reagents designed ready-to-use reaction mixtures such as those commercialized with the TaqMan® brand by Applied Biosystems, Inc (Foster City, CA) Commercial formats have also been able to simplify traditional PCR by automated electrophoresis and the use of hybridization probes.43 The application of PCR for the rapid microbiological analysis of foods has also been limited because of interferences caused by food components that can lead to weak responses and false negatives.3 A wide variety of substances can affect annealing; proteins and fats may inhibit polymerization; and proteinases that can inactivate polymerases are frequently found in food samples.49 In addition to these shortcomings, traditional PCR cannot discriminate between dead and live bacteria Despite the usefulness of PCR in microbiological analysis in research, commercial assays did not become available until the late 1990s.50 The BAX® System from Qualicon, Inc (Wilmington, DE), the Probelia® system from Sanofi Diagnostics Pasteur (Paris, France), and the TaqMan were the first PCR assays marketed for the microbiological analysis of foods.43 The first BAX assays were designed for the detection of Salmonella, Listeria, L monocytogenes and E coli O157:H7 and were based on a semi-automated system that included a thermocycler and prestained gels Probelia formats have been available for detection of Salmonella, Campylobacter, L monocytogenes, and E coli O157:H7, and they relied on a semiautomated thermocycler in which the detection was done by spectrophotometric detection of hybridization of PCR products with reporter probes Initial TaqMan assays included Rapid Analysis Techniques in Food Microbiology 427 protocols for Salmonella and E coli O157:H7, but the detection of PCR products was achieved by a fluorescence system TaqMan took advantage of the 5' → 3' exonuclease activity of Taq polymerase by including specific DNA probes that bind the target DNA downstream of the replication direction These probes have a set of fluorescent reporter and quencher dyes attached, and the fluorescent reporter dye is released as a result of the probe hydrolysis As the PCR reaction proceeds, the amount of free reporter dye increases and the fluorescence increases 16.6.3.3 Real-Time PCR The term “real-time PCR” has been applied to those PCR protocols in which the detection of the amplification products is achieved as they are synthesized The first online measurements of PCR products were conducted using ethidium bromide and UV light with the purpose of measuring PCR reaction kinetics in the early 1990s.43 Since that time several technologies have been developed and now real-time PCR has become a very powerful and popular technique for the detection and identification of bacteria in foods The core of real-time PCR resides in the use of a fluorescent dye system included in the reaction and a thermocycler equipped with fluorescencedetection capability Among the six existing fluorescent systems, the SYBR Green I dye is the only format that uses a nonspecific DNA labeling compound The other five detection protocols use DNA probes and are based on the principle of fluorescence resonance energy transfer (FRET).43 The SYBR Green I dye is capable of binding the minor groove of doublestranded DNA molecules and only fluoresces when attached to DNA As DNA is synthesized, the fluorescent signal increases with SYBR Green I The FRET phenomenon is defined as the ability of certain chemical compounds or moieties to absorb and/or emit the energy produced by an excited fluorophore when the distance between these compounds is approximately 70 to 100 Å.43 In most FRET-based realtime PCR systems, the quencher and the fluorophore are typically incorporated into one primer or probe that does not fluoresce alone, but when the reaction takes place and the target gene is amplified, both moieties are separated and fluorescence is observed To date there are at least five different FRET systems: TaqMan, molecular beacons, Scorpions probes, Amplifluor® probes (Talron Scientific & Medical Products Ltd, Rehovot, Israel) and LightCycler® hybridization probes (Roche Diagnostics, Corp., Basel, Switzerland) (Figure 16.2).51 As described above, the TaqMan system separates the fluorescent dye from the quencher by nuclease hydrolysis Molecular beacons are DNA oligonucleotides designed with complementary sequences at their ends such that they form a hairpin structure with the quencher and the fluorophore in close proximity When the noncomplementary part of the probe binds to the target DNA, the FRET pair is separated and signal is emitted Similar to the molecular beacons, Scorpions and Amplifluor probes employ hairpinforming nucleotides The LightCycler hybridization probes are the only FRET format in which fluorescence is produced when two dyes present in separate probes get in close proximity after specifically binding to the target DNA (Figure 16.2) 428 Methods of Analysis of Food Components and Additives KEY: = double stranded DNA; = fluorophore; SYBR GREEN I = quencher; = target gene; = exciter moiety; TAQMAN =fluorescence; = primer/probe; = excitation light HYBRIDIZATION MOLECULAR PROBES BEACONS FIGURE 16.2 Comparison of four real-time PCR reporter systems: SYBR Green I, TaqMan, hybridization probes, and molecular beacons The commercially available real-time PCR systems for the detection of foodborne pathogens are still only TaqMan, BAX, and Probelia brands.43 TaqMan utilizes its own proprietary probe-hydrolysis technology while BAX assay now utilizes the SYBR Green I dye to obtain real-time measurements The Probelia format licensed to Bio-Rad Laboratories (Hercules, CA) has now been modified to include molecular beacons for detection All of these real-time PCR systems are marketed as a semiautomated system in which enrichment samples are first lyzed and then loaded onto a fluorescence-detecting thermocycler that is connected to a personal computer recording fluorescence after every cycle By using capillary PCR reactors, such as the LightCycler, the reaction time can be reduced by half Despite the fact that total PCR reaction times can be as low as 20 minutes, all available formats still depend on a previous enrichment culture step to detect food-borne pathogens in foods The major advantages of PCR are its extreme sensitivity and specificity, and real-time PCR dramatically reduces analysis time Several researchers have demonstrated that PCR can detect as little as one cell in the reaction mixture, but the limitation for most PCR protocols resides on the ability of the pre-PCR steps to concentrate enough cells To achieve bacterial concentration often includes proprietary enrichment media and DNA extraction with resins.43 Since lyzed culture enrichment extracts of approximately 10 mL are often added to PCR reactions, the theoretical detection limit of PCR is roughly 100 cells per mL Future PCR formats would likely involve automated concentration steps that would be able to reduce this detection limit Rapid Analysis Techniques in Food Microbiology 16.7 429 FUTURE OF RAPID FOOD MICROBIAL TECHNIQUES The development of rapid microbial methods is one of the more active areas in the field of food microbiology The quick determination of the presence of pathogenic bacteria that have been ruled as adulterants, such as E coli O157:H7 and Listeria monocytogenes, continues to be the major driver in the development of detection methods Because of the need for faster detection methods, the potential demand for new rapid techniques will likely continue to be very strong New assay formats will continue to be developed, thanks to the advancement in fields such as molecular biology, immunology, nanotechnology, microarray technology, and bioelectronics Some of the technologies that will soon be commercialized for the detection and identification of food-borne pathogens are biosensors, DNA microchips, microarrays, incorporation of effective separation and concentration steps, and in-packages alert systems.3,4 While the “ultimate” rapid microbiological techniques (as defined in the Introduction section) are developed, the food industry will continue to use traditional and current rapid microbial detection methods in the near future ACKNOWLEDGMENTS The authors would like to thank Drs James Smith (USDA/ARS) and Purnendu C Vasavada (University of Wisconsin-River Falls) for critically reviewing this chapter REFERENCES Johnson, H H and Newsom, S W B., Rapid Methods and Automation in Microbiology Learned Information, Ltd., Oxford, 1976 Weaver, R H., Quicker bacteriological results, Am J Med Technol 20, 14–26, 1954 Feng, P., Development and impact of rapid methods for detection of foodborne pathogens, in Food Microbiology: Fundamentals and Frontiers, Doyle, M P., Beuchat, L R., and Montville, T J ASM Press, Washington, D.C., 2001 Fung, D Y C., Predictions for rapid methods and 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A., Flood, S J A., Livak, K J., Marmaro, J., Knorr, R., and Batt, C A., Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes, Appl Environ Microbiol 61 (10), 3724–3728, 1995 49 Rossen, L., Norskov, P., Holmstrom, K., and Rasmussen, O F., Inhibition of PCR by components of food samples, and DNA-extraction solutions, Intl J Food Microbiol 17, 37–45, 1992 50 Fach, P., Dilasser, F., Grout, J., and Tache, J., Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp in food samples: Probelia Salmonella spp, J Food Prot 62, 1387–1393, 1999 51 Pfaffl, M W., Fluorescence detection chemistry in kinetic RT-PCR, Munchen Technical University, 2003 Index A Accuracy 20–22 Allergic components biosensors 314 detection limits 306 ELISA 309–313 immunochemical analysis 307–309 PCR 314–323 properties 303–306 Antioxidants CE 390 GC 390 HPLC 390 B Benzoic acid CE 383 GC 382 HPLC 382 MS 382 SPE 383 Bioflavonoids analysis 167–171 properties 165 sample preparation 168–169 Biological techniques enzyme and microbial sensors 11, 167, 226, 320, 424–425 immunosensors 11, 90, 167, 307–309, 318–319, 385, 419–421, 425–428 Biotin analysis 167–171 properties 164 sample preparation 168–169 Biphenyl/o-phenylphenol GC 386 HPLC 386 IR 386 MS 386 TLC 386 Boric acid AAS 388 HPLC 388 C Calibration 22–28 Carotenoids HPLC 183–193 MS 184 NMR 183 properties 180–181 sample preparation 181–183 SFC 182 TLC 183 Chlorophylls HPLC 190–193 MS 190 properties 188–189 Choline analysis 167–171 properties 161 sample preparation 168–169 Column chromatography lipids 104–105 vitamins 169 E Erythorbic acid CE 391 electrochemical 391 fluorescence 391 HPLC 391 F Folate, see Folic acid Folic acid analysis 167–171 properties 164 433 434 Methods of Analysis of Food Components and Additives RDA 161 sample preparation 168–169 HPLC, see High performance liquid chromatography G I Gas chromatography antioxidants 390 benzoic acid 382 biphenyl/o-phenylphenol 386 general in drinking water 32–34, 36–49 lipids 99, 107–108 PAH 364–372 pesticide 341, 349–355 polyphenols 244–245 proteins 85 sensory analysis 292–293 trace elements 132–137 vitamins 168, 170–171 GC, see Gas chromatography Genetically modified foods ELISA 318, 322–324 immunochemical analysis 318–319 PCR 320–324 Immunochromatography 422 Inositol analysis 167–171 properties 164 sample preparation 168–169 Ion exchange chromatography proteins 83 vitamins 168 H High performance liquid chromatography antioxidants 390 benzoic acid 382 biphenyl/o-phenylphenol 386 boric acid 388 carotenoids 183–193 chlorophylls 190–193 general 5–6 in drinking water 32–33, 49 lipids 104–109 nisin 387 nitrite/nitrate 386 PAH 365–366 parabens 382 pesticide 341, 349–355 polyphenols 227–242 proteins 84–85 sorbic acid 382 sulfur dioxide/sulfides 385 trace elements 132–138 vitamins 167–172 M Microbiological methods biochemical methods 418 ELISA 421–422 immunoassay 419–423 immunochromatography 422 in drinking water 51–54 PCR 425–428 vitamins 175 MS benzoic acid 382 biphenyl/o-phenylphenol 386 carotenoids 184 chlorophylls 190 general 7–8 in drinking water 32, 49 PAH 365–366, 370–372 parabens 382 pesticide 341, 349–355 polyphenols 229 proteins 91–92 sorbic acid 382 trace elements 119, 126–129, 132–138 vitamins 168, 170–171 N Niacin analysis 167–171 properties 162 RDA 161 sample preparation 168–169 Niacinamide, see Niacin Nisin Index HPLC 387 SPE 387 Nitrite/nitrate CE 386 HPLC 386 O Organic pollutants 31–34 P PABA, see Para-amino benzoic acid PAH, see Polycyclic aromatic hydrocarbons Pantothenic acid analysis 167–171 properties 163 sample preparation 168–169 Paper chromatography polyphenols 244 proteins 80–81 vitamins 169 Para-amino benzoic acid analysis 167–171 properties 164 sample preparation 168–169 Parabens CE 383 HPLC 382 MS 382 SPE 382–383 PCB, see Polychlorinated biphenyls Pesticides CE 349 ELISA 340–341 GC 341, 349–355 HPLC 341, 349–355 in drinking water 43–48 monitoring 332–336 MS 341, 349–355 properties 336–338 sample preparation 339–354 SPE 344, 348–349 Physical techniques electrochemical 9, 391 electrophoresis 9, 85–89, 138, 170–171, 245–246, 349, 383, 385–386, 391 particle analysis 10 435 rheology and texture 10 Polychlorinated biphenyls, in drinking water 43–48 Polycyclic aromatic hydrocarbons GC 364–372 HPLC 365–366 in drinking water 43–48 MS 365–366, 370–372 sample preparation 362–364 SFE 364 Polyphenols CE 245–246 chemometric 221–222 enzymatic analysis 226 extraction 207–210 GC 244–245 HPLC 227–242 HSC chromatography 243 MS 229 NMR 222 paper chromatography 244 properties 200–207 protein precipitation 222–226 TLC 244 UV-VIS analysis 210–220 Proteins hydrolysis 69–70 isolation and purification 61–68 Psychyophysics 275 R Radionucleotides alpha spectrometry 409 gamma spectrometry 409 general 406–408 gross alpha activity 411 RDA, see Recommended daily allowances Recommended daily allowances fat-soluble vitamins 160 water-soluble vitamins 161 S Sensory electronic nose (e-nose) 293–295 evaluation 264–268 GC analysis 292–293 organs 268–275 436 Methods of Analysis of Food Components and Additives responses 277–278 statistics 282–291 SFC, see Supercritical fluid chromatography Solid phase extraction benzoic acid 383 in drinking water 32–34 lipids 104–105 nisin 387 PAH 364 parabens 382–383 pesticides 344, 348–349 sorbic acid 382–383 vitamins 169 Sorbic acid CE 383 HPLC 382 MS 382 SPE 382–383 SPE, see Solid phase extraction Spectroscopic techniques alpha 410 atomic absorption 7, 117, 119–125, 133–138, 388 atomic emission 7, 117, 118–119, 126–127, 133–137 electron spin resonance fluorescence 6, 167, 170–171, 391 gamma 409 infrared 6–7, 91, 170, 386 nuclear magnetic resonance 8, 91, 102, 183, 222 Raman 7, 170 UV-VIS 6, 71–80, 115, 129–130, 172, 174–175, 210–220 Sulfur dioxide/sulfides biosensors 385 CE 385 HPLC 385 Supercritical fluid chromatography carotenoids 182 general lipids 103–104 vitamins 169, 172 T Thin layer chromatography biphenyl/o-phenylphenol 386 carotenoids 183 lipids 104–105, 108 polyphenols 244 proteins 81 vitamins 169 Thresholds 279–282 TLC, see Thin layer chromatography Total fat 101–107 Trace elements AAS 117, 121–126, 134–138 AES 117, 118, 127–128, 134–138 GC 133–138 HPLC 133–138 inductively coupled plasma 117, 119–120, 127, 133–138 mass spectrometry 126–129, 131, 133–136 preparation 114–117 properties 12–113 U–V Uncertainity 16–20 Vitamin A analysis 167–172 properties 165 RDA 160 sample preparation 168–169 Vitamin B1 analysis 167–171 properties 162 RDA 161 sample preparation 168–169 Vitamin B2 analysis 167–171 properties 162 RDA 161 sample preparation 168–169 Vitamin B5, see Pantothenic acid Vitamin B6 analysis 167–171 properties 163 RDA 161 sample preparation 168–169 Vitamin B12 analysis 167–171 properties 163 RDA 161 sample preparation 168–169 Vitamin C Index analysis 167–171 properties 165 RDA 161 sample preparation 168–169 Vitamin D analysis 167–172 properties 166 RDA 160 sample preparation 168–169 Vitamin E analysis 167–172 437 properties 166 RDA 160 sample preparation 168–169 Vitamin K analysis 167–172 properties 166 RDA 160 sample preparation 168–169 Volatile hydrocarbons 40–43 Volatile organohalogen compounds 34–40