Heterocyclic Aromatic Amines in Cooked Meat Products: Causes,Formation, Occurrence,and Risk Assessment

Meat products are sources of protein with high biological value and an essential source of other nutrients, such as vitamins and minerals. Heating processes cause food to become more appetizing with changes in texture, appearance, flavor, and chemical properties by the altering of protein structure and other ingredients. During heat treatment, heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are formed due to the Maillard reaction. The HAAs are classified in at least 2 groups: thermic HAAs (100 to 300 °C) and pyrolytic HAAs (>300 °C). This review focuses on the parameters and precursors which affect the formation of HAAs: preparation, such as the marinating of meat, and cooking methods, including temperature, duration, and heat transfer, as well as levels of precursors. Additionally, factors are described subject to pH, and the type of meat and ingredients, such as added antioxidants, types of carbohydrates and amino acids, ions, fat, and other substances inhibiting or enhancing the formation of HAAs. An overview of the different analytical methods available is shown to determine the HAAs, including their preparation to clean up the sample prior to extraction. Epidemiological results and human daily intake of HAAs obtained from questionnaires show a relationship between the preference for very well‐done meat products with increased HAA levels and an enhanced risk of the incidence of cancer, besides other carcinogens in the diet. The metabolic pathway of HAAs is governed by the activity of several enzymes leading to the formation of DNA adducts or HAA excretion and genetic sensitivity of individuals to the impact of HAAs on human cancer risk.     

Exposure to heterocyclic aromatic amines from the consumption of cooked red meat and its effect on human cancer risk: A review

This review covers the bibliographic data from the last 10 years on the possible carcinogenicity of heterocyclic aromatic amines (HAAs) in humans. Aspects such as red meat intake, cooking methods applied to red meat, and doneness of cooking are discussed from an epidemiological point of view. The role in the carcinogenicity of the HAAs has been assigned to two main factors: first, the very high frequency of consumption of red meat; and, second, very darkly browned meats from cooking. However, there are some uncertainties associated with epidemiological results such as the presence of other carcinogens, co-carcinogens and anti-carcinogens in the diet, analytical results on the content of HAAs in foods, food frequency questionnaires, and mainly genetic susceptibility to HAAs. It is concluded that there is not sufficient scientific evidence to support the hypothesis that human cancer risk is due specifically to the intake of HAAs in the diet.     

Exposure to heterocyclic aromatic amines from the consumption of cooked red meat and its effect on human cancer risk: A review

This review covers the bibliographic data from the last 10 years on the possible carcinogenicity of heterocyclic aromatic amines (HAAs) in humans. Aspects such as red meat intake, cooking methods applied to red meat, and doneness of cooking are discussed from an epidemiological point of view. The role in the carcinogenicity of the HAAs has been assigned to two main factors: first, the very high frequency of consumption of red meat; and, second, very darkly browned meats from cooking. However, there are some uncertainties associated with epidemiological results such as the presence of other carcinogens, co-carcinogens and anti-carcinogens in the diet, analytical results on the content of HAAs in foods, food frequency questionnaires, and mainly genetic susceptibility to HAAs. It is concluded that there is not sufficient scientific evidence to support the hypothesis that human cancer risk is due specifically to the intake of HAAs in the diet.     

Mitigation strategies to reduce the impact of heterocyclic aromatic amines in proteinaceous foods

BackgroundMeat and fish are currently the main sources of proteins necessary for a healthy diet. Cooking proteinaceous food helps reduce biological risks and produce odor-active compounds, but it also generates heat-induced toxicants, of which heterocyclic aromatic amines (HAAs) are probably the most problematic as they are strongly mutagenic and carcinogenic.Scope and approachThis review highlights the most promising strategies for mitigating the impacts of HAAs on human health. These strategies revolve around reducing HAA formation by impacting HAA precursors, controlling the process, adjusting formulations or adapting diets to limit HAA assimilation and metabolism.Key findings and conclusionsIdentifying the different mechanisms of HAA formation and metabolism has made it possible to propose mitigation strategies to limit the risks related to HAA consumption. Various kinds of levers exist. While cooking methods for industrial processed foods can be regulated, it is far more difficult to influence household practices. Mitigation strategies involving other food ingredients are probably more promising from a consumer point of view if pushed by health education campaigns. Efforts to reduce the health risk from HAA consumption should now turn to ingredients like carvacrol that present different concomitant modes of action.     

Analytical methods for the determination of acrylamide in food products: a review

In early 2002, the Swedish National Food Administration reported high acrylamide levels in heat-treated carbohydrate-rich foods. Consequently, intensive activity began examining the many different types of food, and thousands of analyses have been undertaken world wide. Measurement data have been published in many different types of media. Within this flood of publications, there are only a limited number of articles concerned with the technical aspects of the measurements. This review focuses on the state-of-the-art in the analysis of acrylamide in foodstuffs. It covers information on methods from peer-reviewed articles and other sources (e.g. a survey carried out among official and private laboratories of the Member States of the European Union). Alternative methods are presented and discussed alongside the more common measurement techniques for acrylamide in foodstuffs. Special attention is given to sample preparation. The greatest differences between the analytical methods was for acrylamide extraction and clean-up. The influence of different extraction techniques or extraction solvents/solvent mixtures on the measurement results has not yet been fully investigated. There is also a lack of understanding about the sample clean-up. Since both might have a large impact on the results of the analysis, this review should also be considered as a basis for further investigations.     

Effect of Thermal Treatment on Meat Proteins with Special Reference to Heterocyclic Aromatic Amines (HAAs)

Meat is one of the most imperative protein sources available with respect to its production and consumption. It is the richest source of some valuable nutrients like proteins, essential amino acids, polyunsaturated fatty acids, vitamins, and minerals like iron, zinc, and selenium. Thermal treatment produces conformational changes in protein structure as well as flavor, texture, and appearance, and chemical properties of the ingredients are also changed. Heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are formed during the cooking of meat at high temperature. The review paper highlights the effects of various cooking methods, i.e., pan-frying, deep-frying, charcoal grilling, and roasting, on the formation of HAAs. The levels of HAAs produced in cooked meats vary depending upon the cooking method, time of cooking, and the type of meat being cooked. Metabolic behavior of HAAs is very unique, they interfere in the activity of many enzymes, modify the metabolic pathways, and lead to the adduct formation of DNA. The application of black pepper and several other spices during processing may reduce the formation of these (HAAs) mutagenic compounds.     

Factors That Affect the Content of Heterocyclic Aromatic Amines in Foods

Abstract: This review compiles the contents of heterocyclic aromatic amines (HAAs) in foods and beverages, collected from literature data along the period from 1992 up to 2009. Also, it describes the factors that affect the formation of HAAs in foods, such as the cooking method, including temperature, time, and frequency of turning of meat, during cooking. Other factors depending on the type of food and the recipe followed are pH, amounts of HAA precursors, types of amino acids, presence of certain divalent ions, and content of substances with enhancing or inhibiting effects on the formation of HAAs. In addition, there are other factors, which depend on the type of food, such as muscle tissue and the presence of certain genes, since the RN^? allele in pigs increases the glycogen content of muscle. The dispersion of the bibliographic data is evident, and there are scarce data, even no data, referred to individual HAAs. Considering that the diverging results can be due to several causes, possible recommendations are given in order to prevent the dispersion of the results and to achieve more valuable information, applied to determine the HAAs exposure. Although there are not direct indications that HAAs represent a serious health risk to the population, and common cancers are produced by many factors including xenobiotics, all measures to minimize the formation of HAAs should be foreseen, some of which are indicated.     

Effects of traditional chinese cooking methods on formation of heterocyclic aromatic amines in lamb patties

Different amounts of the potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs) are formed in muscle-based foods under different cooking methods. HAAs (9 varieties) in lamb patties cooked using traditional Chinese cooking methods (roasting, frying, panfrying, and stewing in seasonings) were investigated. The total HAAs contents in roasted, fried, pan-fried, and stewed patties were 4.39-123.15 ng/g, 3.59-43.24 ng/g, 0.71-10.05 ng/g, and 51.07-120.32 ng/g, respectively. Amounts of HAAs increased as cooking time increased. 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) was the dominant HAAs in deep roasted and fried samples, while 1-methyl- 9H-pyrido [3,4-b] indole (Harman) and 9H-pyrido [3,4-b] indole (Norharman) were the main HAAs in pan-fried and stewed samples. Types and contents of HAAs formed at different cooking times using different methods are unique. Stewing in seasoning generated a higher HAAs content than the high-temperature cooking methods roasting, frying, and pan-frying.     

MULTI-RESIDUE DETERMINATION AND CONFIRMATION OF TEN HETEROCYCLIC AMINES IN COOKED MEATS

Heterocyclic aromatic amines (HAAs) have been identified in various cooked meat products. HAAs are formed from pyrolysis of proteins and amino acids and are of interest because many of these primary arylamines are mutagens and rodent carcinogens. We developed a screening method for ten HAAs using liquid-liquid extraction, reverse-phase HPLC separation, and tandem electrochemical (ECD) and fluorescence (FLU) detectors. This detection system exhibited greater sensitivity (50-100 fold) for HAAs than the HPLC-UV methods. HPLC-FLU has detection limits of 100 pg to 1 ng for six HAAs, and HPLC-ECD detection limits of four HAAs were 250 pg to 1 ng. We also developed a confirmatory procedure for ten HAAs using HPLC coupled to positive ion APCI-MS. This technique has subnanogram detection limits. These methods were used to quantify HAAs in meat products at levels formed by typical Western cooking methods.     

Effect of cooking methods on the formation of heterocyclic aromatic amines in chicken and duck breast

Heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are pyrolysis formed during the cooking of meat and fish. In the present study, the effects of various cooking methods, pan-frying, deep-frying, charcoal grilling and roasting on the formation of HAAs in chicken breast and duck breast were studied. The various HAAs formed during cooking were isolated by solid-phase extraction and analyzed by high-performance liquid chromatography (HPLC). Results showed that chicken breast cooked by charcoal grilling contained the highest content of total HAAs, as high as 112 ng/g, followed by pan-fried duck breast (53.3 ng/g), charcoal grilled duck breast (32 ng/g), pan-fried chicken breast (27.4 ng/g), deep-fried chicken breast (21.3 ng/g), deep-fried duck breast (14 ng/g), roasted duck breast (7 ng/g) and roasted chicken breast (4 ng/g). For individual HAA, the most abundant HAA was 9H-pyrido-[4,3-b]indole (Norharman), which was detected in charcoal grilled chicken breast at content as high as 32.2 ng/g, followed by 1-methyl-9H-pyrido[4,3-b] indole (Harman) and 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine(PhIP) at 32 and 31.1 ng/g in charcoal grilled chicken breast, respectively. The content of PhIP in pan-fried duck and chicken breast were 22 and 18.3 ng/g, respectively. Generally, the type and content of HAAs in cooked poultry meat varies with cooking method and cooking conditions.     

N ϵ-(carboxymethyl)lysine: A Review on Analytical Methods,Formation, and Occurrence in Processed Food,and Health Impact

Foods are often heat processed and may contain advanced glycation end products (AGE). One of the most widely studied AGE is N ^?-(carboxymethyl)lysine (CML); nevertheless, knowledge on dietary CML is fragmentary. This study aimed to review current scientific knowledge on analytical methods to determine CML contents in food, chemical pathways of CML formation in food, occurrence of CML in food, and health implications of dietary exposure to CML. Chemical analyses of CML in food products are carried out by immunochemical assays and instrumental methods, but the former method may interfere with the food matrix. CML is formed in food through various chemical pathways, depending on food ingredients and processing conditions. The compound is present in many cooked foods, with relatively high concentrations in carbohydrate-rich foods and dairy products. Dietary CML is very likely to impair human health, but full cause-effect evidence is not available yet. More studies on metabolic effects and impact of food-derived CML on human health should be performed. Food production should be optimized to minimize CML concentrations, while maintaining acceptable microbiological safety and organoleptic properties of the final food product. To this end, more insights into effects of food composition and processing conditions on CML formation are necessary.     

烹饪过程中丙烯酰胺的形成与控制

从原料的组成与保藏、煎炸烤制等多种烹调方法以及烹饪工艺条件等方面,阐明丙烯酰胺的形成、食物中的分布规律和控制措施,并扼要讨论了其他相关烹调方法的安全性。     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.     

Polycyclic aromatic hydrocarbons in meat smoked with different types of wood

The influence of the wood used for the smoking of meat on the formation of polycyclic aromatic hydrocarbons (PAH) has been studied. Ten types of wood and charcoal were used for preparation of smoked meat samples. The analytical sample preparation method implied extraction of PAH with cyclohexane, liquid–liquid extraction with N,N-dimethylformamide/water, back extraction with cyclohexane, followed by clean-up on silica solid phase extraction (SPE) column and quantification by gas chromatography–mass spectrometry. It was found that the type of wood has a significant influence on the amount of PAH in smoked meat. The samples smoked with apple-tree and alder contained the smallest PAH concentrations. The samples smoked with spruce had the highest concentrations of PAH. The difference in content of benzo[a]pyrene (from 6.04 till 35.07 μg/kg) and total PAH (from 47.94 till 470.91 μg/kg) indicates that choice of wood for smoking is one of the critical parameter to be controlled in order to diminish the contamination of food products.     

Starch–lipid and starch–lipid–protein complexes: A comprehensive review

Physical interactions often occur between major food components during food processing. These interactions may involve starch, lipids, and proteins forming V‐type starch–lipid complexes or ternary starch–lipid–protein complexes of larger molecular size and greater structural order. Complexes between starch and lipids have been the subject of intensive research for over half a century, whereas the study of starch–lipid–protein complexes is a relatively new field with only a limited amount of knowledge being gained so far. The formation of these complexes can significantly affect the functional and nutritional properties of finished food products in terms of flavor, texture, shelf life, and digestibility. This article provides a comprehensive review of starch–lipid and starch–lipid–protein complexes, including their classification, factors affecting their formation and structure, and preparative and analytical methods. The review also considers how complexes affect the physicochemical and functional properties of starch, including digestibility, and potential applications in the food industry.     

Thermal processing food-related toxicants: a review

Abstract

Heterocyclic aromatic amines, acrylamide, 5-hydroxymethylfurfural, furan, polycyclic aromatic hydrocarbons, nitrosamines, acrolein, chloropropanols and chloroesters are generated toxicants formed in some foodstuffs, mainly starchy and protein-rich food during thermal treatment such as frying, roasting and baking. The formation of these chemical compounds is associated with development of aromas, colors and flavors. One of the challenges facing the food industry today is to minimize these toxicants without adversely affecting the positive attributes of thermal processing. To achieve this objective, it is essential to have a detailed understanding of the mechanism of formation of these toxicants in processed foods. All reviewed toxicants in that paper are classified as probable, possible or potential human carcinogens and have been proven to be carcinogenic in animal studies. The purpose of that review is to summarize some of the most frequent occurring heat-generated food toxicants during conventional heating, their metabolism and carcinogenicity. Moreover, conventional and microwave heating were also compared as two different heat treatment methods, especially how they change food chemical composition and which thermal food toxicants are formed during specific method.     

Solid‐phase extraction and spectrophotometric determination of Allura Red (E129) in foodstuff,soft drink,syrup and energy drink samples: a comparison study

Amberlite XAD‐7 and XAD‐8 resins were used as adsorbents for preconcentration and determination of Allura Red (AR) food dye in aqueous medium. The effects of pH, sample volume, sample and eluent flow rates on the extraction of AR were optimised. The determination of dye was performed at 506.0 nm using spectrophotometry. Interference effects of matrix ions and some dyes were also investigated under optimised conditions. The methods permitted low detection limits which were 1.2 and 0.6 μg L^?1 for XAD‐7 and XAD‐8, respectively. Adsorption behaviours were investigated by adsorption isotherms and zero charge pH experiments. Validations of the method were performed by determination of AR contents in some foodstuffs. AR contents of liquid samples were found between 58 and 440 μg mL^?1. AR concentrations of solid samples were between 416 and 432 μg g^?1. The XAD‐7 and XAD‐8 resins presented a fast and reliable potential to determine AR dye in real samples.     

Novel oil extraction technologies: Process conditions,quality parameters,and optimization

Conventional techniques of extracting oil using organic solvents pose health, safety, and environmental concerns. In modern extraction methods, green solvents such as water, ethanol, ethyl acetate, carbon dioxide, ionic liquids, and terpenes are currently gaining prominence. These green solvents present no signs of pollution and remain in liquid form over a temperature range of 0 to 140 °C. Other techniques covered in this review include microwave‐assisted enzymatic extraction, ultrasound‐assisted extraction, supercritical fluid technology, high pressure–assisted extraction, and pulse electric field–assisted extraction. These techniques are considered environmentally friendly because they exhibit less hazardous chemical synthesis, use renewable feedstock, and reduce the chemical load and emissions generated by organic solvents. Aqueous enzymatic extraction is a novel technique that uses enzymes as the medium for extraction of oil. Selection of the enzymes solely depends on the structure of the oilseed and the composition of the cell wall. Studies reveal an enzyme to substrate ratio of 1% to 8%, the temperature of 40 to 55 °C, and a pH of 4 to 8 to be typical for enzymatic extraction of oil from different oilseeds. Microwave‐assisted extraction has proven to impart significant effects on mass transfer and offers high throughput and extraction efficiency. A microwave power of 275 to 1,000 W and a temperature range of 30 to 60 °C are noticed in the different studies. The review presents a comprehensive account of the modern extraction techniques, the parameters responsible for yield and quality, and their industrial applications. Besides, the review highlights the optimized parameters for oil extraction from different oil‐bearing materials.     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998