Antioxidant spices reduce the formation of heterocyclic amines in fried meat

 Heterocyclic aromatic amines (HAs) are mutagenic compounds that are formed during heating of meat and fish. These substances are reaction products of creatine with amino acids and carbohydrates. It is recommended that exposure to these probable human carcinogens should be minimised. Five heterocyclic aromatic amines which occur in beef were investigated: 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP). Clean-up was done by acid-base partition followed by SPE using blue cotton. HPLC analysis was carried out by using electrochemical detection for IQ- and IQx-type compounds and fluorescence detection for PhIP. The concentrations of the aromatic amines were as follows: IQ, 10.2 μg/kg; MeIQ, 2.46 μg/kg; MeIQx, 13.2 μg/kg; 4,8-DiMeIQx, 2.26 μg/kg; and PhIP, 5.48 μg/kg. The application of spices (rosemary, thyme sage, garlic, brine) reduced the content of the HAs below 60% of the amount found in the control. Received: 23 April 1998     

Dietary exposure to heterocyclic amines in high-temperature cooked meat and fish in Malaysia

The intake of heterocyclic amines is influenced by the amount and type of meat and fish ingested, frequency of consumption, cooking methods, cooking temperature, and duration of cooking. In this study, the dietary intake of heterocyclic amines in Malaysia and their main sources were investigated. Forty-two samples of meat and fish were analysed by high-performance liquid chromatography with photodiode array detector to determine the concentration of the six predominant heterocyclic amines, namely: 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f] quinoline(MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f] quinoxaline (7,8-DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Dietary intake data were obtained using a food-frequency questionnaire when interviewing 600 Malaysian respondents. The level of total heterocyclic amines in food samples studies ranged from not detected to 38.7 ng g^?1. The average daily intake level of heterocyclic amine was 553.7 ng per capita day^?1. The intake of PhIP was the highest, followed by MeIQx and MeIQ. The results reveal that fried and grilled chicken were the major dietary source of heterocyclic amines in Malaysia. However, the heterocyclic amine intake by the Malaysian population was lower than those reported from other regions.     

Tandem solid phase extraction coupled to LC–ESI–MS/MS for the accurate simultaneous determination of five heterocyclic aromatic amines in processed meat products

Carcinogenic heterocyclic aromatic amines are difficult to measure since only trace levels are present in processed meat products. In this study, typical heterocyclic aromatic amines, including 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ), 2-amino-3,8-dimethyli-midazo [4,5-f] quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimi-dazo [4,5-f] quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-ph-enylimidazo [4,5-b]pyridine (PhIP), were studied to develop a sensitive and accurate method for their rapid quantification in animal-derived products, with 2-Amino-3,4,7,8-dimethylimidazo [4,5-f] quinoxalline (TriMeIQx) as an internal standard. Liquid chromatography–electrospray-tandem mass spectrometry conditions were analyzed to enhance detection sensitivity. Diatomaceous earth was employed to extract heterocyclic aromatic amines from meat samples, and the analytes were purified and enriched using tandem solid phase extraction, with siliprep propylsulfonic acid coupled to a C₁₈ cartridge. A number of parameters, including pH, eluent and volume, were carefully optimized to improve the extraction and purification efficiency. Under the optimal experimental conditions, the limits of detection for each analyte within the meat matrix were 0.5 pg (injected). The established method was applied to evaluate commercial meat products. At three spiked levels of 0.2, 1 and 4 μg kg⁻¹, the recoveries and relative standard deviations were measured as 76.4–122.2 and 0.9–23.4%, respectively, suggesting the developed method is promising for the accurate quantification of heterocyclic aromatic amines at trace levels in processed meats.     

Effects of cooking methods on the formation of heterocyclic aromatic amines of two different species trout

Heterocyclic aromatic amines (HAAs) are sometimes formed in meats and fish cooked at high temperatures. In the present study, the effects of cooking methods by deep-fat frying, pan-frying, grilling and barbecuing on the formation of HAAs of fillets of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta fario) were investigated. Barbecued brown trout (1 g) was estimated to contain 0.12 ng of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), 0.02 ng 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline). Grilled rainbow trout (1 g) was estimated to contain 0.02 ng 4,8-DiMeIQx. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline), MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) were not detectable in all cooked fish.     

温和条件下模型体系中烧烤风味及杂环胺形成测定

以美拉德模型体系葡萄糖-肌酸酐-氨基酸（丙氨酸、苏氨酸、甘氨酸）为研究对象,探索3 个体系在130 ℃条件下反应3 h后风味物质及杂环胺的形成。采用固相微萃取-气相色谱-质谱联用方法检测表明,3 种模型均产生烧烤风味物质,主要为烷基吡嗪类。高效液相色谱分析表明,丙氨酸模型（Ⅰ）和甘氨酸模型（Ⅲ）均检测到2-氨基-3,4-二甲基咪唑并[4,5-f]喹啉、2-氨基-3,4,8-三甲基咪唑并[4,5-f]喹喔啉和2-氨基-3,7,8-三甲基咪唑并[4,5-f]喹喔啉3 种杂环胺,苏氨酸模型（Ⅱ）检测到了2-氨基-3,4-二甲基咪唑并[4,5-f]喹啉和2-氨基-3,4,8-三甲基咪唑并[4,5-f]喹喔啉2 种杂环胺;3 种模型体系均未检测到2-氨基-3-甲基咪唑并[4,5-f]喹啉和2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉。130 ℃温和条件下,1.5 h内各模型体系中杂环胺的生成量很少,1.5 h后杂环胺总量随着加热时间的延长逐渐增多,3 h时模型Ⅰ、Ⅱ、Ⅲ中杂环胺总量分别为（375.50±15.80）、（414.00±18.40）、（363.50±12.20） ng/mL。本研究为肉制品加工尤其是烤肉制品加工如何控制加工条件,从而实现美拉德反应定向控制、减少杂环胺的产生提供一定参考。     

Formation and mitigation of heterocyclic aromatic amines in fried pork

Heterocyclic aromatic amines (HAAs) are potent mutagens and carcinogens generated during the heat processing of meat. HAAs, which are abundant in processed meat products, include 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). The content of these three HAAs in fried pork was determined by LC-MS/MS. The effects of frying time and temperature, sample shape, and addition of antioxidants on the generation of HAAs were investigated. The results show that HAAs were produced during frying, and their levels increased with increasing frying time and temperature. Pork patties had the highest concentration of HAAs compared with pork meatballs and pork strips. The addition of antioxidant of bamboo leaves (AOB), liquorice extract, tea polyphenol, phytic acid and sodium iso-ascorbate to pork before frying had an inhibitory effect on HAA generation, with AOB being the most effective antioxidant. Inhibition levels of nearly 69.73% for MeIQx, 53.59% for 4,8-DiMeIQx and 77.07% for PhIP in fried pork were achieved when the concentrations of AOB added were 0.02, 0.01 and 0.10 g kg^?1, respectively.     

Effects of varying degrees of doneness on the formation of Heterocyclic Aromatic Amines in chicken and beef satay

The study was carried out to determine the effect of cooking method on Heterocyclic Aromatic Amines (HAs) concentration in grilled chicken and beef (satay). Six common HAs were investigated: 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2amino 3,4dimethylimidazo [4,5f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8 trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8trimethylimidazo [4,5-f]quinoxaline (7,8-DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Chicken and beef satay samples were grilled to medium and well done level of doneness. Charcoal grilled (treatment A), microwave pre-treatment prior to grilling (treatment B), and microwave-deep fried (treatment C) were applied to beef and chicken satay samples. The satay samples which were microwaved prior to grilling (B) showed significantly (p < 0.05) lower HAs concentration as compared to those charcoal grilled (A). Both medium and well done cooked beef and chicken satay samples that were microwaved and deep fried (C) as an alternative method to grilling were proven to produce significantly lesser HAs as compared to charcoal-grilled (A) and microwaved prior to grilling (B).     

Response surface optimization of effects of some processing variables on carcinogenic/mutagenic heterocyclic aromatic amine (HAA) content in cooked patties

A five-factor Central Composite Orthogonal Design was adopted to study simultaneous effects of some processing variables such as NaCl (0-2%), fat (10-30%), ascorbic acid (0-600 ppm), cooking temperature (150-230°C) and cooking time (5-15 min) on physicochemical properties and heterocyclic aromatic amine (HAA) contents of cooked beef patties. The HAAs analyzed were 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ), 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), as quantified by high-performance liquid chromatography with photo-diode array detection (HPLC-UV/DAD). It was found that ascorbic acid decreased; however, fat, cooking temperature and time levels increased the contents of IQ, MeIQx, MeIQ and PhIP. In addition, estimated ridge analysis was conducted to find values of the processing variables that maximize and minimize the five HAA contents, revealing that the results obtained would be useful for meat industry aiming to decrease HAA content in cooked meat products.     

Determination of Heterocyclic Aromatic Amines in Cooked Commercial Frozen Meat Products by Ultrafast Liquid Chromatography

This paper describes a method for the determination of nine heterocyclic aromatic amines (HCAs) in commercial frozen meat products, which were sold in Turkey by ultrafast liquid chromatography (UFLC) with ultraviolet visible detection. HCAs are separated on a Shim-pack XR-ODS (7.5?×?3 mm, 2.2 μm). Varying levels of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (up to 1.95 ng/g), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) (up to 4.17 ng/g), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (up to 0.69 ng/g), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (up to 0.83 ng/g), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (up to 0.22 ng/g), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) (up to 0.94 ng/g), 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) (up to 4.58 ng/g), 2-amino-9H-pyrido[2,3-b]indole (AαC) (up to 0.57 ng/g), and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) (up to 3.51 ng/g) were detected in these commercial frozen meat products of Turkey. The data obtained show clearly that HCAs could be isolated in a very short time (5 min) by using UFLC.     

Quantitation of heterocyclic aromatic amines in ready to eat meatballs by ultra fast liquid chromatography

Heterocyclic aromatic amines (HCAs) in meatballs ready to eat and sold in restaurants in Turkey were determined. A solid phase extraction method was used to isolate HCAs from meatballs. Various HCAs analysed by ultra fast liquid chromatography (UFLC) were varying levels of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (up to 1.59 ng/g), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) (up to 3.81 ng/g), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (up to 0.66 ng/g), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (not detected or not quantified), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (not detected or not quantified), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) (up to 0.43 ng/g), 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) (up to 1.93 ng/g), 2-amino-9H-pyrido[2,3-b]indole (AαC) (up to 0.35 ng/g), and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) (up to 0.43 ng/g) in cooked meatballs which are consumed in Turkey. Overall average of total HCA amount was 5.54 ng/g. The present study is to prove that HCAs can be isolated in a very short time (5 min) by using UFLC.     

一种检测杂环胺类化合物的酶联免疫检测方法的建立

为了制备一种广谱性杂环胺抗体,并建立一种可以实现多种杂环胺同时检测的快速分析方法。以杂环胺2-氨基-3,4-二甲基咪唑[4,5-f]喹喔啉(MeIQx)为原料,将其与丁二酸单甲酯酰氯(MCO)反应合成杂环胺半抗原,通过活化酯法将半抗原与蛋白偶联制备免疫原进一步制备多克隆抗体,最终建立间接竞争酶联免疫分析方法(ic-ELISA)。该方法灵敏度(IC₅₀,以MeIQx计)为81.16 μg/L,检测限(IC₁₅)为12.07 μg/L。方法对喹啉类杂环胺(IQ、MeIQ)、喹喔啉类杂环胺(IQx、MeIQx、4,8-DiMeIQx、7,8-DiMeIQx、4,7,8-TriMeIQx)以及吡啶类杂环胺(PhIP)具有相同的识别能力,交叉反应率均达到93%以上。油炸牛肉和肉松样品中杂环胺(MeIQx)添加回收率在91.18%~98.64%之间,检测结果与液相色谱串联质谱法(LC-MS/MS)有很好的一致性(R2=0.9927)。本文建立的酶联免疫分析方法可以实现喹啉类、喹喔啉类以及吡啶类杂环胺总量的检测,为热加工肉制品中杂环胺的检测提供了一种简单、准确的快速检测方法。     

Development of a New Extraction Method for Heterocyclic Aromatic Amines Determination in Cooked Meatballs

Heterocyclic aromatic amines (HCAs) are mutagenic and carcinogenic compounds that are produced in meats cooked at high temperature. In this study, 2-amino-3-methylimidazo[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), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-9H-pyrido[3,4-b]indole (harman) were studied. A new extraction method was developed for the determination of heterocyclic amines with high-performance liquid chromatography (HPLC). Linearity for each HCA was observed with a high regression coefficient (r?=?0.9999, P?<?0.01 for IQ; r?=?0.9990, P?<?0.01 for MeIQx; r?=?0.9989, P?<?0.01 for 4,8-DiMeIQx; r?=?0.9934, P?<?0.05 for PhIP; r?=?1.000, P?<?0.01 for norharman; r?=?0.9991, P?<?0.01 for harman). Limits of detection for various HCAs were found between 0.04 and 1.40 ng/g. Limits of quantification were found in the range of 0.13–4.40 ng/g. Recovery rates varied from 68.9 % to 87.8 %. This method was compared with two different common HCA extraction methods in literature. The optimized new extraction method and the other two methods were used for the determination of HCAs in ten different cooked meatball samples. Sample extraction procedures of HCAs were investigated in more detail, and a rapid, accurate, precise, and reliable extraction method was developed.     

Preconcentration and Simultaneous Determination of Heterocyclic Aromatic Amines in Grilled Pork Samples by Ion-Pair-Based Surfactant-Assisted Dispersive Liquid-Liquid Microextraction and High-Performance Liquid Chromatography

An effective ion-pair-based surfactant-assisted dispersive liquid-liquid microextraction combined with high-performance liquid chromatography (HPLC) has been evaluated for the extraction and preconcentration of four heterocyclic aromatic amines (i.e., 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ); 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx); 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); and 1-methyl-9H-pyrido[3,4-B]indole (Harmane)). In the extraction method, sodium dodecyl sulfate (SDS) was used as both ion-pairing and disperser agent and 1-octanol was selected as extraction solvent. The effects of various parameters on the extraction efficiency such as kind and concentration of surfactant, kind and volume of extraction, salt addition, vortex extraction, and centrifugation time were investigated. Under the selected condition, the method provided high enrichment factor in the ranged of 124–145. Good linearity was 0.01–1000 μg kg^?1 with the correlation coefficient (R ²)?>?0.999. The limit of detection was 0.01 μg kg^?1 for all compounds. The matrix match calibration was used for quantitation of the target analytes in grilled pork samples. The proposed method was successfully applied to analysis of heterocyclic aromatic amines in grilled pork samples where good recoveries were obtained in the range of 90 and 106 %.     

杂环胺人工抗原的合成以及多克隆抗体的制备

为建立适用于一类杂环胺的酶联免疫吸附检测方法(enzyme-linked immunosorbent assay,ELISA),利用改良的碳化二亚胺法将在2-氨基-3,4,8-三甲基咪唑[4,5-f]-喹喔啉(2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline,4,8-DiMeIQx)的2-氨基处连接丁二酸单甲酯酰氯制备的半抗原偶联蛋白获得人工抗原。该方法利用适当pH值的缓冲体系,将酯的水解和蛋白偶合一步完成,与传统方法相比,有效地节省了反应时间和成本,且对人工抗原的紫外扫描验证和多克隆抗体效价检测结果都直接和间接地表明偶合是成功的。最终免疫获得4,8-DiMeIQx结构类似物的多克隆抗体,该抗体的最高效价为1∶450 000,且经直接竞争ELISA检测,该抗体对目标物4,8-DiMeIQx的灵敏度(IC50)为24.0μg/L,最低检测限(IC15)为1.8μg/L,对其结构类似物7,8-DiMeIQx、8-Me IQx、2-氨基-3-甲基咪唑[4,5-f]-喹喔啉(IQx)、2-氨基-3,4,7,8-四甲基咪唑[4,5-f]-喹喔啉(Tri Me IQx)的灵敏度分别为35.0、30.0、28.0、40.0μg/L,而其他与目标物结构相差较大的杂环胺,像2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶(Ph IP)、2-氨基-3-甲基咪唑[4,5-f]喹啉(IQ)和2-氨基-3,4-二氨基咪唑[4,5-f]喹啉(2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline,Me IQ),它们几乎不与抗体结合(CR0.01%)。由此可见,可以利用该抗体建立高效快速的杂环胺ELISA检测方法用以定量或定性检测肉类加工食品中的一类杂环胺。     

Analysis of heterocyclic amines (HAs) in pan-fried pork meat and its gravy by liquid chromatography with diode array detection

Aminoazaarenes (heterocyclic amines, HAs) contents were investigated in pan-fried pork meat as well as in gravies generated during frying. The clean-up procedure included alkaline hydrolysis, tandem solid phase extraction on columns filled with Extrelut – diatomaceous earth, cation exchanger (propyl sulfonic acid) and chemically bounded phase – C₁₈. Identification and quantitative analysis of HAs fraction was carried out using a HPLC system with DAD-type detector. Separation was achieved by using TSK-gel ODS 80-T_M column and a mixture of 5% acetonitrile and 95% triethylamine phosphate buffer (pH 3.3) as a mobile phase. Six compounds were determined: 2-amino-1,6-dimethylimidazo[4,5-b]pyridine (DMIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP). Two types of dishes prepared at home according to common recipes used in Poland were investigated. The total content of aminoazaarenes determined in collar was 7.2 and in chop samples 18.0 ng g⁻¹ of cooked meat. The total contents of investigated HAs in gravy samples were 10.2 and 15.1 ng g⁻¹ of cooked meat for collars and chops, respectively.     

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     

Heterocyclic aromatic amines in cooked hamburgers and chicken obtained from local fast food outlets in the Ottawa region

Heterocyclic aromatic amines (HAAs) are formed in protein-rich foods during high temperature cooking such as frying and grilling. Since most HAAs are potent mutagens and almost all are carcinogenic to laboratory animals, their formation in cooked foods is a health concern. In the present study, 31 cooked hamburgers and six chicken preparations were obtained from various fast food outlets in the Ottawa area and analyzed for HAAs. In the developed procedure, ground-up samples were extracted under both acidic and alkaline conditions, cleaned on SPE cartridges, and the concentrations of various HAAs determined using electrospray ionization LC/MS/MS. Deuterium-labelled internal standards of the three most commonly found HAAs (IQ, MeIQx, and PhIP) in such foods were used for quantitation and recovery correction. Varying levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.2–6 μg/kg), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (0.1–3.5 μg/kg), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (0.3–6.9 μg/kg), and 7,8-dimethyl-IQx (2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline) (0.1–2.9 μg/kg) were detected in most hamburgers, whereas our limited data on the chicken samples (wings, drumsticks, and nuggets) indicated the presence of mainly PhIP (0.1–2.1 μg/kg) and MeIQx (0.1–1.8 μg/kg). Traces of 4,7,8-trimethyl-IQx (<0.1 μg/kg), 3-amino-1,4-dimethyl-5H-pyrido[3,4-b]indole (Trp-P-1) (<0.1–0.3 μg/kg), and 3-amino-1-methyl-5H-pyrido[3,4-b]indole (Trp-P-2) (<0.1–0.8 μg/kg) were also detected in some samples of hamburgers but not in any of the chicken analyzed thus far. Since hamburger is a popular meal among Canadians, regular consumption of such items may contribute substantially to one's dietary intake of HAAs.     

Inhibitory effects of pomegranate seed extract on the formation of heterocyclic aromatic amines in beef and chicken meatballs after cooking by four different methods

Beef and chicken meatballs with a 0.5% (w/w) pomegranate seed extract were cooked using four different cooking methods (oven roasting, pan cooking, charcoal-barbecue, and deep-fat frying) and six heterocyclic aromatic amines; IQ, MeIQx, 4,8-DiMeIQx, PhIP, norharman, and harman were observed. In the beef meatballs, the highest inhibitory effects of pomegranate seed extract on heterocyclic aromatic amines formation were 68% for PhIP, 24% for norharman, 18% for harman, 45% for IQ, and 57% for MeIQx. Total heterocyclic aromatic amine formation was reduced by 39% and 46% in beef meatballs cooked by charcoal-barbecue and deep-fat frying, respectively. In the chicken meatballs, the highest inhibitory effects were 75% for PhIP, 57% for norharman, 28% for harman, 46% for IQ, and 49% for MeIQx. When the pomegranate seed extract was added to the chicken meatballs cooked by deep-fat frying, the total heterocyclic aromatic amine formation was inhibited by 49%, in contrast the total heterocyclic aromatic amine contents after oven roasting increased by 70%.     

Influence of natural food ingredients on the formation of heterocyclic amines in fried beef patties and chicken breasts

The effects of natural food ingredients including Korean bramble, onion, and marinade sauce with water extracts of olive and lotus leaf on the formation of 15 heterocyclic amines (HCAs) were evaluated in fried beef patties and chicken breasts. The patties and chicken breasts containing natural food ingredients were fried at 230 and 200°C for 8 min on each side. Addition of 4 g Korean bramble to beef patties reduced the formation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 9H-pyrido [3,4-b]indole (Norharman), and 2-amino-6-methyldipyrido [1,2-a:3′,2′-d]imidazole (Glu-P-1) by 74, 62, and 39%, respectively. Also, when 2 g onion was added to beef patties, the formation of 2-amino-3,4,8-trimethylimidazo [4,5-f]quinoxaline (4,8-DiMeIQx), Glu-P-1, MeIQ, Norharman, and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) was inhibited by 100, 96, 88, 74, and 79%, respectively. When marinade sauce containing 2% water extracts of olive and lotus leaf was added to chicken breasts, most HCAs formation was inhibited. Especially, the formation of Glu-P-1, 2-aminodipyrido [1,2-a:3′,2′-d]imidazole (Glu-P-2), and MeIQ were reduced by 100%.