Chemical analysis and significance of heterocyclic aromatic amines

 Levels of known heterocyclic amines vary from undetectable in many meats sold in fast food restaurants, to over 10 ng/g for meats prepared in restaurants that cook food to order, to hundreds of nanograms per gram for some meats cooked under certain home or laboratory conditions. To simulate the dry reactions that seem to occur at the meat surface we developed a model system to mimic these processes. Mixtures of free amino acids, creatinine and glucose, simulating the composition of beef or chicken, heated at 200  °C, form eight heterocyclic amines. Besides the commonly found 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,6-dimethylimidazo[4,5-b]pyridine, 2-amino-1,5,6-trimethylimidazo[4,5-b]pyridine and 2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine were also found. The calculated risk of consumption of heterocyclic amines is determined by the dietary dose, the extrapolation of carcinogenic potencies from rodents to humans, and the extrapolation of high rodent doses to low human exposures. Results suggest that DNA binding is linear with dose, but that the human DNA forms more adducts per unit dose than that of the rat. Altogether, the risk appears to be equivalent to that for many carcinogens that are regulated. Received: 23 April 1998     

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.     

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.     

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.     

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.     

Analysis of heterocyclic amines and β-carbolines by liquid chromatography–mass spectrometry in cooked meats commonly consumed in Korea

Heterocyclic amines (HAs), which form in meats during heating and cooking, are recognized as mutagenic and carcinogenic compounds. In this study, 13 HAs and 2 β-carbolines (BCs) were analyzed in cooked Korean meat products, including griddled bacon, griddled pork loin, boiled pork loin, boiled chicken meat, chicken meat stock, chicken breast for salad and chicken patty. The samples were either cooked in the laboratory or purchased from local fast-food restaurants. The HAs and BCs in the samples were separated using solid-phase extraction and were analyzed by high performance liquid chromatography–mass spectrometry (HPLC–MS). The most frequently detected HAs and BCs in the cooked meats were harman (1-methyl-9H pyrido[4,3-b]indole; 990.9 ng g^?1), norharman (9H-pyrido[4,3-b]indole; 412.7 ng g^?1) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 258.2 ng g^?1). The griddled pork loin and bacon contained higher levels of norharman, harman and PhIP than the other cooked meats. PhIP, which is classified as a Group 2B carcinogen by the International Agency for Research on Cancer, had levels of 258.2 and 168.2 ng g^?1 in the griddled pork loin and griddled bacon, respectively. The griddled bacon was the only sample containing TriMeIQx (2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline; 79.9 ng g^?1). IQ (2-amino-3-methyl imidazo[4,5-f]quinoline), 7,8-DiMeIQx (2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline) and AαC (2-amino-9H-pyrido[2,3-b]indole) were detected at trace levels in all samples.     

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%.     

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.     

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.     

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.     

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.     

Binding of 14C-Labeled Food Mutagens (IQ, MeIQ, MeIQx) by Dietary Fiber In Vitro

Binding of three mutagens, known to occur in fried or broiled foods, by thirteen different types of dietary fiber was investigated in vitro. Nonspecific binding by other food polymers was minimized by using protease and amylase treatment. Water-insoluble fiber components were responsible for most of the binding capacity. Generally, a slightly larger proportion of 2-amino-3,4-dimethylimidazo [4,5-f]quinoline (MeIQ) than of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo] -4,5-f]quinoxaline (MeIQx) was bound. There was a significant correlation between Klason lignin content and binding of mutagens. Optimum pH for binding was between 4 and 6. Dietary fiber from sorghum had the highest binding capacity, which could be due to the presence of a large Klason lignin fraction.     

Occurrence of heterocyclic amines in cooked meat products

Heterocyclic amines (HCAs), potent mutagens and a risk factor for human cancers, are produced in meats cooked at high temperature. The aim of this study was to determine the HCA content in cooked meat products (beef, chicken, pork, fish) prepared by various cooking methods (pan frying, oven broiling, and oven baking at 170 to 230 °C) that are preferred by U.S. meat consumers. The primary HCAs in these samples were PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine) (1.49-10.89 ng/g), MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline) (not detected-4.0 ng/g), and DiMeIQx (2-amino-3,4,8-trimethyl-imidazo [4,5-f]quinoxaline) (not detected-3.57 ng/g). Type and content of HCAs in cooked meat samples were highly dependent on cooking conditions. The total HCA content in well-done meat was 3.5 times higher than that of medium-rare meat. Fried pork (13.91 ng/g) had higher levels of total HCAs than fried beef (8.92 ng/g) and fried chicken (7.00 ng/g). Among the samples, fried bacon contained the highest total HCA content (17.59 ng/g).     

Effects of precursor composition and water on the formation of heterocyclic amines in meat model systems

Model systems based on pressed meat from ox, pork and chicken were used to study the formation of carcinogenic/mutagenic heterocyclic amines (HAs). The composition of precursors (free amino acids, creatine and glucose) was examined and samples were heated in test-tubes under wet and dry conditions at 175 and 200°C for 30 min. Several HAs were detected, and the formation of DMIP (2-amino-1,6-dimethylimidazo[4,5-b]-pyridine), TMIP (2-amino-1,5,6-trimethyl-imidazo[4,5-b]-pyridine), IFP (2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]-pyridine) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine) was found to be favoured by dry heating conditions. Highest amounts of PhIP and IFP were detected in heated meat juice from chicken breast, while more MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline) was found in heated meat juices from roast beef and pork chop. Norharman (9H-pyrido[3,4-b]-indole) and Harman (1-methyl-9H-pyrido[3,4-b]-indole) were also detected at high levels.     

Effect of enhancement on the formation of heterocyclic amines in cooked pork loins: Preliminary studies

Heterocyclic amines (HCAs) which are produced in meats cooked at high temperature a risk factor for certain human cancers. This study evaluated the effect of enhancement on HCA formation in cooked pork loins. Three samples of pork loin were prepared including non-injected loin, 12% water-injected loin, and 12% salt/phosphate injected loin. The HCAs were identified in all samples: PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine), MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline), and DiMeIQx (2-amino-3,4,8-trimethylimidazo [4,5-f]quinoxaline). Injection of salt/phosphate significantly reduced the level of PhIP by 42.7%, MeIQx by 79.0%, and DiMeIQx by 75.0%. Enhancement with water alone did not reduce HCA formation.     

Formation of heterocyclic amine–amino acid adducts by heating in a model system

Although mutagenic and carcinogenic heterocyclic amines (HCAs) are known to be formed in cooked meat and fish, human HCA exposure and carcinogenic risk have not been elucidated in sufficient detail. In this work, we investigated the formations of HCA–amino acid adducts in a model system by using a liquid chromatography–mass spectrometry to elucidate another source of human HCA exposure. The 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) adduct with glycine was formed easily by heating at 200 °C within 5 min, which is probably based on the dehydration condensation of the amino group of PhIP and carboxyl group of glycine. PhIP and other HCAs such as 2-amino-3-methyl-3H-imidazo[4,5-f]quinolone, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline and 3-amino-1,4-dimethyl-5H-pyrido[3,4-b]indole, also bound with various amino acids by heating. Among these amino acids, proline tends to form adducts with HCAs, but serine, cysteine and lysine hardly bound with HCAs. These results provided a basic understanding of the formation of HCA adducts with amino acids during cooking.     

Formation of amino-imidazo-azaarenes and carbolines in fried beef patties and chicken breasts under different cooking conditions in Korea

The effect of cooking temperature and time on amino-imidazo-azaarenes (AIAs) and carbolines in fried ground beef patties and chicken breast under different cooking conditions in Korea was evaluated. Beef patties were fried at different temperatures (150, 180, and 230°C) for 4, 8, 12, and 16 min per each side and then the amount of AIAs and carbolines was evaluated by solid-phase extraction and HPLC-MS analysis. In fried ground beef patties, formations of 9H-pyrido [3,4-b]indole (Norharman) and 1-methyl-9H-pyrido [3,4-b]indole (Harman) were dramatically increased at 230°C for 16 min. Concentrations of Norhanrman and Harman formed at 230°C for 16 min/side were 12 and 40 times greater than level those of Norharman formed at same cooking condition. In fried chicken breasts, 2-amino-3,7,8-trimethylimidazo[4,5-f] quinoxaline (7,8-DiMeIQx) and 2-amino-3,4,7,8-tetramethylimidazo[ 4,5-f]quinoxaline (Tri-MeIQx) were not found at 150 and 180°C. Norhanrman formed at 230°C for 16 min was approximately 4 times higher than fried chicken breasts at 180°C. These results suggest that increase of cooking temperature and time was directly affected on AIAs and carbolines formation in Korean cooked meat.     

Influence of extra virgin olive oil on the formation of heterocyclic amines in roasted beef steak

In this study, heterocyclic anime (HCA) contents were monitored in commonly consumed pan-fried beefsteak based on the highest level of human exposure. Effect of addition of extra virgin olive oil (EVOO) on HCAs formation in fried beef steaks was evaluated. After EVOO was spread on the meat surface, the raw beef was cooked at 200°C for 5 min on each side. The HCAs were extracted from the meat samples and purified using a solid-phase extraction method and then analyzed by liquid chromatography-mass spectrometry (LC-MS). Among the 15 HCAs, 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), 9H-pyrido [3,4-b]indole (Norharman), 1-methyl-9H-pyrido [3, 4-b]indole (Harman), 2-amino-9H-pyrido [2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido [2,3-b]indole (MeAαC), 2-amino-3,8-dimethylimidazo [4,5-f]-quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) were detected in all of the cooked beefsteaks. HCAs formation was significantly reduced (p<0.05) when the EVOO was added to the beef prior to cooking. The addition of 2 and 4 g of EVOO considerably inhibited HCAs formation in the fried beefsteak. However, adding excess amounts of EVOO promoted some HCAs formation.     

Heterocyclic aromatic amines in fried poultry meat

 Heterocyclic aromatic amines are mutagenic compounds that are formed during heating of meat and fish. These substances are products of the reaction of creatine with amino acids and carbohydrates. It is recommended that exposure to these probable human carcinogens should be minimised. In fried boneless lean turkey breast meat five heterocyclic aromatic amines {2-amino-1-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3,8-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-phenylimidazo[4,5-b]pyridine (PhIP)} were found. The temperature regime which was applied for frying resulted in a surface temperature of about 140°C. Clean-up was done by acid-base partition followed by solid-phase extraction (SPE) using blue cotton. HPLC analysis was carried out using electrochemical detection for IQ- and IQx-type compounds and fluorescence detection for PhIP. The low temperatures used during frying yielded comparably lower amounts of heterocyclic aromatic amines. The concentrations of the aromatic amines were as follows: IQ 1.1 μg/kg, MeIQ 0.9 μg/kg, MeIQx μg/kg, 4,8-DiMeIQx 0.4 μg/kg, and PhIP 3.8 μg/kg. Received: 19 February 1997 / Revised version: 21 April 1997     

Analysis of heterocyclic aromatic amines by high resolution gas chromatography-mass spectrometry: a suitable technique for the routine control of food and process flavours

 Ten heterocyclic aromatic amines (HAA; [2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (1); 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (2); 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (3); 2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoline (4); 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoline (5); 2-amino-3,7,8-trimethyl-3H-imidazo[4,5-f]quinoline (6); 2amino-3,4,7,8-tetramethyl-3H-imidazo[4,5-f]quinoline (7); 2-amino-1-methyl-6-phenyl-imidazo[4,5-f]quinoline (8); 2-amino-6-methyldipyrido[1,2-α : 3′,2′-d]imidazole (9); 2-aminodipyrido[1,2-α : 3′,2′-d]imidazole (10)]) were analysed in commercially available meat products and process flavours. After sample preparation by Extrelut treatment, subsequent solid phase extraction applying propylsulphonic and C₁₈ silica cartridges, as well as derivatization with 3,5-bis-trifluoromethylbenzyl bromide, HRGC-electron-impact-ionization-MS (HRGC-EIMS) analysis in the selected ion monitoring mode was performed. Isotope dilution analysis with 2amino-8-methyl-3-(trideuteromethyl)-3H-imidazo[4,5- f]quinoxaline and 2-amino-1-(trideuteromethyl)-6-phenyl-imidazo[4,5-b]pyridine was used to quantify 4 and 8; for 1–3, 5–7, 9 and 10, standard addition was employed as the determination method. The detection limit of 1 ng/g evaluated for 3–6 and 9 was sufficient for routine analysis, i.e. to obtain an initial insight into the grade of a potential HAA contamination of food or process flavours. To obtain more detailed information, the previously developed, more sensitive technique of HPLC-electrospray-tandem-MS (HPLC-ESI-MS/MS) has to be used, as shown by the comparison of the data obtained by HRGC-MS and HPLC-ESI-MS/MS analyses. Received: 4 March 1999