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     

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).     

Binding of heterocyclic amines by yeast cells and their fractions

The binding of mutagenic pyrolysis products to cells of 50 yeast strains and their cell fractions was investigated. Cells of all yeast strains effectively bound 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Cell walls (CW), and cell wall glucan and mannan (5 mg in each case) showed the highest binding of Trp-P-1 (50 μg ml^?1); glucan adsorbed virtually all of the Trp-P-1. Cytoplasm also showed some binding but was much less effective. Glucans also bound well with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo 4,5-quinoxaline (MeIQX) much more than CW, but 2-amino-5-phenylpyridine (Phe-P-1) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MelQ) were not effectively bound. The quantity of IQ, MeIQ, Phe-P-1 and MeIQX bound was dependent on the strain of yeast. The mutagenic pyrolysis products bound to cells were effectively extracted by aqueous methanol, ammonia (50 g litre^?1) and alcohol, but not by water. The binding was pH dependent and inhibited by metal salts. When yeast cells were heated to 100° for 15 min, the binding of Trp-P-1 decreased by about 30% but Saccharomyces cerevisiae 50 heated to 100° did not differ much from untreated cells in its binding ability.     

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.     

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.     

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     

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。     

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 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     

植物多酚抑制热加工肉制品中杂环胺形成机制研究进展

杂环胺（heterocyclic amines,HAs）是肉制品热加工过程中产生的一类具有极强致畸致癌活性的有毒化合物,其中2-氨基-3-甲基咪唑并[4,5-f]喹啉（2-amino-3-methyl-imidazo[4,5-f]-quinoline,IQ）和2-氨基-3,4-二甲基咪唑并[4,5-f]喹啉（2-amino-3,4-dimethyl-imidazo[4,5-f]-quinoline,MeIQ）,2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine,PhIP）,1-甲基-9H-吡啶并[3,4-b]吲哚（1-methyl-9Hpyrido[3,4-b]indole,Harman）和9H-吡啶并[3,4-b]吲哚（9H-pyrido[3,4-b]indole,Norharman）3类HAs在热加工肉制品中较为常见且分布广泛。本文即围绕上述3类HAs,简要分析其形成的途径及机制,并详细阐述外源添加植物多酚抑制HAs形成的作用机制,最后依据现有研究归纳总结出多酚化合物抑制HAs与其化...     

Lack of formation of heterocyclic amines in fumes from frying French fries

The formation of heterocyclic amines (HAs) in the fumes from frying French fries in soybean oil or lard was studied. A high-pressure liquid chromatography method was used to determine the various HAs in fumes. Results showed that the yields of fumes produced from soybean oil when heated alone for 2 or 4 h were higher than from lard; however, a reversed trend was found when frying French fries in soybean oil and lard. Most fumes from soybean oil and lard while frying French fries were adsorbed onto the condensation apparatus, while the other portions were adsorbed onto the wool and glass beads, which were incorporated in our experimental design for collecting the fumes. The fumes from soybean oil when heated alone were found to contain three HAs, namely, 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), and 1-methyl-9H-pyrido[4,3-b ]indole (Harman), whereas two more HAs, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b ]indole (Trp-P-1), were generated in lard. Lard was more susceptible to the formation of HAs than soybean oil when both were heated alone. No HAs were detected in the fumes from French fries fried in soybean oil and lard.     

Binding of mutagens by fractions of the cell wall skeleton of lactic acid bacteria on mutagens

The binding effect of cells and cell fractions, cell wall skeleton, cytoplasm, whole cells, and cell wall skeleton treated by lysozyme and alpha-amylase at 37 degrees C for 5 h, on Trp-P-1 (3-amino-1,4-dimethyl-[5H]pyrido [4,3-b]indole) and Trp-P-2 (3-amino-1-methyl-[5H]-pyrido[4,3-b]indole) were investigated. The cell and cell wall skeleton of Streptococcus cremoris Z-25 had greater binding activity, but cytoplasm and extract of cell wall skeleton did not bind Trp-P-1 and Trp-P-2. When the cells or cell wall skeleton were treated with lysozyme and alpha-amylase, unbound Trp-P-1 and Trp-P-2 concentrations were greater than that of the untreated control. It is possible that cell walls may be involved in the binding of mutagenic pyrolyzates to lactic acid bacteria. The cell wall skeleton of S. cremoris Z-25, Lactobacillus acidophilus IFO 13951, and Bifidobacterium bifidum IFO 14252 showed binding of Trp-P-1, 2-amino-6-methyldipyrido(1,2-a:3',2'- d)imidazole, 2-amino-5-phenylpyridine, 2-amino-3-methylimidazo(4,5-f)quinoline, 2-amino-3,4-dimethylimidazo(4,5-f) quinoline, and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline. The cell wall skeleton of S. cremoris group and Streptococcus lactis also showed the binding activity with A N-nitrosodimethylamine. The binding of Trp-P-1 to cell walls was very high, and the binding of mutagenic pyrolyzates was variable with different bacterial species. The peptidoglycan complex and polysaccharides liberated from cell wall skeleton of S. cremoris Z-25 showed strong binding of Trp-P-2. Peptidoglycans has a binding effect of about 19.86 micrograms/mg; polysaccharides had a binding effect of 14 micrograms/mg.     

Comparative mutagenicity studies on fried minced beef,grilled sausage and grilled hamburgers

The mutagenicity of the basic fractions of fried beef patties, grilled hamburgers and grilled sausages was determined in the Ames test using activation systems (S9-mix) derived from untreated and Aroclor-pretreated rats and hamsters, and from rats pretreated with 3-methylcholanthrene or phenobarbitone. In order to indicate whether the mutagenic activity was due to the presence of thermally generated amino imidazo aza-arenes of the type previously identified in heated meat products (i.e. 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quin-oxaline), these compounds were assayed under the same conditions. The mutagenicity of the basic fractions of the cooked foods and of the three aza-arenes varied with the activation system used. The activation patterns of the basic fractions from fried beef and grilled sausages resembled that of IQ, whereas that of grilled hamburgers was more similar to that of MeIQ. Interestingly the total mutagenic activity of fried beef patties was greater than that of grilled hamburgers.     

花椒叶提取物对烤牛肉饼杂环胺形成的影响

以牛肉和花椒叶为研究对象,研究花椒叶提取物（Zanthoxylum bungeanum Maxim. leaf extract,ZME）添加量（0.015%、0.030%、0.045%）对烤牛肉饼中杂环胺（heterocyclic amines,HAs）形成的影响。结果表明,与对照组相比,ZME能够显著抑制烤牛肉饼中总HAs的形成（P＜0.05）,对其最大抑制率为39.87%,但对不同种类HAs形成的影响不同。其中,添加0.045% ZME对2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶、2-氨基-3-甲基咪唑并[4,5-f]喹啉、2-氨基-3,4-二甲基咪唑并[4,5-f]喹啉、1-甲基-9H-吡啶并[3,4-b]吲哚和2-氨基-9H-吡啶并[2,3-b]吲哚的抑制率分别达到71.76%、78.02%、49.07%、35.82%和100%;而0.045% ZME却促进2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉和9H-吡啶并[3,4-b]吲哚的形成。进一步分析ZME对烤牛肉饼中HAs前体物的影响,结果显示前体物的消耗随着ZME添加量的增加呈降低趋势,相关性分析表明HAs的形成与多种游离氨基酸的消耗显著相关。上述结果表明ZME能显著抑制烤牛肉饼中HAs的形成,为其提高加工食品安全性及更广泛的应用提供理论依据。     

Effects of beetroot (Beta vulgaris) preparations on the Maillard reaction products in milk and meat-protein model systems

The effects of beetroots (Beta vulgaris) on the formation of Maillard reaction (MR) products possessing health, nutritional and sensory implications were studied. The effect of dried beetroot juice on the formation of N^ε-(carboxymethyl)lysine (CML) and N^ε-(2-furoylmethyl)-L-lysine (furosine) was determined in a milk model system. Beetroot juice reduced furosine formation more than CML, inferring that betalain compounds present in the juice are more effective in reducing the formation of MR products in the early stage than in the advanced stage of MR. Beetroot water extract was fractionated on Sephadex LH20 and obtained three beetroot fractions were used to assess their effect on the formation of heterocyclic amines in a meat-protein model system. Beetroot fraction possessing the highest antioxidant capacity and containing the highest betalain content reduced the amounts of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), 2-amino-3,8-dimethyl-imidazo-[4,5-f]-quinoxaline (MeIQx), and 2-amino-3-methylimidazo-[4,5-f]-quinoline (IQ) by approximately 60, 77 and 87%, respectively. Beetroot preparations were characterized by ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC–Q-TOF–MS/MS). The antioxidant activities of beetroot preparations were also evaluated by 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS⁺) scavenging capacity, oxygen radical absorbance capacity (ORAC) and total phenolic compound assays. Our findings could be useful for creating novel source of functional ingredients exerting anti-carcenogenic and antiglycation activities.     

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.     

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 %.     

FORMATION OF HETEROCYCLIC AMINES IN MEAT EMULSION EXTENDED WITH SOY PROTEIN

Formation of heterocyclic amines (HAs) during heating of meat emulsions at selected temperatures was studied to determine the influence of soy protein and fat contents on yields of HAs. Meat emulsion with two different fat contents, 6% (low) and 19% (high), and with two soy protein contents, 0% (none) and 10%, were used in the study. The emulsions were heated at temperatures of 180 and 200C for times varying from 0 to 40 min. HAs namely IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline), MelQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), and PhIP (2-amino-1-methy-6-phenylimidazo[4,5-b]pyridine) were identified and quantified in the meat emulsions. Concentrations of IQx were from 0 to 8.26 ng/g of emulsion; MeIQx concentrations were from 0 to 14.73 ng/g and PhIP concentrations were from 0 to 2.48 ng/g. Yields of the HAs depended on temperature, time, soy protein and fat contents. Formation of the HAs in the meat emulsions after an initial lag period followed first order kinetics. Increasing soy protein content decreased the maximum concentrations of IQx and MeIQx formed at the different heating condition.     

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.     

Inhibitory effect of mixture herbs/spices on formation of heterocyclic amines and mutagenic activity of grilled beef

ABSTRACT

Natural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p < 0.05) diminished mutagenicity of marinated grilled beef in bacterial Ames test.