Analysis Of Heterocyclic Amines Research Articles

Content, causes and analysis of heterocyclic amines in Chinese traditional braised chicken

ABSTRACTBraised chicken is a popular traditional chicken product in China with a special and complicated culinary method. However, there has been little research focused on the food safety problems caused by hazardous compounds in braised chicken. In this study, the contents of heterocyclic amines (HCAs) in representative braised chicken samples were determined. Total HCAs in braised chicken were relatively high (6.20–27.7 ng g−1), and aminoimidazoazaarenes (AIAs) took a large proportion (54.0–86.4%). 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was found in all samples with a high level (2.72–13.5 ng g−1), whilst 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was low (0.14–0.41 ng g−1). 9H-pyrido[3,4-b]indole (Norharman) and 1-methyl-9H-pyrido[3,4-b]indole (Harman) were main components in aminocarbolines (ACs). So the potential safety risk in braised chicken caused by high HCAs content should be paid attention. Principal component analysis (PCA) was used to analyse and visualise the differences of HCAs levels among braised chicken samples. The result indicated that the differences among these samples were significant, but there were no obvious similarities between samples produced in the same regions. Moreover, there were rare significant positive correlations between chemical components (including moisture, fat, protein, creatine and free amino acids) and hazardous compounds. Considering PCA and correlation analysis results, these parameters may play a major role in the HCAs levels in Chinese traditional braised chicken.

Rapid determination of heterocyclic amines in ruminant meats using accelerated solvent extraction and ultra-high performance liquid chromatograph–mass spectrometry

Cooking techniques such as grilling confer several benefits to meat during food preparation including improved palatability, digestibility, preservation, and safety, as well as enhancing the sensory characteristics and net nutritional gain. However, grilling can lead to the formation of harmful compounds such heterocyclic amines (HCAs). HCAs are potent carcinogenic and mutagenic nitrogen containing compounds produced during certain cooking conditions of protein rich foods. Dietary intake of HCAs is associated with increased risk factors for cancers in humans. As such, there is overwhelming interest in identifying improved methods for rapid and accurate determination of heterocyclic amines in food matrices that is sensitive and avoids exhaustive sample preparation steps. Herein, we describe an approach that involves first extracting HCAs by pressurized accelerated solvent extractor using methanol as solvent, followed by addition of internal standard and quantification of HCAs by ultra-high performance liquid chromatography-high resolution accurate mass spectrometric detection (UHPLC-HRAMS). This method is fast, accurate, reproducible and does not require exhaustive sample pre-treatments prior to UHPLC-HRAMS analysis compared to existing/traditional methods for HCA analysis.•The method is automated, fast and uses tunable pressurized liquid extractor to selectively extract HCAs•Method does not require exhaustive cleanup and preconcentration steps prior to UHPLC/HRAMS analysis of HCAs•Validation showed method to be accurate, precise, and useful for routine multi-sample HCA analyses

Analysis of heterocyclic amines in meat products by liquid chromatography – Tandem mass spectrometry

Heterocyclic amines (HCAs), a class made up of more than 25 compounds, are unintended hazardous substances that are generated by the heating or processing of proteinaceous foods at high temperatures. The International Agency for Research on Cancer (IARC) has classified four such HCAs (IQ, MeIQ, MeIQx, and PhIP) as being probable or possible human carcinogens. In this study, two sample preparation strategies, liquid–liquid extraction (LLE) with solid-phase extraction (SPE) and a rapid, easy, cheap, effective, rugged, and safe extraction (QuEChERS) method, were investigated for the determination of 11 types of HCAs in meat products by LC-MS/MS. The HCAs in the samples were first extracted with acetonitrile by LLE, and followed by SPE. In the case of QuEChERS extraction, acetonitrile is used as the LLME solvent, and PSA, C18EC and MgSO4 were used as the dSPE sorbent. Both methods showed good performance with respect to precision (RSD < 15.15%), accuracy (79.80–117.64%), recovery (52.39–116.88%), limit of quantitation for a spiked meat extract (0.01–10 ppb) and correlation coefficients (>0.993). The QuEChERS extraction strategy provided a better linear dynamic range and superior sensitivity in comparison with the LLE-SPE approach. HCAs were successfully quantified in real samples using the two proposed approaches by LC-MS/MS.

[Advances on sample pretreatment and analytical method for the analysis heterocyclic amines in meat products]

Heterocyclic amines (HAs) are considered as highly potential mutagens and carcinogens in cooked meat products. Efficient sample pretreatment as well as sensitive analytical method is very important for the determination of HAs in complex samples. In this paper, some sample pretreatment methods such as solvent extraction, solid-phase extraction, and solid- phase microextraction as well as analytical methods including liquid chromatography, and liquid chromatography-mass spectrometry are reviewed; The progress of HAs analysis is also prospected. A total of 51 references are cited.

Analysis of heterocyclic amines in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography−tandem mass spectrometry

Mutagenic and carcinogenic heterocyclic amines (HCAs) are formed during heating of various proteinaceous foods, but human exposure to HCAs has not yet been elucidated in detail. To assess long-term exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in hair by automated on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Using a Zorbax Eclipse XDB-C8 column, 16 HCAs were analyzed within 15min. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40μL sample at a flow rate of 200μLmin−1 using a Supel-Q PLOT capillary column as an extraction device. The extracted HCAs were easily desorbed from the column by passage of the mobile phase, with no carryover observed. This in-tube SPME LC–MS/MS method showed good linearity for HCAs in the range of 10–2000pgmL−1, with correlation coefficients above 0.9989 (n=18), using stable isotope-labeled HCA internal standards. The detection limits (S/N=3) of 14 HCAs except for MeAαC and Glu-P-1 were 0.10–0.79pgmL−1. This method was successfully utilized to analyze 14 HCAs in hair samples without any interference peaks, with quantitative limits (S/N=10) of about 0.17–1.32pgmg−1 hair. Using this method, we evaluated the exposure to HCAs in cigarette smoke and the suitability of using hair HCAs as exposure biomarkers.

Plant Extracts, Spices, and Essential Oils Inactivate Escherichia coli O157:H7 and Reduce Formation of Potentially Carcinogenic Heterocyclic Amines in Cooked Beef Patties

Meats need to be heated to inactivate foodborne pathogens such as Escherichia coli O157:H7. High-temperature treatment used to prepare well-done meats increases the formation of carcinogenic heterocyclic amines (HCAs). We evaluated the ability of plant extracts, spices, and essential oils to simultaneously inactivate E. coli O157:H7 and suppress HCA formation in heated hamburger patties. Ground beef with added antimicrobials was inoculated with E. coli O157:H7 (10(7) CFU/g). Patties were cooked to reach 45 °C at the geometric center, flipped, and cooked for 5 min. Samples were then taken for microbiological and mass spectrometry analysis of HCAs. Some compounds were inhibitory only against E. coli or HCA formation, while some others inhibited both. Addition of 5% olive or apple skin extracts reduced E. coli O157:H7 populations to below the detection limit and by 1.6 log CFU/g, respectively. Similarly, 1% lemongrass oil reduced E. coli O157:H7 to below detection limits, while clove bud oil reduced the pathogen by 1.6 log CFU/g. The major heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were concurrently reduced with the addition of olive extract by 79.5% and 84.3% and with apple extract by 76.1% and 82.1%, respectively. Similar results were observed with clove bud oil: MeIQx and PhIP were reduced by 35% and 52.1%, respectively. Addition of onion powder decreased formation of PhIP by 94.3%. These results suggest that edible natural plant compounds have the potential to prevent foodborne infections as well as carcinogenesis in humans consuming heat-processed meat products.

Hollow fibre‐supported liquid membrane extraction and LC‐MS/MS detection for the analysis of heterocyclic amines in urine samples

Heterocyclic amines (HCAs) are potent mutagens/carcinogens to which humans are frequently exposed through the consumption of cooked meat and fish food. The effect of normal intake of HCAs and their role in the aetiology of human cancer is unknown. To some extent, limitations of the existing analytical methods in monitoring the low levels of HCAs in biological samples have hindered obtaining conclusive results. In this study, a method for the analysis of HCAs in human urine has been studied to detect HCAs and metabolites at levels resulting from consumption of food cooked at ordinary conditions. The analytical method consisted of extraction and clean-up by the novel technique liquid-phase microextraction combined with LC-MS/MS. The effect of pH during the extraction and hydrolysis step was examined. High sensitivity was achieved when the extraction was performed in raw urine adjusted to pH 5.5, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine being detected from 2 pg/g urine, levels comparable with a normal exposure. Good reproducibility and repeatability was obtained for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, below 9% using isotopic dilution. The performance of the method on 9H-pyrido[3,4-b]indole, 2-amino-1-methyl-6-(4'-hydroxyphenyl)imidazo[4,5-b]pyridine and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine was also studied.

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.

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 – C 18. 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.

Dietary intake of heterocyclic amines in relation to socio-economic, lifestyle and other dietary factors: estimates in a Swedish population

To estimate the dietary intakes of heterocyclic amines (HCAs), to examine the intakes in relation to socio-economics, lifestyle and other dietary factors and to compare the classification of subjects by intake of HCA versus intake of meat and fish. Cross-sectional analysis within the Malmö Diet and Cancer (MDC) cohort. Data were obtained from a modified diet history, a structured questionnaire on socio-economics and lifestyle, anthropometric measurements and chemical analysis of HCAs. HCA intake was cross-classified against meat and fish intake. The likelihood of being a high consumer of HCAs was estimated by logistic regression analysis. Dietary intakes were examined across quintiles of HCA intake using analysis of variance. Baseline examinations conducted in 1991-1994 in Malmö, Sweden. A sub-sample of 8599 women and 6575 men of the MDC cohort. The mean daily HCA intake was 583 ng for women and 821 ng for men. Subjects were ranked differently with respect to HCA intake compared with intake of fried and baked meat and fish (kappa = 0.13). High HCA intake was significantly associated with lower age, overweight, sedentary lifestyle and smoking. Intakes of dietary fibre, fruits and fermented milk products were negatively associated with HCA intake, while intakes of selenium, vegetables, potatoes, alcohol (among men) and non-milk-based margarines (among women) were positively associated with HCA intake. The estimated daily HCA intake of 690 ng is similar to values obtained elsewhere. The present study suggests that lifestyle factors (e.g. smoking, physical activity, fruit and vegetable intakes, and types of milk products and margarines) may confound associations between HCA intake and disease. The poor correlation between HCA intake and intakes of fried meat and fish facilitates an isolation of the health effects of HCAs.

Analysis of heterocyclic amines in chicken by liquid chromatography with electrochemical detection

The present paper describes a method based on liquid chromatography with electrochemical detection (LC–ECD) for the determination of heterocyclic amines (HAs) in food samples. In order to obtain extracts clean enough to quantify heterocyclic amines by liquid chromatography with electrochemical detection, an additional purification step based on a cationic exchanger (CBA) as sorbent was included in a clean-up performed by several solid-phase extraction steps. Two different mobile phases, acetonitrile–50 mM ammonium acetate (10:90) at pH 5.25, and acetonitrile–50 mM ammonium acetate (30:70) at pH 6.0, were used for the LC separation of polar and less polar amines. Optimum working potentials were +1000 and +950 mV for polar and less polar HAs, respectively. The obtained figures of merit showed detection limits of 5–79 pg for standards, 2–30 ng g −1 for beef extract and 0.3–2.9 ng g −1 for fried chicken sample, and day-to-day precisions of 1.2–7.6% for standards and 2.8–10.4% for fried chicken sample. The applicability of the LC–ECD method has been demonstrated by the determination of the amines in griddled and fried chicken.

Analysis of heterocyclic amines in food products: interlaboratory studies

A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g −1 were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10 ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC–MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples.

ANALYSIS OF HETEROCYCLIC AMINES IN MAINSTREAM CIGARETTE SMOKE USING A NEW NCI GC-MS TECHNIQUE

A new procedure for the analysis of heterocyclic amines (HAs) in cigarette mainstream smoke condensate was developed. The procedure is based on two step derivatization with heptafluorobutyric anhydride (HFBA) and N,N-dimethylformamide dimethyl acetal, followed by GC-MS analysis using negative chemical ionization (NCI) and single ion monitoring (SIM) detection. Using this procedure 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) can be analyzed at levels as low as 0.5 ng/cig. Besides IQ and PhiP, the presence of six other HAs in particulate phase smoke was investigated. The technique is not sensitive enough for the analysis of 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1). The levels of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6- methyldipyrido[1,2-α:3′2′-d]imidazole (Glu-P-1) and 2-amino- dipyrido[1,2-α:3′2′-d]imidazole (Glu-P-2) were below the estimated detection limit of the procedure.

Development of In-tube solid-phase microextraction/Liquid chromatography/electrospray ionization mass spectrometry for the analysis of mutagenic heterocyclic amines

In-tube solid-phase microextraction (in-tube SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was developed for the analysis of mutagenic heterocyclic amines. LC/MS analyses of heterocyclic amines were initially performed by liquid injection onto a LC column. All heterocyclic amines tested in this study gave very simple ESI mass spectra and strong signals corresponding to [M+H]+ were observed for all heterocyclic amines except for Glu-P-1 and Glu-P-2 ([M+NH4−H2O]+ ions were observed from these amines). All heterocyclic amines were well separated with a Supelcosil LC-CN column except for AαC and Glu-P-2 which consistently co-eluted. In order to optimize the extraction of heterocyclic amines several in-tube SPME parameters were examined using five representative compounds. The optimum extraction conditions were as follows: 10 aspirate/dispense steps of 30 μL of sample in 100 mM Tris-HCl (pH 8.5) at the flow the flow rate of 100 μL min−1 with Omegawax 250 capillary column. The heterocyclic amines extracted by the capillary column were easily desorbed by aspiration of 30μL of methanol prior to injection, and carryover of heterocyclic amine was not observed. The calibration curves obtained from the ratios of heterocyclic amine area counts against that of 4,7,8-TriMeIQx as an internal standard were linear in the range of 5 to 200 ng mL−1, with correlation coefficients above 0.9976 (n=18). The detection limits (S/N=3) were 0.2–3.1 ng mL−1. This method was successfully applied to the analysis of food samples.

Analysis of heterocyclic amines as their N-dimethylaminomethylene derivatives by gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method was developed for the determination of heterocyclic amines (HCAs) by gas chromatography (GC). HCAs were converted into their N-dimethylaminomethylene derivatives and measured by GC with nitrogen-phosphorus selective detection using two connected fused-silica capillary columns containing DB-1 and DB-17ht. The derivatives of the 10 HCAs were sufficiently volatile and stable to give single symmetrical peaks. The calibration curves for HCAs in the range 0.5–10 ng were linear with correlation coefficients being above 0.998. The detection limits at a signal-to-noise ratio of 3 were ca. 2–15 pg per injection.