Analysis of nonpolar heterocyclic amines in cooked foods and meat extracts using gas chromatography–mass spectrometry
Introduction
Our diet plays an important role in the prevention but also in the cause of diseases. We are exposed to complex mixtures of compounds which meet nutritional demands as well as some compounds which may constitute health risks. One class of hazardous substances consists of heterocyclic amines, which are predominantly formed during cooking of meat and fish. About 20 years ago, these substances were identified as potent mutagens in the Ames/Salmonella test 1, 2. Many heterocyclic amines have been identified and synthesised and have been shown to cause tumours in animal experiments (for reviews see 3, 4, 5, 6, 7). Based on results from long-term animal studies on mice, rats and nonhuman primates 8, 9, 10, 11, 12several of these compounds have been classified as carcinogens [13]. Heterocyclic amines are primarily found in cooked muscle foods at low ng/g levels (for reviews see 14, 15). The low concentration of heterocyclic amines and the complex sample matrix of cooked foods make the analysis of these compounds very difficult. In most food surveys, only polar heterocyclic amines e.g. MeIQx, DiMeIQx and PhIP have been determined 16, 17, 18, 19, 20, 21. The presence of nonpolar heterocyclic amines (Trp-P-1, Trp-P-2, AαC and MeAαC) is not often reported. These compounds have been thought to be formed exclusively under extreme cooking conditions [22], and consequently not to be found in the Western diet [23]. However, Trp-P-1 and Trp-P-2, have been detected in broiled sardines, beef and chicken, meat extract and bouillon cubes 24, 25, 26, 27, 28, 29. AαC has been detected in barbecued or broiled beef, chicken, mutton, salmon, meat extract and bouillon cubes 12, 26, 28, 30, 31, 32, 33, 34, 35, 36and MeAαC in grilled beef, chicken and mutton [30].
Trp-P-1 and Trp-P-2 have been shown to induce liver tumours in mice and rats 4, 5, 37, 38, and Trp-P-2 is suspected in the etiology of cancer [39]. Furthermore, neonatal mice have been found to be particularly sensitive to heterocyclic amines 11, 40. When Trp-P-1 was administered to pregnant mice, increased levels of Trp-P-1 were observed in the foetal liver and gastrointestinal content, and Trp-P-1 was also bound to the pigmented parts of the foetal eyes [41]. The amino-α-carbolines AαC and MeAαC induce tumours in liver and the vascular system [[5], review], and recently MeAαC was shown to be metabolised by human cyt P4501A2 [42]. Simultaneous administration of five heterocyclic amines, including at least one of the aforementioned compounds, showed a synergistic effect on the carcinogenicity 43, 44, 45. Enhancement of the carcinogenesis in rats was also observed following simultaneous administration of ten heterocyclic amines at low doses [46]. Thus, the presence of nonpolar heterocyclic amines in cooked foods should not be ignored.
In a recently published study, foods prepared under household cooking conditions were analysed for heterocyclic amines using HPLC with UV detection [29]. Several of the food items were shown to contain the nonpolar heterocyclic amines Trp-P-1 and Trp-P-2, and the β-carbolines harman and norharman. AαC and MeAαC were only tentatively identified, as coeluting impurities made reliable identification impossible. These results led us to search more extensively for nonpolar heterocyclic amines in our subsequent experiments and to investigate alternative analytical methods. Here we present data on nonpolar heterocyclic amines in cooked foods and meat extracts using a new gas chromatography–mass spectrometry (GC–MS) method without derivatisation of the compounds.
Section snippets
Chemicals
All commercially available chemicals, reference compounds (Trp-P-1, Trp-P-2, AαC, MeAαC, harman and norharman) and solvents were of analytical or HPLC grade, and have been described previously [27]. According to the manufacturers, the chemical purity of the reference compounds was higher than 99%; this was also confirmed using HPLC with UV detection and multi-component analysis (Varian, PolyView, version 4.0) for each of the reference compounds. The concentrations of the different standard
Results and discussion
A chromatogram from the GC–MS analysis of a standard solution, containing all six nonpolar heterocyclic amines, together with their retention times, is shown in Fig. 1a. The GC–MS method with the detection of negative ions provides high chromatographic efficiency and the analytes can easily be separated from each other. Fig. 1b shows a chromatogram from the analysis of meat extract A. Table 1 presents the averages of duplicate determinations of the concentrations of Trp-P-1, Trp-P-2, AαC,
Abbreviations
Abbreviation Systematic name CAS registry number AαC 2-Amino-9H-pyrido[2,3-b]indole 26148-68-5 DiMeIQx 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline Harman 1-Methyl-9H-pyrido[3,4-b]indole 486-84-0 MeAαC 2-Amino-3-methyl-9H-pyrido[2,3-b]indole 68006-83-7 MeIQx 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline Norharman 9H-Pyrido[3,4-b]indole 244-63-3 PhIP 2-Amino-1-methyl-6-phenylimidazo[4,5-f]pyridine Trp-P-1 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole 62450-06-0 Trp-P-2 3-Amino-1-methyl-5H-pyrido[4,3-b]indole 62450-07-1
Acknowledgements
We thank Dr. T. Nielsen for introducing us to the gas chromatograph. A.S. is grateful to the Swedish Institute for a bilateral scholarship making his stay at Lund University possible. This study was financially supported by the Swedish Cancer Society (1824-B96-15XBB). This study has also been carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific programme, CT96-1080, `Optimisation of the Maillard reaction. A way to improve
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