Mutagenic heterocyclic aromatic amines (HAAs) in 'processed food flavour' samples

Eight samples of 'processed food flavours' (PFFs), chosen from five different categories, were analysed for their mutagenic activity using the Ames Salmonella assay, and also for the presence of eight heterocyclic aromatic amines (HAAs), namely 2-amino-3-(trideuteromethyl)imidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx), 2-amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 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) using liquid chromatography and mass spectrometry (LC/MS). The isolation of HAAs was based on sequential liquid-liquid extraction procedures of samples at both acidic and basic pH values. The recoveries and the clean-up were monitored by introduction of quality control samples and by spiking with three tri-deuterated standards of HAAs. Although the results for the mutagenicity assay were comparable by testing less-purified and highly-purified extracts, the analysis for identification and quantification of HAAs by LC/MS required highly purified concentrates. Four samples had little or no mutagenic activity and these results were in agreement with their LC/MS results: they had no detectable levels (detection limits 1-3 ppb) of any of the HAAs monitored. The mutagenic activity of one sample was in complete agreement with the quantification of HAAs by LC/MS. Two samples produced strong mutagenic responses (3115 and 2664 revertants/g). In one sample, LC/MS analysis revealed the presence of 9.6 ppb IQ, whereas LC/MS of the other could not confirm the presence of any of the eight HAAs monitored. Two samples produced mild mutagenic activity (204 and 160 revertants/g), but relatively elevated concentrations of IQ (6.7 and 6.8 ng/g) by LC/MS. The extracts from all samples were tested for their modifying effects on mutagenicity of four HAAs. The discrepancy between the Ames test and the LC/MS analysis of some samples indicates several possibilities, such as the presence of some other HAAs, of their isomers or of other mutagens. In addition, the presence of mutagen modifiers (inhibitors or synergists) was observed in most samples. The results indicate that although chemical tests (e.g. LC/MS) can provide quantitative data for the HAAs monitored, the Ames mutagenicity test should also be conducted to determine the mutagenic activities of PFFs, in order to assess their health risk potential.