The analysis of heterocyclic aromatic amines using high performance liquid chromatography

The optimum conditions for reversed-phase high performance liquid chromatography analysis of five mutagenic and carcinogenic heterocyclic aromatic amines (aminoazaarenes) were described. The following aminoazaarenes were chosen for the determination: 2-amine-3,4-dimethylimidazo[4,5-f]quinoline, 2-amine-3-methylimidazo[4,5-f]quinoline, 2-amine-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amine-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amine-1-methyl-6-phenylimidazo[4,5-b]pyridine. Three analytical columns: Synchropak RP-8, Vydac C 1 8 -300 and TSK gel ODS 80-T M , as well as several elution systems were tested using isocratic and gradient elution. The columns, mobile phases and elution conditions were assessed by comparing retention factor, separation factor and resolution values. It is demonstrated that the most selective separation of five aminoazaarenes tested was achieved with a TSK gel ODS 80-T M column equipped with a Supelguard Hypersil ODS precolumn and using the triethylamine-phosphate buffer (pH = 3.3) / acetonitrile as the mobile phase. The following gradient elution profile was applied: 5% acetonitrile and 95% buffer for 2 min initially, then linear increase to 25% acetonitrile within 20 min, then to 55% acetonitrile within 10 min and, finally, 55% acetonitrile for 20 min. Use of chemically bonded columns C 8 and C 1 8 did not yield separation of aminoazaarenes as good as in the case of TSK gel column. For these two types of columns the best separation was achieved using a procedure involving isocratic elution with mobile phase consisting of 10% acetonitrile in triethylamine-phosphate buffer (pH = 3.2). The selected optimum conditions of HPLC analysis by use of Vydac C 1 8 -300 and TSK-gel ODS 80-T M columns were applied for qualitative determination of aminoazaarenes fractions isolated from two meat samples: charcoal grilled chicken breast and pork fillet. In both samples the presence of five tested compounds was confirmed.