ANALYSIS OF CARCINOGENIC HETEROCYCLIC AMINES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Separation and determination of mutagenic and carcinogenic heterocycli c amines by a reversed phase isocratic HPLC method is here in describe d. Five of them, namely 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 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-3,4,7,8-te tramethylimidazo[4,5-f] quinoxaline (4,7,8-TriMeIQx) and 2-amino-1-met hyl-6-phenylimidazo[4,5-b] pyridine (PhIP) were encountered in this st udy. Separation was accomplished on a reverse phase HPLC column of Sup elcosil LC-8 equipped with a Supelguard LC-8 precolumn, at a flow rate of 2.0 mL/min. IQ, MeIQx and 4,s-DiMeIQx were resolved using 15% (v/v ) acetonitrile in 50 mM triethylamine-phosphate buffer, pH 3.2, wherea s 4,7,8-TriMeIQx and PhIP using 30% (v/v) acetonitrile in the same buf fer. Detection of the eluted heterocyclic amines was performed at 263 nm for IQ, MeIQx and 4,8-DiMeIQx and at 254 nm for 4,7,8-TriMeIQx and PhIP. Reproducibility tests measuring peak areas gave a relative stand ard deviation of 1.8-4.4%. The calibration graphs for all five amines injected into the column were linear up to approximately 2.0 mu mol an d the detection limits (signal-to-noise ratio 2:1) ranged from 10 to 3 0 pmol. The high sensitivity of the proposed method permits the accura te and reproducible determination in mixtures containing only a few ng /mL of such helerocyclic amines.