Measurement of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) in acid-hydrolyzed urine by high-performance liquid chromatography with flourescence detection

Heterocyclic aromatic amines are carcinogenic combustion products formed during the cooking of meat at moderate to high temperatures. The authors have developed a simplified method to measure the commonly formed heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), in acid-hydrolyzed human urine by HPLC with fluorescence detection after immuno-affinity chromatography. The method was compared with a previously validated method using gas chromatography/mass spectrometry after chemical derivatization. The comparison was performed using 100 urine samples from a study of 10 subjects before, during and after ingesting identical amounts of charbroiled (grilled) meat on 5 consecutive days. The overall correlation between the two assays was r = 0.87 (p < 0.0001). The HPLC method was then used to measure PhIP in acid-hydrolyzed urine collected from 66 subjects participating in a study of fried meat ingestion. Fried meat cooked at high temperature was fed to subjects based on body weight; urines were collected before feeding and 0-12 and 12-24 h after feeding. In a subset of six subjects, urinary PhIP concentration was 3-13 fold higher in the 0-12-h urine than in the 12-24-h urine. In the total study population, a significant correlation (r = 0.61, p < 0.0001) between the amount of fried meat ingested and urinary PhIP was observed. These results indicate that measurement of urinary PhIP by HPLC after immuno-affinity chromatography is a useful alternative to mass spectrometry methods for assessing recent exposure in subjects ingesting moderate to high levels of PhIP.