Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2‑Amino-1-methyl-6-phenylimidazo[4,5‑b]pyridine and Albumin in Humans

2-Amino-1-methyl-6-phenylimidazo­[4,5-b]­pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo­[4,5-b]­pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin in vitro primarily occurs at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography–mass spectrometry method to measure acid-labile albumin–PhIP adducts in human volunteers administered a dietary-relevant dose of 14C-labeled PhIP [Dingley, K. H., et al. (1999) Cancer Epidemiol., Biomarkers Prev. 8, 507–512]. Mild acid treatment of albumin (0.1 N HCl, 37 °C for 1 h) or proteolytic digestion with Pronase [50 mM ammonium bicarbonate buffer (pH 8.5) at 37 °C for 18 h] released similar amounts of covalently bound PhIP, which was characterized by multistage scanning and quantified by Orbitrap mass spectrometry. The amount of [14C]­PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2–21.3% of the [14C]­PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)­phenylimidazo­[4,5-b]­pyridine, a hydrolysis product of the Cys34 S–N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease-treated samples. These findings suggest that a portion of the PhIP bound to albumin in vivo probably occurs as an acid-labile sulfinamide adduct formed at the Cys34 residue.