Extraction and purification of depurinated benzo[a]pyrene-adducted DNA bases from human urine by immunoaffinity chromatography coupled with HPLC and analysis by LC/quadrupole ion-trap MS

In this paper, we describe implementation and testing of an immunoaffinity (IA) column for rapid and selective extraction of 7-(benzo[alpha]pyren-6-yl)adenine (BP-6-N7Ade) and 7-(benzo[alpha]pyren-6-yl)guanine (BP-6-N7Gua) from urine, where BP is benzo[alpha]pyrene. The BP radical cation is a carcinogenic metabolite that reacts with double-stranded DNA, producing depurinated BP-adducted DNA bases excreted in urine. The expected modified nucleobases are BP-6-N7Gua, BP-6-N7Ade, and 8-(benzo[alpha]pyren-6-yl)guanine (BP-6-C8Gua), and they may serve as important biomarkers for DNA damage by PAHs. IA extracts of urine from a cigarette smoker and a nonsmoker contained less than 5% of contaminants present in Sep-Pak extracts and, unlike the latter, were suitable for analytical HPLC. IA extraction achieved 75-95% recovery of BP-6-N7Gua (10 fmol/mL) and BP-6-N7Ade (1 fmol/mL) added to urine samples. Tandem mass spectrometry of IA/HPLC fractions of urine from two coal smoke-exposed women at high risk for lung cancer demonstrated the presence of 20 and 50 fmol BP-6-N7Gua per mL of urine. Unexposed controls were negative. With proposed modifications, the IA-based protocol can achieve a detection limit of 0.1 fmol/mL urine, which is sufficient for routine quantification of BP-adducted bases in urine of cigarette smokers. This procedure may allow screening of persons at risk for lung cancer associated with exposure to PAH in cigarette and other forms of smoke.