Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry.

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.