A gas chromatography-isotope dilution high-resolution mass spectrometry method for quantification of isomeric benzo[a]pyrene diol epoxide hemoglobin adducts in humans

We developed a gas chromatography-isotope dilution high-resolution mass spectrometry (GC-ID-HRMS) method for quantifying isomers of benzo[a]pyrene (BaP) tetrol metabolites resulting from hydrolysis of benzo[a]pyrene-diol-epoxide hemoglobin (BaPDE-Hb) adducts. Acid hydrolysis of BPDE-Hb adducts extracted from human blood samples yielded isomers of benzo[a]pyrene-tetrahydrotetrols, (±)-BaP-r-7,t-8,t-9,c-10-tetrol (BPTI-1), (±)-BaP-r-7,t-8,t-9,t- 10-tetrol (BPTI-2), (±)-BaP-r-7,t-8,c-9,t-10-tetrol (BPTII-1), and (±)-BaP-r-7,t-8,c-9,c-10-tetrol (BPTII-2). The isomeric BaP tetrols were isolated from the matrix by liquid-liquid extraction, and then further purified by solid-phase extraction. Following silylation with N-methyl-N-(trimethylsilyl) -trifluoroacetamide, the analytes were measured by GC-HRMS, using electron ionization. We have found detectable concentrations in the low fmol range for BPTII-1 and BPTI-1 in all donors tested. The mean BaP adduct levels for smoking donors (n = 9) were 0.022 fmol/mg hemoglobin for BPTII-1 and 0.070 fmol/mg hemoglobin for BPTI-1. The mean BaP adduct levels with hemoglobin for non smoking donors (n = 6) was 0.021 fmol/mg hemoglobin for BPTII-1 and 0.105 fmol/mg hemoglobin for BPTI-1.