A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry

The long-range goal of the present study is the development of a general ap proach for in vivo dosimetry of reactive metabolites of polycyclic aromatic hydrocarbons (PAHs), to be used as a tool in cancer risk assessment. With benzo[a]pyrene (BaP) chosen as indicator and a model of PAHs this study aim s at the development of a method for the determination of adducts to histid ine (His) in hemoglobin (Hb) and serum albumin (SA) of reactive metabolites of BaP, The predominantly mutagenic metabolite of BaP has been shown to be a diolepoxide isomer, +(anti)r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetra hydrobenzo[a]pyrene (+BPDE). In comparison with other methods for protein d egradation, hydrazinolysis was found to be sufficiently effective and mild. The His adduct isolated after protein hydrazinolysis, with protection by t ert-butyloxycarbonyl (Boc) of the hydrazide and alpha-amino groups, was sho wn to be N-im-+/-(r-7,t-8,t-9-trihydroxy-7, 8,9,10-tetrahydrobenzo[a]pyren- c-10-yl)-N-alpha,N-2-bis(tert-butyloxycarbonyl)-L-histidinehydrazide., tide , Isomers of this compound, used as references, were synthesized and charac terized by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adduc ts in Hb and SA from in vitro treatment with BPDE were characterized after hydrazinolysis by HPLC-UV/MS, mu HPLC/MS/MS and gas chromatography/mass spe ctrometry (GC/MS), Approximately 70 and 10% of the isolated BPDE adducts fr om SA and Hb, respectively, were His adducts, Other products were released as BaP tetrols and BaP triols, For the purpose of enrichment/purification o f BPDE-His adducts, C-18 and cation exchange solid phase extraction (SPE) w ere utilized. The sensitivity obtained by this new approach, based on hydra zinolysis of protein, enrichment by SPE and analysis with mu HPLC/MS/MS (AP CI), is in the low-fmole range. Copyright (C) 2000 John Wiley & Sons, Ltd.