An LC-MS/MS method for determination of O -benzylguanine and its metabolite O -benzyl-8-oxoguanine in human plasma.

O -benzylguanine (O BG) is an inhibitor of O -alkylguanine-DNA alkyltransferase (AGT). It binds to AGT by transferring its benzyl moiety to the cysteine residue at the active site of the enzyme. O BG synergizes the cytotoxic effects of alkylating agents by halting AGT-mediated DNA repair. O -benzyl-8-oxoguanine (8-oxo-O BG) is a metabolite of O BG, which is an equally potent inhibitor of AGT. In this work, we report the development and validation of an LC-MS/MS method for simultaneous determination of O BG and 8-oxo-O BG in human plasma. O BG and 8-oxo-O BG along with the analog internal standard, pCl-O BG, were extracted from alkalinized human plasma by liquid-liquid extraction using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 ml/min. Quantification was performed using multiple-reaction-monitoring mode with positive ion-spray ionization. The linear calibration ranges of the method for O BG and 8-oxo-O BG were 1.25-250 ng/ml and 5.00-1.00 × 10 ng/ml, respectively, with acceptable assay accuracy, precision, recovery and matrix factor. This method was applied to the measurement of O BG and 8-oxo-O BG in patient plasma samples from a prior phase I clinical trial.