Abstract
O6 -benzylguanine (O6 BG) is an inhibitor of O6 -alkylguanine-DNA alkyltransferase (AGT). It binds to AGT by transferring its benzyl moiety to the cysteine residue at the active site of the enzyme. O6 BG synergizes the cytotoxic effects of alkylating agents by halting AGT-mediated DNA repair. O6 -benzyl-8-oxoguanine (8-oxo-O6 BG) is a metabolite of O6 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 O6 BG and 8-oxo-O6 BG in human plasma. O6 BG and 8-oxo-O6 BG along with the analog internal standard, pCl-O6 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 O6 BG and 8-oxo-O6 BG were 1.25-250 ng/ml and 5.00-1.00 × 103 ng/ml, respectively, with acceptable assay accuracy, precision, recovery and matrix factor. This method was applied to the measurement of O6 BG and 8-oxo-O6 BG in patient plasma samples from a prior phase I clinical trial.
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