Simultaneous determination of O 6-benzylguanine and 8-oxo-O 6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

Abstract

A high-performance liquid chromatographic assay for the quantification of O 6-benzylguanine (O 6BG) in human plasma was modified to include the metabolite, O 6-benzyl-8-oxo-guanine (8-oxo-O 6BG). O 6-( p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C 18 base-deactivated reversed-phase column. Separation was accomplished by gradient elution with mobile phases consisting of acetonitrile and phosphate buffer, pH 3.60. Eluted compounds were observed with diode array detection at 288 nm (O 6BG) and 292 nm (8-oxo-O 6BG). Standard curves were linear from 12.5 ng/ml to 1000 ng/ml, with an average regression coefficient of 0.999 ( n=5) for both compounds. The lowest limit of quantitation was 25 ng/ml, with a signal-to-noise ratio of 8:1. The within-day relative standard deviations for O 6BG quality control samples ( n=18) with concentrations of 735 ng/ml, 305 ng/ml and 38 ng/ml were 2.4%, 4.2% and 5.3%, respectively. The within-day relative standard deviations for 8-oxo-O 6BG quality control samples ( n=18) at concentrations of 735 ng/ml, 420 ng/ml and 42 ng/ml were 2.2%, 4.0% and 7.1%, respectively. The day-to-day relative standard deviations for the same control specimens were 3.1%, 4.8% and 7.1% for O 6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O 6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O 6-benzylguanine. O 6-Benzyl-8-oxo-guanine was identified in patient plasma specimens by liquid chromatography–electrospray mass spectrometry by comparison with spectral data acquired from reference material.