Simultaneous determination of O6-benzylguanine and 8-oxo-O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of O6-benzylguanine (O6BG) in human plasma was modified to include the metabolite, O6-benzyl-8-oxo-guanine (8-oxo-O6BG). O6-(p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C18 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 (O6BG) and 292 nm (8-oxo-O6BG). 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 O6BG 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-O6BG 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 O6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine. O6-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.

Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.