DETERMINATION OF O-6-BENZYLGUANINE IN HUMAN PLASMA BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A high-performance liquid chromatographic assay for O-6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography h as been developed. Plasma samples were alkalinized, extracted into eth yl acetate, evaporated, and the residues were reconstituted and chroma tographed. Separation was accomplished by gradient elution with a mobi le phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. flut ed compounds were detected spectrophotometrically at 280 nm. Sample qu antitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O-6-Benzylguanine peak heights w ere compared to peak heights of O-6-(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 con trol samples were determined with a day-to-day relative standard devia tion of 7 and 8%, respectively (n = 18). The within-day relative stand ard deviations were 2.7 and 3.0% (n = 18) for the high and low concent ration quality control specimens, respectively. Sample quantitation wa s reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This metho d was applied to plasma samples obtained from patients in a clinical t rial of O-6-benzylguanine.