Abstract
A high-performance liquid chromatographic assay for O 6-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 constituted 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. O 6-Benzylguanine peak heights were 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 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 O 6-benzylguanine.
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