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

Tammy L. Stefan, Stephen T. Ingalls, Stanton L. Gerson, James K V Willson, Charles L. Hoppel

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)331-338
Number of pages8
JournalJournal of Chromatography B: Biomedical Applications
Volume681
Issue number2
DOIs
StatePublished - Jun 7 1996

Keywords

  • O-benzylguanine

ASJC Scopus subject areas

  • Chemistry(all)

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