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

J Chromatogr B Biomed Appl. 1996 Jun 7;681(2):331-8. doi: 10.1016/0378-4347(95)00537-4.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetonitriles
  • Antineoplastic Agents / blood*
  • Antineoplastic Agents / pharmacokinetics
  • Buffers
  • Chromatography, High Pressure Liquid / methods*
  • Chromatography, High Pressure Liquid / standards
  • Guanine / analogs & derivatives*
  • Guanine / blood
  • Guanine / pharmacokinetics
  • Humans
  • Hydrogen-Ion Concentration
  • Methanol
  • Phosphates
  • Quality Control


  • Acetonitriles
  • Antineoplastic Agents
  • Buffers
  • Phosphates
  • O(6)-benzylguanine
  • Guanine
  • Methanol
  • acetonitrile