A Sensitive Method for the Simultaneous Determination of Caffeine and its Dimethylxanthine Metabolites in Human Plasma: Application to CYP1A2 Phenotyping

Abstract

A sensitive high performance liquid chromatographic assay was developed for the determination of caffeine and its dimethylxanthine metabolites in human plasma. Caffeine, paraxanthine, theophylline, and theobromine were extracted from 0.5 mL of matrix with methylene chloride. Separation of the analytes was achieved using a mobile phase consisting of 0.1 M sodium acetate pH 4.5, methanol, tetrahydrofuran, and distilled water (10:6.5:1.4:82.1, v/v) and a Microsorb MV C₁₈ analytical column with UV detection at 274 nm. Excellent linearity was observed for each analyte over the concentration range of 20 – 8000 ng/mL. The within- and between-day coefficients of variation were less than 4.1% at all concentrations examined. This method is suitable for pharmacokinetic or pharmacogenetic studies utilizing caffeine as a probe for CYP1A2 activity whether given alone or in combination with other CYP probe drugs.