Simultaneous determination of caffeine and its N-demethylated metabolites in umbilical cord plasma using high-performance liquid chromatography.

A convenient high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Separation is achieved by reversed-phase chromatography using a mobile phase consisting of 0.01 M sodium acetate buffer, pH 5.0-methanol-tetrahydrofuran (95:4:1) in conjunction with a mu Bondapak C18 column protected by a guard column containing Bondapak C18/Corasil. With a flow-rate of 3 ml/min, levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined by ultraviolet detection at 254 nm. The method was used clinically for measuring cord blood samples to provide information regarding fetal exposure to caffeine and its N-demethylated metabolites during late pregnancy.