A Micromethod for Measuring Theophylline and the Metabolites in Serum by Reversed Phase High Pressure Liquid Chromatography

Abstract

We present a micromethod for determining theophylline and its three major metabolites (1 methyl uric acid, 1, 3, dimethyl uric acid, 3 methyl xanthine) in 25 μl serum. Serum sumple was deproteinized by acetonitrile, then concentrated and injected into high pressure liquid chromatograph (HPLC) equipped with ODS reversed phase column run with a mixture of 0.01M sodium acetate (adjusted pH to 5.0): acetonitrile/90: 10 as eluent at flow rate of 0.8 ml/min. Absorption was mas monitored at 275 nm. Theophylline and metabolites were well separated with above chromatographic conditions. Concentrations of three metabolites were about one tenth of theophylline. Accuracy of the method was verified by comparing concentration of theophyline measured by this method with those by enzyme immunoassay (EIA) and fluorescent polarization immunoassay (FPIA). The correlation correlation coefficient (C.C) between this method and EIA was 0.984. The C.C between this and FPIA was 0.987.