Simultaneous determination of quinine and a major metabolite 3-hydroxyquinine in biological fluids by HPLC without extraction

A reverse phase, isocratic HPLC method has been developed for the quantitation of quinine and its major metabolite, 3-hydroxyquinine in human plasma, urine and hepatic microsomal samples. The method involves simple protein precipitation for sample treatment and ion-paired chromatography. The chromatographic separation is accomplished with a mobile phase comprising acetonitrile-aqueous phosphate buffer (40:60, v/v) containing 10 mM sodium dodecyl sulphate and 0.1 mM tetrabutylammonium bromide and adjusted to pH 2.1. The mobile phase is pumped at a flow rate of 0.5 mL/min. A microbore column is used (2 mm I.D. x 100 mm) packed with a C-18 reverse phase material (5 mu m ODS Hypersil). Biological samples (100-500 mu L) were precipitated with two volumes of cold methanol, vortexed and then centrifuged at 1500 g for 10 min. The supernatant (30 mu L) was injected into the HPLC column. The chromatograms were monitored using a fluorescence detector setting with excitation and emission wavelenths of 350 and 450 nm, respectively. Under these conditions, the lower limit of detection was 0.1 mu M (0.034 mu g/mL) for the major metabolite 3-hydroxyquinine, and 0.5 mu M (0.16 mu g/mL) for quinine. The inter- and intra-assay coefficients of variation were found to be less than 7%. The assay procedure is applicable for studying the pharmacokinetics and metabolism of quinine.