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 quan titation of quinine and its major metabolite, 3-hydroxyquinine in huma n plasma, urine and hepatic microsomal samples. The method involves si mple protein precipitation for sample treatment and ion-paired chromat ography. The chromatographic separation is accomplished with a mobile phase comprising acetonitrile-aqueous phosphate buffer (40:60, v/v) co ntaining 10 mM sodium dodecyl sulphate and 0.1 mM tetrabutylammonium b romide and adjusted to pH 2.1. The mobile phase is pumped at a flow ra te of 0.5 mL/min. A microbore column is used (2 mm I.D. x 100 mm) pack ed with a C-18 reverse phase material (5 mu m ODS Hypersil). Biologica l samples (100-500 mu L) were precipitated with two volumes of cold me thanol, vortexed and then centrifuged at 1500 g for 10 min. The supern atant (30 mu L) was injected into the HPLC column. The chromatograms w ere monitored using a fluorescence detector setting with excitation an d emission wavelenths of 350 and 450 nm, respectively. Under these con ditions, 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) fo r quinine. The inter- and intra-assay coefficients of variation were f ound to be less than 7%. The assay procedure is applicable for studyin g the pharmacokinetics and metabolism of quinine.