Validation of an HPLC-FL Method for the Determination of Tepoxalin and its Major Metabolite in Horse Plasma

Abstract

A sensitive, simple, and accurate high-performance liquid chromatographic (HPLC) method has been developed for the determination of tepoxalin, its major metabolite RWJ-20142 and the internal standard RWJ-20294 (Schering-Plough Co.) in horse plasma. The plasma samples (0.5 mL) were directly extracted with acetonitrile 0.9 mL; after high speed centrifugation to precipitate proteins 50 µL were injected into a HPLC. Separation was performed on a Gemini C18 column (250 mm × 4.6 mm i.d., 5 µm particles) with mobile phase A consisting of 0.01 M 1-octane-sulfonic acid in 0.01 M acetic acid aqueous solution and mobile phase B consisting of tetrahydrofuran, using an isocratic elution at 50% and 50% at 1.0 mL/min flow rate. Detection and quantitation were performed by fluorimetric detection at λex = 290 nm and λem = 440 nm. Detection and quantitation limits for tepoxalin and RWJ-20142 were 30 and 50 ng/mL, and 10 and 30 ng/mL, respectively. Recovery values for tepoxalin and RWJ-20142 were near 100%. The linear concentration range for tepoxalin and RWJ 20142 were 50‐5000 and 30‐5000 ng/mL, respectively. The validated reversed-phase HPLC-fluorescence detection method was used for determination of preliminary pharmacokinetic data of the drug in horse plasma.