Development and validation of ultra-high-performance liquid chromatography–mass spectrometry method for the determination of raloxifene and its phase II metabolites in plasma: Application to pharmacokinetic studies in rats

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The aim of this study is to establish a reliable liquid chromatography–mass spectrometry method to simultaneously quantitate raloxifene, and its major metabolites, raloxifene-6-glucuronide, raloxifene-4′-glucuronide, and raloxifene-6-sulfate in rat plasma samples for pharmacokinetic studies. The separation of the analytes was achieved on a Waters BEH C18 column. Water (0.1% formic acid) and acetonitrile were used as the mobile phases for elution. A one-step protein precipitation using a mixture solvent was applied for plasma sample preparation. The method was validated following the FDA guidance. The results showed that the linear range were 1.95–1000 nM for raloxifene-6-glucuronide, and raloxifene-4′-glucuronide, 0.195–100 nM for raloxifene-6-sulfate, and 0.195–200 nM for raloxifene, respectively. The lower limit of quantification was 1.95, 1.95, 0.195, and 0.195 nM for raloxifene-6-glucuronide, raloxifene-4′-glucuronide, raloxifene-6-sulfate, and raloxifene, respectively. Only 20 µl of plasma sample was required since the method is sensitive. The intra- and interday variance is <15% and the accuracy is within 85–115%. The variance of matrix effect and recovery were <15%. The method was successfully applied in a pharmacokinetic study in rats with oral administration of raloxifene.

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