Development and validation of a liquid chromatography–tandem mass spectrometry assay for determination of raloxifene and its metabolites in human plasma

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4′-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC–MS–MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r2 > 0.99) were found from 0.200 to 340 μg/L, from 1.600 to 2720 μg/L and from 0.088 to 60.00 μg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).