Development and Validation of a UPLC-MS/MS Assay for Simultaneous Estimation of Raloxifene and its Metabolites in Human Plasma

Raloxifene ([6-hydroxy-2-(4-hydroxyphenyl)-benzothiophen-3yl][4-[2-(1-piperidyl) ethoxy] phenyl]-methadone) a non-steroidal selective estrogen receptor regulator, is currently applied to both the prevention and treatment of postmenopausal osteoporosis [1,2]. It acts as an estrogen agonist in bone and liver and in this way increases bone mineral density and decreases LDL-cholesterol [3]. Raloxifene is rapidly absorbed from the gastrointestinal tract and undergoes extensive firstpass glucuronidation, predominantly raloxifene-4’-glucuronide (R4G) and raloxifene-6’-glucuronide (R6G) [4-6]. Approximately 60% of an oral dose is absorbed; however, because of extensive presystemic glucuronide conjugation, absolute bioavailability is only 2%. Significant interpatient differences in bioavailability may result from alterations in the rate of glucuronide formation and enterohepatic recycling [7]. Various HPLC and LC-MS/MS methods, validated as effective and selective, have been for the detection of Raloxifene hydrochloride [8-10]. Trontelj et al. developed and validated raloxifene LC-MS/ MS method along with its metabolites and the limit of quantification were 0.0880 to 60.0000 ng/ml, 0.2000 to 340.0000 ng/ml, and 1.6000 to 2720.0000 ng/ml for RAL, R4G and R6G, respectively with 16 min run time [11]. Therefore, the purpose of this study was to develop and validate a rapid and more sensitive UPLC-MS/MS method to quantify RAL, R4G and R6G in human plasma and apply it for the simultaneous determination of RAL, R4G and R6G in a bioequivalence study.