SENSITIVE AND RAPID SIMULTANEOUS QUANTITATION OF RALOXIFENE AND ITS TWO MAJOR ACTIVE METABOLITES 4-GLUCURONIDE RALOXIFENE AND 6-GLUCURONIDE RALOXIFENE IN HEALTHY VOLUNTEERS USING A LIQUID CHROMATOGRAPHY COUPLED WITH TRIPLE QUADRUPLE MASS SPECTROMETER

Simultaneous estimation of parent and its metabolite(s) in a single method poses a threat when one has to monitor one mass transition while running the other compound on liquid chromatography-mass spectrometer. This would again become critical once interference is observed at the retention time of compound of interest in-spite of its absence in the sample. In this paper, a selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of Raloxifene and its two active metabolites, Raloxifene 4-Glucuronide and Raloxifene 6-Glucuronide in human plasma was developed and validated. The method involves a rapid solid-phase extraction from plasma, followed by reversed-phase chromatography with gradient flow condition and mass spectrometry detection. Validation parameters were selected ranging from 0.016 to 1.187 ng/mL for Raloxifene, 3.073 to 700.558 ng/mL Raloxifene 4-Glucuronide and 0.311 to 124.526 ng/ml for Raloxifene 6-Glucuronide. The mean recovery for Raloxifene, Raloxifene 4-Glucuronide, and Raloxifene 6-Glucuronide found to be 85.10, 86.30 & 87.20%, respectively. The peak concentration of Raloxifene, Raloxifene 4-Glucuronide, and Raloxifene 6-Glucuronide were 0.330 ng/mL, 153.047 ng/mL and 27.744 ng/mL respectively for the reference product, in fasted conditions.