Simultaneous Characterization of Sofalcone and Its Metabolite in Human Plasma by Liquid Chromatography

A sensitive and selective method for quantitation of sofalcone and its active metabolite in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/ MS/MS). Plasma samples were transferred into 96-well plate using an automated sample handling system and spiked with 10 μL of 2 μg/mL d₃-sofalcone and d₃-sofalcone metabolite solutions (internal standard), respectively. After adding 0.5 mL of acetonitrile to the 96-well plate, the plasma samples were then vortexed for 30 sec. After centrifugation, the supernatant was transferred into another 96-well plate and completely evaporated at 40 .deg. C under a stream of nitrogen. Dry residues were reconstituted with mobile phase and were injected into a C₁₈ reversed-phase column. The limit of quantitation of sofalcone and its metabolite was 2 ng/ mL, using a sample volume of 0.2 mL for analysis. The reproducibility of the method was evaluated by analyzing 10 replicates over the concentration range of 2 ng/mL to 1000 ng/mL. The validation experiments of the method have shown that the assay has good precision and accuracy. Sofalcone and its metabolite produced a protonated precursor ion ([M+H]⁺) of m/z 451 and 453, and a corresponding product ion of m/z 315 and 317, respectively. Internal standard (d₃-sofalcone and d₃-sofalcone metabolite) produced a protonated precursor ion ([M+H]⁺) of m/z 454 and 456 and a corresponding product ion of m/z 315 and 317, respectively. The method has been successfully applied to a pharmacokinetic study of sofalcone and its active metabolite in human plasma