Simultaneous determination of febuxostat and its three active metabolites in human plasma by liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study in Chinese healthy volunteers

• A new method based on one-step PPT combined with gradient LC–MS/MS was developed.
• Simultaneous analysis of febuxostat and its metabolites in human plasma was achieved.
• The method was validated and applied to pharmacokinetic study of febuxostat in humans.
• The method provides advantages in terms of simplicity, high throughput and low cost.

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of febuxostat and its three active metabolites in human plasma was developed using a ZORBAX SB-C18 column (50 mm × 4.6 mm, 5 μm) and an optimized gradient mobile phase consisting of acetonitrile, water and formic acid. Plasma samples were spiked with the internal standard losartan and then pre-treated using one-step protein precipitation with methanol. Mass spectrometric detection was performed by selective reaction monitoring mode via electrospray ionization source operating in positive ionization mode. The method exhibited good linearity over the concentration range of 10–20,000 ng/mL for febuxostat, 1.0–270 ng/mL for 67M-1 and 67M-2, and 0.8–250 ng/mL for 67M-4, respectively. The intra- and inter-day precisions were less than 14.7% and the accuracy ranged from −4.3% to 5.1%. The method was successfully applied to a clinical pharmacokinetic study of febuxostat in humans after oral administration of a single dose of febuxostat at 40, 80 and 120 mg.

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