Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC–MS/MS

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2   mL of acetonitrile to a 0.1   mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0   min and the elution of pirfenidone was at 1.39   min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m / z 186.2   →   92.1 for pirfenidone and m / z 237.1   →   194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5–2000   ng/mL with a lower limit of quantitation (LLOQ) of 5   ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4–84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0   mg/kg pirfenidone in rats.