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

• First report of the determination of pirfenidone in rat plasma using UPLC–MS/MS.
• The method offered sample preparation with protein precipitation by acetonitrile.
• Run time was only 3.0 min.
• The method was successfully used in a pharmacokinetic study of pirfenidone in rats.

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.