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A simple and rapid analytical method for the simultaneous determination of pirfenidone and its metabolite,5-carboxy-pirfenidone,in human plasma using liquid chromatography-tandem mass spectrometry has been developed and validated.Aliquots of plasma (0.1 ml) containing pirfenidone and 5-carboxy-pirfenidone,as well as deuterium-labeled internal standards (IS),were deproteinized using acetonitrile.An Agilent Zorbax Plus C18 column was used for the chromatography,with isocratic elution.The mobile phase was a mixture of acetonitrile and aqueous ammonium formate solution (5 mM) containing 0.1% formic acid (60:40,v/v).Using multiple reaction monitoring in positive ionization mode,transitions m/z 186.1→65.1,m/z 216.0→77.0,m/z 191.1→65.1,and m/z 221.0→81.0 were chosen to quantify pirfenidone,5-carboxy-pirfenidone and the two ISs,respectively.The analysis time was <3 min.The calibration curve was linear over the concentration ranges 0.005–25 μg/mL for pirfenidone,and 0.005–15 μg/mL for 5-carboxy-pirfenidone.The lower limit of quantification for both analytes was 0.005 μg/mL.The intra- and inter-day precision and relative errors in quality control (QC) samples were between –11.7% and 1.3% for pirfenidone and between –5.6% and 2.5% for 5-carboxy-pirfenidone,with mean recoveries ≥ 90%.The method that has been developed is easy to carry out,sensitive,and rapid,and has been successfully used to investigate the pharmacokinetics of pirfenidone in healthy human volunteers.