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Simultaneous determination of pirfenidone and its metabolite in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

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 (ISs), 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 time of analysis 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 interday precision and relative errors in quality control 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.