An improved LC-MS/MS method for the simultaneous determination of pyrazinamide, pyrazinoic acid and 5-hydroxy pyrazinoic acid in human plasma for a pharmacokinetic study

In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC-MS/MS method. The analytes and their labeled internal standards were extracted from 200 mu L plasma samples by liquid liquid extraction with methyl tert-butyl ether: diethyl ether (90:10, v/v) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100 x 4.6 mm, 3.5 mu m) column using methanol and 0.1% acetic acid (65:35, v/v) as the mobile phase within 4.0 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m/z 124.1 -> 81.1, m/z 125.0 -> 80.9 and m/z 141.0 -> 81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100-30.0 mu g/mL, 0.03-9.00 mu g/mL and 0.002-0.600 mu g/mL for PZA, PA and 5-OH PA respectively with r(2) >= 0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (%CV) across quality controls varied from 93.5-106.7% and 1.10-4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged -6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500 mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples. (C) 2016 Elsevier B.V. All rights reserved.