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

• First LC–MS/MS method for the simultaneous determination of pyrazinamide and its metabolites.
• Most sensitive and rapid method in human plasma compared to all existing methods.
• Chromatographic separation of the analytes within 4 min.
• Consistent and quantitative recovery with minimal matrix interference for all three analytes.
• Successful clinical study with healthy subjects and incurred sample reanalysis.

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 μL 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 × 4.6 mm, 3.5 μm) 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 μg/mL, 0.03–9.00 μg/mL and 0.002–0.600 μg/mL for PZA, PA and 5-OH PA respectively with r2 ≥ 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.