LC–MS/MS method for the simultaneous determination of PA-824, moxifloxacin and pyrazinamide in rat plasma and its application to pharmacokinetic study

• A LC–MS/MS method was first developed and validated for the simultaneous determination of PA-824, moxifloxacin and pyrazinamide in rat plasma. The method has a good specificity, sensitivity and a short run time.
• It is the first pharmacokinetic study concerning co-administration of PaMZ combination in rat.
• Significant differences of Tmax, Cmax, AUC(0–t) and CLz/F were observed between the single and combined groups after equal dose of PA-824 and moxifloxacin administration, which revealed the possibility of drug–drug interaction in the PaMZ combination.

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for simultaneous determination of PA-824, moxifloxacin, and pyrazinamide in rat plasma using metronidazole as internal standard. Sample preparation involved a simple one-step protein precipitation with methanol, followed by centrifugation and evaporation of the organic solvent. The residue was redissolved in mobile phase and analyzed by LC–MS/MS. An Inertsil® ODS3 C18 column (150 mm × 4.6 mm, 5 μm), a mobile phase composed of methanol–0.03% TEA (triethylamine) in water (85:15, v/v), and a flow rate of 0.5 mL/min were employed, and the total run time was 6.0 min. The mass spectrometer was run in positive ion ESI-APCI combined mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. The LLOQ was 1.0 μg/mL for pyrazinamide and 0.1 μg/mL for PA-824 and moxifloxacin. The recoveries obtained for PA-824, moxifloxacin and pyrazinamide were ≥85%. Intra-day and inter-day coefficients of variation were less than 10%. The method had been successfully applied to a pharmacokinetic study of fixed dose administration of PA-824, moxifloxacin, pyrazinamide and their combination in SD rat. Significant differences of Tmax, Cmax, AUC(0–t) and CLz/F were observed between the single and combined groups after equal dose of PA-824 and moxifloxacin administration, which revealed the possibility of drug–drug interaction (DDI) between the PaMZ combination.

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