Simultaneous determination of the potent anti-tuberculosis regimen—Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC–MS/MS method coupled with 96-well format plate

Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 μL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 μm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r(2) > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.