Simple, Selective and High throughput Estimation of Pyrazinamide in Human Plasma using LC-MS/MS

A simple, sensitive and fast throughput high performance liquid chromatography tandem mass spectrometry (HPLCMS/MS) method has been developed for the determination of pyrazinamide. The method involved protein precipitation of pyrazinamide and pyrazinamide-D3 as the internal standard (ISTD) in Acetic Acid in water 1%v/v: Acetonitrile (15:85v/v) from 100μL human plasma. The chromatographic separation is achieved on a Waters Symmetry shield TM RP8 3.5 μm, 4.6x100mm analytical column using isocratic mobile phase, consisting of Acetic acid in water (1.0%v/v) : Acetonitrile (45:55v/v), at a flow-rate of 0.8mL/min. The precursor → product ion transition for pyrazinamide (m/z 124.1/78.90), and IS (m/z 126.90/81.90) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method is validated over a wide dynamic concentration range of 0.500-50.000μg/mL. Matrix effect is assessed by post-column analyte infusion experiment for pyrazinamide. The method is simple, rugged and rapid with a total run time of 2.8 minutes and the same applied to a bioequivalence study 400mg tablet formulation in 36 healthy Indian male subjects under fasting condition.