A Rapid, Sensitive and Validated Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry Method for Determination of Rifampicin in Rat Plasma: Application to Pharmacokinetic Study

Objective: To develop and validate simple, sensitive and selective ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) method for quantification of rifampicin (RIF) in rat plasma and its application to pharmacokinetics study. Methods: Precipitation method was used for the extraction of plasma samples, an aliquot of 25 μl plasma samples was extracted using acetonitrile precipitation technique. Chromatographic separation was performed usingWaters AcquityTM Results: The method had a short chromatography run time of 1.8 min with improved sensitivity over existing methods. Calibration curves been linear over the wide range of 1.97-5047.00 ng/ml. The between and within-batch precision and accuracy of the method was determined by using 4 quality control samples; the highest %CV observed was10.11. The mean recovery values are 74.26, 82.77 and 101.73 at low, medium and highquality control levels; respectively. UPLC columns, BEH C18 (50 mm× 2.1 mm, 1.7 μm) by a gradient mixture of acetonitrile and water (both containing 0.1 % formic acid) as a mobile phase at the flow rate of 0.7 ml/min.The analyte was protonated in the positive ESI (electrospray ionization interface) and detected in MRM (multiple reactions monitoring) modes using the transition m/z 308.60-455.30. Conclusion: It was concluded that the developed and validated UPLC-MS/MS method was sensitive,specific, precise, linear, and rapid. Therefore, the method can be used for quantification of RIFin rat plasma with various advantages over the reported methods. RIF is widely recommended by US-FDAguidance for industry on drug interaction studies and the developed method can be used to explore drug interaction studies in drug discovery and development.