Development and Validation of a Rapid Liquid Chromatography Tandem Mass Spectrometry Method to Quantify Nevirapine in Human Plasma and its Application to Bioequivalence Study in Healthy Human Subjects

Abstract

A simple, accurate and robust liquid chromatography tandem mass spectrometry (LC‐ESI‐MS/MS) method has been developed for estimation of nevirapine in human plasma. Abacavir sulphate was used as an internal standard (IS). The plasma filtrate obtained after solid phase extraction (SPE), using a hydrophilic‐lipophilic balance (HLB) cartridge, was submitted directly to short column liquid chromatography‐tandem mass spectrometric assay, with negligible matrix effect on the analysis. Turbo ion spray, operating in positive mode and multiple reaction monitoring (MRM) scan type was used to quantify nevirapine in human plasma. The extraction procedure yielded extremely clean extracts with a recovery of 91.85 and 97.71% for nevirapine and IS, respectively. The method is simple and reliable with a chromatographic run time of 2.5 min. The lower limit of quantification (LLOQ) found was 25 ng ml−1 with good accuracy and precision. The developed method was validated in the linear dynamic range of 25–5000 ng ml−1 with correlation coefficient (r)≥0.9996. The intra‐and inter‐batch precision for the samples at the LLOQ were 7.04 and 7.71%, respectively. The intra‐ and inter‐batch accuracy ranged from 91.17– 102.17%. The method was successfully applied for bioequivalence studies in 36 healthy Indian human subjects after oral administration of 10 mg ml−1 suspension formulations.