High throughput LC–MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mm × 3.9 mm i.d., 5 μm particle size) column using 5 μL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25–3000 ng/mL for 3TC, 20–2000 ng/mL for d4T and 50–5000 ng/mL for NVP. The absolute recoveries for analytes (≥86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within ±8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC) + 12 (d4T) + 100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.