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

• LC–MS/MS method for simultaneous estimation of TFV, 3TC and NVP.
• Low detection limits of 2 ng/mL for TFV and 10 ng/mL for 3TC and NVP.
• Method utilizes highly selective SPE technique with a sample volume of 0.2 mL.

A selective and high throughput liquid chromatography–mass spectrometry method has been developed and validated for the simultaneous quantification of tenofovir (TFV), lamivudine (3TC) and nevirapine (NVP) in human plasma using emtricitabine (FTC) as internal standard (ISTD). Following solid phase extraction (SPE), the analytes and ISTD were run on Prontosil C18AQ column (100 mm × 4.6 mm, 3 μm) using an isocratic mobile phase consisting of 1 mM ammonium acetate in water (pH 6.5 ± 0.3):acetonitrile (50:50, v/v). The precursor and product ions of the drugs were monitored on a triple quadrupole instrument operated in the negative ionization mode. The method was validated over a concentration range of 2–500 ng/mL for TFV and over a concentration range of 10–4000 ng/mL for 3TC and NVP with relative recoveries ranging from 61 to 85%. The intra and inter batch precision (%CV) across four validation runs was less than 12.2%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) was within ±8.5%, in terms of relative error.