Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids

[Display omitted] •Quantification of tenofovir (TFV) and efavirenz (EFV) was attained for 4–500ngmL−1.•Validated LC–MS/MS method for vaginal tissue, lavage and blood plasma was developed.•Matrix effects were observed for EFV in tissue (+17%) and plasma (−50%) extracts.•LOD and LOQ values were ≤0.4 and ≤0.7ngmL−1 in sample extracts.•PK profile after intravaginal administration showed low systemic exposure. Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry (HPLC–MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3μm, 100×2.1mm) at 45°C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35mLmin−1. Total run time was 9min, with retention time of 2.8 and 4.1min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500ngmL−1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7ngmL−1 in sample extracts, respectively. The method was found to be specific, accurate (96.0–106.0% of nominal values) and precise (RSD