RESUMO
A novel and robust screening method for the determination of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), in human saliva has been developed and validated based on high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges. The analytes were separated by high performance liquid chromatography (Phenomenex Kinetex C18, 150mm×3mm internal diameter, 2.6µm particle size) and detected with tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration was used to elute the analytes, at a flow-rate of 0.4mL/min. The total run time was 8.4min and the retention times for the internal standard (reserpine) was 5.4min and for EFV was 6.5min. The calibration curves showed linearity (r(2), 0.989-0.992) over the concentration range of 3.125-100µg/L. Mean intra- and inter-assay relative standard deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of quantification (LOQ) were 0.46-9.43%, 80-120%, 60% (±7.95), 1.84 and 6.11µg/L respectively. The working range was defined as 6.25-100µg/L. This novel LC-MS/MS assay is suitable for reliable detection of low EFV concentrations in saliva and can be used as a screening tool for monitoring EFV compliance.