Abstract:
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, 2.6m
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 0.4 mL/min. The total run time was 8.4 min and the
retention times for the internal standard (reserpine) was 5.4 min and for EFV was 6.5 min. The calibration
curves showed linearity (r2, 0.989–0.992) over the concentration range of 3.125–100 g/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 6.11 g/L
respectively. The working range was defined as 6.25–100 g/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.
