Direct injection HILIC-MS/MS analysis of darunavir in rat plasma applying supported liquid extraction

A novel bioanalytical method was developed and validated for the quantitative determination of darunavir (DRV) in rat plasma by employing hydrophilic interaction chromatography and tandem mass spectrometry (HILIC-MS/MS) with supported liquid extraction (SLE). Irbesartan (IRB) was used as an internal standard (IS). The analyte in rat plasma (200 µL) was isolated through SLE using ethyl acetate as the eluting solvent. The chromatographic separation was achieved on Luna-HILIC (250 mm×4.6 mm, 5 μm) column with a mobile phase of 0.1% of formic acid in water:acetonitrile (5: 95, v/v), at a constant flow rate of 1.0 mL/min. The MS/MS ion transitions for DRV (548.1→392.0) and IS (429.2→207.1) were monitored on an ion trap mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) was 0.2 ng/mL and quantitation range was 0.2-5000 ng/mL. The method was validated for its selectivity, sensitivity, carryover, linearity, precision, accuracy, recovery, matrix effect and stability. The method was successfully applied to pharmacokinetic study in rats.