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

Abstract

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.

Keywords: Darunavir; HILIC–MS/MS; Rat plasma; Supported liquid extraction.

Fig. 1

(A) ESI-MS signal at different portions of ACN in mobile phase and (B) effect of different solvent types on the extraction efficiency.

Fig. 2

Product ion mass spectra of (A) DRV (m/z 548.1→392.0, scan range 100–700 amu) and (B) IS (m/z 429.3→207.0, scan range 50–500 amu) in positive ion mode.

Fig. 3

Effects of acetonitrile content in the mobile phase on DRV (100 ng/mL) retention time on the HILIC column.

Fig. 4

Representative MRM chromatograms of (A) blank rat plasma, (B) rat plasma spiked with 0.2 ng/mL (LLOQ) DRV, 50 ng/mL IS and (C) a rat plasma sample obtained 1.5 h after an intravenous administration of DRV.

Fig. 5

Mean plasma concentration–time profile of DRV after an oral 50 mg/kg dose of DRV to male Wistar rats.