A validated UPLC–MS/MS assay using negative ionization mode for high-throughput determination of pomalidomide in rat plasma

• UPLC–MS/MS assay for pomalidomide determination in rat plasma.
• Assay suitable for high-throughput determination.
• Assay validated as per USFDA and EMA guideline.
• Assay applied to pharmacokinetic study in rats.

In this study, a sensitive UPLC–MS/MS assay was developed and validated for high-throughput determination of pomalidomide in rat plasma using celecoxib as an internal standard (IS). Liquid liquid extraction using dichloromethane was employed to extract pomalidomide and IS from 200 μL of plasma. Chromatographic separation was carried on Acquity BEH™ C18 column (50 mm × 2.1 mm, 1.7 μm) using an isocratic mobile phase of acetonitrile: 10 mM ammonium acetate (80:20, v/v), at a flow rate of 0.250 mL/min. Both pomalidomide and IS were eluted at 0.66 ± 0.03 and 0.80 ± 0.03 min, respectively, with a total run time of 1.5 min only. A triple quadruple tandem mass spectrometer using electrospray ionization in negative mode was employed for analyte detection. The precursor to product ion transitions of m/z 272.01 → 160.89 for pomalidomide and m/z 380.08 → 316.01 for IS were used to quantify them respectively, multiple reaction monitoring mode. The developed method was validated according to regulatory guideline for bioanalytical method validation. The linearity in plasma sample was achieved in the concentration range of 0.47–400 ng/mL (r2 ≥ 0.997). The intra and inter-day precision values were ≤11.1% (RSD, %) whereas accuracy values ranged from −6.8 to 8.5% (RE, %). In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of pomalidomide in rats.