A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of idelalisib, fludarabine and lenalidomide using tolbutamide as an internal standard. Analytes were recovered by liquid-liquid extraction and separated on a reverse phase C18 column (150mm×4.6mm i.d., 5μm) using methanol:0.1% formic acid buffer (70:30) as mobile phase at a flow rate of 1mL/min in isocratic mode. Selective reaction monitoring was performed using the transitions, i.e. m/z 416.25/176.48, 286.11/154.10, 260.15/149.15, and 271.14/155.06 to quantify idelalisib, fludarabine and lenalidomide and tolbutamide, respectively. The method was validated over the concentration range of 1.15-576.84ng/mL for idelalisib, 0.95-476.25ng/mL for fludarabine and 0.97-486.19ng/mL for lenalidomide. Intra and inter-day accuracy and precision of validated method were within the acceptable limits of <15%. Coefficients of correlation (r(2)) for the calibration curves were >0.998 for all analytes. The method was successfully applied for simultaneous estimation of idelalisib, fludarabine and lenalidomide in a pharmacokinetic study in rats.
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