BMS-753493, a conjugate of the active epothilone moiety, BMS-748285, to folic acid, has been evaluated for the treatment of cancer. The presence of a disulfide bond in BMS-753493 and an ester group in both BMS-753493 and BMS-748285 results in their being unstable in blood or plasma. A stabilization strategy using a cocktail of N-ethylmaleimide and phenylmethanesulfonyl fluoride was developed and applied to stabilize both analytes in human blood during sample collection, processing, and storage. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of BMS-753493 and its active moiety, BMS-748285, in human plasma. The stabilized plasma samples were extracted by protein precipitation. Chromatographic separation was achieved on a Luna C8 analytical column with a gradient elution. Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curves, which ranged from 10.0 to 5000ng/mL for BMS-753493 and from 1.00 to 500ng/mL for BMS-748285, were fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±2.0% CV and inter-assay precision was within ±2.8% CV for both analytes. The assay accuracy was within ±4.6% of the nominal values for both analytes. Assay recoveries were high (∼80% for BMS-753493 and ∼100% for BMS-748285) and internal standard normalized matrix effects were minimal. Both analytes were stable in stabilized human plasma for at least 24h at room temperature, 231 days at -20°C, and following four freeze-thaw cycles. Incurred sample reanalysis played an important role in identifying a potential stability liability of the assay and helped improve the assay quality and robustness. The validated method was successfully applied to sample analysis in Phase I clinical studies.
Keywords: BMS-753493; Epothilone; Epothilone-folate conjugate; ISR failure; Stabilizer.
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