Application of a stabilizer cocktail of N-ethylmaleimide and phenylmethanesulfonyl fluoride to concurrently stabilize the disulfide and ester containing compounds in a plasma LC–MS/MS assay

•Quantified an epothilone-folate conjugate and its epothilone moiety in plasma.•Evaluated the stability of the disulfide and ester containing compounds in plasma.•A cocktail of NEM and PMSF effectively stabilized both analytes in plasma.•ISR helps identifying the minor conversion from BMS-753493 to BMS-748285.•The validated method was successfully applied to support clinical studies. 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/x2 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.