title: 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.
abstract: 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.
authors.affiliation: Bioanalytical Sciences, Analytical & Bioanalytical Development, Bristol-Myers Squibb, Route 206 and Province Line Road, Princeton, NJ 08543, USA. Electronic address: long.yuan@bms.com.
authors.forename: Long
authors.initials: L
authors.lastname: Yuan
authors.forename: Wenying
authors.initials: W
authors.lastname: Jian
authors.forename: Duxi
authors.initials: D
authors.lastname: Zhang
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry; D002853:Chromatography, Liquid; D004220:Disulfides / Q000097:blood* / Q000737:chemistry; D034261:Epothilones / Q000737:chemistry; D004952:Esters / Q000097:blood* / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry*; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D000970:Antineoplastic Agents; C584815:BMS-748285; C584814:BMS-753493; D004220:Disulfides; D034261:Epothilones; D004952:Esters; D010664:Phenylmethylsulfonyl Fluoride; D005492:Folic Acid; D005033:Ethylmaleimide
keywords: BMS-753493; Epothilone; Epothilone-folate conjugate; ISR failure; Stabilizer