title: Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC-MS/MS assay.
abstract: A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (∼90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.
authors.affiliation: Analytical and Bioanalytical Development, Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Electronic address: long.yuan@bms.com.
authors.forename: Long
authors.initials: L
authors.lastname: Yuan
authors.affiliation: Analytical and Bioanalytical Development, Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Yunlin
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Analytical and Bioanalytical Development, Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Duxi
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Yuan-Qing
authors.initials: YQ
authors.lastname: Xia
authors.affiliation: Drug Safety Evaluation, Research and Development, Bristol-Myers Squibb, New Brunswick, NJ 08903, USA.
authors.forename: Qianping
authors.initials: Q
authors.lastname: Peng
authors.affiliation: Analytical and Bioanalytical Development, Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.affiliation: Analytical and Bioanalytical Development, Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D034261:Epothilones / Q000097:blood* / Q000737:chemistry* / Q000493:pharmacokinetics; D005260:Female; D016014:Linear Models; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D034261:Epothilones; D010664:Phenylmethylsulfonyl Fluoride; D002265:Carboxylic Ester Hydrolases; C114026:desoxyepothilone B
keywords: BMS-241027; Epothilone D; Esterase inhibitor; Species different stability; UHPLC–MS/MS