title: High sensitive assay employing column switching chromatography to enable simultaneous quantification of an amide prodrug of gemcitabine (LY2334737), gemcitabine, and its metabolite dFdU in human plasma by LC-MS/MS.
abstract: In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis.
authors.affiliation: Eli Lilly and Company, Indianapolis, IN, USA. enaksha@lilly.com
authors.forename: Enaksha R
authors.initials: ER
authors.lastname: Wickremsinhe
authors.forename: Lisa B
authors.initials: LB
authors.lastname: Lee
authors.forename: Chris A
authors.initials: CA
authors.lastname: Schmalz
authors.forename: John
authors.initials: J
authors.lastname: Torchia
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Ruterbories
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000097:blood* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism; D006801:Humans; D011355:Prodrugs / Q000378:metabolism / Q000493:pharmacokinetics*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; C569272:LY2334737; D011355:Prodrugs; D005467:Floxuridine; D003841:Deoxycytidine; D003857:Deoxyuridine; C084565:2',2'-difluoro-2'-deoxyuridine; D000093542:Gemcitabine
keywords: Column switching; Gemcitabine; LC–MS/MS; Prodrug