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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 932; pp. 117 - 122
• Main Authors: Wickremsinhe, Enaksha R., Lee, Lisa B., Schmalz, Chris A., Torchia, John, Ruterbories, Kenneth J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2013
• Subjects: ambient temperature; Antimetabolites, Antineoplastic - blood; Antimetabolites, Antineoplastic - metabolism; chromatography; Chromatography, Liquid - methods; clinical trials; Column switching; Deoxycytidine - analogs & derivatives; Deoxycytidine - blood; Deoxycytidine - metabolism; Deoxyuridine - analogs & derivatives; Deoxyuridine - blood; Deoxyuridine - metabolism; drugs; Floxuridine - analogs & derivatives; Floxuridine - blood; Floxuridine - metabolism; freeze-thaw cycles; frozen storage; Gemcitabine; Humans; LC–MS/MS; metabolites; Prodrug; Prodrugs - metabolism; Prodrugs - pharmacokinetics; Sensitivity and Specificity; Tandem Mass Spectrometry - methods; Column switching; Prodrug; Gemcitabine; LC–MS/MS
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.06.008

•Quantification of a gemcitabine prodrug, gemcitabine, and dFdU in single LC/MS/MS analysis.•Use of column-switching chromatography.•Using diverse assay ranges within a single LC–MS/MS analysis. 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.