title: Validated procedure for simultaneous trace level determination of the anti-cancer agent gemcitabine and its metabolite in human urine by high-performance liquid chromatography with tandem mass spectrometry.
abstract: A sensitive, accurate and reproducible procedure has been developed for the quantitative determination of gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolite 2',2'-difluorodeoxyuridine (2dFdU) in human urine. The samples (2 mL) were extracted by solid-phase extraction (SPE) and analyzed by reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS), operating in multiple reaction monitoring (MRM mode). This procedure was validated using 2'-deoxycytidine as internal standard (IS). The urine assay was linear over the range 0-50 microg/L, with a limit of quantification (LLOQ) of 0.2 microg/L for gemcitabine and 1.0 microg/L for the metabolite. The respective limits of detection (LODs) for dFdC and 2dFdU were 0.05 and 0.3 microg/L. The precision and accuracy of the assay were determined on three different days. The within-series precision was found to be always less than 8.5 and 12.7% for gemcitabine and 2dFdU, respectively. The overall precision expressed as relative standard deviation (CVr) was always less than 7.1% for both analytes. The recovery of gemcitabine was always greater than 90% with a CVr <6.3%. The measurement uncertainty determined from the validation data assessed the possibility of determining this drug and its metabolite at trace levels in urine, considering that the combined uncertainty of the whole procedure was always less than 30%.
authors.affiliation: Laboratory for Environmental and Toxicological Testing, S. Maugeri Foundation, IRCCS, via Ferrata 8, Pavia, Italy. csottani@fsm.it
authors.forename: Cristina
authors.initials: C
authors.lastname: Sottani
authors.forename: Massimo
authors.initials: M
authors.lastname: Zucchetti
authors.forename: Marco
authors.initials: M
authors.lastname: Zaffaroni
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bettinelli
authors.forename: Claudio
authors.initials: C
authors.lastname: Minoia
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine*; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D003841:Deoxycytidine; C084565:2',2'-difluoro-2'-deoxyuridine; D000093542:Gemcitabine