title: The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry.
abstract: A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Honeywell
authors.forename: A C
authors.initials: AC
authors.lastname: Laan
authors.forename: C J
authors.initials: CJ
authors.lastname: van Groeningen
authors.forename: E
authors.initials: E
authors.lastname: Strocchi
authors.forename: R
authors.initials: R
authors.lastname: Ruiter
authors.forename: G
authors.initials: G
authors.lastname: Giaccone
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D002851:Chromatography, High Pressure Liquid; D017321:Clinical Trials, Phase I as Topic; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D006801:Humans; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014018:Tissue Distribution; D000093542:Gemcitabine
chemical_list: D003841:Deoxycytidine; D000093542:Gemcitabine