The determination of gemcitabine and 2′-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry

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 24 h infusion. The method was developed using 15N3 CdR as an internal standard across the concentration range of 1–500 ng/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 (3.1 mm × 200 mm, 5 μm) with a 50 mM formic acid: acetonitrile (9:1) mobile phase eluted at 1 ml/min. Extracted samples were observed to be stable for a minimum of 48 h after extraction when kept at 4 °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 15N3 CdR (231 m/z; 115 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, ±7.9%, 19 nM; CdR: 100.0%, ±5.3%, 22 nM, linear range LOQ to 2 μM. During 24 h 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.