Preanalytical Stability of Gemcitabine and its Metabolite 2′, 2′-Difluoro-2′-Deoxyuridine in Whole Blood—Assessed by Liquid Chromatography Tandem Mass Spectrometry

Abstract

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines

“…In all cell lines, a long-lasting and strong inhibition of gemcitabine inactivation was achieved with 200 µM THU even at the highest gemcitabine concentrations and at both incubation durations. This is in line with previous studies in human blood performed by our own group ( Bjånes et al, 2015 ) and other researchers ( Bowen et al, 2009 ). dFdU could otherwise be assumed to be derived from the deamination of dFdCMP ( Wong et al, 2009 ), but THU is not known to inhibit gemcitabine-inactivating enzymes other than CDA ( Heinemann and Plunkett, 1989 ).…”

“…Horse serum and sodium pyruvate were bought from Thermo Fisher Scientific (Oslo, Norway), culture flasks and cryotubes from VWR (Oslo, Norway), centrifuge tubes from Sarstedt (Oslo, Norway), and THU from AH diagnostics (Oslo, Norway). All other reagents and equipment used for liquid chromatography tandem mass spectrometry methods have been described previously ( Bjånes et al, 2015 ; Kamčeva et al, 2015 ).…”

“…Quantification of gemcitabine and its metabolites was performed using an Agilent 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany) for chromatographic separation and an Agilent 6410 triple-quad mass spectrometer for mass detection. Gemcitabine and dFdU in culture media samples were quantified as previously described ( Bjånes et al, 2015 ) and optimized with lower limits of quantitation of 0.1 µM for both gemcitabine and dFdU. dFdCTP was analyzed in cell lysates with a modified version of our previously published method ( Kamčeva et al, 2015 ).…”

Ultrasound- and Microbubble-Assisted Gemcitabine Delivery to Pancreatic Cancer Cells

“…Horse serum and sodium pyruvate were obtained from Thermo Fisher Scientific (Oslo, Norway) and tetrahydrouridine (THU) from AH diagnostics (Oslo, Norway). Reagents and equipment used for liquid chromatography tandem mass spectrometric methods (LC-MS/MS) are described elsewhere [24,25].…”

“…Quantification of gemcitabine and its metabolites was performed using an Agilent 1200 series HPLC-system (Agilent Technologies, Waldbronn, Germany) for chromatographic separation and an Agilent 6410 triple-quad mass spectrometer for mass detection. Concentrations of gemcitabine and dFdU in culture media samples were measured according to our previously published method [24], with optimised lower limits of quantitation (LLOQ) of 0.1 µM for both analytes. Gemcitabine triphosphate (dFdCTP) was quantified in cell lysates with a slightly modified version of our previously published method [25].…”