Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

Abstract

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in human plasma. An Oasis((R)) HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 microm, 120 A, 2.0 mm x 150 mm) column. The initial composition of the mobile phase was 2% methanol/98% 5mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3min, followed by a linear gradient over 4 min to 30% methanol/70% 5mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5'-deoxy-5-fluorouridine (5'-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC-H](-), m/z 263 for [dFdU-H](-), and m/z 245 for [5'-DFUR-H](-). The average recoveries for dFdC, dFdU, and 5'-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5-1000 ng/ml for dFdC, and 5-5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were </=3 and </=7% at three concentration levels (50.0, 500, and 5000 ng/ml). The limits of quantitation (defined as 10 times of signal-to-noise ratio) were 3.16 ng/ml for dFdC, and 1.35 ng/ml for dFdU with 50-microl sample injections. This method has been used for measuring plasma concentrations of dFdC and dFdU in samples from adult cancer patients in a Phase I trial of weekly dFdC given as 150 (or lower) mg/(m(2) 24-h) infusion. The average plasma dFdC concentrations at 22- and 23-h into the infusion were 18.3 and 16.8 ng/ml at 150 and 100mg/m(2), respectively; the values for dFdU averaged 2950 and 1372 ng/ml.

Rapid determination of gemcitabine in plasma and serum using reversed‐phase HPLC

“…Protein precipitation with various organic solvents became the standard sample preparation for normal-phase and RP-HPLC [12 -18], whereas TCA was used to remove proteins prior to ion-pair RP-HPLC [19 -22]. Recently, SPE was shown to be suitable for plasma and urine samples analyzed with HPLC coupled to MS, assay formats with a ng/ mL LOQ for gemcitabine and dFdU [23,24]. In these assays, internal standards such as 29,29-difluorothymidine [13], 29-fluorodeoxycytidine [14], floxuridine [16], 29-deoxycytidine [12,18,20,23], and 59-deoxy-5-fluorouridine [24] were employed.…”

Squalenoylation Favorably Modifies the in Vivo Pharmacokinetics and Biodistribution of Gemcitabine in Mice

“…Without delay, plasma was separated by centrifugation at 3000g for 10 min at 4°C. Extraction and quantification of SQdFdC and dFdC in the plasma samples were performed using a validated high-performance liquid chromatography tandem mass spectrometry (Khoury et al, 2007), and dFdU, the metabolite of dFdC, was assayed by a modified LC-MS method described by Xu et al (2004).…”

“…Chromatographic conditions for the quantification of dFdU were adapted from Xu et al (2004) using an Uptisphere C18 column, 3 m, 2-mm i.d. ϫ 100-mm length (Interchim) at a flow rate of 0.2 ml/min.…”

High-performance liquid chromatographic method for the determination of gemcitabine and 2′,2′-difluorodeoxyuridine in plasma and tissue culture media

“…Reversed-phase columns were used by most investigators, although Freeman et al initially employed a normal phase aminopropyl silica column, an approach also used by other investigators [16]. Sottani et al [20] and Xu et al [19] developed an LC-MS methods for gemcitabine and its major metabolite, 2 ,2 -difluorodeoxyuridine. Many of these reported assays require solid-phase [19,20], or liquid-liquid extractions [16].…”

“…Sottani et al [20] and Xu et al [19] developed an LC-MS methods for gemcitabine and its major metabolite, 2 ,2 -difluorodeoxyuridine. Many of these reported assays require solid-phase [19,20], or liquid-liquid extractions [16]. Direct plasma protein precipitation procedures with acids [15] or water-miscible organic solvents [14] have also been used for sample preparation.…”