| The gemcitabine (difluorodeoxycytidine, 2,2'-difluorodeo-xycytidine, dFdC) is a novel deoxycytidine analog with structural and metabolic similarities to cytarabine (arabinosylcytosine, ara-C). However, when compared to ara-C, gemcitabine exhibited stronger activity and broader therapeutic index against a lot of solider tumors, especially to the advanced non-small-cell lung, breast, pancreas. And gemcitabine has significant effect whether single agent or together with other anticancer drugs. Like ara-C, after intravenous administration, gemcitabine was rapidly and widely distributed throughout the body , then it was phosphorylated and actived to be an active dFdC 5'-triphosphate (dFdCTP) in cellular by deoxycytidine kinase (dCK). Elimination of dFdCTP was significantly related to its cellular concentration andexhibited biphasic character with the help of deamination by cytidine deaminase (CDA) in the liver, kidneys and other tissues to a noncytotoxic metabolite, 2,2' -difluorodeo-xyuridine (dFdU).It has been described the determination of gemcitabine and its metabolite in plasma etc. The results were not satisfied by using the methods published to determine dFdC and dFdU in Chinese patients. So a new reversed phase HPLC method was developed to assay gemcitabine and its metabolite in Chinese patients plasma and then to study the pharmacokinetics in Chinese patients.1. Development of analysis for determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatograpyObjective:To establish a RP-HPLC method for the determination of gemcitabine (2,2' -difluorodeoxycytidine, dFdC) and its metabolite(2, 2' -difluorodeoxyuridine , dFdU) in human plasma. Method:1.Prepare the acetate ammonium acetate buffer solution:3.08g ammonium acetate add lOOOmL redistilled water and 1mL glacial acetic acid.2. Assay procedure:3 mL of methanol-acetonitrile (v/v1:9) was added to 1.0mL plasma containing 100L of internal standard.The sample was vortexed for 1 min then centrifuged at 3500 rpm for 10 min. The supernatant was transferred to a disposable tube. This solution was evaporated to dryness at 60癈 in water bath under a gentle stream of nitrogen, and the residues were dissolved in 0.5 mL of mobile phase and centrifuged at 15000 rpm for 10 min. 50 L of the supernatant was injected into the HPLC system. Separation was achieved on a Lichrospher5-C18(4. 6mm 250mm, 5m) column at 25 and detected at 268nm. The mobile phase consisted of 40 mmol L-1 acetate-ammonium acetate(pH5.5) and acetonitrile(v/v 97. 5:2. 5) at a flow rate of 0. 8 mL ?min. 3. Separation and Purification of the metabolite (2,2' -difluorodeoxyuridine , dFdU) in patient's plasma: 4 mL of acetonitrile was added to 2. OmL patient's plasma. The sample was vortexed for 1 min then centrifuged at 3500 rpm for 10 min. The supernatant was transferred to a disposable tube. This solution was evaporated to dryness at 60癈 in water bath under a gentle stream of nitrogen, and the residues were dissolved in 0. 5 mL of mobile phase and centrifuged at 15000 rpm for 10 min. 100 L of the supernatant was injected into the HPLC system. Separation was achieved on a Lichrospher5-C18(4.6mm 250mm, 5m) column at 30 and detected at 253nm. The mobile phase consisted of water andacetonitrile(v/v 90:10) at a flow rate of 1.0 mL min. MS Condition: ionization mode: ESI; drying gas flow: N2, 12L min; drying gas temperature:350;nebulizer pressure:45psi;capillary voltage:3500v;mass range :50-500 m/z; fragmentor voltage: 100v;gain:1;threshold:500. Results:1. The acetate-ammonium acetate buffer solution used for mobile phase has the concentration of 40 mmol L with the pH=5. 5.2. Under the separation conditions, the metabolite dFdU was separated completely from patient's plasma with resolution >3.0. There were not interferences appeared at the peak position . The metabolite was confirmed by MS and compared with MS of the standard reference of gemcitabine.3. Gemcitabine(dFdC), the metabolite(dFdU) and Floxuridine, the internal standard (I.S.) were separated... |
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