Determination of Fluoxetine、Venlafaxine and Their Active Metabolite in Human Serum by HPLC-MS/MS and Its Application
To establish a method for simultaneous determination of fluoxetine(FXT),venlafaxine(VEN)and their active metabolite N-norfluoxetine(N-FXT)and O-desmethylvenlafaxine(O-VEN)in human serum,and apply it to the determination of the metabolism of related drugs in humans.Clinical samples with methol-formic acid as protein precipitating agent,were determined by high performance liquid chromatography-tandem mass spectrometry system(HPLC-MS/MS)after treatment.The chromatographic conditions were as following:Column was Phenomenex Kinetex Biphenyl(2.1 mm×50 mm,2.6 pm),mobile phase consisted of methanol and water containing 0.1％formic acid and 1 mmol/L ammonium formate,flow rate was 0.4 mL· min-1,column temperature was 45 ℃,injection volume was 5 μL.The serum sampleswere detected by electrospray positive ion source in multireaction monitoring mode.The quantitative detection ion pairs of each substance were m/z 310.2→ m/z 44.5(FXT),m/z 315.2→ m/z 44.5(FXT-D5),m/z 296.2→ m/z 30.6(N-FXT),m/z 301.2 m/z 30.6(N-FXT-D5),m/z 278.2→ m/z 58.4(VEN),m/z 284.3→ m/z 64.4(VEN-D6),m/z 264.2→m/z 58.4(O-VEN)、m/z 270.2→ m/z 64.4(O-VEN-D6).FXT,N-FXT,VEN,O-VEN,were not interfered with by internal standards and endogenous substances,the specificity and selectivity of the method were good,the serum concentration had a good linear relationship in the range of 10～1 000,32～3 200,18～1 800,18～1800 ng/mL,respectively.The recovery rate was between 85％～115％,the intra-day RSD and inter-day RSD were both less than 15％.It was stable at room temperature for 24 h,repeated freeze-thaw,long-term storage,the detection accuracy was between 85％～115％and the RSD was less than 15％,and the relative matrix effect was within 20％.The drug concentration of the patients were determined,VEN plus O-VEN,the average value is 263.89±176.67 ng/mL;FXT plus N-FXT,the average value is 139.12±121.3 ng/mL.The developed method exact with low sample consumption and has the advantage of easy processing,high accuracy,strong specificity and good stability,and is suitable for rapid simultaneous determination of the concentration of FXT,VEN and their active metabolites in human serum,and provides reliable methodological support for accurate personalized medicine.
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