超高效液相串联质谱法检测人血浆中华法林及其代谢产物的浓度

Determination of Warfarin and Its Metabolite in Human Plasma by Ultra Performance Liquid Chromatography Tandem Quadrupole Mass Spectrometry(UPLC-MS/MS)

目的：建立快速检测人血浆中华法林及其代谢产物7-羟基华法林浓度的UPLC-MS/MS方法。方法：用乙腈沉淀血浆蛋白的方法处理,运用Waters XEVO TQD三重四级杆液质联用仪,色谱柱为ACQUITY UPLCBEH C18柱（50 mm×2.1mm,1.7μm）;流动相为乙腈-水（含0.1%甲酸）,梯度洗脱,流速为0.4 ml·min-1,柱温为40℃,内标为华法林-d5;质谱条件：电喷雾离子化源（ESI）,正离子检测模式。结果：华法林和代谢产物7-羟基华法林的保留时间分别为1.8 min和1.5 min,线性范围分别为25～2 000 ng·ml-1（r=0.999 3）和5～500 ng·ml-1（r=0.999 6）,最低定量限分别为5,2.5 ng·ml-1,回收率分别为96.9%～105.3%,97.1%～103.3%,日内、日间RSD均〈10%。结论：该法准确可靠,操作简便,重复性好,适于检测人血浆中华法林及其代谢产物7-羟基华法林的浓度。

Objective：To establish an ultra performance liquid chromatography-tandem quadrupole mass spectrometry method for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma. Methods： An ACQUITY UPLC？ BEH C18 （50 mm × 2. 1 mm, 1. 7 μm） column was used as the stationary phase at 40℃. The mobile phase consisted of acetonitrile and water （con-taining 0. 1% formic acid） with gradient elution at a flow rate of 0. 4 ml＆#183;min-1 . Warfarin-d5 was used as the internal standard. The analytes were detected on a triple-quadrupole mass spectrometer equipped with an ESI interface in a positive mode. Results：The reten-tion time of warfarin and 7-hydroxywarfarin was 1. 8 min and 1. 5 min, respectively. Excellent linear calibration curve of warfarin and 7-hydroxywarfarin was obtained within the concentration range of 25-2 000 ng ＆#183; ml-1 （ r =0. 999 3 ） and 5-500 ng ＆#183; ml-1 （ r =0. 999 6）, respectively. The lower limit of quantification of warfarin and 7-hydroxywarfarin was 5 ng＆#183;ml-1 and 2. 5 ng＆#183;ml-1 with the average recovery of 96. 9%-105. 3% and 97. 1% -103. 3%, respectively. The intra-and inter-day standard deviations were both less than 10%. Conclusion： The method is accurate and simple, and suitable for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma.