液相色谱-串联质谱法测定心脏瓣膜置换术患者血浆中华法林的浓度

Determination of warfarin in plasma of patients undergoing cardiac valve replacement by liquid chromatography-tandem mass spectrometry

目的建立一种方便、可靠、灵敏的液相色谱-串联质谱(LC-MS/MS)法,测定心脏瓣膜置换术患者血浆中华法林的含量。方法在Agilent G6410A正离子检测模式下,以尼群地平为内标,色谱柱选择Agilent Poroshell 120 Phenyl Hexyl column(2.1 mm×50 mm,2.7μm),流动相A(0.2%甲酸+1%乙腈水溶液)∶B(0.2%甲酸乙腈溶液)=60∶40(体积比);流速为0.3 mL/min;进样量为10μL;柱温为25℃;每个样品的分析周期为4 min。用于定量分析的检测离子为:华法林质荷比(m/z)309→163,尼群地平m/z 361.2→315.1。结果游离华法林的线性范围为5~500 ng/mL,总华法林的线性范围为50~5000 ng/mL,其相关系数r均>0.990;最低定量限相对标准偏差(RSD)均<15%;日内、日间RSD均<15%;蛋白沉淀法对游离华法林和总华法林的提取回收率分别为78.34%~91.59%和87.33%~89.99%,基质效应为87.50%~104.87%,符合生物样品的检测要求。结论本实验所建立的LC-MS/MS法可以用于测定游离及总华法林的含量,为临床个体化用药提供参考。

Objective To establish a convenient,reliable and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determination of warfarin in plasma of patients undergoing cardiac valve replacement surgery.Methods An Agilent G6410A triple quadrupole mass spectrometer with positive ion mode was applied to carry out all experiments.Nitrendipine was used as internal standard.The chromatographic separation was achieved on an Agilent Poroshell 120 Phenyl Hexyl column(2.1 mm×50 mm,2.7μm)with isocratic elution.The mobile phase contains 0.2%formic acid and 1%acetonitrile in water(A)and 0.2%formic acid in acetonitrile(B),with volume ratio being 60∶40;flow rate was 0.3 mL/min;injection volume was 10μL;column temperature was 25℃;and analytical time for each sample was 4 min.The precursor ion and the product ion used for quantitative analysis were:warfarin mass-to-charge ratio(m/z)309→163,nitrendipine m/z 361.2→315.1.Results The linear concentration ranges of free warfarin and total warfarin were 5-500ng/mL and 50-5000 ng/mL,respectively,with the correlation coefficient(r)both over 0.990.The relative standard deviation(RSD)values of the lowest limit of quantification were less than 15%.The RSD values of the inter-and intra-day precision were less than 15%.The recoveries of free warfarin and total warfarin by protein precipitation were 78.34%-91.59%and 87.33%-89.99%,respectively,and the matrix effect was 87.50%-104.87%.All the items required in pharmacopeia for methodology validation were acceptable.Conclusion This established LC-MS/MS method can be used to determine the concentrations of free and total warfarin,providing a reference for individualized clinical treatment.