利用HPLC-MS/MS联用技术定量测定人血浆中叶酸及5-甲基四氢叶酸浓度的方法学研究

为定量测定人血浆中叶酸及 5 -甲基四氢叶酸浓度 ,建立了利用 HPLC-MS/ MS联用技术的分析方法。血浆样品在弱光环境下 ,经 SPE萃取处理 ,以 5 m mol/ L甲醇与乙酸铵 ( p H6.0 ) ( V(甲醇 ) / V(乙酸铵 ) =2 5∶ 75 )为流动相 ,经 Waters XTerra MS C1 8色谱柱初步分离 ,利用串联质谱多反应离子监测 ( MRM)方式监测叶酸、5 -甲基四氢叶酸和内标的正离子 ,然后进行定量测定。结果表明 :叶酸和 5 -甲基四氢叶酸的最低定量浓度均为 0 .5 ng/ m L;线性范围为 0 .5～ 2 5 ng/ m L,标准曲线相关系数 r≥ 0 .99,精密度和准确度均在± 1 5 %之内。此法专属、灵敏、准确 ,可以用于测定人血浆叶酸和 5 -甲基四氢叶酸的浓度

Quantitative Determination of Folic Acid and 5-Methyltetrahydrofolic Acid in Human Plasma by High Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive and specific HPLC-MS/MS method was developed for the quantitative determination of folic acid and 5-methyltetrahydrofolic acid in human plasma. Plasma samples were prepared through SPE cartridges under subdued laboratory light environment and separated through an Waters XTerra MS C18 column with methanol: 5mmol/L ammonium: acetate (pH6.0) = 25∶75 (v/v) as the mobile phase. Analytes were ionized in the gas-auxiliary electrospray ionization source (ESI) and positive ions were detected by mass analyzer in multiple reaction monitoring mode. Then peak areas were quantitated. The results showed both of the limits of quantitation of folic acid and 5-methyltetrahydrofolic acid were 0.5ng/mL, and the linear ranges were 0.5—25ng/mL. Correlation coefficients of all stadard curve are r≥0.99. Accuracies and precisions of this method are within ±15%. This method is specific, sensitive and accurate and will be applied to the determination of folic acid and 5-methyltetrahydrofolic acid in human plasma.