超高效液相串联质谱测定四棱豆中总叶酸和多聚谷氨酸叶酸的含量

Determination of Total Folate Content and Polyglutamate Folate Distribution in Winged Beans Using UPLC-MS/MS

本文建立了超高液相色谱串联三重四级杆质谱(UPLC-MS/MS)定量测定四棱豆中总叶酸的含量和多聚谷氨酸叶酸分布的方法。四棱豆在缓冲液中蒸煮灭活内源多聚谷氨酸水解酶和破坏叶酸结合蛋白,使结合态叶酸变成游离态,过滤得提取液。将提取的各种形式叶酸转化为5-甲基四氢蝶酰多聚谷氨酸(5MTHFGlun),并经C18萃取小柱除盐后,采用UPLC分离不同形态的5MTHFGlun,电喷雾正离子电离模式(ESI+)和多反应监控扫描模式(MRM)测定不同形态的5MTHFGlun含量。方法采用Pte Glu(1-6)作为内标,5MTHFGlu(1-6)外标进行定量。该方法灵敏度高,最低检测限(LOD)为40~1133 fmol,最低定量限(LOQ)为120~3400 fmol,精密度为0.5%~6.8%,平均回收率为43%。研究发现四棱豆中多聚谷氨酸叶酸的主要形式是蝶酰五谷氨酸,占叶酸总量的65%;其次是蝶酰五谷氨酸占30%;其它形式只占5%;总叶酸含量为1.1μmol/100 g,其含量显著高于菠菜和西兰花等蔬菜。该方法快捷、灵敏、精确,适用于蔬菜中叶酸的分析。

An ultra-high performance liquid chromatography tandem mass-spectrometry(UPLC-MS/MS) method was established to determine the total folate content and polyglutamate folate distribution in winged beans. Coarsely chopped winged beans were boiled to inactivate endogenous polyglutamate hydrolase and degrade folate-binding proteins so that the bound folate was converted to free form. After filtration, the extracted folate in various forms was converted to 5-methyl tetrahydrofolate polyglutamyl(5MTHFGlun), salt was removed using a C18 SPE cartridge, and UPLC was performed to separate the different forms of 5MTHFGlun. The content of 5MTHFGlun in different forms were determined using positive electrospray ionization(ESI+) under multiple reaction monitoring(MRM) mode. Quantitative analysis was conducted using Pte Glu(1–6) as the internal standard and 5MTHFGlu(1–6) as external standards. The method showed high sensitivity, with a limit of detection of 40~1133 fmol and a limit of quantification of 120~3400 fmol. The precision of determination ranged from 0.5% to 6.8% and the average recovery was 43%. The results showed that the predominant form of polyglutamate folate in winged bean was pentaglutamyl folate(65%), followed by 5MTHF, accounting for 29.6%; other forms accounted for only 5.6%. The total folate content in fresh winged beans was 1.1 μmol/100 g, which was significantly higher than that in spinach and broccoli. This method is fast, sensitive, and accurate, and is suitable for determining total folate and polyglutamyl folate distribution in different vegetables.