Rapid method for the determination of total 5-methyltetrahydrofolate in blood by liquid chromatography with fluorescence detection
Rapid method for the determination of total 5-methyltetrahydrofolate in blood by liquid chromatography with fluorescence detection
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DOI：
10.1016/s0378-4347(01)00521-7
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发表时间：
2002-01-25
影响因子：
3
通讯作者：
Ang, CYW
中科院分区：
文献类型：
--
作者：
Luo, WH;Li, H;Ang, CYW
建立了测定全血样品中总5-甲基四氢叶酸（5-MTHF）的液相色谱法。该方法被应用于调查的全血总5-MTHF水平的育龄妇女。为了测定全血总5-MTHF含量，将全血样品冷冻并解冻以破坏红细胞，释放5-MTHF聚谷氨酸盐并通过血浆中的内源性聚谷氨酸盐水解酶水解为5-MTHF单谷氨酸盐。简而言之，将0.1 ml全血样品的等分试样与0.3 ml 57 mmol/l抗坏血酸混合，并在37 ℃下孵育60 min，然后用0.6 ml缓冲液（0.2 mol/l磷酸氢二钾和30 mmol/l巯基乙醇，pH 8.5）稀释。样品经100 ℃加热离心10 min后，上清用反相液相色谱-荧光检测器分析，加标回收率为95 ~ 105%，日内和日间相对标准偏差均小于6.5%。基于三倍噪声水平（峰间），估计检测限为30 mmol/l。应用该法对育龄妇女全血总5-MTHF水平进行了调查，结果表明该法可靠，适用于血总5-MTHF的测定。(C)2002 Elsevier Science B. V.保留所有权利。
A liquid chromatographic method is described for the determination of total 5-methyltetrahydrofolate (5-MTHF) in whole-blood samples. The method was applied to a survey of whole-blood total 5-MTHF levels of women at child-bearing age. To determine whole-blood total 5-MTHF content, a whole-blood sample was frozen and thawed to break red blood cells and the 5-MTHF polyglutamates were released and hydrolyzed into 5-MTHF monoglutamate by endogenous polyglutamates hydrolase in the plasma. In brief, an aliquot of 0.1 ml whole-blood sample was mixed with 0.3 ml 57 mmol/l ascorbic acid and incubated at 37degreesC for 60 min, then diluted with 0.6 ml buffer solution (0.2 mol/l potassium phosphate dibasic and 30 mmol/l mercaptoethanol, pH 8.5). After the sample was heated at 100degreesC for 10 min and centrifuged, the supernatant was analyzed by reversed-phase liquid chromatography with fluorescence detection, The recoveries from spiked samples were from 95 to 105% with within-day and day-to-day relative standard deviations less than 6.5%. The detection limit was estimated to be 30 mmol/l based on three times the noise level (peak to peak). Application of the method to a survey of whole-blood total 5-MTHF levels of women at child-bearing age showed that the method was reliable and suitable for the determination of blood total 5-MTHF. (C) 2002 Elsevier Science B.V. All rights reserved.