采用梯度洗脱高效液相色谱法测定硫胺素及其磷酸酯。

Determination of thiamine and its phosphate esters by gradient-elution high-performance liquid chromatography.

作者信息

Lee B L, Ong H Y, Ong C N

机构信息

Department of Community Medicine, National University of Singapore, Kent Ridge.

出版信息

J Chromatogr. 1991 Jun 14;567(1):71-80. doi: 10.1016/0378-4347(91)80311-y.

DOI:10.1016/0378-4347(91)80311-y

PMID:1918262

Abstract

A high-performance liquid chromatographic method for the determination of thiamine and its phosphate esters using gradient elution is presented. Chromatography was performed on an octadecyl C18 column with post-column derivatization using potassium ferricyanide: thiamine was oxidized to thiochrome, which was detected fluorimetrically. The gradient system avoids the use of a modifier and an ion-pairing reagent. The proposed method enables the separation of thiamine triphosphate, thiamine pyrophosphate, thiamine monophosphate and free thiamine, at concentrations of ca. 2 nM, within 10 min The within-assay precision for blood and serum total thiamine was less than 2 and 5%, respectively, and the between-day variation was less than 3 and 5%, respectively. The method is simple and rapid, and it can be used for screening of thiamine deficiency and for clinical study of various diseases related to vitamin B1 deficiency.

摘要

本文介绍了一种采用梯度洗脱测定硫胺素及其磷酸酯的高效液相色谱法。色谱分析在十八烷基C18柱上进行，柱后衍生使用铁氰化钾：硫胺素被氧化为硫色素，通过荧光法进行检测。该梯度系统避免了使用改性剂和离子对试剂。所提出的方法能够在10分钟内分离浓度约为2 nM的三磷酸硫胺素、焦磷酸硫胺素、单磷酸硫胺素和游离硫胺素。血液和血清中总硫胺素的批内精密度分别小于2%和5%，批间变异分别小于3%和5%。该方法简单快速，可用于硫胺素缺乏症的筛查以及与维生素B1缺乏相关的各种疾病的临床研究。