Iwata H, Matsuda T, Tonomura H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
J Chromatogr. 1988 Oct 26;450(3):317-23. doi: 10.1016/s0021-9673(01)83586-x.
An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.
描述了一种使用柱前衍生反相高效液相色谱法测定动物组织中硫胺素及其磷酸酯的改进方法。硫胺素及其磷酸酯通过碱性溴化氰转化为荧光团，衍生物被应用于ODS填充柱。然后，将酸性流动相得到的流出物与碱性甲醇溶液混合，以增加衍生物的荧光强度，通过荧光分光光度法进行测定。实现了硫胺素及其磷酸酯的完全、快速和定量分离，并且使用酸性缓冲液作为流动相提高了柱稳定性。色谱图上硫色素酯峰的荧光团对用硫胺素三磷酸酶或酸性磷酸酶进行预处理敏感。证明了该方法在测定大鼠各种组织中硫胺素形式的适用性。
