Determination of thiamine and its phosphate esters by gradient-elution high-performance liquid chromatography.
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.
Department of Community Medicine, National University of Singapore, Kent Ridge.
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Lee
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Ong
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Ong
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013050:Spectrometry, Fluorescence; D013831:Thiamine / Q000097:blood*; D013833:Thiamine Monophosphate / Q000097:blood*; D013835:Thiamine Pyrophosphate / Q000097:blood*; D013838:Thiamine Triphosphate / Q000097:blood*
D013838:Thiamine Triphosphate; D013833:Thiamine Monophosphate; D013835:Thiamine Pyrophosphate; D013831:Thiamine