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.
Lee
B L
BL
Department of Community Medicine, National University of Singapore, Kent Ridge.
Ong
H Y
HY
Ong
C N
CN
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