Optimized determination of thiochrome derivatives of thiamine and thiamine phosphates in whole blood by reversed-phase liquid chromatography with precolumn derivatization.
A liquid chromatographic method involving precolumn derivatization for determining thiamine and its phosphate esters in human blood has been optimized. Blood sample stored at - 20 degrees C were haemolysed and deproteinized by perchloric acid. The supernatants of the samples were oxidized by addition of potassium ferricyanide-sodium hydroxide, and phosphoric acid was added to obtain a neutral pH in order to extend the column life. The samples were stable after derivatization for at least 24 h, if protected from light and kept at room temperature. Gradient separation with 140 mmol phosphate buffer (pH 7.0), and methanol, tert-butylammonium hydroxide and dimethylformamide as modifiers, on a 3-microns Chromsphere octadecylsilica column gave an analysis time of 15 min. The method was found to be very suitable for the determination of thiamine components in whole blood. The minimal detectable amount is 0.5 nmol/l and the method is linear to at least 1000 nmol/l. The recovery (98 +/- 3%) and precision are very good.
Brunnekreeft
J W
JW
Department of Clinical Chemistry, Streekziekenhuis Almelo, The Netherlands.
Eidhof
H
H
Gerrits
J
J
D002853:Chromatography, Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D013831:Thiamine / Q000031:analogs & derivatives* / Q000097:blood*; D013833:Thiamine Monophosphate / Q000097:blood*; D013835:Thiamine Pyrophosphate / Q000097:blood*; D013838:Thiamine Triphosphate / Q000097:blood*
D007202:Indicators and Reagents; D013838:Thiamine Triphosphate; D013833:Thiamine Monophosphate; C005793:thiochrome; D013835:Thiamine Pyrophosphate; D013831:Thiamine