Abstract
Publisher Summary This chapter reviews high-performance liquid chromatography (HPLC) methods for the determination of thiamin in food and blood. The HPLC methods used for thiamin are generally based on a reversed-phase column using either ultraviolet (UV) or fluorimetric detection. An external standard is prepared from a thiamin diphosphate working solution. Chromatographic peak areas are calculated and quantitation is carried out by plotting reported values against a standard calibration curve. In techniques involving fluorimetric detection, thiamin and its phosphate esters are quantitatively converted to thiochrome and thiochrome phosphate esters. This reaction can be applied to a HPLC system by using two different approaches. In the first approach, the appropriate reagent is added to the sample containing compounds and the thiochrome derivatives are chromatographed (precolumn derivatization). In the second approach, the reaction is performed after the chromatography. The thiochrome reaction is rapid as performed in highly alkaline solution, where thiochrome and its phosphate esters are stable. Reagents usually used for the alkaline oxidation are potassium hexacyanoferrate, mercuric chloride, and cyanogen bromide.
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