Because thiamine is involved in numerous metabolic processes, proper maintenance of thiamine levels in the human diet is essential. A deficiency of thiamine can produce degenerative disorders such as edema, muscular atrophy, or even beriberi. In addition to foods containing thiamine (e.g., nuts, vegetables, pork, and liver), thiamine is also available in many commercial multiple-vitamin supplements, including vitamin-B complexes and anti-stress preparations.
The determination of thiamine by kinetic fluorescence involves the conversion of thiamine to fluorescent thiochrome, during which changes in fluorescent are monitored. Ryan and Ingle, who developed the fluorometric reaction-rate method for thiamine determination, found Hg2+ to be the most suitable oxidizing agent.1 The reaction is shown in Figure 1.
Ryan and Ingle applied the technique to the determination of thiamine in a synthetic vitamin-mineral preparation. Bowers modified their procedure for a student-laboratory experiment to quantify thiamine in a variety of commercial vitamin preparations.2 The papers by Ryan and Ingle and by Bower contain detailed descriptions of their experimental methods.
For the calibration procedure, four thiamine standards were prepared in concentrations of 2.5, 1.25, 0.625, and 0.313 ppm. Mercuric chloride was dissolved in concentrated HCl and diluted to make a 500-ppm Hg2+ solution, and a phosphate buffer (pH = 12.2) was prepared from Na3PO412H2O) and Na2HPO4.
To 1 mL of the thiamine standard in a sample cuvette was added 1 mL of the Hg2+ solution followed by 1 mL of buffer.