CHEMICAL DETEEMINATION OF VITAMIN B1 I. REACTION BETWEEN THIAMINE IN PURE AQUEOUS SOLUTION AND DIAZOTIZED p-AMINOACETOPHENONE BY DANIEL MELNICK*

In the recent literature several chemical methods for the determination of thiamine are described. Some of these admittedly lack specificity (l-3). The determination of the blue fluorescence in ultraviolet light of thiochrome, one of the oxidation products of thiamine, has been most widely investigated (4-12). Its application to the quantitative determination of thiamine is complicated by similar blue fluorescence given by other substances (4-7, 9), the difficulty in correcting for such non-specific fluorescence (6, 13), the presence in biological materials of substances which may combine with thiochrome to form non-fluorescent compounds (7) or which may affect the intensity of the fluorescence (14, 15), the instability of the fluorescence during measurement (16), and the fact that thiochrome is an intermediate in the oxidation of thiamine (4-8), the yield of which must be influenced by other oxidizing and reducing substances (4, 6-S) as well as the amount of thiamine and reagent present (4-7). In 1934, Kinnersley and Peters (17) reported that the reaction between thiamine and diazotized sulfanilic acid in alkaline solution was made more sensitive by the presence of formaldehyde. This reaction has been repeatedly criticized because of lack of specificity. Their procedures were subsequently improved (18) so that semiquantitative data were obtained when the reaction was applied to the determination of thiamine in biological materials. Prebluda and McCollum (19) modified the reagent using diazotized p-aminoacetophenone; the formaldehyde addition was