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A colorimetric determination of orotic acid reported by Tsuji was examined in detail and improved in several points. Ascorbic acid gave better results than thioglycollate in the step of reduction. Satisfactory results were obtained when the improved method was applied to several multivitamin preparations. The mechanism of the color development was also described. It has been elucidated that bromination of orotic acid results in the production of 5, 5-dibromobarbituric acid and the brominated compound is reduced with ascorbic acid to barbituric acid which condenses with P-dimethylaminobenzaldehyde to yield an orange color, 5-(P-dimethylaminobenzylidene)-barbituric acid.
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Adachi et al. (1963) studied this question.
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