The chemical pathway of the carbamidodiacetyl colorimetric assay was investigated. The experimental variables that were studied include reaction temperature, heating time, and the ratio of the mineral acid mixture to the reactants. Evidence is presented establishing the involvement of either 7- or 8-methyl, or 7,8-dimethyltetrahydroimidazo(4,5-d)imidazole-2,5-diones as chromogens, all three being equally acceptable.
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Veniamin et al. (1970) studied this question.