A Colorimetric Method for the Quantitative Determination of Nitrates and Nitrites in Biologic Fluids

An accurate method for the determination of nitrates in blood and urine is necessary for complete studies of nitrogen metabolism in patients to whom nitrates are given as diuretics. Various calorimetric methods for the determination of nitrates have been described. Caron (1) used diphenylamine, but the results were widely variable. Letts and Rea (2) replaced diphenylamine with diphenylbenzidine. The resulting blue color, however, fades rather rapidly and calorimetric determinations could not be made. In the technique to be described in this paper, the uses of diphenylbenzidine and of sulfuric acid were retained. Otherwise, the method is substantially new. By this method it was possible to get a uniform development of color under conditions that permitted the calorimetric comparison of standard and unknown. The method permits the determination of 0.0003 mg. of nitrate nitrogen for each cc. of material and so is suit.able for biologic fluids, such as blood, urine, edema and ascitic fluids. The essential elements in control of the development of color are prevention of rapid fading of color, control of temperature, and removal of protein. Method.