Preparation and measurement of the purity of the phosphatase reagent, disodium p-nitrophenyl phosphate.

A few years ago one of the authors (0. A. B.) and his colleagues described a relatively simple micromethod for the determination of alkaline phosphatase in serum (1). The method is based on the spectrophotometric measurement of the amount of p-nitrophenol (which absorbs light strongly at 400 mp) liberated from p-nitrophenyl phosphate under certain standard conditions and is simple and rapid to use, owing to the fact that one of the products of phosphatase activity can be measured directly. Because of this feature and the sensitivity of the method, it has found considerable use, especially in studies involving large numbers of determinations, such as dietary surveys. Neither a method of preparation nor a method for determining the purity of the substrate (disodium p-nitrophenyl phosphate) was included in the original publication because the commercial availability of a satisfactory product was anticipated. Since a number of inquiries for a method of preparation continued to be received and since experience has shown the necessity for knowing the purity of the particular specimen in order to prepare properly the phosphatase reagents, there is presented below a simple and satisfactory method for the synthesis of the disodium. salt of p-nitrophenyl phosphate and for determining its purity.