SHORT COMMUNICATIONS Determination of inorganic Phosphate by the Molybdovanadate Method in the Presence of ATP and Some Interfering Organic Bases
A number of spectrophotometric methods have been described for the assay of inorganic phosphates in biological material. These methods employ molybdate to react with phosphate to form molybdophosphate (1, 2). However, during our work with sodium and potassium activated ATPase, it was found that these methods are not applicable when drugs classified as neuro-and psychosedatives, such as chlorpromazine, fhaloperidol, diazepam, and hydroxyzine, are present, These aagents formed turbidity in the presence of inorganic phosphate and molybdate. In the absence of inorganic phosphate, h,owever, the turbidity did not appear. It seems that these drugs form a base-molybdophosphate complex. The extraction of molybdophosphate into organic solvent such as isobutanol or isobutanol-benzene (3) resulted in the transfer of turbidity into the organic phase. The precipitates were soluble in ethanol, but subsequent reactions with reducing agents did not produce the proper color.
We tried several 'organic solvents for the extraction of the neurosedative agents. It was found that these agents could be removed by shaking with chloroform before the addition of molybdate. Satisfactory results were obtained when inorganic phosphate was determined by the molybdovanadophosphate method described by Lecocq and Inesi (4) with modifications.
These authors state that deproteinization can be carried out in a single step by the addition of the coloring reagent, but it is our experience that the developed color precipitates with the protein. We therefore stopped the enzyme reaction by adding trichloroacetic acid to a final concentration of 4% (w/v). Extraction of the interfering agents by chloroform was subsequently carried out. The molybdovanadoph.osphate was found to be soluble in isobutanol in the presence of hydrochloric acid.