HPLC determination of orthophosphate coexisting with large amounts of organophosphates in biological samples

A simple and accurate HPLC method for the determination of orthophosphate in the presence of large amounts of organophosphates is described. The method is based on the formation and separation of the molybdenum orthophosphate complex. In order to prevent the hydrolysis of organophosphates, the sample was deproteinized with silicotungstate in acetate buffer (pH 4.0) under ice-cooling and then treated with ammonium molybdate in maleate buffer (pH 7.0). The sample was injected onto Styragel 60 A column (5 mm ID x 100 mm) with 38% (v/v) acetonitrile containing 0.3 M sulfuric acid as eluent. Detection was at 310 nm. The method was applied to the determination of orthophosphate in liver, kidney, spleen and mouse blood.