Enzymatic determination of phosphate in conjunction with high-performance liquid chromatography.
A selective assay for orthophosphate in complex matrices was developed based on the nucleoside phosphorylase catalyzed conversion of inosine and orthophosphate of hypoxanthine. The enzyme reaction was using only 0.28 units/assay was allowed to proceed for 30 min before quenching. Separation of inosine and hypoxanthine was performed by reversed-phase high-performance liquid chromatography. Quantitation of the hypoxanthine peak was found to be linear with orthophosphate up to 30 micrograms/g. A detection limit of 0.75 ppm could be obtained after dialysis of the commercial enzyme. Interference studies showed that the enzymatic assay unlike the colorimetric molybdate-blue technique was essentially unaffected by complex matrices such as serum, urine, polyphosphates, and phosphoesters.
D002851:Chromatography, High Pressure Liquid; D005189:False Positive Reactions; D006801:Humans; D007042:Hypoxanthines / Q000302:isolation & purification; D008982:Molybdenum; D010710:Phosphates / Q000032:analysis* / Q000097:blood / Q000652:urine; D011683:Purine-Nucleoside Phosphorylase; D013056:Spectrophotometry, Ultraviolet
D007042:Hypoxanthines; D010710:Phosphates; C017541:molybdenum blue; D008982:Molybdenum; D011683:Purine-Nucleoside Phosphorylase