Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of (13)C(5)]glutamate derived from (13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of (13)C(5)]glutamate using (13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of (13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of (13)C(5)]glutamate formed from (13)C(5)]glutamine using amidophosphoribosyltransferase.