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Separation and automated analysis of phosphorylated metabolic intermediates
Authors:Samuel P Bessman  Paul J Geiger  Tsung-Cho Lu  Edward RB McCabe
Institution:Department of Pharmacology, University of Southern California School of Medicine, 2025 Zonal Avenue, Los Angeles, California 90033, USA
Abstract:A chromatographic procedure has been described using a new automated detection system that allows separation at the 10 nmole level of all the glycolytic intermediates as well as AMP, ADP, and ATP in pure samples and samples of skeletal muscle and blood. Separations are carried out on 3 × 500 mm columns packed with modern anion exchange resins of closely sized, fine particles and a linear gradient of ammonium chloride containing borate in order to complex the sugar phosphates. Pressures are moderate, and elutions are complete within 5–8 hr with excellent reproducibility and recovery of each compound. Screening runs can be made in only 90 min with shorter columns and with some sacrifice in resolution for certain compounds.
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