SIMULTANEOUS MEASUREMENT OF ADENOSINE AND HYPOXANTHINE IN HUMAN UMBILICAL-CORD PLASMA USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PHOTODIODE-ARRAY DETECTION AND ONLINE VALIDATION OF PEAK PURITY

A new, robust and sensitive reversed-phase high-performance liquid chr omatographic method was developed for concomitant measurement of plasm a concentrations of the ATP catabolites adenosine and hypoxanthine in human umbilical cord blood. Deproteinized cord plasma was chromatograp hed on Hypersil C-18 columns, using UV photodiode-array detection, spe ctral analysis of peaks and on-line confirmation of peak purity. Eluti on with a gradient of acetonitrile-tetrahydrofuran in ammonium dihydro gen phosphate buffer pH 4.7, yielded sharp, well-resolved peaks of ade nosine and hypoxanthine within 16 min. Peak areas were quantified from external calibration curves and converted to plasma concentrations vi a cord blood hematocrits. In seven deliveries, gestational ages 32-40 weeks, adenosine (range, 0.1-2.1 mu M) was less than hypoxanthine (ran ge, 1.6-18.5 mu M) in the same cord plasma sample. Arteriovenous level s of each purine were similar, except in an abruptio placenta delivery . (C) 1998 Elsevier Science B.V.