Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversed-phase high-performance liquid chromatography with photodiode-array detection and on-line validation of peak purity

A new, robust and sensitive reversed-phase high-performance liquid chromatographic method was developed for concomitant measurement of plasma concentrations of the ATP catabolites adenosine and hypoxanthine in human umbilical cord blood. Deproteinized cord plasma was chromatographed on Hypersil C₁₈ columns, using UV photodiode-array detection, spectral analysis of peaks and on-line confirmation of peak purity. Elution with a gradient of acetonitrile–tetrahydrofuran in ammonium dihydrogen phosphate buffer pH 4.7, yielded sharp, well-resolved peaks of adenosine and hypoxanthine within 16 min. Peak areas were quantified from external calibration curves and converted to plasma concentrations via cord blood hematocrits. In seven deliveries, gestational ages 32–40 weeks, adenosine (range, 0.1–2.1 μM) was less than hypoxanthine (range, 1.6–18.5 μM) in the same cord plasma sample. Arteriovenous levels of each purine were similar, except in an abruptio placenta delivery.