Measurement of adenosine, inosine and hypoxanthine in human term placenta by reversed-phase high-performance liquid chromatography

Abstract

An analytical method was developed for measuring adenosine, inosine and hypoxanthine in freshly delivered human term placentas. Representative freeze-clamped samples were taken from the sub-maternal surface of each placenta. Acid-soluble extracts of the samples were analyzed by reversed-phase high-performance liquid chromatography on columns packed with 10-μm porous octadecylsilica, using gradient elution with a linear increase in methanol concentration in ammonium phosphate buffer. Resolution of hypoxanthine from xanthine and adenosine from adenine, and quantitation of hypoxanthine and adenosine were achieved using 0.05 M ammonium dihydrogen phosphate, pH 6.5, as the low-strength eluent. Resolution of inosine from a prominent peak of β-NAD was optimized using 0.02 M ammonium dihydrogen phosphate, pH 5.6, as low-strength eluent. Recovery of standards was > 90%. Mean contents (± S.D.) of the analytes in placentas from seven normal deliveries were, adenosine 30.6 ± 11.5 nmol/g, inosine 68.0 ± 25.8 nmol/g and hypoxanthine 217 ± 127.5 nmol/g.