Quantitation of adenosine, inosine and hypoxanthine in biological samples by microbore-column isocratic high-performance liquid chromatography

This paper describes a simple and sensitive high-performance liquid chromatographic method for measuring adenosine, inosine and hypoxanthine in cell suspensions. The method involves direct injection of the filtered sample on a microbore C18 reversed-phase column with UV detection at 259 nm. The mobile phase consisted of 125 mM potassium dihydrogenphosphate, 1.0 mM tetrabutylammonium hydrogen-sulfate, 1.5% acetonitrile and 20 mM triethylamine, pH 6.5. The minimum detectable amounts (signal-to-noise ratio of 3:1) were 2.0 pmol of adenosine, 2.5 pmol inosine and 3.5 pmol of hypoxanthine. The limits of quantitation were 2.9 +/- 0.2 pmol for adenosine, 4.2 +/- 0.3 pmol for inosine and 4.9 +/- 0.4 pmol for hypoxanthine. This method was used to quantitate adenosine release by dispersed rat renal outer medullary cells (tubules) under conditions of normoxia and hypoxia.