MEASUREMENT OF ADENOSINE AND INOSINE IN HEART SAMPLES BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

Adenosine and inosine are sensitive indicators of myocardial hypoxia. Previously reported methods for measurement of the low concentrations of these nucleosides have either been cumbersome or require dual wavelength spectrophotometry. With our method concentrations in samples of heart were determined by ultraviolet absorbance following separation by reverse phase high pressure liquid chromatography. Trichloroacetic acid extracts of a minimum of 15 mg of tissue were treated with a strong anion exchange resin (Dowex-1) and then injected directly onto the column. The initial solvent was 4 mm KH2PO4 (pH 7.3) and the nucleosides were sequentially eluted using a linear gradient consisting of 80% CH3OH: 20% H2O against the initial solvent. Absorbance was measured at 260 nm. The nucleosides were quantitated by relating the areas under the peaks to an AMP standard peak of known concentration. This technique combines high nucleoside recovery (> 90% for adenosine and inosine), high sensitivity (20 pmol), and high reproducibility with rapid separation and detection (< 15 min). © 1979.