A simple high-performance liquid chromatography assay for simultaneous measurement of adenosine, guanosine, and the oxypurine metabolites in plasma.

To study the effect of pharmacologic agents on the biologic fate of adenosine, a reversed-phase high-performance liquid chromatography (HPLC) assay coupled with a solid-phase extraction (SPE) method was developed for simultaneous determination of plasma adenosine, hypoxanthine, xanthine, inosine, guanosine, and uric acid. The HPLC system consisted of a reversed phase C18 column, UV detector set at 254 nm, and a mobile phase composed of 0.01 M ammonium phosphate: methanol (9.5 : 0.5) vol/vol with the final pH adjusted to 3.9. The standard curves were linear between 0.1-2 microg/mL for all the analytes (except uric acid 50-400 microg/mL), with r2 > 0.99. The absolute recoveries were >60% and accuracy >85% in almost all cases. The limit of detection was <1 ng based on absolute injection of the analytes. The intraassay variations were <10% and interassay variations <15%. The presence of a wide range of medications in plasma samples did not interfere with the assay. The assay was applied successfully to measure plasma adenosine and the oxypurine metabolites in humans and rats. It was noted that plasma concentrations of adenosine and the oxypurine metabolites can vary considerably depending on the method of blood sample collection, and that species differences are apparent.