Analysis of submicromolar concentrations of adenosine in plasma using reversed phase high-performance liquid chromatography

Abstract

A method is described for the determination of adenosine in small samples of plasma (< 1 ml) using reversed-phase high-performance liquid chromatography (HPLC) in either a simple isocratic or a gradient elution system which gives a clear separation of adenosine from other plasma constituents. Acetone is used to deproteinize plasma and chloroform to remove unwanted lipid soluble material prior to HPLC. 6-Methyladenosine is used as an internal standard for making corrections for changes in concentration during sample processing. Adenosine in plasma could be reliably detected at concentrations lower than its minimum effector concentration as a vasodilator (4 x 10(-8) Mol l(-1) using the isocratic system and 1.9 x 10(-8) Mol l(-1) with gradient elution). The recoveries of adenosine added to blood at concentrations ranging from 2 x 10(-8) Mol l(-1) to 1.4 x 10(-6) Mol l(-1) were from 101.4 +/- 16.9% (n = 4) to 100.0 +/- 3.6% (n = 5). The present method provides a simple, sensitive and selective assay for submicromolar concentrations of adenosine in plasma with good recovery.