Determination of Plasma Adenosine by High-Performance Liquid Chromatography with Column Switching and Fluorometric Detection

Abstract

A new method for the rapid determination of plasma adenosine concentrations was developed by using high-performance liquid chromatography with a column switching technique and fluorometric detection. Several "stop solutions" were used to prevent the enzymatic degradation and cellular uptake and release of adenosine in blood samples. Red blood cells and certain denatured proteins were separated by centrifugation. Subsequently, the supernatant was transferred directly into autosample vials and adenosine was reacted with chloroacetaldehyde to form a strong fluorescent, 1-N6-ethenoadenosine. The adenosine derivative was injected directly and separated on a shielded hydrophobic phase column coupled with a C18 reverse-phase column using a column switching valve. Macromolecules and other interfering substances were excluded by the shielded hydrophobic phase column and bypassed to waste. Then, the adenosine derivative and other retained compounds were switched onto the reverse-phase column for further separation and subsequently to the fluorescence detector. The system reduces the analysis time and contamination of the column and hence allows a shorter cleanup time and a longer column lifetime. Adenosine as low as 30 fmol (signal-to-noise ratio, S/N = 3) can be detected by this method. The percentage of recovery of adenosine in plasma treated with adenosine deaminase was above 90%. This method is very rapid (without tedious sample preparation) and sensitive for determining adenosine in canine blood and should prove to be useful in analyzing the effects of ischemia and reperfusion on arterial and coronary venous adenosine concentrations in blood or perfusate samples released from the ischemic or hypoxic myocardium.