Subtraction method for the high-performance liquid chromatographic measurement of plasma adenosine

The measurement of plasma adenosine with traditional high-performance liquid chromatographic techniques is difficult because of its nanomolar concentration, its short half-life in blood, and because of the difficulty in isolating adenosine from interfering peaks in the chromatogram. To prevent loss of adenosine in the blood sample, a "stop solution" is used to prevent enzymatic degradation and cellular uptake. Peak-shifting techniques on fractionated samples to measure adenosine derivatives have been used in the past to avoid interfering peaks in the chromatogram. A new method has been developed by which nanomolar levels of plasma adenosine can be accurately measured despite co-eluting peaks in the chromatogram. In this method, plasma samples are collected with a stop solution, processed, and divided. Adenosine deaminase is added to part of the sample to form a blank. A computer program subtracts the blank chromatogram from the paired unknown, and the result is compared to adenosine standards prepared from the blank and subtracted in a similar fashion. With this subtraction method, the overall recovery of physiological concentrations of adenosine was 89% from dog blood, and the average coefficient of variation was 12%. In summary, the subtraction method of plasma adenosine measurement offers good recovery, reproducibility, and the ability to quantify low levels of adenosine despite interfering peaks in the chromatogram.