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HPLC PLASMA ADENOSINE ASSAY FOR USE IN CARDIAC STUDIES: 23

To optimize an HPLC assay for adenosine (Ado) in plasma every step in the procedure was examined. The presence in the plasma of several drugs used in the cardiac studies interferred with the usual isocratic HPLC assay of Ado and necessitated the use of a step-gradient. Sample preparation procedures routinely used in biomedical studies were evaluated to determine lability of adenosine prior to chromatographic analysis. Factors investigated included the use of EHNA (an inhibitor of adenosine deaminase), dipyridamole (an inhibitor of nucleoside transport) and EDTA (an inhibitor of ATPase and 5'-nucleotidase). Effects of deprotenization methods, time of analysis, minimum detectable limits and recovery of Ado from both whole blood and plasma were examined. Ado values in samples treated immediately with PCA were much lower than those in which the samples were collected in solutions of EHNA and dipyridamole. Moreover, the latter samples showed substantial intra- and inter-animal variability. However, if EDTA was added to the sample solution, along with the EHNA and dipyridamole, the concentrations of Ado in the plasma samples were similar to those obtained with the PCA. Precision studies showed marked improvement. Our results provide evidence that in the analysis of plasma for Ado, in vitro production as well as the catabolism of Ado must be stopped in order to obtain valid estimates of plasma Ado concentration.