SEPARATION OF FLUORESCENT-1,N6 ETHENODERIVATIVES OF DIADENOSINE POLYPHOSPHATES AND THEIR ENZYMATIC DEGRADATION PRODUCTS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The retention, selectivity and elution order of fluorescent 1,N6-etheno derivatives of diadenosine polyphosphates and their enzymatic degradation products on octadecyl and phenyl-bonded silica columns have been studied as a function of mobile phase pH, ionic strength and organic modifier content. Good separations of the compounds of interest were achieved using mobile phases of around 0.1 M potassium phosphate buffers at neutral pH containing approximately 10 % methanol or 4 % acetonitrile for C18 columns and 5 % methanol or 1.5 % acetonitrile for phenyl columns. The data obtained were used to establish isocratic assays for diadenosine polyphosphate cleaving activities from chromaffin cells using Di(1,N6-ethenoadenosine) polyphosphates as fluorogenicsubstrate analogues followed by fluorescence detection.