Sensitive and selective assay for adenosine using high-pressure liquid chromatography with fluorometry.
A new method for quantitation of adenosine was tested in canine myocardial extracts. The method involves incubation of the extract with chloroacetaldehyde to form the fluorescing adenosine derivative 1, N6-ethenoadenosine. The ethenoadenosine is separated by high-pressure liquid chromatography (HPLC) and quantitated by fluorometry. Experiments demonstrated that 1) the method is selective for adenosine, 2) fluorescence peak height is linearly related to the quantity of ethenoadenosine, and 3) adenosine in the extract is quantitatively converted to ethenoadenosine by the incubation procedure. Also, within the range of adenosine concentrations seen in five extracts, estimates of myocardial adenosine content with the fluorometric method were nearly identical to those using the more routine technique of HPLC with direct detection by ultraviolet (UV) absorption. A primary advantage of the fluorometric method is its greater sensitivity. As little as 0.50 pmol on the column could be quantitated by fluorescence, compared with approximately 20 pmol with UV absorption. Because of the greater sensitivity, the fluorometric method should be more easily applied to samples with smaller adenosine concentrations.
D000225:Adenine / Q000031:analogs & derivatives; D000241:Adenosine / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004285:Dogs; D009206:Myocardium / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
C004981:1,N(6)-ethenoadenine; D000225:Adenine; D000241:Adenosine