AJP - Heart and Circulatory Physiology, Vol 245, Issue 5 887-H890, Copyright © 1983 by American Physiological Society
Sensitive and selective assay for adenosine using high-pressure liquid chromatography with fluorometry
M. K. Jacobson, L. M. Hemingway, T. A. Farrell and C. E. Jones
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.
