Sensitive and selective assay for adenosine using high-pressure liquid chromatography with fluorometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cardiology

Background:

• Adenosine plays a crucial role in myocardial function.
• Accurate quantitation of adenosine is essential for understanding cardiac physiology and pathology.
• Existing methods for adenosine measurement may lack sufficient sensitivity for certain applications.

Purpose of the Study:

• To develop and validate a novel, highly sensitive method for adenosine quantitation.
• To compare the performance of the new method with established techniques.
• To assess the applicability of the method for analyzing adenosine in canine myocardial extracts.

Main Methods:

• Incubation of myocardial extracts with chloroacetaldehyde to form 1,N6-ethenoadenosine.
• Separation of the fluorescent derivative using high-pressure liquid chromatography (HPLC).
• Quantitation of ethenoadenosine via fluorometry.

Main Results:

• The method demonstrated high selectivity for adenosine.
• Linear correlation was observed between fluorescence peak height and ethenoadenosine quantity.
• Quantitative conversion of adenosine to ethenoadenosine was confirmed.
• Adenosine estimates closely matched those obtained by HPLC with UV absorption.
• The fluorometric method exhibited significantly greater sensitivity (0.50 pmol) compared to UV absorption (approx. 20 pmol).

Conclusions:

• The developed fluorometric method provides a sensitive and accurate means for adenosine quantitation.
• This technique is suitable for analyzing adenosine in canine myocardial tissue.
• The enhanced sensitivity makes it ideal for samples with low adenosine concentrations.