Determination of adenosine, inosine and hypoxanthine in human plasma by high performance liquid chromatography

Inosine and hypoxanthine are endogenous low molecular plasma constituents normally found at low concetrations (200-400 ng/mL) in human plasma resulting from dietary and endogenous purine metabolism. Using the mouse model, inosine levels increased from cardiac tissue subjected to cardiac oxidative stress and may serve as potential biomarker indicative of early cardiac ischaemia. We report here development of a new HPLC method for the determination of adenosine, inosine and hypoxanthine using ultra-violet (UV) detection. Before analysis, plasma samples were deproteinized by centrifugal filtration through 10-kDa cut-off membranes. The protein-free ultrafiltrates were analysed on a monolithic reversed phase column Chromolith Performance RP-18e using gradient elution and UV detection at 250nm. Mobile phase consisted of 25 mM acetate buffer and methanol, the flow rate was 1mL/min. Optimal wavelength were confirmed by absorption spectra of inosine and hypoxanthine. Plasma levels of analytes were quantified on the basis of peak area using external standardization.