Determination of Adenosine and Inosine in Sheep Plasma Using Solid Phase Extraction Followed by Liquid Chromatography with UV Detection

Abstract

A simple and sensitive liquid chromatography assay following solid phase extraction was developed for simultaneous determination of adenosine and inosine in sheep plasma. The system consisted of a Symmetry C₁₈ column, a mobile phase composed of acetonitrile, 100 mM sodium dihydrogen phosphate and water, and ultraviolet detection at 254 nm. The method showed good sensitivity (limits of detection for adenosine and inosine were 30 and 50 ng/ml, respectively, in the plasma samples), repeatability, and linearity. The developed method was applied to sheep plasma samples from a study examining the cardio active potential of the combination of adenosine and inosine.

Keywords:

• Adenosine
• Inosine
• Liquid chromatography
• Sheep plasma
• SPE

E. Adams is a post-doctoral fellow of the Fund for Scientific Research (FWO) – Flanders, Belgium.

Notes

Mobile phase A: acetonitrile : 100 mM NaH₂PO₄ buffer : water (5:20:75, v/v/v), Mobile phase B: acetonitrile : 100 mM NaH₂PO₄ buffer : water (50:20:30, v/v/v); Column: Symmetry C18, 5 µm, 150 × 4.6 mm; Column temperature: 30°C; Injection volume: 20 µl; Flow rate: 1.0 ml/min; UV – detection: 254 nm.

R² = determination coefficient, x = concentration in ng/ml, S_y,x = standard error of y-estimate, n_i = number of injections per concentration.