Development and validation of an RP‐HPLC method for the estimation of adenosine and related purines in brain tissues of rats

Abstract A new, rapid and sensitive RP‐HPLC method with UV spectrophotometric detection was developed and validated for the concomitant estimation of adenosine and related purines in rat brain tissue preparations. The HPLC system consisted of C‐18 column with UV–photodiode‐array detection ranging from 210 to 400 nm, facilitating the online confirmation of peak purity. The column temperature was maintained at 30°C and the injection volume was 20 μL. Elution with an isocratic mobile phase consisting of water/methanol/acetonitrile (88:5:7 by volume) at a flow rate of 0.8 mL/min yielded sharp, utmost‐resolved peaks of adenosine (Ade), inosine (Ino), hypoxanthine (Hypoxan) and adenine (Adn) within 10 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity and stability. The method was also employed to estimate the naturally occurring purines in discrete regions of rat brain. A new protocol developed for tissue preparation utilizing H 2 SO 4 and Tris buffer gave well‐resolved peaks and high component recoveries (>96%) which eliminated the need of an internal standard. The results show that the method for the determination of Ade, Ino, Hypoxan and Adn by RP‐HPLC described here has good linearity, accuracy, precision, sensitivity, selectivity and is simple and rapid to perform.