Estimation of adenosine and its major metabolites in brain tissues of rats using high-performance thin-layer chromatography–densitometry

A new, rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method with densitometry was developed and validated for the concomitant estimation of purines like adenosine (Ade) and its major metabolites, inosine (Ino) and hypoxanthine (Hypoxan) in rat brain tissue preparations. The HPTLC method was chosen in order to generate better resolution and evade the tedious and prolonged sample preparation methods necessarily performed with HPLC methods when analyzing biological samples. In this method the planar chromatography was executed on aluminum plates pre-coated with silica gel 60 F254. Elution was performed with a two-step gradient mobile phase consisting of solvent A [n-butanol/water/acetonitrile/10% ammonia solution/glacial acetic acid (5:2:4:1:0.5, v/v)] and solvent B [n-butanol/chloroform/acetonitrile/10% ammonia solution/glacial acetic acid (5:4:2:1:0.5, v/v)]. The quantitative analysis of purines was performed based on the peak areas obtained from the reflectance scanning densitometry, performed at 258 nm. The spectral scan was done from 200 to 300 nm which facilitated the spectral analysis of peaks for purity and spectral matching. The method was validated in terms of linearity, accuracy, precision, sensitivity, selectivity and repeatability. The method was successfully employed to estimate the endogenous purines in discrete regions of rat brain. A novel protocol developed for the tissue preparation utilizing 0.1 M HCl and 0.15 M NaOH solutions made in 60% (v/v) methanol resulted in well-resolved peaks and high component recoveries. The results for the first time show that this method established for the flexible estimation of Ade, Ino and Hypoxan by planar chromatography has good linearity, accuracy, precision, sensitivity, selectivity and is simple, rapid and moreover, economical to produce maximum resolution in brain tissue preparations.