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 degrees C and the injection volume was 20 muL. 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.

Development and Validation of a Specific RP-HPLC Method for the Estimation of γ-Aminobutyric Acid in Rat Brain Tissue Samples Using Benzoyl Chloride Derivatization and PDA Detection

“…The reproducibility of the method was evaluated by repeatability studies of these quality control samples of 1.00, 2.50, 5.00, 25.00, and 100.00 nmol L −1 . The accuracy and precision of the assay results in case inter-run samples (Table III) are shown by DEV and CV values <6.00%, which is acceptable according to established validation protocols [20,21]. …”

On-line electrochemical measurements of cerebral hypoxanthine of freely moving rats

“…K 3 Fe(CN) 6 , FeCl 3 , H 2 O 2 , and glutaraldehyde were all purchased from Beijing Chemical Co. (Beijing, China). Other chemicals were of at least analytical reagent and used as received.…”

“…Briefly, GC electrodes were electrochemically activated at +1.7 V for 180 s in 0.10 M KCl solution buffered with 0.05 M phosphate buffer (pH 7.4). The PB-modified GC electrodes were prepared by dipcoating 10 μL of precursor solution containing 25 mM K 3 Fe(CN) 6 and FeCl 3 in 10 mM HCl onto GC electrodes. After allowed to stand for 20 min under ambient temperature, the electrodes were first sequentially rinsed with 10 mM HCl and doubly distilled water and then heated at 80℃ under vacuum for 3 h. After that, the electrodes were cycled in 0.10 M KCl solution buffered with 0.05 M phosphate buffer (pH 7.4) within a potential range between −0.10 and +0.40 V at a scan rate of 50 mV·s −1 until a stable voltammetric response was obtained (typically after 10 cycles).…”

“…In the central nervous system, as one of the main metabolites in the degradation of adenine nucleotide, HX, on one hand, reflects the catabolism in the brain and, on the other hand, acts as a biomarker in several nervous system disorders, such as brain ischemia and hypoxia [3][4][5] . Current methods employed for the measurements of cerebral HX include the sample preparation from the brain tissue homogenate, sample separation and subsequent detection [6,7] . In spite of their applications in investigations of some physiological and pathological processes associated with HX, those methods are unfortunately technically complicated and time-consuming.…”

Neuroprotective Activity of Curcumin in Combination with Piperine against Quinolinic Acid Induced Neurodegeneration in Rats

“…The mobile phase consisted of water, methanol and acetonitrile (88: 5:7, v/v). Separation and data recording were carried out similarly as discussed above in catecholamines by using standard in the concentration range of 10-100 ng/ml [31] .…”