Simple and fast Development and validation of High Performance Liquid Chromatography Method with UV determination of thiopental in rat plasma

Abstract

Objective: Now days old agent thiopental return to hot-topic of research as new Model of neuropharmacology study. Because of this reason simple, fast and cost benefit method of determination this agent comes an issue in area pharmacokinetic and pharmacodynamics (PK-PD) area of research. Previous study was focus on human plasma and pilot study showed there is differentiation between human and rat plasma. Methods: Separation were perform Nuleodur C18 ec HPLC column (250×4.0×5µm), using a mixture of acetonitrile; potassium; dihydrogen phosphate buffer (10mM, PH 2.7) as mobile phase delivered at flow rate 1.2 ml/minute. 280 nm and room temperature was selected for detection of thiopental and 1-Naphtylamine as internal standard. Plasma sample (100µl) were treated with 180µl precipitation solution and 20µl of internal standard. After the mixture were vortex and centrifuge at 1000 g, finally 40µl of clear supernatant was directly injected into 20µl loop of HPLC apparatus. Calibration curve were fitted by Peak area ratio of thiopental to internal standard. All stage of blood collection was under supervision of Ahvaz ethical committee. Results: Thiopental and internal standard retention time were at 8.3 and 14.5 minute, respectively. 0.5 µg/ml was level of limit of quantification, of our method. 99.4 to 100.3 was range of Accuracy of methods. inter- and intraday precisions were 4-19 % and 6-8%, respectively. A good relationship in the form of a power was found (r2=0.999). Conclusions: the presented simple fast and cost benefit method is great accurate, precise and sensitive for determination of thiopental in plasma of rat.