ANALYTICAL METHOD DEVELOPMENT FOR QUANTIFICATION OF TINIDAZOLE BY REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

A simple and rapid reversed-phase high performance liquid chromatography (HPLC) assay for determination of tinidazole level in human plasma was developed and validated. Hydrochlorothiazide was used as an internal standard (IS). Human plasma (0.4 ml) were deproteinized with 20 µl perchloric acid, vortexed and centrifuged. The supernatant 100 µl was collected and injected into the HPLC system. Analysis was performed using Atlantis dC18 column with a mobile phase composed of 50 mM potassium phosphate, dibasic (pH=3.0) and acetonitrile (80:20, v:v). The eluent was monitored spectrophotometrically at 317 nm. No interference from blank plasma components or commonly used drugs was observed. The relationship of tinidazole concentration and peak area ratio of tinidazole / IS was linear (R² ≥ 0.9902) in the range of 0.05 – 30 µg/ml, the intra and inter-day coefficient of variations were ≤8.9% and ≤8.2%, with a corresponding bias of ±8.2% and ±4.6%, respectively. Mean extraction recovery of tinidazole and the IS were 83% and 95%, respectively. The method was applied to assess the stability of tinidazole under various conditions encountered in the clinical laboratory. Tinidazole stability in processed samples (stored at room temperature for 24 hours or at -20°C 48 hours), unprocessed samples (stored for 24 hours at room temperature or, 8 weeks at -20°C), and after three freeze and thaw cycles was ≥88%, respectively.