Analytical method to evaluate in vitro intestinal absorption of the Acetazolamide by HPLC

MARIA JULIA MORA; ANA LUIZA RIBEIRO DE SOUZA; MARCO VINICIUS CHAUD; MARIA PALMIRA D. GREMIÃO; MARCELA RAQUEL LONGHI; GLADYS ESTER GRANERO

Riberao Preto, San Pablo, Brasil

Congreso; 7 Congreso Internacional de Ciencias Farmaceuticas (CIFARP); 2009

INTRODUCTION: The acetazolamide (ACZ) is a potent and reversible carbonic anhydrase inhibitor, effective in the control of fluid secretion, e. g., glaucomas, in the treatment of certain convulsive disorders, e. g., epilepsy, and in the promotion of diuresis in instances of abnormal fluid retention. It is poorly water- and lipid-soluble drug. Despite this, it is rapidly and almost completely absorbed from the gastrointestinal tract. OBJECTIVE: In this study, a simple HPLC method was developed, in order to monitor the concentration of ACZ in TC-199 buffer in vitro, in the rat everted gut sac absorption model. METHODOLOGY: Varian HPLC system was used for purpose of analysis in the study and it consisted of a ProStar/Dynamax 210/215 Solvent Delivery Module, ProStar 330 UV-VIS PDA Spectrophotometric Detector, and a Rheodine VS 7125, injection valve with a 100 μL loop. The drug was analyzed using 250 x 4.6 mm C18 (Varian - Chromsep) column. The isocratic mobile phase was a mixture of methanol: water pH 4.0 at 10:90 (v/v). The flow-rate was 1 mL min-1 and the quantitative determination was carried out at 265 nm, at 25°C. The calibration curve of ACZ was constructed in TC 199 solution. It was used as solvent medium because this method will be used to monitor concentrations of this drug in in vitro rat intestinal sac absorption experiments. RESULTS: The method was found to be specific, as none of the components of TC-199 or intestinal sac artefacts interfered with the drug peak. The limit of detection was 1,58 g mL-1 and the limit of quantitation was 4,79 g mL-1. The calibration curve was found to be linear in the concentration range of 4.0 ? 40.0 μg mL-1 ACZ, with the correlation coefficient equal to 0.9993. CONCLUSION: The proposed method was found to be rapid and selective and that for can be applied in the monitoring of the absorption of ACZ in in vitro everted gut sac absorption studies.