Development and validation of a method for determining acetazolamide in rat intestinal fluid by HPLC

MORA, M.J., LONGHI, M.R., GRANERO, G.E.

Córdoba

Congreso; 1º Reunión Internacional de Ciencias Farmacéuticas (RICiFa); 2010

Facultad de Ciencias Químicas, UNC

Introduction: Acetazolamide is a potent reversible carbonic anhydrase inhibitor widely used in the medical treatment of glaucoma. It works by reducing the rate of formation of aqueous humor which lowers intraocular pressure in patients treated with this drug. It is also used, either alone or in association with other drugs, in the treatment of various forms of epilepsy, or as a promoter of diuresis in instances of abnormal fluid retention (1-2). The two major problems presented by Acetazolamide are its low aqueous solubility, 0.7 mg/ml, and its low permeability coefficient, 4.1 x 10-6 cm/s (3). The Single Pass Intestinal Perfusion model (SPIP) is commonly used to investigate intestinal drug permeation, and to predict in vivo absorption of drugs in humans. Thus, the aim of this work was developed a specific HPLC method for the determination of ACZ in intestinal perfusion experiments. Materials and methods: The HPLC assays were carried out with a high pressure liquid chromatograph (Agilent 1100 Series) consisting of a standard automatic injector, an isocratic pump, thermostatted column compartment, and diodes and multi-wavelength detector. The chromatographic conditions were: ACN/MeOH/H2O (3:2:95) mobile phase, in an isocratic mode, 25 °C, flow 1.5 ml /min, wavelength 265 nm. We used a reverse phase column 250 x 4.6 mm GraceSmart RP 18 5u (GRACE). The injection volume was 50 µl. The analytical curve was built on rat intestinal fluid obtained by the technique of SPIP. The sample preparation involved protein precipitation with phosphoric acid. The stability of ACZ was tested by their incubation in intestinal fluid at 37ºC for 3 hours. Results: The method developed was specific for Acetazolamide, none of the components of the infusion solution interfered with the peak of the drug. The calibration curve was linear in the concentration range from 0,192 to 2,112 µg/ml of Acetazolamide, with a correlation coefficient of 0,9981. The detection limit was 0,116 µg/ml and the limit of quantification was 0,351 µg/ml. Inter-day and intraday accuracy given by relative error (%RE) of quality control samples were ≤ 10% while inter-day and intra-day precision given by relative standard deviation (%RSD) of quality control samples were ≤ 10%. In stability studies under the test conditions the analyte was found to be stable. The percentage recoveries at low, medium and high concentrations for Acetazolamide were 108,6%. Conclution: This method is simple, reliable and can be routinely used to accurately determine the permeability of Acetazolamide in SPIP studies