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

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.

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 ¡ãC, flow 1.5 ml /min, wavelength 265 nm. We used a reverse

mobile phase, in an isocratic mode, 25 ¡ãC, 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 ¦Ìl. 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.

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 ¦Ìg/ml of Acetazolamide, with a correlation coefficient of 0,9981. The detection limit was

0,116 ¦Ìg/ml and the limit of quantification was 0,351 ¦Ìg/ml. Inter-day and intraday accuracy given by

relative error (%RE) of quality control samples were ¡Ü10% while inter-day and intra-day precision given

by relative standard deviation (%RSD) of quality control samples were ¡Ü10%. 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%.

This method is simple, reliable and can be routinely used to accurately determine the permeability of

Acetazolamide in SPIP studies.