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INTRODUCTION. Systemic exposure studies of a selective carbonic anhydrase II inhibitor, the isoxazole derivative 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (TFISA), require evaluating its pharmacokinetics in whole blood because the compound can accumulate in erythrocytes. Currently, no bioanalytical procedures have been developed to achieve this.AIM. This study aimed to develop a bioanalytical procedure for the determination of TFISA and its metabolites (N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide) in the blood of laboratory animals and compare the pharmacokinetics of TFISA ophthalmic suspension in rats after a single ocular or intraperitoneal administration.MATERIALS AND METHODS. The quantitative determination was performed by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) using rat and rabbit blood samples. The chromatographic separation used a Zorbax Eclipse Plus C18 column (150×3.0 mm, 3.5 µm) and a gradient elution system of 0.1% aqueous formic acid and methanol. The multiple reaction monitoring mass spectrometry mode was used for detection. The pharmacokinetics study was conducted in 2 groups of 6 Wistar rats (3 males and 3 females per group). Group 1 received an instillation of 1% TFISA ophthalmic suspension in each eye at a dose of 3.7 mg/kg. Group 2 received an intraperitoneal injection of the same product at the same dose. Blood samples were collected at baseline and at several intervals after administration.RESULTS. The authors developed a bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals (rabbits and rats). This HPLC-MS/MS procedure was fully validated in accordance with the requirements of the EAEU legislation and the ICH M10 guideline. The analytical ranges in blood were 20–20000 for TFISA, 2–2000 for the N-hydroxy metabolite, and 0.1–100.0 ng/mL for the N-acetyl metabolite. The maximum blood levels after ocular instillation (mean±SD) were 8173±1491 for TFISA, 694±271 for the N-hydroxy metabolite, and 6.33±1.51 ng/mL for the N-acetyl metabolite. The half-lives for this route of administration were 58±10 (TFISA), 70±24 (N-hydroxy metabolite), and 14±3 h (N-acetyl metabolite). The bioavailability of TFISA was 90.18%. CONCLUSIONS. The developed and validated bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals has been successfully applied to samples of rat whole blood. According to the study of ophthalmic suspension pharmacokinetics, TFISA and its metabolites have long halflives and high bioavailability.