Metabolite patterns of the carbonic anhydrase inhibitors brinzolamide and dorzolamide: potential markers for the route of application

Brinzolamide (BA) and dorzolamide (DA) are carbonic anhydrase inhibitors (CAIs), and are commonly used therapeutics for glaucoma treatment via topical application. According to the regulations of the World Anti-Doping Agency (WADA), both drugs are classified under "Diuretics and Masking Agents", prohibiting their use in- and out-of-competition, except for ophthalmic application. Despite ophthalmic use being permitted, systemic absorption occurs, enabling BA and DA detection in blood and urine samples after topical administration. Thus, distinguishing between legitimate (e.g. topical) and prohibited (e.g. oral) drug application routes is critical for sports drug testing result management.

This study investigated the elimination and metabolic profiles of BA and DA following topical and systemic administration in male piglets. Three piglets received BA or DA ophthalmic suspensions, while another three received an oral dose. Urine and blood samples were collected over one week, and all samples were analysed using liquid chromatography-high-resolution tandem mass spectrometry (LCHRMS MS). In vitro experiments yielded five phase I metabolites for DA and BA. After method validation, the approach was confirmed to detect DA and BA, with Limits of Detection (LODs) of 55 pg/mL and 75 pg/mL in urine, 110 pg/mL and 180 pg/mL in red blood cells, and 380 pg/mL and 910 pg/mL in plasma. BA and DA metabolites were primarily found in the red blood cell fraction, with only trace amounts detectable in plasma. N-desethylation was observed as the main metabolic reaction for both drugs, and metabolite-to-parent drug ratios were determined in all collected post-administration samples alongside drug concentration levels. The combined consideration of analyte ratios and drug concentrations appears to be indicative of time and dose of drug use (under the chosen routes of administration), which might assist in sports drug testing result management.