Development and validation of a fast ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determining carbonic anhydrase inhibitors and their metabolites in urine and hair.

A new, rapid, sensitive and comprehensive ultra-high-performance liquid chromatographic-mass spectrometry (UHPLC-MS/MS) method for quantifying diuretics (acetazolamide, brinzolamide, dorzolamide and their metabolites) in human urine and hair was developed and fully validated. Twenty-five milligrams of hair, were incubated with 500 μL M3® buffer reagent at 100°C for 1 h for complete digestion. After cooling, 1 μL supernatant was injected onto chromatography system. Urine samples were simply diluted before injection. The chromatographic run time was short (8 min) through a column with a mobile phase gradient. The method was linear (determination coefficients always higher than 0.99) from limit of quantification (LOQ)-500 ng/mL in urine and from LOQ-10 ng/mg in hair. LOQs ranged from 0.07-1.16 ng/mL in urine and from 0.02-0.15 ng/mg in hair. No significant ion suppression due to matrix effect was observed and process efficiency was always higher than 80%. Intra- and inter-assay precision was lower than 15%. The suitability of the methods was tested with six urine and hair specimens from patients treated with acetazolamide, dorzolamide or brinzolamide for ocular diseases or systemic hypertension. Average urine concentrations were 266.32 ng/mL for dorzolamide and 47.61 ng/mL for N-deethyl-dorzolamide (n=3), 109.27 ng/mL for brinzolamide) and 1.02 ng/mL for O-desmethyl-brinzolamide (n=2) and finally 12.63 ng/mL for acetazolamide. Average hair concentrations were 5.94 ng/mg for dorzolamide and 0.048 ng/mg for N-deethyl-dorzolamide (n=3); 3.26 ng/mg for brinzolamide (n=2) and 2.3 ng/mg for acetazolamide (n=1). The developed method was simple and fast both in the extraction procedure making it eligible in high-throughput analysis for clinical forensic and doping purposes.