Quantification of Carbonic Anhydrase Inhibitors and Metabolites in Urine and Hair of Patients and Their Relatives

Alfredo Fabrizio Lo Faro; Anastasio Tini; Giulia Bambagiotti; Filippo Pirani; Andrea Faragalli; Flavia Carle; Elena Pacella; Artan Ceka; Marco Moretti; Massimo Gottardi; Nicola Vito Lassandro; Michele Nicolai; Marco Lupidi; Cesare Mariotti; Francesco Paolo Busardò; Jeremy Carlier

doi:10.3390/biology11101379

Quantification of Carbonic Anhydrase Inhibitors and Metabolites in Urine and Hair of Patients and Their Relatives

Biology (Basel). 2022 Sep 21;11(10):1379. doi: 10.3390/biology11101379.

Authors

Alfredo Fabrizio Lo Faro¹, Anastasio Tini¹, Giulia Bambagiotti¹, Filippo Pirani¹, Andrea Faragalli², Flavia Carle², Elena Pacella³, Artan Ceka⁴, Marco Moretti⁴, Massimo Gottardi⁵, Nicola Vito Lassandro⁶, Michele Nicolai⁶, Marco Lupidi⁶, Cesare Mariotti⁶, Francesco Paolo Busardò¹, Jeremy Carlier¹

Affiliations

¹ Unit of Forensic Toxicology, Section of Legal Medicine, Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Via Tronto 10/a, 60126 Ancona, AN, Italy.
² Center of Epidemiology Biostatistics and Information Technology, Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Via Tronto 10/a, 60126 Ancona, AN, Italy.
³ Department of Sense Organs, Faculty of Medicine and Dentistry, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, RM, Italy.
⁴ Department of Laboratory Medicine, Reunited Hospitals Torrette of Ancona, Via Conca 71, 60126 Ancona, AN, Italy.
⁵ Comedical srl, Via della Cooperazione 29, 38123 Trento, TN, Italy.
⁶ Eye Clinic, Marche Polytechnic University, Via Tronto 10/a, 60126 Ancona, AN, Italy.

Abstract

Carbonic anhydrase inhibitors (CAIs) are prescription drugs also used in doping to dilute urine samples and tamper with urinalyses. Dorzolamide, brinzolamide, and acetazolamide are prohibited by the World Anti-Doping Agency. Detecting CAIs and their metabolites in biological samples is crucial to documenting misuse in doping. We quantified dorzolamide, brinzolamide, acetazolamide, and their metabolites in the urine and hair of 88 patients under treatment for ocular hypertension or glaucoma. Samples of the patients' relatives were analyzed to assess potential for accidental exposure. After washing, 25 mg hair was incubated with an acidic buffer at 100 °C for 1 h. After cooling and centrifugation, the supernatant was analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urine (100 μL) was diluted and centrifuged before UHPLC-MS/MS analysis. Run time was 8 min through a reverse-phase column with a mobile phase gradient. MS/MS analysis was performed in a multiple-reaction monitoring mode after positive electrospray ionization. Median urinary concentration was 245 ng/mL (IQR: 116.2-501 ng/mL) for dorzolamide, 81.1 ng/mL (IQR: 35.9-125.3 ng/mL) for N-deethyl-dorzolamide, 0.77 ng/mL (IQR: 0.64 ng/mL-0.84 ng/mL) for N-acetyl-dorzolamide, 38.9 ng/mL (IQR: 20.4-79.2 ng/mL) for brinzolamide, and 72.8 ng/mL (IQR: 20.7-437.3 ng/mL) for acetazolamide. Median hair concentration was 0.48 ng/mg (IQR: 0.1-0.98 ng/mg) for dorzolamide, 0.07 ng/mg (IQR: 0.06-0.08 ng/mg) for N-deethyl-dorzolamide, 0.40 ng/mL (IQR: 0.13-1.95 ng/mL) for brinzolamide. Acetazolamide was detected in only one hair sample. Dorzolamide and brinzolamide were detected in the urine of three and one relatives, respectively. Cutoff concentrations of urinary dorzolamide and brinzolamide are necessary to preclude false positives due to contamination or passive exposure. We reported the first concentrations of brinzolamide in hair.

Keywords: acetazolamide; brinzolamide; carbonic anhydrase inhibitor; case report; dorzolamide; hair; liquid chromatography-tandem mass spectrometry; urine.

Grants and funding

I34I19001520001 as CUP/Ministero della Salute