Development and validation of a fast ultra‐high‐performance liquid chromatography tandem mass spectrometry method for determining carbonic anhydrase inhibitors and their metabolites in urine and hair

Abstract

A new, rapid, sensitive, and comprehensive ultra-high-performance liquid chromatography tandem 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) to 500 ng/ml in urine and from LOQ to 10 ng/mg in hair. LOQs ranged from 0.07 to 1.16 ng/ml in urine and from 0.02 to 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 procedures making it eligible in high-throughput analysis for clinical forensic and doping purposes.

Hair testing for acetazolamide as an evidence of the use of a contaminated dietary supplement

“…With respect to the concentrations already published for subjects under treatment with diuretics, 18–20 the measured concentrations are much lower. However, during two anti‐doping violations, Gheddar et al identified hydrochlorothiazide at 6–15 pg/mg in the head hair of a rugby player 21 and at 36 pg/mg in the head hair of a top athlete 20 who challenged his adverse analytical finding, demonstrating that in athletes, the diuretic concentrations can be much lower to the ones measured in clinical patients.…”

“…Identification in hair of acetazolamide has been very recently published by Lo Faro et al 18 The authors measured 2300 pg/mg in the hair of a subject treated daily with acetazolamide for systemic hypertension. However, diuretic detection in hair has been seldom reported.…”

“…Due to the absence of controlled study presenting acetazolamide incorporation into hair, it is not possible to interpret the data in terms of dosage and frequency of consumption. However, the paper of Lo Faro et al 18 is useful to demonstrate the different hair concentration of acetazolamide in chronic consumers assuming a pharmacological treatment and an athlete consuming a dietary supplement.…”

“…In both cases, a single-dose administration was undetectable, and concentrations in the range 12-1845 and 5-1110 pg/mg were measured in subjects under daily treatment with hydrochlorothiazide and furosemide, respectively. Finally, hair concentrations of 230, 1360 and 16,210 pg/mg for dorzolamide (n = 3) and of 290 and 6240 pg/mg for brinzolamide (n = 2) were published by Lo Faro et al 18 in patients treated for ocular diseases or for systemic hypertension, respectively.…”

Adverse analytical finding due to red blood cells transfusion: A rare case involving the diuretic dorzolamide

“…The test was still positive, with an approximate dorzolamide concentration of 0.5 ng/ml. The urine concentration of subjects treated for ocular disease 22 are much higher (a magnitude of 100)…”