title: A simplified procedure for the analysis of formoterol in human urine by liquid chromatography-electrospray tandem mass spectrometry: application to the characterization of the metabolic profile and stability of formoterol in urine.
abstract: Since 1992, formoterol is included in the prohibited list of doping substances and methods, presently reviewed and updated by the World Anti-Doping Agency. Recently a threshold value of 40ng/mL has been established to differentiate between the prohibited (oral) and the permitted (inhalatory) administration of formoterol to athletes. This paper considers the urinary excretion profile of formoterol and its main metabolites after inhalation of different doses of two of the most used medicaments, available in Italy, containing formoterol fumarate bihydrate (12 and 36μg twice a day of Foradil(®) or 9 and 27μg twice a day of Symbicort(®)), focusing also on the effects, on the measured levels of formoterol, of potential alteration processes (thermal and/or microbiological) that may take place after the collection of the urine samples. Urine sample preparation included an enzymatic hydrolysis and a dilution step. Detection of analytes was performed by a newly developed and validated direct LC-ESI-MS/MS procedure, using a triple quadrupole mass spectrometer under positive ion electro-spray ionization conditions and selected reaction monitoring acquisition mode. The results showed the capability and suitability of the direct LC-ESI-MS/MS analysis for the quantitative confirmation analysis of formoterol in urine samples. The data from the analysis of the urine samples obtained in the excretion studies showed that formoterol is excreted mainly as unmodified drug and to a lesser degree as O-demethylated metabolite. The urinary levels of formoterol (40-60%) and its metabolites (O-demethylated metabolite 5-25%; glucuronide metabolites 25-40%) vary significantly depending both on the administered drug formulation and the subject tested. The maximum urinary concentration reached in this study was 15ng/mL (free+glucuronide), that is significantly lower than the threshold value fixed to report an adverse analytical finding. Finally, our results also showed that formoterol is stable for at least 4 weeks in urine samples correctly collected and stored.
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197 Rome, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Mazzarino
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.forename: Ilaria
authors.initials: I
authors.lastname: Fiacco
authors.forename: Chiara
authors.initials: C
authors.lastname: Pompei
authors.forename: Fabiana
authors.initials: F
authors.lastname: Calabrese
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D004300:Doping in Sports; D004355:Drug Stability; D004983:Ethanolamines / Q000737:chemistry / Q000493:pharmacokinetics / Q000652:urine*; D005260:Female; D000068759:Formoterol Fumarate; D006801:Humans; D057230:Limit of Detection; D008297:Male; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004983:Ethanolamines; D000068759:Formoterol Fumarate