title: Development and validation of a ultra high performance liquid chromatography-tandem mass spectrometric method for the direct detection of formoterol in human urine.
abstract: Formoterol is a long acting β(2)-agonist and has proven to be a very effective bronchodilating agent. Hence it is frequently applied therapeutically for the treatment of asthma. Because β(2)-agonists might be misused in sports for the stimulatory effects and for growth-promoting action their use is restricted. Since January 2012, formoterol is prohibited in urinary concentrations higher than 30 ng/mL. The objective of this study was to develop and validate a simple and robust ultra high performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS/MS) method for the direct quantification of formoterol in urine. Sample preparation was limited to an enzymatic hydrolysis step after which 2 μL was injected in the chromatographic system. Chromatography was performed on a C(8)-column using gradient conditions. The mobile phase consisted of water/methanol (H(2)O/MeOH) both containing 0.1% acetic acid (HOAc) and 1mM ammonium acetate (NH(4)OAc). Calibration curve were constructed between 15 and 60 ng/mL. Validation data showed bias of 1.3% and imprecision of 5.4% at the threshold. Ion suppression/enhancement never exceeded 7%. Calculating measurement uncertainty showed proof of applicability of the method. Stability of formoterol was also investigated at 56 °C (accelerated stability test) at pH 1.0/5.2/7.0 and 9.5. At the physiological pH values of 5.2 and 7.0, formoterol showed good stability. At pH 1.0 and 9.5 significant degradation was observed.
authors: LAB DOP-LADETEC, Universidade Federal do Rio de Janeiro, Instituto de Química, Ilha do Fundão, Avenida Athos da Silveira Ramos, 149, 21941-909 Rio de Janeiro, RJ, Brazil.
authors: V F
authors: VF
authors: Sardela
authors: K
authors: K
authors: Deventer
authors: H M G
authors: HM
authors: Pereira
authors: F R
authors: FR
authors: de Aquino Neto
authors: P
authors: P
authors: Van Eenoo
mesh_terms: D000085:Acetates / Q000737:chemistry; D019342:Acetic Acid / Q000737:chemistry; D058666:Adrenergic beta-2 Receptor Agonists / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D001993:Bronchodilator Agents / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D004300:Doping in Sports; D004355:Drug Stability; D004983:Ethanolamines / Q000652:urine*; D000068759:Formoterol Fumarate; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D000432:Methanol / Q000737:chemistry; D058573:Performance-Enhancing Substances / Q000652:urine*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D013696:Temperature; D016482:Urinalysis; D014867:Water / Q000737:chemistry
chemical_list: D000085:Acetates; D058666:Adrenergic beta-2 Receptor Agonists; D015415:Biomarkers; D001993:Bronchodilator Agents; D004983:Ethanolamines; D058573:Performance-Enhancing Substances; D014867:Water; D019342:Acetic Acid; C018824:ammonium acetate; D000068759:Formoterol Fumarate; D000432:Methanol