The adrenergic beta-2 agonists formoterol and salbutamol are used for the treatment of asthma and COPD but are also misused in sports competitions. Therefore, they are included in WADA regulations. Both drugs are mainly excreted in urine after administration via inhalation. A four-armed, double-blind cross-over clinical trial was conducted involving endurance-trained participants (12 females and 12 males). Inhalation dosages of 36 μg formoterol, 1200 μg salbutamol, a combination of both, or a placebo were administered before exercise. Serum and urine were collected after exercise and 3 and 24 h after administration. Here, we show the successful quantitation of formoterol, salbutamol, and its phase II metabolite salbutamol-4′-O-sulfate in all urine and serum samples using ultra-high performance liquid chromatography–tandem mass spectrometry. In the serum analysis, results of up to 14.2 pg/mL formoterol, 10.0 ng/mL salbutamol, and 21.4 ng/mL salbutamol-4′-O-sulfate (calculated as salbutamol equivalent) were found. In urine, maximum concentrations (after deglucuronidation) were 17.2 ng/mL formoterol, 948.5 ng/mL salbutamol, and 2738.5 ng/mL salbutamol-4′-O-sulfate. Sex-specific differences in serum concentrations as well as in urinary excretion were observed. The results pronounce the importance of the implementation and elucidation of phase II metabolites to quantitation methods in antidoping
