Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS

Abstract

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.

Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS

“…The UHPLC-MS/MS method was previously described, and basic validation was performed by Harps et al [5]. Retention time stability, matrix effect (ME), and precision were monitored in this study.…”

“…Additionally, to carry over, as performed by Harps et al [5], basic method validation was successfully performed in terms of retention time stability, matrix effect, recovery, and precision. The method was proven to be suitable for this study.…”

“…In contrast, any administration of enantiopure levosalbutamol is prohibited. To identify and quantify substances or their metabolites by means of mass spectrometric analysis, appropriate reference standards are of great importance for reliable and accurate results [4,5].…”