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.

The misuse of growth-promoting drugs such as beta-2 agonists and steroids is a known problem in farming and sports competitions. Prior to the analysis of biological samples via liquid chromatography (LC)–mass spectrometry (MS) or gas chromatography (GC)–MS, sufficient sample preparation is required to reliably identify or determine the residues of drugs. In practice, broad screening methods are often used to save time and analyze as many compounds as possible. This review was conceptualized to analyze the literature from 2018 until October 2023 for sample preparation procedures applied to animal specimens before LC- or GC-MS analysis. The animals were either used in farming or sports. In the present review, solid phase extraction (SPE) was observed as the dominant sample clean-up technique for beta-2 agonists and steroids, followed by protein precipitation. For the extraction of beta-2 agonists, mixed-mode cation exchanger-based SPE phases were preferably applied, while for the steroids, various types of SPE materials were reported. Furthermore, dispersive SPE-based QuEChERs were utilized. Combinatory use of SPE and liquid–liquid extraction (LLE) was observed to cover further drug classes in addition to beta-2 agonists in broader screening methods.

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4′-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings.

The aim of the study was a comprehensive and quantitative determination of salbutamol and its sulfoconjugated major metabolite in urine samples using achiral ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Therefore, salbutamol-4′-O-sulfate was biosynthesized as a reference using genetically modified fission yeast cells, and the product was subsequently characterized by NMR and HRMS. In competitive sports, salbutamol is classified as a prohibited drug; however, inhalation at therapeutic doses is permitted with a maximum allowance of 600 µg/8 h. In contrast, the enantiopure levosalbutamol is prohibited under any condition. For analytical discrimination, the amount of salbutamol and its main metabolite excreted in the urine was studied. As proof of concept, a longitudinal study in one healthy volunteer was performed in order to investigate excreted amounts and to study potential discrimination using achiral chromatography. Discrimination of administration of racemic salbutamol or the enantiopure levosalbutamol was not achieved by solely analyzing salbutamol as the parent compound. However, a distinction was possible by evaluation of the proportion of salbutamol-4′-O-sulfate in relation to salbutamol. Therefore, reference material of metabolites is of great importance in doping control, especially for threshold substances.

It was the aim of this study to compare the ratio of salbutamol and its major metabolite salbutamol-4’-O-sulfate as well as the quantity excreted renally after oral and inhalative administration. Additionally, the excretion pattern after application of racemic salbutamol opposed to the pure enantiomer levosalbutamol was evaluated. For quantitation of the sulfonated metabolite of salbutamol, reference material was synthesized and characterized by NMR and HRMS. Urine samples were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Salbutamol is mainly metabolized by sulfotransferase 1A3 (SULT1A3), which is most abundantly found in the jejunum. A high first pass effect occurs upon oral administration, which leads to a higher amount of salbutamol-4’-O-sulfate in urine compared to unchanged salbutamol excreted within the first hour post administration. Thus, when administered orally, the proportion of metabolite to total salbutamol was higher than for inhalative administration in the early urinary excretion. Levosalbutamol is the favored enantiomer of SULT1A3 and therefore metabolized at a higher rate whereas racemic salbutamol shows a reduced proportion between metabolite and parent compound. The amount of sulfoconjugate was found higher than that of the parent compound for all urine samples except for those excreted in the first hour after inhalative administration.