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.

Multidrug-resistant (MDR) bacterial infections pose a global public health threat, intensified by the declining efficacy of commonly used antibiotics. The reassessment of neglected antibiotics, such as fosfomycin (FOS), presents a promising strategy against MDR infections. Investigations in in vitro infection models (IVIMs) generate crucial knowledge for treatment optimisation. However, reliable bioanalytical assays for quantification of FOS in Mueller-Hinton broth (MHB), a standard bacterial growth medium used for IVIM experiments, are essential. Therefore, an LC-MS/MS assay for quantification of FOS in MHB was developed and validated according to criteria of the EMA guideline on bioanalytical method validation. The calibration range of 10-2000 mg/L FOS (in original samples; corresponding to 0.04-8 mg/L after sample preparation) covered clinically relevant concentrations. The assay employed a simple and rapid sample preparation consisting of a precipitation and dilution step. Within-run mean accuracy ranging from 91.3 %-108 % and within-run precision with a maximum coefficient of variation of 11.7 % were acceptable across all concentration levels (n = 126). The assay was successfully applied to samples from IVIM experiments using a clinical Escherichia coli isolate. Additionally, thermal stability of FOS in MHB was confirmed under relevant conditions (24 h at 37 °C and 15 min at 50 °C). Overall, the developed assay is best suitable for application in IVIM set-ups, which are a prerequisite for the reassessment of FOS as a promising antibiotic combination treatment option for MDR infections.