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.