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

Abstract

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.

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation

“…The biosynthesized and characterized salbutamol-4′-O-sulfate reference previously described by Jendretzki and Harps et al [14] was analyzed with the developed chiral LC-MS/MS method to determine the enantiomeric purity in the reference. The reference was found to contain almost exclusively (R)-salbutamol-4′-O-sulfate (97.5%) (Figure 7 A).…”

“…To quantitate salbutamol-4 -O-sulfate in urine samples, the substance was biosynthesized as reference using a biochemical approach from Sun et al [10,29] using genetically modified fission yeast strain YN20 expressing SULT1A3. The synthesis, characterization, and quantitation were carried out as described previously by Jendretzki and Harps et al [14]. Racemic salbutamol and pure levosalbutamol were administered to one healthy volunteer as previously described by Jendretzki and Harps et al [14].…”

“…The synthesis, characterization, and quantitation were carried out as described previously by Jendretzki and Harps et al [14]. Racemic salbutamol and pure levosalbutamol were administered to one healthy volunteer as previously described by Jendretzki and Harps et al [14]. Briefly, single doses of 600 µg of racemic salbutamol (SAI, 6 × 100 µg; SalbuHEXAL ® N, Hexal AG, Holzkirchen, Germany) or 630 µg levosalbutamol (LSAI, 14 × 45 µg; Xopenex HFA ® , Sunovion Pharmaceuticals Inc., Marlborough, MA, USA) were applied via inhalation as aerosol.…”

“…An approach to identifying the administration of pure levosalbutamol via achiral chromatography from observation of the proportion of the sulfonated metabolite was reported by Jendretzki et al [14]. Additionally, peroral administration and inhalation were identified in the same manner.…”