Chiral analysis of selected enantiomeric drugs relevant in doping controls

Abstract

Various substances classified by the World Anti-Doping Agency (WADA) as prohibited in sports feature one or more chiral centers. Amongst those, few analytes exist that are so-called threshold substances, for which also enantiomerically pure drugs are available. The commonly employed non-chiral analysis of these compounds does not allow for differentiating between the use of a racemic mixture of e.g. salbutamol and formoterol from their enantiomerically pure analogs. In order to support identifying the exclusive use of the pharmacologically active compound, a multi-analyte chiral chromatography-based analytical approach was developed. The test method considering the β2-agonists fenoterol, formoterol, and salbutamol, the stimulant methamphetamine, the anabolic agent clenbuterol, and the β-blockers metoprolol, pindolol, and propranolol was based on liquid chromatography with a chiral column comprising a stationary phase based on the macrocyclic glycopeptide antibiotic teicoplanin as chiral selector. The liquid chromatograph was interfaced via electrospray ionization to a high resolution/high accuracy mass spectrometer, and urine samples were prepared for analysis following a protocol including enzymatic hydrolysis and subsequent liquid-liquid extraction. For proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of eight selected enantiomeric doping agents, allowing to determine their ratio at urinary concentrations relevant for sports drug testing purposes, i.e. between 0.01 and 2 ng/mL. Enantioselective assays provide the tool to overcome the limitations of non-chiral approaches in routine doping analysis and can offer support in studies where questions of pharmacokinetics and stereoselectivity are to be addressed for result management and decision-making processes.

Keywords

• Sports
• Doping
• Mass spectrometry
• Enantiomers
• Chiral separation
• Enantiomeric ratio