Chiral analysis of selected enantiomeric drugs relevant in doping controls
Ana Rubio, Christian Görgens, Sven Guddat, Thomas Piper, Ann‐Marie Garzinsky, Oliver Krug, Mario Thevis
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 β 2 -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.