Determination and enantioselective separation of zilpaterol in human urine after mimicking consumption of contaminated meat using high-performance liquid chromatography with tandem mass spectrometry techniques.

Abstract

The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5μg or 3μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method. Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5h after ingestion, and maximum levels >5ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.