Presence of β2‐agonist growth promoters in human urine samples: GC‐MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses

β2‐adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri‐TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro‐dose of 2.5 μg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml. β2‐agonists are prohibited in sports. Information is missing on the fate of ractopamine in humans impairing its efficient detection in urine. A confirmation procedure for total ractopamine was developed, involving the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri‐TMS derivative (LOI 0.15 ng/ml). The sulphates form between 85% and 97% of total hydrolysed‐equivalent ractopamine excreted in athletes' samples and in volunteers following the administration of a micro‐dose of 2.5 μg.