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

Abstract

β 2 -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. K E Y W O R D S clenbuterol, GC-MS/MS, ractopamine, zilpaterol, β2-agonists 1 | INTRODUCTION β 2 -adrenergic agonists are banned in sports, listed in section S3 of the World Anti-Doping Code Prohibited List of Substances and Methods. However, clenbuterol, ractopamine, and zilpaterol are specifically considered to be anabolic agents, under section S1.2 other anabolic agents and therefore, are always banned. 1 The abuse of clenbuterol ((RS)-1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino) ethanol, CLEN) in athletes was documented in the 1990s 2 with the first positive case reported in June 1992 marking its inclusion on the IOC prohibited list. Its thermogenic and anabolic effects were suggested by Rothwell and Stock 3 following administration studies in lean and genetically obese rodents. More recently Jessen et al. 4 concluded that CLEN was an 'efficient thermogenic substance' that 'may exert anabolic actions in human skeletal muscle'. CLEN findings are frequently reported; with the implementation of high-sensitivity GC-MS/MS detection methods, the number of cases reported by WADA accredited laboratories went from approximately

Nafion/Silver Nanoparticles as an Electrochemically Sensitive Interface for the Detection of Ractopamine in Pork Liver

“…Currently, analytical methods for detecting Rac in pork are limited to techniques requiring highly sophisticated sample preparation or costly instrumentation, such as enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–tandem mass spectrometry (LC–MS/MS). − ELISA offers a rapid and portable method for detecting Rac; however, its major drawback is its requirements of complicated time-consuming sample pretreatment and preparation because of the complex matrix of animal tissues . Among instrumental techniques, HPLC enables multicomponent and highly sensitive analysis; however, the HPLC instrument is costly and nonportable, which prevents its widespread application .…”

“…Moreover, well-trained personnel are required to operate this instrument. Implementing GC–MS in Rac detection is difficult because of the high boiling point of Rac . To achieve efficient detection, a derivatization reaction must first be performed on Rac, which increases the complexity and uncertainty of the analysis process.…”

“…Implementing GC−MS in Rac detection is difficult because of the high boiling point of Rac. 12 To achieve efficient detection, a derivatization reaction must first be performed on Rac, which increases the complexity and uncertainty of the analysis process. LC−MS/MS has been combined with multiple reaction monitoring for the analysis of Rac.…”

The 40th Manfred Donike workshop on doping analysis

“…propeptide, 2 selective estrogen receptor modulators, and β 2 -aonists. 3 Also, challenges arising from permitted versus prohibited routes and/or time points of drug administration and consumption of dietary products were addressed.…”

“…Central topics of the 2022 conference can be largely subsumed under analytical aspects concerning steroidal substances and erythropoiesis‐stimulating agents as well as blood transfusions, as well as investigations into the detection, characterization, and synthesis of metabolic products of drugs, including, for example, myostatin propeptide, 2 selective estrogen receptor modulators, and β 2 ‐aonists 3 . Also, challenges arising from permitted versus prohibited routes and/or time points of drug administration and consumption of dietary products were addressed.…”