Resolution ofR‐(−)andS‐(+)‐enantiomers of clenbuterol in pharmaceutical preparations and black‐market products using liquid chromatography–tandem mass spectrometry

Abstract

Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, and amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non-Olympic athletes that have adverse analytical findings (AAFs). In some cases, the presence of these substances could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R-(À) and S-(+)-enantiomers of clenbuterol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a chiral column in 15 min with good separation. The method developed can also be used for the analysis of other biological matrices such as urine, serum, and meat. The resolution between two peaks' (R s ) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black-market products has not been reported previously. The information shown could be used by national anti-doping organizations and the athletes with AAFs attributed to clenbuterol.

Evaluation of R‐ (−) and S‐ (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC‐TQ‐MS

“…Comparing resolution, our method showed a higher value of Rs = 1.26 compared to the Rs = 1.0 value reported by Velasco Bejarano. 27 Furthermore, the sensitivity of the present method for each enantiomer included the lowest LOQ reported (12.5 pg/mL) compared to previous methods [27][28][29] (0.025 ng/mL, 50 ng/mL and 2.5 μg/mL). Finally, this work evaluates the effect of thaw-freezing cycles and short-term stability at −80°C after the sample is prepared, which previous studies have not evaluated.…”

“…Additionally, the stability of standards and spiked samples after sample extraction was tested in two conditions; after 7 days of storage at −80°C and 3 freeze-thaw cycles ( Table 3). As observed in Table 3 There are already a few published methodologies to determine clenbuterol enantiomers; however, for most of them the total running analysis time ranged from 24 to 30 minutes, [27][28][29] almost four times more than the present method which last 6.5 minutes.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…A variety of analytical approaches potentially addressing the problem has been studied in the past, among which also the enantiomeric composition of clenbuterol in doping control urine samples was considered as a possible means to identify “animal‐processed” clenbuterol in human urine. The significance of results obtained from these tests was limited, although all investigated pharmaceutical clenbuterol formulations were found to exhibit racemic compositions, and case‐by‐case evaluations of AAFs are still required.…”

“…A variety of analytical approaches potentially addressing the problem has been studied in the past, among which also the enantiomeric composition of clenbuterol in doping control urine samples was considered as a possible means to identify "animal-processed" clenbuterol in human urine. The significance of results obtained from these tests was limited, although all investigated pharmaceutical clenbuterol formulations were found to exhibit racemic compositions,75 and caseby-case evaluations of AAFs are still required. 3 | PEPTIDE HORMONES, GROWTH FACTORS, RELATED SUBSTANCES, AND MIMETICS Erythropoietin-receptor agonists The impact of erythropoietic and non-erythropoietic effects of an illicit use of erythropoietin (EPO) and its derivatives on elite athletes' performances has again been the subject of considerable debate.…”

The 35th Manfred Donike workshop on doping analysis

“…Taken together, this compilation of papers in this, the 2017 Cologne Workshop special issue on advances in sports drug testing, once more mirrors and highlights the multifaceted nature of doping controls and related topics. While expanding test methods plays a major role in anti‐doping research, a trend towards studies aimed at identifying situations that could affect doping controls is recognized …”