Quantification and Stereochemical Composition of R-(−) and S-(+)-Clenbuterol Enantiomers in Bovine Urine by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a β2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10–8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10–10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P < 0.05) using the Wilcoxon test.

Detection of clenbuterol residues in beef sausages and its enantiomeric analysis using UHPLC–MS/MS: A risk of unintentional doping in sport field

“…It is important to note that the clenbuterol molecules showed stability during the cooking process (at 70 C), as some authors had shown previously. 33 In Figure 2 In several countries, including Mexico, 20 China, 27,30 Ecuador, 9 and Italy, 22 beef contaminated with clenbuterol in local markets has been detected, so parts of those contaminated animals may have been used in sausage production. The World Health Organization 11 and European Commission 37 define contaminants as any substance not intentionally added to food or feed for food-producing animals.…”

“…The World Anti‐Doping Agency's (WADA) included the clenbuterol in the list of prohibited substances in sport 17,18 . In the latter, it is used with equines and companion animals, and as a tocolytic agent in bovines 17–21 . It has, however, also been used illegally to promote animal growth because it causes a significant increase in muscle mass and, simultaneously, decreases fat deposits 22,23 .…”

“… 17 , 18 In the latter, it is used with equines and companion animals, and as a tocolytic agent in bovines. 17 , 18 , 19 , 20 , 21 It has, however, also been used illegally to promote animal growth because it causes a significant increase in muscle mass and, simultaneously, decreases fat deposits. 22 , 23 For these reasons, its use is strictly prohibited in the European Union (since 1996), the United States (1991), 24 Mexico (2007), 25 , 26 and many other countries.…”

“… 27 Because the R ‐(−) enantiomer stimulates β2‐receptors, it is recognized as a eutomer. 28 The S ‐(+) enantiomer blocks the effect of β1‐receptors, 29 can accumulate in various areas of an animal's body, 20 , 29 and can be transmitted to humans through contaminated foods. It has the potential to cause unintentional intoxication with clenbuterol.…”

“…Specificity was considered acceptable when the retention time of the target analyte showed that no interfering components were detected. Linearity was evaluated using a fresh calibration curve by spiking samples of organic beef sausages samples with IStd clenbuterol in quadruplicate at five working concentrations(20, 50, 100, 200, & 500 pg g À1 ). The peak area ratio versus the final concentration of the analyte was used.…”