Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography–tandem mass spectrometry
Introduction
Clenbuterol is part of the β2 agonists family, a class of compounds firstly designed for the antiasthmatic properties and tocolytic effect in human and animals. The administration of clenbuterol-like drugs has other physiological effects such as increase of aerobic capacity, blood pressure and oxygen transportation, stimulation of the central nervous system and has an impact on the body fat metabolism rate [1], [2]. The anabolic properties given by the marked increase of skeletal muscles in parallel to the decrease of the body fat mass has forced the World Anti-Doping Agency (WADA) to put clenbuterol in the class S1.2 (other anabolic agents) of the Prohibited List [3]. Because there is no threshold level for clenbuterol in the anti-doping regulations, any of the concentration found in urines of athletes would lead to an adverse analytical finding, leading theoretically to a sanction of the athlete.
Nowadays, clenbuterol has been banned for therapeutic use in humans but is sometimes employed as a growth-promoting agent in food-producing animals [4], [5], [6]. The use of this compound for cattle and bovine breeding is well regulated in the United States of America by the FDA (Directives 96/22/EC and 96/23/EC) as well as in the European Union by the Commission (EC 37/2010). In Europe, Maximum Residue Limits listed for clenbuterol in bovine tissues are the following: 0.1 μg/kg in muscle; 0.5 μg/kg for both liver and kidney and 0.05 μg/kg in milk. Despite these rules, there are still some countries where, after used of clenbuterol for the promotion of animal growth, residues of this compound can be found in the meat [7], [8]. Consumption of such contaminated meat could therefore lead to the presence of traces of this forbidden substance in human urine and then to an anti-doping rule violation for an athlete. Moreover, WADA has introduced since 2004 the strict liability rule in the World Anti-Doping Code meaning that athletes are responsible for themselves and should be cautious about any substances entering his or her body which could cause a doping offence.
In 2011, the Fédération Internationale de Football Association (FIFA) faced several adverse analytical cases containing traces of clenbuterol. The urine samples were collected from athletes living in Mexico or having stayed in this country for a certain period of time. More recently, Guddat et al. published analytical results of urines provided by 28 volunteers after having traveled to China [9]. Clenbuterol was detected in 79% of the analyzed samples with concentrations levels between 1 and 50.5 pg/mL. The authors highlighted the possibility of a general food contamination problem in China which could lead to risks of unintended doping for athletes in sport. A warning was also published on WADA website in November 2011 saying that sufficient evidence has been collected to demonstrate this risk in some countries [10].
Considering these problems and the above-mentioned cases, the development of rapid and robust analytical methods for the detection of urinary traces of clenbuterol is of high interest for anti-doping laboratories [11], [12], [13], [14], [15]. Nowadays, the use of ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) is gaining importance in doping control area where the response time for reporting adverse analytical findings must be reduced as much as possible [16], [17]. Strong data quality and robust results can be obtained with this technique in conjunction with a dedicated sample preparation for the extraction of the compound of interest from biological fluids. Different applications describing the performance and advantages of UHPLC–MS/MS for quantification of prohibited compounds in sports have been published recently. Among them, an interesting work has been reported by Ventura et al. for the quantification of salbutamol in urine samples with a limit of quantification of 200 ng/mL [18], while Guan et al. proposed a method for the identification and quantification of fifty-five anabolic and androgenic steroids in equine plasma after liquid–liquid extraction (LLE) with tert butyl-methyl ether (TBME) [19], [20].
The aim of this study was to develop and validate UHPLC–ESI-MS/MS method for the quantification of clenbuterol in urine samples at the low pg/mL range. After a LLE in basic conditions, the detection and quantification of clenbuterol in urine was demonstrated using an isotope labeled internal standard. The method was validated in compliance with the authority guidelines and applied to real urine samples from eighteen volunteers collecting urines over 6 days after a single intake of clenbuterol-enriched yogurts at three dose levels 1, 5 and 10 μg. These volunteers were part of a clinical trial performed in 2011 and conducted in order to better understand the pharmacokinetics of clenbuterol in human urine.
Section snippets
Chemicals
Clenbuterol hydrochloride, sodium chloride (NaCl) and potassium hydroxide (KOH) were purchased from Sigma–Aldrich (Buchs, Switzerland). Clenbuterol-d9 hydrochloride (Internal Standard, IS) was obtained from NARL (Sydney, Australia). TBME was purchased from Acros Organics (Chemie Brunschwig, Basel, Switzerland) while formic acid and acetonitrile (ACN) of UHPLC–MS grade from Biosolve (Chemie Brunschwig, Basel, Switzerland). Ultra-pure water was provided by a Milli-Q system from Millipore
Method development
The WADA recommendations in terms of Minimum Required Performance Limits (MRPL) for clenbuterol, currently set at 2 ng/mL, will be adjusted down to 200 pg/mL, as described in the Technical Document TD2013MRPL (effective date 1st January, 2013) [33]. The development of the method presented in this paper was therefore focused on sensitivity issue in order to detect and quantify clenbuterol in urine samples in the low pg/mL range. For this purpose, preliminary experiments for the extraction of
Conclusion
Several adverse analytical findings for clenbuterol misuse have been recently reported leading to a sanction of athletes. However, in the last years, the possibility of unintended clenbuterol doping represents a topical debate in the anti-doping area. Indeed, it has been recently reported by several authorities that trace levels of this substance might be found in the meat in some countries. This context has driven the necessity for WADA-accredited laboratories to develop fast and robust
Acknowledgements
The authors wish to thank Dr. Elia Grata for method development as well as Anne Ballif and Sabine Dominé for performing the sample preparation. In addition, the authors are thankful to Alexis Tingirides and all the staff of the Biocheck Clinical Laboratory in Nicosia (Cyprus) for their help and assistance in biological samples collection.
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