An Improved LC–MS–MS Method for the Determination of Clenbuterol in Human Urine

An improved liquid chromatography tandem mass spectrometry (LC–MS–MS) method for the screening and confirmation of clenbuterol in human urine was developed and validated in this study. In addition to the currently used ion transitions (277.1203.0 and 277.1259.1), two new ion transitions (277.1132.0 and 277.1168.0) were utilized for this new screening procedure. Compared with existing methods of clenbuterol screening in human urine, the developed method is of higher sensitivity and reduces the number of false-positive results. The limit of detection (LOD) of the method proposed is 2 pg/mL, which is lower than the minimum required performance limit (MRPL) set by World Anti-Doping Agency (WADA). The success and reliability of the method in doping control analysis was demonstrated for the analysis of a 2011 EQAS (External Quality Assessment Systems) -01 human urine sample obtained from WADA.

Clenbuterol (Figure 1) is a beta-2 adrenergic agonist that stimulates beta-2 adrenergic receptors resulting in relaxation of smooth muscle and dilatation of bronchus, widely used to treat asthma and respiratory disease (1–3). A novel effect of clenbuterol is to slightly increase body temperature, increasing fat metabolism and magnifying the effect of anabolic steroids taken simultaneously (4,5). Therefore, many athletes use clenbuterol to increase their blood pressure and stimulate the heart muscle to maximize their potential. In 2011, clenbuterol was classified within "other anabolic steroids" and listed as prohibited in athletics by the World Anti-Doping Agency (WADA). The minimum required performance limit (MRPL) in and out of competition was set at 2 ng/mL in human urine (6). Following this classification, many publications reported on the two main clenbuterol detection methods: chromatography and immunoassay. Chromatographic techniques to determine clenbuterol include gas chromatography–mass spectrometry (GC–MS) (7–9), high-performance liquid chromatography (HPLC) (10,11) and liquid chromatography–mass spectrometry (LC–MS) (12,13). Immunoassay methods include radio-immunoassay, competitive fluorescence immunoassay and enzyme-linked immunosorbent assays that provide relatively cheap and simple analysis approaches (14–16); however, these immunoassays cannot provide structural information of the molecule, hence they are only suitable for complementary detection.

Figure 1: The chemical structure of clenbuterol.
GC–MS and LC–MS are the two methods currently approved by the WADA for use in doping control, with both the chromatographic retention time and diagnostic mass spectrometric ions required for the identification of a compound (17). However, as GC–MS (or MS–MS) requires time-consuming derivatization steps to enhance the volatility of analytes, liquid chromatography tandem mass spectrometry (LC–MS–MS) has become the main analytical technique for the determination of clenbuterol because of its shorter chromatographic run time and the elimination of time-consuming derivatization procedures.

Many LC–MS–MS methods have been developed for identification of clenbuterol (18–27), but there are few papers on the identification of clenbuterol in human urine. An LC–MS–MS method for determining clenbuterol in equine and human urine has been developed by Mario et al. (28). The pre-treatment procedure included acidic hydrolysis, neutralization and weak basic extraction. The limits of detections (LODs) were 2 ng/mL (human urine) and 10 ng/mL (equine urine), respectively. An LC–MS–MS approach was also reported for the detection of clenbuterol in human urine by using a three-phase solvent bar microextraction (TPSBME) technique for sample pre-treatment (29) and the reported LOD was 7 pg/mL.

It is worth noting that all the reported LC–MS–MS methods for the characterization of clenbuterol employed two abundant transitions at m/z = 277→259 and 277→203 (18–29). These transitions may produce false positive results and so the reported LODs are unreliable. The aim of this study is to develop and validate a more sensitive and specific method for the screening and confirmation of clenbuterol in human urine for doping control purposes.

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