Abstract
A method for the determination of β-agonists was developed by combining the separation of analytes through high-performance liquid chromatography, with a reversed-phase column, coupled to the pulsed amperometric detection at a glassy carbon electrode. Preliminary experiments, using cyclic voltammetry, allowed for an understanding of the electrochemical behavior of clenbuterol, fenoterol, and terbutaline. By analyzing the electrochemical response, the conditions for detecting the analytes and for cleaning the working electrode were identified. The proposed potential-time profile was designed to prevent contamination of the carbon electrode following consecutive analyses, so ensuring a reproducible and sensitive quantitative determination. The waveform electrochemical parameters, including detection and delay times, have been optimized in terms of sensitivity, detection limits, and long-term response stability. The chromatographic separation was carried out using a C8 column in isocratic mode, and a mixture of acetic acid and acetonitrile. The optimized experimental conditions were used for the analysis of standard solutions and real samples. Detection limits, lower than the maximum residue limit set for clenbuterol by European directives, were obtained for all β-agonists investigated. The method validation was performed by evaluating the linearity, selectivity, precision, and recovery. Calf urine samples were used to verify the applicability of the proposed method, analyzing both enriched and naturally contaminated urine samples.
Highlights
The oxidation, attributed to the terbutaline phenolic groups, occurs at a very close potential (1.03 V) of that observed for clenbuterol, showing that glassy carbon electrode (GCE) in acetic/acetonitrile medium is suitable for the simultaneous electrochemical detection of β-agonists
Cyclic voltammetry (CV) experiments evidenced that β-agonists have the advantage/drawback of adsorbing on glassy carbon electrodes
These results suggest that a cleaning before each subsequent electrochemical measurement is necessary, a well-designed triple potential step waveform would be suitable to detect β-agonists following liquid chromatographic separations with organic mobile phases
Summary
Beta-adrenoceptor agonists (β-As) are derivatives of phenylethanolamine with different substituents on the aromatic ring and terminal amino group These compounds have been commonly used as a bronchodilator medicine for the treatment of human chronic obstructive pulmonary disease and asthma [1,2,3]. Some of these compounds may be misused as performance-enhancing drugs in athletes to enhance performance due to their ergogenic potential and anabolic effects [4] Another prohibited application of these substances is their use as veterinary drugs; they have been widely used in livestock to improve the transformation of the level of fat into the production of muscle proteins [5,6]. These illegal uses have harmful effects on human health since beta-agonist residues can accumulate in animal tissues and organs and may have a pharmacological effect in human, such as dizziness, headache, hypokalemia, nausea, tachycardia, cardiac QT interval changes, and effects in the central nervous system such as nervousness and difficulty sleeping [7]
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