Determination of β-agonists in urine samples at low μg/kg levels by means of pulsed amperometric detection at a glassy carbon electrode coupled with RP-LC

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.