Determination of \(\beta\)-Agonists in Urine Samples at Low \(\mu\)g/kg Levels by Means of Pulsed Amperometric Detection at a Glassy Carbon Electrode Coupled with RP-LC  
[PERSON]  
1 Dipartimento di Scienze Agrarie, Degli Alimenti e Dell'ambiente, Universita Degli Studi di Foggia,  
Via Napoli 25, 71122 Foggia, Italy; [EMAIL_ADDRESS] (A.M.); [EMAIL_ADDRESS] (D.C.)  
[PERSON]  
1 Dipartimento di Scienze Agrarie, Degli Alimenti e Dell'ambiente, Universita Degli Studi di Foggia,  
Via Napoli 25, 71122 Foggia, Italy; annalisa. Another prohibited application of these substances is their use as veterinary drugs; in fact, 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]. The administration of \(\upbeta\)-agonists as growth-promoting agents in food-producing animals is banned in many countries [8]. However, ratcopamine and zilpaterol are authorized for production of some animals in a limited number of countries (e.g., the USA, Canada, and Brazil). Maximum residue limits (MRL\({}_{\upbeta}\)) for ratcopamine in cattle and pig muscle (both 10 \(\upmu\)g/kg) have been adopted by the Codex Alimentarius Commission and have been implemented in some countries (e.g., Canada). Others have set alternative limits for ratcopamine (e.g., USA-30 and 50 \(\upmu\)g/kg in cattle and pig muscle). However, many countries have banned meat products containing \(\upbeta\)-agonists. In the EU, \(\upbeta\)-agonists other than clenbuterol (for cattle and horse) are prohibited for use in food producing animals. Therefore, the European Union has improved the official check programs to hinder the use of beta-agonists in stock farming and a maximum residue limit (MRL) of 0.5 \(\upmu\)g/kg has been established for clenbuterol [9]. In the literature there are various analytical methods for the determination of beta agonists, among these are immunoenzymatic methods [10; 11], gas chromatography [12; 13], and HPLC separations, coupled with mass spectrometry [14; 15], UV detection [16; 17], or electrochemical detection [18; 19]. However, these procedures are often lengthy, requiring large volumes of organic solvents and derivatization steps prior to gas chromatography analysis or laborious sample preparation prior to LC-MS determinations [20; 21; 22], in order to minimize interferences and the matrix effect on ion suppression. Electrochemical detection methods (ECDs), and in particular the pulsed amperometric detection, PAD-based ones [23; 24; 25], have attracted much interest as detection methods in HPLC. In this context, the main point to be considered in the development of such methods is the perfect tune between the mobile phase, also acting as the supporting electrolyte, and the electrode material/electrochemical technique used for the detection of analytes. As a consequence, organic mobile phases used in RP-HPLC are not really compatible with ECD at metal-based electrodes. Hydroxy- or amino-phenyl moiety of beta agonists (see Figure 1) implies their electrochemical detection by anodic oxidation, and among the most common electrodes, the carbonaceous ones (including glassy carbon, GCE) were used [26; 27; 28; 29]. With respect to metal electrodes, GCEs possess excellent electrochemical properties in a wide range of working potentials in organic solvents, but they undergo fouling phenomena when operating at constant potential with certain analytes, with a time-dependent deterioration of the electrochemical response. To overcome this limitation, we propose for the first time the proof-of-concept of PAD at GCEs [30].