Determination of clenbuterol and mabuterol in equine plasma by ion-pair liquid chromatography with electrochemical detection. Chromatographic and electrochemical characteristics.

This research developed a method for detecting the presence of the drugs clenbuterol and mabuterol in horse plasma. The technique involves isolating the drugs from the plasma, evaporating the organic phase and dissolving the residue before subjecting it to a particular form of liquid chromatography and quantifying the drugs using an electrochemical detector.

Experimental Procedure

• The researchers began by extracting clenbuterol and mabuterol from alkalinized horse plasma using a process known as liquid-liquid extraction. This involves mixing two liquids that are insoluble to each other (one is usually water or an aqueous solution) and allowing them to separate again. The drug compounds, which were in the alkalinized plasma and dissolved in the liquid phase, could then be isolated.
• The organic phase of this mixture, presumably containing the drug compounds, was then evaporated to a dry state, leaving behind the solid residue of the drugs.
• This residue was then dissolved in what the researchers referred to as a ‘mobile phase’. This liquid would then carry the drug residues through the next step of the process, which was high-performance liquid chromatography.

High-Performance Liquid Chromatography

• High-performance liquid chromatography (HPLC) is a type of column chromatography used to separate, identify, and quantify each component in a mixture.
• In this research, the HPLC was of the ‘reversed-phase’ type – a method used when the substance to be separated is polar and soluble in water.
• Clenbuterol and mabuterol were effectively separated by the HPLC process.

Quantitation Method and Results

• The researchers then had to find out how much of the drugs clenbuterol and mabuterol were in the plasma samples. This was achieved by using a device known as a ‘coulometric detector’ at a specific voltage.
• The routine method they developed resulted in 98% and 95% recoveries for clenbuterol and mabuterol respectively. This means that the method was able to extract almost all of the drug that was present in the plasma.
• The researchers also assessed the influence of pH, the amount of organic modifier, and ion-pairing agent on the retention times.
• They found a linear relationship between peak current and concentration up to 1 microgram/ml for both compounds. This means that as the concentration of the compounds increased, so did the peak current, up to a concentration of 1 microgram/ml.
• The limit of detection was calculated to be 0.5 ng/ml for clenbuterol and 2 ng/ml for mabuterol with a signal-to-noise ratio of 3, indicating that the method is very sensitive in detecting the presence of these drugs in horse plasma.

Implications of the Study

• The method provides a means to detect and quantify these drugs, which is essential for enforcement of regulations in the equestrian sector where these substances may be used for doping.
• Furthermore, understanding the electrochemical properties of these substances and their detection methods can contribute to the development of drug testing in other fields, such as human sport and veterinary medicine.