Detection Of β₂-agonists Residue In Biological Samples By HPLC With Electrochemical Detection

In this thesis, two methods, one for determining salbutamol in human plasma and urine and the other for detecting the residue of clenbuterol in pig liver, were developed by high-performance liquid chromatography with a Coulometric electrode array system. The optimization of extract condition, the component and the pH of mobile phase and the selection of electrode potential were taken into account in the two methods. Salbutamol existed in human plasma and urine was extracted using silica solid-phase extraction cartridges. The mobile phase consisted of 30mmol/L sodium dihydroxy phosphate solution – methanol (90:10, v/v). The sodium dihydroxy phosphate solution was added 0.4% triethylamine and adjusted its pH to 6.0 by adding 20% phosphate acid. The electrodes potential were set at 300mV, 400mV, 550mV and 650mV. The recoveries of salbutamol in plasma and urine were 77.32% and 81.25% and the detection limits were 0.5ng/ml and 2.5ng/ml.After homogenate and centrifugation, the fat existing in liver was removed by diethyl ether. Then, the pH of the remaining aqueous layer was adjusted to 11.6 and clenbutamol was extracted using diethyl ether. The mobile phase component A was 50mmol/L phosphoric acid – 30mmol/L triethylamine and the pH was adjusted to 4.0 with 2mol/L sodium hydroxide. The mobile phase component B was methanol and acetonitrile in the proportion of 30:45 (v/v). An 80:20 mixture of mobile phase components A and B was used in the method and the electrodes potential were set at 450mV, 600mV, 650mV and 680mV. The recovery of the method was 78% and the detection limit of clenbuterol is 1.2 ng/g. In this thesis, the two methods were validated thoroughly. The results showed the two methods developed using HPLC-ECD were reproducible and sensitive enough for biological matrix.