| Beta-adrenoceptor agonists are a class of drugs that are mainly used for the treatment of bronchial asthma and other forms of allergic airway diseases.They also have the effect of increasing the lean body fat rate of animal carcasses and are therefore widely used in animal husbandry production.However,when such veterinary drugs are taken into the body,they will have a considerable amount of residues in the liver,lungs,and eyeballs of the animal's body and further harm the human body through the food chain.The Ministry of Agriculture of the People's Republic of China has issued a series of agricultural bulletins that prohibit the use of 15 beta-agonists,including clenbuterol,salbutamol,and ractopamine,in feed and animal drinking water.However,under stimulation of huge profits,they are still illegally used in animal breeding.Therefore,it is very necessary to establish a sensitive,rapid and simple method to detect residual ?-agonists in animal-source foods.Among different ?-agonists,salbutamol was the first veterinary drug that could be a substitute for clenbuterol,so it is necessary to strengthen the supervision of salbutamol.Considering that salbutamol has a primary alcohol hydroxyl active site,we modified salbutamol by an activated ester method.The obtained salbutamol derivative was coupled with bovine serum albumin(BSA),and monoclonal antibody against salbutamol was obtained in mouse by hybridoma technology.Based on own-produced monoclonal antibody,the corresponding indirect competitive enzyme linked-immunosorbent assay(ic-ELISA)for the detection of salbutamol was established.Under optimal experimental conditions,the IC50 and detection limits of the ELISA for salbutamol were 0.47 ng m L-1 and 0.049 ng m L-1,respectively.The cross-reactivities of the ELISA with some compounds were: 55% for clenbuterol,44% for terbutaline,33% for brombuterol,134% for cimbuterol,2.1% for cimaterol,1.9% for isoprenaline,and 1.2% for clorprenaline.The high ross-reactivity of the ELISA with clenbuterol,terbutaline,brombuterol and cimbuterol may be an advantage of the ELISA to be used for total amount of group-specific ?-adrenoceptor agonists in animal tissues.The swine meat and liver samples spiked with salbutamol were detected by the ELISA.The recovery rates were 76.4%-107.6% and 71.8%-106.8%,with coefficients of variation less than 19.3%.The spiked swine meat and liver samples were also simultaneously detected by the ELISA and HPLC.There was a good correlations(r2>0.9964)between ELISA and HPLC.The result indicated that a sensitive and group-specific ELISA for salbutamol has been successfully developed,which could be served as a rapid screening and feasible quantitative assay for salbutamol analysis in animal tissues.Not only salbutamol,clenbuterol,and ractopamine are also the main ?-agonists detected in China.It is necessary to establish an analytical method that can simultaneously detect the residues of these three kinds of veterinary drugs in foods.Beta-agonists generally have residues in animal tissues or metabolites that are very complex in the matrix.Therefore,the real samples must be purified,otherwise the matrix effect will have a great impact on the detection results.The use of immunoaffinity chromatography combined with LC-MS/MS not only simplifies sample preparation but also improves detection sensitivity.In this study,based on the fact that monoclonal antibody against salbutamol exhibiting 55% of cross-reactivity with clenbuterol,we prepared the mixed immunoaffinity chromatographic(MIAC)gel by immobilizing the monoclonal antibodies against salbutamol and ractopamine on the surface of CNBr-activated agarose 4B.It is expect that MIAC is able to specifically bind with clenbuterol,salbutamol and ractopamine,and can be used as a purification method.The operating conditions of the MIAC gel were optimized.It was found that MIAC gel was able to separately capture salbutamol,clenbuterol,and ractopamine with a maximum capacity of 33,34,and 29 ng/50 ?L gel,respectively.Calibration curves were constructed using a standard solution diluted with blank swine sample matrix.The linear ranges of the calibration curves ranged from 0.2 to 20 ng m L-1 and the coefficients of determination were 0.9946-0.9992(n=3)for three ?-adrenoceptor agonists.In swine meat,liver and urine,the average recoveries of the three targets were 72.2%-76.2%,64.1%-82.4% and 64.8%-92.0%,respectively.Compared with SPE method,it can be clearly seen from the fluorescence chromatograms that the IAC method has a better extraction and purification effect.In summary,the mixed immunoaffinity chromatography combining with LC-MS/MS is a sensitivie method for the detection of salbutamol,clenbuterol and ractopamine in real samples. |
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