| Nowadays, new technologies for enrichment and purification of drug residues in animal-derived food have aroused increasingly interests. Molecularly imprinted solid phase extraction (MISPE) using molecularly imprinted polymers (MIPs) as adsorbent materials, has displayed higher selectivity and better specificity than conventional SPE, especially on the extraction of the target analytes from complex biomatrices. As resulted, MISPE has become one of the most potential applied technologies in the field of food safety detection.Clenbuterol (CLB) is a representative member of artificialβ2-agonists, its abuse and residue in animal-derived food is still very serious, which will threaten human health and related international food trade. Thus, CLB is one of the most prohibited drugs in the current food safety inspection system of China. The methods for detection of clenbuterol included chromatographic analyses, capillary electrophoresis and immunoassay. Unfortunately and realistically, for animal tissue samples, the unavoidable drawbacks of these above methods resulting from the interferences in the complex biomatrices frequently lead to low recovery, long time for sample preparation, low detection sensitivity and false positive or negative results by these detection methods.In our study, we prepare the desirable CLB-MIPs with high specificity and high selectivity, and then establish the combination of MISPE with ELISA to develop a new rapid CLB detection method in tissue samples, which can significantly increase the efficiency of sample pre-treatment and sufficient detection sensitivity.1. We prepared the CLB MIPs, and then focused on development of the post-treatment technology removing the template residue. The GC/MS results demonstrated that the total amount of template bleeding in the CLB MIPs was gradually decreased to only 1.0 ng CLB/20 mg MIPs, which was ten times lower than that of previous report with the lowest value, after a series procedure with Soxhlet extraction, thermal annealing and various washing solutions. Furthermore, chacteristic evaluation of the MIPs confirmed that it had a good effect imprinting (α= 2.363), and the largest adsorption capacity was 9.6μg/60 mg MIPs. At the same time, MIPs towards the target molecule CLB showed the highest specificity (97.1%), while for the analogues of CLB, terbutaline(TER) and salbuterol (SAL), had a moderate cross-reaction (13.0% and 22.1%).2. Based on the obtained MIPs, we established and optimized the MISPE process, and then finalized a standard operating procedure. Firstly, 3 mM phosphate solution (pH 3.4)–MeCN (30:70, v/v) was used as the loading solution, and then washed the column with 1 mL of H2O, 1 mL of MeCN with 2% HOAc, 1 mL of MeCN with 30% H2O, and 1 mL of 100% MeCN, and finally eluted the CLB with 3×1 mL of MeOH: HOAc (9:1, v/v). The obtained recovery was 95.7%.3. We combined the MISPE-ELISA together to develop a new method for CLB detection. Compared to the methods of C18-ELISA and single ELISA, it exhibited high precision and robust accuracy for CLB at all three spiked levels of 0.5, 5.0 and 10.0 ng/g in pig tissue samples, and the recovery was higher than 99.4%. As contrast, the detection sensitivities of the C18-ELISA and single ELISA were both lower than 0.5μg/kg. In conclusion, we developed the combined approach of MISPE-ELISA for the first time as to overcome the existing template residues to detect CLB of animal tissue samples, which had the potential to replace the C18-based conventional column analytical techniques. And more importantly, the established method can also ensure detection sensitivity at 0.5μg/kg CLB in animal tissue samples. Thus it enables to fully meet the existing limited requirements of the national standards. Therefore, the strategy presented in this study could provide a valuable reference for the application in practical uses of MIPs, coupled with immunoassay and other rapid detection methods against other abused drugs or small molecules.
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