| Clenbuterol, which is well known as a series ofβ-agonists with the promotion of animal growth and improve the lean meat, was often added to animal feed illegally. While some other similar chemicals which areβ-blockers are used for animals during transportation to prevent the animal sudden death by stimulus.β-agonists andβ-blockers are prohibited to be used in food animals which lead to residues in animal food origin, There are also potential health risks through consumingβ-agonists orβ-blockers residual animal tissue. Furthermore,β-agonists andβ-blockers are substances listed on the prohibited list of the World Anti-Doping Code by the World Anti-Doping Agency. Therefore, to establish a rapid and efficient early warning system for risk assessment ofβ-agonists andβ-blockers is imperative. This study sets up a full system to make the risk assessment ofβ-agonists andβ-blockers to be possible.The main research works and the main results were listed below:1. Determination of multipleβ-agonists andβ-blockers in animal foods using mixed strong cation exchange SPE and MIP SPE as cleanup by HPLC-LIT-MS3.Two sets of sample extraction and cleanup techniques were used for the determination of 25β-agonists and 23β-blockers by HPLC-LIT-MS3 in animal origin foods. Samples were acid hydrolyzed and extracted with 5% TCA in water, and then cleaned up using MCX SPE cartridge. Another solution is that samples were extracted by acetronile and then hydrolyzed byβ-glucuronidase/arylsufatase, and cleanup by MIP SPE was followed. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, a Waters Atlantis(?)T3 column or a Supelco Ascentis(?) express Rp-Amide column was used for separation. ESI positive ion scan mode was used with MS3 selective reaction monitor.9β-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 25β-agonists and 23β-blockers were 5～200μg/L, the coefficient of correlation was not less than 0.99, and the limit of detection for the analytes in animal origin food was 0.015～0.3μg/kg for MCX procedure and 0.001～0.06μg/kg for MIP procedure. Blank pork, blank liver and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound in the spiked samples were in the range of 40.7%～131.9%, and the relative standard deviations were in the range of 0.9%～30.0%. Incurred samples and positive samples were analysed, and satisfactory resulted were obtained. The developed method is sensitive and specific for the determination ofβ-agonists andβ-blockers in animal foods.2.Determination of multipleβ-agonists andβ-blockers in urine using MSPD and MIP SPE as cleanup by HPLC-LIT-MS3.Based on the work of part 1, a HPLC-LIT-MS3 for the determination of 25β-agonists and 23β-blockers in urine was established. Techniques of MSPD and MIP were used in the extraction and cleanup procedure. The two approaches were described as followings:(1) MSPD:The urine samples were acid hydrolyzed by 5% TCA in water bath for 2 h, and then mixed with ExtrelutTM diatomite and the analytes were eluted by 100 mL Ethyl acetate. (2) MIP:The urine samples were hydrolyzed byβ-glucuronidase/arylsufatase, the extracts were loaded onto the MIP SPE cartridge for cleanup after adjusting the pH value of the hydrolyzed solution to 7.0. The results of method validation were satisfied. The linear ranges of 25β-agonists and 23β-blockers were 5～200μg/L, the coefficient of correlation was not less than 0.99, and the limit of detection for each compound in the muscle tissue were 0.001～0.13μg/L for MSPD method and 0.001～0.06μg/L for MIP method. The recoveries of each compound were in the range of 38.8%～133.4%% at the spike level of 5,10 and 20μg/L using MSPD procedure, and RSD were in the range of 1.5%～38.1%; and the recoveries were 40.4%～125.6% at the spike level of 0.5,1 and 2μg/L for MIP procedure, and RSD were in the range of 1.0%～33.3%. Positive urine samples were analysed, and satisfactory results were obtained. The results demonstrated that the method is easy, fast, sensitive, and suitable for the confirmation and quantitation of 25β-agonists and 23β-blockers in urine samples.3. Supervision ofβ-agonists andβ-blockers in animal foods in Beijing.Supervision ofβ-agonists and P-blockers in animal foods were conducted in Beijing.88 samples including 43 chicken samples,45 pork liver samples, and 6 pork kidney samples were collected from markets of 8 districts in Beijing. The 94 samples and the 4 pooled animal origin food sample of 2009 TDS of Beijing were analyzed using the established method under the satisfied analysis quality control including analysis of blank samples and blank spiked samples. Of all the samples, only salbutamol was detected in 1 pork kidney samples at the level of 31.38μg/kg. The results demonstrate that illeagal use ofβ-agonists on food animals still happened from time to time. Although there were noβ-agonists andβ-blockers in samples of 20, considering thatβ-agonists andβ-blockers are prohibited in the food animal breeding in China and incident of food poison caused byβ-agonists happened from time to time, it is necessary to supervise multiple residues ofβ-agonists andβ-blockers in animal foods.4. Residue level, traceability analysis and dietary exposure estimation ofβ-agonists andβ-blockers in animal food products in 2007 TDSThe concentration level ofβ-agonists andβ-blockers in 48 pooled animal food samples of 2007 TDS were detected by the established HPLC-LIT-MS3 method under the satisfied analysis quality control including analysis of blank and blank spiked samples. In the 48 pooled animal samples, no otherβ-agonists andβ-blockers were detected except for clenbuterol and ractopamine which were simultaneously detected in 2 pooled meat samples from Jiangxi and Shaihai. The concentration level of clenbuterol in the pooled meat sample was higher than that of ractopamine. The residues of clenbuterol and ractopamine were come from pork product, mainly pork meat and pork liver, according to the traceability analysis. It can be inferred that illegal use ofβ-agonists still existed in the industry of food animal breeding.Dietary exposure of clenbuterol and ractopamine were estimated. The average dietary exposure of clenbuterol of Chinese people was 0.0827μg/person d, which is 32.8% of ADI. And the average dietary exposure of ractopamine of Chinese people was relatively low,0.0092μg/person d, which is 0.0015% of ADI, and with this level of dietary exposure of ractopamine, there is not any risk for people's health.However, the total dietary exposure of clenbuterol of Shanghai and Jiangxi were relatively high,0.4764μg/person d and 0.4783μg/person d, and 189.0% and 189.8% of ADI, which post a high risk to people in Shanghai and Jiangxi. The extreme dietary exposure of clenbuterol was also estimated. The results showed that the products with the highest risk for people's health were pork liver. The offal, especially the liver of animal is the organ with the highest level of residue of chemicals. To lower the health risk, it is riot recommended that consuming large amount of animal offal in one meal.
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