食品及生物材料中β-激动剂和β-阻断剂残留检测技术研究及污染评价

【作者】 苗虹；

【导师】 吴永宁； 赵云峰；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2010， 博士

【摘要】 瘦肉精,又称克伦特罗,是一系列β-激动剂,具有促进动物生长和提高瘦肉率的作用,经常被违禁添加在动物饲料中；而另一类化学结构类似的β-阻断剂则用于动物运输过程中,防止动物因应激而造成的突然死亡。食用动物中违禁使用β-激动剂或β-阻断剂会导致其在动物体内的残留,摄入β-激动剂或β-阻断剂残留的动物组织,存在食物中毒的风险或引起潜在的健康危害。β-激动剂和β-阻断剂还是《世界反兴奋剂条例》中规定的禁止在体育赛事中使用的兴奋剂。因此针对β-激动剂和β-阻断剂建立一套快速有效的风险评估预警体系势在必行。本研究建立了由检测、监测、暴露评估以及风险预警的全套技术支撑体系,使得食品安全中亟待解决β-激动剂和β-阻断剂的风险评估预警成为可能。主要研究内容及研究结果如下：1.动物性食品中β-激动剂及β-阻断剂多组分残留的液相色谱-串联质谱检测方法建立经样品制备和检测参数的优化后,分别采用混合型阳离子固相萃取技术和分子印迹(MIP)固相萃取技术进行动物性食品的前处理,以甲醇和含0.1%甲酸的水溶液为流动相,梯度洗脱；采用ESI源正离子模式电离,三级质谱选择离子监测(CRM)模式进行扫描,以9种氘代β-激动剂为内标,建立高效液相色谱-线性离子阱质谱(HPLC-LIT-MS3)测定动物性食品中25种β-激动剂及23种β-阻断剂残留的检测方法。采用AtlantisT3-150 mm(或Supelco Ascentis(?) express Rp-Amide-150 mm)色谱柱进行色谱分离,甲醇和含0.1%甲酸的水溶液为流动相梯度洗脱。测定的线性范围为5～200μg/L,相关系数(r)大于0.99。在动物性食品中两种方法的检出限分别在0.015～0.3μg/kg之间(MCX)和0.001～0.06μg/kg之间。(MIP)。以空白猪肉、猪肝和猪肾样品为代表基质进行了加标回收试验,MCX净化方法的加标水平为5、10、20μg/kg,MIP净化方法的加标水平为1、2、4μg/kg,各化合物的回收率在40.7%～131.9%之间,RSD在0.9%～30.0%,两种方法的精密度及准确度均较好。对阳性样品进行测定,获得良好的确证结果。建立的方法具有很高的灵敏度,而且定性准确、定量可靠,可以用于动物肌肉、肝脏和肾脏组织中β-激动剂和β-阻断剂类药物残留的确证检测。2.生物样品中β-激动剂及β-阻断剂残留的液相色谱-串联质谱检测方法建立在上述工作基础上,分别采用基质固相分散技术(MSPD)和MIP技术建立了尿液中25种β-激动剂及23种β-阻断剂的HPLC-LIT-MS3测定方法。2种不同的样品前处理技术分别为：(1)MSPD技术：尿液样品以三氯乙酸酸解后离心,上清液经ExtrelutTM硅藻土以乙酸乙酯进行洗脱净化；(2)MIP技术：尿液样品经β-葡萄糖醛酸酶/芳基硫酸酯酶酶解后调pH7.0,直接以混合型β-激动剂及β-阻断剂的MIP固相萃取柱净化。进行了方法学验证试验,各化合物定量的线性范围为5～200μg/L,在尿液中两种方法的检出限分别在0.001～0.13μg/L(MSPD)和0.001～0.06μg/L(MIP)。采用MSPD净化的方法的空白尿液加标水平为5、10和20μg/L,各化合物回收率在38.8%～133.4%之间,RSD在1.5%～38.1%；采用MIP净化的方法的空白尿液加标水平为0.5、1和2μg/L,各化合物回收率在40.4%～125.6%之间,RSD在1.0%～33.3%。以阳性尿液样品验证了方法的实用性。建立的检测方法的灵敏度很高,检测限可达ppt级,而且该方法简便快速,完全能满足人或动物尿液中的β-激动剂和β-阻断剂的定性和定量分析。3.北京市动物性食品中β-激动剂和β-阻断剂药物的污染监测及评价采用建立的液相色谱-串联质谱法对北京市场采集的94份鸡肉、猪肝和猪肾样品以及北京市2009年总膳食研究的动物性食品混样样品进行了β-激动剂和β-阻断剂残留的监测。在良好的质量控制保证下,1份市场采集的猪肾样品中检出沙丁胺醇,含量为31.38μg/kg。其他样品中均未检出β-激动剂和β-阻断剂残留。表明仍存在对食品动物违禁使用β-激动剂的情况。北京市2009年总膳食样品中未检出β-激动剂和β-阻断剂残留,说明北京市场动物性食品中不含有β-激动剂和β-阻断剂。但鉴于β-激动剂及β-阻断剂均为我国禁止用于食用动物的兽药,其违禁使用时有发生,为确保我国食品的安全性,因此加强动物性食品中禁用β-激动剂和β-阻断剂的多残留监测实属必要。4.我国动物性食品中β-激动剂和β-阻断剂的残留状况、溯源分析及膳食安全性评价采用建立的液相色谱-串联质谱法,对我国2007年总膳食研究的动物性膳食样品中β-激动剂和β-阻断剂残留进行了检测。在48份动物性膳食混合样品中,有2份样品(江西肉类混样和上海肉类混样)同时检出克伦特罗和莱克多巴胺,克伦特罗的污染水平远高于莱克多巴胺,未检出β-阻断剂的残留。通过溯源分析发现克伦特罗和莱克多巴胺残留主要来源于猪制品-猪肉和猪肝。由此可见,在我国食用动物的饲养中仍存在β-激动剂的违禁使用。以检出的克伦特罗和莱克多巴胺残留量进行膳食暴露评价：克伦特罗全国平均暴露量为0.0827μg/人/天,占ADI的32.8%。莱克多巴胺的全国平均膳食暴露量很低,为0.0055μg/人/天,占ADI的0.009%,因此不会对人体健康产生风险。而对于肉类样品中检出克伦特罗的上海和江西来说,其膳食暴露量较高,分别为0.4764μg/人/天和0.4783μg/人/天,占ADI的189.0%和189.8%,这说明当地居民存在食用β-激动剂和β-阻断剂的健康风险。由克伦特罗的极端暴露量分析可见,极端暴露风险较大的是猪肝制品。由于动物的内脏器官特别是肝脏是β-激动剂如克伦特罗等的体内代谢的主要残留场所,因此为降低β-激动剂和β-阻断剂的健康风险,建议不要一次性大量食用动物内脏。更多还原

【Abstract】 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.更多还原