摘要
β2-受体激动剂是一类苯乙胺类药物,起初主要在临床上用于治疗支气管哮喘、痉挛,阻塞性肺炎等症状,后来发现当把较大剂量的此类药物添加到饲料中时,会大大提高动物的瘦肉转化率,具有营养再分配的作用。若长期食用含有β2-受体激动剂残留的食品就会发生头痛,胸闷,恶心等不良反应,因此欧盟和我国先后都颁布法令禁止在畜禽生产中使用该类药物。本研究旨在建立猪肉中β2-受体激动剂残留的超高效液相色谱串联质谱(UPLC-MS/MS)确证方法和表面等离子生物传感器(SPR-biosensor)的筛选方法。
UPLC-MS/MS方法：称取2.0 g匀质的样品经酶解后,用高氯酸：甲醇(9：1,v/v)混合溶液除蛋白后,经混合型的PCX柱净化,氮气吹干后,使用初始的流动相定容到2.0 mL。最后经过超高效液相色谱电喷雾串联质谱检测。结果表明,本方法有效的减少了离子抑制现象,沙丁胺醇,莱克多巴胺,盐酸克伦特罗三种β2-受体激动剂的平均回收率都在85%以上,相对标准偏差(n=3)在2.3-11%范围内,方法检出限在0.07-0.12μg/kg,定量限在0.22-0.38μg/kg。
SPR生物传感器方法：样品经高氯酸提取,乙酸乙酯净化后,通过分别固定抗原和抗体优化了SPR检测方法,并对缓冲液,再生溶液进行了优化,建立了猪肉中莱克多巴胺的SPR生物传感器抑制免疫残留检测方法,该方法对莱克多巴胺的检出限为0.6μg/kg,在1、2和4μg/kg三个浓度添加水平下,平均回收率为81%-102%,相对标准偏差均在10%以内。本方法简单快速,检测时间为5.0 min,与ELISA方法比较,抗体无需标记。在相同的前处理条件下,与UPLC-MS/MS相比,虽然检测限较高,但回收率和精密度都较好,并且受样品基质的影响小。特别适合大量样品的快速筛选。与国外方法相比,本研究采用中国科学院自行研制的SPR-2004生物传感器及芯片成本低,抗基质干扰能力强,具有广泛的应用前景。
本研究所建立的两种检测猪肉中β2-受体激动剂残留检测方法,样品处理简单,去除基质干扰能力强,回收率、精密度以及方法精密度都较好,UPLC-MS/MS方法,适合样品的确证分析。SPR生物传感器方法,特别适合大量样品的快速筛选工作,而且成本较低,可以用于工厂,超市,集市等场所的质量控制。
β2-agonists are synthetic phenethanolamine compounds as bronchodilatory and obstructive pneumonitis agents in human and veterinary medicine. However, these drugs are often misused as growth promoters for their function in growth rates enhancement and feed efficiently. Meat products coming from treated animals might be a potential threat for consumer health and cause headache, chest tightness, nausea and other adverse reactions. So the use ofβ2-agonists as growth promoters in cattle is banned in the EU and our country. In this research, confirmatory method of ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC-MS/MS) and screening method of surface plasmon resonance (SPR) biosensor method were studied.
UPLC-MS/MS method:After enzymatic hydrolysis, a 2.0 g homogenized pork sample was extracted, with perchloric acid-methanol (9:1, v:v) to precipitate protein, and then cleaned by mixed-mode Bond Elut Plexa PCX solid phase extraction (SPE) cartridges. Finally, the evaporated extract was reconstituted to 2.0 mL with initial mobile phase. The analytes were determinated by ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS-MS). Results indicate that the method is effective to reduce ion suppression phenomena. Recoveries of the threeβ2-agonists in most cases were better than 85% with relative standard deviations between 2.25 and 10.62%. The limits of detection (LOD) were 0.07-0.12μg/kg. The limits of quantitation (LOQ) ranged from 0.22-0.38μg/kg.
SPR biosensor method:After extraction with perchloric acid, pork sample was cleaned by ethyl acetate and analyzed by SPR-2004 biosensor. Rac antibody and Rac derivative were fixed to the chip surface successively in order to optimize SPR detection method. In addition, regeneration solution was also was optimized in order to reduce chip memory effect. The limit of detection (LOD) was 0.60/μg/kg for pork samples. Recoveries of Rac were higher than 80% with relative standard deviations below 10%. This method was simper and rapid. The detection time was only 5 minutes. Compared with ELISA, this method was fast and did not require labeling for sample. Although the same pretreatment was applied for both the UPLC-MS/MS and the biosensor, the biosensor was proved little matrix interference by constructing pure solution and matrix-match calibration curves. Compared with other reports for detection Rac residue using SPR biosensor, SPR-2004 biosensor has minimal matrix interference and cost effective. In conclusion, the proposed biosensor is suitable for use as a practical screening method for the detection of Rac residue in pork and other meat products by food regulatory authorities and food industries.
In conclusion, abilities of resisting matrix effect, the recoveries, precision, and sensitivity of the two residues detection methods ofβ2-agonists in pork in this study were also good. UPLC-MS/MS method was appropriate for confirmatory analysis. SPR biosensor method was perfect for rapid screening for a large number of samples detection in supermarket, food factory and food regulatory authorties.
引文
[1]任廷和,段锡振.β2-受体激动剂的应用[J].亚太传统医药.2009； 5(2)；107-108.
[2]黄茂.支气管哮喘治疗药物临床应用[J].药学与临床研究.2010；18(1)16-19.
[3]戴凌,沙金燕.子宫平滑肌细胞膜的收缩相关蛋白[J].中国妇幼健康研究.2007；18(4)：321-323.
[4]陈建芳.β2-肾上腺素受体激动剂苯乙醇胺类化合物的合成及活性研究[D].沈阳药科大学,005.
[5]李敏艳.猪β2-肾上腺素能受体的克隆,表达及初步分离纯化[D].西北大学,2009.
[6]叶金朝.β2-受体激动剂对某些物质代谢的影响[J].药物流行病学杂志.1994；3(1)：29-30.
[7]蔡秀德.β2-激动剂在治疗哮喘中的不良作用及对策[J].四川医学.1997；18(3)：183-184.
[8]李军,卜秀玲,张鑫.β-受体激动剂治疗哮喘与死亡率增加[J].中国医院药学杂志.1995；15(1).
[9]国家药品不良反应监测中心.药品警戒信息[J].中国药物警戒.2010；7(2)：127-128.
[10]BA C, GD P,阴慧娟.FDA:长效β受体激动剂需限制使用[J].中国处方药.2010；(4)：17.
[11]中岛干夫.β-受体激动剂的正确使用[J].日本医学介绍.1998；19(12)：547-549.
[12]邹如意.东莞市生猪β-肾上腺受体激动剂残留的检测与分析[D].湖南农业大学,2009.
[13]左晓磊,邱伟,韩爱云,邱建琴.菜克多巴胺在畜禽生产中的应用及残留[J].中国畜牧兽医.2007；34(9)：12-14.
[14]Peterla T, Scanes C. Effect of beta-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro[J]. Journal of Animal Science.1990; 68(4):1024.
[15]Haasnoot W, Stouten P, Schilt R, Hooijerink D. A fast immunoassay for the screening of beta-agonists in hair[J]. Analyst.1998; 123(12):2707-2710.
[16]Orcutt AL, Cline TR, Mills SE. Influence of the beta-2-adrenergic agonist clenbuterol on insulin-stimulated lipogenesis in mouse adipocytes [J]. Domestic Animal Endocrinology.1989; 6(1):59-69.
[17]Salem M, Levesque H, Moon TW, Rexroad CE, Yao J. Anabolic effects of feeding [beta] 2-adrenergic agonists on rainbow trout muscle proteases and proteins[J]. Comparative Biochemistry and Physiology-Part A:Molecular & Integrative Physiology.2006; 144(2):145-154.
[18]陶金才.食品安全监督管理体系分析[D].南京农业大学,2006.
[19]毕玉香.瘦肉精的残留危害及监控对策[J].畜牧兽医科技信息.2009；6.
[20]刘军弟.基于产业链视角的猪肉质量安全管理研究[D].南京农业大学,2009.
[21]贾晓飞.超高效液相色谱—串联质谱法测定尿液中9种B2-受体激动剂[D].天津大学,2007.
[22]苗虹.食品及生物材料中B-激动剂和β-阻断剂残留检测技术研究及污染评价[D].中国疾病预防控制中心,2010.
[23]新安传媒网.贪吃蛇吞下“瘦肉精”深圳出现国内首例蛇肉“瘦肉精”致人中毒事[EB/OL]. [cited 2010 09-08]; Available from: http://news.sina.com.cn/c/2010-09-08/084018083369s.shtml.
[24]96/23/EC CD[S].1996.
[25]中华人民共和国农业部第235号公告.动物性食品中兽药最高残留限量[S].2002.
[26]袁雪.保卫公平!保卫荣誉![N].21世纪经济报道.2007-11-28.
[27]袁飚.β2-激动剂构效关系与药理作用[J].西安体育学院学报.2003；20(3)：45-47.
[28]黄佳,赵志刚,奥运违禁药品相关知识介绍[J],临床药物治疗杂志.2008；6(3)：3-8.
[29]埃伦.“兴奋剂”事件席卷美国四大球[N].南方周末,2003-11-20.
[30]美国奥委会爆出兴奋剂丑闻[N].人民日报,2003-04-18.
[31]历届奥运会兴奋剂丑闻之七宗“最”[N].萧山日报,2008-08-13.
[32]阿碧.奥运赛场上的缉毒战[J].科学之友.2008(2)：37-39.
[33]刘婷婷.捍卫神圣的体育净土[J].北京观察.2008(8)：15-17.
[34]DB11/Z 462-2007.奥运会食品安全动物药品使用管理规范[S].
[35]Shao B, Jia X, Zhang J, Meng J, Wu Y, Duan H, et al. Multi-residual analysis of 16 [beta]-agonists in pig liver, kidney and muscle by ultra performance liquid chromatography tandem mass spectrometry[J]. Food Chemistry.2009; 114(3): 1115-1121.
[36]潘云山,聂建荣,朱铭立,周松英,胡翠萍.高效液相色谱串联三重四极杆质谱检测饲料中10种β2-受体激动剂[J].广东农业科学.2010；11.
[37]Shishani E, Chai SC, Jamokha S, Aznar G, Hoffman MK. Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry [J]. Analytica Chimica Acta.2003; 483(1-2):137-145.
[38]孙雷,张骊,朱永林,王树槐,汪霞.超高效液相色谱-串联质谱法检测动物源性食品中残留的9种β-受体激动剂[J].色谱.2008;26(6)：709-713.
[39]卢业玉,尚红霞,余倩,杨迟,方岩雄.饲料中克伦特罗和沙丁胺醇分离方法的研究[J].离子交换与吸附.2003；19(4)：331-336.
[40]Fiori M, Civitareale C, Mirante S, Magaro E, Brambilla G. Evaluation of two different clean-up steps, to minimise ion suppression phenomena in ion trap liquid chromatography-tandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine[J]. Analytica Chimica Acta.2005; 529(1-2): 207-210.
[41]Van Vyncht G, Preece S, Gaspar P, Maghuin-Rogister G, DePauw E. Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of [beta]-agonists in biological matrices[J]. Journal of Chromatography A.1996; 750(1-2):43-49.
[42]Lawrence JF, Menard C. Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection [J]. Journal of Chromatography B:Biomedical Sciences and Applications.1997; 696(2): 291-297.
[43]聂建荣,朱铭立,连槿,潘云山,邓香连,胡翠萍[J].高效液相色谱-串联质谱法检测动物尿液中的15种β-受体激动剂.色谱.2010；8.
[44］王凤美,张鸿伟,庞士平,汤志旭,牛增元,罗忻.超高效液相色谱-串联质谱测定动物源性食品和尿液中4种β-受体激动剂残留[J].分析化学.2008；36(12)：1629-1635.
[45]Williams LD, Churchwell MI, Doerge DR. Multiresidue confirmation of [beta]-agonists in bovine retina and liver using LC-ES/MS/MS[J]. Journal of Chromatography B.2004; 813(1-2):35-45.
[46]刘畅,吴小虎,徐伟东,柴逸峰.LC-MS/MS测定动物源性食品中15种β-受体激动剂残留的研究[J].药物分析杂志.2008；28(12)：2085-2089.
[47]Guy PA, Savoy M-C, Stadler RH. Quantitative analysis of clenbuterol in meat products using liquid chromatography-electrospray ionisation tandem mass spectrometry[J]. Journal of Chromatography B:Biomedical Sciences and Applications.1999; 736(1-2):209-219.
[48]袁倚盛,赵飞浪.免疫法制备色谱样品的研究——血清中沙丁胺醇的分析[J].金陵医院学报.1996；9(4).
[49]王建平,沈建忠.免疫亲和色谱-GC/MS法检测猪尿中的沙丁胺醇与克伦特罗[J].中国兽医杂志.2007；43(4)：61-62.
[50]王建平,史为民,沈建忠.免疫亲和色谱-气相色谱/质谱法(IAC-GC/MS)检测猪肝中的沙丁胺醇与克伦特罗[J].畜牧兽医学报.2007；38(3)：271-275.
[51]Ramos F, Conceicao Castilho M, Irene Noronha da Silveira M, Prates JAM, Dias Correia JHR. Determination of salbutamol in rats at low concentrations using liquid chromatography with electrochemical detection[J]. Analytica Chimica Acta.1993; 275(1-2):279-283.
[52]Machnik M, Geyer H, Horning S, Breidbach A, Delahaut P, Schanzer W. Long-term detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry[J]. Journal of Chromatography B:Biomedical Sciences and Applications.1999; 723(1-2): 147-155.
[53]Shelver WL, Smith DJ. Immunoaffinity column as sample cleanup method for determination of the beta-adrenergic agonist ractopamine and its metabolites[J]. Journal of Aoac International.2002; 85(6):1302-1307.
[54]王培龙,范理,宋荣,高生,苏晓鸥,杨曙明.动物尿液中盐酸克伦特罗的分子印迹固相萃取气相色谱质谱法研究[J].分析化学.2007；7(9).
[55]Van Hoof N, Courtheyn D, Antignac JP, Van de Wiele M, Poelmans S, Noppe H, et al. Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers[J]. Rapid Communications in Mass Spectrometry.2005; 19(19):2801-2808.
[56]Widstrand C, Larsson F, Fiori M, Civitareale C, Mirante S, Brambilla G. Evaluation of MISPE for the multi-residue extraction of [beta]-agonists from calves urine[J]. Journal of Chromatography B.2004; 804(1):85-91.
[57]Kootstra PR, Kuijpers CJPF, Wubs KL, van Doorn D, Sterk SS, van Ginkel LA, et al. The analysis of beta-agonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening[J]. Analytica Chimica Acta.2005; 529(1-2):75-81.
[58]Berggren C, Bayoudh S, Sherrington D, Ensing K. Use of molecularly imprinted solid-phase extraction for the selective clean-up of clenbuterol from calf urine[J]. Journal of Chromatography A.2000; 889(1-2):105-110.
[59]Brambilla G, Fiori M, Rizzo B, Crescenzi V, Masci G. Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices[J]. Journal of Chromatography B:Biomedical Sciences and Applications.2001; 759(1):27-32.
[60]李丽虹,刘岚,罗勇,邓芹英.以分子印迹聚合物为固定相手性拆分1,1’-联-2-萘酚及其衍生物[J].色谱..2006;24(6)：574-577.
[61]王红飞,唐春燕,杨浩,张黎明.分子印迹微凝胶模拟酶的研究[J].高等化学学报.2010；31(12)：2488-2493.
[62]张燕,何佳琪,崔涛,段振娟.禁用兽药氯霉素人工抗体的制备及特异吸附性[J].天津科技大学学报.2007；22(3)：9-11.
[63]王炼,黎源倩,王海波,官艳丽.基质固相分散-超高效液相色谱-串联质谱法同时测定畜禽肉和牛奶中20种兽药残留[J].分析化学.2011；39(2)：203-207.
[64]胡小钟,储晓刚,余建新,李晶,黄鑫,林雁飞,等.基质固相分散和气相色谱-质谱法测定浓缩苹果汁中22种有机氯农药和15种拟除虫菊酯农药的残留量[J].分析测试学报.2004；23(5)：38-42.
[65]洪振涛,张卫锋,何文彪,朱孟丽,聂建荣.快速测定动物组织中B-兴奋剂的新方法[J].广东农业科学.2005；(4)：84-86.
[66]Crescenzi C, Bayoudh S, Cormack PAG, Klein T, Ensing K. Determination of clenbuterol in bovine liver by combining matrix solid-phase dispersion and molecularly imprinted solid-phase extraction followed by liquid chromatography/electrospray ion trap multiple-stage mass spectrometry[J]. Analytical Chemistry.2001; 73(10):2171-2177.
[67]O'Keeffe MJ, O'Keeffe M, Glennon JD. Supercritical fluid extraction (SFE) as a multi-residue extraction procedure for beta-agonists in bovine liver tissue[J]. Analyst.1999; 124(9):1355-1360.
[68]Montrade MP,Le Bizec B, Monteau F, Siliart B, Andre F. Multi-residue analysis for [beta]-agonistic drugs in urine of meat-producing animals by gas chromatography--mass spectrometry[J]. Analytica Chimica Acta.1993; 275(1-2):253-268.
[69]Batjoens P, Courtheyn D, De Brabander HF, Vercammen J, De Wasch K, Logghe M. Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces[J]. Journal of Chromatography A.1996; 750(1-2): 133-139.
[70]Liu W, Zhang L, Wei Z, Chen S, Chen G. Analysis of [beta]-agonists and [beta]-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry[J]. Journal of Chromatography A.2009; 1216(28):5340-5346.
[71]朱馨乐,刘琪,孙雷,王树槐,汪霞.气相色谱-质谱法测定猪尿中莱克多巴胺的研究[J].中国兽药杂志.2008；42(6)：36-39.
[72]朱坚,杨景贤,李波,方晓明.气相色谱-质谱法测定肝,肾和肉中β2-受体激动剂残留量[J].分析测试学报.2004；1.
[73]朱坚,李波,方晓明,陈家华,杨景贤.气相色谱-质谱法测定肝,肾和肉中11种B2-受体激动剂残留量[J].质谱学报.2005；26(3)：129-137.
[74]刘勇军,吴银良,刘素英,单吉浩,尤华.固相萃取气相色谱-质谱联用法测定动物组织中的莱克多巴胺[J].食品科学.2006；27(8)：231-234.
[75]NY/QY421-2003动物尿液中盐酸克伦特罗(瘦肉精)残留的检测-气相色谱/质谱(GC/MS)方法[S].
[76]农业部1031号公告3-2008.猪肝和猪尿中β-受体激动剂残留检测气相色谱-质谱法[S].
[77]农业部1065号公告7-2008.饲料中8种β-受体激动剂检测气相色谱-质谱法[S].
[78]农业部1025号公告16-2008.动物尿液中盐酸克伦特罗残留检测气相色谱-质谱法[S].
[79]NY/T468-2006.动物组织中盐酸克伦特罗的检测-气相色谱/质谱法[S].
[80]DB33/T624-2006.动物组织中特布他林、克伦特罗、沙丁胺醇和莱克多巴胺残留量的测定气相色谱-质谱法[S].
[81]DB33/T623-2006.动物尿液中特布他林、克伦特罗、沙丁胺醇和莱克多巴胺残留量的测定气相色谱-质谱法[S].
[82]Reig M, Batlle N, Navarro JL, Toldra F. Stability of [beta]-agonist methyl boronic derivatives before gas chromatography-mass spectrometry analysis [J]. Analytica Chimica Act.2005; 529(1-2):293-297.
[83]Ramos F, Santos C, Silva A, Noronha da Silveira MI. [beta]2-Adrenergic agonist residues:simultaneous methyl-and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry[J]. Journal of Chromatography B:Biomedical Sciences and Applications.1998; 716(1-2): 366-370.
[84]Damasceno L, Ventura R, Cardoso J, Segura J. Diagnostic evidence for the presence of [beta]-agonists using two consecutive derivatization procedures and gas chromatography-mass spectrometric analysis [J]. Journal of Chromatography B.2002; 780(1):61-71.
[85]Henze MK, Opfermann G, Spahn-Langguth H, Schanzer W. Screening of [beta]-2 agonists and confirmation of fenoterol, orcipr en aline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives [J]. Journal of Chromatography B:Biomedical Sciences and Applications.2001; 751(1):93-105.
[86]Van Rhijn JA, Heskamp HH, Essers ML, van de Wetering HJ, Kleijnen HCH, Roos AH. Possibilities for confirmatory analysis of some [beta]-agonists using two different derivatives simultaneously [J]. Journal of Chromatography B: Biomedical Sciences and Applications.1995; 665(2):395-398.
[87]Rashid BA, Kwasowski P, Stevenson D. Solid phase extraction of clenbuterol from plasma using immunoaffinity followed by HPLC[J]. Journal of Pharmaceutical and Biomedical Analysis.1999; 21(3):635-63.
[88]Liu B, Yan H, Qiao F, Geng Y. Determination of clenbuterol in porcine tissues using solid-phase extraction combined with ultrasound-assisted dispersive liquid-liquid microextraction and HPLC-UV detection[J]. Journal of Chromatography B.2011; 879(1):90-94.
[89]Blomgren A, Berggren C, Holmberg A, Larsson F, Sellergren B, Ensing K. Extraction of clenbuterol from calf urine using a molecularly imprinted polymer followed by quantitation by high-performance liquid chromatography with UV detection[J]. Journal of Chromatography A.2002; 975(1):157-164.
[90]Gigosos PG1, Ferna'ndez TFn, Mari'z OCa, Fente Sampayo CA, Abui'n CF, Sa'ez AC. Rapid and simple determination of clenbuterol residues in retina by high-performance liquid chromatography with ultraviolet detection [J]. Journal of Chromatography B:Biomedical Sciences and Applications.1996; 677(1): 167-171.
[91]Ouyang J, Duan JL, Baeyens WRG, Delanghe JR. A simple method for the study of salbutamol pharmacokinetics by ion chromatography with direct conductivity detection[J]. Talanta.2005; 65(1):1-6.
[92]Mazhar SHRA, Chrystyn H. New HPLC assay for urinary salbutamol concentrations in samples collected post-inhalation[J]. Journal of Pharmaceutical and Biomedical Analysis.2009; 50(2):175-182.
[93]Shelver WL, Smith DJ. Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:Comparative study with HPLC and ELISA[J]. Journal of Agricultural and Food Chemistry.2003; 51(13): 3715-3721.
[94]Hu YL, Liu RJ, Li YW, Li GK. Investigation of ractopamine-imprinted polymer for dispersive solid-phase extraction of trace beta-agonists in pig tissues[J]. Journal of Separation Science.2010; 33(13):2017-2025.
[95]张雪竹.液相色谱—电化学检测器测定生物样品中的痕量β2-受体激动剂[D].天津大学,2003.
[96]邓立刚.阿维菌素(abamectin)在甘蓝和土壤中的残留检测技术及降解规律研究[D].山东师范大学,2009.
[97]苗虹,邹建宏,范赛,甘乐文,赵云峰,吴永宁.高效液相色谱-离子阱质谱法测定尿液中β-受体激动剂及β-受体阻断剂[J].色谱.2010；28(6)：572-578.
[98]吴永宁,苗虹,范赛,赵云峰.高效液相色谱-线性离子阱质谱法测定畜禽肌肉中B-受体激动剂及B-阻断剂类药物残留[J].中国科学：B辑.2009：774-784.
[99]Blanca J, Munoz P, Morgado M,, Mendez N, Aranda A, Reuvers T, et al. Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry[J]. Analytica Chimica Acta.2005; 529(1-2):199-205.
[100]Nielen MWF, Elliott CT, Boyd SA, Courtheyn D, Essers ML, Hooijerink HH, et al. Identification of an unknown beta-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement[J]. Rapid Communications in Mass Spectrometry.2003; 17(14):1633-1641.
[101]Ventura R, Ramirez R, Monfort N, Segura J. Ultraperformance liquid chromatography tandem mass spectrometric method for direct quantification of salbutamol in urine samples in doping control[J]. Journal of Pharmaceutical and Biomedical Analysis.2009; 50(5):886-890.
[102]农业部958号公告-3-2007.动物源性食品中莱克多巴胺残留量的测定高效液相色谱法-质谱法B受体激动剂残留检测液相色谱-串联质谱法[S].
[103]农业部1063号公告-3-2008.动物尿液中11种B-受体激动剂的检测液相色谱-串联质谱法[S].
[104]农业部1063号公告-6-2008.饲料中13中B-受体激动剂的检测液相色谱-串联质谱法[S].
[105]农业部1025号公告-11-2008.猪尿中B-受体激动剂多残留检测液相色谱-串联质谱法[S].
[106]GB/T21313-2007..动物源性食品中B-受体激动剂残留检测方法液相色谱／质谱／质谱法[S].
[107]GB/T22286-2008.动物源性食品中多种β-受体激动剂残留量的测定液相色谱串联质谱法[S].
[108]SN/T1924-2007.进出口动物源食品中克伦特罗,莱克多巴胺,沙丁胺醇,特布塔林残留量的检测方法液相色谱/质谱/质谱法[S].
[109]陈颖.某些β2-受体激动剂及中药有效成分的毛细管电泳及电化学分析研究[D].福州大学,2004.
[110]Chu QC, Geng CH, Zhou H, Ye JN. Fast determination of clenbuterol and salbutamol in feed and meat products based on miniaturized capillary electrophoresis with amperometric detection [J]. Chinese Journal of Chemistry.2007; 25(12):1832-1835.
[111]Wang W, Zhang Y, Wang J, Shi X, Ye J, Determination of [beta]-agonists in pig feed, pig urine and pig liver using capillary electrophoresis with electrochemical detection. Meat Science[J].2010; 85(2):302-305.
[112]Zhou SS, Wang YQ, De Beer T, Baeyens WRG, Fei GT, Dilinuer M, et al. Simultaneous separation of eight beta-adrenergic drugs using titanium dioxide nanoparticles as additive in capillary electrophoresis[J]. Electrophoresis.2008; 29(11):2321-2329.
[113]Anurukvorakun O, Suntornsuk W, Suntornsuk L. Factorial design applied to a non-aqueous capillary electrophoresis method for the separation of [beta]-agonists[J]. Journal of Chromatography A.2006; 1134(1-2):326-332.
[114]Felix FS, Quintino MSM, Carvalho AZ, Coelho LHG, do Lago CL, Angnes L. Determination of salbutamol in syrups by capillary electrophoresis with contactless conductivity detection (CE-C4D)[J]. Journal of Pharmaceutical and Biomedical Analysis.2006; 40(5):1288-1292.
[115]陈丽.胶体金免疫层析法在早孕检测中的应用[J].医学动物防制.2006；22(12)：928-929.
[116]乔伟振,谢平,李琴.应用胶体金免疫层析法检测人群甲型流感病毒抗原阳性率[J].中华医院感染学杂志.2010；(24)：4044-4046.
[117]张正雷,张云涛.甲胎蛋白胶体金检测试剂的研制[J].微生物学免疫学进展.2002；30(3)：46-48.
[118]高以明,吴艳涛,王寿利,张小荣,薛峰,陆承平.检测莱克多巴胺胶体金免疫层析试验的建立[J].中国动物检疫.2009；26：12.
[119］苏存举,陈福生,高志贤,苗颂,刘楠,冯屹.胶体金法与酶联免疫法相结合快速检测克伦特罗[J].解放军预防医学杂志.2008；26(3)：176-179.
[120]Jin J, Lai W, Xiong Y, Chen Y, Liu W. Colloidal gold-based immunochromatographic assay for detection of ractopamine in swine urine samples. Journal of Biotechnology [J].2008; 136:S754.
[121]Zhang GP, Wang XN, Yang JF, Yang YY, Xing GX, Li QM, et al. Development of an immunochromatographic lateral flow test strip for detection of [beta]-adrenergic agonist Clenbuterol residues[J]. Journal of Immunological Methods.2006; 312(1-2):27-33.
[122]张亚峰,张会彩,刘聚祥,李楠,王建平.沙丁胺醇胶体金免疫试纸条的研制[J].中国兽医杂志.2009(9)：59-60.
[123]陈耀强.盐酸克伦特罗、莱克多巴胺兽药残留免疫层析检测方法的研究[D].暨南大学,2010.
[124]DB34/T818-2008.饲料中盐酸克罗的测定-胶体金免疫层析法[S].
[125]DB34/T824-2008.动物组织中盐酸克伦特罗的残留测定-胶体金免疫层析法[S].
[126]申宏丹,窦红,高春平,许丽丽.直接竞争酶联免疫法检测菜克多巴胺研究[J].动物医学进展.2009；30(8)：58-63.
[127]王建平,沈建忠.猪肝和猪尿中沙丁胺醇和克伦特罗残留的酶联免疫吸附检测法研究[J].畜牧兽医学报.2005；36(4)：397-401.
[128]Shelver WL, Smith DJ. Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals[J]. Journal of Agricultural and Food Chemistry.2002; 50(10):2742-2747.
[129]Sawaya WN, Lone K, Husain A, Dashti B, Saeed T. Screening for [beta]-agonists in sheep urine and eyes by an enzyme-linked immunosorbent assay in the state of Kuwait[J]. Food Control.2000; 11(1):1-5.
[130]Sheu SY, Lei YC, Tai YT, Chang TH, Kuo TF. Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan[J]. Analytica Chimica Acta.2009; 654(2):148-153.
[131]Posyniak A, Zmudzki J, Niedzielska J. Screening procedures for clenbuterol residue determination in bovine urine and liver matrices using enzyme-linked immunosorbent assay and liquid chromatography[J]. Analytica Chimica Acta.2003; 483(1-2):61-67.
[132]Shelver WL, Smith DJ, Berry ES. Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine[J]. Journal of Agricultural and Food Chemistry.2000; 48(9): 4020-4026.
[133]孙海新,凌红丽,王雷,王宏华,赵玉惠,董瑞娥.沙丁胺醇残留的酶联免疫检测方法的建立[J].中国动物检疫.2009；26：12.
[134]李艳,张明洲,陈宗伦,刘军,胡华军.猪尿中克伦特罗残留的ELISA检测研究[J].浙江农业学报.2006；18(005)：328-332.
[135]DB34/T823-2008.动物组织中盐酸克伦特罗的残留测定-酶联免疫吸附法[S].
[136]DB34/T827-2008.尿液中莱克多巴胺的残留测定-酶联免疫吸附法[S].
[137]DB34/T825-2008.动物组织中莱克多巴胺的残留测定-酶联免疫吸附法[S].
[138]DB34/T819-2008.饲料中莱克多巴胺的残留测定-酶联免疫吸附法[S].
[139]农业部1025号公告16-2008.动物性食品中莱克多巴胺残留测定酶联免疫吸附法[S].
[140]Silvan G, Martinez-Mateos MM, Blass A, Camacho L, Gonzalez-Gil A, Garcia-Partida P, et al. The effect of long-term exposure to combinations of growth promoters in long evans rats:Part 1:Endocrine adrenal function[J]. Analytica Chimica Acta.2007; 586(1-2):246-251.
[141]Smith D. Total radioactive residues and clenbuterol residues in swine after dietary administration of [14C] clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or seven days[J]. Journal of Animal Science. 2000; 78(11):2903.
[142]Collins S, Okeeffe M, Smyth MR. Multi-residue analysis for beta-agonists in urine and liver samples using mixed-phase columns with determinists with determination by radioiminationassay[J]. Analyst.1994; 119(12):2671-2674.
[143]李旭甡,韩松涛,杨春玫.化学发光酶免疫分析法测定血清前列腺特异性抗原的研究[J].北华大学学报.2008；9(2)：125-127.
[144]李旭甡,张桂杰.化学发光酶免疫分析法测定血清三碘甲状腺原氨酸的建立与研究[J].中国地方病防治杂.2008；23(3)：179-181.
[145]王刘花.建立化学发光酶免疫法检测猪肉中磺胺嘧啶的研究[D].南昌大学；2010.
[146]王照鹏.玉米赤霉烯酮单克隆抗体的制备及ELISA检测方法的建立[D].吉林大学,2009.
[147]胥传来,彭池方,郝凯,金征宇,王武康.化学发光酶免疫方法检测克伦特罗残留[J].分析化学.2005；33(5)：699-702.
[148]徐晓婴,卢庆鋆,徐艳力,卢永红,陈峰.化学发光酶免疫法检测盐酸克伦特罗残留[J].上海交通大学学报.2009；(2)：125-128.
[149]王硕,李细芬,生威,张燕,杨泽琳,方国臻.增强化学发光酶免疫法对猪肉中盐酸克伦特罗的检测[J].分析测试学报.2010；29(3)：215-219.
[150]Roda A, Manetta AC, Piazza F, Simoni P, Lelli R. A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol[J]. Talanta.2000; 52(2):311-318.
[151]徐晓婴.化学发光酶免疫法检测盐酸克伦特罗残留[D].上海交通大学,2009.
[152]Bookout JT, Joaquim TR, Magin KM, Rogan GJ, Lirette RP. Development of a dual-label time-resolved fluorometric immunoassay for the simultaneous detection of two recombinant proteins in potato[J]. Journal of Agricultural and Food Chemistry.2000; 48(12):5868-5873.
[153]胡志刚,刘洁,陈国千,谢国强,黄飚,徐根宝等.时间分辨免疫荧光定量检测乙型肝炎病毒前S1抗原的研究[J].中华检验医学杂志.2006；29(5)：407-410.
[154]谭玉华,谭子军,霍道贵,廖思红.时间分辨荧光免疫法定量测定孕妇血清中甲胎蛋白含量[J].中华临床医师杂志,2010；4(10).
[155]秦卫仕,田蓉.时间分辨荧光免疫分析方法学评价[J].免疫学杂志.2002;18(5)：397-399.
[156]Zhang J, Gao L, Zhou B, Zhu L, Zhang Y, Huanga B. Simultaneous detection of deoxynivalenol and zearalenone by dual-label time-resolved fluorescence immunoassay[J]. Journal of the Science of Food and Agriculture.2011; 91(2): 193-197.
[157]马智鸿,黄飚,屠蔷,高蕾,张珏,王柯.应用生物素-链霉亲和素的时间分辨荧光免疫分析检测玉米赤霉烯酮[J].江苏农业学报.2009；25(4)：905-909.
[158]张艺,黄飚,王柯,张珏,朱岚,马智鸿,等.氯霉素的直接竞争时间分辨荧光免疫检测法[J].安徽农业科学.2009；(27)：12912-12914.
[159]雷红涛,高秀洁,潘科,黄丽,吴英松,孙远明.盐酸克伦特罗时间分辨荧光免疫检测方法的建立[J].中国食品学报.2006；6(1)：6-10.
[160]何明祥.沙丁胺醇时间分辨荧光免疫分析检测技术的研究[J].长江大学学报：农学卷.2009；6(1)：33-37.
[161]樊晓博,杨金易,高秀杰,吴英松,孙远明,雷红涛.Eu-(3+)标抗原盐酸克伦特罗的时间分辨荧光免疫检测[J].食品科学.2010；31(21)：307-310.
[162]缪璐,刘仲明,张水华.SPR生物传感器在临床医学检验中的应用[J].医疗卫生装备.2006；27(8)：36-37.
[163]徐霞,叶尊忠,吴坚,应义斌.表面等离子体共振免疫传感器在蛋白质检测中的应用及其研究进展[J].分析化学.2010；38(007)：1052-1059.
[164]李莹,钟金钢,张永林.基于表面等离子体共振成像的指纹采集[J].中国激光.2006；33(8)：1143-1147.
[165]李莹,钟金钢,张永林,唐勇.基于表面等离子体共振的土霉素残留检测[J].应用激光.2008；28(5)：390-394.
[166]李岩,李宏基,杨国宇,崔莹莹.SPR生物传感器在乳品工业中的应用[J].中国乳品工业.2007；35(7)：37-39.
[167]Caldow M, Stead SL, Day J, Sharman M, Situ C, Elliott C. Development and validation of an optical SPR biosensor assay for tylosin residues in honey[J]. Journal of Agriculture and Food Chemistry.2005; 53(19): 7367-7370.
[168]Cao L, Lin H, Mirsky VM. Surface plasmon resonance biosensor for enrofloxacin based on deoxyribonucleic acid[J]. Analytica Chimica Acta.2007; 589(1):1-5.
[169]李辉,蔡浩原,陈兴,孙建海,张璐璐,崔大付.表面等离子体共振生物传感器连续检测莱克多巴胺[J].分析化学.2010；38(3)：381-384.
[170]曲丽洁.用于动物性食品中莱克多巴胺快速检测的SPR技术研究[D].华中农业大学,2010.
[171]Johansson MA, Hellen s K. Immunobiosensor analysis-of clenbuterol in bovine hair[J]. Food and Agricultural Immunology.2003; 15(3):197-205.
[172]Thompson CS, Haughey SA, TraynorIM, Fodey TL, Elliott CT, Antignac JP, et al. Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry[J]. Analytica Chimica Acta.2008; 608(2):217-225.
[173]Lee H-B, Sarafin K, Peart TE. Determination of [beta]-blockers and [beta] 2-agonists in sewage by solid-phase extraction and liquid chromatography-tandem mass spectrometry[J]. Journal of Chromatography A.2007; 1148(2):158-167.
[174]Antignac J-P, Marchand P, Le Bizec B, Andre F. Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid chromatography-positive electrospray tandem mass spectrometry[J]. Journal of Chromatography B:Analytical Technologies in the Biomedical and Life Sciences.2002; 774(1):59-66.
[175]蔡欣欣,张秀尧.超高效液相色谱三重四极杆质谱法同时快速测定动物源食品中6种B-受体兴奋剂残留[J].中国卫生检验杂志.2008；18(7)：1252-1255.
[176]尹伟伟.莱克多巴胺,氯霉素,三聚氰胺药物残留的酶联免疫检测法的建立[D].山东大学,2007.
