摘要
常山酮(Halofuginone,Hal)是一种应用广泛的抗球虫药，以3mg／kg的浓度添加到饲料中用于预防和治疗家禽的球虫病。本研究合成了常山酮免疫原，制备出针对常山酮的特异性多克隆抗体，建立了鸡组织中常山酮残留检测的ELISA方法和HPLC方法，并研究了鸡组织中常山酮的残留消除。
本研究建立了鸡肝脏和肌肉组织中常山酮残留检测的ELISA方法。用三甲基硅烷咪唑(TMSI)将常山酮结构中的羟基选择性保护，在温和的条件下使常山酮结构中的氨基琥珀酰化。最后在酸性条件下水解保护基团，得到半抗原常山酮琥珀酸衍生物。以NHS活性酯法制备了常山酮免疫原和包被原，偶联比率分别为9∶1和5∶1。ELISA方法的线性检测范围为0.1ng／ml—100ng／ml，以50ng／g、100ng／g和500ng／g的浓度水平添加时，鸡肝脏组织的平均添加回收率范围为74.2％—96.8％，日内变异系数范围为3.8％—12.8％，日间变异系数范围为8.8％—15.9％：鸡肌肉组织的平均添加回收率范围为74.3％-90.0％，日内变异系数为2.6％-16.6％，日间变异系数在5.4％—6.8％。本方法在鸡肝组织和肌肉组织中的检测限分别为23ng／g和15ng／g。
本研究建立了鸡肝脏和肌肉组织中常山酮残留检测的HPLC方法。用胰蛋白酶水解组织样品，乙酸乙酯提取，0.125mol／L乙酸铵缓冲液反提，Oasis~(?) HLB柱净化，HPLC测定。以50ng／g、100 ng／g和500 ng／g浓度水平添加时，鸡肝脏组织的平均添加回收率范围在81.3％-93.3％之间，日内变异系数范围在1.5％-10.9％之间，日间变异系数范围在2.7％-6.1％之间：鸡肌肉组织的平均添加回收率范围在88.4％-103.4％之间，日内变异系数范围在1.4％-11.6％之间，日问变异系数范围在4.6％-12.6％之间。本方法在鸡肝脏和肌肉组织中的检测限分别为19ng／g和23ng／g。
本研究对ELISA方法和HPLC方法进行了比较。ELISA方法和HPLC方法测定鸡肝脏和肌肉组织中常山酮残留有良好的相关性。在添加回收试验中，两种方法在鸡肝脏和肌肉组织中测得常山酮的实测值的相关系数分别为0.9760和0.9344。以3mg／kg剂量的氢溴酸常山酮混饲给予AA肉鸡10天，停药第0天时。ELISA方法测得鸡肝脏组织中常山酮的残留量为105.2ng／g，HPLC方法测得鸡肝脏组织中常山酮的残留量为84.6ng／g；停药第3天后，常山酮的残留量均低于两种方法的检测限。在停药第0天时，鸡肌肉组织中常山酮的残留量就低于检测限。用ELISA方法和HPLC方法检测鸡肝脏和肌肉组织中常山酮残留的结果基本相符。
Halofuginone (Hal) is a widely used anti-coccidiosis agent, which is usually used as a feed additive at the medicated concentration of 3mg/kg for the prevention and treatment of the coccidiosis in poultry. In this study, halofuginone was modified to be linked with carrier protein to obtain a multiclonal antibodies. An ELISA method and a HPLC method for the determination of halofuginone in chicken tissues were developed, and the depletion of halofuginone residue from chicken tissue was investigated.
An ELISA method for determination of halofuginone residue in chicken liver and muscle tissues was developed. The hydroxyl group on halofuginone was protected by N-trimethysilylimidazole, and the amino moiety readily reacted with succinic anhydride to produce the monosubstituted acid succinamide. Finally the trimethyisis moiety was removed by hydrolysis in acid condition to give a succinic acid derivative of halofuginone. This compound was coupled to BSA and OVA by NHS ester procedure,and the coupling ratio were 9.5:1 and 5:1, respectively.The linear detection of the ELISA method ranged from 0.1 ng/ml to 100 ng/ml. In the chicken liver tissue fortified at 50ng/g, 100ng/g, 500ng/g , the average recoveries ranged from 74.2% to 96.8%, with the coefficients of variation within-day were in the range of 3.8%-12.8%, and the coefficients of variation between-days were in the range of 8.8%-15.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 74.3% to 90.0%, with
the coefficients of variation within-day were in the range of 2.6%-16.6%, and the coefficients of variation between-days were in the range of 5.4%-6.8%.The detection limits in chicken liver tissue and in chicken muscle tissue were 23ng/g and I5ng/g, respectively.
A HPLC method for determination of halofuginone residue in chicken liver and muscle tissue was developed. The tissue samples were hydrolyzed with trypsin, extracted with ethyl acetate, and re-extracted with 0.125mol/Lacetamide buffer. The extracts were then subjected to Oasis HLB cartridge for purification, and the elutes were determined by HPLC. In the chicken liver tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 81.3% to 93.3%, with the coefficients of variation within-day were in the range of 1.5%-10.9%, and the coefficients of variation in between-days were in the range of 2.7%-6.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 88.4% to 103.4%, with the coefficients of variation within-day were in the range of 1.4%-11.6%, and the coefficients of variation between-days were in the range of 9.0%-12.6%. The method detection limits in chicken liver tissue and in chicken muscle tissue were 19ng/g and 23ng/g, respectively.
In this study, the ELISA method vs HPLC method for the determination of the halofuginone in chicken liver and muscle were compared. The coefficients of correlation for the detection of halofuginone in chicken liver and muscle by two methods were 0.9760 and 0.9344, respectively. Broiler chickens (AA) were fed at a medicated concentration of 3mg/kg of halofuginone-HBr for 10 days. At
withdrawal time of 0 day, the residue levels of halofuginone in broiler chicken liver tissues detected by ELISA method and HPLC method were 105.2ng/g and 84.6ng/g, respectively. At and after 3 day, the residues were not detected by ELISA method or HPLC method. In the broiler chicken muscles, the residues were not detected by two methods at withdrawal time of 0 day. The ELISA method and the HPLC method for the determination of the halofuginone i n chicken liver and in muscle correlated well.
引文
1 Openshaw H.T. The quinazoline alkaloids, In The Alkaloids, Manske,R.H.E,Holmes,H.L.,Eds;Academic Press;New York, 1953, 3:101-118.
2 Cben C.C. Structural Similarity between febrifugine and chloroquine, J.Theor.Biol, 1976, 59:497-501.
3 Shepard M.L. Stenoroi,in Handbook of Approved New Animal Drug Applications in the United States,R 12-1-1991,Shotwell and Carr Inc., Dalias,TX,USA, 1991,p.3.9.
4 Mcdougald L.R. Control ofcoccidiosis Chemotherapy.In Coccidiosis of Man and Domestic Animals:Long, P.L.,Ed.;CRC Press,Boca Raton,FL, 1992,pp 247-258.
5 朱模忠，《兽药手册》，化学工业出版社，2002．
6 化学工业出版社组织编写，《农用化学品—农药，化肥，农膜，饲料添加剂》，化学工业出版社，1999
7 Anon. Animals: chemical inputs for nutrition and health-overview, in Chemical Economics handbook (Wenzel, A.M.,), SRI International,Menlo Park,CA,USA, 1995,p.201.8002 S
8 Anon. Estimate of U.S. market share for coccidiostats used in 1995-96,A lesding mangment consulting firm in the animal health maret, 1997a
9 Pines M., Vlodavsky I., Nagler, A. Halofiginone:From veterinary Use to Human Therapy, Drug Development Research, 2000, 50:371-378
10 Jang C.S., Fu E.Y., Huang, K.C., et al. Pharmacology of Chang Shan(Dictoa febrifuga),a Chinese antimalarial herb, Nature, 1948, 161:400-401
11 Kuehl EA., Spencer C.E, Foikers,K. Alkaloids of Dictoa febrifuga, Lour, J Am Chem Soc, 1948,70:2091-2093
12 Jang C.S., Fu F.Y., Huang K.C., et al. Ch'ang Shan,a Chinese antimalarial herb, Science, 1946, 103:59.
13 Koepeli J.B., Mead J.E, Brockman J.A. An alkaloid with high antimalarial activity from Dictoa febrifuga, J Am Chem Soc, 1947, 70:1837
14 Baker B.R.,Schaub R.E., McEvoy F.J., et al. An antimalarial alkaloid from Hydrangea, J Org Chem,1952, 17:133-137
15 Uesato H., Kuroda Y., Kato M.,et al.. Confirmation Analysis of Febrifugines and Haiofuginones in Organic Solvents, Chem. Pharm. Bull, 1998, 46:1-5
16 Kantor S.E., Waltezky R.L.,Kennett J.R. The anticoccidial activity of 7-bromo-6-clorofibri-Fugine hydrobromide in chickens. Page 24 in Amer. Soc. Parasitol. University of Arizona, Tusson,Abstr, 1967
17 Ryley J.E, Wilson R.G. Laboratory studies with some recent anticoccidials, Parasitology,1975, 70:203-222
18 Edgar S.A., Flanagan C. Efficacy of_Stenorol (Halofuginone) I.Against Recent Field Isolates of
Six Species of Chicken Coccidia, Poultry Science,1979, 58:1469-1475
19 Edgar S.A., Flanagan C. Efficacy of Stenorol(Haiofuginone) Ⅱ.Plus Roxarsone or Bacitracin MD against Selected Strains of Chicken Eimeria, Poultry Science,1979, 58:1476-1482
20 Dash P.K. Anticoccidial efficacy of monensin and halofuginone in experimental Eimeda tenella infection in broiler chickens.India, Journal of Animal Sciences,1993, 63:918-923
21 Grant R.J. The effect of Stenorol (Halfuginone hydrobromide)against fled isolates of Eimeria species after three years of use in the United States.Coccidia and Intestinal coccidiomorphs Proceedings of the 5th International Coccidiosis conference,Tours(France) 17-20.October 1989,313-316
22 Edgar S.A., Flanagan C.Efficacy of Stenorol (Halofuginone) Ⅲ.For the Control of Coccidiosis in Turkeys, Pouity Science, 1979, 58:1483-1489
23 McDougald L.R.. Halfuginone: Laboratory Titration of Anticoccidial Activity in Young Turkeys,Poulty Science, 1986, 65:280-283
24 McDougald L.R. Anticoccidial Efficacy of Halofuginone in Turkeys Reared to Market Weight,Poulty Science, 1986, 65: 1664-1670
25 Schein E., Voigt W.P. Chemotherapy of bovine theileriosis with Halofuginone, Acta Tropica,1979,36:391-394
26 Morgan D.W T., McHardy N. Comparison of the antitheilerial effect of Weilcome 993C and halfuginone, Research in Veterinary Science,1982, 32:84-88
27 Dolan T.T. Chemotherapy of East Coast fever:Treatment of infections induced by isolates Theileria Parva with halofuginone, Acta Tropica,1986, 43:165-173
28 Njau B.C., Mkonyi P.A., Mleche W.C.H.,et al. Treatment of field cases of East Coast fever with halofuginone.Tropical Animal Health and Production,1985, 17:193-200
29 Kiltz H.H., Hamke R. Bovine theilieriosis in Burundi: chemotherapy with halofuginone lactate.Tropical Animal Health and Production, 1986, 18:139-145
30 Mbwambo H.A., Mkonyl P.A., Chua R.D. Field evaluation of parvaquone against naturally occuring East Coast fever, Veterinary Parasitology, 1987, 23:161-168
31 Chema S., Chumio R.S., Dolan T.T., et al. Clinical trials of halofuginone lactate for the treatment of East Coast fever in Kenya, Veterinary Record,1987, 120:575-577
32 DeVos A.L, Roos J.A. Chemotherapy of Theileria Parva LawTencei infection in cattle with haiofuginone, Onderstepoort Jounnal of Veterinary Research, 1983, 50:33-35
33 Humke R. Halofuginone: toxicology, kenetics, efficacy.in:Irvin,A.D.(ed).Immunization Against Theileriosis in Africa, ILRAD,Nairobi. 1985, 89.
34 Mkonyi P.A., Njau B.C. Proceedings of the first Tanzania Veterinary Association Scientific Conference.,Morogoro. 12th-14th, 1983
35 Kinabo L.D.B. Pharmacology of the New Antitheilerial Drugs:Parvaquone,Buparvaquone and Halofuginone, Veterinary Bulletin,1989, 59(12): 965-968
36 Mehlhorn H., Moltman U., Schein E. Electron microscopic study on the effect ofhalofuginone on
Theileria parva. Tropenmedicin and Parasitologie, 1981, 32:231-233
37 Halofuginone lactate in the treatment of Cryptoridiosia in ruminants. Coccidia and intestinal coccidiomorphs. Proceedings of the V th International coccidiosis conference. Tours(France), 1989,17-20
38 Viilacorta I., Peeters J.E., Vanopdenbosch E.,et al. Efficacy of Halofuginone Lactate against Cryptosporidium parvum in Calves, Antimicrobial Agents and Chemotherapy1991, 283-287.
39 Naciri M., Mancassola R., Yvore P., et al. The effect of halofuginone lactate on experimental Crytospofidium parvum infections in calves, Veterinary Parasitology, 1993, 45:3-4
40 Granot I., Bartov I., Plavnik I., et al. Increased skin tearing in broilers and reduced collagen synthesis in skin in vivo and in vitro in response to the coccidiostat halofuginone, Poulty Science,1991b, 70: 1559-1563.
41 Granot I., Halevy O., Hurwitz S.,et al. Halofuginone: An inhibitor of collagen type I synthesis.Biochim Biophys Acta, 1993, 1156:107-112
42 Levi-schaffer F., Nagler A., Slavin S., et al. Inhibition of collagen sythesis and changes in skin morphology in murine grate-versus-host disease and tight skin mice:effect of halofuginone.J InvestDermatol, 1996, 106(1): 84-88
43 Nagler A., Firman N., Feferman R., et al. Reduction in pulmonary fibrosis in vivo by halofuginone,Am J Resp Criti Care Med, 1996, 154:1082-1086
44 Pines M., Knopov V., Genina O.,et al. Halofuginone, a specific inhibitor of collagen type I sythesis, prevents dimethylnitrosamine-induced liver cirrhosis, J Hepatol, 1997, 26:391-398
45 Nyska M., Nyska A., Rivlin E.,et al.Topically appilied halofuginone,an inhibitor of collagen type I transcription, reduced peritendinous fibrous adhesions following surgery, Connct Tissue Res,1996, 34:97-103
46 Nagler A., Rivkind A.I., Raphael J., et al. Halfuginone-an inhibitor of collagen type I sythesis-prevents postoperative formation of abdominal adhesions, Ann Surg, 1998, 227:575-582
47 Nagler A., Genina O., Lavelin I., et al. Haiofuginone, an inhibitor of collagen type I sythesis-prevents postoperative formation in the rat uterine horn model, Am J Obstet Oynecoi, 1999,180:558-563
48 Nagler A., Gofrit O., Ohana M., et al. The effect of halofuginone, an inhibitor of collagen type I sythesis,on urethral stricture formation:in vivo and in vitro study in a rat model, Urol, 2000, 164:1776-1780
49 Halevy O., Nagler A., Levi-Schaffer F.,et al. Inhibition of collagen type I by skin fibroblasts of graft yersus host disease and scleroderma patients:effect of halofuginone, Biochem Pharmacol, 1996,52:1057-1063
50 Elkin M., Reich R., Nagler A.,et al. Inhibitor of bladder carcioma angiogenesis, stomal support and tumor growth by halofuginone, Cancer Res, 1999, 59:4111-4118
51 Aramovitch R., Dafni H., Neeman M.,et al. Inhibitor ofnevascularization,tumor growth and facilitation of wound repair by halofuginone,n inhibitor of collagen type I sythesis, Neoplasia,
1999,1:321-329
52 Eikin M., Miao H.Q., Nagler A.,et al. Halfuginone: a potent inhibitor of critical steps in angiogenesis progression, FASEB J, 2000, 14:2477-2485
53 Gavish Z., Pinthus J.H., Barak V., et al. Growth inhibition of prostate cancer xenografts by halofuginone, The Prostate,2002, 51: 73-83
54 Keshavarz K., McDougald L.R. Anticoccidial drgs:growth and performance depressing effects in yong chickens, Poultry Science,1982, 61:699-705
55 Morrison. Efficacy of Salinomycin and stenorl against various species of Eimeria and effect on chicken performance, Poulty Science, 1979, 58:1160-1166
56 Ernst R.A., Vohra P., Kratzer F.H., et al.. Effect of halfuginone(Stenorol) on Chukar partridge(Alectoris chukar), Poultry Science,1996, 75:1493-1495
57 Pinion J.L., Bilgili S.F., Eckman M.K.,et al. The effects ofhalofuginone and salinomycin, alone and in combination,on live performance and skin characteristics of broilers, Poultry Science,1995, 74:391-397
58 Angel S., Weinberg Z.G., Polishuk O.,et al. A connection between a dietary coccidiostate and skin tears of female broiler chickens, Poultry Science,1985, 64:294-296
59 Schein E., Voigt W.P. Chemotherapy oftheileriosis in cattle.In:Irvin,A.D.;Cunningham,M.P.;Young A.S(eds). Advances in the Control of Theilerias. Martinus Nijhoff, The Hugue, 1981, 212-214
60 Canton J.H.,Van Esch G.J.. The short-term toxicity of some feed additives to different freshwater organisms, Bulletin of Environmental Contamination & Toxicology, 1976, 152:720-725
61 沈建忠主编，《兽医药理学》，中国农业大学出版社，2000
62 Hamet. Resistance to anticoccidial Drugs in Poultry farms in France from 1975 to 1984, Research in Avian Coccidiosis ,1985, 415-420.
63 Stephen B., Rommel M., Daugschies A., et al. Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains, Vet Parasitol, 1997,69: 19-29.
64 Chapman H.D. Eimeria teneila: stability of resistance to halofuginone,decoquinate and arprinocid in the chicken, Research in Veterinary Science,1986, 40: 139-140,
65 Chapman H.D. Eimeria tenella: Experimental studies on the development of resistance to halofuginone, Veterinary Parasitology,1986, 21:83-90
66 Kinabo L.D.B., Bogan J.A. Determination of halofuginone in bovine plasma by competing-ion high performance liquid chromatography after solid-phase extraction, Biomedical Chromatography,1989,3:136-138
67 Kinabo L.D.B., Mckellar Q.A. Pharmacokinetics of halofuginone in cattle. British Veterinary Journal, 1989, 145:546-551
68 Stecklair K.P., Hamburger D.R., Egorin M.J. Pharmacokinetics and tissue distribution of halofuginone(NSC713250) in CD2F1 mice and Fisher 344 rats, Cancer Chemother Pharmacol,2001,48:375-382
69 Committee for Veterinary Medicinal Products: Halofuginone. The European Agency for the
Evalation of Medicinal Products, Veterinary Medicines and Information Technology, Tune 2000.
70 Broun J. Compound evalation and analytical capability:National Residue Program. 1995;USDA.FSIS,Science and Technology Program: Washington. D.C.1995.
71 Anon. Fedral Register, 1985,50:33718-33719.
72 Anon. Food additive tolerance for halofuginone revised. Food Chem News 1991a(Mar,4).54.
73 农业部文件，《关于发布动物性食品中兽药的最高残留限量(试行)的通知》，2002
74 Anderson A., Christopher D.H., Woodhouse R.N.. Analysis of the anticoccidial drug, halofuginone,in chicken feed using gas-liquid chromatography and high-performance liquid chromatography,Journal of Chromatography,1979,168:471-480(1979).
75 K(?)IV(?)NKOV(?) L., FORET F., BO(?)EK P.Determination of halofuginone in feedstuffs by the combination of capillary isotachophoresis and capillary zone electrophoresis in a column-switch system, Journal of Chromatography, 1991, 545:307-313
76 Anderson A., Goodall E., Bliss G.W., et al. Analysis of the anti-coccidial drug,halofuginone,in chicken tissue and chicken feed using high-performance liquid chromatography,Journal of Chromatography, 1981,212:347-355
77 Analytical Methods Committee: Determination of halofuginone hydrobromide in medicated animal feeds, Analyst, 1983, 108:1252-1256
78 Analytical Methods Committee: Collaborative study of a method for the determination of residues of halofuginone in chicken tissues, Analyst, 1984, 109:171-174
79 USDA,FSIS, Science and Technology, Analytical Chemistry Laboratory GuideBook;Residue Chemistry, R.L.Ellis,Director, Chemistry Division, Room 302,Annex Bldg.,300 12th St.,Washington, DC 20250, Winter 1991,pp. HLF-O-HLF-20
80 Holland D.C., Munns R.K., Roybal J.E.,et al. Liquid chromatographic determination of the antieoceidial drug haiofuginone hydrobromide in eggs, Journal of AOAC international, 1995, 78(1):37-40
81 Yamamoto Y., Kondo E. Determination of halofuginone and Amprolium in Chicken Muscle and Egg by Liquid Chromatography,Journal of AOAC International, 2001, 84(1):43-47.
82 Tillier C., Cagniant E., Devaux P. Determination of halofuginone in poultry feeds by high-performance liquid chromatography, Journal of Chromatography, 1988, 441: 406-416.
83 Beier R.C., Rowe L.D., Abd el-aziz nasr M.I., et al. Extraction and HPLC analysis of halofuginone in chicken serum, Journal of Liquid Chromatography, 1994, 17(13): 2961-2970
84 Rowe L.D., Beier R.C., Elissalde M.H., et al. Facile synthesis of earboxylie linker on halofuginone: A method to derivatize haptens containing both amine and hydroxy functionalities,Synthetic Conmunications, 1993, 23(15): 2191-2197.
85 Rowe L.D., Beier R.C., Elissalde M.H., et al. Production and characterization of monoclonal antibodies against the poultry coccidiostat halofuginone, J Agric Food Chem, 1994, 42: 1132-1137
86 Beier R.C., Rowe L.D., Abd El-Aziz Nasr M.I., et al. Detection of halofuginone residues in chicken serum by a monocional-based immunoassay and high-perfomance liquid chromatography,
Food & Agricultural Immunology, 1996, 8:11-17
87 Beier R.C., Dutko T.J., Buckley S.A., et al. Detection of halofuginone residues in chicken liver tissue by HPLC and monocional-based immunoassay, J Aric Food Chem,1998,46:1049-1054
88 Beier R.C., Feldman S.F., Dutko T.J., et al. Immunoassay and HPLC detection of halofuginone in chicken liver sample obtained from commercial slaughterhouses:A combined study, Food & Agricultural Immunology, 2002, 14:29-40
89 Yakkundi S., Cannavan A., Elliot C.T., et al. Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry, Journal of Chromatography B,2003, 788:29-36
90 Mortier L., Daeseleire E., Delahaut P.Simultaneous detection of five eoeeidiostats in eggs by liquid chromatography-tandem mass spectrometry, Analytica Chimiea Acta,2002, 483:27-27
91 李俊锁，环境中Avermectins残留的分离与检测研究-ELISA,IAC-HPLC-UV，中国农业大学博士学位论文，1995
92 Staros J.V., Wright R.W., Swingei D.M. Enhancement by N-hydroxysulfosuccinimide of water-souible earbodiimide-mediated coupling reactions, Anal Bioehem, 1986, 156:220-222
93 周顺伍主编，<生物化学实验技术》，北京农业大学出版社，1994
94 杨利国等，《酶免疫测定技术》，南京：南京大学出版社，1998
95 李成文，《现代免疫化学技术》，上海科学技术出版社，1992
96 Eilanger, B.F. The Preparation of Antigenic Hapten-Carrier Conjugates: A Survey. Methods in Enzymoiogy, 1980, 70:85-104
97 Engvall, E.; Perlman, P. Ezyme-linked immunosorbent assay (ELISA); Quantitative assay of immunogolobulin G, lmmunoehem, 1971, 8:871-874