臺灣博碩士論文加值系統

分別以卡巴得單一劑量以每公斤體重3.5 mg口服方式及飼料添加卡巴得55 ppm投予豬隻 ( 連續兩週 ) 後，以液相層析連接串聯式質譜法分析豬隻血漿、肌肉、肝臟及腎臟中原型藥物及代謝物quinoxaline-2-carboxylic acid ( QCA ) 分佈及殘留情形。本分析方法其卡巴得及QCA檢測極限分別為0.002及0.180 ng/g，定量極限分別為0.005及0.606 ng/g。卡巴得單一劑量口服後其血中藥物濃度在給藥後2.6小時達到高峰；QCA分別在停藥後5週之肝臟 ( 1.98 ng/g ) 及停藥後6週之腎臟 ( 0.9 ng/g ) 中仍可測得。卡巴得平均穩定狀態分佈體積 ( 4427.39 ± 2070.30 mL/kg ) 及平均曲線下面積 ( 2327.58 ± 580.93 hr ng/mL ) 顯示其自豬隻血中迅速分佈至身體組織中。卡巴得代謝物排除緩慢，故豬隻投藥後供消費者食用前需經較長時間之停藥期。

The tissue distribution and residue depletion of carbadox and its metabolite ( quinoxaline-2-carboxylic acid, QCA ) were investigated in swine after a single oral dose of 3.5 mg/kg body weight of carbadox and multiple dose ( 2 weeks ) in-feed ( 55 ppm ) administration. Plasma, muscle, liver and kidney were sampled pre and post-treatment and subsequently analyzed for carbadox and QCA concentrations using liquid chromatography with tandem mass spectrometry ( LC-MS-MS ). The limits of detection of carbadox and QCA were 0.002 and 0.180 ng/g, the limits of quantitation were 0.005 and 0.606 ng/g for standard solution. Carbadox concentration in plasma was peaked on 2.6 hr after a single oral dose administration, while QCA concentration was still detected in liver ( 1.98 ng/g ) on the fifth week and kidney ( 0.9 ng/g ) on the sixth week after withdrawal. The apparent volume of distribution of carbadox at steady-state ( 4427.39 ± 2070.30 mL/kg ) and areas under the concentration curves ( 2327.58 ± 580.93 hr ng/mL ) indicates that the drug is adequately distributed throughout the body from the blood of pigs. The slow elimination of the carbadox metabolites suggests a need for long withdrawal periods prior to use of dosed swine for human consumption.

目 錄
中文摘要………………………………………………………………Ⅰ
Abstract………………………………………………………………Ⅱ
謝誌……………………………………………………………………Ⅳ
目錄……………………………………………………………………Ⅴ
圖表目錄………………………………………………………………Ⅷ
第1章 前言…………………………………………………………1
第2章 文獻回顧……………………………………………………3
2.1卡巴得之化學特性及藥理作用………………………………3
2.1.1 卡巴得化學結構及理化特性……………………………3
2.1.2 卡巴得之作用機制………………………………………3
2.1.3 卡巴得抗菌劑之藥物動力學及代謝……………………4
2.1.4 卡巴得在獸醫學臨床上之應用…………………………5
2.2卡巴得之毒性及可能造成之危害……………………………6
2.2.1 卡巴得之毒性……………………………………………6
2.2.2 卡巴得在公共衛生上之影響……………………………7
2.3卡巴得及其相關代謝物之檢測方法……………………………8
2.3.1 液相層析法 ( LC )……………………………………..8
2.3.2 薄層色層分析法 ( TLC )………………………………8
2.3.3 高效能液相層分析法 ( HPLC )……………………….9
2.3.4 氣相層析質譜法 ( GC/MS )…………………………..9
2.3.5 液相連接串聯式質譜法 (LC/MS/MS )………………10
2.4台灣及其他國家目前相關法規………………………………11
2.5卡巴得使用情況及殘留研究調查結果之相關文獻…………12
第3章 材料與方法…………………………………………………13
3.1試驗設計………………………………………………………13
3.2試驗用材料……………………………………………………14
3.2.1 試驗用藥品及飼料……………………………………14
3.2.2 試驗用器材……………………………………………15
3.2.3 試驗用設備……………………………………………15

3.2.4 試驗用動物……………………………………………16
3.3藥物動力學試驗………………………………………………16
3.4動物組織殘留試驗……………………………………………17
3.5分析方法………………………………………………………17
3.5.1 標準品配製……………………………………………17
3 . 5 . 2 卡巴得及Quinoxaline-2-carboxylic acid紫外光光
譜………………………………………………………17
3.5.3 實驗試劑配製…………………………………………17
3.5.3.1 0.1 M鹽酸溶液………………………………17
3.5.3.2 1％甲酸溶液：甲醇溶液 ( v/v，50：50 )…17
3.5.3.3 2％偏磷酸：甲醇溶液 ( v/v，80：20 )……18
3.5.3.4 pH 2.5磷酸緩衝溶液…………………………18
3.5.3.5 含0.1％甲酸之20 mM甲酸銨溶液…………18
3.5.3.6 含0.1％甲酸之乙腈溶液………………………18
3.5.4 血清萃取………………………………………………18
3.3.5 組織臟器萃取…………………………………………18
3.3.6 液相層析質譜分析……………………………………19
3.5.6.1 液相層析質譜儀測定條件………………………19
3.3.7 標準曲線製作…………………………………………20
3.3.8 靈敏度試驗……………………………………………20
3.3.9 回收率試驗及精確性試驗……………………………20
3.6藥物動力學數據分析…………………………………………21
第4章 結果…………………………………………………………22
4.1卡巴得及Quinoxaline-2-carboxylic acid紫外光光譜…22
4.2液相層析質譜分析……………………………………………22
4.2.1 卡巴得及Quinoxaline-2-carboxylic acid之質譜.22
4.2.2 標準曲線………………………………………………25
4.2.3 最低檢測極限、最低定量極限………………………25
4.2.4 液相層析質譜分析之滯留時間及再現性……………27
4.2.5 組織臟器回收率………………………………………29
4.3卡巴得之藥物動力學試驗……………………………………29
4.3.1 非室性模式及二室性模式之藥物動力學分析………33
4.4動物組織殘留試驗……………………………………………35
第5章 討論…………………………………………………………37
參考文獻………………………………………………………………44
附錄……………………………………………………………………54
作者簡介………………………………………………………………62

參考文獻
方嘉德 譯。儀器分析。滄海書局，台中，879-881，2003。
行政院農業委員會動植物防疫檢疫局。含藥物飼料添加物之種類及品目，2008。
行政院農業委員會動植物防疫檢疫局。動物用藥使用手冊，2006。
行政院衛生署食品衛生處。動物用藥殘留標準。衛署食字第0970400541號令修正，2008。
呂車鳯。台灣南部市販豬肉中Carbadox之殘留情形。臺灣畜牧獸醫學會會報 60：15-19，1992。
沈振峰、何國榮。液相層析質譜術在藥物分析之應用。藥物食品分析 3：243-258，1995。
李新進、詹益波、楊揚輝。飼料添加物檢驗法—卡巴得 ( carbadox ) 定性試驗研究。臺灣省畜衛試驗所研報 20：111-116，1984。
林士鈺、邱資峰、陳忠松。以高性能液相層析法測定飼料卡巴得。台灣畜牧獸醫學會會報 42：15-23，1983。
陳清、柏崎守、波岡茂郎。厭氧狀況下猪隻由來大腸菌對於Carbadox之感受性。臺灣省畜衛試研報 10：41-45, 1973。
經濟部標準檢驗局。食品中動物用藥殘留量檢驗方法—卡巴得之檢驗。中華民國國家標準：14457，N6333，2000。
經濟部標準檢驗局。飼料中卡巴得、硝基夫喃及富來頓檢驗方法。中華民國國家標準：14680，N4189，2002。
楊繼。動物藥品SPS爭端案例之分析。國際動植物防疫檢疫案例分析研討會，2002。
楊繼、何素鵬 譯。食用動物使用藥物。台灣惠氏股份有限公司，台北，3-10，2003。
劉正雄 譯。應用生物藥劑學與藥物動力學。合記圖書出版社，台北，38，2002。
顏宏逹。養豬新知 (一)飼料營養篇。台灣動物科技研究所，苗栗，42-49，2005。
厚生労働省。食品に残留する農薬、飼料添加物又は動物用医薬品の成分である物質の試験法。食安発第0124001号，2005。
厚生労働省。食品衛生法等の一部を改正する法律による改正後の食品衛生法第11条第3項の施行に伴う関係法令の整備。食安発第1129001号，2005。
堀江正一、村山三徳。LC/MSによる豚筋肉および肝臓中のカルバドックス代謝体（キノキサリン-2-カルボン酸およびデスオキシカルバドックス）の定量。食衛誌 45: 135-140, 2004.
Adams HR. Veterinary pharmacology and therapeutics. 8th. Iowa state university, USA. 889, 2001.
Booth NH, McDonald LE. Veterinary pharmacology and therapeutics. 5th. Iowa state university, USA. 765, 1982.
Chamberlain E, Adams C. Oxidation of sulfonamides, macrolides, and carbadox with free chlorine monochloramine. Water Res 40: 2517-2526, 2006.
Chang ZQ, Oh BC, Kim JC, Jeong KS, Lee MH, Yun HI, Hwang MH, Park SC. Clinical pharmacokinetics of norfloxacin-glycine acetate after intravenous and oral administration in pigs. J Vet Sci 8: 353-356, 2007.

Chen J. Animal models for acquired bone marrow failure syndromes. Clin Med Res 3: 102-108, 2005.
Chen Q, Chen YQ, Qi YT, Hao LH, Tang SS, Xiao XL. Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells. Mutat Res 638: 11-16, 2008.
de Graff GJ, Jager LP, Baars AJ, Spierenburg TJ. Some pharmacokinetic observations of carbadox medication in pigs. Vet Q 10: 34-41, 1988.
de Graff GJ, Spierenburg TJ. Liquid chromatographic determination of carbadox and desoxycarbadox in medicated feeds and in porcine gastrointestinal tract. J Assoc Off Anal Chem 68: 658-660, 1985.
Dewey CE, Cox BD, Straw BE, Bush EJ, Hurd HS. Associations between off-label feed additives and farm size, veterinary consultant use, and animal age. Prev Vet Med 31: 133-146, 1997.
Duhamel GE, Kinyon JM, Mathiesen MR, Murphy DP, Walter D. In vitro activity of four antimicrobial agents against North American isolates of porcine Serpulina pilosicoli. J Vet Diagn Invest 10: 350-356, 1998.
Dunlop RH, McEwen SA, Meek AH, Friendship RA, Clarke RC, Black WD. Antimicrobial drug use and related management practices among Ontario swine producers. Can Vet J 39: 87-96, 1998.
European Commission. Commission Regulation (EC) No 2788/98 of 22 December 1998. Off J Europ Commun L347: 31-32, 1998.
European Union Scientific Committee for Animal Nutrition. Opinion of the Scientific Committee for Animal Nutrition on possible risks for the consumer, the animal and the users (operators) from the use of carbadox and olaquindox as feed additives. 1-25, 1998.

Food and Drug Administration ( FDA ). New animal drug applications. NADA 041-061, 1998.
Food and Drug Administration ( FDA ). Original New animal drug applications. NADA 141-211, 2004.
Griffiths WJ, Jonsson AP, Liu SY, Rai DK, Wang YQ. Electrospray and tandem mass spectrometry in biochemistry. Biochem J 355: 545-561, 2001.
Hamilton R, Hamilton S. Thin Layer Chromatography. Behalf of ACOL, London, 1-10, 1987.
Hathaway MR, Kretchmar DH, Allen CE, Comelius SG, Dayton WR. In vitro muscle cell proliferation and protein turnover as affected by serum from pigs fed antimicrobials. J Anim Sci 68: 3190-3197, 1990.
Hayashi TK, Suenaga I, Narukawa NA, Yamazaki TY. In vitro and in vivo activities of sedecamycin against Treponema hyodysenteriae. Antimicrob Agents Chemother 32: 458-461, 1988.
Health Canada. Progress on the proposal to ban carbadox. Communiqué to Stakeholders 5(1) : 4, 2005.
Hernandez E, Rey R, Puig M, Garcia MA, Solans C, Bregante MA. Pharmacokinetics and residues of a new oral amoxicillin formulation in piglets: A preliminary study. Vet J 170: 237-242, 2005.
Hiasa H, Shea ME. DNA gyrase-mediated wrapping of the DNA strand is required for the replication fork arrest by the DNA gyrase-quinolone-DNA ternary complex. J Biol Chem 275: 34780-34786, 2000.

Hu JG, McDougald LR. The efficacy of some drugs with known antiprotozoal activity against Histomonas meleagridis in chickens. Vet Parasitol 121: 233-238, 2004.
Hutchinson MJ, Young PY, Hewitt SA, Faulkner D, Kennedy DG. Development and validation of an improved method for confirmation of the carbadox metabolite, quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis. Analyst 127: 342-346, 2002.
Hutchinson MJ, Young PB, Kennedy DG. Confirmation of carbadox and olaquindox metabolites in porcine liver using liquid chromatography-electrospray, tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 816: 15-20, 2005.
Hutchinson MJ, Young PB, Kennedy DG. Confirmatory method for the analysis of carbadox and olaquindox in porcine feedingstuffs using LC-electrospray MS–MS. Food Addit Contam 22: 113-119, 2005.
Hutchinson MJ, Young PB, Kennedy DG. Quinoxaline-2-carboxylic acid in pigs: criteria to distinguish between the illegal use of carbadox and environmental contamination. Food Addit Contam 21: 538-544, 2004.
Joint FAO/WHO Expert Committee on Food Additives ( JECFA ). Carbadox. In: Toxicological evaluation of certain veterinary drug residues in food ( WHO Food Additives Series 27 ). Geneva, Switzerland: WHO: 141-73, 1991.
Joint FAO/WHO Expert Committee on Food Additives ( JECFA ). Residues of some veterinary drugs in animals and foods. FAO Food Nutr Pap 41/15: 1-19, 2003a.

Joint FAO/WHO Expert Committee on Food Additives ( JECFA ). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 51, 2003b.
Karlsson M, Fellström C, Gunnarsson A, Lande´n A, Franklin A. Antimicrobial Susceptibility Testing of Porcine Brachyspira (Serpulina) Species Isolates. J Clin Microbiol 41: 2596-2604, 2003.
Kitai K, Kashiwazaki M, Adachi Y, Kunugita K, Arakawa A. In vitro Antimicrobial activity against reference strains and field isolates of Treponema hyodysenteriae. Antimicrob Agents Chemother 31: 1935-1938, 1987.
Köhler B, Karch H, Schmidt H. Antibacterials that are used as growth promoters in animal husbandry can affect the release of Shiga-toxin-2-converting bacteriophages and Shiga toxin 2 from Escherichia coli strains. Microbiology 146: 1085-1090, 2000.
Laine T, Yliaho M, Myllys V, Pohjanvirta T, Fossi M, Anttila M. The effect of antimicrobial growth promoter withdrawal on the health of weaned pigs in Finland. Prev Vet Med 66: 163-174, 2004.
Liu JZ, Fung KF, Chen ZL, Zeng ZL, Zhang J. Pharmacokinetics of florfenicol in healthy pigs and in pigs experimentally infected with Actinobacillus pleuropneumoniae. Antimicrob Agents Chemother 47: 820-823, 2003.
Lowie DM, Teague RT, Quick FE, Foster CL. High pressure liquid chromatographic determination of carbadox and pyrantel tartrate in swine feed and supplements. J Assoc Off Anal Chem 66: 602-605, 1983.
Lynch MJ, Bartolucci SR. Confirmatory identification of carbadox-related residues in swine liver by gas-liquid chromatography/mass spectrometry with selected ion monitoring. J Assoc Off Anal Chem 65: 66-70, 1982.
Lynch MJ, Mosher FR, Schneider RP, Fouda HG. Determination of carbadox-related residues in swine liver by gas chromatography/mass spectrometry with ion trap detection. J Assoc Off Anal Chem 74: 611-618, 1991.
MacIntosh AI, Neville GA. Liquid chromatographic determination of carbadox, desoxycarbadox, and nitrofurazones in pork tissues. J Assoc Off Anal Chem 67: 958-962, 1984.
MacIntosh AI, Lauriault G, Neville GA. Liquid chromatographic monitoring of the depletion of carbadox and its metabolite desoxycarbadox in swine tissue. J Assoc Off Anal Chem 68: 665-671, 1985.
Mathew AG, Robbins CM, Chattin SE, Quigley JD. Influence of galactosyl lactose on energy and protein digestibility, enteric microflora, and performance of weanling pigs. J Anim Sci 75: 1009-1016, 1997.
Mathew AG, Upchurch WG, Chattin SE. Incidence of antibiotic resistance in fecal Escherichia coli isolated from commercial swine farms. J Anim Sci 76: 429-434, 1998.
Miao XS, March RE, Metcalfe CD. A tandem mass spectrometric study of the N-oxides, quinoline N-oxide, carbadox and olaquindox, carried out at high mass a accuracy using electrospary ionization. Int J Mass Spectrom 230: 123-133, 2003.
Molnár L. Sensitivity of strains of Serpulina hyodysenteriae isolated in hungary to chemotherapeutic drugs. Vet Rec 138: 158-160, 1996.
Ohmae K, Yonezawa S, Terakado N. R plasmid with carbadox resistance from Escherichia coli of porcine origin. Antimicrob Agents Chemother 19: 86-90, 1981.

Partanen K, Jalava T, Valaja J, Perttilä S, Siljander-Rasi H, Lindeberg H. Effect of dietary carbadox or formic acid and fibre level on ileal and faecal nutrient digestibility and microbial metabolite concentrations in ileal digesta of the pig. Anim Feed Sci Technol 93: 137-155, 2001.
Pijpers A, Schoevers EJ, Gogh HV, Leengoed LA, Visser IJ, Miert AS, Verheijden JH. The influence of disease on feed and water consumption and on pharmacokinetics of orally administered oxytetracycline in pigs. J Anim Sci 69: 2947-2954, 1991.
Prescott JF, Baggot JD, Walker RD. Antimicrobial therapy in veterinary medicine. 3th. Iowa state university, USA. 2000.
Roybal JE, Munns RK, Shimoda W. Liquid chromatographic determination of carbadox residues in animal feed. J Assoc Off Anal Chem 68: 653-657, 1985.
Savoini G, Bontempo V, Cheli F, Baldi A, Sala V, Mancin G, Agazzi A, Dell’Orto V. Alternative antimicrobials in the nutrition of postweaning piglets. Vet Rec 151: 577-580, 2002.
Sin WM, Chung PK, Lai MC, Siu MP, Tang PO. Determination of quinoxaline-2-carboxylic acid, the major metabolite of carbadox, in porcine liver by isotope dilution gas chromatography-electron capture negative ionization mass spectrometry. Anal Chim Acta 508: 147-158, 2004.
Strock TJ, Sassman SA, Lee LS. Sorption and related properties of the swine antibiotic carbadox and associated N-oxide reduced metabolites. Environ Sci Technol 39: 3134-3142, 2005.
Thorpe VA. Improved rapid colorimetric method for carbadox in feeds. J Assoc Off Anal Chem 61: 88-91, 1978.
Thorpe VA. Sample preparation of carbadox, furazolidone, nitrofurazone, and ethopabate in medicated feeds for high pressure liquid chromatography. J Assoc Off Anal Chem 63: 981-984, 1980.
Uezato K, Kinjo E, Adachi Y. In vitro susceptibility of 21 antimicrobial agents to 37 isolates of Brachyspira hyodysenteriae isolated from pigs in Okinawa prefecture. J Vet Med Sci 66: 307-309, 2004.
van der Molen EJ. Pathological effects of carbadox in pigs with special emphasis on the adrenal. J Comp Pathol 98: 55-67, 1988.
van der Molen EJ, de Graaf GJ, Baars AJ. Persistence of carbadox-induced adrenal lesions in pigs following drug withdrawal and recovery of aldosterone plasma concentrations. J Comp Pathol 100: 295-304, 1989a.
van der Molen EJ, van Lieshout JH, Nabuurs MJ, Derkx FH, Lamers AP, Michelakis AM. Changes in plasma renin activity and renal immunohistochemically demonstrated renin in carbadox treated pigs. Res Vet Sci 46: 401-405, 1989b.
Woodward KN. Veterinary pharmacovigilance. Part 6. Predictability of adverse reactions in animals from laboratory toxicology studies. J Vet Pharmacol Ther 28: 213-231, 2005.
Wu YJ, Wang YL, Huang LL, Tao YF, Yuan ZH, Chen DM. Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using ultrasonic solvent extraction and high-performance liquid chromatography. Anal Chim Acta 569: 97-102, 2006.
Yen JT, Nienaber JA. Influence of carbadox on fasting oxygen consumption by portal vein-drained organs and by the whole animal in growing pigs. J Anim Sci 70: 478-483, 1992.

Yen JT, Nienaber JA, Pond WG, Varel VH. Effect of carbadox on growth , fasting metabolism, thyroid function and gastrointestinal tract in young pigs. J Nutr 115: 970-979, 1985.
Yen JT, Pond WG. Effect of carbadox, copper, or Yucca shidigera extract on growth performance and visceral weight of young pigs. J Anim Sci 71: 2140-2146, 1993.
Yen JT, Pond WG. Effect of carbadox on net absorption of ammonia and glucose into hepatic portal vein of growing pigs. J Anim Sci 68: 4286-4242, 1990.
Yoshimura H. Teratogenic assessment of carbadox in rats. Toxicol Lett 129: 115-118, 2002.
Yuan ZH, Wu YJ, Wang YL, Huang LL, Chen DM, Tao YF. Development of an HPLC-UV method for simultaneous quantification of quinoxaline-2-carboxylic acid and 3-methyl-quinoxaline-2-carboxylic acid in muscle and liver of swine, chicken and fish. J Vet Pharmacol Ther 29 (suppl 1): 142, 2006.
Zhang YL, Huang LL, Chen DM, Fan SX, Wang YL, Tao YF, Yuan ZH. Development of HPLC methods for the determination of cyadox and its main metabolites in goat tissues. Anal Sci 21: 1495-1499, 2005.