山东省出口水产品药物残留分析及代谢动力学研究
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摘要
我国是世界上最大的水产品贸易国,其中养殖水产品占重要比重。在水产养殖快速发展的同时,为了控制各种疾病,渔用药物在水产养殖中应用日益广泛,很多药物对人体有很大危害虽已明令禁止使用,但仍有大量药物残留屡被检出；另外,允许使用的药物残留量超标现象也十分突出。因此,水产品中渔用药物残留问题已成为制约我国水产品进入国际市场的关键问题。对水产品中药品残留检测技术和代谢动力学的系统研究有助于控制养殖过程中渔用药物的使用和减少药物残留,具有十分重要的现实意义。本研究以加工水产品为研究对象,研究了硝基呋喃代谢物残留检测的干扰因素,并建立了应用高效液相色谱(HPLC)同时检测饲料中四种硝基呋喃类原药的方法；以山东省出口的主要养殖水产品-石鲽鱼为研究对象,建立了适用于检测石鲽鱼各组织中磺胺类药物、喹诺酮类药物和四环素类药物残留量的超高效液相色谱串联质谱(UPLC-MS/MS)方法,并对磺胺二甲嘧啶、磺胺甲嗯唑、恩诺沙星、二氟沙星和土霉素5种药物的代谢进行研究,确定了其休药期,并提出了综合防控建议。结果如下：
1.禁用药物硝基呋喃原药及其代谢物残留研究通过实验建立了利用乙腈提取残留药物,经正己烷和酸性氧化铝净化,HPLC检测饲料中四种硝基呋喃原药的检测方法。呋喃西林、呋喃妥因、呋喃它酮三种残留药物的检出限为0.15mg/kg,定量限为0.5mg/kg,呋喃唑酮检出限为0.075mg/kg,定量限为0.25mg/kg,四种药物的平均添加回收率均>75%,相对标准偏差均<10%。另外,对中日硝基呋喃代谢物残留检测方法进行比较,研究水产品加工环节中干扰呋喃西林代谢物(SEM)检测结果的因素。加工环节中使用的次氯酸钠和面粉中含有的偶氮甲酰胺是SEM的主要干扰因素,而检测技术差别和加工过程中使用乙醇对SEM的检测结果无影响。
2.鱼体各组织中27种磺胺类和喹诺酮类药物残留同时检测方法的建立建立了用含1%甲酸的正己烷饱和乙腈溶液提取残留药物,正己烷多次净化,UPLC-MS/MS多反应监测正离子模式下同时检测鱼体各组织中27种磺胺类和喹诺酮药物残留的检测方法。该方法的线性检测范围为10.0～100.0μg/kg,相关系数均大于0.9970。27种药物在石鲽鱼肌肉、血液和肝脏组织的平均回收率为75.1%～110.3%,相对标准偏差均<12%,定量限为6.0～10.7μg/kg。
3.鱼体各组织中4种四环素类药物残留检测方法的建立建立了用酸性缓冲溶液提取,Oasis HLB固相萃取柱净化,UPLC-MS/MS多反应监测正离子模式下测定鱼体各组织中4种四环素类药物残留的检测方法。该方法的线性范围为5.0～200.0μg/kg,相关系数为0.9982-0.9996。4种药物在石鲽鱼肌肉、血液和肝脏组织的平均回收率均>76.0%,相对标准偏差均<11%,定量限为4.3～8.3μg/kg/。
4.限用药物代谢动力学研究在水温15±2℃下以口灌模式和混饲模式两种投药方式,研究了石鲽鱼中磺胺二甲嘧啶、磺胺甲噁唑,恩诺沙星、二氟沙星和土霉素的代谢及消除规律。结果发现,口灌模式下在石鲽鱼中磺胺二甲嘧啶、恩诺沙星、二氟沙星和土霉素的消除速度均为血浆>肌肉>肝脏,磺胺甲噁唑的消除速度为肌肉>肝脏>血浆；混饲模式下在石鲽鱼中磺胺二甲嘧啶、恩诺沙星和二氟沙星的消除速度都为血浆>肌肉>肝脏,磺胺甲噁唑和土霉素的消除速度均为肌肉>血浆>肝脏。根据5种药物药代动力学的研究,制定了混饲模式下对于石鲽鱼的休药期。当磺胺二甲嘧啶、磺胺甲嗯唑、恩诺沙星、二氟沙星和土霉素的给药剂量分别为100mg/kg、100mg/kg、20mg/kg、20mg/kg和50mg/kg鱼体重时,它们的休药期分别为28天、28天、26天、28天和26天。
China is the largest trading country for aquatic products in the world, and a high proportion of aquatic products from aquaculture. However, various drugs applied for aquaculture with an aim to control the diseases of aquatic animal have declined the quality of seafood and reduced the export amount in the international markets. The residue drugs in seafoods have been detected in many aquatic samples including some banned drugs and over standard allowed drugs. It is thus important to set up the detection technology and pharmacokinetics of veterinary drugs to monitor and control the application of drugs in aquaculture processes.
In this study, the nitrofuran metabolite residues in the processed aquatic products were detected and the interfering factors in detection were analyzed; and the methods to analyze four kinds of nitrofurans simultaneously in feeds using high performance liquid chromatography (HPLC) was established. Moreover, the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to detect the residues of sulfonamides, quinolones and tetracyclines in different tissues of stone flounder (kareius bicoeoratas). Then it was studied that the pharmacokinetics for sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline in stone flounder, and the withdrawal periods were determined. Base on these studies, the prevention and control measures were proposed for management. The detailed results were summarized as below:
1. The study on banned drug nitrofurans and its metabolites residues in seafood
A method was established to detect four kinds of nitrofurans in feeds through the experiments. The samples were extracted by acetonitrile, purified by hexane and acidic alumine, and then detected by HPLC. The LOD and LOQ for furacillin, nitrofurantion and furaltadone were0.15mg/kg and0.5mg/kg, and for furazolidone were0.12mg/kg and0.075mg/kg, respectively. The average recoveries of the four nitrofurans were more than75%, with RSD below10%. When the methods using to analyze the metabolites residues of nitrofurans in Japan and China were compared, it was found that the significance interferinge factors determining semlcarbazide hydrochloride (SEM) were the sodium hypochlorite in the aquatic products processing and azodicarbonamide in flour. The difference between detection methods and the use of ethanol in aquatic products processing had no effects.
2. The method for simultaneous determining the residues of27kinds of sulfonamides (SAs) and quinolones (QNs)
The samples were extracted with acetonitrile saturated by hexane containing1%formic acid, cleaned-up by hexane, and then analyzed by UPLC-MS/MS. The mass spectrometer was operated in the positive ion mode using multiple reactions monitoring (MRM) mode for the qualitative and quantitative analysis of27kinds of SAs and QNs at the same time. The standard curves for all compounds were linear over the concentration range of10.0-100.0μg/kg, with correlation coefficients of more than0.997. The average recoveries of27kinds of SAs and QNs in the muscle, blood and liver of stone flounders ranged from75.1%-110.3%with relative standard deviations (RSD) less than12%, and the limits of quantitation (LOQ) were6.0-10.7μg/kg.
3. The method for the simultaneous determining of the residues of4kinds of tetracyclines (TCs)
The samples were extracted with acidic buffer solution, cleaned up by Oasis HLB solid phase extraction column, and then analyzed by UPLC-MS/MS. The mass spectrometer was operated in the positive ion mode using MRM mode for the qualitative and quantitative analysis of4kinds of TCs at the same time. The standard curves for all compounds were linear over the concentration range of5.0-200.0μg/kg and the correlation coefficients ranged from0.9982to0.9996. The average recoveries of4kinds of TCs in the muscle, blood and liver of stone flounders were all more than76.0%with the RSD less than11%and the LOQ were4.3-8.3μg/kg.
4. The pharmacokinetics of restricted-using drugs
The metabolisms of5kinds of restricted-using drugs (sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline) were studied in stone flounders with oral gavage and mixed-feed gavage at15±2℃. The result showed that the metabolism rates of drugs in oral gavage for stone flounders were arranged as the following:plasma>muscle>liver (sulfamethazine, enrofloxacin, difloxacin and oxytetracycline), muscle>liver>plasmare (sulfamethoxazole). The metabolism rates of drugs in mixed-feed gavage were arranged as plasma>muscle>liver(sulfamethazine, enrofloxacin and difloxacin), muscle>plasma>liver (sulfamethoxazole and oxytetracycline). According to these results, the withdrawal periods in mixed-feed gavage for sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline (100mg/kg,100mg/kg,20mg/kg,20mg/kg and50mg/kg body weight) were determined and they were28,28,26,28and26days, respectively.
1.禁用药物硝基呋喃原药及其代谢物残留研究通过实验建立了利用乙腈提取残留药物,经正己烷和酸性氧化铝净化,HPLC检测饲料中四种硝基呋喃原药的检测方法。呋喃西林、呋喃妥因、呋喃它酮三种残留药物的检出限为0.15mg/kg,定量限为0.5mg/kg,呋喃唑酮检出限为0.075mg/kg,定量限为0.25mg/kg,四种药物的平均添加回收率均>75%,相对标准偏差均<10%。另外,对中日硝基呋喃代谢物残留检测方法进行比较,研究水产品加工环节中干扰呋喃西林代谢物(SEM)检测结果的因素。加工环节中使用的次氯酸钠和面粉中含有的偶氮甲酰胺是SEM的主要干扰因素,而检测技术差别和加工过程中使用乙醇对SEM的检测结果无影响。
2.鱼体各组织中27种磺胺类和喹诺酮类药物残留同时检测方法的建立建立了用含1%甲酸的正己烷饱和乙腈溶液提取残留药物,正己烷多次净化,UPLC-MS/MS多反应监测正离子模式下同时检测鱼体各组织中27种磺胺类和喹诺酮药物残留的检测方法。该方法的线性检测范围为10.0～100.0μg/kg,相关系数均大于0.9970。27种药物在石鲽鱼肌肉、血液和肝脏组织的平均回收率为75.1%～110.3%,相对标准偏差均<12%,定量限为6.0～10.7μg/kg。
3.鱼体各组织中4种四环素类药物残留检测方法的建立建立了用酸性缓冲溶液提取,Oasis HLB固相萃取柱净化,UPLC-MS/MS多反应监测正离子模式下测定鱼体各组织中4种四环素类药物残留的检测方法。该方法的线性范围为5.0～200.0μg/kg,相关系数为0.9982-0.9996。4种药物在石鲽鱼肌肉、血液和肝脏组织的平均回收率均>76.0%,相对标准偏差均<11%,定量限为4.3～8.3μg/kg/。
4.限用药物代谢动力学研究在水温15±2℃下以口灌模式和混饲模式两种投药方式,研究了石鲽鱼中磺胺二甲嘧啶、磺胺甲噁唑,恩诺沙星、二氟沙星和土霉素的代谢及消除规律。结果发现,口灌模式下在石鲽鱼中磺胺二甲嘧啶、恩诺沙星、二氟沙星和土霉素的消除速度均为血浆>肌肉>肝脏,磺胺甲噁唑的消除速度为肌肉>肝脏>血浆；混饲模式下在石鲽鱼中磺胺二甲嘧啶、恩诺沙星和二氟沙星的消除速度都为血浆>肌肉>肝脏,磺胺甲噁唑和土霉素的消除速度均为肌肉>血浆>肝脏。根据5种药物药代动力学的研究,制定了混饲模式下对于石鲽鱼的休药期。当磺胺二甲嘧啶、磺胺甲嗯唑、恩诺沙星、二氟沙星和土霉素的给药剂量分别为100mg/kg、100mg/kg、20mg/kg、20mg/kg和50mg/kg鱼体重时,它们的休药期分别为28天、28天、26天、28天和26天。
China is the largest trading country for aquatic products in the world, and a high proportion of aquatic products from aquaculture. However, various drugs applied for aquaculture with an aim to control the diseases of aquatic animal have declined the quality of seafood and reduced the export amount in the international markets. The residue drugs in seafoods have been detected in many aquatic samples including some banned drugs and over standard allowed drugs. It is thus important to set up the detection technology and pharmacokinetics of veterinary drugs to monitor and control the application of drugs in aquaculture processes.
In this study, the nitrofuran metabolite residues in the processed aquatic products were detected and the interfering factors in detection were analyzed; and the methods to analyze four kinds of nitrofurans simultaneously in feeds using high performance liquid chromatography (HPLC) was established. Moreover, the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to detect the residues of sulfonamides, quinolones and tetracyclines in different tissues of stone flounder (kareius bicoeoratas). Then it was studied that the pharmacokinetics for sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline in stone flounder, and the withdrawal periods were determined. Base on these studies, the prevention and control measures were proposed for management. The detailed results were summarized as below:
1. The study on banned drug nitrofurans and its metabolites residues in seafood
A method was established to detect four kinds of nitrofurans in feeds through the experiments. The samples were extracted by acetonitrile, purified by hexane and acidic alumine, and then detected by HPLC. The LOD and LOQ for furacillin, nitrofurantion and furaltadone were0.15mg/kg and0.5mg/kg, and for furazolidone were0.12mg/kg and0.075mg/kg, respectively. The average recoveries of the four nitrofurans were more than75%, with RSD below10%. When the methods using to analyze the metabolites residues of nitrofurans in Japan and China were compared, it was found that the significance interferinge factors determining semlcarbazide hydrochloride (SEM) were the sodium hypochlorite in the aquatic products processing and azodicarbonamide in flour. The difference between detection methods and the use of ethanol in aquatic products processing had no effects.
2. The method for simultaneous determining the residues of27kinds of sulfonamides (SAs) and quinolones (QNs)
The samples were extracted with acetonitrile saturated by hexane containing1%formic acid, cleaned-up by hexane, and then analyzed by UPLC-MS/MS. The mass spectrometer was operated in the positive ion mode using multiple reactions monitoring (MRM) mode for the qualitative and quantitative analysis of27kinds of SAs and QNs at the same time. The standard curves for all compounds were linear over the concentration range of10.0-100.0μg/kg, with correlation coefficients of more than0.997. The average recoveries of27kinds of SAs and QNs in the muscle, blood and liver of stone flounders ranged from75.1%-110.3%with relative standard deviations (RSD) less than12%, and the limits of quantitation (LOQ) were6.0-10.7μg/kg.
3. The method for the simultaneous determining of the residues of4kinds of tetracyclines (TCs)
The samples were extracted with acidic buffer solution, cleaned up by Oasis HLB solid phase extraction column, and then analyzed by UPLC-MS/MS. The mass spectrometer was operated in the positive ion mode using MRM mode for the qualitative and quantitative analysis of4kinds of TCs at the same time. The standard curves for all compounds were linear over the concentration range of5.0-200.0μg/kg and the correlation coefficients ranged from0.9982to0.9996. The average recoveries of4kinds of TCs in the muscle, blood and liver of stone flounders were all more than76.0%with the RSD less than11%and the LOQ were4.3-8.3μg/kg.
4. The pharmacokinetics of restricted-using drugs
The metabolisms of5kinds of restricted-using drugs (sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline) were studied in stone flounders with oral gavage and mixed-feed gavage at15±2℃. The result showed that the metabolism rates of drugs in oral gavage for stone flounders were arranged as the following:plasma>muscle>liver (sulfamethazine, enrofloxacin, difloxacin and oxytetracycline), muscle>liver>plasmare (sulfamethoxazole). The metabolism rates of drugs in mixed-feed gavage were arranged as plasma>muscle>liver(sulfamethazine, enrofloxacin and difloxacin), muscle>plasma>liver (sulfamethoxazole and oxytetracycline). According to these results, the withdrawal periods in mixed-feed gavage for sulfamethazine, sulfamethoxazole, enrofloxacin, difloxacin and oxytetracycline (100mg/kg,100mg/kg,20mg/kg,20mg/kg and50mg/kg body weight) were determined and they were28,28,26,28and26days, respectively.
引文
http://tradeinservices.mofcom.gov.cn/index.shtml?method=view&id=90538山东省水产品国际竞争力分析
http://www.mhlw.go.jp/topics/bukyoku/iyaku/syoku-anzen/zanryu3/san-021.html硝基呋喃代谢物检测方法
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http://www.mhlw.go.jp/topics/bukyoku/iyaku/syoku-anzen/zanryu3/san-021.html硝基呋喃代谢物检测方法
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