输日动物源性蜂产品残留限量及检测技术PPT69(3)

Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,输日动物源性（蜂产品）残留限量及检测技术,报告人：沈崇钰,2006,年,4,月 北京,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,介绍内容,一、日本肯定列表与蜂产品出口,二、蜂产品中各类抗生素残留检测,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,一、日本肯定列表与蜂产品出口,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,2003年5月，日本修订了食品卫生法,依据新修订的食品卫生法，2006年5月29日，实施食品中农业化学品（农兽药及饲料添加剂）残留限量“肯定列表制度”,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,日本将要实施的“肯定列表制度”，对食品、农产品中所有农业化学品残留物都作了明确规定。其中15种农药、兽药禁止使用；797种农药、兽药及饲料添加剂设定了53862个限量标准（包括“现行标准”和“暂定标准”）；对没有设定限量标准的，执行“一律标准”，即含量不得超过0.01毫克/公斤。只有65种天然和化学合成物质（主要是营养剂）做为豁免物质不设限量。从2006年5月29日起，只有符合“肯定列表制度”要求的食品、农产品才能进入日本市场。,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,日本厚生省网站上公布了一批检测方法（176个）,单品种检测方法166个,多残留检测方法6个,大多数为植物源性食品中农药残留检测方法,涉及肯定列表中505种化学物质,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,这176个检测方法中涉及蜂蜜检测的很少,方法陈旧，很多方法都是上世纪80年代的,且大多数不适用（都是单残留检测方法）,日本国内实验室也不使用这些方法,日本国内大多采用多残留检测技术,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,目前日本对蜂产品中残留分类,兽药类,农药类,添加剂类,极少,Animal Plant and Food Inspection Center, JSCIQ,目前日本对蜂产品中动物用药分类,兽药分类,氯霉素类,四环素类,硝基呋喃类,氨基糖苷类,大环内酯类,内酰胺类,苯并咪唑类,硝基咪唑类,阿维菌素类,大环内脂类,同化激素类,苯乙胺类,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,农药分类,菊酯类,有机氯类,有机磷类,其它,杀鼠剂类,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,出口日本蜂蜜现在要求检测的重要兽药项目（高风险项目）,-（双氢）链霉素（氨基糖苷类）,氯霉素,硝基呋喃类,喹诺酮类,泰勒菌素、红霉素（大环内脂类）,内酰胺类,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,二、蜂产品中各类抗生素残留检测技术,（高风险类药物）,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,氯霉素（,Chloramphenicol,简称,CAP,）是一种十分有效的广谱抗生素，自从,1950,年来，广泛的应用于食用动物的疾病的预防和治疗。,一、氯霉素检测,目前限量暂时定为,0.1ug/kg,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,至今为止，氯霉素残留仍然在蜂蜜和王浆产品中大量检出,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,Analytical,Technique,Advantage,Disadvantage,HPLC,Quantitative methodology,LOD 1 5,g / kg,LC-MS/MS,Quantitative /confirmatory,At 0.3,g/kg,Expensive / specialist equipment,ELISA,Cost-effective, high-throughput technique,Reproducibility of kits is often variable,Microbial Inhibition,Cost-effective, little specialist equipment,LOD 50 500,g / kg!,Biosensor,Quantitative, sensitive 20 g/kg,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,卡拉胶是从红藻的角叉菜属,(Chondrus),、麒麟菜属,(Eucheuma),、杉藻属,(Gigartina),及沙菜属,(Hypnea),等品种海藻中提取的海藻多糖的统称。,在食品工业中卡拉胶主要用作凝胶剂、稳定剂和持水剂,广泛使用于鸡肉产品和冰淇淋食品中,卡拉胶,(Carrageenan),Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,SEM in carageenan: processing,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,总结,1,、在一些特定的条件下，硝基呋喃类药物和其代谢物具有非指纹性，,存在非人为污染的可能性,2,、测定硝基呋喃类代谢物的方法，液质联用是唯一的选择,3,、内标法是准确定量的必要条件,4,、已有蜂蜜中硝基呋喃类代谢物检测国标方法，王浆中该类药物残留检测方法已经申报，验证阶段,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,三、链霉素检测技术,无限量规定，暂时定为,10ug/kg,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,放射免疫分析法（,CHARM II）,酶联免疫吸附分析法（,ELISA）,高效液相色谱法（,HPLC）,液相色谱质谱联用分析法(,LC-MS/MS),Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,可以初筛，确认方法，柱后衍生荧光测定方法和气质联用方法,目前有液质方法，但必须使用挥发性含氟酸（三氟乙酸和七氟正丁酸）做为流动相使用，保持峰形。,严重影响负离子测定（例如氯霉素），对正离子测定无影响。,目前已有蜂蜜中链霉素检测国标方法，王浆中链霉素检测方法国标已申报，验证阶段,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,Suitable for use as screening assays,MS-MS,LC/GC-MS,HPLC-F,HPLC-UV,ELISA,Microbiological,Discrimination depends on method used,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,MS-MS,LC/GC-MS,HPLC-F,HPLC-UV,ELISA,Microbiological,Suitable for use as confirmatory assays for MRL compounds,Discrimination depends on method used,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,四、蜂产品中氟喹诺酮类药物测定,喹诺酮类（,quinolones,，,QNs,）,药物是近二十年来迅速发展起来的一类十分重要的光谱抗生素，是继磺胺类药物之后人类在合成抗生素方面最重要的突破,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,由于,QNs,属于高极性、高熔点物质，因此无法用气相色谱（,GC,）,方法进行测定。而液相色谱配合不同检测器是目前测定,QNs,类物质的主要方法。根据,QNs,具有强紫外吸收合荧光性质，因此，紫外检测器合荧光检测器是使用最为广泛的检测器。荧光检测方法检测限一般在,10,g/kg,，,能满足多数测定需求，灵敏度比紫外检测方法高,10,20,倍。紫外方法用于制剂、饲料中,QNs,检测，检测波长,254280,nm,。,随着质谱检测器的普及和应用，高效液相色谱串联质谱方法已经成为所有检测方法中的首选。,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,分析,QNs,中需要注意问题,1、通过调节适合的,pH（4-6），,酸碱,LLE,是提取,QNs,的基本方法,，,可以满足一般分析要求,近些年来，固相萃取，尤其是,SCX（,阳离子交换柱）或者,SAX（,阴离子交换柱）广泛使用，利用,pH,调节,QNs,的电荷状况，可以有效去处某些基质干扰。,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,2、仪器分析容易产生残留记忆效应,选择合适的溶剂,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,已经申请蜂蜜和王浆中喹诺酮检测国家标准,Test amount,1,2,3,4,5,6,7,Enrofloxacin,10,10,10,14.3,10,10,10,Ciprofloxacin,10,10,10,16.4,10,14.1,10,Norfloxacin,13.3,120.4,10,86.3,16.4,13.8,71.8,Ofloxacin,10,10,10,10,10,10,10,市场上某些品牌蜂蜜中喹诺酮类残留情况（,ugkg）,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,五、硝基咪唑类残留检测技术,硝基咪唑类（,nitroimidazoles,）,药物是近些年来迅速发展起来的一类十分重要的抗生素，其具有抗厌氧菌感染作用，用来治疗各种动物的球虫病。,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,目前国内外测定硝基咪唑类的仪器方法有极谱法、气相色谱法、气质联用方法、酶联免疫吸附法和高效液相色谱配合紫外检测器和质谱检测器方法。气相方法需要进行衍生化，过程较为繁琐，酶联免疫吸附方法不适合基质复杂样品，同样液相色谱配合紫外检测器尽管灵敏度可以勉强达到欧盟要求，但是对前处理净化要求严格。而液相色谱配合质谱检测器是目前国外首选方法，抗干扰能力强，定性定量准确，完全可以满足目前国内外要求。,目前已经申请蜂蜜和王浆中该类药物残留的国家标准,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,分析硝基咪唑类药物残留需要注意问题,在动物组织中分析目标化合物会不一致,二甲硝,代谢物,目标化合物,异丙硝唑,目标化合物,代谢物,然而，甲硝唑和洛硝哒唑其在动物体内目标化合物仍然是母体,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,在动物组织中存在不均匀性，蜂产品中是否需要测定代谢物，没有定论,分析硝基咪唑类药物残留需要注意问题,在室温下保存迅速分解，甚至在4,（冷藏）,状态下保存,在冷冻保存状态下稳定,可以选择动物血浆作为测定目标组织,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,六、磺胺类残留检测技术,取代基,取代基,已有蜂蜜中10种和16种磺胺类液相、液质国家标准,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,目前，磺胺类药物残留的检测方法主要有,免疫法,微生物法,理化分析法,气相色谱（,GC）,分光光度法,高效液相色谱（,HPLC,）,联用技术（,GC/MS、LC/MS）,薄层分析法（,TLC）,ELISA,CHARM II,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,磺胺种类多，性质差异大，大部分磺胺药物的,pKa,在5-8之间，提取方法各式各样,A、,单一溶剂提取,B、,混合溶剂提取,C、,酸碱缓冲溶液提取,D、,酸碱缓冲溶液+溶剂提取,对于蜂蜜样品，可以通过缓冲溶液固相萃取方式得到比较好的结果,目前，本实验室可以同时测定蜂蜜中17种磺胺类药物残留,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,六、蜂蜜中多抗生素残留检测技术,兽药种类多，单独检测时间长，成本高，目前蜂蜜已经有多农药残留检测技术。需要开发蜂蜜中多兽药残留技术，满足目前出口日本、欧盟等国家要求。,目前可以测定蜂蜜中共,9,大类，,48,种兽药残留同时检测,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,多残留分类,一、四环素类,(tetracyclines),1,、四环素,(tetracycline) 2,、土霉素,(oxytetracycline) 3,、金霉素,(chlortetracycline).,二、硝基咪唑类,(nitroimidazoles),1,、二甲硝咪唑,(dimetridazole) 2,、甲硝哒唑,(metronidazole) 3,、洛硝哒唑,(ronidazole),三、二氨基嘧啶类,(diaminopyrimidines),1,、三甲氧苄氨嘧啶,(trimethoxypyrimidine),四、大环内酯类,(macrolides),1,、竹桃霉素,(oleandomycin) 2,、泰乐菌素,(tylosin) 3,、红霉素,(erythromycin),5,、罗红霉素,(roxithromycin), 6,、交沙霉素,(josamycin) 7,、替米卡星,(timicosin),8,、北里霉素,(kitasamycin), 9,、螺旋霉素,III(spiramycin III) 10,、螺旋霉素,II(spiramycin II),11,、螺旋霉素,I(spiramycin I),Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,五、林可胺类,(lincosamides),1,、林可霉素,(lincomycin) 2,、氯林可霉素,(clindamycin),六、,-,内酰胺类,(-lactam antibiotics),1,、青霉素,G(benzyl penicillin) 2,、青霉素,V(penicillin V) 3,、乙氧萘青霉素,(nafcillin),4,、苯唑青霉素,(oxacillin) 5,、邻氯青霉素,(cloxacillin) 6,、双氯青霉素,(dicloxacillin),七、磺胺类,(sulfonamides),1,、磺胺嘧啶,(sulfadiazine) 2,、磺胺二甲基嘧啶,(sulfamethazine) 3,、磺胺喹噁啉,(sulfaquinoxaline),4,、磺胺,-6-,甲氧嘧啶,(sulfamonomethoxine) 5,、磺胺甲基嘧啶,(sulfamerazine),6,、磺胺间二甲氧嘧啶,(sulfadimethoxine) 7,、磺胺甲噁唑,(sulfamethoxazole),8,、磺胺噻唑,(sulfathiazole), 9,、磺胺吡啶,(sulfapyridine) 10,、磺胺脒,(sulfaguanidine),11,、磺胺醋酰,(sulfacetamide) 12,、磺胺氯哒嗪,(sulfachloropyridazine),13,、磺胺甲氧哒嗪,(sulfamethoxypyridazine) 14,、磺胺二甲异噁唑,(sulfisoxazole),15,、磺胺甲噻二唑,(sulfamethizole) 16,、磺胺对甲氧嘧啶,(sulfameter),17,、磺胺邻二甲氧嘧啶,(sulfamethoxine),Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,八、氯霉素类,(chloramphenicols),1,、氯霉素,(chloramphenicol),九、喹诺酮类,(quinolones),1,、氧氟沙星,(ofloxacin) 2,、环丙沙星,(ciprofloxacin) 3,、诺氟沙星,(norfloxacin),4,、恩诺沙星,(enrofloxacin),Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,母离子和碎片离子信息,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,母离子和碎片离子信息,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,Internal Standard,13,C6-Sulfamethazine,285.2,162(20),D5-Chloramphenicol,326,157(19),D5-Norfloxacin,325.1,281(18),D3-Dimetridazole,145,99(16),使用内标,内标并不做定量使用,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,内标作用,1,、多残留分析需要进行分时间段进行分析，容易出现保留时间漂移，,需要利用内标进行定位,2,、利用内标来判断每次分析是否在正常变化范围以内,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,Sulfaguanidine,Lincomycin,Ronidazole,Ofloxacin,Norfloxacin,Sulfadiazine,Metronidazole,D,3,-dimetridazole,0,2,4,6,8,10,12,14,16,18,20,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,2.60,5.05,6.12,5.98,6.08,6.59,6.39,6.44,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,0,2,4,6,8,10,12,14,16,18,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,6.47,6.47,6.51,6.44,6.55,6.65,6.75,7.37,Ciprofloxacin,D,3,- dimetridazole,Enrofloxacin,Dimetridazole,Sulfathiazole,Tetracycline,Sulfapyridine,Sulfamerazine,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,0,2,4,6,8,10,12,14,16,18,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,7.44,8.06,8.37,8.69,8.70,9.30,9.10,9.10,Spiramycin III,Timicosin,Sulfamonomethoxine,13,C,6,-Sulfamethazine,Sulfamethazine,Spiramycin II,Spiramycin I,Oxytetracycline,Sulfameter,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,0,2,4,6,8,10,12,14,16,18,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,9.00,9.42,9.83,9.92,10.04,10.02,10.11,11.53,8.70,13,C,6,-Sulfamethazine,Chlortetracycline,Clindamycin,Oleandomycin,Sulfachloropyridazine,Sulfamethiazole,Sulfamethoxazole,Sulfadimethoxine,Sulfamethoxine,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,0,2,4,6,8,10,12,14,16,18,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,10.33,10.50,10.59,10.58,11.42,11.72,11.86,17.68,Trimethoxyprimidine,Roxithromycin,Josamycin,Kitasamycin,Erythromycin,Tylosin,Sulfisoxazole,Sulfaquinoxaline,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,0,2,4,6,8,10,12,14,16,18,Time (min),0,50,100,0,50,100,0,50,100,0,50,100,0,50,100,Relative Abundance,0,50,100,0,50,100,0,50,100,12.17,12.13,14.40,15.04,15.60,15.71,16.09,17.01,Dicloxacillin,Cloxacillin,Chloramphenicol,D,5,-Chloramphenicol,Penicillin G,Penicillin V,Nafcillin,Oxacillin,色谱图,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,基质增强效应,在实际分析中保留时间,8min,以内分析物，发现信号数值容易出现成倍增长，一般为加标数值,2-3,倍。,如何解决？,提取标准曲线,Animal Plant and Food Inspction Center, JSCIQ,Matrix Matched Std,Matrix Extracted,Standard,Animal Plant and Food Inspection Center, JSCIQ,Animal Plant and Food Inspection Center, JSCIQ,如果有任何问题，请与我们联系,Fax: 0086-25-52345189,Direct e-mail:,谢谢！,