1、中华人民共和国出入境检验检疫行业标准SN/T 1736-2006 进出口蜂蜜中黄曲霉毒素的检验方法高效液相色谱法Inspection of afIatoxins in honey for import and export High performance Iiquid chromatography 200601-26发布中华人民共和国国家质量监督检验检疫总局2006皿08-16实施nRn71 Annnnn7 目白自本标准的酣录A为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国河南出人境检验检疫局。本标准主要起草人z杨冀州、刘朝晖、刘亚风、郭俊峰、李全
2、滋。本标准系首次发布的出入境检验检疫行业标准。SN/T 1736-2006 I SN/T 1736-2006 1 寇围进出口蜂蜜中黄曲霉毒素的检验方法高效液相色谱法本标准规定了进出口蜂蜜中黄曲霉毒素检验的抽样、制样和高效液相色谱法的测定方法。本标准适用于进出口蜂蜜中黄曲霉毒素Bl、民、G1和G2的检验。2 规范性引用文件下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注日期的引用文件，其随后所有的修改(不包括勘误的内容)或修订般均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。SN/T 0852 进出口蜂蜜检验方法3 抽样和制样抽样和制样按SN/T0
3、852的规定进行。4 测定方法4. 1 方法提要试样中的黄曲霉毒素Bl、岛、G1和G2用甲薛十水(7十3)提取，提取液经稀释、过滤后，用免疫亲和柱净化。带荧光检测器和柱后衍生装置的高效液相色谱仪测定各种黄曲霉毒素，外标法定寰。4.2 试剂和材料除另有规定外，所用试剂均为分析纯，水为二次蒸锚水。4.2. 1月3事。4.2.2 甲醇，HPLC级。4.2.3 氧化铀。4.2.4 It!事十水(7十3):取700mL甲酶(4.2.1)如300mL水。4.2.5 黄曲霉毒素免疫亲和柱。4.2.6 黄曲霉毒素标准品黄曲霉毒索马、马、G1、G2):纯度二三99%。4.2.7 黄曲霉毒素标准储备液z用甲薛(4
4、.2.2)分别配器tlO. 100 mg/mL的黄曲霉毒素Bl、B2、G1、G2标准储备液，冷藏备用。4.2.8 黄曲霉毒素:昆合标准工作液z准确移取适量的黄曲霉毒素矶、岛、G1、G2标准储备液，用甲薛(4.2.2)稀释成?昆合标准工作液。4.2.9 校后衍生洛液(0.05%腆溶液):称取0.1g碟，溶解于20mL申自事后，加纯水定容至200mL，以0.45m的尼龙滤膜过滤，冷藏避光保存。4.3 仪器和设备4.3. 1 高速均质器。4.3.2 高效液相色谱仪，配有荧光检测器和柱后衍生化装置。4.3.3 玻璃针筒:20mL。4.3.4 空气压力泵。1 SN/T 1736 2006 4.3.5 玻
5、璃纤维滤纸:直径11cm，孔径1.5m。4.3.6 针筒式滤膜:0.45Ino4.4 测定步骤4.4.1 提取称取试样约25g(精确至O.1 g)于250mL均质杯中，加人5g氧化铀及125mL甲喜事十水(4.2.的，以均质器高速搅拌提取5min，取15mL提取攘，加人30mL水，混匀。以玻璃纤维滤纸过滤至滤被澄清。4.4.2 免擅亲和柱净化将免疫亲和柱连接气压力菜与针筒上端连4.4.3 测定4.4.3.1 色谱条件的色谱柱:C18,150 mmX4. 6 b) 流动相z甲薛十水(45c) 流动相流速:0.d) 荧光检测器:激e) f) 一衍生溶液:0: 一一衍生溶液流速:0.2mL/min;
6、 一一反应管温度:700C;一一反应时间:1 mino 4.4.3.2 色谱测定根据样液中黄曲液和样液中黄曲霉毒素岛和样液按上述条件等体间分别约为9.30min、74.4.4 空白试验除不加试样外，均4.5 结果计算和表述用数据处理装置或式中z样)加入玻璃针筒中，将空，再使少量空气通过柱体。1 mL甲醇(4.2. 2)进行洗m滤膜过滤供高效液相.( 1 ) Xx一试样中黄曲霉毒素(B也域也2)含章，单链翠赞菊l每千克g/kg);2 Ax -样液中黄曲霉毒素(B1、B2、G1或G2)的峰高或峰面积，单位为毫米或平方毫米(mm或mmZ); As-一一混合标准工作液中黄曲霉毒素(B1、马、G1或G2
7、)的峰高或峰顶积，单位为主革米或平方毫米(mm或mm2);C一一混合标准工作液中黄曲霉毒素(B1、B2、G1或GZ)的政度，单位为微克每升(g/L);V一一一样液最终定容体积，单位为毫升(mL); 十m一一最终样液所代表的试样量，单位为克(g)。注:计算结果需扣除空白值。5 甜定低限、自收率5. 1 测定低限本方法测定低限:黄曲霉毒素Bl、B2、G1和G2均为0.5g/kgo5.2 黯收率蜂蜜中黄曲霉毒素黄曲霉毒素Bl Bl Bl Bl Bz Bz Bz Bz G1 G1 G1 G1 G, G, G, G, SN/T 1736一2006回收率90.2% 88.6% 92.2% 87.5% 85
8、.3% 88.6% 91. 7% 90.1% 87.2% 86.8% 86.2% 85.0% 89.2% 91. 3% 92.6% 90.5% 3 SN/T 1736 2006 附录(资料性附录标准品色谱圈A O版时附货运叙LU t/min 12 10 Mm税带能组织8 6 剧。假陆明州回去氧2 4 2 0 5.5 4 2.5 3.5 3 5 4.5 黄曲霉毒素B1、B2、G1和G2标准品的液相色谱图回A.14 SN/T 1736-2006 Foreword Annex A of this standard is an informative annex. 了hisstandard was p
9、roposed and managed by Certification and Accreditation Administration of the People s Republic of China. This standard was drafted by Henan Entry幡ExitInspection and Quarantine Bureau. The standard was mainly drafted by Yang jizhou,Liu Zhaohui ,Liu Yafeng ,Guo Junfeng ,Li Quanzi. This standard is a p
10、rofessional standard for ent叩-exitinspection and quarantine promulgated for the first time. 5 SN/T 1736-2006 export-Inspection of aflatoxins in honey for import and High performance liquid chromatography Scope export. 2 Normative SNj丁0852Inspection in 3 Sampling and Sampling and sample 4 Method of 4
11、. 1 Principle The aflatoxin 81、82、G1tion and filtration, the extract is fluorescence detector, using external standard method. 4.2 Reagents and materials determination of aflatoxin 而,82 ,G1 and G2 in honey for import and 十3).After dilu and determined bHPLC with Unless otherwise specified ,all the re
12、agents used should be analytical grade and water is distilled water. 6 阻四四4. 2. 1 Methanol. 4.2.2 Methanol: HPLC gra.de. 4.2.3 Sodium chloride. 4.2.4 Methanol-water 4.2.5 Aflatoxin 4.2.6 4.2.7 G1 and、G2standard , dissolve in spectiveland keep in 4.2.8 Mixed standard working 4. 2. 9 Postcolumn 4.3 In
13、strument and equipment 4. 3. 1 High speed blender. 4.3.2 HPLC equipped 4. 3. 3 Glass syringe: 4.3.4 Air pump. 4.3.5 Glass 4.3.6 4. 4 Procedure 4. 4. 1 Extraction SN/T 1736-2006 amount of aflatoxin 81、民、methanol ,add 200 mL wa-Weigh 25 9 Caccurate to O. 01 g) of the test sample into 250 rilL blender
14、jar and add 5 9 sodium chlo ride nd 125 mL methanol-water, blend 5 minutes at high speed. Pipet 15 mL filtrate mixed with 30 mL water. Filter diluted extract through glass microfiber paper until the filtrate is clear. 7 SN/T 1736-2006 4.4.2 Depuration of immunoaffinity column Connect the immunoaffin
15、itcolumn to a 20 mL glass sringe. Pipet 15 mL second filtrate (equivalent to 1 9 test portion) into syringe reservoir, connect reservoir to pump, push extract through column at flow rate of 1 mL/ min , and pass 2-3 mL ai r through column , then push 10 mL x 2 water through column ,discard water wash
16、ings ,and then pass 2-3 mL air through column. Add 1 mL methanol (4.2. 2) to reservoir,push methanol through column at flow rate of 1 mL/min. Measure 2mL of eluate b HPLC purified watar, and filter it with O. 45m film for HPLC determination. 4.4.3 Determination 4. 4. 3. 1 HPLC operation condition a)
17、 Column: C18 , 150 mm? 4.6 mm，5m. b) Mobile phase: methanol-water (45+55). c) Flow rate: 0.8 mL/min. d) Fluorescence tester: excitation wavelength,360 nm ,emission wavelength ,425 nm. e) Injection volume: 50L f) Postcolumn derivatization sstem: -Derived solution: 0.05% iodine solution. -Flow rate: 0
18、.2 mL/min. -Reaction tube temperature: 70oC. 一-Reactiontime: 1 min. 4. 4. 3. 2 HPLC determination According to the approximate concentration of aflatoxin矶、82、G1and G2 in the sample solution , se lect the standard working solution with similar peak value to that of the sample solution , the respon se
19、s of aflatoxin 81、良、G1and G2 in the standard working solution and the sample solution should be within the liner range of the instrumental detection, the standard working solution should be unevenl injected in唰betweenthe injections of the sample solution of equal volume. Under the above chroma togra
20、phic condition, the retention time of aflatoxin 81、昆、G1and G2 is 9. 3 min ,7. 5 min,6. 6 min ,5. 2 min respectively. See fig A 1 in annex A for chromatogram of the aflatoxin 81、也、G1and G2 standard. 8 SN/T 1736-2006 4. 4 81ank test 8esides the omission of sample addition , the operation of the blank
21、test is the same as. the determi nation procedure described above. 4. 5 Calculation and expression of result The content of aflatoxin 8，、82、G，and G2 in the test sample shall be calculated bHPLC data proces sor or according to formula (1) : V一-m c-iA A一X ( 1 ) Where , X一thecontent of aflatoxin 8，、民、G
22、，and G2 residues in the test sample，g/地;Ax-the peak value of aflatoxin 8，、82、G，and G2 in the sample solution, mm or mm2 ; As一一thepeak value of aflatoxin矶、民、G，and G2 in the standard working solution,mm or mm飞c-the concentration of aflatoxin 昆、82、G，and G2 in the standard working solution，月/L;V-the fin
23、al volume of the sample solution,mL; m-the corresponding mass of the test sample in the sample solution , g. Note: the blank value should be subtracted from the above results of calculation. 5 Lower limits of determination and recovery 5. 1 Lower limit of determination The lower limit of determinati
24、on of this method for aflatoxin 昆、民、G，and G2 is O. 5g/kg respec唰tively. 5. 2 Recovery According to the experimental data , the fortifing concentrations of aflatoxin 8，、民、G，and G2 in hor卜ey and its corresponding recoveries are as following (see Table 1). 9 SN/T 1736-2006 丁able1 Experimental data Afla
25、toxin Fortifying concentrations/ (g/问Recovery 81 0.5 90.2% 日21.0 88.6% 81 2.0 92.2% 81 5.0 87.5% 82 85.3% 82 88.6% 82 91.7% 82 90.1% G1 87.2% G1 86.8% G1 86.2% G1 85.0% G2 89.2% G2 91.3% G2 92.6% G2 90.5% 10 四四川SN/T 1736-2006 Annex A. Clnformative annex) Chromatogram of standards LU 5.5 5 4.5 4 3.5
26、3 2.5 2 . 。呵。旦时。首回闰NOEHO苍白2 4 6 8 10 12 tlmin Figure A. 1 Chromatogram of the aflatoxin 81、良、G1and G2 standard OON|的hFH军中华人民共和国出人撞检验检疫行业标准进出口蜂蜜中黄曲霉毒素的检验方法高效波相色谱法SN/T 1736-2006 9号中国标准出版社出版北京复兴门外三里河北街16号邮政编码，100045网址电话，6852394668517548 中国标准出版社秦皇岛印刷厂印刷* 开本880X 1230 1/16 印张1字数20千字2006年5月第一版2006年5月第一次印刷印数1-2000定价10.00元争夺书号，155066 2-16836 1736 2006
