1、中华人民共和国出入境检验检疫行业标准SN/T 1735-2006 进出口贝类产品中麻痹撑贝类毒素检验方法高效液相色辞辛法Inspection of paralytic shellfish poison in shellfish for import and export High performance Iiquid chromatography 2006皿01-26发布中华人民共和国国家质量监督检验检瘟总局2006-08皿16实施060831000133 b SN/T 1735-2006 前言本标准的附录A为资料性黯录。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
2、和国深圳出入境检验检疫局、中华人民共和国上海出入境检验检疫局。本标准主要起草人:谢丽琪、欧阳姗、周iIE.敏、岳振峰、陈:市金、赵琼晖、方晓明、唐毅峰。本标准系首次发布的出入境检验检疫行业标准。I SN/T 1735-2006 进出口贝类产品中麻痹性贝类毒素检验方法高效液棺色谱法1 括自本标准规定了进出本标准适用于进出2 抽样和制样2. 1 检验批以不超过10000件为一检验批。注:3 测定方法3. 1 方法提要比斗卢/试样中的麻痹性贝类毒素用0.1mol/L的盐酸提取，离心后，将上清敲过C18白相萃取柱净化，再经过10000 D的超滤离心管过滤，滤被用高效液相色谱进行分离，经在线柱后衍生反应
3、后，进行荧光检测，外标法定囊。3.2 试剂租材料除特殊规定外，所用的试剂均为分析纯，7.1.为去离子水。3.2. 1 乙腊z色谱纯。2.2 抽样数最见表10最低抽样数/件冷冻品150及以下35-m 1513 200 3 2011 000 20 2.3 抽样方法按2.2规定的抽样件于4闻，放入清洁容器内2.4 样品的制备从抽取的原始样品的水分，放人搅碎机中2.5 试样保存始样品，原始样品总量不少干净纱布小心地吸去多余祥。密封，并标明标记。1 SN/T 1735-2006 3. 2. 2 100 mmol/L庚烧磺酸铀溶液。3.2.3 6%(体积分数)氨水。3. 2. 4 500 mmol/L磷酸
4、。3. 2. 5 250 mmol/L磷酸氢二抨洛液。3. 2. 6 1. 0 mol/L氢氧化饵溶液。3. 2. 7 500 mmol/L高棋酸。3. 2. 8 1. 0 mol/L乙酸。3.2.9 0.01 mol/L乙酸。3.2. 10 0.1 mol/L氮氧化铀溶液。3.2. 11 标准品:麻痹性员类毒素(PSP): GTX1 ，4、GTX2，3、dcGTX2，3、B1(GTX5) ，neoSTX、STX、dcSTX标准溶液。3.2. 12 标准储备液z将标准榕液用相应的介质溶液稀释成一定浓度的标准储备攘，避光保存于一200C以下，可保存1年。3.2. 13 混合标准溶破:分别准确腹取
5、适量各标准储备液，用0.01mol/L乙酸配成所需放度的海合标准溶蔽。3.3 仪器和设备3.3.1 高效液相色谱仪，配有荧光检翻器，柱后衍生反应装置。3.3.2 离心机，5000 r/min。3.3.3 固相萃取装置。3.3.4 C18国相萃取柱:Sep-PakVac C18 , 3 mLo 3.3.5 10 000 D的超滤离心管。3.3.6 溶剂过滤器和0.45m过滤膜。3.3.7 旋涡振荡器。3.3.8 水浴锅。3.4 击tl定步黯3.4.1 提取准确称取1g试样(精确至0.01g)于10mL具塞离心管中，加入2mL O. 1 mol/L的盐酸榕液，振荡均匀后，用0.1mol/L氮氧化饷
6、调pH至3.0，在调pH时快速搅拌。将离心管置于1000C的沸水浴中，加热5min，冷却后，以5000 r/min离岳10mino 3.4.2 净化将C18圄相萃取柱安装在向相萃取装置上，依次用6mL的申薛和6mL的水活化，取离心得到的上清液过柱，收集流出液，再用约1mL的水洗脱，准确收集2.0mLo取约1mL收集液，于10000D的超滤离心管中离心，滤液供液相色谱测定。3.4.3 测定3.4.3. 1 液相色谱条件2 a) 色谱柱:InertsilC8-3(250 mmX4. 6 mm，5m)，或与之相当。b) 色谱柱温度:30oC。c) 流动相:一一流动相A:3. 0 mmol/L庚皖磺酸
7、铀十8.5mmol/L磷酸镜，pH=7.2:量取15mL 100 mmol/L庚烧磺酸铀溶液、8.5 mL 500 mmol/L磷酸溶液到约450mL水中，用氨水调pH=7.2，用水准确定容到500mL，过0.45m滤膜;一一流动相B:4. 0 mmol/L庚皖磺酸铀十45mmol/L磷酸锻，pH=7.2:量取20mL 100 mmol/L庚烧磺酸铀溶被、45mL 500 mmol/L磷酸溶液到约450mL水中，用氨水调pH=7.2，用水准确定容到500mL，过0.45m滤膜;一一流动相C:乙腊。d) 流动相时间梯度，见表2。表2流动相时间梯度梯度时间/min流动相A/C%)流动相B/C%)流
8、动极c/C%)。100 。20.0 100 。20.5 。93.5 6.5 45.0 。93.5 6.5 45.5 100 。60.0 100 。的柱后衍生条件:SN/T 1735-c-2006 流速/CmL/min)1. 0 1. 0 0.9 0.9 1. 0 1. 0 氧化液:10mmol/L高腆酸十50mmol/L磷酸氧二挥，pH=9.0:量取10mL 500 mmol/L高腆酸、100mL 250mmol/L磷酸氢工锦(K2日P04)溶液，溶解到约450mL水中，用1mol/L氢氧化饵(KOH)溶液调pH至9.0，用水定容到500mL;中和被:1.0 mol/L乙酸。反应温度:50.C
9、;反应液流速梯度(mL/min)，见表3。表3柱后衍生反应搜流速梯度时f /min 反应液。25 25.5 45 45.5 60 氧化液0.4 0.4 0.8 0.8 0.4 0.4 中和液0.4 0.4 0.8 0.8 0.4 0.4 f) 荧光检测:激发波长330nm，发射波长390nm. g) 进样量:10L。3.4.3.2 色谱测定根据样被中麻痹性贝类毒素的含量情况，选定峰面积相近的标准工作截。标准工作夜和样液中麻痹性贝类毒素的响应值均应在仪器检测的线性范围内。标准工作液和样液等体积参插进样测定。在上述色谱条件下，标准品的色谱图参见附录A中图A.1.3.4.4 空白实验除不加试样外，均
10、按上述说!定步骤进行。3.5 结果计算和表述3.5. 1 按式(1)计算样品中各种麻痹性贝类毒素的含最zX=A.c.V 一式中zx一一样品中麻痹性贝类毒素的含量，单位为微克每千克g/kg); A一一一样液中麻痹性贝类毒素的峰面积;As一-标准工作溶液中麻痹性贝类毒素的峰面积;C一标准工作溶液中麻痹性贝类毒素的浓度，单位为微克每升(g/L); V一一样液最终定容体积，单位为毫升(mL);m一一样品的质量，单位为克(g)。注:计算结果需扣除空白值。. ( 1 ) 3 SN/T 1735-2006 3.5.2 毒性转换按照国际惯例，STX毒素的毒性因子被设为1，其他各种麻痹性毒素按照相对于STX的毒
11、性大小来确定毒性因子(见表4所列);样品中麻痹性贝类毒素的含量，按式(2)计算统一转换为STXeq来表示:式中zXi一一各种麻痹性贝ri-一毒性国子。4 部tl定低限和阳收率4. 1 本方法的制定保限为:17.3g/kg;GTX3为6.5STX为14.5g/kg;合4.2 回收率以崩贝和元贝的食用部GTX4浓度在16.7g/五g率在73.6%95. 8%之间。STXeq口I;xi. ( 2 ) 为4.8g/kg;B1为31.9g/kg;dcGTX2为7.3%之间，元贝的因收GTX1浓度在50.7g/kg304. 0g/kg范围，扇贝的因79. 5%91. 3%之间，元贝的回收率在78.3%92
12、. 3%之间。dcGTX3浓度在4.8g/kg-;率在79.2%95. 4%之间。B1浓度在31.9g/kg 在79.8%89. 3%之间。dcGTX2浓度在17.3收率在68.8%98. 2%之GTX3浓度在6.5在71.3%103. 8%之间。GTX2浓度在19.6率在75.2%94. 9%之间。NEO浓度在15.7g/kg 在62.4%96. 8%之间。dcSTX浓度在12.5g/kg75.0 率在78.4%96. 0%之间。92. 4%之间，元贝的回收94.2%之间，元贝的回%之间，元贝的回收率1.8%之间，元贝的因收7.4%之间，元贝的回收率8%96. 0%之间，元贝的回收STX浓度
13、在14.5g/kg87.0g/kg范围，扇贝的回收率在75.2%96. 5%之间，元贝的回收率在74.5%96. 5%之间。4 mV 0 1一一-GTX4;2一一GTXl;3一一-dcGTX3;4一一-Bl;5一一-dcGTX2;6一一-GTX3;7一一-GTX2;8一一-NEO;9一一-dcSTX;lO-STXo 附录A(资料性附录)标准色谱图SN/T 1735-2006 mm 5 SN/T 1735-2006 Foreword Annex A of this standard is an informative annex. This standard was proposed band
14、is under the charge of Certification and Accreditation Administra幽tiOFl of the People s Republic of China. The standard was drafted bShenzhen Entr-Exit Inspection and quarantine Bureau and Shanghai En try而ExitInspection and quarantine Bureau of the People s Republic of China. The main drafters of th
15、is standard are xie Liqi , Ouyang Shan , Zhou Yamin , Yue Zhenfeng , Chen Pei jing, Zhao Qionghui , Fang Xiaoming , Tang Yifeng. This professional standard for entry-exit inspection and quarantine is promulgated for the first time. Note: This English version , translation from the Chinese text , is
16、solely for guidance. 6 SN/T 1735 2006 Inspection of paralytic shellfish poison in shellfish for import and export一High performance liquid chromatography 1 Scope This standard specifies the method of sampling , sample preparation and determination paralytic shell fish poison in shellfish for import a
17、nd export by high performance liquid chromatography. This standard is applicable to the inspection of the paralytic shellfish poison in shellfish products for import and export. 2 Sampling and sample preparation 2. 1 Inspection lot The quantity of an inspection lot should not exceed 10 000 packages.
18、 The characteristics of the cargo within the same inspection I时，suchas packing ,mark,origin,specifi cation and grade etc. should be the sme. 2. 2 Quantity of sample taken See丁able1. Table 1 Quantity of sample taken Nuniber of packages in each inspection lot Minimum number of packages Frozen products
19、 Live or salted products to be taken 150 and below 90 and below 3 151-3200 91-500 5 3201-1 000 501-1 200 13 1201-10000 20 2.3 Sampling procedure A number of packages specif怡din 2. 2 are taken at random and opened one bone . The sample taken as pri-7 SN月1735-2006mary回mplefrom each阳C阳geshould be at le
20、ast 500g. The total weight of all the primary samples should not be less than 4崎.Place in a回mplentainer，seal , label and send to the laboratory in time. 2. 4 Preparation of test sample Take the edible portions from the primary sample. Rinse out filth and sediment. Use filter paper or gauze to suck a
21、wasurplus fc:l:lumidity;Bler:iq-ina-foodrMerldeLa114homogenize thoroughly.Mix well and divide into two equal ples , seal and label. 2. 5 Storage of the test 3.1 Paralytic shellfish poison in the test sample are extracted with O. 1 mol/L hydrochloric acid. Centri-fuge the mixture. The supernatant is
22、cleaned up by umn. The elute is filtrated by 1 with post翩column3. 2 Reagents and Unless otherwise 3.2.1 3.2.2 100 mmol/L 3.2.3 6% ammonia water; 3.2.4 500 mmol/L phosphoric acid solution; 3.2.5 250 mmol/L dipotassium hdrogen phosphate solution; 3.2.6 1.0 mol/L potassium hydroxide solution; 8 solid-p
23、hase extraction C8 col Ifte is determinated by HPLC iI三tandardmethod is used for ater is deionized. 3.2.7 500 mmol/L periodic acid; 3. 2. 8 1. 0 mol/L acetic acid; 3. 2. 9 0.01 mol/L acetic acid; 3.2.10 1.0 mol/L sodium hydroxide; 3. 2. 11 Standards , neoSTX , STX , dcSTX medium solute; 3.2. 13 Stan
24、dard mixture solution: standard solution to confect HAc. 3.3 Apparatus and equi 3. 3. 1 High Performance umn reaction box; 3.3.2 3.3.3 Solid-Phase Extraction 3.3.4 Solid-Phase Extract 3. 3. 5 10 000 Dalton 3.3.6 3.3.7 Vortex mixer; 3.3.8 Water bath boiler. 3. 4 Procedures 3. 4. 1 Sample Extracton SN
25、/T 1735-2006 , 3, B1 (GTX5) , rd solution with relevant reme时，respectively pipette each stock of appropriatencentrations with O. 01 mol/L detector and post-col-Weight accurately 1 9 (to the nearest 0.01 g) of the test sample into a 10 mL centrifuge tube with screw cap. Add 2 mL O. 1 mol/L hydrochlor
26、ic acid into the tube and shake in vortex mixer to mix 9 SN/T 1735 2006 well. Adjust to pH 3. 0 with 1.0 mol/L sodium hdroxide. Put the tube in 100.C water bath for 5 min. After cooling down,centrifuge the mixture at 5 000 r/min for 10 min. 3. 4. 2 Clean-up Place the C8 column onto the Solid-Phase E
27、xtraction apparatus. It is conditioned with 6 mL meth1 al幡cohol and 6 mL water. Pipette the extract solution onto C8 column. Elute the column with about 1 mL water. Collect the effluent and make up the volume to 2 mL with water. Mix well. Pipette about 1 mL of the extraction solution into a 10000 Da
28、lton centrifuge tube. Centrifuge at 5 000 r/min for 10 min. Determinate the filtrate bhigh performance liquid chromatography. 3.4. 3 Determination 3. 4. 3. 1 HPLC conditions a) HPLC column: Inertsil C8-3 (250 mm x 4.6 mm，5m) ,or the equal. b) HPLC column temperature: 30.C. c) Mobile phase: 一一-Mobile
29、phase A: 3.0 mmol/L heptane sulfonic acid sodium salt十8.5mmol/L phosphoric acid , pH = 7. 2: Mix up 15 mL 100 mmol/L heptane sulfonic acid sodium salt solution, 8. 5 mL 500 mmol/L phosphoric acid solution and some water to about 450 mL volume,adjust to pH 7. 2 with ammonia water, make up the volume
30、to 500 mL with water, filter the solution through a 0.45m syringe filter; 一-Mobilephase B: 4.0 mmol/L heptane sulfonic acid sodium salt十45mmol/L phosphoric acid pH = 7. 2: Mix up 20mL 100 mmol/ L heptane sulfonic acid sodium salt solution , 45 mL 500 mmol/ L phosphoric acid solution and some water t
31、o about 450 mL,adjust to pH 7.2 with ammonia wa ter, make up the v91ume to 500 mL with water, filter the solution through a 0.45m syringe fil唰ter; 一一-Mobilephase C: Acetonitrile. d) Gradient time program (see Table 2) Table 2 Gradient time program Gradient time/min Mobile Phase A/(%) Mobile Phase 81
32、 (%) Mobile Phase CI C %) Flow Rate CmL/min) 。100 。1.0 20.0 100 。1.0 20.5 。93.5 6.5 0.9 45.0 。93.5 6.5 0.9 45.5 100 。1.0 60.0 100 。1.0 10 SNjT 1735 2006 e) Conditions of post-column derivatization: Oxidant solution: 10 mmol/L periodic acid十50mmol/L dipotassium hydrogen phosphate: Mix up 10 mL 500 mm
33、ol/L periodic acid , 100 mL 250 mmol/L dipotassium hydrogen phosphate solution and some water to about 450 mL,adjust to pH 9.0 with 1 mol/L potassium hydroxide solution. make up the volume to 500 mL with water; Neutralization solution: 1.0 mol/L HAc; Reaction temperature: 50t ; Gradient time program
34、 of reactant solutin flow rate (mL/min) (see Table 3) : Table 3 Gradient time program of reactant solution flow rate Time/min Solution 。25 25.5 45 45.5 60 Oxidant 0.4 0.4 0.8 solution 0.8 0.4 0.4 Neutralization solution 0.4 0.4 0.8 0.8 0.4 0.4 f) FLD detection: Excitation wavelength 330 nm , Emissio
35、n wavelength 390 nm. g) Injection volume: 10L. 3. 4. 3. 2 HPLC determination According to the .approximate concentration of paraltic shellfish poison in the test sample solution, select the standard working solution with similar peak area to that of the sample solution. The re sponses of paralytic s
36、hellfish poisoning toxin in the standard working solution and sample solution should be within the linear range of the instrumental detection. The standard working solution should be injected randomly in-between the injection of sample solution of equal volume. Under the above chromatographic condit
37、ions,chromatogram of the standard ,is shown as Fig. A. 3. 4. 4 Blank test The operation of the blank test is the same as the described in the method of determination but with the omission of sample addition. 3. 5 Calculation and expression of the result 3.5. 1 Calculate the concentration of paralyti
38、c shellfish poison in sample according to the following equaton: 11 SN/T 1735-2006 Where, X一生二旦二卫一As. m . ( 1 ) X -Concentration of paralytic shellfish poisoning toxin in the test sample，g/kg; A-Peak area of paralytic shellfish poisoning toxin in the test sample; As-Peak area of paralytic shellfish
39、poisoning toxin in working standard solution; C一Concentrationof paralytic shellfish poisoning toxin in working standard solution，问/L;V.，.一Thefinal volume of m一丁hecorresponding 3.5.2 Toxicity Where: X ;-Concentration of r;一丁oxicityfactor. 4 Poisoning Toxins Toxicity factor GTX1 0.99 0.73 4. 1 The lim
40、it of dete GTX1 is 50. 7g/kg; d 6.5g/kg; GTX2 is 19. 4. 2 Recovery According to the experiment corresponding recoveries are as toxin was established to 1.丁heve toxicity to STX (Table 4). The ured as STXeq. Calculation equation: .( 2 ) NEO STX dcSTX 0.92 0.51 for GTX4 is 16. 7g/kg; is 17.3g/kg; GTX3
41、is ; STX is 14.5g/kg. Iytic shellfish poison and their GTX4 between 16. 7g/kg-100.0g/烟，recoveriesin scallop are between 76. 6% -97.3% , recov eries in formosum are between 73. 6% -95. 8%. GTX1 between 50.7g/kg-304.0g/闹，recoveriesin scallop are between 79. 5% -91.3% ，recov峭eries in formosum are betwe
42、en 78. 3% -92.3%. 12 SN/T 1735-2006 dcGTX3 between 4. 8g/kg-28.8g/闸，recoveriesin scallop are between 81.7% -92. 4% , recov eries in formosum are between 79.2% -95.4%. 81 between 31. 9g/kg-191.0g/闸，recoveriesin scallop are between 78. 1 % -90.3% , recoveries in formosum are between 79.8% -89.3%. dcGT
43、X2 between 17. 3g/kg-104.0g/闸，recoveriesin scallop are between 69.4% -94.2% , recoveries in formosum GTX3 between 6. 5 ies in formosum are GTX2 between 19. 6 ies in formosum are dcSTX between 12. 5 eries in formosum are STX between 14.5 ies in formosum are between 74. 5%-96. 5%. 71.7% -96.9% , recov
44、er-75.0% -91.8% , recov-between 67. 5 % - 97. 4% , recover嗣72. 8% -96. 0% , recov-75.2% -96.5% , recover幡13 SN/T 1735-2006 mV T 40 30 20 10 。1一-GTX4;2 -GTX1; 3一一dcGTX3;4一-81;5一-dcGTX2;6一-GTX3;7一一G丁X2;8一NEO;9一-dcSTX;10一一-STX.10 2 34 Annex A Clnformative annex) Chromatogram of standards 5 7 8 20 30 40
45、 50 Figure A. 1 Chromatogram of paralytic shellfish poison standards 14 -一一-口unOONimEH军中华人民共和国出入境检验检疫行业标准进出口贝类产品中麻痹性贝类毒素检验方法高效液相色谱法SN/T 1735一2006争号中国标准出版社出版北京复兴口外三旦河北街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1.25 字数27千字2006年5月第一版2006年5月第一次印刷印数1一2000定价12.00元争夺书号:155066 2-16835
