【商检行业标准(SN)】 进出口贝类产品中麻痹件贝类毒素检验方法高效液相色游法

中华人民共和国出入境检验检疫行业标准SN/T1735—2006
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
Inspecion of paralytic shellfish poison in shelffish for import and export-Highperformanceliquid chromatography2006-01-26发布
中华人民共利国
国家质量监督检验检疫总局
数码防伪
2006-08-16实施
长标准的附录A为资料性附录.
本你滩山国家认证认可股警伴妇委员会据出并：SN/T1735-2006
本尔雅越草中位：中华人民其和压深州出人境检验检疫局中华人民！和国上海出人境份验检局
标准主婴起中人：谢前其，欲朗娜、虎平、乐长峰陈萍令，赵期晖、方晓明、唐毅峰，本标非系首次发有的用人华检验检变行业标准１范围
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
S>/T 1735—2006
本标准规定了进山口贝类产品中麻痹性贝类毒索检验的抽样，制样和液色谱检测方法：本标准适用丁进旧口贝类产品中麻痹性贝类毒索的检验。2抽样和制样
2.1检验批
以不趋过0000件为-检验让
司检验批的鹿品应具有和同的待征，如包装、标识、产地、规格和等级等，2.2抽样数量
见表1
表1抽样数
批量/件
冷沟品
15及以下
1:1--$ 200
$ 20--I t
2.3抽样方法
活品、盐藏品
90及以下
601~-1 230
1 201---10 000
景低样数件
接2.2规定的拥件数随机抽取，邀件开启。从每件中抽取500为原始栏品，原始样品总量不少于4kg·放人清法游器内，加封后，标明标记及时送交实验室，2.4样品的制备
焦油取的原始详品中取可食用部分.用消水冲去污物利泥沙，用滤纸或者净纱布小心地及大多余的水分，放人搅碎机中搅碎至约浆状，均分成两份，装人活的容器内，作为诚样，穿封，并尔叫标记2.5试样保存
将试样”-【8以下避光保在
注：在抛拌和制过程中，必须阴止样品受到污染或发生残留物今量的变化3测定方法
3.1方法提要
试样中的麻察性贝奇素用0.1mo的缺酸提取，离心后.洛上清液其固举取部化：经过1C1的超滤离心管过滤·滤液用高效液性仁谱进分离.终在线杆厅衔么反成而，进行蒙光检测，外标法定量。
3.2试剂和材料
除特殊规定外.所用的试剂均为分析绝，小为去离了水3.2.1乙：色胖纯、
SN/T 1735—2006
3.2.2100 n.mal/1.灰烷磺钠游液。3.2.35述积分数)氢水
3.2.45comma/1.磁酸
3.2.52501m1.谜酸氢—钟溶被
3.2. 61.0 rmo/1. 氢氧化钾溶液，3.2. 7 500 mmo/L 碘酸,
3. 2.81. 0 fmol/1. 乙酸
3. 2. 9 α. 01 mol/L 乙.
3.2.101.1mrl/[.氯氧化钠溶液
3.2.11标准品：联痹性贝美素素(PSP):GTXI-4(TX2.3、dcGTX,3、BI(TX5)、n0SIX,STXtlcsIX标准溶液.
3.2.12标准储备液：将标准落液而相应的介质落液稀释成--定浓度的标准储备液避光保子于一20以下，保存「年。
3.2.13混合标准落液：分别确吸取适量各标准储备液，门0).01m3./1.乙酸配成所诺浓度的混介标准溶液。
3.3仪器和设备
3.3.1高效液相免谱仪.配行荧光检测器戏后衍生反成装置3. 3.2 离心机, 000 /mimg
3.3.3固举取装置
3. 3. 4 C,后,相取社:$ep Pak Var: ml.3.3.510（00D的超离心管
3. 3. 6溶剂过滤幕和0. 415 m过滤膜。3.3.7旋涡振荡器
3.3.8 水浴锅。
3.4测定步骤
3.4.1提取
消确称收1或样精确至0.0g于0ml.具塞离心管中加人2m0.(l/L约动酸游激，振落均匀所.用.1mol/1氧氧花纳调pH至3.C.在调时快速搅半，将离心管置示0nT.的游水浴中,加热5 in.冷部质.以5 0oc r/nin离心19 min。3. 4. 2净化
将（固相翠最托家装在同村萃最装置工，依次用m的印爵和6m.的水活化-最离心得到的清液收集流液.可用约11:.的水洗脱.准确收集2.0mL.约1ml.救集液，一10300的超滤离心管中离心滤液供波相色谱测定3.4. 3测定
3.4.3.1液相色谱条仆
色谱样：netsilc8-3（25mm人4.6mm5μm）.或之相当。色谱柱视度：3C
流动相：
流动相A：3.0mmo/L庭烷磺酸钠18.5mml,1磷酸铵.1H-7.2：吊及15ml1xmmol/1.质烷顽酸钢溶液，8.5nl.500mmol/L磷酸溶液到约=!0ml.水中.川氨水试PH7.2,用水准确是穿到5C0 m过0.43 m滤膜;一流动相B：=.0nmol/1.灰烷破较钠一45mmol/1.磷骏.pll=7.2：量取20m:1.lonr1ol/胰烧碱酸纳辫波，45ml.5ommol/L酸溶液到约0ml.水中-用氢水调pH7.2，用水准确定容到50>ml..过0.15um滤膜；流动相 (., Z.腈。
流动和时间梯度，见表2。
表2流动相时间梯度
度时间irt
e）柱后程生条件：
流动相4/死）
流对相B/(%）
SA/T1735—2006
流速：imlmin
氧化液1cnmol/L高碘酸5ommel/L磷酸氢二钟，pH-9.0：层取1oiml50)n.nol1.高碘度、100ml.250nmmi/.磷胶氢二钮（KHP,)溶报落解到约40ml.水.月1m:/1.餐氧化K()溶没调H9.C.用水定容到500中利液：1.0niolA.乙酸反应温度：50%：
反成液流速筛度（mL/mn），见表3表3柱后衍生反应液流速梯度
反成院
n）茨光检测：激发波长330nm.发射波长390mm。）进样量：10m
3. 4. 3.2色谱测定
问/min
根据样液中麻州贝美再素的含量情况，选定峰向积村近的标准工作液：标准工作液和样液中乐性贝奖能素的响成值均成在仪登检测的我性范画内，标准！作液和样液等体积态拥选样测定，在！述色谱条件下标准品的色谱图参见附录A中图A1。3.4.4空白实验
除不加试样外，巧按「述测定少骤逆行3.5 结果计算和表述
3.5.1按武（1)算样品各种麻痹类素的今量：A.r.V
A，·21
样品中麻痹门灭类声素的含章.中位为微克等于克（gkg）：A
详液中成痹性叫类毒素能峰积
一标准小作溶液中麻蜕洋贝类毒素的峰面积：标准「作溶液中版痹准贝类声素的浓发，单位为微克衍升（）：V—-样液最终容体积.单位为升（ml.)：..
择品的质量.单位为克g)、
注：计算诗求步扣除空、
SN/T 1735--2006
3.5.2毒性转换
按照国际惯例.STX毒案的再性因了被设为I，其他各种麻痹性静素按照相对于STX的母性大小来定性子（见丧1明列）；样品中麻码性贝奖毒繁的含宽，按式（2计算统一转换为$X来表示：
?，各种麻痹性贝类毒素的含量；r
海性菌子
毒性子，
表4麻痹性贝类毒素毒性因子
4测定低限和回收率
4. 1本方法的测定低限为
dGIX3 GTX5(R1)
GTX416.7μg/kg:GTX1为50.7μg/kg;dcX3为4.8\g/kg;11为31.9μg/kg:lcGTX2为17.3 ng/kg:GTX3 为 6.5 μg/kgiGTX2 为 1n. 6 μg/kg:NE() 为 15. 7 μg/kg;desIX 为 12. 5 μg/kg?sTx为1l.5jg/kg+合sXeg为125μg/kg，4.2回收率
以扇贝和元贝的食们部分为基质进行添加回收实验，添加浓度及回收率的实验数据：(Tx1浓度在16.7ug/kg100.0Mg/kg范用，扇贝内可改率在76.6~97.3淤之间，元成的回收率在73.6斤 ~95.8%之间
G1X1浓度在5C.7%/k~-304.0/kg范围，扇贝的回收率在795%~91.3%之间，元贝的同妆率在78.3%~92.3岁之间，
d:GTx3浓度在4.8g/kg~28.8ug/k范围，前贝的回收率在81.7%~92.4%之间，范贝的凹收率在70.295.4次之间.
31浓度在31./kg~191.0g/kg范用，扇贝的回收率在7名.1%～90.3%之间，光以的回收率在79.8岁~89，3次之间
deX2浓度在17.3g/k～104.0g/kg范围，扇贝的可收率在69%~-94.2之间，元贝的网收率在68.8%98.2%之问。
(:TX浓度在6.5/kg～39.0mg/kg范围，扇贝的同收率在71.7%～96.9乐之间，月贝的回收率在71. 3头~. 03. 8%之[,
G1x2浓度在19.6ug/kg~118.0u/k范闹，扇贝的回收率在75.0%~91.8头之间，元贝的团收率在 75. 2%~94. 9 3之间 c
NF0浓度作15.7u/kg~94.2μg/kg范围，扇贝的问收率在67.5%~97.4%之间，元贝的四收率在 62. 4% -~96. 8%之闻 -
de5TX涨在12.5kg~-7.0μgkg范五，扇贝的回收率在72.8岁-96.0为之间·元贝的间孜率在 78.4%-~96. 0端之间,
SX浓度在14.5k87.01g/kg范，最贝的叫收率在75.2%-~96,5%之问，质的回收率在74,5系~96.54之间
3-----i0-X3;
-------deGTX2.
+----t+1x2.
R---.- NED:
- --:deS7X:
附录A
（资料性谢录）
标准色谱图
图 A.1麻性贝类毒素标准色谱图SN/T 1735—2006
SN/T1735--2006
Foreword
Aninex A of this standard is an informative annex.This standard was proposed py and is tinder the charge of Certification and Accrtditation Administra-tion of tho Peaple's Republic of Chiria.The standard was drafted by Shenzhen Entry-Exit Inspection and quarantine Bureau and Shanghai Entry-Exit Inspection and quarantine Bureal of the People's Republic of ChinaThe main drafters of this standard are xie Liqi.Ouyang Shan.Zhou Yamin.Yue Zhenfeng,Chen Pei-jing.Zhao Qionghui.Fang Xiaorning, Tang Yifeng.This professional standard for entry-exit irispection and quarantina is promulgated for the first time.Note: This Engish versican.transiation from the chinese iext, is sololy for cuidarce,1
SN/T 1735-2006
Inspection of paralytic shellfish poison inshellfish for import and export-Highperformanceliguid chromatographyThis standard specifies the method of sampling,samplc prearation and determination paralytic shell-fish poison in shellfishi for import and export by high performance liquid chromatography.This slaridard is applicable to the inspection of the paralytic shellfish poison in shellfish products forimport and export.
Sampling and sample preparation2
2.1Inspection lot
Tnequantityof an inspectionlotshouldnotexceed 10oo0packages.The characteristics uf the cargo within the sane inspection lot.such as packing,mark.origin.specifi-cation and grade etc. should be the same,2.2 Quantity of sample takenSee Table 1.
Table 1 Quantity of sample takenNiumper of packages in eachi inspection lotFrozen products
150 ano oelow
151~3200
3 201 ~ 1 000
2.3 Samplingy procedure
Live ar salted prodicts
90 and below
91~500
501~1200
1 201 -- 10 000
Minirnum nuirnber uf packagesto ae takeri
A number of packages specified in 2. 2 are taken at random and cpened one by one . The samplc takcn as pri-SN/T 1735—2006
mary sample from each package should be at least 50og. The total weight of all the prirrary sarmples shouldnot be less than 4 kg. Place in a sample container, seal. label and send to the laboratory in time.2. 4 Preparation of test sampleTake the edible portions from the primary sample, Rinse out filth and sediment. Use filter paper orgauze to suck away surplus of humidity. Blend in a food blender and homogenize thoroughly. Mix welland divide into two equal portions,place in clean containers as the test samples , seal and label.2, 5 Storage of the test sampleThe test sample shauld be stored below - 18C and avoided direct contact witl light.NOTE : During sampling anid preparation of sample, precautions must be taken to avoid contamination ot any factars thatmay cause the change of residue content.3Method of determination
3. 1Principle of determinationParalytic shellfish poison in the test sample are extracted with o. 1 mol/L hydrochloric acid. Centri-fuge the mixture. The supernatant is cleaned up by passing through solid-phase extraction Cis col-umn. The elute is filtrated by 10 000 Dalton centrifuge tubes. The filtrate is determinated by HPLCwith post-column derivatization and fluorescence detection. External standard method is used forquantitative measurement.
3.2 Reagents and materials
Unless otherwise specified,all reagents used should be of analytical grade. Water is deionized.3.2.1Acetonitrile,HPLC grade;3.2.2100mmol/Lheptane sulfanicacidsodiumsaltsolution;3.2.36%ammoniawater;
3. 2. 4 500 mmal/L phosphoric acid solutian:3. 2. 5 250 mmol/L dipotassium hydrogen phosphate solution;3.2.61.0 mol/L potassium hydroxide solution8
3. 2. 7 500 mmol/ L periodic acid31. 0 mol/ L acetic acid:
3. 2, 9 0, 01 mol/L acetic acid:3. 2. 10 1. 0 mol L sodium hydroxidetSN/T1735—2006
Stardards:paralyticShelfishPoison(PSP):GTX1.4,GTX2.3,dcGTX2.3.B1GTX5)3. 2. 11
neoSTX,STx.desTX standard salution;Stock standard solution: dilute standard solution to stock standard solution with relevant3.2. 125wwW.bzxz.Net
medium soluite:
Standard mixture solution; according to the requirement.respectively pipette each stockstandard solution to corifect stancard mixture solution of appropriate concentrations with o. o1 mol/LHAc.
3. 3Apparatus and equipment
3. 3. 1 High Performance Liquid Chromatogreph equipped with fluorescence detector and post-col.umn reactian box;
Centrifuge, with rotating speed of 5 ooo r/min:3. 3.2［
Solic-Phase Extraction apparatusSolid-Phase Extraction column: Sep-Pak Vac Cia ,3 mlt3.3.4
10 000 Dalton centrifuge tube :Filter Holder.0.45 μm syringe filter:Vortex mixer:
Water bath boiler.
Procedures
Sample Extraction
Weight accurately 1 g (to the nearest 0. 01 g) of the test sample inta a 10 mL centrifuge tube withscrew cap. Add 2 mL 0. 1 mol/L hydrochioric acid into the tube and shake in vortex mixer to mixSN/T 1735—2006
well, Adjust to pH 3, 0 with 1. 0 mol/L sodium hydroxide. Put the tube in 100 C water path for 5 mir1.Aftercoolingdown.centrifugethemixtureat5o0or/minfor10min.3.4.2
Clean-up
Place the Cre column onto the Solid-Phase Extraction apparatus, It is conditioned with 6 mL methyl al-cohol and 6 rmL water. Pipette the extract solution onto C- column. Elute the column with about 1 mlwater. Collect the effluent and make up the volume to 2 ml with water. Mix well. Pipette about 1 miLof the extraction solution into a 10 000 Dalton centrifuge tupe, Centrifuge at 5 0a0 r/min for 10 mirn.Determinate the filtrate by high performance liquid chromatograply3.4.3Determination
3.4.3.1HPLCconditions
a)HPLC column: Iinertsil C8-3 (250 mm × 4. 6 mm.5 μm) ,or the equal.HPLC column temperature: 3o'℃.b)
Mobilephase:
Mobile phase A: 3, 0 mmal/L heptane sulfonic acid sodium salt - 8. 5 mmol/ L phosphoric acid.pH = 7, 2: Mix up 15 mL 100 mmol/L heptane sulfonic acid socdium salt soluition, 8. 5 mL500 mmol/L phosphoric acid solution and some water to about 450 mL volume, adjust to pH 7. 2with ammonia water, make up the volume to 5oo ml with water, filter the solution through a0.45μmsyringefilter:
-Mobile phase B: 4. 0 mmol/L heptane sulfonic acid sodium salt + 45 mmol/L phosphoric acidpH=7.2: Mix up 20mL 100 mmol/L heptane sulfonic acid sodium sall solution.45 rmL 500 mrnof/ Lphosphoric acid solution and some water to about 450 mL.adjust to pH 7. 2 with amrnonia wa-ter,make up the volurme to 5ao rnL with water,filter the solution through a 0.45 μn syringe fil-ter:
Mobile phase C: Acetonitrile.d)
Gradient time program (see Table 2)Table 2
Gradient time program
Gradient time/minMavilePhaseA'(%)MobilePlhaseB:(%)0
Mobile Phase C': % )
Flnw Rate ImLimin)
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