【商检行业标准(SN)】 进出口大豆、油菜籽和食用植物油中赭曲霉素A的检验方法

中华人民共和国出入境检验检疫行业标准SN/T1746—2006
进出口大豆、油菜籽和食用植物油中赭曲霉毒素A的检验方法
Inspection of ochratoxin A in soybean.rapcsecd/canolaand edible vegetable oils for imnport and export2006-01-26发布
数码防伤
中华人民共和国
国家质量监督检验检疫总局
2006-08-16实施
本标雅的陆录A为究容性附录
本标准主国家认证认可监督管理委员会提出并门：SN/T1746-2006
本标雅起草单位：中华人民共利再上海出人境检验检疫局、北京中治维康技术有限公商、工游博尼生物科技有限公司，
本标主安起牌人：褚决华、郭德华、土敏、上雄、沈建英，本标准系百次发布的出人境检验检疫行业标准。rHhttn./afnodmetor
1范围
进出口大克油菜籽和食用植物油中赭曲霉毒素A的检验方法
SV/T 1746—2C06
本标谁规是「进山厂大豆、油莱籽和食用植物油中赭曲霉毒素A含量检验的仙样、制样.以及免亲和杆层析净化商效波相色谱法和免疫亲和柱层析净化荧光光度法测定大，油束打和食用虐物疝中由蒂赫素A的方法。
浓标准适用丁进山大,剂策籽和食川杜物汕中猪曲霉毒素A的检验。2规范性引用文件
下列文伴中的条款过过容标准的引用成为本标准的条款，凡是注川划的州伴，其随而所有的修攻单（不包抵助误的内容)成修收版均木适用于木标，然而·竣厕根据术标准这成协议的各方研究是节可便这些文件的最新版本。凡是不注H期的叫用义件.其最新版本适用十本标GH,T21植物泪脂检验取样、样法S>T(80.）进出口粮油、饲料检验抽样和制样方法3抽样和制样
3.大谢菜籽:按照 SN：「081的规楚决行3.2食币植物消：接照G552的规定热4免疫亲和柱层析净化高效液相色法‘第一法）4.1方法提要
试详丝过甲醇中水提取，提取蔽经过滤、稀释后：滤液经过含有黏函霖导素特只抗任的疫和层析净化·以印醇洗脱，洗聪液供非有荧光检测器的高效液相色谱仪测定，外标洪定量，4.2试剂和溶液
除引有规定外.所埔试剂均为分斯纠水为垂燕水，4.2.1#：鱼谱
4. 2. 2 称--水(2--2):取 80 ml. F醇卵 20 ml水。4.2.3鼠化钠
4.2.4碳梭氧钠。
4.2. 5 (t温 20iTween 20),
浒洗缓冲液：称取25名氯化钠5g碳酸氢钠漆于水中,加人0.1m川祖23,而水称释11.4.2.7乙猎：色谱纯，
4.2.8磷腰钢
1兴基印基激酸铵（a
赭帕察海素A标准：组度大的岛
4.2. 11粘袭素A标滩储备溶液：小.三，配制0.1m.的端册静素A标诸路浚·保存」冰曾备
4.2.12精山存素A你淮作将液：准确移收适量的猪山穿素A东准储备液，间中碗释释成标准SN/T 1746-2006
工作溶液。
4.3器设备
4.3.1童效羧相色谱俊：带有荧光愉测器4.3.2高速巧群: 18 (000 t/n:in--22 000 r/min4.3.3玻璃注东器：10mL..
4. 3.4坡璃试管：占径12 mr,长 75 n.m.无灭光特性4. 3. 5空气压力泵
4. 3. 5微量汗射器;1C0 l.c
4.3.7纹拆登滤纸：
4.3. 8猪曲需萨素 A免疫亲和柱。4. 3.9坡德纤维滤纸：点检1 m,径μm,无荧光特性：4.4分析步骤
4.4.1提取
准价移最试栏0.0（准确到01g）（大豆需要细月粒没2mm）」均质器配置的搅拌宁，圳人5.0g氯化铜（1.2.3）及100）m).币醇（1.2.1)（适月丁油紫籽减.00)ml.干醇-水（8-2)（4,2.2)，以均质器高速搬择提取1min。以槽纹新登恶纸（4.3.7)过滤.准确移取13.0ml.滤较并加入40.0mi水稀释.川皱璃经维纸(4.3.9)过滤全滤液沿清.滤液备市。4.4.2净化
将免疫亲动注(4.3.8逆接于1) ml.瑕璃注射器(4,3,3）下。准确移取).0 mI样品提取波于玻璃注射器巾,将空气压力聚（+,）坡璃注射器相连接，话节压力使溶液以纳ml/nin流速缓慢通差免疫京和壮，白不ml3 ml.空气通过免疫京利机，以0 m 淋缓源液(1.2.5)和ICml 水光所淋就免疫亲和社弃大全部流山液-并复2ml~3m举气道过免疫亲和柱，推确加人5ml.非醇（4.2.1)洗脱.流速为1ml/rin~2ml./mi1：收集全部洗脱液丁皮璃试管（1.3,4)中.供检测用4.4.3测定
4.4.3.1高效液相色谱条件
n)色谱社C性+长 159 mm-内径3. 0 mm,填料当径 5 m);1）流动：乙晴+0.012mml1/1.搅酸钠溶液pH7.（60+40)1g/L1六烷基平基溴骏铵：c）流速:.& mnin:
）荧光检测器激发波长360mm.发射波长120mms）柱温：室温：
f）进样气ul.
4.4.3.2定量
H退样器吸报：011.粘曲需卡素A标准1.作率液（1.2.12)注入高效液相色谱仪，在上述色请条件下测定标准溶液的响垃值（峰高或峰印积）.得划薪曲霉毒索A标准济液高效液相色谱图，谱图参见图A.1
用进样器吸取10.单品洗脱波注人高效获相色谱仪，准上述色谱条外下测定试样的响度值（峰高或峰面积）经过与端曲带萨素A标准裕液谱图比较响应值得到试举中端山毒系A的浓度，4. 4. 4空白试验
除不证试样外，按1述步骤进行
4. 4. 5 结果计算
群站中薪拍莓毒素A的含谨接武（计算：X,=( ).V
*(V+V)
详品中粘曲德毒系A的含昂，单位为微克每千克（g/kg）试样中薪每毒索A的含量，单位为微克每升（/）：空白试验中曲莓毒索A的含量，单位为微克舒（u［）：最终甲醇洗脱液体积单位为掌升（m.）：-最终予化洗脱凌所含的试样质量，单位为克（g）：试样称取的质量的数值，中位为克（）：样品和提取液总体积！单位为毫升（ml.）;稀蜂用样品液体积，单位为膏（ml.)：稀释没体积.单位为毫升（ml)：一逆过免疫亲和杜的样品提收浓体积，单位为暑升（ml.）5测定低限.回收率
测定低限
SN/T 1746—2006
免疫亲和社层析净化高效被相色谱法测定人豆油案籽利食剂挡物洲的低限均为1，k，5. 2回收率
大卫、油案籽和食用植将洱小拷的强毒素A添加浓接及其回收率实验数锯郊下：5.2. 1大豆
±：1.0 g/kg时.回收率为75.053-~96.0;-—-在19.g/kg时收率为8.0~0送在30.0 /kg时.回收率为88系~-109.2对5.2.2油菜籽
：.0/k径时，回收率为84.0%-~106.5降：在10.0#g/kg时回孜率为72.0%--85.1：一0 0kg附-收率为70.1%~83.7%。5.2.3食用植物油
在！.0k[收率为61. 0-~78. 0;
在 .0. 0 μg/kg时.闻收率为 72. 1%~82. 1:0, 0 μg/kg小.间收率为?.7%~2. 1%6免疫亲和柱层析净化荧光光度法（第二法）6.1方法提要
鼠详经过中醇或Ⅲ醇+水提取，提取液经过滤，稀释后，滤液经过含有帖曲带萨索A特杭体的免疫亲称相层析净化，以洗脱溶液洗脱.洗脱液通过费光光度计测定，6.2试剂和溶液
除乃有规笠外，所历试制均为分析纯,水为单蒸水，6.2.1半酵：也谱绝。
6.2.2+醇一水(8+2)取8℃ m. 中醇20 nl 水.6.2.3氯化钠。
6.2.4嵌酸垒钠，
6. 2. 5源 20 Tween 20) .
SN/T 1746---2006
6.2.6淋洗缓浒:称胶 25 氯化钠5 多截酸氢钠溶于水中，加人0.：ml.叶混 2:,而水稀将至1 1. 6.2.7洗税淤(1.1 mml/1.氢氧化链）:称0,4 ”氢氧化钠溶解丁10) rml.水中，6.2.8硫酸。
硫酸游液（0.c5m:l/L)：收2.8ml.浓镜酸.线慢加人造量小中，冷却片定容至10comI.6. 2. 91
璇酸(C..EN,0:·.S2:()
6.2、11荧光光度Ⅱ校准溶液：称取3.4硫俊转宁用.0 mb1/1.硫酸溶液释释举103 L,此溶液的炎光光度计读数和均下36
6.3仪器设备
6.3.1荧光光瘦#
商连均质器：18900/min~22000r/min6.3.2
玻璃注射器：0ml..
6.3.4玻璃试管：自径2mm.长75 mm.无荧光特性：6.3.5空气玉力象，
6.3.6搏纹折盘滤纸，
油毒素A免按亲乱。
台.3.8玻璃纤维滤纸：H径11cn-.孔径1.5m，无荧光特性。6.4分析步骤
6.4.1提取
6. 4.2 净化
将免疫亲和格(6.3.7)推接于10 mI.玻璃注射器（6.3.3)下。准确移取10.0trLV.)样品提取液玻璃注射露中,将审气玉方泉(6.3.)与玻璃注射器相连接，调节压力使济液以约6m/min流遵發慢避过免疫亲和柱占垒2 ml.~3 ml.室通过免疫亲刊莅，以0 1I:L 洗缓冲液(6.2. 6)和 1C n-1.小先后淋洗免疫亲利,弃长全部流出液,并使2 rI.~3In空气通过免疫亲和柱。准确加人1.5 mI(V)洗脱溶波(6.2.7)洗脱.流速为「ml./min～~2ml./min。收集余部洗脱液于玻璃比管（E.3,4)中.供检测用，
6.4. 3测定
6. 4. 3. 1 荧光光度计仪器条件激发波长36mm、发射波长450m
6.4.3.2荧光光度计校准
以0.0 m3./1.硫酸溶液(6.2,9)为空日调零，以荧北光疫卧校准溶液(6.2.11)进行校准：6.4.3.3样液测定
将6.4.2部分的洗脱液立即置于炎光光设计中读取样液中烤曲霉毒素A的浓皮。6. 4. 4 空白试验
除不加试详外，接【逃出骤进行6.4.5结果计算
样品中赭藓素A的舍量按式(3)计算：Y.
-一样品中器也赛毒索A的含脆单你为微克克k！..试样中由群毒案A的含单，单位为微克句（μ/）ti
空血试验黏曲需寿素A的含求，单位为微克有/)：V--最终洗脲液体积.单位为整（ml.)：·最终净化洗脱液所含的试样质晨：单位为克（）；试样称取的质量的数伯，单位为克（g）；-品利提取液总体积，单位为毫（ml.)：V
稀释用样t滤液体积,单位为毫升（mL）；稀释波体积.单位为毫升（mL)
V,--—通过免疫亲和柱的样品提取液体积，单位为毫升(mL)。7测定低限、回收率
测定低限
SN/T 1746—2006
免疫亲利指层析净化炭光光度法测人豆、油菜籽和食用植物油的低限均为1/k7.2回收率
大点、油案籽和食油植物油中鳍曲霉毒素A添加浓度及其回收率实验数错如下：7.2. 1 大豆
在1.0/kg时,国收率为110.0%：
—在1C.0g/k仅时.面收率为90.0%110.0%，在50,0#/kg时，回收率为92.0%~104.0%。7.2.2
油莱籽
-在1.0g/kg时.同收率为110.0%；在10.0g/kg时可收率为8.%--110.0%；-----在50.0#g/k附，回收率为90.0~：06.057.2.3食用植物油
.-存1,0 gg时，国收率为110.0;一—在 :0.0 μg/kg时.国收率为90.0%~-110.0%在50.0g/k，间收率为86.0%106.0%。SN/T 1746—2006
F.D A.F-360,E-410 (T.42:(1O)1
注：举降或素A的保留时间为1.2tit。附录A
（资料性附录）
标准品色谱图
CchaaloinA
图A.1猪曲霉素A标准溶液的液相色谱图http：//foodmate.netnir
Foreword
AnnexAofthisstandard isan infarmativeannex.SN/T1746—2006
This standard is proposed by and is under the charge of tlhe Cartification and Accreditation Adminis.tration of the People's Republic of China.This standard was drafted by Shanghai Entry-Exit Inspection and Quarantine Bureau of the People'sRepublic of China, Beijing Clover Technology Co.Ltd and Shanghai Bioneer Science and TechnologyCo. , Ltd. .
The main drafters of this standard are Chu Qinghua, Guo Dehua. Wang Min, Wang Xiong and ShenJianying.
This standard is a professional standard promulgated far the first tirie.r业网ht+n
SN/T 1746—2006
Inspection of ochratoxin A in soybean, rapeseed/canolaand edible vegetable oils for import and export1Scape
This standard specifies the methods of sampling, sarnple preparation and determination methods onOchratoxin A in soybean, rapeseed/cenola and edible vegetable oils for import and export withmethod of Cleanup by immunaaffinity chramatography and determlnation by high performance liquidchromatography and method of Cleanup by immutroaffinity chramatography and determination by Flu-orometer.
This standard is suitable for determination on ochratoxin A in soybean, rapeseed/canola and edibleails for import and export.
2 Normative reference
The following doclments contain provisions which, through reference in this standard. For datedreferences, all the subsequent revised sheets (not including corrigenda) or revised versions shall notapply to this starctard. However; all the parties raaching an agreement on the basis of this part areencouraged to study the possibility of applying the most recent editions of these documerits. For un-dated references, their iatest editions shall apply to this standard,GB/T 5524 nspection of vegetable pils: methods fot sampling and sample recductionSN/T 0800.1 Inspection of cereals, oils and feadstuffs for import and export-Methods of sam-pling and preparation of samples3Sampling and sample preparation3. 1 Soybean and rapeseed/canola: Follow the instructions given in SN/T 08D0. 1.3. 2 Edible oils:Follow the instructions given in GB/T 55244 Cleanup by immunoaffinity chramatography and determination by high performariceliguid chromatography(the reference method)4.1Principle
The ochratoxin A in the test sample is extracted with methanol or methanolwater solution. The ex-tract is filtrated.diluted with water,and applied to immunoaffinity column for cieanup. Ochratoxin Ais eluted with methanol and is determined by High performance liquid chromatography and is quanti-tated by external standard method.4.2Reagentsandmaterials
SN/T1746—2006
Unless otherwise specified, all the reagents used should be analytically pure; \water\ is redistilledwater.
Methanol:LC grade.
4. 2, 2 Methanol + water (8 + 2):mix 80 ml. methanol with 20 rmL water.4. 2. 3
Sodiumchloride
Sodium bicarbonate.
Tween-20
Mycotoxin wash buffer: waigh 25 g Sodium chloride and 5 g Sodium picarboriate, dissolveand dilute in proper amount of water,add o. 1 mL Tween-2o, dilute to 1 L.4.2.7
Acetonitrile:LC grade.
4.2.8Sodiumphosphate.
4. 2.9 Hexadecyltrimethyl ammorium bromide (C-tab),4.2.10 Ochratoxin A standard.Purity≥99%Ochratoxin A standard stock solution: Weigh an adequate amount of ochratoxin A standard,4. 2. 11
dissoive and dilute with proper amoutnt of methanol (4.2. 1) to prepare the standard stock solutionof0.100mg/mL in concentrationandstoreundar4℃4. 2. 12 Ochratoxin A standard workirig solution:According to the requirement.accurately pipet ade.quate amount of ochratoxin A standard stock solution.mix and dilute with methanol to prepare astaridard working solution of appropriate concentration.4.3Apparatus and equipment
4. 3. t High performance liquid chronatograph: with fluoroscence detector which detection wavelengthis of36onmexcitationand420 nmemission.4. 3. 2High speed blender:18 000 r/min~22 000 r/rminGiass syringe:10mL.
SN/T 1746—2006
Glass test tube:diameter 12 mm, length 75 mm.no fluoroscence4.3.40
4. 3. 5 Air pump.bZxz.net
4.3.6 Micro-syringe:100 μrL4. 3.7 Fluted filter.
4. 3. 8 Ochratoxin A immunoaffinity columnGlass fiber filter: diameter 11 cm,1, 5 μm.no fluoruscence.4.3.9
4. 4 Procedure
4.4.1Extraction
Weigh ca 50 g of the test sample (accurate to 0. 1 g) into the blender (soybean should be ground to2. 0 mm in size ). Add 5 g Sadium chloride <4. 2. 3) and 100 mL methanol (4. 2. 1) (applicable forrapeseed/canola) or 100 mL methanol + water (8+ 2) (4. 2. 2) ( V, ). Blend for 1 min at high speedfor extraction. Filter with flutd fiter (4. 3. 7) and pipet 10. 0 mL V, ) of the filtrate.dilute with40. 0 mL water ( V,) and mix. Filter with glass fiber (4. 3. 9) until the filtrate is clear. Proceed imme-diatelytathecleanup stepwithimmunoaffinity colurnn.4.4.2 Cleanup with immunoaffinity columnConnect the immunoaffinity column (4. 3. 8) to a 10 mL glass syringe (4. 3. 3) .pipet 10 mL clear fil.trate into the syringe. Connect the air pump (4. 3. 5) with the syringe and adjust the pressure in sucha way as to cause the extract passing through the column at a flow rate of ca 6 ml/min and then let2 mL~3 mL of air pass through the column. Wash the column with 10 mL Mycotaxin wash buffer(4.2,6)and 1a mL water respectivelyaru let2mL~3mL pfairpass through thecolumn,Eluteac-curately with 1. 5 mL ( V) methanol at n flaw rate of 1 mL/min--2 rnL'mir. Collect all the eluate in atest tube (4. 3. 4) far quantitation.4. 4. 3Determination
4.4.3.1TestingconditionsofHighperformanceliquidchromatographa
ColumnCo(length15omm.innerdiameter3.0mm.packingdiameter5μm);Mobilephase:Acetonitrile-0.012mol/LSodiuim phosphate solution pH7.5(60+40).1g/Lb)
Hexadecyltrimethyl ammonium bromide :Flow rate:0. 8 mL/min;
Column temperrature: room temperature;Injectionvolume:10μL.
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