【商检行业标准(SN)】 进出口粮谷中呕吐毒素检验方法 液相色谱法(附英文版)

中华人民共和国出入境检验检疫行业标准SN/T 1571—2005
进出口粮谷中呕吐毒素检验方法液相色谱法
Inspection of deoxynivalenol in cereals for import and export-Liquid chromatographic method2005-05-20发布
华人民共和国
国家质量监督检验检疫总局
2005-12-01实施
本标准的附录A为资料性断录，
本标准由区家认证认可监督管理委员会提出并口，SN/T1571—2005
本标翟起萱单位：中华人民共和国辽宁出人境检验检疫同，中华人另共和国黑龙江山人境检验检疫局。
本标主要起草人：林维宣、口苗、庆贺.王孜、曹冬梅、本标准系百次发布的出入境检验检疫行业标准1范围
进出口粮谷中呕吐毒素检验方法液相色谱法
本标准规定了进出「粮谷中岖叶毒素检验的样、制样和液杆色谱检测方本标雅适用：进山口小麦和玉米中呕心毒素的检验。2规范性引用文件
SN/T 1571—2005
下列文件中的条款通过在本标准的引用而戒为本标准的条款：儿是注日期的引用文件,其随后所有的修改单（不包括愈误的内容)或修订版均不适币丁本标准，使用木标准的各方应研究使用下列标注最新版本的可能性。
SN/T0799—1999进出口稳油、何料检验检验般愿则SN/T0800.11999进出口粮洒、衡料检验油栏和制样方汰3抽样和制样
接SV/0800.1执行。在抽样和制样过程中，应防比样品受到污染或发生取让素含量的变化。4测定方法
4.1．方法提要
详品以水或乙腈十水提取,提取液经 LONIes1 HPLC 免疫亲利柱或固相萃取(SPE)柱净化，发相液相色谱杜分肉-紫外检测器22℃nm波长小检测,外标法定量。4.2试剂和材料
4.2.1水：纯水，
4.2.2中腰：色谱纯
4.2.3Z腾：色谱纯。
4.2. 4Z腈+水(84+_6) .
4.2.5区吐毒素标准诺备：称取区吐毒素10,Cmg：而甲醇溶解并定容到190mL容常瓶中，浓度为100以/m（吸证素标准储备液在一20℃冷冻条件下可以烂存4个月）4.2. 6呕叶毒素标准中问储液:标准储备液 1,0 InL于 1CrrL穿量施中,用流动相定穿，浓度为1o.G g/ml.
4.2.7呕吐毒素标准工作液：用上述标准中间液，根据需安配制成适用浓度的标准下作液。所有标准工作腋需在冷条件下贮存（何闹配制）。4.2.8相色谱流动相：甲醇1水（20180），0.5um滤膜过滤。4.2. 9DOV1exIHPI.C 免疫亲和。4.2. 13 固相些取(SPE)杆:MycoScp No. 223,4.2.11繁丙烯塑料管：15man(内径）×85mm，与固相萃取柱（MycoSepNo.225）配套使用。微纤维滤纸：1.5μm直径1=cn。4.2.12
4. 2. 13 聚乙醇 8 00G。
离心：1 rmT.
SN/T 1571—2005
4.2.1510 mI.玻璃汪射器，
4.2.16滋膜：0.45m
4.3仪器和设备
4.3.1高效液相免谱仪：配有紫外检测器。4.3.2泵流控制台。
4.3.3固粘萃敢议。
4.3.4组织均质器：20000t/min
4.3.5涡旋均匀器。
4.4测定步骤
4.4.1提取和净化
4.4.1.1免疫亲和柱净化法
提取：样品经粉碎并通过40月篇子。称取25.0于均质杯中，加人5g聚乙二醇8000mL和l00)ml纯水，盖1均质杯的盖子，高遠均质2mit。静置lCtmit:，将提敢液经槽纹滤纸过滤，拨集滤液。战20 l. 滤液通过微纤维滤纸过滤到50 ml.量杯中净化:将 IXONtest 亲利柱(4,2. 9)的盖帽取下,剪掉帽盖的封I1端,再把帽盖盖「：将 1C nrL 玻璃注射器简(4.2. 15)与亲和拉相连。准确移取「步滤液2. Tml.于玻璃注求器偿中.接「泵流控制器,拨掉亲和杆底帽。控制注力使样液以1滴/：的流遠全部通过亲和杆，占至空气逊人到亲和托中，充去全部流山液。加10ml.纯水以2滴/s的流速淋洗亲和柱，分去全部流凹液，准确加人1.0rml.币醇丁皱璃注射器钉筒巾.以1滴/的流速洗脱，收集部洗脱液于10InL玻璃臀中，氮气欧干，残蜜物加2001甲醇水（4.2.8)溶解，混勾，过滤膜（1.2.16)，逊样分析。4.4.1,2固相萃取(SPE)柱净化法提取：样品经粉碎并通运40月筛子。称取25.0于均质杯中，加人100=nL乙睛1水（4.2.2)·速均质3 rrin。静置10 rrin,将提吸液经滤纸过滤，收集滤液。净化：移8mL提取液丁聚内烯塑料管(4.2.11)，一手持冷化杆，一手持含提妆液的塑料管,把净化柱橡胶法兰一端垂直缓缓推人聚丙烯塑料管中（进行此步操作时不要用三指盖住净化栏1部刀口),净化的提取液进人净化杜「部管内。技集约4 tml.净化的提取液：从净化柱1部准确移取2.0 tnT净化的提取液丁_0 ml.离心管(4.2.11)中，在60℃加热条件下氮气次十（必要时加币醇如快饮十),残留物加20CμL流动相（4.2,8)溶解，过滤膜（4.2.16），进样分。4.4.2测定
4. 4.2. 1
液相色谱条件
色谱柱：Waters 式-MS栏,25t mmX4. mm(内径)，: μm,或相当若;流动相：见4.2.8；
流速：l.0ml/mint
检测波长：220nm
注温：室温；
f）退样量:20 μml
4.4.2.2色谱测定
分别净化后的样液和标准溶液各 2C μL进行HFLC分析,以其标准溶液泽的保留时问为依据进行定性，以其峰面积求山样液中被测物质的含量，供计算，在述色谱条件下，呕吐丧素的保留时问为10. 5 mn 左右.参见附录 A。
4. 4. 3空白试验
除不流样外，接「逆测定步骤进行2
4. 4. 4结果计算和表述
按武（：识管试橙市呕托毒含量：式中：
X=A.xmx(Viv)
试样吡穿衰的含量,单位为毫克年千克(ri/k)；样液亢呕吐毒索的峰面积，单位为方毫米（mm）；标准溶液中呕叶毒索的峰而积.单位为平方毫来(ann)；标准溶液中区吐毒素的浓度，单位为微克每毫Ⅱ（g/mI)；称取的试样量，单位为克(g)；
试样提圾液总体积,单位为亳升(ml.)：净化用提驳液体积,单位为并(rm)：试样净化液最后定容体积，0,2InL注：习算结果底知除突白征，
5测定低限,回收率
5.1测定低限
本方法对吸吡毒索的测定低限为a.0.0mg/kg。5.2回收率
添加浓度设回收率的实验数据：5.2.1小友实验数据见表1
表1小麦实验数据
茶加浓度/(mg/kg）
卡米实验数据见表2，
暴加浓度/(mg/kg)
回改率（免疫亲和柱)/（%）
表 2 玉米实验数据
反政率（免疫京和杆)/（）
SN/T 1571—2005
回收率(相率股栏)《兴)
同收率(固在释取杭)%)
SN/T 1571—205
附录A
(资料性附录）
标准品色谱图
prroo y:
呕吐毒素标准样品液相色谱图
ppegy rx
Foreword
AnnexA of this standard is an informative annexSN/T1571—2005
This standard was proposed by and is under the charge of China National Regulatory Commission forcertification and Accreditalion.This standard was drafted by liaoning Entry-Exit Inspection and Quarantine Bureau of the People'sRepurblic of China and by Hei Longjiang Entry-Exit Inspection and Qurarantine Bureau of the People' sRepublic of China.
The rmain drafters of this standard are Lin Weixuarnl, Tian Miao, Kang Qinghe. Wang Mei, Cao Dong-mei.bzxz.net
This standard is a professional standard promulgated for the first time.SN/T 1571—2095
Inspectionofdeoxynivalenolincerealsforimport and export-Liquid chromatographic method1Scope
This standard specifies the methods of sampling,sample preparation and determination by liquidchromatography of deoxynivalenol in cereals for export and import.This standard is applicable to the determination of deoxynivalenol in wheat and corn for export andimpart.
2Normative references
The following standards contain provisions which, through reference in this text, constitute provi-sions of this standard, Parties to agreements based on this standard are encouraged to investigate thepossibility of applying the most recent editions of the standards indicated below.SN /T 0799 —1999 Inspection of cereals, oils and feedsfuffs for import and export general rulesfor the inspection.
SN /T 0800.1-—1999Inspection of cereals and feedsfuffs for import and export —methods of sam-pling and preparation of sarnples.3Sampling and sample preparationSee sN/T a800. 1. In the caurse of sampling and sample preparation precautian must be taken toavoid contamination or any factors which may cause the change of residue content.4Method of determination
4. 1 Principle
The residues were extracted from the sample with water or acetanitrile and water, purified by DON-test imrrunity affinity cartridges or SPE column.deterrninated by HPLC with UV-detector at 220 nrn.calculated by comparing peak area of the sample with corresponding standard peak area.4. 2 Reagents and materials
4.2.1 Water: Super-pure water.4. 2.2 Methanol: HPLC grade.6
4. 2. 3 Acetonitrile: HPLC grade.4.2. 4 Acetonitrile-water: 84 + 16,SN/T 1571—2005
4.2, 5 Deoxynivalenol standard stock solution: Accurately weigh 10, 0 mg of deoxynivalenol, dissolve with methanol and dilute ta 1ao mL volumetric flask, obtain standard stock solution, with100 μ g/mL (Deoxynivalenol standard stock solution can be stored for 4 months at - 20℃ ,4.2. 6 Deoxynivalenol standard intermediate solution: Pipet 1. 0 mL of standard stock solution into10 mL volumetric flask.and make up to 1o mL with mobile phase.Prepare mixed standard intermedi-ate solution to obtain 10. 0 μg/mL.4. 2.7 Deoxynivalenol standard working solution: Dilute the Deoxynivalenol standard intermediatesolution to the required concentration as the standard working solution. The working solutions can bestored for one week at a'C ~4'℃.4.2.8Mobilephase:methanal+water(20+80),filteredwith0.45μmfiltermembrane.4.2.9DONtestimmunityaffinitycartridges4.2. 10 Solid phase extraction (SPE) cartridges: MycoSep No, 225,4.2. 11 Polypropylene plastic tube: 15 mm(i. d. ) × 85 rmm. together with sPE colurmn(MycoSepNo.225)
4.2.12Microfiberfilterpaper:1.5μm,i.d.11cm4.2. 13Polyethylene glycol 8 000.4.2. 14 Centrifuge tube: 10 mL.4.2. 1510 mL glass syringe.
4.2. 16 Filter film: 0. 45 μm.4.3Apparatus and equipment
4. 3. 1Liquid chromatograph: Equipped with Uv-detector.4.3.2Fluxcontrol instrumentwith pump.4.3. 3SPE instrument.
SN/T 1571—2095
4.3. 4 Tissue homogenizer with roating blades: 20 ao0 r/min.4.3.5Vortexmixer
4.4Procedure
4. 4. 1 Extraction and purification4.4.1. 1Irmrunity affinity cartridgesExtraction: The sample was crushed before it passed through a sieve (40 mesh). The 25. Og of sam-ple,5. 0 g of polyethylene glycol aoo mL and 1o0 mL of pure water were added to a homogenouscup. It was covered and homogenized for 2 min with high speed, Then it was tested for 10 min. Theextraction liquid was filtrated with filter paper and the filtrate was stored up, The 20 mL of filtratewas filtrated with micro fiber filter paper to a graduate (50 mL).Purificatian: The cover cap of DONtest immunity affinity cartridges (4. 2. 9) was taken off and itsseal side was cut off, then the cover cap was covered again. The glass syringe(10mL) (4. 2. 15) wasjoined with immunity affinity cartridges. The 2, 0 mL of filtrate above was exactly transferred to theglass syringe which was joined with a controlled flow pump. The bottom cap of affinity cartridgeswas put off. The sample liquid completely passed through immunity affinity cartridges with 1 d/s bya certain pressure. It didn't throw away the all effluence until the air come into the affinity cartrid-ges, The 10 mL of pure water was added to wash the affinity cartridges with 2 d/s and the all efflul-ence was thrown away. The 1, 0 ml of methanol was exactly added to the glass syringe to elute with1 d/s. The all elurtion liquid was collected to a tube (10 mL) and was blown up with Nz. The 200 μL ofmixture of methanol and water (4. 2. 8) was added to the residual to dissolve. The mixture was hom-ogeneously mixed and was filtrated with filter film,finally was subjected to analysis.4.4. 1. 2 SPE cartridges purificationExtraction: The sample was crushed before it passed through a sieve (40 mesh). The 25. Og of sam-ple and the 100 mL of mixture of acetonitrile and pure water were added to a homogeneous cup. Itwas covered and homogenized for 3 min with high speed, The extraction licuid was filtrated with fil-ter paper and the filtrate was stored up.Purification: The 8 mL of extraction liquid was transferred to a polypropylene plastic tube (4. 2. 11).The purification colurmn was taken by one hand.at the same time the plastic tube contained with ex-traction liquid was taken by the other hand. The rubber flange end of purificatior colurnn was verti-cally pushed into the polypropylene plastic tube by inches (Don't cover the top open of the purifycolumn) , then the purified extraction liquid was come into the upside of purify column. The 4 mL ofpurified extraction liquid was collected. The 2. O mL af purified extraction liquid from the upside ofpurified column was transferred to a 10 mL of centrifugal tube (4.2. 14),and was blown up with N2at 60'c (if necessary the methanol was added to blow up quickly), The 200 μL acetonitrile + water8
SN/T 1571—2005
(84 + 16) (4. 2. 8) was added to the residual to dissolve,then was filtrated with filter film (4. 2. 16) :finally was subjected to analysis.4. 4. 2Determination
4.4.2.1 LC operating conditionsChromatagraphic column: waters 5c8-Ms column,250 mm ×x 4. 6 mm(i. d. ), 1a μm.a)
b)Mobile phase:4.2.8.
c) Flow rate of mobile phase; 1. 0 mL/min.d)
Wave length of UV detector:220 nm.Column temperature: Raorm temperature.e
Sample size: 20 μL.
4. 4. 2. 2 LC determination
LC determination is performed by injecting separately equal volrmn of the derivatized standard work-ing solution and the sample solution into the Lc system. Identify deoxymivalenol by comparing peakretentiari tirrie of the sample with corresponding standard peak reteritior1 time. Calculate the result bycomparing peak area of the sample with corresponding standard peak area,using external standard method, Under the above Lc conditions, the retention time of deoxynivalenol is about 1o, 5 min, Seeannex A.
Blanktest
Perforrn the blank test with the saie procedures as that described in the miethod of determinationbut without of addition of test sample.4, 4. 2. 4Calculation and expression of resultsThe calculation of results is according to formula(1) :X=
Axc,xV
A, ×m×(V2/V,)
the residure content of deoxynivalenol.mg/kg;the peak area of deoxynivalenol of the sample solution,mm? :the peak area af deoxynivalenol of the standard working solutian,mn? ;the concentration of deoxynivalenol in the standard working solution,μg/mLthe mass of test sample,g:
-the total volume of extraction.mL:the volume of purified extraction,mL:the final volurme of sample solution,rml（1）
SN/T 1571—2005
Note: The hlank val.ie shall he surbtraeted from the above result of calculation.5Limitof determinationand recovery5.1Limitofdetemination
Lirmit of determination js 0. 040 rmg/kg.Recovery
According to the experimental data, the fortifying cancentration of deoxynivalenol and its carre- sponding recoveries are:
Wheat of experimental data see Table 1.5.2. 1
Wheatofexpementaldata
Table 1
Addition cancentration?
(mg/kg)
Recovery/(%)
(immunity affinity cartridges)83.8
Corn of experimental data seeTable25.2. 2
Table 2Com of experimental dataAddition cancentration?
(mg/kg)
Recovery/(%
(immunity affinity cartridges)85.0
Recovery?(%)
(SPE certridges)
Recoverv?%)
(SPE cartridges)
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