1、中华人民共和国出入境检验检疫行业标准SN/T 1571一2005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 method 2005-05-20发布2005斗2-01实施中华人民共和国发布国家质量监督检验检疫总局SN/T 1571-2005 前言本标准的附录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z中华人民共和国辽宁出入境检验检疫局、中华人民共和国黑龙江出入境检验检疫局。本标准主要起草人:林维宜、田
2、苗、康庆贺、王玫、曹冬梅。本标准系首次发布的出入境检验检疫行业标准。I 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口粮谷中呕吐毒素检验的抽样、制样和液相色谱检测方法。本标准适用于进出口小麦和玉米中呕吐毒素的检验。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，其随后所有的修改单(不包括勘误的内容)或修订版均不适用于本标准，使用本标准的各方应研究使用下列标准最新版本的可能性。SN/T 0799-1999 进出口粮油、饲料检验检验一般规则SN/T 0800. 1-1999 进出口粮油、饲料检验抽样和制样
3、方法3 抽样和制样按SN/T0800. 1执行。在抽样和制样过程中，应防止样品受到污染或发生呕吐毒素含量的变化。4 测定方法4. 1 方法提要样品以水或乙脯+水提取，提取液经DONtestHPLC免疫亲和柱或固相萃取(SPE)柱净化，反相液相色谱柱分离，紫外检测器220nm波长下检测，外标法定量。4.2 试剂和材料4.2. 1 水:超纯水。4.2.2 甲醇:色谱纯。4.2.3 乙睛:色谱纯。4.2.4 乙睛+水(84+16)。4.2.5 呕吐毒素标准储备液:称取呕吐毒素10.0mg，用甲醇溶解并定容到100mL容量瓶中，浓度为100附/mL(呕吐毒素标准储备液在一200C玲冻条件下可以贮存4个
4、月)。4.2.6 呕吐毒素标准中间储备液:取标准储备液1.0 mL于10mL容量瓶中，用流动相定容，浓度为10.0g/mLo 4.2.7 呕吐毒素标准工作液:用上述标准中间液，根据需要配制成适用浓度的标准工作液。所有标准工作液均需在冷藏条件下贮存(每周配制)。4.2.8 液相色谱流动相:甲醇+水(20+80),0. 45m滤膜过滤。4.2.9 DONtest HPLC免疫亲和柱。4.2.10 固相萃取(SPE)柱:MycoSep No. 2250 4.2. 11 聚丙烯塑料管:15 mm(内径)X85mm，与固相萃取柱(MycoSepNo. 225)配套使用。4.2.12 微纤维滤纸:1. 5
5、m，直径11cm。4.2.13 聚乙二醇8000。4.2.14 离心管:10mL。SN/T 1571-2005 4.2.15 10 mL玻璃注射器。4.2. 16 滤膜:0.45mo4.3 仪器和设备4.3.1 高效液相色谱仪:配有紫外检测器。4.3.2 泵流控制台。4.3.3 固相萃取仪。4.3.4 组织均质器:20000r/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 免瘟亲和柱净化法提取t样品经粉碎并通过40目筛子。称取25.0g于均质杯中，加入5g聚乙二醇8000 mL和100 mL纯水，盖上均质杯的盖子，高速均质2min。静置10min，
6、将提取液经槽纹滤纸过滤，收集惊液。取20mL滤液通过微纤维滤纸过滤到50mL量杯中。净化:将DONtest亲和柱(4.2.9)的盖帽取下.剪掉帽盖的封口端，再把帽盖盖上。将10mL玻璃注射器筒(4.2.15)与亲和柱相连。准确移取上步措液2.0mL于玻璃注射器筒中，接上泵流控制器，拔掉亲和柱底帽。控制压力使样液以1滴/s的流速全部通过亲和柱，直至空气进入到亲和柱中，弃去全部流出液。加10mL纯水以2滴/s的流速淋洗亲和柱，弃去全部流出液。准确加入1.0 mL甲醇于玻璃注射器针筒中，以1滴/s的流速洗脱，收集全部洗脱液于10mL玻璃管中，氮气吹干，残留物加200L甲醇+水(4.2.8)溶解，棍匀
7、，过滤膜(4.2.16)，进样分析。4.4.1.2 固相萃取(SPE)柱净化法提取:样品经粉碎并通过40目筛子。称取25.0g于均质杯中，加入100mL乙睛+水(4.2.4)，高速均质3mino静置10min，将提取液经滤纸过滤，收集滤液。净化:移取8mL提取液于聚丙烯塑料管(4.2.11)中，一手持净化柱，一手持含提取液的塑料管，把净化柱橡胶法兰一端垂直缓缓推入聚丙烯塑料管中(进行此步操作时不要用手指盖住净化柱上部开口)，净化的提取液进入净化柱上部管内。收集约4mL净化的提取液。从净化柱上部准确移取2.0mL 净化的提取液于10mL离心管(4.2.14)中，在600C加热条件下氮气吹干(必要
8、时加甲醇加快吹干)，残留物加200L流动相(4.2.8)榕解，过滤膜(4.2.1时，进样分析。4.4.2 测定4.4.2. 1 液相色谱条件a) 色谱柱:Waters 5Cs-MS柱.250mmX4. 6 mm(内径)，10m，或相当者pb) 流动相:见4.2.8;c) 流速:1. 0 mL/min; d) 检测波长:220nm; e) 柱温:室瘟;f) 进样量:20L。4.4.2.2 色谱测定分别取净化后的样液和标准搭液各20L进行HPLC分析，以其标准榕液峰的保留时间为依据进行定性，以其峰面积求出样液中被测物质的含量，供计算。在上述色谱条件下，呕吐毒素的保留时间为10.5 min左右，参见
9、附录A。4.4.3 空白试验除不加试样外，按上述测定步骤进行。2 4.4.4 结果计算和表述按式(1)计算试样中呕吐毒素含量:式中:X一AX c, X V 3 一A. XmX(VZ/V1) X一一试样中呕吐毒素的含量，单位为毫克每千克(mg/kg); A一一样液中呕吐毒素的峰面积，单位为平方毫米(mmZ);人标准榕液中呕吐毒素的峰面积，单位为平方毫米(mmZ); c，一一一标准溶液中呕吐毒素的浓度，单位为微克每毫升(g/mU;m 称取的试样量，单位为克(g); V1 试样提取液总体积，单位为毫升(mL);Vz一一一净化用提取液体积，单位为毫升(mL); V3一一试样净化液最后定容体积，0.2m
10、L。注:计算结果应扣除空白值。5 测定低限、回收率5. 1 测定低限本方法对呕吐毒素的测定低限为O.040 mg/kg 0 5.2 回收率添加浓度及回收率的实验数据:5.2.1 小麦实验数据见表1。表1小麦实验数据添加浓度/Cmg/kg)回收率(免疫亲和柱)/C%)0.04 83.8 0.5 87.2 1. 0 89.7 5.0 91. 9 5.2.2 玉米实验数据见表20表2玉米实验数据添加浓度/Cmg/kg)回收率(免疫亲和柱)/C %) 0.04 85.0 0.5 91. 1 1. 0 94.2 5.0 90. 7 SN/T 1571-2005 . ( 1 ) 回收率(团相萃取柱)/C%
11、) 82. 5 85.9 93.5 92.3 回收率(固相萃取柱)/C%) 80.0 89. 5 93.3 92.8 3 SN/T 1571-2005 附录(资料性附录)标准晶色谱圈A wm附#ME mAU 4 2 3 。吁.#J ., 4 i叫叫仙，.叫叩申川咛呻叫叫5 7.5 10 12.5 2.5 。川一t/min 呕吐毒素标准样晶液相色谱固圈A.14 SN/T 1571-2005 Foreword Annex A of ths standard is an informative annex. This standard was proposed band is under the c
12、harge of China National Regulatory Commission for certification and Accreditalion. This standard was drafted by liaoning Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China and by Hei Longjiang Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China. T
13、he main drafters of this standard are Li n Weixuan, Tian Miao, Kang Qinghe , Wang Mei , Cao Dong 付1el.This standard is a professional standard promulgated for the first time. 5 SN/T 1571-2005 Inspection of deoxynivalenol in cereals for import and export-Liquid chromatographic method 1 Scope This sta
14、ndard specifies the methods of sampling , sample preparation and determination bliquid chromatography of deoxynivalenol in cereals for export and import. This standard is applicable to the determination of deoxynivalenol in wheat and corn for export and import. 2 Normative references The following s
15、tandards 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 the possibility of applying the most recent editions of the standards indicated below. SN /T 0799 -1999 Inspection o
16、f cereals , oils and feedsfuffs for import and export -general rules for the inspection. SN /T 0800. 1一1999Inspection of cereals and feedsfuffs for import and export methods of sam pling and preparation of samples. 3 Sampling and sample preparation See SN/T 0800. 1. In the course of sampling and sam
17、ple preparation precaution must be taken to avoid contamination or any factors which may cause the change of residue content. 4 Method of determination 4. 1 Principle The residues were extracted from the sample with water or acetonitrile and water, purified by DON test immunity affinity cartridges o
18、r SPE column ,determinated by HPLC with UV-detector at 220 nm , 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 SN/T 1571-2005 4.2.3 Acetonitrile: HPLC grade. 4.2.4 Aceto
19、nitrile-water: 84+ 16. 4.2.5 Deoxnivalenol standard stock solution: Accurately weigh 10.0 mg of deoxnivalenol， dis solve with methanol and dilute to 100 mL volumetric flask , obtain standard stock solution , with 100g/mL (Deoxnivalenol standard stock solution can be stored for 4 months at一20oC.4.2.6
20、 Deoxnivalenol standard intermediate solution: Pipet 1.0 mL of standard stock solution into 10 mL volumetric flask,and make up to 10 mL with mobile phase. Prepare mixed standard intermedi ate solution to obtain 10.0g/mL. 4.2.7 Deoxynivalenol standard working solution: Dilute the Deoxynivalenol stand
21、ard intermediate solution to the required concentration as the standard working solution. The working solutions can be stored for one week at OoC _40C. 4.2.8 Mobile phase: methanol + water(20 + 80) , filtered with 0.45m filter membrane. 4. 2. 9 DONtest immunity affinity cartridges. 4.2.10 Solid phas
22、e extraction (SPE) cartridges: MycoSep No. 225. 4.2.11 Polypropylene plastic tube: 15 mm (i. d. ) x 85 mm, together with SPE column (MycoSep NO.225) 4.2.12 Micro fiber filter paper: 1.5m，i. d. 11 cm. 4.2.13 Polyethylene glcol 8 000. 4. 2. 14 Centrifuge tube: 10 mL. 4.2. 15 10 mL glass syringe. 4.2.1
23、6 Filter film: 0.45m. 4.3 Apparatus and equipment 4.3.1 Liquid chromatograph: Equipped with UV-detector. 4.3.2 Flux control instrument with pump. 4.3.3 SPE instrument. 7 SN/T 1571-2005 4. 3. 4 Tissue homogenizer with roating blades: 20 000 r /min. 4.3.5 Vortex mixer 4. 4 Procedure 4. 4. 1 Extraction
24、 and purification 4. 4. 1. 1 Immunity affinity cartridges Extraction: The sample was crushed before it passed through a sieve (40 mesh). The 25. Og of sam ple ,5.0 9 of polyethylene glycol 8 000 mL and 100 mL of pure water were added to a homogenous cup. It was covered and homogenized for 2 min with
25、 high speed. Then it was rested for 10 min. The extraction liquid was filtrated with filter paper and the filtrate was stored up. The 20 mL of filtrate was filtrated with micro fiber filter paper to a graduate (50 mU. Purification: The cover cap of DONtest immunity affinity cartridges (4. 2. 9) was
26、taken off and its seal side was cut off,then the cover cap was covered again. The glass syringe(10mU (4.2.15) was joined with immunity affinity cartridges. The 2.0 mL of filtrate above was exactly transferred to the glass sringe which was joined with a controlled flow pump. The bottom cap of affinit
27、y cartridges was put off. The sample liquid completely passed through immunity affinity cartridges with 1 d/s by a certain pressure. It didn t throw awathe all effluence until the air come into the affinity cartrid嗣ges. The 10 mL of pure water was added to wash the affinity cartridges with 2 d/s and
28、 the all efflu ence was thrown awa. The 1. 0 mL of methanol was exactly added to the glass syringe to elute with 1 d/s. The all elution liquid was collected to a tube (10 mU and was blown up with N2 The 200L of mrxture of methanol and water (4.2.8) was added to the residual to dissolve. The mixture
29、was hom ogeneously mixed and was filtrated with filter film, finallwas subjected to analysis. 4.4.1.2 SPE臼rtridgespurification Extraction: 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、waterwere added to a
30、homogeneous cup. It was covered and homogenized for 3 min with high speed. The extraction liquid 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 column was taken by
31、 one hand ,at the same time the plastic tube contained with ex traction liquid was taken bthe other hand. The rubber flange end of purification column was verti cally pushed into the pOlypropylene plastic tube binches (Don t cover the top open of the purify colum时，thenthe purified extraction liquid
32、was come into the upside of purify column. The 4 mL of purified extraction liquid was collected. The 2.0 mL of purified extraction liquid from the upside of purified column was transferred to a 10 mL of centrifugal tube (4.2.14) ,and was blown up with N2 at 600C (if necessary the methanol was added
33、to blow up quickly). The 200L acetonitrile + water 8 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) , finallwas subjected to analysis. 4. 4. 2 Determination 4. 4. 2. 1 LC operating conditions a) Chromatographic column: Waters 5C8-MS
34、 column , 250 mm x 4. 6 mm( i. d. ) ，10m. b) Mobile phase:4. 2. 8. c) Flow rate of mobile phase: 1.0 mL/min. d) Wave length of UV detector:220 nm. e) Column temperature: Room temperature. f) Sample size: 20L. 4. 4. 2. 2 LC determination LC determination is performed binjecting separately equal volum
35、n of the derivatized standard work ing solution and the sample solution into the LC system. Identify deoxnivalenol by comparing peak retention time of the sample with corresponding standard peak retention time. Calculate the result by comparing peak area of the sample with corresponding standard pea
36、k area , using external standard method. Under the above LC conditions , the retention time of deoxynivalenol is about 10.5 min. See annex A. 4. 4. 2. 3 81ank test Perform the blank test with the same procedures as that described in the method of determination but wthout of addton of test sample. 4.
37、 4. 2. 4 Calculation and expression of results The calculation of results is according to formula( 1) : _ Ax Cs X V3 ( 1 ) As x m x (V2/ V,) Where X一一-theresidue content of deoxynivalenol , mg/kg; A一-thepeak area of deoxynivalenol of the sample solution , mm勺As一一一thepeak area of deoxynivalenol of th
38、e standard working solution, mm勺Cs一一一theconcentration of deoxynivalenol in the standard working solution，g/mL; m一一一themass of test sample , 9 ; V，一一thetotal volume of extraction, mL; V2一一一thevolume of purified extraction, mL; V3一一-thefinal volume of sample solution, mL. 9 SN/T 1571-2005 Note: The bl
39、ank value shall be subtracted from the above result of calculation. 5 Limit of determination and recover 5. 1 Limit of determination Limit of determination is 0.040 mg/kg. 5. 2 Recovery According to the experimental data. the fortifying concentration of deoxynivalenol and its corre sponding recoveri
40、es are: 5.2. 1 Wheat of experimental data see Table 1. Table 1 Wheat of expermental data Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (immunity affinity cartridges) (SPE cartridges) 0.04 83.8 82.5 0.5 87.2 85.9 1.0 89. 7 93.5 5.0 91.9 92.3 5. 2. 2 Corn of experimental data see T
41、able 2. Table 2 Corn of experimental data Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (immunity affinity cartridges) (SPE cartridges) 0.04 85.0 80.0 0.5 91.1 89.5 1.0 94.2 93.3 5.0 90.7 92.8 10 mAU 4 3 2 。Annex A (informative annex) Chromatogram of standard 融屿，.E叫叩伊脚俨11胁，略咛呵呵呵5
42、 5 7.5 10 12.5 t/mm Fig A. 1 The HPLC chromatogram of Deoxynivalenol standard SN/T 1571-2005 mCON-Fhum-HZm 中华人民共和国出入境检验检疫行业标准进出口粮谷中呕吐毒素检验方法液相色谱法SN/丁1571-2005* 中国标准出版社出版发行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导印张1字数21千字2005年8月第一次印刷开本880X 1230 1/16 2005年8月第-版盼定价10.00元如有印装差错由本社发行中心调换版权专有侵权必究举报电话:(010)68533533书号:155066.2-16318 SN/T 1571
