1、中华人民共和国出入境检验检疫行业标准SN/T 1572-2005 进出口粮谷、饲料中伏马毒素检验方法液相色谱法Inspection of fumonisins in cereals and feedstuffs for import and export Liquid chromatographic method 2005-05-20发布2005-12皿01实施中华人民共和国发布国家质量监督检验检菇总局SN/T 1572-2005 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井归口。本标准起草单位:中华人民共和国辽宁出入境检验检疫局、中华人民共和国吉林出入境检验检疫局
2、。本标准主要起草人:林维宜、田苗、牟俊、赵形彤、郭兰、商明磊、曹冬梅。本标准系首次发布的出入境检验检疫行业标准。1 范围进出口粮谷、饲料中伏马毒素检验方法液相色谱法SN/T 1572一2005本标准规定了进出口粮谷、饲料中伏马毒素Bl、Bz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玉米和玉米饲料中伏马毒素Bl、民的检验。2 规范性引用文件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。凡是不注日期的引用文件，其最新版本适用于本标准。SN/T
3、 0800. 1一1999进出口粮油、饲料检验抽样和制样方法3 抽样和制样按SN/T0800. 1执行。在抽样和制样过程中，必须防止样品受到污染或发生伏马毒素含量的变化。4 测定方法4.1 方法提要样品用甲醇+水提取，经FumoniTest免疫亲和柱或SAX强阴离子固相萃取柱净化，经衍生化，用反相液相色谱柱分离，荧光检测器检测，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 乙腊:色谱纯。4.2.4 乙酸:优级纯。4.2.5 磷酸:优级纯。4.2.6 盐酸:优级纯。4.2.7 磷酸二氢饷:分析纯。4.2.8 磷酸氢二饷:分析纯。4.2.9 磷酸二氢押
4、:分析纯。4.2.10 氯化铀:分析纯。4.2. 11 氯化梆:分析纯。4.2.12 氢氧化铀z分析纯。4.2.13 四棚酸铀z优级纯。4.2.14 琉基乙醇:优级纯。4.2.15 邻苯二甲醒:优级纯。4.2.16 甲醇+水(1十1)。4.2.17 甲醇+水(3十1)。SN/T 1572-2005 4.2.18 甲醇+水(4十1)。4.2.19 乙酸+甲醇(1+99)。4.2.20 伏马毒素标准储备液:称取伏马毒素Bj、B2各10.0mg，分别用乙睛+水0+1)溶解并定容到100 mL容量瓶中，伏马毒素Bj、岛的浓度均为100g/mL。4.2.21 混合标准储备液:取标准储备液Bj、B2以1:
5、 1比例混合，得混合标准储备液，伏马毒素Bj、岛的浓度均为50g/mL。4.2.22 混合标准工作液:取适量混合标准储备液于10mL容量瓶中，用乙腊+水o十1)定容，得伏马毒素Bj、B2的浓度分别为O.2g/mL、1.0g/mL、5g/mL、25g/mL、50/-lg/mL的混合标准工作液(伏马毒素标准储备液和工作液在40C下可贮存6个月)。4.2.23 磷酸盐缓冲榕液:称取磷酸氢二铀1.2 g，磷酸二氢饵0.2g，氧化铀8g，氯化饵0.2g，溶解于990 mL水中，用盐酸调节pH为7.0，用水定容到1L。4.2.24 衍生剂:称取5mg邻苯二甲醒，溶于1mL甲醇，用0.05mol/L四棚酸铀
6、溶液定容到50mL, 加入25L琉基乙醇，混匀，备用(OOC4 oC可保存一周)。4.2.25 液相色谱流动相:甲醇+磷酸二氢铀(0.1mol/L)(77+23)，用磷酸调节pH为3.3m、0.45m滤膜过滤。4.2.26 FumoniTest免疫亲和柱。4.2.27 SAX强阴离子固相萃取(SPE)柱:500mg/10 mL , Varian产品。4.2.28 玻璃纤维滤纸。4.2.29 离心管:50mL。4.2.30 滤膜:0.45m。4.3 仪器和设备4.3. 1 高效液相色谱仪:配有荧光检测器。4.3.2 泵流控制台。4.3.3 固相萃取仪。4.3.4 组织均质器:20 000 r/m
7、in。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 免疲亲和柱净化法提取:样品经粉碎并通过40日筛子。称取25.0g于均质杯中，加入4g氯化饷和50mL甲醇+水(4.2.18) ，高速均质3min，静置10min。将提取液上清液用槽纹滤纸过滤，收集滤破。净化:准确移取上述滤液10.0mL于100mL具塞锥形瓶中，加40mL磷酸盐缓冲榕液(4.2.23), 置于涡旋均匀器上棍匀，经玻璃纤维滤纸过滤，收集捷、液备用。将FumoniTest免疫亲和柱的盖帽取下，剪掉帽盖的封口端，再把帽盖盖上。将10mL玻璃注射器筒与亲和柱相连，准确移取10.0mL上步得到的捕、
8、液于玻璃注射器针筒中，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F山noniTest亲和柱，直至空气进入到亲和柱中。从玻璃注射器上取下FumoniTest亲和柱，将亲和柱上部用磷酸盐缓冲洛液(4.2.23)注满，再把亲和柱连接到玻璃注射器上，用10mL磷酸盐缓冲溶液(4.2.23)以2滴/s的流速淋洗亲和柱，直至空气进入到亲和柱中，弃去全部流出液。再用10mL磷酸盐缓冲榕液(4.2.23)重复淋洗亲和柱一次。准确加入1. 0 mL甲醇以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(4.2.16)洛解，待衍生化。4.4. 1
9、. 2 SAX固相革取柱净化法提取:样品经粉碎并通过40目筛子。称取25.0g于均质杯中，加入50mL甲醇+水(4.2.17)，高2 SN/T 1572一2005速均质3min，静置10min。将提取液上清液经滤纸过滤，收集滤液，用氢氧化铀(1mol/L)调滤液pH为5.86. 5(仅需要1mol/L氢氧化饷榕液2滴3滴)。净化:把SAX固相萃取小柱安装在固相萃取装置上，先用5mL甲醇淋洗小柱，然后用5mL甲醇十水(4.2.17)淋洗小柱。准确移取上述滤液2.0mL，以小于2mL/min的流速通过固相萃取柱，先用5 mL甲醇-7.(4.2.17)，然后用3mL甲醇淋流小柱。用10mL乙酸+甲醇
10、(4.2.19)以小于1mL/min 的流速洗脱伏马毒素，收集洗脱液于20mL玻璃试管中，在600C加热条件下氮气吹干，用1mL甲醇冲洗玻璃试管壁并溶解残留物，再用氮气吹干，确保乙酸已经完全除去。残留物加200L甲醇+水(4.2.16)溶解，待衍生化。注:在液体过柱时，当液体凹液面下端与柱填料上端水平时，停止液体流出，避免SAX柱填料与空气接触。4.4.2 衍生化取样品处理液和标准溶液各50L，各加150L衍生剂(4.2.24)，混匀，在2min内过0.45m滤膜(4.2.30)并进行HPLC分析。4.4.3 测定4.4.3. 1 液相色谱条件a) 色谱柱:Spherisorb Cj8柱，25
11、0mmX4. 6 mm(内径)，10m，或相当者;b) 流动相:见4.2.25; c) 流速:0.8mL/min; d) 荧光检测器波长:Ex335 nm,EM 440 nm; e) 柱温:室温;f) 进样量:20L。4.4.3.2 色谱测定分别取经衍生化的样液和标准溶液20L进行HPLC分析，以其标准溶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，伏马毒素Bj、队的保留时间分别为5.5min和12.6min左右，参见附录A。4.4.4 空白试验除不加试样外，按上述测定步骤进行。4.5 结果计算和表述按式(1)计算试样中伏马毒素含量:式中:x =
12、.A X Cs X 3. . 9 一- As X m X CVz/Vj) X一一为试样中伏马毒素Bj、岛的含量，单位为毫克每千克(mg/kg); A一-为样液中伏马毒素Bj、岛的峰面积，单位为平方毫米(mmZ); As一为标准、溶液中伏马毒素Bj、岛的峰面积，单位为平方毫米(mm2); Cs一一为标准榕液中伏马毒素Bj、岛的故度，单位为微克每毫升(g/mL); m一一称取的试样量，单位为克(g); Vj-一试样提取液总体积，单位为毫升(mL);V2一一净化用提取液体积，单位为毫升(mL);V3-一试样净化液最后定容体积，0.2mL; D一一一净化时稀释因子，免疫亲和柱净化法D=5，固相萃取柱净
13、化法D=l。注:计算结果应扣除空白值。报告结果时以伏马毒素矶、良的总含量(mg/kg)报告结果。( 1 ) 3 SNjT 1572-2005 5 测定低限、回收率5. 1 测定低限本方法对伏马毒素Bj、良的测定低限分别为:Bj o. 050 mg/kg，也0.050mg/kg。5.2 回收率5.2.1 玉米添加浓度/(mg/kg)0.05 o. 5 1. 0 5.0 添加浓度/(mg/kg)0.05 o. 1 0.5 1. 0 5.2.2 玉米饲料添加浓度/(mg/kg)0.05 0.5 1. 0 5.0 添加浓度/(mg/kg)0.05 O. 5 1. 0 5.0 4 L一一表1伏马毒素B1
14、回收率(免疫亲和柱)/(%) 回收率(固相萃取柱)/(%) 73.0 76.0 84.0 83.0 82.5 85.5 84. 9 84. 8 表2伏马毒素B2回收率(免疫亲和柱)/(%) 回收率(固相萃取柱)/(%) 71. 0 74.0 83.0 83. 5 82.2 84.0 82.1 85.4 表3伏马毒素B1回收率(免疫亲和柱)/(%) 回收率(固相萃取柱)/(%) 72.0 74.0 82. 7 86. 1 84.5 83. 5 84. 2 84.5 表4伏马毒素B2回收率(免疫亲和柱)/c%) 回收率(团相萃取柱)/c%)72.0 71. 0 83. 1 83.9 82. 8 8
15、5.9 83. 6 83.3 SN/T 1572-2005 附录(资料性附录)标准晶色谱圈A 5 叫IUEEg3iiL叫NmM响愉町您!jilt一mw, EEHN由.的iiTill-1、，-mMm仰町华l11|11liiJ开53.NA啕叫棋时牛伊阳呻伏马毒素B1、B2标准样晶液相色谱固40 20 圈A.130 SN/T 1572-2005 Foreword Annex A of this standard is an informative annex. This standard was proposed by and is under the charge of China Nationa
16、l Regulatory Commission for certification and Accreditalion. This standard was drafted bliaoning Entry-Exit Inspection and Ouarantine Bureau of the People 5 Republic of China and by JiLi n Entry-Exit Inspection and Ouarantine Bureau of the Peoples Republic of China. The main drafters of this standar
17、d are Li n Weixuan , Tian Miao ,Mu Jun , Zhao Tongtong , Cao Dong 盯lel.This standard is a professional standard promulgated for the first time. 6 SN/T 1572-2005 Inspection of fumonisins in cereals and feedstuffs for import and export-Liquid chromatographic method 1 Scope This standard specifies the
18、methods of sampling , sample preparation and determination by liquid chromatography of fumonisins 息，也incereals and feedstuffs for import and export. This standard is applicable to the determination of fumonisins 凯，也incorn and corn feedstuffs for import and export. 2 Normative references The followin
19、g 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 the possibility of applying the most recent editions of the standards indicated below. SN /T 0800. 1一1999Inspecti
20、on 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. During sampling and sample preparation process , avoid sample contaminated or the amount of fumonisins 8, ，也changed.4 Method of determination 4. 1 P
21、rinciple Fumonisins 8, ，也wereextracted with methanol-water, cleaned up by FumoniTest cartridges or SAX SPE cartridges , and derived , divided with reversed-phase liquid chromatography, detected with FLD detector. Calculate the results use ESTD method. 4. 2 Reagent and materials 4.2. 1 Water: Super-p
22、ure water. 4. 2. 2 Methanol: HPLC grade. 4.2.3 Acetonitrile: HPLC grade. 7 SN/T 1572-2005 4.2.4 Acetic acid: G. R. 4.2.5 H3P04: G. R. 4.2.6 HCI: G. R. 4.2.7 NaH2 P03: A. R. 4.2.8 Na2 HP03: A. R. 4.2.9 KH2 P03: A. R. 4.2.10 NaCI: A. R. 4.2.11 KCI: A.R. 4.2.12 NaOH: A. R. 4.2.13 Sodium tetraborate: G.
23、 R. 4.2.14 2-Mercaptoethanol: G. R. 4.2.15 o-Phthalaldehyde: G. R. 4.2.16 methanol + water (1 + 1). 4.2. 17 methanol + water (3 + 1). 4. 2. 18 methanol + water (4 + 1). 4.2. 19 acetic acid + methanol (1 + 99). 4. 2.20 Fumonisins 81 ，也standardstock solution: Accurately weigh 10.0 mg fumonisins 81 and
24、 fu monisins 82 into 100 mL volumetric flask respectively, dissolve with acetonitrile + water C 1 + 1) to 100 mL,mix. The concentration of fumonisins 81 and fumonisins 82 standard stock solution are all100 g/mL. 4. 2. 21 Mixed standard intermediate solution: Mix above two standard stock solution at
25、ratio 1 : 1 , to prepare mixed standard intermediate solution of each fumonisins 50g/mL. 4.2.22 Mixed standard working solution: Pipet 0.04 mL,O. 20 mL, 1.0 mL,5. 0 mL mixed standard intermediate solution into 10 mL volumetric flask respectively,and make up to 10 mL with acetoni trile + waterC 1 + 1
26、) , in order to prepare mixed standard working solution with O. 2g/mL，1.0g/ 8 SN/T 1572-2005 mL，5g/mL，25g/mL resp. (Fumonisins Mixed standard intermediate solution and mixed standard working solution can be stored for 6 months at 40C ). 4.2.23 Phosphate buffer: Weigh Na2 HP04 1. 2 g, KH2 P04 O. 2 g,
27、 NaCI 8 g, KCI O. 2 g, dissolved with 990 mL water, adjust the pH value to 7.0 with HCI , make up to 1 L with water. 4.2. 24 Derivatization reagent: Weigh 5 mg o-Phthalaldehde， dissolved with 1 mL methanol , make up to 50 mL with 0.05 mol/L Sodium tetraborate solution ,add 25L 2-Mercaptoethanol , mi
28、xed for use. CDerivatization reagent can be store for 1 week at OoC -4C) 4.2.25 LC mobile phase: Methnol-NaH2P04 (0.1 mol/U (77 + 23). Adjust pH value to 3.3 with H3P04 Filtered with 0.45m filter membrane. 4.2.26 FumoniTest immunity affinity cartridges. 4.2.27 SAX super anion solid phase extraction
29、(SPE) cartridges:500 mg/1O mL,product of Varian. 4.2.28 Glass fiber filter paper. 4.2. 29 Centrifuge tube: 50 mL. 4.2.30 Filter membrane: 0.45m. 4.3 Apparatus and equipment 4.3.1 HPLC: Equipped with FLD-detector. 4.3. 2 Flux control instrument with pump. 4.3. 3 SPE instrument. 4.3.4 Tissue homogeniz
30、er: 20000 r/min. 4.3.5 Vortex mixer. 4. 4 Procedure 4. 4. 1 Extraction and clean up 4.4. 1. 1 Method of clean up with immunity affinity cartridges. 9 SN/T 1572-2005 Extraction: Crush sample to make it through 40 mesh sieve. Weigh 25 9 sample in the homogenizer cup ,add 4 9 NaCI and 50 mL methanol +
31、water(4. 2. 18) , homogenize at high speed for 3 min , then wait 10 min. Filter the supernatant of extraction solution with Glass fiber filter paper , collect the fil trate. Clean up: Accurately pipet above filtrate 10 mL to 100 mL taper bottle,add 40 mL Phosphate buffer (4.2.23) ,mix vigorously on
32、vortex mixer, filter with Glass fiber filter paper , collect the filtrate. Re move large top cap of the FumoniTest immunity affinity cartridges,cut bottom 1/8 inch off the end of the top cap with scissors or sharp blade , then put back the cap. Connect a 10 mL glass injector with FumoniTest immunita
33、ffinity cartridges, accuratelpipet above filtrate 10 mL into glass injec tor,connect the Flux control instrumentC4. 3. 2) ,take off the down Cbottom) cap of FumoniTest im munity affinity cartridges. Control the pressure to make the filtrate down through the cartridge at the rate of 1 d/s,until air g
34、o through into the cartridge. Take off the cartridge from the glass injector,add Phosphate bufferC4. 2. 23) into the cartridge, then connect the glass injector again , elute the cartridge with 10 mL Phosphate bufferC4. 2. 23) at rate of 2 d/s,until air go through into the cartridge ,abandon the outf
35、low. Elute the cartridge with 10 mL Phosphate bufferC4. 2. 23) again. Accurateladd 1 mL methanol into the cartridge, elute at the rate of 1 d/ s, collect the eluent in a 10 mL glass tube, evapo rate to dryness with N2 gas stream ,add 200L methanol + waterC4. 2.16) to dissolve the residue , to be der
36、ivatized. 4.4. 1. 2 Clean up with SAX SPE cartridges Extraction: Crush sample to make it through 40 mesh sieve. Weigh 25 9 sample in the homogenizer cup ,add 4 9 NaCI and 50 mL methanol + water(4. 2. 17) , homogenize at high speed for 3 min , then wait 10 min. Filter the supernatant of extraction so
37、lution with Glass fiber filter paper ,collect the fil trate. Adjust the pH value to 5.8-6.5 with NaOH(1 mol/L) (2.3 drop enough). Clean up: Install the SAX SPE cartridges on the SPE instrument , elute the cartridges with 5 mL meth anol , then 5 mL methanol + water(4. 2. 17). Accurately pipet 2. 0 mL
38、 above filtrate, make it go through the SAX SPE cartridges at the rate of no more than 2 mL/min, then respectively elute the cartridges with 5 mL methanol + water(4. 2. 17) and 3 mL methanol. Elute the fumonisins with acetic acid + methanol(4. 2.19) at the rate of no more than 1 mL/min,collect the c
39、luent into 20 mL glass tube,e vaporate to dryness with N2 gas stream at 600C ,wash the tube with 1 mL methanol and dissolve the residue , evaporate to dryness with N2 gas stream again to get rid of any remnant acetic acid. Add 200L methanol + water(4. 2.16) to dissolve the residue , to be derivatize
40、d. Note: Avoid to make the filling of the SAX SPE cartridges contact with air during the liquid go through the cartridges. 4.4.2 Derivatization Respectively pipet 50L sample preparation solution and standard working solution ,add 150L deri vatization reagent , mix, make the mixture go through O. 45m
41、 filter membrane and applto LC with in 2 min. 10 SN/T 1572-2005 4.4.3 Determination 4. 4. 3. 1 LC operating conditions a) Chromatographic column: Spherisorb C8 ,250 mm x 4.6 mm (i. d. )，10m，or equivalent; b) Mobile phase: 4.2.25; c) Flow rate of mobile phase: 0.8 mL/min; d) Wave length of FLD detect
42、or: Ex: 335 nm , EM ;440 nm; e) Column temperature: Room temperature; f) Sample size: 20L. 4. 4. 3. 2 LC determination Inject separately equal volumn of the derivatized standard working solution and the sample solution into the LC sstem. Identify fumonisins 息，82by comparing peak retention time of th
43、e sample with corresponding standard peak tetention time. Calculate the result by comparing peak area of the sam ple with corresponding standard peak area , using external standard method. Under the above condition , the retention time of fumonisins 8, ，也are8, :5. 5 min，也:12.6min，seeappendix A. 4. 4
44、. 4 81ank test The operation of the blank test is the same as that described in the method of determination, but with omission of sample addition. 4.5 Result calculation and description The calculation of fumonisins 鼠，也iscarried out according to the following formula (1) : A X Cs X V3 X 0 X=7一一一( 1
45、) As X m x ( V2/ V,) Where X一一thecontent of fumonisins 息，也insample ,mg/kg; A一一一thepeak area of fumonisins息，82of the sample solution , mm勺As一一-thepeak area of fumonisins 矶，82of the standard solution. mm勺Cs一一-theconcentration of fumonisins 8, , 82 of the standard solution , mm勺m一一-themass of test samp
46、le , 9 ; V，一一二thewhole volume of extraction solution , mL; V2一一-thevolume of extraction solution which has been cleaned Up , mL; 民-一一-thefinal volume of test solut旧n，0.2mL; D一一-dilutionfactor, if use 4. 4. 1. 1 method 0 = 5 11 SN/T 1572-2005 If use 4. 4. 1. 2 method 0 = 1. Note: The blank value shal
47、l be subtracted from the above result of calculation. Result shall be reported as the sum of fu monisins 8, and fumonisins 82 5 Limit of determination and recovery 5. 1 Limit of determination Limit of determination of fumonisins 息，82 are: fumonisins 8,: O. 050 mg/同，fumonisins82: 0.050 mg/kg. 5. 2 Re
48、covery 5.2.1 Corn Table 1 Fumonisins 8, Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (immunity affinity cartridges) (SPE臼rtridges)0.05 73.0 76.0 0.5 84.0 83.0 1.0 82.5 85.5 5.0 84.9 84.8 Table 2 Fumonisins 82 Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (imm
49、unity affinity cartridges) (SPE cartridges) 0.05 71.0 74.0 0.5 83.0 83.5 1.0 82.2 84.0 5.0 82.1 85.4 5. 2. 2 Corn feedstuffs Table 3 Fumonisins 8, Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (immunity affinity cartridges) (SPE cartridges) 0.05 72.0 74.0 0.5 82. 7 86. 1 L一一一一12 SN/T 1572一2005Table 3 (Continued) Addition concentration/ Recovery / ( % ) Recovery / ( % ) (mg/kg) (immunity affinity臼同时啕es)(SPE四川ridges)1.0 84.
