【商检行业标准(SN)】 进出口粮谷、饲料中伏马毒素检验方法 液相色谱法

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 inport 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，
本标准由医家认证认可监督管理委员会提出并口，SN/T1572—2005
本标雅起草单位：中华人民共和国辽之山人境检验检峻局，户华人民共和国古林山入境检验检疫局。
本标主要起草人：林维宣、口苗、牟俊、赵形、郭，离明称、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、饲料中伏马毒素检验方法液相色谱法
SN/T 1572—2005
本标雅规定了逊出口粮夺、饲料中伏马市素检验的抽祥制样和被相色谱检测}方法。本标准适用二逃出口玉米和玉米饲料中伏马寺素B，、B，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波为本标准的条款。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用下列标滩最新版本的叫能性。凡足不注万欺的引用义作，H最新版小适尼丁本标雅，SN/T0800.11999进出口粮油饲料检验油栏和1制样方法3抽样和制样
按SV/T0801执行。在抽栏和制栏过程巾，必领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
群品用甲！水提取，经FrrmariTest 免疫亲和杆或 SAX强阴离子国相萃取杆净化.经衍生化.用反液杆色谱注分离，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乙睛：色谱纯，
4.2.4艺酸：优级纯：
磷酸：优级纯。
盐酸：优级纯。
磷酸一氟纳:分析纯。
磷酸氢二纳：分析统。
磷酸二氢钾：分析绝。
氯化钠分析纯。
氯化钾：分析继。
氧氧化钠：分析纯。
四羧钠：优级纯。
琉甚乙醇：优级纯。
邻苯一中醛：优级纯
干醇1水(1「:)。
半醇+水（3+：）。
SN/T 1572—2005
4.2.18千醇十水（4+：）。
4. 2. 19乙酸十甲醇(1—99)。
4.2.20伏马毒素标准储备液：称取伏马毒素B:、B2各1C.0IIg+分剂用乙腊十水(1十1)溶解并定容到100mL量瓶中，伏马毕烹B的浓度均为100ng/mL，4.2.21派标准储备液;取标储备液B.,1B，以1 ：二比例混合,剂混合标推储备液,伏马寺素 B:、B,的浓度均为50/ml.。
4.2.22浪合标准T作液：取适量混合标准储备液于1CmL容量瓶它，用乙睛十水(1十1)定容，得马毒索B，.B，的浓度分别为0.2μg/mL、.0g/rmL,5μg/mL，25μ/mL,5Cug/mL的混合标准工作液（伏马毒素标雅储备液和工作液在4℃下可贮荐6个月）。4. 2. 23 磷酸盐缓冲溶被:称取磷酸氢一钠 1. 2 g,磷酸一氢钾 0.2 多,氯化钠 8 g,氯化钾 0. 2 +溶解丁990 rml.水巾,用盐酸调节 pH为 7. 0,用水楚容到1I.4.2.24衔生剂:称取5 t1g邻苯二甲醛，溶于1 ml.甲醇,用 0. 05 t:r1/T.四硼酸钠溶液定容到 5ml.,加人25 江统基艺辟,混勾，用(℃℃～2℃可保存周)：4.2.25液相色谱流动相：月醇1磷酸一氮钠(5.1mol/L)（77123），用磷酸调pll为3.3μm、C.45m滤膜过滤。
4.2.26Fumanilest免接亲和柱。4.2. 27
SAX强防离了固杆萃取(SPE)柱：500 mg/0 mL.Varian产品4.2.28
腹璃红维燃纸
离心管，50 mL.e
滤膜：0.45um
4.3仪器和设备
4.3.1良效液相免谱仪：配有荧光检测器。4.3.2泵流控制台，
4.3.3固相举取仪，
4.3.4组织均质器：2G00cr/min，4.3.5涡旋均勾器。
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：样品经粉碎并通造40月筛子。称取25.0g丁均质杯中,加入4g氯化钠和5Gml.甲醇「水（4.2.18），高速均质3rmin，静置[cmn。将提取液上消液用槽纹滤纸过滤，改隽滤液。净化：准确移取1述滤液10.01m.士191.具塞铠形瓶中，如20.酸鼎缓冲落液（4.2.23），置子滑旋均封器「凝每·经玻璃纤维璐红过滤.改集滤液备用：将 Furrlonilest 免疫亲和托的盖帽取下,剪掉帽盖的封口端,再把盖盖l:。将 10 rnL玻璃汗射器筒与亲和托相连确移取1G.0ml.上非得到的滤液丁玻璃注刻器筒中，接上泵流控制器，拔撑亲和柱底帕。控制压力使样液以1滴/s的流速全部通过FurioniTes亲和柱，至空气逊人到亲和柱中。从玻璃注录幕上取下FimoniTes1亲和柱，将亲利柱上部用磷酸盐缓冲落液(4.2.23)注满，再把亲和柱连接到璃注射.1:,月10 rrL磷酸盐缓污溶液(4.2.23)以2滴/s的流邀淋洗亲和柱，直至空气法人到亲和杜中，弃去全部流出被。再用10mI.碰酸盐缓冲溶液（4.2.23)重复淋洗亲和社一次，确加人1.0ml.币醇以1滴/s的流速洗脱，收集全部涝脱液丁10ml,坡璃管中，氮气恢下,残留物加200ul.甲醇十水(4.2.16)溶解，待衍牛化。4.4.1.2SAX固相萃取柱净化法
提取：样品经粉碎并通过40 日筛了。称段25,0于均质杯可，加人50 1=1L中醇十水（4.2.17).高2
SN/T 1572—2005
速均质3 rrin,静背【min。将提取液上清液经滤纸过滤，收集滤液,用氢氧化钠mnl/I.)调滤液pH为 5. 8~-6. 5仪需要 1 t:Ir1/T. 氢氧化钠溶液 2 滴~~3 滴)。净化:把 SAX 固相萃取小柱安装在固相萃取装骨I:,先压5 ImL 平嗨淋洗小柱.然后用可II-L 中1水(1.2._7)淋洗小杆。准硫移取1 述滤液2.0 nL.以小于2 rL/ri1的流逆通过国相辈最栏光用5mL干醇＊(4.2.17),然后用 3ml,币醇淋洗小±：用10mlz酸1尽醇(1.2. 19)以小丁1 mL/min的流速沉脱伏马毒素,收集洗脱液于20ml.玻璃试管中，在60加热条件下氮气吹·1用：ml.甲醇冲洗玻翁试管壁并溶残留物再压氮气次干，确保乙酸已经完企除去。戏留物加2C0 μL中醇十水(4.2.16）溶解.待衍兰化，
汁：在液体过拉时，当液体凹液面下端与杜滨料1端水时，停止液体流白，避免SAX社填料与空气变点，4.4.2衍生化
取样品处理液利标准溶液各 50 μI,冬加150 uL衍生剂(=.2,24),混勾,在2mi=内过 0.45Pm滤膜(4.2.30)并迹行 IIPIC分析，4.4.3测定
4.4.3.1液相色谱条件
谱柱:Sherisarb Cre柱250 miX4,6 内径,l0 μm,或当者a)
流动析：见4.2.25；
说速:G.8 mI/min:
荧光检测器波长：E.335nm，E44Cnm：注温：窄温；
［】送样量:20 μT.。
4.4.3.2色谱测定
分别取经衍生化的样液和标准溶液2CL进行IIPLC分析，以其标准溶液峰的保留时间为依据追行定性.以H峰面科求山样液中被浏物质的含量，供算，在上述色谱条件下，伏马声素B、B，的保留时间分别为5. 5 rin 和 12.6 min左右，签见附录 A4.4.4空白试验
除不加试样外，按「述测定玉骤进行4.5结果计算和表述
按式(:)计算试栏中伏卫毒素含量：AX XV, XI
X=Axmx(VM)
我中：
为试样中伏马每素BB的含量，单位为毫克衔二克（rn/kg）：A
为样液巾伏马毒索B、B的峰而积，单位为方毫米(mm）;为标谁溶液巾伏马毒素B-,13.的降面积,单位为平方亳米(m);为际准溶十马毒素 B、B2的浓度，单位为微京符亳升(ug/ml.):称取的诚样量.位为克()；
试推提最液总休积，单为毫引（m？净化用提妆液体积，单位为毫升（ml.）：试样净化液最后定容体.0.2ml.;净花时稀释因，免疫亲和柱净化法I)一5,固析取柱净化法D一1。注：引算结朵底和除牢：1值。报合统效马毒素B、B的总含旦tI/kg)报告结果-(1)
SN/T 1572—2095
5测定低限、回收率
5. 1测定低限
本方法对伏马每素B、Bz的测定低限分别为：B0.050mg/kg.B0.05Czng/kg2回收率
5.2.1玉米
表 1 伏马毒素 B
恭加浓度/(mg/kg）
泰加浓度/（mg/kg）免费标准下载网bzxz
玉米饲料
添加浓度/(mg/kg)
添加激度/img/kg）
画攻率（免获亲和杜))
表2伏马毒素 B
同攻率（免疫亲利柱)/（%
伏马毒素
同改率免疫亲和柱)
表 4伏马毒素 B,
回改率免疫亲和杜)《)
世收率(固弃率取柱)（路)
同收率（固萃取棕）（%）
回收率（国益率取栏)%）
回收率(相率服栏)《%）
附录A
(资料性附录）
标准品色谱图
伏马毒素 B:、B2标准样品液相色谱图SN/T 1572—2005
SN/T 1572—2035
Foreword
AnnexA of this standard is an informative annexThis 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 JiLin Entry-Exit Inspection and Quarantine Burreau of the People' s Republicof China.
The rnain drafters of this standard are Lin Weixuarni, Tian Miao, Mu Jun,Zhao Tongtong, Cao Dong.mei.
This standard is a professional standard promulgated for the first time.6
SN/T 1572—2005
Inspection of fumonisins in cereals and feedstuffs forimport and export-Liquid chromatographic methodScope
This standard specifies the methods of sampling,sample preparation and determination by liquidchromatography of fumonisins B1 , B, in cereals and feedstuffs for import and export.This standard is applicable to the determination of fumonisins B: , B2 in corn and corn feedstuffs forimpart and expart.
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 0800. 1 —1999 Inspection of cereals and feedsfurffs for import and export -methods of sampling and preparation of samples3Sampling and sample preparationSee SN/T 0800, 1. During sampling and sample preparation process,avoid sample contaminated or theamaunt of fumonisins B, , B, changed.4Methodof determination
4.1Principle
Fumonisins B: ,B, were extracted with methanol-water,cleaned up by FumoniTest cartridges or SAXSPE cartridges,and derived.divided with reversed-phase liquid chromatography, detected with FLD.detector. Calculate the results use EsTD methad4.2 Reagent and materials
4. 2.1 Water: Super-pure water.4.2.2 Methanol:HPLC grade.
4. 2.3 Acetonitrile: HPLC grade.SN/T 1572—2005
Acetic acid: G.R.
H3 PO4 : G, R.
HCI: G. R.
NaH2PO3 : A. R.
NazHPOs : A, R.
KH,PO : A. R.
Nacl: A. R.
NaOH: A. R.
Sodium tetraborate: G, R.
4. 2. 142
2-Mercaptoethanal: G. R.
4.2、15o-Phthalaldehyde:G, R.methanol+water (1+ 1)
methanol+ water (3+ 1).
4. 2. 18 methanol + water (4 + 1)4.2.19acetic acid -methanol (1 +99).4. 2, 20 Fumonisins B: ,B, standard stock solution: Accurately weigh 10, 0 mg fumonisins B, and ful-monisins B, into 10D mL volumetric flask [espectively,dissolve with acetonitrile + water(1 - 1) to100 mL,mix. The concentration of fumonisins B, and fumonisins B2 standard stock solution are all 100g/mL.
4.2.21 Mixed stardard interrnediate solution: Mix above two standard stock solutiorl at ratio 1 : 1.to prepare mixed standard intermediate solution of each fumonisins 5o μg/mL,4. 2. 22 Mixed standard warking solution: Pipet 0.04 mL,0. 20 mL, 1. 0 mL,5. 0 mL mixed standardintermedliate solutioninto10 mLvolumetric flask respectively,andmake up ta 10 mL with acetani-trile+ water(1+ 1),in order to prepare mixed standard working solution with 0.2 μg/mL,1, 0 μg/8
mL,5 μg/mL,25 μg/mL resp.
SN/T 1572—2005
(Fumonisins Mixed standard intermediate solution and mixed standard working solution can bestored for 6 months at 4℃ ).4. 2.23 Phosphate buffer: Weigh NazHPO, 1, 2 g, KH,PO, 0, 2 g,NaCl 8 g.KCI 0, 2 g,dissolved with990 mL water.adjust the pH value to 7. 0 with Hcl,tmiake up to 1 L with water.4. 2. 24 Derivatization reagent: Weigh 5 mg o-Phthalaldehyde, dissolved with 1 mL methanol.makeup to 50mL with 0.05mol/LSodiumtetraborate solution,add25 μL2-Mercaptoethanol,mixed foruse. (Derivatization reagent can be store for 1 week at 0℃ 4℃)4, 2.25 LC mobile phase: Methanol-NaH, PO4 (0. 1 mol/L) (77 + 23). Adjust pH value to 3, 3 withH.POa, Filtered with O. 45 μm filter membrane.4.2.26FumoniTestimmunityaffinitycartridges.4. 2. 279
SAX super anion solid phase extraction (SPE) cartridges: 500 mg/10 mL-product of Varian.4. 2. 28
Glass fiber filter paper
Centrifuge tube: 50 mL.
4.2. 30 Filter membrane: 0. 45 μm.4.3
Apparatus and equipment
4.a. 1HPLC: Equipped with FLD-detector.4.3.2
Flux control instrument with pump.4.3.3
SPE instrument.
Tissue homogenizer:20 000 r/min.4.3.4
5Vortexmixer
4.4Procedure
Extraction and clean up
4. 4. 1, 1 Method of clean up with immunity affinity cartridges.SV/T 1572—2005
Extraction: Crush sample ta make it through 40 mesh sieve. Weigh 25 g sample in the homagenizercup,add 4 g NaCl and 50 mL methanol + water(4. 2. 18),homogenize at high speed for 3 min, thenwait 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 Too mL taper battle,add 40 mL Phosphate buffer(4. 2. 23) ,mix vigorously on vartex nixer,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 endof the top cap with scissors ar sharp blade,then put back the cap. Connect a 10 mL glass injectorwith FumoniTest immunity affinity cartridges,accurately pipet above filtrate 10 mL into glass injec-tor,connect the Flux control instrument(4, 3, 2),take off the down (bottom) cap of FumoniTest im-munity affinity cartridges, Control the pressure to make the filtrate down through the cartridge at therate of 1 d/s.until air go throurgh into the cartridge. Take off the cartridge from the glass injector, addPhosphate buffer(4. 2. 23) into the cartridge, then connect the glass injector again,elute the cartridgewith 10 mL Phosphate buffer(4. 2. 23) at rate of 2 d/s,until air go through into the cartridge,abandonthe outflow. Elute the cartridge with 10 rnL Phosphate buffer(4. 2. 23) again. Accurately add 1 rnLmethanol 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 N, gas stream,add 200 μL methanol+ water(4. 2. 16) to dissolve the residue,tobe derivatized.
4.4.1.2Clean up with SAX SPE cartridgesExtraction1: Crush sarnple to make it through 4 mesh sieve. Weigh 25 g samiple in the hornogenizercup,add 4 g NaCl and 50 mL methanol + water(4. 2. 17), homogenize at high speed for 3 min, thenwait 10 min, Filter the supernatant of extraction solution with Glass fiber filter paper,collect the fil-trate. Adjust the pH value ta 5, 8~6. 5 with NaQH(1 mol/L)(2~~3 drop enough).Clean up: Install the SAX SPE cartridges on the SPE instrument,elute the cartridges with 5 mL methanol, then 5 mL methanol+ water(4. 2. 17). Accurately pipet 2. 0 mL above filtrate.make it go throughthe SAX SPE cartridges at the rate of no more than 2 mL/min, then respectively elute the cartridgeswith 5 mL tmethanol + water(4. 2. 17) and 3 mL methanol. Elute the furronisins with acetic acid +rmethanol(4. 2. 19) at the rate of no miore than 1 rnL/rnin,collect the cluent into 20 ml glass tube,e-vaporate to dryness with N, gas stream at 60'C , wash the tube with 1 mL methanol and dissolve theresidue, evaporate to dryness with N2 gas stream again to get rid of any remnant acetic acid. Add200 μ L methanol - water(4, 2. 16) to dissolve the residue, to be derivatized.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 50 μL sample preparation solution and standard working solution,add 150 μL deri-vatization reagent, mix,make the mixture go through Q. 45 μm filter membrane and apply to Lc within 2 min.
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