SN/T 2426-2010 进出口粮谷中桔霉素含量检测方法 液相色谱法 SN/T2426-2010 标准下载解压密码：www.bzxz.net

中华人民共和国出入境检验检疫行业标准SN/T2426—2010
进出口粮谷中桔霉素含量检测方法液相色谱法
Determination of citrinin contents in cereals for import and export-HPLC method
2010-01-10发布
中华人民共和国
国家质量监督检验检疫总局
2010-07-16实施
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：中华人民共和国湖北出入境检验检疫局。本标准主要起草人：林雁飞、赵晓亚、胡小钟、王鹏、李晶、付晓芳。本标准系首次发布的出入境检验检疫行业标准http：//foodmate.netSN/T2426—2010
1范围
进出口粮谷中桔霉素含量检测方法液相色谱法
本标准规定了进出口粮谷中桔霉素含量液相色谱检测方法。本标准适用于进出口大米、大麦、燕麦、小麦中桔素含量的检测。2方法提要
SN/T2426—2010
用乙睛-异丙醇-水的混合溶液提取试样中桔霉素，Cis固相萃取小柱净化，用配荧光检测器的液相色谱仪测定，外标法定量。
3试剂和材料
除另有规定外，试剂均为分析纯，水为蒸馏水或相当纯度的水3.1异丙醇：色谱纯。
3.2乙睛：色谱纯。
3.3磷酸：优级纯。
3.4提取溶剂：乙睛-异丙醇-水（35+10+55，体积比），用磷酸调pH为1.5。3.5磷酸溶液：取5.6mL磷酸，以水定容至1000mL。3.6流动相：乙腈-异丙醇-0.08mol/L磷酸（35+10+55，体积比）。3.7C18固相萃取柱：500mg，3mL，或相当者。使用前分别用5mL甲醇和5mL水预淋洗并保持柱体湿润。
3.8桔霉素标准物质（citrinin，CiHiO，CAS编号：518-75-2）：纯度大于等于97%3.9桔霉素标准储备液：称取适量桔霉素标准物质，用乙睛溶解并定容至1.0mg/mL，0℃～4℃保存。
3.10桔霉素标准工作液：根据需要用流动相将标准储备液稀释成25ng/mL、50ng/mL、100ng/mL500ng/mL、1000ng/mL的标准工作溶液3.11玻璃纤维滤纸：直径11cm.孔径1.5μm。4仪器和设备
4.1高效液相色谱仪：配有荧光检测器。4.2振荡器。
4.3离心机：4000r/min。
4.4真空固相萃取装置。
4.5氮吹仪。
4.6分析天平。
5试样制备和保存
取有代表性样品500g，用粉碎机粉碎并通过830um圆孔筛，混匀，分成两份装人洁净容器内，密封并标识。在制样的操作过程中，应防止样品受到污染或发生残留物含量的变化，1
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SN/T2426—2010
6测定步骤
6.1提取
称取试样约5g（精确至0.01g）于50mL离心管中，加10mL提取溶剂（3.4）)，在振荡器上振荡提取30min。于3500r/min离心4min，上清液转入另一离心管中。在残渣中再加人5mL提取溶剂（3.4）重复上述操作，合并上清液。在提取液中加水至40mL，并用磷酸调pH为1.5，过玻璃纤维滤纸（3.11)，待净化。
6.2净化
将上述溶液过预淋洗好的C1s固相萃取柱，用5mL水淋洗柱子。待淋洗液全部流出柱子后，减压抽干3min。用10mL甲醇以1.0mL/min的速度洗脱，收集全部洗脱液，在40℃下，N，吹干，再以1.0mL流动相溶解，过0.2μm滤膜，供液相色谱测定。6.3测定
6.3.1液相色谱条件
色谱柱：Cls柱，250mmX4.6mm（内径），粒径5μm，或相当者；b)
流动相：乙睛-异丙醇-0.08mol/L磷酸（35+10+55.体积比）；流速：1.0mL/min;
进样量：50μL；
柱温：28℃；
检测波长：Ex=331nm，Em=500nm。f）
6.3.2色谱测定
根据样液中被测桔霉素含量情况，选定峰面积相近的标准工作溶液。标准工作液和样液中桔霉素响应值均应在仪器检测线性范围内。对标准工作液和样液等体积参插进样测定。在上述色谱条件下，桔霉素保留时间约为9.1min，标准物质色谱图参见附录A中图A.1。6.3.3空白试验
除不加试样外，均按上述操作步骤进行。7结果计算和表述
用色谱数据处理软件或按式（1)计算试样中桔霉素含量，计算结果需将空白值扣除：AXcXV
式中：
试样中桔霉素含量，单位为毫克每千克（mg/kg）；A
样液中桔霉素的峰面积；
标准工作液中桔霉素的峰面积；标准工作液中桔霉素的浓度，单位为微克每毫升（rg/mL）样液最终定容体积，单位为毫升（mL）；最终样液所代表的试样量，单位为克（g）。测定低限、回收率
测定低限
本方法的测定低限为0.01mg/kg。8.2回收率
粮谷中桔霉素检测的添加回收率数据见表1。2
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回收率数据
添加浓度/（mg/kg）
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SN/T2426—2010
回收率/%Www.bzxZ.net
78.9~90.2
88.0~91.4
74.8~96.6
79.1~94.2
74.6~90.5
76.2~89.7
75.9~91.3
77.9~96.2
79.9~93.1
80.4~95.9
SN/T2426—2010
附录A
（资料性附录）
标准物质色谱图
枯酶素
桔霉素标准物质（100ng/mL）的液相色谱图http：//foodmate.net14/mim
Foreword
AnnexAofthisstandardisaninformativeannexSN/T2426—2010
This standard was proposed by and is under the jurisdiction of Certification and Accreditation Admin-istrationofthePeople'sRepublicofChina.This standard is drafted by Hubei Entry-Exit Inspection and Quarantine Bureau of the People's Republicof China.
Main drafters ofthis standard are:LinYanfei,Zhao Xiaoya,HuXiaozhong,WangPeng,Li JingFuXiaofang.
This standard is a professional standard for Entry-Exit inspection and quarantine of the People's RepublicofChinapromulgatedforthefirsttime5
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SN/T2426—2010
Determination of citrinin contents in cerealsfor import and export-HPLC methodScope
This standard specifies the method of determination of citrinin in cereals by high performance liquidchromatography(HPLC).
This standard is applicable to the determination of citrinin contents in rice,barley,oats and wheat forimportandexport.
2Principle
The contents of citrinin in the test sampleis extracted with acetonitrile-isopropanol-water mixed so-lution.Thereafter being cleaned up by the Ci: SPE column,the contents are determined by HPLC withFLD detector,quantified by external standard method.3Reagents and materials
Unless specified,all reagents used should be of analytical grade;\water\is the double distilled water.3.1Isopropanol:Chromatographypure3.2Acetonitrile:Chromatographypure3.3Phosphoricacid:Guaranteedreagent3.4Extraction solution:acetonitrile-isopropanol-water(35+10+55,V/V/V),pH value is adjustedto 1. 5 by phosphoric acid.
3.5HPO solution:pipette 5.6 mL phosphoric acid and dilute to 1 000 mL with water.3.6Mobilephase:acetonitrile-isopropanol-0.08mol/Lphosphoricacid（35+10+55,V/V/V)3.7Cis SPE Cartridge:500 mg,3mL.or equivalent.Condition Cs SPE cartridge with 5mL methanoland5mLwaterbeforeusing.
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SN/T2426—2010
3.8Citrininstandardchemicals（C13H14Os.CASNo:518-75-2),Puritywas97%above.3.9Standard stock solution:Accuratelyweighappropriatecitrinin(3.6),dissolveandquantitative-lywithacetonitrile.Theconcentrationofthesolutionis1.Omg/mL.Thestocksolutionshouldbestoredato℃4℃refrigeratory
Standard working solution:according to the requirement,accurately measure different vol-3.10S
umes of standard stock solution to a 10 mL volumetric flask,dilute with mobile phase to make differ-ent concentration of the standard solution such as 25 ng/mL,50 ng/mL,100 ng/mL,500 ng/mL,1000ng/mL.
GlassMicrofiber：11cm,1.5μm。3.119
Apparatusandequipment
4. 1 High performance liquid chromatography:equipped with fluorescence detector detection.4.2Oscillator.
Centrifuge:4000 r/min
4.4Solidphaseextractionwithvaccumpump4.5 Nitrogen concentrator.
Samplepreparationandstorage
The sample is about 500 g,grind thoroughly and let pass through a 830 μm sieve.Keep the preparedsample into a clean container,seal and label. In the course of sample preparation,precautions shouldbe taken to avoid the contamination or any factors which may cause the change of residue content.6
Procedure
Extractionprocedure
Accurately weight 5 g of the test sample (accurate to0.01 g) into a 50 mL centrifuge tube,add10 mL extracted solution (3.4),mix intensely and extract with ultrasonic extractor 30 min.Thencentrifuge for4min at3500 r/min.The supernatant is taken into other 50 mL centrifuge tube.Anoth-er5mLextracted solution(3.4)is addedand themixture Was extracted again.Combinethe super-natant into thesamecentrifugetubeand add water to 4o mL,adjusted pH value to 1.5byphosphoricacid.
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SN/T2426—2010
Cleanupprocedure
Draw the above solution through a per-conditioned Cis SPE column.Wash the column with 5 mL wa-ter. Then the column is dried by air for at 3 min,and eluted with 10 mL methanol. The eluted solutionis evaporatedtoneardrynessat4o℃anddriedundernitrogenflow,then1.OmLmobilephaseisadded toreconstitute the residue.After beingfiltrated with a 0.2μm filter,the final solution is readyforanalysisbyhighperformanceliquidchromatography6.3
Determination
6.3.1LC operating conditionsLccolumn:Cia.5μm,4.6mm（i.d.）x250mmorequivalent;a）
Mobilephase:acetonitrile-isopropanol-0.08mol/Lphosphoricacid(35+10+55,V/V/V);Flowrate:1.0mL/min;
d)njectionvolume:50μL;
Columntemperature:28℃;
Detectionwavelength：Ex=331nm，Em=500nm.f)
6.3.2HPLC determination
Accordingtotheapproximate concentrationof citrininin thesamplesolution,selectthestandardworking solution with similar peak area to that of the sample solution. The responses of citrinin in thestandard working solution and sample solution should be within the linear range of the instrumentaldetection.Thestandard workingsolution shouldbe randomlyinjectedin-betweentheinjectionsofthe sample solution of equal volume.Under the above operating condition,the retention time of cit-rinin is about 9.1 min. For chromatogram of the standard,see figure A. 1 in annex A.6.3.3
Blanktest
The operation of the blanktest isthe same asthat described in the method of determination,butwith omission of sample addition.CalculationandexpressionoftheresultCalculate the content of citrinin contents in the testsampleby LC data processor oraccording to theformula(1)：
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Where：
X-the content of citrinin in the test sample,mg/kg;A-the peak area of citrinin in the sample solution;A。thepeak areaof citrinin inthestandardworking solution；SN/T2426—2010
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c-the concentration of citrinin in the standardworking solution,μg/mL;V-thefinal volumeofthesamplesolution,mL;m-the corresponding mass of the test sample in the final sample solution.g.8
Limitofdeterminationandrecovery8.1Limit of determination
The limit of determination of this method is 0.01 mg/kg.8.2
Recovery
....(1)
According to the experimental date,the fortifying concentration of citrinin in cereals and its corresponding recoveries seetable1.Table1RecoveriesofspikedsamplesSample
Barley
Levels/(mg/kg)
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Recoveries/%
78.9~90.2
88.0~91.4
74.8~96.6
75.2~96.8
79.1~94.2
75.8~97.6
74.6~90.5
76.2~89.7
75.9~91.3
77.9~96.2
79.9~93.1
80.4~95.9
SN/T2426—2010
AnnexA
(informative)
Chromatogram of the standard粘霉素
14 1/min
Figure A.1-Liquid chromatogram of citrinin (100 ng/mL) standard10
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