【商检行业标准(SN)】 出口禽肉中二氯二甲吡啶酚残留量检验方法 乙酰化-气相色谱法

中华人民共和国进出口商品检验行业标准SN 0283-93
出口禽肉中二氯一甲吡啶酚
残留量检验方法
乙酰化-气相色谱法
Method for the determination of clopidoiresidues in poultry meat for export-Acetylation-gas chromatography1993-12-28发布
1994-05-01实施
中华人民共和国国家进出口商品检验局发布中华人民共和国进出口商品检验行业标准出口禽肉中二氯二甲吡啶酚残留量检验方法乙酰化-气相色谱法
Method for the determination of clopidolresidues in poultry mteat for export-Acctylation-gas chromatography1主题内容与适用范通
SN 028393
本标准规定了出口禽肉中二氯二甲吡啶酚残留量检验的抽样、制样和气相色谱测定方法。本标准适用于出口冻鸡中二氯二甲吡啶酚残留量的检验。2抽样和制样
2.1检验批
以不超过2500件为一检验批。
同一-检验批的商品应具有相同的特征，如包装、标记、产地、规格和等级等。2.2抽样数量
批量，件
26-100
101～250
251~500
501~1 000
1001~2500
2.3抽样方法
最低捆样数，件
按2.2规定的抽样件数随机抽取，逐件开启。每件至少取500品或一袋作为原始样品，原始样品总量不得少于2kg。放入清洁容器内，加封后，标明标记，及时送实验室。2.4试样制备
将所取原始样品缩分出1kg，取可食部分，经组织捣碎机碎均勾，均分成两份，装入洁净容器内，作为试样。密封，并标明标记。2.5试样保存
将试样于一18℃以下冷冻保存。注：在抽样和制样的操作过程中，必须防止样品受到污染或发生残留物含量的变化。3测定方法
3.1方法提要
用甲醇提取样品中二氟二甲吡啶酚，经氧化铝和阴离子交换树脂柱净化。与乙酐反应，转化成相中华人民共和国国家进出口商品检验局1993~12-28批准1994-05-01实施
SN 0283-93
应的酯，用气相色谱电子俘获检测器测定，内标法定最。32试剂和材料
3.2.1甲醇：分析纯，经全玻璃装置重蒸馅，收集64~55℃馏分。3.2.2正已烷：分析纯，经全玻璃装置重蒸馏，收集67~69℃谢分。3.2.3无水硫酸钠：分析纯，经650℃灼烧4l，置于干燥器内备用。3.2.4氧化铝，中性，净化用，100200国；300℃灼烧41，置于干燥器备用。3.2.5阴离子交换树脂，Dowex1-x8100~200目，CI型。3.2.6助滤剂：Celite545，使用前用甲醇清洗。3.2.7乙酸酐;分析纯，重蒸馏。3.2.8吡啶，色谱纯。
3.2.9苯：分析纯。
氢氧化钠：分析纯。
氢氧化钠溶液：1mol/L。取40.0氢氧化钠溶解于1000ml.去离子水中。乙酸钠：分析纯。
乙酸钠溶液：0.5mol/L。溶解68.0名乙酸钠于1000mL去离子水中。四硼酸钠：分析纯。
四硼酸钠溶液：0.1mal/L。取38.1g四硼酸钠溶于1000mL去离子水中冰乙酸;分析纯。
乙酸-甲醇溶液：0.5%。将2.5mL冰乙酸加到477.5mL甲醇中。硫酸钠溶液：2%。取2g硫酸钠溶解于100mL去离子水中。二氮二甲吡啶酚和六氯苯标品，纯度≥99%。3.2.19
二氯二甲吡啶酚标推溶液：推确称取适量的二氟二甲吡啶酚标崔品，用甲醇配成浓度为3. 2. 20
100/mL的标准储备溶液，根据锯要再配成适当浓度的标准工作溶液。3.2.21内标溶液：准确称取适量的六氯苯标难品，用苯配成浓度为100g/ml.的标雄储备溶液，根据需要再用正已烷配成适当浓度的内标工作溶液。3.3仪器和设备
3.3.1气相色谱仪，配有电子俘获检测器。3.3.2组织捣碎机。
3.3.3均质器。
3.3.4离心机。
3.3.5离心管：具磨口塞，5mL。3.3.6氯化铝柱：20cm×20mm3.3.7阴离子交换树脂柱：15crm×10mm(:d)玻璃柱，将明离子交换树脂伴以去离子水装入柱内，高度为2cm，注入100mL氢氧化钠溶液（3.2.10)淋洗，然后用去离子水洗涤至中性。.再用100mlZ酸钠溶液（3.2.12)淋洗，用去离子水洗涤至中性。最后用50mL甲醇水溶液80%(V/V）洗涤后备用（流速为 2 mL/min),
3.3.8微量注射器：10L，100μL。3.4测定步骤
3.4. 1提取
称取试样20g（精确到0.1g）于均质杯中，加入50mL甲醇和3g助滤剂.高速均质3min。在布氏漏斗上敷上2名助滤剂，抽滤均质后的样品，用45ml.甲醇分3次洗涤均质杯并移入布氏溺斗中抽滤。将滤液并入10UmL容量瓶中，用甲醇定容，并混匀。2
3.4.2净化
SN0283—93
将阴离子交换柱置于氧化铝住下，吸取50mL提取液注入氧化铝柱内，控制提取液在阴离子交换柱的流速为1mL/min，用20mL甲醇冲洗两柱。除去氯化锅柱，弃去全部的流出液。用20mL乙酸-甲醇溶液（0.5%）洗脱二氯二甲吡啶酚，洗脱液收集于25nL容地瓶中，用甲醇定容。3.4.3乙酰化
吸取5.0mL洗脱液于离心管中，在5α~60℃水裕中，用氮气吹于。加4mL四硼酸钠溶波（0.1mol/lL.）溶解残渣，离心管置于50C水浴中5min。依次加人1.0mL内标工作溶液（3.2.21）1心=L吡啶和75uL乙酸，加寒，振播1min，2000r/min离心5min。将正已烧相转入另一离心管中，加无水硫酸钠脱水后供气相色谱测定。
3.4.4标准物的乙酰化
取适用浓度的标准工作溶液，用氮气吹干，按3.4.3进行乙酰化反应。3.4.5测定
3.4.5.1色谱条件
a.色谱柱：玻璃柱，2m×3mm（id），填充物为3%0V-17+3.3%QF-1涂于ChromosorbWHP（100～120日）；
色谱柱温度：150℃：
进样口温度：220℃；
检测器温度：250℃
氮气：纯度=99.99%，65ml./min。e.
3.4.5.2色谱测定
分别准确注入5μL经乙酰化后的样液和标准工作溶液于气相色谱仪中，按3.4.5.1的条件进行分析。响应值均应在仪器检测的线性范围之内。在上述色谱条件下，二氯二甲吡啶酚的保留时问约为4.0min，六氯苯约为11min。3.4.6空白试验
除不加试样外，按上述测定步骤进行。3.5结果计算和表述
用色谱数据处理机或按下式计算试样中二氟二甲吡啶酚残留含量：c,.h.hut..m.
Cih, -h.·m
式中X—-试样中氯二甲吡啶酚残留址，mg/kg1c,标准工作溶液中二氯二甲吡啶酚的浓度，pg/mL;标雄工作溶液中六氛苯的浓度，g/mL：Ca
h——样液中二二甲吡啶酚乙酯的峰高，mm；h,一一样液中六氮苯的峰高，mmh-一标工作溶液中二氯二甲吡啶艺酯的峰离，mm.—标推工作溶液中六氯苯的峰高，mm；m;样液中的六氯苯添加苯，g
m-最终样液所代表的样品重量·g。注，计算结果密扣除空白值。
4测定低限、回收率
4.1测定低限
本方法测定低限为 0. c05 mg/kg。SN 0283—93
4.2回收率
回收率的实验数据：二氯二甲吡啶酚添加浓度在0.005～0.05Cmg/kg范圈内，回收率为：77.0%~100.0%。
附加说明：
本标推由中华人民共和国进出口商品检验局提出。本标由中华人民共和国浙江进出口商品检验局负责起草。本标准主要起草人郑自强，杨建民、徐亮。Professional Standard of the People's Republic of Chinafor Import and Export Commodity InspectionMeod for the determination of clopidolresidues in poultry meat for export-Acetylation-gas chromatographyTScope and ficld of applicationSN 0283-93
This standard specifies the methads of sampling,sample preparation and determination by gasrhromatagraphy(GC) of clopidol residues in poultry meat for export.This standard is applicable to the determination of clopidol rcsiducs in chicken meat: for export.2 Sampling and sample preparation2.1 Inspection lot
The quantity of an inspection lot should not be more than 2 500 packagex.The charactcristics of ihe cargo within the same inspection lot,such as acking,mark,origin,grade and specification,should he the same.2.2 Quaritity wf sample takenNumber of packages in
each inspection lot
26-100
101—250
251—500
501—1.000
1 001-2 500
2.3Sampling procedure
Minimum nutnber of packages
to be taken
A numbcr of packages specified in 2. 2 are taken at random and opened one by one. The quantitytaken as the prirmary sample from each package should be at least 5o0 grams or one bag. The totaiweight of all primary samples should not be less than 2 kg the primary sarnple is placed in & elean con-tainer which shall be sealed,labeled and sent to labotratory in time.2.4 Preparation of test sampleThe combined primary sample is reduced to 1 ks ,the edibie portions are blended,and then dividedinto (wo equal portiorns. Each portion is placed in a clean container as the test sample,which is thenApproved by the State Administratlon ofImporl and Export Commodity Inspection ofthe People's Republic of China on Dec. 28, 1993Implemeuted fFrom May. 1, 1994sealed and labeled.
2.5 Storage af test sample
SN 0283—93
The test samples should be stored below -l&'CNote : In the course of sanpling and sample preparation,precaution must he taken tn avnid the cnntaminat:tn at tnyfactors which may cause the change of residue content.3Method af determination
3.1Principle
The rlopidol is extracted from the test sample with methanol,and cleaned up on an alumina col.umn and an anion exchange resin colurnin. Clopidol is acetylated with acetie anhydride,iorming elopidolacetate, which is deterrnined by gas chromstography with electron capture detector, using internalstandard.
3. 2 Reagents and materials
3.2.1 Methanol: Analytical grade, redistill with all glass apparatus,collect the distillate of 64-65℃.
3- 2.2 n-Hexane; Analytical grade,redistill with all glass apparatus,collect the distillate of 67-69℃.
3. 2. 3 Anhydrous sodium sulfate :Analytical grade .ignite at 650 'C far 4 h ,and storc in a desiccator3. 2. 4 Alumina:Neutral,for cleanup,100--200 mesh;ignite at 300C for 4 h and store in a desicca-tot.
3. 2. 5Anion exclhange resin:Dawex 1-x8,100-200 mesh,Cl-type.3. 2.6 Filter aid;Celite 545, wash with rmethanol before use.3. 2.7 Acetic anhydride :Analytical grade,redistill.3.2-8PyridinetChromatographic grade.3.2.9 Benzcne:Analytical grade.3. 2. 10 Sadixm hydroxide;Analytical grade.3-2. 11 Sodium hydroxide solution:1 mol/L. Dissolve 40. 0 g sodium hydroxide in 1 000 mL of deionized water.
3. 2. 12 Sodium acetate :Analytical giade.3. 2. 13 Sodium acetate solutian:0. 5 mol/L. Dissolve 68. 0 g of spdium acetate in 1 000 ml. of de-ion-ized water.
3. 2. 14 Disodium tetr:borate : Analytical grade.3.2. 15 Disodium tetraborate solution:0.1 mol/L, Dissplve 38.1 g of disodium tetraborzte in 1 000ml. of de-ionized water.
3. 2. 16 Glacial acetic acid : Analytical grade.3.2.17 Acetic acid-methanol solution: 0. 5%. Add 2.5 mL of glacial acetic acid tc 477. 5 mL ofmethanol.
3. 2. 18 Sodium sulfate solution:2%. Dissolve 2 g of sodium sulfate in 100 mL of de-ionized water.3.2.19Clopidolstandard,Purity≥99/;Hexachlorobenzene(HCB)standardPurity≥99%3.2.20 Clopidol standard solution:Accutately weigh an adequate amount of clopidol standard,dissolve in mcthanol and prepare a so-lution of 100 μg/mL as the standard stuck sulution. Acrording to the requirement,prepare a standerdworking solution of appropriate concentration.·6
3.2.21Internal standard solution:SN 0283-93
Accurately weigh an adeguate amount of hexachlorobcnzene standatrd,dissolve in benzene and prePare a solution of 1oo μg/mL as the standard stock salution. According to the requirement,preparewith n-hexane an internal standard working solution of apprupriate concentration.3.3Apparatus and equipment
3.3.1 Gas chromatograph:Equipped with the electran capturedetector.3. 3. 2 High speed blender.
3.3.3Homogenizer.
3- 3. 4 Centrifuge.
3.3.5Centrifuge tube:5mL,withground stopper.3. 3. 6 Alumina column:20 cmX20 mm(id) glass column,pack with ca 0. 5 ctn height of absorbentcation at the bottom of the column and fill in 8-g cm height of alurnina by knocking lightly. Rinse thecolumn with methanol before use.3. 3.7 Anion exchange resin column:15 cm X10 mm(id) glass column.fill with Dowex 1-x8 iesin,us-ing de-ionized water to tranafer the resin to the column with a height of 2 cm (after settling down).Rinsc the column with 1oo mL of l mol/L sodium hydroxide solution,next wash with de-ionized wateruntil teulralized. rhen rinse the column with 1o0 ml. pf sodium acetate solution(o. 5 mol/L),nextwash with de-ionized water until neutralized. Finally rinse with 50 ml of methanol-water solution(8+2),keep the flow rate at 2 ml./min and ready for use.3.3.8Micro-syringe:10μL,100μL.3.4Proccdure
3.4.1 Exiraction
Weigh ca 20 g of the test sample(accurate to 0. 1 g) into a hornogenizing bottle. Add 50 mL ofmethanol and 3g of filter aid.Homogenize for 3 min at maximum speed with a homogenizer. Filter thchonogenized sanple through a filter paper padded with 2 g of filter aid on a Buchner funnel. Collcctthe filtrate in a 100 ml. volumetric flask.Wash the bottle and the filter cake with 3 additional 15 mLportions of methanol. Collect the filtrates in the same volumatric flask and dilute to the mark withmethanol and mix well.此内容来自标准下载网
3.4.2 Cleanup
Place the anion exchange column under ihe alutnina column. Pipet 50 ml, of the extract onto alumina column and let elute thraugh both column into a beaker with a rate of 1 mL/tmin. Wash thecolumns with 20 mL of methanol. Remave the alumina column and discard all the efflucnts. Place a 25ml. valumetric flask under the anion exchange column and elute the clopidol with 20 mL of 0.5%aceticacid methanol solution. Collect the eluate in the 25 mL volumetric flask and adjust to mark withmethanal.
3.4.3 Acetylation
Transfer 5. O rnL of the eluate into a 5 ml centrifuge tubeevaporate to dryness in a water-bath at50—60'C under a stream of nitrogen.Disgolve the residue with 4 mL af disodium tetraborate solution(0. I mol/L). Heat gently for 5 min by placing the tube in a 50 C. water bath . Accurately add 1 rnL ofinternal standard working solution,lo μL of pyridine and 75 μl, of acetic anhydride.Shake the mixturefor 1 min and centrifuge for 5 min at 2 oo0r/min. Traasfer the hexane layer into anather tube withstopper. After dehydration with ca 0, 5 g of anhydrous sodium sulfate,the solutian is used for chro-matographic determinatiorn.
3. 4. 4 Acetylation of the standardSN 0283-93
Aceurately pipet the standard working soluticn of suitable concentration to a tube,evaporate todryness under nitrogen current,then proceed the acetylation as described in 3. 4. 3.3.4.5 Determination
3.4. 5. 1 GC operating conditiona.
GC column;Glass,2 mX 3 mm(id),packed with 3% OV-17+3.3%QF-1 an Chranosorb wHP(100--120mesh);
Coiumn temperature,15c'C :
Injection port temperaturc:220'℃;Detector temperature:250'C:
e.Nitrogen:Purity ≥99.99%.65 mL/min.3. 4. 5.2 GC determination
lnject zccurately 5 μL of the acetylated stardard working solution(3,4.4)and sampie solutian(3. 4. 3) separately inta the gas chromatograph ,and cary out the analysis under the condition indicat-ed in 3. 4. 5. 1. The respanses must he within the linear range of the instrurmental detection. Under theabave chromatographic condition,the retention tirne of clopidal acetate is ca 4. 0 min.that of hexachlorobenzene is ca ll min.3.4.6Blank test
The operation of the blank test is thc samc as that described in the method of determination.butwithaut addition of sample.
3.5Calculativn and expression of resultThe residue content of clopidol is calculated by GC data processor or according to the following e-guatian
X=Sh·hami
Ce.-h, .h.-m
X the content of ciopidol in test sample,mg/kgCetheconcentration of clopidol in standard working solution,pg/mL:Ca-the concernitration of hexachlorobenzene in standard working solution,pg/mlththe peak height of clopidol acetate in sample soiution,mm;h,.-the peak height of hexachlorobenzene in sample solution,mm;h.\-ihe peak heighi of clopidal aretate in standard working solution,mnnthithe peak height pf hexachlorohenzene in standard working solution,mmmithe mass of the hexachlorobenzene in the final sample solution μg:m-the corresponding mass of the test sample in the fina! sample solution g-Note: The blank value should be subtracted from the sbove tesult of caleula:ion.4 Limit of determination and recovery4.1 Limit of determination
The limit of determination of this mcthod is 0.005 mg/kg4.2Recovery
SN 0283-93
According to the experimental data,when the fortifying concentration of clopidol is in the range of0.005—0. 050 mg/kg,the recuvery is 77.0%—100.0%.Additional explanations:
This standard was proposed by the State Administration of Import and Export Commodity In-spection of the People's Republic of China.This standard was drafted by the Zhejiang Import and Export Commodity Inspection Bureau ofthePeople's Republic of China.This standard was fnainly drafted by Zheng Ziqiang,Yang Jianmin,Xu Liang.Note: This Fnglish vcrsion, tranglation fram the Chinese xext is solely for guidance.
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