1、:才己中华人民共和国进出口商品检验行业标准SN 0283- 93 出口禽肉中二氯二甲毗咙盼残留量检验方法乙酷化-气相色谱法Method for the determination of .c1opidol residues in pouItry meat for export -Acetylation-gas chromatography 1993-12-28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(京)新登字023号中华人民共和国迸出口商品检验行业标准出口禽肉中二氯二甲毗喧酷残留量检验方法乙酷化-气相色谱法Method for the determination o
2、f c1opidol residues in poultry meat for export - Acetylation-gas chromatography 1 主题内容与适用范围SN 0283-93 本标准规定了出口禽肉中二氯二甲毗睫酣残留量检验的抽样、制样和气相色谱测定方法。本标准适用于出口冻鸡中二氯工甲毗咙酣残留量的检验。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，件最低抽样数，件125 1 26100 5 101250 10 251500 15 50 Ll 000 17 1 00L
3、2 500 20 2.3 抽样方法按2.2规定的抽样件数随机抽取，逐件开启。每件至少取500g或一袋作为原始样品，原始样品总量不得少于2峙。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，经组织捣碎机捣碎均匀，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 试样保存将试样于一18C以下冷冻保存。注z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氯二甲毗睫酣，经氧化铝和阴离子交换树脂柱净化。与乙酸盲平反应，转化成相中华人民共和国国京进出口商品检验局1993
4、-12-28批准1994-05-01实施SN 0283- 93 应的酶，用气相色谱电子俘获检测器测定，内标法定量。3.2 试剂和材料3.2.1 甲醇:分析纯，经全玻璃装置重蒸锢，收集6465C馆分。3.2.2 正己烧z分析纯，经全玻璃装置重蒸锢，收集67690C锢分。3- 2. 3 无水硫酸铀:分析纯，经650C灼烧4h，置于干燥器内备用。3- 2. 4 氧化铝:中性，净化用，100200目;300C灼烧4h，置于干燥器备用。3- 2. 5 阴离子交换树脂:Dowex 1-x8 100200目，Cl型。3.2.6 助滤剂:Celite545，使用前用甲醇清洗。3- 2. 7 乙酸哥:分析纯，重
5、蒸馆。3.2.8 毗睫:色谱纯。3.2.9 苯z分析纯。3.2.10 氢氧化饷z分析纯。3- 2. 11 氢氧化纳溶液:1mol/L。取40.0g氢氧化铀溶解于1000 mL去离子水中。3.2012 乙酸铀:分析纯。3.2.13 乙酸锅溶液:0.5mol/L。溶解68.0g乙酸铀于1000 mL去离子水中。3.2.14 四棚酸饷z分析纯。3.2.15 四棚酸铀溶液:0.1mol/L。取38.1g四跚酸纳溶于1000 mL去离子水中。3.2.16 冰乙酸:分析纯。3.2.17 乙酸甲醇溶液:0.5%。将2.5mL冰乙酸加到477.5mL甲醇中。3.2.18 硫酸锅搭液:2%。取2g硫酸铀溶解于1
6、00mL去离子水中。3.2.19 二氯二甲毗睫盼和六氯苯标准品z纯度99%。3- 2. 20 二氯二甲毗睫酣标准溶液:准确称取适量的二氧二甲毗昵酣标准品，用甲醇配成浓度为100问/mL的标准储备溶液，根据需要再配成适当浓度的标准工作榕液。3.2.21 内标榕液z准确称取适量的六氯苯标准品，用苯配成浓度为100g/mL的标准储备溶液，根据需要再用正己烧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 氧化铝柱:20cmX20 mm(id
7、)玻璃柱，柱底部填约0.5cm高的脱脂楠，装89cm高的氧化铝。用前经甲醇淋洗。3- 3. 7 阴离子交换树脂柱:15 cm X 10 mm(id)玻璃柱，将阴离子交换树脂伴以去离子水装入柱内，高度为2cm，注入100mL氢氧化纳溶液(3.2.10)淋洗，然后用去离子水洗涤至中性。再用100mL乙酸纳溶液(3.2.12)淋洗，用去离子水洗涤至中性。最后用50mL甲醇水榕液80%(V /V)洗涤后备用(流速为2mL/min)。3.3.8 微量注射器:10L、100L。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
8、氏漏斗上敷上2g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漏斗中抽滤。将滤液并入100mL容量瓶中，用甲醇定容，并混匀。2 SN 0283-93 3. 4. 2 净化将阴离子交换柱置于氧化铝柱下，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。除去氧化铝柱，弃去全部的流出液。用20mL乙酸F甲醇溶液(0.5%)洗脱二氯二甲毗睫盼，洗脱液收集于25mL容量瓶中，用甲醇定容。3. 4.3 乙酷化吸取5.0mL洗脱液于离心管中，在5060C水浴中，用氮气吹干。加4mL四棚酸纳溶液(0.1mol/L)溶解残渣，离心管置于
9、50C水浴中5min。依次加入1.0 mL内标工作溶液(3.2.21)、10L毗院和75L乙酸酶，加塞，振摇1min , 2 000 r/min离心5min。将正己烧相转入另离心管中，加无水硫酸纳脱水后供气相色谱测定。3. 4. 4 标准物的乙酷化取适用浓度的标准工作溶液，用氮气吹干，按3.4.3进行乙酷化反应。3. 4. 5 测定3.4.5.1 色谱条件a. 色谱柱z玻璃柱，2mX3 mm (id) ，填充物为3%OV-17十3.3%QF-1涂于ChromosorbW HP (100120目); b. 色谱柱温度:150C;c. 进样口温度:220C;d. 检测器温度:2500C;e. 氮气
10、z纯度二三99.99 % ,65 mL/min。3.4.5.2 色谱测定分别准确注入5L经乙酷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，工氯二甲毗院酣的保留时间约为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氯二甲毗睫酣残留含量:风-m,. -S E町风-h- ., -l LH7n - -k s-s c-c x 式中:X试样中二氯二甲口比睫酣残留量，mg/kg;C，一一标准工作溶液中二氯二甲毗睫盼的浓度
11、问/mL;C.一一标准工作溶液中六氯苯的浓度，g/mL;h一一样液中二氯二甲毗睫酣乙酶的峰高，mm;h，-样液中六氯苯的l峰高，mm;h，一-标准工作溶液中二氯二甲毗睫酣乙醋的峰高，mm;h.-标准工作溶液中六氯苯的峰高，mm;m，一一样液中的六氯苯添加量，g;m一一最终样液所代表的样品重量，g。注:计算结果需扣除空白值。4 测定低限、回收率4.1 测定低限3 SN 0283-93 本方法测定低限为0.005mg/kg。4.2 回收率回收率的实验数据;二氯二甲毗睫酣添加浓度在O.0050. 050 mg/kg范围内，回收率为:77.0%100. 0%。4 附加说明:本标准由中华人民共和国进出口
12、商品检验局提出。本标准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 China for Import and Export Commodity Inspection Method for the determination of c1opidol residues in pouItry meat for export -Acetylation-gas chromatography 1 Scope and field of application SN 028
13、3-93 This standard specifies the rnethods of sarnpling , sarnple preparatiun and deterrnination by gas chromatography(GC) of clopidol residues in poultry meat for export. This standard is applicable to the determination of clopidol residues in chicken meat for export. 2 Sampling and sample preparati
14、on 2. 1 Inspection lot The quantity of an inspection lot should not be more than 2 500 packages. The characteristics of the cargo within the same inspection lot , such as packing , mark , origin , grade and s pecifica tion ,should be the same. 2. 2 Quantity of sample taken Number of packages in Mini
15、mum number of packages each inspection lot to be taken 1一251 26-100 5 101-250 10 251-500 15 501-1 000 17 1 001-2 500 20 2.3 Sampling procedure A number of packages specified in 2. 2 are taken at random and opened one by one. The quantity taken as the primary sample from each package should be at lea
16、st 500 grams or one bag. The total weight of a11 primary samples should not be less than 2 kg , the primary sample is placed in a clean con tainer which sha11 be sealed , labeled and sent to laboratory in time. 2. 4 Preparation of test sample The cumbined primary sample is reduced to 1 kg , the edib
17、le purtiuns are blended ,and then divided into two equal portions. Each portion is placed in a clean container as the test sample , w hich is then Approved by the State Administration of Import and Export Commodity Inspection of the People s Republic of China on Dec. 28 ,1993 Implemented from May. 1
18、 ,1994 5 SN 0283-93 sealed and labeled. 2. 5 Storage of test sample The test samples should be stored below -18 C. Note: 1n the course of samplmg and sample preparatlOn. precaution must be taken to avoid the contammation or any factols which may cause the change of resldue content. 3 Method of deter
19、mination 3. 1 Principle The clopidol is extracted from the test sample with methanol. and cleaned up on an alumina col umn and an anion exchange resin column. Clopidol is acetylated with acetic anhydride , forming clopidol acetate , which is determined by gas chromatography with electron capture det
20、ector, using internal standard. 3. 2 Reagents and materials 3. 2. 1 Methanol: Analytical grade , redistill with all glass apparatus , collect the distillate of 64-65C. 3. 2. 2 n-Hexane: Analytical grade , redistill with all glass apparatus , collect the distillate of 67-69C. 3.2.3 Anhydrous sodium s
21、ulfate:Analytical grade.ignite at 650 C for 4 h ,and store in a desiccator. 3. 2.4 Alumina:Neutral ,for cleanup , 100-200 mesh;ignite at 300C for 4 h and store in a desicca tor. 3.2.5 Anion exchange resin:Dowex 1-x8,100-200 mesh ,Cl-type. 3. 2. 6 Filter aid:Celite 545 ,wash with methanol before use.
22、 3. 2. 7 Acetic anhydride: Analytical grade , redistill. 3. 2. 8 Pyridine :Chromatographic grade. 3. 2. 9 Benzene : Analytical grade. 3. 2. 10 Sodium hydroxide :Analytical grade. 3.2. 11 Sodium hydroxide solution: 1 mol/L. Dissolve 40.0 g sodium hydroxide in 1 000 mL of de ionized water. 3. 2. 12 So
23、dium acetate: Analytical grade. 3. 2. 13 Sodium acetate solution , O. 5 mol/L. Dissolve 68.0 g of sodium acetate in 1 000 mL of de-ion ized water. 3.2.14 Disodium tetraborate : Analytical grade. 3.2. 15 Disodium tetraborate solution: O. 1 mol/L. Dissolve 38. 1 g of disodium tetraborate in 1 000 mL o
24、f de-ionized water. 3. 2. 16 Glacial acetic acid: Analytical grade. 3. 2. 17 Acetic acid-methanol solution: O. 5 %. Add 2. 5 mL of glacial acetic acid to 477. 5 mL of methanol. 3.2. 18 Sodium sulfate solution: 2 %. Dssolve 2 g of sodum sulfate n 100 mL of de-onzed water. 3. 2. 19 Clopidol standard:
25、Purity 9 9 % ; Hexachlorobenzene (HCB) standard: Purity注99%.3.2.20 Clopidol standard solution: Accurately weigh an adequate amount of clopidol standard ,dissolve in methanol and prepare a so lution of 100g/mL as the standard stock solution. According to the requirement , prepare a standard working s
26、olution of appropriate concentration. 6 SN 0283- 93 3. 2. 21 Internal standard solution: Accurately weigh an adequate amount of hexachlorobenzene standard ,dissolve in benzene and pre pare a solution of 100g/mL as the standard stock solution. According to the requirement , prepare with n-hexane an i
27、nternal standard working solution of appropriate concentration. 3. 3 Apparatus and equipment 3. 3. 1 Gas chromatograph: Equipped with the electron capture detector. 3.3.2 High speed blender. 3. 3. 3 Homogenizer. 3. 3- 4 Centrifuge. 3. 3- 5 Centrifuge tube:5 mL ,with ground stopper. 3- 3. 6 Alumina c
28、olumn: 20 cm X 20 mm (id) glass column , pack with ca o. 5 cm height of absorbent cotton at the bottom of the column and fill in 8-9 cm height of alumina by knocking lightly. Rinse the column with methanol before use. 3.3.7 Anion exchange resin column: 15 cmX 10 mm(id) glass column,fill with Dowex 1
29、-x8 resin,us ing de-ionized water to transfer the resin to the column with a height of 2 cm(after settling down). Rinse the column with 100 mL of 1 mol/L sodium hydroxide solution ,next wash with de-ionized water until neutralized. Then rinse the column with 100 mL of sodium acetate solution (0. 5 m
30、ol!L) , next wash with de-ionized water until neutralized. Finally rinse with 50 mL of methanol-water solution(8 十幻，keepthe flow rate at 2 mL/min and ready for use. 3.3.8 Micro-syringe: 10L，100L. 3. 4 Proced ure 3. 4. 1 Extraction Weigh ca 20 g of the test sample (accurate to o. 1 g) into a homogeni
31、zing bottle. Add 50 mL of methanol and 3 g of filter aid. Homogenize for 3 min at maximum speed with a homogenizer. Filter the homogenized sample through a filter paper padded with 2 g of filter aid on a Buchner funnel. Collect the filtrate in a 100 mL volumetric flask. Wash the bottle and the filte
32、r cake with 3 addional 15 mL portions of methanol. Collect the filtrates in the same volumatric flask and dilute to the mark with methanol and mix well. 3. 4. 2 Cleanup Place the anion exchange column under the alumina column. Pipet 50 mL of the extract onto alu mina column and let elute through bot
33、h column into a beaker with a rate of 1 mL/min. Wash the columns with 20 mL of methanol. Remove the alumina column and discard all the effluents. Place a 25 mL volumetric flask under the anion exchange column and elute the clopidol with 20 mL of o. 5 %acetic acd-methanoI soIution. Collect the euate
34、in the 25 mL volumetric fla吕kand adjust to mark with methanol. 3.4. 3 Acetylation Transfer 5.0 mL of the eluate into a 5 mL centrifuge tube ,evaporate to dryness in a water-bath at 50-6.0C under a stream of nitrogen. Dissolve the residue with 4 mL of disodium tetraborate solution (0.1 mol!L). Heat g
35、ently for 5 min by placing the tube in a 50C water自bath. Accurately add 1 mL of internal standard working solution, 10L of pyridine and 75 p.L of acetic anhydride. Shake the mixture for 1 min and centrifuge for 5 min at 2 OOOr /min. Transfer the hexane layer into another tube with stopper. After deh
36、ydration with ca o. 5 g of anhydrous sodium sulfate , the solution is used for chro matographic determination. 7 SN 0283-93 3.4.4 Acetylation of the standard Accurately pipet the standard working solution of suitable concentration to a tube , evaporate to dryness under nitrogen current .then proceed
37、 the cetylation as described in 3.4.3. 3. 4. 5 Determination 3. 4. 5. 1 GC operating condition a. GC column:Glass ,2 mX3 mm(id) ,packed with 3% OV-17十3.3%QF-1 on Chromosorb W HP(lOO-120 mesh); b. Column temperature: 1500C ; c. Injection port temperature: 2 20 oC ; d. Detector temperature: 2500C ; e.
38、 Nitrogen: Purity ;:?;99. 99 % ,65 mL/min. 3.4.5.2 GC determination Inject accurately 5L of the acetylated standard working solution (3. 4. 4) and sample solution (3.4.3) separately into the gas chromatograph ,and carry out the analysis under the condition indicat ed in 3.4.5. 1. The responses must
39、be within the linear range of the instrumental detection. Under the above chromatographic condition , the retention time of clopidol acetate is ca 4. 0 min, that of hex achlorobenzene is ca 11 min. 3. 4. 6 Blank test The operation of the blank test is the same as that described in the method of dete
40、rmination, but without addition of sample. 3. 5 Calculation and expression of result The residue content of clopidol is calculated by GC data processor or according to the following e quatlOn: 风-m- -ES h-h - R ES h-h - -Ea SES C-c x where X一thecontent of clopidol in test sample , mg/kg; c, -the conc
41、entration of clopidol in standard working solution，用/mL;c,-the concentration of hexachlorobenzene in standard working solution，问/mL;h-the peak height of clopidol acetate in sample solution ,mm; h，一thepeak height of hexachlorobenzene in sample solution. mm; h，一thepeak height of clopidol acetate in st
42、andard working solution. mm; h.,-the peak height of hexachlorobenzene in standard working solution .mm; m,-the mass of the hexachlorobenzene in the final sample solution. flg; m-the corresponding mass of the test sample in the final sample solution .g. Note: The blank value should be subtracted from
43、 the above result of calculation. 4 Limit of determination and recovery 4. 1 Limit of determination The limit of determination of this method is 0.005 mg/kg. 8 SN 0283-93 4. 2 Recovery According to the experimental data , when the fortifying concentration of clopidol is in the range of 0.005-0.050 m
44、g/kg,the recovery is 77.0%-00.0%. Additional explanations: This standard was proposed by tle 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 of the Peop!e s Republic of China. This standard was mainly drafted by Zheng Zqiang ,Yang Jianmin.Xu Lang. NotE: This English ven;ion , a translation from the Chinese text ,1S solely for guidance. 2-SNO z 明中国标准出版社北京印刷厂印刷1994年9月第一版书号:155066 2-9505 1994年9月第一次印刷中国标准出版社出版
