1、吕N中华人民共和国出入境检瞌检疫行业标准SN /T 1016. 1 -2001 出口肉及肉制品中卡巴氧残留量检验方法气相色谱法Method for the deermination of carbadox residues in meats and meat p:rodu.cs fo:r export Gas chromatography 2001-12 -30发布2002 - 06 -01实施中华人民凡共和国发布国家质量监督恒验检痊总局SN/T 1016.1-2001 前本标准是按照GB/T1. 11993(标准化工作导则第1单元:标准的起草与表述规则第1部分:标准编写的基本规定及SN/T0
2、001-1995(出口商品中农药、兽药残留量及生物毒素检验方法标准编写的基本规定的要求编写的，其中测定方法是参考国内外有关文献，经研究、改进和验证后制定的。本标准同时制定了抽样和制样方法。测定低限是根据国际上对肉及肉制品中卡巳氧及其代谢物残留量的最高限量和测定方法的灵敏度而制定的。本标准的附录A是提示的附录。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:中华人民共和国浙江出入境检验检疫局。本标准主要起草人:郑自强、唐红芳、朱晓雨、朱宏、丁慧瑛。本标准首次发布。范围中华人民共和国出入境检验检疫行业标准出口肉及肉制品中卡巴氧残留量检验方法气相色谱法Method for the de
3、termination of carbadox residues in meats and meat products for export -Gas chromatography SN/T 1016.1-2001 本标准规定了出口肉及肉制品中卡巴氧残留量检验的抽样、制样和气相色谱测定方法。本标准适用于出口猪肉中卡巴氧代谢物(哇喔琳-2-竣酸)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。2. 2 抽样数量批量/件最低抽样数/件125 1 26100 5 101 250 10 251 500 15 5
4、011000 17 1 001 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。每件中取一袋作为原始样品，其总量不少于2kg。放入清洁容器内，加封后，标明标记，及时送交实验室。如每件中无小包，或有小包装但每袋质量超过2kg者，可用灭菌锋利刀在抽出的包件中，每件割取不少于100g，混合后置于洁净容器内，作为混合原始样。混合原始样的质量不少于2kg。加封后，标明标记，及时送交实验室。2.4 试样制备从所取全部样品中取出有代表性样品约1峙，取可食部分经捣碎机充分捣碎均匀，均分成两份，分别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18C以下冷冻保存。注
5、: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。中华人民共和国国家质量监督检验检疫总局2001-12-30批准2002- 06-01实施l SN/T 1016.1-2001 3 测定方法3. 1 方法提要用碱水解法将卡巴氧代谢物哇喔时中-2竣酸CQCA)从样品中分离出来，接着用乙酸乙醋抽提并用pH 6.0缓冲液反抽提，用离子排斥色谱法分离。用乙酸乙醋抽提柱洗脱液，浓缩提取液，用甲醇化的硫酸使QCA衍生化，所得甲醋衍生物经再抽提后用电子俘获气相色谱法测定，外标法定量。3. 2 试剂和材料除另有规定外，试剂均为分析纯，水为蒸语水。3. 2. 1 甲醇。3. 2. 2 浓硫酸
6、。3. 2. 3 乙酸乙醋。3. 2. 4 拧穰酸:C6H807 H0 3.2.5 氢氧化铀。J 2. 6 浓盐酸。J 2. 7 盐酸溶液:1 mol/L，用水稀释83.3mL被盐酸至100.0 mL。3. 2. 8 氢氧化饷溶液:3 mol/L，溶解120g氢氧化铀于适量水中并稀释至1000 mL。J 2. 9 拧攘酸溶液:1mol/L.梅解21.0 g拧攘酸水合物于1000 mL水中。3.2.10拧穰酸缓冲液(pH6.0): 0.5 mol/L，用5mol/L氢氧化铀恪液(约5.5mL)调节100mL 1 mol/L拧棱酸梅液至pH6.0，加水到200mL。J 2.11 甲醇水(10十90
7、):用水稀释10mL甲醇至100mL。3.2.12 甲醇硫酸(97十3):用经无水硫酸铀平燥过的甲醇稀释3.-0mL浓硫酸至100mL。当天制备，在加入酸以前将甲醇用冰浴玲却。J 2.13 元水硫酸铀:650C灼烧4h，置于干燥器中备用。J 2. 14 AGMP-50树脂:100目200目。3.2.15 喳喔琳2竣酸CQCA)标准品:已知纯度二97%。J 2.16 哇喔琳-2竣酸标准溶液:准确称取适量的喳喔琳2-搓酸标准品，用甲醇配成浓度为100g/mL哇喔咐-2梭酸的标准储备液，根据需要再用甲醇稀释储备液，配成适当浓度的标准工作液。J3 仪器和设备3. J 1 气相色谱仪:配有电子俘获检测器
8、。3. 3. 2 组织捣碎机。3. J 3 离心机。3. 3.4 分液漏斗:125 mL。J J 5 旋转蒸发器，具温控水活。J J6 浓缩瓶:250mL。J J 7 锥形瓶:150 mL。3. 3. 8 离心管:具塞，10mL、25mL。3. 3. 9 离子排斥柱:25cmX10 mm (id)玻璃柱，下端垫一小团玻璃棉。将7.0g AGMP-50 (100目200日)树脂于1mol/L盐酸中调成浆，并将其移入柱中，用一根玻璃棒将柱子装填至10cm高，并用玻璃棉盖住树脂床。保持液面在树脂的上面。3.4 测定步骤3. 4. 1 提取和净化称取约5g样品(精确到0.1g)于150mL三角瓶中，加
9、10.0mL 3 mol/L氢氧化铀恪液，松松加塞，将其置于100C水浴巾，加热30min，浴中水浴面应高于组织试样。2 SN/T 1016.1-2001 将碱性水解产物于冰浴中冷却，用4mL浓盐酸酸化至pHl(至酸碱试纸呈深红色)。于酸化的水解产物中加30mL乙酸乙醋，加塞，振摇提取30S，静置分层，将上层液体滤入一125mL分液漏斗中。待分层后，将下层水相放回样品瓶中，再加20mL乙酸乙醋，加塞，振摇提取30S，静置分层，将上层液体并入分液漏斗中。静置分层，弃去水相。于乙酸乙醋提取液中加5mL O. 5 mol/L拧攘酸缓冲液(pH6.0)，振摇，放置使下层清晰(10mi川。将水相收集于2
10、5mL具塞离心管中。另用5mL pH6. 0的缓冲液再提取乙酸乙醋相一次。使7.)(相清晰，合并水提取液于同一离心管中，加2mL浓盐酸，混匀，供下述柱层析分离用。将酸化的水提取液移入离子排斥柱中。放出提取液使液面至树脂床顶部。用20mL 1 mol/L盐酸淋洗离心管和树脂，经柱放出。另用20mL 1 mol/L盐酸再淋洗柱子，弃去流出液。用75mL甲醇-水(10十90)洗脱，此时可将柱子放净，收集洗脱液并转入一125mL分液漏斗中。流出液的流速约为1. 2 mL/min。洗脱液中加1.0 mL浓盐酸，用3X50mL乙酸乙醋提取。收集乙酸乙醋提取液于250mL圆底烧瓶中，于50C水浴旋转蒸发器中
11、蒸发至干。3. 4. 2 醋化./ 用3mL甲晖将残渣洗入10mL离心管中，在600C水浴氯气流下吹干。加0.2mL新鲜配制的甲醇-硫酸(97+3)，加塞，于55C60.C水浴中保温30min。取出，加1.0mL正己烧涡旋混匀30S，离心(2 000 r/mi口)5 min，取0.1mL正己烧搭液稀释到1.0mL，供气相色谱测定。3. 4. 3 标准物醋化吸取1.0mL喳喔琳-2-竣酸标准工作液置于10mL离心管中，1在60.C水浴氮气流下吹干。加0.2 mL新鲜配制的甲醇硫酸(97十3)，加塞，于550C600C水浴中保温30mino提取和稀释(1: 10)的操作均按3.4. 2步骤进行。醋
12、化标准与醋化样品应同时进行。一 3.4.4 测定3. 4. 4. 1 色谱条件a)色谱柱:HP-5，15mXO.53mm Cid)X1.5m熔融石英毛细管柱或相当的色谱柱;b)柱温:170(8min)旦旦!1200;(lOmin); c)进样口温度:250C; d)检测器温度:280C;e)载气、尾吹气:氮气(纯度二三99.999%);载气流速:6mL/min;尾l吹气流速:40mL/min; f)进样方式:填充柱进样口进样;g)进样量:2L。3. 4. 4. 2 色谱测定根据样液中喳喔琳2竣酸含量的情况，选定峰面积相近的标准工作挥手液。标准工作溶液和样液中哇喔琳2竣酸甲醋的响应值均应在仪器检
13、测的线性范围内。标准工作洛准和样液等体积参插进样测定。在上述色谱条件下l墅喔琳2竣酸甲醋的保留时间约为4min。标准品的色谱图见附录A中图A1。3.4.5 空白试验除不加试样外，均按上述操作步骤进行。3. 4. 6 结果计算和表述用色谱数据处理机或按式(1)计算试样中卡巳氧代谢物(喳喔琳2竣酸)的残留含量:式中:X试样中卡巴氧代谢物(哇喔I琳2竣酸)的残留含量，mg/kg;A 样液中喳喔琳-2-殷酸甲醋的峰面积，mm气A，标准工作液中哇喔琳2一竣酸甲醋的峰面积，mm2;( 1 ) q SN/T 1016.1-2001 C一一标准工作液中喳喔琳-2殷酸的浓度，g/mL;V 样液最终定容体积，mL
14、;m一一最终样液所代表的试样质量，g。注2计算结果需扣除空白值。4 测定低限和回收率4.1 测定低限本方法的测定低限为0.03mg/峙。4.2 回收率4 猪肉中喳喔琳-2-竣酸添加浓度及其回收率的试验数据:在0.03mg/kg添加水平时，回收率为98.3%; 在0.10mg/kg添加水平时，回收率为93.6%; 在0.30mg/kg添加水平时，回收率为93.4%。SN/T 1016.1-2001 附录A(提示的附录)标准晶色谱固卢VN.甘图Al喳喔琳2竣酸标准品衍生物的气相色谱图5 SN/T 1016.1-2001 Foreword This standard was drafted in a
15、ccordance with the requirements of GB/T 1. 1-1993 Directives for the work of standardization-Unit 1: Drafting and presentation of standards-Part 1: General rules for drafting standardsand SN/T 0001-1995General rules for drafting the standard method for the determination of pesticide , veterinary dru
16、g residues and biotoxins in commodities for ex po同The method of determination of this standard was drafted by referring to relevant domestic and foreign literatures throughresearch， modification and verification. Besides , methods of sam pling and sample preparation are also specified in this standa
17、rd.i The limit of determination !in this standard is/defined on the basis of the current international maximum limit for carbadox and its metabolites residues in meats and meat products and the sen sitivity of this method. / Annex A of this standard is n informative one. This standard was proposd by
18、 and is under the charge of China National Regulatory Com mission for certification and Accreditation. / f / This standard was drafted bt/hejiang Ertry-Exit Inspection and Quarantine Bureau of the People s Republic of China. / The main drafters of this lstandard :are Zheng ziqiang , Tang hongfang ,
19、Zhu xiaoyu , Zhu hong and Ding huiying. This standard is promulgaed for the first time. Note: This English Version , a translation from the Chinese text , is solely for guidance. 6 Professional Standard of the People s Republic of China for Entr-Exit Inspection and Quarantine Method for the determin
20、ation of ca由adoxresidues in meats and meat products for export -Gas chromatography SN/T 1016.1-2001 Scope / This standard specifies the methods of sampling, sample preparation and determination by gas chromatography (GC) of carbadox resiqus in meats and meat products for export. This standard is app
21、licable to the de.trmination ofmetabolites of carbadox residues in pork for ex / / port. 2 Sampling and sample preparation 2.1 inspection lot 民-、飞、The quantity of an inspection lot shou咀川otbe l1)fe than 2 5QO packages. / / / The characteristics of the cargo wthin the same inspection lot, such as pac
22、king , mark, origin , speci-fication and grade should be th same. : 2.2 Ouantity of sample taken , Number of packages in an inspction lot 1 - 25 aE 只呵Minimum n.J mber of Packages to be taken 5 10 15 17 20 101 - 250 251 - 500 501 - 1 000 1 001 - 2 500 2.3 Sampling procedure A number of packages speci
23、fied in 2.2 are taken at random and opened one by one. From each package , one bag shall be taken as a primary sample. The total weight of all primary samples should not be less than 2 kg , which shall be placed in a clean container , sealed , labeled and sent to Approved by General Administration o
24、f Ouality Supervision ,lnspection and Ouarantine of the People s Republic of China on 2001-12-30 Implemented from 2002-06-01 SN/T 1016.1一2001laboratory in time. In case the meat-pieces are not contained in each package , or if there are meat-pieces inside but the content of pieces exceeds 2 kg , cut
25、 0仔apart from the meat-pieces in each package not less than 100 9 with a disinfected sharp knife. Mix the pa民sof the meat as the mixed primary sample, which shall not be less than 2 kg. Place in a clean container, seal , label and sent to the laboratory in time. 2.4 Preparation of test sample The mi
26、xed prima叩sampleis reduced to 1 kg. The eatable potions are thoroughly ground and ho mogenized in a meat grinder. Then divide into two equal portions, each portion is placed in a clean container as the test sample, which is sealed and labeled. 2.5 Storage of test sample The test samples should be st
27、ored below - 18C . Note: In the course of sampling and sample preparation , precautions must be taken to avoid the contamination or any factors which may cause the change of residue content. 3 Method of determination 3.1 Principle Metabolites of carbadox quinoxaline-2-carboxylic acid (QCA) is isolat
28、ed from test sample by al kaline hydrolysis, sequential extraction into ethyl acetate and pH6 buffer, and ion exclusion chro matography. The column eluate is extracted with ethyl acetate , the extract is concentrated , and QCA is derivatized with methanolic sulfuric acid. The methyl ester derivative
29、 is extracted and deter mined by electron capture gas chromatography, using external standard method. 3.2 Reagents and materials U nless otherwise specified , all the reagent used should be analytically pure, water is distilled wa ter. 3.2.1 Methanol. 3.2.2 Concentrated sulfuric acid圃3.2 . 3 Ethyl a
30、cetate. 3.2.4 Citric acid: CSHS07 H20. 3.2.5 Sodium hydroxide. 3.2.6 Hydrochloric acid. 8 SN/T 1016.1-2001 3.2.7 Hydrochloric acid solution:1 mol/L, dilute 83.3 mL of concentrated hydrochloric acid to 1 000 mL with water. 3.2.8 Sodium hydroxide solution:3 mol/L, dissolve 120 9 of sodium hydroxide in
31、 enough water to make 1 000 mL. 3.2.9 Citric acid solution: 1 mol/L, dissolve 21.0 9 of citric acid monohydrate in 1 000 mL of wa ter. 3.2.10 Citric acid buffer (pH6.0) :0.5 mol/L, adjust the pH of 100 mL of 1 mol!L citric acid solu tion to pH 6.0 with 5 mol/L sodium hydroxide solution (ca 5.5 mL) .
32、 Adjust the final volume to 200 mL with water. 3.2.11 Methanol-water (10 + 90): Dilute 10 mL of methanol to 100 mL with water. 3.2.12 Methanol-sulfuric acid (97 + 3): Dilute 3.0 mL of concentrated sulfuric acid to 100 mL with methanol which has been dried over anhydrous sodium sulfate. Prepare daily
33、, and use an ice bath to cool the methanol before adding acid. 3.2.13 Anhydrous sodium sulfate: Ignite at 650 C for 4h and keep in an air-tight container. 3.2.14 AGMP-50 resin:100-200 mesh. 3.2.15 Ouinoxaline-2-carboxylic acid (OCA) standard:Purity97%. 3.2.16 Ouinoxaline-2-carboxylic acid standard s
34、olution: Accurately weigh an appropriate amount of Ouinoxaline-2-carboxylic acid standard and dissolve with methanol to prepare a standard stock solution of 100g/mL in concentration. According to the requirement, dilute the stock solution with methanol to prepare a standard working solution of suita
35、ble concentration. 3.3 Apparatus and equipment 3.3.1 Gas chromatography: With electron capture detector. 3.3.2 High speed blender. 3.3.3 Centrifuge. 3.3.4 Separatory funnel: 125 mL. 3.3.5 Rotary vacuum evaporator:With temperature-controlled water bath. 3.3.6 Evaporating bottle:250 mL. 9 SN/T 1016.1一
36、20013.3.7 Conical flask:150 mL. 3.3.8 Centrifuge tube:With glass stopper, 10 and 25 m L. 3.3.9 lon exclusion chromatography:25 cm x 10 mm (id) glass column with a small glass wool plug at the bottom. Slurry 7.0 9 of AGMP-50 (100 - 200 mesh) resin in 1 mol/L hydrochloric acid and transfer to the colu
37、mn. Pack the resin to a height of 10 cm using a glass rod and cap the resin bed with a glass wool plug. Maintain the liquid above the resin. 3.4 Procedure 3.4.1 Extraction and cleanup Weigh ca 5 9 of the test sample (accurate to 0.1 g) into a 150 monical flask.Add 10.0 mL of 3 mol/L sodium hydroxide
38、 solution , stopper lightly, and place/it in a 100C water bath for 30 min utes. The level of water in the bath should exceed that ofthe tissue sample. Cool the alkaline hydrolysate in an ice bath and acidify to pH运1(deep red to alkacid test paper) with 4 mL of concentrated hydrochloric acid. Add 30
39、mL of eth1 acetate to the acidified hy drolysate , stopper and extract by shaking for 30 seconds. Let stand to separate. Filter the upper lay er liquid phase into a 125 mL of seperatory funnel. et sfi:lnd to SE豆parate.Drain the lower a queous layer back into the conical flask. Then add 20 mL of ethy
40、l acetate , stopper and extract bshaking for 30 seconds. Let stand to separate. Combine the upper layer liqllid phase in the same seperatory funnel. Let stand to separate and; the aqueous layer is discarded. Add 5 mL ofO.5 mol/L pH 6.0 citric acid buffer to the ethyLacetate extract , shake.and allow
41、 the lower phase to clarify (ca 10 minutes). Collect the aqueous phase in a 25 mL glass-stoppered cen trifuge tube. Reextract the ethyl acetate、phasewith an additional 5 lTl L of pH 6.0 buffer. Allow the aqueous phase to clarify. Combine the aqueous eracts in the ceqtrifuge tube. Add 2 mL of con cen
42、trated hydrochloric acid. Mix. Save the extracts.for column chmatography stated below. Transfer the acidified aqueous extract to -tne ion exclusion olumn. Drain the extract to the top of the resin bed. Wash the centrifuge tube and resin with 20 mL of 1 mol/L hydrochloric acid solu tim . Drain throug
43、h the column. Rewash the column with an additional 20 mL of 1 mol/L hy drochloric acid so!ution. Discard this and previous effluents from the column. Elute the column with 75 mL of methano!-water (10 + 90) , the column may be allowed to run dry in this step. Collect all the eluants and transfer into
44、 a 250 mL separatory funnel. The flow rate of effluent should be ca 1.2 mL/minute. Add 1.0 mL of concentrated hydrochloric acid to the eluate and extract with three 50 mL portions of ethyl acetate. Collect the extracts in a 250 mL round bottom flask. Evaporate to dryness on a ro tary evaporator at 5
45、0 C in a water bath. 10 SN/T 1016.1-2001 3.4.2 Esterification Transfer the residue to a 10 mL centrifuge tube bwashing the flask with 3 mL of methanol. Evap orate the solvent to dryness under a stream of nitrogen at 600C in a water bath. Add 0.2 mL of freshly prepared methanol-sulfuric acid (97 + 3)
46、 , stopper and heat at 55 C - 600C in a water bath for 30 minutes. Remove the tube from the water bath , add 1.0 mL of hexane , and mix thoroughly on a vortex mixer for 30 seconds. Centrifuge 5 minutes at 2 000 r/min. Dilute 0.1 mL of the hexane extract to 1 .0 mL with hexane. The solution is used f
47、or gas chromatographic determination. 3.4.3 Esterification of the standard Pipet 1.0 mL of standard working solution of Quinoxaline-2-carboxylic acid into a 10 mL centrifuge tube and evaporate to dryness u(nder a stream of nitrogen at60C, in a water bath.Add 0.2 mL of freshly prepared methanol-sulfu
48、ric acid (97 + 3). Stopper and heat at 550C - 60 C in a water bath for 30 minutes. Extract and dilute (1: 10) as directed above steps 3;.4 .2. Esterify standards concur rently with samples. 3.4.4 Determination 3.4圄4.1GC operating conditior:l a) Chromatographic column: HP5， 15 m x 0.53 mm( id) x 1.5m
49、 capillary column of fused silica or equivalent. 10C /min b) Column temperature:170oC (8 min) - -. .200C (10 min). c) Injection port temperature:250C. d) Detector temperature: 280 C . e) Carrier gas and make-up gas: Nitrogen , (purity99. 999%) . FloV rate of carrier gas: 6 mL/min . Flow rate of make-up gas: 40 mL/min. f) Injection mode: Packed column injection. g) Injection volume:2L. 3.4.4.2 GC determination / According to
