SNT13922004进出口肉及肉制品中2甲4氯及2甲4氯丁酸残留量检验方法

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1、SNT 1392-2004 进出口肉及肉制品中2甲4氯及2甲4氯丁酸残留量检验方法r I中华人民共和国出入境检验检疫行业标准SN/T 1392-2004进出口肉及肉制品中2甲4氯及2甲4氯丁酸残留量检验方法Determination of MCPA and MCPBresidues in meat and meat products for import and export2004-06-01发布 2004-12-01实施中 华 人 民 共 和 匡国家质量监督检验检疫总 婿SN/T 1392-2004前 言本标准的附录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。本标准由中华

2、人民共和国天津出人境检验检疫局、天津中医学院第一附属医院起草。本标准主要起草人:林安清、许134 1唐丹舟、垢静。本标准系首次发布的出人境检验检疫行业标准。SN/T 1392-2004进出口肉及肉制品中2甲4氯及2甲4氯丁酸残留量检验方法范围 本标准规定了进出口肉及肉制品中2甲4抓及2甲4抓丁酸残留量检验的抽样、制样和气相色谱-质谱测定方法 。本标准适用于进出口冻分割牛肉中2甲4氯及2甲4氯丁酸残留量的检验。2 抽样和制样2. 1检验批 以不超过2 500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t 抽样数量见表1, 表 1 单位为件批量

3、最少抽样数1-25 一 I26-100 5101-250 一251一 500 15501 -1 000 1700一2 500 一2.3 抽样方法 按2.2规定的抽样件数随机抽取，逐件开启。 肉及肉制品:从每件中取一袋作为原始样品，其总量不少于2 kg，放人清洁容器内，加封后，标明标记，及时送交实验室。 如每件中无小包装或有小包装但每袋重量超过2kg者，则可用锋利刀(用酒精灭菌过)在抽出的包件中，每件割取不少于100 g,混合后置于清洁容器内，作为混合原始样。混合原始样的重量不少于2 kg。加封后，标明标记，及时送交实验室。2.4 试样制备 肉及肉制品:从所取全部样品中取出有代表性样品约1 kg

4、，充分搅碎，混匀，均分成两份，分别装人洁净容器内。密封作为试样，标明标记。在抽样和制样的操作过程中，应防止样品受到污染或发生残留物含量的变化2.5 试样保存 将试样于一918冷冻保存。测定方法3. 1 方法提要 在酸性条件下，用三氯甲烷提取组织中残留的2甲4氯、2甲4氯丁酸及其钠盐，并转移至碱液中，SN/T 1392-2004用有机溶剂洗涤后再将其酸化，2甲4氯,2甲4氯丁酸再用三氯甲烷提取。蒸除溶剂将其甲醋化，用气相色谱一质谱仪检测，外标法定量。3.2 试荆和材料 除另有规定外，试剂均为分析纯，水为蒸馏水。3.2.1 三氯甲烷:重蒸馏。3.2.2 乙醇 。3.2.3 正 己烷 :重蒸馏 。3

5、.2.4 甲醇 。3.2.5 乙醚:重蒸馏。3.2.6 硫酸一水(1+9):用优级纯浓硫酸配制。3.2.7 氢氧化钠:优级纯。3.2.8 氢氧化钠溶液(30 g/L):称取30 g氢氧化钠(3. 2. 7 )溶于1 000 mL蒸馏水。3.2.9 氯化钠:优级纯，于600灼烧4 h.贮于具塞瓶中。3.2. 10 饱和氯化钠溶液:用足量氯化钠(3. 2. 9 )溶解于蒸馏水中直至不溶解。3.2. 11 无水硫酸钠:于650灼烧4h，储于密封容器中备用。3.2. 12 硫酸钠溶液(40 g/L):称取4g无水硫酸钠(3. 2. 11)溶解于100 mL蒸馏水中。3.2. 13 三氟化硼一乙醚溶液:

6、市售，避光保存。3.2. 14 甲酷化溶液:将三氟化硼一乙醚(3. 2. 13)和甲醇，于一15下预冷后，将30 mL冷三氟化硼一乙醚试剂和120 mL冷甲醇混合，于。4储存备用3.2. 15 2甲4氯,2甲4氯丁酸标准品:纯度)99写3.2. 15. 1 2甲4氯,2甲4氯丁酸标准溶液:准确称取适量的2甲4氯,2甲4氯丁酸标准品，用甲醇配制成浓度为。. 1 mg/mL标准储备溶液。再以甲醇稀释成适用浓度的标准工作溶液。3.3 仪器和设备3.3. 1气相色谱一质谱联用仪。3.3.2 高速均质器。3.3.3 心形瓶 :250 mL,3.3.4 密封试管:15 mL，带螺旋盖(配聚四氟乙烯内衬密封

7、垫)。3.3.5 恒温水浴箱 。3.4 测定步骤3.4. 1 提取及净化 称取搅碎混匀的试样约20 g(精确到0.01 g)于锥形瓶中，加15 m1乙醇,5 mL硫酸一水(3.2.6),10 g氯化钠(3.2.9)和100 mL三氯甲烷(3.2.1)。在高速均质器上均质提取5 min。通过快速滤纸过滤，滤液收集于250 m1分液漏斗中。用约50 mL三氯甲烷分三次洗涤锥形瓶及滤渣，合并洗液于分液漏斗中.于上述分液漏斗中加25 mL氢氧化钠溶液(3.2.8)和50 mL蒸馏水，再加人10 mL饱和氯化钠(3.2.10)。此时水相pH应大于12，否则应补加适量氢氧化钠溶液(3.2.8)对于极易乳化

8、的样品可再补加约3g氯化钠(3.2.9),振摇提取2 min。静置分层，弃去三氯甲烷层 用25 mL三氯甲烷洗涤水层两次，弃去三氯甲烷层 再用25 mL乙醚洗涤水层，静置分层，将下层水相转移至另一250 mL分液漏斗中，弃去乙醚层。于上述水相中，加入25 mL硫酸一水(3. 2.6)进行酸化，摇匀。水相pH应小于2，否则应适量补加硫酸一水(3.2.6)，然后分别用50 mL,25 mL,25 mL三氯甲烷提取水相。将三氯甲烷提取液收集于250 mL心形瓶中。于60水浴中通以氮气流将三氯甲烷挥发至约3-5 mL。完全转移至密封试管(3.3. 4 )中。再用氮气流于50水浴中吹干。3.4.2 甲醋

9、化 于上述密封试管中，加1 mL甲醋化溶液(3.2.14)，用螺旋盖密封。充分振动混匀，于70水浴酷SN/T 1392-2004化1h，放冷至室温。以约10 mL硫酸钠溶液(3. 2. 12)将甲醋液转移至25 mL具塞试管中，分别以4 mL正己烷提取两次。用滴管将正己烷层转移至另一25 mL具塞试管中，用2 mL硫酸钠溶液洗涤正己烷层两次，用滴管吸除水层，将正己烷层转移至10 mL容量瓶并以正己烷定容。吸取上述正己烷榕液3-5 mL于10 mL具塞试管中，加约1g无水硫酸钠，振摇脱水。供气相色谱一质谱分析。3.4.3 2甲4氮,2甲4氮丁酸标准工作溶液的制备 准确吸取适用浓度的2甲4氯,2甲

10、4抓丁酸标准溶液1 mL(3. 2. 16)于带螺旋帽盖的试管(3.3.4)中，用氮气流于50水浴中吹干，按3.4.2操作进行甲酷化。3.4.43.4.4. a) b) c) d) e) f)3.4.4.2 a) b) c) d) e) f)测定 色谱条件色谱柱:弹性石英毛细柱DBSMS 30 mX0.32 mm(内径)X0.25 pm或相当者; _ 20'C /ml.服 程序:501: ; 240;C(10 min);汽化室温度:250'C;载气:氦气，纯度99. 999%，流速1.2 mL/min;进样方式:无分流;进样量:1 HL, 质谱条件接口温度:2500C ;离

11、子源:电子轰击源(ED;电子能量:70 eV;离子源温度:2000C;检测方式:SIR;选择离子(m/z)及相对丰度(%):见表2. 表 2 选择离子及相对丰度被侧组分 2甲4氯甲酷 2甲4氛丁酸甲酷选择离子/(m/z) 141 157 214 216 101 107. 142 211相对丰度/(%) 85 20 100 34 100 13 9 83.4.4.3 气相色谱一质谱测定 对样液(3.4.2)及标准工作溶液(3.4.3)等体积参插进样测定。实际应用的标准工作溶液及待测样液中，2甲4氯甲醋,2甲4氯丁酸甲醋的响应值均应在仪器线性范围内。在上述条件下2甲4氯甲酷、2甲4氯丁酸甲酷保留时间

12、分别约为7. 8 min和9. 1 min。标准品气相色谱一质谱图参见附录A.3.4.4.4 空白试验 除不加样品外，按上述相同条件和步骤进行。3.5 结果计算和表述 用色谱数据处理机或按式(1)计算，计算结果需扣除空白值。(1)?、?式中:X 试样中2甲4氯或2甲4氯丁酸含量，单位为毫克每千克(mg/kg) ;A 样液中2甲4氯甲酷或2甲4氯丁酸甲醋的峰面积，单位为毫米(mm) ;A 标准溶液中2甲4氯甲醋或2甲4氯丁酸甲醋的峰面积，单位为毫米(mm) ;c 标准工作溶液中2甲4氯或2甲4氯丁酸浓度，单位为微克每毫升(pg/mL) ;V 样液最终定容体积，单位为毫升(mL) ;SN/T 13

13、92-2004。 最终样液所代表的试样量，单位为克(9)。4 方法的测定低限、回收率4.1 测定低限 本方法测定低限为:0.02 mg/kg.4.2 回收率 牛肉中2甲4氯添加浓度及回收率的实验数据: 在。.02 mg/kg时，回收率为85.5%; 在。，10 mg/kg时，回收率为95,0写; 在0.20 mg/kg时 回收率为92.0%e 牛肉中2甲4氯丁酸添加浓度及回收率的实验数据: 在0. 02 mg/kg时，回收率为90.0%; 在0. 10 mg/kg时，回收率为90.000; 在0.20 mg/kg时，回收率为93.0%SN/T 1392-2004 附 录 A (资料性附录)标准

14、物气相色谱一质谱图M CPA.R1007.802Scan El-TTC9.0R2%7.000 8.000 9.000 10.000图 A. 2甲4氮及2甲4抓丁酸标准衍生物气相色谱一质谱图(TIC)MCPA(7.802 min100 214Scan El266155%12577 216?，5163 741431.54157， 158182?5g 1I3 217167 184?60 80 100 120 160 180 200图A.2 2甲4氮标准衍生物质谱图SN/T 1392-2004MCPB (9.082 min >101100S-2昆59%69 77 107二:.142Jr 211

15、24289 1 _157 19360 80 100 160 180 200 220 240图A.3 2甲4点丁酸标准衍生物质谱图SN/T 1392-2004ForewordAnnex A of this standard is on informative annex.This standard was proposed by andfication and Accreditation.is under the charge of National Regulation Commission for Certi-This standard was drafted by Tianjin Entr

16、y-Exit Inspection and Quarantine Bureau of the People's Re-public of China,No. 1 Preaching Hospital Tianjin University of accessorial Chinese medicine.This standard was mainly drafted by Lin Anging,Xu Hong,Tang Danzhou,GoujingThis standard is a professional standard promulgated for the first ti

17、me.Note:This English version,a translation from the Chine text,is solely for guidanceSN/T 1392-2004 Determination of MCPA and MCPBresidues in meat and meat products for import and export1 ScopeThis standard specifies the methods of sampling, sample preparation and determination of residuesby GC-MS o

18、f (4-cholro-2-methyphenoxy) acetic acid (MCPA) and 4-(4-chloro-o-toloxy) butyric acid(MCPB) residues in meat and meat products for import and export.This standard is applicable to the determination of MCPA and MCPB residues in frozen cuts of beeffor import and export.2 Sampling and sample preparatio

19、n2.1 Inspection lotThe quantity of an inspection lot should not be more than 2The characteristics of the cargo within the same inspectionfication and grade, should be the same.500 packages.lot, such as packing,mark, origin, speci-2. 2 Quantity of sample takenQuantity of sample taken see table 1.Tabl

20、e 1Number of packages in each inspection lot Minimum number of packages to be taken1- 25 26- 100 5101 -250 10251-500 15501-1 000 171 001 -2 500 202.3 Sampling procedureA number of packages specified in 2.2 are taken at random and opened one by one.Meat and meat products: From each, at least one bag

21、shall be taken as a primary sample. The totalweight of all the primary samples should not be less than 2 kg, which shall be placed in a clean con-tainer, sealed, labeled and sent to the laboratory in time.In case the frozen meat-pieces are not contained in small bags inside each package, or if there

22、 are RSN/T 1392-2004small bags inside the package but the content of the bag exceeds 2 kg, cut out a part from the meatin each package of not less than 100 g with a sharp knife (disinfected with alcohol). Mix the parts ofthe mixed primary samples, which shall not be less than 2 kg. Sealed, labeled a

23、nd sent to the labora-tory in time.2. 4 Preparation of test sampleMeat and meat products: The combined primaryand divided into two equal portions, each portionsample is reduced to 1 kg which is blended mixedis placed in a clean vessel as a test sample, whichis then sealed and labeled. In the course

24、of sampling and sample preparation, precaution should betaken to avoid contamination or any factor which may causes the change of residue content.2. 5 Storage of sampleThe test sample should be stored at一18C.3 Method of determination3.1 PrincipleMCPA and MCPB residues and its sodium salts are extrac

25、ted from the tissue with chloroform underacidic condition, then transfer into alkaline solution. The alkaline solution is washed with organic sol-vents and acidified, MCPA and MCPB are then extracted with chloroform and derivatisation after thechloroform has been evaporated. Determination is made by

26、 GC-MS and quantified by using the exter-nal standard.3. 2 Reagents and materialsUnless otherwise specified, all reagents used should be analytically pure, ;water; is distilled water.3.2.1 Chloroform: redistilled.3.2.2 Ethanol3.2.3 n-Hexane: redistilled.3.2.4 Methanol3.2.5 Ether: redistilled3.2.6 Su

27、lfuric acid-water 0 +9) :Prepare the solution with GR grade of sulfuric acid.SN/T 1392-20043.2.7 Sodium hydroxide: GR grade.3.2.8 Sodium hydroxide solution (30 g/L): Weigh 30 g Sodium hydroxide (3. 2.7) into 1 000 mLdistilled H20-3. 2.9 Sodium chloride: GR grade, ignite at 600'C for 4 h, store

28、in a glass jar with stopper.3.2.10tion.Saturated sodium chloride solution: Sodium chloride dissolved in distilled HBO to satura-3.2.11 Anhydrous sodium sulfate: ignite at 6500 for 4 h, and store in air-tight container.3.2. 12 Sodium sulfate solution( 40 g/L: Weigh 4 g of anhydrous sodium sulfate (3.

29、2. 11 )and dis-solve in 100 mL water.3.2.13 Boron trifluoride-ether solution: commercially, kept away from light3. 2. 14 Derivatization reagent: Pre-cool the boron trifluoride-ether solution(3. 2. 13 )and methanolat一15C , add 30 mL cold boron trifluoride-ether into 120 mL cold methanol and mix thoro

30、ughly,store at 0一4.3. 2.15 MCPA and MCPB standard: Purity_>99%.3. 2. 16 MCPA and MCPB standard solution: Prepare 0. 1 mg/mL standard stock solution with meth-anol, then dilute to suitable concentration with methanol as standard working solution.3.3 Apparatus and equipments3. 3. 1 Gas chromatograp

31、h equipped with mass spectrograph (GC-MS)3.3.2 High-speed homogenizer.3.3.3 Heart shape flask: 250 mL.3.3.4 Sealed test tube: 15 mL, with teflon-lined screw cap.3.3.5 Thermostatic water bath3.4 Procedure3.4. 1 Extraction and clean upWeigh ca 20 g (accurate to 0. 1001 g)of test sample in a conical fl

32、ask, add 15 mL ethanol, 5 mL sulfuricSN/T 1392-2004acid-water(3. 2. 6)，10 g sodium chloride (3. 2.9) and 100 mL chloroform (3. 2. 1).Extract 5 minwith high-speed homogenizer. Then filter the extract through rapid filter paper into a 250 mL separa-ting funnel, rinse the conical flask and filter resid

33、ue thrice with ca 50 mL chloroform. Combine allrinsing solutions into the separating funnel. Add 25 mL sodium hydroxide solution (3. 2. 8) and50 mL distilled water. Add 10 mL saturated sodium chloride solution(3. 2. 10). pH of the aqueousphase should be more than 12, otherwise, additional sodium hyd

34、roxide solution(3. 2. 8) is needed.Additional 3 g sodium chloride (3. 2.9) should be added to the easily emulsified sample, shake for2 min. Stand to separate, discard the chloroform layer. Wash the aqueous phase twice with 25 mLchloroform, discard the chloroform layer. And then wash the aqueous phas

35、e with 25 mL ether, standto separate, transfer the below layer to a 250 mL separating funnel, discard the ether layer. The a-bove aqueous phase is acidified with 25 mL sulfuric acid-water (3. 2. 6) and mix thoroughly, pH ofthe aqueous phase should be less than 2, otherwise, additional sulfuric acid-

36、water(3. 2. 6) is needed.Then extract the aqueous phase with 50, 25 and 25 mL chloroform respectively. Collect the extractof chloroform into 250 mL heart shape flask. Evaporate the chloroform to ca 3-5 mL in a water bathat 501C under nitrogen flow. Then transfer entirely to the sealed test tube (3.

37、3. 4)，make the chloro-form to dryness under nitrogen flow in a water bath at 50C.3.4.2 DerivatisationAdd 1 mL derivatization reagent (3. 2. 14) into the above tube and seal tightly, mix thoroughly, placethe tube in water bath for 1 h at 70C. Cool to room temperature, transfer the resulting mixture w

38、ithca 10 mL sodium sulfate solution (3. 2. 12) to a 25 mL tube with stopper. Extract with n-hexanetwice each 4 mL. Transfer the n-hexane layer to another 25 mL tube with stopper. Extract the n-hex-ane layer with each 2 mL of sodium sulfate solution twice and discard the aqueous layer. Using adropper

39、 transfer the n-hexane layer to 10 mL volumetric flask, dilute to volume with n-hexane. Pi-pette 3-5 mL n-hexane layer into a 10 mL tube with stopper, add ca 1 g of anhydrous sodium sulfate(3.2.11)，mix well. The solution is used for GC-MS determination3. 4. 3 Preparation of MCPA and MCPB standard wo

40、rking solutionAccurately pipette 1 mL MCPA and MCPB standard solution of suitable concentration into tube withscrew cap (3. 3.4)，remove the solvent under nitrogen flow in a water bath at 501C，proceed as sec-tion 3. 4. 2.3.4.4 Determination3. 4. 4. 1 GC operating conditionsa. Column: DB5MS 30 m x 0.

41、32(id) mm x 0. 25 um(film thickness) or equivalent;b. Column temperature program: 50Ctot /ruin2400 (10 min);c Injection port temperature: 250C;Carrier Gas; He, Purity>99. 999%;flow rate: 1. 2 mL/min;11SN/T 1392-2004f. I币action mode: Splitless;g. Injection volume: 1 pL.3.4.4. 2 MS operating condit

42、ionsa. Interface temperature: 250;C;b. Ion Source: Electron Impact Ionc. Electron Energy: 70 eV;d. Source temperature: 2001C;e. Detection mode: SIR;f. Selected ions (m/z) and relativeSource (EDintensity(%):see Table 2.Table 2 Selected ions and relative intensityanalyte MCPA methyl ester MCPB methyl

43、esterSelected ions (m/z) 141 157 214 216 101 107 142 242Relative intensity(% ) 85 20 100 34 100 13 9 83. 4. 4. 3 GC-MS determinationThe mix standard working solution should be randomly injected in-between the injections of the sam-ple solution of equal volume. The responses of the MCPA methyl ester

44、and MCPB methyl ester in thestandard working solution and sample solution should be within the linear range of the detector. Theretention time of MCPA and MCPB methyl ester is ca 7. 8 min and 9. 0 min under the above condi-tions. For the chromatogram of the standard, see annex A.3. 4.4.4 Blank testT

45、he operation of blank test is the same as that described in the method of determination, but withomission of sample addition3.5 Calculation and expression of resultCalculate the content of MCPA and MCPB by GC data processor or according to formula;' the blankvalue shall be subtracted from the r

46、esult of calculation. A cV人 二二二 八 ，.m(1)W hereX- the residue content of MCPA or MCPB in test sample, mg/kg;A- the peak area of MCPA methyl ester or MCPB methyl ester in sample solution, mm;A,- the peak area of MCPA methyl ester or MCPB methyl ester in standard solution, mm; c- the concentration of M

47、CPA or MCPB in standard solution, pg/mL;V- the final volume of the sample solution, mL;m- the corresponding mass of test sample in the final sample solution ,g.I2SN/T 1392-20044 Limit of determination and recoveryLimit of determinationlimit of determination of this method is 0.02 mg/kgRecovery?Accor

48、ding to the experimental data, the fortifying concentration of MCPA in beef and its correspond-旧g recoveries are:0.02 mg/kg, recovery 85. 5%;0. 10 mg/ kg, recovery 95.0%;0. 20 mg/kg, recovery 92.0%According to the experimental data, the fortifying concentration of MCPB in beef and its correspond旧g r

49、ecoveries are:0. 02 mg/kg, recovery 90. 0%;0. 10 mg/ kg, recovery 90.0%;0. 20 mg/kg, recovery 93. 0%.13SN/T 1392-2004 Annex A (informative)GC-MS Chromatogram of the standardMCPA卫7.8021009.082%7.000 8.000 9.000 10.000Fig A. 1 GC-MS chromatogram(TIC)of the MCPA methyl ester and MCPB methyl ester stand

50、ardMCPA(7.802 min)100 S- EI214 255141155% ?77143日95163 74 125113 1201271571541581R2?59 ?72860 80 100Fig A. 2 Mass spectrogram of the MCPA methyl ester standard14SN/T 1392-2004MCPB (9.082 mm101100 S-2昆59%69 77 107142211 242144 .157 19360 80 160 180 200 220 240Fig A. 3 Mass spectrogram of the MCPB methyl ester standard