1、叶己中华人民共和国进出口商品检验行业标准SN 0276-93 出口禽肉中氨丙嗜口比q定残留量检验方法薄层色谱法Method for the determination of amprolium residues in poultry meat for export Thin layer chromatography 1993-12-28发布1994-05-01实施中华人民共和国国家迸出口商品检验局发布(京)新登字023号中华人民共和国进出口商品检验行业标准出口禽肉中氨丙嘻E比q定残留量检验方法薄层色谱法Method for the determination of amprolium resi
2、dues in poultry meat for export Thin layer chromatography 1 主题内容与适用范围SN 0276-93 本标准规定了出口禽肉中氨丙略目比睫残留量的抽样、制样和薄层色谱测定方法。本标准适用于出口鸡肉中氨丙嘻毗睫残留量检验。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1._, 25 1 26100 5 101250 10 251500 15 5011 000 17 1 0012 500 20 2. 3 抽样方法按2.2规定的
3、抽样件数随机抽取.逐件开启。每件至少取一袋作为原始样品，原始样品总量不少于2峙。放入洁净容器内.加封后.标明标记.及时送交实验室。2.4 试样制备从每袋原始样品中取出部分有代表性样品，将可食部分放入高速组织捣碎机中捣碎均匀，充分混合，用四分法缩分出不少于500g。装入洁净容器内，加封后标明标记。2.5 试样保存将试样于一180C以下冷冻保存。注:在抽样及制样的操作过程中，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 方法提要用三氯乙酸搭液提取试样中的氨丙唔毗睫。提取液经碱性氧化铝柱净化，用氧化饵盐酸溶液洗脱中华人民共和国国京进出口商品检验局1993臼12-28批准1994-
4、05-01实施1 SN 0276- 9 3 日，在碱性条件下加入硝酸银和铁司化饵溶液，(吏衍生成荧光物质，于薄层板分离.月H障层扫描仪测定。3. 2 试齐IJfil材料所用试剂|涂注明外，均为分析纯.水为蒸榴水。3. 2. 1 之;J乙酸榕城:5%。称取5g :兰提乙酸，溶于100mL水中。3. 2. 2 氧化饵盐酸熔掖:25 5/。科:取25g氯化押.溶于0.1mol/L盐酸使成100mL。如1析出结晶，则加温使之完全洛解。3.2.3 铁氟化神溶液:2叹。称取2g铁钝化饵CK3Fe(CN).溶于100mL水中。转移至棕色瓶中，于冰箱中保存。3. 2. 4 硝酸银榕液:2%。称取2g硝酸银，洛
5、于100mL水中。转移至棕色瓶中，工冰箱中保存。3. 2. 5 氢氧化纳?在液:30%、10%。称取30g , 10 g氢氧化锅，分别溶于100mL水中。3.2.6 异百事:优级纯。3. 2. 7 氧化铝:喊性，层析用，100200日。3.2.8 元水硫酸纳:650C灼烧4h，置干燥器内备用n3. 2. 9 高效薄层板:薄层层析用硅胶G板，豆cmXI0crn、10cmX10 cm，层厚0.25mmo 3.2.10 展开刑:P比院-异丁醇句水(66十17十17)3. 2. 11 延内i骑口比院标准品:纯度二三99%。3.2.12 氨丙晤口比脏标准溶液:精确称取每丙喃自比陀标准:品10.0mg于小
6、烧杯中，用水溶解，转移至500 mL容量瓶中，稀释定容。此做为标准储备溶攘，浓度为20.。g/mL。用移液管移取此洛液5mL于另一容量瓶中，加水陆释歪500mLQ此为标准工作溶液，放度为0.2g/mLo3. 3 仪器和设备3. 3. 1 荧光分光光度计，配有薄层扫描装置，或薄层扫描仪。3.3.2 高速组织捣碎机。3.3.3 振荡器:往复式。3. 3.4 离心机:3000r/mn o 3.3.5 旋转蒸发器。3. 3. 6 紫外灯:10)旧0125W，滤光片365n旧nrr回I盯m3. 3. 7 层析缸。3. 3. 8 自动点样仪。3.3.9 微量注射器:51.，10L、20L、50rL 3.
7、3. 10 锥形瓶:具在，250mL, 3.3. 11 离心智:具塞带刻度，50mLo 3. 3. 12 睦筒:50mL、100mL 3. 3. 13 蒸发皿:50rnL、100mL 3. 3. 14 层中斤柱:20 crn X 1 cm (id )玻璃柱，T具活塞。底部装少许脱脂棉，装约3-5g碱性氧化铝。3.3. 15 恒温水浴锅3.3.16 瓷研钵。3.3. 17 样品瓶:棕色.3mLo 3.4 测定步骤3.4.1 提取和i净化取经绞碎、屁匀的试样20g (精确到O.1 g)于瓷研钵中，加40g无水硫酸纳，研磨脱水，转移至250 mL锥形瓶中o准确加入三氯乙酸溶掖60mL，于振荡器上提取
8、30mino移取上层试液于50mL离心管中，以3000 r/min离心5rnin。准确吸取上i青液15mL，加入氢氧化纳洛液(10%)数滴使之成为碱性，移入层析枪(3.3.14)中。用2 SN 0276 93 水洗海柱子，弃去流出瓶。用氧化饵盐酸j窑液25mL r乱悦性中提I对喘口比睫，使其自然流出，收集洗脱液于50mL带刻度离心管中。于洗脱液中加入4.mL另氧化饷榕掖(30%).旧习3再加入硝酸银溶液0.4mL.摇匀.静置2mino 加入铁氟化梆、溶液2mL.摇匀。加入异r醇12trJL，振摇1min，以3000 r/min离心15min。取上层异T醇J容破9日IL于蒸发皿巾，在6070C水
9、陆r:蒸发近干，并自然挥发干。用O.;lmL异丁醇溶解残渣并转移至样品瓶中，供llj定唱。3.4.2 测定3. 4. 2. 1 i薄层扫描条件a. 光，t: 正灯zb. 激发波长352nm.发射j皮长420nm; C. 橄发狭缝10mm，发射狭缝10mm; d. 也电压7;:;0V; e. 扫描速度60mm/min，纸速5cm/min。3. 4. 2. 2 标准曲线的绘制取0.2，!g/mL司丙略口比院标准E作榕液10mL，按3.1. 1从加入4.mL氢氧化flI.J溶液(30%)开始，主.t.仗测定用操作进行。分别在薄层板t滴加1、2、3、4、5L标rft-1:作恪液，相当于标准量为5、10
10、、15、20、25呵，经薄层展卉后测定。以氨丙略毗睫的含量(ng)对相应的峰面积(或峰高)绘制标准曲线3. 4. 2. 3 薄层包谐测定将自j效薄层板在10510 C活化2h后.置干燥器内备用。如超过一周使用时.省重新陆化。取适宜体积的标准工作榕液和样品溶液在薄层极t距下端1-52 cm :1主线t，参插点样，每，点问隔1cm。将薄层板放入用口比昵异r醇，顶饱和的后析缸中展开101:5 mirJ. 前沿达约10 cm.取i:!fI次干。在紫外灯365nm F观察薄层板结果，氨丙略咆吭呈蓝紫色荧光点。根据标准点的R，值确定样品中氨丙暗毗院位置，标出标记，按薄后扫描;如牛进行/ij!IJ走。3.4
11、.3 ?臼试验:除不加i式样外.均披上述测起步骤进行。3. 5 结果计算相去述按海层扫描测定所得样液中氢丙唔口比腥的峰由在H或峰高).从标准曲线k查出相应的氧丙唔毗盹的量(ng)JH安下式计算试样中氨丙略目比院含量:式中:X一一试样中氨丙暗口lt院含量，mg/kg;X+ tcly. 3. 2.3 Potassium ferncyanide CKYe(CN )6)日olution:2%solution- Dissolve 2 g of K:;Fe(CN)6 in 100 mL of H()， transfer to a brown buule and store in a refrigerato
12、r. 3.2- 4 Silver niuate (AgN03)solution:2% solution. Dissolve 2 g of AgNO; in 10mL of H20 , trans fer to a brown bortle and store in a refrigerator. 3. 2. 5 Sodium hydroxide (NaOH) solution:30% .10%. Dls吕olve30 g and 10 g of NaOH in 100 mL of HzO respec1 ively. 3. 2. 6 lsobutyl alcohol :G. R. grade.
13、 3.2.7 Alumina :Alkaline ,for chromatography , 100-200 mesh. 3.2.8 Anhydrous sodium sulfate: Ignite at 650 C for 4 h ,tore in desiccator. 3.2.9 High performance thin layer v1ate: 5 cm X 10 cm and 10 cm X 10 cm sillca gel G plates , O. 2:; mm thirkness. 3.2.10 Developing reagent :Pyndine-isobuty alco
14、hol-water(66十17十17).3. 2. 11 Amprohum standard: Purity 二万99%.3.2. 12 Amprolium standard solution: Accurately weigh 10.0 mg of amproiium s t111dard into a small beaker、dissolvewitb water and transfer to a 500 mL volumetric flask. Dilu1e to volume and tnix tho roughly to ohtain the standard stock solu
15、tion (20.0g/mL). Pipct 5 mL Il1 to another 500 mL vou metric flask and dilute to 500 mL wlth water , as the standanl workll1g solutlOn. TIH丁concentrationof this standard working soluton is 0.2 fLg/mL. 3. 3 Apparatus and equipments 3. 3. 1 Fluorecence spectrophotometer with thin layer scannin(,( :itt
16、achment or thin layer sr: anning rne-ter. 3.3.2 High speed tissue grinder. 3. 3. 3 Reciproca1mg shaker. 3. 3. 4 Centrifuge : 3 000 r /min. 3. 3. 5 Rotatory evaporator. 3.3.6 UV Fluorescent lamp:100-125 W ,with 365 nm filter. 3.3. 7 Chromatographic jJ r. 6 SN 0276-93 3. 3.8 Auto-spotting meter. 3. 3.
17、 9 Micro-syringe : 5L.10L.20L.50L. 3.3.10 Conical flask:250 mL.with glass stopper. 3. 3. 11 Centrifugc tube: 50 mL. with stopper and graduated. 3. 3. 12 Cylinder: 50 mL. 100 mL. 1 J 13 Evaporating dish: 50 mL, 100 mL 3. 3. 14 Chromatographic column: 20 cm X 1 cm (id) glass column (with stop-cock). p
18、acked with a small piece of absorben cotton at the bottom and ca 3-5 g of alumina (alkaline). 3. 3. 15 Constant temperature water -bath. 3. 3. 16 Porcelain mortar. 3. 3. 17 Sample vial: 3 mL. brown. 3. 4 Procedure 3. 4. 1 Extraction and clean up Wegh ca 20.0 g(accurate to 0.1 g)of the homogenized sa
19、mple into a mortar.add 40 g of anhy drous Na2S0. and grind 10 dehydrate. transfer to a 250 mL conical flask. Add accurately 60 mL of TCA solution .and extract 011 the shaker for 30 min. Transfer the upper layer of the testingsample solutlOn into a 50 mL centrifuge tube and centrifugalize at 3 000 r
20、/min for 5 min. Pip(,t accurately 15 mL of the supernatant .add a few drops of NaOH solution (10 o) to make al kaline.then transfer into the alumma column (3. 3. 14). Wash the column W Ith water and discard the effluept. Elute the arnprolium with 25 mL of KCI-HCl solution .let the solution drain fre
21、ely.collecr the elute in a 50 mL graduated centrifuge tube. Add 4 mL of 30% NaOH soIution to the eluate ,mix well. Add 0.4 mL of AgNO:; solution ,shake well , let stand for 2 min. Add 2 mL K j Fe(CN)6 solution.shake well. Add 12 mL of isobutyl alcohol. shake for 1 min.and then centrifugalize at 3 00
22、0 r/min for 15 min. Pipet 9 mL of the supernatant isobutyl alcohol solution into an evaporating dish , concentrate to near dryness on a water-bath at 60一70Cand Iet evaporate to dryness at room temperature. Dissolve the residue with O. 3 mL of isobutyI alcohol and transfer into a sample vial as t he
23、sample sol1l 1 ion ready for analysis. 3. 4. 2 Determination 3. 4. 2. 1 TLC scanning conditions a. Light suurce: Xenon lamp; b. Wave length of excitation 352 nm. Wave Iength of emission 420 nm; c. Slit for excitation 10 mm , slit for emission 10 mm; d. Light voltage 750 V; e. Rate of scanning 60 mm/
24、mlll. Speed of chart 5 cm/min. 3. 4. 2. 2 Preparation of the standard curve Pipet 10 mL of O. 2g/mL amprolium standard working solution , then proceed as in 3. 4. 1, begm ing from Add 4 mL of 30% NaOH solutiontoready for analyss. Spot the standard working so!u tion 1.2. 3.4，5L respectively.equivalen
25、t to amprolium standard , 5 , 10 , 15.20, 25 ng. Determine by scanning after chromatographic developping. Plot the standard curve by using the amounts of standard (ng)against the value of the peak area(or peak height). 3. 4. 2. 3 TLC Determination Place the high performance thin layer plate at 105-1
26、10 C for 2 h for activating , then place in a 7 SN 0276-93 desiccator ready for use. If the storing time exceeds one week.reactivation is required. The standard working solution is spotted on the TLC plate. 1. 5-2 cm from the baseline and in between with the test sample solution of appropriate volum
27、e. The interval between the spots is 1 cm. Place the plate in a chromatographic jar which is presaturated with pyridine-sobutyl alcohol-wa ter (66+17十17)for developing for 0-15 min. Develop until the front position up to ca 10 cm. Take out and blow to dryness. Observe the plate under UV light at 365
28、 nm. Blue白violetfluorescent spots show the presence of amprolium. Determine the amprolium position of the sample by Rf of the standard spot. Mark the posi tion of the spot and determine according to the scanning condition. 3. 4. 3 Blank test Proceed as above but omitting the addition of the sample.
29、3. 5 Ca!culation and expression of result AECording to the peak area(or peak height)of Jhe spot of sample solution under TLC scanning , obtain the content of amprolium (ng )from the standard curve. Calculate the amprolium content of the test sample by the following formula: X一二五-m Vz where X一一thecon
30、tent of amproliurn in sample ,mg/kg; c一一thecontent of amprolium in the sample solution obtained from the standard curve , ng; V1 -一一thefinal volume of sample solution，L; V2一一寸hevolume of sample solution spotted on the plate，L; m一-thecorresponding mass of sample in the final sample solution , mg. Not
31、e: The blank value should be subtracted from the above result of calculation. 4 Limit of determination and recovery 4. 1 Limit of determination The limit of determination of this method is 0.02 mg/kg. 4.2 RecQvery According to the experimental data. when the fortifying concentration of amprolium is
32、in the range of O. 02一0.10mg/kg.the recovery is 78.0%-91. 6%. AdditionaJ expJanations: This standard was proposed by the State Administration of Import and Export Commodity In spection of the Peoples Republic of ChiH. ThlS standard was drafted by the Tianjin Import and Export Commodity Inspection Bureau of the Peopl，s Republic of China. This standard was mainly drafted by Song Jinfeng , Tang Danzhou. Note: This English vers;on.a translation from the Chmese text ,is solely for guidance. 8 2|KNO Z rJJ 中国标准出版社北京印刷厂印刷1994年10月第一版书号:155066 2-9535 1994年10月第一次印刷中国标准出版社出版
