1、中华人民共和国出入境检验检疫行业标准SN/T 1544-2005 进出口动物源性食品中玉米赤霉醇残留量的检验方法高效液相色谱一质谱/质谱法Inspection of zeranol residues in animal original products for import and export-HPLC-MS/MS method 2005-02-17发布2005-07-01实施中华人民共和国发布国家质量监督检验检疫总局中华人民共和国出入境检验检疫行业标准进出口动物源性食晶中玉米赤霉醇残留量的检验方法高效液相色谱质谱/质谱法SN/T 1544-2005 长中国标准出版社出版发行北京复兴门外三
2、里河北街16号邮政编码:100045网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷* 开本880X1230 1/16 印张1字数23千字2005年5月第一版2005年5月第一次印刷9峙书号:155066.2-16180 定价10.00元如有印装差错由本社发行中心调换版权专有侵权必究举报电话:(010)68533533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。本标准起草单位:中华人民共和国上海出入境检验检疫局。本标准主要起草人:方晓明、陈家华、倪昕路、唐毅锋。本标准系首次发布的出入境检验检疫行业标准。SN/T 1544-2005 I
3、 1 范围进出口动物源性食品中玉米赤霉醇残留量的检验方法高效液相色谱-质谱/质谱法SN/T 1544-2005 本标准规定了动物源性食品中玉米赤霉醇残留量检验的抽样、制样和高效液相色谱-质谱/质i普检测方法。本标准适用于动物组织肝、肾和肌肉中玉米赤霉醇残留量的检验。2 抽样与制样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 抽样数量抽样数量见表1。表1批量最低抽样数125 1 26100 5 101250 10 251500 15 5011000 17 1 0012 500 20 2. 3 抽样方法单位为件按2.2规定的
4、抽样件数随机抽取，逐件开启。从每件中至少取一袋作为原始样品。如每件中无小包装或有小包装，但每袋小包装质量超过2峙，则可用灭菌刀具从每件中割取不少于500g，71昆合后置于清洁容器内作为混合原始样品。混合原始样品的总中质量不少于2峙，加封后，标明标记，及时送交实验室。2.4 试样制备从混合原始样品中取出部分有代表性的样品，充分混匀，用四分法缩分至不少于500g，均分成两份。匀浆后装入清洁容器内作为试样，加封并标明标记。2.5 试样保存将试样置于一180C冷冻保存。3 测定方法3. 1 方法提要匀浆组织样品经葡萄糖昔酸酶水解后，依次用乙酷和氢氧化纳溶液提取玉米赤霉醇。氢氧化铀提取液中和后经C18固
5、相萃取柱净化。甲醇洗脱液经蒸发，定容后供高效液相色谱-质谱/质谱仪测定，外标法定量。SN/T 1544-2005 3.2 试剂和材料除另有规定外，所用试剂均为分析纯，水为二次蒸榴水或相适应的去离子水。3.2. 1 乙睛:液相色谱级。3.2.2 甲醇。3.2.3 无水乙醋。3.2.4 三氯甲烧。3.2.5 氢氧化铀。3.2.6 无水乙酸销。3.2.7 磷酸:纯度85%;密度1.7 g/mL。3.2.8 葡萄糖昔酸酶。3.2.9 玉米赤霉醇(zeranol，zearalanoD标准品:纯度97%。3.2.10 甲醇水溶液(40+60)。3.2. 11 磷酸水榕液:12 mol/L，取69mL浓磷酸
6、(3.2.7)，用水稀释至100mL。3.2. 12 氢氧化铀溶液:1 mol/L。3. 2. 13 o. 05 mol/L乙酸铀缓冲榕液:取4.10g无水乙酸铀，溶解于水中并稀择至1000 mL，用冰乙酸调节至pH4.8。3.2. 14 0.10 mg/mL玉米赤霉醇标准储备液:准确称取适量的玉米赤霉醇标准品(3.2.9)，用乙腊配制成0.10mg/mL的标准储备液。储备液保存于一180C。3.2.15 玉米赤霉醇中间标准液:1. 0g/mL和O.10g/mL，分别取1.0 mL和0.10mL玉米赤霉醇标准储备液(3.2.14)于100mL容量瓶中用乙睛定容至刻度。此溶液保存于一180C。3
7、.2. 16 玉米赤霉醇基质标准工作溶液:根据需要吸取适量玉米赤霉醇中间标准液(3.2.1日，用空白样品提取液稀释成适当浓度的基质标准工作榕液。基质工作溶液在180C保存，可使用一周。3.2.17 C18固相萃取柱:500 mg o 3.2.18 氮气。3.3 仪器和设备3.3. 1 高效液相色谱质谱/质谱仪:配有电喷雾离子源。3.3.2 分析天平:感量0.1mg和0.01g各一台。3. 3. 3 固相萃取装置。3.3.4 恒温氮吹装置。3.3.5 均质机。3.3.6 离心机。3. 3. 7 旋涡混合器。3.3.8 自动加液器:10L500L。3.3.9 离心管:50mL，具塞。3.3.10
8、锥形离心管:5mL, 10 mL和15mL。3.4 测定步骤3.4. 1 提取及净化称取5g试样(精确至0.01g)，置于洁净的50mL具塞离心管中，加入10mL乙酸饷缓冲溶液(3.2.13)和25L葡萄糖昔酸酶，旋涡棍匀，于370C中保温8h14 h。水解结束后，加入10mL无水乙酶，加盖并旋涡棍匀3min，于3500 r/min离心10min后，将上层酷相转移至15mL锥形离心管中，于400C水洛中，氮气流下吹至近干。重复上述操作一次。用1mL 三氯甲皖榕解残渣，加入2X 2.5 mL氢氧化铀溶液(3.2.12)，旋涡1昆匀、抽提，于3500 r/min离心5 min，将上层氢氧化铀溶液转
9、移至10mL锥形离心管中。在合并的氢氧化铀榕液中加入0.5mL磷酸SN/T 1544一2005溶液(3.2.11)，旋涡混匀。将溶液注入C1川柑萃取柱中(C18固相萃取柱预先用3mL甲醇和3mL乙酸铀缓冲洛液淋洗)，依次用2mL乙版纳豆豆冲榕液(3.2.13)和2mL甲醇水溶液(3.2.10)淋洗，弃去流出液。再用2mL甲醇进行洗脱，并收集于5mL锥形离心管中(保持抽气2min，使柱中的液体逐渐干洒)。将洗脱液置于550C水浴中氮气流下吹干。用200L乙腊梅解残渣，供高效掖相色谱质谱/质谱仪测定。3.4.2 测定3.4.2.1 高效液相色谱条件a) 色谱柱:ZorbaxExtendC18 (1
10、 50 mmX 2.1 mm，粒径5m)，或相当者;b) 流动相:乙腊-水溶液(40十60)含0.025%乙酸;c) 流速:0.2mL/min; d) 柱温:220C;e) 进样量:10L。3.4.2.2 质谱条件a) 离子化模式:负离子电喷雾(ESr); b) 锥电压:50V; c) 毛细电压:3kV; d) 碰撞电压:20V; e) 分析器压力t小于3mPa(3X10-5 mbar); f) 碰撞气:氧气;g) 锥气流速:80L/h; h) 喷雾气流速:480L/h; i) 选择反应检测模式:m/z321303，m/z321277。其中m/z321277用于定量测定。3.4.2.3 高效液
11、相色谱质谱/质谱测定按浓度由小到大的顺序，依次分析基质标准工作梅液(3.2.16)，得到浓度与峰面积的工作曲线。样品洛液中玉米赤霉醇的响应值应在工作曲线范围内。在上述高效液相色谱质谱/质谱条件下，玉米赤霉醇的保留时间约8.7min，色质图参见图A.1和图A.203.4.3 空白试验除不加试样外，均按上述操作步骤进行。3.4.4 结果计算和表述试样中玉米赤霉醇残留量利用数据处理系统计算或按式(1)计算:x = cX主z 、，市BA( . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 式中:X 试样中被测组分
12、残留量，单位为微克每千克(g/kg); 正从标准工作曲线得到的被测组分溶液浓度，单位为纳克每毫升(ng/mU;V 试样榕液定容体系，单位为毫升(mL);m 试样溶液所代表试样的质量，单位为克Cg)。注:计算结果应扣除空白值。4 测定低限、回收率4. 1 测定低限本方法的测定低限为1g/kg。3 SN/T 1544-2005 4.2 回收率4 玉米赤霉醇添加浓度及其回收率数据:1g/kg时，回收率72%;一-5g/kg时，回收率74%;一-50g/kg时，回收率80%。100 % 。0.00 100 % 。100 % 0 0.00 100 % 附录A(资料性附录)色质圈321 277 259 I
13、 303 04. ._叫4且，. . I , 1.1飞.rnlz 100125150175200225250275300325 360 2.00 4.00 6.00 8.00 10.00 SN/T 1544-2005 TIC 386 伊宁唱、宁1m12.00 14.00 圄A.1玉米赤霉醇标准物质总离子流固和质谱图(浓度0.02g/mL)2.00 4.00 6.00 8.00 10.00 m/z 321-277 108 mlz 321-303 46 宁嘈tnm 12.00 14.00 图A.2采用选择反应检测模式的玉米赤寓醇标准物质色谱图(浓度0.02g/mL)5 SN/T 1544-2005
14、 Foreword Annex A of this standard is an informative annex. This standard was proposed band is under the charge of Nation Regulatory Commission for Certifi cation and Accreditation. This standard was drafted bShanghai Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic of China. This
15、 standard was mainly drafted by Fang Xiaoming , Chen Jiahua , Ni Xinlu and Tang Yifeng. This standard is promulgated for the first time. 6 SN/T 1544-2005 Inspection of zeranol residues in animal original products for import and export-HPLC-MS/MS 1 Scope This standard specifies the method of sampling
16、 , sample preparation and determination of zeranol in animal original products by high-performance liquid chromatography-tandem mass spectrometry. This standard applies to determiantion of zeranol in animal liver, kidney and meat. 2 Sampling and Sample Preparation 2. 1 Inspection lot Each inspection
17、 lot should not exceed 2 500. The characteristics of the cargo within the same inspection lot, such as packi吨，mark,origin , grade and specification, should be the same. 2.2 Ouantity of sample taken Ouantitof sample taken see table 1. Number of packages in each inspection lot 1-25 26-100 101-250 251-
18、500 501-1 000 1 001-2 500 2.3 Sampling procedure Table 1 Minimum number of packages to be taken 5 10 15 17 20 A number of packages specified in Section 2. 2 are taken at random and opened one by one. The sam ple weight taken as the primary sample from each package should be at least 500 grams. The t
19、otal weight of all primary samples should not be less than 2悔，which shall be sealed , labeled and sent to laboratory in time. 2. 4 Sample preparation Take partition of mixed original sample as representative sample , mixed well. A more than 500 9 of sample was devided in two and homogenized. Package
20、 and label. 7 SN/T 1544-2005 2. 5 Storage of test sample The test sample should be stored at一180C.3 Method of Determination 3. 1 Principle A homogenized tissue sample is hydrolyzed with -glucuronidase and the hydrolysate is extracted with ethyl ether. The supernatant is evaporated to dryness and the
21、 residue is dissolved in chloroform and re-ext阳ctedwith sodium hydroxide solution. After acidification , the extract is cleaned up on a C8 solid-phase extraction cartridge , and analyzed by electrospray HPLC-MS/MS in negative ion mode. 3. 2 Reagents and materials Reagents were of analyticalreagent g
22、rade except for the specific reagents. The water used was dou bly distilled or deionized water. 3.2.1 Acetonitrile: LC grade. 3. 2. 2 Methanol. 3. 2. 3 Diethyl ether. 3. 2. 4 Chloroform. 3. 2. 5 Sodium hydroxide. 3.2.6 Anhdrous sodium acetate. 3.2.7 Phosphoric acid: purity85%; density 1.7 g/mL. 3.2.
23、8日-glucuronidase.3. 2. 9 Zeranol (-zearalanol): purit97%. 3. 2. 10 Methanol喃watersolution (40+60). 3.2.11 Phosphoric acid solution , 12 mol/L: Pipette 69 mL H3 P04 (3. 2. 7) and diluted to 100 mL with water. 3.2. 12 Sodium hydroxide solution , 1 mol/L. 3.2.13 Sodium acetate buffer, 50 mmol/L: Take 4
24、.1 9 of anhdrous sodium acetate , dissolve and 8 SN/T 1544一2005dilute to 1 000 mL with water. Adjust pH to 4.8 with glacial acetic acid. 3.2.14 Zeranol stock solution , O. 10 mg/mL: Accurately weigh proper zeranol (3.2.9) and dis solved with acetonitrile. The stock solution is stored at - 180C. 3. 2
25、. 15 Zeranol Intermediate solutions, 1. 0g/mL and O. 10g/mL: Transfer 1.0 mL and O. 10 mL stock solution (3.2. 14) into 100 mL volumetric flask and dilute to volume with acetonitrile, respec tively. The solutions are stored at - 180C. 3.2.16 Matrix-matched standards for zeranol: to the extract of bl
26、ank sample are added proper zer anol Intermediate solutions (3.2. 15). The matrix-matched standards are stored at - 180C for one week. 3.2.17 C8 solid-phase extraction cartridge, 500 mg. 3.2.18 Nitrogen. 3.3 Apparatus and equipments 3.3. 1 High-performance liquid chromatography- electrospray tandem
27、mass spectrometry. 3.3.2 Balance: sensitivity O. 1 mg and 0.01 g. 3.3.3 Vacuum manifold for solid-phase extraction. 3.3.4 Nitrogen evaporator. 3. 3. 5 Homogeniser. 3. 3. 6 Centrifuge. 3. 3. 7 Vortex mixer. 3.3.8 Pipette:10L.500L. 3.3.9 Polypropylene centrifuge tubes with cap: 50 mL. 3.3.10 Conical c
28、entrifuge tubes:5 mL , 10 mL and 15 mL. 3. 4 Procedure 3. 4. 1 Extraction and Cleanup Weigh 5.0 g:!: 0.1 9 of homogenized tissue sample into a 50 mL centrifuge tube. Add 10 mL of sodi-9 SN/T 1544-2005 um acetate buffer (3. 2. 13) and 25L of -glucuronidase, mix well , and then incubate at 37C for 8 h
29、., 14 h. After hydrolysis, add 10 mL of diethyl ether, cap and vortex for 3 min, and then centrifuge at 3 500 r / min for 10 min. Pipette the upper ether phase into a 15 mL conical centrifuge tube. Evaporate the ether phases nearly to dryness under nitrogen stream at 40oC. Repeat the above extractio
30、n one time. Dissolve the residue in 1 mL of chloroform. Extract zeranol from chloroform using 2 x 2. 5 mL of sodium hydroxide solution (3.2.12). Vortex and centrifuge at 3500 r/min for 5 min. Transfer the upper sodium hydroxide phase into a 10 mL centrifuge tube. Acidify the combined alkaline soluti
31、on with 0.5 mL of phosphoric acid solution (3. 2. 11 ), and load onto a C8 solid-phase extraction car tridge (the cartridge was conditioned with 3 mL of methanol and 3 mL of sodium acetate buffer). After the alkaline solution percolates through , rinse the cartridge with 2 mL of sodium acetate buffe
32、r (3.2.13) and 2 mL of methanol-water solution (3.2.10). Appl2. 0 mL of methanol to the cartridge and collect the eluate into a 5 mL centrifuge tube (draw for 2 min to make the cartridge gradually dry). Evaporate the eluate to dryness under nitrogen stream at 550C. Resuspend the residue with 200L of
33、 acetonitrile for analysis. 3.4.2 Determination 3.4. 2. 1 Liquid Chromatographic Conditions a) Column: Zorbax Extend-C8 column (150 mm x 2. 1 mm , 5m i. d. ), or equivalent. b) Mobile phase: acetonitrile-water (40 + 60) containing 0.025% acetic acid. c) Flow rate: 0.20 mL/min. d) Column temperature:
34、 220C. e) Injection volume: 10L 3. 4. 2. 2 Mass Spectrometric Conditions a) lonization mode: negative electrospray. b) Cone voltage: 50 V. c) Capillary voltage: 3 kV. d) Collision voltage:20 V. e) Analyzer vacuum:3 mPaC3 X 10-5 mbar). f) Collision gas: argon. g) Cone gas flow: 80 L/h. h) Nebuliser g
35、as flow: 480 L/h. i) Multiple reaction monitoring (MRM) mode: m/z 321 303 , m/z 321 277. m/z 321 277 is used for quantitation. 3.4. 2. 3 LC-MS/MS detection Analyze matrix-matched standards (3.2.16) in order of increasing concentration , obtaining the ma trix-matched calibration curve of peak area ag
36、ainst concentration. The detector response of sample should be in the range of calibration curve. Under such LC-MS/MS conditions, the retention time of zeranol is ca. 8.7 min. The mass chromatograms list in appendix A Cappendix A. 1 and A. 2). 10 SN/T 1544一20053. 4. 3 81ank test The operation of the
37、 blank test is the same as that described in the method of determination but without addition of the sample. 3. 4. 4 Calculations The concentration of zeranol in sample is calculated with chromatographic data processor or with the following equation (1): Where: X=cx J(_ m X一一theconcentration of anal
38、te of interest in sample.g/kg; c-the concentration of analyte from calibration curve. ng/mL; V-final volume of sample extract. mL; m一一-weightof sample. g. Note: The blank value should be subtracted from the above result of calculation. 4 Limit of Quantitation and Recover 4. 1 Limit of quantitation T
39、he limit of quantitation of this method is 1g/kg. 4.2 Recover . ( 1 ) According to the experimental data. the fortifying concentration of zeranol and its corresponding re covenes are: 1g/kg. recovery 72%. 5g/kg. recovery 74%. 50g/ kg. recovery 80 % . SON-叮叮巴同军SN/T 1544-2005 Annex A ( informative) ma
40、ss chromatogram TIC 386 321 277 100 100 % 303 创川_.L , 1 P 1，气r m/z 1001251501752旧0225250275300325360 259 % 可mm14.00 12.00 10.00 8. 00 6.00 4.00 2.00 0 0.00 Chromatogram and mass spectrum of zeranol standard(conc. O. 02g/mU mJz 321-277 108 Figure A. 1 100 % m/z 321-303 46 。100 % 申甲_，.甲守.，mm 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0 0.00 Mass chromatograms of zeranol standard using MRM mode(conc. O. 02g/mU Figure A. 2 侵权必究书号:155066.2-16180 定价:7巳10.00 书峰版权专有SN/T 1544-2005
