1、中华人民共和国出入境检验检疫行业标准SN/T 1625-2005 进出口动物源性食品中甲瓷孕酣和醋酸甲搓孕自同残留量的检测方法Determination of medroxyprogesterone and medroxyprogesterone acetate residues in animal original food for import and export 2005-08-18发布中华人民共和国国家质量监督检验检疫总局2006-02-01实施060509000092 SN/T 1625-2005 前吉同本标准的附录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
2、准由中华人民共和国河北出入境检验检疫局起草。本标准主要起草人z郭春海、王凤池、马振栋、陈瑞春。本标准系首次发布的出入境检验检疫行业标准。I SN/T 1625一20051 范围进出口动物源性食品中甲瓷孕丽和醋酸甲握孕酣残留量的检测方法本标准规定了动物源性食品中甲是孕酣和醋酸甲捏孕酣残留量的气相色谱-质谱测定方法。本标准适用于兔肉、鱼肉及猪肾中甲短孕酣和醋酸甲起孕酣残留量的检验。2 测定方法2. 1 方法提要样品中的甲起孕酣和醋酸甲起孕酣经乙酸乙醋提取，提取液过Cts固相萃取柱，用不同比例的甲醇水淋洗和洗脱，洗脱液吹去甲醇后用乙酸乙醋萃取，乙酸乙醋层浓缩至干，用七氟丁酸哥衍生，吹干定容后，气相色
3、谱-质谱仪选择离子检测，外标法定量。2.2 试剂和材料除特殊注明外，所用试剂均为分析纯，水为蒸锢水。2.2. 1 甲起孕酣标准品，二三98%。2.2.2 醋酸甲起孕酣标准品，注97%。2.2.3 标准溶液的配制:2.2.3.1 甲起孕酣标准储备溶液z准确称取适量的甲起孕酣标准品(精确至0.0001 g)，用丙酣配制成浓度为0.1mg/mL标准储备溶液。2.2.3.2 醋酸甲起孕酣标准储备溶液z准确称取适量的醋酸甲捏孕酣标准品(精确至0.0001 g)，用丙酣配制成浓度为0.1mg/mL标准储备溶液。2.2.3.3 甲是孕酣和醋酸甲是孕酣工作溶液准确移取适当体积的甲起孕酣标准储备溶液(2.2.3
4、.1)和醋酸甲起孕酣标准储备溶液(2.2.3.2)，加入乙酸乙醋稀释到一定体积，棍匀，备用。2.2.4 乙酸乙醋(HPLC级)。2.2.5 甲醇。2.2.6 丙嗣(重蒸馆)。2.2.7 七氟丁酸西注98%。2.2.8 无水硫酸铺，650.C灼烧4h，干燥器中放置备用。2.2.9 CI8固相萃取柱，500mg ,3 mL:依次用5mL甲醇、5mL水、3mL甲醇-水(90+10) ,3 mL甲醇水(10+90)预处理，备用。2.2.10 甲醇-水溶液(10+90)，淋洗用。2.2. 11 甲醇-水溶液(90+10)，洗脱用。2.3 仪器和设备2.3. 1 气相色谱-质谱联用仪。2.3.2 高速均质
5、器。2.3.3 超声波清洗器。2.3.4 氮吹仪。2.3.5 固相萃取装置。2.3.6 离心机。1 SN/T 1625-2005 2.3.7 具塞玻璃离心管。2.3.8 撞在涡混合器。2.4 测定步骤2.4. 1 提取称取5g(精确至0.01g)样品于50mL具塞离心管中，加人5g无水硫酸铀，拌匀，加入10mL乙酸乙醋，均质1min，超声10min,2 000 r/min离心5min，取上清液于20mL离心管中。每次加入5mL 乙酸乙醋，按上述步骤提取两次，合并提取液于20mL离心管中，50.C吹干。2.4.2 净化加入1mL的甲醇-水(10+90)，50C加热溶解上述残渣，振荡，-18.C以
6、下冷却5min, 2 000 r/min 离心5min。小心迅速地将非脂肪层转入C18圄相萃取柱(2.2.9)上，用1mL甲醇F水(10+90)洗残渣两次，一180C以下冷却5min ,2 000 r/min离心5min，非脂肪层小心迅速地转移入柱中。待液面接近柱子上部筛板时，加入1mL甲醇-水(10十90)淋洗柱子，流出液全部弃去。加入1mL甲醇-水(90+10)于上述20mL离心管中，加热溶解，振荡，一18.C以下冷却5min，取出后2000 r/min离心5min，转入上述C18固相萃取柱(2.2.9)中，再加入7mL甲醇-水(90+10)，洗脱，收集全部洗脱液。将洗脱液在氮吹仪上50.
7、C吹至0.8mL，补充水至2mL，每次用3mL乙酸乙醋提取，提取3次井离心，合并提取液，在氮吹仪上用氮气流于50C吹干。2.4.3 衍生化残渣加入50L七氟丁酸霄，200L丙酣，60.C衍生40min，在氮吹仪上用氮气流于50.C吹干，正己烧定容至1mL，GC-MS分析。2.4.4 甲孕圈、醋酸甲孕酣衍生物标准溶液准确吸取适用浓度的甲孕酣和醋酸甲捏孕酣混合液(2.2.3.3)1 mL，用氮气流于50C吹干，按照2.4.3进行衍生。2.4.5 测定2.4.5.1 色谱条件a) 色谱柱z弹性石英毛细管柱DB-5MS30 mXO. 25 mm(内径)0.25m膜厚或相当者;30(: min b) 柱
8、温:60.C(1 min)-=-一一一.140.C-一-280.C;c) 进样口:280.C;载气z高纯氮气，纯度二三99.999%; d) 柱流速:1mL/min; e) 进样量:1L;f) 无分流进样。2.4.5.2 质谱条件a) 接口温度:280.C;b) 离子源:230.C;c) 电离电压:70eV; d) 离子源:电子轰击源(EI); e) 检测方式:SIM;f) 选择离子及相对丰度:见表1。表1选择离子及相对丰度被测组分甲经孕翻衍生物醋酸甲短孕嗣衍生物选择离子(m/z)383 461 479 480 423 439 497 582 相对丰度/%)50 18 100 28 28 47
9、 17 100 a 定量离子二一一2 SN/T 1625-2005 2.4.5.3 气相色谱-质谱测定对样品榕液(2.4.3)及标准工作溶液(2.4.4)等体积参差进样测定。实际应用的标准工作溶液及待测样品溶液中，甲经孕酣、醋酸甲起孕酣衍生物的响应值均应在仪器线性范围内。在上述条件下甲起孕酣、醋酸甲起孕酣衍生物的保留时间分别约为17.9min和21.0 min。标准品衍生物的气相色谱-质谱图参见附录A。2.4.5.4 确证试验进行样品测定时，如果检出的质量色谱峰保留时间与标准样品一致，并且在扣除背景后的样品谱图中，各定性离子的相对丰度比与浓度接近的标准溶液谱图一致，则可判断样品中存在对应的被测
10、物。2.4.5.5 空白试验除不加样品外，按上述相同条件和步骤进行。2.5 结果计算和表述用色谱数据处理机或按式(1)计算试样中甲捏孕酣或醋酸甲起孕酣残留量:式中zX=AXcXV 一一一As Xm X一一试样中甲起孕嗣或醋酸甲起孕酣残留量，单位为微克每千克(g/kg);C一一标准工作溶液中甲起孕酣或醋酸甲是孕酣的浓度，单位为纳克每毫升(ng/mL); V 样品溶液最终定容体积，单位为毫升(mL);A 样品溶液中甲起孕酣或醋酸甲是孕酬的峰面积;单位为平方毫米(mm2); As -标准工作榕液中甲起孕酣或醋酸甲是孕酣的峰面积;单位为平方毫米(mm2); m一一最终样液所代表的试样量，单位为克(g)
11、o注:计算结果需扣除空白值3 测定低限和回收率3. 1 测定低限本方法测定低限为1g/kg。3.2 回收率3.2.1 兔肉中甲捏孕酣添加浓度及回收率的数据:在1g/kg、2g/kg、5g/kg时，回收率为88%105%。3.2.2 兔肉中醋酸甲挂孕酣添加浓度及回收率的数据:在1g/kg、2g/kg、5g/同时，回收率为79%102%。3.2.3 鱼肉中甲握孕嗣添加浓度及回收率的数据:在1g/kg、2g/kg、5g/kg时，回收率为88.7%93. 6%。3.2.4 鱼肉申醋酸甲挂孕酣添加浓度及回收率的数据:在1g/kg、2g/kg、5g/kg时，回收率为78%113%。3.2.5 猪肾中甲捏孕
12、嗣添加浓度及回收率的数据1在1g/kg、2g/kg、5g/kg时，回收率为63%78%。3.2.6 猪肾中醋酸甲提孕酣添加浓度及回收率的数据:在1g/kg、2g/kg、5g/同时，回收率为90%113%。. ( 1 ) 3 SN/T 1625-2005 Abundance 7 500 6 000 4 000 2 500 1 000 1一甲瓷孕酣$17.50 2一-醋酸甲经孕阁。附录A(资料性附录)标准物气相色谱质谱固2 18.50 19.50 20.50 21. 50 圈A.1甲握孕圄及醋酸甲握孕酣标准衍生物气相色谱质谱固(TIC)Abundance 6 000 000 479 5 500 0
13、00 5 000 000 4 500 000 383 4 000 000 3 500 000 3 000 000 2 500 000 109 2 000 000 133 1 500 000 1 000 000 169 500 000 423 450 。500 522 545 50 100 150 200 250 300 350 400 450 500 550 固A.2甲握孕酣标准衍生物质谱固4 Abundance 280 000 260 000 240 000 220 000 200 000 180 000 160 000 140 000 120 000 100 000 80 000 60 0
14、00 40 000 20 000 。50 Abundan四3 200 2 800 2 400 2 000 383 1 600 1 200 600 380 SN/T 1625一2005582 479 383 109 439 540 100 150 200 250 300 350 400 450 500 550 圈A.3醋酸甲经孕圄标准衍生物质谱固479 420 450 460 480 圈A.4甲握孕酣标准衍生物选择离子质谱圄5 SN/T 1625-2005 Abundance 582 1 000 800 600 400 439 423 200 497 50 420 440 460 480 500
15、 520 540 560 580 固A.5醋酸甲提孕圄标准衍生物选择离子质谱固6 SN/T 1625-2005 Forword Annex is an informative annex. This standard was proposed by and is under the charge of the National Regulation Commission for Certification and Accreditation. This standard was drafted by Hebei Entr-Exit Inspection and Quarantine Burea
16、u of the Peoples Re public of China. The main drafters of this standard are Guo chunhai , Wang fengchi , Ma zhendong , Chen ruichun and Liu baosheng. This sdandard is a professinal standard for Entry-Exit Inspection and Quarantine published for the first time. Note , This English version , a transla
17、tion from the chanese text , is solely for guidance. 7 SN/T 1625-2005 Determination of medroxyprogesterone and medroxyprogesteroneacetate residues in animal original foodstuff for import and export 1 Scope This standard specifies the determination of medroxyprogesterone and medroxprogesterone acetat
18、e residues in animal original foodstuff by gas chromatography-mass spectrum. This standard is applicable for the determination of medroxyprogesterone and medroxyprogesterone acetate residues in rabbit and its corresponding recoveries , fish and its corresponding recoveries, pig kidney and its corres
19、ponding. 2 Method of determination 2. 1 Principle Follow residues are extracted from the test sample with ethyl acetate. The extraction is passed through C18 SPE column , clean-up and elute the column with method-water of different portion. The elution is blowed out methonal. The water layer is extr
20、acted with ethyl acetate,ethyl acetate layer is blowed to dryness. Adding heptaflurobutyl anhygrous and acetone. Deriviation. Determination is made by GC-MS-SIM and quanyified by using the external standard. 2. 2 Reagents and materials Unless otherwise specified , all reagents used should be analtic
21、ally pure; water is distilled water. 2.2. 1 Medroxyprogesterone standard: purity二98%.2. 2. 2 Medroxyprodresterone acetate: purit注97%.2. 2. 3 Make up of standard solution. 2. 2. 3. 1 Medroxprogesterone standard stock solution: accurately weigh (accurate to 0.000 1 g) adequate amounts of Medroxprogest
22、erone standard in volume flask , dissolved in acetone to make up standard stock solution of O. 1 mg/mL. 2. 2. 3. 2 Medroxyprogesterone acetate standard stock solution: accurately weigh (accurate to O. 000 1 g) adequate amounts of Medroxprogesterone acetate standard in volume flask, dissolved in acet
23、one to make up standard stock solution of O. 1 mg/mL. 8 SN/T 1625-2005 2.2.3.3 Mixture standard working solution of medroxprogesterone and Medroxyprogesterone ac etate: adding adequate each of 2. 2. 3. 1 and 2. 2. 3. 2, diluting with ethI acetate, make up mixture solution. 2. 2. 4 ethyl acetate, HPL
24、C grade. 2. 2. 5 Methanol. 2.2.6 Acetone: redistill. 2.2.7 Heptaflurobutyl anhydride , purity二三98%.2.2.8 Anhydrous sodium sulfate:ignite at 6500C for 4 h,and store in air-tight container. 2.2.9 C18 SPE column:500 mg ,3 mL: by pre-wash with 5 mL of methanol ,5 mL of water,3 mL of methanol-water(90+ 1
25、0)and 3 mL of methanol-water (10+90). 2.2. 10 Methoonal-water( 10 + 90) ,function: rinse. 2.2.11 Methoonal-water(90+ 10) ,function: elute. 2.3 Apparatus and equipments 2.3.1 Gas chromatograph equipped with mass spectrograph(GC-MS). 2.3.2 High-speed homogenizer. 2.3.3 Ultrasonic clean apparatus. 2. 3
26、. 4 Nitrogen evaperator. 2.3.5 Solid phase extract instrument. 2.3.6 Centrifuge,upwards of 3500 r/min. 2. 3. 7 Centrifuge tube with plug. 2. 4 Procedure 2. 4. 1 Extraction Weigh ca 5 g(accurate to O. 01 g)of the test sample into 50 mL centrifuge tube with pl吨，adding 5 9 of anhdrous sodium sulphate ,
27、 mix well , adding 20 mL of ethyl acetate, homogenize 1 min and 9 SN/T 1625-2005 ultrosonic 10 min, centrifuge 5 min at 2)() r/min. Taking the upper liquid into 10 mL centrifuge tube. The lower residues was washed badding 5 mL of ethyl acetate per time, extract twice according to above process . Com
28、bine the all extract solution into the 20 mL centrifuge tube. blow it to dryness with nitrogen at 50 C. 2. 4. 2 Clean up Dissolve the residue with 1 mL of methanol-water (10+90)at 50 C ,shake vigorously,freeze 5 min at -18C, centrifuge 5 min at 2000 r/min ,non-fatty laer was carefully transfer into
29、the pretreated C8 SPE column(2. 2. 9). Washing residue twice in the tube with 1 mL of methanol-water(10+90) , cen trifuge 5 min at 2 000 r/min,transfer into the column according to above process. When the solution arrive to top of the column frits ,adding 1 mL of methanol-water(10+90) wash column ,d
30、iscard all the effluent. Adding 1 mL of methanol-water(90+ 10)to primary 20 mL tube,dissolve the residue in the tube at 50 C , shake vigorously, freeze 5 min at below一18C, centrifuge 5 min at 2 000 r /min, non-fatty layer was carefulltransfer into the C8 SPE column(2. 2.酌，thenintroduce 7 mL of metha
31、 nol-water(90+ 10) to the above column ,and collect the elution in a 20 mL centrifuge tube. evaporate the elution to O. 8 mL,adding water to 2 mL in the tube. Adding 3 mL of ethyl acetate in the tube per time, shake 1 min in shaker , centrifuge 5 min at 2 000 r/min , and combined the extract to anot
32、her tube. Evaporate to dryness under nitrogen flow at 5OC. 2.4.3 Derivation Add 50L of heptaflurobutI anhydride and 200L of anhydrous acetone into the above tube, and seal tightly, mix thoroughly, reaction 30 min at 60C . Evaperate to dryness under nitrogen flow at 50 C . make up to 1 mL with n-hexa
33、ne, mix well. The solution is used for GC-MS determination. 2. 4. 4 Preparation of medroxyprogesterone and medroxyprogesterone acetate derivation mixsture standard working solution Accurately pipette 1 mL mixture standard working solution of medroxyprogesterone and me droxyprogesterone acetate (2. 2
34、. 3.酌，removethe solvent under nitrogen flow at 50 C , proceed as section 2.4. 3. 2. 4. 5 Determination 2. 4. 5. 1 GC operating conditions a. Column: DB-5 MS 30 m X O. 25 mm X O. 25m(film thickness) or equivalent; 30C min 10C min b. Column temperature program: 600C (1 min)一一一一1400C一一一一280C; c. Inject
35、ion port temperature:280oC ; d. carrier gas: He , purity注99.999%;flow rate: 1.0 mL/min; e. Injection volume: 1L; f. Injection mode: splitless. 2. 4. 5. 2 Mass spectrum operation conditions a. Interface temperature:280oC ; 10 SN/T 1625-2005 b. lon source temperature: 230 t ; c. Electron Energy: 70 eV
36、; d. lon source: Electron Impact lon Source(EI); e. Detection mode: SIM; f. Selection ions (m/z) and-relative intensity (%): see table 1. Table 1 Selected ions and relative intensity Analyte Medroxypr，吨esteronedeviation medroxyprogesterone acetate deviation Selected ions(m/z) 383 461 4798 480 423 43
37、9 497 582 Relative intensity / (%) 回18 100 28 28 47 17 100 a quantitative ion. 2. 4. 5. 3 GC-MS determination The mix derivation standard working solution(2. 4. 4) should be randomly injected in-between the in jections of the sample solution(2. 4. 3) of equal volume. The responses of the medroxyprog
38、esterone and medroxyprogesterone acetate deviation in the standard working solution and sample solution should be within the linear range of the detector. The retention time of medroxyprogesterone and medroxyprogesterone acetate deviation is ca 17.9 min and 21.0 min under the above conditions. For t
39、he TIC chromatogram and mass spectrum of the standard , see annex A. 2.4.5.4 Confirmed examination If the retention times of sample chromatogram peaks are consistent with the standards , and after subtracted background noise, the relative intensity ratios of each qualitative ions are also consistent
40、 with similar concentration standards, we can confirm that there are corresponding analyte in the sample. 2. 4. 5. 5 Blank test The operation of blank test is the same as that described in the method of determination, but with ommission of sample addition. 2. 5 calculation and expression of result c
41、alculate the residues content of medroxyprogesterone and medroxprogesterone acetate by GC data processor or according to formula (1): Where, x=x c x V As x m . ( 1 ) X一一一theresidue content of medroxprogesterone or medroxyprogesterone acetate in test sample, mg/kg; A一一-thepeak area of medroxprogester
42、one or medroxyprogesterone acetate in sample solu tlon，阿1m2; c一一-theconcentration of medroxyprogesterone or medroxyprogesterone acetate in standard solution , ng/mL; 11 SN/T 1625-2005 V一一thefinal volume of the sample solution , mL; A.一一-thepeak area of medroxyprogesterone or medroxprogesterone aceta
43、te in sample solu tion, mm2; m一-thecorresponding mass of test sample in the final sample solution, g. Note, The blank value should be subtracted from the above result of臼Iculation.3 Limit of determination and recovery 3. 1 limit of determination The limit of determination of this method is 1g/kg. 3.
44、 2 Recovery according to the experimental data , the fortifying concentrations of medroxyprogesterone or me droxyprogesterone acetate in different test sample and its corresponding recoveries are as follows: 3. 2. 1 The fortifing concentrations of medroxyprogesterone in rabbit ad its corresponding r
45、ecov enes are: at 1g/kg，2g/kg ，5g/ kg , the recovery is 88 % -105 % . 3.2.2 The fortifying concentrations of medroxyprogesterone acetate in rabbit and its correspond mg recovenes are: at 1g/kg，2g/炮，5g/kg，the recovery is 79% -102%. 3.2.3 The fortifing concentrations of medroxyprogesterone in fish and
46、 its corresponding recover les are: at 1g/kg，2g/kg ，5g/kg. the recovery is 88.7%-93.6%. 3.2.4 The fortifying concentrations of medroxyprogesterone acetate in fish and its corresponding recovenes are, at 1g/kg，2g/kg ，5g/kg， the recovery is 78% -113%. 3. 2. 5 The fortifying concentrations of medroxpro
47、gesterone in pig kidney and its corresponding recovenes are, at 1g/kg.2g/kg ，5g/ kg , the recovery is 63 % - 78 %. 3.2.6 The fortifying concentrations of medroxyprogesterone acetate in pig kidney and its corre sponding recoveries are: at 1g/kg，2g/闸，5g/kg.the recovery is 90%-113%. 12 SN/T 1625-2005 A
48、nnex A ( informative) GC-MS Chromatogram of the standard Abundance 7 500 6 000 4 000 2 500 1 000 17.50 18.50 1-一-medroxyprogesteronedeviation; 2一一-medroxyprogesteroneacetate deviation o 19.50 20.50 2 21. 50 Fig A. 1 GC-MS Chromatogram (TIC) of medroxprogesterone and medro:xyprogesterone acetate devi
49、ation Abundance 6 000 000 479 5 500 000 5 000 000 4 500 000 383 4 000 000 3 500 000 3 000 000 2 500 000 109 2 000 000 133 1 500 000 1 000 000 169 500 000 423 450 。50 100 150 200 250 300 350 400 450 500 522 545 500 550 Fig A.2 Mass spectrogram of the medroxyprogesterone deviation 13 SN/T 1625-2005 Abundan出280 000 479 260 000 240 000 220 000 200 000 180 000 160 000 140 000 383 120 000 109 439 100 000 80
