【商检行业标准(SN)】 进出口动物源食品中19-去甲睾酮残留量的测定方法 气相色谱-质谱法

中华人民共和国出入境检验检疫行业标准SN/T1826—2006
进出口动物源食品中19-去甲辜酮残留量的测定方法
气相色谱-质谱法
Determination of 1g-Nortestosterone residues in foodstuff ofanimal origin for import and export-GC-MS
2006-11-10发布
华人民和国
国家质量监督检验检疫总
2007-05-16实施
本标准录A为资料性附求：
本标准由区家认证认可监督管理委员会提出并力口，本标滩山中华人民共和国1海山人境检验检凌局负责起草木称滩主要起草人：韩删李波、郭德华、杨景贤、榭离琴、教本标准系首次发布的出入境检验检疫行业标准。SN/T1826—2006
1范围
进出口动物源食品中19-去甲睾酮残留量的测定方法气相色谱-质谱法
SN/T 1826—2006
本标准舰了进出口动物源食品中肝、舒和肌沟叶19去甲案残留量的测定方法本标准适压：动物源食品中舒、肾和肌肉它-甲率刷残随呆的测定。2测定方法
2.1方法提要
动物凯织中H标花合物在缓冲液中经嗨水解后月巾醇提取，正已烷去脂，提辰液经（i小柱净化衍牛化反成后.用气种色落质谱联用仪测定·外际法定量2. 2 试剂和材料
除方有舰定外，试剂均为分析纯水为蒸馏水。2.2. 1 3 葡较糖营酶:1C0 L./rmT.,2.2.2冰乙胶，
2.2. 3 乙胶钠
2.2. 4中.
2.2.5止凹烷：优级纯.wwW.bzxz.Net
2.2. 6乙酸
2.2.7氧低化钢
足辛烷：优级纯
:氟」腋：
2.2. 151 =1al/L素氧化纳溶液:溶解 1℃ 氧瓦花钠于1水2.2.11乙酸盐缓冲溶液(0,2 1nol/L·pll=5,20.2):16.乙酸钠溶于 9c0 m水.用冰乙酸调节 p1值至 5.20,2.用水容到1L
2.2.1219 去甲罩酮标准品，纯度整99%2.2.1319去中罩酮标准溶液：准确称政适量的19去口辜示准品，用平醇配制成浓度为100/rmL标准诺各溶液：根据需要再用中辟将标准溶液稀释成造用浓度的标准工作液，2.3仪器和设备
2.3.1气相色谱质谱联用仪(GL:MS)2.3.2良速匀质器，
2.3.3离心机:1000 r/=min
2.3.4涡旋视个器，
2.3.5璇转蒸发仪，
2.3.6C固科举联小排：5们） m依次用5ml，中厚利m.水活化备月2.3.7疯吹仪，
2.3.82ml.带旋帽盖的衔牛化小瓶（配繁四氟乙烯内衬密封挚）2.4测定步骤
2.4.1样品的制备
从所取全部动物组织样品中取出有代表准烊品约1k，充分绞碎，得勾，内分戒两份·分别装人洁SV/T 1826—2096
净容器内，密时作为试样，标明标记。制样的操作过程中，应防止样品受到污染成发牛残留物含量的变化：
2.4.2酶解
从试样准确称取10（精矿到3.01）于50塑料离心管加10m乙酸盐缓冲液(2. 2. 11),为质：加50 β葡糖降酸糖件悔,充分约,混合场在87℃保溢18 h。2.4.3提取，净化
详品醇解后加30mL中醇，湿合2min，在60%水浴中放15min，放入-18℃的冰箱它2h~3h.以3500 r/min速变离心5 min,将清液轻轻地倒人刃一50ml塑料离心管凸,斥10 ml,正已烷求取上清液两次，正已烧层齐去，剩余溶液倒人［I链型烧瓶，在℃水浴中旋转蒸发除去甲醇。刻余溶液全邪上样到（固相率收小柱,抽1,用13 ml.止已烷一乙醛溶液(3十70)将月标化介物洗脱并收集：洗脱液经氮气浓缩到7-nl.存右.加1 Tnol/I.的氢氧化钠1ml.,在泻旋混合幕1振荡之mi-,再以2009/min速要离心，将有机层转稳到别·个试管.在10℃水浴下月缓慢的氮气流吹爷近干2.4.4衍生化
将上述示管巾残留物用洗液完企转够到2ml.的衍生瓶巾(2.3.8),继续月氮气吹下加100 uI异卒烷和50 uL七氟」酸,派荡：于80℃的恒温箱中反应30 il取出片冷即至空温再用氮气流于0%水裕中以=，加230 μL的片烷振荡溶解，供相色谱 质谱分析：2. 4.519-去甲睾酮标准工作溶液的制备谁确吸取-定景适用浓度的[9-甲案酮标谁落液（2. 2..3）」带螺旋帽益的衍牛花小瓶巾(3.8）,用氮气流于46℃:水浴巾吹下,接22.4操作进行衔牛化2.4. 6测定
2.4.6.1色谱条件
谱柱：HP-5.MS.33m×3.25mm内约）)×c.2um或租当者；a
柱温程序：80（保持1 min），以1c/rin至170.再以2℃/nin 升温至220℃（保持h）
1min），再以20/min几至280（保持5min）；进样口翌度;2G：
载气：氛气，纯度99.999%，流量：1.0ml/mit:进栏模武:不分流.0.43 min 打开分流阀；F)延样体积:1L。
2. 4. 6. 2
质谱条件
接口温度：280℃；
离源:电『轰士源(EI);
电了能量:70 cV;
离了源温度：230℃；
检测方式:SIM
选泽离了\1/)皮相对三度(%）:见表表1选择离子及相对丰度
被测组分
茂上（m/s
村对丰度（汽）
定量离，
2.4.6.3气相色谱-质谱测定
19-年睾丽 HPFA 衍4:物
其他临测密？
对栏波(2.4.4)及标准:1.作溶液(2.4.:)等本积参进样测定。实际成用的标准:1.作溶液及待测伴2
SN/T 1826—2006
液中19-去甲案酮衍牛物的响应值均应在仪器线性范用内，在上述色谱条件下19-去甲需酮衍牛物的保留时间为34.07 m:in,只气相作谱-质谱图参见附录A。详品小被测场保留时问与标准品，致，四个检测离了均出现,其之问卡度比与标准品十度比的偏准小于1C以此确延择品荐在二9中辜，2. 4. 6. 4空白实验
除不加流样外,按上述测定步骤让行。2.5结果计算和表述
茂武（)计算群品中19-2中辜间线留含量：A·.V
式中：
试样它3-去中率的残留含量，单位为微克每千克（/k)：样液弓二去中萨生物的峰面积：标准二作溶液[9去巾紫酮的淤度，学位为该亢每升（μ/ml):样液最终定容体积,单位为衰引（ml.)：标涯．作溶液户-去甲牢刷衍牛物的峰面积最终样液所代表的激详量，总位为克（）：3测定低限、回收率
3.1测定低限
本方法1S-去中率酮的测定低限为：1.0/k3.2回收率
3.2.1鸡肉中19去中啊添加浓度没儿可收率在1.0g/kg时收率为77.0为89,0为在 3, 0 μg/kg 时,用收率为 83. 7%-.02;在 5. 0 μ/kg 时,间收率为 87.6%~97. 6%。3.2.2鸡所10去中睾酮添小浓虚及其可收率：在1.0/kg吋.回收率为71.5头~-88.4次；在 3.0 1g/kg时.国收率为 79.7次~-92.7：在 5, 0 /kg时,=收率为 75. 2/~~ga. 8/3.2.3猪中19-去中率酮添滚度及其可收率：在1.0g/kg时，回收率为74.7次~91.9准；在3.0/kg时.日收率为82.0%~二01%；在5.0 g/kg时,收率为 85.8%-..023
SV/T1826—2036
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附录A
（资料性附录）
标准品选择离子色谱图
图A.119-去甲睾酮标准品衍生物的选择离子色谱图(TIC)Ahmdine
277..11.17749.531.gtb.b8523
图A.219-去甲率酮标准品衍生物的全扫描质谱图600
Ahurklance
19-去甲睾酮标准品衍生物选择离子质谱图图A.3
SN/T 1826—2006
SN/T 1826—2096
Foreword
Anriex A of this standard is an informative anriex.This standard was proposed by and is under the charge of the Certification and Accreditation Admin-istration of thc Pcoplc's Rcpublic of China.This standard was drafted by ShangHai Entry-Exit Inspection and Quarantine Burea,The standard was mainly drafted by Hanli, Libo, GuoDehua, YangJingxian, YangHuiqin, WangMin.This slandard is a professional slardard for enlry-exit inspeclion and quaranline pramulgaled for Lhefirst tirne.
Nute: This English versiori a Irarnislaliun frurm the Clhinese lexl, is sulely fur guidance6
SN/T 1826—2006
Determination of 19-Nortestosterone residues in foodstuffof animal origin for import and exportGC-MS
1Scope
This standard spccifics thc detcrmination af 19-Nortcstosteronc rosiducs by Gc-Ms in foadstuff ofanimal origin, such as heart. liver and muscleThis slandard is applicable lo the delermination of 19-Nortestosterone residues in foodsluff of animaorigin, such as heart, liver and muscle.2Method of determinatian
2. 1Principle
The analyte ol interest in animal tissues was hydrolyzed by enzyme in the buffer salutian, then ex-tract with methanol and deprivate fat with hexane, The extracted solution was cleaned by SPE-Cis :after derivatization, determination is made by Gc-Ms and quantified by using the external standard.2. 2 Reagents and materials
Unless otherwise specified, all reagents used shall be of analytically pure, “water\ is distilled water.2. 2. 13-glucuroridase: 100 U/mL2. 2. 2
2Glacial acetic acid.
Sodium acetate.
Methanol.
Hexanc. supcrpurc.
sDiethyl ether.
Sodium hydroxide.
SV/T1826—2006
2.2.8 Iso-octane. superpure.2. 2. 9Heptafluorobutyric Arhydride2. 2. 10 1 mol/L sodiun hydraxide solution: Dissolve 40 g sodium dissalve in 1 L water.2. 2. 11 Buffer solution of salt acetate (0. 2 mol/L.pH= 5. 2 10. 2) : 16. 4 g sodium acetate dissolvein 900 mL waler, adjusl pH to 5. 2± 0. 2 with acelic acid. then add waler [o volurme of 1 L.2. 2. 125
Standard af 19-Nortestosterone, purity99%2. 2. 13 Stock standard solution: Weight accurately adequate amount of 19-Nortestosterone stand-ard. dissolve with methanol and prepare a solution of 1ao μg/mL as standard stock solution al 19-Nortestosterone. According to the requiremenit: dilute the staridard stock solution with methanol asthc standard working solution.2.3 Apparatus and equipment
2. 3.1Gas chromatography-Mass spectrorrieter equipment (Gc-Ms)2Tissues homogenizer.
2.3.3Centrifuge，4000r/min.
Vortex mixer.
5Rotarycvaporator.
Cra-SPE cartridges:5oo mg: conditioned with 5 mL methanol and 5 mL water for using.2.3.7 Nitrogen evaparator
3 2 mL little bottle for deriviation with tefolon-lined screw cap.2.3.8
2.4Procedure
Preparation of test sample
The combined primary animal tissuesis reduced to 1 kg which is blended mixed and divided into twoequal portions, each portion is placed in a clean vessel as a [es1 sample, which is then sealed. In thecourse of sample preparation, precaution should be taken to avoid coritarmination or any factor thatmay causcs the changc of rcsiduc contant.S
2.4. 2Enzymolysis
SN/T1826—2006
Weigh 10 g (accurate to 0. a1 g)of test sample in 50 mL plastic centrifuge tubes, add 10 mL buffersolution of salt acetate (2. 2. 11) and mixed well, Add 50 μL -glucuronidase and mixed well again,themixturekeep37℃for18hours.2.4.3Extractionand cleanup
The sample was added 30 rnL methariol after enzymolysis, mixed for 2 min, kept in the 60'c waterbath for 15 min, then kept in the - 18℃ refrigerator for 2 h~3 h, After centrifuging for 5 min at3 500 r/min, transfer the supernatant to another 50 mL clean centrifugal tubes, extract the superna-tant 2 timos with 10 ml hcxanc again and thc phasc of hcxanc was discardcd. Thc residual solutionwas poured int 1oo mL Erlenmeyer flask, and rotary and evaporate the methanol in 4o'c watebath, The residual solution all was loaded to the Cis-SPE cartridge. After purge the cartridges by air.then elute with 13 mL hexane + ether solution (30 + 70). The eluate was concentrated to about 7 mLby nitrogen and added to 1 mL 1 mol/L sodium hydroxide solution, centrifuge at 20oo r/min aftermixed well. Transler the organic phase to the another clean tube and operate to nearly dryness withgentlenilragen in 4oc walerbath.2.4.4Derivatisation
Transfer thc rcsidencc in thc abovc tubc with cluant into 2 ml littlc bottle for dcrivasation (2. 3. 8)completely, continue to blowing to dryness with nitrogen and add 100 μL isa-octane and 50 μL Hep-tafluorobutyric Anhydride. After mixing well. put it into the constant temperature oven at 8o' for3o min. Cool to room temperature after taking it out, then blow to dryness under nitrogen fluent in awater bath at 40℃ and add 200 μL iso-octane dissolve the residence. The solution is used for GC-MSdetermination.
2. 4. 5Preparation of 19-nortestosterane standard working solutionAccurately pipette suitable valume 19-nortestosterane standard solution (2. 2. 13) of suitable con-centration into 2 mL littlc bottlc far derivasation with scrcw cap (2. 3. 8) , blow to dryness under ni-tragen flow in a water bath at 4a'C , proceed as section 2. 4. 4.2.4.6 Determination
2.4.6.1Goperatingcondition
a)Colurnn:HP-5Ms,30 mx0.25 mmci.d.)x0.25 μnifilmthickness)or equivalerit;Column temperature: keep 80'℃ for 1 min, ramp at 10'C/min to 170'C , ramp at 2℃/min tob.
220℃, hold for 1 min, ramp at 20℃ /min to 280℃, hold for 5 min;SV/T 1826—2096
c)Injection port temperature:26oC:d
Carrier gas: high purity Helium, flow rale: 1. o rmL/min;e)Inject mode: splitless. purge after 0.75 min;f) Injcction volumc: 1 μL;
2. 4. 6. 2Ms operating conditionsa) Interface temperature; 280℃ :b）lonSource：ElectronImpactlonSource(El)Electron Energy: 70 eV;
d) Sourcc tampcraturc; 230'c :e
Detection mode: SIM
f)Selected ions(m/z)andrelativeintensity(%)：seeTable1,Selected ions and relative intensityTable 1
Determinate compound
Ratios or m/?
Relativeabundance/(%)
Quantitation ion
2. 4. 6. 3 GC-MS determination19-Nortestosterone HPFA cerivantTha others monitor ions
The mix standard working solution should be randomly injected in-between the injections of the sam-ple salutian of ecual volume.The respanses of the 19-Nortestosterone derivate in the standardworking solution and sample solution should be within the linear range of the detector, The retentiontime of 19-Nortestosterone derivate if ca, 34, 07 min under the above conditions, For the chromato-gram of the standard, see annex A.The presence of analyte of interest in a sample is confirmed if the data agree with the fallowing cri-teria: (1) the peak has the same retention time as the standards; (2) four selected ions are presentas seen in the slandard, and ihe devialion of the abundance ralio belween sample and standard with-in10%
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