SN/T 1826-2006.Determination of 19-Nortestosterone residues in foodstuff of animal origin for import and export-GC-MS.
1范围
SN/T 1826规定了进出口动物源食品中肝、肾和肌肉中19-去甲睾酮残留量的测定方法。
SN/T 1826适用于动物源食品中肝肾和肌肉中19去甲睾酮残留量的测定。
2测定方法
2.1方法提要
动物组织中目标化合物在级冲液中经酶水解后用甲醇提取，正已烷去脂,提取液经C18。小柱净化衍生化反应后,用气相色谱-质谱联用仪测定,外标法定量。
2.2试剂和材料
除另有规定外,试剂均为分析纯水为蒸馏水。
2.2.1β-葡糖醛酸糖昔酶:100U/mL。
2.2.2冰乙酸。
2.2.3乙酸钠。
2.2.4甲醇。
2.2.5正已烷:优级纯。
2.2.6 乙醚。
2.2.7氢氧化钠。
2.2.8异辛烷:优级纯。
2.2.9七氟丁酸酐。
2.2.101 mol/L氢氧化钠溶液;溶解40g氢氧化钠于1 L水中。
2.3仪器和设备
2.3.1气相色谱 质谱联用仪(GC-MS)。
2.3.2高速匀 质器。
2.3.3离心机:4000 r/min。
2.3.4 涡旋混合器
2.3.5旋转 蕪发仪。
2.4.2酶解.
从试样中准确称取10 g(精确到0.01 g)于50 mL塑料离心管中,加10 mL乙酸盐缓冲液
(2.2.11),均质,再加50 μL β-葡糖醛酸糖苷酶,充分混匀,混合物在37℃保温18h。

中华人民共和国出入境检验检疫行业标准SN/T1826-2006
20471427
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 19-Nortestosterone residues in foodstuff ofanimal origin for import and export-GC-MS
2006-11-10发布
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中华人民共和国
国家质量监督检验检疫总局
2007-05-16实施
本标准附录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。本标准由中华人民共和国上海出入境检验检疫局负责起草。本标准主要起草人：韩丽、李波、郭德华、杨景贤、杨惠琴、王敏本标准系首次发布的出人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/T1826-—2006
本标准规定了进出口动物源食品中肝，肾和肌肉中19-去甲辜酮残留量的测定方法。本标准适用于动物源食品中肝肾和肌肉中19-去甲睾酮残留量的测定2测定方法
2.1方法提要
动物组织中目标化合物在缓冲液中经酶水解后用甲醇提取，正已烷去脂，提取液经Ci小柱净化衍生化反应后，用气相色谱-质谱联用仪测定，外标法定量。2.2试剂和材料
除另有规定外，试剂均为分析纯。水为蒸馏水。2.2.1β葡糖醛酸糖苷酶：100U/mL冰乙酸。
乙酸钠。
甲醇。
正已烷：优级纯。
乙醚。
氢氧化钠。
异辛烷：优级纯。
七氟丁酸酐。
1mol/L氢氧化钠溶液：溶解40g氢氧化钠于1L水中乙酸盐缓冲溶液（0.2mol/L.pH—5.2士0.2）：16.4g乙酸钠溶于900mL水中，用冰乙酸调2.2.11
节pH值至5.2士0.2再用水定容到1L。19-去甲睾酮标准品，纯度99%。2.2.12
19-去甲睾酮标准溶液：准确称取适量的19-去甲睾酮标准品，用甲醇配制成浓度为100μg/mL2.2.13
标准储备溶液。根据需要再用甲醇将标准溶液稀释成适用浓度的标准工作液。2.3仪器和设备
气相色谱-质谱联用仪（GC-MS）。2.3.1
2.3.2高速匀质器。
离心机：4000r/min。
涡旋混合器。
旋转蒸发仪。
2.3.6Cs固相萃取小柱，500mg依次用5mL甲醇和5mL水活化，备用。2.3.7
氮吹仪
2.3.82mL带螺旋帽盖的衍生化小瓶（配聚四氟乙烯内衬密封垫）。2.4测定步骤
样品的制备
从所取全部动物组织样品中取出有代表性样品约1kg，充分绞碎，混勾，均分成两份，分别装人洁SN/T1826—2006
净容器内。密封作为试样，标明标记。在制样的操作过程中，应防止样品受到污染或发生残留物含量的变化。
2.4.2酶解
从试样中准确称取10g（精确到0.01g）于50mL塑料离心管中，加10mL乙酸盐缓冲液（2.2.11)，均质，再加50μLβ-葡糖醛酸糖苷酶，充分混勾，混合物在37℃保温18h。2.4.3提取、净化
样品酶解后加30mL甲醇，混合2min，在60℃水浴中放置15min，放人一18℃的冰箱中2h~3h。以3500r/min速度离心5min，将上清液轻轻地倒入另-50mL塑料离心管中，用10mL正已烷萃取上清液两次，正已烷层弃去。剩余溶液倒人100mL锥型烧瓶中，在40℃水浴中旋转蒸发除去甲醇。剩余溶液全部上样到C1固相萃取小柱，抽干，用13mL正已烷+十乙醚溶液（30十70)将目标化合物洗脱并收集。洗脱液经氮气浓缩到7mL左右，加1mol/L的氢氧化钠1mL，在涡旋混合器上振荡2min，再以2000r/min速度离心，将有机层转移到另一个试管中，在40℃水浴下用缓慢的氮气流吹至近干。2.4.4衍生化
将上述试管中残留物用洗脱液完全转移到2mL的衍生瓶中（2.3.8)，继续用氮气吹干，加100μL异辛烷和50μL七氟丁酸酐，振荡，于80℃的恒温箱中反应30min，取出后冷却至室温，再用氮气流于40℃水浴中吹干，加200μL的异辛烷振荡溶解，供气相色谱-质谱分析。2.4.519-去甲睾酮标准工作溶液的制备准确吸取一定量适用浓度的19-去甲睾酮标准溶液（2.2.13）于带螺旋帽盖的衔生化小瓶中（2.3.8），用氮气流于40℃水浴中吹干，按2.4.4操作进行衍生化。2.4.6测定
2.4.6.1色谱条件
色谱柱：HP-5MS，30m×0.25mm内径）×0.25μm或相当者；a）
柱温程序：80℃（保持1min），以10℃/min升至170℃，再以2℃/min升温至220℃（保持b)
1min），再以20℃/min升温至280℃（保持5min）：c)
进样口温度：260℃；
载气：氨气，纯度99.999%，流量：1.0mL/min进样模式：不分流，0.75min打开分流阀；进样体积：1μL。
质谱条件
接口温度：280℃；
离子源：电子轰击源（EI)；
电子能量：70eV：
离子源温度：230℃；
检测方式：SIM
选择离子（m/z）及相对丰度（%）：见表1。表1选择离子及相对丰度
被测组分
荷质比（m/z）
相对丰度/（%）
定量离子
2.4.6.3气相色谱-质谱测定
19-去甲睾酮HPFA衍生物
其他监测离子
对样液（2.4.4)及标准工作溶液（2.4.5)等体积参插进样测定。实际应用的标准工作溶液及待测样2
SN/T1826--2006
液中19-去甲辜酮衍生物的响应值均应在仪器线性范围内。在上述色谱条件下19-去甲辜酮衍生物的保留时间为34.07min，其气相色谱-质谱图参见附录A。样品中被测物保留时间与标准品一致，四个检测离子均出现，其之间丰度比与标推品丰度比的偏差小于10%，以此确证样品中存在19-去甲睾酮。2.4.6.4空白实验
除不加试样外，按上述测定步骤进行。2.5结果计算和表述
按式（1）计算样品中19-去甲睾酮残留含量：X
式中：
试样中19-去甲睾酮的残留含量，单位为微克每干克（μg/kg）；样液中19-去甲睾酮衍生物的峰面积；标准工作溶液中19-去甲睾酮的浓度，单位为微克每毫升（ug/mL）：样液最终定容体积，单位为拿升（mL）：A
标准工作溶液中19-去甲睾酮衍生物的峰面积m
最终样液所代表的试样量，单位为克（g）。测定低限、回收率
测定低限
本方法19-去甲睾酮的测定低限为：1.0ug/kg。3.2回收率
3.2.1鸡肉中19-去甲睾酮添加浓度及其回收率：在1.0μg/kg时，回收率为77.0%89.0%；一在3.0ug/kg时，回收率为83.7%～102%；在5.0ug/kg时，回收率为87.6%~97.6%。3.2.2鸡肝中19-去甲睾酮添加浓度及其回收率：在1.0μg/kg时，回收率为74.5%~88.4%；在3.0ug/kg时，回收率为79.7%~92.7%在5.0ug/kg时，回收率为75.2%~93.8%。3.2.3猪肾中19-去甲睾酮添加浓度及其回收率：在1.0ug/kg时，回收率为74.7%~91.9%；在3.0μg/kg时，回收率为82.0%～101%；在5.0ug/kg时，回收率为85.8%~102%。.........()
SN/T1826-—-2006
Abundance
Abundance
附录A
（资料性附录）
标准品选择离子色谱图
19-去甲睾酮标准品衍生物的选择离子色谱图（TIC）133
477497531555585622
图A.219-去甲晕酮标准品衍生物的全扫描质谱图600
Abundance
图A.319-去甲辜酮标准品衍生物选择离子质谱图600
SN/T1826—2006
SN/T1826—2006
Foreword
Annex A of this standard is an informative annex.This standard was proposed by and is under the charge of the Certification and Accreditation Admin-istration of thePeople's Republic 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 standard is a professional standard for entry-exit inspection and quarantine promulgated for thefirst time.
Note:This English versiona translation fromthe Chinese text,is solelyforguidance6
SN/T1826—2006
Determination of 19-Nortestosterone residues in foodstuffof animal originforimportand export-GC-MS
This standard specifies the determination of 19-Nortestosterone residues by Gc-Ms in foodstuff ofanimal origin,suchas heart,liver andmuscleThis standard is applicable to the determination of 19-Nortestosterone residues in foodstuff of animalorigin,suchasheart,liverandmuscle2Methodof determination
2.1Principle
The analyte of interest in animal tissues was hydrolyzed by enzyme in the buffer solution, 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.2.2.1β-glucuronidase：10ou/mL.2.2.2
Glacial acetic acid.
Sodium acetate.
Methanol.
2.2.5 Hexane, superpure.
Diethyl ether
Sodiumhydroxide.
SN/T1826—2006
2.2.8 Iso-octane, superpure.2.2.9HeptafluorobutyricAnhydride.2.2.10 1 mol/L sodium hydroxide solution:Dissolve 40 g sodium dissolve in 1 L water.2.2.11 Buffer solution of salt acetate (0.2mol/L.pH-5.2±0.2):16.4g sodium acetate dissolvein 900 mL water, adjust pH to 5.2±0.2 with acetic acid, then add water to volume of 1 L.2.2.12 Standard of 19-Nortestosterone, purity≥99%2.2.13 Stock standard solution: Weight accurately adequate amount of 19-Nortestosterone stand-ard, dissolve with methanol and prepare a solution of 10o μg/mL as standard stock solution of 19-Nortestosterone. According to the requirement, dilute the standard stock solution with methanol asthe standard working solution2.3Apparatusand equipment
Gas chromatography-Mass spectrometer equipment (GC-MS)Tissues homogenizer.
Centrifuge,4o0or/min.
Vortex mixer.
Rotaryevaporator
Cr-SPE cartridges:5oo mg,conditioned with5mLmethanol and 5mL water for using.2.3.7 Nitrogen evaporator
2.3.82mL littlebottle for deriviation with tefolon-lined screw cap.2.4 Procedure
2.4.1Preparationof test sampleThe 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 test sample, which is then sealed. In thecourse of sample preparation,precaution should be taken to avoid contamination or any factor thatmay causes the change of residue content.8
2.4.2Enzymolysis
SN/T1826—2006
Weigh10 g (accurate to0.01g)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 β-glucuronidase and mixed well again,the mixture keep 37C for 18 hours.2.4.3Extractionandcleanup
The sample was added 30 mL methanol after enzymolysis. mixed for 2 min, kept in the 60C waterbath for 15 min, then kept in the - 18C 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 times with 1o mL hexane again and the phase of hexane was discarded. The residual solutionwas poured into 100 mL Erlenmeyer flask,and rotary and evaporate the methanol in 4o℃ waterbath. The residual solution all was loaded to the Ci-SPE cartridge. After purge the cartridges by air,then elute with 13 mL hexane+ether solution (30+70),The eluate was concentrated to about 7mLby nitrogen and added to 1 mL 1 mol/L sodium hydroxide solution,centrifuge at 2ooo r/min aftermixed well. Transfer the organic phase to the another clean tube and operate to nearly dryness withgentlenitrogen in 4oc waterbath.2.4.4Derivatisation
Transfer the residence in the above tube with eluant into 2 mL little bottle for derivasation (2.3.8)completely,continue to blowing to dryness with nitrogen and add1oo μL iso-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 40C and add200 μL iso-octane dissolve the residence.The solution is used for Gc-Msdetermination.
2.4.5Preparation of 19-nortestosterone standard working solutionAccurately pipette suitable volume 19-nortestosterone standard solution (2.2.13) of suitable con-centration into 2 mL little bottle for derivasation with screw cap (2.3.8),blow to dryness under ni-trogenflow inawaterbathat4o℃,proceed as section2.4.4.2.4.6Determination
2.4.6.1GCoperatingcondition
Column:HP-5MS,30 m×0.25 mm(i.d.)x0.25 μm(film thickness)or equivalent;a）
Column temperature:keep 80℃ for1min,ramp at 10℃/min to 170℃,ramp at 2℃/min tob)
220℃,holdfor1min,rampat20℃/minto280℃,holdfor5min;9
SN/T1826—2006
Injectionporttemperature:26o℃Carrier gas:high purity Helium, flow rate:1.o mL/min:Injectmode:splitless,purgeaftero.75min;e)
f)Injection volume:1μL;
Ms operating conditions
Interface temperature:280℃
b)lon Source:Electron ImpactlonSource(El);c
Electron Energy:70 eV;
Source temperature:230℃;
Detectionmode:SIM
Selected ions (m/z) and relative intensity (%) : see Table 1.Table 1 Selected ions and relative intensityDeterminate compound
Ratiosofm/z
Relativeabundance/(%)
Quantitation ion
2.4.6.3GC-MS determination
19-Nortestosterone HPFA derivantThe others monitor ions
The mix standard working solution should be randomly injected in-between the injections of the sam-ple solution of equal volume. The responses 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 following cri-teria: (1) the peak has the same retention time as the standards; (2) four selected ions are presentas seen in the standard, and the deviation of the abundance ratio between sample and standard within10%.
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