1、版权所有 禁止翻制、电子传阅、发售中华人民共和国出入境检验检疫行业标准SN/T 1927-2007 进出口水产品中睡赛多残留量的检测方法液相色谱-质谱/质谱法Determination of cyadox residues in aquatic products for import and export LC-MS/MS method 2007-05-23发布2007-12-01实施中华人民共和国发布国家质量监督检验检疫总局版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 目。吕本标准的附录A、附录B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
2、华人民共和国福建出入境检验检疫局、食品安全分析与检测技术教育部重点实验室(福州大学)。本标准主要起草人:杨方、刘正才、黄晓蓉、李耀平、余孔捷、林永辉、陈祥明、陈国南。本标准系首次发布的出入境检验检疫行业标准。版权所有 禁止翻制、电子传阅、发售1 范围进出口水产品中睡赛多残留量的检测方法液相色谱-质谱/质谱法本标准规定了水产品中喳赛多残留量的液相色谱质谱/质谱测定和确证方法。本标准适用虾、鲤鱼及制品、黄鱼中喳赛多残留的测定和确证。2 规范性引用文件SN/T 1927-2007 下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注日期的引用文件，其随后所有的修改单(不包括勘误的内容)或修订版
3、均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。凡是不注明日期的引用文件，其最新版本适用于本标准。GB/T 6682 分析实验室用水规格和试验方法3 方法提要采用酸性乙腊甲醇混合溶剂提取试样中的残留物，提取液经Cl8固相萃取柱净化后，液相色谱质谱/质谱法测定，外标法定量。4 试剂和材料除非另有说明，所用试剂均为分析纯，水为GB/T6682规定的一级水。4.1 乙睛:色谱纯。4.2 甲醇:色谱纯。4.3 甲酸。4.4 二甲亚酬。4.5 0.1%甲酸溶液:移取1mL甲酸，以水定容1Lo 4.6 甲酸十甲醇十乙睛提取液(2十10十邸，体积比)。4.7 0.1%甲
4、酸水溶液十乙睛溶液(7十3，体积比)。4.8 5%甲醇水溶液:移取5mL甲醇，以水定容100mL，由匀。4.9 标准物质:喳赛多标准物质(cyadox，分子式C2比:150CAS号65884-46-0，分子量271.36)，纯度大于等于98%。4.10 标准储备液(100mg/L):准确称取适量的哇赛多标准品，先用少量二甲亚枫溶解后以甲醇定容至100mL，置于40C冰箱中，避光保存1个月。4.11 标准工作液:根据需要取适量标准贮备液，以0.1%甲酸水溶液十乙腊(4.7)稀释成适当浓度的标准工作液，配置过程避光，标准工作液要现配现用。4.12 C18固相萃取小柱:500mg , 3 mL或相当
5、者，用前以3mL甲醇、3mL水活化，保持柱体湿润。4.13 滤膜:0.22m，Jj(系滤膜。5 仪器和设备5.1 液相色谱质谱/质谱联用仪，配有电喷雾(ESD源。5.2 离心机:4 000 r/mino 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 5.3 组织捣碎机。5.4 均质器:转速大于10000 r/mino 5.5 涡旋混合器。5.6 超声波清洗器。5. 7 固相萃取装置。5.8 氮吹仪。6 试样制备和保存6.1 试样制备鲤鱼取连皮的鱼肉，将皮、肉分离后取鱼皮置于微波炉中以中高功率加热30s后连同肉、鲤鱼制品取所有可食部分放入组织捣碎机均质，充分混匀，装入清洁容器内
6、，并标明标记。虾、鱼类取可食部分置于高速组织捣碎机均质，充分由匀，装入清洁容器内，并标明标记。制样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。6.2 试样保存试样于180C以下保存，新鲜或冷冻的组织样品可在20C60C贮存72ho 7 测定步骤7.1 提取称取5g试样(精确至0.01g)，置于50mL聚丙烯离心管中，加入20mL甲酸十甲醇十乙睛混合溶剂(4.的，以均质器于10000 r/min均质提取30s , 4 000 r/min离心5mi口，上清液转移至一支50mL 比色管中。另取一50mL聚丙烯离心管加入15mL甲酸十甲醇十乙睛提取液(4.6) ，洗涤均质器刀头10 s，洗涤
7、液移入前一离心管中，用玻棒搅动残渣，旋涡振荡提取1min，超声波振荡提取5min,4 000 r/min 离心5mi口，合并上清液至50mL比色管，残渣再加入15mL上述提取液，旋涡振荡提取1min,4 000 r/min 离心5mi口，合并上清液至50mL比色管，以上述提取液定容至50.0mL，摇匀后取10.0mL置于一洁净瑛璃离心管中，于350C下水浴氮吹至近干，加入1mL 5%甲醇水溶液(4.的，旋涡振荡30s，待净化。7.2 净化在7.1的提取液中加入1mL正己皖，振荡后弃去正己皖层，下层溶液以约1mL/min的流速全部过C18固相萃取小柱，再以2X3mL 5%甲醇水榕液(4.8)洗涤
8、离心管后淋洗固相萃取小柱，继续抽干约10 mi口，以3mL甲醇洗脱，35C下氮吹至近干，以0.1%甲酸水溶液+乙腊溶液(4.7)定容至1mL，过0.22m滤膜，供测定。7.3 测定7.3. 1 色谱条件a) 色谱柱:YMC-Pack ProCl8柱，5m，150X3.0mm(内径)，或相当者;b) 柱温:30C;c) 流动相:0.1%甲酸溶液十乙腊(7+3，体积tU;d) 流速:400L/mi川巳)进样量:10L。7.3.2 质谱条件:a) 离子源:电喷雾源(ESl)，正离子模式;b) 扫描方式:多反应监测(MRM); 其他参考质谱条件参见附录Ao7.3.3 液相色谱-质谱/质谱测定根据试样中
9、被测物的含量情况，选取响应值适宜的标准工作液进行色谱分析。标准工作液和待测2 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 样液中喳赛多药物的响应值应在仪器线性响应范围内。标准工作液与待测样液等体积进样。上述色谱条件下，哇赛多的参考保留时间为2.5min。标准物质的多反应监测(MRM)色谱图参见附录Bo7.4 定性标准7.4. 1 保留时间待测样品中化合物色谱峰的保留时间与标准溶液相比变化范围应在士2.5%之内。7.4.2 信噪比待测化合物的定性离子的多反应监测(MRM)色谱峰的信噪比应大于等于3(S/N二三刀，定量离子的多反应监测(MRM)色谱峰的信噪比应大于等于10(S/
10、N二三10)。7.4.3 定量离子、定性离子及子离子丰度比待测化合物的质谱定性离子应出现至少应包括一个母离子和两个子离子，而且同一检测批次，对同一化合物，样品中目标化合物的两个子离子的相对丰度比与浓度相当的标准溶液相比，其允许偏差不超过表1规定的范围。表1定性确证时相对离子丰度的最大允许偏差相对离子丰度/c%)允许的相对偏差/c%) 50 20至5010至20+20 +25 8 结果计算和表述用数据处理软件中的外标法，按式(1)计算试样中喳赛多药物的残留量:式中:x= A X C, X芒As m V X 试样中喳赛多残留量的数值，单位为毫克每千克(mg/kg); A 样液中喳赛多的峰面积;人标
11、准工作液中喳赛多的峰面积;Cs 标准工作液中喳赛多的浓度，单位为微克每升(g/U;Vf 样品最终定容体积，单位为毫升(mL); V 测定用分取的样液体积，单位为毫升(mL);V 样品提取液体积，单位为毫升(mL);m 称取试样量，单位为克(g)o9 测定低限、回收率9.1 本方法的测定低限为:10g/kgo9.2 回收率:9.2.1 哇赛多的添加浓度为10g/kg时，回收率的实验数据如下:鲤鱼中喳赛多的回收率在74.9%101. 0%; 烤鲤中喳赛多的回收率在73.0%101. 0%; 虾中喳赛多的回收率在76.1%97. 8%; 黄鱼中喳赛多的回收率在75.2%95. 4%。9.2.2 喳赛
12、多的添加浓度为20g/同时，回收率的实验数据如下:鲤鱼中喳赛多的回收率在78.5%95. 5%; 烤鲤中喳赛多的回收率在79.5%96. 5%; 虾中喳赛多的回收率在78.0%98. 0%; +30 豆豆10+50 . ( 1 ) 3 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 黄鱼中喳赛多的回收率在82.5%97. 0%。9.2.3 哇赛多的添加浓度为40g/kg时，回收率的实验数据如下:4 鲤鱼中喳赛多的回收率在77.8%95. 0%; 烤鲤中喳赛多的回收率在76.8%97. 3%; 虾中喳赛多的回收率在80.5%94. 8%; 黄鱼中喳赛多的回收率在86.0%102.
13、 0%。版权所有 禁止翻制、电子传阅、发售质谱条件:附录Al)(资料性附录)质谱条件a) 雾化气(JEB):12.00 L/min(氮气hb) 气帘气(CUR): 14.00 L/min(氮气hc) 喷雾电压(I曰:3 000 V; d) 去、溶剂温度:450C;e) 去溶剂气流:8.00L/min(氮气); f) 碰撞气(CAD):6.00 mL/min(氮气hg) 驻留时间:150 min; h) 其他质谱参数见表A.10 表A.1唾赛多的主要参考质i晋参数化合物母离子子离子D1 F1 m/z m/z 188. 2 a严D严120 喳赛多272. 2 143.2 55 120 SN/T 1
14、927-2007 CE/ CX1 cV 25 12 45 8 a 为定量离子;对于不同质谱仪器，仪器参数可能存在差异，测定前应将质谱参数优化到最佳。1) 附录A所列质谱条件是在A1l3000型液质联用仪上完成的，此处列出试验用仪器型号仅为提供参考，并不涉及商业目的，鼓励标准使用者尝试不同厂家或型号的仪器。5 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 6 nu ro qd 的且趴nU FD n, 的口-Z350 250 巳FAH【的COHE150 附录B(资料性附录)睡要多标准物质的多反应监测(MRM)色谱图2.50 550 272.0/143.2 450 1岳。.5 1.
15、 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 4. 5 r/min 2.50 272.0/188.2 50 。o.岳1.0 1.岳2.02.岳3.03.岳4.04.岳I/min 固B.1睡要多标准物质的多反应监测(MRM)色谱图版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 Foreword Annex A and Annex B of this standard are informative annexes. This standard is proposed band was under the charge of the Certification
16、and Accreditation admin istration of the People s Republic of China. This standard is drafted by Fujian Entry-Exit Inspection and Ouarantine Bureau of the People s Re public of China ,Key Laboratorof Analysis& Detection Technology for Food Safet(Fuzhou Universi ty) Ministry of Education. The standar
17、d is mainly drafted by Yang Fang , Li u Zhecai , Huang Xiaorong , Li Yaoping , Yu Kongjie , Lin I Yonghui , Chen Xiangming , Chen Guonan. This standard is a professional standard for Entry-Exit Inspection and Ouarantine promulgated for the first time. Note: This Engl ish version is a translation fro
18、m the Chinese text. is solely for guidance. 7 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 Determination of cyadox residues in aquatic products for import and export -LC-MSjMS method 1 Scope The standard specifies the method of determination of cyadox residues in aquatic products for im port and export by liquid
19、 chromatography-tandem mass spectrometry. This standard is applicable to the determination of cyadox residues in shrimp,eel and eel product and yellowtail fish. 2 Normative references The following normative documents contain provisions which , through reference in this text, consti tute provisions
20、of this Professional Standard. For dated references , subsequent amendments to, or revisions of, anof these publications do not apply. However, parties to agreements based in this Professional Standard are encouraged to investigate the possibility of applying the most recent edi tions of the normati
21、ve documents indicated below. For undated references , the latest edition of the normative document referred to applies. GB/T 6682 Water for analytical laboratory use Specification and test methods 3 Principle Extraction of the cyadox residues with acidic acetonitrile-methanol mixed solvents,and the
22、n purifica tion with C18 solid phase extraction (SPE) cartridges , followed by liquid chromatography-tandem mass spectrometranalSIS. 4 Reagents and Materials AII reagents should be of analytical grade unless specified. 4. 1 Acetonitrile: HPLC grade. 4.2 Methanol: HPLC grade. 4.3 Formic acid. 4.4 Dim
23、ethyl sulfoxide 8 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 4.5 0.1% Formic acid solution:Accuratelmeasure 1 mL formic acid into 950 mL water and dilute to the volume with water, then mix well. 4.6 Formic acid + methanol + actonitrile(2 + 10 + 88 , V / V). 4.7 0.1% formic acid solution+ acetonitrile (7+3, V/V
24、). 4. 8 5 % methanol solut ion : Accu ratelmeasure 5 mL methanol into 95 mL water and mix well. 4.9 Standard of cyado元purity关98%.4.10 Stock solutions of cyadox(100 mg/L): Weigh 10.0 mg cyadox standard materials , dissolve with dimethylsulfoxide and then dilute with methanol to a volume of 100 mL, an
25、d store at approxi mately 40C and protect from light for a maximum period of 1 months. 4. 11 Calibration solutions of cyadox for LC-MS/MS: Dilute appropriate volume of stock solutions to a intended concentration with dilute solution and mix well. These solutions should be prepared be fore use. 4.12
26、CB solid phase extraction(SPE)cartridge:500 mg/3 mL, the extraction cartridge is conditioned with 3 mL methanol ,3 mL water before use ,prevent the columns from runing dry. 4.13 Membrane filter:O. 22m. 5 Apparatus 5.1 High Performance Liquid Chromatography-Mass Spectrometer equipment: Equipped with
27、elec trospray (ESI) LC interface. 5.2 Centrifuge:4000 r/min. 5.3 Tissues homogenizer. 5.4 Homogenizer: 10000 r/min or equivalent. 5.5 Vortex mixer. 5.6 Ultrasonic equipment. 5. 7 Solid phase extraction equipment. 5.8 Pressured gas blowing concentrator. 9 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 6 Sample prep
28、aration and storage 6.1 Sample preparation For eel samples , which are combined the muscle tissues and skins heated by microwave oven and mixed well by homogenizer. For eel products , which are combined all the edible portion and mixed well by homogenizer, then sealed in clean containers and marked.
29、 For shrimp samples and other fishes , which are combined the edible portions and mixed well by ho mogenizer, then sealed in clean containers and marked. Precautious measures should be taken to avoid contamination or other factors which macause the change of residues concentration in samples. 6. 2 S
30、ample storage Samples should be stored at一180C,fresh or frozen tissues may be stored at 2 - 60C for 72 h. 7 Method of Determination 7.1 Extraction Weigh 5 9 of the prepared test sample (accurate to 0.01 g) into a 50 mL stoppered polypropylene plastic centrifuge tube , then add 20 mL formic acid + me
31、thanol + actonitrile extract solutions(4. 6) , homogenize at 10 000 r /min for 30 s, then centrifuge at 4 000 r /min for 5min. transfer the superna tant to a 50 mL colorimetric tube. 15 mL extract solutions (4.6) is used to wash the dispersed stick, then added into the former centrifuge tube and mix
32、ed with the solid residues. The tube is vortexed for 1 min and then extracted the analyte with ultrasonic for 5 min , centrifuged at 4 000 r /min for 5 min , the supematant is transferred to the colorimetric tube. Another 15 mL extract solutionsC4. 6) is added to the residues and vortexed for 1 min
33、, cetrifuged at 4 000 r /min for 5 min. Combine the su pernatant to the colorimetric tube and diluteto 50 mL with the extract solutions C 4. 6). Measure 10 mL and evaporate to dryness at 350C under a stream of nitrogen. The residue is redissolved in 1 mL 5% methanol solutionC4. 8). The dissolution i
34、s achieved using vortex for 30 s. 7. 2 Clean-up Load extract through C18 SPE cartridges at a flow rate ca 1 mL/min,discard the elution ,then the car tridges are washed with 2 x 3 mL 5% methanol solutionC4. 8) after which is used to prewash the tube , dried under vacuum , and the cyadox residues are
35、eluted with 3 mL methanol. Concentrate the elution to near dryness at 350C under a stream of nitrogen ,dissolve with solutionC4. 7) and dilute to 1 mL. This solution is filtered with a O. 22m filter prior to LC-MS/MS analysis. 10 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 7.3 Determination 7.3.1 LC operation c
36、onditions a) Column: YMC-Pack Pro C18 column，5m， 150 x 3.0 mm(i. d. ) ,or equivalent. b) Column temperature:30C. c) Mobile phase:0.1% formic acid solutions-acetonitrile (7+3, V/V). d) Flow rate:400L/min. e) Injection volume: 10L. 7.3.2 MS operation conditions a) lon source: ESI , positive ionisation
37、 mode. b) Scan mode: multiple reaction monitoring(MRM) mode. Other reference mass operating conditions are listed in Annex A. 7.3.3 LC-MS/MS analysis Prepare standard solutions containing cyadox at appropriate concentrations according to the analyte in sample extracts. The referenced retention time
38、of cyadox is 2.5 min. Annex B is the multiple re action monitoring ion chromatogram of cyadox standard solution. 7.4 Confirmation test 7.4.1 Retention time The variation range of the retention time of the analyte in the test portion and in the standard work ing solution shall be within the range of士
39、2.5%.7.4.2 Signal-to-noise ratio The signal-to-noise ratio for each diagnostic ion shall be关3门，thesignal-to-noise ratio for quantita tive ion shall be注10: 1. 7.4.3 Relative intensities The qualification ions of the analte must be found , and at least include one precursor ion and two 11 版权所有 禁止翻制、电子
40、传阅、发售SN/T 1927-2007 daughter ions. For the same analsis batch and the same analte， the variation range of the ion ratio between the two daughter ions for the unknown samples and the standard working solutions at the similar concentration can not be out of range of table 1 under the same determinatio
41、n conditions. Table 1-Maximum permitted tolerances for relative ion intensities Relative intensity of base peak/ (%) 50 20 to 50 10 to 20 豆豆10Maximum permitted tol巳rancesfor rela tive ion intensities/ (%) :t 20 :t 25 :t 30 士508 Calculation and expression of result Determination the amount of cyadox(
42、 ing/L) in the aliquot of sample solution injected into LC-MS/ MS system. Calculate the concentration of the cyadox residue in sample according to the formula (1 ) : 弘一机-VA-A X 、，FaEI /h . . . . . . . . . . . . . . . . . . . . . . . where X -cyadox content in the test sample，g/kg; A- the concentrati
43、on of cadox in aliquot of sample solution injected into LC-MS/MS sstem，determined from the calibration curve，g/L: As-the concentration of cadox in aliquot of blank solution injected into LC-MS/MS sstem， deter mined from the calibration curve ,/Lg/L: Vf-final volume of the sa门1pleconstant volu门1e，付1L
44、;V,-aliquot of the sample extracts,mL; V -volume of the sa付1pleextracts，门1L;m-mass of sample,g. 9 Limit of determination and recovery 9. 1 The limit of determination of cadox is 10g/kg. 9. 2 Recovery: 9.2. 1 According to the experimental data , the fortified concentration of cyadox is 10g/kg， the re
45、-12 版权所有 禁止翻制、电子传阅、发售SN/T 1927-2007 covenes are: For eels , the recoveries are 74. 9%-101. 0% , For eel products , the recoveries are 73.0% -101.0% , For shrimps , the recoveries are 76.1 % -97.8% , For yellowail fishes , the recoveries are 75. 2% -95.4%. 9.2.2 According to the experimental data , t
46、he fortified concentration of cyadox is 20g/kg， the re covenes are: For eels , the recoveries are 78. 5% -95.5% , For eel products , the recoveries are 79. 5% -96.5% , For shrimps, the recoveries are 78. 0% -98.0% , For yellowail fishes ,the recoveries are 82.5%-97.0%. 9.2.3 According to the experim
47、ental data , the fortified concentration of cyadox is 40g/kg， the re covenes are: For eels , the recoveries are 77. 8%-95. 0% , For eel products , the recoveries are 76.8% -97.3% , For shrimps , the recoveries are 80. 5% -94.8% , For yellowail fishes ,the recoveries are 86.2%-102.0%. 13 版权所有 禁止翻制、电子
48、传阅、发售SN/T 1927-2007 Reference mass conditions: a) Nebulizer gas: 12.00 mL/min; b) Curtain gas:14. 00 mL/min; c) lon spray voltage: 3 000 V; d) Heat temperature:450oC ; e) Heat gas:8. 00 mL/min; Annex A) ( Informative) Reference mass conditions f) Collision gas:6. 00 mL/min(Nitrogen); g) Dwell time:1
49、50 min; h) Other mass operating conditions are listed in table A. 1. Table A. 1-Main Mass parameters of cydox precursor Monitor Compound DP FP m/z ions m/z 188.2 55 120 Cydox 272.2 143.2 55 120 a Monitor ion with asterisk is used for quantification. CE/eV CXP 25 12 45 8 1) The reference mass parameters in Annex A are accomplished by API 3 000 LC-MS/MS. the equipment and its type involved in th
