1、中华人民共和国出入境检验检疫行业标准SN/T 2269-2009 进出口贝肉中大田软海绵酸的检测液相色谱-串联质谱法Determination of okadaic acid in shellfish meat for import and export LC-MS/MS method 2009-02-20发布2009-09-01实施中华人民共和国发布国家质量监督检验检疫总局目Ij1=1 本标准的附录A、附录B均为资料性附录。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、王敏、唐毅锋、王传现。本标准系首次发布的出入境
2、检验检疫行业标准。标准分享网免费下载SN/T 2269-2009 I SN/T 2269-2009 1 范围进出口贝肉中大田软海绵酸的检测液相色谱-串联质谱法本标准规定了进出口贻贝和蛤唰中大田软海绵酸的制样和液相色谱串联质谱检测方法。本标准适用于进出口贻贝和蛤唰中大田软海绵酸的检测。2 测定方法2.1 方法提要试样中的大田软海绵酸用甲醇/水提取，再用三氯甲皖抽提，经Sep-paksilica固相萃取柱净化后，用C18柱色谱分离，选择电喷雾正离子模式质谱检测，外标法定量。2.2 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。2.2.1 乙1青:液相色谱级。2.2.
3、2 甲醇。2.2.3 甲酸。2.2.4 正己烧。2.2.5 工氯甲烧。2.2.6 丙酬。2.2.7 氮气:纯度99.9%。2.2.8 甲醇水(80+20)。2.2.9 正己烧丙四同水(20+80+0.3)。2.2. 10 甲醇丙酬水(3+97+0.3)。2.2.11 甲醇丙酬水(50+50+0.3)。2.2.12 0.1%甲酸水榕液(含2mmol/L乙酸锻):1L水中加入l.0 mL甲酸和0.156g乙酸镜。2.2.13 乙睛。.1%甲酸水榕液(含2mmol/L乙酸锻)(30+70)。2.2.14 大田软海绵酸(Okadaicacid , C44 H68 013 ，CAS号:78111-17-
4、8)标准物质:纯度大于等于98.0%。2.2.15 大田软海绵酸标准储备液:准确称取适量的大田软海绵酸标准品，用甲醇配制成10g/mL的标准储备液，于18 C中可保存半年。根据需要可将储备液再稀释成适当浓度的中间榕液。2.2.16 大田软海绵酸基质标准工作榕液:根据需要吸取适量大田软海绵酸标准储备液或中间榕液(2.2.15) ，用空白样品提取液稀释成适当浓度的基质标准工作榕液。基质标准工作榕液在18 C保存，可使用1用。2.2.17 Sep-pak silica柱:使用前小柱预先用10mL丙酬(含0.3%水)、10mL甲醇(含0.3%水)和10 mL正己烧丙酬水(20+80+0.3)活化，流速
5、为2mL/mi日。2.3 仪器和设备2.3.1 液相色谱串联质谱仪:配有电喷雾离子源。2.3.2 固相萃取装置。2.3.3 恒温氮吹装置。2.3.4 均质机。1 SN/T 2269-2009 2.3.5 旋涡混合器。2.3.6 离心机:最大转速4000 r/mi日。2.3.7 带刻度离心管:15mL。2.3.8 0.45m有机相针式过滤膜。2.4 测定步骤2.4.1 样晶制备从抽取的原始样品中取可食部分，放入搅碎机中搅碎至匀浆状，均分成两份，装入清洁的容器内，作为试样。密封，并标明标记。将试样于18 C以下冷冻保存。制样操作过程中，应防止样品受到污染或发生残留物含量的变化。2.4.2 提取及净
6、化称取经捣碎成浆的试样约2g(精确至o.01 g)于15mL离心管中，加4.0mL甲醇水(2.2.的，超声提取5min，于3500 r/min离心10mi口，吸取上清液于另一15mL离心管中。残渣用4.0mL甲醇水(2.2.的重复提取一次，合并上清液，最后用甲醇水(3.2.的定容至10.0mL。从定容的搭液中吸取5.0 mL于另一15mL离心管中，加入5mL正己烧，涡旋混匀，静置分层，弃去正己烧层。加入1mL水和6mL三氯甲烧，涡旋混匀，静置分层，吸取水相(上层)于另一15mL离心管中，再加入6mL三氯甲烧，涡旋混匀，静置分层，弃去上层水相，合并三氯甲烧提取液，收集于试管中，在60C中用氮气吹
7、干。用1.0 mL正己烧丙酬水(2.2.9)榕解上述残余物，经过活化的Sep-paksilica柱，分别用1mL正己烧丙酬水(2.2.的和10mL甲醇丙酬水(2.2.10)淋洗，最后用10.0mL甲醇丙酬水(2.2.11)洗脱，收集洗脱液于试管中，于45C中用氮气吹干。残余物用1.0 mL甲醇榕解，过0.45m滤膜，滤液供测试。2.4.3 测定2.4.3.1 液相色谱参考条件a) 色谱柱:巳8柱(150mmX 2.1 mm，粒径3.51时，或相当者;C18预柱(20mmX 3.9 mr丑，粒径5 fJ-m)，或相当者;b) 流动相:乙睛。.1%甲酸水榕液(含2mmol/L乙酸锻)(70+30)
8、; c) 流速:0.2mL/min; d) 柱温:22C; e) 进样量:10fJ-L。2.4.3.2 液相色谱串联质谱测定根据试样中被测物含量情况，选定浓度相近的标准工作榕液，对标准工作榕液与样液等体积参插进样测定，串联质谱参考条件参见附录A。标准工作液和样液中被测物的响应植均应在仪器检测线性范围内。在上述色谱条件下，大田软海绵酸的保留时间约为4min，标准品色谱图和质谱图参见附录B图B.1和图B.2，2.4.4 空白试验除不加试样外，均按上述操作步骤进行。2.5 结果计算和表述2.5.1 定性测定进行样品测定时，如果检出的色谱峰保留时间与标准品的保留时间偏差在士2.5%之内，并且在扣除背景
9、后的样品谱图中，各定性离子的相对丰度与浓度接近的同样条件下得到的标准榕液谱图相比，误差不超过表1规定的范围，则可判断样品中存在对应的被测物。2 相对离子丰度/%允许的相对误差/%表1定性确证时相对离子丰度的最大允许误差50 2050 1020 运10士20士25士30士50标准分享网免费下载SN/T 2269-2009 2.5.2 定量测定用色谱数据处理机或按式(1)计算试样中大田软海绵酸含量，计算结果需将空白植扣除。式中:x 生二三二主A , m X 试样中大田软海绵酸含量，单位为毫克每千克(mg/kg); A 样液中大田软海绵酸的色谱峰面积;A, 标准工作液中大田软海绵酸的色谱峰面积;c
10、标准工作液中大田软海绵酸的浓度，单位为微克每毫升(fJ-g/mL); V 样液最终定容体积，单位为毫升(mL); m 最终样液所代表的试样量，单位为克(g)。3 2月l定低限、回收率3.1 J月l定低限本方法的测定低限为0.1mg/kg。3.2 回收率和精密度3.2.1 贻贝中回收率的实验数据添加浓度在0.1mg/kg时，平均回收率为84%;相对标准偏差7.3%; 添加浓度在0.8mg/kg时，平均回收率为88%:相对标准偏差6.5%; 添加浓度在l.6 mg/kg时，平均回收率为87%;相对标准偏差6.2%。3.2.2 蛤蝴中回收率的实验数据添加浓度在0.1mg/kg时，平均回收率为87%;
11、相对标准偏差7.8%; 添加浓度在0.8mg/kg时，平均回收率为83%:相对标准偏差6.1%; 添加浓度在l.6 mg/kg时，平均回收率为86%;相对标准偏差7.0%。. ( 1 ) 3 SN/T 2269-2009 附录A(资料性附录)API 4000 HPLC-MS/MS仪器参数参考条件API 4000 HPLC-MS/MS仪器参数参考条件:a) 离子源:正离子电喷雾电离(ESI+); b) MRM模式检测:选择离子对m/z 827 723. 6和m/z827 809. 6作为定性离子，其中m/z 827 723. 6作为定量离子;c) 碰撞气压力(CAD):34. 475 kPa(5
12、 psi); d) 气帘气压力(CUR):137.9 kPa(20 psi); e) 雾化气压力(GSl):310. 275 kPa(45 psi); f) 辅助加热气压力(GS2):310. 275 kPa(45 psi); g) 离子源喷雾电压(IS):4500 V; h) 离子源温度(TEM):450 C; i) 去簇电压(DP):160 V; j) 碰撞气能量(CE):60 V; k) 碰撞室出口电压(CXP):20 V。4 标准分享网免费下载SN/T 2269-2009 附录B(资料性附录)大田软海绵酸标准晶色i昔图和质谱图4. 28 1600 1400 1200 f t1000 主
13、800主600400 200 。111/7 827一-723.6900 800 700 600 u 飞之500 三400. : 300 200 100 。飞，2 3 4 6 6 7 8 9 10 11 12 f/ ll1 ltl a) 4.27 h 111/￥827一-809.62 3 4 E、6 7 8 9 10 1 1 1 / f 。I t -b) 图B.l大田软海绵酸标准晶多反应监测(MRM)色谱图5.0c4 4.8e4 4.6e4 4.4e4 4.2c4 4.0c4 :3.8c4 :3.6c4 :3.4c4 :3.2c4 3.0e4 2.8c4 2.6e4 2.4e4 2.2e4 2.
14、0c4 1.8e4 1.6e4 1. 4c4 1. 2c4 1.0e4 8000.0 6000.0 4000.0 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 723.6 827.2 809. 6 岳71.4 ( I11 /Ll/al11l1 图B.2大田软海绵酸的质i昔图5 SN/T 2269-2009 Foreword Annex A and Annex B of this standard are normative annex. This standard was proposed bya
15、nd is under the charged of certification and accreditation administa tion of the Peoples Republic of China. This standard was drafted by Shanghai Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China. Main Drafter of this standard are Fang Xiaomi吨，WangMin , Tang Yife吨，WangChu
16、anxian. This standard is a professional standard for entry-exit inspection and quarantine promulgated for the first time. 6 标准分享网免费下载SN/T 2269-2009 Determination of okadaic acid in shellfish meat for import and export一LC-MSjMSmethod 1 Scope The standard specifies the method of sample preparation and
17、 determination of okadaic acid in mussel and surf clam for import and export by LC-MS/MS method. This standard is applicable to the determination of okadaic acid in mussel and surf clam for import and export. 2 Method of determination 2. 1 Abstract of this method Okadaic acid is extracted with metha
18、nol-water from the sample , and then extracted with chloroform. It is cleaned up with Sep-pak silica column extractor. Finally it is determined by LC-MS/MS. External standard method is used. 2.2 Reagents and materials Unless otherwise specified , all the reagent used should be analytical 9 rade , wa
19、ter is double d isti Iled 叭later.2.2.1 Acetonitrile: HPLC grade. 2.2.2 Methanol: HPLC grade. 2.2.3 Formic acid. 2.2.4 n-Hexane. 2.2.5 Chloroform. 2.2.6 Acetone. 2.2.7 Nitrogen gas:99. 9%. 2.2.8 Methano 1- wate r (80十20).7 SN/T 2269-2009 2.2.9 Hexane-acetone-waterC20十四十0.3).2.2. 10 Methanol-acetone-w
20、aterC3十97十0.3).2.2. 11 Methanol-acetone-waterC50十50十0.3).2.2.12 O. 1 % Formic acid aqueous solution Ccontaining 2 mmol/L ammonium acetate): Dissolve 1.0 mL formic acid and O. 156 9 ammonium acetate in 1 L water. 2.2.13 Acetonitrile-O. 1 % formic acid aqueous solutionCcontaining 2 mmol/L ammonium ace
21、tate) (30十70)。2.2.14 Okadaic acidCC44H6日013,CAS No. :78111-17-8) :Purity二三98.0%.2.2. 15 Stock standard solution of okadaic acid: Accurately weigh okadaic acid standa时，dissolve with methanol , the concentration of solutions are 10g/mL. It should be stored at一18oC for half a year. According to the con
22、centration required , the standard middle solution is prepared from the stock standard solution by methanol. 2.2. 16 Matrix standard working solutions: According to the concentration required , the matrix standard working solution is prepared from the stock standard solution or standard middle solut
23、ion by blank sample extract. It should be stored at一18oC for one week. 2.2.17 Sep-pak silica column: It is conditioned with 10 mL acetoneCcontaining 0.3% water) fol lowed by 10 mL methanolCcontaining 0.3% water)and Hexane-acetone-waterC20十80十O.酌，andthe rate of flow is 2 mL;什2.3 Apparatus and equipme
24、nt 2.3. 1 Liquid chromatograhy with electrospray ionization mass spectrometry. 2.3.2 Vacuum manifold for SPE. 2.3.3 Homoeothermic nitrogen evaporator. 2.3.4 Homogenizer. 2.3.5 Vortex mixer. 2.3.6 Centrifuge:the max speed 4 000 r/min. 2.3.7 Graduated centrifuge tube: 15 mL. 8 标准分享网免费下载SN/T 2269-2009
25、2.3.8 0.45m organic phase filter membrane. 2.4 Procedure 2.4. 1 Preparation of test sample Take the edible part from the whole primary sample ,and it is ground in a blender. Keep the prepared sample into two sample bottles seal and labe l. The test sample is stored in一18oC refrigerator. In the cours
26、e of sample preparation , precautions must be taken to avoid contamination or any fac tors , which may cause the change of residue content. 2.4.2 Extraction and clean up Weigh ca 2 9 of the test sample(accurate to 0.01 g) into 15 mL centrifuge tube, add 4. 0 mL metha nol-water (2. 2.酌，ultrasoundextr
27、act for 5 min. Centrifuge at 3 500 r/min for 10 min. Transfer the supernatant layer into another 15 mL centrifuge tube. Repeat the extraction of the residue in the same way with 4. 0 mL methanol-water and combined the solution ,dilute to 10.0 mL with methanol water. Transfer 5.0 mL of the above solu
28、tion into another 15 mL centrifuge tube,add 5 mL hexane , mix well by vortex minishaker and let stand for separating. Remove the hexane layer. Add 1 mL water and 6 mL chloroforr啊，mixwell by vortex minishaker and let stand for separating. Transfer water layer (upper layer)into another 15 mL centrifug
29、e tube ,add 6 mL chloroform ,mix well by vortex minishaker and let stand for separating. Remove water layer, combine chloroform extract to the test tube, and blow to dryness at 60 oC under light nitrogen. The residue is dissolved with 1.0 mL hexane-acetone-water(20十80十O.3) (2. 2. 9). Transfer the a
30、bove solution into Sep-pak silica column ,add 1 mL hexane-acetone-water(20十80+ 0.3) (2.2. 9)and 10 mL methanol-acetone-water(3十97+ 0.3) (2.2.10) to rinse the column , discard the eluted solu tion. Elute the column with 10.0 mL methanol-acetone-water(50十50十O.3 ) (2. 2. 11) ,collect eluted solution to
31、 test tube and blow to dryness at 45 oC under light nitrogen. The residue is dissolved with 1.0 mL methanol , filtered through 0.45m membrane and ready for LC-MS/MS determination. 2.4.3 Determination 2.4.3. 1 HPLC operating conditon a) Column: C1日150mm x 2.1 mm(i. d.) ,3. 5m particle size , or the e
32、quivalent; b) Mobile phase:acetonitrile-O. 1 % formic acid aqueous solution(containing 2 mmol/L ammonium acetate) (70 + 30) ; c) Flow rate:O. 2 mL/min; 9 SN/T 2269-2009 d) Column temperature: 22 oC ; e) Injectionvolumn:10L。2.4.3.2 LC-MS/MS determination According to the approximate concentration of
33、analyte in sample solution , select the standard work ing solution with similar responses to that of sample solution , MS/MS operating conditon see annex A. The responses of the analyte in the standard working solution and sample solution should be with in the linear range of the instrument detectio
34、n. Under the above LC-MS/MS operating condition , the retention time of Okadaic acid is about 4 min , selected ion chromatograms of the standards see figure B. 1 and figure B. 2 in annex B. 2.4.4 Blank test The operation of the blank test is the same as the described in the method of determination,
35、but with the omission of sample addition. 2.5 Calculation and expression of result 2.5.1 Confirmation If the retention times of sample chromatogram peaks are consistent with the standards , and after bacl咱roundcompensation , the relative ion intensity shall correspond to those of the calibration sta
36、nda时，atcomparable concentrations , measured under the same conditions , within the tolerances listed in table 1 ,and then the corresponding analyte must be present in the sample. Table 1-Maximum permitted tolerances for relative ion intensities while confirmation Relative intensity/% 50 2050 1020 运1
37、0Permitted tolerances/% :t 20 :t 25 :t 30 :t 50 2.5.2 Determination of quantitative Calculation the content of Okadaic acid residues in the test sample by LC-MS/MS data processor of according to the formula(1). The blank value should be subtracted from the above result of calcula tlon. Where: X=A.c.
38、V As m X -the residue content of Okadaic acid residues in the test sample , r丁19/1也;A -the peak area of analyte of the sample solution; As-the peak area of analyte of the standard solution; c-the concentration of Okadaic acid in the standard solution，g/mL; V-the final volume of the sample solution ,
39、mL; 10 标准分享网免费下载. ( 1 ) SN/T 2269-2009 m-the corresponding sample weight of the final solution , 9 。3 Limit of determination and recover 3.1 Limit of determination The limit of determination of this method is O. 1 mg/kg。3. 2 Recovery 3.2. 1 Recovery of fortifying in mussel The average recovery is 84
40、% and RSD is 7. 3% , when fortified at the concentration of O. 1 mg/kg; The average recovery is 88% and RSD is 6. 5% , when fortified at the concentration of O. 8 mg/kg; The average recovery is 87% and RSD is 6. 2% , when fortified at the concentration of 1.6 mg/kg. 3.2.2 Recovery of fortifying in s
41、urf clam The average recovery is 87% and RSD is 7. 8% , when fortified at the concentration of O. 1 mg/kg; The average recovery is 83% and RSD is 6. 1 %, when fortified at the concentration of O. 8 mg/kg; The average recovery is 86% and RSD is 7. 0% , when fortified at the concentration of 1.6 mg/kg
42、. 11 SN/T 2269-2009 Annex A (informative annex) API 4000 HPLC-MS/MS operating conditions API 4000 HPLC-MS/MS operating conditions: a) lon Source Type: ESI , positive mode; b) Mode: multiple reaction monitoring , transitions for confirmation m/z 827 723. 6 and m/z 827 809.6 , transitions for quantifi
43、cation m/z 827 723.6; c) CAD:34.475 kPa(5 psi); d) CUR: 137.9 kPa(20 psi); e) GS1 :310. 275 kPa(45 psi); f) GS2:310.275 kPa(45 psi); g) IS :4 500 V; h) TEM:450 oC; DP: 160 V; j) CE:60 V; k) CXP:20 V。12 标准分享网免费下载SN/T 2269-2009 Annex B (informative annex) Selected ion chromatograms and mass chromatogr
44、am of okadaic acid standard 4. 28 1600 1400 111/7 827一-723.61200 1 000 800 主600400 200 0 2 3 飞4 5 6 7 8 9 10 1 1 1 / f 。-i 1 1 a) 900 800 700 600 u 飞之500 三400. : 300 200 100 。4.27 111/￥827一-809.62 3 4 E、6 7 8 9 10 1 1 1 / f 。I t -b) Figure B. 1-Selected ion chromatograms of okadaic acid standard 5.0
45、c4 4.8e4 4.6e4 4.4e4 4.2c4 4.0c4 :3.8c4 :3.6c4 :3.4c4 :3.2c4 3.0e4 2.8c4 2.6e4 2.4c4 2.2c4 2.0e4 1. 8c4 1. 6c4 1.4e4 1.2e4 1. Oc4 8000.0 6000.0 4000.。岳00520岳40560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 723.6 827.2 809. 6 57 1. 4 (111/刘/alllUFigure B. 2-Mass chromatogram of okadaic acid standard 13 白CON-gNNH军中华人民共和国出入境检验检疫行业标准进出口贝肉中大田软海绵酸的检测i夜相色i昔-串联质谱法SN/T 2269-2009 兴中国标准出版社出版北京复兴门外三里河北街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兴印张1.25 字数24千字2009年5月第一次印刷开本880X 1230 1/16 2009年5月第一版印数1-2000定价21.00元兴书号:155066.2-19657 SN/T 2269-2009 标准分享网免费下载
