DBS22004-2013食品安全地方标准动物源性食品中24种受体激动剂残留量的测定液相色谱-串联质谱法.pdf 12页

'DBS22吉林省地方标准DBS22/004—2013食品安全地方标准动物源性食品中24种受体激动剂残留量的测定液相色谱-串联质谱法2013-10-08发布2013-10-08实施吉林省卫生厅发布 DBS22/004-2013前言本标准的附录A为规范性附录，附录B和附录C为资料性附录。本标准起草单位：吉林省疾病预防控制中心本标准起草人：刘思洁、方赤光、李青、崔勇、姜楠、郭金芝I DBS22/004-2013食品安全地方标准动物源性食品中24种受体激动剂残留量的测定液相色谱-串联质谱法1范围本标准规定了动物源性食品中24种受体激动剂残留量的液相色谱-串联质谱测定方法。本标准适用于动物源性食品猪肉、猪肝、猪肾和牛肉中24种受体激动剂残留量的测定。本标准方法的检出限：马布特罗、克仑潘特、克仑丙罗、沙丁胺醇、克伦特罗、莱卡多巴胺、溴氯布特罗、溴布特罗、奥西那林、利妥特灵、塞布特罗、苯氧丙酚胺、克仑塞罗、妥布特罗、R-美托洛尔、S-美托洛尔、马喷特罗、西马特罗、苯乙醇胺A、非诺特罗、福莫特罗、拉贝特罗、异克仑潘特、丙卡特罗均为0.1μg/kg。2规范性引用文件下列文件对于本文件的应用是必不可少的。凡是注日期的引用文件，仅所注日期的版本适用于本文件。凡是不注日期的引用文件，其最新版本（包括所有的修改单）适用于本文件。GB/T6682分析实验室用水规格和试验方法3原理样品经酶解、高氯酸沉淀蛋白，混合型阳离子交换固相萃取小柱净化和富集，采用液相色谱-串联质谱仪测定24种受体激动剂的残留量，外标法定量。4试剂和材料除另有规定外，所用试剂均为分析纯，水为GB/T6682规定的一级水。4.1甲酸（HCOOH）：色谱纯。4.2乙腈（C2H3N）：色谱纯。4.3乙酸乙酯（C4H8O2）：色谱纯。4.4甲醇（CH3OH）：色谱纯。4.5高氯酸（HClO4）：优级纯。4.6氢氧化钠（NaOH）：优级纯。4.7氨水(NH4OH)：优级纯。4.8乙酸(C2H4O2)。4.9乙酸钠（C2H3NaO2）。4.10乙酸－乙酸钠缓冲溶液（pH=5.2）。4.110.1mol/L高氯酸溶液。1 DBS22/004-20134.125mol/L氢氧化钠溶液。4.130.1mol/L乙酸溶液。4.1445%氨水甲醇溶液。4.150.1%甲酸水溶液。4.1624种受体激动剂标准品：纯度均>95%，见附录A中表A.1。4.17标准储备液：分别称取上述24种受体激动剂10.0mg，置于10mL容量瓶中，用甲醇溶解并定容，于4℃条件下保存。4.18标准工作液：分别吸取上述各储备溶液，用甲醇稀释，配成浓度为1.0μg/mL的混和标准储备溶液。根据需要,用甲醇稀释上述标准储备溶液,配制成不同浓度的标准工作液。4.19β-葡萄糖苷酸酶/芳基硫酸酯酶(β-glucuronidasc/arylsulfatasc)：酶活性≥100,000units/min。4.20混合型阳离子交换固相萃取小柱，(6mL,200mg)或其他等效柱。4.21微孔滤膜：0.22µm，水相。5仪器与设备5.1液相色谱-串联质谱仪。5.2电子分析天平：感量0.1mg。5.3冷冻高速离心机:±20℃，转速15000r/min。5.4氮吹仪。5.5固相萃取装置。5.6超声清洗仪，功率500W。5.7恒温箱。5.8涡旋混合器。6分析步骤6.1样品制备动物组织样品绞碎混合均匀，置于4℃冰箱中保存待测。6.2样品提取准确称取绞碎混合均匀的动物组织样品5.0g,置于50mL离心管中，加入乙酸－乙酸钠缓冲溶液（pH=5.2）(4.10)10mL，加入5μL的β-葡萄糖苷酸酶/芳基硫酸酯酶，50℃水解2h，加高氯酸调节pH值至1.0，在80℃超声提取30min，取出冷却至室温在8000r/min离心10min。沉淀再用10mL0.1mol/L高氯酸溶液（4.11）提取一次，合并两次提取液。用5mol/L的NaOH（4.12）调pH=4.0,以10000r/min冷冻离心，待过混合型阳离子固相萃取小固相萃取柱。6.3净化混合型阳离子交换固相萃取小柱用6mL甲醇、6mL水活化后，加入上述溶液过柱，弃去过滤液。依次用5mL的0.1mol/L乙酸（4.13）、5mL甲醇淋洗,弃去过滤液和淋洗液，再用6mL的5%氨水2 DBS22/004-2013甲醇溶液（4.14）洗脱,收集洗脱液于玻璃小瓶中，氮气吹干，用5mL0.1%甲酸水溶液（4.15）定容，涡漩1min，经0.22μm微孔滤膜过滤液相色谱-串联质谱测定。6.4标准工作曲线的制备称取与试样基质相应的阴性样品5.0g，按6.1和6.2进行处理后得到基质溶液。根据需要,制备不同浓度的基质加标标准工作曲线。6.5测定6.5.1色谱参考条件色谱柱：BEHShieldRP18柱(100mm×2.1mm,1.7μm)或相当色谱柱。柱温：40℃。流动相：A相：含0.1%(体积分数)甲酸水溶液；B相：乙腈。梯度洗脱情况见表1。表1梯度洗脱程序时间（min）流速（ml/min）流动相A流动相B0.00.310001.00.310006.00.390108.50.3802010.00.3703011.00.3010012.00.3010012.50.3100015.00.31000进样量：2μL。6.5.2质谱参考条件离子源：电喷雾电离(ESI(+))，毛细管电压：3.2kV，锥孔电压：35V。射频透镜1(RFLens1)和射频透镜2(RFLens2)电压分别为35.0V和0.5V。离子源温度：110℃；脱溶剂温度：300℃；脱溶剂气流量：600L/h；离子能1和离子能2分别为1.8和1.5。质谱分析优化参数见表2。3 DBS22/004-2013表224种受体激动剂定性定量离子对及锥孔电压、碰撞能量定性/定量离子化合物锥孔电压（v）母离子（m/z）碰撞能量（eV）（m/z）奥西那林30212.2212.2/152.018非诺特罗32304.1304.1/135.125西马特罗24220.2220.2/202.110沙丁胺醇30240.2240.2/147.915塞布特罗28234.2234.2/160.015丙卡特罗25291.2291.2/273.220克仑塞罗30319.2319.2/202.918克仑丙罗22263.0263.0/245.010利妥特灵30288.3288.3/270.214妥布特罗30228.2228.2/153.115克伦特罗30277.3277.3/203.216S-美托洛尔35268.3268.3/116.118R-美托洛尔30267.0267.0/115.015莱卡多巴胺30302.3302.3/164.215溴氯布特罗30323.1323.1/248.915福莫特罗30345.2345.2/149.125溴布特罗30367.1367.1/292.918马布特罗28311.2311.2/237.115克仑潘特28291.1291.1/202.915苯氧丙酚胺28302.3302.3/284.215拉贝特罗28329.2329.2/311.325异克仑潘特25291.1291.1/273.120马喷特罗30325.2325.2/237.015苯乙醇胺A25345.2345.2/150.1206.6测定在上述仪器条件下测定基质加标标准溶液及样品溶液。各检测目标化合物以保留时间和特征离子与定量离子所对应的LC-MS/MS色谱峰面积相对丰度进行定性。要求被测试样中目标化合物的保留时间与4 DBS22/004-2013标准溶液中目标化合物保留时间的相对偏差小于20%；样品特征离子的相对丰度与浓度相当混合标准溶液的相对丰度一致，相对丰度偏差不超过表3的规定，则可判断样品中存在相应的被测物。表3定性测定时相对离子丰度的最大允许偏差相对离子丰度＞50%20%—50%10%—20%＜10%允许的相对偏差±20%±25%±30%±50%6.6.1空白试验除不加试样外，采用完全相同的测定步骤进行平行操作。6.6.2色谱图色谱图参见附录B中图B.1。7结果计算以标准溶液浓度对定量离子峰面积绘制标准曲线，外标法定量。按式（1）计算CV1000X…………………………………(1)m1000式中：X——样品中目标化合物含量，单位为微克每千克（μg/kg）；C——测定浓度，单位为纳克每毫升（ng/mL）；V——定容体积，单位为毫升（mL）；m——最终样液代表的试样量，单位为克（g）。注：计算结果需将空白值扣除。8准确度和精密度8.1准确度本方法添加浓度为0.5μg/kg～10.0μg/kg时，加标回收率见附录C中表C.1。8.2精密度在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的10%。5 DBS22/004-2013AA附录A（规范性附录）24种受体激动剂一览表表A.124种受体激动剂一览表化合物英文名称CAS号化学分子式奥西那林metaproterenol586-06-1C11H17NO3非诺特罗fenoterol13392-18-2C17H21NO4西马特罗cimaterol54239-37-1C12H17N3O沙丁胺醇salbutamol51022-70-9C13H21NO3塞布特罗cimbuterol54239-39-3C13H19N3O丙卡特罗procaterol72332-33-3C16H22N2O3克仑塞罗clencyclohexerol157877-79-7C14H20Cl2N2O2克仑丙罗clenproperol38339-11-6C11H16Cl2N2O利妥特灵ritodrine26652-09-5C17H21NO3妥布特罗tulobuterol41570-61-0C12H18ClNO克伦特罗clenbuterol37148-27-9C12H18ClN2OS-美托洛尔S-metaprolol253-483-7C15H25NO3R-美托洛尔R-metaprolol51384-51-1C15H25NO3莱卡多巴胺ractopamine97825-25-7C18H23NO3溴氯布特罗bromchlorbuterolN/AC12H18BrClN2O福莫特罗formoterol73573-87-2C19H24N2O4溴布特罗brombuterol41937-02-4C12H18Br2N2O马布特罗mabuterol56341-08-3C13H18ClF3N2O克仑潘特clenpenterol37158-47-7C13H21Cl3N2O苯氧丙酚胺isoasuprine579-56-6C18H24ClNO3拉贝特罗labetalol36894-69-6C19H24N2O3异克仑潘特clenisopenterol157664-68-1C13H20Cl2N2O马喷特罗mapenterol54238-51-6C14H21Cl2F3N2O2-（4-nitrophenyl）butan-2-ylamino）-1-苯乙醇胺AN/AC19H24N2O4（4-methoxyphenyl）ethanolhydrochloride6 DBS22/004-2013BB附录B（资料性附录）24种受体激动剂标准色谱图mabuterol105-Jan-2012mabuterol1MRMof1ChannelES+8.91311.2>237.1(mabuterol)906.51e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00clenpen1MRMof1ChannelES+8.96291.1>202.9(clenpent)903.54e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00clenprop1MRMof1ChannelES+5.93263>245(clenprop)901.55e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00salbuMRMof1ChannelES+3.10240.2>147.9(salbut)901.05e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00clenbutMRMof1ChannelES+7.57277.3>203.2(clenbut)906.37e5%-102.003.004.005.006.007.008.009.0010.0011.0012.00ractompSm(Mn,3x2)MRMof1ChannelES+7.92302.3>164.2(ractomp)905.62e5%-10Time2.003.004.005.006.007.008.009.0010.0011.0012.00bromch04-Jan-2012bromchMRMof1ChannelES+8.18323.1>248.9(bromch)901.42e6%11.45-102.003.004.005.006.007.008.009.0010.0011.0012.00brombuMRMof1ChannelES+8.66367.1>292.9(brombu)901.94e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00metap1MRMof1ChannelES+2.17212.2>152(metapro)901.60e5%-102.003.004.005.006.007.008.009.0010.0011.0012.00ritoMRMof1ChannelES+6.11288.3>270.2(rito)907.92e5%-102.003.004.005.006.007.008.009.0010.0011.0012.00cimbuMRMof1ChannelES+3.99234.2>160(cimb)901.61e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00isoxMRMof1ChannelES+9.31302.3>284.2(isox)903.44e6%-10Time2.003.004.005.006.007.008.009.0010.0011.0012.007 DBS22/004-2013clency104-Jan-2012clency1MRMof1ChannelES+5.58319.2>202.9(clency)902.89e5%-102.003.004.005.006.007.008.009.0010.0011.0012.00tulobuterol2MRMof1ChannelES+7.39228.2>153.1(tulobuterol)906.28e4%-102.003.004.005.006.007.008.009.0010.0011.0012.00R-metopMRMof1ChannelES+7.72268.3>116(R-metoprolol)901.34e5%-102.003.004.005.006.007.008.009.0010.0011.0012.00S-metopMRMof1ChannelES+7.67268.3>116(R-metoprolol)908.15e4%-102.003.004.005.006.007.008.009.0010.0011.0012.00mapentMRMof1ChannelES+10.01325.2>237(mapen)906.97e6%-102.003.004.005.006.007.008.009.0010.0011.0012.00cimaMRMof1ChannelES+2.96220.2>202.1(cimaterol)908.57e5%-10Time2.003.004.005.006.007.008.009.0010.0011.0012.00benyichunan14-Feb-2012benyichunanMRMof1ChannelES+10.82TIC(benyichunan)909.00e5%-101.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00fenoternolMRMof1ChannelES-2.63TIC(fenoternol)908.58e3%-101.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00formoterolMRMof1ChannelES+0.84TIC(formoterol)908.559.90e4%1.48-101.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00labetalolMRMof1ChannelES+9.82TIC(labetalol)901.79e4%-101.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00clenisopenterolMRMof1ChannelES+9.84TIC(clenisopenterol)901.79e5%4.534.20-101.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00procaterol1MRMof1ChannelES+0.83TIC(procaterol)907.82e4%0.944.48-10Time1.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.00图B.124种受体激动剂标准色谱图（从上之下依次为马布特罗、克仑潘特、克仑丙罗、沙丁胺醇、克伦特罗、莱卡多巴胺、溴氯布特罗、溴布特罗、奥西那林、利妥特灵、塞布特罗、苯氧丙酚胺、克仑塞罗、妥布特罗、R-美托洛尔、S-美托洛尔、马喷特罗、西马特罗、苯乙醇胺A、非诺特罗、福莫特罗、拉贝特罗、异克仑潘特、丙卡特罗）8 DBS22/004-2013CC附录C（资料性附录）回收率范围表C.124种受体激动剂的不同基质、三个水平添加回收率范围添加水平猪肾/猪肝/牛肉/猪肉化合物（μg/kg）回收率%回收率%回收率%/回收率%0.572.5～88.272.3～86.174.6～90.571.7～85.2奥西那林5.080.5～94.175.9～90.883.8～100.678.8～89.710.074.6～101.180.7～91.282.0～91.584.2～93.30.572.0～80.673.1～86.975.6～89.073.4～87.3非诺特罗5.073.6～83.372.5～87.876.4～101.174.2～92.110.078.2～102.274.6～91.575.0～93.373.6～104.70.580.5～91.482.7～91.880.0～93.481.5～95.2西马特罗5.074.9～90.080.4～94.971.2～93.674.6～95.210.072.7～92.683.8～97.980.5～105.380.4～95.90.572.6～87.874.4～107.282.1～93.472.9～91.7沙丁胺醇5.079.1～95.277.3～93.873.9～96.075.6～95.810.080.8～96.485.3～94.980.8～95.783.5～104.00.573.5～93.179.3～93.686.7～102.572.4～94.7塞布特罗5.085.2～96.875.6～95.878.4～92.485.2～106.210.073.6～94.582.2～96.483.6～95.078.3～96.20.578.1～93.777.7～93.575.2～102.680.0～103.3丙卡特罗5.083.4～96.682.1～95.673.8～93.376.6～94.710.078.5～103.476.7～98.082.8～97.374.2～106.30.578.1～93.675.9～103.781.5～96.480.5～94.6克仑塞罗5.074.9～97.680.5～94.573.7～98.879.7～96.710.082.3～96.973.9～105.082.0～95.678.5～94.80.578.5～104.681.3～95.372.6～105.282.8～96.3克仑丙罗5.074.1～95.080.2～94.983.0～95.681.5～95.910.081.6～96.981.6～105.982.4～98.677.9～97.00.573.8～93.677.3～97.482.2～95.282.7～94.3利妥特灵5.078.6～97.279.2～96.880.6～97.580.6～97.510.084.5～95.883.4～107.378.7～96.673.6～96.40.582.6～97.374.5～95.872.2～103.579.2～97.9妥布特罗5.076.5～95.276.9～97.173.6～96.480.7～98.010.075.9～102.177.7～96.676.2～105.476.9～94.10.575.2～95.274.3～90.983.8～105.281.8～97.1克伦特罗5.073.4～92.983.2～98.184.0～96.971.2～93.610.080.2～103.584.2～95.975.8～95.683.5～98.59 DBS22/004-20130.588.3～106.381.7～94.373.6～89.886.6～95.5S-美托洛尔5.085.1～93.978.4～96.177.5～90.485.8～96.110.082.9～98.485.8～94.880.9～96.184.2～95.90.581.0～108.075.8～95.683.1～96.577.9～94.6R-美托洛尔5.067.9～89.883.1～108.280.8～92.381.1～93.810.075.6～95.378.4～105.890.3～106.485.4～99.00.571.2～93.686.6～95.578.6～93.477.0～92.8莱卡多巴胺5.073.4～92.985.8～96.175.7～93.875.4～107.410.081.8～96.284.2～95.980.3～92.785.6～97.30.570.2～85.168.9～88.775.8～95.666.8～94.2溴氯布特罗5.076.3～90.774.5～91.983.1～108.279.7～98.910.073.5～92.080.3～96.278.4～105.884.5～97.60.581.0～108.073.4～95.879.9～90.376.7～96.1福莫特罗5.074.9～92.876.1～102.676.8～96.184.2～106.610.075.6～95.384.3～93.773.7～89.977.5～96.30.585.2～98.683.8～96.879.9～95.084.3～98.6溴布特罗5.086.4～100.982.9～104.383.7～97.982.5～99.210.083.8～99.085.5～98.787.6～104.480.8～95.70.588.3～106.379.9～94.882.6～96.385.7～97.2马布特罗5.085.1～93.983.9～96.085.8～103.783.6～95.110.082.9～98.486.5～98.279.2～95.975.4～101.10.583.1～96.571.2～93.281.7～94.384.5～104.5克仑潘特5.080.8～92.373.9～91.778.4～96.181.0～95.210.090.3～106.488.2～98.385.8～94.884.5～96.40.580.3～97.178.7～96.579.9～104.382.1～93.9苯氧丙酚胺5.083.5～96.982.4～95.280.6～94.785.3～98.410.085.2～98.281.6～96.886.1～95.884.4～97.50.578.8～95.585.1～95.577.9～94.682.3～93.3拉贝特罗5.083.2～94.984.3～96.181.1～93.885.1～106.610.085.9～95.887.7～105.685.4～99.079.9～93.80.583.4～89.978.4～90.282.0～89.575.5～90.3异克仑潘特5.080.0～92.484.5～95.083.3～107.476.7～91.210.079.7～96.182.6～93.177.7～90.483.8～93.20.576.6～94.371.2～87.270.0～96.778.6～105.3马喷特罗5.070.2～97.579.5～96.378.3～101.271.7～90.910.075.7～92.575.2～94.773.3～92.275.1～91.70.576.4～96.876.2～96.274.6～102.771.2～96.4苯乙醇胺A5.080.1～94.977.4～95.372.5～94.679.6～105.810.086.3～94.675.8～95.977.8～96.072.2～98.5_________________________________10'