免疫亲和柱净化-超高效液相色谱-三重四极杆质谱法高灵敏测定尿液和血浆中3种鹅膏毒肽  

Highly sensitive determination of three kinds of amanitins in urine and plasma by ultra performance liquid chromatography-triple quadrupole mass spectrometry coupled with immunoaffinity column clean-up

作　　者：张秀尧[1] 蔡欣欣[1] 张晓艺[1] 李瑞芬[1] 赵云峰 ZHANG Xiuyao;CAI Xinxin;ZHANG Xiaoyi;LI Ruifen;ZHAO Yunfeng(Wenzhou Municipal Center for Disease Control and Prevention, Wenzhou 325001, China;China National Center for Food Safety Risk Assessment, Beijing 100022, China)

机构地区：[1]温州市疾病预防控制中心,浙江温州325001 [2]国家食品安全风险评估中心,北京100022

出　　处：《色谱》2022年第5期443-451,共9页Chinese Journal of Chromatography

摘　　要：建立了超高效液相色谱-三重四极杆质谱高灵敏测定尿液和血浆中α-鹅膏毒肽、β-鹅膏毒肽和γ-鹅膏毒肽的方法。经过免疫亲和柱净化,尿液样品浓缩20倍、血浆样品浓缩10倍,以Kinetex Biphenyl色谱柱(100 mm×2.1 mm,1.7μm)作为分析柱,甲醇-0.005%(v/v)甲酸水溶液作为流动相进行梯度洗脱分离,电喷雾电离、负离子、多反应监测模式下检测,外标法定量。3种鹅膏毒肽的线性范围为0.1~200 ng/mL,相关系数(r)>0.999。尿液和血浆中3种鹅膏毒肽的基质效应和提取回收率分别为92%~108%和90%~103%,变异系数均小于13%。尿液中3种鹅膏毒肽的准确度为-9.4%~8.0%,重复性和中间精度分别为3.0%~14%和3.5%~18%,当取样量为2.00 mL时,方法的检出限均为0.002 ng/mL;血浆中3种鹅膏毒肽的准确度为-13%~8.0%,重复性和中间精度分别为3.9%~9.7%和5.5%~12%,当取样量为1.00 mL时,方法的检出限均为0.004 ng/mL。该法操作简单、灵敏、准确,已在中毒患者摄入野生蘑菇后138 h的尿液中检出0.0067 ng/mLα-鹅膏毒肽和0.0059 ng/mLβ-鹅膏毒肽。该法已成功解决中毒患者尿液和血浆中超痕量鹅膏毒肽的检测难题,对于疑似中毒病人的早诊断、早治疗、降低死亡率都具有非常重要意义,也为今后开展此类毒素毒理作用及机体代谢规律的研究提供了可靠的技术支撑。Cases of toxic mushroom poisoning occur frequently in China every year.In particular,mushrooms containing amanitins can cause acute liver damage,with high mortality rates.The symptoms of acute liver damage are experienced 9-72 h after consumption of the mushrooms.At this time,the concentration of amanitins in blood and urine is too low to be detected even by the highly sensitive ultra performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS),thus rendering clinical diagnosis and treatment difficult.To this end,a method was developed for the determination ofα-amanitin,β-amanitin andγ-amanitin in urine and plasma by UPLC-MS/MS.Urine and plasma samples were extracted and cleaned up by using an immunoaffinity column.A sample of 2.00 mL urine or 1.00 mL of plasma was diluted with 8.00 mL of phosphate buffer solution(PBS)and then loaded onto the immunoaffinity column at a flow rate of 0.5-1.0 mL/min.After washing the column with 10 mL of PBS and 13 mL of water successively,the bound amanitins were eluted with 3.00 mL of methanol-acetone(1∶1,v/v).The eluent was dried under nitrogen at 55℃.The residue was dissolved in 100μL of 10%(v/v)methanol aqueous solution.The amanitins in urine were concentrated 20 times,while those in plasma were concentrated 10 times.Chromatographic separation was performed on a Kinetex Biphenyl column(100 mm×2.1 mm,1.7μm)with gradient elution using methanol and 0.005%(v/v)formic acid aqueous solution as mobile phases.The three amanitins were detected by negative electrospray ionization tandem mass spectrometry in the multiple reaction monitoring(MRM)mode and quantified by the solvent standard curve external standard method.Method validation was performed as recommended by the European Drug Administration(EMEA).Four levels of quality control(QC)samples were prepared,which covered the calibration curve range,viz.,the limit of quantification(LOQ),within three times the LOQ(low QC),medium QC,and at 85%of the upper calibration curve range(high QC),and used to test the

关 键 词：超高效液相色谱-三重四极杆质谱 免疫亲和柱净化 鹅膏毒肽 尿液 血浆 

分 类 号：O658[理学—分析化学]