生物组织中10种蟾蜍毒素的UPLC-MS/MS检测  

UPLC-MS/MS Detection of 10 Bufotoxins in Biological Tissues

作　　者：董颖 曹振宇 何立 苗丽文 刘剑 张宏民 DONG Ying;CAO Zhenyu;HE Li;MIAO Liwen;LIU Jian;ZHANG Hongmin(Institute of Forensic Science,Ministry of Public Security,Beijing 100038,China;Baotou Public Security Bureau,Baotou 014000,Inner Mongolia,China)

机构地区：[1]公安部鉴定中心,北京100038 [2]包头市公安局,内蒙古包头014000

出　　处：《刑事技术》2024年第5期464-471,共8页Forensic Science and Technology

基　　金：中央级公益性科研院所基本科研业务费专项资金项目(2021JB013)。

摘　　要：本文以肝脏为样本,建立了同时检测生物组织中10种蟾蜍毒素(沙蟾毒精、日蟾蜍它灵、酯蟾毒精、去乙酰华蟾蜍精、酯蟾毒配基、华蟾毒它灵、蟾毒它灵、华蟾酥毒基、蟾毒灵、华蟾毒精醇)的超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UPLC-Q/Exactive MS)的检测方法。取肝脏0.2 g,首先加入0.8 mL乙腈沉淀蛋白,然后用Hybird固相柱进行净化,选用ACQUITY UPLC HSS T3(2.1 mm×100 mm×1.8μm)色谱柱,流动相A相为0.1%甲酸水,B相为乙腈,采用梯度洗脱分离;质谱采用电喷雾离子源,正离子模式进行分析。10种蟾蜍毒素的检出限均在10 ng/mL以内,最低可以达到0.1 ng/mL。10种蟾蜍毒素添加在20~400 ng/mL范围内的线性良好(R^(2)>0.996)。10种蟾蜍毒素的提取回收率在62.9%~93.2%之间,基质效应在90.9%~109.0%之间,日内精密度在9.2%之内,日间精密度在14.6%之内,在基质中的反复冻融稳定性、常温稳定性和提取后稳定性良好,浓度变化均在15%以内。灌胃解剖后在雄鼠体内的分布为:肝>肺>心>肾>生殖器>肌肉,在雌鼠体内的分布为:肺>生殖器>肝>心>肾>肌肉。本研究建立了生物组织中蟾蜍毒素的检测方法,并初步得出毒素在动物体内的分布规律,为蟾蜍毒素的检验和相关检材的选择提供了参考依据。The detection method for simultaneously analyzing 10 bufotoxins(arenobufagin,gammabufotalin,resibufagin,desacetylcinobufagin,resibufogenin,cinobufotalin,bufotalin,cinobufagine,bufalin,cinobufaginol)in biological tissues samples was established using ultra-high performance liquid chromatography-quadrupole/electrostatic fi eld orbital trap high resolution mass spectrometry(UPLC-Q/Exactive MS)with liver as the sample.0.8 mL of acetonitrile was added in 0.2 g of liver to precipitate proteins,and then was purifi ed with a hybrid solid phase column.ACQUITY UPLC HSS T3 column(2.1 mm×100 mm×1.8μm)was selected.The mobile phase A consisted of 0.1%formic acid in water and B consisted of acetonitrile with a gradient elution program for separation.The mass spectrum was analyzed by electrospray ion source with positive ion mode.The detection limit of 10 bufotoxins in liver could be less than 10 ng/mL,and the lowest could reach 0.1 ng/mL.These 10 bufotoxins showed good linearity within the range of 20 ng/mL to 400 ng/mL(R^(2)>0.996).The extraction recoveries of 10 bufotoxins from liver ranged from 62.9%to 93.2%,and the matrix effect ranged from 90.9%to 109.0%.The intraday precision was within 9.2%,and the interday precision was within 14.6%.The stabilities of 10 bufotoxins in the matrix were good under repeated freezing condition,thawing condition and room temperature condition,and after extraction processing,with the RSD of concentration all within 15%.After gastric administration,the distribution order of content in male rats was as follows:liver>lung>heart>kidney>genital organ>muscles;in female rats,the distribution order of content was as follows:lung>genital organ>liver>heart>kidney>muscles.This study established a detection method for bufotoxins in biological tissues and revealed the distribution pattern of bufotoxins in animal bodies tentatively,providing a reference for the examination of bufotoxins and the selection of related specimens.

关 键 词：法医毒物分析 蟾蜍毒素 生物组织 质谱检测 体内分布 

分 类 号：DF795.1[医药卫生—法医学]