生物体液中10种蟾蜍毒素的UPLC-MS/MS检测

Detection of 10 Bufotoxins in Biological Fluids Using UPLC-MS/MS

本文建立了人的血液、尿液中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-四极杆/静电场轨道阱高分辨质谱(UPLC-Q/ExactiveMS)的检测方法。取血液或尿液0.2mL,先加入0.8mL乙腈沉淀蛋白,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。该方法在血液、尿液中对于10种蟾蜍毒素的检出限均可在10 ng/mL以内,最低可以达到0.1 ng/mL。10种蟾蜍毒素的血、尿基质添加在20~400 ng/mL范围内线性良好(R^(2)>0.995)。血、尿中10种蟾蜍毒素的提取回收率在65.0%~97.7%之间,基质效应在90.8%~109.0%之间,日内精密度在9.6%之内,日间精密度在14.7%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,浓度的RSD变化均在15%以内。用本文建立的方法对动物实验中收集到的血液进行分析,结果表明10种蟾蜍毒素均可检出。该方法灵敏度高,适用性广,可以用于蟾蜍毒素中毒类案件的检验鉴定。

A method was developed for the detection of 10 bufotoxins(arenobufagin,gamabufotalin,resibufagin,desacetylcinobufagin,resibufogenin,cinobufotalin,bufotalin,cinobufagin,bufalin,and cinobufaginol)in human blood and urine by ultra-high performance liquid chromatography-quadrupole/electrostaticfield orbital trap high resolution mass spectrometry(UPLC-Q/Exactive MS).0.8 mL of acetonitrile was added in 0.2mL of blood or urine to precipitate proteins,and then was purified with a Hybird solid phase column.A 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 and positive ion mode.The detection limit of 10 bufotoxins in blood and urine can be less than 10ng/mL,and the lowest can reach 0.1 ng/mL.The calibration curves of 10 bufotoxins were in good linear over the range from 20ng/mL to 400ng/mL(R^(2)>0.995)in spiked blood and urine matrix.The extraction recoveries of 10 bufotoxins from blood and urine ranged from 65%to 97.7%,the matrix effect ranged from 90.8%to 109.0%,the intraday precision was within 9.6%,and the interday precision was within 14.7%.The stabilities of 10 bufotoxins in the matrix were good under repeated freezing and thawing condition,room temperature condition,and extraction processing,with the RSD of concentration all within 15%.The method was used to analyze the bodyfluids collected in animal experiments.The results showed that all 10 bufotoxins had been detected.This method has high sensitivity and broad applicability,and can be used for the identification in bufotoxin poisoning cases.