超高效液相色谱串联四极杆飞行时间质谱法同时测定蟾蜍皮中8种蟾蜍毒素

Simultaneous determination of 8 bufo toxins in toad skin by UPLC/Q-TOF-MS

目的建立超高效液相色谱串联四极杆飞行时间质谱法快速定性定量检测中毒患者食用剩余蟾蜍皮中8种蟾蜍毒素。方法样品经甲醇提取后,待测液经过A CQUITY UPLC®HSS T3色谱柱(100 mm×2.1 mm,1.8μm)分离,以0.1%甲酸水溶液为流动相A、甲醇为流动相B进行梯度洗脱,流速0.3 mL/min,柱温35℃,进样量2μL,经ESI正离子模式采集。结果在10.0~1000.0μg/L范围内8种毒素线性关系良好,r^(2)均≥0.990;检出限为0.7~2.1μg/kg,定量限为2.5~7.0μg/kg;三水平加标平均回收率均≥75.79%,相对标准偏差(RSD)均≤9.84%。样品中检测出蟾毒灵、去乙酰华蟾毒精、去乙酰华蟾毒它灵、伪异沙蟾毒精、蟾毒它灵,含量分别为123.8±3.1、5920.4±1.9、680.7±3.2、671.6±9.1、1796.6±74.9μg/kg,和蟾蜍他灵、华蟾毒精与华蟾毒它灵均未检出。结论该方法同时测定蟾蜍毒素中华蟾毒精、蟾毒灵、伪异沙蟾毒精、蟾毒它灵、去乙酰华蟾毒它灵、去乙酰华蟾毒精、和蟾蜍他灵、华蟾毒它灵,有较强实用性、快速、可靠。

Objective To establish a rapid qualitative and quantitative method for the determination of 8 bufo toxins in toad skin residual from poisoned patients by ultra performance liquid chromatography tandem quadrupole time of flight mass spectrometry(UPLC/Q-TOF-MS).Methods After the toad skin samples were pretreated,the sample solution was separated on A Cquity UPLC®HSS T3 column(100 mm×2.1 mm,1.8μm)with 0.1%formic acid solution as mobile phase A and methanol as mobile phase B.The flow rate was 0.3 mL/min,the column temperature was 35℃,and the sample volume was 2μL.Results The linear ranges were 10.0-1000.0μg/L with r^(2)≥0.990.The limits of detection were 0.7-2.1μg/kg,and the limits of quantification were 2.5-7.0μg/kg.The average recoveries were≥75.79%and RSDs were≤9.84%.The contents of bufalin,desacetylcinobufagin,desacetylcinobufotalin,pseudobufarenogin and bufotaline were 123.8±3.1,5920.4±1.9,680.7±3.2,671.6±9.1 and 1796.6±74.9μg/kg,respectively,while gamabufotain,cinobufagin and cinobufotalin were not detected.Conclusion The method is practical,rapid and reliable for the determination of cinobufagin,bufalin,pseudobufarenogin,desacetylcinobufotalin and desacetylcinobufagin,bufotaline,gamabufotain and cinobufotalin in toad skin.