液相色谱-串联质谱-内标法测定尿液中河豚毒素  

Determination of tetrodotoxin in urine by liquid chromatography-tandem mass spectrometry with internal standard calibration

作　　者：徐小民[1] 蔡增轩[1] 许娇娇[1] 郑熠斌 徐美佳 韩见龙[1] Xu Xiaomin;Cai Zengxuan;Xu Jiaojiao;Zheng Yibin;Xu Meijia;Han Jianlong(Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310051,China)

机构地区：[1]浙江省疾病预防控制中心,杭州310051

出　　处：《卫生研究》2024年第1期95-101,共7页Journal of Hygiene Research

基　　金：浙江省医药卫生科技计划(No.2021KY615)。

摘　　要：目的建立液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)-内标法测定尿液中河豚毒素(tetrodotoxin,TTX)的分析方法。方法样品中的TTX经乙酸/甲醇/乙腈(0.005 mL/0.8 mL/1.8 mL)混合溶剂提取,阳离子交换固相萃取(solid phase extraction,SPE)小柱净化,采用含0.3%盐酸的50%乙腈/水洗脱、氨水中和后用XBridgeTMBEH Amide柱(150 mm×3.0 mm,1.7μm)分离,MS/MS测定。通过优化样品提取和SPE净化条件,解决阳离子交换SPE小柱净化尿液基质中TTX回收率偏低、质谱检测基质抑制效应强的难题。结果以春雷霉素为内标定量测定,TTX在0.2~200μg/L范围内线性关系良好,相关系数(r2)0.997,样品基质和纯溶剂中TTX线性相关方程的斜率比为0.95;尿液中TTX检出限和定量限分别为0.1和0.2μg/L;三水平加标(0.2、10.0和200μg/L)平均回收率为89.3%~95.3%,相对标准偏差≤5.1%(n=6)。11例中毒患者尿样中TTX含量为0.4~138μg/L;中毒3天以内获得的尿液中TTX的检出率为100%。结论该方法操作简单、准确、灵敏度高,可以为TTX中毒事件快速处置和毒素体内代谢研究提供可靠的技术支撑。OBJECTIVE An analytical method was developed for tetrodotoxin(TTX)in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS)with internal standard calibration.METHODS TTX in the sample was extracted with the mixture of acetic acid/methanol/acetonitrile(0.005 mL/0.8 mL/1.8 mL),cleaned by solid phase extraction(SPE)with cation exchange cartridge,eluted with 50%acetonitrile/water containing 0.3%hydrochloric acid,and neutralized with ammonia.The extract was separated by a Waters XBridge'N BEH Amide column(150 mm×3.0 mm,1.7μm)and measured by MS/MS.By optimizing sample extraction and SPE cleanup conditions,the problems of low recovery and strong suppression effects of MS signal for TTX in urine were resolved when cleaned with cation exchange cartridge.RESULTS Quantitatively calibrated by the internal standard of Kasugamycin,good linear relationship was found for TTX in urine at the range of 0.2-200μg/L with the correlation coefficient(r?)of 0.997.The limits of detection and quantitation for TTX in sample matrix were 0.1 and 0.2μg/L,respectively.The average recoveries at three spiking levels(0.2,10.0 and 200μg/L)were 89.3%-95.3%with relative standard deviation(n=6)less than 5.1%.The concentrations of TTX in urine from 11 poisoning patients were 0.4-138μg/L.The detection rate was 100%in urine collected within 3 days after poisoning.CONCLUSION The established method was simple,accurate and sensitive.It can provide reliable technical support for the rapid treatment of TTX poisoning events and the study of toxin metabolism in vivo.

关 键 词：液相色谱-串联质谱法 固相萃取 河豚毒素 尿液 食物中毒 

分 类 号：O657[理学—分析化学] R155.5[理学—化学]