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目的 建立了同时测定人尿中烟碱、可替宁、反式-3′-羟基可替宁、烟碱氮氧化物、可替宁氮氧化物、降烟碱、降可替宁、新烟草碱和假木贼碱9种烟草暴露标志物的固体介质液液萃取的液相色谱-质谱分析方法。方法 尿液加入β-葡萄糖醛苷酸酶/芳基硫酸酯酶(1000 units/样品)37℃摇床避光过夜水解后，加入相应的氘代内标，转移至96孔固体介质液液萃取盘中提取，使用1.8 mL异丙醇-二氯甲烷混合溶液(5∶95,V/V)洗脱，氮气吹干，经纯水复溶后使用Gemini?3μm NX-C18 110A(2.0 mm×150 mm, 1.9μm)色谱柱，0.1%(V/V)氨水溶液和甲醇为流动相梯度洗脱，以液相色谱串联质谱，加热电喷雾离子源-平行反应监测-正离子模式扫描测定。结果 9种烟草暴露标志物标准曲线的相关系数(r)在0.9984～0.9999之间，方法检出限为0.02～1.40 ng/mL,方法定量限为0.06～4.60 ng/mL,基质效应范围为85.9%～109%;在低、中、高不同浓度水平下，加标回收率范围为83.5%～115%,相对标准偏差范围为1.0%～16.3%(n=6)。结论 本方法前处理简单通量高，具有高灵敏度、高精密度和高准确度的特点，可同时满足非吸烟和吸烟人群尿液中9种烟草暴露标志物测定的要求。
Abstract：OBJECTIVE To establish a liquid chromatography-mass spectrometry method for the simultaneous determination of nine tobacco exposure biomarkers in human urine, including: nicotine, cotinine, trans-3′-hydroxycotinine, nicotine nitrogen oxide, cotinine nitrogen oxide, nornicotinine, norcotinine, anatabine, and anabasine.METHODS Urine samples were hydrolyzed by adding β-glucuronidase/arylsulfatase(1000 units/sample) and shaking at 37 ℃ overnight in the dark. Deuterium-labeled internal standards were added, and the samples were extracted using a 96-well supported liquid extraction plate. The plate was eluted with 1.8 mL of isopropanol-dichloromethane solution(5∶95, V/V) and dried under a stream of nitrogen. The residues were reconstituted in pure water for analysis. A Gemini? 3 μm NX-C18 110A(2.0 mm×150 mm, 1.9 μm) column was used for separation, with gradient elution using 0.1%(V/V) ammonia solution and methanol as the mobile phase. The method utilized liquid chromatography-tandem mass spectrometry(LC-MS/MS) with a heated electrospray ionization source, employing parallel reaction monitoring and positive ion mode scanning.RESULTS The method exhibited correlation coefficients(r) for the standard curves of the nine tobacco exposure markers ranging from 0.9984 to 0.9999. The detection limits were between 0.02 and 1.40 ng/mL, and the quantitation limits were between 0.06 and 4.60 ng/mL. The matrix effect ranged from 85.9% to 109%. The spiked recoveries ranged from 83.5% to 115%, and the relative standard deviations ranged from 1.0% to 16.3%(n=6).CONCLUSION This method is characterized by high sensitivity, precision, and accuracy, making it suitable for the determination of the nine tobacco exposure markers in the urine of both non-smoking and smoking individuals.
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基本信息:
DOI：10.19813/j.cnki.weishengyanjiu.2025.04.018
中图分类号:R446.12
引用信息:
[1]杨永杰,刘冠年,陆一夫,等.固体介质-液液萃取-液相色谱-质谱法测定人尿中9种烟草暴露标志物[J].卫生研究,2025,54(04):654-662.DOI:10.19813/j.cnki.weishengyanjiu.2025.04.018.
基金信息:
国家自然科学基金(No.82388102)
2024-12-12
2024
2025-01-22
2025
2
2025-07-16
2025-07-16