Yang Yongjie, Liu Guannian, Lu Yifu, Ding Changming, Shi Xiaoming
School of Public Health, Anhui Medical University, Hefei 230032, China.
School of Public Health, Anhui Medical University, Hefei 230032, China China CDC Key Laboratory of Environment and Population Health, National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 100021, China.
Wei Sheng Yan Jiu. 2025 Jul;54(4):654-662. doi: 10.19813/j.cnki.weishengyanjiu.2025.04.018.
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.
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.
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).
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.
建立一种液相色谱 - 质谱法同时测定人尿中9种烟草暴露生物标志物，包括：尼古丁、可替宁、反式 - 3'-羟基可替宁、尼古丁氮氧化物、可替宁氮氧化物、去甲烟碱、去甲可替宁、新烟草碱和假木贼碱。
尿样加入β-葡萄糖醛酸酶/芳基硫酸酯酶（1000单位/样品）水解，于37℃暗处振荡过夜。加入氘代内标，用96孔支持液液萃取板萃取样品。用1.8 mL异丙醇 - 二氯甲烷溶液（5∶95，V/V）洗脱萃取板，氮气吹干。残渣用纯水复溶后进行分析。采用Gemini® 3μm NX - C18 110A（2.0 mm×150 mm，1.9μm）色谱柱分离，以0.1%（V/V）氨溶液和甲醇为流动相进行梯度洗脱。该方法采用液相色谱 - 串联质谱（LC - MS/MS），加热电喷雾电离源，采用平行反应监测和正离子模式扫描。
该方法对9种烟草暴露标志物标准曲线的相关系数（r）在0.9984至0.9999之间。检测限在0.02至1.40 ng/mL之间，定量限在0.06至4.60 ng/mL之间。基质效应在85.9%至109%之间。加标回收率在83.5%至115%之间，相对标准偏差在1.0%至16.3%之间（n = 6）。
该方法具有高灵敏度、精密度和准确度，适用于测定非吸烟和吸烟个体尿液中的9种烟草暴露标志物。
