固相萃取-超高效液相色谱-串联质谱法测定人尿液中4种氧化应激生物标志物  

Determination of four oxidative stress biomarkers in human urine using solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry

作　　者：冯壮壮 林潇 包得军 胡小键[1] 张海婧[1] 朱英[1] 张续[1] FENG Zhuangzhuang;LIN Xiao;BAO Dejun;HU Xiaojian;ZHANG Haijing;ZHU Ying;ZHANG Xu(China CDC Key Laboratory of Environment and Population Health,National Institute of Environmental Health,Chinese Center for Disease Control and Prevention,Beijing 100021,China)

机构地区：[1]中国疾病预防控制中心环境与人群健康重点实验室,中国疾病预防控制中心环境与健康相关产品安全所,北京100021

出　　处：《色谱》2025年第4期317-325,共9页Chinese Journal of Chromatography

基　　金：国家自然科学基金(22106154).

摘　　要：基于超高效液相色谱-串联质谱法(UPLC-MS/MS),建立了人尿液中8-羟基脱氧鸟苷(8-OHdG)、8-羟基鸟苷(8-OHG)、羟基壬烯巯基尿酸(HNEMA)和双酪氨酸(diY)等4种氧化应激生物标志物的测定方法。用水将0.2 mL尿样稀释5倍后,样品经Oasis HLB柱进行富集净化;先用2%(体积分数)甲醇水溶液洗脱diY,再用甲醇洗脱8-OHdG、8-OHG和HNEMA,收集洗脱液后分别进样分析;以甲醇和0.05%(体积分数)乙酸水溶液作为流动相进行梯度洗脱,Acquity UPLC HSS T3色谱柱进行色谱分离。分别在正离子电喷雾(ESI+)和负离子电喷雾(ESI-)多反应监测(MRM)模式下扫描化合物,同位素内标法定量。4种氧化应激生物标志物在0.01~100μg/L范围内线性关系良好,相关系数均≥0.9998;检出限为7~18 ng/L,定量限为22~60 ng/L;4种氧化应激标志物在低、中、高3个水平下的加标回收率分别为103.0%~105.6%(8-OHdG)、100.8%~104.2%(8-OHG)、97.2%~100.2%(diY)和96.9%~106.0%(HNEMA)。采用本方法测定40份尿液样本,结果表明8-OHdG、8-OHG、diY和HNEMA的检出率均为100%;检出质量浓度范围分别为0.52~14.40μg/L、2.75~38.15μg/L、8.92~82.28μg/L和1.74~575.29μg/L,中位值分别为2.89、12.36、37.66和96.92μg/L。该方法操作简单,灵敏度高,精密度好,适用于开展人尿液中4种氧化应激生物标志物的测定。Oxidative stress biomarkers are measurable biological indicators that reflect the balance between the production of reactive oxygen species(ROS)and the body’s ability to neutralize them using antioxidants.Elevated oxidative stress is associated with a number of health effects.Herein,we report the development of a comprehensive and sensitive method for quantifying four typical oxidative stress biomarkers in human urine using solid-phase extraction(SPE)in conjunction with ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The quantified biomarkers include L,L-dityrosine(diY),8-hydroxy-2′-deoxyguanosine(8-OHdG),8-hydroxyguanosine(8-OHG),and 4-hydroxynonenal mercapturic acid(HNEMA),which are markers of oxidative-stress-related damage in proteins,DNA,RNA,and lipids,respectively.To that end,we systematically optimized the MS parameters,SPE cartridge,and elution conditions of the method.Briefly,0.2 mL of a urine sample was mixed with 0.8 mL of pure water,after which an internal-standard mixture was added.The four target analytes were enriched and purified using an HLB SPE cartridge.The diY and the other three compounds were eluted with 2%(volume fraction)methanol aqueous solution and methanol,respectively.The two groups of eluates containing different target analytes were separately injected onto an Acquity UPLC HSS T3 column(100 mm×2.1 mm,1.8μm)and gradient eluted using 0.05%(v/v)acetic acid aqueous solution and methanol.The target analytes were identified using both negative and positive electrospray ionization(ESI-and ESI+)and multiple reaction monitoring(MRM)modes,and quantified using stable-isotope-labeled internal standards.The four typical oxidative-stress biomarkers exhibited good linearities within the mass concentration range of 0.01-100μg/L,with correlation coefficients≥0.9998,and limits of detection(LODs)and limits of quantification(LOQs)of 7-18 and 22-60 ng/L,respectively.The spiked recoveries of the target analytes at three levels(5,10 and 50μg/L)were 103.0%-105.6%(8-OHd

关 键 词：固相萃取 超高效液相色谱-串联质谱法 氧化应激生物标志物 尿液 人体生物监测 

分 类 号：O658[理学—分析化学]