人尿液中8-羟基脱氧鸟苷的超高效液相色谱-串联质谱联用检测方法  

Analysis of 8-hydroxy-2＇-deoxyguanosine in human urine by ultra-high performance liquid chromatography mass spectrometer method

作　　者：刘仲[1] 于志刚[1] 王艳华[2] 慈颖[3] 孙湛[1] 焦海涛[1] 段化伟[2] 

机构地区：[1]济南市疾病预防控制中心理化检验所,250021 [2]中国疾病预防控制中心职业卫生与中毒控制所 [3]中国检验检疫科学研究院卫生检疫所

出　　处：《中华预防医学杂志》2016年第4期357-361,共5页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（81172642）;国家科技支撑计划（2014BA112B02）

摘　　要：目的 建立尿液中8-羟基脱氧鸟苷（8-OHdG ）的超高效液相色谱-串联质谱快速检测方法。方法 配制浓度为0.01~0.1 μg/ml的8-OHdG标准溶液，以7 μl/min的速度导入离子源，找出母离子、子离子质荷比，逐步优化每个离子对的解簇电压、碰撞电压和出口电压等质谱参数。以浓度为10.0 μg/L的加标尿液做样品，分别用1 mol/L甲酸铵溶液和甲酸体积比为5∶1、4∶1、3∶1、2∶1和1∶1的甲酸-甲酸铵溶液调节样品pH值。分别选用HLB、MCX、MAX 3种不同极性的固相萃取柱进行测试。分别测试甲醇和水比例为90∶10、80∶20、50∶50、20∶80、10∶90的洗脱效果，测试乙腈和水比例为90∶10、80∶20、50∶50、20∶80、10∶90的洗脱效果。以标准系列应用液浓度对应化合物定量离子对峰面积的比值绘制标准曲线。以响应值为3倍信噪比对应的8-OHdG浓度计为检出限，以响应值为10倍信噪比对应的8-OHdG浓度计为定量下限。采用空白尿液加标回收的方式来评价精密度和加标回收率。结果 母离子质荷比为284.1，子离子质荷比分别为168.1、140.1、123.0、112.0，定量离子对284.1/168.1的碰撞电压为18 V, 284.1/140.1碰撞电压为42 V, 284.1/123.0碰撞电压为48 V, 284.1/112.0碰撞电压为53 V。由10 ml 1 mol/L甲酸铵溶液、2 ml甲酸、88 ml水混合溶液与样品体积1∶5的比例进行pH调节的方式回收率最高，为87.9%～104.3%。用3 ml甲醇和3 ml水活化的HLB柱进行样品提取净化，用1.0 ml甲醇进行洗脱的方式回收率最高，为87.9%～104.3%。在1.0~100.0 μg/L的浓度范围内，8-OHdG标准应用液检测结果呈良好的线性关系。回归方程y=1.25x＋0.74，相关系数r=0.999 5。方法的检出限是0.2 μg/L，定量下限是0.7 μg/L。方法加标回收率范围在87.9%～104.3%，批内精密度在1.5%～3.7％范围内，批间精密度在1.6%～5.4％范围内。结论 本研究建立的方法灵敏度高，精密度和Objective To develop an ultra-high performance liquid chromatography mass spectrometer method for the rapid determination of 8-hydroxy-2＇-deoxyguanosine （8-OHdG） in human urines.Methods 8-OHdG standard solution with the concentration from 0.01 to 0.1 μg/ml was formulated. The solution was implanted into ion source with a rate of 7 μl/min, the mass-to-charge ratio of parent ion and product ions, and ion mass to charge ratio was identified. The mass spectrum parameters of each Ion pairs, such as DP, EP and EXP, were gradually optimized. The urine sample with a concentration of 10.0 μg/L was detected, and the pH of the sample was adjusted using 1 mol/L ammonium formate and formic acid solution with a volume ratio of 5∶1, 4∶1, 3∶1, 2∶1, and 1∶1. It was tested using three different polarity of SPE, i.e.： HLB, MCX, and MAX. The elution effect of methanol and water mixture with the proportion of 90∶10, 80∶20, 50： 50, 20： 80, and 10： 90 were tested, and then acetonitrile and water mixture with the proportion of 90∶10, 80∶20, 50∶50, 20∶80, 10∶90 were also tested. The standard curve was constructed using the ratio of a standard series application fluid concentration to corresponding compounds quantitative ion liquid concentration of the peak area. The detection limit was determined as 3 times of the signal to noise ratio corresponding to the concentration of 8-OHdG, and the quantitative lower limit was determined as 10 times of the signal to noise ratio corresponding to the concentration of 8-OHdG. The blank urine spiked recovery method was used to evaluate the precision and recovery rate.Results The mass to charge ratio of parent ion was 284.1 and the product ions was 168.1, 140.1, 123.0, and 112.0, respectively. The collision voltage of quantitative ion-pair 284.1/168.1 was 18 V, the 284.1/140.1 collision voltage was 42 V, the 284.1/123.0 collision voltage was 48 V, and the 284.1/112.0 collision voltage was 53 V. The recovery rate was the highest （87.9%-104.3%） when the pH of

关 键 词：脱氧鸟苷 尿 色谱法 超高效液相 氧化损伤 生物标志物 

分 类 号：R446.12[医药卫生—诊断学] O657.63[医药卫生—临床医学]