基于花粉固相萃取和稳定同位素标记的LC-MS/MS方法在尿样中多种苯系物代谢产物同时检测的应用

To assess the exposure of non-occupational population to benzene series, it is necessary for monitoring the metabolites of benzene series (MOBCs) in their urine. However, some disadvantages in previous reports for the analysis of MOBCs were existed, such as few target analytes, high matrix effect, low detection sensitivity and difficulty of obtaining isotope internal standards. To solve the above problems, the current project intends to introduce pollen solid phase extraction and stable isotope labeling technology into MOBCs analysis. First, we employ pollen as SPE adsorbent due to its characteristics of multiple functional groups on the surface, which can selectively separate and purify MOBCs from urine by hydrogen bond, hydrophilic and π-π interaction. Then, the target MOBCs are derivatized by N,N-dimethyl ethylenediamine, (DMED) and d4-DMED, so that the easily ionized tertiary amine group and light/heavy isotope label are introduced into MOBCs to improve detection sensitivity and accuracy in electrospray ionization mass spectrometry analysis. Finally, combined with HPLC-MS/MS analysis, we will develop a new and high sensitive method for the simultaneous determination of various MOBCs in urine. The implementation of this project will provide technical support not only for assessing the exposure of non-occupational population to benzene series in the environment, but also for the scientific development of the comprehensive biological limit value of benzene series.

为了评估非职业接触的普通人群对环境中苯系物的暴露量，有必要对其尿液中苯系物代谢产物（MOBCs）的浓度进行测定。然而现有的测定方法普遍存在目标分析物种类少、基质效应高、检测灵敏度低、同位素内标获取困难等问题。为了解决上述问题，本项目拟将花粉固相萃取和稳定同位素标记技术引入MOBCs分析中。首先利用花粉表面富含多种官能团的性质,将其作为SPE吸附剂，通过氢键、亲水以及π-π相互作用选择性的分离和纯化尿样中的MOBCs；接着采用N,N-二甲基乙二胺（DMED）和 d4-DMED对目标MOBCs进行衍生，从而将质谱易电离的叔胺基团和轻/重同位素标签引入MOBCs，以提高检测灵敏度和准确度；最后结合高效液相色谱串联质谱分析，建立尿样中多种MOBCs高灵敏的同时测定的新方法。本项目的实施可为苯系物环境暴露生物监测以及科学制定综合的苯系物生物限值标准提供技术支撑。

近年来由于室内装修发展迅猛，汽车尾气排放量增大，使得非职业接触的普通人群暴露于苯系物的概率大大提高，因此有必要对普通人群尿样中的苯系物代谢产物（MOBSs）进行生物监测，以综合评价环境中苯系物的污染程度。然而已报导的检测方法通常针对职业暴露人群，其普遍存在目标分析物种类少、基质效应高、检测灵敏度低、同位素内标获取困难等问题。为了解决上述问题，本项目将样品前处理与稳定同位素标记技术引入MOBSs分析中。首先采用盐诱导相分离-微型液液萃取尿样中的MOBSs，以去除尿样基质的干扰；接着采用N,N-二甲基乙二胺（DMED）和 d4-DMED对目标MOBSs进行衍生，从而将质谱易电离的叔胺基团和轻/重同位素标签引入MOBSs，以提高检测灵敏度和准确度；最后结合LC-MS/MS分析，建立尿样中七种MOBSs高灵敏的同时测定的新方法。结果表明，经DMED衍生后，七种MOBSs的检测灵敏度提高了3.4~105.9倍，所建立的方法线性关系良好，线性相关系数R2为0.9978~0.9997，七种MOBSs的检出限在2-9ng/L之间，远远低于文献已报道的方法。在实际尿样中的加标回收率为68.5%-121.7%，日内相对标准偏差小于12.8%，日间相对标准偏差小于14.8%。将建立的方法应用于31名志愿者尿样中MOBSs的检测，每份志愿者尿样均检测出一定含量的MOBSs，说明本方法具有较强的实际应用价值。本项目的实施可为苯系物环境暴露生物监测以及科学制定综合的苯系物生物限值标准提供技术支撑。