| 题名 | 几种新型持久性有机污染物及其蛋白结合物的色质联用分析研究 |
| 作者 | 马强 |
| 学位类别 | 博士 |
| 答辩日期 | 2012 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郭良宏 |
| 关键词 | 持久性有机污染物 persistent organic pollutants 牛血清白蛋白 bovine serum albumin 超高效液相色谱 ultra-performance liquid chromatography 离子淌度质谱 ion mobility-mass spectrometry 电喷雾质谱 electrospray ionization-mass spectrometry |
| 其他题名 | Studies on the Analysis of Several Novel Persistent Organic Pollutants and Their Protein Complex by Liquid Chromatography Coupled with Mass Spectrometry |
| 中文摘要 | 随着社会经济的高速发展，各种化学品的合成和使用正以前所未有的速度增加，导致各种危害环境和人体健康的污染事件时有发生。持久性有机污染物作为通过各种环境介质长距离迁移并长期存在于环境中，对人类健康和环境具有严重危害的有机污染物质，具有长期残留性、生物蓄积性、半挥发性和高毒性，已日益成为备受关注的全球性环境问题。本文针对羟基化多溴联苯醚、甲氧基化多溴联苯醚、烷基酚聚氧乙烯醚等几种新型持久性有机污染物及其蛋白结合物，采用超高效液相色谱、离子淌度质谱、电喷雾质谱等技术，开展了其色质联用分析方法研究。论文主要包括以下几方面研究内容： 羟基化多溴联苯醚的超高效液相色谱-离子淌度质谱全二维分离分析研究。集成了超高效液相色谱技术与离子淌度质谱技术，构建了针对23种羟基化多溴联苯醚（羟基化一溴联苯醚至羟基化八溴联苯醚）的分离分析体系。其中，第一维反相超高效液相色谱分离采用基于亚二微米小颗粒填料的Waters ACQUITY UPLC BEH C18（150 mm × 2.1 mm, 1.7 µm）色谱柱，通过乙腈-水流动相梯度洗脱，流速梯度变化，不同溴原子数目的羟基化多溴联苯醚同系物及其同分异构体按照疏水性差异产生分离；第二维分离采用离子淌度质谱技术，在8.80 ms内完成了不同溴系羟基化多溴联苯醚的快速分离和检测。本研究开发建立的全二维分离系统的正交性相关系数为0.9665，峰分散角为14.87°，实际峰容量达到135，分别为单独应用第一维分离和第二维分离的2倍和15倍，峰容量有显著提高。所构建全二维分离分析体系对于去除质谱背景噪音干扰，提高待测化合物灵敏度和确证可信度，也显示了较好的应用前景。 甲氧基化多溴联苯醚的超高效液相色谱-离子淌度质谱全二维分离分析研究。采用超高效液相色谱-离子淌度质谱联用技术，构建了针对27种甲氧基化多溴联苯醚（甲氧基化二溴联苯醚至甲氧基化八溴联苯醚）的分离分析体系。第一维超高效液相色谱采用基于亚二微米小颗粒填料的Waters ACQUITY UPLC HSS T3（150 mm × 2.1 mm, 1.8 µm）色谱柱，通过甲醇-水流动相梯度洗脱，不同溴原子数目的甲氧基化多溴联苯醚同系物及其同分异构体按照疏水性差异产生分离；第二维离子淌度质谱采用大气压光电电离源，实现了甲氧基化多溴联苯醚系列弱极性组分的有效离子化，并完成了多种类、多组分甲氧基化多溴联苯醚的毫秒级快速分离。通过超高效液相色谱技术和离子淌度质谱技术的偶联，实现了对甲氧基化多溴联苯醚同时基于疏水性差异和离子淌度差异的全面分离。采用飞行时间质谱质量分析器，可完成数据信息的实时快速采集与分析。 烷基酚聚氧乙烯醚的亲水相互作用色谱-离子淌度质谱全二维分离分析研究。构建了集成亲水相互作用色谱技术和离子淌度质谱技术的全二维体系，并应用于辛基酚聚氧乙烯醚和壬基酚聚氧乙烯醚的分离分析。第一维亲水相互作用色谱采用高纯硅胶填料和与电喷雾质谱很好兼容的乙腈-水流动相梯度洗脱，根据烷基酚聚氧乙烯醚乙氧基链的长度，实现了不同聚合度的烷基酚聚氧乙烯醚的理想分离。在保持第一维分离效果的基础上，第二维离子淌度质谱技术，可基于烷基酚聚氧乙烯醚的尺寸、形状和电荷不同，在13.0 ms内实现了烷基酚聚氧乙烯醚的快速分离。所构建的亲水相互作用色谱-离子淌度质谱全二维体系对辛基酚聚氧乙烯醚的正交性相关系数为0.2191，峰分散角为77.34°；对壬基酚聚氧乙烯醚的正交性相关系数为0.1490，峰分散角为81.43°。该全二维分离分析体系的实际峰容量达到972，分别是单独使用第一维分离和第二维分离的7倍和122倍。此外，离子淌度分离技术还可用于烷基酚聚氧乙烯醚的携带不同电荷的同分异质体离子的快速分离，为不同聚合度的烷基酚聚氧乙烯醚的高通量分离和准确测定奠定了坚实基础。 基于电喷雾质谱的羟基化多溴联苯醚与牛血清白蛋白的非共价相互作用研究。羟基化多溴联苯醚对生物体具有多种毒性效应，可造成甲状腺激素转运和代谢紊乱，并可引起神经毒性、细胞毒性和内分泌干扰效应。本研究采用电喷雾质谱法，对具有不同溴原子数目及不同取代位点和不同羟基位点的24种羟基化多溴联苯醚与牛血清白蛋白的非共价相互作用进行了系统研究。结果表明，2种羟基化七溴联苯醚（6-OH-BDE-180和4-OH-BDE-187）和1种羟基化八溴联苯醚（4’-OH-BDE-201）与牛血清白蛋白形成了化学计量比分别1:1和1:2的非共价复合物，并对它们的解离常数进行了计算。本研究的实验结果表明，羟基化多溴联苯醚与血清蛋白具有一定的结合能力，可能干扰甲状腺素与血清蛋白的正常结合，影响其正常生理功能。进一步采用离子淌度质谱技术，对牛血清白蛋白结合羟基化多溴联苯醚前后的构象变化进行了研究，推测羟基化多溴联苯醚在与牛血清白蛋白结合时，可能诱导其构象发生某种变化，使其有利于与羟基化多溴联苯醚的非共价结合。 |
| 英文摘要 | With the rapid development of our social economy, the synthesis and application of chemicals has increased dramastically. The pollution incidents occur from time to time, which bring about considerable hazard and risk to human health and the environment. Persistent organic pollutants (POPs) are organic compounds that are resistant to environmental degradation. Because of this, they have been observed to persist in the environment, to be capable of long-range transport, bioaccumulate in human and environment and to have potential significant impacts on human health and the environment. POPs have been an environmental issue of global concern. In this study, analytical methods based on ultra-performance liquid chromatography (UPLC), ion mobility-mass spectrometry (IM-MS), and electrospray ionization mass spectrometry (ESI-MS) have been developed for the analyses of several novel POPs, including hydroxylated polybrominated diphenyl ethers (OH-PBDEs), methoxylated polybrominated diphenyl ethers (MeO-PBDEs), and alkylphenol ethoxylates (APEOs), and their protein complex. The main contents of the dissertation are listed as follows: Comprehensive two-dimensional separation of OH-PBDEs by UPLC coupled with IM-MS. A comprehensive two-dimensional system, hyphenating UPLC with IM-MS, was constructed for the separation and analysis of 23 OH-PBDE congeners, ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl ether (OH-octaBDE). The first-dimensional reversed-phase UPLC was performed on a sub-2 µm Waters ACQUITY UPLC BEH C18 (150 mm × 2.1 mm, 1.7 µm) chromatographic column using acetonitrile-water gradient elution program with a flow rate ramp. It enabled excellent chromatographic separation for both between-class and within-class OH-PBDEs based on their differences in hydrophobicity. Following the pre-ionization resolution in the first dimension, the second-dimensional IM-MS added an extra post-ionization separation for between-class OH-PBDE congeners on account of their relative mobility disparity during a very short period of 8.80 ms. The orthogonality of the developed two-dimensional system was evaluated with the correlation coefficient of 0.9665 and peak spreading angle of 14.87°. The actual peak capacity of the system was calculated to be 135, approximately 2 and 15 times higher than that of the two dimensions used alone, respectively. The two-dimensional separation plane also contributed to the removal of background interference ions and the enhanced confidence in the characterization of OH-PBDEs of interest. Comprehensive two-dimensional separation of MeO-PBDEs by UPLC coupled with IM-MS. Combining UPLC with IM-MS, a two-dimensional system was constructed for the separation and analysis of 27 MeO-PBDE congners, ranging from methoxylated dibromodiphenyl ether (MeO-diBDE) to methoxylated octabromodiphenyl ether (MeO-octaBDE). The first-dimensional reversed-phase UPLC was performed on a sub-2 µm Waters ACQUITY UPLC HSS T3 (150 mm × 2.1 mm, 1.8 µm) chromatographic column using methanol-water gradient elution program. It enabled excellent chromatographic separation for both between-class and within-class MeO-PBDEs based on their differences in hydrophobicity. An atmospheric pressure photo ionization (APPI) source was utilized, which offered efficient ionization for low-polarity MeO-PBDEs. The second-dimensional IM-MS added an extra post-ionization separation for between-class MeO-PBDE congeners on account of their relative mobility disparity within a millisecond timescale. By virtue of the combination of UPLC and IM-MS, the developed two-dimensional system realized the comprehensive resolution according to both hydrophobicity difference and mobility disparity for MeO-PBDE congeners. The fast-scanning time-of-flight (TOF) mass analyzer enabled the real-time high-speed data acquisition and processing. A comprehensive two-dimensional system coupling hydrophilic interaction chromatography (HILIC) and IM-MS has been developed for the separation and analysis of APEOs. The first-dimensional HILIC was performed on porous silica stationary phase using acetonitrile-water gradient elution, which was readily compatible with ESI, and enabled good chromatographic separation of APEO oligomers on account of their differences in ethoxy chain length. Maintaining the fidelity of pre-ionization resolution in the first dimension, the second-dimensional IM-MS added an orthogonal post-ionization separation for APEOs based on their size, shape and electric charge during a very short period of 13.0 ms. The orthogonality of the developed two-dimensional system was evaluated with the correlation coefficient and peak spreading angle of 0.2191 and 77.34° for octylphenol ethoxylates (OPEOs), and 0.1490 and 81.43° for nonylphenol ethoxylates (NPEOs). A significant enhancement in peak capacity was achieved for the comprehensive two-dimensional plane with the actual peak capacity calculated to be 972, approximately 7 and 122 times higher than that of the two dimensions used alone, respectively. The attractive potential for removing the effects of isobaric interference of APEOs by the rapid and solvent-free ion mobility approach was also highlighted. Study on the noncovalent complexes of OH-PBDEs and bovine serum albumin (BSA) by ESI-MS. There are indications that the exposure to OH-PBDEs may cause thyroid hormone transport and metabolism disorder, neurotoxicity, cytotoxicity, and endocrine disruption. The noncovalent binding of 24 OH-PBDE congeners ranging from OH-monoBDE to OH-octaBDE with BSA was studied by ESI-MS. Among the OH-PBDEs investigated, it was observed that BSA interacted preferentially with 2 hydroxylated heptabromodiphenyl ethers (OH-heptaBDEs) (6-OH-BDE-180 and 4-OH-BDE-187) and 1 OH-octaBDE (4’-OH-BDE-201). The 1:1 and 2:1 binding ratio of the OH-PBDE/BSA complex peaks were observed in the mass spectra. The corresponding dissociation constants were determined. The experimental results therefore revealed that OH-PBDEs may interact with BSA and interfere the binding of thyroxine with BSA and hence its normal physiological function. IM-MS was further utilized to examine the possible conformational change of BSA before and after the binding with OH-PBDEs. Presumably, the conformation of BSA may be induced to change when binding with OH-PBDEs to favor the non-covalent binding. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/35115]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 马强. 几种新型持久性有机污染物及其蛋白结合物的色质联用分析研究[D]. 北京. 中国科学院研究生院. 2012. |
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