芳香环状低聚物组分分布与单体结构关系的研究

根据Jacoson-Stockmayer(J-S)环化理论,应用基质辅助激光解吸离子化飞行时间质谱(MAIDL-TOFMS),对一系列芳香环状低聚物组分分布进行分析,研究了芳香环状低聚物的组分分布与单体结构的关系,结果表明,在芳香聚酯、聚膦酸酯及芳香聚醚环状低聚物系列中,Inc_n与Inn呈良好的线性关系,符合J-S理论分布,在环状聚酯及聚膦酸酯系列中,低聚物的组分分布受双酚单体的中心键角影响,单体的中心键角在100°～120°之间,其中心键角愈小,γ值愈大,而在聚醚系列中,在固定一种单体的前提下,环化物的组分分布受另一单体键角的影响与环状聚酯和聚膦酸酯相一致。

Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.

HPLC与MALDI-TOFMS联用技术分析蛋黄中的磷脂

蛋黄中含有大量磷脂,其中磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)最为丰富。本研究采用高效液相色谱法(HPLC)与基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)联用技术分析了蛋黄中磷脂粗提物。将从蛋黄中提取的多种磷脂通过HPLC预先分离,收集各组分后分别进行MALDI-TOF MS分析得到比较清晰的质谱图。通过质谱图解析确定了蛋黄中磷脂酰胆碱、神经鞘磷脂(SM)的脂肪酸组成。

在线纯化技术应用于MALDI-TOFMS测定溶菌酶的分子量

在用基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)测定蛋白质分子量的过程中,一些盐和蛋白质变性剂经常大大抑制样品信号,产生一些难以解析的离子峰,因此测试前应尽可能去除样品中的添加剂。为此,本研究建立了MALDI—TOFMS测试中在线纯化蛋白质样品的新方法。采用硝酸纤维素膜作为固相载体,将标准蛋白质溶菌酶制成含6 mol/L盐酸胍变性剂、2％SDS表面活性剂的100 mmol/L Tris—HCL溶液进行质谱测定。结果表明:新方法简单、快速,可明显增强离子峰的强度,提高测定蛋白质分子量的灵敏度。

UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.

Accurate mass-based analysis in human sport doping control : Application of liquid chromatography - time-of-flight mass spectrometry

Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.

中药青蒿萜类代谢物谱分析及青蒿素生物合成的研究

青蒿素是从中药青蒿，学名黄花蒿（Artemisia annua L.）植物地上部分分离出的抗疟疾有效单体，为一种倍半萜内酯类化合物，其生物合成途径属于植物类异戊二烯代谢途径。青蒿素生物合成途径及其调控机制仍不完全清楚，本论文采用GC-MS 和GC×GC-TOFMS 方法对青蒿萜类代谢物谱进行检测，用多维统计学方法对检测结果进行整理和比较分析，研究青蒿素生物合成及其与青蒿中其他萜类代谢的关系，取得了以下结果： 一、通过GC×GC-TOFMS 方法对青蒿挥发油成分进行分析，共鉴定出303 种组分。其中挥发油中相对百分含量大于1%的10 种组分中有9 种为萜类化合物，含量接近总挥发油的50%。在相对百分含量大于0.1%的49 种成分中，有30 种萜类化合物。有27 种相对百分含量大于0.1%的成分首次在青蒿挥发油中报道，其中包括10 种萜类化合物。 二、利用GC-MS 方法分析了青蒿001 和SP18 两个青蒿素高产株系不同生长时期萜类代谢物谱，结果表明：青蒿中萜类化合物在不同时期合成和积累是动态变化的，萜类化合物的种类和数量在营养生长期随生长时间的延长而提高，在营养生长后期和现蕾前期达到最高水平，进入生殖生长后随生长时间的延长而迅速降低。通过多维统计PLS-DA（Partial Leasted Square Discriminant Analysis） 分析，确定001 中有17 个化合物的含量在不同生长时期有明显变化，其中15 个为萜类化合物。SP18 中有18 个化合物的含量在不同生长时期有明显变化，其中16 个为萜类化合物。青蒿素，青蒿酸，二氢青蒿酸，青蒿素B 都是含量变化明显的标记物。其中青蒿酸和二氢青蒿酸含量在营养生长后期达到最高水平，进入生殖生长后迅速下降，而青蒿素和青蒿素B 在整个检测时期含量变化相对较小，在营养生长时期含量已经较高，在现蕾前期含量稍有上升，进入现蕾期后有所下降，本研究确定现蕾前期为代谢物谱分析最佳取样时期，并为药材采收提供指导。 三、不同基因型青蒿代谢物谱研究表明，青蒿素高产株系SP18 和001 代谢物表现出一定的差异，通过多维统计PLS-DA 分析，共找出了22 种在两种基因型中差异明显的化合物，其中包括倍半萜化合物12 种，单萜化合物3 种，三萜化合物4 种。SP18 特征化合物为樟脑和两个未鉴定倍半萜化合物，而001 特征化合物是龙脑和β-法呢烯。另外两种基因型中青蒿素及相关前体化合物的积累模式差异明显，SP18 中二氢青蒿酸和青蒿素含量高，而青蒿酸和青蒿素B 含量极低；001 中二氢青蒿酸和青蒿素含量相对SP18 要低，但青蒿素B 和青蒿酸含量比SP18 要高。该结果表明在青蒿素高产株系中，青蒿素含量与二氢青蒿酸的含量呈正相关，结合Brown 等的活体标记研究结果分析，从二氢青蒿酸到青蒿素的转化可能是青蒿素合成的限速步骤。 四、利用GC×GC-TOFMS 方法对转基因青蒿萜类代谢物谱进行了分析，共对200 个左右化合物峰进行PLS-DA 和OSC-PLS (Orthogonal Signal Correction–Partial leasted Square)多维统计分析，结果表明：青蒿萜类代谢物谱在外源基因转入后发生显著变化，与对照株系相比均呈现显著差异。其中过量表达Amorpha-4,11-diene 合酶基因（ads）株系中青蒿素及相关化合物变化最明显，而过量表达FPP 合酶基因（fps）株系中青蒿素及相关化合物变化相对较小，在受到调控而成为差异标记物的化合物中，70%是倍半萜类化合物。 五、考察了外源茉莉酸甲酯对青蒿素生物合成的影响，结果表明：300 μM 外源茉莉酸甲酯能提高青蒿素含量，在处理后第8 天青蒿素含量提高38%。青蒿萜类代谢物谱研究表明，茉莉酸甲酯不仅可以诱导青蒿中青蒿素的合成，还能诱导很多化合物，特别是倍半萜和三萜类的合成。OSC-PLS 分析结果找出了9 个处理后含量明显提高的标记物，其中6 个倍半萜化合物，3 个三萜化合物。标记物鲨烯含量提高了67%，另一个未鉴定出结构的倍半萜提高了60%，这些化合物可能与青蒿素有着类似的调控机制，而外源喷洒茉莉酸甲酯可以作为提高青蒿素产量的有效途径之一。

长白山白眉蝮蛇蛇毒中酶类的纯化和表征

本论文系统研究了长白山白眉蝮蛇蛇毒中四种具有抗栓活性及可以作为基础理论研究的工具酶的分离纯化，并进行了扩大化试验，得到了可直接用于工业生产的分离工艺流程。并采用SDS－聚丙烯酰胺凝胶电泳（SDS－PAGE）、基质辅助激光解吸电离飞行时间质谱（MALDI－TOFMS）等分析手段确认了这四种酶的纯度及分子量，共获得以下几种质谱纯和电泳纯的酶A_2，其分子量为14.0kDa；三种精氨酸酯酶（AEasel、 AEase2和AEase3），它们的分子量分别为29.9kDa、27.5kDa、和28.8kDa；L－氨基酸氧化酶，其分子量为92.8 kDa；以及纤溶酶，其分子量为23.3 kDa。并对几种酶的质谱行为进行了系统研究，考察了酶的浓度等因素对多聚体多电荷离子峰形成的影响，并给出了它们的形成机理。上述酶中，我们按其分子量的大小、亚基组成以及氨基酸残基数目与文献报道的已知的酶相比较，认为L－氨基酸氧化酶、磷脂酶A_2和精氨酸酯酶2(Aease2)和精氨酸酯酶3(AEase3)是酶的新的类型。系统研究了所获得的四种酶的金属离子的含量，指出了磷脂酶A_2和纤溶酶均为Ca~(2+)离子结合蛋白，Ca~(2+)离子是酶分子活性所必需的。精氨酸酯酶和L－氨基酸氧化酶则为含Zn~(2+)酶，每个酶分子中分别含有两个和四个Zn~(2+)离子，它们仅起到稳定蛇毒酶的高级结构的作用，去除后对活性影响不大，并考察了多种金属离子对酶的活性和荧光光谱的作用。本文系统研究了四种酶的荧光光谱，用同步荧光光谱法了解了酶分子中略氨酸和色氨酸残基的微环境；用荧光淬灭方法，研究了其淬灭作用及机理，并讨论了荧光基团所处的环境。另外，还系统表征了几种酶的物理化学性质、酶学性质，得到了许多有意义的结果。

Friedel-crafts反应合成芳香环状齐聚物与开环聚合

芳香环状低聚物的合成是二十世纪八十年代末发展起来的研究领域，其特有的环状结构和可进行可控的开环聚合决定了芳香环状低聚物具有广阔的应用前景。本文从研究单体结构与成环反应的关系出发，开拓了一种合成芳香环状聚醚酮的新方法—改进的Friedel-Crafte反应法，采用该方法成功地合成了一系列新型结构的大环化合物，并首次利用流变仪对开环聚合过程中的流变行为进行了较为系统的观测。根据acoson-Stockmayer环化理论，应用基质辅助激光解吸离子化飞行时间质谱（MAIDL-TOF MS），对一系列芳香环状低聚物组分分布进行分析，研究了芳香环状低聚物的产率、组分分布与单体结构的关系。结果表明：芳香聚醋、聚麟酸醋及芳香聚醚环状低聚物系列中，InCn与1nn呈良好的线性关系，符合J-S理论分布。环状齐聚物的产率与组分分布受单体的中心键角影响，单体的中心键角在100°～120°范围内，其中心键角愈小，γ值愈大。γ值愈大，反应产物中小环化合物的含量越高，而小环化合物的含量的增加是高产率地合成环状齐聚物的前提之一。在此理论的指导下，通过对单体结构的模拟，高选择性地合成了一种新型结构的芳香环状聚硫醋二聚体，对其结构进行了精确的表征，在不同溶剂中得到了该环状二聚体的单晶，单晶X闪ray衍射表明该二聚体为环张力极小的大环化合物。基于上述理论，以有利于成环的邻苯二酞氯为酞基化试剂，对Friedel-Crafts酞基化反应在合成芳香环状齐聚物中的应用进行了系统研究，开拓了一种合成芳香环状预聚体的新方法—改进的Friedel-Crafts酞基化反应法。发现反应体系中Lewis碱的存在有利于选择性地形成环状产物。并进一步确定反应最佳条件为： Lewis碱和催化剂Alcl3与富电单体的摩尔比分别为1.2和3.4; 1,2-二氯乙烷为本反应的最佳溶剂；等当量的反应单体要求缓慢滴加到形成“假高稀”的溶剂体系中；Lewis碱NMP，DMF等都适用于本反应体系。在此优化条件下，以邻苯二酞氯和间苯二酞氯为酞基化试剂，室温下，合成了一系列芳香环状聚醚酮酮、聚醚酮、聚醚矾酮等新型结构的环状齐聚物，利用MALDI-TOF-MS，NMR，GPC，FTIR，DSC，元素分析等手段对环状结构进行了精确的表征；DSC分析表明含邻苯二拨基结构的环状齐聚物为无定型材料；部分产物的产率高达90％。在阴离子引发剂联苯双酚钾存在下，制备的环状齐聚物成功进行了熔融开环聚合，得到了相应结构的高分子量的线性开环聚合产物。其中，含邻苯二拨基结构的环状聚醚酮酮、环状聚醚酮矾的开环聚合产物的比浓粘度分别达到0.42dL／g，0.36 dL／g（0.5％的DMF溶液，25士0.1℃）；四种含间苯二锁基结构的环状齐聚物的开环聚合产物的Tg与常规亲电沉淀反应合成的线性高聚物的Tg相同。含侧甲基的开环聚合产物的Tg比对应的开环聚合的产物的Tg高约5℃。研究结果表明用亲电缩聚方法制备芳香环状聚醚酮与亲核缩聚法相比较，具有成本低廉、反应条件温和丫产率高、易于大规模制备等优势，开拓了一种制备环状化合物的方法。自从美国G.E.公司利用环状聚碳酸酷的开环聚合制备线性聚碳酸醋以来，对芳香环状低聚物的开环聚合过程的研究仅局限在由GPC监测反应某一时刻的产物的分子量，而缺乏对与应用更为接近的开环聚合中的粘度的变化的研究。本文以界面缩聚反应高产率地合成芳香环状双酚A聚酷二聚体为对象，研究了流变仪在开环聚合中的应用。利用流变仪对环状二聚体开环聚合过程进行了较为系统的观测，研究了不同条件下的开环聚合中的流变行为，结果表明，开环聚合存在引发期，而且在引发期，熔融体的粘度低于10Pa·S，超过引发期，粘度呈指数级增长。引发期的长短可以通过引发剂的种类、浓度、开环聚合的温度等条件进行有效地控制。芳香环状聚酷二聚体与环状聚碳酸醋的开环共聚合的流变行为的研究结果表明：开环共聚合可以降低开环聚合的温度，调整引发期，是提高聚合产物的分子量的有效途径。用流变仪对以改进的Friedel-Crafts反应合成的芳香环酮齐聚物的开环聚合中的流变行为进行了监控。在330℃，剪切速率为0.05S-l下，熔融的环状齐聚物的粘度为2.0Pa·S。通过对开环聚合的反应条件的控制，同样实现了开环聚合的可控，通过改变其开环聚合的引发期的长短及粘度的变化规律，可、适应不。条一定为加工设计与成型加工提供理论指导和模型设计，必将进一步推进开环聚合工 艺向应用方向的发展。

Matrix-assisted laser desorption/ionization mass spectrometry using porous silicon and silica gel as matrix

Porous silicon powder and silica gel particles have been applied as inorganic matrices for the analysis of small molecules in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS). In contrast to conventional MALDI-TOFMS, the signal interference of low-molecular analytes by the matrix has been eliminated. Almost no fragmentations of the analytes were observed. Effects of various factors, such as the particle and pore size, the suspending solution, and sample preparation procedures, on the intensity of mass spectra have been investigated. The pore structure of the inorganic matrix and penetration of the analytes into the pores must be optimized for effective desorption and ionization of the analytes. Matrices (DHB and HCCA) were covalently bound to silica gel for improvement of spectrum intensity. Copyright (C) 2001 John Wiley & Sons, Ltd.

利用MALDI-TOF质谱技术研究MPEG-b-PCL两嵌段共聚物的嵌段长度及嵌段分布

利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)结合源后分解(PSD)技术对甲氧基封端的聚乙二醇-b-聚己内酯(MPEG-b-PCL)两嵌段共聚物进行了结构分析.根据得到的MALDI-TOFMS谱图和PSD碎片信息清晰地确定了嵌段共聚物的嵌段长度和嵌段分布.结果表明,采用MALDI-TOFMS结合PSD技术研究这类嵌段共聚物链结构非常有效.这为更好地认识和应用这类嵌段共聚物提供了重要的依据,同时也建立了分析这类嵌段共聚物的方法.

三聚氰胺的基质辅助激光解吸/电离飞行时间质谱研究

Oxidized carbon nanotubes are tested as the matrix for analysis of the melamine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Traditional MALDI matrix are not suit for analysis of the low molecular compounds due to the interference associated to the matrix clusters. Oxidized carbon nanotubes can transfer energy to the analyte under the laser irradiation, which makes analyte well ionized or desorbed. Moreover, the interference of the intrinsic matrix ions can be eliminated. Melamine as the a toxic additive which had been added in the milk powder, then it is necessary to establish a new method for detection of the melamine rapid and sensitive.

基质辅助激光解吸电离飞行时间质谱表征细胞色素C

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF—MS) technique was used for analysis of moleculear weight of cytochrome C.The effects of three kinds of matrix,such as 2,5-dihydroxybenzoic acid(DHB),a-cyano-4-hydroxycinnamic acid(a-CHC) and sinapinic acid(SA),were used to compared and a suitable a-CHC was found.Experimental data showed that this method was properable to analysis of the congeneric biochemical samples.

马鹿茸多肽的提取分离及基质辅助激光解吸飞行时间质谱分析

In this paper,active components of velvet antler polypeptide were extracted and separated.The molecular weight and purity of velvet antler polypeptide were determined by MALDI-TOFMS.The different influence factors such as matrix,sample concentration and laser energy were studied.This method is convenient and suitable for the identification of congener biochemical samples.