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题名:
海洋浮游植物色素UHPLC-MS/MS检测及应用研究
作者: 张晶晶1,2
学位类别: 博士
答辩日期: 2017-05-18
授予单位: 中国科学院大学
授予地点: 北京
导师: 刘东艳
关键词: 浮游植物 ; 特征色素 ; HPLC ; UHPLC-MS/MS ; 碎裂模式 ; 黄海 ; 粒级结构 ; 叶绿素a
学科分类: 地球科学 ; 环境科学技术
中文摘要: 浮游植物是海洋生态系统中最主要的初级生产力,其群落结构及其丰度不仅能够影响上层海洋食物网和生物地球化学循环过程,而且还能够影响气候变化,减轻温室效应带来的不利影响。浮游植物色素是指示其群落结构变化的重要指标,因此,准确检测浮游植物色素是海洋生态技术研究的重要内容。目前,常用的高效液相色谱(HPLC)色素检测方法具有一定的局限性,存在灵敏度与准确度较低的缺点。本研究针对该问题,开发了两种灵敏度高、准确度高的超高效液相色谱串联质谱(UHPLC-MS/MS)方法,可以同时检测多种浮游植物色素,探索了该方法在海水样品浮游植物色素检测中的可行性,并应用于浮游植物粒级分析。主要的研究内容和结果包括如下三个方面:
 
(1)建立了高灵敏度的UHPLC-MS/MS方法,可同时检测14种浮游植物特征色素。
        使用电喷雾离子源正离子模式,采用内标法定量。在优化的色谱质谱条件下,14种色素在15分钟内分离,并且线性关系较好(14种色素线性相关系数范围为0.9915-0.9991)。14种色素的定量限范围为0.005-0.500 μg/L;重复性和重现性均小于15%;在两个添加浓度水平下,平均回收率在62.8-120.2%之间。与以前的方法相比,UHPLC-MS/MS色素检测方法不仅明显提高了检测效率,而且灵敏度高、定量准确度高,可以准确分析复杂海水样品中的痕量浮游植物色素组分。
(2)通过子离子形成的特征碎裂模式,扩展了UHPLC-MS/MS方法可检测的色素种类。
        由于UHPLC-MS/MS方法需要通过色素标准品单标获取色素组分的特征离子对,使得可检测的色素种类受到标准品单标的限制。本研究在电喷雾离子源正离子模式下,分析了38种色素的子离子形成模式。基于色素子离子形成的特征碎裂模式,推导了无标准品色素的特征子离子及其相应的碰撞能。在20分钟内,成功的从DHI混合标准中分离检测到64种色素(包含26种无单标的色素)。该方法在同时检测64种色素时,仍然具有非常高的灵敏度,36种色素的定量限在0.2-0.005 μg/L之间。该方法为使用UHPLC-MS/MS对更多痕量无标准品色素进行分析提供了新的思路。
(3)探讨了UHPLC-MS/MS方法在测定黄海浮游植物粒级分类中应用的可行性。
         2015年8月间,在黄海47个站位取表、中、底三层水样进行了分析,对比了超高效液相色谱(UHPLC)和UHPLC-MS/MS两种方法的海水样品色素检测结果。通过分析发现UHPLC-MS/MS的灵敏度较高,但多数情况下两种方法检测结果吻合度较好,两种方法均适用于浮游植物色素样品的测定。叶绿素a浓度和浮游植物粒级结构空间分析结果显示,山东半岛近岸和长江口东北侧海域,叶绿素a浓度较高,小型浮游植物占优势;黄海冷水团海域,叶绿素a浓度较低,微型和微微型浮游植物比例明显升高。与历史数据相比,空间分布趋势基本一致。尽管叶绿素a绝对浓度和不同粒径浮游植物的贡献率存在一定差异,但这与调查时间、站位以及粒级分析方法的差异有较大关系,需进一步的探索。
 
英文摘要:         Phytoplankton are the most important primary producers in marine ecosystems, and their community and biomass could not only put impact on food web and biogeochemical cycles, but also influence climate change and mitigate the effects of global warming. As an important biological marker for phytoplankton community, phytoplankton pigment detection plays significant role in research of marine ecological technology. High performance liquid chromatography (HPLC), which is commonly used to analyze phytoplankton pigments, still has some limitations (e.g., Low sensitivity and low accuracy). Concerning this issue, this study developed two ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) methods for multiple pigments analyzing with high sensitivity and accuracy, tested their feasibility of detecting phytoplankton pigments in seawater samples, and applied them for analyzing phytoplanktion size fraction. The main research contents and results include the following three aspects:
(1) Development of a sensitive and specific UHPLC–MS/MS method for analyzing 14 marker pigments
        14 marker pigments were determined and validated using an internal standard (IS) method under electrospray ionization (ESI) in positive mode. Under the optimized conditions of chromatogram and mass spectrum, 14 types of pigments were separated in less than 15 min and presented good linearity, with the coefficients ranging from 0.9915 to 0.9991. The limit of quantification (LOQ) attained for 14 pigments were 0.005-0.500 μg/L, which enabled quantification of the pigments in seawater at ppt level. Both intra-day and inter-day precisions were less than 15%. The method was validated using seawater samples and the recovery of the analytes ranged from 62.8% to 120.2% at two spiked concentrations. Compared with previous studies, UHPLC–MS/MS method takes the advantage of improved efficiency of qualitative analysis and significantly decreased detection LOQ on analyzing marker phytoplankton pigments at trace/ultra-trace levels in complex matrices.
(2) Increase of the identifiable pigment types by UHPLC-MS/MS method based on diagnostic fragmentation patterns
        The application of the UHPLC-MS/MS method is limited due to the lack of commercial standards. In this research, the transitions of 38 pigments and their formation patterns during electrospray ionisation (ESI) in positive mode were studied. Based on the diagnostic fragmentation patterns, the transitions and collision energy of more types of pigments were derived, and 64 pigments (including 26 types of pigment without pure standards) were determined from the mixed standards in one injection within 20 min. The method presented high sensitivity even when determined 64 pigments at one injection with the limit of quantitation (LOQ) of 36 types of pigments ranging from 0.2-0.005 μg/L. The developed method expanded the application of UHPLC-MS/MS to pigment without standards, and it can be used for analyzing more pigments with those of known structures.
(3) Feasibility study of UHPLC-MS/MS method for size fraction analysing
        Seawater samples collected from 3 different water layers at 47 sites in the Yellow Sea in August 2015 were analyzed. Comparisons were carried in terms of pigment concentrations acquired by ultra-high performance liquid chromatography (UHPLC) and UHPLC-MS/MS. No significant differences were observed from the result comparisons for most cases although UHPLC-MS/MS were more sensitive, and two methods were both suitable for pigment detection. Based on the pigment results provided by UHPLC-MS/MS, the spatial distribution of chlorophyll a (Chl a) and size fraction were studied. The concentration of Chl a was much higher in the northeast of Yangtze River mouth and coastal waters of Shandong Peninsula, where micro phytoplankton was the dominant size fraction. The concentration of Chl a was much lower in the central Yellow Sea, and the contribution of nano- and pico- phytoplankton increased significantly in this region. Compared with previous studies, the absolute concentrations of Chl a and contribution of size fractions in our study showed some differences, which may be caused by the differences of investigation time, sites or analytical methods. Thus, more comparison study is still needed to further probe the reason behind these differences.
内容类型: 学位论文
URI标识: http://ir.yic.ac.cn/handle/133337/22009
Appears in Collections:中科院烟台海岸带研究所知识产出_学位论文

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作者单位: 1.中国科学院烟台海岸带研究所
2.中国科学院大学

Recommended Citation:
张晶晶. 海洋浮游植物色素UHPLC-MS/MS检测及应用研究[D]. 北京. 中国科学院大学. 2017.
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