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pH对SPE-HPLC-ESI-Q-TOF-MS检测海水中小分子活性物质的影响研究
张倩1,2,3,4, 宋金明1,2,3,4, 李学刚1,2,3,4, 彭全材1,2,3,4, 袁华茂1,2,3,4, 李宁1,2,3,4, 段丽琴1,2,3,4
1.中国科学院海洋生态与环境科学重点实验室 中国科学院海洋研究所 青岛 266071;2.青岛海洋科学与技术国家实验室海洋生态与环境科学功能实验室 青岛 266237;3.中国科学院大学 北京 100049;4.中国科学院海洋大科学研究中心 青岛 266071
摘要:
海水溶解有机物(dissolved organic matter,DOM)中含有的生物活性物质在海洋生态系统中作用巨大,但因缺乏适合的分离提取方法而严重阻碍了对其不同组分在生态系统中作用的探索。固相萃取法对富集提取海水DOM十分有效,在用其提取海水DOM时,海水pH对活性物质提取效果的影响很大,但目前针对海水的这种影响尚存在很大争议。本文以天然近海海水作为基质,探究不同pH条件下用亲水-疏水平衡(hydrophilic-lipophilic balanced,HLB)固相萃取小柱萃取海水中活性分子的提取效率,并使用高效液相色谱-四极杆飞行时间质谱(HPLC-Q-TOF-MS)在负离子(ESI-)模式下检测解析提取物的组成。研究结果表明,当海水样品pH为中性和强酸性时都能获得较好的提取效率,随着pH的降低,提取物质谱的整体响应值降低,但可识别的谱峰数目增加,提取出有机物的分子量和性质差异都更广泛。分析提取物分子在范克雷维伦(van Krevelen)图和质荷比-氢碳比(m/z-H/C)图上的分布发现,中性条件适合提取饱和度较高的小分子化合物,而具有生物活性的带有不饱和基团的化合物及蛋白质、糖类等生物大分子在强酸性提取条件时提取效果和分辨率更好。综合提取效率、有效峰数目和分子组成特征考虑,用HLB固相萃取小柱提取近海海水中的小分子活性物质时,将海水样品pH调节为2较为适宜。
关键词:  有机小分子活性物质  固相萃取分离  海水pH影响  HPLC-ESI-Q-TOF-MS
DOI:10.11693/hyhz20180300056
分类号:P714.4
基金项目:青岛海洋国家实验室鳌山科技创新计划项目,2016ASKJ14号;国家自然科学基金项目,41376092号;中国科学院仪器设备功能开发技术创新项目,HYQY-GNKF2016-1-2号;国家自然科学基金委-山东省联合资助项目,U1606404号。
EFFECT OF pH ON EXTRACTION OF SMALL-MOLECULAR BIOACTIVE SUBSTANCES FROM SEAWATER BY SPE-HPLC-ESI-Q-TOF-MS
ZHANG Qian1,2,3,4, SONG Jin-Ming1,2,3,4, LI Xue-Gang1,2,3,4, PENG Quan-Cai1,2,3,4, YUAN Hua-Mao1,2,3,4, LI Ning1,2,3,4, DUAN Li-Qin1,2,3,4
1.CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Laboratory of Marine Ecology and Environmental Sciences, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China;3.University of Chinese Academy of Sciences, Beijing 100049, China;4.Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
Abstract:
Marine dissolved organic matter (DOM) contains a large number of bioactive substances. However, lacking suitable separation and extraction methods impedes seriously the exploration of what role different components play in the ecosystem. The solid-phase extraction (SPE) is a very effective method for extracting marine DOM from seawater. While extracting marine DOM in the SPE method, the pH of seawater is critical to the extraction efficiency of bioactive molecules. In this paper, HLB (hydrophilic-lipophilic balance) solid-phase extraction column was used to extract the bioactive substance from seawater under 7 different pH values, the extraction efficiency was measured, and detailed molecular composition was detected in HPLC-Q-TOF-MS in ESI (-) mode. The result shows that good extraction efficiency can be obtained when seawater pH is neutral or strongly acidic. When seawater was acidified to pH 5 or 6, the extraction efficiency of marine DOM was very low. With the decrease of seawater pH value, the signal intensities of the mass spectrum reduced, but more spectrum peaks occurred, and the molecular mass and property of extracts were widely dispersed. When the seawater sample was acidified to pH 2, most numbers of spectrum peaks could be identified. As shown in the van Krevelen and m/z-H/C diagrams, the molecular compositions of the extracts differed significantly under different seawater pH values. A neutral pH condition is suitable for the extraction of small molecular and high saturation substances. Strong acidic conditions are good to extract efficiently a variety of substances in high heterogeneity and high resolution, especially those bioactive molecules with unsaturated groups such as acid and ketone, and biological macromolecules such as protein and sugar. When the seawater samples were acidified to pH 2, HPLC-ESI-Q-TOF-MS had the highest response intensity in polar molecules, and this pH condition was especially suitable for extracting bioactive substances from seawater. Therefore, in terms of extraction efficiency, the number of identifiable peaks, and molecular composition of extracts, pH 2 is suggested for the best performance of extracting small molecular bioactive substances by HLB SPE.
Key words:  organic small molecular bioactive substance  solid-phase extraction  seawater pH  HPLC-ESI-Q-TOF-MS
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