Comparison of different buffer systems for separation of 15 nucleosides by capillary electrophoresis

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(bgE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(dEA)为背景电解质,对毛细管区带电泳(CzE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOf/MS)以及胶束电动毛细管电泳(MEkC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CzE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 MMOl/l dEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEkC模式下,以25 MMOl/l磷酸氢二钠(添加70 MMOl/l十二烷基磺酸钠(SdS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOf/MS分析中,以20 MMOl/l乙酸铵(PH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用dEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。The most suitable background electrolytes(BGEs) for simultaneous separation of 15 nucleosides by different modes of capillary electrophoresis(CE) were obtained.Various modes of CE were performed including capillary zone electrophoresis(CZE),capillary electrophoresis-electrospray ionization-time of flight mass spectrometry(CE-ESI-Tof/MS) and micellar electrokinetic chromatography(MEKC).The electrolyte buffers using sodium tetraborate decahydrate,disodium hydrogen phosphate,sodium acetate,sodium bicarbonate,ammonium acetate or 1,2-diamino-ethane(DEA) were tested,and the best of them were systematically optimized.In CZE mode,the nucleosides could not be separated completely with sodium tetraborate decahydrate or disodium hydrogen phosphate as BGEs,demonstrating the limited applicability of the two buffer systems for complex samples.However,with 300 mmol/L DEA(containing 2% acetone) as BGE,15 nucleosides could be separated with good resolution and peak shape,which proved that the DEA buffer was most suitable in CZE.The best buffer system in MEKC mode was 25 mmol/L disodium hydrogen phosphate with 70 mmol/L sodium dodecyl sulfate(SDS),and it was successfully applied for the separation of the nucleosides in Chinese Anthopleura lanthogrammica Berkly.The optimum buffer system for CE-ESI-Tof/MS analysis was 20 mmol/L ammonium acetate(pH 10.0).In the positive ion mode,the MS signals of each compound were better than those in the literature using DEA as BGE.The results of this study demonstrated the applicability of different buffer systems for the simultaneous separation of 15 nucleosides,and were helpful for the development of CE method in complex sample separation.国家自然科学基金项目(20905017);海洋公益性行业科研专项项目(201005034-3);海洋局青年基金项目(2010140);海洋一所基本科研业务专项项目(2010G25);中国科学院实验海洋生物学重点实验室开放基金课

Primary Exploration on Quality Evaluation of Holothurian Sold on the Market by HPLC Fingerprint

以10批不同产地刺参HPlC指纹图谱中的6个共有峰为评价指标,结合相似度分析,对不同刺参质量进行评价和检验。结果表明:不同批次刺参样品的6个色谱峰在指纹图谱分析过程中色谱行为相同,峰面积大,特征性强,能反映刺参的固有化学特征。结合相似度分析,可用于刺参质量评价。An optimized high performance liquid chromatography (HPLC) method was developed for the analysis of holothurian and the HPLC fingerprint was established from 10 batches of the holothurians from different habitats.The HPLC fingerprint showing 6 common characteristic peaks was used to explore the quality evaluation of holothurian and distinguish from the fakes with the similarity analysis.This method is accurate and reliable,providing a scientific basis for the quality control of holothurian and can be used to evaluate the quality of holothurian sold on the market.我国近海海洋综合调查与评价908专项(908-02-05-04):海洋药用生物资源评价和《中华海洋本草》编纂;海洋一所基本科研业务专项(GY-022008T32):海洋绿藻活性成分分析、鉴

Study on the antioxidant activity of extracts from Enteromorpha prolifera

目的通过探讨浒苔提取物体外的抗氧化活性,为浒苔综合开发利用提供科学依据。方法采用AbTS自由基清除法对浒苔不同溶剂提取物的抗氧化活性进行评价,对不同浒苔样品水提物的抗氧化活性进行比较,并以咖啡酸和抗坏血酸作为阳性对照品对浒苔水提物的抗氧化能力进行量化。结果浒苔水提物的抗氧化活性最高,1Ml浒苔水提物溶液(浓度相当于5.0g新鲜浒苔提取后定容至100Ml)的抗氧化能力与1Ml2.55x10--(-3)Mg·Ml--(-1)的咖啡酸或1Ml2.89x10--(-3)Mg·Ml--(-1)的抗坏血酸相当,而提取方法、样品干燥与否、浒苔样品的差异性均对浒苔水提物的抗氧化活性有一定的影响。结论绿潮藻浒苔具有较强的抗氧化活性,可作为潜在的抗氧化剂进行开发利用。Objective To investigate the antioxidant activity of Enteromorpha prolifera by ABTS radical scavenging assay in vitro and provide scientific basis for exploitation of Enteromorpha prolifera.Methods The antioxidant activities of extracts by different solvents from Enteromorpha prolifera and water extracts from different Enteromorpha prolifera samples were investigated by ABTS radical scavenging assay.Moreover,the antioxidant ability of the water extract from Enteromorpha prolifera was evaluated by using caffeic acid and ascorbic acid as positive control standards.Results The water extract from Enteromorpha prolifera provided the highest radical scavenging activity,and the antioxidant capability of 1mL of water extract from Enteromorpha prolifera was equivalent to 1mL of 2.55 10--(-3) mg·mL--(-1) caffeic acid or 1mL of 2.89×10--(-3)mg·mL--(-1) ascorbic acid.However,the extraction methods,dry sample or not and different kinds of Enteromorpha prolifera samples had certain influence on the antioxidant activity of water extract from Enteromorpha prolifera.Conclusion There are abundant antioxidant compounds present in the aqueous extract of Enteromorpha prolifera,and it can be exploited as latent oxidation inhibitor.海洋公益性行业科研专项(200705011;200805039);国家自然科学基金(20602009;20905017);海洋一所基本科研业务专项(GY-022008T32;GY-022008G47)资

CLUSTER ANALYSIS ON FATTY ACID COMPOSITION of ENTEROMORPHA PROLIFERA ofF NORTHERN CHINA COAST

于2008年7月在黄、渤海区域采集了17个漂移浒苔样品,结合3个本地种,对其脂肪酸组成进行了gC-MS分析。结果表明,所有浒苔中优势脂肪酸为十六碳酸,相对含量在27.82%—43.27%之间。藻体中还含有丰富的PufA,主要为9,12,15-十八碳三烯酸、6,9,12-十八碳三烯酸、9,12-十八碳二烯酸、8,11,14-二十碳三烯酸,另外还含有EPA不饱和脂肪酸。以欧式距离平方为距离测量技术,类间距用平均链锁法,对20个不同地理位置的浒苔样品进行聚类分析,结果分为4大类,所有漂浮浒苔聚为第一类,青岛和连云港的本地种分别聚为另三类,说明青岛近海漂浮的浒苔不是青岛本地生浒苔,而是从外海漂移过来的,第一类的聚类结果显示了浒苔的漂移路径。结果表明,藻类脂肪酸的系统聚类分析为海洋绿藻分类与量化评价提供了一种好方法。Fatty acids composition of 20 Enteromorpha prolifera samples from the Yellow Sea and the Bohai Sea were determined with GC-MS.The result indicates that C16 are the predominant fatty acid,taking 27.82% to 43.27%.C18:2(cis,cis-9,12)(n-6),C18:3(gamma)(all cis-6,9,12)(n-6),C18:3(alpha)(all cis-9,12,15)(n-3) and EPA are major polyunsaturated fatty acids(PUFAs) in E.prolifera.The samples were numerically classified in fatty acid composition using Squared Euclidean distance and between-group linkage method as clustering algrithm.The 20 samples were divided into four groups by cluster analysis.All the floating E.prolifera samples are placed in Group I,and other native species in Qingdao and Lianyungang are classified into other three groups.The floating E.prolifera that bloomed in Qingdao coastal area in 2008 were not orininated from local native species but from the open sea.Hierarchical cluster analysis was proved to be a good method of quantitive evaluation and classification of marine green algae.海洋公益性行业科研专项;200805039号;国家海洋局第一海洋研究所基本科研业务费专项资金项目;2008G47号、2008T32号;国家自然科学基金资助项目;20602009号、40776098号;海洋863项目;2007AA09Z435

Determination of chlorophyll a and chlorophyll b in Enteromorpha prolifera by ultrasound-assisted extraction with RP-HPLC

建立了一种反相高效液相色谱法(rP-HPlC)测定浒苔(EnTErOMOrPHA PrOlIfErA)中叶绿素A和b含量。采用的色谱条件:dIAMOnSIl-C18(4.6MMx150MM)柱,流动相为甲醇(含0.2%的乙酸)和丙酮,梯度洗脱,流速为0.8Ml/MIn,进样量10μl,检测波长430nM。在此条件下,叶绿素A和叶绿素b与浒苔中的其他化合物分离良好,叶绿素A、b质量浓度为0.50~500Mg/l具有良好的线性关系(r2=0.9999),能对浒苔提取液中的叶绿素A、b进行准确定量。A convenient and user-friendly reverse phase-high performance liquid chromatography (RP-HPLC) analysis method was established for the determination of chlorophyll a and chlorophyll b in Enteromorpha prolifera.The following HPLC conditions are follows:Diamonsil-C18 (4.6 mm×150 mm) column is used; MeOH-0.2% acetic acid and acetone as the mobile phase with linear gradient elution are used; flow-rate is 0.8 mL/min,detection length of Vis is 430 nm; injection volume is 10 μL.The results indicated that the developed method can be used for the determination of chlorophyll a and b in E.prolifera with a good resolution and linearity between 0.50～500 mg/L (R2=0.999 9).The method is specific,simple,fast,sensitive and feasible for the determination of chlorophyll a and chlorophyll b,which can eliminate the interference of chlorophyll derivatives in E.prolifera.国家自然科学基金项目(20905017;20602009);海洋公益性行业科研专项项目(200705011;200805039);国家海洋局第一海洋研究所基本科研业务专项项目(GY-022008T32;GY-022008G47