基于部分酸水解-亲水作用色谱-质谱的黄芪多糖结构表征

来自中药的水溶性多糖具有广谱治疗和低毒性特点,是天然药物及保健品研发中的重要组成部分。针对中药多糖结构复杂、难以表征的问题,本文以中药黄芪中的多糖为研究对象,采用"自下而上"法完成对黄芪多糖的表征。首先使用部分酸水解方法水解黄芪多糖,分别考察了水解时间、酸浓度和温度的影响。在适宜条件(4 h、1.5 mol/L三氟乙酸、80 ℃)下,黄芪多糖被水解为特征性的寡糖片段。接下来,采用亲水作用色谱与质谱联用对黄芪多糖部分酸水解产物进行分离和结构表征。结果表明,提取得到的黄芪多糖主要为1→4连接线性葡聚糖,水解得到聚合度4~11的葡寡糖。本研究对其他中药多糖的表征具有一定的示范作用。     

低共熔溶剂——新型亲水作用色谱流动相改性剂

低共熔溶剂被用作亲水作用色谱流动相的新型改性剂。选用硅胶柱(150 mm×4.6 mm, 3 μm),以乙腈与低共熔溶剂(氯化胆碱-乙二醇(摩尔比为1:3))的混合溶液为流动相,考察了6个碱基与核苷的色谱分离效果,并讨论了低共熔溶剂在流动相中的比例及温度条件对分离的影响。结果表明,与传统的水相流动相条件相比,在加入低共熔溶剂改性后的流动相条件下,碱基与核苷分离效果得到明显的改善,尤其是胞嘧啶与胞苷能达到完全分离;同时,随着低共熔溶剂在乙腈中浓度的增加,6个碱基与核苷在色谱柱上的保留均有不同程度的减小,其中胞苷的保留减小最为显著;随着柱温的升高,碱基与核苷的保留同样有所减小。本文验证了低共熔溶剂作为亲水作用色谱流动相改性剂的可行性,可在一定程度上解决传统亲水作用色谱分离的困难。     

高效液相色谱法亲水作用模式在亲水化合物分析中的应用

介绍一种新的高效液相色谱法（HPLC）在高极性亲水性化合物分离分析中的应用实例,扩充了仪器分析实验教材中关于HPLC分析对象的范围,使学生在教学过程中更加全面地认识HPLC技术的应用领域。使用Merck公司ZIC®-HILIC色谱柱为分离介质,以典型的医药化工产品胞嘧啶为对象,建立适用其质量控制的测定方法。通过对亲水作用色谱的介绍,实验参数的优选和详尽的方法学验证,以期使读者对该方法有清晰的了解,在生命科学、药学和化工专业的仪器理论和实验教学中增加新的案例。     

人尿液N-糖蛋白/N-糖肽规模化富集鉴定

蛋白质的N-糖基化是真核细胞中一种重要的翻译后修饰,N-糖基化修饰在调控细胞黏附、迁移、信号转导及细胞凋亡等方面扮演着关键角色.蛋白质糖基化修饰的异常变化与多种重要疾病的发生相关.尿液具有蛋白质组复杂程度低和非入侵性等特点,适合大量及连续多时间点采样研究.但由于个体差异和生理条件的影响,尿蛋白丰度的生理波动较大.目前缺...     

Development of analytical methods for the quantification of metabolites of lesogaberan in a MIST investigation

Analytical methods were developed for the determination of six metabolites of lesogaberan to be used in quantitative determinations of metabolites according to the guidelines of Metabolites in Safety Testing. The γ‐amino butyric acid type B receptor agonist lesogaberan and its metabolites are small polar molecules and hydrophilic interaction liquid chromatography was found to be a suitable separation mode. The samples were prepared using protein precipitation and negative electrospray ionization tandem mass spectrometry was used for detection. Initially, exploratory methods for six metabolites were set up for analysis of human plasma samples taken after repeated administration of a high oral dose of lesogaberan. The purpose was to establish which metabolites were present at concentrations significant for further investigation. Four of the six metabolites were then found at clearly detectable concentrations. The analytical methods for these four metabolites were further elaborated and then taken through a qualification procedure, which showed acceptable accuracy (86–114%), precision (<9%) and good linearity in the range 0.03–5 µmol/L. No interferences were seen from endogenous plasma components. Copyright © 2013 John Wiley & Sons, Ltd.     

一种可控离子比例的两性亲水色谱固定相的制备及性能研究

将不同比例的氨基和巯基的硅烷偶联剂键合到硅胶表面，再利用巯基与乙烯基膦酸之间的点击化学反应将膦酸基团引入到硅胶表面，制备了一种可调节正负离子比例的两性亲水色谱固定相。通过测定固定相中C、H、N、P元素的含量，证明了氨基与膦酸基团已成功键合到固定相的表面，同时通过N元素与P元素的质量分数确定固定相表面氨基与膦酸基团的比例。制备了3种不同电荷比例的氨基膦酸固定相，将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中。以一系列经典的极性小分子作为探针，研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对探针分子在3种色谱柱上的保留的影响，结果表明，分析物在固定相上是多重保留机理。最后通过比较核苷、水溶性维生素、碱性化合物、苯甲酸这几类标准物质在3种色谱柱上的保留行为来对比3种不同电荷比例的固定相的分离选择性与色谱性能。结果表明，对于不同的分析物，3种固定相表现出完全不同的分离选择性和色谱行为。可以根据分析物的特征选取不同电荷比例的固定相，表明此种固定相在极性化合物的分离上具有良好的应用前景。     

Separation of urea,hexamethylenetetramine, and their reaction products in sol–gel feed solution by zwitterionic HILIC

Studies were carried out to develop a chromatographic methodology based on hydrophilic interaction liquid chromatography (HILIC) for the separation of various reaction products of hexamethylenetetramine (HMTA) –urea used in the sol–gel process for the preparation of ceramic microspheres. Different chromatographic parameters such as organic modifiers, pH of mobile phase, buffer concentration, column temperature, etc. were studied to arrive at the optimum conditions for separation. Compounds such as urea, monomethylolurea (MMU), dimethylolurea (DMU), and HMTA were separated from a synthetic mixture using a mobile phase consisting of acetonitrile and acetate buffer of pH 6. The methodology developed based on HILIC stationary phase is simple and amicable for integration with electrospray ionization mass spectrometry (ESI-MS) to identify the unknown reaction products. The methodology was applied for the separation of reaction products in pre-boiled and untreated urea–HMTA mixtures used as feed in the sol–gel process.     

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L-arginine and its dimethylated derivatives detection

Asymmetric N(G),-N(G)-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L-arginine (Arg), ADMA, and symmetric N(G),-N(G')-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30 degrees C.     

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism     

Quantitative determination of buprenorphine,naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

A rapid and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0–1000 ng/mL. The intra and inter‐day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra‐ and inter‐day accuracy (bias) ranged from −14.2 to 15.2% and from −9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC–MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine–naloxone combination in rats.