Biological fingerprinting analysis by liquid chromatography/mass spectrometry for evaluation of DNA structural selectivity of multiple compounds in natural products

A method by combination of centrifugal ultrafiltration (CUF) sampling with liquid chromatography-mass spectrometry (LC-MS) analysis was established to evaluate the DNA structure and sequence selectivity of the multiple compounds in a small molecule library. The developed method was applied to analyze the extracts of natural products Coptis chinensis Franch and Rheum palmatum (L.). From the obtained biological fingerprinting chromatograms, 7 compounds in C. chinensis Franch and 11 in R. palmatum (L.) were screened out as DNA binding agents. Most of these compounds were identified by standards and LC-MS analysis after the sample pretreatment with the DNA immobilized cartridge. DNA structural binding preference of the multiple active compounds in these two extracts was then evaluated simultaneously without purification.     

Comprehensive two-dimensional HPLC to study the interaction of multiple components in Rheum palmatum L. with HSA by coupling a silica-bonded HSA column to a silica monolithic ODS column

A mode of comprehensive 2-D LC was developed by coupling a silica-bonded HSA column to a silica monolithic ODS column. This system combined the affinity property of the HSA column and the high-speed separation ability of the monolithic ODS column. The affinity chromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in traditional Chinese medicines (TCMs) with HSA according to their affinity to protein in the first dimension. Then the unresolved components retained on the HSA column were further separated on the silica monolithic ODS column in the second dimension. By hyphenating the 2-D separation system to diode array detector and MS detectors, the UV and molecular weight information of the separated compounds can also be obtained. The developed separation system was applied to analysis of the extract of Rheum palmatum L., a number of low-abundant components can be separated on a single peak from the HSA column after normalization of peak heights. Six compounds were preliminarily identified according to their UV and MS spectra. It showed that this system was very useful for biological fingerprinting analysis of the components in TCMs and natural products.     

大黄蒽醌衍生物在杯[8]芳烃键合固定相上色谱行为的研究

研究了药用掌叶大黄中5种蒽醌衍生物在对-叔丁基杯[8]芳烃硅胶键合固定相上的高效液相色谱行为,并与ODS固定相进行了比较。研究发现这类化合物与杯[8]芳烃固定相之间存在多种相互作用,除疏水作用外,分离过程中还存在与ODS不同的色谱分离机制。杯芳烃键合相与溶质之间的氢键作用、包容络合作用改变了杯芳烃固定相对它们的选择性。     

Preparation of "click" binaphthyl stationary phase and its application for separation of anthraquinones from Rheum palmatum L

An organosilane containing binaphthyl functional group was synthesized by clicking 2,2'-bis(prop-2-ynyloxy)-1,1'-binaphthyl with 3-azidopropyltriethoxysilane (AzPTES). The "click" binaphthyl stationary phase was then synthesized by covalently bonding the organosilane onto silica beads. In this synthetic method, the residue of copper iodide (CuI) catalyst can be controlled at a very low level. Polycyclic aromatic hydrocarbons (PAHs) were well retained on the "click" binaphthyl column, and showed different retention factors (k) and separation ratio (α) values from those on octadecylsilyl (ODS) column due to the π-π interaction between the analytes and the stationary phase. The anthraquinone compounds in Rheum palmatum L. were selectively separated and enriched into a fraction by the "click" binaphthyl column. By using this way, the complexity of the sample was largely reduced. Twelve of anthraquinones were recognized by UPLC-ESI-MS/MS. This stationary phase will be very useful for separating and enriching similar compounds with planar aromatic structures from complex traditional Chinese medicine (TCM) samples.     

基于表面静电排斥/反相混合模式色谱的黄连生物碱分析方法

建立了基于表面静电排斥/反相混合模式色谱的黄连生物碱分析方法。选用实验室自制的C18HCE柱,以乙腈和水为流动相,考察了甲酸、乙酸两种流动相添加剂及其在流动相中的体积分数对黄连生物碱的保留、峰形及选择性的影响。最终确定0.1%（v/v）乙酸作为添加剂能实现黄连生物碱的良好分离,结合质谱和文献对其主要色谱峰进行了识别,分别为黄连碱、表小檗碱、非洲防己碱、药根碱、小檗碱、巴马汀。参考2015版《中国药典》对黄连生物碱的含量测定方法,以盐酸小檗碱进行方法学考察,结果表明,在0.5~100 mg/L范围内线性关系良好,相关系数为0.9996,平均加标回收率为93.74%。利用所建立的方法测定了湖北和重庆两个产地不同批次的黄连样品中各生物碱的含量。该方法简便,重复性好,精密度高,可为其他碱性化合物的分离分析提供参考。     

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

正A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.     

微量热法研究黄连及其主要组分配伍的抑菌作用

基于微量热法,研究黄连、黄连的主要组分小檗碱、药根碱、巴马汀及其配伍模拟方的抑菌作用.以HPLC法测定黄连中小檗碱、药根碱和巴马汀的含量,并根据其含量比值配伍模拟方;微量热法测定黄连、小檗碱、药根碱、巴马汀及其模拟方对痢疾杆菌的生长代谢曲线,得出相应的热动力学参数,并进行对应分析.结果表明黄连、小檗碱、药根碱、巴马汀及其模拟方对痢疾杆菌的生长代谢均有不同程度的抑制作用,黄连作用最强,单体生物碱作用弱,配伍模拟方作用增强,但并未显现明显协同作用,黄连的抑菌作用可能为多种活性成分的综合作用.     

毛细管电泳法测定白头翁汤中黄连和黄柏共煎生物碱的煎出量

 建立了利用毛细管电泳简便、准确地测定白头翁汤中黄连和黄柏共煎生物碱煎出量的方法。采用自组装毛细管电泳装置,采用75μmi.d.×50cm弹性石英毛细管,以0 05mol/LNa2B4O7 CH3OH(体积比为85∶15)溶液作缓冲液,运行电压为14kV,检测波长为232nm。另外,通过实验优化了提取溶剂中乙醇的含量。实验结果表明:以小檗碱、巴马汀提取量为指标,30%(体积分数)的乙醇水溶液是提取白头翁汤中黄连和黄柏共煎生物碱的最佳溶剂。小檗碱和巴马汀的质量浓度分别在15 0mg/L～65 0mg/L、12 5mg/L～50 0mg/L时与其峰面积有良好的线性关系;小檗碱的平均回收率不低于95%。     

Novel Cell Membrane Capillary Chromatography for Screening Active Compounds from Natural Products

Cell membrane chromatography (CMC) is a useful method for the simultaneous isolation and identification of active compounds from natural products. However, it suffers from high cell membrane consumption and is time-consuming to operate. In this study, CMC was performed for the first time with a silica capillary, termed cell membrane capillary chromatography (CMCC). Pancreatic islet cell membranes from a mouse were immobilized onto the capillary inner wall functionalized with aldehyde groups. Scanning electron microscopy observation of the prepared column showed that the cell membrane was evenly coated onto the capillary inner wall. Three model analytes with the pharmacological property of hypoglycemic activity including glibenclamide, glipizide and berberine were tested. They were all retained by the prepared column. Furthermore, the retention factors of the analytes in CMCC correlated well with their pharmacological action. The analytical procedure including washing (to obtain a flat baseline), injection and separation was accomplished within 10 min. The CMCC column was also used for screening active compounds from a natural plant (Coptis chinensis). The hypoglycemia activity of active components such as berberine was verified using the method. The results indicated that CMCC is a viable alternative method for screening active compounds from natural products.     

Novel imidazolium stationary phase for high-performance liquid chromatography

A new imidazolium anion-exchange phase immobilized on silica is synthesized. HPLC separations of common inorganic anions (IO3-, Cl-, NO2-, Br-, NO3-, I-, SCN-) have been performed using a HPLC column (200 mm x 4.6 mm I.D.) packed with this stationary phase, with a phosphate buffer solution as the mobile phase and UV detection at 200 nm. The effects of pH and concentration of eluent on the separation of anions have been studied. Chromatographic parameters are calculated and the results show that the new stationary phase is of significant potential for the analysis of these anions. Successful separations of some ordinary organic anions have also been achieved with the said stationary phase. Meaningfully, organic and inorganic anions can be determined simultaneously and satisfactorily with several neutral compounds using the column. The separation of some organic compounds including hydroxybenzenes, bases and amines by this stationary phase with only water as the eluent has been investigated.     

薄层色谱-荧光法测定黄连生物碱分配系数

建立了薄层色谱和荧光分析法测定黄连生物碱在高速逆流色谱溶剂体系中分配系数的方法.样品在溶剂体系中分配平衡后,取等量的上下相,经点板、展开、刮板,提取后用荧光分光光度法检测样品的荧光值(激发波长365 nm,发射波长409 nm),并计算样品在溶剂体系中的分配系数.在一定的浓度范围内,4种生物碱的含量与荧光强度呈良好的线...     

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.     

Covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography and hydrophilic interaction chromatography

The mixed sulfated/methacryloyl polysaccharide derivative was prepared and successfully immobilized onto the surface of porous silica particles by polymerization. Polysaccharide derivative was calculated as 10.33% in the stationary phase prepared. The new stationary phase (PMSP) showed both hydrophilic interaction (HILIC) and per aqueous liquid chromatography (PALC) characteristics. The effects of column temperature, the water content, pH and ion strength of mobile phase on the retention time of test compounds in highly aqueous eluents were investigated to evaluate the PALC features of PMSP. The column efficiency is about 31,000 plates/m for benzoic acid in water/ACN (97/3, v/v) mobile phase at a flow rate of 1.0 mL/min. Compared with C18 column, the PMSP had shorter retention time for weak polar and non-polar compounds, but also showed stronger retention for strong polar compounds. It indicated that PALC was a suitable mode of chromatography as replacement of HILIC and complementarity of reversed-phase liquid chromatography (RPLC).     

N-methylimidazolium-functionalized monolithic silica column for mixed-mode chromatography

The development of mixed-mode stationary phase to achieve multiple separation capabilities in one column is very important for high performance liquid chromatography. In this paper, a new specific stationary phase based on grafting N-methylimidazolium to a monolithic silica column was successfully prepared for performing capillary liquid chromatography. The characteristics of the column were evaluated by the separation of different types of compounds including inorganic anions, aromatic acids, nucleotides, polycyclic aromatic hydrocarbons, alkylbenzenes, and phenols. The mechanisms for the separation of these compounds were investigated and appeared to involve the mixed interactions including anion-exchange, hydrophilic, π-π, dipole-dipole, and hydrophobic interactions.     

A novel octadecylsilane functionalized graphene oxide/silica composite stationary phase for high performance liquid chromatography

An octadecylsilane functionalized graphene oxide/silica stationary phase was fabricated by assembling graphene oxide onto the silica particles through an amide bond and subsequent immobilization of octadecylsilane. The chromatographic properties of the stationary phase were investigated by reversed-phase chromatography with alkylbenzenes, polycyclic aromatic hydrocarbons, amines, and phenolic compounds as the analytes. All the compounds achieved good separation on the column. The comparison between a C18 commercial column and the new stationary phase indicated that the existence of π-electron system of graphene oxide allows π-π interaction between analyte and octadecylsilane functionalized graphene oxide/silica stationary phase except for hydrophobic interaction, while only hydrophobic interaction presented between analyte and C18 commercial column. This suggests that some analytes can be better separated on the octadecylsilane functionalized graphene oxide/silica column.     

Protein separation using a novel silica-based RPLC/IEC stationary phase modified with N-methylimidazolium ionic liquid

Ionic liquids (ILs) immobilized on silica as novel high performance liquid chromatography (HPLC) stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica (ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.     

原子转移自由基聚合技术制备L-苯丙氨酸手性配体交换色谱固定相及其对手性化合物的拆分

在室温条件下,以甲基丙烯酸环氧丙酯(GMA)为单体,溴异丁酰溴为引发剂,CuCl/2,2′-联吡啶(Bpy)为催化剂,通过原子转移自由基聚合(ATRP)反应,将甲基丙烯酸环氧丙酯聚合在硅胶表面。然后再将L-苯丙氨酸共价键合在硅胶表面的聚合物上,制备了新型手性配体交换色谱固定相,并用该固定相对DL-氨基酸进行分离。用元素分析对其进行了表征;详细考察了固定相的合成过程以及流动相pH值、流动相铜离子浓度、柱温等色谱条件对DL-氨基酸对映体拆分的影响。元素分析得出该固定相表面L-苯丙氨酸接枝密度达到4.32 mg/m2;在手性配体交换分离模式下,流动相为0.05 mol/L KH2PO4-0.1 mmol/L Cu(Ac)2水溶液、流速为1.0 mL/min、柱温为50 ℃和检测波长为223 nm条件下,该色谱固定相可以分离DL-天冬氨酸、DL-天冬酰胺等。同时,流动相pH值、铜离子浓度以及柱温对手性对映体的拆分有较大影响。与传统的在硅胶表面直接键合L-苯丙氨酸制得的固定相相比,所合成的固定相接枝密度高,分离效果好,对DL-天冬氨酸及DL-天冬酰胺实现了基线分离。结果表明,在手性配体交换分离模式下,固定相具有良好的拆分性能。     

醋酸纤维素液相色谱固定相的制备与手性拆分

在均相条件下合成了醋酸纤维素,将其涂渍在修饰硅胶表面,制备了涂渍型高效液相色谱固定相.在40MPa压力下将所得固定相装入色谱柱.在高效液相色谱正相色谱条件下,对包括醇类、羧酸类、胺类、氨基酸类及药物类的16种新型手性化合物进行了拆分试验,结果有6种手性化合物得到了分离,效果良好.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography part I: Evaluation of silanol terminated dimethyl substituted polymers

The thermal stability of silicones can be improved on replacement of certain of the oxygen atoms in the polymer backbone by phenyl groups. Such a polymer has been synthesized and evaluated for use as stationary phase in fused silica capillary gas chromatography; the polymer was dimethyl substituted and silanol terminated. A selectivity was provided by the phenyl groups in the backbone. For comparative purposes, a silanol-terminated dimethylpolysiloxane has also been evaluated. Both stationary phases gave columns of highest separation efficiency and the supporting fused silica surface was deactivated by the stationary phases on thermal treatment. Further, low column bleeding was observed at the maximum temperature tested, 370°C. The phenyl-containing phase could be immobilized to 60% by heat treatment, but the pure dimethylpolysiloxane was 10% immobilized. The influence on immobilization of factors such as nature of the supporting surface, stationary phase silanol content, reaction temperature and atmosphere in the column during reaction has been studied.     

Chemically bonded cationic β-cyclodextrin derivatives as chiral stationary phases for enantioseparation applications

Vinylene-functionalized cationic β-cyclodextrins are co-polymerized with vinylized silica in the presence of AIBN and conjugated monomers. The chemically immobilized cationic β-cyclodextrins were thereafter applied as chiral stationary phases in packed column supercritical fluid chromatography and demonstrated good and reproducible enantioseparations of racemic pharmaceutical compounds.