高效液相色谱法测定金钱菇多糖的单糖组成

采用高效液相色谱法,测定金钱菇多糖的单糖组成.用超声辅助提取金钱菇多糖,通过1-苯基-3-甲基-5-吡唑酮(PMP)衍生水解后的单糖,高效液相色谱法检测衍生物.结果表明：金钱菇多糖由甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、葡萄糖(Glc)、半乳糖(Gal)、木糖(Xyl)组成,其摩尔为1．00∶0．90∶0．91∶28．03∶1．58∶0．11.该方法快速、简便、重现性好,可用于测定金钱菇多糖的单糖组成.     

高效液相色谱法联用蒸发光散射测定薏苡仁多糖的单糖组成

建立高效液相色谱法(HPLC)联用蒸发光散射(ELSD),对薏苡仁多糖的单糖组成进行测定。HPLC色谱条件:Supelco~?APHERA NH_2 Polymer色谱柱(150 mm×2 mm,5μm);流动相:乙腈-0.05 mol/L醋酸铵溶液(体积比为85∶15);流速:0.2 mL/min;进样量:10μL;柱温:35℃;ELSD检测条件:漂移管温度为60℃,载气流量为1.6 L/min。研究结果表明:甘露糖(mannose)、半乳糖(galactose)、葡萄糖醛酸(glucuroaicacid)、鼠李糖(rhamnose)、半乳糖醛酸(galacturonic acid)、葡萄糖(glucose)、阿拉伯糖(arabirose)和岩藻糖(flucose)的进样量对数与峰面积对数在一定范围内均呈现出较好的线性关系(0.999 1～0.999 9),回收率均在97.4%～100.4%,RSD均小于2%。研究表明该方法准确可靠,分离效果好,可用于测定薏苡仁多糖的单糖组成。     

薄芝糖肽的单糖组成分析

目的对灵芝属薄树芝的活性成分薄芝糖肽的单糖进行研究.方法采用薄层色谱法和高效液相色谱-蒸发光散射检测器(HPLC-ELSD)联用技术.结果薄芝糖肽的单糖组成及摩尔比为葡萄糖∶D-甘露糖∶半乳糖∶木糖=1∶0.074∶0.067∶0.0178.结论建立了薄芝糖肽单糖组成的检测方法.     

不同碳源对干酪乳杆菌LC2W胞外多糖组成影响的研究

运用紫外光谱、红外光谱、HPLC和气相色谱对葡萄糖和乳糖两种碳源获得的胞外多糖(EPS)单一组分G1、G2和L1、L2的结构进行了初步分析。结果显示不同碳源对EPS的分子结构、分子量和单糖组成都有影响。紫外光谱分析都不含蛋白和核酸;红外光谱分析G1、G2和L1、L2均呈现多糖的特征峰,但波形、特征吸收波段、波峰强度有些差异;HPLC测定G1、G2、L1和L2的平均分子量分别为6.65×105Da、1.01×104Da、2.25×106Da和1.36×104Da;气相色谱分析G1、G2和L1、L2的主要单糖组成摩尔比分别为:鼠李糖∶葡萄糖∶半乳糖=3.07∶3.29∶1;鼠李糖∶葡萄糖∶半乳糖=2.84∶6.4∶1;甘露糖∶葡萄糖∶半乳糖=17.24∶6.03∶1;甘露糖∶葡萄糖∶半乳糖=28.71∶5.98∶1。     

棉叶中棉酚的快速提取与测定方法研究

棉酚(G)及其相关物甲氧基半棉酚(DHG)、半棉酚酮(HGQ)、半棉酚(HG)、杀实夜蛾素(H1-4)等是棉花中重要的抗虫性萜烯类次生物质.利用高效液相色谱法(HPLC)对棉酚及其相关物进行了分离,测定了棉叶中的棉酚含量,讨论了不同的提取方法和测定条件对结果的影响,给出了一套简便、快速的分析测试方法,同时与紫外-可见分光光度法(苯胺法)的结果进行了对比,认为对于棉花的抗虫性的研究来说,HPLC是比较适宜的方法.     

猪链球菌1、2、14、1/2型荚膜多糖的单糖组成比较

【目的】猪链球菌1、2、14和1/2型间存在单向或双向的交叉抗原性,这种交叉抗原性的产生原因至今未被揭示。【方法】采用Sephacryl S-300凝胶层析柱对猪链球菌14和1/2型荚膜多糖进行分离纯化,经苯酚-硫酸检测和dot-ELISA辅助鉴定,确定荚膜多糖成分。采用高效凝胶渗透色谱法测定14和1/2型猪链球菌荚膜多糖分子量分别为487.38 kDa和512.72 kDa。【结果】经柱前衍生高效液相色谱法、荧光标记液相色谱法和核磁共振测定14和1/2型猪链球菌荚膜多糖单糖组成分别为:Glc/Gal/GlcNAc/Rha/Neu5Ac(1∶2.94∶1.35∶0.24∶0.37)和Glc/Gal/GlcNAc/GalNAc/Rha/Neu5Ac(1∶1.67∶1.05∶0.93∶0.72∶0.7)。并与已知的猪链球菌1、2型荚膜多糖的单糖组成进行比较分析,发现4种血清型荚膜多糖都具有Glc、GlcNAc、Gal和Neu5Ac,但单糖组成和比列并无明显相似性,这种交叉抗原性可能是由于荚膜多糖的空间结构相似性和(或)细胞表面的其他成分引起的。     

野木瓜水溶性多糖的分离纯化及抗氧化活性研究(英文)

采用水提醇沉法获得了水溶性野木瓜粗多糖(CCCPs),经过脱蛋白、脱色及透析,得到了野木瓜精多糖(FCCPs),再经DEAE-纤维素和Sepharose CL-6B柱层析得到均多糖组分(CCP1)。高效液相色谱法测定CCP1的单糖组成为:鼠李糖、阿拉伯糖、果糖、甘露糖、葡萄糖,其摩尔比为0.034∶0.228∶0.045∶0.055∶0.638。体外抗氧化实验结果表明:不同纯度的野木瓜多糖都具有一定的抗氧化活性,并且随着多糖浓度增加,抗氧化活性增强,野木瓜粗多糖(CCCPs)对·OH和O-·2的清除能力比精多糖(FCCPs)和均多糖(CCP1)强。     

桃果肉花色苷遗传多样性及红肉桃判定指标的探讨

运用比色法(UV)和高效液相色谱法(HPLC)测定68份桃品种果肉花色苷含量,比较2种测定结果的一致性,分析桃果肉花色苷的遗传多样性,初步提出红肉桃的判定标准。结果表明,2种方法测定白肉品种花色苷含量接近,可根据实际情况进行选择分析方法,而黄肉品种应尽可能选用高效液相色谱法(HPLC)。HPLC定性定量检测结果表明,48份材料检测出矢车菊-3-葡萄糖苷,是桃果肉中主要的花色苷种类,乌黑鸡肉桃,大果黑桃果肉中同时检测到矢车菊-3-芸香糖苷。根据高效液相色谱法测得花色苷含量将桃划分为5个等级,含量20 mg/100 g定为红肉桃的划分临界点。本研究中共鉴定出5份红肉桃品种,感官判定且风味为甜的红肉桃不符合上述判定标准,需要进一步研究完善。     

雪灵芝多糖的分离纯化及免疫活性评价

为分离纯化雪灵芝(Arenaria kansuensis)多糖,并对纯化组分进行分子量测定、单糖组分分析及免疫活性评价。实验采用水提醇沉法提取雪灵芝粗多糖(Arenaria kansuensis crude polysaccharide, AKCP);以DEAE-52纤维素柱对AKCP进行分离纯化,获得5个雪灵芝多糖组分AKP-1～AKP-5,进一步采用葡聚糖凝胶G-75柱对AKP-2进行分离纯化获得AKP-2a多糖组分。苯酚-硫酸法测定AKCP、AKP-2及AKP-2a的总糖含量分别为52%、70%和79%;凝胶渗透色谱-十八角度激光光散射(GPC-MALS)法检测AKP-2a的重均分子量Mw为2.07×10~5Da、数均分子量Mn为9.838×10~4Da;HPLC法检测AKP-2a是由半乳糖醛酸、甘露糖、核糖、鼠李糖、葡萄糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖、岩藻糖10种单糖组成,其摩尔比为1∶0.25∶0.01∶0.20∶0.11∶0.25∶0.61∶0.07∶0.21∶0.12;以MTT法检测体外培养小鼠脾淋巴细胞增殖,AKCP、AKP-2及AKP-2a各浓度组SI水平,均明显高于对照组(P<0.05)。经NO释放实验及IFN-γELISA检测,AKP-2a各浓度组小鼠腹腔巨噬细胞培养上清中二者的水平,较对照组呈浓度依赖性增高(P<0.01)。综上结果,本研究通过分离纯化,获得了总糖含量较高的雪灵芝多糖AKP-2a组分,初步确定其分子量范围及单糖组成,并证实其具有激活淋巴细胞增殖、促进巨噬细胞功能的生物活性。     

千斤拔多糖的提取纯化及其结构鉴定

利用正交试验考察千斤拔多糖的提取工艺,并比较脱蛋白方法中的Sevag法和三氯乙酸法的纯化效果,总糖含量测定采用苯酚-硫酸法,蛋白质含量测定采用考马斯亮蓝法;采用DEAE-52纤维柱法来分离多糖,并运用HPLC色谱来分析千斤拔多糖中的单糖成分。结果表明:经正交试验得出千斤拔多糖的最佳提取条件为时间2.5 h,料液比为1∶30,温度80℃,其多糖得率为8.558%。对比两种脱蛋白的方法,Sevage法萃取3次时蛋白的脱除效果最好。经DEAE-52纤维柱来分离多糖共分得7个组分。经HPLC色谱鉴定出有葡萄糖,甘露糖和阿拉伯糖,主要单糖成分为葡萄糖。     

Analysis of the monosaccharide composition of purified polysaccharides in Ganoderma atrum by capillary gas chromatography

Introduction – Ganoderma, one of the best‐known traditional Chinese medicines, has attracted considerable attention owing to the fact that dozens of polysaccharides isolated from it have shown diverse and potentially significant pharmacological activities. However, no work has been reported on the analysis of monosaccharide composition of polysaccharide isolated from the aqueous extract of Ganoderma atrum yet. Objective – To develop a simple and sensitive GC‐based method for the analysis of monosaccharide composition of purified polysaccharides in Ganoderma atrum. Methodology – The polysaccharide was first hydrolysed to give the constituent monosaccharides, which were subsequently derived into acetylated aldononitriles and analysed by gas chromatography using a capillary column packed with a (5%phenyl) methylpolysiloxane stationary phase with the addition of acetyl inositol as the inner standard. High‐performance liquid chromatography was also used for comparison. Results – The stable derivatives of the most common monosaccharides could be separated and reproducibly determined with high sensitivity. The limits of detection and quantification were 0.013 and 0.043 mg/mL, respectively. The intermediary precision values (expressed as the RSD) were less than 10%. The mean recovery of the method was 100 ± 3%, with RSD values of less than 5%. The results obtained from GC and HPLC methods were found to be close to each other within acceptable error ranges. Conclusion – This study demonstrated that the developed method could be applied as an accurate method for the compositional analysis of monosaccharides in the field of biological and biochemical study. Copyright © 2009 John Wiley & Sons, Ltd.     

Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids

Quantitative separation of ceramide, sphingoids (dihydrosphingosine, sphingosine, psychosine), and glycosphingolipids as individual fractions was achieved with silicic acid, Dowex column chromatography, and specific solvent mixtures that have not been previously described. Purified ceramide, resolved as a single band, was assayed by thin-layer chromatography (TLC) followed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Sphingoids, purified by Dowex, were assayed by GC and HPLC without mild alkaline hydrolysis, which reduces the yield by interfering with the free amino group of psychosine and dihydrosphingosine. Several less polar (than cerebroside) alkali-/acid-labile glycosphingolipids that elute with galactosylceramide were also identified. Neutral and acidic glycosphingolipids, quantitatively recovered and purified to homogeneity, were resolved by TLC. We used these techniques to determine sphingolipids and sphingoids of vertebrate central nervous system (CNS) tissue, using as little as 30-50 mg (wet weight) of tissue. In addition, phosphatidylcholine and sphingomyelin, relevant to ceramide metabolism, were quantitatively recovered in pure form and resolved by TLC. This method, used to study CNS sphingolipid content, may well be applicable to determine the sphingolipid composition of other tissues and cell culture, but further experiments are necessary to ascertain this.     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

薄层色谱和高效液相色谱联用测定暗纹东方鲀肌肉中肌肽和谷胱甘肽的含量

通过薄层色谱(TLC)与高效液相色谱(HPLC)联用技术鉴定了暗纹东方鲀(Takifugu obscurus)肌肉中存在肌肽和谷胱甘肽,同时利用高效液相色谱法测定了肌肽和谷胱甘肽(GSH)的含量。薄层层析采用的展开剂为正丁醇:乙酸:水(4∶2∶1),层析板为硅胶板。高效液相色谱利用Kromasil C18反相柱分析,流动相为10%的纯乙腈和90%含有0.05%三氟乙酸的超纯水。结果表明:通过薄层色谱和高效液相色谱鉴定了暗纹东方鲀肌肉中肌肽和谷胱甘肽,其中暗纹东方鲀肌肉中肌肽含量约213μg/g(鲜重),还原性谷胱甘肽含量约211μg/g(鲜重)。本法样品无需衍生,操作简便,适合于暗纹东方鲀肌肉中肌肽和谷胱甘肽的测定。本文为生物体内肌肽和谷胱甘肽的研究提供借鉴意义。     

Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC). A streamlined and more sensitive method for the measurement of esterified plasma F2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 microL) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF2alpha being identified as a major isomer. With this procedure, esterified plasma F2-isoprostanes were found to be 8.3-fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF2alpha was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay.     

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.     

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision. This revised version was published online in June 2006 with corrections to the Cover Date.     

阿魏蘑多糖理化性质及免疫活性研究*

以阿魏蘑Pleurotus ferulae Lanzi子实体和菌丝体为试验材料,采用水浸法提取阿魏蘑多糖,分别得到子实体粗多糖A和菌丝体粗多糖B。将A经Sevag法去蛋白、透析、CTAB络合、乙醇沉淀、NaCl溶液溶解、透析,得到多糖A1。紫外光谱分析鉴定多糖A1为均一组分。苯酚—硫酸法测得多糖A1糖含量为82.9%。凝胶渗透色谱法测得多糖A1数均分子量Mn=141088,重均分子量Mw=142897。气相色谱分析多糖A1单糖组成及其摩尔比为Xyl∶Gla∶Glc=1∶1.102∶2.899。巨噬细胞吞噬作用试验、迟发型变态反应试验、白细胞介素-2（IL-2）的诱生与检测试验测得粗多糖A、粗多糖B具有免疫活性。     

Purification and spectroscopic properties of pyrene fatty acids

Pyrene fatty acids are routinely purified by silica based column chromatography and analyzed on thin-layer silica plates (H.-J. Galla et al., Chem. Phys. Lipids, 23 (1979) 239-251). Although pyrene decanoic acid runs as a single spot on thin-layer chromatography (TLC), gas-liquid chromatography (GC) of the methyl ester derivatives of a representative sample revealed four separate peaks with the major component only 92% of the total. High performance reverse phase liquid chromatography (HPLC) was used to purify pyrene decanoic acid and separate the contaminants. After two passes on a C18 reverse phase HPLC column, pyrene decanoic acid is 99.98% pure by GC analysis. Absorption, fluorescence, and NMR spectra were recorded for pyrene decanoic acid and the major impurities. The results indicate that one impurity is a C10 fatty acid with an altered aromatic moiety. Two other impurities are pyrene derivatives but their acyl chains probably are not decanoic acid.