The stereochemical resolution of enantiomeric free and derivatized amino acids using an HPLC chiral stationary phase based on immobilized alpha-chymotrypsin: chiral separation due to solute structure or enzyme activity

The stereochemical separation of free and derivatized amino acids on active alpha-chymotrypsin bonded to silica is governed by two mechanisms based on the structure of the solutes or on the enzymatic activity of the enzyme. The deactivation of the hydrolytically active site of the enzyme demonstrated that a significant portion of the retention on this support is due to hydrophobic interactions at other sites. These sites appear to be stereoselective for the ester derivatives of amino acids but not for the other studied solutes.     

Interactions of chiral molecules with an (r)-n-(3,5-dinitrobenzoyl) phenylglycine HPLC stationary phase

Forty different chiral molecules were studied by liquid chromatography with a Pirkle-type, (R)-N-(3,5-dinitrobenzoyl) phenylglycine (DNBPG), chiral stationary phase column. The dramatic effect of a small molecular change on chiral recognition was demonstrated using DL-amino acid derivatives. The inductive effect on chiral recognition was also studied using trifluoro-, trichloro-, dichloro-, monochloroacetyl, and acetyl derivatives of four different chiral amines. The study of the enantiomer separation of 11 different crown ethers of 2,2′-binaphthyldiyl showed that the rigidity of the chiral center can be an additional parameter in chiral recognition for the DNBPG phase but not for a β-cyclodextrin bonded chiral phase. It is apparent from this study that steric effects, inductive effects, and molecular rigidity play important roles in chiral recognition with DNBPG chiral stationary phases.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

"Molded" rods of macroporous polymer for preparative separations of biological products

A continuous rod of porous poly(glycidy1 methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 16-mm-i.d. glass column. The epoxide groups of the rod have been modified in situ by their reaction with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydroxypropyl functionalities that are required for the ion-exchange chromatographic mode. The bimodal pore size distribution curve typical for other molded separation media also prevail for the preparative-size rod. The column has been used successfully for the chromatographic separation of a mixture of standard proteins and yeast enzymes. The column exhibits a dynamic capacity that exceeds 420 mg of bovine serum albumin at a flow velocity of 60 cm/h. (c) 1995 John Wiley & Sons, Inc.     

The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity

The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site.     

两种水溶性抗菌活性物质的分离提取

对水溶性、不解离的极性物质分离时,一般采用吸附层析和凝胶层析等途径。实验通过硅胶柱层析、葡聚糖凝胶柱层析以及硅胶ＧＦ２５４制备型薄板层析,从发酵液样品中分离出两种有抗菌活性的纯物质。薄层层析的展开剂为二氯甲烷－四氢呋喃－甲醇－水（２５：３０：２）,分离出的组分中Ｒｆ＝０．７和Ｒｆ＝０．８两种物质有抗菌活性。硅胶柱层析洗脱过程为梯度洗脱,先用１５０ｍｌ上述展开剂洗脱,再用二氯甲烷－甲醇（２０：８０）     

双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组

采用荧光染料(Cy5)标记中国辽宁蛇岛蝮(Gloydius shedaoensis shedaoensis,GSS)蛇毒(snake venom,SV,GSS-SV)蛋白质,获得了该蛇毒的双向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(2D SDS-PAGE)图谱,经DeCyder软件分析,分辨出1000多个蛋白点,分子量范围在10～150ku间,等电点在4～7的蛋白质点占78.8%。凝胶后染色(post-staining)采用蛋白荧光染料Deep Purple,选取的5个蛋白点经胶内酶解,产生的肽段经高效液相色谱-电喷雾串联质谱(high performance liquid chromatography/HPLC-electro-spray ionization tandem mass spectrometry/ESI-MS/MS,HPLC-ESI-MS/MS)进行序列测定,质谱数据经Sequest Bioworks软件分析,为蛇毒L-氨基酸氧化酶、金属蛋白酶、类凝血酶、纤溶酶原激活物和磷脂酶A2的同源蛋白。本研究采用的荧光标记2DSDS-PAGE结合HPLC-ESI-MS/MS的技术适于高通量研究蛇毒蛋白组成。     

Lack of metabolic racemisation of ropivacaine,determined by liquid chromatography using a chiral AGP column

Ropivacaine hydrochloride monohydrate (ropivacaine) is a new local anaesthetic agent which is administered exclusively as the (-)-(S)-form. The aim of the study was to determine whether metabolic racemisation of (-)-(S)-ropivacaine occurs. This was tested in man, rat, dog, and sheep after different routes of administration. The enantiomers of ropivacaine and two of the major metabolites, 3-hydroxy-ropivacaine and 2′,6′-pipecoloxylidide (PPX), were determined in urine samples by liquid chromatography on a Chiral AGP column after liquid–liquid extraction. It was possible to detect <1% of the (+)-(R)-enantiomer of both ropivacaine and the two major metabolites. In the samples examined, no trace of metabolic racemisation was observed. In pharmacokinetic, pharmacodynamic, toxicological, and metabolic studies, therefore, nonchiral assays are considered to be adequate. © 1995 Wiley-Liss, Inc.     

Xterra RP18柱高效液相色谱法快速分离测定氨基酸

建立了一种用XterraRP1 8色谱柱快速分离测定水解氨基酸的方法。所采用的色谱条件是 :WatersAlliance系统 ,柱温 5 6℃ ,流速 1 .8ml/min ,检测波长 2 4 8nm ,梯度分离 ,运行周期 2 5min,柱反压低于 2 0 0 0Psi。在 1 7.5min内分离了包括AMQ、NH3 和牛磺酸在内的 2 1种氨基化合物 ,适应于复合氨基酸注射液、含牛磺酸的氨基酸口服液及水解氨基酸样品的分析测定     

Thermospray liquid chromatography/mass spectrometry for the analysis of l-carnitine and its short-chain acyl derivatives

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.     

Isolation,Purification, and Resolution of the Extracellular Proteinase Complex of Aspergillus ochraceus513 with Fibrinolytic and Anticoagulant Activities

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus513 was isolated, purified, and resolved by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodonsnake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

油橄榄叶中橄榄苦苷的分离纯化

考察了6种不同大孔树脂对橄榄苦苷的吸附,筛选出吸附选择性好的D101大孔吸附树脂为实验材料.得到的D101大孔树脂的最佳吸附条件:试样上样质量浓度41.06[J].,流速5 mL/min,50％乙醇溶液洗脱,洗脱液用量260mL,树脂可以反复使用.经大孔树脂富集后,橄榄苦苷的纯度可达55％以上,再经葡聚糖凝胶精纯化后,可达76％以上.