Preparation and Characterization of 61,6n-DI-O-(α-D-Galactopyranosyl)Cyclomaltooctaoses

ABSTRACT

Four positional isomers of 6¹,6ⁿ-di-O-(D-galactopyranosyl)cyclomaltooctaoses (cG₈s, γ-cyclodextrins) (n = 2?5) were chemically synthesized using the trichloroacetimidate method. The desired compounds having two α-(1→6)-linkages were isolated from a mixture of configurational isomers by HPLC, and their structures were confirmed by ¹³C NMR spectroscopy and FAB-high resolution mass spectra (HRMS). The elution behavior of their four positional isomers on an ODS column by HPLC is discussed.     

Analyses of a Mixture of Glucosyl-Cyclomaltoheptaoses Prepared on an Industrial Scale

ABSTRACT

A mixture of glucosyl-cyclomaltoheptaoses (β-cyclodextrins, βCDs) was prepared by glucoamylolysis of a mixture of maltosyl-βCDs which was produced on an industrial scale from maltose and β CD through the reverse action of Klebsiella pneumoniae pullulanase. Glucosyl-βCDs in the mixture were separated by HPLC on a reversed phase column and their molecular weights were measured by FAB-MS. In addition, the number of side-chains in each molecule was confirmed by methylation analysis and it was proved that the mixture comprised mainly of a monoglucosyl-βCD [G-β CD] and diglucosyl-β-CDs [(G)₂-βCDs], and as a minor component triglucosyl-β CDs [(G)₃-βCDs], and that G-, (G)₂-, and (G)₃-β CDs were produced in the ratios of 50%:45%:5%. The structures of three positional isomers of (G)₂-β CD were established by HPLC analysis of partial hydrolyzates, ¹³C NMR spectroscopy, and chemical synthesis. Four regioisomeric (G)₃-β CDs which could be isolated were characterized by ¹³C NMR spectroscopy.     

Cyclodextrins with heterocyclic substitution as GC stationary phases

Summary Two new cyclodextrin (CD) derivatives, heptakis{2,6-di-O-pentyl-3-O-[3′-(2″-chloro-4″,5″-dioxylmethene)-phenyl-5′-iso-oxazolylmethyl]}-β-CD (CD I) and heptakis{2,6-di-O-methyl-3-O-[3′-(2″-chloro)-phenyl-5′-iso-oxazolylmethyl]}-β-CD (CD II) were synthesized and coated on fused-silica capillary columns. Their chromatographic characteristics, including column efficiency, polarity, selectivity and phase transition were studied and compared with similar β-CD stationary phases. It was found that the heterocycle group has a significant effect on the selectivity of the CD stationary phases. Both stationary phases can be successfully used to separate many di- and trisubstituted benzene positional isomers and show stronger separation ability in separating low-polarity benzene positional isomers than other β-CD stationary phases.     

Liquid chromatography analysis of monosubstituted sulfobutyl ether-beta-cyclodextrin isomers on porous graphitic carbon

The retention behaviour of the three positional isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was investigated on a porous graphitic carbon (PGC) column. The influence of the mobile phase composition (nature and concentration of organic and electronic modifiers) was studied as well as the effect of column temperature. These hydrophilic and anionic analytes were highly retained on the PGC stationary phase compared to octadecyl bonded phases. The retention is mainly governed by a reversed-phase mechanism with electronic interaction playing a secondary role. An increase in solute retention and efficiency with temperature was observed. Successful isocratic separation with satisfactory baseline resolution of the three isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was achieved at 75 degrees C on a Hypercarb column by using ammonium acetate as electronic modifier in water-acetonitrile (83:17). The chromatographic methodology developed can be easily used for relative quantification of each isomer within a mixture and can be applied for semi-preparative purification of each one. The evaporative light scattering detector allows the detection of these non UV-visible absorbing molecules.     

缬氨霉素作为毛细管气相色谱新型固定相的性能研究

本文研究了缬氨霉素的气相色谱特性, 考察了它们对一些难分离物质对、位置异构体以及一些手性化合物的分离效果. 除文献[3]外, 目前尚未见到有关将环状肽作为色谱固定相的报道.     

A LC Separation and Quantification of o‐Toluene Sulphonamide and its Related Positional Isomers Using a Cellulose tris(3,5‐dimethyl phenylcarbamate)

A simple, rapid, selective and sensitive high performance liquid chromatographic method was developed for the separation and quantification of o‐Toluene Sulphonamide and its positional isomers which is an intermediate of Zafirlukast drug substance using a chiral column. Elution time was below 10 min in normal phase mode and ultra violet detection was carried out at 220 nm. Efficient separation was achieved on a Chiralcel OD‐H column using of n‐Hexane and 2‐Propanol mixture (50:50 v/v) as isocratic at 1.0 mL min⁻¹ flow rate. Resolutions between m‐Toluene Sulphonamide and p‐Toluene Sulphonamide isomers of o‐Toluene Sulphonamide were found to be >; 2.5. The calibration graphs for m‐Toluene Sulphonamide and p‐Toluene Sulphonamide isomers of o‐Toluene Sulphonamide were linear (R² > 0.999) when ranging from the limit of quantitation to 0.5%. The method is found to be selective, precise, linear, accurate and also robust. It was used for not only Quality assurance but also monitoring the synthetic reactions involved in the process development of Zafirlukast. The liquid chromatographic method is found to be specific and reliable for the determination of unreacted levels of positional isomers in reaction mass. This method was successfully validated according to the International Conference Harmonization (ICH) guidelines (Validation of Analytical Procedures: Test and Methodology Q2).     

Fused silica capillary GC separation of alkene halohydrins

The use of a fused silica capillary column for the gas chromatographic analysis of alkene halohydrin positional isomers is described. This method is illustrated with the complete resolution of the positional isomers for propylene bromohydrin, propylene iodohydrin, and allyl bromide bromohydrin. Comparison with the resolution obtained on a packed column is also provided.     

Analysis of fentanyl derivatives by ultra high performance liquid chromatography with diode array ultraviolet and single quadrupole mass spectrometric detection

Fentanyl has become pervasive as a drug of abuse and as adulterant in seized drugs. Positional isomers analyzed by gas chromatography with mass spectrometry can follow the same fragmentation pathway and therefore may not be differentiated. Additionally, electron ionization leads to lack of discernible molecular ion for most fentanyl related compounds. Liquid chromatography may be used as an orthogonal identification technique with diode array ultraviolet and mass spectrometric detection. Here we provide a chromatographic method for the separation of 20 different fentanyl analogues, homologues and positional isomers using ultra high performance liquid chromatography with photodiode array ultraviolet and mass spectrometry detection. Five different columns were investigated utilizing reverse phase chromatography and hydrophilic interaction chromatography. Chromatographic systems were evaluated to determine which could separate the most compounds overall, as well as the most positional isomers. We found that isocratic elution, with a methanol modifier (35%) and formic acid (0.1%) as an additive, on a C18 column at a temperature of 25°C could resolve 10/20 compounds overall and 16/20 positional isomers. Using electrospray ionization, compounds with different masses could easily be distinguished based on their pseudo molecular ions. Ultraviolet detection facilitated differentiation of positional isomers that could not be distinguished by either electron ionization or electrospray ionization mass spectrometry alone.     

基于LC-MS/MS的咖啡酰奎宁酸异构体鉴别方法研究

该文对单咖啡酰奎宁酸和二咖啡酰奎宁酸的位置异构体分别进行液相色谱分析和不同碰撞能下的二级质谱分析,并对其质谱裂解规律进行了研究。结果发现,单咖啡酰奎宁酸的母离子377和子离子163在不同碰撞能下其强度发生显著变化,通过377/163的强度比值可区分其位置异构体。377/163比值大小依次为3-O-咖啡酰奎宁酸、4-O-咖啡酰奎宁酸、5-O-咖啡酰奎宁酸。二咖啡酰奎宁酸的母离子539和子离子377、163在不同碰撞能下其强度发生显著变化,通过539/377、377/163的强度比值可区分其位置异构体。539/377比值大小依次为4,5-O-二咖啡酰奎宁酸、3,4-O-二咖啡酰奎宁酸、3,5-O-二咖啡酰奎宁酸。377/163比值大小依次为3,5-O-二咖啡酰奎宁酸、4,5-O-二咖啡酰奎宁酸、3,4-O-二咖啡酰奎宁酸。基于Agilent Poroshell 120 SB-Aq C_(18)色谱柱,不同梯度下咖啡酰奎宁酸位置异构体的洗脱顺序均为5-O-咖啡酰奎宁酸、4-O-咖啡酰奎宁酸、3-O-咖啡酰奎宁酸、3,4-O-二咖啡酰奎宁酸、3,5-O-二咖啡酰奎宁酸和4,5-O-二咖啡酰奎宁酸。通过色谱保留特征和质谱裂解规律对金银花中咖啡酰奎宁酸的位置异构体实现了准确鉴别。     

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.     

Synthetic study of a moss-produced oxylipin and its structural revision

The proposed structure for a moss-produced acetylenic oxylipin (10-keto-type structure) was synthesized from a known glycidol derivative by an 11-step sequence involving epoxide ring opening with a terminal acetylene and enzymatic hydrolysis of a methyl ester intermediate. The NMR spectra of the proposed structure was, however, different from those of the natural oxylipin, which prompted us to synthesize its 9-epi-isomer and two diastereomeric 12-keto-type positional isomers. Comparison of the NMR spectra of the three isomers with those of the natural oxylipin indicated that the natural oxylipin was actually a mixture of the two diastereomeric positional isomers.     

Characterisation of the surface Lewis acid-base properties of poly(butylene terephthalate) by inverse gas chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were applied to separate 3- and 4-sulfophthalic acid from a mixture. Conventional HSCCC was useful for the separation of up to several hundred milligram quantities of these positional isomers, while pH-zone-refining CCC was implemented successfully to separations at the multigram level. The conventional HSCCC separations were performed with a standard J-type HSCCC system that has a superior resolution but a lower level of retention of the stationary phase of the biphasic solvent system used (acidified n-butanol-water). The pH-zone-refining CCC separations were performed with an X-type HSCCC system (a cross-axis system) that has a higher capability for retention of the stationary phase. The purified positional isomers (over 99% pure as determined by HPLC) were characterized by 1H NMR and negative ion electrospray ionization mass spectrometry.     

Preparation of a polyhedral oligomeric silsesquioxanes hybrid monolith via a single‐step ring‐opening polymerization for pressurized capillary electrochromatography

An organic‐silica hybrid monolith was prepared by a single‐step ring‐opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS‐epoxy), polyethylenimine (PEI), and β‐cyclodextrin (β‐CD) in a ternary porogenic solvent consisting of polyethylene glycol, 1,4‐butanediol, and 1‐propanol. The framework of POSS‐PEI hybrid monolith could offer well‐defined 3D skeleton, while β‐CD with the ability of forming a host‐guest inclusion complexes with a variety of compounds could show an ability of specific selection. The obtained hybrid monoliths were successfully applied for separation of phenols, benzoic acids, and nucleobases. Especially due to the introduction of β‐CD, positional isomers including hydroquinone and resorcinol, o‐nitrophenol and p‐nitrophenol, as well as p‐chlorophenol and o‐chlorophenol were baseline separated and the column efficiency reached 82 300 plates/m for hydroquinone.     

Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.     

A chiral porous organic cage for molecular recognition using gas chromatography

Molecular organic cages as shape-persistent organic molecules with permanent and accessible cavities have attracted a lot of interest because of their importance as host-guest systems. Herein, we report a chiral porous organic cage (POC) CC9 diluted with a polysiloxane OV-1701 to fabricate a CC9-coated capillary column, which was used for the high-resolution gas chromatographic separation of organic compounds, including positional isomers and racemates. On the CC9-coated capillary column, a large number of racemic compounds such as chiral alcohols, esters, ethers and epoxides can be resolved without derivatization. By comparing the chiral recognition ability of the CC9-coated column with the commercially available β-DEX 120 column and the POC CC3-R coated column recently reported by our group, the CC9-coated column offered good resolution during the separation of some racemates, that were not separated using the β-DEX 120 column or POC CC3-R coated column. Therefore, the CC9-coated column can be complementary to the β-DEX 120 column and CC3-R coated column. The results indicated that the CC9-coated column exhibited great potential for application in the separation of positional isomers and enantiomers with great selectivity, high resolution and good reproducibility.     

Characterisation of sorbate geometrical isomers

trans,trans Isomers of sorbic acid, its potassium salt and ethyl sorbate isomerise under UV irradiation. All four geometrical isomers of the acid, salt and ester were separated using high-performance liquid chromatography on a nonpolar reversed-phase column (C18) and the ester also by gas chromatography on a VOCOL capillary column. The limit of detection and the interval of linearity were determined for all chromatographic methods. Individual isomers were identified with NMR analysis. Resolved chemical shifts of protons adjacent to the double bonds enabled qualitative and quantitative determination of isomers in the mixture. Antimicrobial activity of potassium sorbate isomers was tested on yeast Saccharomyces cerevisiae. Results show that the pure trans,trans isomer has a higher antimicrobial activity than the mixture of isomers.     

Non-aqueous capillary electrophoresis of the positional isomers of a sulfated monosaccharide

A non-aqueous capillary electrophoresis (NACE) method coupled to indirect absorbance detection has been developed for the separation of the three positional isomers of monosulfated fucose. The optimized electrolyte was composed of 12 mM ethanolamine, 2 mM trimesic acid buffer in a methanol-ethanol (1:1, v/v) mixture. As the retained electrolyte entails no separating agent other than the pH buffer, the NACE separation of the positional isomers has been ascribed mainly to selective ion-pairing with the electrolyte counter-ion and the possibility of a selective solvation effect in the alcohol mixture. In the absence of pure isomeric standards, peak identification was completed by MS and NMR spectroscopy and selective enzymatic desulfation. This method should be of interest for the structure elucidation of monosulfated fucose-based polysaccharides and for the screening of sulfoesterase of unknown activity.     

苯并芘DNA加合物anti-BPDE-N^2-dG四种立体异构体的合成及色谱分离

体外合成了苯并芘DNA加合物-邻二醇环氧苯并芘-脱氧鸟苷加合物(anti-BPDE-N2-dG)四种立体异构体(两对手性异构体)。通过优化体外反应条件,anti-BPDE-N2-dG四种异构体的合成产量较现有合成方法提高了2倍多,为定量检测生物体中anti-BPDE-N2-dG提供了标准品。并首次将五氟苯基色谱柱应用于该立体异构体的色谱分离提纯,通过优化色谱条件,采用常规的五氟苯基色谱柱(250 mm×4.6 mm,5μm),以乙腈-0.1%甲酸水(22.5∶77.5)为流动相,流速1.2 mL/min条件下,45 min内即可分离提纯四种立体异构体。该方法与常规C18柱(250 mm×4.6 mm,5μm)需要160 min,苯基柱(250 mm×4.6 mm,5μm)需要85~100 min才能将四种立体异构体实现色谱分离相比,缩短了分离时间,提高了提纯效率。通过紫外吸收光谱、质谱、圆二色谱对四种立体异构体进行表征,确定出峰顺序为trans(-)、trans(+)、cis(+)、cis(-)-anti-BPDE-N2-dG。此外,利用高效液相色谱-串联质谱(HPLC-MS/MS)检测anti-BPDE-N2-dG四种立体异构体标准品时,使用常规的五氟苯基色谱柱可在30 min内完成分离检测,与相同规格的苯基柱需要60 min相比提高了检测效率。     

HPLC separation of triacylglycerol positional isomers on a polymeric ODS column

A polymeric ODS column was applied to the resolution of triacylglycerol positional isomers (TAG-PI), i.e. 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) and 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), with a recycle HPLC system. To investigate the ODS column species and the column temperatures for the resolution of a TAG-PI pair, a mixture of OPO and OOP was subjected to an HPLC system equipped with a non-endcapped polymeric, endcapped monomeric, endcapped intermediate, or non-endcapped monomeric ODS column at three different column temperatures (40, 25, or 10 degrees C). Only the non-endcapped polymeric ODS column achieved the separation of OPO and OOP, and the lowest column temperature (10 degrees C) showed the best resolution for them. The other pair of TAG-PI, a mixture of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) and 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (PPO) was also subjected to the system equipped with a non-endcapped polymeric or monomeric ODS column at five different column temperatures (40, 32, 25, 17, and 10 degrees C). Thus, POP and PPO were also separated on only the non-endcapped polymeric ODS column at 25 degrees C. However, no clear peak appeared at 10 degrees C. These results would indicate that the polymeric ODS stationary phase has an ability to recognize the structural differences between TAG-PI pairs. Also, the column temperature is a very important factor for separating the TAG-PI pair, and the optimal temperature would relate to the solubility of TAG-PI in the mobile phase. Furthermore, the recycle HPLC system provided measurements for the separation and analysis of TAG-PI pairs.