手性逆流色谱的研究进展

总结了手性逆流色谱的5个特点,系统地介绍了逆流色谱的手性分离以及高速逆流色谱手性分离中氨基酸衍生物、环糊精衍生物、手性有机酸、多糖衍生物、牛血清白蛋白等手性选择剂的应用。     

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1 beta-hydroxylated and unsaturated bile acids in serum and urine

12 bile acids, including 1 beta-hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 microL of serum and 50 microL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.     

胆汁酸的双敏感探针苯并[b]吖啶酮-5-乙基对甲苯磺酸酯:荧光检测和APCI-MS鉴定

在HypersilC18色谱柱上,利用新型荧光试剂苯并[b]吖啶酮-5-乙基对甲苯磺酸酯(BAETS)作柱前衍生化试剂,采用梯度洗脱对10种胆汁酸衍生物进行了优化分离.在二甲亚砜溶剂中,以碳酸钾作催化剂,95℃,45min后获得稳定的荧光产物.衍生物在稳态荧光条件下,在乙腈和甲醇水溶液中的百分离子化δ值在0%～88.83%和0%～89.15%范围内.最大激发和发射波长为λex/λem=272/505nm.采用大气压化学电离源(APCI)正离子模式实现了胆汁中胆汁酸的定性定量测定.线性回归系数均在0.9995以上,线性范围宽,检出限为0.76～1.62ng/mL.     

Optimization of solid-phase extraction and solid-phase microextraction for the determination of alpha- and beta-endosulfan in water by gas chromatography-electron-capture detection

The electrophoretic behaviour of ionizable and neutral alkylxanthines commonly used in pharmaceutical preparations was studied. The performance of various separation modes including capillary zone electrophoresis (CZE), cyclodextrin electrokinetic chromatography, and micellar electrokinetic chromatography (MEKC) with either sodium dodecyl sulfate (SDS) or bile salts as surfactants, was assessed. CZE in an alkaline medium successfully separates ionizable xanthines and dyphylline. The addition of carboxymethyl-β-cyclodextrin to the background electrolyte allows only partial resolution of neutral xanthines. Based on MEKC results, bile salts exhibit more discrimination ability than SDS to separate similar xanthines. The best results are provided by taurodeoxycholic acid, which ensures baseline separation of xanthines.     

Retention behavior of cardiac steroids using cyclodextrin in the mobile phase in high-performance liquid chromatography

The retention behaviour of twenty cardiac steroids and four fluorescent derivatives was examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The addition of a suitable cyclodextrin improved the separation of isomeric cardiac steroids. The steroid A/B ring junction is the most important factor in the choice of the optimum cyclodextrin to be added; the C/D ring junction is less important. The hydroxyl group at the 3- or 12-position of the steroid enhanced the changes in retention times of these compounds. The effect of an unsaturated lactone ring at the 17 beta-position on the retention in the presence of cyclodextrin was also determined with cardenolide (five-membered ring) and bufadienolide (six-membered ring) but little difference was observed. Isomeric cardiac steroids, whose separation has not been done by the conventional method, were clearly separated by this method. The fluorescence intensity of 3-(1-anthroyl)-cardiac steroid was enhanced by the addition of cyclodextrin to the mobile phase.     

Enantioseparation of acetyltropic acid by countercurrent chromatography with sulfobutyl ether‐β‐cyclodextrin as chiral selector

Acetyltropic acid is an important synthetic intermediate for preparation of tropane alkaloid derivatives, which can be used as anticholinergic drugs, deliriants, and stimulants. In the present work, acetyltropic acid was successfully enantioseparated by countercurrent chromatography using sulfobutyl ether‐β‐cyclodextrin as chiral selector. A biphasic solvent system composed of n‐butyl acetate/n‐hexane/0.1 mol/L citrate buffer at pH = 2.2 containing 0.1 mol/L of sulfobutyl ether‐β‐cyclodextrin (7:3:10, v/v) was selected, which produced a suitable distribution ratio D_S = 1.14, D_R = 2.31 and a high enantioseparation factor α = 2.03. Baseline separation was achieved for preparative enantioseparation of 50 mg of racemic acetyltropic acid. A method for chiral analysis of acetyltropic acid by conventional reverse phase liquid chromatography with hydroxylpropyl‐β‐cyclodextrin as mobile phase additive was established, and formation constants of inclusion complex were determined. It was found that different substituted β‐cyclodextrin should be selected for enantioseparation of acetyltropic acid by countercurrent chromatography and reverse phase liquid chromatography.     

胆汁酸的双敏感探针苯并[b]吖啶酮-5-乙基对甲苯磺酸酯: 荧光检测和APCI-MS鉴定

在Hypersil C₁₈色谱柱上, 利用新型荧光试剂苯并[b]吖啶酮-5-乙基对甲苯磺酸酯(BAETS)作柱前衍生化试剂, 采用梯度洗脱对10种胆汁酸衍生物进行了优化分离. 在二甲亚砜溶剂中, 以碳酸钾作催化剂, 95 ℃, 45 min后获得稳定的荧光产物. 衍生物在稳态荧光条件下, 在乙腈和甲醇水溶液中的百分离子化δ值在0%～88.83%和0%～89.15%范围内. 最大激发和发射波长为λ_ex/λ_em＝272/505 nm. 采用大气压化学电离源(APCI)正离子模式实现了胆汁中胆汁酸的定性定量测定. 线性回归系数均在0.9995以上, 线性范围宽, 检出限为0.76～1.62 ng/mL.     

Determination of serum metabolic profiles of bile acids by microcolumn liquid chromatography/laser-induced fluorescence

A method for the determination of individual free and conjugated bile acids in serum using microcolumn liquid chromatography coupled with a laser-induced fluorescence detector is described. Bile acids are separated into free/glycine-conjugate and taurine-conjugate fractions using a Sep-Pak SIL cartridge. The taurine-conjugated bile acid fraction is subjected to enzymatic hydrolysis. Subsequently, free and conjugated bile acids are labeled using 4-(bromomethyl)-7-methoxycoumarin as a fluorogenic reagent, producing stable derivatives that can be excited by the 325 nm line of a He/Cd laser. Prior to their fluorimetric detection, the individual components of a bile acid serum profile are separated by reversed-phase microcolumn liquid chromatography.     

Improved method for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography

An improved method has been developed for the determination of the major neutral steroids (cholesterol and 5 beta-cholestan-3 beta-ol) and unconjugated bile acids (deoxycholic acid and lithocholic acid) in human faeces, using capillary gas chromatography with flame ionization detection. The freeze-dried faecal sample was subjected to a two-stage Soxhlet extraction followed by an aqueous alkali-organic solvent partition step to separate neutral steroids from bile acids. The neutral steroids were analysed as their trimethylsilyl ether derivatives on an OV-1 capillary column. The bile acids were further purified on a Sep-Pak C18 cartridge and then fractionated on a Sep-Pak SIL cartridge. Unconjugated bile acids were analysed as their methyl ester-trimethylsilyl ether derivatives also on an OV-1 capillary column. Quantitation of neutral steroids and unconjugated bile acids was achieved by reference to appropriate internal standards, added to the faecal extract immediately after the Soxhlet extraction stage. The method is being used in a study of the effect of diet on the metabolic activity of human gut flora.     

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated gamma-cyclodextrin (CM-gamma-CD) as pseudostationary phase mixed with beta-cyclodextrin (beta-CD) or permethylated beta-cyclodextrin (PM-beta-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-gamma-CD, 10 mM beta-CD or 20 mM PM-beta-CD, and 2 M urea at a temperature of 45 degrees C and an applied voltage of 20 kV.     

Separation and quantitation of fatty acids, sterols and bile acids in feces by gas chromatography as the butyl ester-acetate derivatives

A system allowing the separation and quantitation of individual species of fecal fatty acids, sterols and bile acids in a single chromatographic step is described. The system is based on the butylation of carboxyl groups and acetylation of free hydroxyls of the compounds in fecal lipid extracts, followed by their resolution by temperature-programmed gas chromatography. As the butyl ester-acetate derivatives, fatty acids, sterols and bile acids elute separately and with no overlap on a variety of chromatographic columns, obviating the need for prior separation of each class by thin-layer or column chromatography. All common bile acids, a wide variety of sterols and keto-steroids, as well as saturated and unsaturated fatty acids may be routinely resolved. Quantitation is facilitated by the addition of the internal standards, heptadecanoic acid and nor-deoxycholic acid to each sample. With an automatic sample injector, the rapid assessment of a wide range of potential risk factors for colorectal cancer may be carried out in large numbers of samples.     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.     

手性选择剂在色谱分析中的应用

本文综述近年来在光学异构体分离领域中所涉及的主要技术,如高效液相色谱、电色谱、气相色谱及其他色谱法中所使用的常见手性选择性。它们包括环糊精、冠醚、抗菌素、蛋白质、多糖、氨基酸、表面活性剂和分子模板高聚物及其相关的衍生物。     

Bile acids and their oxo derivatives: Potential inhibitors of carbonic anhydrase I and II,androgen receptor antagonists and CYP3A4 substrates

Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico‐chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal‐phase thin‐layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure–activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α ‐hydroxy‐12‐oxo‐5β ‐cholanoic and 3α ‐hydroxy‐7‐oxo‐5β ‐cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α ‐hydroxy‐12‐oxo‐5β ‐cholanoic, 3α ‐hydroxy‐7‐oxo‐5β ‐cholanoic, 3,7,12‐trioxo‐5β ‐cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.     

Gas chromatographic enantiomer separation of agrochemicals using modified cyclodextrins

The enantiomers of phenoxypropionic acid type herbicides have been resolved by capillary gas chromatography employing modified cyclodextrins as chiral stationary phases. Excellent separations were obtained with columns containing a 1:1 mixture of per-O-pentylated and per-O-methylated γ-cyclodextrin. The enantiomers of the methyl esters of mecoprop and dichlorprop were also resolved on octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. On this phase the order of elution of the enantiomers was temperature-dependent, the elution order being reversed as the temperature passed through the isoenantioselective temperature. This is the first time such behavior has been observed with cyclodextrin derivatives. The enantiomers of the polychlorinated polycyclic pesticides cis- and trans-chlordane, oxychlordane, heptachlor, heptachlorepoxide, and three chiral organophosphorus pesticides could be resolved using selectively derivatized cyclodextrin derivatives.     

Capillary gas chromatography separation of pyrethroic acid methyl esters using four acylated cyclodextrin derivatives as chiral stationary phases

Summary Four cyclodextrin derivatives (CDs) were synthesized by substituting 3-OH of 2,6-di-O-pentyl-β-cyclodextrin with four different chain lengths of acyl groups (butyryl, valeryl, heptanoyl, octanoyl). The chromatographic properties of the four CD derivatives as stationary phases of capillary gas chromatography (CGC) were investigated. These CDs exhibit a wide range of application. Not only five pairs of enantiomers of pyrethroic acid methyl esters were separated on the four CDs, but also some other racemic compounds. Among the four CDs, 2,6-di-O-pentyl-3-O-butyryl-β-CD possesses better enantiomer separation abilities to the studied enantiomers of pyrethroic acid methyl esters than the other studied CDs. The extension of chain length of the acyl groups in 3-position of CDs cannot improve the enantiomer separation abilities of the CD derivatives.     

Determination of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid and related bile acids in human serum by gas chromatography-mass spectrometry

The glycine, taurine and sulphate conjugates of 3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid were synthesized as authentic samples for the analysis of this unusual bile acid. A highly sensitive and specific quantitative assay of the bile acid and related compounds has been developed by selected-ion monitoring in gas chromatography-mass spectrometry of their methyl ester-trimethylsilyl ether derivatives, using the deuterium labelled internal standards: [2H6]3 beta,12 alpha-dihydroxy-5-cholen-24-oic acid, [2H5]3 beta-hydroxy-5-cholen-24-oic acid, [2H7]cholic acid and their sulphates. Calibration curves for these bile acids were linear over the range 0.01-100 micrograms/ml in human serum. Recoveries of the bile acids and their conjugates ranged from 95 to 103% of the added amounts of their standard samples. The unsaturated bile acid was identified in a significant amount of 25.2 micrograms/ml in serum of an infant with liver disease, and its sulphate comprised 55.1% of the amount of the bile acid.     

Two-way enantioselective control in the epoxidation of alkenes with the keto bile acid-Oxone® system

A number of 3-keto bile acid derivatives has been prepared and evaluated in the asymmetric epoxidation of unfunctionalized olefins with Oxone. The control of the enantioselectivity with the production of both enantiomers is strictly regulated by the bile acid inductor, as a function of substitution at carbons C(7) or C(12). Up to 98% ee has been achieved. The stereochemical outcome of the reaction may be rationalized in terms of spiro transition state model.     

Studies on steroids. CCXXVIII Trace analysis of bile acids by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A suitable derivatization method for the trace analysis of bile acids by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to the positive ion current. A characteristic carboxylate anion [M - 181]- was produced as the most abundant ion by the loss of the PFB group in NICI. The PFB esters were further derivatized to the dimethylethylsilyl (DMES) ethers, whereby lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and cholic acid were distinctly separated by GC on a cross-linked methyl silicone fused-silica capillary column. The detection limit for the PFB-DMES derivatives of dihydroxylated bile acids was 2 fg when the fragment ion was monitored at m/z 563 in the NICI mode using isobutane as a reagent gas.     

Improved enantioselectivity in the epoxidation of cinnamic acid derivatives with dioxiranes from keto bile acids

The asymmetric epoxidation of substituted cinnamic acids has been obtained in the presence of different keto bile acid derivatives as optically active carbonyl inducers and Oxone as oxygen source. Predominant or almost exclusive formation of both enantiomeric epoxides is obtained (ee up to 95%) depending on the specific substitution at carbons C(7) and C(12) of the bile acid.