羧甲基-β-环糊精为手性选择剂拆分托吡卡胺等4种手性药物对映体

目的以羧甲基-β-环糊精(CM-β-CD)为手性选择剂,建立毛细管区带电泳法分离托吡卡胺、文拉法辛、美托洛尔和瑞格列奈4种药物对映体。方法采用未涂壁熔融石英毛细管柱,磷酸盐缓冲液作为背景电解质溶液,分离电压为20 kV。考察了缓冲溶液的pH值、环糊精质量浓度、缓冲盐浓度对对映体分离的影响。结果在优化的分离条件下,4种手性药物均能达到完全分离。结论CM-β-CD适用于上述4种药物的对映体分离。     

Synthesis,resolution and characterization of ring substituted phenylalanines and tryptophans

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (Nα-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.     

Separation of vinca alkaloid enantiomers by capillary electrophoresis applying cyclodextrin derivatives and characterization of cyclodextrin complexes by nuclear magnetic resonance spectroscopy

In this work, the enantiomeric separation of three vinca alkaloid enantiomers (vincamine, vinpocetine and vincadifformine) has been investigated in an aqueous capillary electrophoresis (CE) system using cyclodextrins (CDs). The investigated CDs were the native α-, β-, and γ-CDs and their hydroxypropylated, randomly methylated, carboxymethylated and sulfobutylated derivatives. The first part of this study consisted of the determination of the apparent averaged complex stability constants with the selected CDs. Several parameters, such as the nature and the concentration of the CD, were studied and were found to have a significant effect on the enantiomeric resolution for all studied compounds. All three vinca alkaloids were successfully enantioseparated with CDs where different migration orders were observed in case of several CDs depending on the cavity size or substituent of the host. Chiral separation and determination of the stability constants were also performed with NMR spectroscopy which confirmed the CE results. Averaged stoichiometries of the complexes were determined using the Job plot method resulting in a 1:1 complex irrespective of the alkaloid enantiomers or cyclodextrin derivative. The structures of the inclusion complexes were elucidated using 2D ROESY NMR spectroscopy. On the basis of NMR results reversal of enantiomer migration order was clarified in various cases.     

氧氟沙星对映体的毛细管电泳手性分离

用高效毛细管电泳和二极管阵列检测器,以环糊精为拆分试剂对氧氟沙星进行手性分离(最大分离度可达2.18)。发现衍生化环糊精有较好的分离效果,用含DM-β-CD40mmol·L^-1,KH₂PO₄70mmol·L^-1,pH2.5的缓冲液,在25℃电泳,20kV分离电压下得到优化的分离结果。     

The indirect UV detection in the analysis of ursodeoxycholic acid and related compounds by HPCE

A high-performance capillary zone electrophoretic (HPCE) assay has been developed for the determination of ursodeoxycholic acid (UDCA) and its usual impurities. Considering the low molecular absorptivity of UDCA and its related compounds indirect UV detection was used. The electrophoretic capillary was filled with a background electrolyte (BGE) containing an UV absorbing ion: benzoic acid (BA) or 5,5-diethylbarbituric acid (DBA). To enhance the selectivity of the assay diimethyl-β-cyclodextrines (D-β-CDs) or trimethyl-β-cyclodextrines (T-β-CDs) have been added to the running buffer together with methylcellulose or urea. All considered impurities were well resolved with two buffers studied, with the exception of methylursodehoxycholate, a neutral compound.     

Development and validation of a capillary electrophoresis method for the enantiomeric purity determination of RS86017 using experimental design

A selective capillary electrophoresis method for determination of enantiomeric purity of RS86017, a new antiarrhythmic agent with two chiral centers, was developed and validated using sulfobutyl ether-β-cyclodextrin as chiral selector. The concentration of the chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature, and applied voltage were systematically optimized by using orthogonal design and concentration of chiral selector was further optimized. The optimal conditions included 25mM phosphate buffer at pH 8.0, containing 28mg/mL sulfobutyl ether-β-cyclodextrin and 20% acetonitrile as running buffer, an applied voltage of 22kV, and a temperature of 20°C. The detection wavelength was 206nm. The obtained method was capable of separating RS86017 from its potential chiral impurities, the S,R-enantiomer, the R,R-diastereomer and the S,S-diastereomer with a short analysis time of 10min. The separation was validated with respect to its selectivity, repeatability, linearity, precision, accuracy, limits of detection (LOD), limits of quantitation (LOQ) and robustness testing. The LODs and LOQs were 0.8μg/mL and 2.5μg/mL for all isomers of RS86017, respectively. Finally, the method was used to investigate the chiral purity of RS86017 in bulk samples.     

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.     

Development and validation of a GC method for quantitative determination of enantiomeric purity of a proline derivative

Enantioselective HPLC, SFC and GC methods were evaluated for separation and quantitative determination of chiral purity of (2R,4R)-1-(1-tert-butoxyvinyl)-4-methoxypyrrolidine-2-carboxylic acid [(2R,4R)-TBMPCA], a common building block in organic synthesis. All three separation methods can provide baseline resolution of (2R,4R)-TBMPCA and its enantiomer (2S,4S)-TBMPCA; however, both enantioselective HPLC and SFC are unsuitable for quantitation of low levels of the undesired enantiomer in (2R,4R)-TBMPCA. Comparatively, the enantioselective GC method not only separates the derivatized enantioselective pair with resolution as high as 4, but also was shown to be sufficiently linear, precise, and accurate to enable quantitation of derivatized (2S,4S)-TBMPCA down to 2.4 μg/ml (0.04% of nominal concentration). The sample derivatization procedure is simple, and no sample clean-up is needed before injecting samples for enantiomeric GC analysis. Compared to the enantioselective HPLC and SFC methods, the enantioselective GC method is advantageous because of its high efficiency and high sensitivity.     

Comparison of three chiral stationary phases with respect to their enantio- and diastereoselectivity for cyclic beta-substituted alpha-amino acids.

Three chiral stationary phases were examined for the enantio- and diastereoseparation of cycloaliphatic beta-substituted alpha-quaternary alpha-amino acids. Resolution of diastereomeric analytes is feasible with a chiral crown ether based column, whereas the separation of enantiomers, except for one pair of amino acids, could not be achieved. The two chiral stationary phases with the glycopeptide antibiotic teicoplanin and with the copper(II)-D-penicillamine complex, respectively, are, however, both very potent in the separation of the enantiomeric, as well as of the diastereomeric amino acids. A baseline separation of all four stereoisomeric forms in one chromatographic run was possible with the exception of one type of amino acid. The results of the method development are presented in this paper.     

A method for measurement of tranylcypromine in rat brain regions using gas chromatography with electron capture detection

A gas chromatography method, using electron capture detection, is described for measurement of the monoamine oxidase inhibitor tranylcypromine (TCP) in rat brain regions. The analytical method involves extraction of the drug from tissue homogenate with a liquid ion exchange resin and back extraction into acid. TCP is then acetylated and derivatized with pentafluoropropionic anhydride or trifluoroacetic anhydride for gas chromatographic analysis. Conditions for analysis on both packed and capillary columns are described. The method has been used to quantify TCP in rat brain regions after three different dosage/time schedules of TCP administration. The presence of N-acetyl-TCP has been demonstrated in brain tissue from rats treated with TCP.     

Recent trends in the determination of polyphenols by electromigration methods

An overview mapping recent trends in the determination of polyphenols of natural origin (mostly flavonoids) and their synthetic derivatives by electromigration methods is presented. The overview (covering the period of the recent 5 years and comprising 61 references) is focused on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with various detection methods. Techniques comprising on-line pre-separation such as isotachophoresis (ITP)-CZE and flow-injection-CZE, chiral separations and CZE evaluation of antioxidation activity are also discussed.     

Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum

Trichorzianines A, membrane active peptides of the peptaibol class, were isolated from cultures of the mould Trichoderma harzianum. Trichorzianines A were separated into pure components by HPLC on octadecyl bonded and SiO₂ phases successively. Nine trichorzianines A (IIa, IIIa, IIIb, IIIc, IVb, Vb, VIa, VIb and VII) were isolated from the complex microheterogeneous mixture. Their N-terminal amino acid is acetylated, the C-terminal amino alcohol is either tryptophanol or phenylalaninol, 7 to 8 of the 19 residues are α-aminoisobutyric acid. Gas chromatography on a chiral phase showed isovaline to have the d -configuration and all the other optically active amino acids and amino alcohols to have the l -configuration. The amino acid sequences were determined from their positive ion FAB mass spectra which exhibited the preferential cleavage of the Aib 12-Pro 13 amide bond as a main fragmentation. The resulting fragments subsequently underwent amide bond ruptures that generated two series of abundant acylium ions which enabled direct determination of the 1–19 sequence. The relative position of the isomeric amino acids in the sequence of trichorzianine AVII was assigned from analysis of the N- and C-terminal oligopeptides yielded by its selective acidic hydrolysis. The microheterogeneity of trichorzianines A results mainly from single or multiple substitution of amino acids at the specific positions 5, 14, 16 and 19.     

Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.     

Determination of the enantiomeric purity of S-ropivacaine by capillary electrophoresis with methyl-beta-cyclodextrin as chiral selector using conventional and complete filling techniques.

Capillary electrophoresis (CE) methods based on the conventional and complete filling techniques for determination of the enantiomeric purity of S-ropivacaine are described. The complete filling technique is a separation method which can be used instead of the partial filling technique in order to reduce the total analysis time, when the chiral selector solution does not absorb UV light. In the complete filling technique the total length of the capillary is filled with the chiral selector solution, prior to application of the analyte. During the run both ends of the capillary are connected to the background electrolyte, i.e. without chiral agent. An interlaboratory study was performed to validate the method. The limit of detection and quantification for R-ropivacaine were found to be about 0.6 and 1.6 microg/ml, respectively, corresponding to 0. 1 and 0.25% enantiomeric purity of S-ropivacaine. Good performances were demonstrated for the repeatability and linearity. The consumption of the chiral selector was about 160 times lower with the complete filling technique compared with the conventional CE technique.     

Distinguishing the cyanobacterial neurotoxin β-N-methylamino-l-alanine (BMAA) from other diamino acids

β-N-methylamino-l-alanine (BMAA) is produced by diverse taxa of cyanobacteria, and has been detected by many investigators who have searched for it in cyanobacterial blooms, cultures and collections. Although BMAA is distinguishable from proteinogenic amino acids and its isomer 2,4-DAB using standard chromatographic and mass spectroscopy techniques routinely used for the analysis of amino acids, we studied whether BMAA could be reliably distinguished from other diamino acids, particularly 2,6-diaminopimelic acid which has been isolated from the cell walls of many bacterial species. We used HPLC-FD, UHPLC-UV, UHPLC-MS, and triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) to differentiate BMAA from the diamino acids 2,6-diaminopimelic acid, N-2(amino)ethylglycine, lysine, ornithine, 2,4-diaminosuccinic acid, homocystine, cystine, tryptophan, as well as other amino acids including asparagine, glutamine, and methionine methylsulfonium.     

Resolution of Optical Isomers by Thin-Layer Chromatography: Enantiomeric Purity of Methyldopa. Dünnschichtchromatographische Enantiomerentrennung: Enantiomere Reinheit von Methyldopa

Just 20 % of chiral synthetic drugs are used as pure stereoisomers¹⁾, although it is known from numerous examples that optical isomers may have quite different biological effects²⁾. Therefore the enantiomeric purity is an important probe for monitoring the quality of drugs. The availability of reliable analytical techniques for the correct determination of enantiomeric compositions is therefore becoming increasingly important. Such a method should allow us to deal successfully with the determination of small enantiomeric impurities.     

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of l-β-ODAP by increasing the oxidative stress: Studies on a motor neuron cell line

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea (Lathyrus sativus L.) containing l-β-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of l-β-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of l-β-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. l-β-ODAP further lowered the content in the deprived media to be 32-44% of the controls compared to normal media being 62-74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by l-β-ODAP under sulfur amino acid-deficient conditions.     

Selectivity manipulation in micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) permits the separation of electrically neutral analytes by chromatographic principles in a capillary electrophoresis system. The most effective way to obtain high resolution in MEKC is to increase the separation factor, as in conventional chromatography. The separation factor in MEKC depends on the molecular structure of the micelle and hence on the surfactant used, the pH of solution, and the nature of any additives to the micellar solution. The hydrophilic moieties of surfactant molecules generally affect selectivity more than do the hydrophobic moieties. Chiral surfactants enable the enantiomeric separation of mixtures of chiral solutes to be achieved. Mixed micelles consisting of ionic and nonionic surfactants display different selectivity from that of single ionic micelles. Additives such as cyclodextrins, ion-pair reagents, urea, organic solvents and metals can also serve as useful modifiers of the micellar solution for improving separation. In particular, cyclodextrins are useful for the separation of aromatic isomers and enantiomers. A general introductory guide to the design of successful separations by MEKC is proposed, based primarily on the author's work.     

Enantiomeric separation of fluoxetine derivatives on polysaccharide-based chiral columns

A high performance liquid chromatography (HPLC) method employing amylose-based chiral columns (Chiralpak AD-RH and Chiralpak AD) and cellulose-based chiral columns (Chiralcel OD) as chiral stationary phases have been developed for the enantiomeric separation of fluoxetine (FLX) derivatives. The FLX was derivatized with 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) and 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), respectively. Influence of the mobile phase composition and column temperature on the enantioseparation was discussed during the separation. On the basis of separation of derivatized FLX enantiomers, the paper also discussed the separation mechanism on the chiral stationary phases used.     

Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography

Hesperetin (HT) is a flavanone abundantly found in citrus fruits. It has been reported that HT possesses significant antioxidant, anticancer, anti-inflammatory and analgesic activities. This explains the necessity of developing new methods more powerful and sensitive for analyzing HT in biological fluids. Taking into account the chiral nature of HT, the study of the stereospecific kinetics of in vitro and in vivo metabolism and tissue distribution could be a useful tool for further understanding stereoselective biotransformations in human body. A simple nano-liquid chromatographic method for the determination of the enantiomeric composition of hesperetin in human urine was developed. Chiral separation was achieved using a 100 μm I.D. capillary, packed with phenyl-carbamate-propyl-β-cyclodextrin stationary phase, employing a mobile phase composed by a mixture of triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) at room temperature. The detection was done by using on-column UV detector at 205 nm. Calibration curves were linear in the studied concentration range from 0.25 to 25 μg/mL (r² > 0.999). Precision assay was <4.5% and was within 3% at the limit of quantification (0.5 μg/mL). The recovery of 7-ethoxycoumarin (IS), R- and S-hesperetin was greater than 82.48%, utilizing a liquid–liquid extraction procedure. The developed method was successfully applied to the determination of hesperetin enantiomers in urine samples obtained from a male volunteer, after the ingestion of 1 L of a commercial blood orange juice.