Enantioselective separation
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Enantioselektivní TLC je dosud se vyvíjející oblastí analýzy léčiv. V posledních letech byla podobně, jako je tomu v HPLC, publikována především přímá dělení enantiomerů bez jejich předchozí derivatizace. Přednostně k nim byly použity chirální stacionární fáze, dělení s chirálním selektorem v mobilní fázi byla méně častá. K širšímu využití enantioselektivní TLC v budoucnosti by mohl přispět větší výběr chirálních stacionáreních fází komerčně dostupných.
Enantioselective TLC represents a still developing field of analysis of therapeutic agents. In recent years, similarly as in HPLC, mainly direct separations of enantiomers without their preceding derivatization have been published, preferentially using chiral stationary phases, separations with a chiral selector in the mobile phase being less frequent. In the future, a larger choice of commercially available chiral stationary phases could contribute to a more widespread use of enantioselective TLC.
Derivatized polysaccharide-based columns have high enantiodiscrimination potential mainly in normal phase separation mode. In this work chiral recognition ability of four immobilized polysaccharide-derived chiral stationary phases was evaluated under reversed phase conditions. A set of 30 chiral compounds, particularly drugs possessing various functional groups was used for testing. Baseline enantioseparation was achieved for 17 of them. In general, amylose-based chiral stationary phases showed higher enantioselectivity than the cellulose-based ones, mainly for acidic and bifunctional compounds. The influence of the type and pH of the aqueous mobile phase constituents as well as the role of the organic modifier on the enantioselective separation ability of the stationary phases were also investigated and compared. Complementary separations were obtained on the amylose- and cellulose-based columns. The immobilized polysaccharide-based chiral stationary phases were shown to be useful tool for the enantioseparation of a broad spectrum of chiral analytes in reversed phase separation mode.
In the present investigation, eleven human adipose tissue samples, two seal blubber samples and two pelican muscles samples were analyzed with regard to their concentrations of PCB parent compounds as well as to the respective chiral methylsulfonyl metabolites 3-MeSO2-CB 91, 4-MeSO2-CB 91, 3-MeSO2-CB 95, 4-MeSO2-CB 95, 3-MeSO2-CB 149, 4-MeSO2-CB 149, 3-MeSO2-CB 132, 4-MeSO2-CB 132, 3-MeSO2-CB 174, and 4-MeSO2-CB 174 and the achiral metabolites 3-MeSO2-CB 49, 4-MeSO2-CB 49, 3-MeSO2-CB 101, 4-MeSO2-CB 101, 3-MeSO2-CB 110, 4-MeSO2-CB 110 and 3-MeSO2-DDE. In order to verify enantioselective transformation processes and to compare the different enzymatic transformation pathways in birds and mammals, the enantioselective excesses of the chiral PCB-metabolites were determined by enantioselective gas chromatography with electron capture and mass spectrometric detection using modified cyclodextrin phases, including heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-)-beta-cyclodextrin/OV1701 (1:1) for the parent PCBs and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-)-beta-cyclodextrin/SE52 (1:4) for the metabolites, respectively.
- MeSH
- cyklodextriny metabolismus MeSH
- dichlordifenyldichlorethylen analýza metabolismus MeSH
- látky znečišťující životní prostředí analýza metabolismus MeSH
- lidé MeSH
- methansulfonáty analýza metabolismus MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- polychlorované bifenyly analýza metabolismus MeSH
- stereoizomerie MeSH
- svaly chemie MeSH
- tuková tkáň chemie MeSH
- tuleňovití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Application of the superficially porous particles (SPPs) grafted with chiral selectors can substantially improve resolution in chromatographic techniques. In this work, we carried out a deeper study on supercritical fluid chromatography systems with 2.7 µm SPPs bonded with teicoplanin and vancomycin. Fast separations of the majority of enantiomers of phytoalexins, substituted tryptophans, and ketamine derivatives, as representatives of important biologically active and structurally diverse chiral compounds have been achieved. The chromatographic behavior of the structurally different analytes served to characterize these separation systems. The influence of separation conditions, namely mobile phase composition, i.e. type of co-solvent and additive on retention, enantioselective resolution and enantioselectivity was examined. The success rate of baseline and partial separations in individual groups of compounds differed with the chiral stationary phase and also with mobile phase composition. The best, baseline separations for the phytoalexins were achieved on the TeicoShell column using methanol as a co-solvent and trifluoroacetic acid as an additive if used. Mostly partial separations were achieved on the vancomycin-based column for all groups of analytes. Complementary separation behavior of these CSPs was confirmed for the majority of the chiral compounds examined in this work.
- MeSH
- alkaloidy chemie MeSH
- ketamin chemie MeSH
- kyselina trifluoroctová chemie MeSH
- poréznost MeSH
- rozpouštědla chemie MeSH
- seskviterpeny chemie MeSH
- stereoizomerie MeSH
- superkritická fluidní chromatografie metody MeSH
- teikoplanin chemie MeSH
- vankomycin chemie MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
The market share of single-enantiomer drugs is steadily increasing. The pharmacodynamics and pharmacokinetics of individual enantiomers can differ considerably. Thus, their characteristics have to be addressed as early as possible in the development process of new pharmaceuticals. Capillary electrophoresis is a promising technique for enantioselective drug metabolism studies due to highly effective separations, minuscule consumption of sample and reagents, compatibility with a variety of detection techniques, high-throughput via automation, and the implementation of online procedures. An online method comprised of the diffusion-based mixing of cytochrome P450 3A4 with racemic ketamine, incubation of the enzyme reaction, separation of the reaction products S- and R-norketamine, and their quantification is presented in this chapter. Since diffusion is an inherent property of all molecules, the method enables the addition of virtually any compound to the reaction mixture without the need for additional optimization of the mixing conditions, and thus can be favorably used for the rapid screening of putative cytochrome P450 3A4 inhibitors.
- MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- elektroforéza kapilární metody MeSH
- inhibitory cytochromu P450 CYP3A farmakologie MeSH
- ketamin analogy a deriváty farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- plošný screening metody MeSH
- stereoizomerie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Enantioseparation performance of two teicoplanin-based chiral stationary phases, Chirobiotic T and Chirobiotic T2, with different teicoplanin coverage and distinct linkage chemistry was compared. Three structurally diverse groups of analytes, amino alcohols (beta-blockers), chlorophenoxypropionic acids, and branched-chain amino acids, in various mobile phase compositions/separation modes were examined. The amino alcohols showed better enantioresolution on Chirobiotic T2 in reverse-phase, hydrophilic interaction chromatography, and polar-ionic mode separation systems. The best results with these analytes were obtained in the polar-ionic mobile phase. In contrast, the chlorophenoxypropionic acids and the branched-chain amino acids yielded an improved enantioresolution on the chiral stationary phase with lower amount of teicoplanin in the hydrophilic interaction chromatography system. The comparison of separation of the chlorophenoxypropionic acids enantiomers in the reverse-phase and hydrophilic interaction chromatography environments showed completely opposite results. While better enantioresolution of chlorophenoxypropionic acids was achieved on Chirobiotic T2 in mobile phases with low methanol content, high methanol concentration was needed to reach baseline enantioseparation on Chirobiotic T.
- MeSH
- aminoalkoholy chemie izolace a purifikace MeSH
- aminokyseliny chemie izolace a purifikace MeSH
- časové faktory MeSH
- molekulární struktura MeSH
- propionáty chemie izolace a purifikace MeSH
- stereoizomerie MeSH
- teikoplanin chemie MeSH
- vysokoúčinná kapalinová chromatografie metody přístrojové vybavení MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight difference in estimated free energy of binding and inhibition constant between two enantiomers. Although studies with other pesticides demonstrated strong enantioselective differences in toxicity, the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity mechanism will require further research.
- MeSH
- androgenní receptory metabolismus MeSH
- buněčné linie MeSH
- dihydrotestosteron metabolismus farmakologie MeSH
- hexachlorcyklohexan chemie metabolismus farmakologie MeSH
- isomerie MeSH
- lidé MeSH
- luciferasy MeSH
- reportérové geny MeSH
- transfekce MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A sensitive and specific high performance liquid chromatography method for the separation and determination of tapentadol enantiomers has been developed and validated. Ten different chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 2.5 for all enantiomers was achieved on Chiralpak AD-H using mixture of heptane-propan-2-ol-diethylamine (980:20:1, v/v/v). The detection was carried out using fluorescence detector at excitation wavelength of 295 nm and emission wavelength of 273 nm. The influence of mobile phase composition, namely organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including linearity, limit of detection, limit of quantification, precision, accuracy and selectivity was performed according to the International Conference on Harmonization guidelines. The advantage of the method is a good enantioseparation, short analysis time (less than 20 min) and therefore this method is suitable for routine determination of chiral purity of (R,R)-tapentadol in enantiopure active pharmaceutical ingredient.
