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In this study, the attention was focused on quizalofop-ethyl, a chiral herbicide whose formulation has recently been marketed as quizalofop-P-ethyl, i.e. the (+)-enantiomer exhibiting herbicidal activity. To verify the real enantiomeric purity of this product as well as to study its environmental fate, the enantioselective separation of the P- and M- enantiomers of quizalofop-ethyl was achieved on Lux Cellulose-2 column (3­chloro,4-methylphenilcarbamate cellulose) under isocratic conditions in polar organic mode. Once established that the commercial formulation contains ˜ 0.6% (enantiomeric fraction) of M as an impurity, an HPLC-MS/MS method was developed, validated and applied to the analysis of soil, carrots and turnips treated with the herbicide. A simple solid-liquid extraction allowed recoveries greater than 70%; limits of detections of P and M enantiomers were below 5 ng g-1. The analyses of the real samples showed a modification of the enantiomeric fraction of quizalofop-M-ethyl between the commercial formulation (EFM = 0.63 ± 0.03%) and the analysed matrices (EFM = 7.6 ± 0.1% for carrots; EFM = 0% for the other matrices). This outcome highlighted the occurrence of an enantioselective biotic dissipation, responsible for a greater persistency of the distomer in carrots. On the other hand, since screening analyses revealed the occurrence of residues of the metabolite quizalofop-acid with the same EFs as the ester precursor, it was concluded that the hydrolytic conversion was an abiotic process.

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A new approach for the evaluation of chiral purity of serine esterification products bearing long-chain alkyl substituents was developed. The compounds were simply converted to aryl-substituted oxazolines which: (i) facilitates effective chromatographic enantioseparation and (ii) enables direct detection using ultraviolet absorption. The method employs a polysaccharide-based chiral stationary phase and allows enantioseparation of highly stable oxazoline products in less than 6 min using a simple binary mobile phase. As opposed to the previously used normal phase method the developed method was performed in the reversed-phase mode. Aside from the benefits of switching to less hazardous solvents with regard to the principles of Green Chemistry, this has also led to a reduction in the analysis time. In comparison with known serine chromophores, the best enantioseparation of aryloxazoline rigid structure may be achieved only based on non-polar interactions with the chiral stationary phase. In contrast, the substitution of the chromophore moiety with hydroxyl substituent affected intra and intermolecular interactions that caused enantioseparation differences. Concurrently, we found high chirality retention of (R)- and (S)-configuration oxazoline standards (≥99% enantiomeric excess) during the introduction of the ultraviolet label. The method is suitable for rapid injection of the mixture containing the ultraviolet absorption marker without prior purification.

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The immobilization of cellulose 3,5-dimethylphenyl carbamate and amylose 3,5-dimethylphenyl carbamate on silica gel carrier was achieved by using photochemical and thermal processes. Both approaches provide an easy access to materials which were applied as chiral stationary phases (CSPs) for the chromatographic resolution of racemic molecules. The influence of parameters such as irradiation time and solvent on immobilization effectiveness was investigated. For the thermal processes, azo-bis-isobutyrontrile and di-tert-butyl peroxide were evaluated as radical initiators. The influence of parameters such as amount of radical initiator, solvent, temperature, and further handling operations on the immobilization rate was examined. The chiral recognition ability and the overall performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto packed HPLC columns. As there is almost no limitation of organic solvent types that can be used as mobile phases with the immobilized CSPs, they can be applied under chromatographic conditions which are prohibited with the corresponding non-bonded CSPs. This extended applicability considerably broadens the options for improving enantioselectivity and resolving chiral compounds which are not or only poorly soluble in the conventional mobile phases.

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Racemic ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate is a key synthon for the design of promising therapeutic drugs. It is mainly synthesized from racemic ethyl 2,3-dibromopropionate and 2-aminophenol in presence of K2 CO3 in refluxed acetone. Unfortunately, synthesis of (R)- and (S)-enantiomers using the enantioselective version of this reaction, which should normally be performed with a double SN 2 mechanism, is unsuitable due to a racemization process involving the dehydrobromination of enantiopure ethyl 2,3-dibromopropionate into ethyl 2-bromoacrylate. For the first time, the enantioselective version is studied (ee ≈ 55-66%), and the percentage of racemization process has estimated to around 34-46% after determination of the optimal experimental conditions for analytical HPLC enantioseparation of racemate. The influence of the experimental and purification conditions on the racemization rate is also studied. The results indicate that racemization occurs faster at the beginning of the reaction but the initiation of the double SN 2 process takes place more faster to limit the racemization rate. The study of the influence of experimental conditions (reaction times, temperature, solvent or type of base, etc.) on the degree of racemization of the (R)- enantiomer is performed and shows that despite changes in the experimental conditions, the synthesis of the (R)- enantiomer is always accompanied by a racemization rate which is difficult in reducing. In parallel, (R)- and (S)-enantiomers are obtained in high enantiopurity (ee > 99.5%) by preparative HPLC enantioseparation of racemate on multigram scale and characterized by optical rotation measurements, ECD and UV spectra.

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Racemic ethyl 2,3-dibromopropionate, commercially available at low price, is a key intermediate used in the synthesis of several heterocycle fragments, which are present in many biologically active compounds. Surprisingly, the enantiomers are not commercially available and have never been described in the literature. In this work, we undertook two different strategies to obtain these enantiomers, which are enantioselective synthesis and preparative HPLC enantioseparation of commercially available racemate on multigram scale. The first strategy has proved inadequate because racemization occurred during the synthesis (ee ≈ 9-50%). Conversely, the second strategy produced a very good enantioseparation of commercially available racemate (ee > 99.5% for both enantiomers) on multigram scale.

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A new platform technology for the preparation of stable chiral stationary phases was successfully optimized. The chiral selector tert-butylcarbamoylquinine was firstly covalently connected to the polymer poly(3-mercaptopropyl)methylsiloxane by thiol-ene click reaction. Secondly, the quinine carbamate functionalized polysiloxane conjugate was coated onto the surface of vinyl modified silica particles and cross-linked via thiol-ene click reaction. The amount of polysiloxane, chiral selector, radical initiator, reaction solvent (chloroform and methanol), reaction time, and pore size of the supporting silica particles were varied and systematically optimized in terms of achievable plate numbers while maintaining simultaneously enantioselectivity. The optimization was based on elemental analysis data, chromatographic results, and H/u-curves (Van Deemter) of the resultant chiral stationary phases. The results suggest that better chromatographic efficiency (higher plate numbers) at equal enantioselectivity can be achieved with methanol (a poor solvent for the polysiloxane that is dispersed rather than dissolved) and a lower film thickness of quinine carbamate functionalized polysiloxane. In this study, chiral stationary phases based on 100 Å silica slightly outperformed 200 Å silica particles (each 5 µm). The optimized two step material exhibited significantly reduced mass transfer resistance compared to the one step material and equal performance as a brush-type chiral stationary phase.

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Chiral 2-hydroxycarboxylic acids are compounds that have been linked to particular diseases and are putative biomarkers with some diagnostic potential. The importance of identifying whether a particular enantiomer is related to certain diseases has been encouraged recently. However, in many cases it has not yet been elucidated whether there are stereochemical implications with respect to these biomarkers and whether their enantioselective analysis provides new insights and diagnostic potential. In this study 13 disease-related chiral 2-hydrocarboxylic acids were studied for their chiral separation by high-performance liquid chromatography on three cinchona alkaloid-derived chiral stationary phases. From a subgroup of eight 2-hydroxymonocarboxylic acids, baseline resolution could be achieved and inversion of elution order by exchanging tert-butylcarbamoyl quinidine chiral stationary phase (Chiralpak QD-AX) for the corresponding quinine analogue (Chiralpak QN-AX) is shown for seven of them. Furthermore, conditions for chiral separation of the 2-hydroxydicarboxylic acids, citramalic acid, 2-isopropylmalic acid, and 2-hydroxyadipic acid are reported and compared to the previous reported conditions for 2-hydroxyglutaric acid and malic acid.

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This review article is aimed at providing an overview of the current market of chiral drugs by exploring which is the nowadays tendency, for the pharmaceutical industry, either to exploit the chiral switching practice from already marketed racemates or to develop de novo enantiomerically pure compounds. A concise illustration of the main techniques developed to assess the absolute configuration (AC) and enantiomeric purity of chiral drugs has been given, where greater emphasis was placed on the contribution of enantioselective chromatography (HPLC, SFC and UHPC). Afterwards, we focused our study on the cohort of 45 new drugs that have been approved by the US Food and Drug Administration (FDA) in 2015. We extracted the chemical structure of the new drugs from the FDA approval chemistry reviews available on the database of the agency's Center for Drug Evaluation and Research (CDER), and we selected a subgroup (i.e., 44% of the cohort) of small-molecule active pharmaceutical ingredients (APIs) containing one or more chirality centers. On the basis of the FDA dossiers examined, it emerged that all the chiral drugs approved by the FDA in 2015 are enantiomerically pure compounds with a well-defined AC, with the exception of one, namely lesinurad, which has been licensed as the racemate of two enantiomeric atropoisomers, arising because of the hindered rotation around the single C-N bond in the naphthalene ring. Finally, none of the previously developed racemates has been switched to the single-enantiomer version in 2015.

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Laccase mediator systems are important biodegradation agents as the rate of reaction could be enhanced in the presence of redox mediators. In the present study the commercial enzyme laccase from Trametes versicolor and the redox mediator 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were used for the biotransformation of the synthetic fragrances 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8,-tetramethyl-2-naphthyl)ethan-1-one (Iso-E-Super, OTNE), 1,3,4,6,7,8,-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[g]-2-benzopyran (Galaxolide, HHCB), 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphtalene (Tonalide, AHTN) and the transformation product of HHCB, 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[g]-2-benzopyran-1-one (Galaxolidone, HHCB-lactone) in water. A particular focus was to assess the effects of the enzyme laccase from Trametes versicolor in the enantioselective degradation of the target compounds, for this reason gas chromatography with an enantioselective column was used as separation technique followed by mass spectrometry detection. In addition, as enantioselective degradation of musk fragrances was observed in wastewater, sewage sludge and fish samples, enantiomeric fractions of selected compounds were studied during composting. In a period of 144h, the target fragrances could be effectively removed by the enzyme laccase with removal percentages greater than 70%, except AHTN with a removal percentage of 42%. However, the degradation process prompted by the enzyme laccase was shown to be non-enantioselective as no significant differences were observer between the enantiomeric fractions calculated at the beginning and at the end of the degradation process. Meanwhile, the composting process was shown to be enantioselective.

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Chiral short chain aliphatic hydrocarboxylic acids (HCAs) are common compounds being part of different biological processes. In order to control and understand these processes is of pivotal importance to determine the identity of the involved enantiomer or their enantiomeric ratio. In this study the capacity of quinine- and quinidine-derived chiral stationary phases to perform the enantioseparation of eight chiral HCAs (tartaric acid, isocitric acid, malic acid, glyceric acid, 2-hydroxyglutaric acid, 2-hydroxybutyric acid, lactic acid and 3-hydroxybutyric acid) was evaluated. MS-compatible conditions consisting of ACN/MeOH mixtures as eluents with formic acid, acetic acid and/or their ammonium salts as additives, temperatures between 10 and 25°C (except for -20°C for 3-hydroxybutyric acid) and a flow rate of 1.00mL/min yielded full baseline resolution for all studied HCAs. Elution order for the HCA enantiomers was determined revealing different behaviors between the studied compounds.

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A process to immobilize para-methylbenzoyl cellulose (PMBC) on silica gel has been developed and applied to prepare chiral stationary phases (CSPs) for enantioselective chromatography. The immobilization was achieved by simple irradiation of the polysaccharide derivative with ultraviolet light after coating on a silica gel support. The influence of parameters such as irradiation time and solvent on immobilization effectiveness were investigated. The performance of the prepared immobilized phases were evaluated by injection of a series of racemic compounds onto the packed columns and determination of their chiral recognition ability. By contrast to the classical coated phase, the immobilized CSP can be used under various chromatographic conditions without limitation of organic solvent types as the mobile phase. This extended applicability permits to improve selectivity and to resolve chiral compounds which are not or only poorly soluble in the mobile phases which are compatible with the non-immobilized PMBC stationary phase.

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A photochemical method for immobilizing polysaccharide derivatives on silica gel has been developed and applied to 4-methylbenzoyl cellulose (PMBC). The photochemically immobilized materials have been used as chiral stationary phases (CSPs) for the chromatographic separation of the stereoisomers of chiral molecules. Through to the immobilization which makes the chromatographic material insoluble in almost all organic solvents, there no restriction regarding the kind of solvent used in the mobile phase. This feature permits to considerably extend the possibilities to improve the selectivity of the separations and or the solubility of the solute in the mobile phase. The influence of various parameters such as immobilization process, cross-linker type and amount on the chromatographic properties and chiral recognition ability of the resulting CSPs has been investigated using a set of chiral molecules. The impact of the amount of coated polysaccharide material on chiral recognition ability was also examined.

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d- and l-2-Hydroxyglutaric acid (d- and l-2-HG, respectively) are metabolites related to some diseases (2-hydroxyglutaric aciduria, cancer), which make their identification and analysis crucially important for diagnostic purposes. Chiral stationary phases (CSP) based on tert-butylcarbamoyl-quinine and -quinidine (Chiralpak QN-AX and QD-AX), and the corresponding zwitterionic derivatives (Chiralpak ZWIX(+) and Chiralpak ZWIX(-)) were employed in a weak anion-exchange mechanism to perform the enantiomer separation of d- and l-2-HG without derivatization. QD-AX CSP showed the most promising separation and therefore optimization of eluent, additives, and temperature, required for the baseline separation of solutes was carried out. Depending on experimental conditions resolution values ranged up to 2.0 with run times <20min and MS-compatible conditions. Inversion on the elution order of d- and l-2-HG was possible by using the pseudo-enantiomeric QN-AX CSP.

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A panel of methods of general suitability for complete structural elucidation of the stereochemistry of cyclopeptides, depsipeptides and lipopeptides is presented and described in detail. The suitability of the proposed methods was exemplified on the lipopeptide poaeamide from Pseudomonas poae. Amino acid configurations have been assigned by direct LC enantiomer separation with Chiralpak ZWIX(+) and were confirmed by GC enantiomer separation on Chirasil L-Val. 3-Hydroxydecanoic acid absolute configuration was analyzed on Chiralpak ZWIX(+) and confirmed by injection on ZWIX(-) which showed opposite elution order. Plenty of d-amino acids have been found in this lipopeptide. It contained in total 5 Leu residues of which one had d-configuration. The position of the d-Leu in the peptide sequence was determined by pepsin and chemical digestions in combination with isolation of diagnostic peptide-fragments and subsequent identification of absolute configurations of the Leu residues. This allowed pinpointing the position of the d-amino acid. The complementarity of the peptide retention profiles on Chiralpak ZWIX column as compared to both RPLC and HILIC suggests its great utility as an alternative peptide separation tool.

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The occurrence of d-amino acids (D-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing D-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)(®) and ZWIX(-)(®) chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(-) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS(2) measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, (18)O/(16)O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible ß-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.

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This review aims to present the issues associated to enantioseparation of chiral pharmaceuticals in biological and environmental matrices using chiral stationary phases (CSP). Thus, it related some enantioselective methods in liquid chromatography (LC) and compares the importance given to chiral separation in biomedical and environmental fields. For that the most used CSP, the enantioselective chromatographic methods, their advantages and drawbacks were swiftly revised and compared. The recent advances and the limitations of chiral analytical methods in LC were also discussed.

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