Direct chiral liquid chromatography determination of aryloxyphenoxypropionic herbicides in soil: deconvolution tools for peak processing

In this paper, the enantiomeric separation of two aryloxyphenoxypropionic esters (fluazifop-butyl and quizalofop-ethyl) and a safener herbicide (mefenpyr-diethyl), which is widely used for protecting crop plants, has been studied by direct liquid chromatography (LC) with UV detection on an α₁-acid glycoprotein as chiral stationary phase. Optimization of separation conditions was done by factorial experimental design. Experimental factors and ranges selected were propanol (5–10%), phosphate buffer pH (6.5–7.0), and column temperature (15–25 °C). Responses were expressed in terms of enantioresolution (R _s) and adjusted retention time of the second eluted enantiomer (t _r2′). The chemometric method used to explore data was response surface analysis. Multiple response analyses were carried out to determine the combination of experimental factors which simultaneously optimize experimental responses. Under optimum conditions for enantioseparation of each herbicide, partially overlapped or fully resolved enantiomers were obtained. Deconvolution tools were employed as an integration method to fit chromatographic data and to achieve a more precise enantiomeric ratio (ER) and enantiomeric fraction (EF) values. Applicability of both direct chiral LC and peak deconvolution methods was evaluated in spiked soil samples at different R/S enantiomeric ratios. Acceptable and reproducible recoveries between 71% and 96% with precision in the range 1–6% were achieved for herbicide-spiked levels from 0.50 to 9.0 μg g^–1. In addition, parameters such as R _s, ER, and EF were calculated and compared with values obtained using the common valley drop integration method.     

Validated Chiral LC Method for the Enantiomeric Separation of β-Amino-β-(3-Methoxyphenyl) Propionic Acid

A chiral liquid chromatographic method for enantiomeric resolution of β-amino-β-(3-methoxyphenyl) propionic acid was developed and validated. The “hybrid” π-electron donor–acceptor based stationary phase (R,R) Whelk-01 was found to be enantiomerically selective for (R) and (S) enantiomers of β-amino-β-(3-methoxyphenyl) propionic acid with a resolution greater than 2.0. The effects of isopropyl alcohol and ethanol on enantioselectivity and resolution of enantiomers were evaluated. Calibration curves were linear over the range of 0.10–1.00, with a regression coefficient (r) of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were 300 and 1,000 ng mL⁻¹ respectively for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 2.8. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 102. The test solution was observed to be stable up to 24 h after the preparation. The developed normal phase chiral LC method can be used for the enantiomeric purity evaluation of R-β-amino-β-(3-methoxyphenyl) propionic acid.     

A new approach to the chiral separation of novel diazenes

Two new types of potential liquid‐crystalline azo compounds were synthesized in the form of racemic mixtures and as the individual S enantiomers. Both materials consisting of two substituted aromatic rings in the molecular core and one chiral center at the aliphatic moiety showed mesomorphic behavior. The separation of the R and S enantiomers by chiral high‐performance liquid chromatography was unsuccessful when the azo compounds were in their natural E state. However, the irradiation of the compounds in the solution by UV light led to an almost quantitative transition to their Z forms. The chromatographic behavior of the compounds in their Z forms was significantly different, and partial separation of the individual enantiomers on chiral polysaccharide‐based stationary phases was obtained. Thus, the proposed procedure represents a novel approach to the enantioseparation of chiral diazenes.     

Development of a high‐performance liquid chromatography method for the simultaneous determination of chiral impurities and assay of (S)‐clopidogrel using a cellulose‐based chiral stationary phase in methanol/water mode

A simple reversed‐phase high‐performance liquid chromatography method for the chiral separation of the active pharmaceutical ingredient (S)‐clopidogrel has been developed on the cellulose‐based Chiralcel OJ‐RH chiral stationary phase. The S enantiomer was baseline resolved from its R impurity (impurity C) with a mobile phase consisting of methanol/water (100:15) without any interference coming from the other two potential chiral impurities A and B. The enantio‐ and chemoselective method was partially validated and compared with that reported in the United States Pharmacopoeia for the drug product. The versatility of the Chiralcel OJ‐RH allowed separating the enantiomers of the impurity B also under normal phase and setting up an efficient strategy to convert the racemic sample into the enantiomeric S form on a semipreparative scale.     

Gas Chromatographic Separation of Enantiomers on Optically Active Metal-Complex-Free Stationary Phases. New Analytical Methods (24)

If a stationary phase A employed in gas chromatography possesses a chemical affinity for substance B, which is to be separated, then the retention behavior is not only determined by the normal physical equilibrium between the gas and liquid phases but also by the chemical equilibrium A + B ? AB. If A and B are chiral and A is present in optically active form while B is a racemic mixture, then it is possible to achieve a gas chromatographic enantiomer resolution without the isolation of diastereomers: the energetically different diastereomeric associates A_R B_R and A_R B_S are formed rapidly and reversibly. This enantiospecific resolution principle was first demonstrated in 1966 by the quantitative resolution of racemic amino acid derivatives on optically active peptide phases in analogy to the well-known stereospecificity of enzymes. The anchoring of the chiral resolving agent to thermally stable polysiloxanes together with the employment of high resolution capillary columns and the use of appropriate derivatization strategies has led to the development of enantiomer resolution into a routine modern method for many classes of substances. The demonstration of enantiospecificity in the gas chromatographic separation process is of fundamental interest, and its systematic study can result in a significant contribution to the understanding of the molecular mechanism of “chiral recognition”. The gas chromatographic separation of enantiomers has also proven to be an accurate and sensitive method for the determination of the enantiomeric composition of natural products and products of enantioselective transformations (asymmetric syntheses, “chiral pool” transformations, kinetic resolutions, biomimetic reactions) and for the quantification of racemization, e.g. in the synthesis and hydrolysis of peptides. In any research program devoted to the phenomenon of chirality, the gas chromatographic separation of the enantiomers of volatile compounds constitutes an indispensable modern instrumental technique.     

A Validated LC Method for Determination of the Enantiomeric Purity of Montelukast Sodium in Bulk Drug Samples and Pharmaceutical Dosage Forms

A new and accurate chiral liquid chromatographic method has been developed for determination of the enantiomeric purity of montelukast sodium (R enantiomer) in bulk drugs and dosage forms. Normal phase chromatographic separation was performed on an immobilized amylose-based chiral stationary phase with n-hexane–ethanol–1,4-dioxane–trifluoroacetic acid–diethylamine 65:25:10:0.3:0.05 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. The elution time was approximately 15 min. The resolution (R _S) between the enantiomers was >3. The mobile phase additives trifluoroacetic acid and diethylamine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. Limits of detection and quantification for the S enantiomer were 0.07 and 0.2 μg, respectively, for a test concentration of montelukast sodium of 1,000 μg mL^?1 and 10 μL injection volume. The linearity of the method for the S enantiomer was excellent (R ² > 0.999) over the range from the LOQ to 0.3%. Recovery of the S enantiomer from bulk drug samples and dosage forms ranged from 97.0 to 103.0%, indicative of the high accuracy of the method. Robustness studies were also conducted. The sample solution stability of montelukast sodium was determined and the compound was found to be stable for a study period of 48 h.     

Synthesis of Both Enantiomers of Flavanone and 2‐Methylchromanone

An efficient enantiospecific synthesis of the (R)‐ and (S)‐enantiomers of flavanone and 2‐methylchromanone is described. The key steps are a C,C‐bond formation by ring opening of a chiral epoxide with a dithiane anion, followed by a Mitsunobu cyclization. The products obtained have high enantiomeric purity.     

A Validated LC Method for Determination of the Enantiomeric Purity of Darifenacin in Bulk Drug and Extended Release Tablets

A new and accurate chiral liquid chromatographic method has been developed for the determination of enantiomeric purity of darifenacin [(S)-enantiomer] in bulk drugs and extended release tablets. Normal phase chromatographic separation was performed on an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with n-hexane:ethanol:diethylamine (50:50:0.3, v/v/v) as mobile phase at a flow rate of 1.0 mL min⁻¹. The elution time was ~15 min. The resolution (R _s ) between the enantiomers was greater than four and interestingly the (R)-enantiomer was eluted prior to darifenacin in the developed method. The limit of detection (LOD) and limit of quantification (LOQ) for the (R)-enantiomer were 0.02 μg and 0.07 μg, respectively, for a 10 μL injection volume. The method was extensively validated in terms of linearity, precision and accuracy and satisfactory results were obtained. Robustness studies were also conducted. The sample solution stability of darifenacin was determined and the compound was found to be stable for a study period of 48 h.

     

Chiral HPLC and physical characterisation of orthoconic antiferroelectric liquid crystals

New orthoconic antiferroelectric liquid crystalline materials were synthesised and characterised in their racemic forms and as (S) enantiomers. The materials possess oligo-methylene spacers of different lengths in semi-fluorinated achiral chains and lateral substitution by fluorine at two different positions of the molecular core. For comparison purposes, analogical materials without fluorine lateral substitutions were also prepared. Polysaccharide chiral stationary phases based on two different chiral selectors were used for the separation of the enantiomers of the individual racemic mixtures by high-performance liquid chromatography. A baseline separation of (S) and (R) enantiomers was obtained for four of the six studied liquid crystalline materials. Two of the materials were partially separated under the optimised separation conditions. The elution order of the individual enantiomers in the racemic mixtures was successfully assigned, as pure (S) enantiomers of all the studied materials were available. Both the position of the fluorine atom within the molecular core and the size of the achiral moiety had significant effects on the separation of the individual enantiomers of the studied compounds. Moreover, it was also found that the structure of the chiral stationary phase selector significantly influenced the enantiomeric resolution.     

Determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycines by chiral capillary electrophoresis

Summary A fast and practical chiral capillary electrophoretic method has been developed for determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG) compounds. In particular, the isomer PCCG-13, a potent selective and competitive antagonist at phospholipaseD-coupled metabotropic glutamate receptors (mGluRs), was completely resolved from its enantiomer PCCG-15 by use of dimethyl-β-cyclodextrin (DMCD) as chiral selector. pH 9.0 running buffer containing 2-amino-2-methyl-1,3-propanediol (AMPD; 100mM) was a suitable medium enabling resolution of the enantiomers in a short (32.5 cm total length) poly(vinyl alcohol) (PVA)-coated capillary. Because of the suppression of the electroosmotic flow and the good peak shape, baseline resolution of the enantiomers was obtained by use of the optimum concentration of chiral selector. For quantitative purposes at impurity levels, high sample loading was required and adequate separation was obtained in the presence of 80 mM DMCD. This CE method enabled quantification of 0.3% m/m of undesired enantiomer in PCCG-13; the samples analyzed, obtained from enantioselective synthesis, proved to be of high enantiomeric purity.     

Direct Resolution of the Enantiomers of Betaxolol and Related Intermediates,and Its Application

An OJ-H chiral column has been used for direct resolution of the enantiomers of betaxolol and related intermediates in the preparation of (S)-betaxolol. The enantiomers can be excellently resolved (R _S > 2) within 9.4 min with high peak symmetry. The enantiomers of some acetylated intermediates, which cannot be resolved on an OD-H column, can also be resolved. The method is simple and suitable for routine determination of ee values in the preparation of (S)-betaxolol.

     

Enantiomeric separation of six beta-adrenergic blockers on Chiralpak IB column and identification of chiral recognition mechanisms by molecular docking technique

Enantiomeric separation of six β-adrenergic blockers was systematically studied for the first time on a polysaccharide-based chiral stationary phase, i.e. Chiralpak IB, under the normal-phase mode. The effect of alcohol modifiers, alcohol content and basic additive on enantiomeric separation was evaluated and optimized. Under the optimal conditions, the enantiomers of atenolol, bevantolol, cartelol, esmolol, metoprolol and propranolol were all baseline resolved with resolutions of 1.50, 8.56, 2.05, 2.11, 3.56 and 4.02, respectively. Additionally, molecular docking was tested to explain chiral recognition mechanisms of this set of the drug enantiomers on Chiralpak IB. The details of the various interactions affecting enantiomeric separation were confirmed from the molecular level and the modeling data were in agreement with the chromatographic results concerning the enantioselectivity.     

Chiral ligand-exchange separation and resolution of extremely rigid glutamate analogs: 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids

Owing to their chelation ability, a series of fully constrained l-Glu analogs formed by the spiro-union of two cyclopropane rings (1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids, ASPED A–D), was submitted to chiral ligand-exchange chromatographic (CLEC) analysis. As the initial step, two methodologically different chiral devices were evaluated. A chiral stationary phase (CSP) obtained by dynamic coating of C₁₈ chains with the S-trityl-(R)-cysteine ((R)-STC) was used first with this objective. The lack of separation of the enantiomers of ASPED C and D prompted us to utilize the chiral mobile phase (CMP) prepared from O-benzyl-(S)-serine ((S)-OBS). The latter afforded complete separation of the four pairs of enantiomers. For all the pairs, quantum mechanical investigations shed light on the main features responsible for the different enantiomer recognition mechanism with (S)-OBS. The validated analytical method was then fruitfully adopted for semi-preparative-scale isolation of the enantiomers of ASPED C.     

Identification and characterization of an anomalous racemate

Direct crystallization is a convenient and generally efficient method to increase the optical purity of partially resolved enantiomers. The design of chiral purification processes based on crystallization requires measurement of solubility of all stable phases, i.e. enantiomers and racemic mixtures, in the process solvent. Enantiomeric systems have been classified by the nature of the solid phases formed as racemic compounds, conglomerates or solid solutions. In rare cases, however, enantiomers form crystalline addition compounds with a stoichiometry other than 1(S):1(R). These structures are generally known as “anomalous racemates”. The solubility and melting point diagrams of each type of enantiomeric system exhibit characteristic features which have significant implications on the efficiency of the crystallization to enhance optical purity. In this study, we report the formation of a non-equimolar 3:1 addition compound in the R/S 3-hydroxy-4-(2,4,5-trifluorophenyl) butanoic acid.     

Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors

CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3‐hydroxypropan‐2‐yl)‐1H‐1,2,3‐triazol‐4‐yl]phosphonic acid, 2‐[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2?(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6‐diaminopurine, uracil, and 5‐bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with a 2‐(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51–2.94, within a reasonable time, 13–28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL β‐cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with 2‐(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium β‐cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range.     

Resolution and absolute configuration of 1,2,3,5,6,10bβ-hexahydro-6α-phenylpyrrolo[2,1-α]isoquinoline (McN-4612-Z). A problem of determination of the enantiomeric purity for a tertiary amine

Details for the resolution of 1,2,3,5,6,10bβ-hexahydro-6α-phenylpyrrolo[2,1-α]isoquinoline (1) , a potent antidepressant-like compound, into its enantiomers with di-p-toluoyltartaric acid ( 2) are reported. Enantio-merically-enriched R-(+)-2-phenylpyrrolidine was transformed into enantiomerically-enriched 1 to determine enantiomeric purity and absolute stereochemistry for the resolved amines 1 . Thus, we ascertain that samples of (+)- and (—)- 1 with an enantiomeric purity of ≥99% were prepared, and that bioactive (+)- 1 possesses the 6S,10bR absolute configuration. The enantiomeric purity of ≥99% was confirmed by 360-MHz ¹H nmr examination of 1:1 diastereomeric salts formed from 1 or (—)- 1 and (+)-Mosher's acid (MTPA). The maximal optical rotation reported (ref 8) for (+)- 3 (100% o.p.) was shown to correspond to 100% e.e.     

Preparation and characterization of phenyl carbamate derivative β-cyclodextrin bonded phase for chiral HPLC

A partially substituted β-cyclodextrin chiral stationary phase was prepared by the reaction of phenyl isocyanate. The enantiomers of a series of O,O-diethyl(p-methylbenzenesulfonamido)-aryl(or alkyl)-methylphosphonates were studied on the prepared phenyl carbamate derivative β-cyclodextrin bonded phase and a commercial (S)-(+)-1–(1-naphthyl) ethylcarbamate derivative β-cyclodextrin bonded phase on normal phase chromatographic condition. Results show that the prepared phenyl carbamate derivative β-cyclodextrin bonded phase has better enantiomeric selectivity to the series of compounds. A chiral recognition mechanism was suggested for the separation of these novel organic phosphorus enantiomers.     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

Chiral discrimination of aliphatic amines and amino alcohols using NMR spectroscopy

Two methods are compared for analyzing the enantiomeric purity of aliphatic amines and amino alcohols using NMR spectroscopy. The first employs (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as a chiral NMR solvating agent in methanol‐d₄. The second involves a derivatization scheme in which the amine is reacted with naphtho[2,3‐c]furan‐1,3‐dione to form the corresponding amide. The naphthyl amide is then mixed with a chiral calix[4]resorcinarene in deuterium oxide. The crown ether only produces sufficient enantiomeric discrimination to determine enantiomeric purity for three of the nine substrates studied. The system with the naphthyl amide and a calix[4]resorcinarene produces enantiomeric discrimination of sufficient magnitude to determine enantiomeric purity for all nine substrates. The H1 and H4 resonances of the naphthyl ring are especially suitable to monitor for enantiomeric discrimination. The order of the (R)‐ and (S)‐enantiomers of the H1 and H4 resonances exhibit specific trends for aliphatic amines and amino alcohols that correlate with the absolute configuration. Copyright © 2012 John Wiley & Sons, Ltd.     

Asymmetric synthesis of optically active α-substituted α-amino-H-phosphinates through resolution of 1,1-diethoxyethyl(aminomethyl)phosphinates

Both enantiomers of 1,1-diethoxyethyl(aminomethyl)phosphinates were prepared through chromatographic separation of a diastereomeric mixture derived from (S)-phenylethylamine and 1,1-diethoxyethyl-H-phosphinate. The individual enantiomer was transformed into α-substituted α-amino-H-phosphinate with high enantiomeric purity by a highly diastereoselective alkylation at the α-carbon on the basis of our previously developed method.