Determination of enantiomeric compositions of pharmaceutical products by near-infrared spectrometry

A new method for the determination of enantiomeric compositions of a variety of drugs including propranolol, atenolol, and ibuprofen has been developed. The method is based on the use of the near-infrared technique to measure diastereomeric interactions between an added carbohydrate compound and both enantiomeric forms of a drug followed by evaluation of the data by partial least square analysis. The fact that the method works well with all three macrocyclic carbohydrates with different cavity sizes (i.e., alpha-, beta-, and gamma-cyclodextrin) and with sucrose, which is a linear carbohydrate, clearly demonstrates that it is not necessary to have inclusion complex formation to produce effective diastereomeric interactions. Rather a simple adsorption of the drug onto a carbohydrate is sufficient. Since inclusion complex formation is not a requisite, this method is not limited to the three drugs evaluated in this study but is rather universal as it can, in principle, be used for the sensitive and accurate determination of enantiomeric compositions of many different types of drugs with only about 1.5mg/mL concentration and enantiomeric excess as low as 0.80%, in water or in a mixture of water with organic solvent. Furthermore, it does not rely on the use of rather expensive carbohydrates such as cyclodextrins but is equally as effective even with a simple and inexpensive carbohydrate such as sucrose.     

Evaluation of the stereoselective metabolism of the chiral analgesic drug etodolac by high-performance liquid chromatography

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reversed-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1′-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.     

Polarimetric detection in high-performance liquid chromatography

Chiroptical detection for HPLC is particularly useful as a selective detection method for chiral molecules, and in enantiomeric purity determination with partial chiral separation or without chiral separation. The recent development of laser-based polarimeters with microdegree sensitivity has increased the applicability of optical rotation detection in HPLC. The detection limit of these instruments is submicrogram on-column for many chiral compounds in analytical HPLC. A variety of applications of the selective detection of optically active molecules are reviewed. The use of polarimetric detection with partial chiral separation is considered, both as an aid to method development and for enantiomeric purity determination. Finally applications to enantiomeric purity determination without chiral separation are reviewed, with the dual use of nonchirally selective and chiroptical detectors to determine the total amount and optical purity of the analyte. Determinations of chiral purity for samples of high enantiomeric excess are described, which with laser-based instrumentation may give accuracies of better than +/- 1% with sample loadings of 50 micrograms on an achiral column. Applications to the study of enantioselective reactions are also considered, with determination of enantiomeric excess in near-racemates to better than +/- 0.1%.     

Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans

The prochiral carbonyl group of fenofibrate (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propionate) is reduced during its metabolism giving rise to a chiral secondary alcohol, "reduced fenofibric acid." Chiral and diastereomeric HPLC methods have been developed for the determination of its enantiomeric composition and these have been applied to the measurement of the "reduced fenofibric acid" enantiomers in urine of rats, guinea pigs, dogs, and human volunteers given [14C]fenofibrate. In the three animal species, the reduction is markedly enantioselective for the (-)-isomer, the enantiomeric ratios (-/+) being 95:5. This was not due to differences in the excretion of the enantiomers, since when racemic "reduced fenofibric acid" was given to rats it was recovered in the urine with the same enantiomeric composition as the dose form. In humans the ratio was 52:48 showing the lack of stereoselectivity of reduction in this species.     

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.     

A versatile chiral selector for determination of enantiomeric composition of fluorescent and nonfluorescent chiral molecules using steady-state fluorescence spectroscopy

A fluorescent chiral molecular micelle (FCMM), poly (sodium N-undecanoyl-L-phenylalaninate) (poly-L-SUF), was developed as a chiral selector for enantiomeric recognition and determination of enantiomeric composition of four fluorescent and four nonfluorescent chiral molecules by use of steady-state fluorescence spectroscopy. The influence of FCMM concentration, buffer pH and complexation medium on FCMM-analyte host-guest complexation, and the emission spectral properties of the resulting complexes were investigated. The chiral interactions of the analytes,1,1'-binaphthyl-2,2'-diamine, 1-(9-anthryl)-2,2,2-trifluoroethanol, propranolol, naproxen, chloromethyl menthyl ether (CME), citramalic acid, tartaric acid, and limonene (LIM), in the presence of poly-L-SUF were based on diastereomeric complex formation. The figures of merit obtained from the partial-least-squares regression modeling of the calibration samples suggested good prediction ability for the validation of six of the eight chiral analytes. Better host-guest complexation of the more hydrophobic molecules, CME and LIM, were obtained in methanol/water mixtures, resulting in better predictability of the regression models. Prediction ability of the models was evaluated by use of the root-mean-square percent relative error (RMS%RE) and was found to range from 1.77 to 15.80% (buffer), 1.26 to 7.95% (25:75 methanol/water), and 1.21 to 4.28% (75:25 methanol/water).     

Syntheses and highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions using chiral oxacalix[2]arene[2]bisbinaphthes

The triazine-based bisbinaphthyl crown ethers oxacalix[2]arene[2]bisbinaphthes R-1, R-2, R-3 and S-1, S-2, S-3 were synthesized. The interactions of these compounds with various α-aminocarboxylic acid anions were studied. The crown ethers were found to carry out highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions. It is observed that within a certain concentration range, one enantiomer of the chiral α-aminocarboxylic acid anions can increase the fluorescence intensity of the crown ethers by fivefold to sixfold, whereas the other enantiomer scarcely enhances the fluorescence. Such unusually high enantioselective responses make these crown ethers very attractive as fluorescent sensors in determining the enantiomeric composition of α-aminocarboxylic acid anions.     

Determination of the enantiomeric excess of an M3 antagonist drug substance by chemometric analysis of the IR spectra of different guest-host complexes

A novel approach for the potential on-line determination of the enantiomeric excess (ee) of an M3 antagonist drug substance combining attenuated total reflectance infrared (ATR-IR) spectroscopy, guest-host complexes, and chemometric data analysis is described. Chiral recognition through a formation of diastereomeric complexes was measured by ATR-IR. Small changes on the IR spectra reflect the interaction between the guest (M3) and host (chiral selector). These changes are measured as a function of M3 enantiomer excess. The standard error of prediction is 1.3 ee%. The prediction results based on the IR method were in good agreement with the gravimetric method. The robustness of the calibration model was evaluated by varying the concentration of the chiral selector, the pH of the solution, and the organic solvents. The stability of the calibration model was also demonstrated through measuring different sets of samples on different days.     

Elucidation of the chromatographic enantiomer elution order for praziquantel: An experimental and theoretical assessment

In this work, we have studied both experimentally and theoretically the praziquantel (PZQ) chiral discrimination. According to the main results, the enantioseparation of PZQ was efficiently optimized by HPLC on the reverse phase from the Chiralpak IB column, which has cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) as a chiral selector. The thermodynamic and structural parameters obtained via density functional theory (DFT) calculations pointed out the chiral discrimination as well as the enantiomeric elution order of PZQ, thus elucidating the experimental data and validating our proposed method. Finally, the hydrogen bonds and π-π stacking interactions played a key role in the discrimination between the PZQ diastereomeric complexes formed.     

Evaluation of enantioselective gas chromatography for the determination of minute deviations from racemic composition of alpha-amino acids with emphasis on tyrosine: accuracy and precision of the method

Whereas the determination of high enantiomeric fractions (EF) of chiral compounds is very well established, the accurate determination of small deviations from racemic compositions has not yet received much attention despite its relevance to studies dealing with the origin of homochirality, where only small initial enantiomeric bias is expected. Racemic samples of representative alpha-amino acids were derivatized as N-(O,S)-trifluoroacetyl/ethylesters and analyzed by enantioselective gas chromatography (GC) on fused silica capillaries coated with the chiral stationary phases (CSPs) Chirasil-D-Val, Chirasil-L-Val, and Lipodex E with GC/FID and GC/MS detection. The validation (accuracy and precision) of the determination of the enantiomeric fraction EF of the D-enantiomer in racemic or near-racemic compositions for 10 DL-alpha-amino acids obtained from commercial sources has been carried out. Emphasis is given to DL-tyrosine, the enantiomers of which have recently been claimed to show different crystallization properties. Values of EF obtained from GC measurements using CSPs were compared with those from CE using chiral mobile phase additives. While the precision of the GC method is generally better than 0.08% for all DL-alpha-amino acids studied, accuracy (trueness) of determination of amino acids with polar side chains is poorer than expected from the precision as a result of systematic errors. The accuracy determined relied on measurements on two oppositely configurated CSPs.     

Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phosphodiester ether lipid derivatives.

Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.     

Configurational assignment of optically active hydroperoxy homoallylic alcohols and the corresponding diols by circular dichroism and multidimensional gas chromatography

Allylic hydroperoxides are a class of compounds of versatile synthetic utility. Optically active diastereomeric hydroperoxy homoallylic alcohols and their corresponding diols are easily available through horseradish peroxidase (HRP)-catalyzed kinetic resolution of racemic hydroperoxides. Here we describe the assignment of the absolute configuration of the optically active products and substrates obtained after HRP-catalysis by the circular dichroism exciton chirality method. Moreover, the analytical-scale separation of the enantiomers based on multidimensional gas chromatography on chiral columns is presented. Since the enantiomeric elution order on the ciral columns was constituted, the absolute stereochemistry of optically active allylic diols can easily be deduced by their retention times on β-cyclodextrins. Chirality 9:69–74, 1997. © 1997 Wiley-Liss, Inc.     

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

The two enantiomers of ethyl 3‐hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)‐3‐hydroxybutyrate. Herein, we also functionally characterized one novel salt‐tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)‐3‐hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio‐selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.     

Enantiopure O‐Ethyl Phenylphosphonothioic Acid: A Solvating Agent for the Determination of Enantiomeric Excesses

An improved method, which is highly reproducible, was developed for the enantioseparation of racemic O‐ethyl phenylphosphonothioic acid ( 1a ) with brucine by introducing seeding to a supersaturated solution of the diastereomeric salt mixture. The present method gave both diastereomeric salts in high yields with a diastereomeric ratio of >99.5:0.5 upon choosing the crystallization solvent (MeOH for the ( (R)-1a salt and MeOH/H₂O for the ( (S)-1a salt). The enantiopure acid (R)-1a , (S)-1a showed a good chirality recognition ability for not only strong bases, such as amines and amino alcohols, but also weakly basic alcohols and was applicable as a solvating agent to the ¹H NMR determination of the enantiomeric excess of chiral amines, amino alcohols, and alcohols, including aliphatic substrates. Chirality 26:614–619, 2014. © 2014 Wiley Periodicals, Inc.     

Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples

The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-beta-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (+/-25.8degmldm(-1)g(-1) at 20 degrees C and 589nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (-)-VX could be quantified up to 720min.     

Synthesis of the first sulfur-35-labeled hERG radioligand

The synthesis of the first high specific activity S-35-labeled hERG radioligand, [(35)S]MK-0499, for use in HTS assays of drug candidates for hERG interaction is described. The radioligand is prepared by [(35)S]sulfonylation of a high diastereomeric excess (de) aniline precursor prepared from unlabeled MK-0499.     

A NMR shift method for determination of the enantiomeric composition of hydroperoxides formed by lipoxygenase.

A method is presented for determination of the enantiomeric composition of hydroxyperoxides formed by enzymic oxygenation of unsaturated fatty acids. After reduction of the hydroperoxy group with NaBH4, and esterification, the positional isomers of the resulting hydroxy compounds are separated by high performance liquid chromatography. The latter are subsequently subjected to a chiral derivatization to form diastereomeric alpha-methoxy-alpha-trifluoromethylphenylacetate esters. Determination of the diastereomeric composition by a NMR shift experiment furnishes the enantiomeric composition of the parent hydroperoxides. The method has been applied to the hydroperoxides formed by incubation of linoleic acid by corn germ or soybean lipoxygenase. Our results indicate that under the conditions used the hydroperoxides are mainly enantiospecifically formed.     

Bicyclic glutamic acid derivatives

For the second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids via Strecker reaction of chiral ketimines, the cyanide addition as the key stereodifferentiating step produces mixtures of diastereomeric alpha-amino nitrile esters the composition of which is independent of the reaction temperature and the type of the solvent, respectively. The subsequent hydrolysis is exclusively achieved with concentrated H(2)SO(4) yielding diastereomeric mixtures of three secondary alpha-amino alpha-carbamoyl-gamma-esters and two diastereomeric cis-fused angular alpha-carbamoyl gamma-lactams as bicyclic glutamic acid derivatives, gained from in situ stereomer differentiating cyclisation of the secondary cis-alpha-amino alpha-carbamoyl-gamma-esters. Separation was achieved by CC. The pure secondary trans-alpha-amino alpha-carbamoyl-gamma-esters cyclise on heating and treatment with concentrated H(2)SO(4), respectively, to diastereomeric cis-fused angular secondary alpha-amino imides. Their hydrogenolysis led to the enantiomeric cis-fused angular primary alpha-amino imides. The configuration of all compounds was completely established by NMR methods, CD-spectra, and by X-ray analyses of the (alphaR,1R,5R)-1-carbamoyl-2-(1-phenylethyl)-2-azabicyclo[3.3.0]octan-3-one and of the trans-alphaS,1S,2R-2-ethoxycarbonylmethyl-1-(1-phenylethylamino)cyclopentanecarboxamide.     

Enantiomeric resolution,thermodynamic parameters,and modeling of clausenamidone and neoclausenamidone on polysaccharide‐based chiral stationary phases

The aim of the paper is to describe a new synthesis route to obtain synthetic optically active clausenamidone and neoclausenamidone and then use high‐performance liquid chromatography (HPLC) to determine the optical purities of these isomers. In the process, we investigated the different chromatographic conditions so as to provide the best separation method. At the same time, a thermodynamic study and molecular simulations were also carried out to validate the experimental results; a brief probe into the separation mechanism was also performed. Two chiral stationary phases (CSPs) were compared with separate the enantiomers. Elution was conducted in the organic mode with n‐hexane and iso‐propanol (IPA) (80/20 v/v) as the mobile phases; the enantiomeric excess (ee) values of the synthetic R‐clausenamidone and S‐clausenamidone and R‐neoclausenamidone and S‐ neoclausenamidone were higher than 99.9%, and the enantiomeric ratio (er) values of these isomers were 100:0. Enantioselectivity and resolution (α and Rs, respectively) levels with values ranging from 1.03 to 1.99 and from 1.54 to 17.51, respectively, were achieved. The limits of detection and quantitation were 3.6 to 12.0 and 12.0 to 40.0 ug/mL, respectively. In addition, the thermodynamics study showed that the result of the mechanism of chiral separation was enthalpically controlled at a temperature ranging from 288.15 to 308.15 K. Furthermore, docking modeling showed that the hydrogen bonds and π‐π interactions were the major forces for chiral separation. The present chiral HPLC method will be used for the enantiomeric resolution of the clausenamidone derivatives.     

Assignment of absolute configuration and optical purity determination of (R)- and (S)-econazole nitrate by enantioselective HPLC: Method development and application

A method is described for the synthesis and optical purity determination of (?)-(R)- and (+)-(S)-econazole via the optically pure intermediates, (R)- and (S)-imidazolylethanol, which are available by chromatographic resolution or by fractional crystallization of diastereomeric O,O′-disubstituted (R*;R*)- or (S*;S*)-tartaric acid monoesters of the parent imidazolylethanol racemate. Furthermore, this method allows the chromatographic assignment of the absolute configuration of the chiral center of the imidazolylethanol enantiomers and consequently of econazole enantiomers. In addition, a direct liquid chromatographic enantioseparation method for the determination of the optical purity of (R)- and (S)-econazole and other chiral imidazoles on a protein type CSP (OVM) is described and applied to confirm chromatographically the absolute configuration evaluations. © 1994 Wiley-Liss, Inc.