Enantiomeric separation by HPLC of 1,4-dihydropyridines with vancomycin as chiral selector

The macrocyclic antibiotics represent a relatively new class of chiral selectors in CE, HPLC, and TLC. We have examined the use of the macrocyclic antibiotic vancomycin as a chiral selector in HPLC for the separation of 1,4-dihydropyridines (DHPs) calcium antagonists (CAs). Chromatographic data of six 1,4-dihydropyridine calcium channel blockers obtained on the vancomycin chiral stationary phase (Chirobiotic V) were compared with those obtained on an alpha(1)-acid glycoprotein (AGP) HPLC stationary phase. Optimization of pH and organic modifier was carried out in order to modulate the retention properties of each system. All chiral neutral DHPs were resolved on the AGP column, whereas on Chirobiotic V only basic DHPs showed a split peak. The analytical chromatographic procedure on Chirobiotic V proved suitable for semipreparative separation, since the separation factor on the analytical column was high enough to obtain pure enantiomers with high yields.     

Resolution of terfenadine enantiomers by beta-cyclodextrin chiral stationary phase high-performance liquid chromatography.

Enantiomers of terfenadine were resolved by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) column packed with beta-cyclodextrin (beta-CD) covalently bound to silica. Separation was achieved in both the reverse phase and normal phase modes. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analysis.     

Stereoselective HPLC analysis of tertatolol in rat plasma using macrocyclic antibiotic chiral stationary phase

Enantiomeric resolution of teratolol was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase known as Chirobiotic V with UV detection set at 220 nm. The polar ionic mobile phase (PIM) consisted of methanol-glacial acetic acid-triethylamine (100:0.01:0.015, v/v/v) has been used at a flow rate of 0.8 ml min(-1) . The calibration curves in plasma were linear over the range of 5-500 ng ml(-1) for each enantiomer with detection limit of 2 ng ml(-1) . The proposed method was validated in compliance with the international conference on harmonization (ICH) guidelines. The developed method applied for the trace analyses of tertatolol enantiomers in plasma and for the pharmacokinetic study of tertatolol enantiomers in rat plasma. The assay proved to be suitable for therapeutic drug monitoring and chiral quality control for tertatolol formulations by HPLC.     

Enantiomeric separation of drugs and herbicides on a beta-cyclodextrin-bonded stationary phase.

A chemically bonded beta-cyclodextrin chiral stationary phase for HPLC was prepared in a "one pot" process by the reaction of a phenylated beta-cyclodextrin with silica gel. Various racemic analytes such as drugs (aminoalcohol adrenergic beta-blockers, benzodiazepine anxiolytics, arylpropionic acid antiinflammatory agents) and herbicides (aryloxypropionic acids and esters) were separated on the prepared material. The column showed good chiral recognition ability for most of the solutes tested when using heptane and either 2-propanol or chloroform as organic mobile phase modifiers.     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity

The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site.     

Enantiomeric resolution of chiral sulfoxides on polysaccharide phases by HPLC

The enantiomeric resolution of chiral sulfoxides was investigated on amylose (S)-α-methylbenzyl carbamate phase coated on aminopropylated 7 μm silica with 500Å diameter pores. This was shown to be very successful in the separation of alkyl/aryl, aryl/aryl, and non-aromatic sulfoxides. The effect of pore size using naked silica was also investigated, demonstrating that the pore size does not affect the resolution. © 1996 Wiley-Liss, Inc.     

First synthesis of the two enantiomers of alpha-methyldiphenylalanine [(alphaMe)Dip] by HPLC resolution.

A strategy for the preparation of enantiomerically pure (R)- and (S)-alpha-methyldiphenylalanine, constrained phenylalanine analogs, is described. A racemic precursor was prepared in high yield from easily available starting products and subjected to HPLC resolution on a noncommercial chiral stationary phase. More than 600 mg of each enantiomer was isolated in optically pure form by using a 150 x 20 mm ID column containing mixed 10-undecenoate/3,5-dimethylphenylcarbamate of cellulose covalently bonded to allylsilica gel and a mixture of n-hexane/2-propanol/acetone as the mobile phase.     

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride

Besifloxacin is a unique chiral broad‐spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R‐form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S‐isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high‐performance liquid chromatography (HPLC) method for determination of R‐besifloxacin and S‐besifloxacin (BES impurity A) was developed and validated for in‐process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD‐H (250 x 4.6 mm, 5 μm) column using n‐heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S‐isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal‐to‐noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 μg/mL, while the LOQ was 0.90 μg/mL. The calibration curves were linear in the range of 0.9–7.5 μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628–632, 2016. © 2016 Wiley Periodicals, Inc.     

Separation of Nadolol Stereoisomers by Chiral Liquid Chromatography at Analytical and Preparative Scales

The separation of the four nadolol stereoisomers on Chiralpak® AD by chiral liquid chromatography was carried out at both analytical and preparative scales. A screening of possible mobile‐phase compositions was performed using different alcohol–hydrocarbon mixtures. The results obtained confirm the use of 20:80:0.3 ethanol‐hexane‐diethylamine reported by McCarthy (1994) but introduce other possibilities for the complete resolution of the four nadolol stereoisomers at analytical scale, namely, the mixtures 30–40:70–60:0.3 ethanol‐heptane‐diethylamine. Additionally, this work describes how retention and resolution depend on the ethanol content in hexane and heptane mixtures. The separation of nadolol stereoisomers is also carried out at preparative scale and different alcohol–hydrocarbon compositions are proposed, depending on the target component to be obtained. Particularly, this work presents the experimental separation of the more retained nadolol stereoisomer (RSR‐nadolol) by simulated moving bed (SMB) chromatography using an 80:20:0.3 ethanol‐heptane‐diethylamine mobile phase. For a 2 g/l feed concentration, RSR‐nadolol is 100% recovered at the extract outlet stream, 100% pure, and with a system productivity of 0.65 g_{RSR‐nadolol}/(l_bed^.h) and a solvent consumption of 9.6 l_solvent/g_{RSR‐nadolol}. Chirality 25:197–205, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioselective pharmacokinetics of ethotoin in humans following single oral doses of the racemate.

Racemic ethotoin (1000 mg) was administered orally as a single dose to six healthy adult volunteers. Blood samples were collected at appropriate times for 120 h following the dose. Ethotoin was quantified enantio-selectively in plasma using a novel chiral column HPLC procedure. One of the enantiomers of the chiral metabolite, 5-phenylhydantoin, was also quantified in the HPLC method. The Cmax and AUC0-infinity values for (+)-(S)-ethotoin were significantly greater than those for (-)-(R)-ethotoin (ratio of mean AUC0-infinity values 0.88), but the elimination half-lives of the isomers were virtually identical [12.35 +/- 5.15 h for (-)-(R)-ethotoin; 12.28 +/- 5.34 h for (+)-(S)-ethotoin]. Parameters derived from AUC0-infinity (Cl0/F and V(area)/F) also differed slightly between the isomers. The data were interpreted as indicating a small difference in the absorption of the two isomers; it seemed unlikely, in terms of the identical elimination rates, that their metabolic profiles would differ greatly. The 5-phenylhydantoin was eliminated with a significantly longer half-life (18.69 +/- 6.11 h) than that of ethotoin. Enantioselectivity in the pharmacokinetics of ethotoin is therefore a minor issue.     

Separation of Nadolol Stereoisomers Using Chiralpak IA Chiral Stationary Phase

Chiralpak IA adsorbent is used for both analytical and preparative chromatographic separation of nadolol stereoisomers. The results include a complete screening of the mobile phase composition for both the baseline resolution of all four nadolol stereoisomers (analytical separation) and the simulated moving bed (SMB) pseudo‐binary separation of the most retained stereoisomer. The experimental results show that analytical baseline resolution of nadolol stereoisomers can be achieved using alcohol/hydrocarbon and alcohol/acetonitrile solvent mixtures. The 10%ethanol/90%acetonitrile mixture is presented as the one that presents baseline resolution with lower retention. For the preparative pseudo‐binary separation, pure ethanol, pure methanol, alcohol/acetonitrile, and alcohol/tetrahydrofuran mixtures proved to allow good separation results. The 100%methanol/0.1%diethylamine solvent composition was selected to perform the experimental SMB separation. Using a 10 g/L total feed concentration, the more retained stereoisomer was recovered at the extract outlet stream with 99.5% purity, obtaining a system productivity of 1.98 gL^?1 h^?1 and requiring a solvent consumption of 3.13 L/g of product. Comparing these results with the ones recently presented by Ribeiro et al. (2013), this work shows that the Chiralpak IA chiral adsorbent is an interesting alternative to Chiralpak AD for the separation of nadolol stereoisomers at both analytical and preparative scales. Chirality 28:399–408, 2016. © 2016 Wiley Periodicals, Inc.     

Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.     

Chiral HPLC method for chiral purity determination of paroxetine drug substance

This article describes a chiral HPLC method for (+) trans isomer of paroxetine in a paroxetine drug substance. The method development was performed to establish a suitable HPLC system in order to separate both enantiomers. It was found that a system based on a Chiralpak AD column was suitable for the analysis. Proper column maintenance and the optimized eluent composition allowed good reproducibility and sensitivity for the method. The method was also checked on a number of different columns using different HPLC equipment and gave both reproducible chromatography and reproducible quantitative results.     

Enantiomeric Separations of Pyriproxyfen and its Six Chiral Metabolites by High‐Performance Liquid Chromatography

Pyriproxyfen is a chiral insecticide, and over 10 metabolites have been identified in the environment. In this work the separations of the enantiomers of pyriproxyfen and its six chiral metabolites were studied by high‐performance liquid chromatography (HPLC). Both normal phase and reverse phase were applied using the chiral columns Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralcel OD‐RH, Chiralpak AY‐H, Chiralpak AD‐H, Chiracel OJ‐H, (R,R)‐Whelk‐O 1, and Lux Cellulose‐3. The effects of the chromatographic parameters such as mobile phase composition and temperature on the separations were investigated and the enantiomers were identified with an optical rotation detector. The enantiomers of these targets could obtain complete separations (resolution factor Rs > 1.5) on Chiralpak IA, Chiralpak IB, Chiralcel OD, Chiralpak AY‐H, or Chiracel OJ‐H under normal conditions. Chiralcel OJ‐H showed the best chiral separation results with n‐hexane as mobile phase and isopropanol (IPA) as modifier. The simultaneous enantiomeric separation of pyriproxyfen and four chiral metabolites was achieved on Chiralcel OJ‐H under optimized condition: n‐hexane/isopropanol = 80/20, 15°C, flow rate of 0.8 ml/min, and UV detection at 230 nm. The enantiomers of pyriproxyfen and the metabolites A , C , and D obtained complete separations on Chiralpak IA, Chiralpak IC, and Lux Cellulose‐3 under reverse phase using acetonitrile/water as the mobile phase. The retention factors (k) and selectivity factors (α) decreased with increasing temperature, and the separations were better under low temperature in most cases. The work is of significance for the investigation of the environmental behaviors of pyriproxyfen on an enantiomeric level. Chirality 28:245–252, 2016. © 2016 Wiley Periodicals, Inc.     

Enantiomeric separation of vilanterol trifenatate by chiral liquid chromatography

Vilanterol trifenatate is a novel chiral long‐acting β2‐agonist developed. Vilanterol combined with inhaled corticosteroids can treat COPD and asthma. A simple liquid chromatographic method is developed for the quantitative determination of R‐vilanterol and S‐vilanterol (impurity S). HPLC separation was achieved on Chiralpak ID (250 × 4.6 mm; particle size 5 μm) column using hexane‐ethanol‐ethanolamine (75:25:0.1, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The resolution is greater than 3.3. Ethanolamine in the mobile phase is vital to enhance chromatographic efficiency and resolution between the isomers. The method was validated with respect to accuracy, specificity, precision, LOD, LOQ, linearity, and robustness as ICH guidelines.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

Chiral HPLC analysis of milnacipran and its FMOC-derivative on cellulose-based stationary phases

The HPLC enantioseparation of the last generation antidepressive drug milnacipran (+/-)-1 was investigated on different cellulose-based chiral stationary phases (CSPs). On carbamate-type columns, Chiralcel OD and OD-H (+/-)-1 could be separated with alpha value about 1.20 but the resolution was quite low because of the tailing of the peaks. Direct determination of (+/-)-1 with high selectivity and resolution was obtained on Chiralcel OJ in normal phase mode elution. Precolumn derivatization of milnacipran with Fmoc-Cl gave compound (+/-)-2 which was enantioseparated on all the investigated CSPs and allowed enhanced UV or fluorimetric detection. The Chiralpak IB, that could be considered the immobilized version of Chiralcel OD-H, was found completely ineffective in the chiral recognition of (+/-)-1 and moderately efficient in the separation of (+/-)-2.     

Chirality in the Absence of Rigid Stereogenic Elements: Steric and Electronic Effects on the Configurational Stability of C3 Symmetric Residual Tris‐Aryl Phosphanes

Residual stereoisomers result whenever closed subsets of appropriately substituted interconverting isomers (the residual stereoisomers) are generated from a full set of stereoisomers under the operation of a favored stereomerization mechanism. In the case of the three‐bladed propellers, differentiation of the edges of the blades and strict correlation in the motion of the rings are the prerequisites for the existence of residual stereoisomers. In these systems, the two‐ring flip mechanism is the lowest energy process. It does not interconvert all possible conformational stereoisomers generated by helicity and the three‐blade‐hub rotors. In the case of C₃ symmetric systems, two noninterconverting subgroups (the residual stereoisomers) are generated, each one constituted of quickly interconverting diastereoisomers. A series of tris‐aryl phosphanes, structurally designed for existing as residual enantiomers or diastereoisomers, bearing substituents differing in size and electronic properties on the aryl rings, were synthesized and characterized. The configurational stability of residual phosphanes, evaluated by dynamic ¹H‐ and ³¹P‐NMR analysis and by dynamic enantioselective high‐performance liquid chromatography (HPLC), was found 10 kcal mol^‐1 lower than that shown by the corresponding phosphane‐oxides. In accordance with the calculations, an unexpectedly low barrier for phosphorus pyramidal inversion was invoked as responsible for the scarce configurational stability of the residual tris‐arylphosphanes. Chirality 26:601–606, 2014. © 2014 Wiley Periodicals, Inc.     

Analysis of stereoisomers of chiral drug by mass spectrometry

Chiral molecules are of great importance in the life science since individual enantiomers may differ in biological activity, mechanism, and toxicity, making it necessary to explore efficient chiral analysis methods. Chromatography approaches are often used to differentiate enantiomers while mass spectrometry (MS) was thought to be blind in chiral analysis. With the development of MS technique, it began to play a more and more crucial part in chiral observation. In this review, we will give a detailed introduction of the analysis methods related to MS for chiral drugs, including its mechanism, applications, and future development.