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.     

Enantioseparation of racecadotril using polysaccharide‐type chiral stationary phases in polar organic mode

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide‐type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile) at 5 different temperatures (5–40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector—and mobile‐phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R‐enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.     

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.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Enantioseparation of chiral perfluorooctane sulfonate (PFOS) by supercritical fluid chromatography (SFC): Effects of the chromatographic conditions and separation mechanism

Perfluorooctane sulfonate (PFOS) is one of the most frequently detected perfluoroalkyl substances in environmental and human samples. Previous studies have shown that nonracemic PFOS in biological samples can be used as a marker of PFOS exposure sources. In recent years, supercritical fluid chromatography (SFC) has emerged as a powerful method to separate chiral compounds. In this study, a method of perfluoro‐1‐methylheptane sulfonate (1 m‐PFOS) enantioseparation by SFC was established. The optimal separation was obtained using a Chiralpak QN‐AX column with CO₂/2‐propanol (70/30, v/v) as the mobile phase with a flow rate of 1 mL/min, column temperature was 32°C, and BPR pressure was 1800 psi. The resolution (Rs) and retention time were 0.88 and 130 minutes, respectively. This method is more economic and greener than HPLC. Modifier pH and column temperature were determined to be significant factors of SFC chiral separation. Modifier pH is negatively correlated with the retention factors and Rs. Adsorption thermodynamics were used to explain the influence of temperature change, and it was concluded that the transfer of two enantiomers from the mobile phase to the stationary phase is enthalpy‐driven. Enantioseparation of 1 m‐PFOS by SFC follows the same rules of ion exchange as those for the chiral separation by HPLC.     

Enantiomeric Separation and Simulation Studies of Pheniramine,Oxybutynin, Cetirizine,and Brinzolamide Chiral Drugs on Amylose‐Based Columns

Solid phase extraction ( SPE)‐chiral separation of the important drugs pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C₁₈ cartridge and AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in human plasma. Pheniramine, oxybutynin, cetirizine, and brinzolamide were resolved using n‐hexane‐2‐PrOH‐DEA (85:15:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (80:20:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (70:30:0.2, v/v), and n‐hexane‐2‐propanol (90:10, v/v) as mobile phases. The separation was carried out at 25 ± 1 ºC temperature with detection at 225 nm for cetirizine and oxybutynin and 220 nm for pheniramine and brinzolamide. The flow rates of the mobile phases were 0.5 mLmin^‐1. The retention factors of pheniramine, oxybutynin, cetirizine and brinzolamide were 3.25 and 4.34, 4.76 and 5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively. The separation factors of these drugs were 1.33, 1.18, 1.09 and 1.20 while their resolutions factors were 1.09, 1.45, 1.63 and 1.25, and 1.15, respectively. The absolute configurations of the eluted enantiomers of the reported drugs were determined by simulation studies. It was observed that the order of enantiomers elution of the reported drugs was S‐pheniramine > R‐pheniramine; R‐oxybutynin > S‐oxybutynin; S‐cetirizine > R‐cetirizine; and S‐brinzolamide > R‐brinzolamide. The mechanism of separation was also determined at the supramolecular level by considering interactions and modeling results. The reported SPE‐chiral high‐performance liquid chromatography ( HPLC) methods are suitable for the enantiomeric analyses of these drugs in any biological sample. In addition, simulation studies may be used to determine the absolute configuration of the first and second eluted enantiomers. Chirality 26:136–143, 2014. © 2014 Wiley Periodicals, Inc.     

Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N‐protected α‐amino acids and their ester derivatives

Liquid chromatographic enantiomer separation of several N‐benzyloxycarbonyl (CBZ) and N‐tert‐butoxycarbonyl (BOC) α‐amino acids and their corresponding ethyl esters was performed on covalently immobilized chiral stationary phases (CSPs) (Chiralpak IA and Chiralpak IB) and coated‐type CSPs (Chiralpak AD and Chiralcel OD) based on polysaccharide derivatives. The solvent versatility of the covalently immobilized CSPs in enantiomer separation of N‐CBZ and BOC‐α‐amino acids and their ester derivatives was shown and the chromatographic parameters of their enantioselectivities and resolution factors were greatly influenced by the nature of the mobile phase. In general, standard mobile phases using 2‐propanol and hexane on Chiralpak IA showed fairly good enantioselectivities for resolution of N‐CBZ and BOC‐α‐amino acids and their esters. However, 50% MTBE/hexane (v/v) for resolution of N‐CBZ‐α‐amino acids ethyl esters and 20% THF/hexane (v/v) for resolution of N‐BOC‐α‐amino acids ethyl esters afforded the greatest enantioselectivities on Chiralpak IA. Also, liquid chromatographic comparisons of the enantiomer resolution of these analytes were made on amylose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IA and Chiralpak AD) and cellulose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IB and Chiralcel OD). Chiralpak AD and/or Chiralcel OD showed the highest enantioselectivities for resolution of N‐CBZ‐α‐amino acids and esters, while Chiralpak AD or Chiralpak IA showed the highest resolution of N‐BOC‐α‐amino acids and esters. Chirality 2009. © 2008 Wiley‐Liss, Inc.     

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors,potential adrenergic ligands,by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n‐hexane‐alcohol (ethanol, 1‐propanol, or 2‐propanol) in various proportions, and silica‐based amylose (tris‐3, 5‐dimethylphenylcarbamate) Chiralpak AD and (tris‐(S)‐1‐phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n‐hexane‐alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra‐violet (UV) and evaporative light‐scattering detection (ELSD). Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Enantiomeric separation of prothioconazole and prothioconazole‐desthio on chiral stationary phases

Prothioconazole is a type of broad‐spectrum triazole thione fungicide developed by the Bayer Company. Prothioconazole‐desthio is the main metabolite of prothioconazole in the environment. In our study, enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on various chiral stationary phases (CSPs) by high‐performance liquid chromatography (HPLC). It was found that polysaccharide CSPs showed better ability than brushing CSPs in enantiomeric separation. The successful chiral separation of prothioconazole could be achieved on self‐made Chiralcel OD, commercialized Chiralcel OJ‐H and Lux Cellulose‐1. Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralpak AY‐H, Chiralpak AZ‐H, and Lux Cellulose‐1 realized the baseline separation of prothioconazole‐desthio enantiomers. Simultaneous enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on Lux Cellulose‐1 using acetonitrile (ACN) and water as mobile phase. In most cases, low temperature favored the separation of two compounds. The influence of the mobile phase ratio or type was deeply discussed. We obtained larger Rs and longer analysis time with a smaller proportion of isopropanol (IPA) or ethanol and more water content at the same temperature. The ratio of ACN and water had influences on the outflow orders of prothioconazole‐desthio enantiomers. This work provides a new approach for chiral separation of prothioconazole and prothioconazole‐desthio with a discussion of chiral separation mechanism on different CSPs.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

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.     

Comparison of polysaccharide-based and protein-based chiral liquid chromatography columns for enantioseparation of drugs

Two different columns—Lux Cellulose-1 and Chiralpak CBH—were evaluated for their chiral recognition abilities for eight drugs comprising three β-blockers, one antacid, and four cathinones in polar-organic elution mode and reversed-phase elution mode, respectively. The factors that affected the enantioseparation were tested and optimized to develop a suitable chiral separation method whose LC conditions are compatible with MS detection. In polar-organic elution mode with the Lux Cellulose-1 column, methanol and acetonitrile were tested as the main components of the mobile phase. In addition, the effects of adding isopropanol as organic modifier, acidic additives (formic acid), and basic additives (diethylamine) were evaluated. In reversed-phase elution mode with the Chiralpak CBH column, the effect of type and concentration of organic modifier (isopropanol, acetonitrile, and methanol), the mobile phase pH (6.4 and 5.0), and buffer concentration (1mM-20mM ammonium acetate) were evaluated. The best enantioseparation was achieved with the Chiralpak CBH column with a mobile phase composed of 5mM ammonium acetate aqueous (pH = 6.4)/methanol (95/5, v/v) at a flow rate of 0.1 mL/min and a temperature of 30°C. Under these conditions, six of eight chiral drugs were baseline separated.     

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide‐fipronil and acid‐fipronil) were investigated by high‐performance liquid chromatography (HPLC) with a CHIRALPAK^® IB chiral column. The mobile phase was n‐ hexane or petroleum ether with 2‐propanol or ethanol as modifier at a flow rate of 1.0 mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide‐fipronil, and acid‐fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n‐ hexane with 2‐propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy‐driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.     

Development of a Validated LC Method for Separation of Process‐Related Impurities Including the R‐enantiomer of S‐Pramipexole on Polysaccharide Chiral Stationary Phases

Despite the availability of a few methods for individual separation of S‐pramipexole from its process‐related impurities, no common liquid chromatography (LC) method is reported so far in the literature. The present article describes the development of a single‐run LC method for simultaneous determination of S‐pramipexole and its enantiomeric and process‐related impurities on a Chiralpak AD‐H (150 x 4.6 mm, 5μm) column using n‐hexane/ethanol/n‐butylamine (75:25:0.1 v/v/v) as a mobile phase in an isocratic mode of elution at a flow rate of 1.2 ml/min at 30°C. The chromatographic eluents were monitored at a wavelength of 260 nm using a photodiode array detector. Excellent enantioseparation with good resolutions (Rs ≥ 2.88) and peak shapes (A_s ≤ 1.21) for all analytes was achieved. The proposed method was validated according to International Conference Harmonization (ICH) guidelines in terms of accuracy, precision, sensitivity, and linearity. Limits of quantification of impurities (0.25–0.55 μg/ml) indicate the highest sensitivity achievable by the proposed method. The method has an advantage of selectivity and suitability for routine determination of not only chiral impurity but also all possible related substances in active pharmaceutical ingredients of S‐pramipexole. Chirality 27:430–435, 2015. © 2015 Wiley Periodicals, Inc.     

Optimum operational conditions for chiral separation of tryptophan enatiomers using ligand exchange liquid chromatography

A simple and precise method for chiral separation of tryptophan enantiomers using high performance liquid chromatography with aligand exchange mobile phase was developed. Chiral separation was performed on a conventional C₁₈ column, using a mobile phase that consisted of a water-methanol solution (88∶12, v/v) containing 10 mmol/Ll-leucine and 5 mmol/L copper sulfate as a chiral ligand additive at a flow rate of 1.0 mL/min. This method allowed baseline separation of two enantiomers with a resolution of 1.84 in less than 30 min. The effect of various conditions, including concentration, type of ligand, organic modifier, pH, flow rate, and temperature, on enantioseparation were evaluated and chiral recognition mechanisms were investigated. Thermodynamic data (ΔΔH and ΔΔS) obtained by van't Hoff plots revealed that enantioseparation is an enthalpy-controlled process.     

Enantioseparation and determination of flumequine enantiomers in multiple food matrices with chiral liquid chromatography coupled with tandem mass spectrometry

The present work firstly described the enantioseparation and determination of flumequine enantiomers in milk, yogurt, chicken, beef, egg, and honey samples by chiral liquid chromatography‐tandem mass spectrometry. The enantioseparation was performed under reversed‐phase conditions on a Chiralpak IC column at 20°C. The effects of chiral stationary phase, mobile phase components, and column temperature on the separation of flumequine enantiomers have been studied in detail. Target compounds were extracted from six different matrices with individual extraction procedure followed by cleanup using Cleanert C18 solid phase extraction cartridge. Good linearity (R²>0.9913) was obtained over the concentration range of 0.125 to 12.5 ng g^‐1 for each enantiomer in matrix‐matched standard calibration curves. The limits of detection and limits of quantification of two flumequine enantiomers were 0.015‐0.024 and 0.045‐0.063 ng g^‐1, respectively. The average recoveries of the targeted compounds varied from 82.3 to 110.5%, with relative standard deviation less than 11.7%. The method was successfully applied to the determination of flumequine enantiomers in multiple food matrices, providing a reliable method for evaluating the potential risk in animal productions.     

Enantiomeric separation of type I and type II pyrethroid insecticides with different chiral stationary phases by reversed‐phase high‐performance liquid chromatography

The enantiomeric separation of type I (bifenthrin, BF) and type II (lambda‐cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose‐1, Lux Cellulose‐3, and Chiralpak IC chiral columns was investigated by reversed‐phase high‐performance liquid chromatography. Methanol/water or acetonitrile/water was used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase composition, column temperature, and thermodynamic parameters on enantiomer separation were carefully studied. Bifenthrin got a partial separation on Lux Cellulose‐1 column and baseline separation on Lux Cellulose‐3 column, while LCT enantiomers could be completely separated on both Lux Cellulose‐1 and Lux Cellulose‐3 columns. Chiralpak IC provided no separation ability for both BF and LCT. Retention factor (k) and selectivity factor (α) decreased with the column temperature increasing from 10°C to 40°C for both BF and LCT enantiomers. Thermodynamic parameters including ?H and ?S were also calculated, and the maximum R_s were not always obtained at lowest temperature. Furthermore, the quantitative analysis methods for BF and LCT enantiomers in soil and water were also established. Such results provide a new approach for pyrethroid separation under reversed‐phase condition and contribute to environmental risk assessment of pyrethroids at enantiomer level.     

High performance liquid chromatographic enantioseparation of chiral bridged polycyclic compounds on chiralcel OD-H and chiralpak OT(+)

The HPLC enantiomeric separation of 29 racemic bridged polycyclic compounds was examined on commercially available Chiralcel OD-H and Chiralpak OT(+) columns. The separations were evaluated under normal-phase mode (hexane containing mobile phase) for Chiralcel OD-H and under normal-phase as well as under reversed-phase mode (pure MeOH, temperature 5 degrees C) for Chiralpak OT(+). Almost all compounds were resolved either on Chiralcel OD-H or on Chiralpak OT(+), in some cases on both. The use of trifluoroacetic acid (TFA), as modifier of the hexanic mobile phase, had a beneficial effect on the enantioseparation of some polar and acidic compounds on Chiralcel OD-H. The influence of small chemical structural modifications of the analytes on the enantioseparation behavior is discussed. A structure-retention relationship has been observed on both stationary phases. This chromatographic evaluation may provide some information about the chiral recognition mechanism: in the case of Chiralcel OD-H, hydrogen bonding, pi-pi and distereoselective repulsive are supposed to be the major analyte-CSP interactions. In the case of Chiralpak OT(+), a reversed-phase enantioseparation could take place through hydrophobic interactions between the aromatic moiety of the analytes and the chiral propeller structure of the CSP. The synthesis of some unknown racemic bromobenzobicyclo[2.2.1] analytes is also described.     

Enantioseparation of nine indanone and tetralone derivatives by HPLC using carboxymethyl‐β‐cyclodextrin as the mobile phase additive

High‐performance liquid chromatography (HPLC) is a powerful method in the area of chiral separation. In this study, a method of HPLC using carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) as chiral selector was developed for enantioseparation of nine indanone and tetralone derivatives. The separation was performed on a conventional C₁₈ column. The optimal mobile phase was a mixture of methanol and 0.05 mol/L phosphate buffer at pH 1.8 (55:45, v /v) containing 22.9 mmol/L CM‐β‐CD. Under such conditions, the resolutions of all analytes were over 1.8 except for Compound F. The results of the study indicate the presence of a complex with 1:1 stoichiometry of the inclusion complex. In addition, it can be inferred from thermodynamic analysis that the behavior of formation of the inclusion complex and enantioseparation occurred simultaneously, while they were driven by different forces. The effect of analyte structure is also discussed.     

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.