Chiral Liquid Chromatographic Method Research Articles

Separation and quantitation of valacyclovir enantiomers using stability‐indicating chiral liquid chromatography method with a chiral stationary phase of amylose tris‐(3,5‐dimethylphenylcarbamate)

AbstractThe present research developed and validated a new stability‐indicating technique for the stereo‐selectively enantiomers of the antiviral nucleoside analog Valacyclovir hydrochloride (VAL). The chiral separation was performed using normal‐phase high‐performance liquid chromatography (HPLC) with a chiral stationary phase consisting of amylose tris(3‐chloro‐5‐methylphenylcarbamate) and a mobile phase of “n‐hexane, methanol, ethanol, and diethylamine”, flow rate of 0.60 mL/min, column temperature of 30°C, injection volume of 10‐μL, detection wavelength of 254‐nm, and run time of 25‐min. The enantiomers (S‐enantiomer, L‐isomer, R‐enantiomer, and D‐isomer) of Valacyclovir were separated with a resolution of 4.8 and no interference. The validation parameters verified for the proposed method, linearity in a range of 0.1002–24.3486 μg/mL (0.02–4.86%) with a regression coefficient of 0.999, and the accuracy was determined with excellent recoveries ranging from 94.38%–109.97%. The concentrations established for the detection limit and quantitation limit were 0.01% and 0.02%, respectively. The forced degradation experiments were used to assess the stability‐indicating qualities. D‐Valacyclovir impurity was successfully evaluated in release and stability samples of VAL in drug substance and tablet dosage forms using the proposed normal phase chiral HPLC approach.

Valbenazine isomers and enantiomer determination by chiral normal phase liquid chromatography.

A novel, simple, specific, rapid, enantioselective normal phase chiral high-performance liquid chromatographic method with amylose-based Chiral Pak IG-3(250 × 4.6 mM) 3.0 μM column was developed and validated for separation and quantification of isomers and enantiomer of Valbenazine. The mobile phase composed of n-Heptane, isopropyl alcohol, dichloromethane, ethanol, and diethylamine in the ratio of 70:10:15:5:0.1 (V/V/V/VV) with a gradient flow rate was applied. The injection volume was 10 μl, and detection was carried out using a photodiode array detector at 282 nM. The column compartment was set at 35°C. The resolution between the enantiomer and isomers was found to be more than 2.0. The method was linear over the concentration range of limit of quantitation to 250% for isomers and enantiomers. The method was found to be robust with column temperature. The proposed chiral method is applicable for the determination of isomers and enantiomer of Valibenazine and was successfully used in the quality control of bulk drug manufacturing and pharmaceuticals.

Enantioseparation and a comprehensive spectroscopic analysis of novel synthetic cathinones laterally substituted with a trifluoromethyl group

Recently, the number of structural modifications of synthetic cathinones has been growing making them the second largest group of new psychoactive substances in Europe. Although they are abused because of their various psychoactive effects, some compounds from this group also serve as pharmaceuticals. Since synthetic cathinones are chiral molecules with one chiral center, their biological, toxicological, and pharmacological properties may significantly differ according to their absolute configuration and enantiomeric excess. In this study, we have synthesized two substances bearing a pharmacologically interesting trifluoromethyl group and developed a chiral liquid chromatography method using a polysaccharide chiral stationary phase to separate the corresponding enantiomers of both these drugs. Subsequently, we utilized molecular spectroscopic methods including chiroptical (electronic circular dichroism and vibrational circular dichroism) and non-polarizable (infrared and ultraviolet absorption) spectroscopies. In combination with density functional theory calculations, we have obtained stable conformers of selected enantiomers in solution and their relative abundances, which we used to simulate their spectra. The experimental and calculated data have been used to elucidate the 3D structure of the enantiomerically pure compounds and assign the absolute configuration of all prepared compounds.

Validated Chiral Ultra Fast Liquid Chromatographic Method for Quantitative Analysis of Enantiomeric Vildagliptin

A rapid, accurate, and precise chiral Ultra fast liquid chromatography (UFLC) method was developed and validated for enantiomeric separation of racemic vildagliptin and S-vildagliptin according to the guidelines of the International Conference on Harmonization (ICH). The chiral chromatographic separation was achieved with a mobile phase consisting of 20 mM borax buffer (pH 9.0 ± 0.05), ACN, and 0.1% Triethylamine (50:50:0.1, v/v/v) at a flow rate of 1 ml/min using a chiralcel OD-RH column, tris(3,5-dimethyl phenyl carbamate) (250 mm × 4.6 mm, 5 μm) column. The UFLC analysis was monitored at 210 nm. The method showed good linearity with a regression coefficient (r2) of 0.999 in the range of 1 - 12 μg/ml for S-vilda. The detection limit (LOD), quantitation limit (LOQ), and the average percentage recovery for S-vilda were found to be 0.024, 0.075 μg/mL, and 99.19% to 100.4%, respectively. The percentages of relative standard deviation (% RSD) for intra- and inter-day precision were found to be 0.346% and 0.364%, respectively. The developed method proved to be reproducible as % RSD was S-vilda was found to be 99.19 w/w. The proposed chiral method can be put in application for the enantiomeric purity determination of S-vilda formulations.

Evaluation of Chiral Liquid Chromatographic Method for Separation and Quantification of Isomers of Brivaracetam.

A selective, sensitive and robust chiral analytical method was developed for the quantification of Brivaracetam (BRV) and its three isomers. Systematic chiral chromatographic elution process was executed in different modes on chiral columns of polysaccharide based to attain the finest condition. The analytical method was developed by utilizing immobilized polysaccharide chiral column (CHIRALPAK IG-U) with reversed phase under isocratic condition containing acetonitrile and 10 mM ammonium bicarbonate in the proportion of 40:60 (v/v). The mobile phase flow rate and column temperature were monitored at 0.3 mLmin-1 and 25°C with a resolution of more than 2.0. The eluted components from the column were processed at 212 nm UV detection. The limit of detection and limit of quantification values of BRV, 2R, 4S-Isomer, 2R, 4R-Isomer and 2S, 4S-Isomers were found to be 0.0066/0.02, 0.0035/0.0107, 0.0036/0.0109 and 0.005/0.0152 µgml-1 respectively. Precision, linearity, accuracy and robustness were conducted according to ICH guidelines and the findings were within the acceptable limits. The proposed analytical method was found to be precise, accurate and specific for the quantification of enantiomer and its diastereomers for drug product and drug substance of BRV.

Reverse-phase chiral high-performance liquid chromatography for separation of a diastereomer in alalevonadifloxacin: A novel antibacterial agent.

Alalevonadifloxacin (ALA) is a novel antibacterial drug, recently launched in India to treat infections caused by Gram-positive bacteria. In present work, a chiral high-performance liquid chromatographic method was developed and validated for the quantification of a diastereomeric impurity (DI) in ALA. The separation was achieved on Pirkle type (R,R) Whelk-O1 chiral stationary phase, using ammonium formate buffer and acetonitrile in gradient fashion at a flow rate of 1.5 ml/min. The method was extensively validated for the quantification of DI in ALA. The detector response for DI was linear over the concentration range of 0.24-4.78 μg/ml. Limit of quantitation and limit of detection for DI were 0.24 and 0.07 μg/ml respectively. The mean recovery of the DI was 103.47 ± 5.14%. The impact of column temperature on the chiral separation was evaluated. The method was employed for controlling diastereomeric impurity in the batches of ALA used in preclinical studies.

Utilization of the hysteresis phenomenon for chiral high-performance liquid chromatographic method selection in polar organic mode

Polysaccharide-based chiral stationary phases (CSPs) are outstandingly suitable to play a key role in chiral HPLC method selection strategies, since they provide high success rates. One reason for this ability is that they adopt a diversity of higher order structures in various eluents, resulting in versatile chiral environments.A potential to extend this versatility further was expected and examined in the present study, based on the recently discovered hysteretic behavior of a widely used chiral selector (CS), amylose tris(3,5-dimethylphenylcarbamate). The hindered transitions of its structure, which are behind the history dependence of its separation ability, were used as a tool to identify distinct states of the chiral selector in order to exploit an extended selectivity space. The identification was carried out using a single diagnostic compound, as opposed to the common approach where testing a library of compounds is required. Eluent mixtures consisting of 2-propanol and either methanol or ethanol were scrutinized in terms of stability and robustness of the observed retentions. The solvent mixtures that were eligible for practical application in these respects were used to construct a screening sequence, including identical compositions combined with different column pretreatment. The gain achievable by using the proposed sequence was then evaluated using 15 enantiomer pairs with focus on resolution, enantiomer elution order and chemoselectivity.

A Validated LC Method for the Enantiomeric Separation of EAI045 on Chiral Stationary Phase.

A simple and accurate chiral liquid chromatographic method was developed for enantiomeric resolution and determination of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide (EAI045). The enantiomers of EAI045 were baseline resolved on a Chiralpak AD-H (250mm×4.6mm, 5μm) column using a mobile phase system containing n-hexane: 2-propanol (75: 25v/v) at a flow rate of 1mLmin-1 at 30°C. The eluted analytes were subsequently detected with an ultraviolet detector at 254nm. The effects of organic modifiers and temperature on the enantioselectivity and resolution of the enantiomers were evaluated. The calibration curves were plotted within the concentration range between 2 and 600μgmL-1 (n=11), and recoveries between 98.74% and 101.52% were obtained, with relative standard deviation<1.4%. The limit of detection and limit of quantitation for R-enantiomer were 0.94 and 3.07μgmL-1 and for S-enantiomer were 0.86 and 2.84μgmL-1, respectively. The validated method was found to be suitable for enantiomeric separation and sufficiently accurate for the determination of enantiomeric purity of EAI045 in bulk drugs.

Validated chiral liquid chromatographic method for the enantiomeric separation of Tenofovir Disoproxil drug substance and its dosage forms

Validated chiral liquid chromatographic method for the enantiomeric separation of Tenofovir Disoproxil drug substance and its dosage forms

Liquid Chromatographic Separation and Thermodynamic Investigation of Ethyl Nipecotate Enantiomers on Immobilized Amylose-Based Chiral Stationary Phase.

Ethyl nipecotate enantiomers are widely used as chiral building blocks in the synthesis of drug substances. An efficient and economic chiral high-performance liquid chromatographic method for determination of enantiomeric purity of ethyl nipecotate is developed and validated. Chiral separation was achieved on immobilized amylose-based stationary phase using a mixture of n-hexane: ethanol: diethylamine (80:20:0.1, v/v/v) as mobile phase. Resolution between R-(-)-ethyl nipecotate (REN) and S-(+)-ethyl nipecotate (SEN) peaks was found to be 3.59. The detector response of SEN exhibited an excellent linearity over the concentration range of 4.5-120μgmL-1. The limit of detection and limit of quantification for SEN were 0.016 and 0.045μg and REN were 0.015 and 0.043μg, respectively. The influence of column oven temperatures on chiral retention and separation was studied in the range of 25°C to 50°C; the thermodynamic parameters ΔH°, ΔS° and ΔG° were evaluated from van't Hoff plots and used to explain the strength of interaction between analyte and stationary phase.

Enantiomeric separation of Midodrine hydrochloride in bulk and pharma-ceutical dosage form by chiral HPLC

A simple, sensitive chiral liquid chromatographic method was developed for the separation and quantification of enantiomers Midodrine. A chiral PAK IG-3 (150 x 4.6 mm) 3µm column was used for the separation of the enantiomers. The mobile phase consists of 10 mM ammonium bicarbonate in water and acetonitrile in the ratio of 95:5, v/v with a flow rate of 0.7 ml/min. The detection was done at 290 nm with column temperature maintained at 40°C. The method linear ranged between 10 – 110 μg/ml and 5 – 100 μg/ml for (+) and (-) Midodrine enantiomers. The recovery of the method was found to be in the range of 99.1 to 101.2 %. The detection limit for the (+) and (-) enantiomers was found to be 4 μg/ml and 1 μg/ml, respectively. A simple validated chiral HPLC method with reverse elusion is described for the separation and quantification of the enantiomers of Midodrine in bulk and formulation.

Chiral separation and thermodynamic investigation of WCK 3023: A novel oxazolidinone antibacterial agent, application to pre-clinical pharmacokinetic study.

A chiral liquid chromatographic method was developed and validated for the quantification of R-enantiomer impurity (RE) in WCK 3023 (S-enantiomer), a new drug substance. The separation was achieved on Chiralpak IA (amylose-based immobilized chiral stationary phase), using a mobile phase consisting of n-hexane-ethanol-trifluoroacetic acid (70:30:0.2, v/v/v) at a flow rate of 1.0 mL/min. The method was extensively validated for the quantification of RE in WCK 3023 and proved to be robust. For RE the detector response was linear over the concentration range of 0.11-5 μg/mL. The limit of quantitation and limit of detection for RE were 0.11 and 0.04 μg/mL respectively. Average recovery of the RE was in the range of 98.11-99.55%. The developed method was specific, sensitive, precise and accurate for quantitative determination of RE in WCK 3023. The impact of thermodynamic parameters on the chiral separation was evaluated. The method was employed for controlling the enantiomeric impurity in the lots of WCK 3023 used for pre-clinical studies. The method was successfully applied to evaluate the possible conversion of WCK 3023 to RE in rat serum samples during pre-clinical pharmacokinetic studies.

Development and Validation of the Chiral Liquid Chromatography Method for Separation of Enantiomeric Impurity in Novel Oxazolidinone Antibacterial Agent WCK 4086.

A highly stereo-specific liquid chromatographic method was developed and validated for the quantification of enantiomeric impurity (R-enantiomer) in novel oxazolidinone antibacterial agent (WCK 4086), a drug substance. The separation was achieved on Chiralpak AD-H (amylose-based chiral stationary phase) using a mobile phase consisting of n-hexane:2-propanol:methanol:trifluoroacetic acid (80:10:10:0.4, v/v/v/v) at a flow rate of 1.0 mL min-1. Chromatographic resolution between two enantiomers was found to be more than 2.0. Method was extensively validated for the quantification of R-enantiomer in WCK 4086 and proved to be robust. Method was found to be highly specific as all other related impurities were separated from the enantiomers. The calibration curve for R-enantiomer showed an excellent linearity over the concentration range of 1-5 μg mL-1. Limit of quantitation (LOQ) and limit of detection (LOD) for R-enantiomer were 0.009 μg and 0.003 μg, respectively. Average recovery of the R-enantiomer was in the range of 94.55-109.67%. Analytical solutions were found to be stable up to 70 h at room temperature. Developed method was found to be specific, sensitive, precise and accurate for quantitative determination of R-enantiomer in WCK 4086 and useful for controlling the enantiomeric impurity in drug substance used for preclinical studies.

ENANTIOSELECTIVE ANALYSIS OF GUAIFENESIN IN BULK AND PHARMACEUTICAL DOSAGE FORMS BY CHIRAL REVERSE PHASE HPLC-PDA METHOD

A novel, accurate and precise chiral reverse-phase high pressure liquid chromatographic method was developed for enantioselective analysis of guaifenesin (GFN) in bulk and tablet dosage forms. Chiral separation was achieved on phenomenex Lux Cellulose-4 column (250×4.6mm, 5μ) using 0.02% formic acid: acetonitrile (90:10 V/V) as the mobile phase at a flow rate of 1mL/min at 230nm. The retention times of GFN enantiomers A and B was 15 and 16 minutes, respectively, with good peak resolutions and showing good linearity in the concentration range of 10-50 μg/mL (R2 &gt; 0.999). The developed method was validated as per the International Conference on Harmonization guidelines and the results were well within the acceptable limits. The percentage assay in tablet dosage form was found to be 98.8 and 98.2 respectively, for enantiomers A and B and was with in the compendial specifications, demonstrating the suitability of developed method for enantioselective analysis of guaifenesin racemic mixture.

Enantioseparation and determination of asenapine in biological fluid micromatrices by HPLC with diode array detection

Asenapine is a recent drug approved in the European Union for the treatment of bipolar disorder. An original approach has been developed for asenapine analysis in patients treated with the drug, including miniaturized microsampling procedures, separation and quantitation of drug enantiomers. An original enantioselective method based on high-performance liquid chromatography with diode array detection was developed and applied to the determination of asenapine enantiomer levels in innovative haematic samples: four micromatrices have been tested, two based on dried matrix spots (dried blood spots and dried plasma spots) and two based on volumetric absorptive microsampling (from blood and plasma). Chiral separation was achieved on a cellulose-tris(3,5 dimethylphenylcarbamate) column, with a mobile phase containing bicarbonate buffer and acetonitrile. The method was validated with satisfactory results of linearity and precision on all matrices that showed also a significant performance in terms of stability, feasibility and reliability, when compared to fluid plasma sampling, handling and processing. Among micromatrices, both volumetric absorptive microsampling types were superior to dried matrix spots in terms of data reproducibility and correspondence with plasma levels. The bioanalytical approach proposed herein provides for the first time a chiral high-performance liquid chromatographic method for the determination of asenapine enantiomers, coupled to a very effective microsampling strategy.

DEVELOPMENT AND VALIDATION OF RP-CHIRAL HPLC METHOD FOR QUANTIFICATION OF (S)-ISOMER IN TENOFOVIR DISOPROXIL FUMARATE

Objective: The main objective of present study was to develop and validate a reverse phase enantioselective chiral high performance liquid chromatographic method was developed for enantiomeric resolution of Tenofovir disoproxil fumarate; it decreases the HIV infection in human body. The method is specific, rapid, precise and accurate for the separation and determination of (S)-isomer in tenofovir disoproxil fumarate drug substance form.Methods: The S-Isomer of Tenofovir disoproxil fumarate was resolved on a Chiral AGP (150 × 4.0 mm, 5 µm) column (L-41) using a mobile phase system containing 0.1 M ammonium acetate in water pH 6.8 with ammonia solution and methanol in the ratio of (85:15 v/v). The mobile phase was set at a flow rate of 0.8 ml/min and the volume injected was 10μl for every injection. The detection wavelength was set at 260 nm and the column temperature was set at 15 °C.Results: The proposed method was productively applied for the quantitative determination of (S)-isomer in Tenofovir disoproxil fumarate drug substance form. The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.125 to 3.75 µg/ml for (S)-isomer, 0.125-3.75 µg/ml for Tenofovir disoproxil fumarate. The mean values of the correlation coefficient were 0.999 and 0.999 for (S)-isomer and Tenofovir disoproxil fumarate. The method was validated as per the ICH guidelines. The detection limit (LOD) was about 0.05 µg/ml and quantitation limit (LOQ) was about 0.125 µg/ml for (S)-isomer and Tenofovir disoproxil fumarate. The relative standard deviation was found to be 0.78 % for (S)-isomer in Tenofovir disoproxil fumarate.Conclusion: The developed and validated HPLC method and the statistical analysis showed that the method is repeatable and selective for the estimation of the (S)-isomer of the Tenofovir disoproxil fumarate drug substance.

A Validated Chiral Liquid Chromatographic Method for the Enantiomeric Separation of Orphenadrine Citrate in Pharmaceutical Dosage Form

Introduction: Orphenadrine citrate is chemically (±)-N, N-Dimethyl-2-[(o-methyl-a-phenyl benzyl) oxy] ethylamine citrate. It is used to treat painful muscle spasms (spasm of skeletal muscles), as well as the treatment of Parkinson’s disease. The salts of orphenadrine are available in two varieties, namely hydrochloride and citrate. The hydrochloride salt is used for the treatment of Parkinson’s disease while the citrates form as a skeletal muscle relaxant. Method: In the present study, an isocratic reverse phase-HPLC method was developed and validated as per ICH guidelines. The chromatographic separation was achieved on Phenomenex® Lux Cellulose 1 (250 mm x 4.6 mm i.d, 5 µm particle size) column using mobile phase system containing acetonitrile: 0.02M ammonium bicarbonate (75:25 v/v) at the flow rate of 1.0 mL/min, and UV detection was carried out at 241 nm. The described method was linear over the range of 2 – 10 µg/mL for (±) orphenadrine enantiomers with a correlation coefficient (r2 = 0.999). The limits of detection and quantification were found to be 0.50 µg/mL and 2.00 µg/mL respectively, for 20µL injection volume. The recovery study of orphenadrine from tablet formulation was found to be 102.21%. Orphenadrine standard solution and mobile phase were found to be stable for at least 48h. The orphenadrine enantiomers were well resolved with mean retention times of about 4.78 min and 6.00 min respectively. Conclusion: The developed method was extensively validated and proved to be robust, accurate, precise and suitable for analysis of orphenadrine enantiomers in tablet dosage form and stability studies of orphenadrine. Keywords: Chirality, ICH, orphenadrine citrate, RP-HPLC, validation.

Development and validation of a chiral liquid chromatography method for the determination of MP 3950 enantiomers, a high selective 5-HT4 receptor agonist, in rat plasma and its application to stereoselective pharmacokinetic study.

MP 3950 is an active metabolite of mosapride which exhibits high 5-HT4 receptor agonistic effect. The present paper describes an enantioselective chiral HPLC method for separation and quantification of MP 3950 enantiomers in rat plasma. The plasma samples were prepared by liquid-liquid extraction with ethyl acetate and the baseline chromatographic separation was achieved on a Chiralcel OJ-H column with a mobile phase consisting n-hexane/ethanol/methanol/diethylamine (85:5:10:0.4, v/v/v/v) at the flow rate of 1.0mL/min. The ultraviolet detection was performed at 276nm. The calibration curve was linear in the range from 0.100 to 5.00μg/mL with the lower limit of quantification of 0.100μg/mL for each enantiomer. The intra- and inter-day precisions were not more than 14.7%. The accuracy was from -6.4% to 14.0%. The validated method was successfully applied to chiral inversion and stereoselective pharmacokinetic study in rats after oral administration of MP 3950 racemate. The results indicated that no stereochemical inversion was occurred in rats. And the plasma concentrations and area under plasma concentration-time curve of (S)-MP 3950 were all significantly higher than those of (R)-MP 3950 in both male and female rats, which indicated that the disposition of MP 3950 in rats might be stereoselective.

Validation of a Chiral Liquid Chromatographic Method for the Degradation Behavior of Flumequine Enantiomers in Mariculture Pond Water.

In this work, flumequine (FLU) enantiomers were separated using a Chiralpak OD-H column, with n-hexane-ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6 mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detection, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high-performance liquid chromatography (HPLC) method were determined. The developed method was then applied to investigate the degradation behavior of FLU enantiomers in mariculture pond water samples. The results showed that the degradation of FLU enantiomers under natural, sterile, or dark conditions was not enantioselective. Chirality 28:649-655, 2016. © 2016 Wiley Periodicals, Inc.

Development and Validation of a Chiral Liquid Chromatographic Method for Quantitative Determination of (+)-OTX015 in (−)-OTX015

A simple, rapid and robust enantioselective method was developed and validated for the quantitation of OTX015 enantiomers [(−)-OTX015 and (+)-OTX015] with ultrahigh-performance liquid chromatography (UHPLC) as per ICH guidelines. The active [(−)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using methanol consisting of 0.1 % diethyl amine at a flow rate of 1.0 mL min−1. The resolution between the enantiomers was found to be more than 3.7 in the optimized method. The developed method was extensively validated and proven to be robust. The calibration curve for (+)-OTX015 showed excellent linearity over the concentration range of 10–100 µg mL−1. The limit of detection and the limit on quantitation for (+)-OTX015 were 5 and 10 µg mL−1, respectively. The recovery for (+)-OTX015 ranged between 100.7 and 102.5 % in the bulk drug sample of (−)-OTX015. The proposed method was found to be suitable and accurate for quantitative determination of (+)-OTX015 in bulk drug.