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

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

The Effect of Enantiomer Elution Order on the Determination of Minor Enantiomeric Impurity in Ketoprofen and Enantiomeric Purity Evaluation of Commercially Available Dexketoprofen Formulations

In a recent study, opposite enantiomer elution order was observed for ketoprofen enantiomers on two amylose-phenylcarbamate-based chiral columns with the same chemical composition of the chiral selector but in one case with coated while in the other with an immobilized chiral selector. In the present study, the influence of this uncommon effect on method validation parameters for the determination of minor enantiomeric impurity in dexketoprofen was studied. The validated methods with two alternative elution orders for enantiomers were applied for the evaluation of enantiomeric impurity in six marketed dexketoprofen formulations from various vendors. In most of these formulations except one the content of enantiomeric impurity exceeded 0.1% (w/w).     

Enantiomeric LC Separation of Epinephrine on Amylose based sorbent

A simple, rapid, and robust chiral HPLC method has been developed and validated for separation of the enantiomers of epinephrine, l-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanol, an antihypertensive drug, in the bulk drug. The enantiomers were resolved on an amylose-based stationary phase with n-hexane–2-propanol–methanol–trifluoroacetic acid–diethylamine 90:05:05:0.2:0.2 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. In the optimized method resolution between the enantiomers was not less than 3.0. The trifluoroacetic acid and diethylamine in the mobile phase were important for enhancing chromatographic efficiency and hence the resolution of the enantiomers. The method was extensively validated and proved to be robust. The calibration plot for the d enantiomer was highly linear over the concentration range 100–2,000 μg mL^?1. The limits of detection and quantification for the d enantiomer were 0.15 and 0.45 μg mL^?1, respectively. Recovery of the d enantiomer from bulk drug samples of epinephrine ranged between 99.5 and 101.5%. Epinephrine sample solution was stable for up to 48 h. The method was suitable for accurate quantitative determination of the d enantiomer in the bulk drug substance     

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

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

A Validated LC Method for the Determination of Chiral Purity of (1S)-6,11-Dioxo-1,2,3,4,6,11-Hexahydropyridazino[1,2-b]Phthalazine-1-Carboxylic Acid: A Key Intermediate of Cilazapril

A simple and accurate normal phase liquid chromatographic method was developed for the determination of chiral purity of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, S-enantiomer used as key intermediate in the manufacturing of cilazapril bulk drug. Chromatographic separation between (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, and its opposite enantiomer (1R)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, R-enantiomer was achieved using a Chiralpak AD-H column using a mobile phase containing hexane, isopropyl alcohol and tri-fluoro acetic acid (80:20:0.1 v/v/v). The resolution between the two enantiomers was found to be more than 3.2. The limit of detection (LOD) and limit of quantitation (LOQ) of the R-enantiomer was 0.15 and 0.5 μg mL^?1, respectively, for 10 μL injection volume. The percentage recoveries of the R-enantiomer ranged from 96.5 to 105.3 in the bulk samples of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid. The test solution and mobile phase was observed to be stable up to 24 h after the preparation. The developed method was validated as per International Conference on Harmonization guidelines in terms of LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

Determination of Enantiomeric Purity of GSK962040 Based on Liquid Chromatography Using Chiral Stationary Phase Combined with a Pre-column Derivatization Procedure

A sensitive and accurate LC method was developed and further validated for the determination of enantiomeric purity of GSK962040. Before separation, a pre-column derivatization procedure was performed. Baseline separation with a resolution higher than 1.9 was accomplished within 15 min using a Chiralpak AD-H (250 × 4.6 mm; particle size 5 μm) column, with n-hexane: 2-propanol (85:15 v/v) as mobile phase at a flow rate of 1 mL min^?1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within the concentration range between 4 and 200 μg mL^?1 (n = 8), and recoveries between 98.15 and 101.48% were obtained, with relative standard deviation (RSD) lower than 1.42%. The LOD and LOQ for the Boc-GSK962040 were 1.23 and 4.15 μg mL^?1 and for its enantiomer were 1.38 and 4.76 μg mL^?1, respectively. The developed method was also evaluated and validated by analyzing bulk samples with different enantiomeric ratios of GSK962040. It was demonstrated that the method was accurate, robust and sensitive, and also had practical utilities for real analysis.     

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL⁻¹ (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL⁻¹; those for its enantiomer were 1.49 and 4.94 µg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Development and Validation of the Chiral HPLC Method for Daclatasvir in Gradient Elution Mode on Amylose-Based Immobilized Chiral Stationary Phase

A selective and specific high-performance liquid chromatography method for the determination of daclatasvir enantiomers has been developed and validated. Various immobilized polysaccharide-based chiral stationary phases were used to define a separation strategy utilizing normal-phase and polar organic chromatography modes. Excellent resolution between daclatasvir and its enantiomer was achieved on amylose tris (3-chlorophenylcarbamate) stationary phase, namely CHIRALPAK ID-3, using binary gradient containing acetonitrile:diethylamine and methanol:diethylamine as the mobile phase. The flow rate of the mobile phases was maintained at 1.0 mL min⁻¹ while the column oven temperature was maintained at 40 °C. The column effluent was monitored by UV detection at 315 nm. In comparison with isocratic method, the binary gradient method offered excellent peak shape and improved resolution between daclatasvir and its enantiomer while maintaining the specificity with diastereomers. The method was found to be precise, accurate, and linear (R ² > 0.999). Limit of detection and limit of quantitation of the enantiomer were found to be 0.083 µg mL⁻¹ as and 0.25 µg mL⁻¹, respectively. Recovery of the enantiomer was found to be in the range of 90 to 112 %.

     

Determination of AR-42 enantiomeric purity by HPLC on chiral stationary phase

A sensitive and accurate liquid chromatographic method for the determination of AR-42 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.9 was accomplished within 10 min using a CHIRALPAK AD column (250 mm × 4.6 mm; particle size 5 μm) and n-hexane/2-propanol/diethylamine (75:25:0.1 v/v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 260 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.001 and 0.5 mg mL^?1 (n = 10), and the recoveries between 98.23 and 101.87% were obtained, with relative standard deviation lower than 1.31%. Limit of detection and limit of quantitation for AR-42 were 0.39 and 1.28 μg mL^?1 and for its enantiomer were 0.36 and 1.19 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AR-42, especially for the analysis of bulk samples.     

Direct TLC Resolution of (±)-Ketamine and (±)-Lisinopril by Use of (+)-Tartaric Acid or (−)-Mandelic Acid as Impregnating Reagents or Mobile Phase Additives. Isolation of the Enantiomers

Direct resolution of the enantiomers of the racemic drugs ketamine and lisinopril has been achieved by TLC. Enantiomerically pure tartaric acid and mandelic acid were used as chiral impregnating reagents and as mobile phase additives. When (?)-mandelic acid was used as chiral impregnating reagent use of ethyl acetate–methanol–water 3:1:1 (v/v) as mobile phase enabled successful resolution of the enantiomers of both compounds. For lisinopril, the mobile phase acetonitrile–methanol–water–dichloromethane 7:1:1:0.5 (v/v) was successful when (+)-tartaric acid was used as impregnating agent. When (+)-tartaric acid was used as mobile phase additive the mobile phase acetonitrile–methanol–(+)-tartaric acid (0.5% in water, pH 5)–glacial acetic acid 7:1:1.1:0.7 (v/v) enabled successful resolution of the enantiomers of lisinopril. The effects on resolution of temperature, pH, and the amount of chiral selector were also studied. The separated enantiomers were isolated and identified. Spots were detected with iodine vapour. LODs were 0.25 and 0.27 μg for each enantiomer of ketamine with (+)-tartaric acid and (?)-mandelic acid, respectively, whereas for lisinopril LODs were 0.14 and 0.16 μg for each enantiomer with (+)-tartaric acid (both conditions) and (?)-mandelic acid, respectively.     

Simultaneous Enantiomeric Separation of Carfentrazone-Ethyl Herbicide and Its Hydrolysis Metabolite Carfentrazone by Cyclodextrin Electrokinetic Chromatography. Analysis of Agrochemical Products and a Degradation Study

The different activity and toxicity that the enantiomers of agrochemicals may have requires the development of stereoselective analytical methodologies enabling the individual determination of each enantiomer. The aim of this work was to develop the first Electrokinetic Chromatography methodology enabling the simultaneous enantiomeric separation of carfentrazone-ethyl herbicide and its hydrolysis metabolite carfentrazone. The use of an anionic cyclodextrin as chiral selector (captisol at 2.5% (w/v)) in a 25 mM acetate buffer, at a temperature of 30 °C, and an applied voltage (reverse polarity) of −30 kV, allowed the simultaneous separation of the four enantiomers of the two compounds studied in 6.8 min with enantiomeric resolutions of 5.0 for carfentrazone-ethyl and 5.1 for carfentrazone. Analytical characteristics of the developed method were evaluated and found adequate to achieve the quantitation of carfentrazone-ethyl and carfentrazone. Analysis of a commercial herbicide formulation showed the potential of the method for the quality control of these agrochemical products. Degradation studies for carfentrazone-ethyl revealed that no significant degradation took place in cleaned sand samples while a significant but not stereoselective degradation took place in soils for the whole period of time considered (seven days).     

A Validated Chiral LC Method for the Separation and Quantification of (S,R,S)-Enantiomer and (R,R,R)-Isomer of Aprepitant

A new and accurate chiral liquid chromatographic method has been developed for the separation and quantification of (S,R,S)-enantiomer (unwanted enantiomer) and (R,R,R)-isomer (key intermediate) of aprepitant in bulk drug and formulation samples of apprepitant. The elution time was approximately 20 min using an immobilized amylose-based chiral stationary phase (Chiralpak-IA). The mobile phase was n-hexane and ethanol (90:10, v/v) and was delivered at a flow rate of 1.0 mL min^?1. Detection was carried out with a wavelength set to 220 nm. The resolution factor between enantiomers was found to be greater than five. Limit of detection for both (S,R,S) enantiomer and (R,R,R) isomer of aprepitant was 0.035 µg, and limit of quantification for both (S,R,S) enantiomer and (R,R,R) isomers of aprepitant was 0.1 µg, for a 10 µL injection. The developed method showed excellent linearity (r > 0.999) for both isomers. When the method was applied to bulk drug samples and in pharmaceutical formulations recoveries were obtained ranging from 97.2 to 103.1%. Aprepitant sample solutions were found to be stable when characterized over a period of 48 h.     

Enantiomeric Separation of Two Antiparkinsonian Drugs by Electrokinetic Chromatography Using Dextran Sulfate

Anionic polysaccharide dextran sulfate (DxS) was successfully employed as chiral selector for the enantioseparation of two antiparkinsonian drugs, including rotigotine and trihexyphenidyl (THP), by electrokinetic chromatography (EKC). The enantioseparation was performed under normal and reversed polarity modes and reversed enantiomer migration order was achieved under two modes. The parameters affecting the chiral separation, such as buffer pH, DxS concentration, organic additive, and temperature were investigated and optimized. Reversed polarity mode provided better separation for the two drugs. The optimized conditions for the enantioseparation under reversed polarity mode were 2.0% (w/v) DxS, 10 mM phosphate buffer, pH 2.5 with an applied voltage of ?30 kV at 25 °C. Direct UV detection was performed at 200 nm. Under the optimal conditions, rotigotine and THP enantiomers were enantioresolved in 40 min with the resolution of 2.0 and 5.8, respectively. The analytes could be enantioseparated using DxS of molecular mass 1,000,000 or 500,000. It was inferred that the electrostatic, hydrophobic, and steric interactions may be involved in the chiral separation mechanism in this study.     

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

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

A Validated Chiral LC Method for the Enantiomeric Separation of Cinacalcet Hydrochloride

A rapid isocratic chiral LC method has been developed for the separation of (S)-cinacalcet from (R)-cinacalcet. Good resolution with R _S  > 3 was obtained using a Chiralpak-IA column (250 × 4.6 mm, particle size 5 μm) and n-hexane, ethanol and trifluoroacetic acid as the mobile phase (95:5:0.1, v/v) at ambient temperature. Flow rate was kept at 1.0 mL min^–1 and elution was monitored by UV detection at 223 nm. This method was further used to determine the presence of (S)-cinacalcet in enantiopure pharmaceutical formulations containing (R)-cinacalcet. This method allowed for the detection and quantitation of (S)-cinacalcet of levels at 0.04 and 0.16 μg mL^–1, respectively. The method was validated following ICH guidelines.     

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

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

     

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane:2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL⁻¹ (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL⁻¹; those for its enantiomer were 0.70 and 2.34 μg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Enantiomeric resolution of albuterol sulfate by preferential crystallization

Albuterol is a β₂-adrenoceptor agonist prescribed for the treatment of bronchial asthma; it exists as a racemate and its bronchodilator activity resides in the (R)-isomer or levalbuterol. The aim of this study was to determine a methodology that would separate the enantiomers of albuterol by preferential crystallization after a conglomerate is identified within its derivatives. We found that albuterol sulfate behaves as a conglomerate showing the characteristic α_x-value = 2 (mole fraction solubility ratio of racemate vs enantiomer), the V-shaped ternary phase diagram and the preferential crystallization by seeding with the pure enantiomer. On the basis of these characteristics, we separated the enantiomers by entrainment, and crystallizing out a saturated methanolic solution of albuterol sulfate at 15 °C.     

A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R _s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min^?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL^?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

A Validated Stability Indicating LC Method for Mitotane in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of mitotane, its impurity in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved using a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at a wavelength of 230 nm. The mobile phase consisted of buffer and acetonitrile (25:75, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 2.5 by orthophosphoric acid. In the developed LC method the resolution (R _s ) between mitotane and its impurity namely Imp-1 was found to be greater than 2.5. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for mitotane and its impurity. This method was capable to detect the impurity of mitotane at a level of 0.003% with respect to test a concentration of 0.2 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for mitotane and its impurity was found to be within 2.0% RSD. The method has shown good and consistent recoveries for mitotane in bulk drugs (99.2–101.5%), pharmaceutical dosage forms (98.2–103.1%) and for its impurity (99.7–102.1%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in basic stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.97%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.