A Validated Chiral LC Method for the Determination of Enantiomeric Purity of Pemetrexed Disodium on an Amylose-Based Chiral Stationary Phase

A simple and new isocratic normal phase chiral HPLC method has been developed for the determination of enantiomeric purity of pemetrexed disodium (l-enantiomer) in bulk drugs with a short run time of about 20 min. Chromatographic separation of l and d-enantiomers of pemetrexed disodium was achieved on an amylose based chiral stationary phase using a mobile phase consists of hexane, ethanol and trifluoro acetic acid. The resolution between the enantiomers was found to be more than 2.0. The system precision and method precision were found to be within 5% RSD for the distomer (d-enantiomer) at its specification level (i.e. not more than 1.0% w/w). The limit of detection and limit of quantification of distomer were 1.6 and 5 μg mL⁻¹, respectively for 10 μL injection volume. The percentage recovery of distomer was ranged from 90.6 to 105.7 in bulk drug samples. The test solution was found to be stable in the diluent for 48 h. The method was found to be specific for the enantiomers of pemetrexed disodium and can be conveniently used for the quantification of undesired d-enantiomer present in the bulk drug samples of pemetrexed disodium.     

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (R_s > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.     

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 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 Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

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.     

Direct Enantiomeric Purity Determination of Acetyl-L-carnitine by LC with a Ligand-Exchange Chiral Stationary Phase

A direct HPLC method for the determination of acetyl-D-carnitine in acetyl-L-carnitine was investigated. The enantiomers were successfully separated on a SUMICHIRAL OA-6100 column, which has a ligand-exchange type of chiral moiety. The mobile phase consisted of an aqueous solution of copper (II) sulfate and sodium perchlorate. Successful enantioseparation seems to be achieved through the formation of not only the complex with copper (II) ion but also by ion-pairing with the perchlorate ion, because no enantioseparation was observed with the usual copper (II) mobile phase alone. Employing 2 mM aqueous CuSO₄ solution containing 500 mM NaClO₄, the enantiomers of acetylcarnitine eluted in the order of D- and L-forms within 15 min with R _s = 1.92 and α = 1.11. The obtained LOQ and LOD values were 0.15 and 0.1%, respectively. The validated results were satisfactory for a practical quality control method for the enantiomeric purity determination of acetyl-L-carnitine.     

Validated Chiral LC Method for the Enantiomeric Separation of Palonosetron Hydrochloride

A new and accurate chiral liquid chromatographic method has been developed for the separation of palonosetron hydrochloride (PALO) and its (R,R)-enantiomer in bulk drug samples with an elution time of about 20 min. The chromatographic separation was carried out by normal phase chromatography using an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with a mobile phase composed of n-hexane:ethanol:1,4 dioxane:trifluoroacetic acid:diethylamine (65:30:5:0.3:0.3, v/v) pumped at a flow rate of 1.0 mL min⁻¹. The resolution (R _s ) between the enantiomers was found to be greater than 3.0 and interestingly the (R,R)-enantiomer was eluted prior to the (S,S)-enantiomer (PALO) in the developed method. Mobile phase additives, trifluoroacetic acid and diethylamine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. The limit of detection (LOD) and limit of quantification (LOQ) of the (R,R)-enantiomer were found to be 0.03 and 0.1 μg respectively for 10 μL injection volume. The developed method shows excellent linearity (r ² > 0.999) over a range of LOQ to 0.3% for the (R,R)-enantiomer. The percentage recovery of the (R,R)-enantiomer in bulk drug samples ranged from 97.2 to 102.3 revealing good sensitivity of the developed method. Robustness studies were also carried out on the developed method.

     

A Validated chiral LC Method for the Enantiomeric Separation of Galantamine

A rapid isocratic chiral HPLC method has been developed for the separation of R-galantamine from S-galantamine. Good resolution viz. Rs > 3 between R- and S- forms of galantamine was achieved with chiralpak AD-H (250 × 4.6 mm) column using n-hexane, 20% propionic acid in isopropanol and diethyl amine in the ratio of 80:20:0.2 (v/v) as mobile phase at ambient temperature. Flow rate was kept as 0.8 mL min⁻¹ and the elution was monitored at 289 nm. This method was further used to determine the amount of R-galantamine in pure and pharmaceutical formulations of S-galantamine. This method is capable to detect and quantitate R-galantamine to the levels of 0.21 and 0.84 μg mL⁻¹, respectively. The method was validated as per International Conference on Harmonization (ICH) guidelines in terms of limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, specificity, robustness and ruggedness.     

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.     

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.

     

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.     

在多糖衍生物类色谱柱上手性拆分乙炔基氮杂环丙烷类化合物

在以正己烷－异丙醇为移动相的体系中，用ＣｈｉｒａｌｐａｋＡＤ和ＣｈｉｒａｌｃｅｌＯＤ作为手性固定相对１５种乙炔基氮杂环丙烷类化合物对映体进行了ＨＰＬＣ手性拆分。这些化合物至少在一支柱上能基线级分离。     

核磁共振技术测定联萘酚的对映体纯度

手性联萘酚((±)-BINOL)制备是国内高校常开设的一个实验,其产品的对映体纯度测试是实验的重要一环。以2-甲酰基苯硼酸和(S)-(?)-1-苯乙胺的混合物作为手性试剂与联萘酚发生Bull-James Assembly反应,以此设计了核磁共振氢谱(¹H NMR)测定联萘酚对映体纯度的实验。实验结果表明,通过反应生成的对映体混合物的核磁信号,能准确地计算出(±)-BINOL的对映体纯度。以选定的苯环氢核信号计算出的(R)-BINOL含量与理论ee值有着良好的线性关系(R²=0.9999)。此实验方法能够快速完成大量学生样品的测量,同时大大减少实验废液的产生量。学生通过此实验可对核磁共振技术有更进一步的了解。     

A Validated LC Method for Determination of Trace Impurities in Technical Triadimefon

A simple and sensitive liquid chromatography with ultraviolet detection (LC?CUV) method was developed for the determination of three impurities with a content over 0.1% (w/w) in technical triadimefon. A Gemini C₁₈ column (5 ??m, 250 mm × 4.6 mm i.d.) was used for the chromatographic separations. The samples were separated by gradient elution with water (solvent A) and methanol (solvent B) using the following conditions: 70% A isocratic for 12 min, linear to 0% A within 8 min, and isocratic for 10 min at 0% A with a flow rate of 1.0 mL min^?1. Chromatograms were recorded at an absorption wavelength of 280 nm. The chromatographic resolutions between triadimefon and its potential impurities A, B, and C were greater than 3. The developed LC method was validated with respect to linearity, accuracy, precision, and robustness. This method was successfully applied to analyze the impurities in commercial technical triadimefon. In addition, the structures of the three impurities were identified to be (A) 4-chlorophenol, (B) 1-(2,4-dichlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, and (C) 1,1-bis(4-chlorophenoxy)-3,3-dimethyl-2-butanone.