A validated chiral LC method for the enantiomeric separation of AT13148 on polysaccharide-based chiral stationary phase

A sensitive and accurate LC method for the determination of AT13148 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.8 was accomplished within 15 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane: 2-propanol: diethylamine (85:15:0.1, v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 254 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 7 and 500 μg mL^?1 (n = 11), and the recoveries between 98.24 and 100.99% were obtained, with relative standard deviation lower than 1.32%. LOD and LOQ for AT13148 were 2.46 and 7.38 μg mL^?1 and for its enantiomer were 2.54 and 7.49 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AT13148, especially for the analysis of bulk samples.     

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.     

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 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.

     

An Improved and Validated LC Method for Resolution of Panthenol

An improved LC method was developed and validated for determination of enantiomeric purity of panthenol in bulk drugs. The method is based on derivatization of panthenol with 3,5-dinitrobenzoyl chloride. Baseline separation with resolution >2.7 was achieved within 20 min on Kromasil CHI-DMB (250 × 4.6 mm) column using n-hexane:ethanol (95:5 v/v) as mobile phase at a flow rate of 1.5 mL min^?1. The analytes were detected by their UV absorbance at 265 nm. The effects of ethanol, 2-propanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was extensively validated and proved to be robust. The recoveries were between 98.3 and 101.4% with <1.6% relative standard deviation. The regression equations for the derivatives of d-panthenol and l-panthenol were y ₁ = 18.01x ₁ ? 32.56 (r ₁ ²  = 0.9984) and y ₂ = 17.855x ₂ ? 28.16 (r ₂ ²  = 0.9990), respectively. The LOD and LOQ for the derivative of d-panthenol were 10.6 and 37.4 μg mL^?1 and for the derivative of l-panthenol were 12.1 and 40 μg mL^?1, respectively. The improved method was found to be simple, rapid, and sensitive for the determination of enantiomeric purity of panthenol in bulk drugs.     

Reversed-Phase Liquid Chromatographic Method for Determination of Daclatasvir Dihydrochloride and Study of Its Degradation Behavior

A simple and selective reversed-phase stability-indicating liquid chromatographic method has been developed and validated for the determination of daclatasvir in drug substance and drug product. Daclatasvir was subjected to acidic, alkaline, oxidative, thermal and photo-degradation study. The LC method was based on isocratic elution of daclatasvir and its degradation products on a reversed-phase C₁₈ Hypersil column using a mobile phase consisting of phosphate buffer (10 mM, 1 mL triethylamine L^?1): acetonitrile (60:40 v/v) at a flow rate of 2 mL min^?1. Quantitation was achieved with UV detection at 312 nm. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 0.75–120 μg mL^?1, with regression coefficient value of 0.9999, and with limit of detection and quantitation of 0.148 and 0.447 μg mL^?1, respectively. Peak purity was checked for principle drug and its alkali induced degradation product, and the pathway of alkaline hydrolysis of daclatasvir was suggested by LC/MS.     

Simultaneous Determination of Clobetasol Propionate and Calcipotriol in a Novel Fixed Dose Emulgel Formulation by LC-UV

A new, rapid, selective, cheap and simple RP-LC method has been developed and validated for the simultaneous determination of clobetasol propionate and calcipotriol mixtures in bulk drugs (raw materials) and in a novel-fixed dose emulgel formulation. Separation was carried out using a NovaPak C18 column with methanol:water (74:26 v/v) as mobile phase for isocratic elution at a flow rate of 1.0 mL min^?1. The column temperature was set at 25 °C. Calibration curves were established ranging between 0.5 and 20 μg mL^?1 and 0.5 and 10 μg mL^?1 for clobetasol propionate and calcipotriol, respectively. Limit of detection and limit of quantification values of the method was found as 0.16 and 0.48 μg mL^?1 for clobetasol propionate and 0.10 and 0.30 μg mL^?1 for calcipotriol, respectively. The method was validated in accordance with ICH guidelines and obtained results proved that the proposed method was precise, accurate, selective and sensitive for the simultaneous analysis of clobetasol propionate and calcipotriol. The proposed method can be easily applied for the simultaneous determination of clobetasol propionate and calcipotriol in prepared emulgel formulations. The obtained validation results showed that the RP-LC method is suitable for routine quantification of clobetasol propionate and calcipotriol in emulgel formulations with high precision and accuracy.     

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 Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL^?1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL^?1 and a limit of quantitation at 0.02 μg mL^?1, respectively.     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL^?1 of ENL, 0.260–399 μg mL^?1 of HCZ for HPLC system and 0.270–399 μg mL^?1 of ENL and 0.065–249 μg mL^?1 of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL^?1 and 31.477 ng mL^?1 for HCZ, 2.804 ng mL^?1 for ENL and 2.943 ng mL^?1 for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.     

Ion-Pair LC Method for Simultaneous Determination of Aliskiren Hemifumarate,Amlodipine Besylate and Hydrochlorothiazide in Pharmaceuticals

A rapid and precise LC method was developed for the simultaneous determination of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) using acetonitrile:25 mM octane sulfonic acid sodium salt monohydrate in water (60:40 v/v) as the mobile phase. The flow rate was maintained at 1.2 mL min^?1 on a stationary phase composed of Supelco, Discovery^® HS (C18) column (25 cm × 4.6 mm, 5 μm). Isocratic elution was applied throughout the analysis. Detection was carried out at λ _max (232 nm) at ambient temperature. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 32–320, 2–44 and 4–64 μg mL^?1 for ALS, AML and HCZ, respectively. LOD and LOQ were estimated and found to be 0.855 and 2.951 μg mL^?1, respectively, for ALS, 0.061 and 0.202 μg mL^?1, respectively, for AML as well as 0.052 and 0.174 μg mL^?1, respectively, for HCZ. The method was successfully applied for the determination of the three drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in pharmaceutical preparations.     

Stability-Indicating UPLC Method for the Determination of Bisoprolol Fumarate and Hydrochlorothiazide: Application to Dosage Forms and Biological Sample

A sensitive, selective and accurate ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate and hydrochlorothiazide in their combined dosage forms and as well as in spiked human urine samples. The separation was achieved on an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column, at 40 °C with mobile phase consisting of acetonitrile:phosphate buffer (20 mM) at pH 3.0 with a gradient elution at 225 nm. Bisoprolol fumarate and hydrochlorothiazide were well separated in <1.5 min with good resolution and without any tailing and interference of excipients. The method was fully validated according to ICH guidelines in terms of accuracy, precision, linearity and specificity. A linear response was observed over the concentration range 0.5–150 μg mL^?1 for hydrochlorothiazide and 0.5–250 μg mL^?1 for bisoprolol fumarate. Limit of detection and limit of quantitation for hydrochlorothiazide were calculated as 0.01 and 0.03 μg mL^?1, respectively, and for bisoprolol fumarate were 0.07 and 0.21 μg mL^?1, respectively. Moreover, bisoprolol fumarate and hydrochlorothiazide were subjected to degradation conditions such as hydrolytic, oxidative and thermal stress conditions to evaluate the ability of the proposed method for the separation of bisoprolol fumarate and hydrochlorothiazide from their degradation compounds.     

Development and Validation of an Accurate LC Method for the Quantitative Determination of Voriconazole in a New Emulsion Formulation

A simple, sensitive and accurate liquid chromatographic method with UV detection was developed and validated to determine voriconazole in a new emulsion formulation. Chromatographic separation was achieved on a Diamonsil C₁₈ column (250 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile-water-acetic acid (40:60:0.25, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection wavelength was set at 256 nm. The linear calibration curves were obtained in the concentration range of 1.00–100 μg mL^?1 with the limit of quantification of 1.00 μg mL^?1. The within- and between-run precisions in terms of percentage relative standard deviation were lower than 7.4 and 7.1%, respectively. The accuracy in terms of percentage relative error ranged from ?1.5 to 1.4%. This validated method was successfully applied to the determination of the content of voriconazole in a new emulsion formulation.     

Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations

A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracetamol in tablets and in microsphere formulations has been developed and validated. The mobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UV detection was at 276 nm. The method was linear over the concentration ranges 16–24 and 80–120 μg mL^?1 for aceclofenac and paracetamol, respectively, with recovery in the range 100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and 0.1120 μg mL^?1, respectively; those for PCM were 0.0631 and 0.1911 μg mL^?1, respectively.     

Rapid Determination of Polyphenols in Tobacco by MLC

A rapid, simple and reliable analytical procedure has been developed for the determination of polyphenols, including chlorogenic acid, rutin and scopoletin, in tobacco samples. After extraction, the polyphenols were analyzed by micellar liquid chromatography coupled with UV detection at 340 nm without sample preparation step. The compositions of the micellar mobile phase for the separation of these polyphenols were selected adequately by a chemometric method. The optimal resolution and satisfactory peak shapes were achieved with 0.022 mol L^?1 sodium dodecyl sulfate and 0.45% (volume fraction) 1-propanol solution at pH 5.0 using Eclipse XDB-C18 (250 mm × 3.0 mm, 5 μm) column. The limits of detection for chlorogenic acid, rutin and scopoletin were 0.15, 0.29 and 0.22 μg mL^?1, respectively. This validated method was successfully applied to qualitatively and quantitatively analyze the polyphenols in tobacco in less than 10 min.     

Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL^?1 for ascorbic acid, 0.084–0.250 mg mL^?1 for chlorogenic acid, 0.053–0.360 mg mL^?1 for caffeic acid, and 0.016–0.250 mg mL^?1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL^?1 for ascorbic acid, 18 and 54 μg mL^?1 for chlorogenic acid, 37 and 112 μg mL^?1 for caffeic acid, and 11 and 34 μg mL^?1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.     

Simultaneous Determination of Benzodiazepines and Ketamine from Alcoholic and Nonalcoholic Beverages by GC-MS in Drug Facilitated Crimes

A GC-MS method with HP-5MS capillary column was developed for the simultaneous determination of underivatized flunitrazepam, clonazepam, alprazolam, diazepam and ketamine from drinks by extraction with chloroform: isopropanol 1:1 (v/v). All linearity ranges were between 50 and 1,000 μg mL^?1 for all compounds both in beer and in peach juice. Limit of detection was between 1.3 and 34.2 μg mL^?1, limit of quantification was between 3.9 and 103.8 μg mL^?1, the range of recoveries was 73.0 and 112.6% for all drugs in both beverages. The reported method was sensitive, rapid, and suitable for the analysis of the spiked drinks as evidence of sexual assault and robbery phenomena.     

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.