A Validated Stability-Indicating LC Method for Zonisamide

A simple, isocratic, rapid, and accurate reversed-phase high-performance liquid chromatographic method has been established for quantitative determination of zonisamide. The method is also applicable to determination of related substances in the bulk drug. Chromatographic separation was achieved on a 250 mm × 4.6 mm, 5-μm particle, C₁₈ column; the mobile phase was a 70:30 (v/v) mixture of 0.1% (v/v) aqueous triethylamine, adjusted to pH 2.5 with dilute orthophosphoric acid, and acetonitrile. Chromatographic resolution of zonisamide from its potential impurity, A, was found to be >2. The limits of detection and quantification of zonisamide and impurity A were 0.04 and 0.12 μg mL^?1, respectively, for 20 μL injection volume. Recovery of zonisamide ranged from 98.5 to 101.2% and recovery of impurity A from a sample of zonisamide ranged from 97.4 to 102.7%. The method was validated for linearity, accuracy, precision, and robustness.     

A Validated Stability-Indicating LC Method for Zonisamide

A simple, isocratic, rapid, and accurate reversed-phase high-performance liquid chromatographic method has been established for quantitative determination of zonisamide. The method is also applicable to determination of related substances in the bulk drug. Chromatographic separation was achieved on a 250 mm × 4.6 mm, 5-μm particle, C₁₈ column; the mobile phase was a 70:30 (v/v) mixture of 0.1% (v/v) aqueous triethylamine, adjusted to pH 2.5 with dilute orthophosphoric acid, and acetonitrile. Chromatographic resolution of zonisamide from its potential impurity, A, was found to be >2. The limits of detection and quantification of zonisamide and impurity A were 0.04 and 0.12 μg mL⁻¹, respectively, for 20 μL injection volume. Recovery of zonisamide ranged from 98.5 to 101.2% and recovery of impurity A from a sample of zonisamide ranged from 97.4 to 102.7%. The method was validated for linearity, accuracy, precision, and robustness.

     

A Validated and Stability-Indicating LC Assay Method for Valdecoxib

A gradient reversed-phase liquid chromatographic assay was developed for the quantitative determination of the non-steroidal anti-inflammatory drug valdecoxib. The developed method was also applicable to the determination of related substances in the bulk drug. Forced degradation studies were performed on bulk valdecoxib using acid (2.0 N hydrochloric acid), base (2.0 N sodium hydroxide), oxidation (6.0% v/v hydrogen peroxide), water hydrolysis, heat (60 °C) and photolysis. Mild degradation was observed using alkaline conditions and considerable degradation observed during oxidative stress. Chromatographic separation of process-related impurities and degradation products was achieved using a 5 micron Zorbax SB-CN LC column. The mobile phase consisted of aqueous potassium dihydrogen phosphate (pH 3.0) and acetonitrile. Stressed samples were assayed using the developed LC method and determination of the mass balance accounted for 99.5%, thus indicating the suitability of this stability-indicating method. Linearity, accuracy, precision and robustness have also been evaluated.     

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, Stability-Indicating, LC Method for Rabeprazole Sodium

We present a simple and reliable method for simultaneous determination of voriconazole and its main metabolite resulting from N-oxidation (UK-121,265), in human plasma. The work-up procedure used acetonitrile and potassium salts to precipitate plasma proteins. No internal standard was used. The chromatographic system used a LiChroCART^® 250-4 cartridge packed with LiChrospher^® 100 RP-8 (diameter particules, 5 μm). The UV monochromatic detector was set on 260 nm. The mobile phase consisted of a 60/40 (v/v) mixture of acetonitrile and water. The flow rate was 1 mL min^?1. The retention times for voriconazole and its metabolite were 8.98 and 4.02 min respectively, and total run time was 12 min. The linearity of the method was investigated from 0.31 to 10.0 mg L^?1; the lowest limit of quantification was 0.30 mg L^?1. Precision ranged from 2.41% to 6.32% for voriconazole and 0.80% to 11.6% for the N-oxide voriconazole metabolite. Accuracy was between 93.0% and 101% for voriconazole and 90.0% and 101% for the N-oxide voriconazole metabolite. This rapid and accurate method could be interesting to investigate metabolite/voriconazole ratio with respect to CYP2C19 genetic status and CYP3A4 activity changes.     

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 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.     

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.

     

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.     

Validated RP–LC Method for Standardization of Ashokarishta: A Polyherbal Formulation

A rapid LC-photodiode array method for the separation and identification of secondary plant metabolites especially phenolic compounds belonging to different types in a run (35 min) has been developed. The method has been optimized and validated using the selectivity, precision, recovery and robustness parameters with an aim for standardization of herbal drugs. Almost all the compounds have linearity with an R ² more than 98% and an RSD of less than 5% (in terms of variation of retention time). Inter-day and intra-day variability was also found to be less than 5%. The method thus developed has been successfully applied in identification and quantification of phenolic compounds present in polyherbal ayurvedic formulation (Ashokarishta). The results indicate that the method developed is rapid, accurate and robust for the analysis of different classes of phenolic compounds and can be successfully applied in the quality control and standardization of herbal drugs as well as polyherbal formulations.     

A Validated LC Method for Simultaneous Determination of Three Major Components in Entada phaseoloides

A high performance liquid chromatography method was developed for the simultaneous determination of three major active constituents in Entada phaseoloides, namely phaseoloidin (1), entadamide A (2), entadamide A-β-d-glucopyranoside (3), respectively. The samples were separated on an Aglient Eclipse XDB-C₁₈ column with gradient elution of acetonitrile and 0.3% phosphoric acid (v/v) at a flow rate of 1.0 mL min^?1 and detected at 280 nm. The three target compounds were completely separated within 10 min. All calibration curves showed good linearity (r > 0.9999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and RSD values were less than 1.04%. The recoveries were between 97.15 and 101.95%. The method was successfully applied to the analysis of three compounds in 22 commercial samples of E. phaseoloides. The results indicated that the developed LC assay was readily utilized as a quality control method for E. phaseoloides.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

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.

     

A Simple and Validated LC Method for the Simultaneous Determination of Three Compounds in Mikania laevigata Extracts

The Mikania genus is widely known as guaco and is used to treat fever, rheumatism, influenza and respiratory diseases. This article deals with the simultaneous quantification of three commercially available phenolic markers (o-coumaric acid, coumarin and syringaldehyde) in M. laevigata extracts, through LC-PDA. The validation data show that the method is specific, accurate, precise and robust, and also indicative of the stability of guaco extract. The method was linear, over a range of 1.25–20.0 μg mL⁻¹ for o-coumaric acid, 2.5–40.0 μg mL⁻¹ for coumarin, and 0.25–4.0 μg mL⁻¹ for syringaldehyde. The range of recovery was 94.3–96.4% for all the components, at a level of 100%.

     

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.     

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 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.