Stability-indicating Reversed-phase HPLC Method Research Articles

A Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation and In Vitro and In vivo Evaluation: Curcumin and Naringin Co-amorphous System

Curcumin (CUR) is a phytochemical widely used in food industries, cosmetics, and in the treatment of various ailments. It is a polyphenol derived from turmeric and is often considered the golden spice. CUR has a low solubility of less than 1 µg/ml and poor oral bioavailability which can be improved by co-amorphization with naringin (NRG). Analytical method to simultaneously quantify CUR and NRG is not reported in literature. This study aimed to develop a stability-indicating reverse phase HPLC method in gradient mode to simultaneously quantify CUR and NRG in co-amorphous system. The co-amorphous system of CUR and NRG in molar ratios 1:1 and 1:2 was prepared by quench cooling technique. The separation was attained on a Genesis C18, (4.6 mm × 150 mm, 4 µm) column with the mobile phase comprising of methanol and a 0.1% acetate buffer pH 3.8 at a single wavelength, 289 nm. CUR and NRG eluted at 5.1 and 11.1 min, respectively. For both the molecules, the linearity range was 0.125–16 µg/ml with LOD and LOQ of 0.063 and 0.125 µg/ml. The method developed was validated as per International Conference on Harmonization (ICH) guidelines for linearity, accuracy, precision, and robustness. The method was used to estimate CUR and NRG content in co-amorphous mixture and for in vitro evaluation.Graphical

Stability-Indicating Reversed Phase-HPLC Method Development and Validation for Estimation of Phenylephrine in Bulk and Tablet

Stability-Indicating Reversed Phase-HPLC Method Development and Validation for Estimation of Phenylephrine in Bulk and Tablet

A stability-indicating reverse phase-HPLC method development and validation for newly approved drug, Belzutifan in bulk and pharmaceutical dosage forms

A stability-indicating reverse phase-HPLC method development and validation for newly approved drug, Belzutifan in bulk and pharmaceutical dosage forms

Characterization of Degradation Products of Lifitegrast by Mass Spectrometry: Development and Validation of a Stability-indicating Reversed Phase HPLC Method

Lifitegrast API was subjected to forced degradation studies under various conditions of hydrolysis (acidic, alkaline, and neutral/water), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A short and simple reversed phase HPLC method was developed. The method was developed on a Shimadzu Ultra C18, (100 x 2.1) mm, 3.0 μm column. The gradient method consisted of 0.1% orthophosphoric acid as mobile phase A and mixture of acetonitrile and methanol in the ratio of 50:50 (v/v) as mobile phase B. The flow rate of the mobile phase was 0.8 mL/min. The developed method was validated using ICH Q2 (R1) guidelines. The parameters considered for method validation were solution stability, specificity, DL/QL, linearity, accuracy, precision and robustness. The drug showed significant degradation in acidic and alkaline conditions while slight degradation was observed in oxidative condition. The drug was found stable in water, photolytic and thermal conditions. The characterization of three major degradation products (DP1, DP2, and DP3) was done with LC-Q-TOF-MS/MS in combination with accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed on the basis fragmentation pattern.

A novel stability-indicating method for known and unknown impurities profiling for diltiazem hydrochloride pharmaceutical dosage form (tablets)

BackgroundA novel gradient, high-sensitive and specific stability-indicating reverse-phase HPLC method was developed and validated for quantitative purpose of known, unknown and degradant impurities profiling for diltiazem hydrochloride tablets. The impurities were separated on the Zorbax RX C8 column (150 mm × 4.6 mm, 5 μm) with mobile phase-A consisting of a mixture of 0.05 M sodium dihydrogen phosphate monohydrate buffer pH 3.0 and methanol in the ratio 800:200v/v and mobile phase-B consisting of acetonitrile with a flow rate of 1.0 mL min−1. The column compartment was maintained at 35 °C, and the detection wavelength was 240 nm. Diltiazem hydrochloride, its known impurities and unknown impurities have been well resolved from each other.ResultsThe linearity of the method has been demonstrated across the concentration range of 0.18 to 5.65 µg mL−1 for EP impurity-F with correlation coefficient R2 greater than 0.99. Recovery of method was proved from LOQ to 150% for known and unknown impurities with respect to test concentration and found in between 80 and 120%. Forced degradation study and specificity experiment results with mass balance proved the stability-indicating nature of the method and separated all known, unknown impurities and degradants from each other as well as from main drug component (diltiazem hydrochloride). The mass balance for stress study was found in between 95 and 105%.ConclusionNewly developed analytical method was validated as per ICH Q2 (R1) guidelines “Validation of analytical procedure” and found linear, accurate, specific, robust and precise in the established working range.

RP-HPLC stability-indicating method for simultaneous determination of sodium valproate, methylparaben and propylparaben in oral solution

Abstract A new, sensitive, stability-indicating reversed-phase HPLC method was validated and applied for the simultaneous quantitation of sodium valproate and two paraben preservatives; methylparaben, and propylparaben in the liquid dosage form. Stability tests were carried out through exposure of the analyte's solution to stress conditions. Separation of the analytes was achieved on (waters) C18 Column (150 mm × 3.9 mm, 5 μm). A mixture of 0.05 M monobasic potassium phosphate pH 3.5 and acetonitrile (50:50; v/v) was applied at 1.5 ml/min flow rate and UV detection wavelength at 210 nm. The degradation products and the analytes were completely separated. The linearity was performed in the range of 50–150 % from a target concentration of 10 μg/ml propylparaben, 90 μg/ml methylparaben, and 2.88 mg/ml sodium valproate with a coefficient correlation (R2) 1.0 for methylparaben, propylparaben and sodium valproate. The validation results of the suggested method were in a good agreement with ICH guidelines. Application of the proposed method for analysis of liquid dosage forms was successfully carried out in the routine quality control process.

A RP-HPLC Method for the Analysis of Neostigmine Methylsulfate and Process-Related Impurities, Forced Degradation Studies, in the Injection Formulation.

Neostigmine methylsulfate is an anticholinesterase agent and is clinically used for treating myasthenia gravis. It is also used for reversing nondepolarising neuromuscular blocking agents. Neostigmine methylsulfate may be administered by intravenous, intramuscular, or subcutaneous injection. In this research paper, a distinct stability-indicating reverse phase HPLC method was developed and validated for the quantitative determination of related impurities and degradation impurities in neostigmine methylsulfate API and injection formulation. The specific objective was to improve the resolution between European Pharmacopoeia listed impurity A and impurity B and degradation impurity of neostigmine methylsulfate API and injection formulation. The analysis was performed using Kromasil C18 column at 30°C of column oven temperature with phosphate-buffer/acetonitrile in a gradient mode. The RP-HPLC method was developed and validated for in-house neostigmine methylsulfate synthesis process sample and injection formulation. The injection formulation sample was studied for accelerated stability, temperature cycling stability, and photostability. The validation studies for neostigmine methylsulfate synthesis process API were studied using impurity A, impurity B, and impurity C. The analytical method validation parameters studied were specificity, precision, linearity, limit of detection, limit of quantitation, accuracy, and robustness. The API and the injection formulation were subjected to forced degradation under acid, alkali, oxidation, and photolytic and thermal conditions. The proposed method showed a significantly improved RRT (Relative Retention Time) of impurity A and impurity B with a resolution greater than 1.5. The developed method eliminates the use of an ion-pairing agent and thereby a good performance of column was established.

Development and Validation of a Stability-Indicating HPLC Method for Assay of Milbemycin Oxime and Estimation of Its Related Compounds

A stability-indicating reversed-phase HPLC method for assay of milbemycin oxime (MO) and estimation of its related compounds has been developed and validated as per VICH and ICH method validation guidelines. The stability-indicating capability of this method has been demonstrated by adequate separation of all process-related impurities including all potential degradation products of milbemycin oxime (MO) that were generated using stress degradation conditions that are prescribed in ICH guideline. The stability-indicating capabilities of this method were further challenged by analyzing aged stability samples of MO active pharmaceutical ingredient (API). Chromatographic separation of MO and its related compounds was achieved using an isocratic elution at a flow rate of 0.5 mL/min on a Supelco Ascentis Express C18 column (100 mm × 3.0 mm, 2.7 µm particle size, 90 A pore size) maintained at 50 °C. The isocratic mobile phase was composed of 30% v/v of 0.05% phosphoric acid in aqueous solution and 70% v/v of a mixture of methanol and acetonitrile (6:4, v/v). UV detection at 244 nm was employed to monitor the analytes.

Validation of Stability-Indicating Reverse Phase HPLC Method for the Determination of Related Substances in Dapagliflozin Drug Substance

Validation of Stability-Indicating Reverse Phase HPLC Method for the Determination of Related Substances in Dapagliflozin Drug Substance

Quantitative determination of clobetasone butyrate in bulk and cream formulation by a validated stability-indicating reversed-phase HPLC method

A simple isocratic reversed-phase HPLC method for quantification of clobetasone in bulk and cream dosage forms has been developed. Chromatographic analysis was accomplished on an C18 column utilizing a mixture of methanol and water (84:16 v:v, pH = 6.0) as mobile phase. An effluent flow rate of 1 mL/min was adjusted and the detection was made at 240 nm wavelength. The method was evaluated according to ICH guidelines Q2 R1 for linearity, specificity, sensitivity, precision and accuracy. The method exhibited good linearity with correlation coefficient (r2) of 0.9993 over the concentration range from 5 to 50 μg/mL. The recoveries of the test drug from the cream sample was found to be 98.56 to 99.51% and the limit of detection and quantification were calculated as 0.85 and 2.83 μg/mL, respectively, suggesting the accuracy and sensitivity of the developed method. The precision was demonstrated by a low percentage of relative standard deviation (<1%) from six independent assay analysis performed for the cream formulation. Stability indicating property of the proposed method was demonstrated by performing the analysis of forced degradation samples. The developed method can be used for estimation of the clobetasone butyrate in bulk and pharmaceutical formulations for routine analysis in the quality control laboratories.

Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate

ABSTRACTAbiraterone acetate is a prodrug of Abiraterone widely used for the treatment of metastatic castration resistant prostate cancer. In this study, a simple, sensitive, and rapid stability-indicating reverse phase HPLC method was developed and validated for the determination of Abiraterone acetate in bulk and its pharmaceutical formulation. The method was developed by HPLC using a Hypersil ODS C-18 (150 mm × 4.6 mm, 5 µm) column in a isocratic mode with mobile phase constituted by potassium phosphate buffer and acetonitrile (40:60, v/v%) flow rate was 1.0 mL min−1, column temperature of 30°C, UV detection wavelength 235 nm, and injection volume of 20 µL. The validated parameters were in accordance with FDA and ICH specifications, assay exhibited a linear range of 25–250 µg mL−1 with regression (r2) coefficient 0.9998. The limits of detection and quantification were 0.23 and 0.70 µg mL. Accuracy was between 99.34 and 100.07%. The drug was subjected to various stress conditions like acidic, base hydrolysis, oxidation, thermal, and photolytic degradation. Stress study Abiraterone acetate was found susceptible to degrade under hydrolytic (acid and base) conditions. The proposed method has stability indicating the resolution of the main peak from their degradation peaks. The validated method is suitable for quality control application and reduced analysis time.

A stability indicating RP-HPLC assay method for simultaneous estimation of abacavir, Lamivudine, Nevirapine and Zidovudine in pharmaceutical dosage form

A stability-indicating reverse phase HPLC method has been developed and validated for the simultaneous Assay estimation of Abacavir, Lamivudine, Nevirapine and Zidovudine in pharmaceutical dosage form. The chromatographic separation was achieved on a simple Inertsil ODS C18, 250 x 4.6 mm, 5 µm (Make: GL Sciences). The method employed a simple isocratic elution mode with a ternary mixture of mobile phase. The detection wavelength was set at 270 nm. The proposed method was extensively validated as per ICH guidelines. The specificity of the method was proved by performing forced degradation studies along with peak purity. This method is found to be simple, fast and economical Hence this validated method can be used in routine quality testing of individual dosage forms and combination dosage forms of Abacavir, Lamivudine, Nevirapine and Zidovudine.

A stability-indicating HPLC method for simultaneous determination of morphine and naltrexone

This study developed a stability-indicating reversed-phase HPLC method for the simultaneous determination of morphine sulfate and naltrexone hydrochloride content in bulk, Solid dosage forms and in-vitro dissolution samples to support product development and quality control efforts. Chromatographic separation of the pharmaceutical compound was achieved on a perfectSil™ MZ C18 column (250×4.6mm, 5μm) with an isocratic mobile phase composed of a mixture of acetate buffer (10mM, pH 4, containing 0.1% of 1-heptanesulfonic acid sodium salt) and acetonitrile with 80/20 at a flow rate of 1.5mlmin(-1). Both analytes were quantified using a photodiode array detector set at a wavelength of 280nm and column temperature was set to 30°C. naltrexone, morphine and a mixture of the two were subjected to thermal, peroxide, acid, base and photolytic degradation and their peak homogeneity was obtained using a photodiode array detector, demonstrating the specificity of method. These pharmaceuticals were spiked in biological fluid to examine method selectivity. The method was validated for system suitability, linearity, accuracy, precision, detection and quantification limits and robustness and was found it is acceptable in range of 2-250μgml(-1) for morphine and 4-100μgml(-1) for naltrexone.

Stability-indicating reversed-phase HPLC method development and characterization of impurities in vortioxetine utilizing LC–MS, IR and NMR

The current study reports the development and validation of a stability-indicating reversed phase HPLC method for the separation and identification of potential impurities in vortioxetine, a recently developed antidepressant. The structures of a new compound and four process-related impurities formed during the synthesis were characterized and confirmed by NMR, MS, and IR spectroscopy analyses. The most probable formation mechanisms of the impurities identified were proposed. Based on the characterization data, the new compound was proposed to be 1-[4-[(2,4-dimethylphenyl)thio]phenyl]-piperazine. In addition, an efficient chromatographic method was optimized to separate and quantify the impurities, which were obtained in the 0.05–0.75μg/mL range. The developed HPLC method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantitation.

Development and validation of a stability-indicating reversed phase HPLC method for the quality control of Zolpidem in bulk and tablet dosage forms

An isocratic stability-indicating reversed phase liquid chromatography (RP-HPLC) method for quantitative determination of Zolpidem in the presence of its stressed degradation products in bulk and commercial tablets was developed and validated. Forced degradation studies were carried out on bulk samples and tablet dosage forms of Zolpidem using acid, base, H2O2, heat, and UV light as described by International Conference on Harmonization (ICH) for stress conditions to demonstrate the stability indicating power of the method. A Perfectsil® Target ODS column (3–5 μm, 125 × 4 mm) was used for chromatographic separation. The mobile phase consisted of methanol-50 mM ammonium acetate buffer (pH 3.7, 40: 60, v/v). The flow rate was 0.7 mL/min and UV detection was optimized at 254 nm. The present method linearity for Zolpidem was investigated in the range of 1–20 μg/mL (r = 0.9998). The limits of detection (LOD) and quantification (LOQ) were 400 and 1000 ng/mL respectively. The method selectivity was evaluated by peak purity test using a diode array detector. There was no interference with detection of Zolpidem and its stressed degradation products.

A New Simultaneous Determination of Rosuvastatin Calcium and its Lactone Impurity by Reverse Phase HPLC method

A reliable, sensitive and stability-indicating reverse phase HPLC method was developed for the determination of Rosuvastatin calcium and its lactone impurity in drug substance and pharmaceutical dosage form. Rosuvastatin calcium was fourth highest selling drug in the United States, accounting approximately $5.2 billion in the year of 2013, which is used for the treatment of hypercholesterolemia. The resolution between Rosuvastatin and lactone impurity was good with resolution factors more than 10.0. The chromatographic separation was achieved on a sunfire column C18 (250 x 4.6 mm, 5 μm) with mobile phase containing a gradient mixture of solvent-A (10 mM ammonium acetate) and solvent-B (acetonitrile: methanol (50: 50 v/v)). The eluted compounds were monitored at 242 nm and the total run time was 15 minutes. Degradation behavior of the Rosuvastatin calcium was studied under various degradation stress conditions. The Limit of detection and limit of quantitation of lactone impurity was found to be 0.01μg/mL and 0.04μg/mL, respectively, for 10μL injection volume. The sample solution and mobile phase were stable for at least 48 hours. The proposed accurate method can be useful for quantification of Rosuvastatin calcium and its lactone impurity in the bulk drug substance and also dosage form.

A novel validated stability indicating high performance liquid chromatographic method for estimation of degradation behavior of ciprofloxacin and tinidazole in solid oral dosage

Objective:The objective of current investigation was to study the degradation behavior of Ciprofloxacin and Tinidazole. The study was performed as per International Conference on Harmonization recommended stress condition. A novel stability-indicating reverse phase HPLC method was developed for the determination of Ciprofloxacin and Tinidazole purity in the presence of its impurities and forced degradation products. This method is also capable to separate placebo peaks as well in pharmaceutical dosage forms. The solid oral dosage form was subjected to the stress conditions such as oxidative, acid, base hydrolysis, heat and photolytic degradation.Materials and Methods:The method was developed using Waters symmetry shield, Reverse Phase (RP) C18, 250mm x 4.6mm, 5μ as a stationary phase. The mobile phase containing a gradient mixture of solvent A and B. 10mM phosphate buffer, adjusted pH 3.0 with phosphoric acid was used as a buffer. Buffer pH 3.0 was used as solvent A and buffer pH 3.0: Acetonitrile in the ratio of 20: 80 v/v were used as solvent B. The eluted compounds were monitored 278 nm (Ciprofloxacin), 317 nm (Tinidazole). The run time was 50 minute.Results:In the precision study the % RSD for the result of Ciprofloxacin, Tinidazole and its impurities was below 10%. The method was linear with the correlation coefficient greater than 0.997. The percentage recoveries were calculated and observed from 93.0% to 106.7%. The peak purity of Ciprofloxacin, Tinidazole peak had not shown any flag, thus proved the stability-indicating power of the method.Conclusion:The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.

Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in Combination by Using Validated RP-HPLC Method

A fixed-dose combination of atorvastatin and aspirin is widely used for the treatment of myocardial infarction. The present work describes a comprehensive study of the stress degradation behavior of atorvastatin and aspirin alone as well as in combination of 1:1 and 1:7.5 ratios, respectively, as per ICH guidelines. The degradation products of aspirin as well as atorvastatin were successfully separated by a developed simple, selective, and precise stability-indicating reversed-phase HPLC method. Chromatographic separation was achieved on the Phenomenex Luna analytical column, 150 mm × 4.6 mm, 5μm. The mobile phase consisted of 0.1% glacial acetic acid in water and acetonitrile in the ratio of 50:50 v/v at a flow rate of 1.0 ml/min. UV detection was performed at 246 nm. The extent of degradation was significantly influenced when both of the drugs were present in combination. Stress degradation behavior of atorvastatin was highly influenced by aspirin under acid hydrolysis, thermal degradation, and oxidative stress conditions. Similarly, the stress degradation behavior of aspirin was affected by atorvastatin especially under neutral hydrolysis, thermal degradation, and oxidative stress conditions. Additionally, the combination ratio of aspirin and atorvastatin also influenced the percentage degradation of each other. A mixture of aspirin and atorvastatin was also analyzed after a one-month stability study at 40 °C and 75% RH. All the results indicate chemical incompatibility of both aspirin and atorvastatin if present in combination.

STABILITY-INDICATING HPLC METHOD FOR DETERMINATION OF OFLOXACIN IN BULK DRUG AND TABLETS USING TRIFLUOROACETATE AS COUNTER ANIONS

A stability-indicating reversed-phase HPLC method for the quantitative determination of ofloxacin in bulk samples and tablet formulation has been developed and validated. The separation was performed on a C18 column (4.6 × 250 mm, 5 µm) using an isocratic mobile phase comprising of acetonitrile and buffer pH 3.3 (16: 84, v/v) with a diode array UV/Vis detection at 294 nm. The buffer was composed of ammonium acetate (50 mM) and ion-pairing reagent, trifluoroacetic acid (20 mM). Forced degradation studies were performed on bulk sample as per ICH prescribed stress conditions using acid hydrolysis, alkaline hydrolysis, neutral hydrolysis, oxidative, thermal stress, and photolytic degradation to show the stability indicating power of the method. The proposed method provided linear responses (r2=0.9999) over the concentration range of 10–30 μg mL−1. LOD and LOQ values for the active substance were 0.13 and 0.45 μg mL−1, respectively. The method validation showed good results for specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, and robustness. The proposed method was successfully employed for quantification of ofloxacin in tablets.

Separation and Quantification of Octahydro-1h-Indole-2-Carboxilic Acid and Its Three Isomers by HPLC using Refractive Index Detector

Octahydro-1H-indole-2-carboxylic acid is a key starting material for the synthesis of Perindopril and Trandolapril. A rapid, economical, simple, sensitive and reliable stability-indicating reverse phase HPLC method developed and validated for the quantitative determination of all isomers related to Octahydro-1H-indole-2-carboxylic acid and its related substances. The compound is non chromophoric, it has three chiral centers and there is a possibility of four pairs of enantiomers. Refractive index detector was used for the quantification of all its isomers. Optimized mobile phase is 10 mM potassium phosphate buffer with pH-3.0. The C18 column (Inertsil ODS-4, 250 mm×4.6 mm×5 μm) is used as the stationary phase with a mobile phase flow rate of 1.5 mLmin–1 and column temperature maintained at 35°C. The developed method was validated as per ICH guidelines for Accuracy, linearity, range, precision, ruggedness, robustness, solution stability, limit of quantification, and limit of detection. A linear range from LOQ to 150% performed for the analyte and its three isomers. The correlation coefficient obtained for all the isomers was more than 0.999. The recoveries obtained for all isomers were found in between 93.9% and 107.9%. The detection limit for all the isomers were about 0.006 mg/mL and the quantification limits were in between 0.022 mg/mL to 0.024 mg/mL respectively. The proposed method can be successfully applied for the quantification of the three isomers in Octahydro-1H-indole-2-carboxylic acid and provides a simple and cost effective quality control tool for routine analysis.