Stress Degradation Studies Research Articles

STABILITY INDICATING RP-HPLC METHOD FOR SEPARATION AND QUANTIFICATION OF RELATED SUBSTANCES OF TICLOPIDINE IN BULK AND PHARMACEUTICAL FORMULATIONS

A simple, specific, accurate and stable reverse phase liquid chromatographic method was developed for the simultaneous determination of ticlopidine and its related impurities A and B in bulk drug and tablet dosage forms. The analysis has been performed on XTerra C18 column (250 mm×4.6 mm; 5 µ id) and mobile phase containing of methanol and pH 6.8 phosphate buffer in the ratio of 80:20 (V/V). The detection was carried at 228 nm with a flow rate of 1.0 mL/min. The retention times were found to be 8.9, 5.98 and 4.62 min for ticlopidine, impurities A and B, respectively. The method was validated according to ICH guidelines. The method was validated for specificity, precision, linearity, accuracy and robustness. The linearity range of 50-200 µg/mL for ticlopidine and 0.5-2.0 µg/mL for impurity A and B. The recoveries of ticlopidine and impurities were found to be within the range of 98-102 and the % RSD in each spiked level was found to be less than 2. The stress degradation studies confirmed that the method was effectively separate the degradation products and impurities formed in the stress studies and hence the method was found to be stability indicating method. The method can effectively quantify the standard drug ticlopidine and its impurities A and B in bulk drug and pharmaceutical formulations.

STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ANTIDIABETIC DRUGS, DAPAGLIFLOZIN AND SAXAGLIPTIN

For simultaneous measurement of Dapagliflozin (DAPA) and Saxagliptin (SAXA) in combination, as well as quantification of their major degradation products, a simple reversed-phase high-performance liquid chromatographic technique was devised and validated. The separation was accomplished using an ODS C18 column (250 mm 4.6 mm, i.d.5 m) with isocratic elution at room temperature. The optimum mobile phase consists of an aqueous phase (sodium acetate buffer, pH 4) and an organic phase (acetonitrile: methanol, 40:40) at a ratio of (20:80). The flow rate was set to 1.0 ml min-1, and effluents were measured using a 228 nm diode array detector. This method showed good linearity over a range of 2.0-12 μg ml-1 and 1-6 μg ml-1 for DAPA and SAXA respectively. The retention times of samples were 2.31 and 2.90 minutes for DAPA and SAXA respectively. According to the International Conference Harmonization (ICH) criteria, the drugs in combination were submitted to different stress degradation studies. The suggested approach is appropriate for stability investigations, according to the results of the stress degradation experiments. The proposed method was simple, rapid and precise for the study of a new formulation that has been approved for the cure of diabetes mellitus.

VALIDATED AND QUANTIFIED STABILITY INDICATING STRESS DEGRADATION STUDY OF ORAL ANTI-DIABETIC AGENT CANAGLIFLOZIN BY RP-HPLC METHOD

Objective: The present investigation is aimed to develop and validate, a simple, consistent and sensitive stability-indicating reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of oral anti-diabetic drug Canagliflozin in bulk and pharmaceutical dosage form as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH-Q2 (R1)). Methods: The chromatographic separation was achieved by using Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) ZORBAX C18 (250 mm x 4.6 mm, 5μm particle size) with a mobile phase consisting of Acetonitrile: Water in a ratio of 53:47% v/v at a flow rate of 1 ml/min with an injection volume of 20 μl. Results: The Retention time of the drug Canagliflozin was found to be 2.36±0.05 min and detected at 214 nm UV wavelength. The linear regression equation was found to be y = 60702x–2156.2 with a correlation coefficient 0.9999. Stress degradation studies were performed by exposing the Canagliflozin into acidic, alkaline, oxidative, thermal and photolytic stress conditions with active samples withdrawn at different time intervals as per ICH guidelines. Conclusion: The proposed Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was found to be robust, precise and specific for estimation of Canagliflozin in pharmaceutical dosage forms.

Rapid aggregation of therapeutic monoclonal antibodies by bubbling induced air/liquid interfacial and agitation stress at different conditions

Degradation of therapeutic monoclonal antibodies (mAb) due to interfacial agitation through air bubbling was investigated. Samples containing mAb in phosphate buffered saline were subjected to rapid bubbling by using a peristaltic pump at an air flow rate of 11.5 mL/min. Samples were analyzed by visual observation, UV–Vis, fluorescence, circular dichroism and infrared spectroscopy, size-exclusion chromatography (SEC), dynamic light scattering, microscopy, and cell-based activity assays. The stressed samples showed increasing turbidity with bubbling time, with mAb1 showing a protein loss of 53% in the supernatant at the latest time point (240 min), indicating formation of sub-visible and visible aggregates. Aggregate rich samples exhibited altered secondary structure and higher hydrophobicity with 40% reduction in activity. The supernatants of the stressed samples showed unchanged secondary and tertiary structure without the presence of any oligomers in SEC. Furthermore, the impact of various factors that could affect aggregation was investigated and it was found that the extent of aggregation was affected by protein concentration, sample volume, presence of surfactants, temperature, air flow rate, and presence of silicone oil. In conclusion, exposure to air/liquid interfacial stress through bubbling into liquid mAb samples effectively generated sub-visible and visible aggregates, making air bubbling an attractive approach for interfacial stress degradation studies of mAbs.

Development and Validation of a Stability-Indicating HPTLC Method for the Estimation of Eszopiclone in Pharmaceutical Dosage Forms

Objective: A simple, sensitive, selective, precise repeatable and stability-indicating high-performance thin layer chromatographic method was developed and validated for Eszopiclone in bulk drug and in formulation. Method: Silica gel 60 F-254, TLC precoated aluminium plates was used as the stationary phase for analyzing Eszopiclone and its degradation products, using mobile phase consisting toluene: ethyl acetate: methanol (6: 4: 2 v/v/v). Result: This mobile phase gave compact spots for Eszopiclone with Rf value of 0.52 ± 0.02. Eszopiclone was exposed to hydrolysis, oxidation, neutral and photolytic conditions for conducting stress degradation study. The peak of Eszopiclone and the degradation product was well resolved from each other with a significantly different Rf value. Densitometric estimation of Eszopiclone was performed at 304nm. A good linear plot was obtained in the concentration range of 150-300ng/spot. The method was validated for precision, accuracy (recovery) and robustness study. The limit of detection (LOD) and limit of quantitation (LOQ) was found to be 130ng/spot and 150ng/spot, respectively. Conclusion: The developed HPTLC method can separate Eszopiclone from its degradation products, hence stability studies can be performed using this method.

Development and validation of a new stability indicating RP-UFLC method for the estimation of Bosentan

A new stability-indicating RP-UFLC method has been developed for the estimation of Bosentan in pharmaceutical dosage forms and the method was validated. Bosentan is used to lower the high blood pressure in lungs (pulmonary arterial hypertension). Bosentan acts by blocking the actions of endothelin -1and thereby lowers the blood pressure. Mobile phase mixture consisting of sodium acetate (pH 5.0) buffer solution and acetonitrile (50: 50 v/v) with flow rate 0.7 mL/min were the optimized chromatographic conditions (Detection wavelength 254 nm) for the present study. Linearity was observed in the concentration range of 0.1–100 μg/mL (R2 = 0.9998) with regression equation y = 126698 x – 392.49. The LOQ was found to be 0.08964 µg/mL and the LOD was found to be 0.02913 µg/mL. Stress degradation studies such as acidic, alkaline, oxidation, photolysis and thermal degradations were performed by exposing Bosentan and finally the proposed method was validated as per ICH guidelines. The assay of Bosentan was conducted by applying the proposed method to the marketed formulations. The proposed method is simple, specific, precise, and accurate and can be applied for the estimation of pharmaceutical formulations.

A stability-indicating HPLC method for estimation of doxylamine succinate in tablets and characterization of its major alkaline stress degradation product

BackgroundA new selective rapid RP-HPLC-DAD method was developed and evaluated for the quantification of doxylamine succinate (DOX) in bulk and pharmaceutical dosage form. The separation of DOX at different degradation conditions was achieved with a Kromasil C18 (4.6 × 250 mm, 5-μm particle size). The mobile phase employed comprised of phosphate buffer (pH 3.5) and methanol in the ratio of 45:55 v/v. The flow rate was kept maintained at 1.0 ml/min and eluents were detected at 262 nm. The drug was subjected to different stress conditions like acid, base, neutral, hydrolysis, oxidation, photolysis, and thermal degradation. The analytical performance of the proposed HPLC method was thoroughly validated in terms of linearity, precision, accuracy, specificity, robustness, detection, and quantification limits.ResultsThe method produces linear responses that were found in the range of 10–50 μg/ml. The regression equation was found to be Y = 42984x − 10260. The correlation coefficient was found to be 0.9998. The LOD and LOQ for DOX were found to be 0.96 and 3.28 μg/ml, respectively. The short-term solution stability of DOX (100 μg/ml) was evaluated under (25 ± 2°C) storage condition and found to be 98.82 to 101%. The percentage recovery for DOX was in the range of 99.73 to 99.91%. The obtained results of the stress degradation study and peak purity data indicate the potential of the developed HPLC method to resolve degradants from DOX peak. The major alkaline degradation product was isolated using preparative chromatographic technique and extensive FT-IR was performed to ascertain the structure of the alkaline degradant.ConclusionIt was concluded that the proposed method was simple, sensitive, accurate, cost-effective, and less time-consuming for the quantification of DOX. This method was successfully utilized for stability testing of commercially available DOX tablets. Hence, the proposed method can be applied for routine quality control of DOX in bulk drug as well as in marketed formulations.

DEVELOPMENT AND VALIDATION OF UV SPECTROSCOPIC METHOD OF PALIPERIDONE PALMITATE IN BULK AND ITS APPLICATION FOR DRUG LOADING AND ENTRAPMENT EFFICIENCY ANALYSIS OF LONG-ACTING INJECTABLE MICROSPHERES

Objective: The objective of the current study was to develop simple, precise and cost-effective ultraviolet (UV) spectroscopic method for estimation of paliperidone palmitate (PP) as a bulk and in the polymeric depot dosage form.
 Methods: UV spectrophotometer with quartz sample cells and spectral manager software was used for analysis samples. 20 % Methanol in Mili-Q-Water was used as a solvent for the sample and standard preparations. Method validation was carried out as per ICH Q2 (R1) guidelines. Stress degradation studies were carried out to check the stability-indicating performance of the developed method. The validated method was applied for the estimation of the PP content of long-acting injectable microspheres of PP.
 Results: The wavelength of maximum absorption was found to be 283 nm. The proposed method was validated in the range of 5-30 µg/ml of PP. The mean recovery for 80%, 100% and 120% standard solution was found to be 99.03%, 99.21% and 99.35% respectively with less than 2% relative standard deviation (RSD). Intra-day and inter-day method precision ranged from 0.54 to 1.62% and 0.67 and 1.24% respectively. Limit of detection (LOD) and limit of quantitation (LOQ) were estimated to be 0.03 and 0.10 µg/ml respectively. The developed method was found to be robust and rugged. Stress degradation studies were carried out under acidic, alkaline, hydrolytic and oxidative and photo stress. The proposed method was capable to detect changes in assay due to stress conditions.
 Conclusion: The developed method was successfully employed for routine analysis of drug loading in long-acting injectable microspheres of PP.

Method Development, Validation and Stress Degradation Study of Teneligliptin by RP-HPLC

Method Development, Validation and Stress Degradation Study of Teneligliptin by RP-HPLC

Design of Copper Microfabricated Potentiometric Sensor for in‐line Monitoring of Neostigmine Degradation Kinetics

AbstractThe quality of pharmaceutical products is critical for human health. Drug development requires tools to assess the presence of degradation products and contaminants during the manufacturing and storage processes. Accelerated stress degradation and kinetic studies play a vital role in predicting final product stability. This work describes the design of potentiometric sensor based on copper microfabricated electrodes for in‐line tracking the degradation kinetics of neostigmine. The proposed electrochemical technique provides a continuous profile for the hydrolysis of NEO under different temperatures and pH. The hydrolysis activation energy was found to be 18.88 kcal mol−1 which was aligned with the reported hydrolysable ester values. Consequently, the kinetic data analysis is crucial to predict the optimum analysis and storage conditions.

Characterization of degradation products of celiprolol hydrochloride using hyphenated mass and NMR techniques

Stress degradation studies were carried out on celiprolol hydrochloride under the ICH prescribed hydrolysis (acidic, basic and neutral), photolytic, oxidative and thermal conditions. Maximum degradation was observed upon hydrolysis, especially in the basic condition. In oxidative condition, the drug degraded only upon severe exposure to H2O2, but it remained stable when challenged with AIBN. It also degraded significantly under photolytic conditions. However, the drug was stable to thermal stress. A total of seven degradation products were formed, whose separation was successfully achieved on an Inertsil ODS-3V C-18 HPLC column employing a gradient mobile phase. A comprehensive mass fragmentation pattern of the drug was initially established through the support of high resolution mass spectrometry (HR-MS), multi-stage tandem mass spectrometry (MSn) and on-line H/D exchange MS data. The same approach was then extended to characterization of the degradation products. Additionally, two degradation products were isolated and subjected to 1D/2D NMR studies for their structural confirmation. One of the degradation products showed instability during isolation, therefore, it was subjected to LC-NMR studies for its structural confirmation.

Optimization and Validation of Rp-Hplc Stability-Indicating Method for Determination of Efavirenz and its Degradation Products

Stability is considered one of the most important criteria in pharmaceutical quality control. With this objective a stability-indicating high performance liquid chromatographic (HPLC) method has been established for analysis of Efavirenz in the presence of the degradation products generated in the stress degradation study. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal decomposition. Extensive degradation was found to occur in alkaline medium and under thermal stress. Minimum degradation was observed under acidic medium, in the photolytic conditions and oxidative stress. Successful separation of drug from degradation products formed under stress conditions was achieved on a C-8 column using acetonitrile: potassium dihydrogen phosphate (pH 2.9, 25mM) - (60:40% v/v) as the mobile phase. The flow rate was 1 mL min -1 and the detector was set at in a range of wavelength be- tween 220nm to 390nm. The method was validated for linearity, range, precision, accuracy, limit of quantification and limit of detection. Because the method effectively separates the drugs from their degradation products, it can be used as stability-indicating method.

Comparison of static and dynamic mode in the electrochemical oxidation of fesoterodine with the use of experimental design approach

Electrochemical conversion of fesoterodine to one of its oxidation products was evaluated with the application of the wall-jet flow cell. A traditional, “static” mode of electrolysis was compared with the “dynamic” mode of cell performance. For statistical assessment of the data, experiments were planned and performed with the application of design of experiments approach, namely Taguchi L18 design. After screening phase, the experimental settings were broadened or adjusted according to the results and optimization was performed. All of the samples were electrolysed with the use of chronoamperometric method in a three electrode system. The electrolysed samples were analysed using UHPLC-PDA-QDA method. The chromatographic run was performed in gradient elution with the application of C8 column. The response was expressed as % area of the main peak found with the PDA detection method whereas QDA detector was used in positive SIM mode for structural confirmation. All data obtained for both screening and optimization were treated together and linear models were adjusted. The use of large-surface glassy carbon electrode along with pH~7 were found to be the most significant factors influencing electrochemical oxidation of fesoterodine in both modes. The major differences were identified in terms of voltage applied to the electrodes which yielded the highest amounts of oxidation product. Evolution of electrochemical methods may serve as complementary technique in stress degradation studies in pharmaceutical industry.

Quality-by-Design Based Development and Validation of Stability Indicating Method by UPLC Method for Impurities of Simvastatin from Drug and Pharmaceutical Dosage Form

A rapid, robust and accurate ultra-high performance liquid chromatographic method was developed and validated for determination of impurities of simvastatin in drug and its pharmaceutical formulation. A systematic Quality-by-design approach was used for method development with the Fusion AETM software, to screen and optimize the column, mobile phase, column temperature, gradient time and other chromatographic conditions. The optimized method uses Waters Acquity Charged Surface Hybrid, Octadecylsilane C18 (1.7μm×2.1 mm×100 mm) column with gradient elution. Orthophosphoric acid pH was adjusted to 4.5 with triethylamine and acetonitrile with a ratio of (80:20) was used as mobile phase A, whereas methanol:acetonitrile (20:80) as mobile phase B, with a flow rate of 0.35 ml/min. Ultra Violet detector was programmed at 238 nm with a runtime of 13 min, wherein all the impurities were well resolved and were separated from main peak of simvastatin. The method was validated for accuracy, repeatability, reproducibility and robustness. All the validation parameters were in acceptable range. The linearity, limit of detection and limit of quantitation was established for all the impurities and for simvastatin. The method was also applied suitably for determining the degradation products of simvastatin using stress degradation studies

Stress degradation study of bortezomib: effect of co-solvent, isolation and characterization of degradation products by UHPLC-Q-TOF-MS/MS and NMR and evaluation of the toxicity of the degradation products

Bortezomib (BTZ) is a first-in-class, potent reversible inhibitor of proteasome used in the treatment of multiple myeloma, the second most common hematological cancer.

A novel rp-hplc method for determination of esomeprazole in pharmaceutical dosage forms in human plasma: A pilot stress degradation study

Esomeprazole, an proton pump inhibitor used for the remedy of acid reflux disorders including healing and maintenance of erosive esophagitis, gastroesophageal reflux disease and peptic ulcer disease. It is utilized in a prolonged treatment of pathological hypersecretory conditions such as Zollinger -Ellison syndrome. In this study, a novel RP-HPLC method was developed for the determination of esomeprazole both in API and tablets (Esmoz (40mg)), Phenomenex Luna, 5μm C18 100 A° LC column (250X4.6mm) was used at 40°C. Isocratic elution was utilized with acetonitrile: water (96:4v/v) mixture. The flow rate was 1.0mL min-1 and UV detection was at 270nm. The RT value was observed at 3.713 minutes respectively. The developed method was validated according to ICH guidelines and found to be linear within the range of 100-500μg/mL. In similar lines, linearity was achieved by spiking esomeprazole in human plasma within the range of 20-80μg/mL. The developed method was accurate, precise, robust, and rapid. Hence it can be applied for both stability studies and routine quality control analyses.

Method Development, Validation and Stability Indicating Studies for Simultaneous Estimation of Anti-Hypertensive Drugs from Pharmaceutical Formulation by RP-HPLC

Objective: Method development, validation & stability indicating studies for simultaneous estimation of Anti-Hypertensive drugs, Benidepine (BEN) and Metoprolol (MET) from pharmaceutical formulation by RP-HPLC.
 Methods: For present work, reverse phase chromatography was selected as its suggested use for ionic and moderate to non-polar compounds. Reverse phase chromatography is simple, suitable, better regarding efficiency, stability, and reproducibility. C18 packed column, a 100 X 2.1mm. ID column of 5.0 μm particle packing, was selected for separation of BEN and MET. Different solvent systems were tried and optimized in combinations as mobile phase. BEN (4 μg/ml) and MET (50 μg/ml) in 15mM ammonium formate-Methanol (15:85 v/v) was developed as it was showing good peak shapes and a significant amount of resolution. The mobile phase was flowed at 1.2 ml/min with detection of BEN analytes at 236 nm and MET analytes at 225 nm respectively.
 Result: Method development was done. Specificity, linearity, accuracy, precision, robustness, limit of detection and limit of quantitation were used to accomplish validation. The method was found linear from 32.5 – 500 µg.ml-1 for both BEN and MET individually. The percentage recovery of BEN when placed for period of 12 hours was found to 100% in 0.1N/M NaOH at 60˚C and Thermal (60˚C); 12 % degradation in 0.1N/M HCl at 60˚C; Oxidation (3-6% H2O2) at room temperature whereas for MET was 100 % in 0.1N/M NaOH, 0.1N/M HCl at 60˚C, at thermal (60˚C) as well as oxidation by 3-6% H2O2 at room temperature. 
 Conclusion: Developed analytical method for the simultaneous estimation of Benidipine (BED) and Metoprolol (MET) in both bulk and tablet formulation has obliged the ICH guidelines including, tailing factor (T), separation factors (α), theoretical plates (N), capacity factor (k’), resolution (R) and RSD (%). The validated stress degradation studies under thermal, oxidative, alkali and acid ascertained few degradation products for Benidipine whereas the Metoprolol was unaffected with forced degradation studies.
 Keywords: Benidipine, Metoprolol, Reverse-Phase High Performance Liquid Chromatography, Stability indicating method.

Stability Indicating HPTLC Method for Sofosbuvir and Daclatasvir in Combination

Direct acting antiviral agents represent the major advance in treatment of hepatitis C virus (HCV) infection. Daclatasvir with sofosbuvir that are co-administrated once per day oral dose has been reported to achieve a high rate of virological response in patients with HCV genotype 1, 2 or 3. So, the basic objective of a research involved development and validation of stability indicating HPTLC Method for simultaneous estimation of Sofosbuvir and Daclatasvir available in market in the form of combination tablet. Samples were applied on HPTLC aluminium plates precoated with silica gel 60 F254 (250μm thickness). Mobile phase consists of ethyl acetate: isopropanol in the ratio 9:1v/v. A good resolution was observed between peaks of both the drugs. The retention factor for Sofosbuvir is about 0.51 ±0.02. And for Daclatasvir is about 0.30 ± 0.02. Deuterium lamp as a source of radiation at the wavelength of 260 and 318 nm for analysis of Sofosbuvir and Daclatasvir, respectively. The proposed method was validated according to ICH guidelines and the results were acceptable for linearity and range, accuracy, precision, robustness, detection limit and quantitation limit. The calibration curves were linear over a wide range of 200-1000 ng/band (r2= 0.991) for Sofosbuvir and 45-225 ng/band (r2=0.990) for Daclatasvir. The limit of detection was found to be 21.17 ng/band and 4.38 ng/band for SOF and DAC and limit of quantitation was 64.18 ng/band and 13.28 ng/band for SOF and DAC respectively. During stress degradation study, it was observed that the Daclatasvir is degraded less in thermal and photolytic condition but more in basic hydrolysis condition. Sofosbuvir was found to be sensitive to all stress conditions except the fluorescent light. The suggested method was successfully applied for analysis of both drugs and excellent recovery results were obtained. Being simple, fast, robust, and economic, the method could be applied to the quality control and routine stability monitoring of Sofosbuvir and Daclatasvir.

A New HPTLC Method for Analysis of Artemisinin Derivatives (Artemether and Lumefantrine) in Bulk Drug and Liposomal Formulation: Stress Degradation Study

A New HPTLC Method for Analysis of Artemisinin Derivatives (Artemether and Lumefantrine) in Bulk Drug and Liposomal Formulation: Stress Degradation Study

Review on Chemistry, Analysis and Pharmacology of Teneligliptin: A Novel DPP-4 Inhibitor.

This article provides comprehensive and collective facts about teneligliptin. Teneligliptin is a dipeptide peptidase-4 (DPP-4) inhibitor that belongs to the third generation, used in the management of type 2 diabetes. It inhibits human DPP-4 enzyme activity. This drug falls under class 3; it interacts with S1, S2, and S2E extensive sub-sites. Teneligliptin and its metabolites are mainly determined in the human plasma matrix by hyphenated chromatographic methods. These developed methods could be foreseen for their clinical applications. Moreover, the stress degradation studies of Teneligliptin under different stress conditions provide an insight into degradation pathways and help in the elucidation of the structure of the degradation products by liquid mass spectroscopy. These methods are also used for routine quality control analysis of teneligliptin in pharmaceutical dosage forms.