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SUMMARY Aim: A new stability-indicating liquid chromatography method was developed and validated for the quantitative determination of luliconazole. Materials and methods: Preliminary forced degradation study demonstrated an additional peak of the degradation product at the same retention time to the drug, due to this, the method was developed optimizing the chromatographic conditions to provide sufficient peak resolution (R ≥ 2). The experimental design was evaluated to assess the robustness and the best chromatographic conditions to be used for the validation. Methodology: Luliconazole solutions were exposed to various stress conditions to evaluate the method indication stability, in which the degradation product (DP-1) formed was isolated, identified, and evaluated in silico to predict degradation pathway and toxicity. The procedure was validated by robustness, selectivity, linearity, precision, and accuracy. Liquid chromatography was performed in a Phenomenex® RP-18 column with a mixture of acetonitrile and 0.3% (v/v) triethylamine solution as a mobile phase in isocratic elution. Results and conclusions: The method demonstrated robustness, good recovery, precision, linear response over a range from 5.0 to 40.0 μg.mL-1- and to be stability indicating. The alkaline stress condition resulted in the formation of DP-1. HRMS studies identified this product as an hydroxyacetamide derivative, and in silico studies did not show toxic potential.
RESUMEN Objetivo: Un nuevo método indicativo de estabilidad por cromatografía líquida fue desarrollado y validado para la determinación cuantitativa de luliconazol. Materiales y métodos: Estudios preliminares de degradación forzada demostraron un pico adicional en el mismo tiempo de retención del fármaco. El método desarrollado para optimizar las condiciones cromatográicas proporcionó una adecuada resolución (R ≥ 2). El diseño experimental fue evaluado para verificar su robustez y la mejor condición cromatográica para validación. Metodología: Las soluciones de luliconazol fueron expuestas a diferentes condiciones de estrés para evaluar la indicación de estabilidad del método, el aislamiento del producto de degradación formado (DP-1), su identificación y análisis in silico para predecir su ruta de degradación y toxicidad. El procedimiento se validó por robustez, selectividad, linealidad, precisión y exactitud. Las condiciones cromatográficas incluyeron una columna Phenomenex® RP-18, como fase móvil una mezcla de acetonitrilo y solución 0,3% (v/v) de trietilamina en elución isocrática. Resultados y conclusiones: El método mostró ser robusto, con buena recuperación, precisión, respuesta lineal en el rango de 5,0 a 40,0 μg.mL-1 e indicativo de la estabilidad. La condición de estrés alcalina resultó en la formación de DP-1. Estudios por HRMS identificaron este producto como un derivado hidroxiacetamida y los estudios in silico no mostraron potencial de toxicidad.
RESUMO Objetivo: Um novo método indicativo de estabilidade por cromatograia líquida foi desenvolvido e validado para a determinação quantitativa de luliconazol. Materiais e métodos: Estudos preliminares de degradação forçada demonstraram um pico adicional no mesmo tempo de retenção do medicamento. O método desenvolvido para otimizar as condições cromatográficas proporcionou resolução adequada (R ≥ 2). O delineamento experimental foi avaliado para verificar sua robustez e a melhor condição cromatográica para validação. Metodologia: Soluções de luliconazol foram expostas a diferentes condições de estresse para avaliar a indicação da estabilidade do método, o isolamento do produto de degradação formado (DP-1), sua identificação e análise in silico para predizer sua rota de degradação e toxicidade. O procedimento foi validado quanto à robustez, seletividade, linearidade, precisão e exatidão. As condições cromatográficas incluíram uma coluna Phenomenex® RP-18, como fase móvel uma mistura de acetonitrila e solução de trietilamina 0,3% (v/v) em eluição isocrática. Resultados e conclusões: O método mostrou-se robusto, com boa recuperação, precisão, resposta linear na faixa de 5,0 a 40,0 μg.mL-1 e indicativo de estabilidade. A condição de estresse alcalino resultou na formação de DP-1. Os estudos da HRMS identificaram este produto como um derivado da hidroxiacetamida e os estudos in silico não mostraram nenhum potencial de toxicidade.
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The discovery of new direct-acting antiviral drugs gave rise to a leap forward in the treatment of hepatitis C viral infections. For the first time since 1998, the Food and Drug Administration (FDA) approved interferon-free oral treatment paradigms. Among the new treatment regimens, the combinations of Sofosbuvir and Velpatasvir became ideal treatment regimens for being potent, highly tolerated and used once daily. Hence an accurate, precise, selective and sensitive stability indicating method for simultaneous estimation of Sofosbuvir and Velpatasvir by High-Performance Thin Layer Chromatography has been developed and validated. Chromatographic separation was achieved on TLC plates coated with silica gel 60 F254 as stationary phase. Ethyl acetate: iso-propyl alcohol (9:1 v/v) was used as mobile phase.Densitometric scanning was carried out at 260 and 302 nm for Sofosbuvir and Velpatasvir, respectively. The method was successfully validated as per the ICH Guideline. The linear concentration range was100- 2000 ng/band (r2= 0.991) and 100-500 ng/band (r2 = 0.991) for SOF (Sofosbuvir) and VEL (Velpatasvir) respectively. The LOD were 25.16 ng/band and 9.96 ng/band for SOF and VEL, LOQ were 76.25 ng/band and 30.19 ng/band for SOF and VEL.The method could be applied to the quality control and routine analysis of Sofosbuvir and Velpatasvir in their pure forms and pharmaceutical formulations.
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Objective::To study the degradation of salvianolate lyophilized injection (SLI) and establish a stability-indicating analysis method. Method::UPLC-Q-TOF-MS/MS was used to conduct a qualitative study on the main components of SLI, and a stability-indicating analysis method was established for simultaneous determination of the original components of SLI and its degradation products. The stability of SLI were systematically assessed under physicochemical conditions of high temperature, oxidation, metal ions. Result::Totally 13 main active ingredients in SLI were identified, and a semi-quantitative analysis was performed. Under the conditions of high temperature, oxidation, light, trivalent ion and divalent ion, 6, 4, 3, 4 and 1 new degradation products were added respectively. The established stability-indicating analysis method can simultaneously determine the degradation products of the main components and their active components in SLI, with a good separation effect. Conclusion::According to the degradation mechanism of the main ingredients in SLI, macromolecular polyphenol acid compounds are degraded into small molecular compounds, such as tanshinol and protocatechu aldehyde by a series of reactions, like benzofuran open-loop, hydrolysis of ester bond and removal of DSS. The stability-indicating analysis method can be used for the stability quality control of traditional Chinese medicine Salvianolate Lyophilized Injection (SLI).
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We developed and validated a stability-indicating assay method for the simultaneous determination of enrofloxacin and piroxicam in combination and in the presence of degradation products. Reverse-phase high-performance liquid chromatography analyses were carried out on a Vertisep C18 column and acetonitrile-water (48:52 v/v, pH 3.0) mobile phase with a 1.00 mL min−1 flow rate. The efficient chromatographic separation of these drugs and their forced degradation products was achieved in less than 5min with a peak purity match factor higher than 950. The method used showed linearity in the concentration ranges of 0.25 to 16.0 µg mL−1 for enrofloxacin (r = 0.9997) and 0.125 to 8.0 µg mL−1 for piroxicam (r = 0.9999) as well as precision (relative standard deviation lower than 2%), accuracy (mean recovery 100 ± 2%), and robustness, according to ICH (International Conference on Harmonization) and AOAC (Association of Official Analytical Chemists) guidelines. This method can simultaneously determine the combination of these drugs in a veterinary formulation and separate the drug peaks from their forced degradation products. Additionally, its optimized chromatographic conditions can contribute to the quality control of this formulation in pharmaceutical manufacturing plants and minimize waste from the organic solvent.
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Objective: To develop and validate simple, sensitive stability indicating HPTLC (High performance thin layer chromatography) method for apixaban. Methods: The chromatographic separation was performed on aluminium plates precoated with silica gel 60 F254 using toluene: ethyl acetate: methanol (3:6:1 v/v/v) as mobile phase followed by densitometric scanning at 279 nm. Results: The chromatographic condition shows sharp peak of apixaban at Rf value of 0.38±0.03. Stress testing was carried out according to international conference on harmonization (ICH)Q1A (R2) guidelines and the method was validated as per ICH Q2(R1) guidelines. The calibration curve was found to be linear in the concentration range of 100-500 ng/band for apixaban. The limit of detection and quantification was found to be 11.66ng/bandand35.33ng/band, respectively. Conclusion: A new simple, sensitive, stability indicating high performance thin layer chromatographic (HPTLC) method has been developed and validated for the determination of apixaban.
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Standard parenteral nutrition solutions are mixtures comprising interacting components that may de-grade themselves over time. The objective of this study was to investigate the physicochemical and microbiological stability of a hospital preparation for parenteral nutrition in neonatology. The analyses were performed throughout the storage of the preparations at 2–8 °C (up to 4 months). The extent of stability was based on the determination of amino acids dosage, visual and physicochemical properties (glucose and electrolytes concentrations, pH and osmolality measurements, particle counting) and mi-crobiological analysis (sterility test). A thermal degradation of ascorbic acid was conducted to evaluate the antioxidant properties of the parenteral mixture. Physicochemical and microbiological controls were found to comply with the specifications. Amino acids showed a good stability throughout the 4months storage except for cysteine, which was progressively degraded to cystine, conferring a yellow coloration to parenteral solutions. Parenteral nutrition standards solutions remain stable for 4 months at 2–8 °C, ensuring safe administration in preterm infants.
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ABSTRACT A stability indicating HPLC method to determine diltiazem hydrochloride (DTZ) in tablets and compounded capsules was developed and validated according to Brazilian and the International Conference on Harmonization (ICH) guidelines. The separation was carried out on a Purospher Star® C18 (150 x 4.6 mm i.d., 5 µm particle size, Merck Millipore) analytical column. The mobile phase consisted of a 0.05% (v/v) trifluoroacetic acid aqueous solution and a 0.05% trifluoroacetic acid methanolic solution (44:56, v/v). The flow rate was 1.0 mL.min-1 with a run time of 14 minutes. The detection of DTZ and degradation products (DP) was performed at 240 nm, using a diode array detector. The method proved to be linear, precise, accurate, selective, and robust, and was adequate for stability studies and routine quality control analyses of DTZ in tablets and compounded capsules.
Subject(s)
Diltiazem/therapeutic use , Chromatography, High Pressure Liquid/methods , Validation Study , Tablets/pharmacology , Capsules/pharmacologyABSTRACT
A new institutional clinical trial assessed the improvement of sleep disorders in 40 children with autism treated by immediate-release melatonin formulation in different regimens (0.5 mg, 2 mg, and 6 mg daily) for one month. The objectives of present study were to (i) prepare low-dose melatonin hard capsules for pediatric use controlled by two complementary methods and (ii) carry out a stability study in order to determine a use-by-date. Validation of preparation process was claimed as ascertained by mass uniformity of hard capsules. Multicomponent analysis by attenuated total reflectance Fourier transformed infrared (ATR-FTIR) of melatonin/microcrystalline cellulose mixture allowed to identify and quantify relative content of active pharmaceutical ingredients and excipients. Absolute melatonin content analysis by high performance liquid chromatography in 0.5 mg and 6 mg melatonin capsules was 93.6% ± 4.1% and 98.7% ± 6.9% of theoretical value, respectively. Forced degradation study showed a good separation of melatonin and its degradation products. The capability of the method was 15, confirming a risk of false negative < 0.01%. Stability test and dissolution test were compliant over 18 months of storage with European Pharmacopoeia. Preparation of melatonin hard capsules was completed manually and melatonin in hard capsules was stable for 18 months, in spite of low doses of active ingredient. ATR-FTIR offers a real alternative to HPLC for quality control of high-dose melatonin hard capsules before the release of clinical batches.
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A novel stability-indicating Reverse Phase High Pressure Liquid Chromatography (RP-HPLC-PDA) method was developed and validated for quantitative determination of Camptothecin (CPT) in bulk, formulation and in dissolution samples using Inertsil-C18 (250mm x 4.6mm, 5μm) column with mobile phase combination of 15mM Ammonium acetate and acetonitrile (60:40) at a flow rate of 1mL/min. Eluents were monitored at a wavelength of 254 nm with an injection volume of 20µL. CPT was completely degraded in oxidative and base hydrolysis conditions and around 37% in acidic conditions and no degradation of CPT was observed with thermal, thermal/humidity and photo conditions. CPT showed linearity over a concentration range of 2-10μg/mL with a regression coefficient (R2) of 0.994 and correlation coefficient (R) of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) values for CPT were 0.025μg/mL and 0.077μg/mL respectively. The developed method was validated as per ICH guidelines. The method was also successfully applied to dissolution testing of controlled release formulation.
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En este trabajo, fue desarrollado y validado un método indicador de estabilidad por cromatografía líquida, para ser aplicado al estudio cinético de Rosuvastatina Cálcica en diferentes valores de pH y temperatura. Las condiciones cromatográficas seleccionadas fueron: columna C18, 50 x 4.6 mm y 3,5 µm de tamaño de partícula; fase móvil MeOH: Agua-0.1%TFA, temperatura de la columna de la columna 25 ° C, y velocidad de flujo 1 mL/min. El método validado presentó una adecuada repetibilidad y precisión intermedia y una recuperación superior al 98%. Por otra parte, el método fue lineal en el rango de 10 a 150 ppm. En condiciones ácidas, fueron identificados tres posibles productos de degradación como: Rosuvastatina Lactona, Rosuvastatina Anti-isómero y Rosuvastatina Lactona Anti-isómero y en condiciones de degradación con la luz se identificaron dos posibles productos mayoritarios. El método validado puede ser empleado en estudios de estabilidad y de degradación cinética del fármaco.
In this work, a liquid chromatography stability-indicating method was development and validated to be applied at study the hydrolytic behavior of Rosuvastatin Calcium in different pH values and temperatures. The selected chromatographic conditions were a column C18 50 x 4.6 mm and 3.5 µm. The mobile phase was Methanol: Water-1%TFA; 25°C column temperature and flow rate 1 mL/min. The validation method exhibited an adequate repeatability and intermediate precision and a recovery higher than 98%. Furthermore, the method was lineal in range of 10 to 150 ppm. Under acidic conditions, three degradation products possible were identified as Rosuvastatin lactone, Rosuvastatin anti-isomer and Rosuvastatin lactone anti-isomer, with light were identified two major decomposition products. The validated method can be used in the stability studies and drug degradation kinetics.
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Forced degradation is a degradation of new drug substance and drug product at conditions more severe than accelerated conditions. It is required to demonstrate specificity of stability indicating methods and also provides an insight into degradation pathways and degradation products of the drug substance and helps in elucidation of the structure of the degradation products. Forced degradation studies show the chemical behavior of the molecule which in turn helps in the development of formulation and package. In addition, the regulatory guidance is very general and does not explain about the performance of forced degradation studies. Thus, this review discusses the current trends in performance of forced degradation studies by providing a strategy for conducting studies on degradation mechanisms and also describes the analytical methods helpful for development of stability indicating method.
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A simple, specific, accurate and stability-indicating high performance liquid chromatographic method was developed and validated for the determination of Epinastine Hydrochloride in pharmaceutical dosage form. The chromatographic conditions comprised of a reverse-phase, C18 column (150×4.6 mm), 5μm with a mobile phase consisting of a mixture of aqueous phase (3.8g of sodium pantanesulphonate monohydrate and 4.0g of potassium dihydrogen orthophosphate was dissolved in 1L of water and pH of solution was adjusted to 4.5 with o-phosphoric acid) and organic phase (acetonitrile and methanol was mixed in the ratio of 4:1 v/v) in the ratio of 60:40 v/v at a flow rate of 1.0ml/min. Detection was carried out at 220nm. The retention time of Epinastine Hydrochloride was found to be 3.5 min. The calibration curve was found linear between 2-200μg/ml. The percentage recoveries of Epinastine Hydrochloride were found to be in the range of 99.05-100.50%. The method was validated for accuracy, linearity, precision, detection limit, quantitation limit and robustness. The drug was subjected to acidic hydrolysis, basic hydrolysis, neutral hydrolysis, oxidation, photochemical and thermal degradation. All the peaks of degraded product were resolved from the active pharmaceutical ingredient with significantly different retention time. As the method could effectively separate the drug from its degradation product, it can be employed as a stability indicating one.
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O diabetes mellitus (DM) é uma síndrome na qual o metabolismo de hidratos de carbono, gorduras e proteínas está alterado, por falta de secreção de insulina ou por diminuição da sensibilidade tissular a este hormônio. O DM pode ser do tipo I, também denominado diabetes mellitus insulinodependente (DMID), caracterizado pela falta de secreção da insulina, e do tipo II, também denominado diabetes mellitus não insulinodependente (DMNID), caracterizado pela menor sensibilidade dos tecidos efetores às ações metabólicas da insulina. Embora ainda não haja cura definitiva, há vários tratamentos disponíveis que proporcionam qualidade de vida para o paciente portador, como a administração de hipoglicemiantes orais. Nesta pesquisa, objetivou-se desenvolver métodos indicativos de estabilidade para a glibenclamida e o cloridrato de metformina. Assim, foram desenvolvidos métodos de rastreamento ortogonais utilizando a cromatografia líquida de alta eficiência (HPLC) e a eletroforese capilar (CE), metodologias que foram desafiadas com os estudos da degradação forçada. Realizou-se, também, a identificação do principal produto de degradação da glibenclamida utilizando a cromatografia líquida acoplada à espectrometria de massa (LC-MS). O método por HPLC teve o melhor desempenho no monitoramento dos produtos de degradação da glibenclamida, apresentando boa linearidade nas concentrações entre 0,210 e 0,360 mg/mL; com coeficiente de correlação maior de 0,99. A precisão calculada como desvio padrão relativo (DPR) foi menor de 3%, exatidão do método comprovada mediante teste de recuperação, obtendo-se valores de 100±3,0%. No teste de especificidade, foram detectados três potenciais produtos de degradação, com os seguintes tempos de retenção relativos (TRR) de 0,33; 0,46 e 0,83. O método CZE teve o melhor desempenho no monitoramento dos produtos de degradação do cloridrato de metformina, apresentando boa linearidade nas concentrações entre 0,210 e 0,360 mg/mL, com coeficiente de...
Diabetes mellitus (DM) is a syndrome which alters the metabolism of carbohydrates, fats and proteins by lack of insulin secretion or a decrease in tissue sensitivity to insulin. Type 1 DM is also known as insulin-dependent diabetes mellitus is characterized by lack of insulin secretion. The second type of diabetes known as Type II, also called non-insulin dependent, is characterized by the reduced sensitivity of target tissues to the metabolic actions of insulin. Although there is no definitive cure, there are several treatments available that provide quality of life for the patient how treatment with oral hypoglycemic agents. This study had as objective to developed stability-indicating methods for glibenclamide and metformin hydrochloride. Thus, the techniques used in this study involved high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), and both were challenged with forced degradation studies. The identification of degradation products of glibenclamide was also performed, utilizing the mass spectrometry (MS) technique. However, the HPLC technique had the best performance for monitoring the degradation products of glibenclamide, which showed a good linearity in concentrations between 0.210 - 0.360 mg/ml, with a correlation coefficient greater than 0.99. The precision was calculated as a relative standard deviation (RSD) which was less than 3%; the accuracy of the method calculated as the percent recovery was obtained with the following values: 100 ± 3.0%. On the specificity were detected three potential products of degradation utilizing forced degradation, with the following relative retention times (RRT) 0.33, 0.46 and 0.83. The CZE method had the best performance to monitoring the degradation products of metformin hydrochloride, which showed good linearity of concentrations between 0.210 and 0.360 mg/ml, with a correlation coefficient greater than 0.99. The intra-day and inter-day precision calculated as DPR was less than...
Subject(s)
Diabetes Mellitus , Drug Stability , Chemistry, Pharmaceutical/methods , Chromatography, High Pressure Liquid/methods , Electrophoresis, Capillary/methodsABSTRACT
A simple, specific, accurate and stability-indicating high performance liquid chromatographic method was developed and validated for the determination of Epinastine Hydrochloride in pharmaceutical dosage form. The chromatographic conditions comprised of a reverse-phase, C18 column (150×4.6 mm), 5μm with a mobile phase consisting of a mixture of aqueous phase (3.8g of sodium pantanesulphonate monohydrate and 4.0g of potassium dihydrogen orthophosphate was dissolved in 1L of water and pH of solution was adjusted to 4.5 with o-phosphoric acid) and organic phase (acetonitrile and methanol was mixed in the ratio of 4:1 v/v) in the ratio of 60:40 v/v at a flow rate of 1.0ml/min. Detection was carried out at 220nm. The retention time of Epinastine Hydrochloride was found to be 3.5 min. The calibration curve was found linear between 2-200μg/ml. The percentage recoveries of Epinastine Hydrochloride were found to be in the range of 99.05-100.50%. The method was validated for accuracy, linearity, precision, detection limit, quantitation limit and robustness. The drug was subjected to acidic hydrolysis, basic hydrolysis, neutral hydrolysis, oxidation, photochemical and thermal degradation. All the peaks of degraded product were resolved from the active pharmaceutical ingredient with significantly different retention time. As the method could effectively separate the drug from its degradation product, it can be employed as a stability indicating one.
ABSTRACT
The present paper describes development of stability- indicating RP- HPLC method for the simultaneous determination of Ofloxacin and Satranidazole in presence of its degradation products, generated from forced degradation studies. Ofloxacin and Satranidazole and their combination drug product were exposed to acid, base, neutral hydrolysis; oxidation, dry heat, photolytic stress conditions and the stressed samples were analyzed by proposed method. The proposed HPLC method utilizes HiQ sil C18W column (250mm × 4.6mm i.d., 5μm) of KYA TECH, Corporation and a mobile phase comprising of acetonitrile: phosphate buffer (pH3) in ratio of 35:65v/v with flow rate of 1ml/min. The retention time of OFLX and STZ was found to be 2.85min and 6.25min respectively. Quantitation was achieved with UV detection at 296nm for OFLX and 320nm for STZ. The method has been validated for ofloxacin and satranidazole in terms of accuracy, precision, linearity, LOD, LOQ and robustness. The developed validated stability-indicating HPLC method was found to be simple, specific, accurate and reproducible for the determination of instability of these drugs in bulk and commercial products.