A Stability Indicating Reverse Phase High Performance Liquid Chromatography Method for Related Substances of Sofosbuvir in Tablet Dosage Form

Sofosbuvir is an antiviral drug that is used to treat hepatitis C. A present investigation deals with simple, sensitive, rapid, precise and accurate reverse phase high-performance chromatography (RP-HPLC) method developed and validated for related substances of sofosbuvir in tablet dosage form. The chromatographic separation was achieved on a Kromasil 100 C18 (250 × 4.6 mm, 5 ?) column. A sofosbuvir and its impurities were extracted by composed mixture of Mobile Phase A: buffer solution: acetonitrile (97.5:2.5% v/v) and Mobile Phase B: acetonitrile, isopropyl alcohol, methanol and purified water (60:20:10:10 % v/v/v/v) using with flow rate was 1-mL/min, column temperature was 25°C, injection volume was 10 ?L, Vial thermostat temperature was 10°C. The UV detection was carried out at 263 nm. The retention time of sofosbuvir, methyl ester and ethyl ester were 54.28, 36.31 and 43.77, respectively. The method shows linearity with correlation coefficient of sofosbuvir and its impurity was 0.999 over the 0.5–7.5 ppm range. The average recovery was found to be 90.2–113.9%. The LoD and LoQ for sofosbuvir and its impurities were found to be 0.1 and 0.5 ?g/mL, respectively. The method was validated as per ICH guidelines. The developed method was precise, accurate, novel and detectable towards sofosbuvir and its impurity. This method is efficient in separating the sofosbuvir and its impurity. Hence, the proposed method can be utilized for the determination of related substances in routine analysis in quality control department of pharmaceutical Industry.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR PIMAVANSERIN TARTRATE

Objective: The aim of current research work was to investigate degradation behavior of Pimavanserin tartrate upon exposure to stress conditions recommended by ICH Q1A (R2) and Q1B guidelines.Methods: Chromatographic separation was achieved on Merck’s TLC aluminum plates pre-coated with silica gel G 60 F254 as stationary phase and Methanol: Chloroform (2:8 v/v) as mobile phase. Densitometry scanning was carried out at 224 nm.Results: The retardation factor (Rf) was observed to be 0.56±0.02. Pimavanserin tartrate showed degradation in all stress conditions, but no degradation product was found in any stress condition. Peak purity was found to be 0.999 indicating no interference by degradation products to drug peak. The developed HPTLC method was successfully validated as per ICH Q2 (R1) guideline. Method was found to be linear within the range of 400-2000 ng/band with correlation coefficient R2= 0.9982. % RSD for intra-day and inter-day precision were found to be 1.35 and 1.78 % and % recovery was found to be in range 98-102 %. LOD and LOQ were found to be 17.58 ng/band and 53.27 ng/band respectively.Conclusion: A simple, economic stability indicating high performance thin layer chromatography method has been developed and validated for Pimavanserin tartrate. It is used for the treatment of delusions and hallucinations in Parkinson’s disease.

Selective Separation and Mass Spectral Characterization of Degradants in Viloxazine by LC-MS/MS

This research describes a novel technique for the selective separation of degradants from API employing HPLC and online coupling of a triple quadrupole mass analyzer and PDA detector with a SCIEX QTRAP 5500 mass spectrometer. Chromatography was used to separate all degradants on the column Agilent eclipse XDB (150 mm x 4.6 mm, 3.5 ?) with mobile phase ACN: 0.1% TEA (40:60) %v/v. The highest absorption was found to occur at 220 nm, which allows for simultaneous detection without being impacted by the placebo matrix. According to the general ICH recommendations, the suggested RP-HPLC method was accepted. All of the metrics- specificity, linearity, LoD, LoQ, accuracy, precision and robustness of validation were deemed sufficient. The proposed method exhibits strong correlation and great linearity over the range of (12.5–75 ?g/mL). The accuracy trials produced consistent recoveries (95–105%), while the precision experiments' percent RSD was less than 2%. The intrinsic stability of the drug molecules in the current formulation could be ascertained by conducting forced degradation studies to assess the degradation products produced under various stress settings. The degradants produced were well separated and further characterized by MS/MS studies. The newly devised approach was demonstrated to be stable and sensitive to all degradants during validation tests. Validation trials demonstrate that the newly developed method was also accurate, precise, resilient, selective, and linear within the necessary operating range.

Characterization of Degradation Products of Guanfacine Hydrochloride API: Development and Validation of a Stability-indicating Reversed Phase UHPLC Method

Guanfacine Hydrochloride API was subjected to forced degradation studies under various conditions of hydrolysis (acidic, alkaline, and neutral/water), oxidation, photolysis, and thermal. A short and simple, reverse phase UHPLC method was developed on a Shimadzu, Shimpack GIST, C18, (100 × 2.1) mm, 3.0 ?m column. The gradient method consisted of 0.1% orthophosphoric acid as mobile phase A and acetonitrile as mobile phase B. The flow rate of the mobile phase was 0.3 mL/min. The method was validated using ICH guidelines considering the parameters solution stability, specificity, DL/QL, linearity, accuracy, precision and robustness. The drug was found highly sensitive to alkaline conditions and showed significant degradation. The drug was found sensitive to acidic degradation conditions. Slight degradation was observed in oxidative and water conditions. The drug was found to be stable in thermal and photolytic conditions. The mass compatible UHPLC method was prepared by simply substituting the orthophosphoric acid with formic acid in the mobile phase. Characterization of two major degradation products (DPs) was done. DP1 was characterized with LC–Q-TOF-MS/MS in combination with accurate mass measurements. DP2 was isolated and characterized with NMR, IR and HRMS spectroscopic techniques. The mechanisms of the formation of DPs were proposed.

Aplicação da eletroforese capilar - espectrometria de massas para identificação dos produtos de degradação do aripiprazol em medicamentos / Application of capillary electrophoresis - mass spectrometry to identify aripiprazole degradation products in drugs

Diretrizes internacionais e nacionais como a FDA (Food and Drug Administration), ICH (International Council for Harmonisation) e ANVISA (Agência Nacional de Vigilância Sanitária) estabelecem a exigência de testes de estabilidade para entender melhor a qualidade de um medicamento. O estudo de estabilidade deve ser realizado usando métodos indicativos de estabilidade que possam qualificar e quantificar os insumos farmacêuticos do medicamento, bem como as impurezas e produtos de degradação nele contidos. O aripiprazol é um antipsicótico atípico de segunda geração aprovado para o tratamento de esquizofrenia, transtorno bipolar, depressão e transtornos do espectro do autismo. Os métodos oficiais descritos nas farmacopeias para avaliar o aripiprazol e suas impurezas utilizam a cromatografia líquida de alta eficiência (HPLC) como técnica principal. Nesta pesquisa, objetivou-se desenvolver um método indicativo de estabilidade por eletroforese capilar de zona (CZE) para o aripiprazol na forma farmacêutica de comprimidos, e identificação dos produtos de degradação por espectrometria de massas. O estudo de degradação forçada e a optimização do método desenvolvido por CZE foram realizados utilizando o conceito de delineamento de experimentos (DoE). A separação do aripiprazol de seus produtos de degradação foi conseguida usando uma coluna capilar de sílica fundida (30,2 cm x 75 µm ID), eletrólito de formiato de amônio 6 mmol/L (pH 3) com 5% de metanol sob um potencial de 15 kV e detecção em 214 nm. A capacidade indicativa de estabilidade do método foi investigada pela análise do aripiprazol após ser submetido a condições de estresse ácido, alcalino, térmico, fotolítico e oxidativo, de acordo com as diretrizes ICH. A oxidação foi a principal via de degradação entre as condições de estresse avaliadas. O aripiprazol foi separado dos seus produtos de degradação oxidativa em tempo de corrida abaixo de 5 minutos. O método por CZE mostrou ser linear na faixa de 60 - 140 µg/mL, R2 = 0,9980, precisão calculada como desvio padrão relativo (DPR) menor que 2% e exatidão calculada como recuperação média de 100,93 ± 0,77%. Os resultados obtidos demonstram que o método por HPLC-RP em modo gradiente, separou o aripiprazol e seus produtos de degradação em um tempo de corrida de 30 minutos. Quatro produtos de degradação foram detectados pelo método LC-MS e o principal produto de degradação oxidativo foi identificado. O aripiprazol mostrou-se suscetível à oxidação no grupo piperazina, gerando principalmente o composto aripiprazol-1-N-óxido

International and national guidelines such as the FDA (Food and Drug Administration), ICH (International Council for Harmonization) and ANVISA (National Health Surveillance Agency) establish the requirement for stability tests to better understand quality of a medicine. The stability study must be carried out using stability indicating methods that can qualify and quantify the pharmaceutical ingredients of the drug, as well as the impurities and degradation products contained therein. Aripiprazole is a second-generation atypic antipsychotic drug approved for the treatment of schizophrenia, bipolar disorder, depression, and autism spectrum disorders. The official method described in the pharmacopoeias to evaluate aripiprazole and its impurities is high performance liquid chromatography (HPLC) as the main technique. In this research, the objective was to develop an indicative method of stability by capillary zone electrophoresis (CZE) for aripiprazole in the pharmaceutical form of tablets, and identification of degradation products by mass spectrometry. The forced degradation study and the optimization of the method developed by CZE were carried out using the concept of design of experiments (DoE). The separation of aripiprazole from its degradation products was achieved using a fused silica capillary column (30,2 cm x 75 µm ID), 6 mmol/L ammonium formate electrolyte (pH 3) with 5% methanol under a potential of 15 kV and detection at 214 nm. The indicative stability of the method was investigated by analyzing aripiprazole after being subjected to acid, alkali, thermal, photolytic and oxidative stress conditions, according to the ICH guidelines. Oxidation was the main degradation pathway among the stress conditions evaluated. Aripiprazole was separated from its oxidative degradation products at run times below 5 minutes. The CZE method proved to be linear in the range of 60 - 140 µg/mL, R2 = 0,9980, precision calculated as a relative standard deviation (DPR) of less than 2% and accuracy calculated as a mean recovery of 100,93 ± 0,77%. The results obtained demonstrate that the HPLC-RP method in gradient mode separated aripiprazole and its degradation products in a run time of 30 minutes. Four degradation products were detected by the LC-MS method and the main oxidative degradation product was identified. Aripiprazole was shown to be susceptible to oxidation in the piperazine group, generating mainly the compound aripiprazole-1-N-oxide

Luliconazole: Stability-indicating LC method, structural elucidation of major degradation product by HRMS and in silico studies / Luliconazol: método por LC indicativo de estabilidad, elucidación estructural del producto de degradación mayoritario por HRMS y estudios in silico / Luliconazol: método LC indicativo de estabilidade, elucidação estrutural do principal produto de degradação por HRMS e estudos in sílico

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.

Taxifolin stability:In silico prediction and in vitro degradation with HPLC-UV/UPLC-ESI-MS monitoring / 药物分析学报

Taxifolin has a plethora of therapeutic activities and is currently isolated from the stem bark of the tree Larix gmelinni(Dahurian larch).It is a flavonoid of high commercial interest for its use in supplements or in antioxidant-rich functional foods.However,its poor stability and low bioavailability hinder the use of flavonoid in nutritional or pharmaceutical formulations.In this work,taxifolin isolated from the seeds of Mimusops balata,was evaluated by in silico stability prediction studies and in vitro forced degradation studies(acid and alkaline hydrolysis,oxidation,visible/UV radiation,dry/humid heating)monitored by high performance liquid chromatography with ultraviolet detection(HPLC-UV)and ultrahigh perfor-mance liquid chromatography-electrospray ionization-mass spectrometry(UPLC-ESI-MS).The in silico stability prediction studies indicated the most susceptible regions in the molecule to nucleophilic and electrophilic attacks,as well as the sites susceptible to oxidation.The in vitro forced degradation tests were in agreement with the in silico stability prediction,indicating that taxifolin is extremely unstable(class 1)under alkaline hydrolysis.In addition,taxifolin thermal degradation was increased by humidity.On the other hand,with respect to photosensitivity,taxifolin can be classified as class 4(stable).Moreover,the alkaline degradation products were characterized by UPLC-ESI-MS/MS as dimers of taxifolin.These results enabled an understanding of the intrinsic lability of taxifolin,contributing to the development of stability-indicating methods,and of appropriate drug release systems,with the aims of preserving its stability and improving its bioavailability.

Forced degradation study of efonidipine HCl ethanolate, characterization of degradation products by LC-Q-TOF-MS and NMR

Efonidipine HCl Ethanolate is an antihypertensive drug with 1,4 dihydropyridine and phosphinane derivative. Forceddegradation study was performed in Efonidipine as per the guidelines by International Conference on Harmonization(ICH) Q1A (R2). Extensive degradation and slight degradation were observed in alkaline and photolytic conditions,respectively, whereas acidic, oxidative, and thermal conditions did not show any degradation. Degradation productswere separated on Thermo Hypersil BDS C18 column (250 × 4.6 mm, 5 µ), mobile phase in gradient mode usingammonium acetate buffer and acetonitrile with detection at a wavelength of 254 nm. Six degradation products in alkalinecondition and four degradation products in photolytic condition were identified by HPLC and characterized by massspectrometry using LC-Q-TOF-MS, and degradation pathway was proposed. This is the typical case of degradation,where co-solvent methanol reacts with Efonidipine to form pseudo degradation products such as DP1, DP4, DP5, andDP6. Three degradation products DP1, DP3, and DP4 in alkaline condition were isolated by preparative HPLC andwere characterized by LC-Q-TOF-MS, 1H/13C NMR, and IR techniques. By characterization with these techniques,DP1 is characterized as 3-2-(N-benzylanilino)ethyl 3-oxo-2,2-dimethylpropyl hydrogen 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridin-3-yl-3-phosphonate, DP3 is characterized as 2-(N-benzyl-N-phenylamino) ethanol, and DP4is characterized as 3-methoxy-2,2-dimethylpropyl hydrogen 1,4-dihydro-2,6-dimethyl-5-methyloxycarbonyl-4-(3-nitro)phenylpyridin-3-yl-3-phosphonate. The developed method was validated as per guidelines by ICH with respectto linearity, accuracy, precision, limit of detection, and robustness.

Development And Validation Of A Stability Indicating Rp-Hplc Method For The Determination Of Prucalopride Succinate In Bulk And Tablet

A very simple, precise, economical, accurate, robust, and reproducible reverse phase-high-performance liquid chromatography method along with stability indicating attributes has been developed for estimating of prucalopride succinate (PRU) in both bulk and tablet formulation (PRUVICT 2). The estimation of the solutes was performed on a Grace C18 column of dimension 150 mm × 4.6 mm, 5 μm. PRU was eluted with acetonitrile: 0.02 M potassium dihydrogen phosphate in the ratio of 20:80 v/v in a 10 min isocratic mode at a flow rate of 1 ml/min at 30°C column temperature and monitored at a wavelength of 277 nm. The retention time of PRU was found to be 5.416 minutes. The Q2b validation of the analytical method revealed good linearity over the concentration range 2–12 μg/mL for IVA with r2 of 0.999. The mean recovery % over the three tested ranges of 50%, 100%, and 150% were found to be 100.173%, 99.077%, and 98.575%, respectively. In intra-day variability study, the % RSDs was detected to be 0.754, 1.032, and 0.482 whereas the inter-day variability study demonstrated % RSDs of 0.797, 0.559, and 0.524, respectively. The acid, alkali, boiled water, hydrogen peroxide, dry heat, and UV radiations based stress studies presented the formation of a variety of characteristic degradation products. The developed analytical method may be employed for the routine analysis of PRU in bulk and tablet formulations.

Development and validation of a stability indicating UHPLC method for Sacubitril/Valsartan complex in the presence of impurities and degradation products

Sacubitril/Valsartan (SAC/VAL) is a combination drug used for the treatment of heart failure. In the present work,novel and rapid, sensitive, specific, and robust ultra high-performance liquid chromatography method was developedand validated for the simultaneous estimation of SAC/VAL in presence of their seven related impurities anddegradation products. The chromatographic separation was achieved on Accucore XL C8, (100 × 4.6) mm; 3 μmreverse phase column maintained at 30°C. The peaks were eluted using tetrahydrofuran (THF) and 0.1% perchloricacid in water (8:92, %v/v) as a mobile phase A and THF:water:acetonitrile (5:15:80, %v/v/v) as mobile phase B in agradient mode. The flow rate was set at 0.6 ml/minute and the analytes were monitored in the range of 200–400 nmusing a Photo Diode Array (PDA) detector for 21 minutes run time. The method was validated as per ICH Q2 (R1)guideline and all the validation parameters were found within the acceptance criteria. The forced degradation studyfor SAC/VAL showed that the drugs were prone to acidic, alkaline, and neutral hydrolytic as well as oxidative stressconditions. All the degradation products were separated from each other, SAC/VAL and their impurities showing thestability indicating power of the method. The newly developed method can be used for estimation of assay and relatedsubstances from bulk or their finished products with good efficiency.

Stability-indicating HPLC method for determination of amiodarone hydrochloride and its impurities in tablets: a detailed forced degradation study

Resumo Amiodarone hydrochloride is one of the most important drugs used to treat arrhythmias. The USP monograph for amiodarone hydrochloride describes an HPLC method for the quantification of seven impurities, however, this method shows problems that result in unresolved peaks. Moreover, there is no monograph for tablets in this compendium. Thus, a stability indicating HPLC method was developed for the determination of amiodarone, its known impurities and degradation products in tablets. A detailed forced degradation study was performed submitting amiodarone API, tablets and placebo to different stress conditions: acid and alkaline hydrolysis, oxidation, metal ions, heat, humidity, and light. Amiodarone hydrochloride API was susceptible to degradation in all stress conditions. The tablets also showed degradation in all environments, except in acidic condition. The analytes separation and quantification were achieved on an Agilent Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 3.5 µm). The mobile phase was composed of 50 mM acetate buffer pH 5.5 (A) and a mixture of methanol-acetonitrile (3:4, v/v) (B) in gradient elution. The method was validated in the range of 350-650 µg/mL for assay and 10-24 µg/mL for impurities determination. Therefore, this method can be used both for stability studies and routine quality control analyses.

Quantification of rasagiline mesylate by stability indicating RP-HPLC method: Development and validation

A novel reversed phase-high-performance liquid chromatography (HPLC) method for the estimation of rasagilinemesylate (RM), a potent anti-Parkinson drug in Active Pharmaceutical Ingredient (API), and tablet dosage form wasdeveloped and validated in the present research. HPLC instrument (Shimadzu) comprises ultraviolet detector andphenomenex 100 C18 (250 × 4.6 mm, 5 µm) column was utilized in the study. A mixture of acetonitrile and waterin the ration of 50:50, adjusted to pH 3.0 ± 0.05 with ortho-phosphoric acid was used as the mobile phase. Thechromatographic conditions; flow rate 0.8 ml/minute, run time 6.0 minutes, injection volume 50 µl, and detectionwavelength 268 nm were maintained during the study at room temperature. To ensure performance of novel methoddeveloped, it was validated according to International conference on harmonization guidelines. The developed methodwas subjected to the forced degradation studies to find out the stability indicating nature of the method by quantifyingthe RM in presence of its degradation products and the peaks which were responsible for the degraded products werenot interfered with the API principle peak. The proposed stability indicating newly developed and validated methodcan be adopted for the quantification of RM in bulk and pharmaceutical formulations.

Development and validation of a stability-indicating RP-HPLC method of cholecalciferol in bulk and pharmaceutical formulations: Analytical quality by design approach

The present article utilized analytical quality by design (AQbD) methodology to optimize chromatographic conditionsfor the routine analysis of Cholecalciferol (CHL). Taguchi orthogonal array design and Box–Behnken designwere employed to screen and optimize critical method parameters for augmenting the method performance. Theoptimal chromatographic separation was attained on Eurosphere® 100-5, C8 (250 × 4.6 mm i.d., 5 μm) column in anisocratic elution mode using methanol:acetonitrile (50:50, % v/v) as mobile phase at a flow rate of 1.0 ml/minutesand photodiode array detection at 265 nm. The optimized chromatographic method was successfully validated asper International Council for Harmonisation Q2 (R1) guidelines. The method was found to be linear (r2 = 0.9993)in the range of 20–100 IU/ml. Limit of detection and limit of quantitation were found to be 10 and 20 IU/ml. Theprecision, robustness, and ruggedness values were within the acceptance limits (relative standard deviation < 2). Thepercent recovery of in-house developed 400 IU mouth dissolving tablets and marketed Tayo 60k tablets were foundto be 99.89% and 101.46%, respectively. The forced degradation products were well resolved from the main peaksuggesting the stability-indicating the power of the method. In conclusion, the AQbD-driven method is highly suitablefor analysis of CHL in bulk and pharmaceutical formulations

Advancing USP compendial methods for fixed dose combinations: A case study of metoprolol tartrate and hydrochlorothiazide tablets / 药物分析学报

The current United States Pharmacopeia–National Formulary (USP–NF) includes more than 250 mono-graphs of fixed dose combinations (FDCs), and some of them need to be updated due to incompleteness of impurity profiles and obsolescence of analytical methodologies. A case study of metoprolol tartrate and hydrochlorothiazide tablets is presented to summarize challenges encountered during the USP monograph modernization initiative of FDCs and to highlight an "adoption and adaptation" approach employed for method development. To this end, a single stability-indicating HPLC method was devel-oped to separate the two drug substances and eight related compounds with resolution 2.0 or higher between all critical pairs. Chromatographic separations were achieved on a Symmetry column (C18, 100 mm × 4.6 mm, 3.5 μm) using sodium phosphate buffer (pH 3.0; 34 mM) and acetonitrile as mobile phase in a gradient elution mode. The stability-indicating capability of this method has been demon-strated by analyzing stressed samples of the two drug substances. The developed HPLC method was validated for simultaneous determination of metoprolol tartrate and hydrochlorothiazide and relevant impurities in the tablets. Moreover, the developed method was successfully applied to the analysis of commercial tablet dosage forms and proved to be suitable for routine quality control use. The case study could be used to streamline USP's monograph modernization process of FDCs and strengthen compendial procedures.

Novel degradation products of argatroban:Isolation, synthesis and extensive characterization using NMR and LC-PDA-MS/Q-TOF / 药物分析学报

Forced degradation study of argatroban under conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH Q1A (R2), was accomplished. The drug showed sig-nificant degradation under hydrolysis (acidic, alkaline) and oxidation (peroxide stress) conditions. The drug remained stable under thermal and photolytic stress conditions. In total, seven novel degradation products (DP-1 to DP-7) were found under diverse conditions, which were not reported earlier. The chemical structures of these degradation products were characterized by 1H NMR, 13C NMR, 2D NMR, Q-TOF-MSn and IR spectral analysis and the proposed degradation products structures were further confirmed by the individual synthesis.

Structural confirmation of sulconazole sulfoxide as the primary degradation product of sulconazole nitrate / 药物分析学报

Sulconazole has been reported to degrade into sulconazole sulfoxide via sulfur oxidation; however, structural characterization data was lacking and the potential formation of an N-oxide or sulfone could not be excluded. To clarify the degradation pathways and incorporate the impurity profile of sulconazole into the United States Pharmacopeia–National Formulary (USP–NF) monographs, a multifaceted approach was utilized to confirm the identity of the degradant. The approach combines stress testing of sulco-nazole nitrate, chemical synthesis of the degradant via a hydrogen peroxide-mediated oxidation reaction, semi-preparative HPLC purification, and structural elucidation by LC―MS/MS and NMR spectroscopy. Structural determination was primarily based on the comparison of spectroscopic data of sulconazole and the oxidative degradant. The mass spectrometric data have revealed a McLafferty-type rearrange-ment as the characteristic fragmentation pathway for alkyl sulfoxides with aβ-hydrogen atom, and was used to distinguish the sulfoxide from N-oxide or sulfone derivatives. Moreover, the generated sulco-nazole sulfoxide was utilized as reference material for compendial procedure development and valida-tion, which provides support for USP monograph modernization.

Developed and validated stability indicating HPLC method for the determination of epirubicin in bulk drug, marketed injection and polymeric nanoparticles

Present work is aimed to develop a simple, sensitive, robust and reliable HPLC method for routine quality control of epirubicin (EPI) in bulk drug, marketed injections and polymeric nanoparticles. Separation was carried out by C18 column. Isocratic elution was carried out using mobile phase A: 0.16% o-phosphoric acid solution, B: acetonitrile and methanol mixture (80:20, v/v) in the ratio of 60:40 (A: B) while the flow rate was maintained at 1mL/min. Analyses were performed at 233.5 nm using PDA detector. Excellent linear relationship was observed between peak-area versus drug concentration in the range of 1.0-100.0 µg/mL (r2, 0.999). Developed method was found to be sensitive (Limits of detection and quantification were found to be ~8 ng/mL and ~25 ng/mL, respectively), precise (RSD <1.0%, for repeatability and <2.0% for intermediate precision, within acceptable ranges of precision), accurate (recovery in different dosage form, 94.65 -100.26%, within acceptable range, 80-120%), specific and robust (% RSD <2, for system suitability parameters). Stress-induced degradation studies demonstrated that method can suitability be applied in the presence of degradants. Developed method has been successfully applied for the determination of entrapment efficiency, drug loading, in vitro release profile, in vitro permeation studies as well as stability assessment of polymeric nanoparticles

Evaluation of the influence of fluoroquinolone chemical structure on stability: forced degradation and in silico studies

ABSTRACT Fluoroquinolones are a known antibacterial class commonly used around the world. These compounds present relative stability and they may show some adverse effects according their distinct chemical structures. The chemical hydrolysis of five fluoroquinolones was studied using alkaline and photolytic degradation aiming to observe the differences in molecular reactivity. DFT/B3LYP-6.31G* was used to assist with understanding the chemical structure degradation. Gemifloxacin underwent degradation in alkaline medium. Gemifloxacin and danofloxacin showed more degradation perceptual indices in comparison with ciprofloxacin, enrofloxacin and norfloxacin in photolytic conditions. Some structural features were observed which may influence degradation, such as the presence of five member rings attached to the quinolone ring and the electrostatic positive charges, showed in maps of potential electrostatic charges. These measurements may be used in the design of effective and more stable fluoroquinolones as well as the investigation of degradation products from stress stability assays.

Forced degradation of gliquidone and development of validated stability-indicating HPLC and TLC methods

Forced degradation studies of gliquidone were conducted under different stress conditions. Three degradates were observed upon using HPLC and TLC and elucidated by LC-MS and IR. HPLC method was performed on C18 column using methanol-water (85:15 v/v) pH 3.5 as a mobile phase with isocratic mode at 1 mL.min-1 and detection at 225 nm. HPLC analysis was applied in range of 0.5-20 µg.mL-1 (r =1) with limit of detection (LOD) 0.177 µg.mL-1. TLC method was based on the separation of gliquidone from degradation products on silica gel TLC F254 plates using chloroform-cyclohexane-glacial acetic acid (6:3:1v/v) as a developing system with relative retardation 1.15±0.01. Densitometric measurements were achieved in range of 2 -20 µg /band at 254 nm (r = 0.9999) with LOD of 0.26 µg /band. Least squares regression analysis was applied to provide mathematical estimates of the degree of linearity. The analysis revealed a linear calibration for HPLC where a binomial relationship for TLC. Stability testing and methods validation have been evaluated according to International Conference on Harmonization guidelines. Moreover, the proposed methods were applied for the analysis of tablets and the results obtained were statistically compared with those of pharmacopeial method revealing no significant difference about accuracy and precision.

Development and validation of a stability-indicating LC-UV and LC-MS/MS methods for quantitative analysis of anisomycin and identification of degradation products

ABSTRACT Multifunctional drug anisomycin was subjected to forced degradation in accordance with International Conference on Harmonisation (ICH) guidelines for the first time. The drug was exposed to the recommended stress conditions of hydrolysis (acidic, alkaline and neutral), oxidation, thermal stress and photolysis, in order to investigate its stability. Optimized LC-MS/MS method was validated as recommended by ICH Q2(R1) guideline with respect to the specificity, accuracy, precision, limits of detection and quantitation, linearity and robustness. Anisomycin exhibited high instability under alkaline and thermal (neutral hydrolysis) conditions. It showed moderate stability under acidic, neutral, oxidative, thermal (acidic hydrolysis) and photolytic conditions, with the lowest degradation level observed in the case of light and oxidation stress. Formation of the same degradation product, identified as deacetylanisomycin, was observed under all applied stress conditions.