Empagliflozin: Validation of Stability-Indicating LC Method and in silico Toxicity Studies.

A new stability-indicating liquid chromatography method was developed for the quantification of empagliflozin and two synthetic impurities. The chromatographic conditions included Spherisorb® RP-18 column (150 × 4.6 mm, 5 µm) with a PDA detector, using acetonitrile and formic acid (pH 4.0) as mobile phase in gradient elution and flow-rate of 1.2 mL·min-1. The gradient increasing from 51 to 100% acetonitrile until 11.00 min, followed by decreasing the solvent from 100% to the initial ratio from 11.01 to 15.00 min. The method was validated according to International Council of Harmonization guidelines. The LOD and LOQ values for impurities A and B were 35 and 15 ng·mL-1, respectively, (for LOD) and 115 and 35 ng.mL-1, respectively (for LOQ). The method was linear in the range of 80-140, 115-1150 and 35-350 ng·mL-1 for EMPA, impurities A and B, respectively, and the correlation coefficient were > 0.999 in all situations, indicating the method good linearity. The developed method showed a good recovery for empagliflozin and added impurities. The method has proven to be precise, demonstrated values less than 2.0% to empagliflozin and 5.0% to synthetic impurities, robust and selective with no interference from other products in the determination of analytes. The in silico toxicity prediction suggested that the impurities do not present any toxicity risk for the parameters evaluated.

A Review of Characteristics, Properties, Application of Nanocarriers and Analytical Methods of Luliconazole.

Luliconazole is an imidazole agent, used for the treatment of fungi infection, especially dermatophytes. The mechanism of action of the drug consisting in inhibits sterol 14α-demethylase which interferes with ergosterol biosynthesis. Due to low aqueous solubility and highly lipophilic, there is a need to develop drug delivery systems (nanocarriers) capable to increase the solubility, permeability, and skin retention of luliconazole, and promote a better therapeutic effect. In this context, this review presents characteristics, properties, nanocarriers, and analytical methods used for luliconazole. From the analyzed studies, the majority reports the use of RP-HPLC techniques for luliconazole determination, but also are cited spectrophotometric UV methods. The luliconazole has been qualitatively and quantitatively analyzed in different matrices, such as raw material and pharmaceutical formulations, however, in this review, only one study was found with the luliconazole quantification biological matrix, demonstrating the lack of studies related to the quantification of the drug in biological matrices. The drug quantification in different matrices by analytical methods is of great importance since they assist in the control of the quality, efficacy, and safety of the medicine.

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.

Bilastine: Quantitative Determination by LC with Fluorescence Detection and Structural Elucidation of the Degradation Products Using HRMS.

BACKGROUND: A liquid chromatography (LC) stability-indicating method was developed and validated for the quantitative determination of bilastine in coated tablets. OBJECTIVE: The procedure was validated for specificity, linearity, robustness, precision, and accuracy. Plackett-Burmann experimental design was used to determine the robustness of the method. METHOD: Chromatographic separation was performed on a Shim-pack® RP-18 column with fluorescence detection. The degradation products formed under oxidative conditions were isolated and identified using high-resolution mass spectrometry (HRMS). In silico prediction of degradation products and in silico toxicity studies were also performed. RESULTS: The LC method presented good recovery and precision (intraday and interday), the response was linear in a range of 0.20 to 0.70 µg mL-1, and the results demonstrated the robustness of the analytical method under the evaluated conditions. CONCLUSIONS: The degradation products were identified as benzimidazole (DP1) and amine N-oxide of bilastine (DP2). The results for the toxicity studies demonstrated the high mutagenic potential of DP1 and hepatotoxicity and hERG I inhibitor effects of DP2. HIGHLIGHTS: Bilastine degradation products were identified as benzimidazole and amine N-oxide using HRMS.

The use of Brazilian vegetable oils in nanoemulsions: an update on preparation and biological applications

ABSTRACT Vegetable oils present important pharmacological properties, which gained ground in the pharmaceutical field. Its encapsulation in nanoemulsions is considered a promising strategy to facilitate the applicability of these natural compounds and to potentiate the actions. These formulations offer several advantages for topical and systemic delivery of cosmetic and pharmaceutical agents including controlled droplet size, protection of the vegetable oil to photo, thermal and volatilization instability and ability to dissolve and stabilize lipophilic drugs. For these reasons, the aim of this review is to report on some characteristics, preparation methods, applications and especially analyze recent research available in the literature concerning the use of vegetable oils with therapeutic characteristics as lipid core in nanoemulsions, specially from Brazilian flora, such as babassu (Orbignya oleifera), aroeira (Schinus molle L.), andiroba (Carapa guaianiensis), casca-de-anta (Drimys brasiliensis Miers), sucupira (Pterodon emarginatus Vogel) and carqueja doce (Stenachaenium megapotamicum) oils.

Simple HPLC-UV for the quantification of a new leishmanicidal candidate (E)-1-4(trifluoromethyl) benzylidene)-5-(2-4-dichlorozoyl) carbonylhydrazine (LASSBio-1736) in rat plasma for pharmacokinetics assessment.

In this study, a sensitive HPLC-UV assay was developed and validated for the determination of LASSBio-1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid-liquid extraction using acetonitrile was employed to extract LASSBio-1736 and IS from 100 µL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb(®) S5 ODS2 C18 column (150 × 4.6 mm, 5 µm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio-1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 µg/mL and linearity between 0.2 and 4 µg/mL was obtained, with an R(2) > 0.99. The accuracy of the method was >90.5%. The relative standard deviations intra and interday were <6.19 and <7.83%, respectively. The method showed the sensitivity, linearity, precision, accuracy and selectivity required to quantify LASSBio-1736 in preclinical pharmacokinetic studies according to the criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2016 John Wiley & Sons, Ltd.

Structural elucidation of gemifloxacin mesylate degradation product.

Gemifloxacin mesylate (GFM), chemically (R,S)-7-[(4Z)-3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid methanesulfonate, is a synthetic broad-spectrum antibacterial agent. Although many papers have been published in the literature describing the stability of fluorquinolones, little is known about the degradation products of GFM. Forced degradation studies of GFM were performed using radiation (UV-A), acid (1 mol L(-1) HCl) and alkaline conditions (0.2 mol L(-1) NaOH). The main degradation product, formed under alkaline conditions, was isolated using semi-preparative LC and structurally elucidated by nuclear magnetic resonance (proton - (1) H; carbon - (13) C; correlate spectroscopy - COSY; heteronuclear single quantum coherence - HSQC; heteronuclear multiple-bond correlation - HMBC; spectroscopy - infrared, atomic emission and mass spectrometry techniques). The degradation product isolated was characterized as sodium 7-amino-1-pyrrolidinyl-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylate, which was formed by loss of the 3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl ring and formation of the sodium carboxylate. The structural characterization of the degradation product was very important to understand the degradation mechanism of the GFM under alkaline conditions. In addition, the results highlight the importance of appropriate protection against hydrolysis and UV radiation during the drug-development process, storage, handling and quality control.

Development and validation of discriminating method of dissolution for fosamprenavir tablets based on in vivo data.

The aim of this work is to develop and validate a dissolution test for fosamprenavir tablets (Telzir(®)) based on in vivo data. The appropriate conditions were determined after testing sink conditions in dissolution medium, rotation speed and stability of the drug. In vivo release profiles were obtained from the literature. The fraction (and percentage) of dose absorbed (FA) was calculated by deconvolution, using the Wagner-Nelson method. For this formulation, the best dissolution conditions were achieved using a USP apparatus 1 900 ml of medium containing HCl 0.01 M at a rotation speed of 75 rpm. Under these conditions a significant linear relationship between fraction of drug absorbed versus dissolved was obtained (R(2)=0.984) and a level-A IVIVC was established. The in vitro dissolution samples were analyzed using a HPLC method and the validation was performed according to USP protocol. The method showed accuracy, precision, linearity and specificity within the acceptable range. The discriminatory power of the dissolution method was challenged. The kinetics of dissolution was determined using model-dependent methods. The dissolution profiles were best described by the Hixson-Crowell model. The dissolution test was validated and could be applied to evaluate the dissolution profile of fosamprenavir tablets.

Morfoanatomia das folhas de Dodonaea viscosa Jacq., Sapindaceae / Morph-anatomy of the leaves of Dodonaea viscosa Jacq., Sapindaceae

Dodonaea viscosa Jacq., Sapindaceae, é uma planta tradicionalmente utilizada como antifebril, anti-reumática e antimicrobiana. Neste trabalho foram determinados parâmetros morfo-anatômicos, por análise macro e microscópicas, das folhas de Dodonaea viscosa, com o objetivo de auxiliar sua diagnose como insumo farmacêutico. Macroscopicamente as folhas apresentam limbo com forma lanceolada, margem inteira, consistência áspera e venação eucamptódroma. O pecíolo é curto, reto e em seção transversal é triangular com os ângulos arredondados. Microscopicamente destaca-se a cutícula com formações lenticulares, os tricomas glandulares com quatro células na base, os tricomas tectores unicelulares de ápice afilado, os estômatos higrofíticos dispostos apenas na face abaxial da epiderme, o parênquima paliçádico com até três camadas de células e os feixes vasculares com xilema envolto por floema e cordões de parênquima que ligam um maciço central de células de parênquima a uma bainha de esclerênquima. Estas características morfo-anatômicas, quando analisadas em conjunto, contribuem no controle botânico de qualidade das folhas de Dodonaea viscosa como insumo farmacêutico.

Dodonaea viscosa Jacq., Sapindaceae, is a plant traditionally used as anti fever, anti- rheumatic and antimicrobial. This work determined morpho-anatomical parameters, by macro and microscopic analysis of Dodonaea viscosa leaves, aiming to reach their diagnosis as pharmaceutical input. Macroscopically, the leaves have a lanceolate shape limb, full margin, rough consistence and venation eucamptodromous. The petiole is short, straight and in transversal section it is triangular with round angles. Microscopically, the lenticullar formation cuticle is seen with prominence, the glandular thricomes are seen with four cells on the base, the non-glandular unicellular thricomes are seen with pointed apex, the higrophitic stomatas are disposed in the lower surface epidermis only, the palisade parenchyma show up to three-cell layers and vascular bundles with xylem involved by phloem and parenchyma lines which bound a central mass of parenchyma cells to a sclerenchyma sheath. This morph-anatomical characteristic, when analyzed in group, contributes to the botanical quality control of Dodonaea viscosa leaves as pharmaceutical input.

Development and validation of a discriminating in vitro dissolution method for a poorly soluble drug, olmesartan medoxomil: comparison between commercial tablets.

A dissolution test for tablets containing 40 mg of olmesartan medoxomil (OLM) was developed and validated using both LC-UV and UV methods. After evaluation of the sink condition, dissolution medium, and stability of the drug, the method was validated using USP apparatus 2, 50 rpm rotation speed, and 900 ml of deaerated H(2)O + 0.5% sodium lauryl sulfate (w/v) at pH 6.8 (adjusted with 18% phosphoric acid) as the dissolution medium. The model-independent method using difference factor (f(1)) and similarity factor (f(2)), model-dependent method, and dissolution efficiency were employed to compare dissolution profiles. The kinetic parameters of drug release were also investigated. The obtained results provided adequate dissolution profiles. The developed dissolution test was validated according to international guidelines. Since there is no monograph for this drug in tablets, the dissolution method presented here can be used as a quality control test for OLM in this dosage form, especially in a batch to batch evaluation.

Determination of amlodipine in pharmaceutical dosage forms by liquid chromatography and ultraviolet spectrophotometry.

A liquid chromatography (LC) method and an ultraviolet (UV) spectrophotometric method were developed and validated for quantitative determination of amlodipine in tablets and compounded capsules. The isocratic LC analyses were performed on an RP18 column using a mobile phase composed of 0.1% (v/v) ortho-phosphoric acid (pH 3.0) -acetonitrile (60 + 40, v/v) at a flow rate of 1.0 mL/min. The UV spectrophotometric method was performed at 238 nm. The analytical methods were validated according to International Conference on Harmonization Guidelines. The calibration graphs were linear [correlation coefficient (r) > 0.999] in the studied concentration range of 10-30 microg/mL for LC and 10-35 microg/mL for UV spectrophotometry. The relative standard deviation values for intraday and interday precision studies were less than 2%, and the accuracy was greater than 98% for both methods. The specificity of the LC method was proved using forced degradation. Statistical analyses showed no significant difference between the results obtained by the 2 methods. The proposed methods are precise and accurate and can be applied directly and easily to the oral pharmaceutical preparations of amlodipine.

Liquid chromatographic determination of cetirizine in oral formulations.

The development and validation of a reversed-phase liquid chromatographic (LC) method for the determination of cetirizine dihydrochloride in oral formulations are described. An isocratic LC analysis was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size). The mobile phase was 1% orthophosphoric acid solution, pH 3.0-acetonitrile (60 + 40, v/v), pumped at a constant flow rate of 1.0 mL/min. Measurements were made at a wavelength of 232 nm. The calibration curves were linear over the range of 10-30 microg/mL (r2 = 0.9999). The relative standard deviation (RSD) values for intraday precision were 0.94 and 1.43% for tablets and compounded capsules, respectively. The RSD values for interday precision were 0.13 and 0.82% for tablets and compounded capsules, respectively. Recoveries ranged from 97.7 to 101.8% for tablets and from 98.4 to 102% for compounded capsules. No interferences from the excipients were observed. Because of its simplicity and accuracy, the method is suitable for routine quality-control analysis for cetirizine in tablets and compounded capsules.

Determination of omeprazole in bulk and injectable preparations by liquid chromatography.

An accurate, simple, reproducible, and sensitive liquid chromatographic method was developed and validated for the determination of omeprazole in powder for injection and in pellets. The analyses were performed at room temperature on a reversed-phase C18 column of 250 x 4.6 mm id, 5 microm particle size. The mobile phase, composed of methanol-water (90 + 10, v/v), was pumped at a constant flow rate of 1.5 mL/min. Detection was performed on a UV detector at 301 nm. The method was validated in terms of linearity, precision, accuracy, and ruggedness. The response was linear in the range 32-48 microg/mL (r2 = 0.9976). The relative standard deviation values for intra- and interday precision studies were 1.22 and 1.56% for injectable and 2.13 and 2.45% for pellets, respectively. Recoveries ranged between 95.81 and 100.48%.