Análise de impurezas orgânicas no cloridrato de besifloxacino por cromatografia líquida de alta eficiência com eluição isocrática e gradiente

A análise de impurezas é uma etapa importante no controle de qualidade do insumo farmacêutico e do produto final. Provenientes da síntese do medicamento ou dos excipientes, mesmo em pequenas concentrações, as impurezas podem afetar a eficácia e a segurança. No presente trabalho foram desenvolvidos e validados dois métodos empregando cromatografia líquida de alta eficiência (CLAE) para a avaliação do besifloxacino e sua impureza de síntese, um com eluição isocrática e outro com eluição gradiente. As análises por CLAE com modo de eluição isocrática foram executadas utilizando coluna Ciano, mantida a 25 °C. A fase móvel foi constituída por 0,5% de trietilamina (pH 3,0) : acetonitrila (88:12 v/v), eluída na vazão de 1,0 mL/min com detecção a 330 nm. O método com eluição gradiente foi conduzido com a mesma coluna e componentes da fase móvel modificando apenas as proporções entre fase orgânica e aquosa durante as análises. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados dentro dos limites aceitáveis. Os métodos apresentaram-se lineares na faixa de 140 a 260 μg/mL para o besifloxacino e de 0,3 a 2,3 μg/mL para a impureza A. Com volume de injeção de 20 μL, os limites de detecção e quantificação foram, respectivamente, 0,07 e 0,3 μg/mL. A precisão foi alcançada para todas as análises realizadas, obtendo DPR inter-dia igual a 6,47 e 6,36 para a impureza A com eluição isocrática e gradiente, respectivamente. A exatidão foi superior a 99% e a robustez apresentou resultados satisfatórios. No método isocrático obteve-se tempo de análise de 25 min e no gradiente de 15 min. O número de pratos teóricos no modo isocrático foi na ordem de 5000 enquanto no modo gradiente foi na ordem de 45000, ou seja, obteve-se maior eficiência da coluna com alteração da composição da fase móvel durante a eluição. No insumo besifloxacino e no produto farmacêutico utilizados neste trabalho, impurezas relacionadas estavam presentes, mas a impureza A não foi detectada. Os métodos propostos, considerando-se o limite de quantificação, podem ser aplicados na determinação quantitativa da impureza A na análise da matéria prima do besifloxacino, assim como na suspensão oftálmica.Analysis of impurities is an important step in quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. In the present study two chromatographic methods were developed and validated for high-performance liquid chromatography (HPLC) for the assessment of besifloxacin and its synthesis impurity, one with isocratic and another with gradient elution. Analyses by HPLC in isocratic elution mode were performed using Cyano column maintained at 25 °C. Mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 ml/min with detection at 330 nm. The method with gradient elution was carried out with the same column and mobile phase components modifying only proportion between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, obtaining results within acceptable limits. The methods presented a linear response from 140-260 μg/mL for besifloxacin and from 0.3 to 2.3 mg/mL for impurity. With the injection volume of 20 μL, the limit od detection and limit of quantitation were, respectively, 0.07 and 0.3 μg/mL. Precision was achieved for all analyses, obtaining inter-day RSD equal to 6.47 and 6.36 for impurity A with isocratic and gradient elution, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method was obtained analysis time 25 min and 15 min gradient. The number of theoretical plates in the isocratic mode was of the order of 5000 while in the gradient mode was of the order of 45000, that is, gave greater efficiency of the column by changing the mobile phase composition during elution. In raw material of besifloxacin and pharmaceutical product used in this study, related impurities were present but the impurity A was not detected. The proposed methods, considering the limit of quantitation, can be in quantitative determination of impurity A in the analysis of besifloxacin raw material, as well as in ophthalmic suspension

Development and validation of a dissolution test for empagliflozin in film-coated tablets

The present study proposes a validated dissolution method for empagliflozin (EMPA) in film coated tablets. A gradual in vitro dissolution profile for this formulation was obtained using 900 mL of hydrochloric acid 0.01 M at 37 °C ± 0.5 °C as dissolution medium and USP apparatus 2 (paddle) at 50 rpm. The dissolved percentage of EMPA was quantified by ultraviolet spectrophotometric method to obtain cost technique and produce little residual solvents. Validation parameter for dissolution methodology such as the specificity, linearity, accuracy and precision were evaluated according to the international guidelines, giving results within the acceptable range. The method is linear in the range of 1 - 40 µg/mL, precise, with RSD value less than 2.62%, accurate (mean recovery 106.97%) and robust. Therefore, since no official method has been described, the proposed dissolution conditions represent a relevant contribution to evaluate the dissolution profile of coated tablet containing 25 mg of EMPA

Simultaneous analysis of dapagliflozin and its three related impurities by stability-indicating UPLC method and in vitro toxicity evaluation

A new stability- indicating analytical method by UPLC was developed for the simultaneous determination of dapagliflozin and three of its synthesis impurities. A Waters® Acquity UPLC H- Class model was used for method development and validation. The separation was achieved in a Zorbax phenyl column (50 x 3.0 mm, 1.8 μm), using a mixture of acetonitrile: water (70:30, v/v) as mobile phase in isocratic mode. All the peaks were well detected by a photodiode array detector (PDA) at 230 nm. The method was properly validated according ICH guidelines with respect to linearity, specificity, precision, accuracy and robustness. The calibration curves of each analyte showed determination coefficients (r2) > 0.99 and the method was linear at the concentrations range 30-70 μg/mL for dapagliflozin and 1-10 μg/mL for the impurities. Lastly, this UPLC method presented low limits of detection (LOD) and quantification (LOQ) for both dapagliflozin and impurities, being a technique with high sensitivity. The toxicity evaluation of dapagliflozina and its related impurities were evaluated using 3T3 cells. MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, mitochondrial membrane potential (ΔψM), measurement of intracellular reactive oxygen and DNA damage (measured by comet assay) were evaluated. The impurity 3 showed significant damage in cytotoxicity tests at a concentration of 0.5 µM, being even more expressive at higher concentrations. On the other hand, under the conditions tested, DNA damage was not detected and the compounds tested do not induce significant cell death

In vitro toxic evaluation of two gliptins and their main impurities of synthesis

Background: The presence of impurities in some drugs may compromise the safety and efficacy of the patient’s treatment. Therefore, establishing of the biological safety of the impurities is essential. Diabetic patients are predisposed to tissue damage due to an increased oxidative stress process; and drug impurities may contribute to these toxic effects. In this context, the aim of this work was to study the toxicity, in 3 T3 cells, of the antidiabetic agents sitagliptin, vildagliptin, and their two main impurities of synthesis (S1 and S2; V1 and V2, respectively). Methods: MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, DNA damage (measured by comet assay), intracellular free radicals (by DCF), NO production, and mitochondrial membrane potential (ΔψM) were evaluated. Results: Cytotoxicity was observed for impurity V2. Free radicals generation was found at 1000 μM of sitagliptin and 10 μM of both vildagliptin impurities (V1 and V2). A decrease in NO production was observed for all vildagliptin concentrations. No alterations were observed in ΔψM or DNA damage at the tested concentrations. Conclusions: This study demonstrated that the presence of impurities might increase the cytotoxicity and oxidative stress of the pharmaceutical formulations at the concentrations studied

Aperfeiçoamento e validação de método por cromatografia líquida de alta eficiência para determinação de minociclina na análise da matéria-prima

A Minociclina é um antibiótico semi-sintético de segunda geração de administração oral. Usado no tratamento de infecções genitourinárias, infecções na pele e tecidos moles, pneumonia atípica, entre outros. As farmacopeias americana (USP) e britânica (BP) preconizam o doseamento da matéria-prima minociclina por cromatografia líquida de alta eficiência (CLAE) empregando solventes como tetrahidrofurano (THF), dimetilformamida (DMF) e oxalato de amônio. O objetivo deste trabalho foi propor alterações cromatográficas nos métodos propostos visando substituir modificador orgânico e otimizar o tempo de retenção do fármaco, de modo que o método possua especificidade para emprego em estudos de estabilidade do fármaco. Para isso diversas condições foram testadas e a condição a qual utilizou-se coluna Bridge C8 (250x4,6 mm, 5,0 μm), à temperatura de 40 °C, com fase móvel composta de tampão fosfato de potássio pH 7,0, acetonitrila e trietilamina (TEA) (75:25:0,5; v/v), a uma vazão de 1,0 mL/min foi a que se mostrou mais simples e reprodutível. As amostras foram preparadas na concentração de 200 μg/mL em fase móvel. O método desenvolvido foi validado e apresentou ser preciso (DPR= 1,79); específico frente às condições de estresse a que a amostra foi submetida; linear na faixa de concentração de 25,0 a 500,0 μg/mL, com coeficiente de correlação igual a 0,9999; exato e robusto, indicando ser adequado para o controle de qualidade da matéria prima de minociclina.Minocycline is a second-generation semisynthetic antibiotic of oral administration. It is used in the treatment of genitourinary infections, skin and soft tissue atypical pneumonia and others. The American (USP) and British (BP) pharmacopoeia recommend the determination of minocycline raw material by high performance liquid chromatography (HPLC) using solvents such as tetrahydrofuran (THF) , dimethylformamide (DMF) and ammonium oxalate . The aim of this study was propose changes in the chromatographic method intended to replace the organic modifier and optimize the drug retention time, so that the method has specificity for use in stability studies. For that different conditions were tested and the one that uses Bridge C8 (250x4, 6 mm , 5.0 mM) column at 40 ° C with a mobile phase consisting of potassium phosphate buffer pH 7.0 acetonitrile and triethylamine (TEA) (75:25:0,5 , v/v) at a flow rate of 1.0 ml/min, proved to be the simpler and more reproducible condition. The samples were prepared in a concentration of 200 μg/ml in mobile phase. The method was validated and showed to be precise (RSD = 1.79) ; specific due to the conditions of stress which the sample was subjected ; linear in the concentration range from 25.0 to 500.0 mg/mL , with a coefficient of correlation coefficient of 0.9999, exact and robust , indicating that it is suitable for quality control of minocycline raw material

Development and validation of a dissolution test for empagliflozin in film-coated tablets

The present study proposes a validated dissolution method for empagliflozin (EMPA) in film coated tablets. A gradual in vitro dissolution profile for this formulation was obtained using 900 mL of hydrochloric acid 0.01 M at 37 °C ± 0.5 °C as dissolution medium and USP apparatus 2 (paddle) at 50 rpm. The dissolved percentage of EMPA was quantified by ultraviolet spectrophotometric method to obtain cost technique and produce little residual solvents. Validation parameter for dissolution methodology such as the specificity, linearity, accuracy and precision were evaluated according to the international guidelines, giving results within the acceptable range. The method is linear in the range of 1 - 40 µg/mL, precise, with RSD value less than 2.62%, accurate (mean recovery 106.97%) and robust. Therefore, since no official method has been described, the proposed dissolution conditions represent a relevant contribution to evaluate the dissolution profile of coated tablet containing 25 mg of EMPA

Simultaneous analysis of dapagliflozin and its three related impurities by stability-indicating UPLC method and in vitro toxicity evaluation

A new stability- indicating analytical method by UPLC was developed for the simultaneous determination of dapagliflozin and three of its synthesis impurities. A Waters® Acquity UPLC H- Class model was used for method development and validation. The separation was achieved in a Zorbax phenyl column (50 x 3.0 mm, 1.8 μm), using a mixture of acetonitrile: water (70:30, v/v) as mobile phase in isocratic mode. All the peaks were well detected by a photodiode array detector (PDA) at 230 nm. The method was properly validated according ICH guidelines with respect to linearity, specificity, precision, accuracy and robustness. The calibration curves of each analyte showed determination coefficients (r2) > 0.99 and the method was linear at the concentrations range 30-70 μg/mL for dapagliflozin and 1-10 μg/mL for the impurities. Lastly, this UPLC method presented low limits of detection (LOD) and quantification (LOQ) for both dapagliflozin and impurities, being a technique with high sensitivity. The toxicity evaluation of dapagliflozina and its related impurities were evaluated using 3T3 cells. MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, mitochondrial membrane potential (ΔψM), measurement of intracellular reactive oxygen and DNA damage (measured by comet assay) were evaluated. The impurity 3 showed significant damage in cytotoxicity tests at a concentration of 0.5 µM, being even more expressive at higher concentrations. On the other hand, under the conditions tested, DNA damage was not detected and the compounds tested do not induce significant cell death

The application of quality by design in the development of the liquid chromatography method to determine empagliflozin in the presence of its organic impurities

Analysis of impurities is an important step in the quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. The method was developed following Quality by Design (QbD) for the analysis of the antidiabetic empagliflozin. The concept of QbD is used as a tool for the development of methods and formulations. Through predefined objectives and risk analysis, robust methodologies and reduced solvent consumption are developed. A simple HPLC method was developed and validated for the quantitative determination of empagliflozin and its organic impurities from the synthesis process. The method was carried out in a Shim-pack phenyl column with a mobile phase consisting of an acetonitrile/ water mixture (72 : 28), with isocratic elution and the detector wavelength was 230 nm. The validation process, in accordance with international guidelines, shows that the method was linear, precise and accurate for empagliflozin, impurity 1 and impurity 2. Limits of detection (0.01, 0.02 and 0.01 mg mL 1) and quantification (0.10, 0.10 and 0.05 mg mL 1) were determined for EMPA, IMP1 and IMP2, respectively. The HPLC method for impurity determination in empagliflozin was linear, precise, accurate and robust. It can be successfully applied in the quality control of empagliflozin and the synthesis of impurities, being adequate for routine analysis

The application of quality by design in the development of the liquid chromatography method to determine empagliflozin in the presence of its organic impurities

Analysis of impurities is an important step in the quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. The method was developed following Quality by Design (QbD) for the analysis of the antidiabetic empagliflozin. The concept of QbD is used as a tool for the development of methods and formulations. Through predefined objectives and risk analysis, robust methodologies and reduced solvent consumption are developed. A simple HPLC method was developed and validated for the quantitative determination of empagliflozin and its organic impurities from the synthesis process. The method was carried out in a Shim-pack phenyl column with a mobile phase consisting of an acetonitrile/ water mixture (72 : 28), with isocratic elution and the detector wavelength was 230 nm. The validation process, in accordance with international guidelines, shows that the method was linear, precise and accurate for empagliflozin, impurity 1 and impurity 2. Limits of detection (0.01, 0.02 and 0.01 mg mL 1) and quantification (0.10, 0.10 and 0.05 mg mL 1) were determined for EMPA, IMP1 and IMP2, respectively. The HPLC method for impurity determination in empagliflozin was linear, precise, accurate and robust. It can be successfully applied in the quality control of empagliflozin and the synthesis of impurities, being adequate for routine analysis