Simple method for simultaneous quantification of two new derivatives of betulinic and ursolic acids with antimalarial activity by RP-HPLC-UV-CAD

Endemic in more than 90 countries and territories, malaria is the most widely, populational, and geographically, parasitic disease in the world. Plasmodium sp. resistance to available drugs is one of the biggest problems for malaria eradication. In this study, we develop a method for the simultaneous determination of two new derivatives of betulinic and ursolic acids with antimalarial activity designated 3-O-butanoylbetulinic and 3-O-butanoylursolic acids. An analytical method was developed by high-performance liquid chromatography coupled, in series, to ultraviolet (UV) and charged aerosol (CAD) detectors. The chromatographic system, operated isocratically by reversed-phase, consisted in a mobile phase composed of acetonitrile: water pH 3.0 adjusted with formic acid (85:15, v/v), flow rate of 1.2 mL/min and a PhenoSphere Next octadecylsilane column (250 mm x 4.6 mm, 5 μm particle size). Chromatograms were recorded simultaneously in UV and CAD, at a concentration of 50 µg mL-1, an injection volume of 20 μL at a controlled temperature of 50 °C. The method was found to be selective, linear (r > 0.99), precise (RSD < 2.0%), accurate, and robust for both analytes, and considered statistically validated, and can be applied to the identification and quantification of these new drug candidates

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

Desenvolvimento de método analítico para avaliação de impurezas orgânicas de ticagrelor

O ticagrelor é um novo fármaco destinado para a prevenção de síndromes coronarianas agudas (SCA) caracterizadas pela formação de placas ateroscleróticas que se rompem no interior das artérias. Atua de forma diferenciada na inibição da ação plaquetária, inibindo de forma reversível e não competitiva o receptor P2Y12, o qual atua diretamente na agregação das plaquetas. Este medicamento está registrado no Brasil desde 2011, sob nome comercial de Brilinta®, na forma de comprimidos revestidos de 90 mg, e, até o presente momento, são poucos os estudos publicados a respeito do controle de impurezas e produtos de degradação do fármaco. No presente trabalho foi desenvolvido um método por cromatografia líquida de alta eficiência (CLAE) adaptado para a análise de impurezas orgânicas do ticagrelor. O método por CLAE foi estabelecido utilizando equipamento Agilent 1200 Series, acoplado a detector de arranjo de diodos (DAD) em 270 nm, com coluna cromatográfica Zorbax Plus C8 (150 x 4,6 mm, 5,0 μm), fluxo de 0,7 ml/min, volume de injeção de 20 μl e temperatura constante de 25ºC. A fase móvel foi composta de acetonitrila: acetato de amônio 50 mM (57:43, v/v), pH 8,2. O método demonstrou ser específico, linear, preciso, exato e robusto, sendo adequado para a finalidade a que foi proposto. Além disso, foram elucidados os principais produtos de degradação originados quando as amostras dos comprimidos de ticagrelor em solução foram expostas à radiação UVC por duas horas, com base em um estudo preliminar de estabilidade do mesmo. Os mecanismos envolvidos na degradação fotolítica do ticagrelor também foram propostos.Ticagrelor is a new drug intended for the prevention of acute coronary syndromes (ACS) characterized by the formation of atherosclerotic plaques that rupture inside the arteries. Ticagrelor reversibly binds to P2Y12 receptor, which acts directly on platelet aggregation. The drug was approved in Brazil in 2011, under the comercial name Brilinta® in the form of coated tablets of 90 mg, and until now has few published studies about the impurities control and degradation products. In this study, it was developed a method by high-performance liquid chromatography (HPLC) for the analysis of organic impurities of ticagrelor. The HPLC method was established using Agilent 1200 Series equipment coupled to photodiode array detector (PDA) at 270 nm with a Zorbax Plus C8 column (150 x 4.6 mm, 5.0 μm), flow rate of 0.7 ml/min, injection volume of 20 μL, and a constant temperature of 25°C. The mobile phase consisted of acetonitrile: ammonium acetate 50 mM (57:43 v/v), pH 8.2. The method demonstrated to be specific, linear, precise, accurate and robust, being suitable for the intended purpose. Besides, the mayor degradation products formed when samples of ticagrelor tablets in solution were exposed to UVC radiation for two hours were elucidated based on a preliminary stability study. The mechanisms involved in photolytic degradation of ticagrelor have also been proposed

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

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