Análise químico-farmacêutica de cloridrato de cefepima em pó liofilizado para solução injetável
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Data
2017-05-19
Autores
Rodrigues, Danilo Fernando [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O surgimento de micro-organismos resistentes a um grande número de antimicrobianos clinicamente aprovados vem sendo cada vez mais frequente, o que limita as opções para o tratamento das infecções bacterianas. Como estratégia, fármacos com elevadas atividades antimicrobianas encontram-se em evidência. Destaca-se uma classe de antimicrobianos, a das cefalosporinas, tendo como quarta geração a cefepima (CEF), um produto semi-sintético que apresenta atividade contra várias bactérias Gram-positivas (ex. Staphylococcus aureus sensível à oxacilina) e Gram-negativas (ex. Pseudomonas aeruginosa) aeróbias. Seu mecanismo de ação é similar ao dos outros β-lactâmicos, ou seja, inibe a síntese da parede celular bacteriana, ligando-se a uma ou mais proteínas de ligação das penicilinas (penicillin-binding proteins - PBPs). Nesse contexto, pesquisas inovadoras envolvendo métodos analíticos são de fundamental importância e altamente relevantes para otimizar sua análise na indústria e garantir a qualidade do produto já comercializado. Análises qualitativas foram utilizadas e desenvolvidas durante o trabalho, a fim de promover a identificação do fármaco. Nesta pesquisa também foram desenvolvidos e validados métodos para quantificação de CEF em pó liofilizado para solução injetável. Dentre eles, o ensaio microbiológico pelo método turbidimétrico foi validado utilizando a cepa Staphylococcus aureus ATCC 6538, sendo linear na faixa de 36,0 a 81,0 μg/mL, onde apresentou potência de 102,46% e exatidão de 99,92%. O método por cromatografia líquida de alta eficiência (CLAE) foi validado utilizando como fase móvel somente água purificada e etanol (45:55, v/v), no comprimento de onda () de 258 nm, apresentando faixa linear de 10 a 70 μg/mL e tempo de retenção (tR) igual a 4,99 minutos. O doseamento apresentado por CLAE foi de 106,31% e a exatidão obteve um valor de 100,08%. Ensaios por espectrofotometria com absorção nas regiões do ultravioleta (UV) e do visível (VIS) tiveram seus parâmetros validados para a quantificação de CEF, empregando água purificada como solvente nos comprimentos de onda de 258 e 740 nm, respectivamente. O método por UV apresentou linearidade na faixa de 10 a 20 μg/mL, com teor de 103,81% e exatidão de 100,80%. O método no VIS foi desenvolvido em escala miniaturizada, diminuindo o uso de materiais e solventes, bem como o tempo de preparo das análises. A linearidade do método no VIS foi apresentada na faixa de 15 a 40 μg/mL e o valor de teor obtido foi de 104,26%. Por fim, outro método desenvolvido e validado foi por espectrofotometria de absorção na região do infravermelho (IV), o qual não utilizou solventes orgânicos para os ensaios, característica importante na redução de resíduos. O teor de CEF obtido pelo método de IV foi de 103,86%, dentro da faixa linear apresentada nas concentrações de 0,2 a 0,6 mg/pastilha de brometo de potássio (KBr) e com valor resultante da exatidão de 100,83%. Análises de variância (ANOVA) foram realizadas para os métodos validados, comprovando a segurança e a confiabilidade dos resultados obtidos. Análises de espectrometria de massas foram realizadas para detectar possíveis produtos após a degradação de CEF. Estudos de estabilidade de curta e longa duração de CEF em pó liofilizado também foram realizados, resultando em perdas nas porcentagens dos teores, ficando dentro do limite descrito na literatura. Assim, todos os métodos desenvolvidos e validados neste trabalho são voltados à química verde e recomendados para o controle de qualidade de CEF em formulação farmacêutica.
The emergence of resistant microorganisms to a large number of clinically approved antimicrobials has been increasingly used, which limits the options for the treatment of bacterial infections. As a strategy, drugs with high antimicrobial activities are in evidence. Stands out a class of antimicrobial, the cephalosporins, having as fourth generation cefepime (CEF), a semi- synthetic product which has activity against various Gram-positive bacteria (e.g. oxacillin resistant Staphylococcus aureus) and Gram-negative (e.g. Pseudomonas aeruginosa) aerobic. Its mechanism of action is similar to other β-lactams, that mean, inhibits bacterial cell wall synthesis by binding to one or more penicillin-binding proteins - PBPs. In this context, innovative research involving analytical methods present fundamental importance and it is highly relevant to optimize its analysis in routine pharmaceutical industry and ensure quality of product already marketed. Qualitative analyzes were developed and used during work in order to promote the identification of the drug. In this research were also developed and validated methods for the quantification of CEF in lyophilised powder for solution for injection. Among them, microbiological test by turbidimetric method was validated using Staphylococcus aureus ATCC 6538 strain, being linear in the range from 36.0 to 81.0 μg.mL-1, which has potency of 102.46% and 99.92% accuracy. The method using high performance liquid chromatography (HPLC) was performed using as mobile phase only purified water and ethanol (45:55, v/v) at a wavelength () of 258 nm, showed the linear range in 10 to 70 μg.mL-1 and retention time (tR) equal to 4.99 minutes. The determination shown by HPLC was 106.31% and the accuracy obtained a value of 100.08%. Assays by spectrophotometry with absorption in the regions of ultraviolet (UV) and of visible (VIS) had their validated parameters to quantification CEF using purified water as solvent in the wavelengths of 258 and 740 nm, respectively. The method by UV showed linearity in the range of 10 to 20 μg.mL-1, with content of 103.81% and accuracy of 100.80%. The VIS method was developed on miniaturized scale, reducing the use of materials and solvents, as well as the time of prepare of the analyzes. The linearity of the VIS method was presented in the range of 15 to 40 μg.mL-1 and the content value obtained was of 104.26%. Lastly, another method was developed and validated by spectrophotometry with absorption in the region of infrared (IR), which did not use organic solvents for the analysis, important feature in reducing residues. The CEF content obtained by the IR method was 103.86%, within the linear range shown in concentrations of 0.2 to 0.6 mg/pellet of potassium bromide (KBr) and with value resulting of accuracy of 100.83%. Analysis of variance (ANOVA) were performed for validated methods, proving the safety and reliability of the results obtained. Analyses of mass spectrometry were performed to detect possible products after CEF degradation. The accelerated and long term stability studies of CEF in lyophilized powder also were performed, decreasing the percentages of the contents, but in according with the limit described in the literature. Thus, all the methods developed and validated in this work are turned on green chemistry and recommended for the quality control of CEF in pharmaceutical formulation.
The emergence of resistant microorganisms to a large number of clinically approved antimicrobials has been increasingly used, which limits the options for the treatment of bacterial infections. As a strategy, drugs with high antimicrobial activities are in evidence. Stands out a class of antimicrobial, the cephalosporins, having as fourth generation cefepime (CEF), a semi- synthetic product which has activity against various Gram-positive bacteria (e.g. oxacillin resistant Staphylococcus aureus) and Gram-negative (e.g. Pseudomonas aeruginosa) aerobic. Its mechanism of action is similar to other β-lactams, that mean, inhibits bacterial cell wall synthesis by binding to one or more penicillin-binding proteins - PBPs. In this context, innovative research involving analytical methods present fundamental importance and it is highly relevant to optimize its analysis in routine pharmaceutical industry and ensure quality of product already marketed. Qualitative analyzes were developed and used during work in order to promote the identification of the drug. In this research were also developed and validated methods for the quantification of CEF in lyophilised powder for solution for injection. Among them, microbiological test by turbidimetric method was validated using Staphylococcus aureus ATCC 6538 strain, being linear in the range from 36.0 to 81.0 μg.mL-1, which has potency of 102.46% and 99.92% accuracy. The method using high performance liquid chromatography (HPLC) was performed using as mobile phase only purified water and ethanol (45:55, v/v) at a wavelength () of 258 nm, showed the linear range in 10 to 70 μg.mL-1 and retention time (tR) equal to 4.99 minutes. The determination shown by HPLC was 106.31% and the accuracy obtained a value of 100.08%. Assays by spectrophotometry with absorption in the regions of ultraviolet (UV) and of visible (VIS) had their validated parameters to quantification CEF using purified water as solvent in the wavelengths of 258 and 740 nm, respectively. The method by UV showed linearity in the range of 10 to 20 μg.mL-1, with content of 103.81% and accuracy of 100.80%. The VIS method was developed on miniaturized scale, reducing the use of materials and solvents, as well as the time of prepare of the analyzes. The linearity of the VIS method was presented in the range of 15 to 40 μg.mL-1 and the content value obtained was of 104.26%. Lastly, another method was developed and validated by spectrophotometry with absorption in the region of infrared (IR), which did not use organic solvents for the analysis, important feature in reducing residues. The CEF content obtained by the IR method was 103.86%, within the linear range shown in concentrations of 0.2 to 0.6 mg/pellet of potassium bromide (KBr) and with value resulting of accuracy of 100.83%. Analysis of variance (ANOVA) were performed for validated methods, proving the safety and reliability of the results obtained. Analyses of mass spectrometry were performed to detect possible products after CEF degradation. The accelerated and long term stability studies of CEF in lyophilized powder also were performed, decreasing the percentages of the contents, but in according with the limit described in the literature. Thus, all the methods developed and validated in this work are turned on green chemistry and recommended for the quality control of CEF in pharmaceutical formulation.
Descrição
Palavras-chave
CLAE, Cloridrato de cefepima, Controle de qualidade, Ensaio microbiológico, Espectrofotometria, Estabilidade, Métodos analíticos, Produtos de degradação, Validação, Analytical methods, Cefepime hydrochloride, Degradation products, HPLC, Microbiological assay, Quality control, Spectrophotometry, Stability, Validation