Análise de subprodutos de degradação forçada do antibiótico cefalexina
Data
2021-06-01
Autores
Lourenço, Cecília 
Orientadores
da Silva, Emerson Rodrigo
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A cefalexina é um antibiótico beta-lactâmico pertencente à primeira geração de cefalosporinas. Trata-se de um composto com ampla aceitação clínica, eficaz contra uma variedade de infecções bacterianas, cuja comercialização anual supera 13 milhões de unidades apenas no Brasil. A degradação forçada desse medicamento leva a misturas complexas de subprodutos cujo conhecimento é essencial para avaliação de possíveis efeitos sobre a saúde humana e o meio ambiente. Objetivos: Nesta dissertação, o objetivo foi elaborar uma metodologia para análise de subprodutos de cefalexina gerados sob condições extremas de estresse físico-químico. Buscou-se desenvolver e validar um protocolo cromatográfico (HPLC) para dosagem do antibiótico em amostras submetidas à degradação, além da avaliação de métodos de espectrometria de massas (MS) e de ressonância magnética nuclear (RMN) para determinação das espécies geradas. Métodos: Soluções de cefalexina foram submetidas a seis condições de degradação: hidrólise em meio ácido e alcalino, oxidação induzida por H2O2 e CuSO4, termodegradação e fotodegradação. As amostras foram analisadas por LC-MS com ionização via eletrospray para determinação das concentrações da molécula íntegra, e uma listagem de compostos relacionados à cefalexina e aos excipientes usados na formulação de comprimidos foi levantada a partir de dados da literatura. As estruturas moleculares desses compostos foram analisadas computacionalmente para determinar suas massas moleculares e simular os respectivos espectros de RMN de prótons. As amostras foram investigadas experimentalmente por HPLC-MS e RMN e os espectros obtidos foram confrontados com as simulações teóricas. Resultados: O método cromatográfico calibrado permitiu a detecção de concentrações de cefalexina a partir 0.250 mg/ml com precisão de 5.57%. Essas análises indicaram taxas extremamente altas de transformação da molécula, com valores entre 50 e 100% de degradação, tanto em amostras contendo apenas o Insumo Farmacêutico Ativo (IFA) quanto em formulações preparadas com comprimidos comercialmente disponíveis. Análises de MS e RMN permitiram identificar alguns dos principais subprodutos formados, incluindo o ácido cefalosporínico e suas versões descarboxiladas, o grupo ácido 7-desacetoxiaminocefalosporânico, além das formas oxidada e dimérica da cefalexina. Conclusões: Em suma, neste trabalho conseguimos desenvolver e validar um protocolo cromatográfico capaz de dosar concentrações de cefalexina em amostras submetidas a condições extremas de estresse. Além disso, o levantamento de uma listagem com as estruturas moleculares de subprodutos relacionados ao antibiótico mostrou-se uma abordagem inicial bastante útil para auxiliar na identificação de compostos em misturas complexas como as geradas sob condições de degradação forçada.
Introduction: Cephalexin is a beta-lactam antibiotic of the first generation of cephalosporins. It is a compound with wide clinical acceptance, highly efficient against a variety of bacterial infections, with more than 13 million units traded every year in Brazil. The forced degradation of this antibiotic may lead to complex mixtures of by-products which must be known in detail to evaluate potential side effects on both human health and environment. Objectives: In this work, the main goal was to elaborate a methodology to analyze by-products from cephalexin degradation in samples submitted to extreme physicochemical stress. We aimed to develop and validate a chromatographic protocol (HPLC) to quantify the antibiotic concentration in degraded samples. In addition, we evaluated the use of mass spectrometry (MS) and nuclear magnetic resonance (NMR) to determine the species generated upon degradation. Methods: Cephalexin solutions were submitted to six stressing conditions: acid and alkaline hydrolysis, degradation by H2O2 and Cu2+ ions, thermal degradation and photodegradation. Samples were analyzed by LC-MS with electrospray ionization to determine the concentration of cephalexin and a non-exhaustive list of cephalexin-related compounds (alongside excipient products used in the formulation of pills) was collected from data available in literature. The molecular structures of these compounds were computationally analyzed to determine their molecular masses and simulate the corresponding proton NMR spectra. Finally, the samples were experimentally investigated by HPLC-MS and NMR, and the obtained spectra were compared with those from theoretical simulations. Results: The calibrated chromatographic method allowed to detect cephalexin concentration from 0.250 mg/ml with precision of 5.57%. These analyzes indicated extremely high transformation ratios for cephalexin, with values between 50% and 100%, either for samples containing only the API or in formulation prepared with commercially available pills. MS and NMR data allowed to identify a few of the main by products, including cephalosporinic acid and its decarboxylated form, the 7- aminodesacetoxycephalosporanic acid group, and oxidized and dimeric forms of cephalexin. Conclusions: Summarizing, we successfully developed a valid method which was able to quantify cephalexin in samples submitted to extreme stressing conditions. In addition, the production of a non-exhaustive list with the molecular structure of by-products is a potential approach to assist the identification of compounds in complex by-product mixtures such as those generated in forced degradation assays.
Introduction: Cephalexin is a beta-lactam antibiotic of the first generation of cephalosporins. It is a compound with wide clinical acceptance, highly efficient against a variety of bacterial infections, with more than 13 million units traded every year in Brazil. The forced degradation of this antibiotic may lead to complex mixtures of by-products which must be known in detail to evaluate potential side effects on both human health and environment. Objectives: In this work, the main goal was to elaborate a methodology to analyze by-products from cephalexin degradation in samples submitted to extreme physicochemical stress. We aimed to develop and validate a chromatographic protocol (HPLC) to quantify the antibiotic concentration in degraded samples. In addition, we evaluated the use of mass spectrometry (MS) and nuclear magnetic resonance (NMR) to determine the species generated upon degradation. Methods: Cephalexin solutions were submitted to six stressing conditions: acid and alkaline hydrolysis, degradation by H2O2 and Cu2+ ions, thermal degradation and photodegradation. Samples were analyzed by LC-MS with electrospray ionization to determine the concentration of cephalexin and a non-exhaustive list of cephalexin-related compounds (alongside excipient products used in the formulation of pills) was collected from data available in literature. The molecular structures of these compounds were computationally analyzed to determine their molecular masses and simulate the corresponding proton NMR spectra. Finally, the samples were experimentally investigated by HPLC-MS and NMR, and the obtained spectra were compared with those from theoretical simulations. Results: The calibrated chromatographic method allowed to detect cephalexin concentration from 0.250 mg/ml with precision of 5.57%. These analyzes indicated extremely high transformation ratios for cephalexin, with values between 50% and 100%, either for samples containing only the API or in formulation prepared with commercially available pills. MS and NMR data allowed to identify a few of the main by products, including cephalosporinic acid and its decarboxylated form, the 7- aminodesacetoxycephalosporanic acid group, and oxidized and dimeric forms of cephalexin. Conclusions: Summarizing, we successfully developed a valid method which was able to quantify cephalexin in samples submitted to extreme stressing conditions. In addition, the production of a non-exhaustive list with the molecular structure of by-products is a potential approach to assist the identification of compounds in complex by-product mixtures such as those generated in forced degradation assays.