Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/21439 |
Resumo: | Rivaroxaban (RIV) is an oral anticoagulant with the mechanism of action based on the inhibition of the factor Xa of the coagulation cascade. It is clinically used for the prophylaxis and treatment of thromboembolic diseases and nonvalvular atrial fibrillation. In the present research, a stability-indicating micellar electrokinetic capillary chromatography (MEKC) method was validated for the analysis of RIV in tablets dosage forms, and the results were compared to those of the anti-factor Xa bioassay and the reversed-phase high performance liquid chromatography (RP-HPLC) method. Besides, the chiral-HPLC was optimized to separate the enantiomers. The MEKC method was performed on a fused-silica capillary (effective length 40 cm and 50 μm i.d.), maintained at 25ºC, and the separation voltage was 30 kV. The background electrolyte solution consisted of 75 mM MES buffer and 25 mM sodium dodecyl sulphate (SDS) solution at pH 2.0. Injections were carried out using a pressure mode at 50 mbar for 60 s, with detection by a photodiode array detector set at 202 nm. The migration time was 2.81 min and the method was linear over the concentration range of 0.5 – 50 μg/mL (r² = 0.9991). The detection limit (DL) and quantitation limit (QL) were 0.16 μg/mL and 0.54 μg/mL, respectively. Specificity and stability-indicating capability of the method were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.67% with bias lower than 1.60%. The proposed method was applied to the quantitative analysis of RIV in tablet dosage forms and the results were correlated to those of the anti-factor Xa assay and the validated RP–HPLC method showing values 0.66% higher and 0.59% lower, respectively, with non-significant differences (p > 0.05). The results show that the MEKC could constitute an alternative for the analysis of the tablets. Then, the chiral-HPLC was performed to separate the enantiomers, which were subjected to the anti-factor Xa assay and cytotoxicity test, showing low activity for the R-enantiomer and lower toxicity. The results of the physicochemical methods were compared to those of the anti-factor Xa assay, which showed non-significant differences (p > 0.05). Besides, the analytical capability of each method was emphasized, and only the chiral RP-HPLC was able to detect and quantify the enantiomeric forms. In this context, the present research represents a contribution to guarantee the quality, to assure the safety and therapeutic efficacy of the pharmaceutical products, and to establish bases for advances in the study of the generic drug containing RIV. |
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Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabanaStudy of chromatographic, eletroforetic methods and in vitro bioassays for the analysis of rivaroxabanRivaroxabanaCromatografia eletrocinética micelarCromatografia líquida quiralEnantiômerosCromatografia liquida em fase reversaBioensaiosRivaroxabanMicellar electrokineticCapillary chromatographyChiral liquid chromatographyEnantiomersReversed-phase liquid chromatographyBioassayCNPQ::CIENCIAS DA SAUDE::FARMACIARivaroxaban (RIV) is an oral anticoagulant with the mechanism of action based on the inhibition of the factor Xa of the coagulation cascade. It is clinically used for the prophylaxis and treatment of thromboembolic diseases and nonvalvular atrial fibrillation. In the present research, a stability-indicating micellar electrokinetic capillary chromatography (MEKC) method was validated for the analysis of RIV in tablets dosage forms, and the results were compared to those of the anti-factor Xa bioassay and the reversed-phase high performance liquid chromatography (RP-HPLC) method. Besides, the chiral-HPLC was optimized to separate the enantiomers. The MEKC method was performed on a fused-silica capillary (effective length 40 cm and 50 μm i.d.), maintained at 25ºC, and the separation voltage was 30 kV. The background electrolyte solution consisted of 75 mM MES buffer and 25 mM sodium dodecyl sulphate (SDS) solution at pH 2.0. Injections were carried out using a pressure mode at 50 mbar for 60 s, with detection by a photodiode array detector set at 202 nm. The migration time was 2.81 min and the method was linear over the concentration range of 0.5 – 50 μg/mL (r² = 0.9991). The detection limit (DL) and quantitation limit (QL) were 0.16 μg/mL and 0.54 μg/mL, respectively. Specificity and stability-indicating capability of the method were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.67% with bias lower than 1.60%. The proposed method was applied to the quantitative analysis of RIV in tablet dosage forms and the results were correlated to those of the anti-factor Xa assay and the validated RP–HPLC method showing values 0.66% higher and 0.59% lower, respectively, with non-significant differences (p > 0.05). The results show that the MEKC could constitute an alternative for the analysis of the tablets. Then, the chiral-HPLC was performed to separate the enantiomers, which were subjected to the anti-factor Xa assay and cytotoxicity test, showing low activity for the R-enantiomer and lower toxicity. The results of the physicochemical methods were compared to those of the anti-factor Xa assay, which showed non-significant differences (p > 0.05). Besides, the analytical capability of each method was emphasized, and only the chiral RP-HPLC was able to detect and quantify the enantiomeric forms. In this context, the present research represents a contribution to guarantee the quality, to assure the safety and therapeutic efficacy of the pharmaceutical products, and to establish bases for advances in the study of the generic drug containing RIV.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESA rivaroxabana (RIV) é um anticoagulante de uso oral que apresenta mecanismo de ação baseado na inibição do fator Xa (FXa) da cascata de coagulação. É indicada para prevenção e tratamento da trombose venosa profunda (TVP) e para pacientes com fibrilação arterial não-valvar. No presente trabalho foi desenvolvido e validado método por cromatografia eletrocinética micelar (MEKC) para avaliação de RIV em comprimidos, e os resultados foram comparados com aqueles fornecidos pelo bioensaio da atividade antifator Xa e pelo método por cromatografia líquida em fase reversa (CL-FR). Além disso foi otimizado método por cromatografia líquida quiral (CL-quiral) para separação dos enantiômeros. O método por MEKC foi executado com capilar de sílica fundida (40 cm de comprimento efetivo x 50 μm d.i.), mantido a temperatura de 25ºC e a tensão aplicada foi de 30 kV. A solução eletrolítica foi composta de ácido morfolinoetano sulfônico (MES) 75 mM e dodecilsulfato de sódio (SDS) 25 mM, em pH 2,0. O tempo de injeção foi de 60s com pressão de 50 mBar, e detecção por arranjo de diodos (DAD) a 202 nm. O tempo de migração da RIV foi de 2,81 min e a linearidade do método determinada na faixa de concentração de 0,50 − 50 μg/mL (r2 = 0,9991). Os limites de detecção e quantificação foram 0,16 e 0,54 μg/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, e demonstrou-se também, que não houve interferência dos excipientes. A exatidão foi de 100,67%, com erro relativo inferior a 1,60%. A média dos teores encontrados pela aplicação do método proposto foi 0,66% maior e 0,59% menor, em relação aos resultados do bioensaio da atividade antifator Xa e do método por CL-FR, respectivamente, com diferenças não significativas (p > 0,05). Demonstrou-se que o método por MEKC representa alternativa para a análise de comprimidos de RIV. Executaram-se, então, estudos por CL-quiral e os enantiômeros separados foram submetidos a avaliação da atividade biológica e citotoxicidade, observando-se que a forma S é ativa e a R apresenta baixa atividade e menor citotoxicidade. Os resultados obtidos pelos métodos físico-químicos foram comparados com os fornecidos pelo ensaio biológico do antifator Xa, demonstrando que não apresentam diferença significativa (p > 0,05). Além disso, destacou-se a capacidade analítica de cada método, e que somente a cromatografia quiral possibilita a análise qualitativa e quantitativa das formas enantioméricas. Neste contexto, a pesquisa representa contribuição analítica para assegurar a qualidade, contribui para garantir a segurança e eficácia terapêutica dos produtos farmacêuticos, e estabelece bases para avanços no estudo de medicamento genérico contendo RIV.Universidade Federal de Santa MariaBrasilAnálises Clínicas e ToxicológicasUFSMPrograma de Pós-Graduação em Ciências FarmacêuticasCentro de Ciências da SaúdeDalmora, Sergio Luizhttp://lattes.cnpq.br/4505166045049607Malesuik, Marcelo DonadelBajerski, LisianeSilva, Carine VianaManfron, Melânia PalermoWalter, Maurício Elesbão2021-07-14T20:30:41Z2021-07-14T20:30:41Z2019-08-21info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttp://repositorio.ufsm.br/handle/1/21439porAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSM2021-07-27T20:20:31Zoai:repositorio.ufsm.br:1/21439Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2021-07-27T20:20:31Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana Study of chromatographic, eletroforetic methods and in vitro bioassays for the analysis of rivaroxaban |
title |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
spellingShingle |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana Walter, Maurício Elesbão Rivaroxabana Cromatografia eletrocinética micelar Cromatografia líquida quiral Enantiômeros Cromatografia liquida em fase reversa Bioensaios Rivaroxaban Micellar electrokinetic Capillary chromatography Chiral liquid chromatography Enantiomers Reversed-phase liquid chromatography Bioassay CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
title_full |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
title_fullStr |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
title_full_unstemmed |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
title_sort |
Estudo de métodos cromatográficos, eletroforético e biológicos in vitro para análise de rivaroxabana |
author |
Walter, Maurício Elesbão |
author_facet |
Walter, Maurício Elesbão |
author_role |
author |
dc.contributor.none.fl_str_mv |
Dalmora, Sergio Luiz http://lattes.cnpq.br/4505166045049607 Malesuik, Marcelo Donadel Bajerski, Lisiane Silva, Carine Viana Manfron, Melânia Palermo |
dc.contributor.author.fl_str_mv |
Walter, Maurício Elesbão |
dc.subject.por.fl_str_mv |
Rivaroxabana Cromatografia eletrocinética micelar Cromatografia líquida quiral Enantiômeros Cromatografia liquida em fase reversa Bioensaios Rivaroxaban Micellar electrokinetic Capillary chromatography Chiral liquid chromatography Enantiomers Reversed-phase liquid chromatography Bioassay CNPQ::CIENCIAS DA SAUDE::FARMACIA |
topic |
Rivaroxabana Cromatografia eletrocinética micelar Cromatografia líquida quiral Enantiômeros Cromatografia liquida em fase reversa Bioensaios Rivaroxaban Micellar electrokinetic Capillary chromatography Chiral liquid chromatography Enantiomers Reversed-phase liquid chromatography Bioassay CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
Rivaroxaban (RIV) is an oral anticoagulant with the mechanism of action based on the inhibition of the factor Xa of the coagulation cascade. It is clinically used for the prophylaxis and treatment of thromboembolic diseases and nonvalvular atrial fibrillation. In the present research, a stability-indicating micellar electrokinetic capillary chromatography (MEKC) method was validated for the analysis of RIV in tablets dosage forms, and the results were compared to those of the anti-factor Xa bioassay and the reversed-phase high performance liquid chromatography (RP-HPLC) method. Besides, the chiral-HPLC was optimized to separate the enantiomers. The MEKC method was performed on a fused-silica capillary (effective length 40 cm and 50 μm i.d.), maintained at 25ºC, and the separation voltage was 30 kV. The background electrolyte solution consisted of 75 mM MES buffer and 25 mM sodium dodecyl sulphate (SDS) solution at pH 2.0. Injections were carried out using a pressure mode at 50 mbar for 60 s, with detection by a photodiode array detector set at 202 nm. The migration time was 2.81 min and the method was linear over the concentration range of 0.5 – 50 μg/mL (r² = 0.9991). The detection limit (DL) and quantitation limit (QL) were 0.16 μg/mL and 0.54 μg/mL, respectively. Specificity and stability-indicating capability of the method were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.67% with bias lower than 1.60%. The proposed method was applied to the quantitative analysis of RIV in tablet dosage forms and the results were correlated to those of the anti-factor Xa assay and the validated RP–HPLC method showing values 0.66% higher and 0.59% lower, respectively, with non-significant differences (p > 0.05). The results show that the MEKC could constitute an alternative for the analysis of the tablets. Then, the chiral-HPLC was performed to separate the enantiomers, which were subjected to the anti-factor Xa assay and cytotoxicity test, showing low activity for the R-enantiomer and lower toxicity. The results of the physicochemical methods were compared to those of the anti-factor Xa assay, which showed non-significant differences (p > 0.05). Besides, the analytical capability of each method was emphasized, and only the chiral RP-HPLC was able to detect and quantify the enantiomeric forms. In this context, the present research represents a contribution to guarantee the quality, to assure the safety and therapeutic efficacy of the pharmaceutical products, and to establish bases for advances in the study of the generic drug containing RIV. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-08-21 2021-07-14T20:30:41Z 2021-07-14T20:30:41Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/21439 |
url |
http://repositorio.ufsm.br/handle/1/21439 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Análises Clínicas e Toxicológicas UFSM Programa de Pós-Graduação em Ciências Farmacêuticas Centro de Ciências da Saúde |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Análises Clínicas e Toxicológicas UFSM Programa de Pós-Graduação em Ciências Farmacêuticas Centro de Ciências da Saúde |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
collection |
Manancial - Repositório Digital da UFSM |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
atendimento.sib@ufsm.br||tedebc@gmail.com |
_version_ |
1805922099036946432 |