FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Foram desenvolvidos e validados trÃs mÃtodos robustos para a determinaÃÃo de amoxicilina, norfloxacino, oxcarbazepina (OXC) e 10,11-dihidro-10-hidroxicarbamazepina (MHD) em plasma, utilizando cromatografia lÃquida de alta eficiÃncia em fase reversa (RP-HPLC) com detecÃÃo ultravioleta, fluorescÃncia e espectrometria de massa, respectivamente. Os mÃtodos envolveram extraÃÃo lÃquido-lÃquido, com diclorometano (amoxicilina), acetonitrila (amoxicilina e norfloxacino), Ãter etÃlico-diclorometano (60:40 v/v, oxcarbazepina), utilizando cefadroxil, ciprofloxacino e d10-carbamazepina como padrÃes interno (PI). SeparaÃÃes cromatogrÃficas foram realizadas utilizando colunas Gemini C18 5 Âm (150 X 4,6 mm), Synergi RP-MAX 4 Âm (150 X 4,6 mm) e Luna C18 Âm (150 X 4,6 mm), com sistemas de eluiÃÃo constituÃdos por mistura de tampÃo fosfato de 0,01 M (pH 3,5)/acetonitrila (95:5 v/v), acetonitrila-tampÃo fosfato (85:15, v/v) e acetonitrila-Ãgua (50:50 v/v) + 20 mM Ãcido acÃtico, respectivamente. As curvas de calibraÃÃo foram lineares, nas faixas de 0,5 a 40 Âg/mL, 30 a 3500 ng/mL, 20 a 5250 ng/ml e 40 a 10500 ng/ml. As recuperaÃÃes nas concentraÃÃes de 1,5, 15 e 30 Âg/ml foram de 59,4%, 60,5% e 67,1% para amoxicilina; 90, 1400 e 2800 ng/mL foram de 103,5%, 100,2% e 100,2% para norfloxacino, 60, 2000 e 4000 ng/ml foram de 105,4, 89,2 e 92,8% para OXC e 120, 4000 e 8000 ng/ml foram de 88,4, 88,7 e 90,6% para MHD, respectivamente. Os mÃtodos validados incluÃram avaliaÃÃo de precisÃo e exatidÃo intra e interlote, assegurando que estes estavam dentro de limites admissÃveis. Os mÃtodos foram entÃo aplicados com sucesso em estudos de bioequivalÃncia, administrando 500 mg ou 400 mg das formulaÃÃes referÃncia/teste de amoxicilina e norfloxacino, e em estudos farmacocinÃticos com formulaÃÃo de oxcarbazepina suspensÃo (6%), em voluntÃrios sadios. / A robust method for the determination of amoxicillin, norfloxacin, oxcarbazepine (OXC) and its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine (MHD) in human plasma, using reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet, fluorescence and mass spectrometry detection, respectively, have been developed and valited. The methods involve precipitation of plasma protein with dichloromethane (amoxicillin), acetonitrile (amoxicillin and norfloxacin) and diethyl etherâdiclhoromethane (60:40 v/v, oxcarbazepine), using cefadroxil, ciprofloxacin and deuterade carbamazepine (d10-carbamazepine) as internal standard (IS). Chromatographic separations were performed on a column Gemini C18 5 Âm (150 X 4.6 mm), Synergi MAX-RP 4 Âm (150 X 4.6 mm) and Luna C18 5 Âm (150mm X 4.6 mm) with an elution system consisting of a mixture of 0.01 M buffer phosphate (pH 3.5)/acetonitrile (95:05 v/v), phosphate bufferâacetonitrile (85:15, v/v) and acetonitrile/water (50:50 v/v) + 20mM acetic acid, respectively. The calibration curve was linear, in the range of 0.5 to 40 Âg/mL, 30 to 3500 ng/mL, 20 to 5250 ng/mL and 40 to 10,500 ng/mL. The recoveries at concentrations of 1.5, 15 and 30 Âg/mL were foram 59.4%, 60.5% and 67.1% for amoxicillin; 90, 1400 and 2800 ng/mL were 103.5%, 100.2% and 100.2% for norfloxacin, 60, 2000 and 4000 ng/mL were 105.4, 89.2 and 92.8% for OXC and 120, 4000 and 8000 ng/mL were 88.4, 88.7 and 90.6% for MHD, respectively. The statistical evaluation of the developed method was conducted by examining within-batch and between-batch precision data, which were within the required limits. The methods were successfully applied in bioequivalence studies given 500 or 400-mg of the reference formulation / test amoxicillin and norfloxacin, and pharmacokinetic studies with formulation of oxcarbazepine suspension (6%) in healthy volunteers.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:2964 |
| Date | 29 July 2009 |
| Creators | Ismael Leite Martins |
| Contributors | Maria Elisabete Amaral de Moraes, Maria Bernadete de Sousa Maia, Gilmara Silva de Melo Santana, Gisela Costa CamarÃo, Francisco Vagnaldo Fechine Jamacaru |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Farmacologia, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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