RESUMO
Foram desenvolvidos e validados três métodos robustos para a determinação de amoxicilina, norfloxacino, oxcarbazepina (OXC) e 10,11-dihidro-10-hidroxicarbamazepina (MHD) em plasma, utilizando cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) com detecção ultravioleta, fluorescência e espectrometria de massa, respectivamente. Os métodos envolveram extração líquido-líquido, com diclorometano (amoxicilina), acetonitrila (amoxicilina e norfloxacino), éter etílico-diclorometano (60:40 v/v, oxcarbazepina), utilizando cefadroxil, ciprofloxacino e d10-carbamazepina como padrões interno (PI). Separações cromatográficas foram realizadas utilizando colunas Gemini C18 5 μm (150 x 4,6 mm), Synergi RP-MAX 4 μm (150 x 4,6 mm), com sistemas de eluição constituídos 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-água (50:50, v/v) + 20 mM ácido acético, respectivamente. A curvas de calibração foram lineares, nas faixas de 0,5 a 40 μg/mL, 30 a 3500 ng/mL, 20 a 5250 ng/mL e 40 a 10500 ng/ml. As recuperações nas concentrações de 1,5, 15 e 30 μg/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 métodos validados incluíram avaliação de precisão e exatidão intra e interlote, assegurando que estes estavam dentro de limites admissíveis. Os métodos foram então aplicados com sucesso em estudos de bioequivalência, administrando 500 mg ou 400 mg das formulações referência/teste de amoxicilina e norfloxacino, e em estudos farmacocinéticos com formulação de oxcarbazepina suspensão (6%), em voluntários sadios...
Assuntos
Amoxicilina , Cromatografia , FarmacocinéticaRESUMO
A robust method for the determination of norfloxacin in human plasma, using reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection, has been developed. The method involves precipitation of plasma protein with acetonitrile and the use of ciprofloxacin as internal standard (IS). Chromatographic separations were performed on a Synergi MAX-RP 150 x 4.6-mm, 4-micro column with an elution system consisting of a mixture of phosphate buffer-acetonitrile (85:15, v/v). The calibration curve was linear, in the range of 30 to 3500 ng/mL. The recoveries at concentrations of 90, 1400, and 2800 ng/mL were 103.5%, 100.2%, and 100.2%, respectively. The quantification limit for norfloxacin was 30 ng/mL per 10-microL injection employing fluorescence detection with excitation and emission set at 300 and 450 nm, respectively. The method validation included examining the within-run and between-run precision and accuracy and ensuring that these were within accepted limits; in summary, the precision was <8.6% and accuracy ranged from 95.8% to 104.1% for concentration from 90 to 2800 ng/mL. The precision and accuracy for the lowest calibration standard (30 ng/mL) was well within accepted limits for lower limit of quantification. The method was then applied in a bioequivalence study in healthy volunteers given 400-mg doses of reference and test formulations of norfloxacin in random order and including a 7-day washout phase.
Assuntos
Antibacterianos/farmacocinética , Norfloxacino/farmacocinética , Antibacterianos/administração & dosagem , Proteínas Sanguíneas/metabolismo , Calibragem , Cromatografia Líquida de Alta Pressão , Ciprofloxacina/administração & dosagem , Ciprofloxacina/farmacocinética , Estudos Cross-Over , Método Duplo-Cego , Medicamentos Genéricos , Humanos , Indicadores e Reagentes , Norfloxacino/administração & dosagem , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Equivalência TerapêuticaRESUMO
A fast and sensitive method to quantify oxcarbazepine (OXC) and its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine (MHD) in human plasma using HPLC-MS/MS has been developed. The method involved liquid-liquid extraction (LLE), with diethyl ether-diclhoromethane (60:40v/v) using deuterade carbamazepine (d10-carbamazepine) as internal standard (IS). The analytes and IS were separated using an isocratic mobile phase (acetonitrile/water (50:50v/v)+20 mM acetic acid) on the analytical column Phenomenex Luna C18 5 microm (150 mm x 4.6 mm) at room temperature. Detection was performed by a Micromass Quatro LC mass spectrometer in the reaction monitoring mode using positive electrospray ionization (ESI+). The MS-MS ion transition monitored were m/z 253>208 for OXC, m/z 255>194 for MHD and m/z 247>204 for IS. Over the range 20-5250 ng/ml for OXC and 40-10,500 ng/ml for MHD, the calibration curves were defined by the following equations: y = 0.00568 + 0.00296x -5.70e - 8x(2) and y = 0.00749 + 0.00178x - 5.70e - 8x(2) for OXC and MHD, respectively. All coefficient of determination (r(2)) were close to unity (0.9986-0.9994). The lower limits of quantification obtained as a result of the LLE procedure was 20 ng/ml for OXC and 40 ng/ml for MHD. 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 suitability of the assay for pharmacokinetics studies was determined by measuring OXC and MHD concentration after administration of a single 10 ml of OXC oral suspension (6%) in plasma human of healthy volunteers.