Desenvolvimento e validação de método bioanalítico por claeuv para determinação da doxiciclina em plasma: Farmacocinética pré-clínica da doxiciclina em coelhos
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Data
2023-12-15
Autores
Gouvea, Igor Edmar Martim
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A doxiciclina (DOX) é um antibiótico bastante utilizado em medicina humana e
veterinária que, em geral, aplica extrapolação empírica dos regimes posológicos em
humano para animais. Esta prática pode levar ao uso inadequado do medicamento,
uma vez que não são consideradas as características fisiológicas próprias da espécie,
contribuindo para o desenvolvimento de resistência bacteriana. No presenteestudo,
foi realizado o desenvolvimento e validação de um método bioanalítico por
cromatografia líquida de alta eficiência (CLAE) para a determinação de DOX em
plasma e realizado um estudo piloto de farmacocinética em coelhos de diferentes
formulações de DOX, a saber: um grupo recebeu DOX em dose única (5 mg/kg) por
IV bolus (n= 3), um grupo recebeu uma nova formulação contendo DOX em dose única
pela via oral na forma de comprimido (25 mg; n=3) e um terceiro recebeu a formulação
disponível no mercado, contendo DOX, em dose única, pela via oral na forma de
comprimido (50 mg; n=3). Foi utilizado um sistema de cromatografia líquidade alta
eficiência (CLAE) com detector ultravioleta-visível (UV-Vis), operando a 357 nm, uma
coluna SunFire C18, fase móvel constituída de solução aquosa de ácido oxálico 5 mM
(pH 2,2), acetonitrila e metanol (65:20:15) com vazão de 0,9 mL/min em modo
isocrático. O volume de injeção foi 40 μL, tempo de corrida de 10 min e a oxitetraciclina
(OXI) foi usada como padrão interno (PI). O processamento do plasmafoi realizado
por extração líquido-líquido com acetato de etila e a validação de acordo com a RDC
nº 27, de 17 de maio de 2012. Os tempos de retenção do PI e fármaco foram de 3 e 6
min, respectivamente. A curva de analítica foi linear na faixa de concentração de
0,003125 – 1,000000 μg/mL, o limite inferior de quantificação (LIQ) foi de 0,003125
μg/mL, com precisão e exatidão intra-dia e inter-dia adequados. Os estudos indicaram
estabilidade do analito em todas as condições avaliadas. Embora não tenham sido
aplicadas as comparações estatísticas em decorrência do pequeno número de
animais estudados, os resultados observados sugerem que a nova formulação deve
melhorar a biodisponibilidade da DOX, demonstrando aspectos promissores para a
continuidade do desenvolvimento do produto com potencial aplicação terapêutica e
eventual liberação de venda no mercado.
Doxycycline (DOX) is an antibiotic widely used in human and veterinary medicine that, in general, applies empirical extrapolation of human dosage regimens to animals. This practice can lead to inappropriate use of the drug, since the physiological characteristics of the species are not considered, contributing to the development of bacterial resistance. In the present study, the development and validation of a bioanalytical method by high performance liquid chromatography (HPLC) for the determination of DOX in plasma and a pilot pharmacokinetic study in rabbits of different DOX formulations were carried out, one group received DOX in dose (5 mg/kg) by IV bolus (n=3), one group received a new formulation containing DOX in a single dose orally in the form of a tablet (25 mg; n=3) and one group received the formulation available in the market containing DOX, in a single oral dose in the form of a tablet (50 mg; n=3). A high performance liquid chromatography (HPLC) system with an ultraviolet-visible detector (UV-Vis) was used, operating at 357 nm, a SunFire C18 column, mobile phase consisting of an aqueous solution of 5 mM oxalic acid (pH 2.2), acetonitrile and methanol (65:20:15) with a flow rate of 0.9 mL/min in isocratic mode. The injection volume was 40 μL, run time was 10 minutes and oxytetracycline (OXI) was used as the internal standard (PI). Plasma processing was performed by liquid-liquid extraction with ethyl acetate and validation according to RDC No. 27, of May 17, 2012. PI and drug retention times were 3 and 6 minutes, respectively. The calibration curve was linear in the concentration range of 0.003125 - 1 μg/mL, the lower limit of quantification (LIQ) was 0.003125 μg/mL, with adequate intra-day and inter-day precision and accuracy. The studies indicated analyte stability under all conditions evaluated. Although statistical comparisons were not applied due to the small number of animals studied, the results observed suggest that a new formulation should improve the bioavailability of DOX, demonstrating promising aspects for the continuity of product development with potential therapeutic application and eventual release for sale. in the market.
Doxycycline (DOX) is an antibiotic widely used in human and veterinary medicine that, in general, applies empirical extrapolation of human dosage regimens to animals. This practice can lead to inappropriate use of the drug, since the physiological characteristics of the species are not considered, contributing to the development of bacterial resistance. In the present study, the development and validation of a bioanalytical method by high performance liquid chromatography (HPLC) for the determination of DOX in plasma and a pilot pharmacokinetic study in rabbits of different DOX formulations were carried out, one group received DOX in dose (5 mg/kg) by IV bolus (n=3), one group received a new formulation containing DOX in a single dose orally in the form of a tablet (25 mg; n=3) and one group received the formulation available in the market containing DOX, in a single oral dose in the form of a tablet (50 mg; n=3). A high performance liquid chromatography (HPLC) system with an ultraviolet-visible detector (UV-Vis) was used, operating at 357 nm, a SunFire C18 column, mobile phase consisting of an aqueous solution of 5 mM oxalic acid (pH 2.2), acetonitrile and methanol (65:20:15) with a flow rate of 0.9 mL/min in isocratic mode. The injection volume was 40 μL, run time was 10 minutes and oxytetracycline (OXI) was used as the internal standard (PI). Plasma processing was performed by liquid-liquid extraction with ethyl acetate and validation according to RDC No. 27, of May 17, 2012. PI and drug retention times were 3 and 6 minutes, respectively. The calibration curve was linear in the concentration range of 0.003125 - 1 μg/mL, the lower limit of quantification (LIQ) was 0.003125 μg/mL, with adequate intra-day and inter-day precision and accuracy. The studies indicated analyte stability under all conditions evaluated. Although statistical comparisons were not applied due to the small number of animals studied, the results observed suggest that a new formulation should improve the bioavailability of DOX, demonstrating promising aspects for the continuity of product development with potential therapeutic application and eventual release for sale. in the market.
Descrição
Palavras-chave
Antibiótico, Doxiciclina, HPLC, Farmacocinética