Enantioselective analysis of fluoxetine and norfluoxetine in plasma samples by protein precipitation and liquid chromatography with fluorescence detection

Abstract

Neste trabalho, o método de precipitação de proteínas e cromatografia líquida de alta eficiência com detecção de fluorescência (PP/CLAE-fluorescência) foi desenvolvido para a análise enantioseletiva da fluoxetina e norfluoxetina em amostras de plasmas. Diferentes agentes precipitantes, como solventes orgânicos, ácidos e sais, em diferentes proporções, foram avaliados. Segundo testes calorimétricos de Bradford, dentre todos os agentes precipitantes avaliados, a acetonitrila apresentou maior eficiência, considerando o número de etapas requeridas e a porcentagem de precipitação de proteínas (99,7%), quando adicionado na proporção de 3:1 (agente precipitante: plasma, v/v). O método PP/CLAE-fluorescência apresentou limite de quantificação de 30 ng mL −1 e intervalo linear de 30 a 1000 ng mL −1 com coeficientes de regressão linear acima de 0,9973 para os quatro enantiômeros. Em conclusão, o método PP/CLAE-fluorescência padronizado e validado proposto neste estudo foi aplicado com sucesso na análise de amostras de plasma de pacientes em terapia com a fluoxetina.In this study, the enantioselective analysis of fluoxetine and norfluoxetine in plasma samples was performed by the protein precipitation method and high performance liquid chromatography with fluorescence detection (PP/LC-FD). Different precipitating agents -organic solvents, acids, and salts -in several proportions were available. The Bradford colorimetric method employed for evaluation of the efficiency of protein precipitation, has shown that for the sake of simplicity and percentage of protein precipitation (99.7%), acetonitrile was most effective when added at a ratio of 3:1 (acetonitrile/plasma, v/v). The quantification limit of the PP/LC-FD method was 30 ng mL −1 for the four enantiomers. The response of the proposed method was linear over a dynamic range from LOQ to 1000 ng mL −1 , with correlation coefficients higher than 0.9973. In conclusion, the PP/LC-FD method can be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine.

Analytical methodologies for the stereoselective determination of fluoxetine: An overview

“…Bueno, Silvia, and Quieroz () performed an enantioselective analysis method of FLX and NFLX in human plasma samples using protein precipitation method and an HPLC method with fluorescence detection. Separation was carried out by RP‐LC, using Chiralcel OD‐R as chiral stationary phase.…”