Recebido em 26/3/12; aceito em 17/7/12; publicado na web em 7/12/12 A sensitive, accurate and simple method using HPLC-MS/MS was developed and validated for levodopa quantitation in human plasma. Analysis was achieved on a pursuit ® C18 analytical column (5 µm; 150 x 4.6 mm i.d.) using a mobile phase (methanol and water , 90:10, v/v) containing formic acid 0.5% v/v, after extracting the samples using a simple protein plasma precipitation with perchloric acid. The developed method was validated in accordance with ANVISA guidelines and was successfully applied to a bioequivalence study in 60 healthy volunteers demonstrating the feasibility and reliability of the proposed method.
Desenvolvimento de metodologia baseada em HPLC/MS/MS para a determinação de 3-O-metildopa em plasma humano e sua aplicação a estudos de bioequivalência ABSTRACT A simple, sensitive and specific HPLC/MS/MS methodology was developed and it was validated for determining 3-O-methyldopa, the major metabolite of dopamine, in human plasma. The separation was achieved on Atlantis T3 C18 analytical column (5 μm; 150 x 4.6 mm i.d.) using a mobile phase consisted of a solution of water and methanol (85:15, v/v) and containing formic acid 0.05 %. The extraction from the analyte and the internal standard sample was performed using a simple protein plasma precipitation with perchloric acid. The detection was conducted on a triple quadrupole tandem mass spectrometer with a positive multiple reaction monitoring mode (MRM). The monitored fragmentation transitions were m/z 212.0 m/z 166.0 for 3-O-methyldopa and m/z 227.10 m/z 181.0 for carbidopa (internal standard). The calibration curves were linear in the range of 50-4000 ng/mL for 3-O-methyldopa. The methodology presented a good precision and accuracy in accordance to the criteria for biomedical analysis. And it was successfully applied to the bioequivalence study of two formulations levodopa + benserazide (200 + 50 mg) in plasma samples from healthy human volunteers, following the ANVISA guidelines. RESUMOUma metodologia específica, simples e sensível baseado em HPLC/MS/MS foi desenvolvida e validada para realizar a determinação de 3-o-metildopa, o principal metabolito da dopamina, em plasma humano. A separação foi realizada em coluna analítica Atlantis T3 C18 (5 μm; 150 x 4,6 mm i.d.), utilizando-se uma fase móvel composta por uma solução de água e metanol (85:15, v/v), e contendo 0,05 % de ácido fórmico. A extração do analito e da amostra padrão interno foi executada utilizando-se uma simples precipitação proteica no plasma com ácido perclórico. A detecção foi realizada por meio de espectrômetro de massa triplo quadrupolo, em modo de monitoramento de reações múltiplas (MRM) positivo. A transição monitorizada foi m/z 212,0 m/z 166,0 para 3-O-metildopa e m/z 227,1 m/z 181,0 para carbidopa (padrão interno). As curvas de calibração foram lineares na faixa de 50 a 4000 ng/mL para 3-O-metildopa. A metodologia apresentou boa precisão e exatidão de acordo com os critérios para análises biomédicas e esta foi aplicada com sucesso no estudo de bioequivalência de duas formulações contendo levodopa + benserazida (200 + 50 mg) em amostras de plasmas humanos. Palavras-chave. 3-O-metildopa, HPLC-ESI-MS/MS, bioequivalência, plasma, farmacocinética.
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