Development of a HPLC/MS/MS methodology for determining 3-O-methyldopa in human plasma and its application in a bioequivalence study

Abstract

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.

Analysis of 2′-hydroxyflavanone (2HF) in mouse whole blood by HPLC–MS/MS for the determination of pharmacokinetic parameters

“…In this study, naringenin was used as an internal standard (IS). 2HF and Naringenin belong to the same flavonoid class, with similar chemical structures, and demonstrated similar HPLC–MS/MS parameters and retention times, 3.76 and 2.23 min for naringenin and 2HF, respectively ( Martins et al, 2000 ; 2013 ).…”

Development and Validation of a Quantification Method for L-DOPA in Plants and Pharmaceutical Materials

“…Therefore, it is recommended that doses of L-Dopa be modified according to the patient's individual needs based on clinical response and adverse event profile [1]. Several analytical methods for the determination of L-Dopa and its metabolites in biological matrices have been identified in the literature using high-performance liquid chromatography and various detection techniques such as electrochemical detection [8][9][10][11][12][13][14][15][16], tandem mass spectrometry (MS-MS) [17][18][19][20][21][22], and fluorescence [23,24]. Most of the above-mentioned methods have a common low sensitivity limitation and time consuming chromatographic separation.…”