Determination of amitriptyline and its main metabolites in human plasma samples using HPLC-DAD: application to the determination of metabolic ratios after single oral dose of amitriptyline

Abstract

Foi desenvolvido e validado um método simples e rápido, empregando CLAE-DAD, para a determinação simultânea de amitriptilina, nortriptilina, E-10-hidroxiamitriptilina, Z-10-hidroxiamitriptilina, E-10-hidroxinortriptilina, Z-10-hidroxinortriptilina e desmetilnortriptilina em amostras de plasma humano. O método utiliza extração líquido-líquido em duas etapas e separação isocrática em fase reversa. A precisão apresentou D.P.R. % menor que 12.1% e a precisão esteve na faixa entre 93,1 e 102,5%. O limite de detecção foi 5 ng mL -1 para todos os analitos. Foram avaliadas razões metabólicas de desmetilação da amitriptilina em indivíduos genotipados para CYP2C19, com nítidas diferenças entre os valores obtidos nos voluntários com zero ou dois alelos ativos. O método é adequado para o monitoramento terapêutico de pacientes em terapia com amitriptilina, permitindo também a indicação da atividade de CYP2C19.A simple and sensitive HPLC-DAD method for the simultaneous determination of amitriptyline, nortriptyline, E-10-hydroxyamitriptyline, Z-10-hydroxyamitriptyline, E-10-hydroxynortriptyline, Z-10-hydroxynortriptyline and desmethylnortriptyline in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation with isocratic elution. Precision assays showed R.S.D % lower than 12.1% and accuracy was in the range of 93.1 to 102.5%. Lowest Limit of detection was 5 ng mL -1 for all analytes. Metabolic ratios of amitriptyline demethylation were evaluated in individuals genotyped for CYP2C19, with clear differences between volunteers with zero or two active alleles. The method is suitable for therapeutic drug monitoring of patients under amitriptyline treatment, also allowing the indication of CYP2C19 activity.

Relation between CYP2C19 phenotype and genotype in a group of Brazilian volunteers

“…Another alternative is the use of metabolic ratios obtained at routine therapeutic drug monitoring analyses, when the correlation of these rations and the phenotype of CYP2C19 are well known (Van der Weide et al, 2005). Recent studies demonstrated the correlation of CYP2C19 phenotype with amitriptyline demethylation metabolic ratios, a reaction also catalyzed by CYP2C19 (Linden et al, 2008).…”

Practical liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of amitriptyline, nortriptyline and their hydroxy metabolites in human serum

“…Levels of AMI and NOR in biofluids have also been studied through other systems, such as gas chromatography (Gupta, Shah, & Hwang, ), but the prevalent procedures in published methods encompass HPLC (Khatoon, Mohd‐Setapar, Ahmad, & Nian‐Yian, ; Plenis & Bączek, ; Samanidou, Nika, & Papadoyannis, ). Linden and colleagues () described a method for the determination of AMI, NOR, desmethylnortriptyline, E‐OH AMI and Z‐OH AMI, and E‐OH NOR and Z‐OH NOR in human plasma samples using HPLC coupled with a diode array detector. The lower limit of quantification (LLOQ) for all compounds evaluated was 5 ng/mL with complete separation achieved in around 22 min and the CYP2C19 genotype correlating with AMI demethylation ratios in healthy volunteers.…”

A novel strategy for spectrophotometric simultaneous determination of amitriptyline and nortriptyline based on derivation with a quinonoid compound in serum samples

“…The proposed method is of great value in determination of the mentioned drugs, because of their improved simplicity and sensitivity, low cost, and free from dependence on expensive instruments and hazardous analytical reagents. [3] …”

“…The formation of the E-10-hydroxy metabolites is dependent on the activity of CYP2D6, with stereospecificity to the (−)-EHAT and (−)-EHNT metabolites. The main metabolic pathways of AT and NT in blood are shown in Scheme 1 [3].…”