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

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.