Abstract
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.
Highlights
Amitriptyline (AT), even after 40 years of its introduction still is a leading drug in the treatment of depression, specially in developing countries.[1,2] AT is metabolized mainly by demethylation forming nortriptyline (NT) (Figure 1), and by hydroxylation, leading to the formation of E-10-hydroxy (EHAT) and Z-10hydroxyamitriptyline (ZHAT)
Therapeutic drug monitoring (TDM) in AT pharmacotherapy is usually based on the determination of plasma concentrations of AT and its main active
To the best of our knowledge, there is only one report on simultaneous determination of AT, NT, NNT, EHAT, ZHAT, EHNT and ZHNT in biological samples, which was done by LC-MS/MS.[17]
Summary
Amitriptyline (AT), even after 40 years of its introduction still is a leading drug in the treatment of depression, specially in developing countries.[1,2] AT is metabolized mainly by demethylation forming nortriptyline (NT) (Figure 1), and by hydroxylation, leading to the formation of E-10-hydroxy (EHAT) and Z-10hydroxyamitriptyline (ZHAT). Determination of Amitriptyline and its Main Metabolites in Human Plasma Samples Several analytical methods have been developed for the determination of AT and some of its metabolites in human plasma using either gas,[13,14] or liquid chromatography.[15,16] To the best of our knowledge, there is only one report on simultaneous determination of AT, NT, NNT, EHAT, ZHAT, EHNT and ZHNT in biological samples, which was done by LC-MS/MS.[17] there is no report of a low-cost HPLC-UV method to simultaneously determine AT and its 6 main metabolites.
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