Fast and sensitive LC–MS/MS assay for quantification of nortriptyline and its active metabolites E- and Z-10-hydroxynortriptyline in human plasma

Abstract

Background: A fast and sensitive validated assay for nortriptyline, E-10-hydroxynortriptyline and Z-10-hydroxynortriptyline in plasma following a single oral dose of nortriptyline 25 mg was needed to support a clinical study. Results: Plasma samples were prepared by protein precipitation, separated on a C18 column with a mobile phase consisting of 0.1% formic acid in an acetonitrile gradient over 6 min and detected by ESI in the positive mode and MS/MS. Mean recoveries of at least 90% were achieved. The LLOQ was 0.2 ng/ml for nortriptyline and 0.5 ng/ml for the metabolites. The standard curve was linear within LLOQ to 40 ng/ml (r2 ≥ 0.997), precision was under 7.1% coefficient of variance (<16% at LLOQ) and accuracy was 92–114%. Conclusion: A fast and sensitive assay for nortriptyline, E- and Z-10-hydroxynortriptyline in plasma was developed and validated. It has been applied successfully to a clinical study.