Development and validation of a liquid chromatography–tandem mass spectrometry assay for hair analysis of atomoxetine and its metabolites: Application in clinical practice

Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate.A procedure based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine – 4 hydroxyATX – and N-desmethylatomoxetine – des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2 ml 1 M NaOH at 45 °C. Then, analytes were extracted from alkaline solution with two different 2 ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water–60% 5 mM ammonium acetate, 50 mM formic acid, 4 mM trifluoroacetic acid in acetonitrile–water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring.The method was linear over the concentration range 0.2–50 ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0 ng/mg hair and from 0.3 to 1.0 ng/mg, respectively.Notwithstanding the absence of any dose–hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.