Please cite this article in press as: A. de Castro et al., J. Chromatogr. A (2007), doi:10.1016/j.chroma.2007.01.137 ARTICLE IN PRESS +Model CHROMA-347277; No. of Pages 10 Journal of Chromatography A, xxx (2007) xxx–xxx High-throughput on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma Ana de Castro a , Maria del Mar Ram´ ırez Fernandez b,∗ , Marleen Laloup b , Nele Samyn b , Gert De Boeck b , Michelle Wood c , Viviane Maes d , Manuel L ´ opez-Rivadulla a a Forensic Toxicology Service, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain b Federal Public Service Justice, National Institute of Criminalistics and Criminology, Brussels, Belgium c Waters Corporation, MS Technologies Centre, Manchester, UK d Department of Clinical Chemistry-Toxicology, Academic Hospital, Free University of Brussels, Brussels, Belgium Abstract A rapid, sensitive and fully automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 L of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 g/L and the r 2 > 0.99 over the range investigated (10–1000 g/L). Limits of quantification (LOQs) were estimated to be 10 g/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD). © 2007 Elsevier B.V. All rights reserved. Keywords: Antidepressant; Plasma; On-line SPE; LC–MS/MS 1. Introduction Major depressive disorder (MDD) is a condition characterized by a prolonged depression of mood or by a marked loss of interest or pleasure. Depression has received increased attention owing to the growing recognition of its prevalence. For many years, the prevailing hypothesis has been that the condition is caused by (or associated with) a deficiency ∗ Corresponding author at: Federal Public Service Justice, National Institute of Criminalistics and Criminology, Chauss´ ee de Vilvorde 100, 1120 Brussels, Belgium. Tel.: +32 2 240 05 00; fax: +32 2 241 61 05. E-mail address: mariadelmar.ramirezfernandez@just.fgov.be (M.d.M.R. Fernandez). of the monoamines, notably noradrenaline and serotonin; current theories also acknowledge that other factors may be involved in the pathogenesis of depression. Pharmacological treatment for depression has advanced greatly since the development of the first therapies in the 1950s, with the introduction of monoamine oxidase inhibitors (MAOIs) and tricyclic antidepressants (TCAs) [1]. Since the late 1980s, a whole new generation of chemically and neuropharmacologi- cally unrelated agents have been introduced which appear to be safer and better tolerated [2]. These include: selective sero- tonin reuptake inhibitors (SSRIs), serotonin and noradrenaline reuptake inhibitors (SNaRIs), noradrenergic and specific sero- tonergic antidepressants (NaSSAs) and noradrenaline reuptake inhibitors (NaRIs) [3]. 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.01.137