JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2007; 42: 1342–1347 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jms.1288 Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry Claudete Alves, Alvaro J. Santos-Neto, Christian Fernandes, Jos ´ e C. Rodrigues and Fernando M. Lan ¸ cas ∗ Universidade de S ˜ ao Paulo, Instituto de Qu ´ ımica de S˜ ao Carlos, Laborat ´ orio de Cromatografia, Av. Trabalhador S ˜ aocarlense, 400, 13560-970, S˜ ao Carlos – SP – Brasil Received 6 September 2006; Accepted 16 July 2007 Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptylin and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 µm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 °C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 ° C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96–98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm × 2.1 mm, 5 µm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plas Copyright  2007 John Wiley & Sons, Ltd. KEYWORDS: LC-MS; SPME; tricyclic antidepressants; plasma; factorial planning INTRODUCTION Tricyclicantidepressants (TCAs) have been widely pre- scribed in the treatment of psychiatric disorders such as depression,mainly endogenous majordepression.These drugs have also been frequently encountered in emergency toxicology screening, drug abuse, and forensic medical exam- inations. TCAs act inhibiting the reuptake of norepinephrine (as is the case of desipramine, nortriptyline, and protripty- line secondary amines) and serotonin (as isthe case of amitriptyline, imipramine, clomipramine, and doxepine ter- tiary amines) in the central nervous system. 1 – 7 The recentliterature describes several studies on the improvementof analyticalsensitivity forthe determina- tion of drugs,including TCAs in biological fluids. High- performance liquid chromatography and high-resolution gas chromatography (HPLC and HRGC) are regarded as the ref- erence techniques for that application. The hyphenation of a mass spectrometer to a chromatograph has been widely exploited once it combines the advantages of chromatogra- phy (high selectivity and separation efficiency) with those Ł Correspondence to: Fernando M. Lan¸ cas, Universidade de S ˜ ao Paulo, Instituto de Qu ´ ımica de S ˜ ao Carlos, Laborat ´ orio de Cromatografia, Av. Trabalhador S ˜ aocarlense, 400, 13560-970, S ˜ ao Carlos – SP – Brasil. E-mail: flancas@iqsc.usp.br of mass spectrometry (structural information and further increase in selectivity). This combination, especially LC-MS, has been successfully applied to analyze pharmaceuticals in biological matrices. Additionally,LC-MS with electrospray ionization (LC-ESI-MS) has become a powerful analytical tool for the determination ofpolar and/or thermolabile compounds. 8 – 10 Sample preparation – which includes extraction,pre- concentration and cleanup – is mostly required fordrug analysis in biofluids. Liquid–liquid extraction (LLE), solid- phase extraction (SPE), column switching approach and, more recently, solid-phase microextraction (SPME) and stir bar sorptive extraction (SBSE) have been adopted for that purpose. 10 – 17 SPME, introduced by Pawliszyn’s group in 1990, presents some advantages when compared with other sample prepa- ration techniques, such as savings in solvent consumption, ease of operation, and speed.Moreover,it allows reusing the fibers and obtaining cleaner extracts. In SPME, the thin polymeric layer coated on the fused-silica fiber is exposed into the sample or its headspace. During exposure, the ana- lytes present in the sample matrix and with chemical affinity for the SPME fiber coating are sorbed and retained on the fiber. 18 – 26 The on-line coupling of SPME and LC-MS permits higher sensitivity when compared to the off-line desorption. Copyright  2007 John Wiley & Sons, Ltd.