Research Article On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed- phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites An assay for the simultaneous analysis of pharmaceutical compounds and their meta- bolites from micro-whole blood samples (i.e.5 mL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat. Keywords: Drug metabolism / HILIC/RP system / LC/MS/MS / On-line DBS / Whole blood DOI 10.1002/jssc.200900593 1 Introduction Simultaneous analysis of polar and non-polar compounds remains a challenge to many applications, such as in the biomedical or pharmaceutical fields. Profiling of phase I and phase II metabolites is an important aspect of drug development since biotransformation can lead to unwanted effects such as enhanced clearance, formation of active or reactive metabolites and drug–drug interactions [1]. Conse- quently, numerous methods have been developed and applied for the simultaneous analysis of drugs and their metabolites using LC/MS [2, 3]. Although reversed-phase (RP) LC is still the most widely used approach, analytical experiments should consider that phase I and II reactions can lead to increased parent drug polarity, which can be problematic because of the non- retention of the more polar metabolites on conventional RP chromatography [4, 5]. However, hydrophilic interaction chromatography (HILIC) provides a good alternative to RP separation since it offers orthogonal separation for polar and ionic compounds [6–9]. In fact, HILIC was shown to be particularly attractive for the separation of phase II meta- bolites such as glucuronide or certain polar drugs [10–13]. Inversely, the problem is that parent drug and phase I metabolites are often too hydrophobic to be retained on these columns, but one interesting approach is to couple these two orthogonal phases into a single system where the polar compounds are eluted on an HILIC column and then the apolar ones are separated on an RP column [14, 15]. LC/MS is the method of choice for drug metabolism analysis due to its high selectivity, sensitivity and efficiency [1], especially with triple-quadruple mass spectrometers. These instruments offer high sensitivity for the quantitative approach (MRM mode) and a powerful tool for compounds Aure ´ lien Thomas 1,4Ã,Ãà Julien De ´ glon 1,4à Thierry Steimer 2 Patrice Mangin 1 Youssef Daali 3 Christian Staub 1,4 1 Unit of Toxicology, University Center of Legal Medicine, Geneva, Switzerland 2 Unit of Clinical Psychopharmacology, Geneva University Hospitals, Geneva, Switzerland 3 Division of Clinical Pharmacology and Toxicology, Geneva University Hospitals, Geneva, Switzerland 4 Swiss Center of Applied Human Toxicology, University of Geneva, Geneva, Switzerland Received September 15, 2009 Revised November 4, 2009 Accepted November 5, 2009 Abbreviations: CH 3 COONH 4 , ammonium acetate; DBS, dried blood spot; HILIC, hydrophilic interaction chromatography à These two authors contributed equally to this work. Ãà Additional corresponding author: Aure ´lien Thomas E-mail: aurelien.thomas@hcuge.ch Correspondence: Christian Staub, Unit of Toxicology, University Center of Legal Medicine, 1 rue Michel Servet, 1211 Gene `ve 4, Switzerland E-mail: Christian.staub@hcuge.ch Fax:141-22-7892417 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2010, 33, 873–879 873