Journal of Chromatography A, 1443 (2016) 152–161 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma “Heart-cut” bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system Umberto M. Battisti a,b,1 , Cinzia Citti c,d,1 , Martina Larini a , Giuseppe Ciccarella c,d , Natalia Stasiak a , Luigino Troisi c , Daniela Braghiroli a , Carlo Parenti a , Michele Zoli e , Giuseppe Cannazza a,d,∗ a Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy b Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, United States c Department of Biological and Environmental Sciences and Technologies, University of Salento, Via per Monteroni, 73100 Lecce, Italy d CNR-NANOTEC, Campus Ecotekne of the University of Salento, Via per Monteroni, 73100 Lecce, Italy e Department of Biomedical, Metabolic and Neural Sciences, Via Campi 287, 41125 Modena, Italy a r t i c l e i n f o Article history: Received 31 December 2015 Received in revised form 8 March 2016 Accepted 11 March 2016 Available online 14 March 2016 Keywords: Microdialysis Chiral chromatography Bidimensional chromatography Endogenous neurotransmitters a b s t r a c t A “heart-cut” two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrom- etry method (LC–LC–MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4- benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro bio- logical activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the “heart-cut” two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d 4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accu- racy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabo- lites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euth- anizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal. © 2016 Elsevier B.V. All rights reserved. ∗ Corresponding author. E-mail address: giuseppe.cannazza@unimore.it (G. Cannazza). 1 The authors contributed equally to the work 1. Introduction Pharmacokinetic (PK) characterization and in vivo pharmaco- logical properties of new pharmaceutical candidates are relevant components during lead compoundselection and optimization in the drug discovery process. Accordingly, there is a growing inter- http://dx.doi.org/10.1016/j.chroma.2016.03.027 0021-9673/© 2016 Elsevier B.V. All rights reserved.