title: "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.
abstract: A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry 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 biological 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-d4 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, accuracy 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 metabolites 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 euthanizing 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.
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy; Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, United States.
authors.forename: Umberto M
authors.initials: UM
authors.lastname: Battisti
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Via per Monteroni, 73100 Lecce, Italy; CNR-NANOTEC, Campus Ecotekne of the University of Salento, Via per Monteroni, 73100 Lecce, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Citti
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Larini
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Via per Monteroni, 73100 Lecce, Italy; CNR-NANOTEC, Campus Ecotekne of the University of Salento, Via per Monteroni, 73100 Lecce, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ciccarella
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy.
authors.forename: Natalia
authors.initials: N
authors.lastname: Stasiak
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, Via per Monteroni, 73100 Lecce, Italy.
authors.forename: Luigino
authors.initials: L
authors.lastname: Troisi
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Braghiroli
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Parenti
authors.affiliation: Department of Biomedical, Metabolic and Neural Sciences, Via Campi 287, 41125 Modena, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Zoli
authors.affiliation: Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 103, 41125 Modena, Italy; CNR-NANOTEC, Campus Ecotekne of the University of Salento, Via per Monteroni, 73100 Lecce, Italy. Electronic address: giuseppe.cannazza@unimore.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cannazza
mesh_terms: D000109:Acetylcholine / Q000737:chemistry; D000818:Animals; D001581:Benzothiadiazines / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002490:Central Nervous System / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D016503:Drug Delivery Systems; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017551:Microdialysis; D015203:Reproducibility of Results; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry
chemical_list: D001581:Benzothiadiazines; C006709:2H-1,2,4-benzothiadiazine-1,1-dioxide; D000109:Acetylcholine
keywords: Bidimensional chromatography; Chiral chromatography; Endogenous neurotransmitters; Microdialysis