title: Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography.
abstract: The profiling analysis of catecholamines and their metabolites in brain tissue offers a crucial key to understand their functions in the body and the opportunity to follow up neural diseases. A rapid and simple liquid chromatography-fluorescence detection (LC-FLD) method was developed and validated for simultaneously measuring several catecholamines and endogenous related compounds in the rat brain tissue samples. The target analytes measured in this bioanalytical assay were levodopa (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-O-methyldopa (3-O-MD), and homovanillic acid (HVA), being the 3,4-dihydroxybenzylamine (DHBA) used as internal standard (IS). The six analytes (L-DOPA, DA, NE, E, 3-O-MD and HVA) can be determined in a single chromatographic run of less than 12min, and all the compounds (analytes and IS) were detected using their native fluorescence and monitored at excitation/emission wavelengths of 279nm/320nm, respectively. The chromatographic and detection conditions were experimentally optimized and then several validation parameters (linearity, limits of quantification and detection, precision and accuracy, recovery, stability and selectivity) were examined. In accordance with the international guidelines of the Food and Drug Administration and European Medicines Agency the method described herein exhibited limits of quantification in the range of 2-25ngmL-1, linearity in wide concentration ranges (r2≥0.994), and acceptable precision (coefficient variation ≤8.76%) and accuracy (bias ±14.65%) levels. Since the bioanalytical procedure does not involve pre-purification or derivatization of the sample, the absolute recovery was found to be around 100%. Moreover, the developed LC-FLD method was successfully applied for the determination of the compounds of interest in tissue samples of different rat brain regions (cerebellum, amygdala, cortex, hippocampus, striatum, mesencephalon, medulla oblongata, substantia nigra and ventral tegmental area). Hence, this assay represents a valuable bioanalytical tool to support several pre(non)clinical studies in the broad field of neurosciences, requiring the quantitative analysis of these bioamines and their metabolites.
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: Beatriz M
authors.initials: BM
authors.lastname: Fonseca
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal; UDI-IPG-Research Unit for Inland Development, Polytechnic Institute of Guarda, 6300-749 Guarda, Portugal.
authors.forename: Márcio
authors.initials: M
authors.lastname: Rodrigues
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Cristóvão
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Pharmacology Department, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Gonçalves
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Pharmacology Department, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Fortuna
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: Liliana
authors.initials: L
authors.lastname: Bernardino
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Pharmacology Department, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Amílcar
authors.initials: A
authors.lastname: Falcão
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal; CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal. Electronic address: gilberto@fcsaude.ubi.pt.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Alves
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002395:Catecholamines / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D002395:Catecholamines
keywords: Bioanalytical method validation; Brain tissue; Catecholamines; Native fluorescence