title: Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC-MS/MS.
abstract: Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.
authors.affiliation: Department of Drug Abuse Research, Domain of Forensic Sciences, Norwegian Institute of Public Health, Oslo, Norway. Electronic address: Marianne.Skov.Skov.Bergh@fhi.no.
authors.forename: Marianne Skov-Skov
authors.initials: MS
authors.lastname: Bergh
authors.affiliation: Department of Drug Abuse Research, Domain of Forensic Sciences, Norwegian Institute of Public Health, Oslo, Norway.
authors.forename: Inger Lise
authors.initials: IL
authors.lastname: Bogen
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway.
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.affiliation: Department of Drug Abuse Research, Domain of Forensic Sciences, Norwegian Institute of Public Health, Oslo, Norway.
authors.forename: Åse Marit Leere
authors.initials: ÅML
authors.lastname: Øiestad
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045604:Extracellular Fluid / Q000737:chemistry*; D057230:Limit of Detection; D008297:Male; D008810:Mice, Inbred C57BL; D018377:Neurotransmitter Agents / Q000032:analysis*; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018377:Neurotransmitter Agents
keywords: Brain tissue; Extracellular fluid; Metabolites; Neurotransmitters; Protein precipitation; UHPLC–MS/MS