Sensitive and Fast Detection of Monoamines and Their Metabolites by High-Performance Liquid Chromatography Coupled with an Electrochemical Detector (HPLC-ECD) Under Isocratic Conditions: Application to Intracerebral Microdialysis in Mice Treated by Fluoxetine and Atomoxetine

Abstract

Neurotransmitters (NTs) play a crucial role in brain function and associated with various neurological and neuropsychiatric disorders. This study devised an optimized analytical method, using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD), to concurrently assess homovanillic acid (HVA), epinephrine (E), norepinephrine (NE), serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA), melatonin (MT), and dopamine (DA) in mouse brain tissue. The mobile phase composition was fine-tuned to achieve efficient separation of these compounds, with optimal conditions involving 5% acetonitrile, 10% methanol, and 85% aqueous phase containing phosphate buffer, citric acid, sodium dodecyl sulfate (SDS) and ethylenedinitrilotetraacetic acid (EDTA). The pH of the mobile phase was adjusted to 3.2. An amperometric module was employed for electrochemical detection, with potential optimization to enhance sensitivity. The developed method exhibited excellent linearity and sensitivity, with limit of detection (LOD) and limit of quantification (LOQ) values lower than nmol L⁻¹. The method was applied to an intracerebral microdialysis experiment in mice hippocampus, demonstrating the capability to monitor changes in NTs and their metabolites in response to systemic fluoxetine/atomoxetine administration. This study presents a reliable and sensitive analytical approach to investigating NTs dynamics, which could contribute to a deeper understanding of neurotransmission under normal and pathological conditions.