Development and Full Validation of a Novel Liquid Chromatography Electrochemical Detection Method for Simultaneous Determination of Nine Catecholamines in Rat Brain

INTRODUCTION: Chemical neurotransmission, managed by neurotransmitters, has a crucial role in brain processes such as fear, memory, learning, and pain, or neuropathologies such as schizophrenia, epilepsy, anxiety/depression, and Parkinson’s disease. The measurement of these compounds is to elucidate the disease mechanisms and evaluate the outcomes of therapeutic interventions. However, this can be quite difficult due to various matrix effects and the problems of chromatographic separation of analytes. In the current study; for the first time, an optimized and fully validated fully according to FDA and EMA Bioanalytical Validation Guidance HPLC-EC method was developed for the simultaneous analysis of nine neurotransmitter compounds which are dopamine (DA), homovanilic acid (HVA), vanilmandelic acid (VA) serotonin (SER), 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG), norepinephrine (NE), 3,4 dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) and simultaneously determined in rat brain samples. METHODS: The separation was achieved with 150 mm × 4.6 mm, 2.6 μm F5 Kinetex (Phenomenex, USA) column isocratically, and analysis was carried out HPLC equipped with DECADE II electrochemical detector. RESULTS: The method exhibited good selectivity and correlation coefficient values for each analyte’s calibration curves were >0.99. The detection and quantification limits ranged from 0.01 to 0.03 ng/mL and 3.04 to 9.13 ng/mL, respectively. The stability of the analytes and method robustness were also examined in detail in the study, and the obtained results are also presented statistically. DISCUSSION AND CONCLUSION: The developed fully validated method has been successfully applied to real rat brain samples and important results have been obtained. In the rat brain sample analysis, the least amount of SER and the highest amount of NA were found.