A New, Fast, and Sensitive UPLC‐ESI‐MS/MS Method for the Simultaneous Analysis of 13 Neurotransmitters in Rat Brain

Neurotransmitters are crucial for the chemical signaling in the central nervous system. The present work proposes a novel UPLC-ESI-MS/MS method for simultaneously determining 13 neurotransmitters in the amygdala brain region in rats. A Zorbax SB C18 column and a gradient mobile phase consisting of a mixture of water and acetonitrile comprising 0.2% heptafluorobutyric acid and 0.1% formic acid were used. By the ICH guideline, outstanding linearity of the method (R² > 0.99), detectors' sensitivity, and specificity are demonstrated with low LOD (0.011 pg/mL for endorphins to 2.69 ng/mL for serotonin) and LOQ (0.06 for endorphins to 14 ng/mL for serotonin) values. The recovery ranged from 94% for anandamide to 98% for fatty acid amide hydrolase. Following the static test validation process, aspects of the method such as accuracy, precision, matrix effect, and stability were confirmed, with each neurotransmitter assigned retention times ranging from 1 to 4.3 min (GABA and endorphins, respectively). In the context of brain regions, the technique allows comprehensive neurochemical analysis in the amygdala and is a step forward for neurochemistry understanding. It opens up a brand-new approach to studying brain disorders, providing brain operation and its pathologies in an elaborate matrix in an extremely short time.