Development and validation of a method for the quantitative determination of monoamine neurotransmitters and their metabolites in rat brain tissue using HPLC-MS/MS

Abstract

Background. Determining changes in the content of monoamine neurotransmitters and their metabolites in brain structures is a necessary part of studying the pharmacodynamics of  antiparkinsonian drugs. A  method for  the  joint determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue has not previously been developed.The aim of the study. To develop and to validate a method for the quantitative determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue using high-performance liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS).Materials and  methods. A method for  determining monoamine mediators and their metabolites was developed using the HPLC-MS/MS method. Brain tissue homogenates were prepared using a  mechanical hand-operated homogenizer. The  effect of  various antioxidants on  the  stability of  norepinephrine, adrenaline, dopamine and 3,4-dihydroxyphenylacetic acid in the test samples was studied.Results. Chromatographic separation of sample components was carried out using two Synergi Max RP (20 × 2.0 mm, 2.5 µm) and Synergi Fusion RP 80Å (250 × 4.6 mm, 4 µm) chromatographic columns. Elution was carried out in a gradient mode using a  mobile phase based on  methanol and  a  0.1% solution of  formic acid in  water. To prepare homogenate batches, the samples were diluted with a solution of internal standards in methanol. A 5% aqueous solution of ascorbic acid was chosen as an antioxidant stabilizer.Conclusion. The  developed methodology has been fully validated and meets the requirements of Russian and international guidelines. The chosen stabilization method allows samples of brain homogenates to be stored for 30 days after collection.