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

Authors :
A. L. Khokhlov
I. I. Yaichkov
M. K. Korsakov
I. N. Kagramanyan
N. N. Volkhin
S. S. Petukhov
V. E. Zaikova
Source :
Acta Biomedica Scientifica, Vol 9, Iss 1, Pp 177-191 (2024)
Publication Year :
2024
Publisher :
Scientific Сentre for Family Health and Human Reproduction Problems, 2024.

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.

Details

Language :
Russian
ISSN :
25419420 and 25879596
Volume :
9
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Acta Biomedica Scientifica
Publication Type :
Academic Journal
Accession number :
edsdoj.09aee0671c34e3a8eb3a3cf56e928e8
Document Type :
article
Full Text :
https://doi.org/10.29413/ABS.2024-9.1.18