THE USE OF HPLC-MS/MS FOR THE QUANTITATIVE ANALYSIS OF NEUROACTIVE AMINO ACIDS IN RAT BRAIN HOMOGENATES AFTER DERIVATIZATION WITH 9-FLUORENYLMETHYL CHLOROFORMATE

DOI: https://doi.org/10.29296/25877313-2022-05-06
Issue: 
5
Year: 
2022

N.S. Popov
Ph.D. (Pharm.), Head of Research Laboratory, Tver State Medical University (Tver, Russia)
V.Yu. Balabanyan
Dr.Sc. (Pharm.), Associate Professor, Leading Research Scientist, Laboratory of Translational Medicine,
Faculty of Fundamental Medicine, M.V. Lomonosov Moscow State University (Moscow, Russia)
M.B. Petrova
Dr.Sc. (Biol.), Professor, Head of Department of Biology,
Tver State Medical University (Tver, Russia)
E-mail: education@tvgmu.ru
N.Yu. Kolgina
Ph.D. (Med.), Associate Professor, Head of Department of Pharmacology and Clinical Pharmacology,
Tver State Medical University (Tver, Russia)
G.A. Petrov
Ph.D. (Med.), Associate Professor, Department of Pharmacology and Clinical Pharmacology,
Tver State Medical University (Tver, Russia)

Relevance. The effect on the metabolism of neuroactive amino acids is the most important component of the mechanism of action of psychotropic drugs. Variation of substance of amino acids in the structures of the rat brain can act as a pharmacodynamic marker diagnostic sign in the study of the pathogenesis of diseases of the central nervous system. Purpose of the study. development of HPLC-MS / MS method for quantitative determination of neuroactive amino acids in rat brain homogenates after derivatization with 9-fluorenylmethylchloroformate. Material and methods. For the detection of amino acids from the rat brain was used a Potter-Elvehjem homogenizer. Deproteinization and derivatization were performed by adding a solution of 9-fluorenylmethylchloroformate in acetonitrile to the samples. Amino acid derivatives were detected using a Sciex 3200 mass spectrometer. For chromatographic separation was used an Agilent 1260 Infinity II HPLC. Elution was carried out with a mixture of acetonitrile and water in a gradient mode. Results. Sample preparation includes mixing 100 μl of rat brain tissue homogenate, 100 μl of borate buffer, 20 μl of 1 mM norvaline solution and 250 μl of 12 mM Fmoc-Cl solution in acetonitrile, followed by centrifugation for 10 minutes. For the separate Fmoc-derived amino acids was used hromatographic column InfinityLab Poroshell 120 EC-C18 4.6 × 100 mm, 2.7 μm. The total time of chromatographic analysis was 10 minutes, the retention time of Fmoc derivatives of glycine, GABA, aspartic and glutamic acids, asparagine and glutamine was 6.7; 6.8; 6.4; 6.4; 6.2 and 6.1 minutes, respectively. The analytical range of the method for each amino acid was from 0.05 to 50 nmol in 1 ml of homogenate. The method was tested by analyzing the amino acid content in the brain of 6 intact Wister rats. Conclusion. A chromatography-mass spectrometric method for the quantitative determination of glutamine, asparagine, glycine, GABA, glutamic and aspartic acids in rat brain homogenates has been developed. Precolumn derivatization of amino acids with 9-fluorenylmethylchloroformate was carried out to increase the sensitivity of the analysis.

Keywords: 
HPLC-MS/MS
chromatography
mass-spectrometry
neuroactive amino acids.

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