Перейти
на сайт журнала "Врач" |
Перейти на сайт журнала "Медицинская сестра"
|
Перейти на сайт журнала "Фармация"
|
Перейти на сайт журнала "Молекулярная медицина"
|
Перейти на сайт журнала "Вопросы биологической, медицинской и фармацевтической химии"
|
Журнал включен в российские и международные библиотечные и реферативные базы данных
ВАК (Россия)
|
РИНЦ (Россия)
|
Эко-Вектор (Россия)
|
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
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.
References:
- Federal'nyj zakon ot 12 aprelja 2010 g. № 61 «Ob obrashhenii lekarstvennyh sredstv». M. 56 s. V red. ot 11.06.2021.
- Rukovodstvo po provedeniju doklinicheskih issledovanij lekarstvennyh sredstv. FGBU «NCJeSMP» Minzdravsocrazvitija Rossii. M.: Grif i K, 2012. 944 s.
- Chen X., Broeyer F., de Kam M., Baas J., Cohen A., van Gerven J. Pharmacodynamic response profiles of anxiolytic and sedative drugs. British journal of clinical pharmacology. 2017; 83(5): 1028–1038. doi.org/10.1111/bcp.13204
- Sousa A., Dinis-Oliveira R.J. Pharmacokinetic and pharmacodynamic of the cognitive enhancer modafinil: Relevant clinical and forensic aspects. Substance abuse. 2020; 41(2): 155–173. doi.org/10.1080/08897077.2019.1700584
- Mankar S.S., Turan S.P., Mankar S.S., Shelke, P.A. Antidepressant in animal models of depression and study of cognitive property. GSC Biological and Pharmaceutical Sciences. 2019; 7(3): 64–76. doi.org/10.30574/gscbps.2019.7.3.0069
- Bourin M. Mechanisms of Action of Anxiolytics. Psychiatry and Neuroscience Update. Springer, Cham. 2021: 195–211. doi.org/ 10.1007/978-3-030-61721-9_18
- Piras F., Vecchio D., Assogna F., Pellicano C., Ciullo V., Banaj N. Spalletta G. Cerebellar gaba levels and cognitive interference in parkinson’s disease and healthy comparators. Journal of Personalized Medicine. 2021; 11(1): 16. doi.org/10.3390/jpm11010016
- Li J., Chen L., Guo F., Han X. The Effects of GABAergic System under Cerebral Ischemia: Spotlight on Cognitive Function. Neural Plasticity. 2020. doi.org/10.1155/2020/8856722
- Procter A. W., Bowen D. M. Cerebral Biopsy in the Neurochemical Study of Alzheimer Disease. Alzheimer Disease. CRC Press. 2020: 279–294.
- Srinivasa Rajagopalachari N.K., Shanmugasundaram P. Analytical method validation for the determination of Ninhydrin Positive Substances in amino acids by High Performance Liquid Chromatography. Annals of the Romanian Society for Cell Biology. 2021; 25(6): 4323–4330.
- Uutela P., Ketola R. A., Piepponen P., Kostiainen R. Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry. Analytica chimica acta. 2009; 633(2): 223–231. doi.org/10.1016/j.aca.2008.11.055
- Ziegler J., Hussain H., Neubert R. H. Abel S. Sensitive and selective amino acid profiling of minute tissue amounts by HPLC/electrospray negative tandem mass spectrometry using 9-fluorenylmethoxycarbonyl (Fmoc-Cl) derivatization. Amino Acid Analysis. Humana, New York. 2019: 365–379. doi.org/10.1007/978-1-4939-9639-1_27
- Peng M. Z., Cai Y. N., Shao Y. X., Zhao L., Jiang M. Y., Lin Y. T., Liu L. Simultaneous quantification of 48 plasma amino acids by liquid chromatography-tandem mass spectrometry to investigate urea cycle disorders. Clinica Chimica Acta. 2019; 495: 406-416. doi.org/10.1016/j.cca.2019.05.011
- Minkler P., Stoll M., Ingalls S., Yang S., Kerner J., Hoppel C. Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization-mass spectrometry. Clin. Chem. 2008; 54: 1451–1462. doi.org/10.1373/clin-chem.2007.099226