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Analysis of Content of 2-Oxoacids in Rat Brain Extracts Using High-Performance Liquid Chromatography

Vadim N. Tashlitsky1, Artem V. Artiukhov2,3, Natalia V. Fedorova2, Maxim A. Sukonnikov1, Alexander L. Ksenofontov2,a*, Victoria I. Bunik2,3,4, and Ludmila A. Baratova2,b*

1Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

2Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

3Department of Biochemistry, Sechenov University, 119991 Moscow, Russia

4Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

* To whom correspondence should be addressed.

Received March 17, 2022; Revised March 19, 2022; Accepted March 20, 2022
2-Oxoacids are involved in a number of important metabolic processes and can be used as biomarkers in some human diseases. A new optimized method for quantification of 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids using high-performance liquid chromatography was developed based on available techniques for quantification of 2-oxoacids in mammalian brain. The use of the 2,4-dinitrophenylhydrazine derivatives of 2-oxoacids was shown to be more advantageous in comparison with the previously used phenylhydrazine derivatives, due to a high chemical stability of the former. Here, we determined the concentrations of pyruvate, glyoxylate, 2-oxoglutarate, 2-oxomalonate, and 4-methylthio-2-oxobutyrate in the methanol/acetic acid extracts of the rat brain using the developed method, as well discussed the procedures for the sample preparation in analysis of mammalian brain extracts. The validation parameters of the method demonstrated that the quantification limits for each of the analyzed of 2-oxoacids was 2 nmol/mg tissue. The developed method facilitates identification of subtle changes in the tissue and cellular content of 2-oxoacids as (patho)physiological biomarkers of metabolism in mammalian tissues.
KEY WORDS: HPLC, 2-oxoacids, rat brain extract, 2,4-dinitrophenylhydrazine, method validation

DOI: 10.1134/S0006297922040058