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Research article2023Peer reviewedOpen access

Structural elucidation of 3-nitrophenylhydrazine derivatives of tricarboxylic acid cycle acids and optimization of their fragmentation to boost sensitivity in liquid chromatography-mass spectrometry

Hodek, Ondrej; Henderson, John; Argemi-Muntadas, Lidia; Khan, Adnan; Moritz, Thomas

Abstract

Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore, there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collision-induced dissociation have not been fully studied before. In our study, we elucidated how annotation of most abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 mu L of human plasma and the targeted method was validated in the same matrix. The same methodology with a modified gradient elution was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling identification of 29 medium-to long-chain fatty acids in human plasma. The TCA carboxylates were also quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quan-tification of the carboxylates in less than 105 cells.

Keywords

Carboxylic acid; Derivatization; Liquid chromatography-mass spectrometry; 3-nitrophenylhydrazine

Published in

Journal of Chromatography B
2023, Volume: 1222, article number: 123719
Publisher: ELSEVIER