김민주 (한국과학기술연구원)

이재익 (한국과학기술연구원)

김민주 (한국과학기술연구원)

강민식 (한국과학기술연구원)

이민규 (한국과학기술연구원)

이지현 (한국과학기술연구원)

오한빈 (서강대학교)

차상원 (동국대학교)

이재익 (한국과학기술연구원)

Di- and tri-carboxylic acids containing metabolites are important targets in central carbon and organic acid metabolism, but LC–MS analysis is often limited by poor retention and asymmetric peaks. This study evaluated the effects of column type, mobile-phase additives and a metal chelator on the chromatographic behavior of these metabolites.

Nine dicarboxylic acids and three tricarboxylic acids were analyzed using a stainless-steel reversed-phase column and a PEEK monolithic column. Mobile phases containing 2 mM ammonium acetate, ammonium formate, or ammonium fluoride and 0.2% acetic acid or formic acid were compared based on retention, peak width, and tailing factor. Column performance depended on mobile-phase composition. Ammonium salt conditions generally produced sharp peaks for dicarboxylic acids but showed greater tailing for tricarboxylic acids, whereas acidic conditions produced broader but more symmetric peaks. Subsequently, ammonium fluoride and formic acid with 5 µM medronic acid (MDP) as metal chelator were investigated. Effects of MDP depended on analyte structure and mobile phase composition.In conclusion, ammonium fluoride produced sharp but early-eluting peaks, while formic acid with medronic acid provided a better balance between retention and peak symmetry. These findings provide fundamental chromatographic information for establishing LC–MS conditions for organic acid metabolites in biological samples.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-NR076398).