Method development of amine-containing metabolite analysis using di-tert-butyl dicarbonate derivatization coupled with liquid chromatography-mass spectrometry

Abstract

Amine metabolites play crucial roles in biological systems and serve as biomarkers for various diseases. In this study, we developed a cost-effective derivatization method based on N-tert-butyloxycarbonylation (Boc), which is the most affordable among previously reported amine derivatization methods. This environmentally friendly approach enhances the retention and detection of amine metabolites in reversed-phase liquid chromatography-mass spectrometry, with Boc derivatives demonstrating excellent stability for at least 20 days at 4 degrees C and 60 days at -20 degrees C. Importantly, Boc derivatization does not affect the signal intensity or the number of non-amine metabolites, ensuring the method's compatibility with untargeted metabolomics workflows. The effectiveness of this method was validated using immature rice seed extracts from japonica and indica cultivars. Boc derivatization enabled the signal detection of 54 amine metabolites, with comparative analysis identifying 11 significantly differentiated metabolites between the two rice varieties, including histidine, methionine, pipecolate, and GABA. The specificity and robustness of this method confirm its utility for discovering biologically relevant metabolites in complex biological and agricultural samples. With its simplicity, catalyst-free workflow, stability, and improved sensitivity, the Boc derivatization method represents a valuable tool for amine metabolite screening in metabolomics research, offering a cost-effective and efficient solution for untargeted analysis.