High-coverage identification of hydroxyl compounds based on pyridine derivatization-assisted liquid chromatography...

Hydroxyl compounds are widely present in plants and play essential roles in plant growth and development. High-coverage detection of hydroxyl compounds is crucial for understanding the physiological processes of plants. Despite the prevalence of chemical derivatization-assisted liquid chromatography-high resolution mass spectrometry (CD-LC-HRMS) in high-coverage detection of compounds with diverse functional groups, the confident identification of these compounds after derivatization remains a significant challenge. Herein, a novel method was developed for the identification of pyridine (PY)-derivatized hydroxyl compounds by comparing the MS/MS similarity of derivatized and corresponding underivatized compounds. Fragmentation rules of standards were summarized, and theoretical calculations have demonstrated the MS/MS similarity of PY-derivatized hydroxyl compounds with their underivatized counterparts. The effectiveness of the developed method was demonstrated by identifying PY-derivatized authentic standards. A total of 90 hydroxyl compounds were putatively identified in maize using the proposed method. This method can significantly enhance ionization efficiency with minimal impact on the quality of the MS/MS spectra, enabling the effective utilization of mass spectra databases for the identification of hydroxyl compounds.