The measured precursor masses of carboxylic-EDC (O-acylisourea) and carboxylic-BNMA conformed to the corresponding theoretical masses and molecular structures (Table and Figure ). Both labeling products are highly sensitive in positive ESI mode because the tertiary amine structure gives a permanent positive charge. The carboxylic-EDC intermediates (m/zcarboxylic-EDC) were detected without the 4-BNMA labeling step, confirming the successful EDC activation. By adding 4-BNMA, the intermediates were transformed completely to carboxylic-BNMA, and no carboxylic-EDC was detected. In AIF mode, the carboxylic-BNMA precursor ions produced the product ion pair of m/z 168.965 and 170.963 in high abundance, which agrees with the fragmentation of biological carboxylic acids obtained by Bryce et al. Based on the 4-BNMA molecular structure, m/z 168.965 and 170.963 were assigned to phenyl bromine ions 79 BrC7H6 + and 81 BrC7H6 + , respectively. The chromatographic retention time of those 4-BNMA derivatives was also determined by analysis of the acid standards mixture using LC-Orbitrap MS (Table , Figure ).