Derivatization agents for LC-MS analysis of amino acids: effects of core structure and functional groups

Abstract The analysis of amino acids in biological samples is challenged by their high polarity, low ionization efficiency, and lack of chromophores, which limit detection by LC-UV or LC-MS. Derivatization is widely used to improve retention, sensitivity, and detection. In this study, derivatization agents based on pyridine, quinoline, and isoquinoline positional isomers scaffolds with COCl, SO₂Cl, and NHS ester reactive groups were systematically evaluated using deuterium-labeled amino acids to assess stability, derivatization efficiency, chromatographic behavior, and MS response. NHS ester-based agents were found to exhibit superior stability, maintaining activity for over 1 year, whereas carbonyl chlorides and sulfonyl chloride–based agents were highly reactive but less stable. NHS ester of isoquinoline-6-carboxylic acid (6-CiQ-NHS) was identified as the most effective agent, combining rapid derivatization, strong MS signal, and stability. LC-MS analysis demonstrated excellent linearity ( R 2 ≥ 0.995), low nanomolar detection limits (0.23–6.33 nM), and separation of isomeric amino acids (e.g., isoleucine/leucine). 6-CiQ-NHS is proposed as a practical derivatization approach for amino acid quantification and provides a framework for the rational design of future agents. Graphical Abstract