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.