Improving LC-MS-Based Marfey’s Analysis on a Single-Quadrupole Platform through Reagent Evaluation and SIM/Scan Acquisition

Abstract Marfey’s method is a widely used approach for determining the absolute configuration of amino acids in peptides and natural products. However, its broader application is often limited by challenges in resolving amino acid epimers and the relatively large amount of analyte typically required for analysis. In this study, we evaluated three commonly used Marfey’s reagents (l-FDAA, l-FDVA, and l-FDLA) for their ability to resolve amino acid diastereomers and investigated whether synchronous selected ion monitoring (SIM)/scan mode in liquid chromatography–mass spectrometry (LC-MS) could improve the sensitivity of LC-MS analysis. Using the cyanobacterial peptide micropeptin 982 (d-Gln) as a representative case study, we evaluated three acquisition modes on a widely available single-quadrupole LC-MS platform and found that synchronous SIM/scan, particularly with time-segmented SIM groups, substantially improved practical sensitivity while preserving scan-mode information. Limits of detection were achieved in the range of 11–16 pmol, and absolute configuration assignments were accomplished using as little as 50 μg of analyte. This method provides a practical strategy for stereochemical analysis of scarce peptide natural products, especially when material needs to be prioritized for downstream biological research.