Quantification of Disaccharide Isomers via Porous Graphitic Carbon Chromatography Coupled with Microdroplet Chemistry with Online Tandem Mass Spectrometry

Abstract The structural characterization of isomeric disaccharides remains analytically challenging due to their high polarity, low ionization efficiency, and lack of specificity in fragmentation patterns. Herein, we report a method based on porous graphitic carbon chromatography (PGCC) coupled with contained electrospray ionization (cESI), which enabled online microdroplet chemistry to be performed on disaccharides eluting from the PGCC system to form chloride-saccharide adducts. The adducts were subsequently subjected to tandem mass spectrometry (MS/MS) in real time. This PGCC–cESI–MS/MS platform integrates high-resolution chromatographic separation with specific fragmentation patterns to enable confident differentiation and quantification of native disaccharide isomers in complex mixtures. The platform enabled 15 structurally related (linkage, constitution, and anomeric isomers) disaccharides to be resolved and characterized based on complementary retention-time and diagnostic MS/MS fragmentation patterns. The method demonstrated wide quantitative linearity (R2 = 0.9999), low limits of detection (0.16 μM), good recoveries (101.3–109.1%), accuracy (96.1–104.3%), and high precision (CV ≤ 9.9%) with minimal sample preparation. Application to complex matrices, including mono- and polyfloral honey, beer, apple, milk, and soft drinks, enabled the identification and quantification of known disaccharides, as well as the detection of multiple unknown isomeric species. The integration of chromatographic separation with microdroplet adduction and MS/MS enables rapid, robust, and comprehensive analysis of disaccharides in complex matrices.