Ultrasensitive LC-MS analysis of saccharides using 2-hydrazinyl-6-methoxy-1,3-benzothiazole as a novel derivatization reagent

Background Monosaccharides and disaccharides are central components of carbohydrate metabolism, and fluctuations in their abundances are closely associated with disease onset, progression, and diagnostic outcomes. However, accurate analysis of trace-level saccharides in complex biological matrices remains challenging due to their limited chromatographic retention, modest ionization efficiency, and limited MS/MS fragmentation. Derivatization has been widely explored to improve analytical performance in LC-MS-based saccharide analysis; nevertheless, further development of simple, sensitive, and broadly applicable strategies is desirable, particularly for small-volume and low-abundance biological samples. This work presents an efficient derivatization-LC-MS approach enabling ultrasensitive quantification of saccharides under microscale analytical conditions. Results A derivatization strategy based on 2-hydrazinyl-6-methoxy-1,3-benzothiazole (HMBT) was developed to enhance LC-MS analysis of monosaccharides and disaccharides. The mild reaction generated stable derivatives directly compatible with LC-MS. HMBT tagging significantly improved chromatographic retention and separation on conventional C18 columns and markedly enhanced ionization efficiency. The derivatives produced high-intensity precursor ions and a dominant, highly responsive product ion, enabling ultrasensitive MRM detection on a triple-quadrupole system. Using nine representative saccharides, the method achieved excellent quantitative linearity down to 10 pg·mL −1 . Application to mouse serum enabled the quantification of glucose at single-cell-equivalent levels, and spiked galactose showed a linear range of 0.05–100 ng·mL −1 . Importantly, the workflow enabled quantification of five endogenous saccharides in individual HeLa cells. Significance The HMBT derivatization workflow provides an ultrahigh-sensitivity LC-MS platform for saccharide analysis, enabling robust MRM-based quantification at pg·mL −1 levels. Its operational simplicity, minimal sample handling, and full compatibility with conventional LC-MS instrumentation facilitate practical implementation. The demonstrated applicability to true single-cell measurements highlights its potential for glycometabolic studies in microscale biological systems.