Utility of Authentic ¹³C‐Labeled Disaccharide to Calibrate Hyaluronan Content Measurements by LC‐MS

Hyaluronan (hyaluronic acid, HA), a key glycosaminoglycan in the extracellular matrix, plays crucial roles in various physiological and pathological processes, including development, tissue hydration, inflammation, and tumor progression. Traditional methods for HA quantification, such as ELISA-like assays, often have limitations in sensitivity and specificity, particularly for lower molecular weight HA. In this work, we introduce a coupled liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method that employs a chemoenzymatically synthesized ¹³C-labeled lyase-derived authentic HA disaccharide calibrant for quantification of HA at the nanogram level. The method was validated against three HA polysaccharides with the sizes of ~33, 210, and 540 kDa. We applied this quantification technique to mouse tissues and plasma from both healthy and acetaminophen-induced acute liver injury mice. Our data revealed a ~75-fold increase in HA concentration in the liver of acetaminophen-injured mice with a concomitant depletion from plasma. Overall, our method offers a robust, universal, and highly sensitive tool for HA analysis in diverse biological samples that will advance the investigation of the roles of this polysaccharide in human disease conditions.