Exploring the In Vivo Fate of β-1, 3/1, 6-Glucan Using Quantitative Tandem Mass Spectrometry Based on a Structure-Specific Fragment

β-glucan, a promising drug candidate for immuno-antitumor therapy, holds tremendous potential for clinical applications. However, the absence of highly sensitive quantitative methods for polysaccharides, attributed to their complicated chemical structures and susceptibility to endogenous interference, has posed significant challenges for their clinical development. Here, we report a highly sensitive and reliable analytical strategy for quantifying β-1, 3/1, 6-glucan derived from <i>Durvillaea antarctica</i> (BG136) in various biological matrices. This approach integrates targeted depolymerization and derivatization, followed by oligosaccharide isomer fingerprinting using ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). The absolute quantification of BG136 relied on the abundance of the structure-specific trisaccharide (Glc-β1, 6-Glc-β1, 3-Glc) generated. This methodology not only facilitates prototype-based BG136 administration but also exhibits remarkable sensitivity. Following method optimization and validation, we successfully explored the in vivo fate of BG136 across multiple models, including cellular uptake and release kinetics, as well as preclinical and clinical pharmacokinetics. These achievements provide insight into the "black box" of BG136 from administration to elimination in vivo. This work marks the first practical application of this strategy in complex biological matrices, offering methodological support for the successful execution of the BG136 Phase I clinical trial.