(P09-020-24) Absolute Quantification of Polysaccharides in Mushrooms Using LC-MS/MS Platform

Objectives: In this study, we applied FITDOG (Fenton’s initiation toward defined oligosaccharide groups) and a rapid-throughput LC-MS/MS platform to analyze polysaccharide composition in over 100 mushroom samples from 11 cultivars and 20 producers.

Methods: Mushroom samples were lyophilized and homogenized into a fine powder. Sample stock solutions (25 mg/mL) were prepared in nanopure water, precipitated with 80% ethanol, and spiked with isotope-labeled starch as an internal standard. Subject were then subjected to FITDOG reaction, which was an oxidative method using ferric ion and H₂O₂, to depolymerize polysaccharides into oligosaccharides, which serve as a representative of the parent polysaccharide. The oligosaccharide fragments preserve the glycosidic linkage information and act as a fingerprint for the parent polysaccharide. The oligosaccharides then underwent reduction by NaBH₄ and purification by C-18 and PGC cartridges. Oligosaccharides were separated on a PGC column and analyzed via UPLC-dMRM-MS.

Results: A UPLC-dMRM MS method was developed to quantify polysaccharides in a rapid-throughput, 96-well plate format. Nine targeted standards including linear and branched polysaccharides were combined to construct external calibration curves. The coefficient of variation (CV) and bias ranged from 5 to 25 % for triplicates of biological control samples. Standard addition was also evaluated to decrease the matrix effect.

This platform was then applied to over 100 mushroom samples. Amylose and ß-glucan were found to be the top two most abundant polysaccharides present in all mushroom varieties. All data are reported in g/100g fresh weight. The polysaccharide composition of Shiitake mushroom includes: 1.11g amylose, 0.67g ß-glucan, 0.038g cellulose, 0.015g chitin, and 0.010g mannan. Out of 11 cultivars, King Oyster mushroom had the highest average amount of ß-glucan at 4.4g, while Lion’s Mane mushroom had the highest average amount of cellulose at 0.0394g. The amount of amylose ranged from 0.46 to 1.19g across all samples. Analysis like these could be used to provide guidance to incorporate mushroom dietary fiber and aid in the development of new functional foods.

Conclusions: A rapid-throughput LC-MS/MS platform was developed to characterize polysaccharide composition in mushrooms.

Funding Sources: USDA (U.S. Department of Agriculture)