High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biomedical Science

Background:

• Glycosaminoglycans (GAGs) are crucial heteropolysaccharides with complex structures dictating biological functions.
• Accurate, high-throughput quantification of GAGs in biofluids is vital for biomedical research and diagnostics.
• Current GAG quantification methods are often hindered by complex sample preparation, low throughput, and insufficient accuracy due to structural variability.

Purpose of the Study:

• To develop and validate a streamlined, high-throughput method for extracting and quantifying diverse GAGs in biofluids.
• To enable precise measurement of 17 disaccharides from major GAG classes, including chondroitin sulfate, heparan sulfate, and hyaluronic acid.
• To provide a scalable and reproducible protocol suitable for clinical laboratory settings.

Main Methods:

• Enzymatic depolymerization of GAGs from biofluid samples.
• Derivatization of released GAGs using 2-aminoacridone for enhanced detection.
• Quantification via ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-MS/MS) on a 96-well plate format.

Main Results:

• The developed UHPLC-MS/MS method allows for the simultaneous quantification of 17 GAG-derived disaccharides.
• The protocol is designed for 96-well plates, processing up to 82 samples per run with high precision and reproducibility.
• Sample preparation to MS analysis completion takes approximately 1.5 days, plus 15 minutes of MS runtime per sample, fitting standard working hours.

Conclusions:

• This UHPLC-MS/MS protocol offers a robust, scalable, and accurate solution for high-throughput GAG analysis in biofluids.
• The method's efficiency and precision make it a valuable tool for clinical laboratories aiming to utilize GAGs as biomarkers.
• The streamlined workflow overcomes limitations of existing methods, facilitating broader application in biomedical research and diagnostics.