Development of an Accurate Double Isotopic Standard LC-MS/MS Method for Hyaluronic Acid Quantification in Biological...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biomaterials Science

Background:

• Hyaluronic acid (HA) is vital for tissue hydration, repair, and cell signaling.
• Accurate HA quantification in biological samples is difficult due to its endogenous nature and low detectability by mass spectrometry.
• Existing methods often struggle with matrix effects and variability.

Purpose of the Study:

• To develop and validate a robust LC-MS/MS method for quantifying hyaluronic acid in complex biological matrices.
• To overcome challenges associated with HA's endogenous presence and poor mass spectrometric detectability.
• To provide a reliable tool for clinical and pharmaceutical applications.

Main Methods:

• Enzymatic hydrolysis of HA using recombinant hyaluronidase to generate quantifiable Δ4-mer oligomers.
• Utilized dual ¹³C-labeled hyaluronic acid internal standards (100% and 50%) to correct for enzymatic digestion and MS detector variability.
• Incorporated the standard addition method to effectively mitigate matrix effects in biological samples like bovine vitreous humor and human synovial fluid.

Main Results:

• The developed method demonstrated excellent linearity (r² > 0.99) and high recovery rates (>90%).
• Achieved low limits of detection (LOD) and quantification (LOQ), suitable for physiological concentrations.
• Significant matrix effects were observed, validating the necessity of the standard addition technique.

Conclusions:

• The validated LC-MS/MS strategy provides a reliable and accurate method for hyaluronic acid quantification in challenging biological samples.
• This method addresses key limitations of previous approaches, enabling more precise measurements.
• The findings support the application of this method in clinical diagnostics and pharmaceutical research involving hyaluronic acid.