# Development of an Accurate Double Isotopic Standard LC–MS/MS Method for Hyaluronic Acid Quantification in Biological Matrices

## TL;DR

Researchers developed a precise method to measure hyaluronic acid in biological samples using isotopic standards and mass spectrometry.

## Contribution

A novel LC–MS/MS method with dual 13C-labeled internal standards for accurate hyaluronic acid quantification in complex matrices.

## Key findings

- The method achieved excellent linearity (r² > 0.99) and low detection limits in bovine vitreous humor and human synovial fluid.
- High recovery (>90%) and accuracy were confirmed, with significant matrix effects controlled via the standard addition method.
- Measured hyaluronic acid concentrations aligned with physiological ranges, validating the method's reliability.

## Abstract

Hyaluronic acid (HA)
plays key roles in tissue hydration, repair,
and cellular signaling. Its quantification in biological matrices
is crucial but challenging due to its endogenous nature and poor mass
spectrometric detectability. We developed a robust method based on
enzymatic hydrolysis, dual 13C-labeled internal standards,
and the standard addition method combined with LC–MS/MS analysis.
Samples from bovine vitreous humor and human synovial fluid were depolymerized
with recombinant hyaluronidase to generate Δ4-mer oligomers,
quantified using 100%- and 50%-1
3C-labeled HA
as internal standards to correct the variabilities of the enzymatic
digestion and MS detector. The standard addition method was used to
control the matrix effects. The method showed excellent linearity
(r
2 > 0.99), low estimated LOD (0.147
± 0.007 μg/mL for bovine vitreous humor and 0.143 ±
0.028 μg/mL for human synovial fluid) and LOQ (0.491 ±
0.022 μg/mL for bovine vitreous humor and 0.475 ± 0.093
μg/mL for human synovial fluid) values, high recovery (>90%),
and suitable accuracy. Significant matrix effects were detected, reinforcing
the need for the standard addition method. HA concentrations measured
were consistent with physiological ranges. This validated strategy
offers a reliable tool for HA quantification in complex biological
samples, supporting both clinical and pharmaceutical applications.