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

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%-¹³C-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 (<i>r</i>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.