Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography.

Luis Antonio Suárez-Hernández,Manuel Reinhart Kirchmayr,Eduardo Padilla-Camberos,Enrique Arriola-Guevara,Guadalupe María Guatemala-Morales,Rosa María Camacho-Ruíz,Rosa Isela Corona-González

doi:10.3390/molecules26175360

Abstract

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100–1000 mg/L and 0.011–2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

Highlights

Hyaluronic acid (HA) is a linear polysaccharide composed of a repeated disaccharide formed by D-glucuronic acid and N-acetyl glucosamine, linked by β-(1,4) and β-(1,3) bonds [1]
The concentration and molecular weight (MW) of hyaluronic acid (HA) vary according to the type of animal tissue used for extraction or culture conditions used for microbial production
These results demonstrate that the method complies with the international acceptance criteria that establish a coefficient of variation (CV) < 5%, which means that the method allows identifying and quantifying HA reliably

Summary

Introduction

Hyaluronic acid (HA) is a linear polysaccharide composed of a repeated disaccharide formed by D-glucuronic acid and N-acetyl glucosamine, linked by β-(1,4) and β-(1,3) bonds [1]. The most used methods to estimate HA concentration are based on hydrolysis of the polymer by acid, alkaline, or enzymatic hydrolysis (indirect methods) [8,9,10,11,12,13], which determine the presence of one of its monomers (usually D-glucuronic acid) by photometric methods These methods are susceptible to interference by residual carbohydrates and proteins from the source tissues or microbial processes, and the results are unreliable [14,15]. It is usually connected to a chromatographic system, and it is essential that it is connected to a concentration detector (refractive index or UV) since it does not quantify HA Other methods, such as viscosimetry, gel, capillary electrophoresis, molecular exclusion chromatography, etc., are available to assess the molecular distribution of HA and are recognized as relative methods as they usually require a calibration process [22,23,24,25,26,27,28]. NMR-DOSY, NIR and Rayleigh scattering resonance techniques have been described to determine the molecular distribution of HA [29,30,31], but the equipment is poorly accessible due its availability and high cost [32]

Methods

Results

Conclusion