2022 Volume 69 Issue 1 Pages 17-29
Hyaluronic acid (HA), a high-molecular-weight (MW) polysaccharide, has attracted considerable attention from the public because of its multifunctional health effects. Although the MW of HA is typically over 1 000 kDa, HA of less than 100 kDa (low-MW-HA) is currently manufactured and used in health foods to increase absorbability in humans. However, this has caused confusion in the definition of HA and has made accurate quantification difficult. Thus, a new so-called enzyme-HPLC method focusing on low-MW-HA was developed. Recombinant hyaluronidase was prepared and used to generate HA-disaccharides for quantitative accuracy optimization for low-MW-HA. Additionally, both saturated and unsaturated HA disaccharides were prepared as analytical standards. Further, a post-column-derivatization HPLC method was improved to simultaneously determine the two HA disaccharides following enzyme-hydrolysis of HA. A validation study was carried out by spiking a high-MW-HA standard material to a processed food. The resulting parameters were satisfactory: repeatability 0.8 %; intermediate precision 3.9 %; recovery 93.1 %. The lower quantification limit was inferred to be 0.01 g / 100 g. We also report new findings on the non-hyaluronic partial structures of an artificial low-MW-HA material.
