Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples

The present study describes the development of a fast, affordable and reliable method for hyaluronic acid detection in complex samples. The method involves three principle steps. The first is the separation of hyaluronic acid (HA) from interfering glycosaminoglycans as well as mono- and oligosaccharides by cetyltrimethylammonium bromide fractioning. The second is subsequent digestion of HA with Streptococcus pneumoniae hyaluronate lyase to 4,5-unsaturated disaccharides (Delta HA2). The third is the reaction of Delta HA2 with 3-methyl-2-benothiazolinonehydrazone (MBTH) resulting in an intense blue-colored product. The extinction coefficient of Delta HA2-MBTH product is 34,735 mol(-1) at 654 nm. The theoretical sensitivity of the assay is 0.07-0.09 mg/1 HA. The practical sensitivity is 0.3 mg/1; the highest repeatability was achieved in the range of 3-2000 mg/1 HA (r(2) = 0.9994). The analysis took 25-60 min depending on sample complexity. The described method was evaluated in an industrial setting for online monitoring of HA losses during downstream processes and for HA determination in veterinary products. It was positively rated by users and was introduced to routine laboratory practices.