Publication detail

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

PEPELIAEV, S. HRUDÍKOVÁ, R. SMIRNOU, D. ČERNÝ, Z.

Original Title

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

English Title

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

Type

journal article in Web of Science

Language

en

Original Abstract

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.

English abstract

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.

Keywords

Hyaluronic acid assay; Colorimetry; Glycosaminoglycan precipitation; Streptococcus pneumoniae hyaluronan lyase

Released

14.09.2017

Publisher

PERGAMON-ELSEVIER SCIENCE LTD

Location

OXFORD

ISBN

0014-3057

Periodical

European Polymer Journal

Year of study

94

Number

94

State

GB

Pages from

460

Pages to

470

Pages count

11

URL

Documents

BibTex


@article{BUT165012,
  author="Stanislav {Pepeliaev} and Radka {Hrudíková} and Dzianis {Smirnou} and Zbyněk {Černý}",
  title="Colorimetric enzyme-coupled assay for hyaluronic acid determination in complex samples",
  annote="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.",
  address="PERGAMON-ELSEVIER SCIENCE LTD",
  chapter="165012",
  doi="10.1016/j.eurpolymj.2017.07.036",
  institution="PERGAMON-ELSEVIER SCIENCE LTD",
  number="94",
  volume="94",
  year="2017",
  month="september",
  pages="460--470",
  publisher="PERGAMON-ELSEVIER SCIENCE LTD",
  type="journal article in Web of Science"
}