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Microdetermination of hyaluronic acid in human urine by high performance liquid chromatography
Author(s) -
Akiyama Hiroshi,
Toyoda Hidenao,
Yamanashi Shigeyuki,
Sagehashi Yoshinori,
Toida Toshihiko,
Imanari Toshio
Publication year - 1991
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130050502
Subject(s) - chemistry , chromatography , disaccharide , detection limit , high performance liquid chromatography , hyaluronidase , calibration curve , chondroitin , acetonitrile , hyaluronic acid , urine , enzyme , biochemistry , glycosaminoglycan , biology , genetics
A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (ΔDi‐HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of ΔDi‐HA and ΔDi‐OS, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows–column: a stainless steel tube (4 mm i.d. × 250 mm) packed with TSKgel NH 2 ‐60; eluent: a mixture of acetonitrile and 0.1 M Tris‐HCI buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2‐cyanoacetamide in alkaline medium was used for post‐column detection. The calibration curve for ΔDi‐HA was linear in the range 5 pmol‐5 nmol with a practical detection limit of 2 pmol. The assay coefficients of variation ( n = 5) at 200 pmol for ΔDi‐HA and ΔDi‐OS were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.