Akiyama H, Toyoda H, Yamanashi S, Sagehashi Y, Toida T, Imanari T
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Biomed Chromatogr. 1991 Sep;5(5):189-92. doi: 10.1002/bmc.1130050502.
A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (delta Di-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of delta Di-HA and delta Di-0S, 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. x 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCl 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 delta Di-HA was linear in the range 5 pmol-5nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for delta Di-HA and delta Di-0S were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.
描述了一种高效液相色谱（HPLC）系统，用于通过用透明质酸酶SD消化来测定人尿中源自透明质酸（HA）的不饱和二糖（δDi-HA）。研究了洗脱液对δDi-HA和δDi-0S分离的影响，δDi-0S源自软骨素与该酶的反应。建立的色谱条件如下——色谱柱：填充有TSKgel NH2-60的不锈钢管（内径4 mm×250 mm）；洗脱液：乙腈与含0.1 M硼酸和10 mM硫酸钠的0.1 M Tris-HCl缓冲液的混合物，pH 7.0（64:36，v/v）。在碱性介质中与2-氰基乙酰胺反应后不饱和二糖的强荧光用于柱后检测。δDi-HA的校准曲线在5 pmol - 5 nmol范围内呈线性，实际检测限为2 pmol。δDi-HA和δDi-0S在200 pmol时的测定变异系数（n = 5）分别为1.7%和1.5%。该HPLC系统已应用于人尿中HA的测定。
