Akiyama H, Shidawara S, Mada A, Toyoda H, Toida T, Imanari T
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
J Chromatogr. 1992 Sep 2;579(2):203-7. doi: 10.1016/0378-4347(92)80383-2.
A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.
已开发出一种灵敏的化学发光高效液相色谱法，用于测定透明质酸、硫酸软骨素和硫酸皮肤素，其方法是将它们作为不饱和二糖-丹磺酰肼衍生物，并采用有效的样品净化系统。由硫酸软骨素酶ABC和/或硫酸软骨素酶ACII作用于透明质酸、硫酸软骨素和硫酸皮肤素得到的不饱和二糖的丹磺酰腙，使用含0.1M醋酸钠缓冲液（pH 6.0）和80%乙腈的混合物，在填充有酰胺-80硅胶珠（直径5微米）的柱（250毫米×4.0毫米内径）上通过反相色谱法进行分离。在化学发光系统中用于柱后洗脱的是，乙腈中的1mM双[2-(3,6,9-三氧杂癸酰氧基羰基)-4-硝基苯基]草酸酯和3mM过氧化氢。每种糖胺聚糖的检测限为100飞摩尔。该方法适用于测定大鼠腹膜肥大细胞中透明质酸、硫酸软骨素和硫酸皮肤素的水平。
