Whitfield D M, Stojkovski S, Pang H, Baptista J, Sarkar B
Research Institute, Hospital for Sick Children, Toronto, Ontario.
Anal Biochem. 1991 May 1;194(2):259-67. doi: 10.1016/0003-2697(91)90228-l.
A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.
采用带脉冲安培检测（PAD）的高效液相色谱（HPLC）法测定从生物源分离得到的含艾杜糖醛酸寡糖的酸水解产物。将该HPLC - PAD法与气相色谱（GLC）法进行了比较。由于寡糖的酸水解会产生多种产物，通过化学合成制备了几种糖醛酸衍生物。这些经过充分表征的标准品结合质谱法可鉴定出大多数糖胺聚糖的甲醇解或水解产物，其中包括硫酸软骨素A和B（硫酸皮肤素）、肝素和透明质酸。发现对于GLC，在100℃下于甲醇中用（4 M）HCl处理24小时是最佳条件，而对于HPLC - PAD，在100℃下用1 M盐酸水溶液处理4小时是最佳条件。使用这两种技术中的任何一种，都可以在单个色谱步骤中分别鉴定所有的单糖、氨基己糖和糖醛酸。良好的分离度、高灵敏度（低微克级样品）和快速分析使得这些方法对于测定从生物源分离的样品中少量的糖胺聚糖和其他糖缀合物特别有用。这两种技术经过专门设计，可对碳水化合物组成和含量完全未知的样品进行碳水化合物含量和组成的定性测定。
