Volpi Nicola
Dipartimento di Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy.
Electrophoresis. 2003 Mar;24(6):1063-8. doi: 10.1002/elps.200390123.
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.
本文描述了一种快速、高度灵敏且可重复的高效毛细管电泳（HPCE）方法（十二烷基硫酸钠电动色谱法），用于测定致病性大肠杆菌K4细菌（05:K4:H4）多糖及其去果糖基化产物中存在的二糖。在对K4及其衍生物进行软骨素酶消化后，两种二糖，即K4的ΔHexAFrc-GalNAc和去果糖基化K4的δHexA-GalNAc，在未涂层熔融石英毛细管上于20 kV的正常极性下，230 nm波长处检测，20分钟内即可分离并轻松测定。通过在等度强阴离子交换HPLC中分离这两种二糖进行了比较。发现这两种不饱和二糖在很宽的浓度范围内呈线性关系，高效液相色谱（HPLC）约为0.5至5μg，HPCE约为0.06至0.3μg。HPCE分离的检测灵敏度比HPLC高（约高10倍）。所描述的方法用于评估在剧烈酸性条件下K4的去果糖基化过程。两种技术在不饱和二糖的量上具有良好的对应关系。
