第三章螺旋藻多糖的单糖组成分析 - 仪器信息网.doc

摘 要 高效液相色谱（HPLC）已经成为糖类物质分析的重要工具，但由于糖类物质没有紫外或荧光吸收，需对样品进行柱前衍生使它带上紫外或荧光基团，从而可通过紫外或荧光检测器对其进行检测。由于已有的各种衍生试剂都有衍生反应难控制或质谱检测灵敏度不高的等局限性，不能很好地满足当前糖生物学分析的需要。因此，继续研究开发新的高灵敏衍生化试剂及方法有着重要意义。本论文重点研究了一种新型糖类物质柱前衍生化方法，首次以3-氨基-9-乙基咔唑（3-amino-9-ethylcarbazole，AEC）作为衍生化试剂对糖类物质进行柱前衍生化HPLC分离和MALDI-TOF-MS分析。对反应条件和色谱条件进行了优化，对方法进行了考察，并用该衍生化方法对不同生物样品进行了分析，取得了很好的效果。本研究主要取得了以下的研究成果和结论： 1. 首次将AEC试剂用于标准单糖的柱前衍生化，利用HPLC和MALDI-TOF-MS分析对反应结果进行检测和分析。对反应条件进行了优化，并考察了方法的稳定性、灵敏度、检测限等。结果显示：衍生化方法反应温和（70℃反应60 min）0.06 ng·mL-1；稳定性好，存储5天的样品的相对标准偏差（RSD）3%样品的平均回收率大于98%关键词：糖，柱前衍生，，高效液相色谱，基质辅助激光解析电离质谱Abstract High performance liquid chromatography (HPLC) has become an essential analytical tool of carbohydrates analysis, due to the lack of fluorescent or ultraviolet group, carbohydrates need to be derivatized before HPLC analysis. A lot of derivatization reagents have been used to saccharide analysis, however, some of these derivatizations suffered from the problems including tedious reaction procedures or a signal decreasing by biological mass spectrometry detection that did not meet the demands of modern trace level analyses with regard to sensitivity and/or selectivity. Hence, it’s significant for us to employ a high sensitive derivatization reagent for saccharide analysis in actual work. In our paper, we established a new pre-column derivatization method for saccharides analysis, employing 3-amino-9-ethylcarbazole as a derivatizing reagent. The derivatives were separated by reverse-phase HPLC and identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. We optimized the derivatization reaction as well as the HPLC conditions and validated the described method. Many samples including monosaccharide and oligosaccharides were successfully analyzed by the described method. Finally, we have got these results and achievements as fellows: The standard monosaccharides were detected and analyzed by HPLC and MALDI-TOF-MS, previously derivatized with AEC. We have optimized th