以高效能液相層析串聯質譜儀分析半乳寡醣之結構特徵

Abstract

半乳寡醣(Galacto-oligosaccharides, GOS)為一種益生質(Prebiotic)，其結構上由數個半乳糖以β構型鍵結形成，聚合度(Degree of polymeration, DP)為二至八。由轉醣反應(Transgalactosylation)而來的半乳寡醣為一系列之鍵結異構物(Linkage isomers)，因為分析方法的困難以致不同鍵結的半乳寡醣對其益生質特性的相關性尚無法建立，因此發展不同結構之半乳寡醣分析方法是瞭解其生物性質的基礎。論文先期以分子篩層析法與2-胺基吡啶(2-Aminopyridine)標記衍生化配合毛細管電泳技術，分析6種市售半乳寡醣的聚合度分佈，發現樣品中聚合度小於6的寡醣，其中有3種樣品不含有五醣與六醣，所有樣品均以三醣為主要的寡醣。 本論文選擇對胺基苯甲酸乙酯(p-Aminobenzoic acid ethyl ester, ABEE)對半乳寡醣進行發色團標記衍生化(Chromophore-labeling derivatization)，除了使寡醣帶有紫外光吸收特性，亦能幫助寡醣於液相層析的分離。在半乳寡醣之鍵結異構物的定性上以質譜之碰撞引致裂解(Collision-induced dissociation, CID)反應，在負離子偵測模式下分別以m/z 368和410、m/z 260和398、m/z 263、m/z 236和266為二醣之1-2、1-3、1-4及1-6的特徵離子斷片，根據特徵離子斷片之重建離子層析圖可判別半乳寡醣之二醣鍵結異構物。以建立之液相層析串聯質譜之方法針對市售六種半乳寡醣產品進行定性與定量，結果發現市售半乳寡醣之二醣異構物介於2-4種，大部分以1-2鍵結為主要的二醣結構、其次為1-4鍵結之二醣，1-6鍵結之二醣結構相對較少。本論文的發現將可做為半乳寡醣生產的品管指標。

Galacto-oligosaccharides (GOS) are prebiotic, consist ofβ-linked galactose. Degree of polymerization (DP) is from two to eight. GOS are formed by transgalactosylation from lactose, result in several linkage isomers. Due to lack of analytical method for those linkage isomers, correlation between prebiotic effect and GOS structures were not well-developed. Developing analytical method for structure elucidation of GOS is fundamental for further bioactive researches. First part of this study utilized size exclusion chromatography and 2-aminopyridine labeling technique followed by capillary electrophoresis to investigate DP distributions of 6 commercial GOS products. DP up to 6 was found, but three of six products contained no DP5 and DP6. DP3 were the major oligosaccharides for all GOS products. Further study derivatized GOS with p-aminobenzoic acid ethyl ester (ABEE). Chomophore-labeling derivatization enabled oligosaccharides not only with UV absorption but help them with better separation on liquid chromatography. For linkage isomers determination, collision-induced dissociation (CID) by mass spectrometry was utilized. In negative-ion mode, m/z 368 and 410, m/z 260 and 398, m/z 263, m/z 236 and 266 were the characteristic ions for characterizing 1-2, 1-3, 1-4 and 1-6 linkage isomers of GOS disaccharides. According to the study, linkage isomers of GOS DP2 could be categorized into 2-4. 1-3 linkage was the dominant disaccharide for most of the samples, followed by 1-4 linkage isomers. 1-6 linkage made minor contribution to DP2 structures of GOS. Above all, this method could be the quality control index for GOS production.