Oligosaccharides derivatization procedures

Kallin, E., Lonn, H., Norberg, T., and Elofsson, M. (1989) Derivatization procedures for reducing oligosaccharides, Part 3 Preparation of oligosaccharide glycosylamines, and their conversion into oligosaccharide-acrylamide copolymers./. Carbohydr. Chem. 8, 597-611. 

Because of the lack of a chromophore, oligosaccharides are difficult analytes in conventional LC procedures. They can only be detected via amperometiy or refractive index detection. Many derivatization procedures have been developed directed at the introduction of chromophores for UV detection or a fluorophore for fluorescence detection [49]. Often, derivatization also facihtates the separation of the derivatized ohgosaccharides by means of RPLC. Because oligosaccharides are also not very efficiently ionized by ESI, various derivatization procedures are directed at improving the response in ESI and/or at directing the fragmentation in MS-MS. The mass increments due to some of the derivatization reactions are given in Table 20.2. 

Despite the development of ESI and MALDI modes of ionization, GC/MS is still a popular method for determination of the monosaccharide composition of carbohydrate chains [13]. This method involves breaking the oligosaccharide into constituent monosaccharides by hydrolysis or methanolysis. Following derivatization, the released monosaccharides are analyzed by GC/MS to identify sugar residues through their retention times and mass spectral profile. Common derivatization procedures are methylation, acetylation, and trimethylsilylation. 

Columns of dextran have been prepared with a more elaborate procedure.13 It is claimed that the EOF has been eliminated and that columns are very stable under very harsh conditions, e.g., 0.05 M NaOH with 5% SDS or 1 M HC1. Cellulose-coated capillaries have been prepared by coupling 2-hydroxyethyl methacrylate (HEMA)-derivatized hydroxypropyl cellulose (HPC) to columns derivatized with 7-oct-l-enyltrimethoxysilane.The coating was stable for several weeks in the pH range from 2 to 10 and diminished the EOF, and it has been found useful for separation of peptides, glycoproteins, and derivatized oligosaccharides. 

Mass Spectrometry (MS). MS is one of the key techniques used in structure determination of carbohydrates and analyses via electron impact (E.I.) and chemical ionization (C.I.) methods are performed routinely on low molecular weight permethylated or peracetylated carbohydrates. Recently, MS procedures have found wider application in the structure elucidation of less volatile higher molecular weight oligosaccharides as a result of instrumental developments (in particular, desorption methods of ionization, based on fast atom bombardment (FAB) (93), field desorption (FD), laser desorption (LD), plasma desorption (PD), and secondary ion (SI) mass spectrometry) and improvements in derivatization techniques. For example, a series of malto-oligosaccharides, starch and other glycans have been examined with LD FD-MS (94,95) whilst FAB techniques have been employed for studies of cello- and malto-oligosaccharides (96) and branched cyclo-dextrins (97). 

---