CHARACTERIZATION OF THE CHEMICAL-STRUCTURE OF SULFATED GLYCOSAMINOGLYCANS AFTER ENZYMATIC DIGESTION - APPLICATION OF LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY WITH AN ATMOSPHERIC-PRESSURE INTERFACE

Pneumatically assisted electrospray was demonstrated to be a powerful ionization source for the analysis of oligosaccharides. A mass spectro meter was interfaced to an HPLC system, using this interface, to deter mine oligosaccharides from the enzymatic digestion of heparin separate d on a reversed-phase column. To set up the technique, and particularl y to clarify the ionization process, purified disaccharides, from enzy matic digestion of chondroitin sulphates, were measured. The use of a suitable counter ion in the mobile phase, tetrapropylammonium (TPA), t o optimize the HPLC separation, gave, with sulphated di- and oligosacc harides, adducts [M + nTPA - (n + m)H]m-, which were unexpectedly stab le to fragmentation; molecular ions [M - (n + 1)H]n-, in the presence of the counter ion, were observed only with desulphated or monosulphat ed disaccharides. The stability of the adducts and the use of a deuter ated ion-pair reagent permitted an exact evaluation of the molecular m asses of disaccharides and oligosaccharides of unknown structure. Spec tra obtained in the absence of the counter ion contained singly or mul tiply charged molecular ions and fragmentation ions mainly from loss o f the sulphate groups; under these ionization conditions the exact mas s determination and interpretation of the spectra were difficult. Afte r removal of the counter ion, tandem mass spectra could be obtained wi th some interesting data for the characterization of these molecules. Complete spectral analyses were performed with amounts of samples of 5 0 mug but, using microbore columns, one twentieth of this amount may g ive good spectra.