HPLC and on-line MS detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone.

In this study, we developed an on-line reverse-phase (RP)-HPLC-ESI-MS separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC) providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC-disaccharide was found to be linear from 3 to 500 ng with very high coefficients of correlation values due to the high efficiency of separation and to the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition and minor oligomer species identified as GalNAc-SO4 at the non-reducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-Fp-ESI-MS.