HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY COUPLED WITH MASS-SPECTROMETRY FOR THE DETERMINATION OF CARBOHYDRATES

Methodology has been developed to couple high-performance anion-exchange chromatography (HPAE) with mass spectrometry utilizing the ion spray liquid chromatography/mass spectrometry (LC/MS) interface. Anion micro-membrane suppression (AMMS) has been used to remove the high concentrations of NaOH and NaOAc (10-400 mM total [Na+]) necessary for the separation of mixtures of monosaccharides and oligosaccharides. Post-suppressor addition of CH3CN/H2O solutions containing NH4OAc or LiOAc provided low-nanomole detection of the monosaccharides by selected ion monitoring of the cationized adducts. Maltooligosaccharide mixtures (three to seven residues) were separated and detected by the HPAE/AMMS LC/MS system in the full-scan mode. Low declustering potentials (35 V) in the LC/MS API source afforded intact singly and doubly charge ammoniated and diammoniated adducts of the sugars. Higher declustering potentials (65 V) produced abundant fragmentation of the ammoniated adducts. The corresponding lithiated and dilithiated species produced intact molecule ion species at higher declustering potentials. The endo H-released oligomannose species from RNase B were determined by the HPAE/AMMS LC/MS system as ammoniated adducts and resulting fragment ions with a high declustering potential (95 V) in the full-wan mode.