Quantitative determination of disaccharide content in digested unfragmented heparin and low molecular weight heparin by direct-infusion electrospray mass spectrometry.

摘要

Heparins and low molecular weight heparins (LMWHs) are heterogeneous glycosaminoglycans derived from natural sources that are prescribed as anticoagulants. In this work, a direct-infusion electrospray ionization mass spectrometry (ESI-MS) method was applied to the quantitative analysis of known disaccharides in various native heparins and LMWHs after digestion with heparinase enzymes. Disaccharide deltaUA2S-->GlcNS6S was found to compose the majority of all samples analyzed (81-88%). The values were significantly higher than those reported by previously published methods. The disaccharide isomer pair deltaUA-->GlcNS6S/deltaUA2S-->GlcNS was also detected in all samples at lower levels (11-19%). While digestion with heparinases I and II revealed a limited number of disaccharides, the addition of heparinase III to digests led to the detection of disaccharide deltaUA2S-->GlcNAc6S in native porcine heparin. This result indicated the importance of utilizing all three heparinases to gain maximum information when analyzing heparin and LMWH digests. This method displayed good between-day (4-6%) and between-digest (1-2%) reproducibility in separate experiments. To determine if the digestion matrix was suppressing the signal of low-abundance disaccharides, several disaccharides were exogenously added at low levels (1-10 pmol/mg) to a quenched digest reaction. Analysis revealed that low level disaccharides were detectable in this matrix above the limits of detection (0.1-0.2 pmol/mg) and quantitation (0.2-0.7 pmol/mg). While this method was unable to distinguish between disaccharide isomers, it utilized simple mass spectrometry instrumentation to provide useful quantitative data for characterizing preparations of native heparin and LMWH, which could be used to compare various marketed preparations of these popular anticoagulants.