Fingerprinting analyses of low molecular weight heparin with an orthogonal MHC 2D LC-MS system

In this work, a synthetical glycan fingerprinting strategy using a multiple heart-cut two-dimensional liquid chromatography system linked to mass spectrometry (MHC 2D LC-MS) was developed to analyze enoxaparin, a widely used low molecular weight heparin (LMWH). Glycans from an enoxaparin standard were prepared offline based on size, and the derived tetra-, hexa-, octa- and decasaccharides were profiled using a qualitative analytical platform. Strong anion exchange chromatography (SAX) was employed as the first-dimensional chromatography (¹D) to separate glycans of the same size but with different charges or sequences, while size exclusion chromatography (SEC) was used in the second dimension (²D) for desalting before MS analysis. The retention times (RTs), accurate masses and structural compositions of the glycans were fully characterized. Real samples were analyzed using the same platform but with ¹D and ²D exchanged: SEC was used to separate enoxaparin glycans by size, followed by SAX using the same parameters as in the qualitative platform to separate glycans by charge or sequence. Glycans in real samples were identified by matching their RTs to those assigned in the qualitative analysis and semi-quantitated after normalization of peaks in each SAX chromatogram of glycans of different sizes. The analyses were performed automatically and robustly using this synthetical platform, enabling the fingerprinting and differentiation of enoxaparins from various sources. This platform could serve as a powerful tool for structural analysis, quality control, and heparin-related drug development.