Liquid Chromatography-mass Spectrometry For Compositional Analysis Of Low Molecular Weight Heparins

Heparin is a heterogeneous polysaccharide in terms of variable chain length, sulfo and N-acetyl substitutions, and epimerization, which are found on the cell surface and extracellular matrix. It consists of repeating disaccharide units of β-D-glucuronic acid or a-L-iduronic acid (1-4) linked to D-glucosamine. The hexuronic acid may be2-0-sulfated, and glucosamine may be6-0-sulfated, N-sulfated, N-acetylated, or (infrequently)3-0-sulfated. For a long time, heparin has been used extensively as a clinical anticoagulant drug for prevention and treatment of thrombotic diseases. However, the negative effects of heparin, including bleeding and inducing thrombocytopenia and osteoporosis, limit its clinical application.Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and widely used as anticoagulant drugs. LMWH have advantages with lower molecular weight, high antithrombotic effect, high bioavailability and long plasma half-time. With the emergence of low molecular weight heparin generics, compositional analysis is indispensable to demonstrate the fine compositional structure of generic and innovators’LMWH products. To analyze the structure and composition of LMWH, identification and quantitation of their natural and modified building blocks are indispensable.We established a novel reversed phase (RP) high performance liquid chromatography (HPLC)-diode array detection (DAD)-electrospray ionization-mass spectrometry (ESI-MS) approach for compositional analysis of LMWH. After exhaustively digested and labeled with2-aminoacridone (AMAC), the structural motifs constructing LMWH, including17components from dalteparin and15components from enoxaparin, were well separated, identified, and quantified. Besides the8natural heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures from the reducing end (RE) and non-reducing end (NRE),3-O-sulfated tetrasaccharides, as well as trisaccharides, have been unambiguously identified based on their retention time and mass spectra. Comparing to the traditional heparin compositional analysis methods, the approach described herein is not only robust, but also comprehensive because it is capable to identify and quantify nearly all components from lyases digests of LMWH.