Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization

Abstract

The invention relates to a reverse-phase chromatography and mass-spectrometry combined detection method for a complete low-molecular-heparin degradation product through precolumn derivatization. All components of the complete low-molecular-heparin degradation product are detected by combining reversed-phase chromatography and a high-resolution mass spectrum; all the components in a sample are separated through the reversed-phase chromatography, and a precision molecular weight can be obtained through the high-resolution mass spectrum; besides detection of eight common heparin disaccharides, tail end modification structures and special structures such as 3-O-tetrose-hydrosulfates relevant to anticoagulation and trisaccharides obtained through peeling reaction can be also detected; therefore, the structure of each complete low-molecular-heparin degradation product can be precisely represented. According to the reverse-phase chromatography and mass-spectrometry combined detection method, the problem that only the eight common heparin disaccharides can be detected in the prior art is solved; the reverse-phase chromatography and mass-spectrometry combined detection method has an extremely large practical value in research, development and production control of low-molecular-heparin imitating drugs and guarantee of the safety of the drugs.

Description

technical field [0001] The invention relates to a combined detection method of low-molecular-weight heparin complete degradation products combined with pre-column derivatization by reversed-phase chromatography and high-resolution mass spectrometry, and belongs to the technical field of medicine, raw material medicine and raw material detection. Background technique [0002] Heparin is a heterogeneous glycosaminoglycan with anticoagulant effect and can be used as a clinical anticoagulant drug for the prevention and treatment of thrombotic diseases. However, as an anticoagulant drug, heparin can easily cause side effects such as bleeding, osteoporosis and thrombocytopenia, which limits its clinical application. Low-molecular-weight heparin is a new type of anticoagulant drug prepared by controlled enzymatic and chemical degradation of heparin. Due to different production processes, each low-molecular-weight heparin has a different terminal modification structure, making its s...

AI Technical Summary

Problems solved by technology

The non-reducing end of dalteparin sodium is a saturated uronic acid, which does not have characteristic UV absorption and cannot be detected by traditional analytical methods, but it can be combined with a derivatizing reagent by pre-column derivatization to carry a fluorescent group or Has characteristic UV absorption; and the reducing end structures of dalteparin sodium and enoxaparin sodium do not have reducing hemiacetal hydroxyl groups, so they cannot be combined with derivatization reagents through pre-column derivatization methods, but the reducing end structures of dalteparin sodium and heparinase After degradation, there is an unsaturated double bond at its non-reducing end, so that the reducing end structure has a characteristic absorption at 232nm; therefore, only UV detection or fluorescence detection cannot be used for qualitative analysis of all components of low molecular weight heparin.

Images