Method for quickly detecting heparin disaccharide content in heparin and/or low-molecular heparin

Abstract

The invention provides a method for quickly detecting a heparin disaccharide content in heparin and / or low-molecular heparin. The method comprises S1) mixing heparin and / or low-molecular heparin solution with heparinase to react to obtain an enzymolysis product; S2) utilizing reversed-phase ion pair chromatography to detect the enzymolysis product and recording an HPLC spectrogram, wherein a mobile phase of the reversed-phase ion pair chromatography includes a mobile phase A and a mobile phase B, the mobile phase A is an organic solvent and water mixed solution containing reversed-phase ion pairs, the mobile phase B is an organic solvent and water mixed solution containing reversed-phase ion pairs and alkali metal chlorine salt, and the reversed-phase ion pairs are quaternary ammonium salt. Compared with the prior art, the method disclosed by the invention can obtain main heparin disaccharide composition and / or small fragments like trisaccharide and tetrasaccharide units by completelydegrading the heparin and / or the low-molecular heparin through the heparinase; a reversed-phased ion pair reagent and a reversed-phase chromatography are combined to quickly separate and quantitatively analyze the main heparin disaccharide composition and the small fragments; furthermore, the method has very good durability and repeatability.

Description

technical field [0001] The invention belongs to the technical field of drug detection, in particular to a method for rapidly detecting the content of heparin disaccharide in heparin and / or low molecular weight heparin. Background technique [0002] Enoxaparin sodium belongs to low-molecular-weight heparin. With the increase of generic drug manufacturers, the diversity of low-molecular-weight heparin products emerges endlessly. FDA has strengthened the monitoring of low-molecular-weight heparin generics entering the US market. [0003] On July 23, 2010, the FDA formulated a systematic and rigorous method based on five criteria, the so-called generic drug consistency guidelines, including the following five points: 1. Physical and chemical properties; 2. Heparin source and degradation method ; 3. Sequence identity of disaccharide and oligosaccharide fragments; 4. Biochemical equivalence; 5. Pharmacodynamic equivalence in vivo. In addition to this, the FDA used in vivo, in vit...

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Problems solved by technology

[0006] The Chinese patent with the publication number CN104792896A discloses a heparinase composition and its application that can completely and specifically enzymatically hydrolyze enoxaparin sodium. The heparinase composition provided by it is heparinase II and heparinase III The mixture can thoroughly and specifically enzymatically hydrolyze heparin sodium, but in this patent, the HPLC detection of the reduction product after enzymolysis using this heparanase composition requires the use of a guard column, and the mobile phase needs to adjust the pH and the detection time is also longer

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