Capillary electrophoresis method for fine structural analysis of enoxaparin sodium

摘要

A capillary electrophoresis method for quantitatively analyzing characteristic oligosaccharide present in enoxaparin sodium is provided in this invention. The method may be used for quantitatively determining the contents of disaccharides, trisaccharides, tetrasaccharides and in particular oligosaccharides having a 1,6-anhydro ring, which are unique compounds for enoxaparin sodium, within an exhaustively digested enoxaparin sodium sample with a mixture of heparinase I, II, and III, so as to quantitatively determine the molar percentage of oligosaccharides having 1,6-anhydro ring in enoxaparin sodium. The method may be used for the pharmaceutical quality control of enoxaparin sodium during the manufacturing process.

主权项

1. A capillary electrophoresis (CE) method for fine structural analysis of enoxaparin sodium, comprising:
(1) digesting an enoxaparin sodium sample exhaustively with a mixture of heparin degrading enzymes; (2) separating oligosaccharides in the digested enoxaparin sodium sample by capillary electrophoresis, wherein the oligosaccharides include disaccharides, trisaccharides, tetrasaccharides and oligosaccharides with 1,6-anhydro structure, wherein the capillary electrophoresis is conducted under the following conditions; (a) a fused silica capillary having a total length ranging from 50 to 100 cm and an inner diameter ranging from 25 to 75 μm; (b) a running buffer including NaH2PO4—H3PO4, Tris-H3PO4 or LiH2PO4—H3PO4, or any combinations thereof, in a concentration ranging from 150 to 300 mM, and in a pH ranging from 1.5 to 4.0; the running buffer further comprises MgCl2 or ZnCl2 in a concentration ranging from 1 to 5 mM, and polyethylene glycol (PEG) having a molecular weight from 5000 to 100,000 Da in a concentration ranging from 0.1% to 5% (m/v); (c) a separation voltage ranging from −15 to −30 kV; (d) an injection pressure ranging from 1 to 100 mbar and the injection time ranges from 1 to 60 seconds; (e) a capillary temperature ranging from 10 to 40° C.; and (f) a UV detection wavelength ranging from 230 to 235 nm; wherein:after elution of sulfated disaccharide ΔIIA of the structure: an alternative pressure ranging from 5 to 150 mbar is applied to push disaccharide ΔIVA of the structure: to a detection window for detection; (3) after said elution step (2) pairing peaks present in an electropherogram from the capillary electrophoresis to the oligosaccharides in the digested enoxaparin sodium sample according to the linear relationship between electrophoretic mobilities and charge-to-mass ratio of the oligosaccharides; and (4) quantitatively determining the percentage of each oligosaccharide in total oligosaccharides in the digested enoxaparin sodium sample by using a measured normalized chromatographic peak area.