Identification method of haina polysaccharide by nuclear magnetic resonance spectroscopy

An identification method of a Haina polysaccharide (HPS) by nuclear magnetic resonance (NMR) spectroscopy, comprising the steps of:

• mixing a sample to be tested, an internal standard, and deuterium oxide to obtain a test solution;
• subjecting the test solution to NMR spectroscopy test, thereby obtaining a hydrogen nuclear magnetic resonance (1H NMR) spectrum;
• and
• analyzing the 1H NMR spectrum, and identifying whether the sample to be tested is a qualified product of the HPS based on an obtained analysis result;
• wherein
• the NMR spectroscopy test is conducted under conditions comprising:
• a frequency not less than 600 MHz, a pulse angle of 90°, an acquisition time not less than 2 seconds (s), a relaxation delay not less than 12 s, a number of scans not less than 16, and a spectral width covering at least a range from 10 ppm to 0 ppm;
• a standard for the 1H NMR spectrum of the qualified product of the HPS comprises:
• in the 1H NMR spectrum of the qualified product of the HPS, there are signal peaks of fucose anomeric hydrogen, N-acetyl methyl hydrogen, and fucose methyl hydrogen;
• relative to a methyl signal of the internal standard, a signal peak of the fucose anomeric hydrogen has a relative chemical shift of 5.68 ppm±0.03 ppm, a signal peak of the N-acetyl methyl hydrogen has a relative chemical shift of 2.08 ppm±0.03 ppm, and a signal peak of the fucose methyl hydrogen is a doublet centered at a relative chemical shift of 1.38 ppm±0.03 ppm;
• the signal peak of the N-acetyl methyl hydrogen in the 1H NMR spectrum has a signal-to-noise ratio not less than 2000:1;
• in relative chemical shifts of 0.2 ppm to 0.8 ppm and 6.5 ppm to 10 ppm of the 1H NMR spectrum, no unidentified signals greater than 8% of an intensity of the signal peak of the fucose methyl hydrogen are found;
• in a relative chemical shift of 3 ppm to 6 ppm of the 1H NMR spectrum, no unidentified signals greater than 120% of the intensity of the signal peak of the fucose methyl hydrogen are found;
• and the intensity of the signal peak of the fucose methyl hydrogen is expressed as an average intensity of the doublet centered at the relative chemical shift of 1.38 ppm±0.03 ppm;
• and
• an integral area ratio value of the signal peak of the N-acetyl methyl hydrogen to the signal peak of the fucose methyl hydrogen in the 1H NMR spectrum is in a range of 1.2 to 1.8, wherein the signal peak of the N-acetyl methyl hydrogen is integrated in a relative chemical shift range of 1.8 ppm to 2.4 ppm, and the signal peak of the fucose methyl hydrogen is integrated in a relative chemical shift range of 1.1 ppm to 1.6 ppm;
• and
• the qualified product of the HPS is a fucosylated chondroitin sulfate polysaccharide;
• and the HPS has a weight-average molecular weight of 90,000 to 130,000, and is composed of monosaccharides comprising glucuronic acid, N-acetyl-D-galactosamine, and fucose, wherein a molar ratio of the glucuronic acid, the N-acetyl-D-galactosamine, and the fucose is in a range of 1:0.8-1.2:0.5-0.8;
• and a sulfate ester group in the HPS has a mass percentage of 25% to 40%.