CN114252546A - Method for determining content of low-molecular-weight fucosylated glycosaminoglycan - Google Patents
Method for determining content of low-molecular-weight fucosylated glycosaminoglycan Download PDFInfo
- Publication number
- CN114252546A CN114252546A CN202011010607.XA CN202011010607A CN114252546A CN 114252546 A CN114252546 A CN 114252546A CN 202011010607 A CN202011010607 A CN 202011010607A CN 114252546 A CN114252546 A CN 114252546A
- Authority
- CN
- China
- Prior art keywords
- solution
- low
- molecular
- fucosylated glycosaminoglycan
- glycosaminoglycan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920002683 Glycosaminoglycan Polymers 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 37
- 238000012360 testing method Methods 0.000 claims description 31
- 239000013558 reference substance Substances 0.000 claims description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 11
- 239000012488 sample solution Substances 0.000 claims description 9
- 239000012085 test solution Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 6
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 2
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 229940097043 glucuronic acid Drugs 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims 1
- 238000006116 polymerization reaction Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000002270 exclusion chromatography Methods 0.000 abstract 1
- 239000000825 pharmaceutical preparation Substances 0.000 abstract 1
- 238000005259 measurement Methods 0.000 description 10
- 238000000691 measurement method Methods 0.000 description 7
- 150000004676 glycans Chemical class 0.000 description 6
- 229920001282 polysaccharide Polymers 0.000 description 6
- 239000005017 polysaccharide Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 3
- SHZGCJCMOBCMKK-PQMKYFCFSA-N L-Fucose Natural products C[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O SHZGCJCMOBCMKK-PQMKYFCFSA-N 0.000 description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 3
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 241000251511 Holothuroidea Species 0.000 description 2
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 241000258955 Echinodermata Species 0.000 description 1
- 241000258195 Holothuria Species 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000002337 glycosamines Chemical class 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- MBLBDJOUHNCFQT-OSMVPFSASA-N n-[(2r,3r,4r,5r)-3,4,5,6-tetrahydroxy-1-oxohexan-2-yl]acetamide Chemical compound CC(=O)N[C@@H](C=O)[C@@H](O)[C@@H](O)[C@H](O)CO MBLBDJOUHNCFQT-OSMVPFSASA-N 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/78—Detectors specially adapted therefor using more than one detector
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
- G01N2030/885—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds involving polymers
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a content determination method of low-molecular weight fucosylated glycosaminoglycan, which adopts molecular exclusion chromatography for detection, wherein the chromatographic condition is Shodex Ohpak SB-804HQ gel chromatographic column; 0.1mol/L sodium chloride is used as a mobile phase, and an Ultraviolet (UV) detector and a differential detector (RI) are connected in series. The method is simple and efficient, has high accuracy, can realize the rapid determination of the content of the low-molecular-weight fucosylated glycosaminoglycan in the pharmaceutical preparation, and is suitable for large-scale application of pharmaceutical production.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a content determination method of low-molecular-weight fucosylated glycosaminoglycan.
Background
LFG (Low Molecular Weight fused Glycosaminoglycan, hereinafter referred to as "LFG") is obtained by extracting a marine organism belonging to the genus Holothuria, further refining, and semi-synthesizing. Fucosylated glycosaminoglycans (FGAG) are a class of Glycosaminoglycan derivatives with sulfated fucose side chain substitutions obtained from the body wall of echinoderms. The common denominator of FGAG from different sources is mainly represented by the constituent monosaccharides including D-2-acetamido-2-deoxygalactose (D-GalNAc), D-glucuronic acid (D-GlcUA) and L-fucose (L-Fuc) and their sulfates. Wherein D-GlcUA and D-GalNAc are alternately linked by beta- (1-3) and beta- (1-4) glycosidic linkages to form a glycosaminoglycan backbone and L-Fuc is linked to the backbone in the form of side chains (Ricardo P.et al, JBC,1988,263 (34): 18176; Kenichiro Y.et al, Tetrahedron Lett,1992,33(34): 4959). FGAG from various sea cucumbers has strong anticoagulant activity and has important medicinal value for preventing and clinically treating thrombotic diseases.
The determination of sugar content is an essential step in the research of polysaccharide drugs, and is also an important content for quality control and product standard drawing, and because polysaccharide drugs have various sources and different structure types, the determination methods of polysaccharide are various. The commonly used glycosyl groups in polysaccharide drugs comprise neutral hexose, pentose and deoxyhexose, negative-charge uronic acid, sialic acid, electropositive aminosugar and the like, and can be determined by a method aiming at the difference of the chemical structures of the polysaccharides or establishing specificity for hydrolyzing the polysaccharides into monosaccharides. Since LFG is a glycosaminoglycan-type drug composed of a series of complex oligosaccharide chains of different molecular weights, it is not suitable for conventional content measurement methods (such as color development). And LFG only has ultraviolet absorption at short wavelength, and the absorption wavelength is at about 232nm of short wavelength, and an ultraviolet detector can detect the LFG, but has more influence factors and larger interference. The differential detector is a universal detector and has wide applicability, so that SEC (size exclusion chromatography) is applied, the ultraviolet detector is connected with the differential detector in series for testing, the detection results of the two detectors can be mutually referenced and compared, and finally the data measured by the differential detector is used as the standard. In addition, in consideration of the polydispersity of LFG, the main peak is not completely symmetric, and cannot be evaluated completely by the chromatographic evaluation method of small molecule chemical drugs, so the content measurement result and evaluation criteria can be relaxed appropriately.
Disclosure of Invention
The invention aims to provide a method for measuring the content of low-molecular-weight fucosylated glycosaminoglycan, which is used for monitoring the quality of low-molecular-weight fucosylated glycosaminoglycan, ensuring the stability and controllability of the quality of the low-molecular-weight fucosylated glycosaminoglycan product and promoting the application of the low-molecular-weight fucosylated glycosaminoglycan in the fields of medicines, foods and the like, and the specific technical scheme is as follows:
a low molecular weight fucosylated sugar amine is polymerized from fucose, N-acetylgalactosamine and glucuronic acid, has a molecular weight of 4000-6000, and comprises the following steps:
(1) preparation of control solutions:
taking a low-molecular-weight fucosylated glycosaminoglycan pure product which is dried by phosphorus pentoxide to constant weight as a reference substance, and adding 0.1mol/L sodium chloride solution to dissolve the low-molecular-weight fucosylated glycosaminoglycan pure product to prepare a reference substance solution of 1-20 mg/ml; preferably 10 mg/ml.
(2) Preparation of a test solution:
taking a proper amount of a sample to be detected containing low molecular weight fucosylated glycosaminoglycan, and adding 0.1mol/L sodium chloride solution to dissolve the sample to be detected to prepare 1-20 mg/ml sample solution; preferably 10 mg/ml.
(3) And (4) HPLC detection: respectively sucking the reference substance solution and the test substance solution obtained in the step (1) and the step (2), and respectively injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph for determination;
wherein, the chromatographic condition and system applicability test is a chromatographic column using hydrophilic spherical high polymer as a filler; taking 0.1mol/L sodium chloride as a mobile phase; the detection wavelength is 232 nm; the flow rate was 0.5ml per minute; the column temperature was 35 ℃; the temperature of the differential detector is 35 ℃; ultraviolet (UV) and differential detectors (RI) were connected in series. Weighing appropriate amount of glucose and dextran D2000, dissolving with mobile phase respectively, diluting quantitatively to obtain solution containing 2.5mg per lml, injecting 20 μ l into liquid chromatograph, recording chromatogram, and measuring retention time tTAnd t0Retention time t of main peak in chromatogram of test solution and control solutionRShould be at tTAnd t0And the number of theoretical plates is not less than 2000 calculated according to glucose peak.
In one embodiment, the pure fucosylated glycosaminoglycan with low molecular weight is prepared into a series of standard solutions with different concentrations, HPLC chromatography detection is carried out on the obtained standard solutions with different concentrations, the concentration is an abscissa, and the corresponding chromatographic peak area integral value is an ordinate, so as to draw a standard curve; wherein, the standard curve obtained by the ultraviolet detector is in the concentration range of 1.0mg/ml to 20.0mg/ml, and the linear correlation coefficient is 0.9998; the linear correlation coefficient of the standard curve obtained by the differential detector in the concentration range of 1.0 mg/ml-20.0 mg/ml is 0.9999.
The method is suitable for low molecular weight glycoglycosaminoglycan aqueous solution for injection or powder freeze-dried powder for injection, and the prepared sample keeps stable for 0, 3, 6, 10, 15, 18 and 24 hours at room temperature.
The method disclosed by the invention is simple to operate, economic and efficient, can realize rapid detection of the content of the low-molecular-weight fucosylated glycosaminoglycan in the low-molecular-weight fucosylated glycosaminoglycan product, is high in accuracy, and has wide application in the aspects of pharmaceutical production, quality control and the like.
Drawings
FIG. 1 shows a linear plot of a standard curve of a difference detector.
FIG. 2 is a linear plot of a calibration curve for an ultraviolet detector.
Detailed Description
The following examples are presented to illustrate embodiments of the present invention and to further understand the advantages and features of the present invention and should not be construed as limiting the scope of the invention.
The instruments and reagents used in the following examples are all generally commercially available unless otherwise specified.
The relevant test instruments and reagents used in the following examples are as follows:
primary device information
Table 1 main device information table
Primary reagent information
TABLE 2 Main reagent information Table
Reference information
TABLE 3 reference information sheet
Information of test article
The LFG test sample used in the examples was extracted from sea cucumber according to the method disclosed in Chinese patent 201410007855.
TABLE 4 information sheet of test articles
Example 1 method for measuring the content of Low molecular weight fucosylated glycosaminoglycan
1. Preparation of the solution
(1) Preparing a reference substance solution:
taking a low-molecular-weight fucosylated glycosaminoglycan control which is dried by phosphorus pentoxide to constant weight, and adding 0.1mol/L sodium chloride solution to dissolve the low-molecular-weight fucosylated glycosaminoglycan control to prepare a control solution containing 10mg of the low-molecular-weight fucosylated glycosaminoglycan in each ml;
(2) preparing a test solution:
taking a proper amount of the fucosylated glycosaminoglycan with low molecular weight to be tested, adding 0.1mol/L sodium chloride solution to dissolve the fucosylated glycosaminoglycan to be tested to prepare a test solution containing about 10mg of fucosylated glycosaminoglycan in each L of ml, and shaking up;
2. and (4) HPLC detection:
chromatographic conditions are as follows: using Shodex Ohpak SB-804HQ gel column as chromatographic column; taking 0.1mol/L sodium chloride as a mobile phase; the injection volume is 20 mu L; the detection wavelength is 232 nm; the flow rate is 0.5 mL/min; the column temperature was 35 ℃; the temperature of the differential detector is 35 ℃; ultraviolet (UV) and differential detector (RID) in series. The peak area was calculated by external standard method.
Example 2 methodological investigation procedure and verification content
1 System suitability test
(1) Measurement method
Sample introduction precision: control solutions were prepared according to the assay of example 1, and the relative standard deviation of the control peak areas was calculated by running 6 consecutive control solutions under chromatographic conditions for HPLC detection.
The specificity is as follows: weighing appropriate amount of glucose and dextran D2000, respectively adding mobile phase for dissolving and diluting to obtain solution containing 2.5mg per l ml, respectively taking 20 μ l per each of HPLC detection chromatogram according to chromatographic conditions of example 1, recording chromatogram, and measuring retention time tTAnd t0Retention time t of the main peak in the chromatogram of the LFG control solutionRShould be at tTAnd t0In the meantime.
Theoretical plate number: the theoretical plate number calculated by glucose should not be less than 2000.
(2) Test results and conclusions
The sample injection precision and specificity test results are shown in tables 5-6.
TABLE 5 table of measurement results of applicability of system
TABLE 6 tables of results of specificity measurement
According to the experimental result, the LFG reference substance is continuously injected into 6 needles, and the RSD value of the peak area of the LFG reference substance is less than 1.0 percent in both 0.27 percent (RID) and 0.45 percent (UV); the LFG main peak-off time is 18.750min, and the LFG main peak-off time is between dextran D2000 Rt 14.287min and glucose Rt 22.405min, and has no interference with a blank solvent, thereby indicating that the method has good system applicability and specificity.
2. Linear relationship and range
(1) Measurement method
Control stock solutions: an appropriate amount of LFG reference substance is precisely weighed, and a mobile phase is added for dissolution and dilution to a solution with the concentration of 20mg/ml to be used as a stock solution.
Standard solutions of series concentration: appropriate amount of the reference stock solution is measured respectively and diluted into 1.0, 5.0, 10.0, 15.0 and 20.0mg/ml solution by mobile phase.
20. mu.L of each concentration level of the linear solution was precisely measured under the chromatographic conditions of HPLC detection in example 1, and injected into a liquid chromatograph, and the chromatogram was recorded. And performing linear regression by taking the concentration as an abscissa and the peak area as an ordinate, and calculating a linear correlation coefficient of the linear regression.
(2) Test results and conclusions
The results of the linear tests are shown in Table 7.
TABLE 7 results of linear measurement
The linear relationship is shown in fig. 1 and 2.
According to the experimental result, the LFG is in the concentration range of 1.0 mg/ml-20.0 mg/ml, the peak area measured by the ultraviolet and differential detector and the LFG reference substance concentration are both linear, and the linear correlation coefficient R2The values were 0.9999 (RID) and 0.9998(UV), respectively, and the linearity was good.
3. Precision (repeatability test)
(1) Measurement method
2 parts of control solution and 6 parts of test solution are prepared in parallel according to the content determination method in the example 1;
according to the chromatographic conditions of HPLC detection in example 1, 20. mu.l of each of the reference solution and the sample solution is precisely measured, injected into a liquid chromatograph, and the chromatogram is recorded. The relative standard deviation of the content (calculated as dry product) was calculated by external standard method.
(2) Test results and conclusions
The results of the specific tests of the reproducibility test are shown in Table 8.
TABLE 8 repeatability test results table
As can be seen from the test results, the RSD value of LFG content was 1.03% (RID) and 0.99% (UV), and the reproducibility of the content measurement was good.
4. Stability of solution
(1) Measurement method
Preparing a test solution according to the content determination method in the example 1;
according to the chromatographic conditions of HPLC detection in example 1, 20 μ L of the sample solution respectively placed for 0, 3, 6, 10, 15, 18 and 24 hours at room temperature is precisely measured, injected into a liquid chromatograph, the peak area of the chromatogram is recorded, and the relative standard deviation of the peak area of the sample solution at different storage times is calculated.
(2) Test results and conclusions
The results of the solution stability test are shown in Table 9.
TABLE 9 table of the results of the stability measurement of the test solutions
According to the test results, the LFG test sample solution is respectively placed for 0, 3, 6, 10, 15, 18 and 24 hours at room temperature, the RSD values of the peak areas of the two detectors are respectively 1.09% (RID) and 0.35% (UV) which are both less than 2.0%, and the good stability of the sample solution within 24 hours at room temperature is proved.
5. Accuracy (recovery rate with standard)
(1) Measurement method
An appropriate amount of LFG reference substance dried to constant weight by phosphorus pentoxide is dissolved by adding a mobile phase and quantitatively diluted to prepare a solution containing about 100mg of LFG per liter of ml, and 2 parts of the LFG reference substance solution is prepared in parallel. 40, 50 and 60mg of LFG test sample (batch number: 20170623) with known content are precisely weighed, about 40, 50 and 60mg of LFG reference sample are added, the mixture is placed into a 10mL volumetric flask, and the mobile phase is added for dissolution and is diluted to scale to be used as test sample solution, and 3 parts are prepared in parallel.
According to the chromatographic conditions of HPLC detection in example 1, 20. mu.L of each of the reference solution and the sample solution is precisely measured, injected into a liquid chromatograph, recorded in a chromatogram, and calculated for the recovery rate of the added standard.
(2) Test results and conclusions
The results of the standard recovery tests are shown in Table 10.
TABLE 10 Table of results of recovery measurements with standard addition
From the test results, the standard addition yields of the samples were 101.1% (RID) and 110.9% (UV), indicating that the assay method had high accuracy and the detection result using the RID detector had higher accuracy.
6. Detection limit
(1) Measurement method
LFG reference substance 40.26mg is precisely weighed and placed in a 10mL measuring flask, the mobile phase is added for dissolution and is diluted to a scale mark, the mixture is injected into a liquid chromatograph according to the chromatographic conditions detected by HPLC in example 1, the chromatogram is recorded, and the minimum detection quantity of each detector is measured.
(2) Test results and conclusions
Under the above conditions, the minimum detection amount of the RID detector of LFG was 401.6ng, and the minimum detection amount of the DAD detector was 301.2 ng. The content determination method is high in sensitivity.
EXAMPLE 3 measurement results of three test samples
Three batches of samples to be tested were tested according to the method described in example 1. Among them, the results of content measurement by RID measurement are shown in Table 11.
TABLE 11 table of results of contents of three LFG test samples
The detection method disclosed by the invention has the advantages of simplicity in operation, strong specificity, high sensitivity, accuracy and precision, capability of realizing rapid and efficient quantification, obvious separation effect and the like, and has a better application prospect in industrial production.
Claims (6)
1. A method for measuring the content of low molecular weight fucosylated glycosaminoglycan, wherein the low molecular weight fucosylated glycosaminoglycan is formed by polymerization of fucose, N-acetylgalactosamine and glucuronic acid, and the molecular weight is 4000-6000;
the method is characterized by comprising the following steps:
(1) preparation of control solutions:
taking a low-molecular-weight fucosylated glycosaminoglycan pure product which is dried by phosphorus pentoxide to constant weight as a reference substance, and adding 0.1mol/L sodium chloride solution to dissolve the low-molecular-weight fucosylated glycosaminoglycan pure product to prepare a reference substance solution of 1-20 mg/ml;
(2) preparation of a test solution:
taking a proper amount of a sample to be detected containing low-molecular-weight fucosylated glycosaminoglycan, and adding 0.1mol/L sodium chloride solution to dissolve the sample to be detected to prepare a sample solution of 1-20 mg/ml;
(3) and (4) HPLC detection: respectively sucking the reference substance solution and the test substance solution obtained in the step (1) and the step (2), and respectively injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph for determination;
wherein, the chromatographic condition is a chromatographic column which uses hydrophilic spherical high polymer as a filling agent; taking 0.1mol/L sodium chloride as a mobile phase; the injection volume is 20 mu L; the detection wavelength is 232 nm; the flow rate was 0.5ml per minute; the column temperature was 35 ℃; the temperature of the differential detector is 35 ℃; ultraviolet and differential detectors were connected in series.
2. The assay method according to claim 1, wherein in the step (1), the pure low molecular weight fucosylated glycosaminoglycan is dissolved in 0.1mol/L sodium chloride solution to prepare a control solution of 10 mg/ml; in the step (2), a sample to be tested of the low molecular weight fucosylated glycosaminoglycan is added with the mobile phase to be dissolved to prepare a test solution of 10 mg/ml.
3. The method according to claim 1, wherein the column is Shodex Ohpak SB-804HQ gel column.
4. The measuring method according to claims 1 to 3, wherein the low molecular weight fucosylated glycosaminoglycan pure product is prepared into a series of standard solutions with different concentrations, HPLC chromatography is performed on the obtained standard solutions with different concentrations, the concentration of the standard solution is an abscissa, and the corresponding chromatographic peak area integral value is an ordinate, and a standard curve is drawn.
5. The method according to claim 4, wherein the standard curve obtained by the UV detector has a good linear relationship in the concentration range of 1.0mg/ml to 20.0 mg/ml; the standard curve obtained by the differential detector has good linear relation in the concentration range of 1.0 mg/ml-20.0 mg/ml.
6. The method for determining the content of low-molecular-weight fucosylated glycosaminoglycan according to claim 1, wherein the sample to be tested for the low-molecular-weight fucosylated glycosaminoglycan is a low-molecular-weight glycoglycosaminoglycan aqueous solution for injection or a powder injection lyophilizate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011010607.XA CN114252546A (en) | 2020-09-23 | 2020-09-23 | Method for determining content of low-molecular-weight fucosylated glycosaminoglycan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011010607.XA CN114252546A (en) | 2020-09-23 | 2020-09-23 | Method for determining content of low-molecular-weight fucosylated glycosaminoglycan |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114252546A true CN114252546A (en) | 2022-03-29 |
Family
ID=80788662
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011010607.XA Pending CN114252546A (en) | 2020-09-23 | 2020-09-23 | Method for determining content of low-molecular-weight fucosylated glycosaminoglycan |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114252546A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735336A (en) * | 2009-11-06 | 2010-06-16 | 深圳海王药业有限公司 | Oligomeric fucosylated glycosaminoglycan and preparation method thereof |
| US20120270834A1 (en) * | 2009-11-25 | 2012-10-25 | Shenzhen Neptunus Pharmaceutical Co., Ltd. | Depolymerized glycosaminoglycan from thelenota ananas and preperation method thereof |
| CN103214591A (en) * | 2013-04-12 | 2013-07-24 | 中国科学院昆明植物研究所 | 2,5-anhydrated talose or derivatives thereof terminal low-molecular-weight glycosaminoglycan derivative |
| CN103869002A (en) * | 2012-12-11 | 2014-06-18 | 深圳海王药业有限公司 | Analysis method for determining oligomerization thelenota ananas glycosaminoglycan content |
-
2020
- 2020-09-23 CN CN202011010607.XA patent/CN114252546A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735336A (en) * | 2009-11-06 | 2010-06-16 | 深圳海王药业有限公司 | Oligomeric fucosylated glycosaminoglycan and preparation method thereof |
| US20120270834A1 (en) * | 2009-11-25 | 2012-10-25 | Shenzhen Neptunus Pharmaceutical Co., Ltd. | Depolymerized glycosaminoglycan from thelenota ananas and preperation method thereof |
| CN103869002A (en) * | 2012-12-11 | 2014-06-18 | 深圳海王药业有限公司 | Analysis method for determining oligomerization thelenota ananas glycosaminoglycan content |
| CN103214591A (en) * | 2013-04-12 | 2013-07-24 | 中国科学院昆明植物研究所 | 2,5-anhydrated talose or derivatives thereof terminal low-molecular-weight glycosaminoglycan derivative |
Non-Patent Citations (2)
| Title |
|---|
| XIXI LIU 等: "Characterization of the Hydrolysis Kinetics of Fucosylated Glycosaminoglycan in Mild Acid and Structures of the Resulting Oligosaccharides", 《MARINE DRUGS》, vol. 18, no. 286, 31 May 2020 (2020-05-31), pages 1 - 18 * |
| 赵龙岩 等: "岩藻糖化糖胺聚糖的确切化学结构研究", 《云南省植物学会第十二届会员大会暨61周年学术年会论文集》, 31 March 2017 (2017-03-31), pages 53 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Restaino et al. | A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate | |
| EP1319183B1 (en) | Methods and products related to low molecular weight heparin | |
| Volpi et al. | Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations | |
| Stone | Optical rotary dispersion of mucopolysaccharides III. Ultraviolet circular dichroism and conformation al specificity in amide groups | |
| CN102329397B (en) | Fucosylated glycosaminoglycan derivative and preparation method thereof | |
| Keire et al. | Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches | |
| CN102323344B (en) | Method for quantitatively detecting hyaluronic acid fragment structure change | |
| CN103675144B (en) | Method for chemically degrading heparin and detecting composition of heparin disaccharide | |
| EP2985298B1 (en) | Low-molecular-weight fucosylated glycosaminoglycan derivative containing terminal 2,5-anhydrated talose or derivative thereof | |
| Han et al. | Monosaccharide compositions of sulfated chitosans obtained by analysis of nitrous acid degraded and pyrazolone-labeled products | |
| CN104558064A (en) | Preparation method of iron sucrose | |
| CN114252546A (en) | Method for determining content of low-molecular-weight fucosylated glycosaminoglycan | |
| Dinges et al. | Affinity capillary electrophoresis for the determination of binding affinities for low molecular weight heparins and antithrombin‐III | |
| CN110736796B (en) | Method for detecting molecular weight of pneumococcal capsular polysaccharide | |
| Monteiro et al. | Translational diffusion of dilute aqueous solutions of sugars as probed by NMR and hydrodynamic theory | |
| CN103575835B (en) | Method for determining 2-furfural in dextran-40 sodium chloride injection | |
| CN103869002B (en) | Analysis method for determining oligomerization thelenota ananas glycosaminoglycan content | |
| Grant et al. | Infrared spectroscopy of chemically modified heparins | |
| CN109459503B (en) | Method for measuring weight average molecular weight and content of heparin drugs | |
| CN102980951A (en) | Detection method of sodium hyaluronate and molecular weight in preparation of sodium hyaluronate | |
| CN108845044A (en) | The detection method of sodium content in a kind of sulfobutyl ether betadex sodium | |
| Song et al. | Absolute pharmacokinetics of heparin in primates | |
| Li et al. | Techniques for Detection of Clinical Used Heparins | |
| CN109030445B (en) | Method for measuring content of chitosan oligosaccharide | |
| CN113588817A (en) | Method for simultaneously determining content of atropine sulfate and EDTA-2Na in atropine eye drops |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |