CN112505166A - Content determination method of glucosamine hydrochloride capsule - Google Patents
Content determination method of glucosamine hydrochloride capsule Download PDFInfo
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- CN112505166A CN112505166A CN202011013465.2A CN202011013465A CN112505166A CN 112505166 A CN112505166 A CN 112505166A CN 202011013465 A CN202011013465 A CN 202011013465A CN 112505166 A CN112505166 A CN 112505166A
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- CBOJBBMQJBVCMW-BTVCFUMJSA-N (2r,3r,4s,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;hydrochloride Chemical compound Cl.O=C[C@H](N)[C@@H](O)[C@H](O)[C@H](O)CO CBOJBBMQJBVCMW-BTVCFUMJSA-N 0.000 title claims abstract description 46
- 229960001911 glucosamine hydrochloride Drugs 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000002775 capsule Substances 0.000 title abstract description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- 230000005284 excitation Effects 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 2
- XXZSQOVSEBAPGS-DONVQRBFSA-L cisatracurium besylate Chemical class [O-]S(=O)(=O)C1=CC=CC=C1.[O-]S(=O)(=O)C1=CC=CC=C1.C1=C(OC)C(OC)=CC=C1C[C@H]1[N@+](CCC(=O)OCCCCCOC(=O)CC[N@+]2(C)[C@@H](C3=CC(OC)=C(OC)C=C3CC2)CC=2C=C(OC)C(OC)=CC=2)(C)CCC2=CC(OC)=C(OC)C=C21 XXZSQOVSEBAPGS-DONVQRBFSA-L 0.000 claims 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 claims 1
- 229950002863 cisatracurium besilate Drugs 0.000 claims 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 14
- 239000007788 liquid Substances 0.000 abstract description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 abstract description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 abstract description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 abstract description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 abstract description 7
- 239000000945 filler Substances 0.000 abstract description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 2
- 238000001917 fluorescence detection Methods 0.000 abstract 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 abstract 1
- 239000000377 silicon dioxide Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 76
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 53
- 239000013558 reference substance Substances 0.000 description 49
- 239000012224 working solution Substances 0.000 description 40
- 239000000047 product Substances 0.000 description 35
- 239000012085 test solution Substances 0.000 description 35
- 238000012360 testing method Methods 0.000 description 35
- 238000002156 mixing Methods 0.000 description 31
- 238000002360 preparation method Methods 0.000 description 28
- 238000001212 derivatisation Methods 0.000 description 26
- 239000012490 blank solution Substances 0.000 description 25
- 229910021538 borax Inorganic materials 0.000 description 25
- 239000004328 sodium tetraborate Substances 0.000 description 25
- 235000010339 sodium tetraborate Nutrition 0.000 description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 239000011550 stock solution Substances 0.000 description 20
- 239000000523 sample Substances 0.000 description 18
- 239000012088 reference solution Substances 0.000 description 17
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 15
- 238000005303 weighing Methods 0.000 description 14
- 239000012488 sample solution Substances 0.000 description 9
- 238000000926 separation method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 229960002442 glucosamine Drugs 0.000 description 3
- 201000008482 osteoarthritis Diseases 0.000 description 3
- 210000001612 chondrocyte Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000836261 Homo sapiens U4/U6.U5 tri-snRNP-associated protein 2 Proteins 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 102100027243 U4/U6.U5 tri-snRNP-associated protein 2 Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000004263 amino monosaccharides Chemical class 0.000 description 1
- 230000002926 anti-osteoarthritic effect Effects 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 210000000544 articulatio talocruralis Anatomy 0.000 description 1
- 238000013096 assay test Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000004394 hip joint Anatomy 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000323 shoulder joint Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000003857 wrist joint Anatomy 0.000 description 1
Classifications
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- 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
-
- 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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—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
- G01N30/62—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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
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- 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 Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a glucosamine hydrochloride capsule content determination method, which adopts a liquid phase-fluorescence detection method, a chromatographic column with octadecylsilane chemically bonded silica as a filler, a phosphate solution (4.08 g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, a flow rate of 1.3-1.7 ml/min, a column temperature of 30-45 ℃ and a fluorescence detector as a detector.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining the content of glucosamine hydrochloride capsules by using liquid chromatography.
Background
Glucosamine hydrochloride is a natural amino monosaccharide, is a precursor substance for synthesizing proteoglycan, can stimulate chondrocytes to generate proteoglycan with a normal polymer structure, can improve the repair capability of the chondrocytes, inhibit enzymes damaging articular cartilage, and promote the repair and reconstruction of ointment matrix. Tablets and capsules come into the market in the world. The glucosamine hydrochloride capsule is mainly used for treating and preventing osteoarthritis of all parts of the whole body, including knee joints, shoulder joints, hip joints, wrist joints, neck joints, spinal joints, ankle joints and the like. Can relieve and eliminate pain and swelling of osteoarthritis, and improve joint movement function.
Glucosamine hydrochloride preparations are available on the market in capsules, tablets and granules in the global range, and are not available on the market for a while because of development and research of new formulations such as glucosamine hydrochloride ointments, oral liquid, gels and the like; wherein the capsule is only available in hong Kong region and China, and the market specification is 0.75g and 0.24 g. Currently, glucosamine hydrochloride becomes an anti-osteoarthritis medicament widely used clinically, is used for treating and preventing osteoarthritis of all parts of the whole body, has definite clinical curative effect and low toxic and side effects, and is widely applied in domestic and foreign markets. The chemical name of glucosamine hydrochloride capsules is poly (allylamino-co-N, N' -diallyl-1, 3-diamino-2-hydroxypropane) carbonate, and the structural formula of the glucosamine hydrochloride capsules is shown as the following formula:
（a）
the original national standard WS1- (X-090) -2005Z of glucosamine hydrochloride capsules adopts an ultraviolet visible spectrophotometry, the specificity and repeatability of the method are poor, USP39 adopts HPLC (high performance liquid chromatography) to measure, the detection wavelength is 195nm, the system interference is large under the wavelength condition, the influence of a solvent is large, the repeatability result is poor, aiming at the problems, the derivatization reaction product is detected by a liquid phase-fluorescence method, and the method has good specificity, sensitivity and stable result.
Disclosure of Invention
The invention aims to provide a content measuring method of glucosamine hydrochloride capsules.
The purpose of the invention is realized by the following technical scheme, and the method comprises the following steps:
the chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.5ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a blank solution: taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1ml of 0.1mol/L hydrochloric acid and 1.0ml of derivatization reagent solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and then measuring.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute the product to a scale, and shaking the product uniformly to obtain a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Under the chromatographic conditions, precisely absorbing 10 mu l of blank solution, reference solution and test solution, injecting into a liquid chromatograph, recording the chromatogram, and calculating the glucosamine hydrochloride content by peak area according to an external standard method.
The test sample of the present invention contains glucosamine C6H13O5N.HCl should be 95% -105% of the indicated amount.
The preferred method for measuring the content of glucosamine hydrochloride capsules comprises the following steps: the chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.5ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a blank solution: taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1ml of 0.1mol/L hydrochloric acid and 1.0ml of derivatization reagent solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and then measuring.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute the product to a scale, and shaking the product uniformly to obtain a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Precisely absorbing 10 mu l of blank solution, reference solution and test solution under the chromatographic conditions, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the test solution has the same peak at the same retention time as that of the reference solution; the separation degree of the main component and the adjacent chromatographic peak in the reference solution is more than 1.5. No peaks appear in the blank solution identical to the glucosamine hydrochloride control.
Chromatographic conditions are as follows: the chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.4ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a blank solution: taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1ml of 0.1mol/L hydrochloric acid and 1.0ml of derivatization reagent solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and then measuring.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute the product to a scale, and shaking the product uniformly to obtain a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Precisely absorbing 10 mu l of blank solution, reference solution and test solution under the chromatographic conditions, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the test solution has the same peak at the same retention time as that of the reference solution; the separation degree of the main component and the adjacent chromatographic peak in the reference solution is more than 1.5. No peaks appear in the blank solution identical to the glucosamine hydrochloride control.
The test sample of the present invention contains glucosamine C6H13O5N.HCl should be 95% -105% of the indicated amount.
Chromatographic conditions are as follows: the chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.6ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a blank solution: taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1ml of 0.1mol/L hydrochloric acid and 1.0ml of derivatization reagent solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and then measuring.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute the product to a scale, and shaking the product uniformly to obtain a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Precisely absorbing 10 mu l of blank solution, reference solution and test solution under the chromatographic conditions, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the test solution has the same peak at the same retention time as that of the reference solution; the separation degree of the main component and the adjacent chromatographic peak in the reference solution is more than 1.5. No peaks appear in the blank solution identical to the glucosamine hydrochloride control.
The test sample containing glucosamine C6H13O5N.HCl is 95-105% of the labeled amount.
The glucosamine hydrochloride capsule is prepared by the following method: taking glucosamine hydrochloride as a main drug and microcrystalline cellulose as a filling agent, granulating, mixing, filling capsules and packaging to obtain the oral liquid.
The quality control method of the invention shows that the repeatability is good, the linear relation is good, the precision is good, the solution stability is good, the recovery rate is good, and the linear range is wide according to the test result.
The present invention will be described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples. It is intended that all such alterations and modifications that come within the spirit and scope of the invention are desired to be embraced therein without departing from the spirit and scope of the invention as defined by the appended claims.
Example 1 specificity test
The chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.5ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a blank solution: taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1ml of 0.1mol/L hydrochloric acid and 1.0ml of derivatization reagent solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and then measuring.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain a reference stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution, adding 1.0ml of derivatization test solution, and carrying out the same operation under the section of 'preparation of blank solution' to obtain the product.
Under the chromatographic conditions, precisely sucking 10 μ l of blank solution, reference solution and test solution, injecting into a liquid chromatograph, and recording chromatogram. A chromatogram of the test solution having the same peak at the same retention time as the control solution; the separation degree of the main component and the adjacent chromatographic peak in the reference solution is not less than 1.5. The same peak should not appear in the blank solution as in the glucosamine hydrochloride control.
The results show that: in the chromatogram of the test solution, the same peak is present at the same retention time of the chromatogram of the reference solution, the separation degree of the main component in the reference solution and the adjacent chromatographic peak is not less than 1.5, and the blank solution has no peak same as the glucosamine hydrochloride reference, namely the blank does not interfere with the glucosamine hydrochloride reference.
Example 2 Linear relationship test
The control stock solution (2 mg/ml) was diluted with 0.1mol/L hydrochloric acid to give control solutions of different concentrations, 4. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 30. mu.g/ml and 40. mu.g/ml, as control working solutions. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, respectively adding 1ml of reference substance working solution with different concentrations, adding 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, respectively sucking 10ul of each reference solution, and injecting into a liquid chromatograph. The linear equation and regression coefficient of the concentration and peak area values were calculated, and the results are shown in Table 1.
TABLE 1 Linear relationship test data sheet
| Numbering | 1 | 2 | 3 | 4 | 5 |
| Concentration (μ g/ml) | 0.40 | 1.00 | 2.00 | 3.00 | 4.00 |
| Peak area (mAU) | 175685 | 254400 | 368735 | 496561 | 620774 |
The regression equation: y = 122921.2392 x + 127299.1463, and the correlation coefficient R = 0.9997.
The test result shows that the linear relation between the solution concentration value and the corresponding peak area value is good in the range of 0.40 mu g/ml to 4.00 mu g/ml.
Example 3 precision test
The control solution (20. mu.g/ml) was precisely aspirated (10 ul) and injected into the chromatograph, and the measurement was repeated 6 times to calculate the precision, the results of which are shown in Table 2.
TABLE 2 precision test data sheet
The test result shows that the precision is good.
Example 4 stability test
Accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. 2.0ml of 0.037mol/L borax solution is taken, 1.0ml of water is added, 1.0ml of reference substance working solution is added, 1.0ml of derivatization test solution is added, vortex mixing is carried out for 20 seconds, after standing for 3 minutes, 5ml of acetonitrile-water (1: 1) is added, mixing is carried out, after standing for 2 hours, 10 mul of solution is precisely absorbed and injected into a chromatograph for 0 hour, 2 hours, 4 hours, 8 hours and 10 hours respectively, and the result is shown in Table 3.
TABLE 3 stability test data sheet
| Control solution | Peak area | Change value (%) |
| 0h | 3685653 | N.A |
| 2h | 3620385 | 98.2 |
| 4h | 3591314 | 97.4 |
| 8h | 3566408 | 96.8 |
| 10h | 3526408 | 95.7 |
The test results show that the solution is stable within 10 hours.
EXAMPLE 5 repeatability test
Accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain a reference stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution and 1.0ml of derivatization sample solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours to serve as sample solution, and repeating the method to prepare 6 parts of sample. Taking 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution and 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours to serve as reference substance solution, and precisely sucking 10 mu L of reference substance solution and sample solution to be injected into a chromatograph, wherein the results are shown in Table 4.
TABLE 4 repeatability test data sheet
Test results show that the method has good repeatability.
Example 6 recovery test
The control stock solution (2 mg/ml) was diluted with 0.1mol/L hydrochloric acid to give control solutions of different concentrations, 16. mu.g/ml, 20. mu.g/ml and 24. mu.g/ml, as control working solutions. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, respectively adding 1ml of reference substance working solution with different concentrations, adding 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours, and preparing 3 parts of each concentration in parallel to serve as a test solution. Taking 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution and 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, and standing for 2 hours to serve as a reference substance solution. Respectively sucking 10 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph. And calculating the recovery rate. The relative standard deviations were examined and the results are shown in table 5.
TABLE 5 recovery test data sheet
Test results show that the method has good recovery rate.
EXAMPLE 7 durability test
And evaluating the bearing degree of the measurement result which is not influenced when the measurement condition is slightly changed by changing the proportion of the mobile phase, the flow speed and the column temperature. Under each condition, 100mg of the product is precisely weighed, placed in a 50ml measuring flask, diluted to scale by adding 0.1mol/L hydrochloric acid solution, and shaken uniformly to be used as a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution and 1.0ml of derivatization sample solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, and standing for 2 hours to obtain the sample solution. Taking 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution and 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, standing for 2 hours to serve as reference substance solution, precisely absorbing 10 mu L of reference substance solution and sample solution, injecting the reference substance solution and the sample solution into a chromatograph, recording a chromatogram, wherein the separation degree of main components in the reference substance solution and adjacent chromatographic peaks is not less than 1.5. The results are shown in Table 6.
TABLE 6 durability test data sheet
The test result shows that the method has good durability.
EXAMPLE 8 assay test
The chromatographic column Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm, phosphate solution (taking 4.08g of monopotassium phosphate and 2.08g of phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, the flow rate of 1.5ml/min, the column temperature of 35 ℃, the detector as a fluorescence detector, the excitation wavelength of 390nm, the emission wavelength of 475nm and the collection time of 30 minutes.
Preparation of a test solution: accurately weighing 100mg of the product, placing the product in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute the product to a scale, and shaking the product uniformly to obtain a stock solution of a test sample. Precisely measuring 1ml, and placing in a 100ml measuring flask to obtain a working solution of a test article. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of sample working solution and 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, and standing for 2 hours.
Preparation of control solutions: accurately weighing 100mg of glucosamine hydrochloride reference substance, placing the glucosamine hydrochloride reference substance in a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution to dilute to scale, and shaking up to obtain reference substance stock solution. Precisely measuring 1ml, and placing in a 100ml measuring flask as a reference working solution. Taking 2.0ml of 0.037mol/L borax solution, adding 1.0ml of water, adding 1.0ml of reference substance working solution and 1.0ml of derivatization test solution, vortex mixing for 20 seconds, standing for 3 minutes, adding 5ml of acetonitrile-water (1: 1), mixing uniformly, and standing for 2 hours. Precisely sucking 10 μ l of reference solution and sample solution, injecting into chromatograph, recording chromatogram, and evaluating the result. The results are shown in Table 7.
The content of three samples was measured using the above test method and the results are shown in Table 7.
TABLE 7 glucosamine hydrochloride Capsule content measurement results
| Batch number | Content (%) |
| 20200418 | 98.5% |
| 20200426 | 99.2% |
| 20200521 | 99.5% |
Claims (4)
1. The method for detecting the content of glucosamine hydrochloride is characterized in that the detection adopts high performance liquid chromatography.
2. The method for detecting glucosamine hydrochloride content according to claim 1, wherein a fluorescence detector is used, and the excitation wavelength is 390nm and the emission wavelength is 475 nm.
3. The method for detecting cisatracurium besilate enantiomer according to claim 1, characterized in that the mobile phase is phosphate solution-acetonitrile in a ratio of 60: 40.
4. The method for detecting the enantiomers of cisatracurium besilate according to claim 1, characterized in that an Agilent Eclipse XDB-C18, 4.6X 150mm, 5 μm column was used.
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| CN115248256A (en) * | 2021-04-26 | 2022-10-28 | 武汉伯睿科医药科技有限公司 | Method for determining content of glucosamine sulfate capsule |
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| CN115248256A (en) * | 2021-04-26 | 2022-10-28 | 武汉伯睿科医药科技有限公司 | Method for determining content of glucosamine sulfate capsule |
| CN113325101A (en) * | 2021-05-27 | 2021-08-31 | 南京海纳医药科技股份有限公司 | Method for detecting dissolution rates of glucosamine hydrochloride preparation in different media |
| CN113325101B (en) * | 2021-05-27 | 2022-09-16 | 南京海纳医药科技股份有限公司 | Method for detecting dissolution rates of glucosamine hydrochloride preparation in different media |
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