CN116773701A - Method for detecting related substances of desmopressin acetate oral solution - Google Patents
Method for detecting related substances of desmopressin acetate oral solution Download PDFInfo
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- CN116773701A CN116773701A CN202310751710.7A CN202310751710A CN116773701A CN 116773701 A CN116773701 A CN 116773701A CN 202310751710 A CN202310751710 A CN 202310751710A CN 116773701 A CN116773701 A CN 116773701A
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- Prior art keywords
- desmopressin acetate
- impurity
- solution
- mobile phase
- oral solution
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- 108010000437 Deamino Arginine Vasopressin Proteins 0.000 title claims abstract description 80
- FIEYHAAMDAPVCH-UHFFFAOYSA-N 2-methyl-1h-quinazolin-4-one Chemical compound C1=CC=C2NC(C)=NC(=O)C2=C1 FIEYHAAMDAPVCH-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 229960002845 desmopressin acetate Drugs 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 42
- 229940100688 oral solution Drugs 0.000 title claims abstract description 39
- 239000000126 substance Substances 0.000 title claims abstract description 37
- 239000012535 impurity Substances 0.000 claims abstract description 103
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 55
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 238000010828 elution Methods 0.000 claims abstract description 25
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 239000000741 silica gel Substances 0.000 claims abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 3
- 238000002347 injection Methods 0.000 claims description 15
- 239000007924 injection Substances 0.000 claims description 15
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 2
- BXWLVQXAFBWKSR-UHFFFAOYSA-N 2-methoxy-5-methylsulfonylbenzoic acid Chemical compound COC1=CC=C(S(C)(=O)=O)C=C1C(O)=O BXWLVQXAFBWKSR-UHFFFAOYSA-N 0.000 abstract description 25
- 239000000463 material Substances 0.000 abstract description 16
- 239000012467 final product Substances 0.000 abstract description 13
- 238000002360 preparation method Methods 0.000 abstract description 11
- 239000002904 solvent Substances 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 56
- 239000000523 sample Substances 0.000 description 20
- 239000013558 reference substance Substances 0.000 description 18
- 239000012488 sample solution Substances 0.000 description 17
- 238000007865 diluting Methods 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000012360 testing method Methods 0.000 description 14
- 239000003085 diluting agent Substances 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- 239000012490 blank solution Substances 0.000 description 10
- 230000006378 damage Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 239000012085 test solution Substances 0.000 description 8
- 239000000377 silicon dioxide Substances 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 7
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 229960004281 desmopressin Drugs 0.000 description 4
- NFLWUMRGJYTJIN-NXBWRCJVSA-N desmopressin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSCCC(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(N)=O)=O)CCC(=O)N)C1=CC=CC=C1 NFLWUMRGJYTJIN-NXBWRCJVSA-N 0.000 description 4
- 238000012494 forced degradation Methods 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 3
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 3
- 229960002216 methylparaben Drugs 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000003255 drug test Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 2
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 2
- 229960003415 propylparaben Drugs 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 101800001144 Arg-vasopressin Proteins 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 208000019505 Deglutition disease Diseases 0.000 description 1
- 201000003542 Factor VIII deficiency Diseases 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000027276 Von Willebrand disease Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002686 anti-diuretic effect Effects 0.000 description 1
- 229940124538 antidiuretic agent Drugs 0.000 description 1
- 239000003160 antidiuretic agent Substances 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 201000010064 diabetes insipidus Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000008832 photodamage Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002947 procoagulating effect Effects 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000012137 von Willebrand disease (hereditary or acquired) Diseases 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention discloses a method for detecting substances related to desmopressin acetate oral solution, which separates and detects desmopressin acetate and the substances related to the desmopressin acetate oral solution by adopting a high performance liquid chromatography; wherein the chromatographic conditions include: the chromatographic column is octadecylsilane chemically bonded silica gel column; the volume ratio is (90-100): 0.01 to 0.07mol/L buffer solution and acetonitrile mixed solution of (0 to 10) is a mobile phase A, acetonitrile is a mobile phase B, and gradient elution is carried out; the detection wavelength is 220nm; the column temperature is 25-40 ℃; the flow rate is 0.8-1.5 mL/min. The detection method provided by the invention can effectively separate the main peak, the impurity I, the impurity II, the impurity III, the impurity IV and other related impurities at the same time, and the blank solvent and the blank auxiliary material have no interference, so that the detection method can be used for the preparation process of the desmopressin acetate oral solution and the quality control of the final product.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and relates to a method for detecting substances related to desmopressin acetate oral solution.
Background
Desmopressin acetate is a structural analogue of natural arginine vasopressin, has antidiuretic and procoagulant effects, and is clinically used for treating hemophilia A, I type von willebrand disease, diabetes insipidus and the like. Desmopressin acetate is usually prepared into injection, injection (powder), tablet and nasal spray, in 2013, desmopressin acetate oral solution developed by GP Pharma company is marketed in European batches, and compared with injection and tablet, the oral solution dosage form is more suitable for children and dysphagia people, and has accurate dosage and better compliance.
The prescription of desmopressin acetate oral solution is composed of raw materials, methylparaben and propylparaben, and is prepared by adjusting pH with hydrochloric acid. At present, the pharmacopoeia of each country does not record the method for measuring the related substances of the desmopressin acetate oral solution, and the method for measuring the related substances of the desmopressin acetate bulk drug recorded in the United states pharmacopoeia and European pharmacopoeia can strongly interfere with peak emergence of preservative, so that a novel method for measuring the related substances of the desmopressin acetate oral solution needs to be developed.
Disclosure of Invention
The invention aims to provide a method for detecting substances related to desmopressin acetate oral solution. The method for detecting the substances related to the desmopressin acetate oral solution can effectively separate the main peak, the impurity I, the impurity II, the impurity III, the impurity IV and other related impurities at the same time, and the blank solvent and the blank auxiliary material have no interference, so that the method can be used for the preparation process of the desmopressin acetate oral solution and the quality control of the final product.
In the present invention, "related substances" refer to impurities (including organic impurities such as intermediates, byproducts, etc.) which may be generated and degraded during the preparation and storage of drugs, pharmaceutical preparations, etc. Wherein, the desmopressin acetate oral solution related substances comprise impurities I, impurities II, impurities III, impurities IV and other related impurities, and the impurities I, the impurities II, the impurities III and the impurities IV are as follows in sequence:
impurity I: mercaptopropionyl-L-tyrosyl-L-phenylalanyl-L-glutamyl-L-aspartyl-L-cysteinyl-L-prolyl-D-arginyl-L-glycylamide (1- > 6-disulfide ring);
impurity II: mercaptopropionyl-L-tyrosyl-L-phenylalanyl-L-glutamyl-L-aspartyl-L-cysteinyl-L-prolyl-D-arginyl-L-glycylamide (1→6-disulfide ring);
impurity III: mercaptopropionyl-L-tyrosyl-L-phenylalanyl-L-glutamyl-L-aspartyl-L-cysteinyl-L-prolyl-D-arginyl-L-glycine (1- > 6-disulfide ring);
impurity IV: mercaptopropionyl-L-tyrosyl-L-phenylalanyl-L-glutamyl-L-aspartyl-L-cysteinyl-L-prolyl-D-arginyl-glycylamine (intermolecular cyclic 1. Fwdarw.6, 1. Fwdarw.6-disulfide bond).
According to one aspect of the present invention, there is provided a method for detecting substances associated with desmopressin acetate oral solution, comprising the steps of: separating and detecting desmopressin acetate and related substances in desmopressin acetate oral solution by adopting high performance liquid chromatography; wherein the related substances comprise impurities I, II, III, IV and other related impurities; the chromatographic conditions of the high performance liquid chromatography include:
the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler;
the mobile phase comprises a mobile phase A and a mobile phase B, and gradient elution is adopted; wherein the mobile phase A is a mixed solution of buffer solution and acetonitrile (buffer solution-acetonitrile), and the volume ratio of the buffer solution to the acetonitrile is (90-100): (0-10), the concentration of the buffer solution is 0.01-0.07 mol/L; mobile phase B is acetonitrile;
the detection wavelength is 220nm; the column temperature of the chromatographic column is 25-40 ℃; the flow rate of the mobile phase is 0.8-1.5 mL/min.
In some embodiments, the column may be 4.6mm by 250mm in size, 5 μm in diameter, i.e., 4.6mm in inside diameter, 250mm in length, and 5 μm in filler particle size.
In some embodiments, the buffer may be a phosphate buffer, the components of which include potassium dihydrogen phosphate, disodium hydrogen phosphate, and water, prepared primarily by dissolving anhydrous disodium hydrogen phosphate, potassium dihydrogen phosphate in water, at a pH of approximately 7.0.
In some embodiments, the phosphate buffer may have a concentration of 0.0335mol/L, and the preparation method thereof includes the steps of: taking 2.37g of anhydrous disodium hydrogen phosphate and 2.28g of monopotassium phosphate, and adding 1000mL of water to dissolve the anhydrous disodium hydrogen phosphate. Thus, the problem that the detection result is affected by salting out occurring in the detection process due to too high buffer concentration can be avoided.
In some embodiments, the volume ratio of buffer to acetonitrile in mobile phase a may be 95:5.
In some embodiments, the column temperature may be 35 ℃.
In some embodiments, the sample volume may be 50 to 100 μl.
In some embodiments, the mobile phase flow rate may be 1.0mL/min.
In some embodiments, the procedure for mobile phase gradient elution may be:
under the elution procedure, the blank auxiliary materials and the blank solution have no interference on detection, and the main peak of desmopressin acetate and specific impurities (impurity I, impurity II, impurity III and impurity IV) are better separated from adjacent peaks.
Compared with the prior art, the invention has the beneficial effects that:
(1) Under the chromatographic conditions of high performance liquid chromatography for measuring the content of the desmopressin acetate oral solution, which are established by the invention, the blank auxiliary materials and the blank solution have no interference on detection, all related substances can be effectively detected, and the separation degree of the main peak of desmopressin acetate, the related substances and the adjacent peaks is more than 1.5, so that the separation is better; can be used for quality control of desmopressin acetate oral solution, and is beneficial to ensuring the quality and clinical curative effect thereof;
(2) The method for detecting substances related to the desmopressin acetate oral solution provided by the invention has good linear relation between the concentration of desmopressin acetate, the impurity I, the impurity II, the impurity III and the impurity IV and the peak area thereof in a certain concentration range, and the method has good sensitivity, repeatability and durability; has important significance for evaluating the synthesis of desmopressin acetate and the quality of the preparation process, controlling the quality of the final product and detecting the stability sample;
(3) The method for detecting the substances related to the desmopressin acetate oral solution is convenient, efficient, time-saving and accurate in detection.
Drawings
FIGS. 1 to 4 are chromatograms of a blank solution, a blank auxiliary material solution, a test sample solution and a system applicability solution measured by the condition 5 in sequence;
FIGS. 5-7 are chromatograms of a system applicability experiment; wherein fig. 6 and 7 are partial sectional views of fig. 5;
fig. 8 is a chromatogram obtained by the detection method of desmopressin acetate raw material drug recorded in european pharmacopoeia.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments. The examples are for illustration only and are not intended to limit the invention in any way. The raw materials and reagents used in the examples were conventional products which were obtained commercially, unless otherwise specified; the experimental methods for which specific conditions are not specified in the examples are generally in accordance with the conditions conventional in the art or in accordance with the manufacturer's recommendations.
Test example 1 construction of method for detecting substances related to desmopressin acetate oral solution
1. Instrument and reagent
The main instrument is as follows: waters E2695-2998 high performance liquid chromatograph, sidoris electronic balance.
The main reagent comprises: acetonitrile, chromatographic purity; anhydrous disodium hydrogen phosphate, analytically pure; potassium dihydrogen phosphate, analytically pure; sodium hydroxide, analytically pure.
2. Sample information
Desmopressin acetate control, content: 90.1%, source: chinese food and drug testing institute;
impurity I reference substance, content: N/A, source: chinese food and drug testing institute;
impurity II reference, content: 99.2%, source: guangzhou Jia Tu science and technology Co Ltd
Impurity III reference, content: 99.2%, source: impurity IV reference substance of Guangzhou Jia Tu technology Co., ltd.) content: 88.0% of source: suzhou Tianma pharmaceutical group Co Ltd
3. Solution preparation
Blank solvent/diluent: 0.0335mol/L phosphate buffer-acetonitrile (95:5, v/v);
impurity control stock: respectively precisely weighing impurity I, impurity II, impurity III, and impurity IV reference substance 10mg in 25mL measuring flask, adding diluent, dissolving, diluting to scale, and shaking to obtain the final product;
system applicability solution: precisely weighing desmopressin acetate 10mg, placing into a 25mL measuring flask, adding a proper amount of diluent to dissolve, adding 0.25mL of impurity reference substance stock solution, diluting to scale with the diluent, and shaking uniformly to obtain the final product;
desmopressin acetate control solution: precisely weighing about 10mg of desmopressin acetate reference substance, placing in a 25mL measuring flask, dissolving with solvent, diluting to scale, and shaking to obtain stock solution of desmopressin acetate reference substance; precisely measuring the stock solution of the desmopressin acetate reference substance in a 1 mL-100 mL measuring flask, diluting to a scale with a diluent, and shaking uniformly to obtain a desmopressin acetate reference substance solution;
sensitivity solution: precisely measuring 1mL of desmopressin acetate reference substance solution, placing in a 20mL measuring flask, diluting to scale with diluent, and shaking to obtain the final product;
test solution: taking desmopressin acetate oral solution (specification: 0.36 mg/mL) to obtain;
blank auxiliary material solution: taking blank auxiliary material solution to obtain the product.
4. Chromatographic conditions
Condition 1:
chromatographic column: octadecylsilane chemically bonded silica column (Xitaamate-C18, 4.6 mm. Times.250 mm,5 μm;
buffer solution: 3.4g of potassium dihydrogen phosphate and 2.0g of sodium 1-heptanesulfonate were dissolved in 1000mL of water; adjusting pH to 4.50+ -0.05 with phosphoric acid or sodium hydroxide as required;
mobile phase: acetonitrile-buffer (22:78, v/v);
flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: isocratic elution.
Condition 2:
chromatographic column: octadecylsilane chemically bonded silica column (Ultimate-XB-C18), 4.6mm.times.150mm, 3 μm;
mobile phase: taking 0.067mol/L phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B; wherein, the preparation method of the phosphate buffer solution with the concentration of 0.067mol/L comprises the following steps: weighing 9.4g of disodium hydrogen phosphate and 9.1g of monopotassium phosphate, adding 2000mL of water for dissolution, adding 1mol/L of NaOH for regulating the pH to 7.0, shaking uniformly, and carrying out suction filtration to obtain the compound;
flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: gradient elution, elution procedure is:
| time (min) | A％ | B％ |
| 0 | 88 | 12 |
| 4 | 88 | 12 |
| 18 | 79 | 21 |
| 35 | 74 | 26 |
| 40 | 88 | 12 |
| 50 | 88 | 12 |
Condition 3:
chromatographic column: octadecylsilane chemically bonded silica column (Ultimate-XB-C18), 4.6mm.times.150mm, 3 μm;
mobile phase: taking 0.0335mol/L phosphate buffer as a mobile phase A and acetonitrile as a mobile phase B; wherein, the preparation method of the phosphate buffer solution with the concentration of 0.0335mol/L comprises the following steps: taking 2.37g of anhydrous disodium hydrogen phosphate and 2.28g of monopotassium phosphate, and adding 1000mL of water to dissolve the anhydrous disodium hydrogen phosphate to obtain the aqueous potassium dihydrogen phosphate;
flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: gradient elution, elution procedure is:
| time (min) | A％ | B％ |
| 0 | 82 | 18 |
| 30 | 82 | 18 |
| 40 | 60 | 40 |
| 50 | 60 | 40 |
| 50.01 | 82 | 18 |
| 60 | 82 | 18 |
Condition 4:
chromatographic column: octadecylsilane chemically bonded silica column (Ultimate-XB-C18), 4.6mm.times.150mm, 3 μm;
mobile phase: taking 0.0335mol/L phosphate buffer as a mobile phase A and acetonitrile as a mobile phase B;
flow rate: 1.0mL/min; column temperature: 35 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: gradient elution, elution procedure is:
| time (min) | A(％) | B(％) |
| 0 | 82 | 18 |
| 30 | 82 | 18 |
| 40 | 60 | 40 |
| 50 | 60 | 40 |
| 50.01 | 82 | 18 |
| 60 | 82 | 18 |
Condition 5:
chromatographic column: octadecylsilane chemically bonded silica column (Horizon-C18), 4.6 mm. Times.250 mm,5 μm;
mobile phase: taking 0.0335mol/L phosphate buffer solution-acetonitrile (95:5, v/v) as a mobile phase A and acetonitrile as a mobile phase B;
flow rate: 1.0mL/min; column temperature: 35 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: gradient elution, elution procedure is:
| time (min) | A％ | B％ |
| 0 | 89 | 11 |
| 5 | 89 | 11 |
| 15 | 80 | 20 |
| 25 | 67 | 33 |
| 25.01 | 60 | 40 |
| 30 | 60 | 40 |
| 30.01 | 89 | 11 |
| 40 | 89 | 11 |
6. Measurement
And precisely measuring a blank solution, a blank auxiliary material solution, a system applicability solution, a sensitivity solution and a sample solution respectively, injecting the solutions into a chromatograph, and recording a chromatogram.
Suitable detection conditions should meet the following requirements: the blank solution and the blank auxiliary material solution do not interfere detection, the impurity II, the impurity I, the impurity III, the desmopressin acetate and the impurity IV in the system applicability solution chromatogram sequentially show peaks, the separation degree of the desmopressin acetate peak and the impurity III peak is more than or equal to 1.5, and the signal to noise ratio of the desmopressin peak in the sensitivity solution is more than or equal to 10.
7. Results
The chromatogram obtained in the condition 1 shows that under the chromatographic condition, 2 preservatives (methylparaben and propylparaben) all interfere with the peak, and the interference degree is large;
the chromatogram obtained by the measurement under the condition 2 shows that under the chromatographic condition, the blank solvent interferes with the main peak to form a peak, and the buffer salt concentration is high and easy to salt out;
the chromatogram measured under the condition 3 shows that under the chromatographic condition, the impurity III cannot be separated from the base line, the peak of the impurity IV is interfered, and the main peak and the adjacent impurity peak are not well separated;
the chromatogram measured under the condition 4 shows that under the chromatographic condition, the impurity II and the impurity I are not well separated, and partial impurities are early in peak emergence;
the chromatograms of the blank solution, the blank auxiliary material solution, the sample solution and the system applicability solution which are measured by the condition 5 are sequentially shown in figures 1 to 4, and the blank auxiliary material and the blank solution have no interference on detection under the chromatographic condition, and the main peak, the specific impurity and the adjacent peak are well separated; therefore, condition 5 was used as a condition for detecting desmopressin acetate and related substances in desmopressin acetate oral solutions.
Example 1 desmopressin acetate oral solution related substance detection method
The method comprises the following steps:
(1) Solution preparation
Preparation of a control solution: precisely weighing about 10mg of desmopressin acetate reference substance, placing into a 25mL measuring flask, dissolving with diluent (mobile phase A), diluting to scale, and shaking; precisely measuring the solution in a measuring flask with 1mL to 100mL, diluting to a scale with a diluent, and shaking uniformly to obtain the product.
Test solution: taking desmopressin acetate oral solution (specification: 0.36 mg/mL).
(2) Chromatographic conditions:
chromatographic column: octadecylsilane chemically bonded silica column (Horizon-C18), 4.6 mm. Times.250 mm,5 μm; or other chromatographic column of comparable performance;
mobile phase: taking 0.0335mol/L phosphate buffer solution-acetonitrile (95:5, v/v) as a mobile phase A and acetonitrile as a mobile phase B; wherein, the preparation method of the phosphate buffer solution with the concentration of 0.0335mol/L comprises the following steps: taking 2.37g of anhydrous disodium hydrogen phosphate and 2.28g of monopotassium phosphate, and adding 1000mL of water to dissolve the anhydrous disodium hydrogen phosphate to obtain the aqueous potassium dihydrogen phosphate;
flow rate: 1.0mL/min; column temperature: 35 ℃; detection wavelength: 220nm; sample injection volume: 50. Mu.L;
elution mode: gradient elution, elution procedure is:
(3) Precisely measuring 50 mu L of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, recording the chromatograms, and calculating the contents of main peaks and impurities in the sample solution according to an external standard method.
Test example 2 examination of the method for detecting substances related to desmopressin acetate oral solution
1. Forced degradation experiment
After the desmopressin acetate oral solution (hereinafter referred to as "the product") was destroyed by severe conditions such as high temperature, acid, alkali, oxidation, light, etc., the relevant substances were measured in the same manner as in example 1 to examine whether the selected chromatographic conditions could detect degradation products which may be generated by the product, as follows:
(1) Nondestructive test solution: taking desmopressin acetate oral solution (specification: 0.36 mg/mL) to obtain;
(2) Acid damages the test solution: precisely measuring 1mL of 5mol/L HCl solution, placing into a 10mL measuring flask, adding 5mL of sample solution, standing for 2h, adding 1mL of 5mol/L NaOH solution, diluting to scale with the sample solution, and shaking uniformly to obtain the final product;
(3) Alkaline destruction of the test solution: precisely measuring 1mL of 0.5mol/L NaOH solution, placing into a 10mL measuring flask, adding 5mL of sample solution, standing for 10min, adding 1mL of 0.5mol/L HCl solution, diluting to scale with the sample solution, and shaking uniformly to obtain the final product;
(4) Oxidative destruction of the test solution: precise measuring 3%H 2 O 2 1mL of the solution is put into a 10mL measuring flask, diluted to a scale by the solution of the sample, shaken uniformly and stood for 6 hours to obtain the solution;
(5) High temperature damage to the test solution: placing the sample solution at 100deg.C for 6 hr to obtain the final product;
(6) Light damages the test solution: placing the sample solution in an illumination box (4500+ -500 Lux) for 3 days to obtain the final product.
The forced degradation test results are shown in table 1 below:
TABLE 1 forced degradation test results
The result shows that the product is stable under high temperature and illumination damage conditions, and no unknown impurities which are larger than the limit (1.0%) of single impurities are detected. Under the condition of acid destruction, the main degradation generates impurity I (detection amount is 4.23%), impurity II (detection amount is 1.02%), and impurity III (detection amount is 3.32%); mainly degrading under alkaline destruction conditions to produce unknown impurities of RRT 1.13 (detected 1.01%); the main degradation under oxidative destruction conditions resulted in unknown impurities of RRT 0.38 (detected 1.40%) and RRT0.84 (detected 2.33%), and furthermore, no impurities above the single impurity limit (1.0%) were detected.
Under each damage condition, the blank solution and the blank auxiliary materials have no interference on detection, the purity angle of a main peak is smaller than a purity threshold value, the minimum separation degree between a desmopressin peak and an adjacent impurity peak is 3.91, the separation degree is larger than 1.5, the ratio (material balance) of the total peak area before and after forced degradation is 99.8-106.8%, and the material conservation indicates that each degradation product of the product can be effectively detected under the condition of the related substance detection method of the product.
2. Quantitative limit and detection limit
Desmopressin acetate control stock: precisely weighing desmopressin acetate reference substance about 10mg, placing into 25mL measuring flask, dissolving with solvent, diluting to scale, and shaking.
Mixing the reference substance solution: precisely measuring desmopressin acetate reference substance stock solution, respectively 5mL of the impurity reference substance stock solution prepared in test example 1 in 50mL measuring flask, diluting to scale with diluent, and shaking uniformly to obtain the final product.
Quantitative limiting solution: precisely measuring the mixed reference substance solution in a measuring flask with 1mL to 200mL, diluting to a scale with a diluent, and shaking uniformly to obtain the final product.
Detection limit solution: precisely measuring 5mL of quantitative limiting solution, placing in a 10mL measuring flask, diluting to scale with a diluent, and shaking uniformly to obtain the final product.
The chromatographic conditions were followed by separate sample injection and recording of the chromatograms.
The results are shown in tables 2-3.
TABLE 2 quantitative limit results
| Compounds of formula (I) | Concentration (μg/mL) | Concentration ratio with test article (%) | S/N |
| Desmopressin acetate | 0.1823 | 0.05 | 21.0 |
| Impurity II | 0.1984 | 0.06 | 32.2 |
| Impurity I | 0.2072 | 0.06 | 28.6 |
| Impurity III | 0.2046 | 0.06 | 24.9 |
| Impurity IV | 0.1760 | 0.05 | 16.8 |
Conclusion: the concentration of the quantitative limiting solutions of desmopressin acetate, the impurity I, the impurity II, the impurity III and the impurity IV are respectively 0.18 mug/mL, 0.20 mug/mL and 0.18 mug/mL, and the signal to noise ratios are respectively 21.0, 28.6, 32.2, 24.9 and 16.8, which are approximately equivalent to 0.05%, 0.06% and 0.05% of the concentration of the solution of the test sample, so that the quantitative limiting solutions can be accurately quantified, and the method has good sensitivity.
TABLE 3 limit of detection results
| Compounds of formula (I) | Concentration (μg/mL) | Concentration ratio with test article (%) | S/N |
| Impurity II | 0.0992 | 0.03 | 18.9 |
| Impurity I | 0.1036 | 0.03 | 18.2 |
| Impurity III | 0.1023 | 0.03 | 18.5 |
| Desmopressin acetate | 0.0911 | 0.03 | 17.9 |
| Impurity IV | 0.0880 | 0.02 | 8.3 |
Conclusion: the detection limit solution concentrations of desmopressin acetate, impurity I, impurity II, impurity III and impurity IV are respectively 0.09 mug/mL, 0.10 mug/mL and 0.09 mug/mL, and the signal to noise ratios are respectively 17.9, 18.2, 18.9, 18.5 and 8.3, which are approximately equivalent to 0.03%, 0.03% and 0.02% of the solution concentration of the sample, so that the method has good sensitivity.
3. Repeatability experiments
Adding a labeled test sample solution: precisely measuring 5mL of the impurity reference substance stock solution prepared in the test example 1, placing the stock solution in a 50mL measuring flask, diluting to a scale with a diluent, and shaking uniformly; precisely measuring the solution in a measuring flask with 2mL to 20mL, diluting the solution to the scale with the solution of the sample, and shaking the solution evenly to obtain the product.
6 parts of the same sample solution to be added with the standard are prepared, sample introduction is carried out respectively, detection is carried out according to the chromatographic conditions of the embodiment 1, a chromatogram is recorded, and the content of 6 parts of the sample and the RSD thereof are calculated according to an external standard method. The results are shown in Table 4.
TABLE 4 results of repeatability experiments
| Impurity% | 1 | 2 | 3 | 4 | 5 | 6 | RSD％ |
| Impurity II | 0.90 | 0.90 | 0.90 | 0.90 | 0.91 | 0.89 | 0.7 |
| Impurity I | 1.02 | 1.02 | 1.02 | 1.02 | 1.02 | 1.00 | 0.7 |
| Impurity III | 0.97 | 0.97 | 0.97 | 0.96 | 0.97 | 0.95 | 0.8 |
| Impurity IV | 0.77 | 0.77 | 0.77 | 0.74 | 0.73 | 0.73 | 2.7 |
| Maximum unknown single impurity | 0.07 | 0.07 | 0.07 | 0.09 | 0.07 | 0.07 | 0.02 (extremely poor) |
| Total impurities | 3.73 | 3.72 | 3.72 | 3.71 | 3.71 | 3.63 | 1.0 |
The results show that in 6 parts of the standard-added sample solution, the RSD of the impurity II content is 0.7%, the RSD of the impurity I content is 0.7%, the RSD of the impurity III content is 0.8%, the RSD of the impurity IV content is 2.7%, the maximum unknown single impurity range is 0.02%, and the total impurity content RSD is 1.0%, which meet the requirements, and the method is good in repeatability.
4. System applicability experiment
System applicability requirements: in the system applicability solution, the impurity II, the impurity I, the impurity III, the desmopressin acetate and the impurity IV sequentially show peaks, and the separation degree of the desmopressin acetate peak and the impurity III peak is more than 1.5.
A system applicability solution was prepared according to the method of test example 1, 50. Mu.L of the system applicability solution was precisely measured and injected into a chromatograph, and sample injection detection was performed according to the chromatographic conditions of example 1, and chromatograms were recorded as shown in FIGS. 5 to 7.
The results are shown in Table 5 below.
Table 5 results of System suitability experiments
| Impurity II | Impurity I | Impurity III | Desmopressin acetate | Impurity IV | |
| Retention time/min | 15.490 | 15.725 | 16.079 | 16.932 | 22.474 |
| Degree of separation | - | 1.15 | 1.63 | 3.63 | 25.84 |
Conclusion: in the system applicability solution, the impurity II, the impurity I, the impurity III, the desmopressin acetate and the impurity IV sequentially form peaks, and the separation degree of the desmopressin acetate peak and the impurity III peak is 3.63 and is more than 1.5, so that the specification is met.
5. Linear experiments
Taking the mixed reference substance solution prepared in the steps of '2, quantitative limit and detection limit', quantitatively diluting, preparing 6 parts of solutions with different concentrations, respectively carrying out sample injection detection according to the chromatographic conditions of the embodiment 1, and recording a chromatogram. And calculating a regression equation and a correlation coefficient by using the concentration of the reference substance solution as an abscissa and the peak area as an ordinate and using a least square method.
The results are shown in Table 6.
TABLE 6 Linear experiment results
| Names of Compounds | Linear range μg/ml | Linear equation | Linear correlation coefficient |
| Desmopressin | 0.1823～7.2909 | y＝49773.3296x-680.3369 | 0.9999 |
| Impurity I | 0.2072～8.2880 | y＝43411.0681x-311.2907 | 1.0000 |
| Impurity II | 0.1984～7.9360 | y＝40517.8613x-627.9289 | 0.9999 |
| Impurity III | 0.2046～8.1820 | y＝42134.6289x-397.3510 | 0.9999 |
| Impurity IV | 0.1760～7.0400 | y＝43152.9080x-5571.0940 | 0.9999 |
The results show that under the detection method of the invention, desmopressin acetate, impurity I, impurity II, impurity III and impurity IV can all have good linear relationship within a certain concentration range.
6. Solution stability
Taking the labeled sample solution prepared in the '3 and repeatability experiments', inspecting at room temperature, sampling at different time points (0 h,19h,42h and 76 h), respectively sampling and detecting according to the chromatographic conditions of the example 1, and recording a chromatogram.
The results are shown in Table 7.
TABLE 7 stability test results
Conclusion: placing the standard sample solution at room temperature for 76 hours, wherein the ratio of the peak area of the impurity II to 0h measured at each time point is 1.00; the ratio of the area of the impurity I peak to 0h is 1.00; the ratio of the peak area of the impurity III to 0h is 1.00; the ratio of the peak area of the impurity IV to 0h is 1.05-1.06, the ratio of the peak area of desmopressin to 0h is 1.00-1.01, no new degradation impurity is generated, and the stability of the standard sample solution is good in 76h under the condition of room temperature.
7. Durability test
The column temperature + -1 ℃ and the relative value change of the flow velocity + -0.1 mL/min and different brands of chromatographic columns are respectively inspected, and durability experiments are carried out. Precisely measuring a blank solution, a blank auxiliary material solution and a system applicability solution, injecting the blank solution, the blank auxiliary material solution and the system applicability solution into a liquid chromatograph, respectively carrying out sample injection detection according to the chromatographic conditions of the embodiment 1, recording a chromatogram, and observing the influence of each condition on a detection result. The results are shown in Table 8.
Table 8 durability test results
The results show that under all conditions (different column temperatures + -1 ℃, different chromatographic columns and different flow rates + -0.1 mL/min), the system applicability meets the regulations, and the method has good durability. .
Comparative example
The detection method of desmopressin acetate raw material medicine recorded in European pharmacopoeia is used for detecting related substances of desmopressin acetate oral solution, and chromatographic conditions are as follows:
chromatographic column: octadecylsilane chemically bonded silica column (Ultimate-XB-C18), 4.6mm.times.150mm, 3 μm;
mobile phase: taking 0.067mol/L phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B; flow rate: 1.0mL/min; column temperature: 25 ℃; detection wavelength: 220nm; sample injection volume: 100. Mu.L;
elution mode: gradient elution, elution procedure is:
| time (min) | A％ | B％ |
| 0 | 76 | 24 |
| 5 | 76 | 24 |
| 22.5 | 58 | 42 |
| 43.75 | 48 | 52 |
| 50 | 76 | 24 |
| 60 | 76 | 24 |
The results are shown in FIG. 8, where methylparaben interferes with the main peak to peak.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.
Claims (9)
1. The method for detecting substances related to desmopressin acetate oral solution is characterized by comprising the following steps of: separating and detecting desmopressin acetate and related substances in desmopressin acetate oral solution by adopting high performance liquid chromatography; wherein the related substances comprise impurities I, II, III and IV; the chromatographic conditions of the high performance liquid chromatography include:
the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler;
the mobile phase comprises a mobile phase A and a mobile phase B, and gradient elution is adopted; wherein the volume ratio of the mobile phase A is (90-100): the mixed solution of the buffer solution and acetonitrile, wherein the concentration of the buffer solution is 0.01-0.07 mol/L; the mobile phase B is acetonitrile;
the detection wavelength is 220nm; the column temperature of the chromatographic column is 25-40 ℃; the flow rate of the mobile phase is 0.8-1.5 mL/min.
2. The method for detecting substances related to desmopressin acetate oral solution according to claim 1, wherein the buffer solution is phosphate buffer solution, and the components of the buffer solution comprise potassium dihydrogen phosphate, disodium hydrogen phosphate and water.
3. The method for detecting substances related to desmopressin acetate oral solution according to claim 2, wherein the concentration of the phosphate buffer is 0.0335mol/L.
4. The method for detecting substances related to desmopressin acetate oral solution according to claim 1, 2 or 3, wherein the volume ratio of the buffer solution to the acetonitrile in the mobile phase A is 95:5.
5. The method for detecting substances associated with desmopressin acetate oral solution according to claim 4, wherein the chromatographic column has a specification of an inner diameter of 4.6mm, a length of 250mm and a filler particle diameter of 5 μm.
6. The method for detecting substances associated with desmopressin acetate oral solution according to claim 5, wherein the column temperature of the chromatographic column is 35 ℃.
7. The method for detecting substances associated with desmopressin acetate oral solution according to claim 5 or 6, wherein the flow rate of the mobile phase is 1.0mL/min.
8. The method for detecting substances related to desmopressin acetate oral solution according to claim 1, wherein the gradient elution procedure is as follows:
9. The method for detecting substances related to desmopressin acetate oral solution according to claim 1, wherein the sample injection volume is 50-100 μl.
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