CN114609268B - Detection method for related substances in sodium rabeprazole bulk drug - Google Patents
Detection method for related substances in sodium rabeprazole bulk drug Download PDFInfo
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- 229960004157 rabeprazole Drugs 0.000 title claims abstract description 73
- 239000000126 substance Substances 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 239000003814 drug Substances 0.000 title claims abstract description 34
- 229940079593 drug Drugs 0.000 title claims abstract description 30
- YREYEVIYCVEVJK-UHFFFAOYSA-N rabeprazole Chemical compound COCCCOC1=CC=NC(CS(=O)C=2NC3=CC=CC=C3N=2)=C1C YREYEVIYCVEVJK-UHFFFAOYSA-N 0.000 title abstract description 60
- 239000012535 impurity Substances 0.000 claims abstract description 230
- 238000000034 method Methods 0.000 claims abstract description 41
- 238000010828 elution Methods 0.000 claims abstract description 26
- 238000012937 correction Methods 0.000 claims description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 18
- -1 3-methoxypropoxy Chemical group 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 13
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 10
- 229910052708 sodium Inorganic materials 0.000 claims description 10
- 239000011734 sodium Substances 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
- 150000003457 sulfones Chemical class 0.000 claims description 7
- SILNNFMWIMZVEQ-UHFFFAOYSA-N 1,3-dihydrobenzimidazol-2-one Chemical compound C1=CC=C2NC(O)=NC2=C1 SILNNFMWIMZVEQ-UHFFFAOYSA-N 0.000 claims description 3
- YHMYGUUIMTVXNW-UHFFFAOYSA-N 1,3-dihydrobenzimidazole-2-thione Chemical compound C1=CC=C2NC(S)=NC2=C1 YHMYGUUIMTVXNW-UHFFFAOYSA-N 0.000 claims description 3
- XPYNCLYLFSMFQE-UHFFFAOYSA-N 2-(chloromethyl)-4-(3-methoxypropoxy)-3-methylpyridine Chemical compound COCCCOC1=CC=NC(CCl)=C1C XPYNCLYLFSMFQE-UHFFFAOYSA-N 0.000 claims description 3
- JWYUFVNJZUSCSM-UHFFFAOYSA-N 2-aminobenzimidazole Chemical compound C1=CC=C2NC(N)=NC2=C1 JWYUFVNJZUSCSM-UHFFFAOYSA-N 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 150000003462 sulfoxides Chemical class 0.000 claims description 3
- GWXQTTKUYBEZBP-UHFFFAOYSA-N 1h-benzimidazol-1-ium-2-sulfonate Chemical compound C1=CC=C2NC(S(=O)(=O)O)=NC2=C1 GWXQTTKUYBEZBP-UHFFFAOYSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- KRCQSTCYZUOBHN-UHFFFAOYSA-N rabeprazole sodium Chemical compound [Na+].COCCCOC1=CC=NC(CS(=O)C=2[N-]C3=CC=CC=C3N=2)=C1C KRCQSTCYZUOBHN-UHFFFAOYSA-N 0.000 claims 12
- 239000008186 active pharmaceutical agent Substances 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 229940088679 drug related substance Drugs 0.000 claims 1
- 238000012856 packing Methods 0.000 claims 1
- 230000003595 spectral effect Effects 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 24
- 230000014759 maintenance of location Effects 0.000 abstract description 9
- 230000004044 response Effects 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 70
- 239000000523 sample Substances 0.000 description 50
- 239000000243 solution Substances 0.000 description 35
- 238000012360 testing method Methods 0.000 description 29
- 239000002904 solvent Substances 0.000 description 24
- GRONZTPUWOOUFQ-UHFFFAOYSA-M sodium;methanol;hydroxide Chemical compound [OH-].[Na+].OC GRONZTPUWOOUFQ-UHFFFAOYSA-M 0.000 description 22
- 239000007788 liquid Substances 0.000 description 20
- 239000012488 sample solution Substances 0.000 description 19
- 239000011259 mixed solution Substances 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000011084 recovery Methods 0.000 description 14
- 238000004587 chromatography analysis Methods 0.000 description 12
- 239000013558 reference substance Substances 0.000 description 12
- 238000005259 measurement Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 230000006399 behavior Effects 0.000 description 7
- 239000000543 intermediate Substances 0.000 description 7
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000007689 inspection Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000007857 degradation product Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- YREYEVIYCVEVJK-RUZDIDTESA-N dexrabeprazole Chemical compound COCCCOC1=CC=NC(C[S@@](=O)C=2NC3=CC=CC=C3N=2)=C1C YREYEVIYCVEVJK-RUZDIDTESA-N 0.000 description 2
- 229960000646 dexrabeprazole Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- RAPCQINSRSZSKF-UHFFFAOYSA-N 2-[(4-chloro-3-methylpyridin-2-yl)methylsulfinyl]-1h-benzimidazole Chemical compound CC1=C(Cl)C=CN=C1CS(=O)C1=NC2=CC=CC=C2N1 RAPCQINSRSZSKF-UHFFFAOYSA-N 0.000 description 1
- HZNKDLUKNUEOOX-UHFFFAOYSA-N 2-[(4-methoxy-3-methylpyridin-2-yl)methylsulfanyl]-1h-benzimidazole Chemical compound COC1=CC=NC(CSC=2NC3=CC=CC=C3N=2)=C1C HZNKDLUKNUEOOX-UHFFFAOYSA-N 0.000 description 1
- ITARWNDPMUQOCB-UHFFFAOYSA-N 2-[(4-methoxy-3-methylpyridin-2-yl)methylsulfinyl]-1h-benzimidazole Chemical compound COC1=CC=NC(CS(=O)C=2NC3=CC=CC=C3N=2)=C1C ITARWNDPMUQOCB-UHFFFAOYSA-N 0.000 description 1
- BSXAHDOWMOSVAP-UHFFFAOYSA-N 2-[[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulfanyl]-1h-benzimidazole Chemical compound COCCCOC1=CC=NC(CSC=2NC3=CC=CC=C3N=2)=C1C BSXAHDOWMOSVAP-UHFFFAOYSA-N 0.000 description 1
- ZGDLVKWIZHHWIR-UHFFFAOYSA-N 4-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl]morpholine Chemical compound O1C(C)(C)C(C)(C)OB1C1=CC=C(N2CCOCC2)N=C1 ZGDLVKWIZHHWIR-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 238000009658 destructive testing Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 210000001711 oxyntic cell Anatomy 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 229960001778 rabeprazole sodium Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAZLUNIWYYOJPC-UHFFFAOYSA-M sulfenamide Chemical compound [Cl-].COC1=C(C)C=[N+]2C3=NC4=CC=C(OC)C=C4N3SCC2=C1C QAZLUNIWYYOJPC-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 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
- G01N30/26—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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- 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
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- 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)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Plural Heterocyclic Compounds (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a detection method of related substances in a sodium rabeprazole bulk drug, which selects a specific chromatographic column, optimizes elution gradient proportion and column temperature, monitors various impurity types and number, has good separation degree between impurities and between the impurities and main components, has higher retention capacity of the main components and the impurities, has higher response of the components, ensures that the content of 13 related impurities in the sodium rabeprazole bulk drug can be presented in one chromatographic behavior, and can rapidly and accurately monitor the related substances in the sodium rabeprazole bulk drug, and the method is simple and easy to implement and has high accuracy and precision.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of related substances of a sodium rabeprazole bulk drug.
Background
Dexrabeprazole is a Proton Pump Inhibitor (PPI) which is secreted by cells on the surface of the stomach wall (H + ,K + ) The 2 sites of the atpase system produce covalent binding, the last step in inhibition of gastric acid production. Sodium dexrabeprazole, similar to sodium rabeprazole, is a prodrug that is protonated at the parietal cells to form the active sulfenamide. For short-term treatment of gastric, duodenal ulcers and gastroesophageal reflux disease (GERD).
The related substances are starting materials, intermediates, side reaction products, degradation impurities and the like which are brought in the drug synthesis production process, and the quality and the safety of the drug can be controlled by detecting the related substances. The detection method of related substances of the sodium rabeprazole is not recorded in the current domestic and foreign pharmacopoeias, and the detection method of related substances of the sodium rabeprazole is recorded in Chinese pharmacopoeias, EP, USP and JP pharmacopoeias, wherein the elution conditions of the Chinese pharmacopoeias and the JP pharmacopoeias adopt an isocratic elution mode, and impurities with smaller polarity are difficult to elute, so that the accuracy of analysis results is affected; the elution mode of the mobile phase in USP pharmacopoeia is complex, the mobile phase is mixed in double terms, the number of the researched impurities is small (4), and the detection requirement cannot be met; the mobile phase in the EP pharmacopoeia is three-phase gradient elution, and the gradient elution is when three-phase mixing, easily produces the bubble and influences the baseline steadily, disturbs the effective detection of certain impurity, and the three-phase mixing is higher to the instrument precision requirement simultaneously, easily produces great error when the mobile phase elution proportion is little change.
Chinese patent CN112834628A discloses a method for measuring sodium rabeprazole and impurities thereof by high performance liquid chromatography, wherein phosphate buffer solution and acetonitrile are used as mobile phases for gradient elution detection, the sodium rabeprazole and 9 common impurities are contained, the types of analysis impurities are fewer, the sensitivity and the accuracy are lower, and the quality monitoring requirement in the production process of the sodium rabeprazole bulk drug still cannot be met.
In order to ensure the safety and effectiveness of the medicament, the related substances in the medicament need to be researched, detected and monitored. The related substances are mainly process byproducts and degradation products, and the impurity spectrum of the medicine changes in the placing process, so that a proper analysis method is required to be established according to different synthetic routes, production processes and storage conditions, and the accurate and effective detection and monitoring of the related substances of the sodium rabeprazole bulk drug are achieved.
Disclosure of Invention
The invention aims to provide a detection method for related substances of a sodium rabeprazole bulk drug, which can detect 13 related impurities in one chromatographic behavior, is convenient for controlling the quality of products in the production process, and has the advantages of simplicity, easiness, high accuracy and precision and good reproducibility.
The technical scheme of the invention is as follows:
the detection method adopts high performance liquid chromatography to carry out qualitative or quantitative detection on the sodium rabeprazole and related substances, and the high performance liquid chromatography conditions comprise: the chromatographic column is Shimadzu Wondasil C 18 -a WR pillar; adopting a mobile phase A and a mobile phase B as mixed mobile phases for gradient elution, wherein the mobile phase A is 15-25 mmol/L potassium dihydrogen phosphate buffer solution, and regulating the pH value to 6.0-8.0; mobile phase B was methanol.
The initial proportion of the mobile phase A to the mobile phase B in the gradient elution process is 73-77:27-23; the volume ratio of the mobile phase A to the mobile phase B is kept unchanged within 0-8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 55:45 at a constant speed within 8-18 minutes; the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45 within 18-33 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 55:45 to 25:75 at a constant speed within 33-75 minutes; the volume ratio of mobile phase A to mobile phase B remains 25:75 unchanged within 75-95 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 25:75 to the initial ratio at a constant speed within 95-96 minutes; the volume ratio of mobile phase a to mobile phase B remained the same during 96-106 minutes.
For the purposes of the present invention, the initial ratio of mobile phase A to mobile phase B is controlled to be 73-77:27-23 during gradient elution, and may be, but is not limited to 73:27, 74:26, 75:25, 76:24 or 77:23, in a preferred embodiment, the initial ratio of mobile phase A to mobile phase B is 75:25.
In a preferred embodiment, when the initial ratio of mobile phase A to mobile phase B is 75:25; the gradient elution comprises the following steps: the volume ratio of mobile phase A to mobile phase B remains 75:25 unchanged within 0-8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 55:45 at a constant speed within 8-18 minutes; the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45 within 18-33 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 55:45 to 25:75 at a constant speed within 33-75 minutes; the volume ratio of mobile phase A to mobile phase B remains 25:75 unchanged within 75-95 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 25:75 to 75:25 at a constant speed within 95-96 minutes; the volume ratio of mobile phase A to mobile phase B remained 75:25 during 96-106 minutes. The specific gradient elution procedure is shown in table 1 below:
TABLE 1 gradient elution Process
When the invention adopts high performance liquid chromatography to detect, the mobile phase A is 15-25 mmol/L potassium dihydrogen phosphate buffer solution, and the pH value is regulated to 6.0-8.0; mobile phase B was methanol. In a preferred embodiment, mobile phase A is 20mmol/L potassium dihydrogen phosphate buffer solution and its pH is adjusted to 6.8-7.2. Particularly preferably, mobile phase A is a 20mmol/L potassium dihydrogen phosphate buffer solution, the pH of which is adjusted to 7.0.
In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, selectionGood selectivity, high analysis speed, etc. The invention adopts high performance liquid chromatography to qualitatively or quantitatively detect the sodium rabeprazole and related substances, and the chromatographic column is Shimadzu Wondasil C 18 The WR column monitors various impurity types and quantity in the chromatographic analysis process, has good separation degree between impurities and between the impurities and the main component, and can rapidly and accurately monitor related substances in the sodium rabeprazole. Under the condition of not affecting the detection effect, the length of the chromatographic column is preferably 250mm, the diameter is 4.6mm, the particle size of the filler is 5 mu m, namely the chromatographic column adopted by the invention is Shimadzu Wondasil C 18 -WR column (250 x4.6mm,5 μm). During the course of experiments, it was found that using other similar chromatographic columns, such as Inertsil ODS-3, the separation of some impurities from the main component was poor, and even a baseline separation could not be achieved during the chromatographic analysis.
For the invention, 13 different impurities including intermediate impurities, process impurities and degradation impurities need to be detected, the types of the impurities are more, the column temperature in the chromatographic analysis process needs to be strictly controlled in the chromatographic analysis process, and when the column temperature selected by the chromatographic analysis is higher or lower, the separation degree between certain impurities is easily lower than 1.5, the separation degree is poor, and the baseline separation is not easy to achieve. In the present invention, the column temperature is controlled to 18 to 22℃and may be, but not limited to, 18℃19℃20℃21℃or 22℃and preferably 20 ℃.
Further, the high performance liquid chromatography conditions include: the detection wavelength is 200-400nm, preferably 280nm.
Further, the flow rate is 0.5-1.5 ml/min; preferably 1.0ml/min.
Further, the sample injection amount is 5 to 20. Mu.l, and can be, but not limited to, 5. Mu.l, 10. Mu.l, 15. Mu.l or 20. Mu.l; preferably, the amount of sample introduced is 10. Mu.l.
The related substances in the sodium rabeprazole bulk drug comprise the following substances: impurity 1: 2-chloromethyl-4- (3-methoxypropoxy) -3-methylpyridine; impurity 2: 2-mercaptobenzimidazole; impurity 3:2- [ [4- (3-methoxypropoxy) -3-methylpyridin-2-yl ] -methylthio ] -1H-benzimidazole; impurity 4:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole sodium salt; impurity 5: 2-aminobenzimidazole; impurity 6:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfoxide ] -1H-benzimidazole nitroxide; impurity 7: 2-hydroxy benzimidazole; impurity 8:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole nitroxide; impurity 9: benzimidazole-2-sulfonic acid; impurity 10:1- (1H-benzoimidazol-2-yl) -1, 4-dihydroxy-3-methyl-4-oxo-2-pyridinecarboxylic acid sodium salt; impurity 20:2- [ [ (4-chloro-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole; impurity 21:2- [ [ (4-methoxy-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole; impurity 22:2- [ (4-methoxy-3-methylpyridin-2-yl) methylsulfanyl ] -1H-benzimidazole.
The structural formulae of the above-mentioned related substances are as follows:
the invention adopts high performance liquid chromatography to screen and optimize from the aspects of chromatographic conditions, respectively, draws out related substance methods, performs methodology verification, performs qualitative research on impurity 1 (SMA), performs quantitative research on impurity 2 (SMB), impurity 3 (intermediate 1), and impurities 4-10 and 20-22 (process impurities and degradation impurities), provides a complete verification scheme, and has simple operation and good stability and reproducibility. Wherein, the correction factor of impurity 2 is 0.57, the correction factor of impurity 3 is 0.88, the correction factor of impurity 4 is 1.13, the correction factor of impurity 5 is 0.71, the correction factor of impurity 6 is 0.67, the correction factor of impurity 7 is 0.74, the correction factor of impurity 8 is 0.88, the correction factor of impurity 9 is 0.93, the correction factor of impurity 10 is 0.69, the correction factor of impurity 20 is 0.76, the correction factor of impurity 21 is 0.81, and the correction factor of impurity 22 is 0.81.
In a preferred scheme, the detection method of related substances in the sodium rabeprazole bulk drug provided by the invention comprises the following operation steps:
(1) Preparing a solution:
and taking a proper amount of a sodium rabeprazole sample, dissolving with a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio is 40:60)) and quantitatively diluting to prepare a solution containing about 1.0mg of sodium rabeprazole per 1ml serving as a test sample solution. 1.0ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale by adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] and uniformly shaken to serve as a control solution.
(2) Respectively injecting the sample solution and the impurity reference substance solution into a liquid chromatograph, recording the chromatogram, calculating according to an external standard method and peak area, wherein the chromatographic conditions are as follows:
chromatographic column: shimadzu Wondasil C 18 -WR pillars (250 x4.6mm,5 μm);
the column temperature is: 18-22 ℃, preferably 20 ℃;
detection wavelength: 200-400nm, further, the wavelength is 280nm;
the flow rate is: 0.5-1.5 ml/min; preferably 1.0ml/min;
sample injection amount: 5-20 mul; preferably 10 μl;
mobile phase a:20mmol/L potassium dihydrogen phosphate buffer solution, and adjusting the pH value to 6.0-8.0; preferably, the pH is adjusted to 6.8-7.2; more preferably, the pH thereof is adjusted to 7.0;
mobile phase B: methanol was gradient eluted as follows:
by adopting the technical scheme of the invention, the advantages are as follows:
according to the detection method for related substances in the sodium rabeprazole bulk drug, provided by the invention, a specific chromatographic column is selected, the elution gradient proportion and the column temperature are optimized, the monitored impurity types and the monitored impurity numbers are multiple, the separation degree between the impurities and the main component is good, the main component and the retention capacity of the impurities are high, and the response of the components is high, so that the content of 13 related impurities in the sodium rabeprazole bulk drug can be presented in one chromatographic behavior, and the related substances in the sodium rabeprazole bulk drug can be monitored rapidly and accurately, so that the method is simple and easy to implement, and has high accuracy and precision.
Drawings
FIG. 1 is a chromatogram of a mixed solution of impurities and a sample;
FIG. 2 is a chromatogram of a test solution of the present invention;
FIG. 3 is a linear plot of impurities 2-10, impurities 20-22, and sodium dextral rabeprazole;
FIG. 4 is a chromatogram of a mixed solution of impurities and a sample in comparative example 1;
FIG. 5 is a chromatogram of a mixed solution of impurities and a sample in comparative example 2;
FIG. 6 is a chromatogram of a mixed solution of impurities and a sample in comparative example 3.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific examples described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: HPLC detection method for related substances of dextral rabeprazole sodium
1. Experimental materials and instruments
1. Medicine and reagent: sodium dextral rabeprazole (Nanjing Haina medical technologies Co., ltd.), 2-chloromethyl-4- (3-methoxypropoxy) -3-methylpyridine (impurity 1, kanji Hawa chemical Co., ltd.), 2-mercaptobenzimidazole (impurity 2, nanji Kang Manlin biomedical technologies Co., ltd.), 2- [ [4- (3-methoxypropoxy) -3-methylpyridin-2-yl ] methylthio ] -1H-benzimidazole (impurity 3, nanjin sea medical technologies Co., ltd.), 2- [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole sodium salt (impurity 4, STD), 2-aminobenzimidazole (impurity 5, STD), 2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfoxide ] -1H-benzimidazole (impurity 6, STD), 2-hydroxy benzimidazole (impurity 7, TRC), 2- [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole (impurity 4, STD), 2- [ [4- (3-methoxypropoxy) -3-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole (impurity 9, STD) Sodium 1- (1H-benzimidazol-2-yl) -1, 4-dihydroxy-3-methyl-4-oxo-2-pyridinecarboxylate (impurity 10, USP), 2- [ [ (4-chloro-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole (impurity 20, STD), 2- [ [ (4-methoxy-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole (impurity 21, STD), 2- [ (4-methoxy-3-methylpyridin-2-yl) methylthio ] -1H-benzimidazole (impurity 22, STD), methanol (chromatographic purity, shanghai Stark high purity solvent Co., ltd.), potassium dihydrogen phosphate (analytical purity, national pharmaceutical Congress chemical Co., ltd.), sodium hydroxide (analytical purity, shanghai-Tetana technologies Co., ltd.), ultrapure water (self-made, millipore).
2. Instrument: the names and specifications of the specific instruments are shown in Table 2 below.
Table 2 names and specifications of specific instruments
| FA124 parts per million balance | SHANGHAI SUNNY HENGPING SCIENTIFIC INSTRUMENT Co.,Ltd. |
| XPE204 ten thousandth balance | Meite Teler |
| AUW 120D one ten thousandth balance | Shimadzu Japan |
| SQP ten thousandth balance | Sidoriscom instruments Co Ltd |
| XP6I038 hundredOne ten thousandth balance | Meite Teler |
| pHS-3C digital acidimeter | SHANGHAI INESA SCIENTIFIC INSTRUMENT Co.,Ltd. |
| Agilent1100 high performance liquid chromatograph | Agilent |
2. Conditions of liquid chromatography
The chromatographic column adopts Shimadzu Wondasil C 18 -WR pillars (250 x4.6mm,5 μm); gradient elution was performed by using 20mmol/L potassium dihydrogen phosphate buffer solution (pH adjusted to 7.0 with sodium hydroxide) as mobile phase A and methanol as mobile phase B according to Table 1 below; the flow rate is 1.0ml/min; column temperature 20 ℃; the detection wavelength is 280nm, 10 μl of the sample solution is precisely measured, and the sample solution is injected into a liquid chromatograph to record the chromatogram.
3. Experimental procedure
1. Detection of related substances in sodium rabeprazole bulk drug
The sample (batch number: 180923421) was weighed precisely, dissolved in a solvent [0.01mol/L sodium hydroxide-methanol (40:60) ] and diluted quantitatively to prepare a solution containing about 1mg of sodium dextro-rabeprazole per 1ml as a sample solution. 1.0ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale by adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] and uniformly shaken to serve as a control solution. Measuring 10 μl of control solution, and injecting into a liquid chromatograph, and adjusting detection sensitivity according to the liquid chromatographic conditions to make the peak height of the main component chromatographic peak about 15% -20% of full range; then, 10 μl of each of the sample solution and the control solution was measured precisely, and the samples were injected into a liquid chromatograph, and the chromatograms were recorded, as shown in fig. 1. The known impurities are quantified by self-contrast with correction factors, and the unknown impurities are measured by self-contrast. The test results are shown in Table 3. Samples (lots 181024421 and 181105421) were tested according to the test methods described above and the results are shown in Table 3.
TABLE 3 test results for different batches of test samples
2. Methodological verification
2.1 specificity
A right amount (about 10 mg) of a sodium rabeprazole sample (batch number: 180923421) is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale with a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio: 40:60)) and shaken uniformly to obtain a sample solution. Taking proper amounts of impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22, respectively dissolving with a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] and preparing a solution containing about 100 mug of each 1ml as impurity mother liquor; and weighing about 10mg of a sodium rabeprazole sample, placing the sodium rabeprazole sample into a 10ml measuring flask, adding a proper amount of the impurity mother liquor, adding a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to dilute the mixture to a scale, so that impurities 2, 3, 5, 6, 7, 8, 9, 10, 20, 21 and 22 are 1 mug/ml (wherein, the impurity 1 is 10 mug/ml, the impurity 4 is 5 mug/ml, and the concentration of a main component is 1 mg/ml), and shaking the mixture evenly to obtain a mixed solution of the impurities and the sample.
About 10mg of the sodium rabeprazole sample is weighed, placed in a 10ml measuring flask, dissolved and diluted to scale by adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to serve as a sample solution.
And respectively taking a proper amount of each impurity mother solution, and diluting with a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to prepare a solution with a certain concentration as a single impurity positioning solution.
And precisely measuring 10 mu l of each of the solvent, the mixed solution of the impurities and the sample, the solution of the test sample and the single impurity positioning solution, injecting into a liquid chromatograph, recording the chromatograms, and obtaining the results shown in Table 4 and the related spectrograms shown in figures 1-2.
TABLE 4 results of specificity experiments
| Impurity and sample mixed solution | Retention time min | Number of theoretical plates | Degree of separation |
| Impurity 9 | 8.180 | 8149 | / |
| Impurity 10 | 9.039 | 6587 | 2.12 |
| Impurity 5 | 11.260 | 6452 | 4.41 |
| Impurity 7 | 16.566 | 30797 | 11.29 |
| Impurity 2 | 17.593 | 35437 | 2.73 |
| Impurity 8 | 31.728 | 26743 | 24.55 |
| Impurity 6 | 33.218 | 20960 | 1.76 |
| Impurity 21 | 40.362 | 35624 | 8.05 |
| Impurity 4 | 45.191 | 61183 | 6.08 |
| Impurity 20 | 47.479 | 66497 | 3.11 |
| Main component | 49.966 | 86523 | 3.51 |
| Impurity 1 | 54.760 | 99371 | 6.97 |
| Unknown impurity 1 | 55.774 | 116608 | 1.50 |
| Unknown impurity 2 | 58.082 | 153539 | 3.70 |
| Unknown impurity 3 | 59.083 | 167287 | 1.71 |
| Impurity 22 | 60.124 | 136277 | 1.69 |
| Impurity 3 | 65.324 | 192971 | 8.33 |
| Unknown impurity 4 | 67.829 | 212086 | 4.23 |
The results show that: under the chromatographic condition, the baseline is stable, the blank solvent has no interference to the measurement of the product, the theoretical plate number of the main peak of the sodium rabeprazole of the dextral is 88655, and the main peak is well separated from adjacent impurities. In the mixed solution of the impurity samples, the peak shape of each substance is good, the separation degree of the main peak and the adjacent impurities is more than 1.5, and the minimum separation degree of each impurity is 1.50, which indicates that the specificity is good.
2.2 destructive testing
In order to examine whether degradation products possibly generated by the sodium rabeprazole can be detected under the selected chromatographic conditions, the degradation products are destroyed by high temperature, acid, alkali, oxidation, illumination and other severe conditions, a destroyed sample is dissolved by a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to prepare a test solution, 10 μl of each solution is precisely measured respectively, and the solution is injected into a liquid chromatograph to record the chromatogram, wherein the specific method is shown in Table 5. The solvents mentioned in Table 5 are [0.01mol/L sodium hydroxide-methanol (volume ratio 40:60) ].
TABLE 5 results of failure test
The result shows that under the conditions of acid, alkali, high temperature, oxidation and illumination, the product has good separation degree from the main peak, and the purity of the main peak of each destroyed sample meets the requirement. The material balance is calculated by taking the total peak area/sample weighing of the undamaged sample as 100%, and the total peak area/sample weighing and undamaged ratio of other destroyed samples are calculated, and the material conservation is between 95% and 105%. The condition is suitable for detecting the related substances of the product.
2.3 determination of quantitative limit and detection limit
The sodium rabeprazole reference substance and the impurities 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22 are respectively taken, a sample with a certain concentration is prepared, 10 μl is precisely measured after gradual dilution, the sample is injected into a liquid chromatograph, a chromatogram is recorded, and the measurement is carried out according to the signal to noise ratio S/N=3 and S/N=10, and the result is shown in table 6.
TABLE 6 limit of detection and quantitative limit results
The result shows that under the condition of the concentration and the chromatographic condition of the related substances of the product, the sodium rabeprazole and all known impurities have proper detection sensitivity, and all impurities with the content of 0.05 percent can be accurately quantified.
2.4 sample solution stability test
And taking a proper amount of a sodium rabeprazole sample, dissolving the sodium rabeprazole sodium sample with a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio is 40:60)) to prepare a solution containing 1mg of sodium rabeprazole per 1ml serving as a sample solution, and respectively placing the solution at room temperature after preparation for 0h, 4h, 8h, 12h, 16h, 20h and 24h for sample injection analysis to examine the content of each impurity and main component in the sample solution, wherein the results are shown in Table 7 according to an area normalization method.
TABLE 7 sample solution stability test (Room temperature 15 ℃ C.)
The results show that: the sample was stable at room temperature (15 ℃) for 24 hours in the sample solution.
2.5 linearity
2.5.1 preparation of impurity stock solution
And dissolving and diluting the sodium rabeprazole reference substance and the impurities 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22 with a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to prepare a solution containing about the impurities 2, 3, 5, 6, 7, 8, 9, 10, 20, 21 and 22 in 1ml, wherein the impurities 4 are 10 mug, the sodium rabeprazole reference substance is 20 mug, and the solution is taken as a reference substance stock solution and is stored at the temperature of 2-8 ℃.
2.5.2 Linear
Respectively precisely sucking a proper amount of control stock solution, and diluting with solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to obtain a series of solutions with a certain concentration. And precisely sucking 10 mu l of each of the series of gradient concentration solutions, sequentially carrying out sample injection analysis from quantitative limit to high concentration, recording a chromatogram, taking the concentration C (mu g/ml) of the impurity reference substance solution as an abscissa, taking the peak area of the impurity reference substance as an ordinate, and carrying out linear regression to obtain a regression equation, wherein the results are shown in Table 8 and FIG. 3.
Table 8 linearity investigation result (n=5)
2.6 sample injection precision test
Accurately weighing the impurities and a proper amount of the sodium rabeprazole reference substance, adding a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio is 40:60)) to dilute the impurities and the sodium rabeprazole reference substance to prepare a mixed solution containing 10 mug/ml of each impurity (5 mug/ml of impurity 4) and 10 mug/ml of sodium rabeprazole in each 1ml, continuously sampling and measuring for 6 times, and observing the change condition of peak area and retention time. The results are shown in Table 9.
TABLE 9 sample injection precision test results
The result shows that the peak area RSD of the sodium rabeprazole and each impurity is less than 2.0%, the retention time RSD is less than 1.0%, and the instrument sample injection precision is good.
2.7 stability of Mixed control solution
The sodium rabeprazole reference substance and the impurity reference substances (impurities 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22) are taken as proper amounts, and dissolved by a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to prepare a mixed solution of about 10 mug/ml of each impurity (impurity 4 is 5 mug/ml) and 10 mug/ml of sodium rabeprazole as reference substance solution, and the mixed solution is placed at room temperature for sample injection analysis for 0h, 4h, 8h, 12h, 16h, 20h and 24h after preparation, and the results are shown in Table 10.
TABLE 10 stability test results of impurity control solution (Room temperature 15 ℃ C.)
The results show that the impurity control solution is stable for 24 hours at room temperature after formulation.
2.8 repeatability test
Right amount of sodium rabeprazole sample is taken, 6 parts of sodium rabeprazole sample is precisely weighed, and dissolved in a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio is 40:60)) to prepare solutions containing 1mg of sodium rabeprazole in each 1ml of solution as a sample solution. Precisely measuring 10 μl of each solution, respectively injecting into a liquid chromatograph, calculating the content of each known impurity in the sample according to the external standard method by using the peak area, and calculating the content of the unknown impurity in the sample solution according to the main component control method, wherein the measurement results are shown in Table 11.
TABLE 11 repeatability test results
Note that: "-" indicates undetected, content less than 0.02% and RSD value was not calculated.
2.9 recovery test
Nine parts of a sodium rabeprazole sample are precisely weighed, about 10mg of each part is added into a 10ml volumetric flask, 80%, 100% and 120% of impurity control with limited amount of impurities are respectively added, the mixture is diluted to scale by adding a solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ], 10 mu L of each part is precisely measured, and the mixture is injected into a liquid chromatograph for recovery rate measurement, and the results are shown in tables 12-23.
TABLE 12 results of impurity 2 recovery test
TABLE 13 results of impurity 3 recovery test
TABLE 14 results of impurity 4 recovery test
TABLE 15 results of impurity 5 recovery test
TABLE 16 results of impurity 6 recovery test
TABLE 17 results of impurity 7 recovery test
TABLE 18 results of impurity 8 recovery test
TABLE 19 results of impurity 9 recovery test
TABLE 20 results of impurity 10 recovery test
TABLE 21 results of impurity 20 recovery test
TABLE 22 results of impurity 21 recovery test
TABLE 23 results of impurity 22 recovery test
2.10 intermediate precision test
Taking 6 parts of a sodium rabeprazole sample, respectively adding a solvent (0.01 mol/L sodium hydroxide-methanol (volume ratio is 40:60)) to dissolve and dilute the sodium rabeprazole sample to prepare a solution containing 1mg of sodium rabeprazole per 1ml serving as a test solution. Precisely measuring 10 μl, respectively injecting into liquid chromatograph, and recording chromatogram. The content of each known impurity in the sample solution was calculated as peak area according to the external standard method, and the content of the unknown impurity in the sample solution was calculated according to the main component control method, and the results are shown in Table 24.
Table 24 results of intermediate precision test
Note that: instrument 1: high performance liquid chromatograph 1100, numbered: 100141a; instrument 2: high performance liquid chromatograph 1100, numbered: 100129A.
The test results show that: the method has good intermediate precision.
2.11 correction factor determination
On 2 liquid chromatographs, 3 columns were used, P 3 2 The total of 6 samples were prepared by combining the samples in a total of 6 times, and the peak areas were regressed with the concentrations of the components and the correction factors of the impurities relative to the sodium dextral rabeprazole were calculated within 200% of the prescribed limiting concentration, and the results are shown in table 25.
Table 25 determination of correction factors
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Note that: chromatographic column 1: wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:7K5707-21;
chromatographic column 2: wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:5F5701-11;
chromatographic column 3: wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:6J5702-14;
instrument a: agilent1100 high performance liquid chromatograph, instrument number 100137a;
instrument B: agilent1100 high performance liquid chromatograph, instrument number 100141a.
The test results show that: the correction factor of impurity 2 was 0.57, the correction factor of impurity 3 was 0.88, the correction factor of impurity 4 was 1.13, the correction factor of impurity 5 was 0.71, the correction factor of impurity 6 was 0.67, the correction factor of impurity 7 was 0.74, the correction factor of impurity 8 was 0.88, the correction factor of impurity 9 was 0.93, the correction factor of impurity 10 was 0.69, the correction factor of impurity 20 was 0.76, the correction factor of impurity 21 was 0.81, and the correction factor of impurity 22 was 0.81.
2.12 durability inspection
To examine the degree of tolerance of the method to slight changes in conditions, a durability test was performed. The factors under investigation include: the initial ratio of the organic phase is changed by 25+/-2%, the pH of the mobile phase is 7.0+/-0.2, the column temperature is 20+/-2 ℃ and different chromatographic columns are tested. The examination indexes comprise retention time of main components in the test sample, separation degree (minimum separation degree) of main peaks and adjacent peaks, theoretical plate number, number of detected impurity peaks and impurity content (known impurities are calculated according to a main component control plus correction factor method), and the measurement results are shown in tables 26-29.
2.12.1 variation of the initial organic phase proportion of the mobile phase
The initial organic phase (methanol) ratios were examined for durability at 23%, 25% and 27%, respectively, and the measurement results are shown in Table 26.
Table 26 durability inspection (Mobile phase initial organic phase comparison)
The results show that: when the ratio of the initial organic phase (methanol) is changed within the range of 23% -27%, the retention time of the main peak, the theoretical plate number and the separation degree are obviously changed, and other chromatographic behaviors and sample detection results are not obviously changed.
2.12.2 pH change of mobile phase
The durability of the mobile phase at pH 6.8, 7.0 and 7.2 was examined, and the measurement results are shown in Table 27.
Table 27 durability inspection (mobile phase pH)
The results show that: when the pH value of the mobile phase is changed within the range of 6.8-7.2, the retention time of the main peak, the theoretical plate number and the separation degree are obviously changed, and other chromatographic behaviors and sample detection results are not obviously changed.
2.12.3 column temperature variation
To examine the effect of column temperature on chromatographic behavior, the durability at 18℃and 20℃and 22℃were examined, respectively, and the measurement results are shown in Table 28.
Watch 28 durability inspection (column temperature)
The results show that: when the column temperature is changed within the range of 18-22 ℃, the retention time of the main peak, the theoretical plate number and the separation degree are obviously changed, and other chromatographic behaviors and sample detection results are not obviously changed.
2.12.4 chromatographic column variation
The durability of the different columns was examined and the measurement results are shown in Table 29.
Table 29 durability inspection (different chromatographic columns)
Note that: column 1 is a Wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:6J5702-14;
column 2 is a Wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:5F5701-11.
Column 3 is a Wondasil C18-WR column (250 mm. Times.4.6 mm,5 μm), S/N:7K5707-21;
the number of impurities 12 in columns 1 and 3 was not included in the calculation range because the content of impurity 2 was less than 0.005%.
The results show that: the chromatographic columns of different batches have no influence on chromatographic behaviors and sample detection results.
In summary, the invention adopts high performance liquid chromatography to screen and optimize from the aspects of chromatographic conditions, and the like, draws out related substance methods, performs methodology verification, performs qualitative study on impurity 1 (SMA), and performs quantitative study on impurity 2 (SMB), impurity 3 (intermediate 1), impurity 4-10 and impurity 20-22 (process impurity and degradation impurity). The detection method provided by the invention has the advantages of good linear relation between the sodium rabeprazole and related substances, good accuracy and precision, strong specificity and high stability. The detection method has good reproducibility, can meet the detection requirements of substances related to the sodium rabeprazole bulk drug, and can be used for quality control of the sodium rabeprazole bulk drug.
Comparative example 1 influence of different gradients on detection results during chromatography
Liquid chromatography conditions: the chromatographic column adopts Shimadzu Wondasil C 18 -WR pillars (250 x4.6mm,5 μm); gradient elution was performed using 20mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 7.0 with sodium hydroxide) as mobile phase A and methanol as mobile phase B according to Table 30; the flow rate is 1.0ml/min; column temperature 20 ℃; the detection wavelength was 280nm.
TABLE 30 gradient elution procedure
And respectively taking proper amounts of impurity 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22 and sodium rabeprazole samples, placing the samples into a 10ml measuring flask, adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to dilute the samples to scale, so that the impurity 2, 3, 5, 6, 7, 8, 9, 10, 20, 21 and 22 is 1 mug/ml (wherein the impurity 1 is 10 mug/ml, the impurity 4 is 5 mug/ml and the concentration of the main component is 1 mg/ml), and shaking the samples uniformly to obtain a mixed solution of the impurity and the sample.
10. Mu.l of the above solution was poured into a liquid chromatograph, and a chromatogram was recorded.
TABLE 31 influence of different gradient elution conditions on chromatographic results
As can be seen from fig. 4 and table 31, in the gradient elution process, in the mixed solution of the impurity and the sample, the impurity and the main component can be well separated, but the impurity 1 at 44.425min and the unknown impurity 1 (at 44.718 min) have mutual interference, the separation degree is 0.78, the separation degree of the unknown impurity 2 at 45.993min and the unknown impurity 3 at 46.314min is 0.83, and the baseline separation is not achieved.
Comparative example 2 influence of different column temperatures on detection results during chromatography
Liquid chromatography conditions: the chromatographic column adopts Shimadzu Wondasil C 18 -WR pillars (250 x4.6mm,5 μm); gradient elution was performed using 20mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 7.0 with sodium hydroxide) as mobile phase A and methanol as mobile phase B according to Table 30; the flow rate is 1.0ml/min; column temperature 25 ℃; the detection wavelength was 280nm. The specific elution procedure is shown in Table 1.
And respectively taking proper amounts of impurity 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22 and sodium rabeprazole samples, placing the samples into a 10ml measuring flask, adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to dilute the samples to scale, so that the impurity 2, 3, 5, 6, 7, 8, 9, 10, 20, 21 and 22 is 1 mug/ml (wherein the impurity 1 is 10 mug/ml, the impurity 4 is 5 mug/ml and the concentration of the main component is 1 mg/ml), and shaking the samples uniformly to obtain a mixed solution of the impurity and the sample.
10. Mu.l of the above solution was poured into a liquid chromatograph, and a chromatogram was recorded.
TABLE 32 influence of different column temperatures on chromatographic results
As can be seen from fig. 5 and table 32, the degree of separation between the unknown impurity 3 (53.038 min) and the impurity 22 (53.815 min) in the mixed solution of the impurity and the sample was 1.36 under the conditions of the column temperature, and the baseline separation was not achieved.
Comparative example 3 influence of different chromatography columns on detection results during chromatography
Liquid chromatography conditions: the chromatographic column adopts an Shimadzu Inertsil ODS-3 column (250×4.6mm,5 μm); gradient elution was performed using 20mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 7.0 with sodium hydroxide) as mobile phase A and methanol as mobile phase B according to Table 30; the flow rate is 1.0ml/min; column temperature 20 ℃; the detection wavelength was 280nm. The specific elution procedure is shown in Table 1.
And respectively taking proper amounts of impurity 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21 and 22 and sodium rabeprazole samples, placing the samples into a 10ml measuring flask, adding solvent [0.01mol/L sodium hydroxide-methanol (volume ratio is 40:60) ] to dilute the samples to scale, so that the impurity 2, 3, 5, 6, 7, 8, 9, 10, 20, 21 and 22 is 1 mug/ml (wherein the impurity 1 is 10 mug/ml, the impurity 4 is 5 mug/ml and the concentration of the main component is 1 mg/ml), and shaking the samples uniformly to obtain a mixed solution of the impurity and the sample.
10. Mu.l of the above solution was poured into a liquid chromatograph, and a chromatogram was recorded.
TABLE 33 influence of different chromatography columns on the results of chromatography
As can be seen from fig. 6 and table 33, the degree of separation of the impurity 20 (33.563 min) from the main peak (34.076 min) in the mixed solution of the impurity and the sample was 1.19 under the conditions of the column, and the baseline separation was not achieved.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.
Claims (11)
1. A detection method of related substances in a sodium rabeprazole bulk drug is characterized in that the detection method adopts high performance liquid chromatography to carry out qualitative or quantitative detection on sodium rabeprazole and related substances, and the high performance liquid chromatography is adopted to carry out the qualitative or quantitative detectionThe spectral conditions include: the chromatographic column is Shimadzu Wondasil C 18 -a WR column with a length of 250mm, a diameter of 4.6mm and a packing particle size of 5 μm; the column temperature is 18-22 ℃; gradient elution is carried out by adopting a mobile phase A and a mobile phase B as mixed mobile phases, wherein the mobile phase A is 20mmol/L potassium dihydrogen phosphate buffer solution, and the pH value of the buffer solution is regulated to 6.8-7.2; the mobile phase B is methanol; the initial ratio of the mobile phase A to the mobile phase B in the gradient elution process is 73-77:27-23; the volume ratio of the mobile phase A to the mobile phase B is kept unchanged within 0-8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 55:45 at a constant speed within 8-18 minutes; the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45 within 18-33 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 55:45 to 25:75 at a constant speed within 33-75 minutes; the volume ratio of mobile phase A to mobile phase B remains 25:75 unchanged within 75-95 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 25:75 to the initial ratio at a constant speed within 95-96 minutes; the volume ratio of mobile phase A to mobile phase B remains unchanged within 96-106 minutes;
wherein the related substances comprise the following substances: impurity 1: 2-chloromethyl-4- (3-methoxypropoxy) -3-methylpyridine; impurity 2: 2-mercaptobenzimidazole; impurity 3:2- [ [4- (3-methoxypropoxy) -3-methylpyridin-2-yl ] -methylthio ] -1H-benzimidazole; impurity 4:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole sodium salt; impurity 5: 2-aminobenzimidazole; impurity 6:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfoxide ] -1H-benzimidazole nitroxide; impurity 7: 2-hydroxy benzimidazole; impurity 8:2- [ [ [4- (3-methoxypropoxy) -3-methyl-2-pyridinyl ] methyl ] sulfone ] -1H-benzimidazole nitroxide; impurity 9: benzimidazole-2-sulfonic acid; impurity 10:1- (1H-benzoimidazol-2-yl) -1, 4-dihydroxy-3-methyl-4-oxo-2-pyridinecarboxylic acid sodium salt; impurity 20:2- [ [ (4-chloro-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole; impurity 21:2- [ [ (4-methoxy-3-methyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole; impurity 22:2- [ (4-methoxy-3-methylpyridin-2-yl) methylsulfanyl ] -1H-benzimidazole.
2. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 1, wherein the initial ratio of mobile phase a to mobile phase B in the gradient elution process is 75:25; the volume ratio of mobile phase A to mobile phase B remains 75:25 unchanged within 0-8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 55:45 at a constant speed within 8-18 minutes; the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45 within 18-33 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 55:45 to 25:75 at a constant speed within 33-75 minutes; the volume ratio of mobile phase A to mobile phase B remains 25:75 unchanged within 75-95 minutes; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 25:75 to 75:25 at a constant speed within 95-96 minutes; the volume ratio of mobile phase A to mobile phase B remained 75:25 during 96-106 minutes.
3. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 1, wherein the high performance liquid chromatography conditions comprise: the column temperature was 20 ℃.
4. The method for detecting related substances in the sodium rabeprazole bulk drug according to claim 1, wherein the mobile phase A is 20mmol/L potassium dihydrogen phosphate buffer solution, and the pH value is adjusted to 7.0.
5. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 1, wherein the high performance liquid chromatography conditions comprise: the detection wavelength is 200-400 nm.
6. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 5, wherein the high performance liquid chromatography conditions comprise: the detection wavelength was 280nm.
7. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 1, wherein the high performance liquid chromatography conditions comprise: the sample injection amount is 5-20 mu l.
8. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 7, wherein the high performance liquid chromatography conditions comprise: the sample injection amount is 10 mu l.
9. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 1, wherein the high performance liquid chromatography conditions comprise: the flow rate is 0.5-1.5 ml/min.
10. The method for detecting related substances in a sodium rabeprazole bulk drug according to claim 9, wherein the high performance liquid chromatography conditions comprise: the flow rate was 1.0ml/min.
11. The method for detecting related substances in a sodium rabeprazole drug substance according to claim 1, wherein the correction factor of impurity 2 is 0.57, the correction factor of impurity 3 is 0.88, the correction factor of impurity 4 is 1.13, the correction factor of impurity 5 is 0.71, the correction factor of impurity 6 is 0.67, the correction factor of impurity 7 is 0.74, the correction factor of impurity 8 is 0.88, the correction factor of impurity 9 is 0.93, the correction factor of impurity 10 is 0.69, the correction factor of impurity 20 is 0.76, the correction factor of impurity 21 is 0.81, and the correction factor of impurity 22 is 0.81.
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