CN112305125A - Method for analyzing omeprazole by high performance liquid chromatography - Google Patents
Method for analyzing omeprazole by high performance liquid chromatography Download PDFInfo
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- CN112305125A CN112305125A CN202011195927.7A CN202011195927A CN112305125A CN 112305125 A CN112305125 A CN 112305125A CN 202011195927 A CN202011195927 A CN 202011195927A CN 112305125 A CN112305125 A CN 112305125A
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- SUBDBMMJDZJVOS-UHFFFAOYSA-N 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole Chemical compound N=1C2=CC(OC)=CC=C2NC=1S(=O)CC1=NC=C(C)C(OC)=C1C SUBDBMMJDZJVOS-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 229960000381 omeprazole Drugs 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000004471 Glycine Substances 0.000 claims abstract description 7
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical group OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims abstract description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000010828 elution Methods 0.000 claims abstract description 3
- 239000000945 filler Substances 0.000 claims abstract description 3
- 239000012046 mixed solvent Substances 0.000 claims abstract description 3
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical compound CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000004007 reversed phase HPLC Methods 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
- 239000012085 test solution Substances 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 12
- 239000003085 diluting agent Substances 0.000 claims description 9
- CDMADVZSLOHIFP-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane;decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 CDMADVZSLOHIFP-UHFFFAOYSA-N 0.000 claims description 9
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 9
- 230000009977 dual effect Effects 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 2
- 239000012535 impurity Substances 0.000 description 98
- 239000011550 stock solution Substances 0.000 description 44
- 238000012360 testing method Methods 0.000 description 34
- 239000002904 solvent Substances 0.000 description 28
- 238000007865 diluting Methods 0.000 description 21
- 239000013558 reference substance Substances 0.000 description 21
- 239000000523 sample Substances 0.000 description 17
- 238000011084 recovery Methods 0.000 description 16
- 239000000843 powder Substances 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 238000009210 therapy by ultrasound Methods 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000012937 correction Methods 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000005303 weighing Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 4
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 3
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 3
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- -1 2, 4-dimethyl-1-hydroxymethyl-4 methoxypyridine Chemical compound 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 208000007107 Stomach Ulcer Diseases 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 208000000718 duodenal ulcer Diseases 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 201000005917 gastric ulcer Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 206010063655 Erosive oesophagitis Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- MPBVHIBUJCELCL-UHFFFAOYSA-N Ibandronate Chemical compound CCCCCN(C)CCC(O)(P(O)(O)=O)P(O)(O)=O MPBVHIBUJCELCL-UHFFFAOYSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 239000003485 histamine H2 receptor antagonist Substances 0.000 description 1
- 229960005236 ibandronic acid Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 208000000689 peptic esophagitis Diseases 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000001612 separation test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 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
- G01N30/04—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
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention provides a method for analyzing omeprazole by high performance liquid chromatography, which adopts a reversed-phase high performance liquid chromatography column, uses octyl silane-based bonded silica gel as a chromatographic column filler, adopts a diode array detector, and uses a glycine solution as a mobile phase A: the concentration is 0.01-0.05mol/L, the pH value is 8.5-11.0, the mobile phase B is acetonitrile-methanol mixed solvent with the volume ratio of 85: 15, gradient elution is adopted, the gradient elution program is listed in the table, and a specific elution program is adopted.
Description
Technical Field
The invention relates to the field of drug analysis, and particularly relates to a method for detecting ibandronate sodium by ion chromatography.
Background
Omeprazole is a proton pump inhibitor which is effective in inhibiting gastric acid secretion. It also has inhibitory effect on pepsin secretion, has no obvious change on blood flow of gastric mucosa, and has no influence on body temperature, arterial blood pressure, venous hemoglobin, arterial oxygen partial pressure, carbon dioxide partial pressure and arterial blood pH. Can be used for treating gastric and duodenal ulcer, reflux or erosive esophagitis, etc., and also has therapeutic effect on gastric and duodenal ulcer with H2 receptor antagonist.
Omeprazole contains a plurality of impurities which are introduced in the production and storage processes, and whether the impurities are effectively controlled or not can substantially influence the medical effect and clinical safety of the medicament. CN103592379A adopts high performance liquid chromatography to detect related substances in omeprazole bulk drug, and the method aims at impurity A, C, D, E, I. CN110609109A adopts high performance liquid chromatography to detect related substances in omeprazole bulk drug, and the method aims at impurity A, B, C, D, E, F, G, H, I. CN107957458A adopts high performance liquid chromatography to detect related substances in omeprazole enteric-coated capsules, and the method aims at A, B, C, D, E, F, G, H, I impurities, and the structures of the impurities are shown in Table 1.
Table 1 known impurity structural formula of omeprazole
In the prior art, no detection result and analysis method is given for 2, 4-dimethyl-1-hydroxymethyl-4 methoxypyridine which is an impurity in omeprazole.
Disclosure of Invention
The invention provides a method for analyzing omeprazole by high performance liquid chromatography, which adopts the following detection conditions:
adopting a reversed-phase high-performance liquid chromatography column, using octyl silane-based bonded silica gel as a chromatographic column filler, adopting a diode array detector, and using a glycine solution as a mobile phase A: the concentration is 0.00-0.05mol/L, the pH value is 8.5-11.0, the mobile phase B is acetonitrile-methanol mixed solvent with the volume ratio of 85: 15, gradient elution is adopted, and the gradient elution program is listed in the following table. By adopting the method, various impurities in the omeprazole can be stably detected with high separation degree, and particularly, the impurity 8 can be detected.
In a preferred embodiment, dual wavelength detection is used, with wavelengths of 300-. .
In a preferred embodiment, the column temperature is from 23 to 27 deg.C, preferably 25 deg.C.
In a preferred embodiment, the sample is taken in an amount of 10-20. mu.l, preferably 20. mu.l.
In a preferred embodiment, the diluent for preparing the test solution is an aqueous solution wherein the ethanol concentration is 130-180g/L, the sodium tetraborate decahydrate concentration is 1.0-5.0g/L, and the disodium ethylenediaminetetraacetate concentration is 0.2-1.0g/L, pH value is 10.5-11.5.
In a preferred embodiment, the diluent for preparing the test solution is a solution having an ethanol concentration of 157.8g/L, a sodium tetraborate decahydrate concentration of 3.8g/L, and an ethylenediaminetetraacetic acid disodium salt 0.5g/L, pH value of 11.0. + -. 0.1.
Detailed Description
To obtain the experimental data of the present invention, the inventors used commercially available or self-made samples, the information of which is shown in table 2.
TABLE 2 Experimental control and sample information
Preparation of test and reference solutions
To prepare the test and control solutions, the present invention uses a specific diluent. The diluent is preferably an aqueous solution in which the ethanol concentration is 130-180g/L, the sodium tetraborate decahydrate concentration is 1.0-5.0g/L, and the disodium ethylenediaminetetraacetate concentration is 0.2-1.0g/L, pH value is 10.5-11.5.
The inventor compares the diluent with the pH of 7.0, and respectively carries out ultrasonic treatment for 15min and standing for 0 hour and 24 hours without ultrasonic treatment on two systems containing samples, and the result shows that the sample solution adopting the diluent is treated by ultrasonic treatment for 15min, the solution is stable when the sample solution is placed at 4 ℃ for 24 hours, and no degraded impurity peak appears after the main peak. And the impurity of the test solution (ultrasonic 15 minutes) prepared by the phosphate buffer solution system with pH7.0 is increased after 0 hour main peak compared with that of the test solution after ultrasonic 1 minute, and degradation peaks appear at a plurality of positions after the test solution is placed for 20 hours at 4 ℃, so that the stability of the solution is poor, therefore, the test solution with pH11.0 as the solvent is ensured to have good stability.
In a typical example, to obtain the diluent, 7.6g of sodium tetraborate decahydrate and 1.0g of disodium ethylenediamine tetraacetic acid are weighed, 800ml of water is added to dissolve, the pH value is adjusted to 11.0 +/-0.1 by using 50% sodium hydroxide solution, 400ml of absolute ethyl alcohol is added, the mixture is diluted to 2000ml by using water, and the mixture is mixed uniformly. The obtained diluted solution had ethanol concentration of 157.8g/L, sodium tetraborate decahydrate concentration of 3.8g/L, and disodium ethylenediaminetetraacetate concentration of 0.5g/L, pH of 11.0 + -0.1. Unless otherwise specified, dilutions (also referred to as blank solvents) used to demonstrate the analytical effect below were obtained using the methods described herein.
Test solution: taking 20mg of omeprazole, putting the omeprazole into a 20ml measuring flask, adding a proper amount of solvent, carrying out ultrasonic treatment for 15 minutes to dissolve the omeprazole, diluting the omeprazole with the solvent to a scale, shaking up the omeprazole, and filtering to obtain a subsequent filtrate.
When the test article is an omeprazole formulation, such as an enteric coated tablet, the amount of the drug taken is preferably an amount containing about 20mg of the omeprazole drug substance. In order to eliminate interference during the analysis of the preparation, a blank auxiliary material test solution needs to be prepared, and the typical method is as follows: weighing an appropriate amount of auxiliary materials (about equal to 20mg of omeprazole) according to the formula ratio, placing the auxiliary materials into a 20ml measuring flask, adding an appropriate amount of solvent, carrying out ultrasonic treatment for 15 minutes, diluting the auxiliary materials with the solvent to a scale, shaking up, filtering, and taking the subsequent filtrate to obtain the omeprazole oral liquid.
Adding a standard test solution: accurately weighing appropriate amounts of A, B, C, D, E, H, I, 8 and F/G mixed reference substances, placing in different 25ml measuring bottles, adding solvent, diluting to scale, and collecting the reference substance as reference substance stock solution. And taking a proper amount of impurity F/G reference substance stock solution, and heating at 55 ℃ for 30 minutes to obtain F/G conversion impurity stock solution. And precisely weighing a proper amount of the fine powder (about 20mg, 180402 batches of omeprazole) of the product, placing the fine powder into a 20ml measuring flask, precisely adding a proper amount of impurities A, B, C, D, E, H, I, 8 and F/G into a stock solution of the converted impurities, carrying out ultrasonic treatment for 15 minutes to dissolve the impurities, diluting the mixture with a solvent to prepare a mixed solution containing about 2 mu G of the impurities A, B, C, E, H, I, 8 and F/G and about 5 mu G of the impurities D in each 1ml of the mixed solution, and filtering to obtain a subsequent filtrate as a sample solution for adding a standard.
Chromatographic conditions
In a typical embodiment of the invention, the chromatographic conditions are:
the instrument comprises the following steps: waters high performance liquid chromatograph (e2695-2998)
A chromatographic column: waters Xbridge C8，4.6×150mm，5μm
Mobile phase A: weighing 6.0g glycine, adding 1500ml water for dissolving, adjusting pH to 9.0 with 50% sodium hydroxide solution, adding water for diluting to 2000ml
Mobile phase B: acetonitrile-methanol (85: 15)
Flow rate: 1.0ml/min
Wavelength: dual wavelength, 305nm (detection impurity A, B, C, D, E, F, G, H, I)/264nm (detection impurity 8)
Column temperature: 25 deg.C
Sample introduction amount: 20 μ l
Solvent: weighing 7.6g of sodium tetraborate decahydrate and 1.0g of disodium ethylene diamine tetraacetate, adding 800ml of water for dissolving, adjusting the pH value to 11.0 +/-0.1 by using 50% sodium hydroxide solution, adding 400ml of absolute ethyl alcohol, diluting to 2000ml by using water, and uniformly mixing to obtain the sodium tetraborate decahydrate.
Gradient program:
the present invention, using the above conditions, can separate well all impurities found in the prior art and can quantitatively detect newly found impurities 8.
Specificity test
In order to ensure the specificity of the detection method of related substances and understand the main degradation path and potential degradation products of the main medicine components of the product, the product is taken to carry out destructive tests of acid, alkali, oxidation, high temperature and illumination, the conditions of the related substances of a sample are inspected, and the peak purity of the main peak is detected. The results show that the samples destroyed by acid, alkali, oxidation, high temperature and illumination are detected by the method of the related substances, the samples destroyed by alkali are found to be relatively stable, the samples destroyed by acid, oxidation, solution high temperature and illumination conditions are degraded in different degrees, the blank auxiliary materials are destroyed and do not interfere the detection of the samples, impurity peaks generated by the samples after being destroyed can be well separated, the impurity peaks can be separated from a main peak to a base line, the purity angle of the omeprazole main peak is smaller than the purity threshold, and the related substances of the omeprazole can be effectively detected under the chromatographic conditions.
Durability test
Blank auxiliary materials: weighing an appropriate amount of auxiliary materials (about equal to 20mg of omeprazole) according to the formula ratio, placing the auxiliary materials into a 20ml measuring flask, adding an appropriate amount of solvent, carrying out ultrasonic treatment for 15 minutes, diluting the mixture to a scale with the solvent, shaking up, and filtering to obtain the omeprazole.
Durability test solution 1 (separation test solution): precisely measuring an appropriate amount of impurity F/G mixed reference stock solution, diluting with a solvent to obtain a solution containing about 20 μ G of impurities per 1ml, heating at 55 ℃ for 30min to obtain a reference stock solution 1, and diluting with a solvent to obtain a mixed solution containing about 20 μ G of impurities A, B, C, E, H, I and 8 and about 50 μ G of impurities D per 1ml to obtain a reference stock solution 2. Precisely weighing 10mg of omeprazole reference substance, placing the omeprazole reference substance into a 10ml measuring flask, adding a proper amount of solvent to dissolve the omeprazole reference substance, precisely weighing 1ml of reference substance stock solution 1 and 1ml of reference substance stock solution 2 respectively, placing the reference substance stock solution and the reference substance stock solution into the same 10ml measuring flask, diluting the omeprazole reference substance stock solution and the reference substance stock solution to scales by using the solvent, shaking the omeprazole reference substance stock solution and the reference substance stock solution uniformly to obtain durability test solution.
Durability test solution 2 (impurity control solution): precisely measuring 2ml of each of the reference substance stock solution 1 and the reference substance stock solution 2, placing the reference substance stock solutions in a 20ml measuring flask, and diluting the measuring flasks to the scale with a solvent to obtain a durability test solution 2.
Durability test solution 3 (test solution): taking a proper amount of fine powder (about equal to 20mg of omeprazole in 180402 batches) of the product, putting the fine powder into a 20ml measuring flask, adding a proper amount of solvent, carrying out ultrasonic treatment for 15 minutes to dissolve the fine powder, diluting the fine powder to a scale with the solvent, and shaking up the solution to obtain a durability test solution 3.
Mu.l of each of the 3 solutions was measured under the conditions shown in the following table, and the chromatogram was recorded. When the influence on the measurement result is examined when the chromatographic conditions slightly change, the content of each known impurity in the durability test solution 3 is calculated according to an external standard method under each chromatographic condition, the range difference of the single impurity and the total impurity content is less than 20 percent of the limit, the detection results of related substances in the durability test solution 3 are basically consistent, and the results are shown in the following table.
Table 3: durability of related substance inspection HPLC method
Table 4: test results at different flow rates
Table 5: test results under different column temperature conditions
Table 6: test results under different glycine concentrations
Table 7: test results under different mobile phase pH values
Table 8: test results under different batch number chromatographic column conditions
The results show that: the flow rate, the column temperature, the concentration of the glycine solution, the pH value and the batch number of the chromatographic column under the chromatographic conditions are slightly changed, the measurement result is not influenced, the separation degree of each peak in the durability test solution 1 is more than 1.5, the detection results of related substances in the durability test solution 3 are basically consistent, and the range of single impurity and total impurity content is less than 20 percent of the limit.
Linear relation test
For known impurities, 6 concentration points were taken in the range from the quantitative limit concentration to the 250% limit concentration and studied, and the sample amount (ng) was linearly regressed with each peak area measured. For unknown impurities, the linear relation of single unknown impurities is investigated by replacing omeprazole.
The result shows that the peak area of the impurity A has a good linear relation in the range of 0.36 ng-98.22 ng of sample injection amount, and the correction factor is 0.3. The peak area of the impurity B has good linear relation in the range of the sample injection amount of 2.18 ng-98.70 ng, and the correction factor is 0.9. The peak area of the impurity C has good linear relation in the range of the sample injection amount of 1.72 ng-98.62 ng, and the correction factor is 1.0. The area of the impurity D peak has good linear relation in the range of 1.52 ng-250.74 ng of the sample injection amount, and the correction factor is 1.0. The peak area of the impurity E has good linear relation in the range of the sample amount of 1.08 ng-100.06 ng, and the correction factor is 1.0. The peak area of the impurity F/G is in good linear relation in the range of 0.80 ng-99.92 ng of sample injection amount, and the correction factor is 0.7. The peak area of the impurity H has good linear relation in the range of the sample injection amount of 2.74 ng-99.54 ng, and the correction factor is 1.0. The area of the impurity I peak has good linear relation in the range of 0.74 ng-98.59 ng of sample injection amount, and the correction factor is 1.1. The peak area of the impurity 8 has good linear relation in the range of 8.16 ng-99.96 ng of sample injection amount, and the correction factor is 1.7. The area of the omeprazole peak has good linear relation in the range of 1.76 ng-99.26 ng of sample injection amount.
Limit of measure test
Diluting the impurity stock solution with known concentration to a mixed solution with low concentration, detecting according to the related substance measuring method, taking 20 mul to inject into a liquid chromatograph, recording a chromatogram, taking each peak response signal as a signal with 10 times of noise as a quantitative signal (S/N is approximately equal to 10), calculating the quantitative limit, and obtaining the result shown in the following table.
Table 9: quantitative Limit test measurement 1
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | Mean value of | RSD(％) |
| Impurity F/G | 3533 | 3426 | 3377 | 3109 | 3455 | 3187 | 3348 | 4.92 |
| Impurity A | 2365 | 2283 | 2366 | 2561 | 2521 | 2355 | 2409 | 4.48 |
| Impurity I | 1935 | 2021 | 2007 | 1886 | 1683 | 1805 | 1890 | 6.82 |
| Impurity E | 2556 | 2554 | 2741 | 2696 | 2784 | 2624 | 2659 | 3.63 |
| Impurity D | 4252 | 3747 | 3590 | 4057 | 3681 | 4235 | 3927 | 7.41 |
| Impurity B | 5560 | 5614 | 6324 | 5237 | 6381 | 5712 | 5805 | 7.81 |
| Omeprazole | 5970 | 6150 | 7106 | 5987 | 6645 | 6306 | 6361 | 6.95 |
| Impurity H | 6759 | 6577 | 6645 | 6805 | 6760 | 6457 | 6667 | 2.00 |
| Impurity C | 4667 | 4129 | 4927 | 4692 | 4586 | 4159 | 4527 | 7.01 |
| Impurity 8 | 5350 | 5398 | 5157 | 5202 | 5189 | 5015 | 5219 | 2.65 |
Table 10: quantitative Limit test measurement 2
Precision survey
(1) Repeatability test
And because the detected amount of the impurities in the test sample is small, the repeatability of the method is inspected by adding the known impurities into the test sample solution. Precisely measuring an appropriate amount of impurity F/G mixed reference stock solution, dissolving and diluting the impurity F/G mixed reference stock solution by adding a solvent to prepare a solution containing about 20 mu G of impurities in each 1ml, heating the solution at 55 ℃ for 30min to obtain an F/G converted impurity stock solution 1, precisely measuring an appropriate amount of impurity A, B, C, D, E, H, I and impurity stock solutions 8, diluting the impurity F/G converted impurity stock solution by adding the solvent to prepare a mixed solution containing about 20 mu G of impurities A, B, C, E, H, I and 8 and about 50 mu G of impurities D in each 1ml to obtain an impurity stock solution 2. Precisely measuring 2ml of each of impurity F/G conversion peak stock solution 1 and impurity stock solution 2, placing in a 20ml measuring flask, diluting with solvent to scale, and shaking up to obtain impurity reference substance solution. Taking a proper amount of fine powder (about 20mg, 180402 batches of omeprazole) of the product, placing the fine powder into a 20ml measuring flask, adding a proper amount of solvent, precisely adding 2ml of impurity F/G conversion peak stock solution and 2ml of impurity stock solution respectively, dissolving by ultrasonic treatment for 15 minutes, diluting the mixture to a scale by using the solvent, shaking up the mixture to be used as a test solution, preparing 6 parts by the same method, preparing a test solution without adding the F/G conversion peak stock solution and the impurity stock solution, measuring, and deducting impurities contained in the test solution when calculating the recovery rate. And respectively and precisely measuring 20 mu l of the solution, injecting the solution into a liquid chromatograph, recording a chromatogram, and inspecting the recovery rate of each impurity according to an external standard method, wherein the results are shown in the table below.
Table 11: results of repeated investigation
The result shows that the method has good repeatability.
(2) Intermediate precision test
The influence of random variation factors on precision is examined, another analyst uses different instruments to measure on different dates, 6 test sample solutions are prepared again, the preparation method is the same as the repeatability test, and the results are shown in the table.
Table 12: intermediate precision investigation results
And (4) conclusion: the results of 12 times of measurement of the comprehensive repeatability test and the intermediate precision test show that the method has good precision.
Accuracy test
Precisely measuring an appropriate amount of impurity F/G mixed reference stock solution, dissolving and diluting with a solvent to prepare a solution containing about 20 mug of impurities per 1ml, heating at 55 ℃ for 30min to obtain an F/G converted impurity stock solution 1, and further taking an appropriate amount of impurity A, B, C, D, E, H, I and 8 impurity stock solutions, diluting with a solvent to prepare a mixed solution containing about 20 mug of impurities A, B, C, E, H, I and 8 and about 50 mug of impurities D per 1ml as an impurity stock solution 2. Precisely measuring 2ml of the F/G conversion impurity stock solution 1 and 2ml of the impurity stock solution respectively, placing the F/G conversion impurity stock solution and 2ml of the impurity stock solution in a 20ml measuring flask, and diluting the F/G conversion impurity stock solution to a scale by using a solvent to serve as an impurity reference substance solution. Precisely weighing an appropriate amount of fine powder (about 20mg, 180402 batches of omeprazole) in 9 parts, respectively placing the fine powder into a 20ml measuring flask, adding an appropriate amount of solvent, precisely adding 1.6ml, 2.0ml and 2.4ml of F/G conversion impurity stock solution 1 and impurity stock solution 2 respectively according to three concentrations of low (80%), medium (100%) and high (120%), carrying out ultrasonic treatment for 15 minutes to dissolve the fine powder, diluting the fine powder to a scale by using the solvent, and shaking up to obtain a sample solution. And preparing a test sample solution without adding F/G conversion peak stock solution and impurity stock solution for measurement, and deducting impurities contained in the test sample when calculating the recovery rate. Precisely measuring the impurity reference substance solution and the sample solution, respectively injecting into a liquid chromatograph with 20 μ l each, and recording chromatogram. The recovery rate is calculated by peak area according to an external standard method, the recovery rate is required to be between 90% and 108%, and the result is shown in the following table.
Table 13: results of F/G impurity recovery test
Table 14: test results on recovery rate of impurity A
Table 15: test results on recovery of impurity I
Table 16: test results on recovery of impurity E
Table 17: test results on the recovery rate of impurity D
Table 18: test results on the recovery rate of impurity B
Table 19: test results on the recovery rate of impurity H
Table 20: test results on recovery rate of impurity C
Table 21: experimental results on the recovery rate of impurity 8
The result shows that the recovery rates of the impurities F/G, A, B, C, D, E, H, I and 8 under three concentrations are between 90% and 108%, and the method has the advantages of good recovery rate and high accuracy.
Claims (8)
1. A method for analyzing omeprazole by high performance liquid chromatography is characterized by comprising the following detection conditions:
adopting a reversed-phase high-performance liquid chromatography column, using octyl silane-based bonded silica gel as a chromatographic column filler, adopting a diode array detector, and using a glycine solution as a mobile phase A: the concentration is 0.02-0.05mol/L, the pH value is 8.5-11.0, the mobile phase B is acetonitrile-methanol mixed solvent with the volume ratio of 85: 15, gradient elution is adopted, and the gradient elution program is listed in the following table:
2. the method according to claim 1, wherein the concentration of glycine in mobile phase A is 0.04mol/L and the pH value is 11.0.
3. The method as claimed in claim 1, wherein the dual wavelength detection is adopted, and the wavelengths are 300-315nm and 255-270nm, respectively.
4. The process according to claim 1, wherein the column temperature is 23 to 27 ℃, preferably 25 ℃.
5. The method according to claim 1, wherein the sample amount is 10-20 μ l, preferably 20 μ l.
6. The method as set forth in claim 1, wherein the diluent for preparing the test solution is an aqueous solution having an ethanol concentration of 130-180g/L, a sodium tetraborate decahydrate concentration of 1.0-5.0g/L, and an ethylenediaminetetraacetic acid concentration of 0.2-1.0g/L, pH value of 10.5-11.5.
7. The method of claim 1, wherein the diluent for preparing the test solution is a solution having an ethanol concentration of 157.8g/L, a sodium tetraborate decahydrate concentration of 3.8g/L, and an ethylenediaminetetraacetic acid disodium salt 0.5g/L, pH value of 11.0 ± 0.1.
8. A method according to claim 3, characterized in that a dual wavelength detection is used, the wavelengths being preferably 305nm and 264nm, respectively.
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