CN117129599A - Method for measuring impurity F content in piracetam by high performance liquid phase method - Google Patents
Method for measuring impurity F content in piracetam by high performance liquid phase method Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 97
- GMZVRMREEHBGGF-UHFFFAOYSA-N Piracetam Chemical compound NC(=O)CN1CCCC1=O GMZVRMREEHBGGF-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 41
- 229960004526 piracetam Drugs 0.000 title claims abstract description 40
- 239000007791 liquid phase Substances 0.000 title claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 73
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000012488 sample solution Substances 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 239000013558 reference substance Substances 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 9
- 239000012071 phase Substances 0.000 claims abstract description 9
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims abstract description 8
- 239000000523 sample Substances 0.000 claims description 23
- 239000007924 injection Substances 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 11
- 238000007865 diluting Methods 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 239000012088 reference solution Substances 0.000 claims description 2
- 238000004364 calculation method Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000002904 solvent Substances 0.000 description 23
- 238000012360 testing method Methods 0.000 description 20
- 239000000047 product Substances 0.000 description 18
- 230000006378 damage Effects 0.000 description 10
- 238000011084 recovery Methods 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000002585 base Substances 0.000 description 3
- 239000012490 blank solution Substances 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000000197 pyrolysis Methods 0.000 description 3
- 239000003513 alkali Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001066 destructive effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 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 1
- 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 1
- 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 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000005978 brain dysfunction Effects 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical class NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 229940100688 oral solution Drugs 0.000 description 1
- 230000008832 photodamage Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for measuring the content of impurity F in piracetam by a high-performance liquid phase method, and belongs to the technical field of medicines. The method comprises the following steps: 1) Preparing a sample solution and a reference substance solution; 2) Setting high performance liquid phase detection conditions; the chromatographic column is YMC-Triart C18; the mobile phase is a mixed solution of sodium heptanesulfonate solution and acetonitrile; 3) And respectively taking a sample solution and a reference substance solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating the content of the impurity F. The invention can separate and detect the impurity F rapidly, effectively, accurately and reliably, is beneficial to better controlling the product quality of piracetam and improving the medication safety of patients.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for measuring the content of impurity F in piracetam by a high-efficiency liquid phase method.
Background
Piracetam, which is 2-oxo-1-pyrrolidinylacetamide, belongs to brain metabolism improving medicine and is a ring derivative of g-aminobutyric acid, and the existing preparation variety comprises oral solution, tablet, injection and capsule, has the functions of resisting brain function injury caused by physical factors and chemical factors, can promote ATP in brain, promote acetylcholine synthesis and enhance nerve excitation conduction, and has the function of promoting brain metabolism. Can enhance memory and improve learning ability, and is suitable for hypomnesis and light and moderate brain dysfunction caused by various reasons, and also for intelligent hypoevolutism of children.
At present, a Kjeldahl nitrogen determination method is adopted for measuring the content of related substances of piracetam, the sample treatment is complex and complicated in the test process, the nitrogen digestion process time is long, and the heating temperature and time in the test process have great influence on the reaction end point. The impurity F is the impurity possibly generated in the synthesis process, but is easy to ignore because of low ultraviolet response, and in order to better control the safety and effectiveness of the piracetam raw material, the invention establishes a method for quickly, effectively, accurately and reliably separating and detecting the impurity F, thereby being beneficial to better controlling the product quality of the piracetam and improving the medication safety of patients.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for measuring the content of the impurity F in the piracetam by a high-efficiency liquid phase method, which can quickly, effectively, accurately and reliably separate and detect the impurity F and improve the product quality and the medication safety of patients.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for measuring the content of impurity F in piracetam by a high performance liquid phase method comprises the following specific steps:
1) Preparing a sample solution and a reference substance solution;
2) Setting high performance liquid phase detection conditions; the chromatographic column is YMC-Triart C18; the mobile phase is a mixed solution of sodium heptanesulfonate solution and acetonitrile;
3) And respectively taking a sample solution and a reference substance solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating the content of the impurity F.
Further, in step 1), the preparation method of the sample solution comprises the following steps: precisely weighing piracetam, placing in a volumetric flask, adding acetonitrile-water to dilute to a constant volume to obtain 10mg of piracetam-containing solution per 1mL, and uniformly mixing to obtain a sample solution; the preparation method of the reference substance solution comprises the following steps: precisely weighing the impurity F, and diluting with acetonitrile-water to obtain a reference substance solution containing 10 mug of the impurity F per 1mL, wherein the volume ratio of acetonitrile to water is 5:95.
further, in the step 2), the column YMC-Triart C18 was 250mm long, 4.6mm in inner diameter, and 5 μm in filler particle diameter.
Further, in step 2), the concentration of the sodium heptanesulfonate solution was 1.5g/L, and the pH was adjusted to 4.5 with sulfuric acid.
Further, in the step 2), the volume ratio of the sodium heptanesulfonate solution to the acetonitrile is 86-88: 14-12.
Further, in the step 2), the column temperature of the chromatographic column is 38-42 ℃.
Further, in the step 2), the flow rate of the mobile phase is 0.95-1.05 mL/min.
Further, in step 2), the sample injection amounts of the sample solution and the control solution are 20. Mu.L.
Further, in the step 3), the detection wavelength of the liquid chromatograph was 205nm.
Further, in the step 3), the calculating method is an external standard method, and the impurity F content in the sample solution is calculated to be less than or equal to 0.1%.
The beneficial effects are that: compared with the prior art, the invention has the advantages that:
(1) The high performance liquid chromatography provided by the invention can be used for rapidly, effectively, accurately and reliably separating and detecting the impurity F in the piracetam bulk drug, and the blank solvent has no tiny inverted peak interference, and the solvent peak does not interfere with the determination of the impurity F.
(2) According to the invention, after peak purity detection, the spectrum purity of the destroyed sample is more than 990 under each condition, impurities generated by destruction under each condition can be well separated from a main peak, the detection specificity of related substances is good, and the impurity F can be well separated from unknown impurities.
(3) The invention has no influence on the separation and inspection results of related substance impurity F in piracetam when the chromatographic condition parameters are slightly changed, and has good durability.
(4) The invention can rapidly, effectively, accurately and reliably separate and detect the impurity F in the piracetam, is beneficial to improving the product quality of the impurity F and improves the medication safety of patients.
Drawings
FIG. 1 is a test chromatogram under chromatographic conditions of the present invention;
fig. 2 is a diagram of the applicability of the piracetam system;
fig. 3 is a standard F graph of piracetam impurity.
Detailed Description
The invention is further described below in connection with specific embodiments.
Example 1
1. A method for measuring the content of impurity F in piracetam by adopting a high performance liquid phase method comprises the following steps:
1) Preparing a control solution and a test solution: about 100mg of piracetam is taken and put into a 10mL volumetric flask, and the solvent is diluted to a constant volume and mixed uniformly; preparation of a control solution: and taking a proper amount of impurity F, and adding a solvent to dilute the mixture into a reference substance solution with the impurity F content of about 10 mug per 1 ml. The sample injection amounts of the sample solution and the reference solution are 20 mu L. The solvents in the reference substance solution and the test substance solution consist of acetonitrile-water, and the volume ratio of acetonitrile to water is 5:95.
2) Setting high performance liquid phase detection conditions; detection conditions: the column was YMC-Triart C18, 25 cm. Times.0.46 cm,5 μm, mobile phase consisting of 1.5g/L sodium heptanesulfonate solution (pH adjusted to 4.5 with sulfuric acid) and acetonitrile in a volume ratio of 87:13; column temperature 40 ℃, flow rate 1.0mL/min; the detection wavelength of the high performance liquid chromatograph detector is 205nm.
3) Taking 20 μl of each of the sample solution and the control solution, injecting into a liquid chromatograph, and recording the chromatogram (figure 1).
FIG. 1 shows a test chromatogram under the chromatographic conditions of the present invention, in which the impurity F is not interfered by the blank solvent, and the method is suitable.
Example 2
The detection method of the embodiment 1 is subjected to method verification, and the verification is respectively carried out from the aspects of system applicability, destructive test, quantitative limit, detection limit, linear relation, precision, accuracy, solution stability and the like.
1. System applicability determination:
taking the impurity reference substances of the piracetam and a proper amount of the piracetam reference substances, precisely weighing, adding acetonitrile for dissolving and diluting to prepare about 0.5mg of each impurity stock solution containing each impurity per 1 mL. And (3) transferring a proper amount of each impurity stock solution, and adding a solvent to prepare each impurity positioning solution with about 10 mug of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H and piracetam in each 1 mL.
Accurately weighing a proper amount of a sample to be tested in a proper measuring flask, adding a proper amount of each impurity stock solution in the same measuring flask, and dissolving and diluting with a solvent to prepare a mixed solution which contains about 10mg of piracetam and 10 mug of other impurities per 1mL, wherein the mixed solution is used as a system applicability solution.
And respectively taking 20 mu L of each of the blank solution, each of the impurity positioning solution and the system applicability solution, injecting the solution into a high performance liquid chromatograph, recording a chromatogram, inspecting the separation condition of each component, and recording the chromatogram (figure 2), wherein the result is shown in the table 1 below.
TABLE 1 impurity localization and separation degree
Fig. 2 shows the applicability of the piracetam system, and it is known that neither the known impurity nor the main component interfere with the determination of impurity F, and that the chromatographic conditions are applicable.
Table 1 shows the impurity positioning and separation degree table, and it is clear from the table that the impurity F has a good separation degree from the adjacent impurities, and other known impurities do not interfere with the measurement of the impurity F.
2. Destructive test
Unbroken solution: about 100mg of the product is taken, placed in a 10mL volumetric flask, dissolved and diluted to a scale with a solvent, and shaken well to serve as a solution before destruction.
Acid breaking solution: about 100mg of the product is taken and placed in a 10mL volumetric flask, 1mL of 0.1mol/L hydrochloric acid solution is added, the product is destroyed for 4 hours at room temperature, 1mL of 0.1mol/L sodium hydroxide solution is added for neutralization, the product is dissolved and diluted to scale by a solvent, and the product is shaken uniformly to be used as an acid destruction solution.
Base destruction solution: about 100mg of the product is taken and placed in a 10mL volumetric flask, 1mL of 0.1mol/L sodium hydroxide solution is added, the product is destroyed for 20 minutes at room temperature, 1mL of 0.1mol/L hydrochloric acid solution is added for neutralization, the product is dissolved and diluted to scale by a solvent, and the product is uniformly shaken to be used as an alkali destruction solution.
Oxidative destruction solution: about 100mg of the product is taken and placed in a 10mL volumetric flask, 1mL of 3% hydrogen peroxide is added, the product is destroyed for 4 hours at room temperature, the product is dissolved and diluted to scale by a solvent, and the product is uniformly shaken to be used as an oxidative destruction solution.
Pyrolysis destruction solution: about 100mg of the product is taken and placed in a 10mL volumetric flask, a proper amount of solvent is added for dissolution, heating and destroying is carried out for 4 hours at 100 ℃, cooling is carried out to room temperature, solvent dissolution is used for diluting to scale, and shaking is carried out uniformly, thus obtaining pyrolysis destroying solution.
High temperature destruction solution: about 100mg of the product is taken and placed in a 10mL volumetric flask, heated and destroyed for 4 hours at 100 ℃, cooled to room temperature, dissolved and diluted to scale by a solvent, and shaken uniformly to be used as a high-temperature destroyed solution.
Light damage solution: the undamaged solution was subjected to irradiation with light at an ultraviolet wavelength of 254nm and 365nm for 1 hour to obtain an irradiation-damaged solution.
Acid base blank: taking 1mL of 0.1mol/L hydrochloric acid, placing in a 10mL measuring flask, adding 1mL of 0.1mol/L sodium hydroxide for neutralization, adding a solvent for dilution to a scale, and shaking uniformly to obtain an acid-base blank solution.
Oxidation blank: taking 1mL of 3% hydrogen peroxide, placing in a 10mL measuring flask, adding a solvent to dilute to a scale, and shaking uniformly to obtain an oxidation blank solution.
Taking 20 mu L of each solution, injecting the solution into a chromatograph, recording the chromatogram, calculating the measured sample of each impurity peak by an area normalization method, respectively destroying the measured sample by acid, alkali, oxidation, high temperature, pyrolysis and illumination conditions, examining the peak purity and the change condition of each impurity, wherein the sample is not degraded to obtain the impurity F under each destroy condition, and the degradation product does not interfere with the measurement of the impurity F.
3. Quantitative limit and detection limit test
Taking a proper amount of the impurity F reference substance, precisely weighing, adding a solvent for dissolving and diluting to prepare a stock solution containing 10 mug of impurity F per 1 mL. The quantitative limit (S/N. Gtoreq.10) and the detection limit (S/N. Gtoreq.3) were measured by stepwise dilution, and the results are shown in Table 2.
Table 2 shows quantitative limits and detection limits, and it is clear from the tables that the quantitative limits and the detection limits meet the detection requirements.
TABLE 2 quantitative limits and detection limits
4. Linear relationship
And (3) taking a proper amount of impurity F, precisely weighing, dissolving by adding a solvent, and diluting to prepare a solution containing 0.5mg of impurity F per 1mL serving as a linear stock solution. The above solutions were diluted with solvents in the following table to prepare linear solutions of each concentration. A linear solution was prepared as in Table 3.
TABLE 3 Linear solution formulation Table
The 20. Mu.L of each solution was measured precisely and injected into a high performance liquid chromatograph, the chromatogram was recorded, the peak area was measured, linear regression was performed with the peak area A as the ordinate and the concentration C as the abscissa, and the results are shown in Table 4.
As is clear from the results of linear measurement of impurity F in Table 4, impurity F was in the range of 0.5312 to 21.2464. Mu.g/mL, and the peak area and the measured concentration exhibited a good linear relationship.
TABLE 4 Linear determination of impurity F
Fig. 3 is a standard graph of piracetam impurity F, and shows that the impurity F peak area and concentration have a good linear relationship (r=1.0000) in the range of 0.5312 to 21.2464 μg/mL.
5. Precision of sample injection
And (3) taking a proper amount of the impurity F reference substance, and diluting with a solvent to prepare a mixed solution containing about 10 mug of impurity F per 1mL, wherein the mixed solution is used as a sample injection precision solution. 20 mu L of the sample was injected into a liquid chromatograph, the sample was continuously injected for 6 times, and the peak area was recorded, and the results are shown in Table 5.
Table 5 shows the results of the sample injection precision test, and the sample injection precision is good by continuously injecting 6 needles and the target peak area RSD% is less than or equal to 2.0%.
TABLE 5 sample injection precision test results
| Precision of sample injection | IMP-F |
| 1 | 25.676 |
| 2 | 25.830 |
| 3 | 25.739 |
| 4 | 25.792 |
| 5 | 25.726 |
| 6 | 25.770 |
| RSD | 0.21％ |
6. Solution stability test
And respectively testing the solution stability of the reference substance solution and the test sample solution.
(1) Stability of control solution:
samples with precision of sample injection are taken and injected respectively at 0 hour, 3 hours, 7 hours, 13 hours, 17 hours, 25 hours, 32 hours and 52 hours, and the daily stability is examined, and the results are shown in the table 6 below.
Table 6 results of impurity control solution stability As shown in the following Table, impurity F was found to have good solution stability (RSD. Ltoreq.2.0%) within 52 hours.
TABLE 6 stability results of impurity control solutions
| Time | IMP-F |
| 0H | 25.280 |
| 3H | 25.288 |
| 7H | 25.193 |
| 13H | 25.268 |
| 17H | 25.225 |
| 25H | 25.163 |
| 32H | 25.219 |
| 52H | 25.231 |
| RSD | 0.17％ |
(2) Test article solution stability:
according to the proposed related substance method, the sample is taken and prepared into related substance test sample solutions, and the sample solutions are sampled at 0 hour, 3 hours, 7 hours, 13 hours, 17 hours, 25 hours, 32 hours and 52 hours respectively, so that the daily stability of the test sample solutions is examined, and the results are shown in the table 7 below.
Table 7 shows the stability results of the sample solutions, and it is clear that the impurity F was not detected in the sample solutions within 52 hours, and the sample solutions were stable.
TABLE 7 test solution stability results
| Time | IMP-F |
| 0H | Not detected |
| 3H | Not detected |
| 7H | Not detected |
| 13H | Not detected |
| 17H | Not detected |
| 25H | Not detected |
| 32H | Not detected |
| 52H | Not detected |
7. Repeatability test
Taking a piracetam test sample, and measuring related substances according to a planned method. The measurement was repeated for 6 parts. The results are shown in Table 8.
Table 8 shows the results of the repeatability test, and as can be seen from the table, no impurity F was detected in each of the 6 repeatability samples, and the repeatability was good.
TABLE 8 repeatability measurement results
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 |
| Impurity F | Not detected | Not detected | Not detected | Not detected | Not detected | Not detected |
8. Accuracy test (sample recovery rate)
The impurities are taken and dissolved by adding a solvent to prepare a mixed solution with each impurity of 100 mug per 1mL, and the mixed solution is taken as a recovery rate stock solution.
The above mixed stock solution was precisely aspirated and diluted to prepare 50%, 100% and 200% solutions according to the following table, 3 parts of each concentration was prepared, and the recovery rate solution was prepared according to the following table 9.
TABLE 9 recovery rate solution formulation table
And sucking 20 mu L of each sample, recording the peak area of each known impurity, and calculating the recovery rate and RSD of each known impurity. The results are shown in Table 10.
Table 10 shows the experimental results of the recovery rate of the impurity F, and the table shows that each recovery rate and the average recovery rate of the impurity F are in the range of 90-108%, and the RSD% of the recovery rate is less than or equal to 10.0%.
TABLE 10 Experimental results on recovery of impurity F
9. Intermediate precision
The same operator performs isomer content inspection on the same batch of samples at different times, by different operators, by the same operator and by different instruments according to the repeatability test method. The results are shown in Table 11.
Table 11 shows the results of the intermediate precision measurement, and it is clear from the results that the intermediate precision of the present inspection method is good.
TABLE 11 results of intermediate precision measurements
| Conditions (conditions) | Repeatability of | Different instruments | Different dates of day | Different people |
| Impurity F | Not detected | Not detected | Not detected | Not detected |
10. Durability test
Piracetam-related substance method durability was mainly verified from four aspects of different flow rates, different column temperatures, different initial ratios of mobile phases, and different chromatographic columns.
The experiment was performed under the conditions of Table 12, and the results are shown in Table 13.
Table 13 shows the durability results, and it is clear from the table that the impurity F was not detected under the conditions of small changes in column temperature, flow rate, mobile phase ratio, pH and changing different chromatographic columns, and the intrinsic chromatographic conditions were good.
Table 12 durability conditions
TABLE 13 durability results
Under the conditions of small changes of column temperature, flow rate, mobile phase proportion and pH and changing of different chromatographic columns, the impurity F in the system applicability solution is not interfered by other impurities, the separation degree is good, the blank solvent does not interfere with the measurement of the impurity F, and the impurity F is not detected in the sample.
Claims (10)
1. A method for measuring the content of impurity F in piracetam by a high performance liquid phase method is characterized by comprising the following specific steps:
1) Preparing a sample solution and a reference substance solution;
2) Setting high performance liquid phase detection conditions; the chromatographic column is YMC-Triart C18; the mobile phase is a mixed solution of sodium heptanesulfonate solution and acetonitrile;
3) And respectively taking a sample solution and a reference substance solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating the content of the impurity F.
2. The method for determining the content of impurity F in piracetam by using the high performance liquid phase method according to claim 1, wherein in the step 1), the preparation method of the sample solution is as follows: precisely weighing piracetam, placing in a volumetric flask, adding acetonitrile-water to dilute to a constant volume to obtain 10mg of piracetam-containing solution per 1mL, and uniformly mixing to obtain a sample solution; the preparation method of the reference substance solution comprises the following steps: precisely weighing the impurity F, and diluting with acetonitrile-water to obtain a reference substance solution containing 10 mug of the impurity F per 1mL, wherein the volume ratio of acetonitrile to water is 5:95.
3. the method for determining the impurity F content in piracetam by high performance liquid chromatography according to claim 1, wherein in step 2), the chromatographic column YMC-Triart C18 has a length of 250mm, an inner diameter of 4.6mm and a filler particle size of 5 μm.
4. The method for determining the content of impurity F in piracetam according to claim 1, characterized in that in step 2), the concentration of the sodium heptanesulfonate solution is 1.5g/L, and the pH is adjusted to 4.5 with sulfuric acid.
5. The method for determining the content of impurity F in piracetam by high performance liquid phase method according to claim 1, characterized in that in step 2), the volume ratio of the sodium heptanesulfonate solution to acetonitrile is 86-88: 14-12.
6. The method for determining the content of impurity F in piracetam according to claim 1, characterized in that in step 2), the column temperature of the chromatographic column is 38 to 42 ℃.
7. The method for determining the content of impurity F in piracetam according to claim 1, characterized in that in step 2), the flow rate of the mobile phase is 0.95 to 1.05mL/min.
8. The method for determining the impurity F content in piracetam by high performance liquid chromatography according to claim 1, wherein in step 2), the sample injection amounts of the sample solution and the reference solution are 20 μl.
9. The method for determining the content of impurity F in piracetam according to claim 1, characterized in that in step 3), the detection wavelength of the liquid chromatograph is 205nm.
10. The method for determining the impurity F content in piracetam by using the high performance liquid chromatography according to claim 1, wherein in the step 3), the calculation method is an external standard method, and the impurity F content in the sample solution is calculated to be less than or equal to 0.1%.
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