CN114354803A - Method for detecting related substances of sodium valproate oral solution - Google Patents
Method for detecting related substances of sodium valproate oral solution Download PDFInfo
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- NIJJYAXOARWZEE-UHFFFAOYSA-N di-n-propyl-acetic acid Natural products CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 229940084026 sodium valproate Drugs 0.000 title claims abstract description 49
- AEQFSUDEHCCHBT-UHFFFAOYSA-M sodium valproate Chemical compound [Na+].CCCC(C([O-])=O)CCC AEQFSUDEHCCHBT-UHFFFAOYSA-M 0.000 title claims abstract description 49
- 229940100688 oral solution Drugs 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims description 39
- 239000000126 substance Substances 0.000 title claims description 15
- 239000012535 impurity Substances 0.000 claims abstract description 123
- 238000000605 extraction Methods 0.000 claims abstract description 60
- 238000001514 detection method Methods 0.000 claims abstract description 53
- 239000007788 liquid Substances 0.000 claims abstract description 7
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 198
- 239000000243 solution Substances 0.000 claims description 78
- 239000000523 sample Substances 0.000 claims description 43
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 36
- 239000012085 test solution Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims description 17
- 238000002347 injection Methods 0.000 claims description 15
- 239000007924 injection Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 10
- RXFAGYKLLRXFDX-UHFFFAOYSA-N 3-propylhexan-2-one Chemical compound CCCC(C(C)=O)CCC RXFAGYKLLRXFDX-UHFFFAOYSA-N 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- QUMITRDILMWWBC-UHFFFAOYSA-N nitroterephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C([N+]([O-])=O)=C1 QUMITRDILMWWBC-UHFFFAOYSA-N 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- ADNADZOSMJDVIS-UHFFFAOYSA-N 2-methylpropyl pentanoate Chemical compound CCCCC(=O)OCC(C)C ADNADZOSMJDVIS-UHFFFAOYSA-N 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 4
- WUWPVNVBYOKSSZ-UHFFFAOYSA-N 2-ethyl-2-methyl valeric ccid Chemical group CCCC(C)(CC)C(O)=O WUWPVNVBYOKSSZ-UHFFFAOYSA-N 0.000 claims description 2
- ODPKTGAWWHZBOY-UHFFFAOYSA-N 2-propan-2-ylpentanoic acid Chemical compound CCCC(C(C)C)C(O)=O ODPKTGAWWHZBOY-UHFFFAOYSA-N 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims description 2
- 229940005605 valeric acid Drugs 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 238000002360 preparation method Methods 0.000 abstract description 21
- 238000000926 separation method Methods 0.000 abstract description 17
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000004945 emulsification Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 206010015037 epilepsy Diseases 0.000 abstract description 3
- 230000009885 systemic effect Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 24
- 238000007865 diluting Methods 0.000 description 23
- 239000011550 stock solution Substances 0.000 description 21
- 238000012360 testing method Methods 0.000 description 21
- 239000000047 product Substances 0.000 description 20
- 239000002904 solvent Substances 0.000 description 17
- 230000015556 catabolic process Effects 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- 238000006731 degradation reaction Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 239000003755 preservative agent Substances 0.000 description 9
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 8
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 8
- 238000005464 sample preparation method Methods 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 230000006378 damage Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 229960003415 propylparaben Drugs 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 239000012490 blank solution Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- QQZOPKMRPOGIEB-UHFFFAOYSA-N 2-Oxohexane Chemical compound CCCCC(C)=O QQZOPKMRPOGIEB-UHFFFAOYSA-N 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 229960000604 valproic acid Drugs 0.000 description 4
- 208000034308 Grand mal convulsion Diseases 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 3
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 3
- 229960002216 methylparaben Drugs 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 206010010904 Convulsion Diseases 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000001961 anticonvulsive agent Substances 0.000 description 2
- 229960003965 antiepileptics Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 230000001066 destructive effect Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000012494 forced degradation Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- ZUQOBHTUMCEQBG-UHFFFAOYSA-N 4-amino-5-hydroxynaphthalene-1,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC(O)=C2C(N)=CC=C(S(O)(=O)=O)C2=C1 ZUQOBHTUMCEQBG-UHFFFAOYSA-N 0.000 description 1
- JCSTXMWOSJYBFK-UHFFFAOYSA-N C1(=CC=CC=C1)C(C(=O)OO)C.[Na] Chemical compound C1(=CC=CC=C1)C(C(=O)OO)C.[Na] JCSTXMWOSJYBFK-UHFFFAOYSA-N 0.000 description 1
- 208000003078 Generalized Epilepsy Diseases 0.000 description 1
- 101000620451 Homo sapiens Leucine-rich glioma-inactivated protein 1 Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 102100022275 Leucine-rich glioma-inactivated protein 1 Human genes 0.000 description 1
- 208000036572 Myoclonic epilepsy Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 208000037158 Partial Epilepsies Diseases 0.000 description 1
- 206010061334 Partial seizures Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 201000009028 early myoclonic encephalopathy Diseases 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 201000007186 focal epilepsy Diseases 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- RIMIDGSPFGJFCN-UHFFFAOYSA-N hydroxymethyl benzoate Chemical compound OCOC(=O)C1=CC=CC=C1 RIMIDGSPFGJFCN-UHFFFAOYSA-N 0.000 description 1
- 201000001993 idiopathic generalized epilepsy Diseases 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000005297 material degradation process Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
Sodium Valproate Oral Solution (Sodium Valproate Oral Solution) is a red clear viscous liquid used for the treatment of systemic, partial or other types of epilepsy. At present, in the detection of sodium valproate oral solution, the extraction emulsification phenomenon in the sample preparation process is seriously difficult to separate or the separation time is too long, the impurity detection is incomplete under the chromatographic condition of the prior art, and the separation degree is poor. The invention scientifically screens and researches the preparation mode and the chromatographic condition of the sample, and provides an analysis method which has proper extraction time and complete detection of each component.
Description
Technical Field
The invention belongs to the field of medicine detection, and particularly relates to a method for detecting related substances of sodium valproate oral solution.
Background
Sodium Valproate Oral Solution (Sodium Valproate Oral Solution) is red clear viscous liquid and is used for treating generalized epilepsy, partial epilepsy or other types, Sodium Valproate which is a main component is a nitrogen-free broad-spectrum antiepileptic drug, is quickly and completely absorbed by Oral administration, is effective on various types of epilepsy such as various types of seizures, myoclonic epilepsy, local seizures, grand mal and mixed epilepsy, and is the first choice drug for primary grand mal and absence grand mal.
At present, only the indian pharmacopoeia and the british pharmacopoeia in the domestic and foreign pharmacopoeias have collected the standard of sodium valproate oral solution and the domestic approved national drug standard (YBH01302004-2014Z), wherein the preparation principles of related substance sample solutions are acidification by adding acid, and the detection is carried out after the extraction by using a proper solvent (dichloromethane or n-heptane), and the detection process is found by referring to the technology reported by the prior literature: the extraction emulsification phenomenon in the sample preparation process is seriously difficult to separate or takes too long to separate, the impurity detection is incomplete under the chromatographic condition of the prior art, and the separation degree is poor. The invention scientifically screens and researches the preparation mode and the chromatographic condition of the sample, and provides an analysis method which has proper extraction time and complete detection of each component.
Disclosure of Invention
The sodium valproate oral solution is used as an important broad-spectrum antiepileptic drug and widely applied to children, and the quality of the sodium valproate oral solution directly affects the health of patients, so that the sodium valproate oral solution is particularly important to quality control. The invention aims to provide a sodium valproate oral solution related substance sample preparation method and a detection technology, which are simple and convenient to operate, complete in impurity detection, scientific and controllable, and aim to overcome the defects in the prior art.
The invention is realized by adjusting the solution preparation method and the chromatographic conditions during detection, and particularly comprises the following steps:
firstly, when preparing a test solution, after n-heptane extraction or extraction of acidified sodium valproate oral solution, water washing is not needed;
secondly, during detection, the temperature rise program is adjusted as follows: the initial temperature is 55-65 ℃, the temperature is maintained for 4-6 minutes, the temperature is increased to 140-160 ℃ at the rate of 5-8 ℃ per minute, the temperature is increased to 180-200 ℃ at the rate of 2-4 ℃ per minute, the temperature is increased to 220-240 ℃ at the rate of 4-6 ℃ per minute, and the temperature is maintained for 20-40 minutes; so as to achieve the purposes of complete impurity detection and complete peak appearance.
The acid used in the sample preparation method is one of hydrochloric acid, sulfuric acid and nitric acid.
The concentration of the acid is 1-10 mol/l; further, the concentration of the acid was 2.0 mol/l.
In the technical scheme of the invention, the traditional manual extraction is replaced by a separating funnel vertical oscillator extraction mode during extraction, so that the extraction efficiency is improved, and the popularization and the use are facilitated.
When the separating funnel is extracted by a vertical oscillator, the shaking frequency is 100-350 times/min; further, the shaking frequency is 220-280 times/min; furthermore, the shaking frequency is 260-280 times/min.
When the separating funnel is extracted by a vertical oscillator, the single extraction time is 0.5-5 min; further, the single extraction time is 2-5 min.
The concentration of the test solution prepared by the method is 1-10 mg/ml; further, the concentration of the sample solution was 5 mg/ml.
In the invention, the temperature of the detector is 235-265 ℃; further, the detector temperature was 250 ℃.
In the invention, the temperature of a sample inlet is 200-230 ℃; further, the injection port temperature was 220 ℃.
In the technical scheme of the invention, the capillary column is a strong polarity column, and includes but is not limited to a capillary column taking nitroterephthalic acid modified polyethylene glycol as stationary liquid.
In the detection method, the flow rate is 1-3 ml per minute; further, the flow rate was 1.5ml per minute.
In the detection method, the sample injection volume is 1-10 mu l; further, the injection volume was 1. mu.l.
The split ratio in the detection method is 10-1: 1; further, the split ratio was 5: 1.
In the technical scheme of the invention, n-heptane is selected as the extraction solvent, compared with the prior art that dichloromethane is used as the extraction solvent, the problems that the extraction emulsification phenomenon is seriously difficult to separate or the separation time is too long in the sample preparation process are solved, and the comparative example 2 shows that the time required by standing and layering after once extraction is shortened by about 3 times, so that the sample processing time is greatly shortened.
According to the invention, the shaking frequency and the single extraction time are optimized, so that the sufficient extraction of related substances in the sodium valproate oral solution is ensured, and a reliable basis is provided for the accuracy of a detection result.
According to the technical scheme, n-heptane is selected as a solvent, and after n-heptane layers are combined, water washing is not needed, and n-heptane is adopted for direct dilution. Compared with the existing sample preparation method (adopting water washing, concentration and dilution after extraction), the existing sample preparation steps are more complicated, and impurity loss or other impurities which are not introduced into the product may exist in the water washing and concentration processes, the comparison and investigation of the water washing and concentration steps (comparative example 1) shows that the water washing and concentration are unnecessary operations, the water washing step in the sample preparation process is removed, the sample preparation is more convenient, the impurity loss and the introduction of other impurities in the water washing and filtering processes are avoided, and the real impurity level in the sodium valproate oral solution can be reflected better.
The preparation method of the control solution comprises the following steps: precisely measuring a proper amount of the test solution, and quantitatively diluting with n-heptane to obtain a solution containing about 5 μ g of sodium valproate per 1 ml.
In one embodiment of the present invention, the chromatographic conditions (chromatographic condition 3) are: a capillary column using nitroterephthalic acid modified polyethylene glycol as stationary liquid; the initial temperature is 60 ℃, the temperature is maintained for 5 minutes, the temperature is increased to 150 ℃ at the rate of 7 ℃ per minute, then the temperature is increased to 190 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 230 ℃ at the rate of 5 ℃ per minute, and the temperature is maintained for 30 minutes; the temperature of the detector is 250 ℃; the temperature of a sample inlet is 220 ℃; flow rate was 1.5ml per minute; the sample injection volume is 1 mu l; the split ratio is 5: 1.
In one embodiment of the present invention, the chromatographic conditions (chromatographic condition 2) are: a capillary column using nitroterephthalic acid modified polyethylene glycol as stationary liquid; the initial temperature is 60 ℃, the temperature is maintained for 5 minutes, the temperature is raised to 200 ℃ at the rate of 7 ℃ per minute, the temperature is maintained for 5 minutes, the temperature is raised to 230 ℃ at the rate of 20 ℃ per minute, and the temperature is maintained for 25 minutes; the temperature of the detector is 250 ℃; the temperature of a sample inlet is 220 ℃; the flow rate was 2.0ml per minute; the sample injection volume is 1 mu l; the split ratio is 5: 1.
In another specific embodiment of the present invention, the chromatographic conditions (chromatographic condition 1) are: a capillary column using nitroterephthalic acid modified polyethylene glycol as stationary liquid; the initial temperature is 60 ℃, the temperature is maintained for 5 minutes, the temperature is raised to 150 ℃ at the rate of 7 ℃ per minute, then the temperature is raised to 190 ℃ at the rate of 3 ℃ per minute, the temperature is maintained for 9 minutes, then the temperature is raised to 230 ℃ at the rate of 20 ℃ per minute, and the temperature is maintained for 30 minutes; flow rate was 1.5ml per minute; the sample injection volume is 1 mu l; the split ratio is 5: 1.
In the technical scheme of the invention, the impurities in the sodium valproate oral solution comprise: impurity A, impurity C, impurity K, 3-propyl-2-hexanone, 2-methyl propyl valerate and two unknown impurities, wherein impurity A is valeric acid; impurity C is (2RS) -2- (1-methylethyl) pentanoic acid; the impurity K is (2RS) -2-ethyl-2-methyl pentanoic acid.
The chromatographic conditions 1-3 can separate and detect the 8 impurities, and can ensure that preservatives methyl hydroxybenzoate and propyl hydroxybenzoate in the sodium valproate oral solution have complete peak appearance in one needle.
The invention has the beneficial effects that:
1. according to the invention, sodium valproate oral solution is taken as a detection object, n-heptane is taken as an extraction solvent, and compared with the prior art (taking dichloromethane as the extraction solvent), the sodium valproate oral solution is easier to layer during extraction, the layering time is shortened by about 3 times, and the separation time is shorter.
2. According to the invention, the traditional manual extraction is replaced by a separating funnel vertical oscillator extraction mode, the extraction conditions such as single extraction time and shaking frequency are optimized, the related substances in the sodium valproate oral solution are extracted as far as possible, the extraction efficiency is improved, and the method is more beneficial to popularization and use.
3. The invention examines the specificity of the method, in the degradation specificity experiment of example 4, no degradation impurity is generated under the destruction conditions of acid, alkali, oxidation and illumination, 1 degradation impurity is generated under the destruction condition of high temperature, the content of the degradation impurity is 0.02%, the separation degree of a main peak and an adjacent impurity peak under each forced degradation condition is more than 1.5, and the degradation product does not interfere with the detection of each known impurity; compared with the non-destructive test sample, the material balance of each degraded test sample is between 90.0 percent and 108.0 percent, which meets the requirement.
4. The detection method of the invention has good linearity: when the concentration of the impurity K is measured in the range of 0.9774 mu g/ml-14.6610 mu g/ml, the peak area and the concentration have good linear relation, the linear equation is that y is 0.0455x-0.0012, and R is20.9989, correlation coefficient r is 0.9994; when the concentration of sodium valproate is measured in the range of 1.0157 mu g/ml-101.5700 mu g/ml, the peak area and the concentration have good linear relation, and the linear equation is that y is 0.0401x +0.0108, R20.9993, the correlation coefficient r is 0.9996.
5. The detection method of the invention has good accuracy: in the accuracy test of example 4, the recovery rate of the impurity K was 106.0% on average and RSD was 0.8% < 2.0%, which met the relevant detection standards.
6. The detection method of the invention has good weight: in 6 times of repeated detection experiments, the content of unknown impurities in 6 samples is 0.03 percent, and the range is 0.
7. In the method, the stability of the solution is good, the RSD of the valproic acid peak area in the contrast solution is 1.7 percent and less than 2.0 percent, the impurity content of the test solution is 0.03 percent, and the range is 0, which indicates that the test solution and the contrast solution are stable after being placed for 50 hours at room temperature.
In conclusion, the technology of the invention is based on gas chromatography, and on the basis of the technology disclosed by the prior document, aiming at the defects of long sample processing time, serious emulsification phenomenon, incomplete impurity separation, incomplete peak inclusion and peak emergence and the like in the prior art, the method combines an automatic extraction device and adopts a new temperature rise program to ensure that impurities can be completely separated and all components have complete peaks emergence, scientifically screens and researches the sample processing technology, greatly shortens the sample processing time, solves the problems in the prior art, and results of the method provided by the invention are in an acceptable range through the research and verification of methodologies such as specificity and accuracy, and shows that the method can more effectively control the related substances of the sodium valproate oral solution.
Drawings
Fig. 1 is a gas chromatogram of chromatographic condition 1.
Fig. 2 is a gas chromatogram of chromatographic condition 2.
Fig. 3 is a gas chromatogram of chromatographic condition 3.
FIG. 4 is an extraction time screening spectrum.
FIG. 5 is a graph of a frequency spectrum of shaking.
FIG. 6 is a chart of the detection of n-heptane as the extraction solvent.
FIG. 7 is a chart of detection using methylene chloride as an extraction solvent.
FIG. 8 is a BP2020 detection spectrum.
FIG. 9 is a detection spectrum of the method of the present invention.
FIG. 10 is a detection spectrum under the chromatographic conditions of patent CN 113252803A.
Fig. 1 to 3 are attached with the following reference numerals: 2-hexanone, 2-methyl propyl valerate, 3-propyl-2-hexanone, unknown 1, impurity A, unknown 2, impurity C, impurity K, main component, hydroxy methyl benzoate,
Propyl hydroxybenzoate.
Detailed Description
The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.
Aiming at the sample preparation mode and the detection method, the inventor carries out a large number of experiments and scientifically screens and verifies the extraction mode and the chromatographic condition, and the method comprises the following specific steps:
first, an embodiment
1) Example 1: screening of chromatographic conditions
Table 1 comparison with prior art process
Compared with the prior art, the method of the prior art is inspected by adopting an impurity standard-adding solution, the national drug standard (YBH01302004-2014Z) is basically consistent with the IP2010 method, 6 known impurities are added, 2 impurities are actually detected, more impurity peaks are not separated, and the preservative does not peak; in the BP2020 method, 5 of the known impurity a and the unknown impurity are not separated, the preservative peak does not appear in one injection time and remains in the next injection, and similarly to the BP2020 method, the preservative cannot be detected in one injection time by the method of patent CN 11323824 a.
Further optimized chromatographic conditions:
TABLE 2 optimization of chromatographic conditions
TABLE 3 optimization results
Note: the added standard impurities 8 are 6 known impurities +2 auxiliary materials.
Figures 1, 2 and 3 in the attached drawings of the specification show that under three chromatographic conditions, the separation degree of main components and impurities and the separation degree of the impurities meet the requirements, and preservatives can normally peak and can be used for detecting related substances of sodium valproate oral solution.
2) Example 2: investigation of single extraction time
Test solution: weighing 12.5ml of sodium valproate oral solution, placing in a separating funnel, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, each time for 20ml, shaking for 0.5, 1, 2, 3, 4 and 5 minutes respectively each time by using a separating funnel vertical oscillator, combining the extracting solutions (n-heptane layers), placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking uniformly to obtain the sodium valproate oral solution.
The test solutions prepared at different extraction times were tested under the chromatographic conditions 3 in example 1.
TABLE 4 Single extraction time screening
And (4) conclusion: within the range of 0.5-5 minutes of single extraction time (figure 4 in the attached figure), the peak area of each component is increased along with the increase of the extraction time, when the extraction time is within the range of 2-5 minutes, the peak area of each component is the largest and has no obvious difference, which indicates that the extraction is complete, and the single extraction time is determined to be 2-5 minutes.
3) Example 3: investigation of vibration frequency
Test solution: weighing 12.5ml of sodium valproate oral solution, placing in a separating funnel, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking, extracting with n-heptane for 3 times, 20ml each time, mixing extractive solutions (n-heptane layers), placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking to obtain the final product. The shaking parameters of the vertical oscillator were performed according to the following table parameters 1, 2, 3, 4, and 5, respectively.
TABLE 5 Shake parameter screening
Test solutions prepared at different shaking frequencies were taken and tested under chromatographic conditions 2 in example 1, respectively.
TABLE 6 results of screening shaking parameters
And (4) conclusion: in the range of the shaking frequency of 220-280 times/min (figure 5 of the attached drawing), the peak areas of all components in the test solution increase along with the increase of the frequency, which shows that the extraction effect increases along with the increase of the shaking frequency, when the shaking frequency is in the range of 260-280 times/min, the peak areas of all components reach the maximum and have no obvious difference, the extraction effect is the best, and the shaking frequency is determined to be 260-280 times/min.
4) Example 4: validation of analytical methods
4.1) solution preparation:
blank solution: n-heptane.
Test solution: weighing 12.5ml of the test sample, placing in a separating funnel, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking, extracting with n-heptane for 3 times, 20ml each time, mixing n-heptane extract, placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking.
Control solution: precisely measuring 1.0ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to the scale with n-heptane, shaking up, precisely measuring 1.0ml of the test solution, placing the test solution in a 10ml measuring flask, diluting the test solution to the scale with n-heptane, and shaking up.
Blank adjuvant solution: weighing blank adjuvants 12.5ml, and preparing according to the method of test solution.
Stock solutions of various impurities: respectively taking appropriate amount of impurity A, impurity C, 3-propyl-2-hexanone, impurity K, 2-hexanone, and 2-propylmethyl valerate as reference substances, and diluting with n-heptane to obtain solution containing 0.5mg per 1 ml.
Sodium valproate stock solution: taking appropriate amount of sodium valproate control, placing in a centrifuge tube, adding appropriate amount of water to dissolve, adding dilute sulfuric acid, acidifying, shaking, extracting with n-heptane for 3 times, mixing n-heptane layers, and diluting with n-heptane to obtain solution containing about 5mg per 1 ml.
Each impurity localization solution: precisely measuring 1.0ml of each impurity stock solution, respectively placing into 50ml measuring flasks, diluting with n-heptane to scale, and shaking.
Methylparaben positioning solution: an appropriate amount of methylparaben control was diluted with n-heptane to make a solution containing about 80. mu.g of methylparaben per 1 ml.
Propyl hydroxybenzoate positioning solution: an appropriate amount of propylparaben control was diluted with n-heptane to make a solution of about 30 μ g per 1 ml.
Impurity spiking solution: weighing 12.5ml of the product, placing the product in a separating funnel, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, placing in a 100ml measuring flask, respectively adding 2ml of each impurity stock solution and 2ml of each positioning solution of methyl hydroxybenzoate and propyl hydroxybenzoate, diluting to scale with n-heptane, and shaking uniformly to obtain the product.
4.2) System suitability and impurity positioning test
And (3) taking a blank solution, a blank auxiliary material solution, a component positioning solution, an impurity labeling solution and a sample solution, and respectively carrying out sample injection analysis under the chromatographic condition 3 in the embodiment 1.
As a result: the blank solution and the blank auxiliary material solution are not interfered at the peak positions of the components, and the unknown impurity peak in the sample solution is not interfered with the detection of the known impurities. The peak-out time of each component in the impurity standard-adding solution is consistent with the retention time of the positioning solution, and the separation degree among the components meets the requirement.
4.3) degradation specificity test
Alkali destruction test solution: precisely measuring 12.5ml of the product, placing the product in a 20ml headspace bottle, adding 1.0ml of 2mol/L sodium hydroxide solution, sealing by a cover, shaking, breaking the product at 40 ℃ for 24 hours, neutralizing by using 2mol/L hydrochloric acid solution with the same amount, transferring to a separating funnel, rinsing the headspace bottle by using 8ml of water for 3 times, transferring all the headspace bottles to the separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting by using n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, placing in a 100ml measuring flask, diluting to the scale by using n-heptane, and shaking uniformly.
Acid destruction test solution: precisely measuring 12.5ml of the product, placing the product in a 20ml headspace bottle, adding 1.0ml of 2mol/L hydrochloric acid solution, sealing the bottle with a cover, shaking the bottle, breaking the bottle at 40 ℃ for 24 hours, neutralizing the bottle with 2mol/L sodium hydroxide solution with the same amount, transferring the bottle to a separating funnel, rinsing the headspace bottle with 8ml of water for 3 times, transferring the bottle to the separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking the bottle uniformly, extracting the bottle with n-heptane for 3 times, 20ml each time, combining the n-heptane extracting solutions, placing the bottle in a 100ml measuring bottle, diluting the bottle with n-heptane to the scale, and shaking the bottle uniformly.
Oxidative destruction of test article solution: precisely measuring 12.5ml of the product, placing the product in a 20ml headspace bottle, adding 1.0ml of 30% hydrogen peroxide solution, sealing the bottle with a cover, shaking the bottle, destroying the bottle at room temperature for 24 hours, rinsing the headspace bottle with 9ml of water for 3 times, completely transferring the bottle to a separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking the bottle uniformly, extracting the bottle with n-heptane for 3 times, each time for 20ml, combining the n-heptane extracting solutions, placing the bottle in a 100ml volumetric bottle, diluting the bottle with n-heptane to the scale, and shaking the bottle uniformly.
Destroying the test solution at high temperature: precisely measuring an appropriate amount of the product, placing the product in a 20ml headspace bottle, covering and sealing, breaking the headspace bottle in an oven at 105 ℃ for 24h, cooling to room temperature, measuring 12.5ml of the sample, rinsing the headspace bottle with 10ml for 3 times, transferring the headspace bottle to a separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining the n-heptane extracting solutions, placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking uniformly.
Irradiating to destroy the test solution: precisely measuring an appropriate amount of the product, placing into a 20ml headspace bottle, sealing with a cover, breaking in a 4500Lx +/-500 Lx illumination box for 7d, measuring 12.5ml of the sample, rinsing the headspace bottle with 10ml for 3 times, transferring into a separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking, extracting with n-heptane for 3 times, each time 20ml, combining the n-heptane extract, placing into a 100ml volumetric bottle, diluting with n-heptane to scale, and shaking.
Blank auxiliary material degradation solution: the blank auxiliary materials are used for replacing the test sample and are prepared by respectively destroying the test sample solution by acid, alkali, oxidation, high temperature and illumination.
The blank solution, the sample solution and the respective disrupting solutions under 4.1) were collected and examined under the chromatographic conditions 3 in example 1.
As a result: under each degradation condition, the blank auxiliary material solution has no interference on the detection of the main peak and each impurity, no degradation impurity is generated under the conditions of acid, alkali, oxidation and illumination damage, 1 degradation impurity is generated under the condition of high-temperature damage, the content of the degradation impurity is 0.02 percent, the separation degree of the main peak and the adjacent impurity peak under each forced degradation condition is more than 1.5, and the degradation product does not interfere with the detection of each known impurity; compared with the non-destructive test sample, the material balance of each degraded test sample is between 90.0 percent and 108.0 percent, which meets the requirements and is detailed in Table 7.
TABLE 7 degradation specificity test results
4.4) testing of detection and quantitation limits
Quantitative limiting solution: accurately measuring appropriate amount of sodium valproate and each impurity stock solution under 4.1, and gradually diluting until the signal-to-noise ratio (S/N) is about 10.
Detection limiting solution: accurately measuring sodium valproate under 4.1 and appropriate amount of each impurity stock solution, and gradually diluting until the signal-to-noise ratio (S/N) is about 3.
The results of the detection limit and quantification terms are detailed in table 8:
TABLE 8 test results of detection limit and quantitation limit
4.5) Linear test
Linear mixing stock solutions: precisely measuring 1.5ml of impurity K stock solution and 1.0ml of sodium valproate stock solution under the condition of 4.1), placing the impurity K stock solution and the sodium valproate stock solution into the same 10ml measuring flask, adding a solvent to dilute to the scale, and shaking up uniformly.
Taking the quantitative limit solution of the impurity K and the sodium valproate under the test items of 4.4) detection limit and quantitative limit as a linear solution 1.
Respectively and precisely measuring 0.2ml, 0.3ml, 0.5ml, 0.6ml and 1.0ml of linear mixed stock solution, placing the linear mixed stock solution into a 5ml measuring flask, fixing the volume to the scale with n-heptane, and shaking up to obtain linear solutions 2, 3, 4, 5 and 6.
And respectively injecting the solution, recording peak areas, and taking the peak areas as vertical coordinates and the concentration as horizontal coordinates to obtain a linear regression equation.
Under the condition of the method, when the concentration of the impurity K is measured in the range of 0.9774 mu g/ml-14.6610 mu g/ml, the peak area and the concentration have good linear relation, the linear equation is that y is 0.0455x-0.0012, R is20.9989, correlation coefficient r is 0.9994; when the concentration of sodium valproate is measured in the range of 1.0157 mu g/ml-101.5700 mu g/ml, the peak area and the concentration have good linear relation, and the linear equation is that y is 0.0401x +0.0108, R20.9993 and a correlation r of 0.9996, as detailed in table 9.
TABLE 9 results of the Linear test
4.6) accuracy test
Impurity K accuracy stock solution: an appropriate amount of impurity K was taken and diluted with n-heptane to make a solution containing about 0.75mg per 1 ml.
Impurity K control solution: precisely measuring 1.0ml of an impurity K accurate stock solution, placing the impurity K accurate stock solution into a 100ml measuring flask, adding n-heptane to dilute to a scale, and shaking up to obtain the product.
50% recovery solution: precisely measuring 12.5ml of a sample, placing the sample in a separating funnel, precisely measuring 0.5ml of impurity K accuracy stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining organic layers, placing the organic layers in a 100ml measuring flask, diluting with n-heptane to scale, and shaking uniformly.
100% recovery solution: precisely measuring 12.5ml of the product, placing the product in a separating funnel, precisely measuring 1.0ml of impurity K accurate stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining organic layers, placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking uniformly.
150% recovery solution: precisely measuring 12.5ml of the product, placing the product in a separating funnel, precisely measuring 1.5ml of impurity K accurate stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining organic layers, placing in a 100ml measuring flask, diluting with n-heptane to scale, and shaking uniformly.
The blank solution, the sample solution and the impurity K reference solution and the recovery solution under the item 4.1) are respectively injected, the recovery rate is calculated according to an external standard method, and the results are shown in a table 10.
TABLE 10 recovery test results
4.7) precision test of the instrument
Instrument precision solution: taking about 15mg of the impurity K reference substance, precisely weighing, placing in a 100ml measuring flask, adding n-heptane to dilute to a scale, and shaking up to obtain an impurity K stock solution. Taking 50mg of sodium valproate reference substance, precisely weighing, placing in a centrifuge tube, adding 1ml of water for dissolving, adding 0.5ml of dilute sulfuric acid, shaking up, extracting with n-heptane for three times, 2ml each time, combining n-heptane layers, placing in a 10ml measuring flask, adding 0.5ml of impurity K stock solution, diluting with n-heptane to scale, and shaking up to obtain the final product.
Sample introduction was performed 6 times in succession according to the chromatographic conditions 3 in example 1, chromatograms were recorded, and RSD of the retention time and peak area of the impurity K and valproic acid peaks were calculated.
As a result: RSD of the retention time of the impurity K peak is 0.02 percent and less than 1.0 percent, and RSD of the peak area is 1.5 and less than 2.0 percent; the RSD of the valproic acid peak retention time is 0.02 percent to less than 1.0 percent, the RSD of the peak area is 1.4 percent to less than 2.0 percent, and the precision of the instrument is good.
4.8) repeatability test
Taking 6 parts of test solution and control solution prepared in parallel under the item 4.1), carrying out sample injection detection according to the chromatographic condition 3 in the example 1, recording a chromatogram, and calculating the content of impurities.
As a result: 6 samples, the content of unknown impurities is 0.03 percent, the range is 0, and the repeatability of the method is good.
4.9) solution stability test
Taking the sample solution and the control solution under the item 4.1), respectively standing at room temperature for 0h, 6h, 13h, 26h, 38h and 50h, performing sample injection detection according to the chromatographic condition 3 in the example 1, recording a chromatogram, and calculating the impurity content and RSD of the peak area of the control solution.
As a result: the RSD of the area of the valproic acid peak in the control solution is 1.7 percent and less than 2.0 percent, the impurity content of the test solution is 0.03 percent, and the range is 0, which indicates that the test solution and the control solution are stable after being placed at room temperature for 50 hours.
Second, comparative example
In order to show that the method can effectively control the related substances of the sodium valproate oral solution, the beneficial effects of the method are proved by comparative experiments.
1) Comparative example 1: comparison of sample preparation methods
In the prior art, the sample preparation method adopts extraction, then water washing, concentration and dilution, the sample preparation steps are complicated, and impurity loss or other impurities which are not in the product can exist in the water washing and concentration processes, the water washing and concentration steps are compared and investigated, the water washing and concentration are shown to be unnecessary operations, the water washing step in the sample preparation process is removed, the sample preparation is more convenient, the loss of the impurities and the introduction of other impurities in the water washing and filtering processes are avoided, the real impurity level in the sodium valproate oral solution can be reflected, and the specific comparison is as follows:
TABLE 11 comparative sample preparation
Taking sodium valproate oral solution, preparing test solution according to the above preparation method, detecting according to chromatographic condition 3 in example 1, and the results are shown in table 12:
TABLE 12 comparative sample preparation methods
Under the condition of converting into the same absolute sample amount, the sample preparation method has better extraction effect on all impurities, main components and preservatives than the prior art, and the peak areas of all components are higher than that of the prior art, so that the sample preparation method has the advantages of more sufficient extraction, more contribution to control of related substances of the sodium valproate oral solution, removal of a water washing step in the sample preparation process, more convenience and rapidness in sample preparation, avoidance of introduction of other impurities in the water washing and filtering processes, and better reflection of the real impurity level in the sodium valproate oral solution.
2) Comparative example 2: comparison of extraction solvents
Test solution: weighing 12.5ml of sodium valproate oral solution, placing in a separating funnel, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking, extracting with n-heptane and dichloromethane for 3 times, 20ml each time, mixing extractive solutions, placing in a 100ml measuring flask, diluting to scale with corresponding extraction solvent, and shaking.
Control solution: precisely measuring 1.0ml of the test solution, placing in a 100ml measuring flask, diluting to scale with corresponding extraction solvent, and shaking.
The test solutions and the control solutions were prepared from n-heptane and dichloromethane, respectively, and tested under the chromatographic conditions 3 in example 1, and the results are shown in Table 13:
TABLE 13 solvent comparison results
And (4) conclusion: when dichloromethane is used as an extraction solvent (figure 7), blank and auxiliary material impurity peaks are more, when n-heptane is used as the extraction solvent (figure 6), the response of extraction impurities and main components is higher, although the peak of the preservative is smaller than the peak area when dichloromethane is used as the extraction solvent, the preservative is used as an auxiliary material and is not controlled as related substances, and when n-heptane is used as the extraction solvent, the standing and layering time after extraction is shorter, the sample preparation time is greatly shortened, the effect of extraction impurities is higher, and the method is more suitable for popularization and use.
3) Comparative example 3: comparison of impurity detection Capacity
The impurities of the examples were added to the standard solution and detected according to BP2020 and chromatographic condition 3 of example 1, with the results shown in table 14:
TABLE 14 comparison of impurity detectability results
And (4) conclusion: the 3-propyl-2-hexanone and the unknown impurities are not completely separated under the British pharmacopoeia method (figure 8), the impurity C is overlapped with the impurity K, no separation trend exists, and the other impurities are well separated; the auxiliary material sodium hydroxy phenylpropionate does not peak in one collection time and remains to the next needle. Under the method (figure 9) of the invention, each component has complete peak separation, impurities and impurities, and impurities and main peaks are well separated, and the impurity separation and detection amount are obviously improved compared with the prior art, and related substances of the sodium valproate oral solution can be effectively controlled.
4) Comparative example 4: comparison of chromatographic conditions
Taking the impurity adding standard solution and the sample solution under the condition of the example, respectively detecting according to the patent CN113252803A and the chromatographic condition 3 of the example 1, wherein the chromatographic conditions are as shown in the following table 15:
TABLE 15 comparison of chromatographic conditions
The comparison results are shown in Table 16:
TABLE 16 detection results of CN113252803A under chromatographic conditions
Note: the separation degree is the separation degree between the current chromatographic peak and the next adjacent chromatographic peak.
And (4) conclusion: comparing and inspecting an impurity standard solution (the added components are 6 known impurities and 2 preservatives) (figures 9-10), effectively detecting the added components under the method, ensuring that the separation degree and the peak shape of each component are good (the symmetry factor is 0.8-1.1), and detecting 2 unknown impurities in a test solution; the added components are not completely detected under the method of the comparison document CN113252803A, wherein, the propyl hydroxybenzoate does not peak within one needle, and only 1 unknown impurity is detected in the sample solution when the propyl hydroxybenzoate remains to the next needle, and the impurity A and the main peak have poor peak shape (the impurity A peak symmetry factor is 2.5, and the main peak symmetry factor is 1.6). The method has the advantages that the peaks of the components are complete, the peak shape is better, the impurity detection of the sample is more complete, no residual peak exists, the method has obvious advantages compared with the chromatographic condition of the patent CN113252803A, and the related substances of the sodium valproate oral solution can be effectively controlled.
The above description is further intended to describe the present invention in detail with reference to specific embodiments, and it should not be construed that the specific embodiments of the present invention are limited to these descriptions. It will be apparent to those skilled in the art that various changes, substitutions and alterations can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for detecting related substances of sodium valproate oral solution comprises the following steps:
(1) preparing a solution: taking sodium valproate oral solution, adding acid solution for acidification, extracting with n-heptane, combining n-heptane layers, and directly adding n-heptane for dilution to prepare a test solution without washing with water;
(2) the temperature rise procedure during detection is as follows: the initial temperature is 55-65 ℃, the temperature is maintained for 4-6 minutes, the temperature is increased to 140-160 ℃ at the rate of 5-8 ℃ per minute, the temperature is increased to 180-200 ℃ at the rate of 2-4 ℃ per minute, the temperature is increased to 220-240 ℃ at the rate of 4-6 ℃ per minute, and the temperature is maintained for 20-40 minutes.
2. The assay of claim 1 wherein the temperature ramp is initiated at 60 ℃ for 5 minutes, ramped at 7 ℃ per minute to 150 ℃, ramped at 3 ℃ per minute to 190 ℃, ramped at 5 ℃ per minute to 230 ℃ for 30 minutes.
3. The detection method according to claim 1, wherein the temperature of the detector is 235-265 ℃ and the temperature of the sample inlet is 200-230 ℃; further, the detector temperature was 250 ℃ and the injection port temperature was 220 ℃.
4. The detection method according to claim 1, wherein the chromatographic column during detection is a capillary column using nitroterephthalic acid modified polyethylene glycol as a stationary liquid, the flow rate is 1-3 ml per minute, the sample injection volume is 1-10 μ l, and the split ratio is 1-10: 1; further, the flow rate was 1.5ml per minute, the injection volume was 1. mu.l, and the split ratio was 5: 1.
5. The detection method according to claim 1, wherein the acid used is sulfuric acid, and the concentration of the sulfuric acid is 1 to 3 mol/l.
6. The detection method according to claim 1, wherein the extraction is performed 3 to 5 times by using a vertical oscillator of a separating funnel, the shaking frequency is 220 to 280 times/min, and the single extraction time is 0.5 to 5 min; furthermore, the extraction times are 3 times, the shaking frequency is 260-280 times/min, and the single extraction time is 2-5 min.
7. The detection method according to claim 1, wherein the concentration of the prepared test solution is 1-10 mg/ml; further, the concentration of the sample solution was 5 mg/ml.
8. The assay of any one of claims 1 to 7, wherein the impurities in the oral solution of sodium valproate comprise: impurity A, impurity C, impurity K, 3-propyl-2-hexanone, 2-methyl propyl valerate and two unknown impurities, wherein impurity A is valeric acid; impurity C is (2RS) -2- (1-methylethyl) pentanoic acid; the impurity K is (2RS) -2-ethyl-2-methyl pentanoic acid.
9. The detection method according to any one of claims 1 to 8, wherein the temperature raising condition comprises: the initial temperature was 60 ℃ for 5 minutes, at a rate of 7 ℃ per minute to 150 ℃, at a rate of 3 ℃ per minute to 190 ℃, for 9 minutes, at a rate of 20 ℃ per minute to 230 ℃, for 30 minutes.
10. The detection method according to any one of claims 1 to 8, wherein the temperature raising condition comprises: the initial temperature was 60 ℃ for 5 minutes, at a rate of 7 ℃ per minute to 200 ℃ for 5 minutes, and at a rate of 20 ℃ per minute to 230 ℃ for 25 minutes.
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| CN113203824A (en) * | 2021-04-23 | 2021-08-03 | 海南倍特药业有限公司 | Detection method of sodium valproate related substances |
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