CN114354803B - Method for detecting related substances of sodium valproate oral solution - Google Patents
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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 52
- 229940084026 sodium valproate Drugs 0.000 title claims abstract description 44
- AEQFSUDEHCCHBT-UHFFFAOYSA-M sodium valproate Chemical compound [Na+].CCCC(C([O-])=O)CCC AEQFSUDEHCCHBT-UHFFFAOYSA-M 0.000 title claims abstract description 44
- 229940100688 oral solution Drugs 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims description 42
- 239000000126 substance Substances 0.000 title description 14
- 239000012535 impurity Substances 0.000 claims abstract description 122
- 238000000605 extraction Methods 0.000 claims abstract description 56
- 238000001514 detection method Methods 0.000 claims abstract description 49
- 239000007788 liquid Substances 0.000 claims abstract description 7
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 201
- 239000000243 solution Substances 0.000 claims description 68
- 239000000523 sample Substances 0.000 claims description 45
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000012488 sample solution Substances 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 17
- 239000003755 preservative agent Substances 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 10
- 230000002335 preservative effect Effects 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
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution 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
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 4
- -1 2-propyl methyl Chemical group 0.000 claims description 3
- HNBDRPTVWVGKBR-UHFFFAOYSA-N n-pentanoic acid methyl ester Natural products CCCCC(=O)OC HNBDRPTVWVGKBR-UHFFFAOYSA-N 0.000 claims description 3
- 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
- IXMINYBUNCWGER-UHFFFAOYSA-M sodium;4-propoxycarbonylphenolate Chemical compound [Na+].CCCOC(=O)C1=CC=C([O-])C=C1 IXMINYBUNCWGER-UHFFFAOYSA-M 0.000 claims description 2
- 229940005605 valeric acid Drugs 0.000 claims description 2
- 235000010268 sodium methyl p-hydroxybenzoate Nutrition 0.000 claims 1
- PESXGULMKCKJCC-UHFFFAOYSA-M sodium;4-methoxycarbonylphenolate Chemical group [Na+].COC(=O)C1=CC=C([O-])C=C1 PESXGULMKCKJCC-UHFFFAOYSA-M 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 19
- 238000000926 separation method Methods 0.000 abstract description 16
- 238000012216 screening Methods 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000004945 emulsification Methods 0.000 abstract description 4
- 206010015037 epilepsy Diseases 0.000 abstract description 4
- 230000009885 systemic effect Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 23
- 238000007865 diluting Methods 0.000 description 20
- 238000012360 testing method Methods 0.000 description 20
- 239000012085 test solution Substances 0.000 description 19
- 239000002904 solvent Substances 0.000 description 17
- 239000011550 stock solution Substances 0.000 description 17
- 239000000047 product Substances 0.000 description 16
- 239000000463 material Substances 0.000 description 14
- 230000015556 catabolic process Effects 0.000 description 13
- 238000006731 degradation reaction Methods 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 11
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 11
- 230000006378 damage Effects 0.000 description 10
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 8
- 238000005464 sample preparation method Methods 0.000 description 8
- 238000002474 experimental method Methods 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
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 229960003415 propylparaben Drugs 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 239000012490 blank solution Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 5
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 5
- 229960002216 methylparaben Drugs 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- QQZOPKMRPOGIEB-UHFFFAOYSA-N 2-Oxohexane Chemical compound CCCCC(C)=O QQZOPKMRPOGIEB-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 229960000604 valproic acid Drugs 0.000 description 4
- 239000003513 alkali Substances 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
- 239000012467 final product Substances 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000012044 organic layer Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 3
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 3
- 239000001961 anticonvulsive agent Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000012494 forced degradation Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- ADNADZOSMJDVIS-UHFFFAOYSA-N 2-methylpropyl pentanoate Chemical compound CCCCC(=O)OCC(C)C ADNADZOSMJDVIS-UHFFFAOYSA-N 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 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
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 229960003965 antiepileptics Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000004817 gas chromatography 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
- 229930014626 natural product Natural products 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000009659 non-destructive testing Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000008832 photodamage Effects 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
- 238000013112 stability test Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
Sodium valproate oral solution (Sodium Valproate Oral Solution) is a red clear viscous liquid for the treatment of systemic, partial or other types of epilepsy. At present, in the detection of sodium valproate oral solution, the extraction and emulsification phenomenon is seriously difficult to separate or takes too long to separate in the sample preparation process, the impurity detection is incomplete under the chromatographic condition in the prior art, and the separation degree is poor. The invention provides an analysis method with proper extraction time and complete detection of each component by scientifically screening and researching the preparation mode and chromatographic conditions of the sample.
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
The sodium valproate oral solution (Sodium Valproate Oral Solution) is red clear viscous liquid, is used for treating general, partial or other types of epilepsy, the sodium valproate as a main component is a nitrogen-free broad-spectrum anti-epileptic drug, is fast and complete to be orally absorbed, is effective on various types of epilepsy of people, such as various small-onset, myoclonus epilepsy, localized onset, large-onset and mixed-type epilepsy, and is a first-choice drug for primary large-onset and low-loss seizures.
At present, only Indian pharmacopoeia and British pharmacopoeia in the domestic and foreign pharmacopoeia receive the standard of sodium valproate oral solution, and domestic approved national drug standard (YBH 013302004-2014Z), wherein the preparation principle of related substance sample solution is acidification by adding acid, detection is carried out after extraction by a proper solvent (dichloromethane or n-heptane), and the detection process is found by referring to the technology reported in the prior literature: the extraction and emulsification phenomenon is seriously difficult to separate or the time for separating is too long in the sample preparation process, and the impurity detection is incomplete and the separation degree is poor under the chromatographic condition in the prior art. The invention provides an analysis method with proper extraction time and complete detection of each component by scientifically screening and researching the preparation mode and chromatographic conditions of the sample.
Disclosure of Invention
As an important broad-spectrum antiepileptic, the quality of the compound is directly affected on the health of patients and is widely applied to children, so that the quality control of the sodium valproate oral solution is particularly important. 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 and scientific in impurity detection and controllable, aiming at the defects in the prior art.
The invention is realized by adjusting the solution preparation method and the chromatographic conditions during detection, and specifically comprises the following steps:
firstly, when preparing a sample solution, water washing is not needed after n-heptane is used for extraction or the acidified sodium valproate oral solution is extracted;
secondly, the temperature rise program is adjusted during detection: the initial temperature is 55-65 ℃, the temperature is maintained for 4-6 minutes, the temperature is raised to 140-160 ℃ at the rate of 5-8 ℃ per minute, the temperature is raised to 180-200 ℃ at the rate of 2-4 ℃ per minute, the temperature is raised 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 formation.
The acid used in the sample preparation method of the invention 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 2.0mol/l.
In the technical scheme of the invention, the traditional manual extraction is replaced by the extraction mode of the separating funnel vertical oscillator during extraction, so that the extraction efficiency is improved, and the popularization and the use are facilitated.
When the separating funnel vertical oscillator is used for extracting, the shaking frequency is 100-350 times/min; further, the shaking frequency is 220-280 times/min; further, the shaking frequency is 260 to 280 times/min.
When the separating funnel vertical oscillator is used for extracting, the single extraction time is 0.5-5 min; further, the single extraction time is 2-5 min.
The concentration of the sample solution prepared by the method is 1-10 mg/ml; further, the concentration of the test solution was 5mg/ml.
The temperature of the detector is 235-265 ℃; further, the detector temperature was 250 ℃.
The temperature of the sample inlet is 200-230 ℃; further, the sample inlet temperature was 220 ℃.
In the technical scheme of the invention, the capillary column is a strong polar column, including but not limited to a capillary column using nitro terephthalic acid modified polyethylene glycol as a fixing liquid.
The flow rate in the detection method is 1-3 ml per minute; still further, the flow rate was 1.5ml per minute.
In the detection method, the sample injection volume is 1-10 mu l; further, the sample volume was 1. Mu.l.
In the detection method, the split ratio is 10-1:1; further, the split ratio was 5:1.
In the technical scheme of the invention, n-heptane is selected as an 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 comparative example 2 shows that the time required for standing and layering after one time of extraction is shortened by about 3 times, and the sample processing time is greatly shortened.
According to the invention, through optimizing the shaking frequency and the single extraction time, the full 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.
In the technical scheme of the invention, n-heptane is selected as a solvent, and after the n-heptane layers are combined, the n-heptane is directly diluted without washing. Compared with the existing sample preparation method (adopting water washing for re-concentration and dilution after extraction), the existing sample preparation method has more complicated steps, and impurities possibly lost or introduced into other non-natural products in the water washing and concentration process, the invention (comparative example 1) performs comparison investigation on the water washing and concentration steps, which 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 loss of impurities 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 more.
The preparation method of the control solution comprises the following steps: the sample solution was precisely measured and diluted with n-heptane to give a solution containing about 5. Mu.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 nitro terephthalic acid modified polyethylene glycol as a fixing 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, the temperature is raised to 190 ℃ at the rate of 3 ℃ per minute, the temperature is raised to 230 ℃ at the rate of 5 ℃ per minute, and the temperature is maintained for 30 minutes; the detector temperature was 250 ℃; the temperature of the sample inlet is 220 ℃; the flow rate is 1.5ml per minute; sample volume 1 μl; the split ratio was 5:1.
In one embodiment of the present invention, the chromatographic conditions (chromatographic condition 2) are: a capillary column using nitro terephthalic acid modified polyethylene glycol as a fixing 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 detector temperature was 250 ℃; the temperature of the sample inlet is 220 ℃; the flow rate is 2.0ml per minute; sample volume 1 μl; the split ratio was 5:1.
In another specific embodiment of the present invention, the chromatographic conditions (chromatographic condition 1) are: a capillary column using nitro terephthalic acid modified polyethylene glycol as a fixing liquid; the initial temperature is 60 ℃, maintained for 5 minutes, heated to 150 ℃ at a rate of 7 ℃ per minute, heated to 190 ℃ at a rate of 3 ℃ per minute, maintained for 9 minutes, heated to 230 ℃ at a rate of 20 ℃ per minute, and maintained for 30 minutes; the flow rate is 1.5ml per minute; sample volume 1 μl; the split ratio was 5:1.
In the technical scheme of the invention, impurities in the sodium valproate oral solution comprise: impurity A, impurity C, impurity K, 3-propyl-2-hexanone, 2-propyl methyl valerate and two unknown impurities, wherein impurity A is valeric acid; impurity C is (2 RS) -2- (1-methylethyl) pentanoic acid; impurity K is (2 RS) -2-ethyl-2-methyl pentanoic acid.
The chromatographic conditions 1-3 can separate and detect the 8 impurities, and ensure that the preservative methylparaben and propylparaben in the sodium valproate oral solution have complete peaks in one needle.
The invention has the beneficial effects that:
1. the invention takes the sodium valproate oral solution as a detection object, takes n-heptane as an extraction solvent, and compared with the prior art (taking methylene dichloride as the extraction solvent), the sodium valproate oral solution is easier to delaminate during extraction, the delamination time is shortened by about 3 times, and the separation time is shorter.
2. In the invention, the traditional manual extraction is replaced by the extraction mode of the separating funnel vertical oscillator, the extraction conditions of single extraction time, shaking frequency and the like are optimized, related substances in the sodium valproate oral solution are extracted as much as possible, the extraction efficiency is improved, and the popularization and the use are facilitated.
3. In the degradation specificity experiment of the embodiment 4, 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%, 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 products do not interfere with detection of each known impurity; compared with the nondestructive testing, the material balance of each degradation test sample is between 90.0% and 108.0%, and meets the requirements.
4. The detection method provided by the invention has good linearity: the impurity K has good linear relation between peak area and concentration in the range of 0.9774-14.6610 mug/ml, the linear equation is y=0.0455x-0.0012, R 2 =0.9989, the correlation coefficient r is 0.9994; the peak area and the concentration of the sodium valproate are in good linear relation within the range of 1.0157-101.5700 mug/ml of the measured concentration, and the linear equation is y=0.0401x+0.0108, R 2 =0.9993, and the correlation coefficient r is 0.9996.
5. The detection method has the advantages of good accuracy: in the accuracy experiment of example 4, the recovery rate of impurity K was 106.0% on average, RSD was 0.8% <2.0%, and the relevant detection criteria were met.
6. The detection method has the advantages of good weight: in 6 repeated detection experiments, the content of unknown impurities in 6 samples is 0.03%, and the extreme difference is 0.
7. In the method, the stability of the solution is good, the RSD of the peak area of valproic acid in the control solution is less than 2.0%, the impurity content of the test solution is 0.03%, the extremely poor is 0, which indicates that the test solution and the control solution are stable when being placed for 50 hours at room temperature, and the solution prepared according to the technical scheme of the invention has good stability, so that the test result is more accurate and reliable.
In summary, the technology of the invention is based on gas chromatography, on the basis of the technology disclosed in the prior document, the defects of long sample processing time, serious emulsification phenomenon, incomplete impurity separation, incomplete peak inclusion and peak emission and the like in the prior art are overcome, an automatic extraction device is combined, a novel heating program is adopted, impurities can be completely separated, the peak emission of each component is complete, the sample processing technology is scientifically screened and researched, the sample processing time is greatly shortened, the problems of the prior art are solved, and the method provided by the invention is researched and verified by methodologies such as specificity, accuracy and the like, and the results are all in an acceptable range, so that the method can more effectively control related substances of 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 diagram of a shaking frequency screen.
FIG. 6 is a graph of an extraction solvent assay using n-heptane.
FIG. 7 is a graph of the detection of extraction solvent with methylene chloride.
FIG. 8 is a BP2020 method detection spectrum.
FIG. 9 is a graph of the detection spectrum of the method of the present invention.
FIG. 10 is a spectrum of the detection under the chromatographic conditions of patent CN 113252803A.
Fig. 1 to 3 show the following signs: (1) 2-hexanone, (2)2-methyl propyl valerate, (3)3-propyl-2-hexanone, (4) unknown 1, (5) impurity A, (6) unknown 2, (7) impurity C, (8) impurity K, (9) main component, methyl hydroxy benzoate, and (4) impurity A,Propyl hydroxybenzoate.
Detailed Description
The technical scheme of the present invention is further defined below in conjunction with the specific embodiments, but the scope of the claimed invention is not limited to the description.
Aiming at the sample preparation mode and the detection method, the inventor performs a great deal of experiments, and performs scientific screening and verification on the extraction mode and the chromatographic conditions, and the method specifically comprises the following steps:
1. examples
1) Example 1: screening of chromatographic conditions
Table 1 comparison with prior art methods
Comparing and inspecting the prior art method by adopting an impurity adding standard solution, wherein the national drug standard (YBH 013302004-2014Z) is basically consistent with the IP2010 method, adding 6 known impurities, actually detecting 2 impurities, wherein more impurity peaks are not separated, and no preservative is shown; the BP2020 method actually detects 5, the known impurity a and the unknown impurity are not separated, the preservative peak does not appear in one needle time, and the preservative is remained in the next needle, and the CN 11323824A method is similar to the BP2020 method, and the preservative cannot be detected in one needle time.
Further optimized chromatographic conditions:
table 2 optimizing chromatographic conditions
TABLE 3 optimization results
Note that: the 8 marked impurities are 6 known impurities and 2 auxiliary materials.
The figures 1, 2 and 3 of the specification show that under three chromatographic conditions, the separation degree of the main component and the impurity and the separation degree of the impurity and the impurity all meet the requirements, and the preservative can normally show peaks and can be used for detecting related substances of the sodium valproate oral solution.
2) Example 2: investigation of the Single extraction time
Test solution: measuring 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, 20ml each time, shaking for 0.5, 1, 2, 3, 4 and 5 minutes each time by using a separating funnel vertical oscillator, mixing the extracting solutions (n-heptane layers), placing in a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly to obtain the product.
Test solutions prepared at different extraction times were each tested under chromatographic condition 3 in example 1.
Table 4 single extraction time screening
Conclusion: in the single extraction time range of 0.5-5 minutes (figure 4), the peak area of each component increases with the increase of the extraction time, and when the extraction time is in 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 shaking 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 uniformly, extracting with n-heptane for 3 times, 20ml each time, mixing the extractive solutions (n-heptane layers), placing in a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly to obtain the final product. The shaking parameters of the vertical oscillators were respectively set forth in the following table parameters 1, 2, 3, 4, and 5.
Table 5 shaking parameter screening
Test solutions prepared at different shaking frequencies were taken and tested under chromatographic condition 2 in example 1, respectively.
TABLE 6 shaking parameter screening results
Conclusion: in the shaking frequency range of 220-280 times/min (figure 5), the peak area of each component in the sample solution increases with the increase of frequency, which shows that the extraction effect increases with the increase of the shaking frequency, when the shaking frequency is in the range of 260-280 times/min, the peak area of each component reaches the maximum without obvious difference, the extraction effect is optimal, and the shaking frequency is determined to be 260-280 times/min.
4) Example 4: verification of analytical methods
4.1 Solution preparation:
blank solution: n-heptane.
Test solution: taking 12.5ml of the sample, 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, 20ml each time, mixing n-heptane extract, placing in a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly to obtain the final product.
Control solution: precisely measuring 1.0ml of the sample solution, placing in a 100ml measuring flask, diluting to the scale with n-heptane, shaking uniformly, precisely measuring 1.0ml, placing in a 10ml measuring flask, diluting to the scale with n-heptane, and shaking uniformly.
Blank auxiliary material solution: weighing 12.5ml of blank auxiliary materials, and preparing according to a test sample solution method.
Each impurity stock solution: and respectively taking a proper amount of each of the impurity A, the impurity C, the 3-propyl-2-hexanone, the impurity K, the 2-hexanone and the 2-propyl methyl valerate reference substances, and respectively adding n-heptane to dilute each 1ml of the reference substances to prepare a solution containing about 0.5 mg.
Sodium valproate stock solution: taking a proper amount of sodium valproate reference substance, placing into a centrifuge tube, adding a proper amount of water to dissolve, adding dilute sulfuric acid to acidify, shaking uniformly, extracting with n-heptane for 3 times, combining n-heptane layers, and adding n-heptane to dilute to prepare a solution containing about 5mg of n-heptane per 1 ml.
Each impurity localization solution: precisely measuring 1.0ml of each impurity stock solution, respectively placing into 50ml measuring flask, diluting to scale with n-heptane, and shaking.
Methylparaben positioning solution: a proper amount of methylparaben reference substance is taken, and n-heptane is added for dilution to prepare a solution containing about 80 mug of the methylparaben reference substance in each 1 ml.
Propyl paraben positioning solution: the propyl hydroxy benzoate reference substance is taken in proper amount, and the mixture is diluted by adding n-heptane to prepare a solution with about 30 mug in each 1 ml.
Impurity adding and marking solution: weighing 12.5ml of the product, 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, respectively adding 2ml of each impurity stock solution, 2ml of each positioning solution of methylparaben and propylparaben, diluting to scale with n-heptane, and shaking.
4.2 System applicability and impurity positioning test
Taking a blank solution, a blank auxiliary material solution, a positioning solution of each component, an impurity adding standard solution and a sample solution to be tested, and respectively carrying out sample injection analysis under the chromatographic condition 3 in the embodiment 1.
Results: the blank solution and the blank auxiliary material solution have no interference at the peak positions of all components, and the unknown impurity peaks in the sample solution do not interfere with the detection of all known impurities. The peak time of each component in the impurity adding standard 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
Alkaline destruction of the test solution: precisely measuring 12.5ml of the product, placing the product into a 20ml headspace bottle, adding 1.0ml of 2mol/L sodium hydroxide solution, capping, sealing, shaking, destroying for 24 hours at 40 ℃, neutralizing with an equal amount of 2mol/L hydrochloric acid solution, transferring to a separating funnel, rinsing the headspace bottle with 8ml of water for 3 times, transferring to the separating funnel completely, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, each time for 20ml, combining n-heptane extract, placing the product into a 100ml measuring flask, diluting with n-heptane to scale, shaking uniformly.
Acid damages the test solution: precisely measuring 12.5ml of the product, placing the product into a 20ml headspace bottle, adding 1.0ml of 2mol/L hydrochloric acid solution, capping, sealing, shaking, destroying for 24 hours at 40 ℃, neutralizing with an equal amount of 2mol/L sodium hydroxide solution, transferring to a separating funnel, rinsing the headspace bottle with 8ml of water for 3 times, transferring to the separating funnel completely, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extract, placing the product into a 100ml measuring flask, diluting with n-heptane to scale, shaking uniformly.
Oxidative destruction of the test solution: precisely measuring 12.5ml of the product, placing into a 20ml headspace bottle, adding 1.0ml of 30% hydrogen peroxide solution, capping, sealing, shaking, breaking for 24 hours at room temperature, rinsing the headspace bottle with 9ml of water for 3 times, transferring all 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 n-heptane extract, placing into a 100ml measuring bottle, diluting to scale with n-heptane, shaking uniformly.
High temperature damage to the test solution: precisely measuring the proper amount of the product, placing the product into a 20ml headspace bottle, sealing by a cover, breaking the headspace bottle in an oven at 105 ℃ for 24 hours, cooling to room temperature, measuring 12.5ml of the sample, rinsing the headspace bottle with 10ml for 3 times, transferring all the samples into a separating funnel, adding 10ml of sulfuric acid solution with the concentration of 2mol/L, shaking the mixture uniformly, extracting the mixture with n-heptane for 3 times, extracting 20ml each time, combining n-heptane extract, placing the mixture into a 100ml measuring bottle, diluting the mixture with n-heptane to a scale, shaking the mixture uniformly.
Light damages the test solution: precisely measuring the proper amount of the product, placing into a 20ml headspace bottle, sealing with a cover, breaking for 7d in a 4500 Lx+/-500 Lx illumination box, measuring 12.5ml of the sample, rinsing the headspace bottle with 10ml for 3 times, transferring all the sample into a separating funnel, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extract, placing into a 100ml measuring flask, diluting with n-heptane to scale, shaking uniformly.
Blank auxiliary material degradation solution: and (3) replacing the test sample with blank auxiliary materials, and respectively preparing the test sample solution by means of acid, alkali, oxidation, high temperature and illumination.
The blank solution, the test sample solution and the destruction solutions of item 4.1) were taken and tested according to chromatographic condition 3 in example 1.
Results: under each degradation condition, the blank auxiliary material solution has no interference to the detection of a 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%, 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 products do not interfere with the detection of each known impurity; the material balance of each degradation test sample is between 90.0% and 108.0% compared with the material balance of the degradation test sample without damage, and the material balance meets the requirements, and is shown in Table 7 in detail.
TABLE 7 degradation specific test results
4.4 Detection limit and quantitative limit test
Quantitative limiting solution: and precisely measuring sodium valproate and a proper amount of each impurity stock solution under the condition of 4.1 items, and gradually diluting until the signal to noise ratio (S/N) is about 10.
Detection limit solution: and precisely measuring sodium valproate and a proper amount of each impurity stock solution under the condition of 4.1 items, 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 detection limit and quantitative limit test results
4.5 Linear test)
Linear mixing stock solution: precisely weighing 1.5ml of impurity K stock solution and 1.0ml of sodium valproate stock solution under the condition of 4.1), placing into a same 10ml measuring flask, adding solvent to dilute to scale, and shaking uniformly.
Taking 4.4) quantitative limit solution of impurity K and sodium valproate under the detection limit and quantitative limit test items as a linear solution 1.
Precisely measuring 0.2ml, 0.3ml, 0.5ml, 0.6ml and 1.0ml of the linear mixed stock solution respectively, placing into a 5ml measuring flask, fixing the volume to the scale by using n-heptane, and shaking uniformly to obtain linear solutions 2, 3, 4, 5 and 6.
And (3) respectively injecting the solutions, recording peak areas, and obtaining a linear regression equation by taking the peak areas as ordinate and the concentrations as abscissa.
Under the condition of the method, the impurity K has good linear relation between peak area and concentration within the range of 0.9774-14.6610 mug/ml, the linear equation is y=0.0455x-0.0012, R 2 =0.9989, the correlation coefficient r is 0.9994; the peak area and the concentration of the sodium valproate are in good linear relation within the range of 1.0157-101.5700 mug/ml of the measured concentration, and the linear equation is y=0.0401x+0.0108, R 2 =0.9993, the correlation coefficient r is 0.9996, see table 9 for details.
TABLE 9 Linear test results
4.6 Accuracy test)
Impurity K accuracy stock solution: a proper amount of impurity K is taken, and n-heptane is added for dilution to prepare a solution containing about 0.75mg of the impurity K in each 1 ml.
Impurity K control solution: precisely measuring 1.0ml of impurity K accurate stock solution, placing into a 100ml measuring flask, adding n-heptane to dilute to scale, and shaking to obtain the final product.
50% recovery solution: precisely measuring 12.5ml of a sample, placing in a separating funnel, precisely measuring 0.5ml of an impurity K accuracy stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting 3 times with n-heptane for 20ml each time, combining organic layers, placing in a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly.
100% recovery solution: precisely measuring 12.5ml of the product, placing into a separating funnel, precisely measuring 1.0ml of impurity K accuracy stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting 3 times with n-heptane for 20ml each time, combining organic layers, placing into a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly.
150% recovery solution: precisely measuring 12.5ml of the product, placing into a separating funnel, precisely measuring 1.5ml of an impurity K accuracy stock solution, adding 10ml of water, shaking, adding 10ml of 2mol/L sulfuric acid solution, shaking uniformly, extracting 3 times with n-heptane for 20ml each time, combining organic layers, placing into a 100ml measuring flask, diluting to scale with n-heptane, and shaking uniformly.
And 4.1) feeding the blank solution, the sample solution, the impurity K reference solution and the recovery rate solution under the condition of the sample solution, respectively, and calculating the recovery rate according to an external standard method, wherein the results are shown in Table 10.
TABLE 10 recovery test results
4.7 Precision test of instrument
Instrument precision solution: about 15mg of impurity K reference substance is taken, precisely weighed, placed in a 100ml measuring flask, diluted to scale by adding n-heptane, and shaken uniformly to serve as impurity K stock solution. Taking 50mg of sodium valproate reference substance, precisely weighing, placing into a centrifuge tube, adding 1ml of water, dissolving, adding 0.5ml of dilute sulfuric acid, shaking uniformly, extracting with n-heptane three times, 2ml each time, combining n-heptane layers, placing into a 10ml measuring flask, adding 0.5ml of impurity K stock solution, diluting to scale with n-heptane, and shaking uniformly to obtain the product.
The chromatographic conditions 3 in example 1 were followed by 6 successive injections, the chromatograms were recorded, and the RSD of impurity K with valproic acid peak retention time and peak area was calculated.
Results: the RSD of the retention time of the impurity K peak is 0.02 percent and less than 1.0 percent, and the RSD of the peak area is 1.5 percent and less than 2.0 percent; the RSD of the retention time of the valproic acid peak is 0.02 percent and less than 1.0 percent, the RSD of the peak area is 1.4 percent and less than 2.0 percent, and the instrument precision is good.
4.8 Test for repeatability
6 parts of test solution and control solution are prepared in parallel under the condition of 4.1), sample injection detection is carried out according to chromatographic condition 3 in the embodiment 1, a chromatogram is recorded, and the impurity content is calculated.
Results: the content of unknown impurities in 6 samples is 0.03%, the extremely bad is 0, and the method repeatability is good.
4.9 Solution stability test
Taking the sample solution to be tested under the condition of 4.1) and the control solution, respectively standing for 0h, 6h, 13h, 26h, 38h and 50h at room temperature, performing sample injection detection according to chromatographic condition 3 in example 1, recording a chromatogram, and calculating the impurity content and the RSD of the peak area of the control solution.
Results: the RSD of the valproic acid peak area 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 extremely poor is 0, which indicates that the test solution and the control solution are stable at room temperature for 50 hours.
2. Comparative example
In order to show that the method can more effectively control related substances of the sodium valproate oral solution, the beneficial effects of the invention are proved by the following comparative experiments.
1) Comparative example 1: comparison of sample preparation modes
The sample preparation method in the prior art adopts water washing and re-concentration and dilution after extraction, has complicated sample preparation steps, and can cause impurity loss or introduce other impurities in non-products in the water washing and concentration processes, the invention carries out contrast investigation on the water washing and concentration steps, which shows that the water washing and concentration are unnecessary operations, the water washing steps in the sample preparation process are removed, the sample preparation is more convenient, the loss of impurities and the introduction of other impurities in the water washing and filtration processes are avoided, and the real impurity level in the sodium valproate oral solution can be reflected more, and the specific contrast is as follows:
table 11 sample preparation method comparative experiment
Sample solutions were prepared in the above-described sample preparation methods, and the test solutions were tested according to chromatographic condition 3 in example 1, and the results are shown in table 12:
table 12 sample preparation mode comparison experiment results
Under the condition of being converted into the same absolute sample injection amount, the sample preparation method has better extraction effect on each impurity, main component and preservative than the prior art, and the peak area of each component is higher than that of the prior art, which shows that the sample is extracted more fully, is more beneficial to controlling related substances of the sodium valproate oral solution, removes the washing step in the sample preparation process, is more convenient to prepare, avoids the introduction of other impurities in the washing and filtering processes, and can reflect 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 uniformly, respectively extracting with n-heptane and dichloromethane for 3 times, 20ml each time, mixing the extractive solutions, placing in a 100ml measuring flask, diluting to scale with corresponding extraction solvent, and shaking uniformly.
Control solution: precisely measuring 1.0ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with corresponding extraction solvent, and shaking.
Test solutions and control solutions were prepared from n-heptane and dichloromethane, respectively, and tested according to chromatographic condition 3 in example 1, and the results are shown in table 13:
TABLE 13 solvent comparative results
Conclusion: when dichloromethane is used as the extraction solvent (figure 7), the blank and auxiliary materials have more miscellaneous peaks, when n-heptane is used as the extraction solvent (figure 6), the response of the extracted impurities and main components is higher, although the preservative peak is smaller than the peak area when dichloromethane is used as the extraction solvent, the preservative is used as the auxiliary material and is not used as related substances for control, and when n-heptane is used as the extraction solvent, the standing layering time after extraction is shorter, the sample preparation time is greatly shortened, the effect of the extracted impurities is higher, and the popularization and the use are more utilized.
3) Comparative example 3: comparison of impurity detection capability
The impurity addition standard solution under the example was taken and tested according to BP2020 and chromatographic condition 3 of example 1, and the results are shown in Table 14:
TABLE 14 comparison of impurity detection Capacity and results
Conclusion: 3-propyl-2-hexanone is not completely separated from unknown impurities (figure 8) under the British pharmacopoeia method, impurity C is overlapped with impurity K, no separation trend exists, and other impurities are well separated; the auxiliary material sodium propylparaben does not show a peak in one collecting time and remains until the next needle. The method (figure 9) has the advantages that the peak of each component is completely separated, the separation between impurities and the main peak between the impurities is good, the separation and detection quantity of the impurities are obviously improved compared with the prior art method, and the related substances of the sodium valproate oral solution can be more effectively controlled.
4) Comparative example 4: comparison of chromatographic conditions
The impurity addition standard solution and the sample solution in the example were taken and detected according to the patent CN113252803a and the chromatographic condition 3 in example 1, respectively, and the chromatographic conditions were as shown in table 15:
TABLE 15 comparison of chromatographic conditions
The comparison results are shown in Table 16:
TABLE 16 detection results under the chromatographic conditions of CN113252803A patent
Note that: the above-mentioned degree of separation is the degree of separation between the current chromatographic peak and the subsequent adjacent chromatographic peak.
Conclusion: comparing and inspecting (drawing figures 9-10) by adopting impurity adding standard solution (the adding components are 6+2 preservative agents of known impurities), effectively detecting each adding component in the method, ensuring good separation degree and peak shape (symmetry factor 0.8-1.1) among each component, and detecting 2 unknown impurities in the sample solution; the components added in the method of comparative document CN113252803a are not completely detected, wherein the propyl hydroxybenzoate does not have a peak in one needle time, remains until the next needle, only 1 unknown impurity is detected in the sample solution, and the impurity a and the main peak have a peak shape difference (impurity a peak symmetry factor 2.5, main peak symmetry factor 1.6). The method has the advantages that the peak of each component is complete, the peak shape is better, the detection of sample impurities is more complete, no residual peak exists, and compared with the chromatographic condition of patent CN113252803A, the method has obvious advantages, and can more effectively control related substances of sodium valproate oral solution.
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to those specific embodiments. It will be apparent to those skilled in the art that several deductions or substitutions may be made without departing from the spirit of the invention, and these shall be considered to be within the scope of the invention.
Claims (12)
1. The method for detecting impurities and preservatives of the sodium valproate oral solution comprises the following steps:
(1) Preparing a solution: taking sodium valproate oral solution, adding acid liquor for acidification, extracting with n-heptane, combining n-heptane layers, directly adding n-heptane for dilution without water washing to prepare a sample solution;
(2) The temperature rise program at the time of detection is: the temperature of the detector is 235-265 ℃, the temperature of the sample inlet is 200-230 ℃, the initial temperature is 55-65 ℃, the temperature is maintained for 4-6 minutes, the temperature is raised to 140-160 ℃ at the rate of 5-8 ℃ per minute, the temperature is raised to 180-200 ℃ at the rate of 2-4 ℃ per minute, the temperature is raised to 220-240 ℃ at the rate of 4-6 ℃ per minute, and the temperature is maintained for 20-40 minutes;
the chromatographic column in detection is a capillary column taking nitro terephthalic acid modified polyethylene glycol as a fixing liquid;
the impurities in the sodium valproate oral solution are as follows: impurity A, impurity C, impurity K, 3-propyl-2-hexanone, 2-propyl methyl valerate and two unknown impurities, wherein impurity A is valeric acid; impurity C is (2 RS) -2- (1-methylethyl) pentanoic acid; impurity K is (2 RS) -2-ethyl-2-methylpentanoic acid; the preservative is sodium methylparaben and sodium propylparaben.
2. The method of claim 1, wherein the temperature programming is initiated at 60 ℃ for 5 minutes, at a rate of 7 ℃ per minute to 150 ℃, at a rate of 3 ℃ per minute to 190 ℃, and at a rate of 5 ℃ per minute to 230 ℃ for 30 minutes.
3. The method of claim 1, wherein the detector temperature is 250 ℃ and the sample inlet temperature is 220 ℃.
4. The method according to claim 1, wherein the flow rate is 1-3 ml/min, the sample volume is 1-10 μl, and the split ratio is 1-10:1.
5. The method of claim 1, wherein the flow rate is 1.5ml per minute, the sample volume is 1 μl, and the split ratio is 5:1.
6. The method according to claim 1, wherein the acid is sulfuric acid, and the concentration of sulfuric acid is 1 to 3mol/l.
7. The detection method according to claim 1, wherein the extraction is performed 3 to 5 times by using a separating funnel vertical oscillator, the shaking frequency is 220 to 280 times/min, and the single extraction time is 0.5 to 5min.
8. The method according to claim 7, wherein the number of extractions is 3, the shaking frequency is 260 to 280 times/min, and the single extraction time is 2 to 5min.
9. The method according to claim 1, wherein the concentration of the prepared sample solution is 1-10 mg/ml.
10. The method according to claim 9, wherein the concentration of the sample solution is 5mg/ml.
11. The detection method according to claim 1, wherein the temperature-increasing condition or includes: the initial temperature was 60℃for 5 minutes, at a rate of 7℃per minute to 150℃and then at a rate of 3℃per minute to 190℃for 9 minutes, and then at a rate of 20℃per minute to 230℃for 30 minutes.
12. The detection method according to claim 1, wherein the temperature-increasing condition or includes: the initial temperature was 60℃for 5 minutes, raised to 200℃at a rate of 7℃per minute for 5 minutes, and raised to 230℃at a rate of 20℃per minute for 25 minutes.
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