CN115015457B - Method for analyzing bromoacetic acid impurity in selapage by high performance liquid chromatography - Google Patents
Method for analyzing bromoacetic acid impurity in selapage by high performance liquid chromatography Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 35
- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 239000012535 impurity Substances 0.000 title claims abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 72
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000012064 sodium phosphate buffer Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 31
- 239000013558 reference substance Substances 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 239000012085 test solution Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- DFIWJEVKLWMZBI-UHFFFAOYSA-M sodium;dihydrogen phosphate;phosphoric acid Chemical compound [Na+].OP(O)(O)=O.OP(O)([O-])=O DFIWJEVKLWMZBI-UHFFFAOYSA-M 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 231100000024 genotoxic Toxicity 0.000 abstract description 2
- 230000001738 genotoxic effect Effects 0.000 abstract description 2
- 239000000377 silicon dioxide Substances 0.000 abstract description 2
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 239000000945 filler Substances 0.000 abstract 1
- 238000004007 reversed phase HPLC Methods 0.000 abstract 1
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 27
- 239000000523 sample Substances 0.000 description 14
- 238000011084 recovery Methods 0.000 description 11
- 238000007865 diluting Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VSTXCZGEEVFJES-UHFFFAOYSA-N 1-cycloundecyl-1,5-diazacycloundec-5-ene Chemical compound C1CCCCCC(CCCC1)N1CCCCCC=NCCC1 VSTXCZGEEVFJES-UHFFFAOYSA-N 0.000 description 2
- -1 5, 6-diphenylpyrazin-2-yl Chemical group 0.000 description 2
- VUGNCPVAXWZTOL-UHFFFAOYSA-N 5-chloro-2,3-diphenylpyrazine Chemical compound C=1C=CC=CC=1C1=NC(Cl)=CN=C1C1=CC=CC=C1 VUGNCPVAXWZTOL-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000009079 Epoprostenol Receptors Human genes 0.000 description 1
- 108010073099 Epoprostenol Receptors Proteins 0.000 description 1
- 206010050661 Platelet aggregation inhibition Diseases 0.000 description 1
- 206010064911 Pulmonary arterial hypertension Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 125000004106 butoxy group Chemical group [*]OC([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- QXWZQTURMXZVHJ-UHFFFAOYSA-N selexipag Chemical compound C=1C=CC=CC=1C1=NC(N(CCCCOCC(=O)NS(C)(=O)=O)C(C)C)=CN=C1C1=CC=CC=C1 QXWZQTURMXZVHJ-UHFFFAOYSA-N 0.000 description 1
- 229960003841 selexipag Drugs 0.000 description 1
- 230000024883 vasodilation Effects 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
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Abstract
The invention provides a method for analyzing bromoacetic acid in selapage by high performance liquid chromatography, which adopts a reversed-phase high performance liquid chromatography column, adopts octadecylsilane chemically bonded silica as a chromatography column filler, adopts phosphoric acid-sodium phosphate buffer with pH value of 2.1-2.3 as a mobile phase A, adopts acetonitrile as a mobile phase B, and adopts gradient elution. The method can detect bromoacetic acid by an ultraviolet detector, can conveniently monitor the content of the genotoxic impurity in the selapage by using a common instrument, and has better specificity, stability and accuracy.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a method for evaluating medicine quality by using high performance liquid chromatography.
Background
Selapag, english name Selexipag, chemical name 2- {4- [ (5, 6-diphenylpyrazin-2-yl) (propane-2-yl) amino ] butoxy } -N- (methylsulfonyl) acetamide, is an excellent PGI2 receptor agonism, has various effects such as platelet aggregation inhibition, vasodilation, bronchomyoexpansion, lipid precipitation inhibition, leukocyte activation inhibition, etc., has excellent effect in treating pulmonary arterial hypertension, and has been marketed in china in 2018 with large demand.
The prior national standard HJ758-2015 adopts a gas chromatography to measure bromoacetic acid in water quality, the method firstly uses sulfuric acid acidified methanol solution to derive bromoacetic acid, and then carries out gas chromatography analysis, and the derivation step is time-consuming and has poor convenience.
CN 108828081A is based on liquid chromatography mass spectrometry, separation is performed by anion exchange chromatography, and bromoacetic acid is detected by multi-reaction monitoring (MRM) in negative ion mode. The method has high requirements on instruments, high detection cost and troublesome operation process.
Disclosure of Invention
The invention provides a high performance liquid chromatography analysis method for monitoring the content of bromoacetic acid which is a genotoxic impurity in selapage, and the method uses a common ultraviolet detector for detection, is simple, accurate, efficient and easy to operate, and is not interfered by other various organic impurities and residual solvent components in the selapage.
According to the invention, the following detection conditions are used:
chromatographic column: adopting octadecylsilane chemically bonded silica gel column;
mobile phase: taking phosphate-sodium phosphate buffer solution (pH value is 2.1-2.3) as a mobile phase A, and acetonitrile as a mobile phase B; gradient elution was used, the gradient elution procedure was as follows:
TABLE 1
In a preferred embodiment, the preparation of the test solution is: 200mg of selapage is taken, placed in a 10ml measuring flask, added with 2ml of acetonitrile, heated in a water bath at a temperature of not more than 65 ℃ and preferably 60 ℃ until the acetonitrile is dissolved, then added with mobile phase A, shaken while being diluted to a scale, and then placed still after shaking uniformly, centrifuged, the supernatant is taken out, and the supernatant is taken out after centrifugation again.
In a preferred embodiment, the preparation of the control solution comprises: taking a proper amount of bromoacetic acid reference substance, precisely weighing, adding a solvent to dissolve and dilute the bromoacetic acid reference substance to prepare a solution containing 9-10 mug per lml, wherein the solvent is a mixed solution of acetonitrile and mobile phase A according to a volume ratio of 20:80.
In a preferred embodiment, the detection is carried out using an ultraviolet detector, the detection wavelength being 190-250nm, preferably 205nm. The flow rate is in the range of 0.9 to 1.1ml/min, preferably 1.0ml/min.
In the present invention, the column temperature of the chromatographic column is 38 to 42 ℃, preferably 40 ℃.
The detector used in the present invention may employ an ultraviolet detector having a detection wavelength of 190-400nm.
Drawings
FIG. 1 is a superposition of specificity tests under the conditions of the present invention.
Detailed Description
The technical scheme of the invention is described in detail below with reference to specific embodiments. Table 2 lists details of the materials used in the examples.
Table 2 control and sample information for experiments
Preparation of test and control solutions
The inventor compares the preparation modes of different samples, and shows that the recovery rate of the method 3 is good from the result, namely the test sample is heated to be dissolved by adopting a water bath at 60 ℃, then the mobile phase A is added, the test sample is shaken while being diluted to the scale, the test sample is kept stand for 2 hours after being shaken uniformly, the solution is transferred into a centrifuge tube, the centrifugation is carried out for 10 minutes at 5000 revolutions per minute, the supernatant is taken, and the test sample solution is obtained after the supernatant is centrifuged again by adopting the same method.
Table 3: comparison of recovery rates of different sample preparation modes
Unless otherwise indicated, the methods for preparing test solutions used hereinafter to demonstrate the effect of the assay were all obtained using the methods described herein.
Test solution: 200mg of the product is precisely weighed, placed in a 10ml measuring flask, added with 2ml of acetonitrile, heated in a water bath at 60 ℃ until the acetonitrile is dissolved, then added with mobile phase A, shaken while being diluted to a scale, kept stand for 2 hours after shaking uniformly, transferred into a centrifuge tube, centrifuged for 10 minutes at 5000 revolutions per minute, and the supernatant is centrifuged again in the same way and is obtained.
Control solution: taking a proper amount of bromoacetic acid reference substance, precisely weighing, adding a solvent for dissolving and diluting to prepare a solution containing about 9.4 mug of bromoacetic acid in each lml.
Chromatographic conditions
In an exemplary embodiment of the invention, the chromatographic conditions are:
chromatographic column: octadecylsilane chemically bonded silica column Kromasil100-5C18,4.6 ×250mm,5 μm;
mobile phase a:0.0025mol/L sodium phosphate buffer (pH adjusted to 2.2 with phosphoric acid) as mobile phase A;
mobile phase B: acetonitrile;
flow rate: 1.0ml/min;
wavelength: 205nm;
column temperature: 40 ℃;
sample injection amount: 80 μl, and
gradient procedure: TABLE 1
Specificity test
SLPZ31 positioning solution: 1.175mg of SLPZ31 reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by acetonitrile, precisely removed from the measuring flask after shaking, placed in a 10ml measuring flask by 0.8ml, diluted to a scale by solvent, and shaking to obtain the product.
SLPZ32 positioning solution: 1.175mg of SLPZ32 reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by acetonitrile, precisely removed from the measuring flask after shaking, placed in a 10ml measuring flask by 0.8ml, diluted to a scale by solvent, and shaking to obtain the product.
Intermediate 1 positioning solution: 1.25mg of intermediate 1 reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by acetonitrile, precisely removed from the measuring flask after shaking, placed in a 10ml measuring flask by 1.6ml, diluted to the scale by solvent, and shaking to obtain the product.
Intermediate 3 positioning solution: 1.5mg of intermediate 3 reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by acetonitrile, precisely removed from the measuring flask after shaking, placed in a 10ml measuring flask, diluted to the scale by solvent, and shaking uniformly to obtain the product.
5-chloro-2, 3-diphenylpyrazine positioning solution: taking 1.25mg of 5-chloro-2, 3-diphenyl pyrazine reference substance, precisely weighing, placing into a 10ml measuring flask, dissolving with acetonitrile, diluting to scale, shaking, precisely removing 1.6ml from the measuring flask, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
Imidazole positioning solution: taking 1.25mg of imidazole reference substance, precisely weighing, placing into a 10ml measuring flask, dissolving with acetonitrile, diluting to scale, shaking, precisely transferring 1.6ml from the measuring flask, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking.
1, 8-diazabicyclo undec-7-ene (DBU) localization solution: taking 50mg of 1, 8-diazabicyclo undec-7-ene (DBU) reference substance, precisely weighing, placing into a 50ml measuring flask, dissolving with acetonitrile, diluting to scale, shaking, precisely removing 0.5ml from the measuring flask, placing into a 25ml measuring flask, diluting to scale with solvent, and shaking.
Adding a standard solution to a test sample: about 200mg of selapage is taken, precisely weighed, placed in a 10ml measuring flask, added with 2ml of acetonitrile, heated in a water bath at 60 ℃ until the acetonitrile is dissolved, precisely added with 1.0ml of reference stock solution, then added with mobile phase A, shaken while being diluted to a scale, kept stand for 2 hours after shaking, transferred into a centrifuge tube, centrifuged for 10 minutes at 5000 revolutions per minute, and the supernatant is centrifuged again in the same way, and the supernatant is taken.
Precisely measuring each positioning solution, reference substance solution, test substance solution, and test substance adding solution by 1 needle, injecting into liquid chromatograph, and recording chromatogram, with the results shown in figure 1 and table 4.
Table 4: results of specificity investigation
The results indicate that the blank solution is undisturbed and that each known impurity does not interfere with the determination of bromoacetic acid. In the chromatogram of the sample adding standard solution, the retention time of bromoacetic acid is consistent with that of bromoacetic acid in the reference solution, the minimum separation degree of bromoacetic acid peak and adjacent peak is 8.035, which is not less than 1.5, and the specificity meets the requirements.
Durability test
Taking 80 μl of each of the control solution and the sample addition solution, measuring under the conditions described in the following table, and recording chromatograms. And calculating the standard adding recovery rate of bromoacetic acid according to an external standard method. When the chromatographic conditions were examined for a slight change, the effect on the measurement results was examined.
Table 5: method for preparing bromoacetic acid content in selapage
The results were examined as follows:
table 6: results of durability test (Fine tuning column temperature, flow Rate, mobile phase pH)
Table 7: determination method durability RSD results
The method is characterized in that the durability of a column temperature, a flow rate and a mobile phase pH value inspection method in a fine adjustment method are adopted, the minimum separation degree of bromoacetic acid and an adjacent peak in a sample adding standard solution under different durability test conditions is 12.278, the minimum separation degree is not less than 1.5, and the RSD of the bromoacetic acid adding standard recovery rate is not more than 2.7 percent and not more than 30.0 percent; the minimum separation degree of bromoacetic acid and adjacent peaks is 12.667 and is not less than 1.5 by changing the durability of the chromatographic column inspection method, and the ratio of bromoacetic acid addition recovery rate in the test sample addition standard solution measured by using different chromatographic columns is 106%. The above results indicate that the process is robust.
Test of Linear relation
The linear and range studies of bromoacetic acid were performed by taking 5 concentration points over a range corresponding to 200% of the quantitative limit concentration to the bromoacetic acid limit concentration.
80 μl of each linear solution was precisely measured and injected into 1 needle, and the linear regression was performed on the sample injection concentration (μg/ml) with respect to the measured peak area by a liquid chromatograph. The results show that the bromoacetic acid peak area has good linear relationship in the range of 0.3828 mu g/ml to 19.1400 mu g/ml. The correlation coefficient r=1.0000, which is not less than 0.999; intercept deviation = 1%, no greater than 20%.
Quantitative limit test
The control stock solution with known concentration was diluted to a low concentration solution, and the solution was detected by the above-mentioned measurement method, 80. Mu.l was injected into a liquid chromatograph, the chromatogram was recorded, and the quantitative limit was calculated by taking the response signal of each peak as a quantitative signal (S/N. Apprxeq.10) 10 times the noise, and the results were shown in the following table.
Table 8: quantitative limit test measurement results
Precision investigation
(1) Repeatability test
Since no bromoacetic acid was detected in the test sample, the reproducibility of the method was examined by adding known bromoacetic acid to the test sample solution. About 200mg of the product is precisely weighed, placed in a 10ml measuring flask, added with 2ml of acetonitrile, heated in a water bath at 60 ℃ until the acetonitrile is dissolved, precisely added with 1.0ml of reference stock solution, then added with mobile phase A, shaken while being diluted to a scale, shaken uniformly and then stood for 2 hours, the solution is transferred into a centrifuge tube, centrifuged for 10 minutes at 5000 revolutions per minute, and the supernatant is centrifuged again in the same way, and the supernatant is obtained. 80 μl of each of the obtained products was measured and injected into a liquid chromatograph, the chromatogram was recorded, and the recovery rate of bromoacetic acid was examined by an external standard method, and the results were shown in the following table.
Table 9: repeatability investigation results
The result shows that the method has good repeatability.
(2) Intermediate precision test
And (3) examining the influence of random variation factors on precision, and measuring by another analyst on different dates by using different instruments, and re-preparing 6 samples of the sample to be tested and adding the standard solution, wherein the preparation method and the repeatability test are the same, and the results are shown in the table below.
Table 10: results of intermediate precision investigation
Conclusion: the method has the advantages of comprehensive repeatability test and intermediate precision test, total measurement results of 12 times, and the results show that the method has good precision.
Accuracy test
Taking about 23.5mg of bromoacetic acid reference substance, precisely weighing, placing into a 250ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking to obtain reference substance stock solution. Precisely transferring 1.0ml of the reference substance stock solution, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking to obtain reference substance solution. About 200mg of the product is precisely weighed, 9 parts are taken, the total weight is respectively placed in a 10ml measuring flask, 2ml of acetonitrile is added, water bath heating is performed at 60 ℃ until the mixture is dissolved, 0.8ml, 1ml and 1.2ml of reference stock solution are respectively precisely added according to three concentrations of low (80%), medium (100%) and high (120%), mobile phase A is added, shaking is performed while shaking and dilution is performed to a scale, standing is performed for 2 hours after shaking, the solution is transferred into a centrifuge tube, centrifugation is performed for 10 minutes at 5000 revolutions per minute, supernatant is taken, and centrifugation is performed again in the same way, so that supernatant is taken. Precisely measuring 80 μl of each of the control solution and the sample solution, respectively, and recording the chromatograms. The recovery rate was calculated as peak area according to the external standard method, and the recovery rate was required to be between 85% and 110%, and the results are shown in Table 10.
Table 11: bromoacetic acid recovery test results
The result shows that the recovery rate of bromoacetic acid in 3 concentration levels (80%, 100%, 120%) of the standard test sample solution is between 99.3% and 101.7%, and the method has better recovery rate and high accuracy.
Claims (7)
1. The method for analyzing bromoacetic acid impurities in selapage by high performance liquid chromatography is characterized by comprising the following detection conditions:
the chromatographic column adopts octadecylsilane chemically bonded silica gel column;
mobile phase: taking phosphate-sodium phosphate buffer solution with pH value of 2.1-2.3 as mobile phase A and acetonitrile as mobile phase B; detecting by an ultraviolet detector, wherein the detection wavelength is 190-250nm; column temperature is 38-42 ℃; gradient elution was used and the gradient elution procedure is as set forth in the following table:
。
2. the method of claim 1, wherein the preparation of the test solution is: 200mg of selapage is taken and placed in a 10ml measuring flask, 2ml of acetonitrile is added, water bath heating is carried out at the temperature of not more than 65 ℃ until the acetonitrile is dissolved, then mobile phase A is added, shaking is carried out while shaking is carried out, the mobile phase A is diluted to a scale, shaking is carried out, standing is carried out after shaking, centrifugation is carried out, supernatant is taken, and the supernatant is taken for centrifugation again, thus obtaining the solid.
3. The method of claim 1, wherein the preparing of the control solution comprises: taking a proper amount of bromoacetic acid reference substance, precisely weighing, adding a solvent to dissolve and dilute the bromoacetic acid reference substance to prepare a solution containing 9-10 mug per lml, wherein the solvent is a mixed solution of acetonitrile and mobile phase A according to a volume ratio of 20:80.
4. The method according to claim 1, wherein the flow rate is 0.9 to 1.1ml/min.
5. The method according to claim 1, wherein the chromatographic column is Kromasil100-5c18,4.6mm×250mm,5 μm.
6. The method according to claim 1, wherein the detection wavelength is 205nm.
7. The method according to claim 1, wherein the flow rate is 1.0ml/min, the column temperature is 40℃and the buffer pH is 2.2.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279047A (en) * | 2015-05-13 | 2017-01-04 | 上海适济生物科技有限公司 | A kind of preparation method of prostacyclin receptor agonist |
| CN106324141A (en) * | 2016-08-30 | 2017-01-11 | 山东京卫制药有限公司 | HPLC (high-performance liquid chromatography) detection method for escitalopram oxalate related substances |
| WO2018008042A1 (en) * | 2016-07-05 | 2018-01-11 | Maithri Drugs Private Limited | Novel process for the preparation of 2-{4-[(5,6-diphenyl pyrazin-2-yl)(isopropyl)amino]butoxy}-n-(methylsulfonyl)acetamide and novel polymorphs thereof |
| CN112939877A (en) * | 2019-12-11 | 2021-06-11 | 南京理工大学 | Synthesis method of diphenylpyrazine derivative |
| CN114324699A (en) * | 2021-12-30 | 2022-04-12 | 湖南方盛制药股份有限公司 | Method for analyzing 4- (isopropylamino) butanol by gas chromatography |
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- 2022-05-29 CN CN202210597165.6A patent/CN115015457B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279047A (en) * | 2015-05-13 | 2017-01-04 | 上海适济生物科技有限公司 | A kind of preparation method of prostacyclin receptor agonist |
| WO2018008042A1 (en) * | 2016-07-05 | 2018-01-11 | Maithri Drugs Private Limited | Novel process for the preparation of 2-{4-[(5,6-diphenyl pyrazin-2-yl)(isopropyl)amino]butoxy}-n-(methylsulfonyl)acetamide and novel polymorphs thereof |
| CN106324141A (en) * | 2016-08-30 | 2017-01-11 | 山东京卫制药有限公司 | HPLC (high-performance liquid chromatography) detection method for escitalopram oxalate related substances |
| CN112939877A (en) * | 2019-12-11 | 2021-06-11 | 南京理工大学 | Synthesis method of diphenylpyrazine derivative |
| CN114324699A (en) * | 2021-12-30 | 2022-04-12 | 湖南方盛制药股份有限公司 | Method for analyzing 4- (isopropylamino) butanol by gas chromatography |
Non-Patent Citations (2)
| Title |
|---|
| "肺动脉高压药物Selexipag含量及有关物的测定";马慧丽;《科学中国人》(第30期);第69页 * |
| "超高效液相色谱串联质谱法检测饮用水中卤乙酸";李宗来 等;《环境化学》;第30卷(第02期);第574-576页 * |
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