CN112763623A - Method for detecting peramivir trihydrate by reversed-phase high-performance liquid chromatography - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 9
- 238000004007 reversed phase HPLC Methods 0.000 title claims abstract description 9
- RFUCJKFZFXNIGB-ZBBHRWOZSA-N (1s,2s,3s,4r)-3-[(1s)-1-acetamido-2-ethylbutyl]-4-(diaminomethylideneamino)-2-hydroxycyclopentane-1-carboxylic acid;trihydrate Chemical compound O.O.O.CCC(CC)[C@H](NC(C)=O)[C@@H]1[C@H](O)[C@@H](C(O)=O)C[C@H]1N=C(N)N RFUCJKFZFXNIGB-ZBBHRWOZSA-N 0.000 title claims description 33
- 239000012535 impurity Substances 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 27
- 238000012360 testing method Methods 0.000 claims abstract description 14
- 239000000523 sample Substances 0.000 claims abstract description 11
- 238000010521 absorption reaction Methods 0.000 claims abstract description 5
- 239000012488 sample solution Substances 0.000 claims abstract description 4
- 238000010606 normalization Methods 0.000 claims abstract description 3
- 238000005303 weighing Methods 0.000 claims description 17
- 239000012085 test solution Substances 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 2
- -1 alkyl silica gel Chemical compound 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 229960001084 peramivir Drugs 0.000 abstract description 29
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 4
- XRQDFNLINLXZLB-CKIKVBCHSA-N peramivir Chemical compound CCC(CC)[C@H](NC(C)=O)[C@@H]1[C@H](O)[C@@H](C(O)=O)C[C@H]1NC(N)=N XRQDFNLINLXZLB-CKIKVBCHSA-N 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 35
- 239000002904 solvent Substances 0.000 description 19
- 239000012452 mother liquor Substances 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 9
- 239000003814 drug Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 description 2
- 229960003752 oseltamivir Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229960001028 zanamivir Drugs 0.000 description 2
- ARAIBEBZBOPLMB-UFGQHTETSA-N zanamivir Chemical compound CC(=O)N[C@@H]1[C@@H](N=C(N)N)C=C(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO ARAIBEBZBOPLMB-UFGQHTETSA-N 0.000 description 2
- OWHFMAAYSUJJCO-UHFFFAOYSA-N 3,7-ditert-butyl-1,5-dinitro-3,7-diazabicyclo[3.3.1]nonane Chemical compound C1N(C(C)(C)C)CC2([N+]([O-])=O)CN(C(C)(C)C)CC1([N+]([O-])=O)C2 OWHFMAAYSUJJCO-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940124393 anti-influenza virus drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 208000037798 influenza B Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 238000003359 percent control normalization Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 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
- G01N30/26—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
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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
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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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Abstract
The invention discloses a method for detecting peramivir by using reverse-phase high performance liquid chromatography, which comprises the following steps of preparing a test sample solution, detecting the test sample by using the reverse-phase high performance liquid chromatography, and calculating the contents of single impurities and total impurities in the test sample according to an area normalization method; by the method, the Palamivir and each impurity chromatographic peak are completely separated by changing the mobile phase and the proportion, the wavelength of an ultraviolet absorption detector and the like; the invention has the advantages of high specificity, accuracy and sensitivity, fast peak-off time, short detection time, accurate and stable detection result, simple detection method and good linear relation, and can carry out more accurate detection and quality control on the peramivir.
Description
Technical Field
The invention relates to an analysis method of peramivir, in particular to a reversed phase high performance liquid chromatography analysis method of peramivir trihydrate.
Background
Peramivir is a novel cyclopentane-type anti-influenza virus drug, and is another novel influenza virus NA inhibitor after Zanamivir (Zanamivir) and Oseltamivir (Oseltamivir) have been successfully developed and marketed in 1999. In 2013, 4 and 5, the national food and drug administration approves an anti-influenza drug peramivir sodium chloride injection, and the existing clinical test data prove that the injection is effective to influenza A and B.
The peramivir synthesis finished product is (1S,2S,3R,4R) -3- ((S) -1-acetamide-2-ethylbutyl) -4-guanidine-2-hydroxycyclopentane-1-carboxylic acid, and the molecular formula is C15H28N4O4·3H2O, molecular weight 382.46, structural formula as follows:
at present, manufacturers supplying peramivir in the market have more products, large quality difference and some impurity compounds, and the quality and the medication safety of peramivir are seriously affected by the existence of some impurities, so that the peramivir needs to be detected and controlled. Peramivir solubility and multiple pKa exist, presenting great difficulty for screening of mobile phase pH.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for detecting peramivir trihydrate by using a reverse phase high performance liquid chromatography with simple method and high specificity, accuracy and sensitivity.
The technical scheme is as follows: the detection method of peramivir comprises the following steps:
(1) preparing a test solution: weighing peramivir, dissolving with water and diluting to obtain a test solution with the concentration of 1-3 mg/ml;
(2) the stationary phase of the chromatographic column is long alkyl silica gel embedded with polar amide groups; an ultraviolet absorption detector is adopted; the mobile phase A is phosphate buffer solution, and the mobile phase B is phosphate buffer solution-acetonitrile mixed solution; the elution mode is gradient elution;
(3) detecting the sample solution by reversed-phase high performance liquid chromatography, injecting sample, and performing gradient elution;
(4) the contents of the individual impurities and the total impurities were calculated by area normalization.
Preferably, in the step (2), the phosphate in the mobile phase a is potassium dihydrogen phosphate.
Preferably, in step (2), the specification of the chromatographic column: the inner diameter is 3.0 to 5.0mm, the length is 100 to 250mm, and the particle size of the filler is 3 to 5 μm. Further, the specification of the chromatography column: the inner diameter was 4.6mm, the length was 250mm, and the filler particle size was 3.5. mu.m.
Further, the chromatographic column is an agent Zorbax Box RP.
Preferably, the column temperature of the column is 20 to 50 ℃, and further, the column temperature of the column is preferably 30 ℃.
Preferably, in the step (2), the concentration of the phosphate in the mobile phase A is 5-15mmol/l, and more preferably 10 mmol. Further, the concentration of phosphate in the mobile phase B is 5-15mmol/l, and more preferably 5 mmol.
Preferably, in the step (2), the ratio of the phosphate buffer solution to the acetonitrile mixed solution in the mobile phase B is a fixed value of 40: 60.
Preferably, in step (2), the pH of mobile phase A phosphate buffer A is 4-6, preferably 4.5.
Preferably, in the step (3), the amount of the sample is 5 to 100. mu.l, and more preferably 10. mu.l. Further, the flow rate is 0.9 to 1.1ml/min, preferably 1.0 ml/min.
Further, the ultraviolet absorption detector has a wavelength of one of 205nm, 210nm, and 215nm, preferably 210 nm.
Further, the concentration ratios of the gradient elution mobile phase are shown in table 1;
TABLE 1 concentration ratio of gradient elution mobile phase
| T(min) | A(％) | B(％) |
| 0 | 90 | 10 |
| 5 | 90 | 10 |
| 20 | 50 | 50 |
| 25 | 50 | 50 |
| 25.01 | 90 | 10 |
| 40 | 90 | 10 |
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the method can effectively separate 3 impurity peaks in the crude product sample; the invention has the advantages of fast peak-producing time, short detection time, accurate and stable detection result, simple detection method and good linear relation, simultaneously ensures the specificity, accuracy and sensitivity of detection, and can carry out more accurate detection and quality control on the peramivir.
Drawings
FIG. 1 is an HPLC chromatogram of a blank solvent of the present invention;
FIG. 2 is an HPLC chromatogram of a solution suitable for use in the system of the present invention, wherein the marker chromatographic peak is peramivir;
FIG. 3 is an HPLC chromatogram of a test sample of the present invention.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
TABLE 2 instruments and reagents used in the assay
| Name (R) | Model/specification | Manufacturer of the product |
| Liquid chromatograph | U3000 | Thermo |
| Electronic balance | MSA125P-1CE-DU | Sadoris sp |
| Dipotassium hydrogen phosphate | Super grade pure/500 g/bottle | SINOPHARM CHEMICAL REAGENT Co.,Ltd. |
| Phosphoric acid | HPLC grade/500 mL/bottle | TEDIA |
TABLE 3 control used in the test method
| Name (R) | Batch number | Content/% | |
| Impurity | |||
| 1 | A0433-200515-0101 | 86.3 | Nanjing Zhengji Pharmaceutical Research Co.,Ltd. |
| Impurity 2 | S0405-191106-0201 | 96.8 | Nanjing Zhengji Pharmaceutical Research Co.,Ltd. |
| Impurity 3 | S0409-191204-0101 | 95.5 | Nanjing Zhengji Pharmaceutical Research Co.,Ltd. |
| Peramivir | 101260-201902 | 85.1 | Chinese food medicine inspectionResearch institute of Ding |
Example 1: specificity test
Solvent: mobile phase a-mobile phase B ═ 92:8(V: V)
Peramivir mother liquor: weighing 8mg of peramivir reference substance, precisely weighing, placing in a 10mL measuring flask, adding a solvent to dissolve and dilute to scale, shaking up, and preparing into a solution with the peramivir concentration of 0.6 mg/mL.
Impurity stock solution: transferring 1.00mL of each of the impurity 1 mother liquor, the impurity 2 mother liquor and the impurity 3 mother liquor, placing the mother liquor in a 50mL measuring flask, adding a solvent to dissolve and dilute the mother liquor to a scale mark, shaking up, and preparing the solution with the concentrations of the impurity 1, the impurity 2 and the impurity 3 being 12 mu g/mL.
System applicability solution: weighing 15mg of the peramivir sample, precisely weighing, placing in a 10mL measuring flask, adding the impurity stock solution to dissolve and dilute to scale, shaking up, and preparing into a solution with the concentration of the peramivir being 1.5mg/mL, the concentration of the impurity 1, the concentration of the impurity 2 and the concentration of the impurity 3 being 12 mu g/mL.
Test solution: weighing 15mg of the product, precisely weighing, placing in a 10mL measuring flask, dissolving with a solvent, diluting to scale, shaking up, and preparing into a solution with the peramivir concentration of 1.2 mg/mL.
Control solution: precisely transferring 1.00mL of the test solution, placing the test solution in a 100mL measuring flask, diluting the test solution to a scale with a solvent, and shaking up. Transferring 1.00mL of the solution, placing the solution into a 10mL measuring flask, diluting the solution to a scale mark by using a solvent, and shaking the solution uniformly to prepare a solution with the peramivir concentration of 1.2 mu g/mL.
Positioning solution: precisely measuring 1.0mL of the mother solution, placing into 10mL measuring bottles, and diluting with solvent to scale to obtain positioning solution.
TABLE 4 specificity test
Chromatograms were recorded and the results are shown in table 5 and figures 1-3.
TABLE 5 results of the specificity experiments
| Order of appearance | Retention time (min) | Degree of separation | Number of theoretical plate | Asymmetry factor | 0.1% control signal-to-noise ratio |
| Peramivir | 11.487 | 2.07 | 39033 | 1.22 | 49.8 |
| |
12.697 | 3.25 | 45224 | - | - |
| |
12.970 | 1.89 | 64136 | - | - |
| |
15.337 | 15.82 | 66023 | - | - |
As can be seen from Table 5 and FIGS. 1 to 3, the blank solution was not interfered at the main peak retention time in the test and control solutions; the separation degree between the impurities and the main components is more than or equal to 1.5, which indicates that the specificity of the detection method meets the quality control requirement.
Example 2: sensitivity test
Impurity quantitative limiting solution: taking the impurity 1, the impurity 2 and the impurity 3 under the special item, respectively placing the impurities in different 50mL measuring bottles, adding a solvent to dilute the impurities to the scales, shaking the measuring bottles evenly, respectively precisely measuring 3mL of the impurities to place the measuring bottles in the same 100mL measuring bottle, adding the solvent to dilute the impurities to the scales, and shaking the measuring bottles evenly. And precisely measuring 3mL of the solution, placing the solution into a 5mL measuring flask, and adding a solvent to dilute the solution to a scale.
Impurity detection limiting solution: precisely measuring 3mL of limiting solution, placing the limiting solution into a 10mL measuring flask, and adding a solvent to dilute the limiting solution to a scale.
Peramivir quantitative limiting solution: weighing 16mg of peramivir reference substance, precisely weighing, placing in a 20mL measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking up. Precisely measuring 1mL, placing in a 50mL measuring flask, adding a solvent to dilute to a scale, shaking up, precisely measuring 3mL, placing in a 100mL measuring flask, adding a solvent to dilute to a scale, and shaking up. And precisely measuring 3mL of the solution, placing the solution into a 5mL measuring flask, and adding a solvent to dilute the solution to a scale.
Respectively and precisely measuring 10 μ l of the above solutions, injecting into a liquid chromatograph, continuously injecting 6 needles of quantitative limiting solution, injecting 1 needle of detection limiting solution, and recording chromatogram, wherein the results are shown in Table 2.
TABLE 6 results of quantitative limit and detection limit
As can be seen from Table 6, the detection limit sensitivity of the detection method of the present invention is 0.01% (0.1. mu.g/ml), and the quantitative limit sensitivity is 0.03% (0.4. mu.g/ml), indicating that the specificity of the detection method of the present invention meets the quality control requirements.
Example 3: linear test
Diluting the peramivir and the impurity reference substance solution with a diluent to prepare a series of reference solutions with a series of concentrations, injecting the reference solutions into a liquid chromatograph, and recording a chromatogram, wherein the results are shown in tables 3-6.
Table 7 standard curve for impurity 1
TABLE 8 impurity 2 linearity
TABLE 9 impurity 3 linearity
TABLE 10 Peramivir linearity
As can be seen from tables 7-10, the detection method of the present invention has a good linear relationship in the range of 0.2-3 μ g/ml.
Example 4: repeatability test
Weighing peramivir raw materials, dissolving and diluting the peramivir raw materials by using a diluent to obtain a test solution with the concentration of 1.2 mg/ml; 6 parts of test sample solution is prepared in parallel, injected into a liquid chromatograph, the chromatogram is recorded, and the content of each impurity and the total impurity in the test sample is calculated, and the result is shown in table 7.
TABLE 11 results of repeated measurements
As can be seen from Table 11, the impurity content in the mixed solution fluctuates within. + -. 0.01% in 6 solutions prepared in parallel, which indicates that the method has good repeatability.
Claims (10)
1. A method for detecting peramivir trihydrate by reversed-phase high performance liquid chromatography is characterized by comprising the following steps:
(1) preparing a test solution, weighing peramivir trihydrate, dissolving with water and diluting to obtain the test solution with the concentration of 1-3 mg/ml;
(2) the chromatographic column stationary phase adopts long alkyl silica gel embedded with polar amide groups as a filling agent, the mobile phase A is phosphate buffer solution, the mobile phase B is phosphate buffer solution-acetonitrile mixed solution, and the detector is an ultraviolet absorption detector;
(3) detecting the sample solution by reversed-phase high performance liquid chromatography, injecting sample, and performing gradient elution;
(4) and calculating the contents of single impurities and total impurities in the test sample according to an area normalization method.
2. The detection method according to claim 1, wherein the phosphate in the mobile phase A in the step (2) is potassium dihydrogen phosphate.
3. The detection method according to claim 1, wherein the molar concentration of phosphate in the mobile phase A in the step (2) is 5-15 mmol/l.
4. The detection method according to claim 1, wherein the molar concentration of phosphate in the mobile phase B in the step (2) is 5-15 mmol/l.
5. The detection method according to claim 1, wherein the phosphate buffer solution-acetonitrile ratio in the mobile phase B in the step (2) is a fixed value.
6. The detection method according to claim 1, wherein the pH of the mobile phase A phosphate buffer solution in the step (2) is 4 to 6.
7. The detection method according to claim 1, wherein the column temperature of the chromatographic column in the step (2) is 20 to 50 ℃.
8. The detection method according to claim 1, wherein the wavelength of the ultraviolet absorption detector in the step (2) is one of 205nm, 210nm and 215 nm.
9. The detection method according to claim 1, wherein the sample is injected in the step (3) in an amount of 5 to 100. mu.l.
10. The detection method according to claim 1, wherein the concentration ratio of the gradient elution mobile phase in the step (3) is as follows:
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| CN114295747A (en) * | 2021-12-30 | 2022-04-08 | 苏州正济药业有限公司 | Analysis method of peramivir starting material and impurities |
| CN115791999A (en) * | 2022-10-31 | 2023-03-14 | 海南葫芦娃药业集团股份有限公司 | Method for detecting impurity C of peramivir injection |
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| CN114295747A (en) * | 2021-12-30 | 2022-04-08 | 苏州正济药业有限公司 | Analysis method of peramivir starting material and impurities |
| CN114295747B (en) * | 2021-12-30 | 2023-10-20 | 苏州正济药业有限公司 | Analysis method of Parami Wei Qishi material and impurities |
| CN115791999A (en) * | 2022-10-31 | 2023-03-14 | 海南葫芦娃药业集团股份有限公司 | Method for detecting impurity C of peramivir injection |
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