CN112763623B - Method for detecting peramivir trihydrate by reversed-phase high-performance liquid chromatography - Google Patents
Method for detecting peramivir trihydrate by reversed-phase high-performance liquid chromatography Download PDFInfo
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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 synthetic finished product of peramivir is (1S, 2S,3R, 4R) -3- ((S) -1-acetamide-2-ethylbutyl) -4-guanidine-2-hydroxycyclopentane-1-carboxylic acid, and the molecular formula is C 15 H 28 N 4 O 4 ·3H 2 O, molecular weight is 382.46, and the structural formula is as follows:
at present, the number of manufacturers supplying peramivir in the market is large, the quality difference is large, some impurity compounds exist, 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) And calculating the contents of single impurities and total impurities according to 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 3.0-5.0 mm, the length is 100-250 mm, and the grain diameter of the filler is 3-5 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 step (2), the phosphate concentration in mobile phase A is 5 to 15mmol/l, more preferably 10mmol. Further, the phosphate concentration in the mobile phase B is 5 to 15mmol/l, and more preferably 5mmol.
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.
Preferably, in step (2), the pH of the 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 1.0ml/min.
Further, the ultraviolet absorption detector has a wavelength of one of 205nm, 210nm, and 215nm, preferably 210nm.
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 method 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 detection method
| 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 Group Chemical Reagent Co., Ltd. |
| Phosphoric acid | HPLC grade/500 mL/bottle | TEDIA |
TABLE 3 control used in the test method
| Name(s) | 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 | China Institute for food and drug control |
Example 1: specificity test
Solvent: mobile phase a-mobile phase B =92 (V: V)
Peramivir mother liquor: weighing 8mg of peramivir reference substance, accurately weighing, placing in a 10mL measuring flask, adding a solvent to dissolve and dilute to a 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 peramivir, 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 fig. 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. 1-3, the blank solution did not interfere at the main peak retention times 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 the quantitative limiting solution, placing the quantitative limiting solution into a 10mL measuring flask, and adding a solvent to dilute the quantitative limiting solution to the 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.
Precisely measuring 10 μ l of the above solutions, respectively, injecting into a liquid chromatograph, continuously injecting 6 needles of limit solution, 1 needle of limit 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 (7)
1. The method for detecting the peramivir trihydrate by using the reversed-phase high performance liquid chromatography is characterized by comprising the following steps of:
(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 stationary phase of the chromatographic column adopts long alkyl silica gel embedded with polar amide groups as a filling agent, the mobile phase A is phosphate buffer solution, the pH is 4.5, the mobile phase B is mixed solution of phosphate buffer solution and acetonitrile, the proportion is a fixed value of 40;
(3) Detecting the sample solution by reversed-phase high performance liquid chromatography, injecting sample, and performing gradient elution;
the concentration ratio of the gradient elution mobile phase is as follows:
(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 5-15mmol/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 5-15mmol/l.
5. The detection method according to claim 1, wherein the column temperature of the chromatographic column in the step (2) is 20 to 50 ℃.
6. 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.
7. The detection method according to claim 1, wherein the sample is introduced in the step (3) in an amount of 5 to 100. Mu.l.
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