Detection method of ketoprofen macrogol ester impurities
The separation and quantification of ketoprofen polyethylene glycol ester impurities through high-performance liquid chromatography solves the problem of detection of ketoprofen polyethylene glycol ester in drugs, and achieves efficient control and safety guarantee of drug quality.
Patent Information
- Application Number
- CN202410123811.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-30
- Publication Date
- 2025-08-01
AI Technical Summary
There is a lack of effective methods in the prior art to detect and quantify ketoprofen polyethylene glycol ester impurities, affecting the quality and safety of the drug.
High performance liquid chromatography was used, and octaalkylsilane bonded silica gel was used as filler, 0.08%~0.12% aqueous phosphoric acid solution and 0.08%~0.12% acetonitrile solution of phosphoric acid were mobile phases, with gradient elution, detection wavelength was 255~257 nm, and ketoprofen polyethylene glycol ester impurities were separated and quantified.
It realizes efficient, simple and accurate detection of ketoprofen polyethylene glycol ester impurities, ensures the quality and safety of drugs, and provides effective quality control means.
Smart Images
Figure CN120404959A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of chemical analysis, and particularly relates to a method for detecting impurities of ketoprofen polyethylene glycol ester. Background Art
[0002] Ketoprofen, whose chemical name is 2-(3-benzoylphenyl)-propionic acid, has a molecular formula of C 15 H 22 O2. Ketoprofen belongs to carboxylic acid compounds and has a carboxyl group (-COOH) and a benzene ring.
[0003] Polyethylene glycol is a family of water-soluble linear polymers formed by the addition reaction of ethylene oxide and an equivalent amount of ethylene glycol, and its molecular formula is represented by HO(CH2CH2O)nH. Here, "n" is the average number of repeating oxyethylene groups. For example, polyethylene glycol 600 is composed of a polymer distribution with different molecular weights having an average molecular weight of 600, and the average value of the corresponding repeating groups ("n") is approximately 13. Polyethylene glycol is a common pharmaceutical excipient.
[0004] In pharmaceutical preparations, when ketoprofen and polyethylene glycol coexist, the carboxyl group of ketoprofen and the hydroxyl group of polyethylene glycol undergo an esterification reaction to form ketoprofen polyethylene glycol ester impurities, which affect the quality of the preparation.
[0005] The structural formulas of ketoprofen, polyethylene glycol, and ketoprofen polyethylene glycol ester are shown in the following table: .
[0006] Taking the Lornoxicam Trometamol Gel Patch as an example, polyethylene glycol 400 is used as an excipient, which can generate corresponding polyethylene glycol ester impurities with the drug active ingredient, and n is about 8.
[0007] There is no report on the detection of ketoprofen polyethylene glycol ester. Therefore, it is of great significance to provide a method for detecting ketoprofen polyethylene glycol ester impurities in drugs. Summary of the Invention
[0008] The purpose of the present invention is to provide a high performance liquid chromatography detection method for ketoprofen polyethylene glycol ester, which can effectively determine the content of ketoprofen polyethylene glycol ester, and has the characteristics of simplicity, specificity, accuracy, and high sensitivity, and is applicable to the detection of ketoprofen polyethylene glycol ester impurities in drugs.
[0009] To achieve the above purpose, the present invention is realized through the following technical solutions: A method for detecting impurities of ketoprofen polyethylene glycol ester, using high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel, mobile phase A is the aqueous phase, mobile phase B is the organic phase, and gradient elution is carried out; The structural formula of the ketoprofen polyethylene glycol ester is as follows: .
[0010] Further, the mobile phase A is an aqueous phosphoric acid solution with a concentration of 0.08% - 0.12%.
[0011] Further, the mobile phase B is an acetonitrile solution of phosphoric acid with a concentration of 0.08% - 0.12%.
[0012] Further, the specifications of the chromatographic column are 4.6 * 150 mm, 5 µm.
[0013] Further, the initial stage of the gradient elution is a linear gradient. The initial proportion of the organic phase in the linear gradient is 3% - 5%, the ending proportion is 50% - 55%, and the slope of the linear gradient is 1.5 - 3.0.
[0014] Further, the chromatographic conditions further include one or more of the following (1) - (4): (1) The flow rate of the mobile phase is 0.6 ± 0.2 ml / min; (2) The temperature of the chromatographic column is 25°C ± 2°C; (3) The injection volume is 10 - 20 µl; (4) The detection wavelength is 255 - 257 nm.
[0015] Further, the high performance liquid chromatography method includes the following steps: Prepare a test sample into a test solution, inject the test solution for high performance liquid chromatography analysis, record the chromatogram, and calculate the amount of ketoprofen polyethylene glycol ester by the self - reference method of the main component.
[0016] Further, for the high performance liquid chromatography method, the chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6 * 150 mm, 5 µm; the mobile phase A is an aqueous solution of 0.1% phosphoric acid, and the mobile phase B is an acetonitrile solution of 0.1% phosphoric acid; the flow rate is 0.6 - 0.8 ml / min; the column temperature is 25°C; the detection wavelength is 255 - 257 nm; the injection volume is 10 - 20 μl; Perform gradient elution according to the following table: .
[0017] Further, for the high performance liquid chromatography method, the chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6*150mm, 5μm; 0.1% phosphoric acid aqueous solution is used as mobile phase A, and 0.1% phosphoric acid acetonitrile solution is used as mobile phase B; the flow rate is 0.6 ml / min; the column temperature is 25°C; the detection wavelength is 257nm; the injection volume is 10 μl; Perform gradient elution according to the following table: [[ID=##]] 。
[0018] Furthermore, for the high performance liquid chromatography method described above, the chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6*150mm, 5μm; 0.1% phosphoric acid aqueous solution is used as mobile phase A, and 0.1% phosphoric acid acetonitrile solution is used as mobile phase B; the flow rate is 0.6 ml / min; the column temperature is 25°C; the detection wavelength is 257nm; the injection volume is 10 μl; Perform gradient elution according to the following table: 。
[0019] The high performance liquid chromatography method described in the present invention includes the following steps: Take the drug preparation to be tested and prepare it into a test solution. Inject the test solution for high performance liquid chromatography analysis, record the chromatogram, and calculate the content of related substances in the test sample according to the self-control method of the main component. The calculation formula is as follows: 。
[0020] Where: Ai is the peak area of ketoprofen polyethylene glycol ester in the test solution; As is the peak area of the main peak in the control solution; f is the correction factor of ketoprofen polyethylene glycol ester and dexketoprofen.
[0021] Advantages of the present invention: 1. The method of the present invention detects the content of ketoprofen polyethylene glycol ester impurities in drug preparations by high performance liquid chromatography, and has the characteristics of simplicity, specificity, accuracy and high sensitivity, and is suitable for the detection of polyethylene glycol ester impurities in drugs.
[0022] 2. The present invention has screened out the best chromatographic analysis conditions through a large number of experiments. Experimental verification shows that the high performance liquid chromatography detection method for ketoprofen polyethylene glycol ester impurities provided by the present invention has high resolution and good stability, can effectively separate ketoprofen and polyethylene glycol ester impurities and accurately quantify them, helps to objectively, accurately and comprehensively evaluate the quality of drugs, provides technical guarantee for ensuring drug safety, and has important practical significance for product quality control. Description of the Drawings
[0023] Figure 1 It is the chromatogram of the blank solution in Example 1; Figure 2 It is the chromatogram of the blank excipient solution in Example 1; Figure 3 It is the chromatogram of the reference substance solution in Example 1; Figure 4 It is the chromatogram of the system suitability solution in Example 1; Figure 5 It is the chromatogram of the control solution in Example 1; Figure 6 It is the chromatogram of the test solution in Example 1; Figure 7 It is the chromatogram of the test solution in Example 2 (injection volume: 20 μl); Figure 8 It is the chromatogram of the test solution in Example 3 (injection volume: 10 μl); Figure 9 It is the chromatogram of the test solution in Example 4; Figure 10 It is the chromatogram of the test solution in Example 5; Figure 11 It is the chromatogram of the test solution in Example 6; Figure 12 It is the chromatogram of the reference substance solution in Comparative Example 1. Detailed implementation mode
[0024] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail through examples below. The following examples are only used to explain the present invention and do not represent the scope of the claimed rights of the present invention. The scope of the claimed rights of the present invention shall be subject to the claims.
[0025] The reagents, raw materials and excipients used in the present invention are all commercially available.
[0026] Example 1 The method for detecting impurities of ketoprofen polyethylene glycol ester of the present invention includes the following steps: 1. Solution preparation Preparation of the blank solution: Methanol.
[0027] Preparation of the blank excipient solution: Take 1 patch of the blank excipient sample of ketoprofen trometamol gel patch (without active ingredient), cut out the middle part of 70 cm 2 , cut it into pieces, place it in a 150 ml brown stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonically treat it for 40 minutes, let it cool, weigh it again, add methanol to make up the lost weight, filter it, and take the subsequent filtrate to obtain the solution.
[0028] Reference solution: Weigh an appropriate amount of ketoprofen polyethylene glycol ester reference substance accurately, dissolve it in methanol and quantitatively prepare a solution containing about 10 μg of ketoprofen polyethylene glycol ester per 1 ml.
[0029] System suitability solution: Weigh an appropriate amount of ketoprofen polyethylene glycol ester reference substance and tromethamine dexketoprofen reference substance accurately, dissolve them in methanol and quantitatively dilute to prepare a mixed solution containing about 10 μg of each of ketoprofen polyethylene glycol ester and tromethamine dexketoprofen per 1 ml.
[0030] Test solution: Take 1 patch of tromethamine dexketoprofen gel patch sample, cut out the middle part of 70 cm 2 (equivalent to about 30 mg of dexketoprofen), cut it into small pieces and place it in a 150 ml brown stoppered conical flask. Accurately add 50 ml of methanol, weigh it, ultrasonically treat for 40 minutes, let it cool, weigh it again, add methanol to make up the lost weight, filter, and take the continuous filtrate, that is obtained.
[0031] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml brown volumetric flask, dilute it to the mark with methanol, and shake well, that is obtained.
[0032] [[ID=?]]2. Chromatographic conditions Use octadecylsilane chemically bonded silica gel as the filler; use 0.1% phosphoric acid aqueous solution as mobile phase A and 0.1% phosphoric acid acetonitrile solution as mobile phase B; the flow rate is 0.6 ml / min; the column temperature is 25 °C; the detection wavelength is 257 nm; perform gradient elution according to the following table; the injection volume is 10 μl.
[0033] .
[0034] 3. Determination method Accurately measure the blank solution, blank excipient solution, reference solution, system suitability solution, control solution, and test solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.
[0035] 4. Calculation method Calculate the impurity content by the self-control method of the main component.
[0036] The chromatographic data are shown in the following table: .
[0037] ]>It can be seen from the test results that the blank solution and the blank excipient solution do not interfere with the detection of ketoprofen polyethylene glycol ester; the resolution between the ketoprofen polyethylene glycol ester peak and the main peak (tromethamine dexketoprofen) in the system suitability solution is 2.109, and the number of theoretical plates meets the requirements; the retention time of the ketoprofen polyethylene glycol ester peak in the test solution is consistent with the retention time of the ketoprofen polyethylene glycol ester peak in the reference solution and the system suitability solution.
[0038] Example 2 The solution preparation and elution gradient were the same as those in Example 1. Compared with Comparative Document 1, the flow rate was 0.8 ml / min, the detection wavelength was 255 nm, and the injection volume was 20 μl.
[0039] Precisely measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.
[0040] The chromatographic data are shown in the following table: 。 Example
[0041] The solution preparation and elution gradient were the same as those in Example 1. Compared with Comparative Document 1, the flow rate was 0.8 ml / min, the detection wavelength was 255 nm, and the injection volume was 10 μl.
[0042] Precisely measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.
[0043] The chromatographic data are shown in the following table: 。
[0044] The chromatographic conditions of Examples 2 and 3 were basically the same, and the only difference was the injection volume. The resolution between the ketoprofen polyethylene glycol ester peak and the main peak (dexketoprofen trometamol) in Examples 2 and 3 was greater than 1.5. Compared with an injection volume of 20 μl, the resolution was improved with an injection volume of 10 μl, and the peak area was in a proportional relationship with the injection volume.
[0045] Example 4 The solution preparation was the same as that in Example 1. Compared with Comparative Document 1, the only difference was the elution gradient. The elution gradient of Example 4 is shown in the following table: 。
[0046] Precisely measure the test solution separately and inject it into the liquid chromatograph, and record the chromatogram.
[0047] The chromatographic data are shown in the following table: 。
[0048] The resolution between the ketoprofen polyethylene glycol ester peak and the main peak (dexketoprofen trometamol) was greater than 1.5.
[0049] Example 5 The solution preparation was the same as that in Example 1. Compared with Comparative Document 1, the only difference was the elution gradient. The elution gradient of Example 5 is shown in the following table: 。
[0050] Precisely measure the test solution separately and inject it into the liquid chromatograph, and record the chromatogram.
[0051] The chromatographic data are shown in the following table: 。
[0052] The resolution between the ketoprofen polyethylene glycol ester peak and the main peak (right ketoprofen trometamol) is greater than 1.5.
[0053] Example 6 1. Solution preparation Test solution: Take 1 patch of the right ketoprofen trometamol gel patch sample, take the middle part of 70 cm 2 (equivalent to about 30 mg of right ketoprofen), cut it into small pieces and place it in a 150 ml brown stoppered conical flask. Precisely add 50 ml of methanol, weigh it, ultrasonically treat it for 40 minutes, let it cool, add methanol to make up the lost weight, filter, and take the subsequent filtrate, that is, obtain it.
[0054] 2. Chromatographic conditions Use octadecylsilane-bonded silica gel as the filler; use 0.1% phosphoric acid aqueous solution as mobile phase A and 0.1% phosphoric acid acetonitrile solution as mobile phase B; the flow rate is 0.8 ml / min; the column temperature is 25 °C; the detection wavelength is 255 nm; the injection volume is 20 μl, and gradient elution is carried out according to the following table: 。
[0055] 3. Determination Precisely measure 20 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.
[0056] 。
[0057] The resolution between the ketoprofen polyethylene glycol ester peak and the main peak (right ketoprofen trometamol) in the test solution is greater than 1.5.
[0058] The chromatograms of Examples 1 to 6 are shown in Figures 1 to 11 。From the chromatograms and relevant data, it can be seen that in the system suitability solution, the ketoprofen polyethylene glycol ester impurity and the main component can be effectively separated, and the resolution is greater than 1.5, and the system suitability meets the requirements. The resolution between the ketoprofen polyethylene glycol ester impurity and the main component in the test solution is high, and the solvent and excipients do not interfere with the detection, and this method meets the requirements.
[0059] The results show that under the chromatographic conditions of the present invention, the resolution between the ketoprofen polyethylene glycol ester impurity and the main component is high, and the separation and quantification of the ketoprofen polyethylene glycol ester impurity can be achieved.
[0060] Comparative Example 1 1. Solution preparation Reference solution: Weigh an appropriate amount of ketoprofen polyethylene glycol ester reference substance accurately, dissolve it in methanol and quantitatively dilute it to prepare a solution containing 0.1 mg of ketoprofen polyethylene glycol ester reference substance per 1 ml.
[0061] 2. Chromatographic conditions Use octadecylsilane-bonded silica gel as the filler; use 0.1% phosphoric acid aqueous solution as mobile phase A and 0.1% phosphoric acid acetonitrile solution as mobile phase B; the flow rate is 0.8 ml / min; the column temperature is 25 °C; the detection wavelength is 255 nm; the injection volume is 20 μl; perform gradient elution according to the following table: .
[0062] 3. Determination method Determination: Accurately measure 20 μl of the reference solution, inject it into the liquid chromatograph, and record the chromatogram.
[0063] 4. Calculation method Calculate the impurity content by the self-control method of the main component.
[0064] The chromatographic data are shown in the following table: .
[0065] The results show that the peaks of ketoprofen polyethylene glycol ester and the main peak (ketoprofen) in the reference solution can be basically separated, but the resolution is <1.5, and the resolution is relatively low.
[0066] To further illustrate the beneficial effects of the present invention, the following test examples are provided in the present invention.
[0067] Test example 1 Taking the detection method of Example 1 as a representative, some linear test data are shown.
[0068] Determine the f value in the calculation formula.
[0069] Ketoprofen polyethylene glycol ester stock solution: Accurately weigh 4.05 mg of ketoprofen polyethylene glycol ester reference substance in a 100 ml brown volumetric flask, and dilute it to the mark with methanol.
[0070] Ketoprofen polyethylene glycol ester linear solution: Accurately measure 0.6 ml, 1.5 ml, 3 ml, 6 ml, and 9 ml of the ketoprofen polyethylene glycol ester stock solution into a 20 ml brown volumetric flask respectively, dilute it to the mark with methanol, and shake well to obtain 20%, 50%, 100%, 200%, and 300% linear solutions.
[0071] Dexketoprofen stock solution: Accurately weigh 6.019 mg of dexketoprofen trometamol reference substance in a 100 ml brown volumetric flask, and dilute it to the mark with methanol. The conversion coefficient of dexketoprofen trometamol to dexketoprofen is 0.6773.
[0072] Dexketoprofen linear solution: Accurately pipette 0.6 ml, 1.5 ml, 3 ml, 6 ml, and 9 ml of the dexketoprofen stock solution into separate 20-ml brown volumetric flasks. Dilute to the mark with methanol and mix well to obtain 20%, 50%, 100%, 200%, and 300% linear solutions.
[0073] Pipette 10 μl of each of the above linear solutions and inject into the liquid chromatograph. Record the chromatogram.
[0074]
[0075]
[0076] Test Example 2 The impurity concerned in the present invention is dexketoprofen polyethylene glycol ester produced by the reaction of dexketoprofen with the preparation excipient polyethylene glycol 400. If not monitored and controlled, it will affect the effectiveness and safety of the drug.
[0077] The dexketoprofen polyethylene glycol ester impurity reference substance used in the test was prepared by the company from dexketoprofen and polyethylene glycol. The purity detected by high performance liquid chromatography was 99.86%, and its structure was confirmed by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.
[0078] (1) Liquid chromatography - mass spectrometry Instrument model: Agilent 1290 - 6530 Chromatographic conditions: 0.1% formic acid in water (A) - acetonitrile (B) Gradient elution (0 - 10 min, 95% A - 5% A) Mass spectrometry conditions: ESI positive ion mode.
[0079] The source voltage is 5.5 kV, nitrogen is the drying gas, the ion source Gas1 / Gas2 is 50 psi, the curtain gas is 35, the CAD gas is 7, the temperature is 500 °C, and the scanning range is 10 - 2000.
[0080] Since polyethylene glycol is a mixture and the target product is also a mixture, and the monomers differ from each other by one or more hydroxyethyl units, its quasi-molecular ion peak consists of a series of continuous and regular peaks. As shown Figure 3 in the appendix, the m / z of [M + H] + can be observed to be 651.3491, and the m / z of the adduct ion peak [M + NH4] is 668.3763; the m / z of [M + H] + can also be observed to be 563.2959, 607.3229, 695.3738, 703.4006 and the signals of their corresponding adduct ion peaks [M + NH4] + .
[0081] The exact molecular weight is 651.3491, and the deduced corresponding molecular formula is C 34 H 50 O 12 , indicating a degree of polymerization of 414; the exact molecular weight is 607.3229, and the deduced corresponding molecular formula is C 32 H 46 O 11 , indicating a degree of polymerization of 370; the exact molecular weight is 695.3738, and the deduced corresponding molecular formula is C 36 H 54 O 13 , indicating a degree of polymerization of 458. The molecular weight composition of the mixture is approximately normally distributed, in which C 34 H 50 O 12 has the highest relative abundance of the exact molecular weight, and the result corroborates that the degree of polymerization of the raw material polyethylene glycol is 400.
[0082] Further combined with high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy analysis, the structure of the target product is identified as: polyethylene glycol 2(3-benzoylphenyl)propionate.
[0083] The above content is a further detailed description of the present application in combination with specific embodiments, and it cannot be determined that the specific implementation of the present application is only limited to these descriptions. For those of ordinary skill in the technical field to which the present application belongs, without departing from the concept of the present application, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present application.
Claims
1. Detection method for impurities of ketoprofen polyethylene glycol ester, characterized in that, The method uses high performance liquid chromatography. The chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, mobile phase A is the aqueous phase, mobile phase B is the organic phase, and gradient elution is performed. The structural formula of ketoprofen polyethylene glycol ester is as follows: 。 2. The high performance liquid chromatography method according to claim 1, wherein The mobile phase A is an aqueous solution of 0.08% - 0.12% phosphoric acid.
3. The high performance liquid chromatography method according to claim 1, wherein The mobile phase B is an acetonitrile solution of 0.08% - 0.12% phosphoric acid.
4. The high performance liquid chromatography method according to claim 1, characterized in that, The specifications of the chromatographic column are 4.6*150mm, 5μm.
5. The high performance liquid chromatography method according to claim 1, wherein The initial stage of the gradient elution is a linear gradient. The initial proportion of the organic phase in the linear gradient is 3% - 5%, the ending proportion is 50% - 55%, and the slope of the linear gradient is 1.5 - 3.
0.
6. The high performance liquid chromatography method according to any one of claims 1 to 5, characterized in that, The chromatographic conditions also include one or more of the following (1) - (4): (1) The flow rate of the mobile phase is 0.6 ± 0.2 ml / min; (2) The temperature of the chromatographic column is 25°C ± 2°C; (3) The injection volume is 10 - 20 μl; (4) The detection wavelength is 255 - 257 nm.
7. The high performance liquid chromatography method according to any one of claims 1 to 6, characterized in that, It includes the following steps: Take the sample to be tested and prepare a test solution. Inject the test solution for high performance liquid chromatography analysis, record the chromatogram, and calculate the amount of ketoprofen polyethylene glycol ester by the self - control method of the main component.
8. The high performance liquid chromatography method according to any one of claims 1 to 7, characterized in that, The chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6*150mm, 5μm; 0.1% aqueous phosphoric acid solution is used as mobile phase A, and 0.1% acetonitrile solution of phosphoric acid is used as mobile phase B; the flow rate is 0.6 - 0.8 ml / min; the column temperature is 25°C; the detection wavelength is 255 - 257 nm; the injection volume is 10 - 20 μl; Perform gradient elution according to the following table: 。 9. The high performance liquid chromatography method according to any one of claims 1 to 7, characterized in that The chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6*150mm, 5μm; 0.1% aqueous phosphoric acid solution is used as mobile phase A, and 0.1% acetonitrile solution of phosphoric acid is used as mobile phase B; the flow rate is 0.6 ml / min; the column temperature is 25°C; the detection wavelength is 257 nm; the injection volume is 10 μl; Perform gradient elution according to the following table: 。 10. The high performance liquid chromatography method according to any one of claims 1 to 7, characterized in that, The chromatographic conditions are as follows: The chromatographic column is packed with octadecylsilyl silica gel, Inersil C8, 4.6*150mm, 5μm; 0.1% aqueous phosphoric acid solution is used as mobile phase A, and 0.1% acetonitrile solution of phosphoric acid is used as mobile phase B; the flow rate is 0.6 ml / min; the column temperature is 25°C; the detection wavelength is 257 nm; the injection volume is 10 μl; Perform gradient elution according to the following table: 。