A method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde

Abstract

The application provides a method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-formaldehyde, which comprises the following steps: mixing a sample to be tested with a solvent to obtain a test solution; mixing a related substance standard with a solvent to obtain an impurity control positioning solution; then performing liquid chromatography detection on the test solution and the impurity control positioning solution, and obtaining the content of the related substances in the sample to be tested according to the detection result; the chromatographic column filler for the liquid chromatography detection is pentafluorophenyl silane bonded silica gel; the related substances include impurity I 11 and impurity I 12 . The method provided by the application can effectively separate impurity I 11 , impurity I 12 , realizes accurate control of specific impurity I 11 , impurity I 12 , has the characteristics of strong specificity, significantly improved sensitivity, significantly improved accuracy, good repeatability, good durability and the like, and meets the quality control requirements for the two impurities in drug registration evaluation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, specifically relating to a method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde. Background Technology

[0002] 5-(2-Fluorophenyl)-1H-pyrrole-3-carboxaldehyde is a pharmaceutical intermediate, generally used as a starting material for synthesizing active pharmaceutical ingredients (APIs). Impurities in the starting material can affect the quality of the finished API.

[0003] Related substances (impurities) are direct indicators of drug purity, and controlling their content is crucial for ensuring drug quality. Based on the synthesis process of the starting materials, 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde may contain specific impurities. 11 I 12 Since impurities are known, strict quality control of the related substances of the starting material 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde is required to ensure the quality of the finished drug product.

[0004] The starting material is 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde (SM1), whose structural formula is: The SM1 synthesis process may introduce several known impurities into the finished product, especially known impurity I. 11 and I 12 Due to their extremely similar molecular structures, differing in molecular weight by only two H atoms, and similar properties, these two impurities are extremely difficult to separate and quantify under conventional C18 column conditions using related substance methods for quality control. Currently, no effective quality control methods for these two specific impurities are reported in existing literature. Therefore, there is an urgent need to provide a separation method for determining these two specific impurities in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde.

[0005] The existing methods include the following: Reference 1: CN116953129A discloses a method for the simultaneous determination of twelve impurities in vonoprazan fumarate by high performance liquid chromatography. The chromatographic conditions and method use a C18 column. Mobile phase A is a mixture of 0.025 mol / L phosphate buffer, acetonitrile, and methanol at a volume ratio of 70:5:25. Mobile phase B is methanol. Quality control studies were conducted on various known impurities present in the synthesis and preparation of the active pharmaceutical ingredient. However, these impurities do not include 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde (SM1) or known impurity I present in the starting material process. 11 and I 12 .

[0006] Reference 2: CN118817887A discloses a method for detecting related substances in vonoprazan fumarate, comprising eighteen impurities. The liquid chromatography conditions used are: an Agilent Poroshell 120ECC18 4.6*150mm 2.7μm column; mobile phase A: 12.5mmol / L potassium dihydrogen phosphate solution, adjusted to pH 6.50 with potassium hydroxide; and mobile phase B: acetonitrile. This chromatographic method can perform quality control on 18 known impurities during the synthesis and preparation of the active pharmaceutical ingredient, and determine the related substances of these known impurities. 11 I 12 This is completely different, and the method uses chromatographic conditions with salt systems, resulting in very high column pressure, which is not as good as the method for determining salt-free systems.

[0007] Reference 3: CN117924141A discloses a method for preparing key impurities A and B of fumarate vonoprazan, which uses high performance liquid chromatography to determine specific impurity I. 11 Chromatographic conditions: Liquid chromatography conditions: Waters XBridge C18 4.6mm × 250mm, 5μm column; mobile phase A: 0.02mol / L dipotassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid); mobile phase B: acetonitrile; detection wavelength: 245nm; column temperature: 30℃. This method uses chromatographic conditions with a salt system, resulting in excessively high column pressure, which prevents complete and simultaneous separation of impurity I. 11 and I 12 The method for determining the salt-free system is not as good as the method for determining the salt-free system.

[0008] The above literature describes methods for separating and detecting related substances in active pharmaceutical ingredients using HPLC, but none of these methods can achieve the separation and detection of I... 11 I 12 Effective separation and detection. Because 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde contains known impurity I... 11 and I 12 With a molecular weight difference of 2 and only a slight difference in the number of methylene and imine bonds, and almost no difference in polarity, these materials are difficult to completely and simultaneously separate in a typical octadecylsilane-bonded silica gel packing system. 11 and I 12 Impurities, due to the hydrophobic separation mechanism of conventional C18 columns, are difficult to distinguish between the two, resulting in complete overlap or severe tailing of the two impurity peaks, making accurate quantification impossible. To date, no effective method has been found to separate and simultaneously determine I in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde. 11 and I 12 This report describes analytical methods for these two structurally similar impurities. Therefore, there is an urgent need for a method that can effectively separate impurity I from 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde. 11 and I 12This method enables quality control of 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, overcoming the shortcomings of existing liquid chromatography separation techniques. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde. The method provided by this invention can effectively separate impurity I. 11 I 12 This enables the targeting of specific impurities I 11 I 12 The system offers precise control with high specificity, significantly improved sensitivity, significantly enhanced accuracy, good repeatability, and good durability, meeting the quality control requirements for these two impurities in drug registration review.

[0010] To achieve this objective, the present invention employs the following technical solution: This invention provides a method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, the method comprising the following steps: The sample to be tested is mixed with a solvent to obtain a test solution; the relevant substance standard is mixed with a solvent to obtain an impurity reference standard positioning solution; then the test solution and the impurity reference standard positioning solution are subjected to liquid chromatography detection, and the content of relevant substances in the sample to be tested is obtained according to the detection results. The column packing material for the liquid chromatography detection is pentafluorophenylsilane-bonded silica gel; The relevant substances include impurity I. 11 and impurity I 12 ; The impurity I 11 It is bis((5-(2-fluorophenyl)-1H-pyrrole-3-yl)methyl)amine, wherein the impurity I 12 It is (E)-1-(5-(2-fluorophenyl)-1H-pyrrolo-3-yl)-N-((5-(2-fluorophenyl)-1H-pyrrolo-3-yl)methyl)methylimine. Specifically: The above method can effectively separate impurity I 11 I 12 This enables the targeting of specific impurities I 11 I 12 The system offers precise control with high specificity, significantly improved sensitivity (significantly reduced limits of quantitation and detection, with some impurities having limits of quantitation as low as 0.005 μg / mL and detection limits as low as 0.001 μg / mL), significantly improved accuracy, good repeatability (range of known impurities is 0.01), and good robustness (detection results for known impurities are basically consistent).11 with I 12 It has the characteristics of a separation degree of over 4.0, which meets the quality control requirements for these two impurities in drug registration review.

[0011] In the above method, pentafluorophenylsilane-bonded silica gel is used to selectively separate and target structurally very similar I... 11 and I 12 Effective separation was achieved. In addition to hydrophobic interactions, the pentafluorophenyl stationary phase provides multiple retention mechanisms, including π-π interactions, hydrogen bonding, dipole-dipole interactions, and shape selectivity, enabling it to sensitively identify I... 11 with I 12 The subtle structural differences between them (such as the difference in electron cloud density between amino and imine groups, and the difference in molecular spatial conformation) enable the effective separation of the two.

[0012] The above method uses impurity reference standard positioning solutions for qualitative analysis of related substances, and combines the main component self-reference standard method for quantitative analysis.

[0013] Preferably, the solvent includes anhydrous methanol.

[0014] Preferably, the chromatographic column used for liquid chromatography detection includes any one of Phenomenex Kinetex® F5 100Å, Ailent InfinityLab Poroshell 120Å PFP, or Netw xcsep® PFP 120Å.

[0015] Preferably, the chromatographic column used for liquid chromatography detection is a Phenomenex Kinetex® F5 100Å.

[0016] Preferably, the chromatographic column used for liquid chromatography detection has a size of 4.6 mm × 150 mm and a packing particle size of 2.6 μm.

[0017] The specific chromatographic column described above can effectively separate impurity I. 11 I 12 Other impurity peaks before and after the main peak of the test sample were well separated from the main peak, with a resolution greater than 1.5, achieving complete baseline separation and meeting the requirements for the determination of related substances.

[0018] Preferably, the mobile phase for detection by liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid and mobile phase B is a methanol-acetonitrile mixed solution.

[0019] Preferably, the mass fraction of the phosphoric acid aqueous solution is 0.05-0.12%, such as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, or 0.12%, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0020] Preferably, in the methanol-acetonitrile mixed solution, the volume ratio of methanol to acetonitrile is (0.8-1.2):(0.8-1.2).

[0021] Preferably, the liquid chromatography detection employs a gradient elution program, which is specifically as follows: From 0 to 5 minutes, the volume fraction of mobile phase A changes uniformly from 88-92% to 63-67%, with the remainder being mobile phase B; From min 5 to min 15, the volume fraction of mobile phase A was 63-67%, with the remainder being mobile phase B; Between 15 and 25 minutes, the volume fraction of mobile phase A changed uniformly from 63-67% to 53-57%, with the remainder being mobile phase B. Between 25 and 35 minutes, the volume fraction of mobile phase A changes uniformly from 53-57% to 38-42%, with the remainder being mobile phase B. From 35 to 35.1 min, the volume fraction of mobile phase A changed uniformly from 38-42% to 88-92%, with the remainder being mobile phase B; After 35.1 min, the volume fraction of mobile phase A was 88-92%, with the remainder being mobile phase B.

[0022] Preferably, the liquid chromatography detection uses an ultraviolet detector with a detection wavelength of 265-275 nm, such as 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm or 275 nm, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0023] Preferably, the column temperature for liquid chromatography detection is 20-30℃, such as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, but not limited to the values ​​listed above. Other values ​​not listed above are also applicable, with 23-27℃ being the preferred value.

[0024] Preferably, the flow rate of the mobile phase for liquid chromatography detection is 0.9-1.1 mL / min, such as 0.9 mL / min, 0.95 mL / min, 1 mL / min, 1.05 mL / min or 1.1 mL / min, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0025] Preferably, the injection volume for liquid chromatography detection is 10-25 μL, such as 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, or 25 μL, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, which can effectively separate impurity I. 11 I 12 This enables the targeting of specific impurities I 11 I 12 The accurate control of these impurities exhibits characteristics such as high specificity, significantly improved sensitivity (significantly reduced limits of quantitation and detection, with some impurities having limits of quantitation as low as 0.005 μg / mL and detection limits as low as 0.001 μg / mL), significantly improved accuracy, good repeatability (the range of known impurities is 0.01), and good robustness (the detection results for known impurities are basically consistent), meeting the quality control requirements for these two impurities in drug registration review. Specifically: 1) Using the method of this invention, the separation degree of the characteristic peaks of the two known impurities is not less than 1.5, indicating good separation. 2) The limits of quantitation and detection for the two known impurities mentioned above both showed a significant decreasing trend, with SM1 and impurity I showing the same trend. 11 I 12 The limits of quantitation are all no higher than 0.005 μg / mL, and the limits of detection are all no higher than 0.003 μg / mL; The limit of quantitation (LOQ) can be as low as 0.005 μg / mL, which is only 0.002% of the concentration of the test solution (0.25 mg / mL * 0.002% = 0.005 μg / mL), equivalent to about one percent of the limit concentration. The limit of detection (LOD) can be as low as 0.003 μg / mL, which is only 0.001% of the concentration of the test solution (0.25 mg / mL * 0.001% = 0.003 μg / mL), equivalent to one hundred and fiftyth of the limit concentration, resulting in extremely high detection sensitivity. 3) Within the specified concentration range, 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde reacts with impurity I 11 I 12 The characteristic absorption peak area and its corresponding concentration showed a good linear relationship, with a linear correlation coefficient r > 0.999. Compared with the linear correlation coefficient r > 0.990 required by general standards, the accuracy of its detection method is higher. Experimental results show that SM1 exhibits good linearity in the range of 0.005–1.016 µg / mL; impurity I 11 It exhibits good linearity in the range of 0.005–1.007 µg / mL; impurity I 12 It exhibits good linearity in the range of 0.005–1.012 µg / mL; 4) The recovery rates of all the known impurities were maintained between 90% and 108%, and the maximum RSD of the recovery rate of the 9 samples was 2.1%, with all RSDs less than 5%, indicating good method accuracy; 5) The detection method of the present invention performs repeatability tests on parallel samples, and the range of the detection results for each known impurity is less than 0.02%, demonstrating good repeatability; 6) Within the conditions described in this invention, good and accurate detection results can be achieved, and the measurement results exhibit good and significant robustness within the aforementioned conditions. Furthermore, no related direct and simultaneous separation and detection methods have been found for specific impurities I in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde. 11 I 12 Therefore, this control method is worth promoting.

[0027] This method can accurately control the quality of 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, which is beneficial for the quality control of the final product, the active pharmaceutical ingredient. Attached Figure Description

[0028] Figure 1 This is the chromatogram of the blank solvent in the specificity test of Example 1 of the present invention; Figure 2 This is a chromatogram of the solution used in the specificity test system of Embodiment 1 of the present invention. Figure 3 This is the chromatogram of the test sample solution for the specificity test in Example 1 of the present invention; Figure 4 Specific impurity I in Embodiment 1 of the present invention 12 Positioning chromatogram; Figure 5 Specific impurity I in Embodiment 1 of the present invention 11 Positional chromatogram. Detailed Implementation

[0029] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0030] Example 1: A method for the separation and detection of related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde and specificity test: Blank solvent (diluent): anhydrous methanol.

[0031] Test solution: Take an appropriate amount of the 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde sample to be tested, accurately weigh it, dissolve it in anhydrous methanol and dilute it to prepare a solution containing 0.25 mg per 1 mL.

[0032] Reference solution: Accurately measure 1 mL of the test solution and place it in a 50 mL volumetric flask. Dilute to the mark with solvent and mix well. Then accurately measure 2 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well. This solution is used for quantitative analysis of the main component using the self-reference method.

[0033] Impurity reference standard stock solution: Take impurity I separately 11 Impurity I 12 Accurately weigh appropriate amounts of each reference standard, dissolve and dilute them separately in solvent to prepare a solution containing impurities I per 1 mL. 11 Impurity I 12 50 μg of each reference stock solution.

[0034] Impurity reference standard positioning solution: Accurately measure impurity I separately 11 Impurity I 12 Take 1 mL of each reference stock solution and place them in a 100 mL volumetric flask. Dilute to the mark with solvent and shake well to obtain (0.5 μg / mL) solution for qualitative analysis.

[0035] System suitability solution: Take 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, impurity I 11 Impurity I 12 Accurately weigh an appropriate amount of reference standard, dissolve and dilute it in anhydrous methanol to prepare a solution containing 0.25 mg, 0.5 μg, and 0.5 μg of 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde and impurity I per mL. 11 Impurity I 12 The solution.

[0036] Chromatographic conditions: Instrument: High-performance liquid chromatograph; Chromatographic column: Pentafluorophenylsilane-bonded silica gel as the packing material (Phenomenex Kinetex® 2.6 µm F5100 Å 150 mm × 4.6 mm); Mobile phases: Mobile phase A is a 0.1% aqueous solution of phosphoric acid (v / v), and mobile phase B is a methanol-acetonitrile mixed solution (1:1). Flow rate: 1.0 mL / min; Detection wavelength: 270nm; Injection volume: 20 μL; Column temperature: 25℃; Elution gradient: Specificity experimental procedures and conclusions: Accurately measure 20 μL of each of the above solutions and inject them into the high-performance liquid chromatograph. Record the chromatograms and results. The results are shown in Table 1. Related results are shown in […]. Figure 1-5 .

[0037] Table 1. Results of specificity experiments Conclusion: High-performance liquid chromatography (HPLC) was used to separate the above samples. The experimental results show that the blank solvent does not interfere with the determination of the main peak and known impurities. In the mixed solution, the resolution between the main peak and the adjacent known impurity peak is greater than 1.5; the resolution between the known impurity peak and the main peak is good, and the theoretical plate number is not less than 5000.

[0038] As can be seen from the above experimental results, the determination method provided by the present invention can simultaneously and effectively separate two extremely difficult-to-separate specific impurities, and the chromatographic peaks of 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde and the two known impurities show good separation effect and good specificity.

[0039] Example 2: Selection of Chromatographic Column System suitability solution: Same as in Example 1; Referring to the test conditions of Example 1, the type of chromatographic column was changed: Replace the Phenomenex Kinetex® 2.6 µm F5 100 Å 150 mm × 4.6 mm column in Example 1 with the following: Method 1: The chromatographic column was an Ailent InfinityLab Poroshell 120Å PFP; Method 2: The chromatographic column was a Netw xcsep® PFP 120Å column; Method 3: The chromatographic column used was a Waters XBridge C18. Other chromatographic conditions were the same as in Example 1. 20 μL of system suitability solution was accurately measured for analysis and detection. The test results are shown in Table 2.

[0040] Table 2. Effect of column type on system suitability solution test results When using the Phenomenex Kinetex® F5 100Å column of Example 1 of this invention to separate system suitability solutions (see attached...) Figure 2 Impurity I 11 and impurity I 12 The resolution between the two peaks was 4.1. Other impurity peaks before and after the main peak of the test sample were well separated from the main peak, with a resolution greater than 1.5, achieving complete baseline separation and meeting the requirements for the determination of related substances.

[0041] Other brands and types of chromatographic columns were used, such as the Ailent Infinity LabPoroshell 120Å PFP 4.6mm × 150mm, 2.6μm column used in Example 2-Method 1, with impurity I. 11 and impurity I 12 The chromatographic peaks overlapped, and the method used in Example 2-Method 2 was employed. The chromatogram was Netw xcsep® PFP 120Å, 4.6 × 150 mm, 2.6 μm, with impurity I. 11 and impurity I 12 The chromatographic peaks overlapped. Using Method 3 of Example 2-3 (Waters XBridge C18, 4.6mm × 250mm, 5.0μm), impurity I could not be detected. 11 and impurity I 12 Complete separation was achieved, with two specific impurity peaks coinciding. The separation results were not as good as those achieved using Phenomenex Kinetex® F5 100Å, 2.6μm.

[0042] Example 2-Method 3 uses a chromatographic column different from the pentafluorophenylsilane-bonded silica gel packing. The C18 packing type chromatographic column was used to determine two specific impurities, but it could not completely separate them. The separation effect was significantly different from that of the pentafluorophenyl column, so it is not suitable.

[0043] Example 3 Selection of mobile phase A concentration System suitability solution: Same as in Example 1; Referring to the test conditions of Example 1, the concentration of phosphoric acid in the mobile phase aqueous phase was changed (V / V), and the 0.1% phosphoric acid aqueous solution (v / v) of Example 1 was replaced with the following: Method 1: 0.05% phosphoric acid aqueous solution was used as mobile phase A, and methanol-acetonitrile mixed solution (1:1) was used as mobile phase B; Method 2: 0.12% phosphoric acid aqueous solution was used as mobile phase A, and methanol-acetonitrile mixed solution (1:1) was used as mobile phase B; Other chromatographic conditions were the same as in Example 1. 20 μL of system suitability solution was accurately measured for analysis and detection. The test results are shown in the table below.

[0044] Table 3. Effect of mobile phase concentration on system suitability solution test results Conclusion: When using the mobile phase chromatographic conditions of Example 1 of this invention (aqueous phase, for determining the suitability of the solution for the system), impurity I... 11 and impurity I 12 The resolution between the two peaks was 4.1. Other impurity peaks before and after the main peak of the test sample were well separated from the main peak, with a resolution greater than 1.5, achieving complete baseline separation and meeting the requirements for the determination of related substances.

[0045] Example 3: Mobile phase aqueous concentration of methods 1 and 2, impurity I 11 and impurity I 12 The toxins were well separated, achieving complete baseline separation. However, considering that the acidity was too high with a 0.12% phosphoric acid ratio, and that impurity I was present with a 0.05% phosphoric acid ratio... 12 Given that the resolution of the previous peak is less than 1.5, and considering all other factors, the preferred mobile phase in Example 1 is a phosphoric acid-proportioned aqueous phase, namely a 0.1% phosphoric acid aqueous solution.

[0046] Example 4 Destructive Testing Forced degradation tests are conducted under relatively harsh conditions, such as strong light irradiation, high temperature, high humidity, acid and alkali degradation, hydrolysis, and oxidation, to accelerate sample degradation. The purpose is to evaluate the effectiveness and applicability of the analytical method by examining the separation of degradation products, main peaks, and known impurities. Simultaneously, a photodiode array is used for peak purity checks: in the spectra obtained from the degradation experiment, if the peak purity angles of impurities and the main peak are less than the purity threshold, the assay method is considered to meet the requirements.

[0047] Solution preparation: Solvent (diluent): Anhydrous methanol.

[0048] Undegraded solution: Weigh 12.5 mg of 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the solution; Acid destruction: Accurately weigh 12.5 mg of this product and place it in a 50 mL volumetric flask. Add 1 mL of 1 mol / L hydrochloric acid solution and incubate at 60°C for 4 hours. Neutralize with 1 mL of 1 mol / L sodium hydroxide solution, dilute to the mark with solvent, and shake well. Prepare the acid-base blank solvent using the same method.

[0049] Alkali destruction: Weigh 12.5 mg of this product accurately, place it in a 50 mL volumetric flask, add 1 mL of 1 mol / L sodium hydroxide solution, place at 60 °C for 4 h, add 1 mL of 1 mol / L hydrochloric acid to neutralize, dilute to the mark with solvent, and shake well to obtain the product.

[0050] Oxidative degradation: Accurately weigh 12.5 mg of this product and place it in a 50 mL volumetric flask. Add 1 mL of 30% hydrogen peroxide and let it stand at room temperature for 4 hours. Dilute to the mark with solvent and shake well to obtain the product. Prepare the blank solvent for oxidation using the same method.

[0051] High-temperature destruction: Weigh 25 mg of this product accurately, place it in a 50 mL volumetric flask, place it at 105 °C for 8 hours, remove it, cool it, dissolve it in solvent and dilute it to the mark, shake it well, and use it as the high-temperature destruction sample solution.

[0052] Photodegradation: Accurately weigh 25 mg of this product and place it in a 50 mL volumetric flask. Expose the flask to light at 4500 x l ± 500 x l for 48 hours. Remove the flask, dissolve and dilute it to the mark with solvent, and shake well to obtain the photodegradation sample solution.

[0053] Experimental steps and conclusions: Accurately measure 20 μL of each of the above solutions and inject them into the high-performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. Record the chromatograms and results. The results are shown in the table below.

[0054] Table 4 Destructive Test Results Conclusion: The destructive testing results show that the blank solvent does not interfere with the detection of various impurities. In the chromatograms of the destructive solutions of this product under acid, alkali, oxidation, high temperature, and light-induced destructive conditions, the minimum resolution between the main peak and adjacent peaks is 5.9, and greater than 1.5; the minimum resolution between impurity peaks and adjacent impurity peaks is 4.0, and not less than 1.2; the material balance is between 97.2% and 102.7%, and within the range of 90.0% to 110.0%, the purity of the main peak is 1000, greater than 990, meeting the requirements for the determination of related substances in this product.

[0055] The above results indicate that this analytical method is effective and applicable under various degradation conditions.

[0056] The above content describes the separation of two known impurities in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, with all impurity peaks being separable. Furthermore, the following content describes the quantitative detection of specific impurities involved, enabling the assessment and calculation of the content of specific impurities in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde.

[0057] Example 5 Linearity Test Solution preparation: Solvent (diluent): Anhydrous methanol.

[0058] SM1 reference stock solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing 0.1 mg per 1 mL, shake well, and you will get (100 μg / mL). Impurity I 11 Reference stock solution 1: Take impurity I 11 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Impurity I 12 Reference stock solution 2: Take impurity I 12 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Linear stock solution of mixed impurity reference standard: Accurately measure SM1 reference standard and impurity I separately. 11 Reference standard, impurity I 12 1 mL each of the reference standard and the reference standard stock solution are placed in a 20 mL volumetric flask, diluted to the mark with solvent, and shaken well to obtain the final product. Preparation of linear solutions: Prepare linear solutions of the corresponding concentrations using the linear stock solution according to the table below, and mix thoroughly.

[0059] Table 5. Related substances method validation - preparation of linear solutions Experimental steps and conclusions: Accurately measure 20 μL of each of the above linear solutions and inject them into the high-performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. Record the chromatograms and results. The results are shown in the table below.

[0060] Table 6. Results of linear range determination by related substances method - SM1 Table 7 Results of linear range determination by related substances method - Impurity I 12 Table 8 Results of linear range determination by related substances method - Impurity I 11 in conclusion: Analysis of the above linear solutions revealed the following: Within the concentration range of 0.005 μg / mL to 1.016 μg / mL (equivalent to 1% to 200% of the test sample solution concentration), linear regression was performed with concentration as the abscissa and peak area as the ordinate. The linear regression equation was y = 90.20 x + 0.03, r = 1.0000, indicating a good linear relationship.

[0061] I 12 Within the concentration range of 0.005 μg / mL to 1.012 μg / mL (equivalent to 1% to 200% of the test sample solution concentration), a linear regression was performed with concentration as the abscissa and peak area as the ordinate. The linear equation was y = 115.88 x + 0.16, r = 0.9999, which is greater than 0.990; the linear relationship was good.

[0062] I 11 Within the concentration range of 0.005 μg / mL to 1.007 μg / mL (equivalent to 1% to 200% of the test sample solution concentration), a linear regression was performed with concentration as the abscissa and peak area as the ordinate. The linear equation was y = y = 85.08 x + 0.12, r = 0.9999, which is greater than 0.990; the linear relationship was good.

[0063] The above results indicate that 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde (SM1) and impurity I 12 I 11 Within the linear range, the linear correlation coefficients r were all greater than 0.990, indicating a good linear relationship between peak area and concentration. The optimal linear correlation coefficient r reached 1.0000; compared to the general standard requirement of r > 0.990, the accuracy of the determination was significantly improved.

[0064] Example 6: Limit of Quantitation and Limit of Detection Test Solution preparation: Take 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde (SM1) and impurity I. 12 I 11 The reference standard was prepared into a reference solution, and 20 μL was accurately measured and injected into the liquid chromatograph using a stepwise dilution method. The chromatogram and results were recorded, and the signal-to-noise ratio was calculated. The limit of quantitation was defined as the signal-to-noise ratio of approximately 10 times the baseline noise, and the limit of detection was defined as the signal-to-noise ratio of not less than 3 times the baseline noise.

[0065] Experimental steps and conclusions: Accurately measure 20 μL of each of the above solutions and inject them into the high performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. Record the chromatograms and results. The results are shown in the table below.

[0066] Table 9 Related Substances Method Validation - Limit of Quantitation and Limit of Detection Results - SM1 Table 10 Related Substances Method Validation - Limit of Quantitation and Limit of Detection Results - I 12 Table 11 Related Substances Method Validation - Limit of Quantitation and Limit of Detection Results - I 11 Conclusion: Using the separation conditions described above, the limits of quantitation and detection of this method are as follows: The limits of quantitation and detection for the two known impurities mentioned above showed a significant decreasing trend. 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde and impurity I... 12 I 11 The limits of quantitation for all three substances were not higher than 0.005 μg / mL, specifically 0.005, 0.005, and 0.005 μg / mL; 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde and impurity I 12 I 11 The detection limits were all no higher than 0.003 μg / mL, specifically 0.003, 0.003, and 0.003 μg / mL, respectively. The limit of quantitation (LOQ) can be as low as 0.005 μg / mL, which is only 0.002% of the concentration of the test solution (0.002% of the test solution concentration = 0.25 mg / mL * 0.002% = 0.005 μg / mL), equivalent to about one percent of the limit concentration; the limit of detection (LOD) can be as low as 0.003 μg / mL, which is only 0.001% of the concentration of the test solution (0.003% of the test solution concentration = 0.25 mg / mL * 0.001% = 0.003 μg / mL), equivalent to one hundred and fiftyth of the limit concentration, resulting in very high detection sensitivity. Within a defined concentration range, 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde reacts with known impurity I. 12 I 11 The characteristic absorption peak area and its corresponding concentration exhibit a good linear relationship, with a linear correlation coefficient r > 0.999, where impurity I... 12 I 11 The linear correlation coefficient (r) of SM1 reached 0.9999, and the linear correlation coefficient of SM1 reached 1.0000. Compared with the general standard requirement of a linear correlation coefficient (r) > 0.990, the accuracy of its detection method is higher. The experimental results show that this method can meet the requirements for simultaneous separation and quality control of known impurities in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, with extremely high sensitivity.

[0067] Example 7 Recovery Test Solution preparation: Solvent (diluent): Anhydrous methanol.

[0068] Preparation of background solution: Weigh 12.5 mg of this product accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the solution; Impurity I 11 Reference stock solution 1: Take impurity I 11 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Impurity I 12 Reference stock solution 2: Take impurity I 12 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Impurity reference standard mixed stock solution: Take impurity I separately 11 I 12 Transfer 5 mL of the stock solution to a 20 mL volumetric flask, dilute to the mark with solvent, and mix well to obtain (25 μg / mL). (Divide into two parallel dilutions) Impurity reference solution: Accurately measure 1 mL of the impurity mixed stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with solvent and mix well. (Prepare two parallel portions) Preparation of R1-30% solution Accurately weigh 12.5 mg of this product and place it in a 50 mL volumetric flask. Then, accurately add 0.3 mL of the mixed impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. (Prepare 3 parallel portions) Preparation of R2-100% solution Accurately weigh 12.5 mg of this product and place it in a 50 mL volumetric flask. Then, accurately add 1 mL of the mixed impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. (Prepare 3 parallel portions) Preparation of R3-150% solution Accurately weigh 12.5 mg of this product and place it in a 50 mL volumetric flask. Then, accurately add 1.5 mL of the mixed impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. (Prepare 3 parallel portions) System suitability solution: Weigh 12.5 mg of this product accurately and place it in a 50 mL volumetric flask. Accurately measure 1 mL of the impurity mixed stock solution and place it in the same 50 mL volumetric flask. Dilute to the mark with solvent and shake well to obtain the solution. Experimental steps and conclusions: Accurately measure 20 μL of each of the above-mentioned accuracy test solutions and inject them into the high-performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. Record the chromatograms and results. The results are shown in the table below.

[0069] Table 12 Determination of related substances I 12 Recovery rate test results Table 13 Determination of Related Substances I 11 Recovery rate test results Conclusion: By measuring 50%, 100%, and 150% accuracy solutions (equivalent to limit concentrations), impurity I was found. 12 I 11 The average recovery rates within and between groups were both between 96.49% and 100.33%, with impurity I... 12 I 11 The RSDs for the recoveries were all less than 1.5%, far lower than the standard detection requirements for each impurity (RSD < 10%). The experiments demonstrate that this method is suitable for detecting the aforementioned I... 12 I 11 The accuracy is good.

[0070] Example 8 Precision Test Six sample solutions were repeatedly measured by different technicians on different dates using different instruments. The repeatability test results for the six sample solutions were calculated. An intermediate precision test was performed using the same method, and the test results were calculated. The precision of the 12 data sets was statistically analyzed.

[0071] Solution preparation: Blank solvent (diluent): anhydrous methanol.

[0072] Test solution: Weigh 12.5 mg of this product accurately, place it in a 50 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0073] Self-control solution: Accurately measure 1 mL of the test solution and place it in a 50 mL volumetric flask. Dilute to the mark with solvent and mix well. Then accurately measure 2 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well.

[0074] System suitability solution: Take 12.5 mg of this product and place it in a 50 mL volumetric flask. Accurately add impurity I. 12 I 11 1 mL of the reference stock solution (25 μg / mL) was dissolved and diluted with solvent to a concentration of SM1 and impurity I per mL. 12 I 11The solution is obtained by mixing 0.25 mg, 0.5 μg, and 0.5 μg of the solution respectively.

[0075] Experimental steps and conclusions: Accurately measure 20 μL of each of the above sample solutions and inject them into the high performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. Record the chromatograms and results. The results are shown in the table below.

[0076] Table 14 Results of Precision Test for Related Substances Determination Conclusion: 6 repeatable samples, impurity I. 12 Impurity I 11 The content was between 0.1% and 1.0%, with RSDs of 1.2% and 1.5%, both less than 3%; the intermediate precision was achieved in 6 samples, with impurity I... 12 Impurity I 11 The content was between 0.1% and 1.0%, with RSDs of 0.54% and 0.52%, respectively, both less than 3%; precision was achieved in 12 samples, with impurity I... 12 Impurity I 11 With a content between 0.1% and 1.0%, the RSDs were 3.9% and 2.5%, respectively, both less than 6%, meeting the acceptable standard. This method has good precision.

[0077] Example 9 Solution stability test Test solution: Weigh 12.5 mg of this product accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and use it as the test solution; Control solution: Accurately measure 1 mL of the test solution and place it in a 50 mL volumetric flask. Dilute to the mark with solvent and mix well. Then accurately measure 2 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well to obtain the control solution. Spiked test solution: Take the sample with the highest recovery rate (100%) as the test solution; The above sample solution was placed at 5°C for refrigeration and measured at different time points. 20 μL was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in the table below.

[0078] Table 15 Results of SM1 related substance solution stability tests Table 16 Stability results of spiked test solutions under refrigeration at 5℃ Table 17 Stability results of the self-control solution under refrigeration at 5℃ Conclusion: The results show that after the test solution was placed at 5℃ for 47 hours, the change in the peak area of ​​the main component compared with 0h was the largest at 3.0% and less than 5.0%, while the change in the peak area of ​​the impurities compared with 0h was the largest at 15.9% and less than 20.0%. This indicates that the test solution has good stability after being placed at 5℃ for 47 hours.

[0079] When the spiked test solution was stored at 5°C for 42 hours, the maximum change in peak area at each time point compared to 0h was 1.8%, and all were less than 5.0%, indicating that the spiked test solution had good stability when stored at 5°C for 42 hours.

[0080] The self-control solution, after being stored at 5°C for 47 hours, showed the largest change in peak area at each time point compared to 0h, which was 3.9%, less than 5.0%; indicating that the control solution exhibited good stability after being stored at 5°C for 47 hours.

[0081] Example 10 Durability Test The system's suitability and tolerance to changes in measurement results were examined by modifying method parameters. The robustness of this product was investigated by varying the column temperature (±5°C), flow rate (±0.1 mL / min), wavelength (±2 nm), and different batches of columns from the same manufacturer, based on the detection conditions of Example 1, on the results and specific impurities. Solution preparation and other chromatographic conditions were the same as in Example 1, and the robustness results are shown below.

[0082] Solution preparation: Blank solvent (diluent): anhydrous methanol.

[0083] Test solution: Weigh 12.5 mg of this product accurately, place it in a 50 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0084] Self-control solution: Accurately measure 1 mL of the test solution and place it in a 50 mL volumetric flask. Dilute to the mark with solvent and mix well. Then accurately measure 2 mL of the solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and mix well.

[0085] Impurity I 11 Reference stock solution 1: Take impurity I 11 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Impurity I 12 Reference stock solution 2: Take impurity I 12 Weigh an appropriate amount of the reference standard accurately, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing 0.1 mg per 1 mL. Shake well to obtain (100 μg / mL). Impurity reference standard mixed stock solution: Take impurity I separately 11 I 12 Take 5 mL of the stock solution and place it in a 20 mL volumetric flask. Dilute to the mark with solvent and shake well to obtain (25 μg / mL).

[0086] System suitability solution: Take 12.5 mg of this product and place it in a 50 mL volumetric flask. Accurately add impurity I. 12 I 11 1 mL of the reference stock solution (25 μg / mL) was dissolved and diluted with solvent to a concentration of SM1 and impurity I per mL. 12 I 11 The solution is obtained by mixing 0.25 mg, 0.5 μg, and 0.5 μg of the solution respectively.

[0087] Table 18 Changes in robustness parameters for related substances method validation Table 19 Durability - Results of Mixed Solution Separation Table 20 Results of the test on the spiked test solution for durability Conclusion: Robustness tests of columns with varying flow rates, column temperatures, and wavelengths, and using different batches of columns, showed that impurity I... 12 and impurity I 11 The minimum resolution of the impurity peak is 1.9, which is greater than 1.2. In the test solution, impurity I... 12 and impurity I 11 The maximum RSD of the content is 10.2%, which is less than 15.0%, meeting the acceptable standard for robustness testing. This indicates that the method disclosed herein for determining two specific, extremely difficult-to-separate impurities in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde exhibits good robustness.

[0088] Comparative Example 1 Referring to the detection method in the prior art, two impurities (Impurity I) in the test solution and the mixed impurity reference solution of the present invention were tested. 12 and impurity I 11 Separation and determination were performed.

[0089] Prior art document 1: Publication patent CN116953129A; Prior art document 2: Publication patent CN118817887A; Reference document 3: Publication patent CN117924141A.

[0090] Table 21 Comparison of chromatographic conditions between this application and prior art documents Table 22 Comparison of chromatographic conditions between this application and prior art documents Referring to the detection conditions and methods of comparative documents 1, 2 and 3, the two specific impurities in Example 1 of the present invention were analyzed and detected, and the results are shown in the table below.

[0091] Table 23 Comparison of test results between this application and prior art documents Conclusion: Under the detection conditions of Example 1 of the present invention, two specific impurities (Impurity I and Impurity II) with extremely similar properties can be completely separated simultaneously. 11 Impurity I 12 The resolution is greater than 1.5, the resolution with unknown impurities is not less than 1.5, the resolution between the main peak and adjacent impurity peaks is good, and all known impurities can be eluted under these conditions and can be effectively separated and detected.

[0092] Under the detection conditions of comparative documents 1, 2, and 3, impurity I 11 Impurity I 12 The impurities overlap, and at wavelengths of 230 nm and 245 nm, impurity I... 11 Impurity I 12 The response values ​​are all less than those at a wavelength of 270 nm, making it impossible to accurately quantify the two specific impurities simultaneously. Therefore, directly using the chromatographic methods and mobile phase conditions of comparative documents 1, 2, and 3 cannot accurately quantify the two specific impurities I. 11 Impurity I 12 .

[0093] summary: Compared to other detection methods, only the detection method of this invention (e.g., Example 1) can distinguish the main peak from other impurities, as well as two known impurities (I 11 Impurity I 12 The quantitative detection of ) showed good separation effect.

[0094] Conversely, using other detection methods with conditions closest to these (Comparison File 1, Comparison File 2, Comparison File 3) cannot separately detect the two known impurities (I... 11 Impurity I 12 Quantitative detection of ).

[0095] The applicant declares that this invention illustrates the method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for separating and detecting related substances in 5-(2-fluorophenyl)-1H-pyrrole-3-carboxaldehyde, characterized in that, The separation and detection method includes the following steps: The sample to be tested is mixed with a solvent to obtain a test solution; the relevant substance standard is mixed with a solvent to obtain an impurity reference standard positioning solution; then the test solution and the impurity reference standard positioning solution are subjected to liquid chromatography detection, and the content of relevant substances in the sample to be tested is obtained according to the detection results. The column packing material for the liquid chromatography detection is pentafluorophenylsilane-bonded silica gel; The relevant substances include impurity I. 11 and impurity I 12 ; The impurity I 11 It is bis((5-(2-fluorophenyl)-1H-pyrrole-3-yl)methyl)amine, wherein the impurity I 12 It is (E)-1-(5-(2-fluorophenyl)-1H-pyrrolo-3-yl)-N-((5-(2-fluorophenyl)-1H-pyrrolo-3-yl)methyl)methyleneimine.

2. The separation and detection method according to claim 1, characterized in that, The solvent includes anhydrous methanol.

3. The separation and detection method according to claim 1 or 2, characterized in that, The chromatographic column used for the liquid chromatography detection includes any one of Phenomenex Kinetex® F5 100Å, Ailent InfinityLab Poroshell 120Å PFP, or Netw xcsep® PFP 120Å.

4. The separation and detection method according to claim 3, characterized in that, The chromatographic column used for the liquid chromatography detection was a Phenomenex Kinetex® F5 100Å.

5. The separation and detection method according to claim 4, characterized in that, The chromatographic column used in the liquid chromatography detection has a size of 4.6 mm × 150 mm and a packing particle size of 2.6 μm.

6. The separation and detection method according to any one of claims 1-4, characterized in that, The mobile phase for the liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid and mobile phase B is a methanol-acetonitrile mixed solution; Preferably, the mass fraction of the phosphoric acid aqueous solution is 0.05-0.12%; Preferably, in the methanol-acetonitrile mixed solution, the volume ratio of methanol to acetonitrile is (0.8-1.2):(0.8-1.2).

7. The separation and detection method according to claim 6, characterized in that, The liquid chromatography detection employs a gradient elution program, which is detailed below: From 0 to 5 minutes, the volume fraction of mobile phase A changes uniformly from 88-92% to 63-67%, with the remainder being mobile phase B; From min 5 to min 15, the volume fraction of mobile phase A was 63-67%, with the remainder being mobile phase B; Between 15 and 25 minutes, the volume fraction of mobile phase A changed uniformly from 63-67% to 53-57%, with the remainder being mobile phase B. Between 25 and 35 minutes, the volume fraction of mobile phase A changes uniformly from 53-57% to 38-42%, with the remainder being mobile phase B. From 35 to 35.1 min, the volume fraction of mobile phase A changed uniformly from 38-42% to 88-92%, with the remainder being mobile phase B; After 35.1 min, the volume fraction of mobile phase A was 88-92%, with the remainder being mobile phase B.

8. The separation and detection method according to any one of claims 1-7, characterized in that, The liquid chromatography detection uses an ultraviolet detector with a detection wavelength of 265-275 nm.

9. The separation and detection method according to any one of claims 1-8, characterized in that, The column temperature for the liquid chromatography detection is 20-30℃, preferably 23-27℃; Preferably, the flow rate of the mobile phase for liquid chromatography detection is 0.9-1.1 mL / min.

10. The separation and detection method according to any one of claims 1-9, characterized in that, The injection volume for the liquid chromatography detection is 10-25 μL.

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