Method for separating and determining melogabalin besylate key intermediate and related impurities thereof by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining melogabalin besylate key intermediates and related impurities by HPLC (High Performance Liquid Chromatography), gradient elution is carried out by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler, a perchloric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and detection is carried out in a detector. According to the method, the melogabalin besylate key intermediate and related impurities can be effectively separated, and the method has the advantages of high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable result, and is of great significance for realizing melogabalin besylate drug quality control.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and relates to a method for detecting a melogabalin besylate intermediate, in particular to an HPLC method for separating and determining a key melogabalin besylate intermediate and related impurities. Background Art

[0002] Mirogabalin besylate is a GABA analog that acts on the α2-δ1 subunit of the voltage-sensitive calcium channel complex (widely present in the nervous system that mediates pain transmission and processing throughout the body), thereby playing a role in treating chronic neuropathic pain. In January 2019, the product was first approved for marketing in Japan for the treatment of peripheral neuropathic pain (PNP). Later, Daiichi Sankyo expanded its indications to central neuropathic pain (CNP) in March 2022. In June 2024, Mirogabalin besylate tablets were approved for marketing in my country. The chemical name of Mirogabalin besylate is (1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-ene-6-acetic acid benzenesulfonate, and its chemical structure is as follows:

[0003]

[0004] The compound of formula I is an important chiral intermediate for the synthesis of melogabalin benzenesulfonate, and its chemical name is 2-((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl)acetic acid tert-butyl mandelate. The compound of formula I is subjected to esterification to remove the tert-butyrate group and then to salt formation to obtain melogabalin benzenesulfonate. Therefore, the control of related substances of the compound of formula I is one of the key factors for the quality control of impurities in the final product melogabalin benzenesulfonate. If the related impurities of the intermediate are not controlled, they will remain in the finished product or derive related impurities of melogabalin. If good technical means are not used to control the related substances of the intermediate, the quality of the finished product will be affected. The chemical structure of the compound of formula I is as follows:

[0005]

[0006] The analysis and research of the key intermediates of melogabalin benzenesulfonate plays a vital role in the control of the reaction process, the control of impurities and the improvement of the quality of the finished melogabalin benzenesulfonate product.

[0007] Chinese patent CN202111622394.0 reported that 0.05% phosphoric acid aqueous solution-acetonitrile was used as the mobile phase, with a ratio of 60:40, for gradient elution, which was used to separate the starting material (1R, 5S)-3-ethylbicyclo[3.2.0]hept-3-ene-6-one and its enantiomers in the synthesis process of melogabalin. Chinese patent CN202410259310.9 reported that normal phase chromatography was used, with a chiral column coated with a polysaccharide derivative as the chromatographic column, and a mixed solution of n-hexane and alcohol solution as the mobile phase, to separate and detect (1R, 5S)-3-ethylbicyclo[3.2.0]hept-3-ene-6-one and its enantiomers. Chinese patent CN202111310371.6 reports a method for detecting melogabalin and its enantiomeric impurities by high performance liquid chromatography, using ammonium acetate buffer solution-isopropanol with a concentration of 5-30 mMol / L as the mobile phase for isocratic elution. However, so far, no literature has reported a separate detection method for the melogabalin intermediate shown in formula I, and no public method has reported a method for simultaneously separating and determining the melogabalin intermediate shown in formula I and its related substances. Therefore, it is necessary to establish a simple and effective method for quality control of the intermediate.

[0008] Generally speaking, the total impurity content of a drug should be less than 1.0%, and the content of a single impurity should be less than 0.1%. Impurities generated during the preparation of melogabalin besylate or related substances introduced must be strictly controlled in the raw materials. 2-((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl)acetic acid tert-butyl mandelate is a key intermediate of melogabalin besylate. The quality control of this intermediate plays an important role in the preparation of high-quality melogabalin besylate raw materials. Therefore, developing an efficient and accurate analytical method to analyze the quality of the key intermediate of melogabalin besylate is of great significance for the subsequent preparation of high-purity melogabalin besylate products. Summary of the invention

[0009] In view of this, the object of the present invention is to provide a method for separating and determining the key intermediate of melogabalin besylate and its related impurities by HPLC; the method of the present invention can effectively separate the key intermediate of melogabalin besylate and its related impurities, and the method has high sensitivity and separation degree, good repeatability and durability, simple operation, and stable and reliable results.

[0010] To achieve the above purpose, the applicant has conducted a large number of experiments and determined that the technical solution of the present invention is:

[0011] A method for separating and determining a key intermediate of melogabalin besylate and its related impurities by HPLC, wherein the chromatographic column used in the method is filled with octadecylsilane bonded silica gel, and mobile phase A and mobile phase B are used for gradient elution, and the mixture is entered into a detector for detection; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H, and the specific impurity names are as follows:

[0012]

[0013]

[0014] The mobile phase A is a perchloric acid aqueous solution, and the mobile phase B is an organic solvent;

[0015] The key intermediate of melogabalin benzenesulfonate is 2-((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl)acetic acid tert-butyl mandelate, which is a key chiral intermediate for synthesizing melogabalin benzenesulfonate, and its chemical formula is C 16 H 27 NO2·C8H8O3, with the structural formula as follows, is also referred to as "the compound of formula I" or "the key intermediate of melogabalin besylate" in the present invention:

[0016]

[0017] Further, the concentration of perchloric acid in the perchloric acid aqueous solution is 0.3% to 0.5%;

[0018] As a preferred embodiment, the perchloric acid concentration in the perchloric acid aqueous solution is 0.4%;

[0019] Further, the pH value of the perchloric acid aqueous solution is adjusted to 2.3-2.7 with sodium hydroxide solution;

[0020] As a preferred embodiment, the pH value of the perchloric acid solution is adjusted to 2.5 with sodium hydroxide solution;

[0021] Further, the mobile phase B is one or more of acetonitrile, ethanol and methanol;

[0022] Furthermore, the mobile phase B is acetonitrile.

[0023] The flow rate of the mobile phase for elution is 1.0-1.4 ml / min, preferably 1.2 ml / min.

[0024] The gradient elution was set up as follows:

[0025]

[0026] As a preference, the gradient elution is set as follows:

[0027]

[0028] Furthermore, the filler particles of the octadecylsilane bonded silica gel have a particle size of 2-5 μm; and the column temperature of the chromatographic column is 30-40°C.

[0029] As a preference, the filler particles of the octadecylsilane bonded silica gel have a particle size of 3.5 μm; and the column temperature of the chromatographic column is 35° C.

[0030] Furthermore, the detection wavelength of the detector is 205±5 nm.

[0031] Preferably, the detection wavelength of the detector is 205 nm.

[0032] Further, the method for separating and determining the key intermediate of melogabalin besylate and its related impurities by HPLC, wherein the related impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, specifically comprises the following steps:

[0033] 1) Prepare the test solution: dissolve the test sample in the diluent to make a solution containing about 1 mg per 1 ml to obtain the test solution;

[0034] 2) Prepare control solution: Take an appropriate amount of the test sample solution and dilute it with a diluent to make a solution containing about 10 μg per 1 ml as the control solution.

[0035] 3) Preparation of system suitability solution: Take appropriate amounts of the key intermediate of melogabalin besylate and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, accurately weigh them, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare a solution containing about 1.0 mg of the compound of formula I, about 5 μg of impurity A, about 2 μg of impurities B, impurity C, impurity D, impurity E and impurity F, and about 1.5 μg of impurities G and impurity H per 1 ml;

[0036] 4) Inject the system suitability solution described in step 3) and perform high performance liquid chromatography analysis, record the chromatogram, determine the retention time of the key intermediate of melogabalin besylate and its impurities, and then respectively inject the test solution described in step 1) and the control solution described in step 2), and calculate the content of impurities in the key intermediate of melogabalin besylate in the test solution according to the principal component self-reference method.

[0037] 5) The linear correlation coefficients of the key intermediate of melogabalin besylate and its impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H are shown in the following table:

[0038] name Concentration range (μg / ml) Linear equations Correlation coefficient (r) Compounds of formula I 0.3103～14.9180 y=10.6481x+0.4608 0.9999 Impurity A 0.0662～14.6953 y=22.9890x+0.4217 1.0000 Impurity B 0.0962～5.5508 y=25.7557x-0.0433 0.9999 Impurity C 0.1261～4.8508 y=16.0527x+0.4323 0.9998 Impurity D 0.1424～5.4777 y=9.7175x+0.0650 1.0000 Impurity E 0.0736～5.6652 y=19.7843x+0.7186 1.0000 Impurity F 0.0743～5.7117 y=27.6326x+1.2515 0.9999 Impurity G 0.0873～4.1959 y=14.5799x+0.2110 1.0000 Impurity H 0.0785～4.5260 y=19.7765x+0.1906 1.0000

[0039] The solvent is a solution of acetonitrile-water (volume ratio is 50:50).

[0040] The second object of the present invention is to provide a reagent composition for solid-liquid separation and determination of key intermediates of melogabalin besylate and its impurities, which is composed of the following reagents:

[0041] Reagent A: aqueous perchloric acid solution;

[0042] Reagent B: organic solvent;

[0043] The relevant impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H;

[0044] The perchloric acid concentration in the perchloric acid aqueous solution is 0.3% to 0.5%; the organic solvent is one or more of acetonitrile, ethanol and methanol.

[0045] Further, the pH value of the perchloric acid aqueous solution is adjusted to 2.3-2.7 with sodium hydroxide solution;

[0046] As a preferred embodiment, the perchloric acid concentration in the perchloric acid aqueous solution is 0.4%; the pH value of the perchloric acid aqueous solution is adjusted to 2.5 with sodium hydroxide solution; and the organic solvent is acetonitrile.

[0047] The reagent composition for solid-liquid separation and determination of a key intermediate of melogabalin besylate and its impurities provided by the present invention can effectively separate the key intermediate of melogabalin besylate and its impurities, and is of great significance for achieving quality control of the key intermediate of melogabalin besylate and melogabalin besylate.

[0048] The beneficial effects of the present invention are:

[0049] 1) The present invention provides a method for separating and determining a key intermediate of melogabalin besylate and its related impurities by HPLC. The method of the present invention can effectively separate the key intermediate of melogabalin besylate and its impurities AH, and the separation degree between the main peak and the adjacent peak and the separation degree between each impurity are greater than 1.5. At the same time, the method has high sensitivity and separation degree, good repeatability and durability, simple operation, stable and reliable results.

[0050] 2) It has been found through research that the impurities C, D, E and G of the key intermediate of formula I are very similar in structure to the key intermediate. If they are not controlled at this step, they will be difficult to remove if they are brought into the raw material drug during the continued reaction. Therefore, each impurity needs to be strictly monitored in this step of the reaction. That is, the analysis and research of the key intermediate of melogabalin benzenesulfonate in the present invention plays a vital role in the control of the synthesis reaction and the improvement of the quality, and also directly affects the quality of the melogabalin benzenesulfonate finished product. Therefore, this method is of great significance for achieving the quality control of the key intermediate of melogabalin benzenesulfonate and melogabalin benzenesulfonate. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The chromatogram of the system suitability solution under the conditions of Example 1 of the present invention shows that the order of peaks is impurity A (retention time Rt12.317), impurity B (retention time Rt14.250), compound of formula I (retention time Rt14.817), impurity C (retention time Rt15.399), impurity D (retention time Rt16.518), impurity E (retention time Rt18.885), impurity F (retention time Rt19.241), impurity G (retention time Rt19.927), and impurity H (retention time Rt20.601);

[0052] Figure 2 System suitability solution chromatogram under the conditions of Control Example 1;

[0053] Figure 3 System suitability solution chromatogram under the conditions of Control Example 2;

[0054] Figure 4 System suitability solution chromatogram under the conditions of Control Example 3. DETAILED DESCRIPTION

[0055] The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods for which specific conditions are not specified in the preferred embodiments are usually carried out under conventional conditions. The examples are provided to better illustrate the contents of the present invention, but the contents of the present invention are not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the embodiments according to the above-mentioned invention contents, which still fall within the scope of protection of the present invention. The key intermediate of melogabalin besylate in this embodiment refers to a compound of formula I.

[0056] Embodiment 1

[0057] 1Chromatographic conditions:

[0058] Chromatographic column: Waters Xbridge C18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: 0.4% perchloric acid aqueous solution (pH adjusted to 2.5 with sodium hydroxide solution), mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0059]

[0060] Flow rate: 1.2 ml / min, column temperature: 35°C, detection wavelength: 205 nm, injection volume: 10 μl, solvent: acetonitrile-water (50:50).

[0061] 2 Methods and Results

[0062] 2.1 Solution preparation

[0063] Solvent: acetonitrile-water (50:50)

[0064] Test solution: Take an appropriate amount of the key intermediate of melogabalin besylate, accurately weigh it, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 1.0 mg per 1 ml.

[0065] Control solution: Accurately measure an appropriate amount of the test sample solution and dilute it with solvent to make a solution containing approximately 10 μg per 1 ml.

[0066] 2.2 Exclusivity

[0067] System suitability solution: Take appropriate amounts of the key intermediate of melogabalin besylate and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, accurately weigh them, place them in the same volumetric flask, add solvent to dissolve and dilute them to make a solution containing about 1.0 mg of the compound of formula I, about 5 μg of impurity A, about 2 μg of impurity B, impurity C, impurity D, impurity E and impurity F, and about 1.5 μg of impurity G and impurity H per 1 ml.

[0068] Accurately measure 10 μl of system suitability solution and inject it into the liquid chromatograph. Record the chromatogram. The results of system suitability solution are shown in the attached Figure 1 In the system suitability solution, the minimum resolution of the key intermediate of melogabalin besylate and its adjacent impurity peaks was 2.0, and the retention time Rt was 14.817.

[0069] 2.3 Repeatability

[0070] Spiked test solution: Take an appropriate amount of the key intermediate of melogabalin benzenesulfonate and its impurity reference substance, dissolve in solvent and dilute to make a solution containing about 1.0 mg of the compound of formula I, about 5 μg of impurity A, about 2 μg of impurities B, C, D, E and F, and about 1.5 μg of impurities G and H per 1 ml. (Prepare 6 portions in parallel)

[0071] Control solution: Accurately measure 1 ml of the spiked test solution and place it in a 100 ml volumetric flask, and dilute it with solvent to make a solution containing about 10 μg of the compound of formula I per 1 ml.

[0072] Accurately measure the above-mentioned spiked test solution and control solution and inject them separately, and record the chromatogram. Calculate the relative standard deviation (RSD) of the content of each related substance in the 6 spiked test solutions by the principal component self-reference method with correction factor. The RSD of the content of each related substance in the 6 spiked test solutions is less than 10.0%, which meets the requirements of high performance liquid chromatography for the detection of related substances.

[0073] 2.4 Linearity

[0074] Take an appropriate amount of the key intermediate of melogabalin besylate and its impurity reference substance, dissolve and dilute with a solvent to make a solution containing about 100 μg of the compound of formula I, about 100 μg of impurity A, about 40 μg of impurity B, about 40 μg of impurity C, about 40 μg of impurity D, about 40 μg of impurity E, about 40 μg of impurity F, about 30 μg of impurity G, and about 30 μg of impurity H per 1 ml as a linear stock solution. Measure 0.5 ml of the stock solution into 20 ml, 2 ml into 50 ml, 1 ml into 20 ml, 2 ml into 20 ml, and 3 ml into 20 ml volumetric bottles, dilute to the scale with solvent, and shake well; take another quantitative limit solution as a linear test solution. Linear regression is performed on the concentration using the peak area to obtain a linear equation (Table 1). The key intermediate of melogabalin besylate and its impurities have a good linear relationship within the linear range.

[0075] Table 1 Linear determination results

[0076]

[0077]

[0078] 2.5 Limit of detection and limit of quantification

[0079] Take appropriate amounts of the key intermediate of melogabalin besylate and its impurity reference substance to prepare a series of solutions. When S / N ≥ 10, it is used as the quantitative limit solution, and when S / N ≥ 3, it is used as the detection limit solution. The quantitative limit and detection limit results of the key intermediate of melogabalin besylate and its impurities are shown in Table 2.

[0080] Table 2 Results of limit of quantitation and limit of detection

[0081]

[0082] 2.6 Accuracy

[0083] Take appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, dissolve and dilute with solvent to make a solution containing about 50μg of impurity A, about 20μg of impurity B, about 20μg of impurity C, about 20μg of impurity D, about 20μg of impurity E, about 20μg of impurity F, about 15μg of impurity G and about 15μg of impurity H per 1ml, as impurity mixed reference substance stock solution; take appropriate amount of key intermediate of melogabalin besylate, accurately measure 1ml of impurity mixed reference substance stock solution into 100ml, 0.5ml into 10ml, 1ml into 10ml, 1.5ml into 10ml volumetric flask, dilute to scale with solvent, shake well, as recovery test solution, and conduct 3 replicates for each concentration. Calculate the recovery rate and RSD of each impurity by the principal component self-control method with correction factor. The results showed that the recovery rates of each impurity at each concentration were between 80.0% and 120.0%, and the RSDs were all less than 10.0%, which met the requirements of HPLC for the detection of related substances.

[0084] 3 Conclusion:

[0085] Under the chromatographic conditions, the key intermediate of melogabalin besylate and its impurities can be completely separated. This method has strong specificity, good accuracy, high sensitivity, good repeatability, and good system applicability. It meets the technical requirements of drug quality research standards, and the results obtained are stable and reliable.

[0086] Comparative Example 1:

[0087] 1Chromatographic conditions:

[0088] Chromatographic column: Waters Xbridge C18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: 0.2% perchloric acid aqueous solution (pH adjusted to 4.0 with sodium hydroxide solution), mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0089]

[0090] Flow rate: 1.0 ml / min, column temperature: 40°C, detection wavelength: 205 nm, injection volume: 10 μl, solvent: acetonitrile-water (50:50).

[0091] 2 Method: Take appropriate amounts of the key intermediate of melogabalin besylate and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, accurately weigh them, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a solution containing about 1.0 mg of the compound of formula I, about 5 μg of impurity A, about 2 μg of impurity B, impurity C, impurity D, impurity E, impurity F, and about 1.5 μg of impurity G and impurity H per 1 ml, as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the system suitability solution chromatogram is attached. Figure 2 .

[0092] 3 Conclusion: Under the chromatographic conditions, impurity B overlaps with the key intermediate of melogabalin besylate (retention time Rt17.594), which affects the quantitative result and qualitative judgment of impurity B. Impurity F (retention time Rt24.774, 24.956) and impurity G (retention time Rt27.515, 27.672) have poor peak shapes and cannot be quantified.

[0093] Comparative Example 2:

[0094] 1Chromatographic conditions:

[0095] Chromatographic column: Waters Xbridge C18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: 0.5% perchloric acid aqueous solution (pH adjusted to 9.0 with sodium hydroxide solution), mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0096]

[0097] Flow rate: 1.0 ml / min, column temperature: 40°C, detection wavelength: 205 nm, injection volume: 10 μl, solvent: acetonitrile-water (50:50).

[0098] 2 Method: The method is the same as that of control example 1. The system suitability solution chromatogram is attached. Figure 3 .

[0099] 3 Conclusion: Under the chromatographic conditions, impurity C overlaps with the key intermediate of melogabalin besylate (retention time Rt18.011), which affects the quantitative result and qualitative judgment of impurity C. Impurity F (retention time Rt24.62, 24.801) and impurity G (retention time Rt27.359) have poor peak shapes and cannot be quantified.

[0100] Comparative Example 3:

[0101] 1Chromatographic conditions:

[0102] Chromatographic column: Waters Xbridge C18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: 0.3% perchloric acid aqueous solution (pH adjusted to 4.0 with sodium hydroxide solution), mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0103]

[0104] Flow rate: 1.0 ml / min, column temperature: 40°C, detection wavelength: 205 nm, injection volume: 10 μl, solvent: acetonitrile-water (50:50).

[0105] 2 Method: Take appropriate amounts of the key intermediate of melogabalin besylate and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and impurity H reference substances, accurately weigh them, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a solution containing about 1.0 mg of the compound of formula I, about 5 μg of impurity A, about 2 μg of impurity B, impurity C, impurity D, impurity E, impurity F, and about 1.5 μg of impurity G and impurity H per 1 ml, as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the system suitability solution chromatogram is attached. Figure 4 .

[0106] 3 Conclusion: Under the chromatographic conditions, the separation degree between impurity B (retention time Rt17.609) and the key intermediate of melogabalin besylate (retention time Rt17.92) was less than 1.5, and the baseline separation was not achieved, which affected the quantitative result and qualitative judgment of impurity B. The peak shapes of impurity F (retention time Rt24.904, 25.087) and impurity G (retention time Rt27.664) were poor and could not be quantified.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting a key intermediate of melogabalin benzenesulfonate, the method is a method for separating and determining the key intermediate of melogabalin benzenesulfonate and its related impurities by HPLC, the chromatographic column used is octadecylsilane bonded silica gel as a filler, mobile phase A and mobile phase B are used for gradient elution, and the mixture enters a detector for detection; the related impurities include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H, and the specific impurity names are as follows: The mobile phase A is a perchloric acid aqueous solution, and the mobile phase B is an organic solvent; The key intermediate of melogabalin benzenesulfonate is 2-((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl)acetic acid tert-butyl mandelate, which is a key intermediate for synthesizing melogabalin benzenesulfonate, and its chemical formula is C 16 H 27 NO2·C8H8O3, the structural formula is as follows: Formula (I), which is also referred to as "the compound of Formula I" or "the key intermediate of melogabalin besylate" in the present invention: The gradient elution was set up as follows: The flow rate of the mobile phase for elution is 1.0-1.4 ml / min.

2. The method according to claim 1, characterized in that The mobile phase A is a perchloric acid aqueous solution, and the mobile phase B is acetonitrile.

3. The method according to claim 2, characterized in that The concentration of perchloric acid in the perchloric acid aqueous solution is 0.3% to 0.5%.

4. The method according to claim 3, characterized in that: The concentration of perchloric acid in the perchloric acid aqueous solution is 0.4%.

5. The method according to claim 2, characterized in that: The pH value of the perchloric acid aqueous solution is adjusted to 2.3-2.7 with sodium hydroxide solution.

6. The method according to claim 3, characterized in that The pH value of the perchloric acid aqueous solution was adjusted to 2.5 with sodium hydroxide solution.

7. The method according to claim 1, characterized in that The gradient elution was set up as follows:

8. The method according to claim 1, characterized in that: The filler particles of the octadecylsilane bonded silica gel have a particle size of 2-5 μm, and the column temperature of the chromatographic column is 30-40° C.

9. The method according to claim 1, characterized in that: The detection wavelength of the detector is 205±5 nm.

10. The method according to claim 1, characterized in that The relevant impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H reference substances, and specifically include the following steps: 1) Prepare the test solution: dissolve the test sample in the diluent to make a solution containing about 1 mg per 1 ml to obtain the test solution; 2) Prepare control solution: Take an appropriate amount of the test solution and dilute it with a diluent to make a solution containing about 10 μg per 1 ml as the control solution; 3) Preparation of system suitability solution: Take the key intermediate of melogabalin besylate and its impurities A, B, C, D, E, F, G and H, and dissolve and dilute them with a diluent to prepare a system suitability solution; 4) sampling the system suitability solution described in step 3), performing high performance liquid chromatography analysis, recording the chromatogram, determining the retention time of the key intermediate of melogabalin besylate and its impurities, and then respectively sampling the test solution described in step 1) and the control solution described in step 2), and calculating the content of impurities in the key intermediate of melogabalin besylate in the test solution by the principal component self-reference method; The diluent is a solution of acetonitrile-water (volume ratio is 50:50).

Images