A method for determining isomers in carfilzomib and its preparations by reversed phase liquid chromatography

Abstract

The present invention provides a method for determining isomers in carfilzomib and its preparations by reverse phase liquid chromatography, and belongs to the technical field of drug analysis. The present invention adopts high performance liquid chromatography to determine isomers in carfilzomib and its preparations, and the chromatographic conditions are as follows: chromatographic column: octylsilane bonded silica gel as filler; mobile phase: sulfobutyl-β-cyclodextrin sodium aqueous solution-acetonitrile as mobile phase. The chromatographic column used in the present invention is a conventional chromatographic column, which is inexpensive and has a longer life; the chiral separation agent added to the mobile phase has a lower cost than special chiral reagents. Through systematic methodological verification, the results show that the method of the present invention has good specificity, sensitivity, accuracy, repeatability, durability, etc., and is suitable for the detection of chiral isomers in carfilzomib and its preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining isomers in carfilzomib and a preparation thereof by reversed-phase liquid chromatography. Background Art

[0002] Carfilzomib is a tetrapeptide epoxyketone-based irreversible inhibitor of the chymotrypsin-like activity of the 20S proteasome. Carfilzomib, a formulation of carfilzomib prepared from carfilzomib, is indicated in combination with dexamethasone for the treatment of adult patients with relapsed or refractory multiple myeloma who have received at least two prior therapies, including a proteasome inhibitor and an immunomodulatory agent.

[0003] Carfilzomib is poorly soluble in water, but has good solubility in a variety of organic solvents. The key to developing carfilzomib into an injection is to solve the water solubility problem. Sulfonbutyl-β-cyclodextrin sodium is an anionic β-cyclodextrin derivative prepared from β-cyclodextrin. It has high water solubility and low nephrotoxicity. It can encapsulate poorly soluble carfilzomib in its hydrophobic cavity to increase the solubility of the raw material. Sulfonbutyl-β-cyclodextrin sodium is used as a solubilizer in drugs such as posaconazole, voriconazole, carbamazepine, amiodarone, melphalan, and remdesivir.

[0004] Carfilzomib preparations may produce enantiomers and diastereomers during production and storage. Isomer impurities may affect the quality of the drug and produce adverse safety effects. Therefore, it is necessary to develop appropriate methods for the study and control of isomers in the quality research of carfilzomib preparations. There are two common methods for the separation and analysis of chiral isomers: ① Normal phase chromatography: the stationary phase is a chiral chromatographic column, and the mobile phase is n-hexane-alcohol solvents; ② Reverse phase chromatography: the stationary phase is a chiral chromatographic column, and the mobile phase is a buffer solution-acetonitrile / methanol or the stationary phase is a common chromatographic column (C8, C18, etc.), and the mobile phase is a buffer solution (with chiral reagents added)-acetonitrile / methanol.

[0005] In the carfilzomib preparation, carfilzomib is encapsulated in the cavity of cyclodextrin, and the solubility of the lyophilized product is similar to that of sodium sulfobutyl-β-cyclodextrin, that is, it is easily soluble in water and difficult to dissolve in organic solvents. Considering the solubility characteristics of the carfilzomib preparation, only reverse phase chromatography can be used to separate and analyze the chiral isomer impurities in the carfilzomib preparation.

[0006] There are currently two patents for the separation and analysis of carfilzomib enantiomeric impurities using reverse phase chromatography. From an economic and practical perspective, these two invention patents are not superior. Patent CN116559354A uses a common solvent system without chiral additives as the mobile phase, and a chiral column with amylose-tris (3-chloro-5-methylphenylcarbamate) covalently bonded to the surface of silica gel as the chromatographic column for separation and analysis, but this chiral column is generally expensive and has a short life. Patent CN109406685A uses 1-Boc-4-aminopyrrolidine-2-carboxylic acid methyl ester as a chiral additive as the mobile phase, and uses an ordinary C18 column as the chromatographic column for separation and analysis, but it is not economical to use this special chiral reagent as a mobile phase additive.

[0007] In summary, it is of great significance to develop an economical and practical method for the detection of enantiomers in carfilzomib preparations. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention aims to provide a method for determining isomers in carfilzomib by liquid chromatography. The present invention is applicable to the detection and analysis of isomers in carfilzomib raw materials, and is also applicable to the detection and analysis of isomers in carfilzomib preparations. Carfilzomib preparations include carfilzomib for injection, or other compositions containing carfilzomib.

[0009] The isomers of carfilzomib are particularly preferably enantiomers (472-1A) and diastereomers (472-1V).

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A method for determining isomers in carfilzomib by reversed phase liquid chromatography, which uses high performance liquid chromatography to determine isomers in carfilzomib and its preparations, characterized in that reversed phase liquid chromatography is used, and the chromatographic conditions are as follows:

[0012] Chromatographic column: reverse phase bonded silica gel is used as filler.

[0013] The reverse phase bonded silica gel is selected from octyl bonded silica gel (C8) and / or octadecyl bonded silica gel (C18), preferably C8; the length of the chromatographic column is 100mm-250mm, preferably 150mm; the inner diameter of the chromatographic column is 2.1mm-4.6mm, preferably 3.9mm.

[0014] Mobile phase: sulfobutyl-β-cyclodextrin aqueous solution-organic solvent was used as the mobile phase.

[0015] The sulfobutyl-β-cyclodextrin includes sodium salt, potassium salt, etc. of sulfobutyl-β-cyclodextrin, preferably sodium sulfobutyl-β-cyclodextrin. The organic solvent in the mobile phase includes commonly used chromatographic solvents, such as methanol, acetonitrile, etc., preferably acetonitrile.

[0016] The lower the proportion of water phase in the mobile phase, the faster the elution time of carfilzomib and its isomers, but the separation effect becomes worse due to the lower concentration of cyclodextrin; the higher the proportion of water phase in the mobile phase, the shorter the life of the chromatographic column, and the slower the elution time of carfilzomib and its isomers. The volume ratio of the sulfobutyl-β-cyclodextrin sodium aqueous solution to acetonitrile is 60-90:10-40; the preferred volume ratio is 70-75:25-30; more preferably, the volume ratio is 72:28.

[0017] When the pH value of the aqueous phase in the mobile phase is low, the silica gel filler matrix of the chromatographic column is easy to hydrolyze, and the column efficiency of the chromatographic column will be significantly reduced; the presence of the sulfonic acid group in the sulfobutyl-β-cyclodextrin molecule makes it negatively charged in a wide pH range, and when the pH value of the aqueous phase in the mobile phase is high, the N in the carfilzomib molecule is not easy to be protonated, so the electrostatic force with sulfobutyl-β-cyclodextrin is weakened, and the separation effect is poor. The preparation method of the sodium sulfobutyl-β-cyclodextrin aqueous solution is: take sodium sulfobutyl-β-cyclodextrin, add water, stir to dissolve, and adjust the pH value to 2.0-5.0 with phosphoric acid; the preferred pH value is 2.5-4.0; more preferably, the pH value is 3.1.

[0018] The concentration of the sulfobutyl-β-cyclodextrin aqueous solution has a significant effect on the separation of isomers. At low concentrations, the host-guest inclusion complex is unstable and the chiral recognition effect is weak. At high concentrations, the chiral separation effect is better, but there is a risk of incompatibility between high concentrations of sulfobutyl-β-cyclodextrin and the organic phase. The concentration of the sulfobutyl-β-cyclodextrin sodium aqueous solution is 10g / L-100g / L, preferably 35g / L-80g / L, and more preferably 68.1g / L.

[0019] As some preferred embodiments, the flow rate in the method is 0.5-3.0 mL / min, preferably 1.0-2.0 mL / min, and more preferably 1.5 mL / min.

[0020] Carfilzomib and its isomers have no obvious maximum absorption wavelength, and have stronger absorption at the ultraviolet end. Considering the baseline interference at low wavelength and the lower response of the analyte at high wavelength, the detection wavelength in the described method is 200-220 nm, preferably 210 nm.

[0021] Under the condition of high column temperature, the stability of carfilzomib and its isomers and cyclodextrin inclusion complex is low, thereby reducing the chiral recognition effect; while when the column temperature is low, the column pressure increases significantly, affecting the life of the chromatographic column. Therefore, the column temperature in the method described is 15-40°C; preferably 20-30°C, and more preferably 25°C.

[0022] As a preferred embodiment, the elution condition in the method is isocratic elution.

[0023] As a preferred embodiment, the chromatographic conditions adopted are as follows:

[0024] Chromatographic column: Symmetry C8, 3.9 mm × 150 mm, 5 μm;

[0025] Mobile phase: sulfobutyl-β-cyclodextrin sodium aqueous solution-acetonitrile was used as the mobile phase; the volume ratio of the mobile phase was 72:28;

[0026] Flow rate: 1.5 mL / min;

[0027] Detection wavelength: 210nm;

[0028] Column temperature: 25°C;

[0029] Injection volume: 20 μL;

[0030] Elution conditions: isocratic elution;

[0031] The preparation method of the sodium sulfobutyl-β-cyclodextrin aqueous solution is as follows: taking a certain amount of sodium sulfobutyl-β-cyclodextrin, adding 1000 mL of water, stirring to dissolve, and adjusting the pH value to 3.1 with phosphoric acid to obtain the aqueous solution.

[0032] The structural formulas of carfilzomib, enantiomer (472-1A), diastereomer (472-1V) and impurity F described in the present invention are:

[0033]

[0034] .

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) Using conventional reversed-phase columns as stationary phase instead of chiral columns commonly used for isomer detection, which is cheaper and has a longer lifespan;

[0037] (2) Use lower-cost sodium sulfobutyl-β-cyclodextrin (after inquiry: McLean 35 yuan / 5g, Aladdin 58 yuan / 5g) as the chiral separation agent in the mobile phase instead of special chiral reagents such as 1-Boc-4-aminopyrrolidine-2-carboxylic acid methyl ester (after inquiry: McLean 3114 yuan / 5g, Aladdin 3129 yuan / 5g). The cost of sodium sulfobutyl-β-cyclodextrin is about 1.5% of that of special chiral reagents.

[0038] Through systematic methodological verification, the results showed that the method of the present invention has good specificity, sensitivity, accuracy, repeatability, durability, etc., and is suitable for the detection of chiral isomers in carfilzomib. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The detection spectrum of the method described in Example 1;

[0040] Figure 2 for Figure 1 A partial enlarged view of

[0041] Figure 3 Investigate chromatographic overlap for isomer specificity;

[0042] Figure 4 for Figure 2 A partial enlarged view of

[0043] Figure 5 It is the linear fitting diagram of impurity 472-1A;

[0044] Figure 6 It is the linear fitting diagram of impurity 472-1V;

[0045] Figure 7 The detection spectrum of the method described in Comparative Example 1;

[0046] Figure 8 for Figure 7 A partial enlarged view of

[0047] Fig. 9 This is the interference spectrum of the known impurity F on the isomeric impurity described in Comparative Example 1;

[0048] Fig.10 for Fig. 9 A partial enlarged view of . DETAILED DESCRIPTION

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0051] Example 1 A method for determining isomers in a carfilzomib preparation by reverse phase liquid chromatography

[0052] The steps include:

[0053] (1) Preparation of mixed reference solution: Take appropriate amount of impurity 472-1A and impurity 472-1V reference substances, add mobile phase and ultrasonically dissolve and dilute to make a mixed solution containing about 3.3 μg of impurity 472-1A and impurity 472-1V per 1 mL. This solution is used to locate isomeric impurities.

[0054] (2) Preparation of spiked test solution - fully mixed: Take 1 bottle of carfilzomib preparation, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, place in a 10 mL volumetric flask, add appropriate amount of impurity 472-1A, impurity 472-1V, impurity F and other known impurities stock solution, dilute to scale with mobile phase, shake well, and prepare a mixed solution containing approximately 1 mg of carfilzomib, 3.3 μg of impurity 472-1A, 3.3 μg of impurity 472-1V, 3.3 μg of impurity F, and other known impurities at the approximate limit concentration per 1 mL. This solution is used to investigate the specificity of the isomer detection method.

[0055] (3) Preparation of spiked test solution: Take 1 bottle of carfilzomib preparation, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, place in a 10 mL volumetric flask, add appropriate amount of impurity 472-1A and impurity 472-1V stock solution, dilute to scale with mobile phase, shake well, and prepare a mixed solution containing approximately 1 mg of carfilzomib, 3.3 μg of impurity 472-1A, and 3.3 μg of impurity 472-1V per 1 mL. This solution is used to investigate the effect of the main component on the detection of isomeric impurities.

[0056] (4) Preparation of test solution: Take 1 bottle of carfilzomib preparation, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, place in a 10 mL volumetric flask, dilute to the mark with mobile phase, and shake well. This solution is used for the detection of isomers in carfilzomib preparation.

[0057] (5) Preparation of impurity F localization solution: Take an appropriate amount of impurity F reference substance, add mobile phase and ultrasonically dissolve and dilute to make a solution containing about 3.3 μg of impurity F per 1 mL. This solution is used to localize impurity F.

[0058] (6) The isomers in carfilzomib preparations were determined by high performance liquid chromatography. The chromatographic conditions used were as follows:

[0059] Chromatographic column: Symmetry C8, 3.9 mm × 150 mm, 5 μm;

[0060] Mobile phase: sulfobutyl-β-cyclodextrin sodium aqueous solution-acetonitrile was used as the mobile phase; the volume ratio of the mobile phase was 72:28;

[0061] Flow rate: 1.5 mL / min;

[0062] Detection wavelength: 210nm;

[0063] Column temperature: 25°C;

[0064] Injection volume: 20 μL;

[0065] Elution conditions: isocratic elution;

[0066] The preparation method of the sodium sulfobutyl-β-cyclodextrin aqueous solution is as follows: 68.10 g of sodium sulfobutyl-β-cyclodextrin is added with 1000 mL of water, stirred to dissolve, and the pH value is adjusted to 3.1 with phosphoric acid to obtain the aqueous solution.

[0067] The test results are shown below Figure 1-2 .

[0068] according to Figure 1-2 The detection results show that the main peak appears at around 36 minutes, the impurity F peak is well separated from the impurity 472-1V peak, and the impurity peaks of related substances have no interference with the impurity 472-1V peak and the impurity 472-1A peak.

[0069] Effect detection:

[0070] 1. Exclusive inspection

[0071] Reference substance stock solution: Accurately weigh about 10 mg of each impurity 472-1A reference substance and impurity 472-1V reference substance, place in different 100 mL volumetric flasks, add a small amount of acetonitrile to dissolve and quantitatively dilute with mobile phase to make a stock solution containing about 0.1 mg of each reference substance per 1 mL.

[0072] Blank auxiliary material positioning solution: Take 1 bottle of blank auxiliary material, accurately add 29mL of water, shake to dissolve, accurately measure 5mL, place in a 10mL volumetric flask, dilute to the scale with mobile phase, and shake well.

[0073] 472-1A positioning solution: Take an appropriate amount of impurity 472-1A reference substance stock solution and dilute it with mobile phase to make a solution with a concentration of about 3.3 μg / mL.

[0074] 472-1V positioning solution: Take an appropriate amount of the impurity 472-1V reference substance stock solution and dilute it with mobile phase to make a solution with a concentration of about 3.3μg / mL.

[0075] Mixed solution: Take 1 bottle of this product, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, and place it in a 10 mL volumetric flask, and take appropriate amounts of impurity 472-1A reference substance stock solution and impurity 472-1V reference substance stock solution, dilute to the scale with mobile phase, shake well, and prepare a mixed solution containing approximately 1 mg of carfilzomib, and approximately 3.3 μg of impurity 472-1A and impurity 472-1V per 1 mL.

[0076] The test results are shown in Table 1 and Figure 3-4 .

[0077] Table 1 Results of known component positioning tests

[0078]

[0079] The results showed that: (1) there was no interference in the blank solvent and blank excipient solution, and the retention time of each component in the fixed solution and the mixed solution was basically the same; (2) in the specific mixed solution, the separation degree between the main peak and the 472-1A peak was 3.1, and the separation degree between the main peak and the 472-1V peak was 6.5. The specificity of the analytical method was good.

[0080] 2. Investigation of limit of quantification and limit of detection

[0081] Accurately measure the stock solutions of impurity 472-1A and impurity 472-1V reference substances, dilute them step by step with mobile phase to a certain concentration (see Table 2), shake well, accurately measure the above solutions respectively, and inject them into the liquid chromatograph. The signal-to-noise ratio is not less than 3:1, and the detection limit is obtained; the signal-to-noise ratio is not less than 10:1, and the quantification limit is obtained; the quantification limit solution is injected continuously for 6 injections, and the detection limit solution is injected continuously for 3 injections. The results are shown in Table 2-4 below.

[0082] Table 2 Detection limit and quantification limit solution dilution process

[0083]

[0084] Table 3 Quantitation limit test results

[0085]

[0086] Table 4 Detection limit test results

[0087]

[0088] The results showed that: (1) the limit of quantification of impurity 472-1V was 1.0119 μg / mL (0.1012%), the RSD of the peak area for 6 consecutive injections was 4.4%, and the signal-to-noise ratio was 22.5-40.7; the detection limit was 0.3036 μg / mL (0.0304%), and the signal-to-noise ratio was 6.6-8.0; (2) the limit of quantification of impurity 472-1A was 0.9932 μg / mL (0.0993%), the RSD of the peak area for 6 consecutive injections was 8.1%, and the signal-to-noise ratio was 13.5-24.1; the detection limit was 0.2980 μg / mL (0.0298%), and the signal-to-noise ratio was 3.9-5.1.

[0089] 3. Linearity and range investigation

[0090] Prepare linear solutions of impurity 472-1A and impurity 472-1V with different concentration gradients, inject them into liquid chromatography according to the proposed method, and draw linear curves based on the test results. Figure 5-6 .

[0091] Combination Figure 5-6 It can be seen that: (1) The peak area and concentration of impurity 472-1A are linearly related in the concentration range of 0.9932-6.7535 μg / mL: the correlation coefficient is 0.9997; the linear equation is y=25.227x-0.9567. (2) The peak area and concentration of impurity 472-1V are linearly related in the concentration range of 1.0119-6.8812 μg / mL: the correlation coefficient is 0.9999; the linear equation is y=23.469x+2.7503. The analytical method has a good linear relationship.

[0092] 4. Repeatability test

[0093] Repeatability solution: Take 1 bottle of this product, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, put it in a 10 mL volumetric bottle, then add appropriate amount of impurity 472-1A and impurity 472-1V reference substance stock solution, dilute to scale with mobile phase, shake well, and prepare a mixed solution containing about 3.3 μg of impurity 472-1A and impurity 472-1V in each 1 mL, and prepare 6 copies in parallel. The test results are shown in Table 5 below.

[0094] Table 5 Repeatability test results

[0095]

[0096] The results showed that among the six reproducible solutions, the RSDs of the test results of 472-1V and 472-1A were 3.8% and 5.4% respectively. The analytical method had good reproducibility.

[0097] 5. Accuracy test

[0098] Quantitative limit accuracy solution: Take 1 bottle of this product, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, put it in a 10 mL volumetric bottle, add appropriate amount of impurity 472-1A and impurity 472-1V reference substance stock solution, dilute to scale with mobile phase, shake well, and prepare a solution with impurity 472-1A and impurity 472-1V spiked in 1 mL at the agreed quantitative limit concentration. Prepare 3 copies in parallel.

[0099] 100% limit accuracy solution: Take 1 bottle of this product, accurately add 29 mL of water, shake to dissolve, accurately measure 5 mL, put it in a 10 mL volumetric flask, add appropriate amount of impurity 472-1A and impurity 472-1V stock solutions, dilute to the scale with mobile phase, shake well, and prepare a mixed solution with about 3.3 μg of impurity 472-1A and impurity 472-1V spiked in each 1 mL. Prepare 3 copies in parallel.

[0100] 150% limit accuracy solution: Take 1 bottle of this product, accurately add 29mL of water, shake to dissolve, accurately measure 5mL, put it in a 10mL volumetric bottle, add appropriate amount of impurity 472-1A and impurity 472-1V stock solution, dilute to scale with mobile phase, shake well, and prepare a mixed solution with impurity 472-1A and impurity 472-1V spiked in 1mL of about 5μg, and prepare 3 copies in parallel. The test results are shown in Table 6-7 below.

[0101] Table 6 Accuracy test results - Impurity 472-1V

[0102]

[0103] Table 7 Accuracy test results - Impurity 472-1A

[0104]

[0105] The results showed that the recovery rate of impurity 472-1V was between 92.83% and 103.06%, with an RSD of 2.6%. The recovery rate of impurity 472-1A was between 91.74% and 99.02%, with an RSD of 0.9%. The analytical method had good accuracy.

[0106] 6. Durability inspection

[0107] The durability of the analytical method was investigated under different mobile phase ratios, different cyclodextrin concentrations, different mobile phase pH values, different column temperatures, different flow rates, etc. The test results are shown in Table 8 below.

[0108] Table 8 Durability test results

[0109]

[0110] According to the above test results, the analytical method has good durability under the conditions of mobile phase ratio 70% aqueous phase-75% aqueous phase, flow rate 1.0mL / min-2.0mL / min, column temperature 20℃-30℃, buffer pH 2.5-4.0, and sulfobutyl-β-cyclodextrin sodium aqueous solution concentration 35g / L-80g / L, and the separation degree between the carfilzomib peak and the isomer peak is not less than 1.5.

[0111] Comparative Example 1:

[0112] The difference from Example 1 is that: mobile phase: sulfobutyl-β-cyclodextrin sodium aqueous solution-acetonitrile is used as the mobile phase; the volume ratio of the mobile phase is 68:32, the detection wavelength is 210nm, the flow rate is 1.2mL / min, the injection volume is 20μL, the column temperature is 20°C, and the others are the same as Example 1.

[0113] Prepare the spiked test solution (main component about 1 mg / mL, impurities 472-1A and 472-1V about 3.3 μg / mL), inject the sample for detection, and the detection spectrum is as follows Figure 7-8 .

[0114] according to Figure 7 and Figure 8 It can be seen that the main peak appears at about 18 minutes, and the separation between the main peak and 472-1V and 472-1A is 5.9 and 2.6 respectively. The overall separation is good and the impurity response is improved. However, this product has multiple known impurities in related substances. In order to investigate the impact of related substance impurities on isomer impurity detection, the specific interference of isomer detection is further investigated. Fig. 9 and Fig.10 It can be seen that under this method, impurity F and isomer impurity 472-1V in the relevant substance are not well separated, which has a certain impact on the isomer detection.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining isomers in carfilzomib and its preparations by reverse phase liquid chromatography, which uses high performance liquid chromatography to determine isomers in carfilzomib and its preparations, characterized in that: Using reverse phase liquid chromatography, the structural formulas of enantiomers, diastereomers and impurity F are: The chromatographic conditions were as follows: Chromatographic column: reverse phase bonded silica gel as filler; Mobile phase: sulfobutyl-β-cyclodextrin aqueous solution-organic solvent as mobile phase; The volume ratio of the sulfobutyl-β-cyclodextrin aqueous solution to the organic solvent is 70-75:25-30; The pH value of the sulfobutyl-β-cyclodextrin aqueous solution is 2.0-5.0; The reverse phase bonded silica gel is octyl bonded silica gel; The organic solvent in the mobile phase is selected from methanol, acetonitrile or a mixture of the two in any proportion; The reverse phase liquid chromatography method adopts an ultraviolet detector with a detection wavelength of 200nm-220nm.

2. The method according to claim 1, characterized in that: The length of the chromatographic column is 100mm-250mm; the inner diameter of the chromatographic column is 2.1mm-4.6mm.

3. The method according to claim 2, characterized in that: The length of the chromatographic column is 150 mm, and the inner diameter of the chromatographic column is 3.9 mm.

4. The method according to claim 1, characterized in that: The concentration of the sulfobutyl-β-cyclodextrin aqueous solution is 10g / L-100g / L.

5. The method according to claim 1, characterized in that: The flow rate in the described method is 0.5-3.0 mL / min.

6. The method according to claim 1, characterized in that: The column temperature in the described method is 15-40°C.

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