Methylprednisolone dimer impurity and separation and purification method thereof

Abstract

The invention discloses a methylprednisolone dimer impurity and a separation and purification method thereof. The invention provides a compound as shown in a formula I and / or a compound as shown in a formula II. The two methylprednisolone dimer impurities provided by the invention are not reported, are detected in the product, and are of great significance to product quality control.

Description

TECHNICAL FIELD

[0001] The present application relates to methylprednisolone dimer impurities and a method for separating and purifying the same. BACKGROUND

[0002] Methylprednisolone is 11β, 17, 21-trihydroxy-6α-methylpregna-1, 4-diene-3, 20-dione, the structure of which is shown as follows:

[0003]

[0004] It is an artificial synthesis of moderate glucocorticoid, has strong anti-inflammatory, immunosuppressive, anti-allergic, anti-shock and other pharmacological effects, less side effects, often used in respiratory diseases, endocrine disorders, rheumatic diseases, blood diseases, skin diseases, allergic states, nervous system diseases, etc., can also be used as emergency medicine for critical illness, and is widely used in clinical application.

[0005] The dosage form of methylprednisolone is mainly tablet and injection, among which tablet is most commonly used. Methylprednisolone tablet was first marketed in the United States by Pfizer in 1957, and has been marketed in many countries and regions around the world such as the United States, Europe, Japan, China, etc. In 1999, Pfizer introduced methylprednisolone tablet into China, with the trade name of Medrol.

[0006] Impurities in drugs can reduce efficacy, affect the stability of the drug, and some are even harmful or have other toxic side effects on human health, so impurity research is an important part of drug development, which not only provides a basis for process quality control of raw materials and preparations, but also helps to improve the reliability of product quality standards. With the increasing intensity of drug evaluation in recent years, the requirements for impurity research and testing of finished products are becoming higher and higher. For methylprednisolone preparations, the existing technology for impurity research mainly targets the impurities disclosed in the European Pharmacopoeia, and there is currently no literature reporting the existence of methylprednisolone dimer impurities. Polymer impurities can trigger an immune response in the human body, leading to allergy, inflammation, and even severe allergic reactions, for example, polymer impurities in β-lactam antibiotics can easily trigger allergic reactions and even anaphylactic shock, threatening patient safety (HUC Q, JIN S H. Progress in polymer impurities in β-lactam antibiotics and their analytical techniques [J]. World Notes Antibiot, 1996, 17(5): 336-343). We found that the dimer impurities of methylprednisolone will increase significantly after long-term storage of concentrated solution, which directly affects the quality and safety of the drug, therefore, using the dimer as a reference substance to control the purity and quality of methylprednisolone raw materials and related preparations can further ensure the quality of the drug, and thus ensure the safety of clinical medication and the health of patients. Therefore, further research will help the development and utilization of methylprednisolone and quality control. SUMMARY

[0007] The technical problem solved by the present application is to provide two kinds of methylprednisolone dimer impurities and a separation and purification method thereof. The two kinds of methylprednisolone dimer impurities provided by the present application have not been reported and are detected in the product, which is of great significance for product quality control.

[0008] The present application solves the above technical problems by the following technical solutions.

[0009] The present application provides a compound as shown in formula I, and / or a compound as shown in formula II,

[0010]

[0011]

[0012] The present application provides a separation method of a compound as shown in formula I and / or a compound as shown in formula II according to any one of the present application, which comprises the following steps:

[0013]

[0014] Step one: separating the test product to obtain test product A by liquid chromatography;

[0015] The test product contains one or more of methylprednisolone, a compound as shown in Formula I, or a compound as shown in Formula II;

[0016] In the liquid chromatography, the chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column;

[0017] The mobile phase is acetonitrile and an aqueous solution, and the aqueous solution contains phosphoric acid, and the volume ratio of the phosphoric acid to the water is 0.2:100;

[0018] The volume ratio of the acetonitrile to the aqueous solution is (30%-40%):(70%-60%); "%" refers to the volume percentage; the sum of the volume percentages of the acetonitrile and the water is 100%;

[0019] Step two: separating the test product A to obtain a compound as shown in Formula I, and / or a compound as shown in Formula II by liquid chromatography;

[0020] The liquid chromatography conditions are scheme A or scheme B: the separation conditions of the liquid chromatography of scheme A include:

[0021] The chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column;

[0022] The elution mode is isocratic elution;

[0023] The mobile phase is acetonitrile and an aqueous solution, and the aqueous solution contains phosphoric acid, and the volume ratio of the phosphoric acid to the water is 0.2:100;

[0024] The volume ratio of the acetonitrile to the aqueous solution is (5%-29%):(95%-71%); "%" refers to the volume percentage; the sum of the volume percentages of the acetonitrile and the water is 100%;

[0025] The separation conditions of the liquid chromatography of scheme B include:

[0026] The chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column;

[0027] When the elution mode is gradient elution,

[0028] The mobile phase A is water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100;

[0029] The mobile phase B is acetonitrile;

[0030] The elution program is:

[0031] Time (min) Volume % of mobile phase A Volume % of mobile phase B 0→5 X1 100％-X1 5→45 X1→X2 (100%-X1)→(100%-X2) 45→50 X2→X1 (100%-X2)→(100%-X1) 50→55 X1 (100％-X1)

[0032] wherein X1 is 93%-97%; X2 is 48%-52%;

[0033] wherein, for example, "0→5min, volume of mobile phase A is X1" means that in the range of 0 to 5 minutes, the volume of mobile phase A (the percentage of the volume of mobile phase A in the total volume of mobile phase) is fixed at X1; "5→45min, volume of mobile phase A is X1→X2" means that in the range of 5 to 45 minutes, the volume of mobile phase A (the percentage of the volume of mobile phase A in the total volume of mobile phase) is linearly changed from X1 to X2.

[0034] In some embodiments of the present application, the test product is a conventional methylprednisolone product in the art, including tablets or injections.

[0035] In some embodiments of the present application, the preparation of the test product comprises the following steps: mixing methylprednisolone tablets with an alcoholic solvent, and filtering (preferably placing for 1-50 days (for example, 20 days or 30 days)) to obtain a test product solution.

[0036] In some embodiments of the present application, the alcoholic solvent is methanol.

[0037] In some embodiments of the present application, the mass-volume ratio of the methylprednisolone tablets to the alcoholic solvent is 10 mg / mL-20 mg / mL, for example, 12 mg / mL or 13 mg / mL.

[0038] In some embodiments of the present application, the filtering can be filtered by conventional methods in the art, for example, membrane filtration, preferably 10 μm or 0.8 μm membrane filtration.

[0039] In some embodiments of the present application, the placing is at a temperature of 25°C-45°C.

[0040] In some embodiments of the present application, in step one, the volume ratio of the acetonitrile to the aqueous solution is 35%:65% or 40%:60%.

[0041] In some embodiments of the present application, in step one, the flow rate of the mobile phase can refer to the conventional flow rate in the art when detecting by chromatography, preferably 20-50 mL / min, for example, 30 mL / min or 40 mL / min.

[0042] In some embodiments of the present application, in step one, the retention time of the test product A is 8.0 min-16.0 min, preferably 10.0 min-15.5 min or 8.5 min-12.2 min.

[0043] In some embodiments of the present application, in scheme A, the volume ratio of the acetonitrile to the aqueous solution is 25%:75%.

[0044] In some embodiments of the present application, in the method A, the flow rate of the mobile phase can refer to the conventional flow rate in the field of chromatography, preferably 10-20 mL / min, for example 14 mL / min.

[0045] In some embodiments of the present application, in the method A, the retention time of the compound of formula I is 20.0 min-23.0 min, preferably 20.1 min-22.5 min.

[0046] In some embodiments of the present application, in the method A, the retention time of the compound of formula II is 26.0 min-29.0 min, preferably 26.7 min-28.8 min.

[0047] In some embodiments of the present application, in the method B, the elution procedure is:

[0048] Time (min) Volume % of mobile phase A Volume % of mobile phase B 0→5 95 5 5→45 95→50 5→50 45→50 50→95 50→5 50→55 95 5

[0049] For example, "0→5 min, mobile phase A volume is 95%" means that the mobile phase A (the percentage of the volume of mobile phase A in the total volume of mobile phase) is fixed at 95% in the range of 0 to 5 minutes; "5→45 min, mobile phase A volume is 95→50" means that the mobile phase A (the percentage of the volume of mobile phase A in the total volume of mobile phase) is linearly changed from 95% to 50% in the range of 5 to 45 minutes.

[0050] In some embodiments of the present application, in the method B, the flow rate of the mobile phase can refer to the conventional flow rate in the field of chromatography, preferably 5-10 mL / min, for example 7 mL / min.

[0051] In some embodiments of the present application, in the method B, the retention time of the compound of formula I is 18.0 min-20.0 min, preferably 18.0 min-19.1 min.

[0052] In some embodiments of the present application, in the method B, the retention time of the compound of formula II is 28.0 min-30.0 min, preferably 28.4 min-29.8 min.

[0053] In some embodiments of the present application, the particle size of the packing of the chromatographic column is 5-20 μm, for example 10 μm.

[0054] In some embodiments of the present application, the length of the chromatographic column can be 100-300 mm, preferably 250 mm or 150 mm.

[0055] In some embodiments of the present application, the inner diameter of the chromatographic column can be 20-50 mm, preferably 30 mm or 22.1 mm.

[0056] In some embodiments of the present application, the chromatographic column is Kromasil C18; preferably the chromatographic column is Kromasil C18, 30 mm x 250 mm, 10 μm or Kromasil C18, 22.1 mm x 150 mm, 10 μm.

[0057] In some embodiments of the present application, the column temperature of the chromatographic column is 25-40 °C, preferably 30 °C.

[0058] In some embodiments of the present application, the detector of the liquid chromatography is an ultraviolet detector; the detection wavelength of the detector is preferably 200-300 nm, for example 247 nm.

[0059] In some embodiments of the present application, the injection volume of the liquid chromatography can refer to the conventional injection volume of chromatography detection analysis in the art, preferably 100-1000 μL, for example 500 μL or 1000 μL.

[0060] In some embodiments of the present application, the separation method satisfies the following conditions:

[0061] (1) in the first step, the mobile phase is 35% acetonitrile, 65% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100; and

[0062] (2) in the second step, the mobile phase is 25% acetonitrile, 75% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100.

[0063] In some embodiments of the present application, the separation method satisfies the following conditions:

[0064] (1) in the first step, the mobile phase is 40% acetonitrile, 60% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100; and

[0065] (2) in the second step, the elution program is:

[0066] Time (min) Volume % of mobile phase A Volume % of mobile phase B 0→5 95 5 5→45 95→50 5→50 45→50 50→95 50→5 50→55 95 5 .

[0067] The present application also provides a use of a compound as shown in Formula I and / or a compound as shown in Formula II in the detection of methylprednisolone impurities.

[0068] Those skilled in the art can understand that the compound represented by formula I and / or the compound represented by formula II provided by the present application exist widely in methylprednisolone products, and are not limited to the tablets produced by Jiulong Renfu and Pfizer.

[0069] On the basis of not violating the common sense of the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0070] The reagents and raw materials used in the present application are commercially available.

[0071] The positive progress effect of the present application is that the present application provides two new methylprednisolone dimmer impurities and provides a method for separating and purifying the same. The two methylprednisolone dimmer impurities provided by the present application are new impurities that have not been disclosed, and are detected in products, which is of great significance for product quality control. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 HSQC spectrum of impurity 1 1 H- 1 H COSY spectrum;

[0073] Figure 2 HSQC spectrum of impurity 1

[0074] Figure 3 HMBC spectrum of impurity 1

[0075] Figure 4 HSQC spectrum of impurity 2 1 H- 1 H COSY spectrum;

[0076] Figure 5 HSQC spectrum of impurity 2

[0077] Figure 6 HMBC spectrum of impurity 2. DETAILED DESCRIPTION

[0078] The present application will be further described by way of examples below, but the present application is not limited to the scope of the examples described. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0079] The main experimental instruments used in the examples are shown in Table 1:

[0080] Table 1

[0081] Name Manufacturer Model High performance liquid chromatograph SHIMADZU LC 2030C Preparative liquid chromatograph Beijing Innovative Tongheng Science and Technology Co., Ltd. LC3000 Liquid chromatograph-mass spectrometer Thermo Vanquish-Q Exactive Nuclear magnetic resonance spectrometer Agilent 400MR Freeze dryer Zhengzhou Ansheng Science and Technology Instrument Co., Ltd. AS-LGJ-18A Rotary evaporator BUCHI R-300

[0082] The sources of the samples involved in the examples are shown in Table 2:

[0083] Table 2: Sample Consumable Information Table

[0084]

[0085] Example 1: Isolation and purification of methylprednisolone dimer

[0086] Take methylprednisolone tablets (batch: Z03210301) 150 g, slowly add 0.5 L of methanol and stir to make methylprednisolone fully dissolved, the dissolved solution is filtered through 10 μm, 0.8 μm filter membrane in turn, to obtain a clear high concentration solution 0.5 L, the concentration is about 12 mg / mL, place at (40±2℃) for 20 days, to obtain methylprednisolone sample solution.

[0087] Use the preparation column (Kromasil C18, 30 mm x 250 mm, 10 μm), the mobile phase is 35% acetonitrile-65% water (containing 0.2% phosphoric acid, 0.2% is the volume percentage of phosphoric acid in water), the flow rate is 30 mL / min, the column temperature is 30℃, the detection wavelength is 247 nm, the injection volume is 1000 μL. After equilibrating the preparation column with 2 column volumes of mobile phase, take the methylprednisolone sample solution, collect the fractions according to the online detection spectrum, the fraction collection time is: 10.0 min-15.5 min. The collected eluent is concentrated under reduced pressure using a rotary evaporator to obtain about 10 mL of concentrated solution.

[0088] Take the concentrated solution obtained in the previous step to prepare impurity monomers; use the preparation column (Kromasil C18, 30 mm x 250 mm, 10 μm), the mobile phase is 25% acetonitrile-75% water (containing 0.2% phosphoric acid, 0.2% is the volume percentage of phosphoric acid in water), the column temperature is 30℃, the flow rate is 14 mL / min, the detection wavelength is 247 nm, the injection volume is 500 μL. After equilibrating the preparation column with 2 column volumes of mobile phase, take the concentrated solution, collect the fractions according to the online detection spectrum, the fraction collection time is: 20.1 min-22.5 min (impurity 1), 26.7 min-28.8 min (impurity 2). The impurity 1 fraction and the impurity 2 fraction with HPLC purity greater than 95% are collected respectively, and then freeze-dried after concentration under reduced pressure, to obtain a total of 47 mg of impurity 1 with a chromatographic purity of 95.5%, and a total of 59 mg of impurity 2 with a chromatographic purity of 96.8%.

[0089] Example 2: Isolation and purification of methylprednisolone dimer

[0090] Take methylprednisolone tablets 500 g (batch: Z03210301), slowly add 1 L of methanol and stir to make methylprednisolone fully dissolved, the dissolved solution is filtered through 10 μm, 0.8 μm filter membrane in turn, to obtain a clear high concentration solution 1 L, the concentration is about 13 mg / mL, place at (30±2℃) for 30 days, to obtain methylprednisolone sample solution.

[0091] A preparative column (Kromasil C18, 22.1 mm x 150 mm, 10 μm) was used with a mobile phase of 40% acetonitrile-60% water (containing 0.2% phosphoric acid, 0.2% being the volume percentage of phosphoric acid in water), a column temperature of 30°C, a flow rate of 40 mL / min, a detection wavelength of 247 nm, and an injection volume of 1000 μL. After equilibrating the preparative column with 2 column volumes of the mobile phase, the methyiprednisolone solution was loaded, and fraction collection was performed according to the online detection spectrum, with a fraction collection time of 8.5 min to 12.2 min. The collected eluent was concentrated under reduced pressure using a rotary evaporator to obtain 10 mL of a concentrated solution.

[0092] The concentrated solution obtained in the previous step was used to prepare the impurity monomer; a preparative column (Kromasil C18, 22.1 mm x 150 mm, 10 μm) was used with a mobile phase A of water (containing 0.2% phosphoric acid, 0.2% being the volume percentage of phosphoric acid in water) and a mobile phase B of acetonitrile, with a gradient elution program as follows:

[0093] Time (min) Volume % of mobile phase A Volume % of mobile phase B 0→5 95 5 5→45 95→50 5→50 45→50 50→95 50→5 50→55 95 5

[0094] A flow rate of 7 mL / min, a column temperature of 30°C, a detection wavelength of 247 nm, and an injection volume of 500 μL. After equilibrating the preparative column with 2 column volumes of the mobile phase, the concentrated solution was loaded, and fraction collection was performed according to the online detection spectrum, with a fraction collection time of 18.0 min to 19.1 min (impurity 1) and 28.4 min to 29.8 min (impurity 2). The impurity 1 fraction and the impurity 2 fraction with a HPLC purity of greater than 95% were combined, and then concentrated under reduced pressure and lyophilized to obtain 178 mg of impurity 1 with a chromatographic purity of 98.5%, and 218 mg of impurity 2 with a chromatographic purity of 99.1%.

[0095] Example 3: Application of the methyiprednisolone dimer (formula I and formula II) impurities

[0096] The method for detecting related substances in the quality standard for methyiprednisolone (METHYLPREDNISOLONE) in EP 11.0 was used, with the following specific method parameters:

[0097] Chromatographic column: octadecylsilane-bonded silica gel as the filler (4.6 mm x 15 cm, 3 μm)

[0098] Flow rate: 1.5 mL / min Column temperature: 45°C

[0099] Injection volume: 10 μl Ultraviolet detection wavelength: 247 nm

[0100] Phase A: phosphoric acid: tetrahydrofuran: acetonitrile: water (0.1: 1.5: 10: 90)

[0101] Phase B: Phosphoric acid: Tetrahydrofuran: Acetonitrile (0.1: 1.5: 100)

[0102] The gradient elution was performed according to the following table procedure:

[0103] Time (min) Mobile phase A Mobile phase B 0-14 83 17 14-30 83→52 17→48 30-35 52→83 48→17 35-40 83 17

[0104] Solvent: Acetonitrile: Water: Phosphoric acid (50: 50: 0.1)

[0105] Test solution: Take methylprednisolone tablets (batch: Z03210301, 56211203, EF9413) each 10 tablets, grind, and accurately weigh methylprednisolone tablet powder sample about 300 mg (equivalent to methylprednisolone 12 mg) in a 20 mL volumetric flask, add solvent appropriately, ultrasonic for 15 min, cool, dilute to the mark with solvent, shake well, filter with 0.45 μm filter membrane, and take the filtrate as the test solution (0.6 mg / mL).

[0106] Control solution: accurately measure 1 mL of test solution in a 100 mL volumetric flask, dilute to the mark with solvent, shake well, and obtain (0.006 mg / mL).

[0107] Calculation formula:

[0108]

[0109] In the formula: A 杂 represents the area of the impurity peak in the test solution; f represents the impurity correction factor;

[0110] A 对 represents the peak area of the main peak in the control solution.

[0111] The methodological linearity and range verification was carried out using the methylprednisolone dimer (formula I and formula II) impurity reference substance obtained in Example 1, and the dimer impurity 1, 2 correction factors are shown in Table 3:

[0112] Table 3

[0113] Component Regression equation Correlation coefficient R 2 ]] Slope Correction factor Methylprednisolone y=0.2575x+0.0044 1.0000 0.2575 - Impurity 1 (Formula I) y=0.2142x+0.0034 0.9998 0.2142 1.20 Impurity 2 (Formula II) y=0.1988x-0.0036 1.0000 0.1988 1.30

[0114] The self-developed and reference commercial preparations were quantitatively detected, and the detection results are shown in Table 4:

[0115] Table 4

[0116] Impurity % Impurity 1 % Impurity 2 % 56211203 long-term 18 months 0.02 0.04 EF9413 short-term 0.01 0.13 Z03210301 long-term 31 months 0.05 0.10

[0117] Example 4: Structure confirmation of the two methylprednisolone dimer impurities obtained in Example 1

[0118] 1. Impurity 1 structure confirmation Figure 1-3 ​

[0119] LC-HRMS parameters are shown in Table 5:

[0120] Table 5

[0121]

[0122] Results: The sample was analyzed by mass spectrometry using electrospray ionization (HESI) in positive ion mode for full scan MS1, and the quasi-molecular ion peak [M+H] of the sample was m / z 747.40826 + C 44 H 58 O 10 (M+H) was m / z 747.40826, and the molecular formula of the sample was calculated as C + Theoretical value: m / z 747.41027, relative mass deviation 2.69ppm).

[0123] NMR analysis parameters are shown in Table 6:

[0124] Table 6

[0125]

[0126] Results: The sample was analyzed by mass spectrometry using electrospray ionization (HESI) in positive ion mode for full scan MS1, and the quasi-molecular ion peak [M+H] of the sample was m / z 747.40826 1 There were 58 effective hydrogen signals in the H-NMR (400MHz, DMSO-d6) spectrum of the sample; 13 There were 22 groups of carbon signals in the C-NMR (100MHz, DMSO-d6) spectrum of the sample, which represented 44 effective carbon signals due to complete structural symmetry.

[0127] Based on the analysis of the 1 H- 1 H COSY, HSQC and HMBC spectra, the carbon and hydrogen data of the sample were assigned, as shown in Table 7; the impurity is a dimer impurity formed by the connection of two methylprednisolone 22 carbon atoms.

[0128] Based on the above LC-HRMS and NMR data analysis, the structure of the sample can be confirmed as shown in formula I:

[0129]

[0130] Table 7 Assignment of 1D and 2D NMR data of the sample (400 and 100MHz, DMSO-d6)

[0131]

[0132] 2. Impurity 2 structure confirmation Figure 4-6 )

[0133] LC-HRMS parameters are shown in Table 8:

[0134] Table 8

[0135]

[0136] Result: The sample was analyzed by mass spectrometry with electrospray ionization (HESI) in positive ion mode, and the quasi-molecular ion peak [M+H] of the sample was m / z 731.41583. The high resolution mass spectrum calculated the molecular formula of the sample as C + H 44 H 58 O9 (its [M+H] + Theoretical value: m / z 731.41536, relative mass deviation 0.64ppm).

[0137] The NMR analysis parameters are shown in Table 9:

[0138] Table 9

[0139]

[0140] Result: The sample was analyzed by mass spectrometry with electrospray ionization (HESI) in positive ion mode, and the quasi-molecular ion peak [M+H] of the sample was m / z 731.41583. The high resolution mass spectrum calculated the molecular formula of the sample as C 1 H-NMR (400MHz, DMSO-d6) spectrum gave a total of 53 effective hydrogen signals; 13 C-NMR (100MHz, DMSO-d6) spectrum detected 37 groups of carbon signals, due to the characteristics of the structure, some carbon signals overlapped, representing a total of 44 effective carbon signals.

[0141] Based on the analysis of the LC-HRMS and NMR data, the structure of the sample was confirmed as shown in the following formula II: 1 H- 1 H COSY, HSQC and HMBC spectra, the carbon and hydrogen data of the sample PI-2311009 were assigned, as shown in Table 10; the impurity is a dimer impurity formed by the connection of two methylprednisolone 22 carbon and the removal of hydroxyl group at 17.

[0142] Based on the above LC-HRMS and NMR data analysis, the structure of the sample was confirmed as shown in the following formula II:

[0143]

[0144] Table 10 1D and 2D NMR data assignment of the sample (400 and 100MHz, DMSO-d6)

[0145]

Claims

1. A compound as shown in Formula I, and / or, a compound as shown in Formula II, 2. A method of isolating a compound of formula I and / or a compound of formula II according to claim 1, characterized in that comprising the following steps: Step one: separating the test sample by liquid chromatography to obtain test sample A; the test sample comprises one or more of methylprednisolone, a compound as shown in Formula I, or a compound as shown in Formula II; in the liquid chromatography, the chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column; the mobile phase is acetonitrile and an aqueous solution, the aqueous solution comprising phosphoric acid, the volume ratio of the phosphoric acid to the water being 0.2:100; the volume ratio of the acetonitrile to the aqueous solution is (30%-40%):(70%-60%); "%" refers to volume percentage; the sum of the volume percentages of the acetonitrile and the water is 100%; Step two: separating test sample A by liquid chromatography to obtain a compound as shown in Formula I, and / or, a compound as shown in Formula II; the liquid chromatography conditions are scheme A or scheme B: the separation conditions of the liquid chromatography of scheme A comprise: the chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column; the elution mode is isocratic elution; the mobile phase is acetonitrile and an aqueous solution, the aqueous solution comprising phosphoric acid, the volume ratio of the phosphoric acid to the water being 0.2:100; the volume ratio of the acetonitrile to the aqueous solution is (5%-29%):(95%-71%); "%" refers to volume percentage; the sum of the volume percentages of the acetonitrile and the water is 100%; the separation conditions of the liquid chromatography of scheme B comprise: the chromatographic column is an octadecyl-bonded silica gel reversed-phase chromatographic column; the elution mode is gradient elution, the mobile phase A is water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100; the mobile phase B is acetonitrile; the elution program is: wherein, X1 is 93%-97%; X2 is 48%-52%.

3. The separation method of claim 2, wherein, the preparation of the test sample comprises the following steps: mixing methylprednisolone tablets with an alcoholic solvent, filtering to obtain a test sample solution.

4. The separation method of claim 3, wherein, it satisfies one or more of the following conditions: (1) the alcoholic solvent is methanol; (2) the mass-volume ratio of the methylprednisolone tablets to the alcoholic solvent is 10 mg / mL-20 mg / mL, for example 12 mg / mL or 13 mg / mL; (3) the filtering is performed using a filter membrane, preferably a 10 μm or 0.8 μm filter membrane; (4) after the filtering, it further comprises standing for 1-50 days, for example 20 days or 30 days; preferably, (4) the standing condition is 25°C-45°C.

5. The separation method of claim 2, wherein, it satisfies one or more of the following conditions: (1) in step one, the volume ratio of the acetonitrile to the aqueous solution is 35%:65% or 40%:60%; (2) in step one, the flow rate of the mobile phase is 20-50 mL / min; (3) in step one, the retention time of test sample A is 8.0 min-16.0 min; (4) in scheme A, the volume ratio of the acetonitrile to the aqueous solution is 25%:75%; (5) in scheme A, the flow rate of the mobile phase is 10-20 mL / min; (6) in the scheme A, the retention time of the compound as shown in formula I is 20.0 min-23.0 min; (7) in the scheme A, the retention time of the compound as shown in formula II is 26.0 min-29.0 min; (8) in the scheme B, the elution procedure is: ； (9) in the scheme B, the flow rate of the mobile phase is 5-10 mL / min; (10) in the scheme B, the retention time of the compound as shown in formula I is 18.0 min-20.0 min; (11) in the scheme B, the retention time of the compound as shown in formula II is 28.0 min-30.0 min; (12) the particle size of the packing material of the column is 5-20 μm; (13) the length of the column is 100-300 mm; (14) the inner diameter of the column is 20-50 mm; (15) the column temperature is 25-40 °C; (16) the detector of the liquid chromatography is an ultraviolet detector; (17) the injection volume of the liquid chromatography is 100-1000 μL.

6. The separation method of claim 5, wherein, It meets one or more of the following conditions: (1) in the step one, the flow rate of the mobile phase is 30 mL / min or 40 mL / min; (2) in the step one, the retention time of the test sample A is 10.0 min-15.5 min or 8.5 min-12.2 min; (3) in the scheme A, the flow rate of the mobile phase is 14 mL / min; (4) in the scheme A, the retention time of the compound as shown in formula I is 20.1 min-22.5 min; (5) in the scheme A, the retention time of the compound as shown in formula II is 26.7 min-28.8 min; (6) in the scheme B, the flow rate of the mobile phase is 7 mL / min; (7) in the scheme B, the retention time of the compound as shown in formula I is 18.0 min-19.1 min; (8) in the scheme B, the retention time of the compound as shown in formula II is 28.4 min-29.8 min; (9) the particle size of the packing material of the column is 10 μm; (10) the length of the column is 250 mm or 150 mm; (11) the inner diameter of the column is 30 mm or 22.1 mm; (12) the column temperature is 30 °C; (13) the detection wavelength of the detector is 200-300 nm, for example, 247 nm; (14) the injection volume of the liquid chromatography is 500 μL or 1000 μL.

7. The separation method of claim 2, wherein, The column is Kromasil C18; preferably, the column is Kromasil C18, 30 mm x 250 mm, 10 μm or Kromasil C18, 22.1 mm x 150 mm, 10 μm.

8. The separation method of claim 2, wherein, The separation method meets the following conditions: (1) in the step one, the mobile phase is 35% acetonitrile, 65% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100; and (2) in the second step, the mobile phase is 25% acetonitrile, 75% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:

100.

9. The separation method of claim 2, wherein, The separation method meets the following conditions: (1) in the first step, the mobile phase is 40% acetonitrile, 60% water and phosphoric acid; the volume ratio of the phosphoric acid to the water is 0.2:100; and (2) in the second step, the elution procedure is:

10. Use of a compound as shown in Formula I and / or a compound as shown in Formula II in the detection of methylprednisolone impurities. 。 ​

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