A method for detecting an atrasentan enantiomer

Abstract

The application discloses a method for detecting atrasentan enantiomers, and particularly relates to a method for detecting and / or separating compound I and / or II, which comprises the following steps: using a high performance liquid chromatography method, using a mobile phase to elute the to-be-detected substance in a chromatographic column, and then the method can be completed. The method can detect the purity of the compound I, and has good system adaptability, and can effectively separate the compound I and the compound II. Further, the preferred scheme of the application has the advantages of extremely low detection limit and quantitative limit, good linearity, high repeatability, good precision, high accuracy, strong stability and preferable recovery rate and the like.

Description

Technical Field

[0001] This invention relates to a method for detecting atrasentan enantiomers. Background Technology

[0002] Compound (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (Ⅰ) is a selective endothelin antagonist. During its synthesis, it produces the enantiomer (2R,3R,4R)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (Ⅱ), which corresponds to (Ⅰ). If this enantiomer is not controlled effectively, it will significantly affect the quality of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid.

[0003]

[0004] Therefore, it is crucial to develop a detection method that can detect enantiomer II in I, so as to achieve quantitative detection of this enantiomer. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for detecting atrasentan enantiomers, thereby improving the quality of atrasentan. This method can detect the purity of compound I and has good system adaptability, effectively separating compound I and compound II. Furthermore, the preferred embodiment of this invention has advantages such as extremely low limits of detection and quantitation, good linearity, high repeatability, good precision, high accuracy, strong stability, and excellent recovery rate.

[0006] This invention provides a method for detecting and / or separating compounds I and / or II, comprising the following steps:

[0007]

[0008] High-performance liquid chromatography (HPLC) is used, in which the analyte is eluted in a chromatographic column using a mobile phase.

[0009] The analyte comprises compound I and / or II;

[0010] The chromatographic column is a chiral chromatographic column of polysaccharide derivatives;

[0011] The mobile phase comprises the following components by volume percentage: 70%~95% mobile phase A and 5%~30% mobile phase B; the volume percentage represents the percentage of the volume of each component in the mobile phase.

[0012] The mobile phase A includes alkane solvents, and the mobile phase B includes alcohol solvents and organic acids.

[0013] In a preferred embodiment, the chromatographic column is a polysaccharide derivative-bonded chiral chromatographic column or a polysaccharide derivative-coated normal-phase chiral chromatographic column, for example, a polysaccharide derivative-bonded chiral chromatographic column, or for example, a cellulose tris(3,5-dichlorophenylcarbamate)-bonded silica gel chromatographic column.

[0014] In a preferred embodiment, the particle size of the packing material of the chromatographic column is 1.5μm to 10μm, for example 2.5μm to 7.5μm, or even 5μm.

[0015] In a preferred embodiment, the length of the chromatographic column is 100mm to 500mm, for example 100mm to 300mm, or for example 250mm.

[0016] In a preferred embodiment, the inner diameter of the chromatographic column is 2 mm to 7 mm, for example 3 mm to 5 mm, or 4.6 mm.

[0017] In a preferred embodiment, the chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column with dimensions of 250 mm * 4.6 mm and 5 μm, for example, YMC CHIRAL ART Cellulose-SC.

[0018] In a preferred embodiment, the mobile phase A is an alkane solvent.

[0019] In a preferred embodiment, the alkane solvent in the mobile phase A is n-hexane.

[0020] In a preferred embodiment, the alcohol solvent in the mobile phase B is methanol or ethanol, for example, ethanol.

[0021] In a preferred embodiment, the organic acid is trifluoroacetic acid.

[0022] In a preferred embodiment, the volume percentage of the organic acid in the mobile phase B is 0.05% to 5%, for example 0.1% to 1%, or for example 0.25%.

[0023] In a preferred embodiment, the mobile phase B is composed of an alcohol solvent and an organic acid, such as ethanol and trifluoroacetic acid.

[0024] In a preferred embodiment, the mobile phase is composed of the following components by volume percentage: 75% to 90% mobile phase A and 10% to 25% mobile phase B; for example, 80% to 85% mobile phase A and 15% to 20% mobile phase B; or, for example, 80% mobile phase A and 20% mobile phase B.

[0025] In a preferred embodiment, the mobile phase consists of 80% mobile phase A and 20% mobile phase B, wherein mobile phase A is n-hexane, and mobile phase B is ethanol and trifluoroacetic acid, wherein the volume percentage of trifluoroacetic acid in the total volume of mobile phase B is 0.25%.

[0026] In a preferred embodiment, the analyte includes compound I, for example, compound I and compound II.

[0027] In a preferred embodiment, the analyte is injected in the form of an analyte solution, and preferably, the solvent in the analyte solution is an alcohol solvent.

[0028] In a preferred embodiment, the alcohol solvent in the analyte solution is methanol or ethanol, such as ethanol.

[0029] In a preferred embodiment, the mass ratio of the analyte to the volume of the alcohol solvent in the analyte solution is 0.01 to 3 mg / ml; for example, 0.025 mg / ml or 2.5 mg / ml.

[0030] The injection volume of the high performance liquid chromatography method is the conventional injection volume for chromatographic detection in this field, for example, 1~50 μL, or 3~20 μL, preferably 5 μL.

[0031] The flow rate of the mobile phase is the conventional flow rate for chromatographic detection in this field, preferably 0.3~1.2 mL / min; for example 0.5~1.1 mL / min, or even 0.5 mL / min or 0.7 mL / min.

[0032] In a preferred embodiment, the column temperature of the chromatographic column is 25~40℃, for example 30~38℃, or for example 30℃ or 35℃.

[0033] In a preferred embodiment, the detector in the high-performance liquid chromatography is a DAD and / or UV detector, for example, a DAD or UV detector.

[0034] In a preferred embodiment, the detection wavelength in the high-performance liquid chromatography method is 200 nm to 400 nm, preferably 227 nm.

[0035] In a preferred embodiment, the elution time is 7-24 min, for example 9-17 min or 12-23 min, or for example 11 min, 15 min, 16 min or 22 min, wherein the elution time is the difference between the elution end time and the elution start time of the elution stage.

[0036] In a preferred embodiment, the elution conditions for the high-performance liquid chromatography (HPLC) are as follows: Option A or Option B:

[0037] Scheme A: The analytes are compound I and compound II;

[0038] The chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column;

[0039] The chromatographic column has dimensions of 250mm*4.6mm and a diameter of 5μm.

[0040] The mobile phase consists of the following components by volume percentage: 80% mobile phase A and 20% mobile phase B;

[0041] The mobile phase A is n-hexane;

[0042] The mobile phase B is composed of ethanol and trifluoroacetic acid; wherein the trifluoroacetic acid accounts for 0.25% of the total volume of the mobile phase B.

[0043] The flow rate of the mobile phase is 0.7 mL / min;

[0044] The column temperature of the chromatographic column is 35℃;

[0045] In the high-performance liquid chromatography method described above, the detection wavelength is 227 nm;

[0046] In the high-performance liquid chromatography method described above, the injection volume is 5 μL.

[0047] Better place,

[0048] The elution time is 16 minutes.

[0049] Scheme B: The analytes are compound I and compound II;

[0050] The chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column;

[0051] The chromatographic column has dimensions of 250mm*4.6mm and a diameter of 5μm.

[0052] The mobile phase consists of the following components by volume percentage: 80% mobile phase A and 20% mobile phase B;

[0053] The mobile phase A is n-hexane;

[0054] The mobile phase B is composed of ethanol and trifluoroacetic acid; wherein the trifluoroacetic acid accounts for 0.25% of the total volume of the mobile phase B.

[0055] The flow rate of the mobile phase is 0.5 mL / min;

[0056] The column temperature of the chromatographic column is 30℃;

[0057] In the high-performance liquid chromatography method described above, the detection wavelength is 227 nm;

[0058] In the high-performance liquid chromatography method described above, the injection volume is 5 μL.

[0059] Better place,

[0060] The elution time is 22 minutes.

[0061] In this invention, the detection method may be an external standard method, a self-comparison method, or an area normalization method. The external standard method calculates the content of isomers based on peak area. The self-comparison method calculates the content of isomers based on peak area of ​​the sample. The area normalization method calculates the isomer content by dividing the peak area of ​​the isomer in the chromatogram of the test solution by the sum of the peak areas of the isomer and the sample.

[0062] In this invention, the term "external standard method" refers to a method of quantification by comparing the response signals of the isomer in the reference substance and the pure isomer in the sample with those of the reference substance.

[0063] In this invention, the term "self-comparison method" refers to a quantitative method that compares the response signal of the main component in a self-comparison solution with the response signal of the isomer in the sample.

[0064] In this invention, the term "area normalization method" refers to a quantitative method that compares the response signal of the isomer in the sample with the sum of the response signals of the isomer in the sample and the sample.

[0065] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0066] The reagents and raw materials used in this invention are all commercially available.

[0067] The positive and progressive effects of this invention are as follows: The detection method for the (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid isomer provided by this invention can realize the detection and control of isomer (II), thus ensuring the quality of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid. Detailed Implementation

[0068] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0069] Sample Name: (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (I) (Source: Nanjing Yaotan Biotechnology Co., Ltd.; Batch No.: CD24051-30-J-01; Content: 99.78% on anhydrous basis)

[0070] Sample Name: (2R,3R,4R)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid isomer (II) (Source: Nanjing Yaotan Biotechnology Co., Ltd.; Batch No.: CD24051-30-J-02; Content: 99.13% on anhydrous basis)

[0071] Example 1:

[0072] Weigh 25 mg of (2R,3R,4R)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid isomer (II), place it in a 100 mL volumetric flask, dissolve and dilute to the mark with ethanol, shake well, then accurately measure 1 mL of this solution, place it in a 10 mL volumetric flask, dilute to the mark with ethanol, shake well, and use as the final volume. Stock solution of isomer (II): Weigh 25 mg of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (I), place it in a 10 mL volumetric flask, transfer 1 mL of stock solution of isomer (II) into it, add ethanol to dissolve and dilute to the mark, shake well, and use it as the system suitability solution.

[0073] The chromatographic column used was a YMC CHIRAL ART Cellulose-SC (250 mm * 4.6 mm, 5 μm), the column temperature was 35 °C, the detection wavelength was 227 nm, and a DAD detector was used.

[0074] The mobile phase was n-hexane-ethanol (0.05% trifluoroacetic acid) (80:20), the flow rate was 0.7 mL / min, and the injection volume was 5 μL.

[0075] In the chromatogram of the system suitability solution, (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (Ⅰ) and (2R,3R,4R)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl) The enantiomer (II) of pyrrolidine-3-carboxylic acid elutes sequentially. The retention time of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (I) is approximately 10.001 min, and the retention time of enantiomer (II) is approximately 15.226 min (relative retention time approximately 1.52). The resolution between the two is not less than 1.5, and the theoretical plate number based on the isomer peak is not less than 10,000.

[0076] The test results are shown in the table below:

[0077]

[0078] Example 2:

[0079] (1) Limit of quantitation and limit of detection

[0080] 1.1 Solution Preparation

[0081] Isomer (II) stock solution: Weigh 25 mg of isomer (II), place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, shake well, and use as isomer (II) stock solution.

[0082] Main component stock solution: Weigh 25 mg of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (I), place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, shake well, and use as the main component stock solution.

[0083] Accurately measure 2 mL each of the stock solution of isomer (II) and the stock solution of the main component, place them in a 20 mL volumetric flask, add ethanol to dilute to the mark, shake well, and use as the mixed stock solution ①.

[0084] Take 1 mL of the mixed stock solution ①, place it in a 100 mL volumetric flask, add ethanol to dilute to the mark, shake well, and use it as the limit of quantitation solution.

[0085] Accurately measure 3 mL of the limit of quantitation solution, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain the limit of detection solution.

[0086] 1.2 Verification Procedure

[0087] Inject samples in the order shown in the table below:

[0088]

[0089] 1.3 Acceptable Standards

[0090] In the chromatogram of the limit of detection solution, the signal-to-noise ratio of the isomer (II) peak is not less than 3; in the chromatogram of the limit of quantitation solution, the signal-to-noise ratio of the isomer (II) peak is not less than 10, and the relative standard deviation of the peak area of ​​the isomer (II) for six consecutive injections is not greater than 10%.

[0091] 1.4 Verification Results

[0092]

[0093] 1.5 Verification Conclusion

[0094] The limit of quantitation is 0.25 μg / ml, equivalent to 0.01% of the sample concentration, and the limit of detection is 0.075 μg / ml, equivalent to 0.003% of the sample concentration.

[0095] In the chromatogram of the limit of quantitation solution, the signal-to-noise ratio of the peak of isomer (II) is 18.7, and the relative standard deviation of the peak area of ​​isomer (II) for six consecutive injections is 0.93%. In the chromatogram of the limit of detection solution, the signal-to-noise ratio of the peak of isomer (II) is 5.3, which meets the requirements.

[0096] (2) Linearity and Range

[0097] 2.1 Solution Preparation

[0098] Accurately measure 1 mL of the mixed stock solution ①, place it in a 100 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain linear solution 1.

[0099] Accurately measure 1 mL of the mixed stock solution ①, place it in a 50 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain linear solution 2.

[0100] Accurately measure 1 mL of the mixed stock solution ①, place it in a 20 mL volumetric flask, add ethanol to dilute to the mark, shake well, and use it as linear solution 3.

[0101] Accurately measure 1 mL of the mixed stock solution ①, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, shake well, and use it as linear solution 4.

[0102] Accurately measure 2 mL of the mixed stock solution ①, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, shake well, and use it as linear solution 5.

[0103] 2.2 Verification Procedure

[0104]

[0105] 2.3 Acceptable Standards

[0106] The correlation coefficient r of the linear equation is not less than 0.995, and the ratio of the absolute value of the intercept of the linear equation to the peak area at 100% concentration is not greater than 20%.

[0107] 2.4 Verification Results

[0108]

[0109] 2.5 Verification Conclusion

[0110] Within the concentration range of 0.2500–5.0010 μg / mL, the principal component showed a linear correlation between concentration and response signal. The linear equation for principal component (I) was y = 8.6463x + 0.4871, with a correlation coefficient r = 0.9999. The ratio of the absolute value of the intercept to the peak area at 100% concentration was 2.20%. Within the concentration range of 0.2484–4.9683 μg / mL, isomer (II) also showed a linear correlation between concentration and response signal. The linear equation for isomer (II) was y = 8.7124x + 0.7945, with a correlation coefficient r = 0.999. The ratio of the absolute value of the intercept to the peak area at 100% concentration was 3.54%.

[0111] The ratio of the slopes of the linear equations for principal component (Ⅰ) and isomer (Ⅱ) is 0.99, and the external standard method can be used to determine the isomers.

[0112] (3) Repeatability

[0113] 3.1 Solution Preparation

[0114] Stock solution of isomer (II): Weigh 25 mg of isomer (II), place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well. Then accurately measure 1 mL of this solution, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain the stock solution of isomer (II).

[0115] 100% spiked test solution: Weigh 25 mg of main component (I), place it in a 10 mL volumetric flask, accurately add 1 mL of stock solution of isomer (II), add ethanol to dissolve and dilute to the mark, shake well, and make 6 parallel portions.

[0116] Unspecified test solution: Weigh 25 mg of the main component (I), place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, shake well, and make 6 parallel portions.

[0117] 3.2 Verification Procedure

[0118] Inject samples according to the injection procedure in the table below:

[0119]

[0120] 3.3 Acceptable Standards

[0121] The content of isomer (II) in the test solution is less than 0.1%, and the relative standard deviation of the content of isomer (II) in 6 samples shall not exceed 10%; the content of isomer (II) is between 0.1% and 0.5%, and the relative standard deviation of the content of isomer (II) in 6 samples shall not exceed 5%.

[0122] 3.4 Verification Results

[0123]

[0124] 3.5 Verification Conclusion

[0125] The average value of the 6 samples with 100% spiked solution was 0.134%, with a relative standard deviation of 0.82%; the average value of the 6 samples without spiked solution was 0.031%, with a relative standard deviation of 2.42%, both of which meet the requirements.

[0126] (4) Intermediate precision

[0127] 4.1 Solution Preparation

[0128] The test personnel were changed to conduct the test at different times according to the repeatability test requirements.

[0129] 4.2 Verification Procedure

[0130] See item 3.2 under the same repetition item.

[0131] 4.3 Acceptable Standards

[0132] The content of isomer (II) in the test solution is less than 0.1%, and the relative standard deviation of the content of isomer (II) in 12 samples shall not exceed 10%; the content of isomer (II) is between 0.1% and 0.5%, and the relative standard deviation of the content of isomer (II) in 12 samples shall not exceed 5%.

[0133] 4.4 Verification Results

[0134]

[0135] 4.5 Verification Conclusion

[0136] The relative standard deviation of 12 samples with 100% spiked sample was 1.08%; the relative standard deviation of 12 samples without spiked sample was 2.85%, which meets the requirements.

[0137] (5) Accuracy

[0138] 5.1 Solution Preparation

[0139] Stock solution of isomer (II): Weigh 25 mg of isomer (II), place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well. Then accurately measure 1 mL of this solution, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain the stock solution of isomer (II).

[0140] 50% spiked test solution: Weigh 25 mg of main component (I), place it in a 10 mL volumetric flask, accurately add 0.5 mL of isomer stock solution, add ethanol to dissolve and dilute to the mark, shake well, and make 3 parallel portions.

[0141] 100% spiked test solution: Weigh 25 mg of main component (I), place it in a 10 mL volumetric flask, accurately add 1.0 mL of stock solution of isomer (II), add ethanol to dissolve and dilute to the mark, shake well, and make 3 parallel portions.

[0142] 150% spiked test solution: Weigh 25 mg of main component (I), place it in a 10 mL volumetric flask, accurately add 1.5 mL of isomer (II) stock solution, add ethanol to dissolve and dilute to the mark, shake well, and make 3 parallel portions.

[0143] Control solution: Accurately measure 1 mL of the above isomer (II) stock solution, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, shake well, and make two parallel portions.

[0144] 5.2 Verification Procedure

[0145] Inject samples according to the injection procedure in the table below:

[0146]

[0147] 5.3 Acceptable Standards

[0148] The recovery rate ranges from 92% to 105%, and the relative standard deviation must not exceed 5%.

[0149] 5.4 Verification Results

[0150]

[0151] 5.5 Verification Conclusion

[0152] The recoveries at all concentrations were in the range of 92% to 105%, with a relative standard deviation of 0.85%, which met the requirements.

[0153] (6) Solution stability

[0154] 6.1 Solution Preparation

[0155] Stock solution of isomer (II): Weigh 25 mg of isomer (II), place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well. Then accurately measure 1 mL of this solution, place it in a 10 mL volumetric flask, add ethanol to dilute to the mark, and shake well to obtain the stock solution of isomer (II).

[0156] Main component (I) solution: Accurately measure 25 mg of main component (I) and place it in a 10 mL volumetric flask. Accurately measure 1 mL of the above isomer (II) stock solution and place it in the above 10 mL volumetric flask. Add ethanol to dilute to the mark and shake well.

[0157] 6.2 Verification Procedure

[0158]

[0159] 6.3 Acceptable Standards

[0160] In the chromatograms of the main component (I) solution at each time point, the ratio of the peak area of ​​the main component (I) to the peak area at 0 hours should be between 95% and 105%, and the ratio of the peak area of ​​the isomer (II) to the peak area at 0 hours should be between 90% and 110%.

[0161] 6.4 Verification Results

[0162]

[0163] 6.5 Verification Conclusion

[0164] Within 14 hours, in the chromatograms of the main component (I) solution at each time point, the ratio of the peak area of ​​the main component (I) to the peak area at 0 hours was between 95% and 105%. In the chromatogram of the test sample solution, the ratio of the peak area of ​​the isomer (II) to the peak area at 0 hours was between 90% and 110%.

[0165] (7) Conclusion

[0166] The method for detecting enantiomers of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid provided by this invention can accurately quantify the isomers of a sample. This method has high accuracy and precision, and can strictly control the quality of (2R,3R,4S)-4-(1,3-benzodioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid.

[0167] Example 3

[0168] The sample selection and preparation were the same as in Example 1, and the detection was performed using the chromatographic conditions shown below:

[0169]

[0170] The test results are shown in the table below:

[0171]

[0172] Under these chromatographic conditions, the separation of compound I and compound II was satisfactory.

[0173] Comparative Example 1

[0174] The sample selection and preparation were the same as in Example 1, and the detection was performed using the chromatographic conditions shown below:

[0175]

[0176] The test results are shown in the table below:

[0177]

[0178] Under these chromatographic conditions, compound I and compound II could not be separated.

[0179] Comparative Example 2

[0180] The sample selection and preparation were the same as in Example 1, and the detection was performed using the chromatographic conditions shown below:

[0181]

[0182] The test results are shown in the table below:

[0183]

[0184] Under these chromatographic conditions, the separation of compound I and compound II was not satisfactory.

[0185] Comparative Example 3

[0186] The sample selection and preparation were the same as in Example 1, and the detection was performed using the chromatographic conditions shown below:

[0187]

[0188] The test results are shown in the table below:

[0189]

[0190] Under these chromatographic conditions, the separation of compound I and compound II was not satisfactory.

[0191] Comparative Example 4

[0192] The sample selection and preparation were the same as in Example 1, and the detection was performed using the chromatographic conditions shown below:

[0193]

[0194] The test results are shown in the table below:

[0195]

[0196] Under these chromatographic conditions, the separation of compound I and compound II was not satisfactory.

Claims

1. A method for detecting and / or separating compounds I and / or II, characterized in that, It includes the following steps: ； High-performance liquid chromatography (HPLC) is used, in which the analyte is eluted in a chromatographic column using a mobile phase. The analyte comprises compound I and / or II; The chromatographic column is a chiral chromatographic column of polysaccharide derivatives; The mobile phase comprises the following components by volume percentage: 70%~95% mobile phase A and 5%~30% mobile phase B; the volume percentage represents the percentage of the volume of each component in the mobile phase. The mobile phase A includes alkane solvents, and the mobile phase B includes alcohol solvents and organic acids.

2. The detection and / or separation method as described in claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The chromatographic column is a polysaccharide derivative bonded chiral chromatographic column or a polysaccharide derivative coated normal-phase chiral chromatographic column; (2) The particle size of the packing material of the chromatographic column is 1.5μm~10μm; (3) The length of the chromatographic column is 100mm~500mm; (4) The inner diameter of the chromatographic column is 2 mm to 7 mm; (5) In the mobile phase A, the alkane solvent is n-hexane; (6) In the mobile phase B, the alcohol solvent is methanol or ethanol; (7) The organic acid mentioned is trifluoroacetic acid; (8) In the mobile phase B, the volume percentage of the organic acid in the total volume of the mobile phase B is 0.05% to 5%; (9) The analyte includes compound I; (10) The analyte is injected in the form of an analyte solution; (11) The flow rate of the mobile phase is 0.3~1.2 mL / min; (12) The column temperature of the chromatographic column is 25~40℃; (13) In the high performance liquid chromatography method described above, the detector is a DAD and / or a UV detector; (14) In the high performance liquid chromatography method described above, the detection wavelength is 200 nm to 400 nm; (15) The elution time is 7~24 min.

3. The detection and / or separation method as described in claim 2, characterized in that, It satisfies one or more of the following conditions: (1) The chromatographic column is a polysaccharide derivative bonded chiral chromatographic column, such as a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica gel chromatographic column; (2) The particle size of the packing material of the chromatographic column is 2.5 μm to 7.5 μm; (3) The length of the chromatographic column is 100mm~300mm; (4) The inner diameter of the chromatographic column is 3mm~5mm; (5) In the mobile phase B, the alcohol solvent is ethanol; (6) In the mobile phase B, the volume percentage of the organic acid in the total volume of the mobile phase B is 0.1% to 1%; (7) The analytes include compound I and compound II; (8) The solvent in the analyte solution is an alcohol solvent; (9) The injection volume of the analyte solution is 1~50 μL; (10) The flow rate of the mobile phase is 0.5~1.1 mL / min; (11) The column temperature of the chromatographic column is 30~38℃; (12) The detector is a DAD or UV detector; (13) The detection wavelength is 227 nm; (14) The elution time is 9~17 min or 12~23 min.

4. The detection and / or separation method as described in claim 3, characterized in that, It satisfies one or more of the following conditions: (1) The particle size of the packing material of the chromatographic column is 5 μm; (2) The length of the chromatographic column is 250 mm; (3) The inner diameter of the chromatographic column is 4.6 mm; (4) The volume percentage of the organic acid in the total volume of the mobile phase B is 0.25%; (5) In the analyte solution, the alcohol solvent is methanol or ethanol; (6) In the analyte solution, the ratio of the mass of the analyte to the volume of the alcohol solvent is 0.01~3 mg / ml; (7) The injection volume is 3~20 μL; (8) The flow rate of the mobile phase is 0.5 mL / min or 0.7 mL / min; (9) The column temperature of the chromatographic column is 30℃ or 35℃; (10) The elution time is 11 min, 15 min, 16 min or 22 min.

5. The detection and / or separation method as described in claim 4, characterized in that, It satisfies one or more of the following conditions: (1) In the analyte solution, the alcohol solvent is ethanol; (2) In the analyte solution, the ratio of the mass of the analyte to the volume of the alcohol solvent is 0.025 mg / ml or 2.5 mg / ml; (3) The injection volume is 5 μL.

6. The detection and / or separation method according to any one of claims 1-5, characterized in that, It satisfies one or two of the following conditions: (1) The mobile phase A is an alkane solvent; (2) The mobile phase B is composed of alcohol solvents and organic acids, such as ethanol and trifluoroacetic acid.

7. The detection and / or separation method according to any one of claims 1-5, characterized in that, The mobile phase is composed of the following components by volume percentage: 75% to 90% mobile phase A and 10% to 25% mobile phase B; for example, 80% to 85% mobile phase A and 15% to 20% mobile phase B; or, for example, 80% mobile phase A and 20% mobile phase B.

8. The detection and / or separation method as described in claim 7, characterized in that, The mobile phase consists of 80% mobile phase A and 20% mobile phase B. Mobile phase A is n-hexane, and mobile phase B is ethanol and trifluoroacetic acid. The volume percentage of trifluoroacetic acid in mobile phase B is 0.25%.

9. The detection and / or separation method according to any one of claims 1-5, characterized in that, The chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column with dimensions of 250mm*4.6mm and 5μm, for example, YMC CHIRAL ART Cellulose-SC.

10. The detection and / or separation method as described in claim 1, characterized in that, The elution conditions for the high-performance liquid chromatography method are as follows: Scheme A or Scheme B: Scheme A: The analytes are compound I and compound II; The chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column; The chromatographic column has dimensions of 250mm*4.6mm and a diameter of 5μm. The mobile phase consists of the following components by volume percentage: 80% mobile phase A and 20% mobile phase B; The mobile phase A is n-hexane; The mobile phase B is composed of ethanol and trifluoroacetic acid; wherein the trifluoroacetic acid accounts for 0.25% of the total volume of the mobile phase B. The flow rate of the mobile phase is 0.7 mL / min; The column temperature of the chromatographic column is 35℃; In the high-performance liquid chromatography method described above, the detection wavelength is 227 nm; In the high-performance liquid chromatography method described above, the injection volume is 5 μL; Better place, The elution time is 16 minutes; Scheme B: The analytes are compound I and compound II; The chromatographic column is a cellulose tris(3,5-dichlorophenylcarbamate) bonded silica chiral column; The chromatographic column has dimensions of 250mm*4.6mm and a diameter of 5μm. The mobile phase consists of the following components by volume percentage: 80% mobile phase A and 20% mobile phase B; The mobile phase A is n-hexane; The mobile phase B is composed of ethanol and trifluoroacetic acid; wherein the trifluoroacetic acid accounts for 0.25% of the total volume of the mobile phase B. The flow rate of the mobile phase is 0.5 mL / min; The column temperature of the chromatographic column is 30℃; In the high-performance liquid chromatography method described above, the detection wavelength is 227 nm; In the high-performance liquid chromatography method described above, the injection volume is 5 μL; Better place, The elution time is 22 minutes.

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