Detection method for isomer impurities in novel dopamine agonists

Abstract

This invention discloses a novel method for detecting isomer impurities in dopamine agonists. The method employs high-performance liquid chromatography (HPLC), using a cyclodextrin-packed column, with sodium perchlorate buffer as mobile phase A and acetonitrile as mobile phase B. The test solution is injected into the HPLC system for isocratic elution. This invention overcomes the technical bias of requiring forward chromatography for isomer detection and separation, creatively exploring a method for analyzing and detecting isomer impurities in pramipexole hydrochloride tablets using reverse chromatography. The method offers advantages such as high specificity, high accuracy, good durability, and low reagent hazard, effectively filling a gap in the quality standards for pramipexole hydrochloride tablets. It provides a technical reference for the quality control of pramipexole hydrochloride tablets, ensuring medication safety.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a novel method for detecting isomer impurities in dopamine agonists. Background Technology

[0002] Pramipexole dihydrochloride, chemically named (S)-2-amino-4,5,6,7-tetrahydro-6-propylamine-benzothiazole dihydrochloride monohydrate, CAS number: 191217-81-9, molecular formula: C 10 H 17 N3S·2HCl·H2O, with a molecular weight of 302.26, has the following structural formula:

[0003]

[0004] Pramipexole hydrochloride is a novel dopamine receptor agonist used to treat all stages of Parkinson's disease (PD). This drug effectively improves symptoms in PD patients and can be used alone or in combination with levodopa. It is currently a first-line treatment recommended by the American Academy of Neurology, the Movement Disorders Society of America, and PD treatment guidelines in various countries. It is also used for the symptomatic treatment of moderate to severe idiopathic restless legs syndrome. Regarding efficacy, in vivo and in vitro pharmacodynamic studies have shown that pramipexole has a high affinity for the dopamine D2 receptor subfamily and a preferential affinity for the dopamine D3 receptor, and it has a protective effect against dopamine and levodopa-induced neuronal damage. Regarding safety, comprehensive toxicological tests, including general toxicity, acute toxicity, reproductive toxicity, mutagenicity, and carcinogenicity tests, have shown that pramipexole has no teratogenic, carcinogenic, or mutagenic effects at clinical doses, demonstrating a good safety profile.

[0005] There is no publicly available data on the research of isomers of pramipexole hydrochloride extended-release tablets, either domestically or internationally, and relevant legal standards for extended-release or regular tablets are not included. For isomer research, the industry standard method is normal-phase chromatography. However, this product contains nearly 50% high-viscosity hydroxypropyl methylcellulose and carbomer, making normal-phase chromatography more difficult to extract the main components from the sample solution. Furthermore, the reagents used in normal-phase chromatography, such as isopropanol and n-hexane, have certain toxicity and are harmful to the environment and laboratory personnel.

[0006] If reverse chromatography is used, the aqueous solution of the test sample prepared directly without pretreatment will form a gel, making it impossible to inject accurately or clogging the chromatographic system after injection. The relevant substances and contents included in the import registration standard of sustained-release tablets use cellulase and amylase to hydrolyze the excipients to avoid gelation, which has a destructive effect on the chromatographic column packing material to be used in this invention and is not applicable to this invention. Summary of the Invention

[0007] The main objective of this invention is to provide a novel method for detecting isomer impurities in dopamine agonists.

[0008] The isomers to be detected in this invention are isomer impurities generated during the production of pramipexole hydrochloride sustained-release tablets, with the specific structure as follows:

[0009]

[0010] The aforementioned isomer impurities are commonly present in the production of active pharmaceutical ingredients (APIs) and finished drug products, and they have a serious impact on the final purity and quality of pramipexole hydrochloride formulations. Therefore, it is necessary to detect isomer impurities to strictly control product quality.

[0011] To achieve the above objectives and solve the problems existing in the current analysis of pramipexole hydrochloride sustained-release tablets, this application adopts the following technical solution:

[0012] A novel method for detecting isomer impurities in dopamine agonists is disclosed. The method employs high-performance liquid chromatography (HPLC), using a cyclodextrin-packed column, with mobile phase A being sodium perchlorate buffer and mobile phase B being acetonitrile. The test solution is injected into the HPLC system for isocratic elution. This method is applicable to pramipexole hydrochloride sustained-release tablets. The pH of mobile phase A is 1.5–2.5, preferably 2. The volume ratio of mobile phase A to mobile phase B is 85–95:15–5, preferably 90:10.

[0013] Furthermore, the sodium perchlorate buffer in mobile phase A is prepared by dissolving sodium perchlorate monohydrate in water and then adjusting the pH with perchloric acid. The concentration of the sodium perchlorate buffer is generally selected to be 0.1–0.5 mol / L.

[0014] Furthermore, the preparation method of the test solution is as follows: take pramipexole hydrochloride sustained-release tablets, add disintegrant to disintegrate the tablets, then dilute quantitatively with diluent, filter, and take the filtrate to obtain the test solution.

[0015] Furthermore, the disintegrant is a methanol-acetonitrile-phosphoric acid (60-70:39-29:1, volume ratio) mixed solution. Disintegrants within this concentration range can dissolve pramipexole hydrochloride sustained-release tablets. Preferably, the disintegrant is a methanol-acetonitrile-phosphoric acid (66:33:1) mixed solution.

[0016] Furthermore, the diluent is one or a mixture of several of the following: aluminum sulfate, potassium chloride, zinc sulfate, and magnesium chloride solution. Preferably, the concentration of the diluent is 0.01–1 g / ml, more preferably 0.01 g / ml. When preparing the test solution, using an inorganic salt solution as the diluent can solve the problem of detection difficulties caused by the easy gelation of pramipexole hydrochloride sustained-release tablets during test solution preparation. Simultaneously, it can achieve complete extraction of the main component, pramipexole hydrochloride, and its isomers, thereby improving detection accuracy.

[0017] Furthermore, the chromatographic column used was a Chiral CD-Ph.

[0018] Furthermore, a guard column can be added before the chromatographic column, preferably a Chiral CD-Ph10mm×4.0mm, S-5 model. The guard column, positioned before the chromatographic column, allows small amounts of gelatinous substances in the sample solution to deposit within it, thus extending the column's lifespan and reducing analytical costs.

[0019] Furthermore, the analytical conditions for liquid chromatography in the technical solution of the present invention include one or more of the following (i) to (vi):

[0020] (i) The chromatographic column has dimensions of 250 mm × 4.6 mm and a diameter of 5 μm.

[0021] (ii) The chromatographic column should be Chiral CD-Ph or a column with equivalent performance.

[0022] (iii) The protective post has the specifications of 10mm × 4.0mm, S-5.

[0023] (iv) The guard column should be a Chiral CD-Ph column or a column with equivalent performance.

[0024] (v) The column temperature is 40±5℃.

[0025] (vi) Range of initial mobile phase ratio variation: ±5%,

[0026] (vii) The flow rate is 0.7–0.9 ml / min.

[0027] (ⅷ) The injection volume is 50–100 μl.

[0028] (ⅸ) The detection wavelength is 262nm.

[0029] During the exploration of this technical solution, several technical difficulties arose. Firstly, there were no precedents for reference, requiring the R&D personnel to explore the methods independently. Secondly, given the characteristics of this project, pramipexole hydrochloride sustained-release tablets contain nearly 50% high-viscosity excipients. Without pretreatment, the resulting aqueous solution would form a gel, making injection impossible or clogging the chromatographic system. The import registration standards for sustained-release tablets use cellulase and amylase to hydrolyze the excipients, while this invention uses a cyclodextrin-filled column. Residual enzymes in the sample solution would damage the column, making enzymatic pretreatment unsuitable. Using other chromatographic columns, however, would fail to effectively detect and separate isomers in the pramipexole hydrochloride sustained-release tablets, thus failing to achieve the intended purpose.

[0030] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0031] (1) This technical solution fills the gap in the detection of isomers in pramipexole hydrochloride sustained-release tablets and is a brand-new method for isomer determination.

[0032] (2) Using a chromatographic column filled with cyclodextrin, the main peak and the isomer peak can be completely separated at the baseline, and the detection repeatability and accuracy are good.

[0033] (3) This technical solution overcomes the technical prejudice that isomer detection requires normal phase chromatography. The mobile phase only needs to be acetonitrile and sodium perchlorate aqueous solution, and it is isocratic elution. The reagents are simple and easy to obtain. Compared with normal phase reagents such as n-hexane and diethylamine, it is less harmful to the environment and laboratory personnel. Attached Figure Description

[0034] Figure 1 This is the chromatogram of the blank solvent in Example 1.

[0035] Figure 2 This is a chromatogram of the system suitability solution in Example 1.

[0036] Figure 3 This is the chromatogram of the reference solution in Example 1.

[0037] Figure 4 This is the chromatogram of the test solution in Example 1. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of this application.

[0039] The main instruments, reagents, and materials used in the following examples are as follows:

[0040] High-performance liquid chromatograph (Acquity Arc, Waters), electronic balance (MS205DU, XPR2, Mettler Toledo), pH meter (FE20, Mettler Toledo), water bath constant temperature shaker (SHA-B, Jiangsu Jinyi Instrument Technology Co., Ltd.).

[0041] Pramipexole hydrochloride sustained-release tablets (specification: 0.375mg, batch number: X200601, Hunan Jiudian Pharmaceutical Co., Ltd.), Pramipexole hydrochloride sustained-release tablet blank excipient (batch number: KF200601, Hunan Jiudian Pharmaceutical Co., Ltd.), Pramipexole hydrochloride working reference standard (batch number: PLK-19092501, content: 99.8%, Hunan Jiudian Pharmaceutical Co., Ltd.), Pramipexole hydrochloride isomer reference standard (batch number: PLK-D-18092901-R1, content: 97.8%, Hunan Jiudian Pharmaceutical Co., Ltd.).

[0042] Sodium perchlorate (monohydrate) (analytical grade, Sinopharm), perchloric acid (superior grade, Sinopharm), methanol (chromatographic grade, Sigma), acetonitrile (chromatographic grade, Sigma), phosphoric acid (chromatographic grade, Tianjin Kemeio Chemical Reagent Co., Ltd.), aluminum sulfate octahydrate (analytical grade, Sinopharm).

[0043] Example 1: Detection Method Example

[0044] 1. Solution preparation

[0045] Disintegrant: 660ml methanol, 330ml acetonitrile, 10ml phosphoric acid, mix well.

[0046] Diluent: Weigh 10g of aluminum sulfate octadechydrate and add 1000ml of water to dissolve it.

[0047] Blank solvent: Take 10 ml of disintegrant and place it in a 100 ml volumetric flask, then dilute to the mark with diluent.

[0048] System suitability solution: Take appropriate amounts of pramipexole hydrochloride working reference standard and isomer reference standard, add appropriate amount of disintegrant to dissolve, and dilute quantitatively with diluent to prepare a solution containing 15 μg of pramipexole hydrochloride and 0.075 μg of isomer per 1 ml.

[0049] Reference solution: Take an appropriate amount of pramipexole hydrochloride isomer reference standard, add an appropriate amount of disintegrant to dissolve it, and dilute quantitatively with diluent to prepare a solution containing 0.075 μg of isomer per 1 ml.

[0050] Test solution: Weigh 4 tablets of pramipexole hydrochloride sustained-release tablets (specification: 0.375mg) accurately, place them in a 100ml volumetric flask, add 10ml of disintegrant, shake for 20 minutes to completely disintegrate the sample, add an appropriate amount of diluent, sonicate for 20 minutes, dilute to the mark with diluent, filter, and collect the filtrate.

[0051] 2. Experimental Methods

[0052] Chromatographic column: Chiral CD-Ph 250mm×4.6mm, 5μm (guard column: Chiral CD-Ph 10mm×4.0mm, S-5);

[0053] Mobile phase: Sodium perchlorate buffer (preparation method: weigh 14.05 g of sodium perchlorate monohydrate, add 1000 ml of water to dissolve, adjust the pH to 2.0 with perchloric acid, and filter) - acetonitrile (90:10); mix well and sonicate to obtain the mobile phase;

[0054] Detection wavelength: 262 nm; flow rate: 0.8 ml / min; column temperature: 40 ℃; injection volume: 100 μl (absorption rate: 25 μl / min).

[0055] Detection: Inject 100 μl each of blank solvent, system suitability solution, reference solution, and test solution into the high performance liquid chromatograph, perform isocratic elution, and record the chromatogram.

[0056] 3. Experimental Results

[0057] The chromatogram was obtained after testing. Figures 1-4 As shown, Figure 1 This is a blank solvent chromatogram. Figure 2 Chromatogram of the solution for system suitability. Figure 3 This is the chromatogram of the reference solution. Figure 4 The image shows the chromatogram of the test sample solution. Figure 2 As can be seen, the retention time of the isomer in the system suitability solution is 24.193 min, and the separation degree between the isomer peak and the main peak is 1.53.

[0058] The experimental results show that under these chromatographic conditions, the isomers of pramipexole hydrochloride sustained-release tablets can be well separated, and peaks can be eluted and effectively separated within a suitable retention time.

[0059] Example 2: Exploring Pretreatment Conditions

[0060] The test solutions were prepared using 0.1 g / ml potassium chloride solution, 0.1 g / ml zinc sulfate solution, 0.1 g / ml magnesium chloride solution, 1 g / ml aluminum sulfate octahydrate solution, 0.1 g / ml aluminum sulfate octahydrate solution, 0.05 g / ml aluminum sulfate octahydrate solution, and 0.01 g / ml aluminum sulfate octahydrate solution as diluents, according to the conditions in Example 1. The extraction rate of the main component was determined using the content determination method in the imported registered standard. The experimental results are shown in Table 1.

[0061] Table 1. Results of extraction efficacy of pramipexole hydrochloride principal components by different salt solutions.

[0062]

[0063]

[0064] Experimental results show that potassium chloride, zinc sulfate, magnesium chloride, and aluminum sulfate octahydrate solutions of different concentrations can all completely extract the main components from pramipexole hydrochloride sustained-release tablets.

[0065] Example 3: Methodological Validation

[0066] 1. Chromatographic conditions

[0067] Chromatographic column: Chiral CD-Ph 250mm×4.6mm, 5μm (guard column: Chiral CD-Ph 10mm×4.0mm, S-5);

[0068] Mobile phase: Mobile phase A is sodium perchlorate buffer (weigh 14.05g of sodium perchlorate monohydrate, add 1000ml of water to dissolve, adjust the pH to 2.0 with perchloric acid, and filter), and mobile phase B is acetonitrile, with a volume ratio of 90:10.

[0069] Disintegrant: 660ml methanol, 330ml acetonitrile, 10ml phosphoric acid, mix well;

[0070] Diluent: Aluminum sulfate octahydrate solution;

[0071] Detection wavelength: 262 nm; flow rate: 0.8 ml / min; column temperature: 40 ℃; injection volume: 100 μl (absorption rate: 25 μl / min).

[0072] 2. Solution preparation

[0073] Disintegrant: 660ml methanol, 330ml acetonitrile, 10ml phosphoric acid, mix well.

[0074] Diluent: Weigh 10g of aluminum sulfate octadechydrate and add 1000ml of water to dissolve it.

[0075] Blank solvent: Take 10 ml of disintegrant and place it in a 100 ml volumetric flask, then dilute to the mark with diluent.

[0076] Test solution: Weigh 4 tablets of pramipexole hydrochloride sustained-release tablets (specification: 0.375mg) accurately, place them in a 100ml volumetric flask, add 10ml of disintegrant, shake for 20 minutes to completely disintegrate the tablets, add an appropriate amount of diluent, sonicate for 20 minutes, dilute to the mark with diluent, filter, and collect the filtrate.

[0077] Reference solution: Weigh 15 mg of the isomer reference standard into a 100 ml volumetric flask, dissolve and dilute to the mark with disintegrant, and shake well; accurately measure 1 ml into a 20 ml volumetric flask, dilute to the mark with disintegrant, and shake well to obtain the isomer stock solution; accurately measure 1 ml into a 100 ml volumetric flask, add 9 ml of disintegrant, dilute to the mark with diluent, and shake well to obtain the final solution.

[0078] System suitability solution: Weigh 15 mg of pramipexole hydrochloride reference standard into a 50 ml volumetric flask, dissolve and dilute to the mark with disintegrant, and shake well; measure 5 ml into a 100 ml volumetric flask, add 1 ml of isomer stock solution and 4 ml of disintegrant, dilute to the mark with diluent, and shake well to obtain the solution.

[0079] Limit: The content of isomers shall not exceed 0.5%.

[0080] 3. Methodological Validation

[0081] The above analytical method was validated according to the requirements of the Chinese Pharmacopoeia, and the validation results are shown in Table 2. The validation results show that the analytical method of the present invention is reasonable and feasible.

[0082] Table 2. Methodological Validation Results

[0083]

[0084] 3.1 Exclusivity

[0085] Blank solvent, blank excipient solution, test solution, system suitability solution, mixed solution (containing the main component and known impurities, with the concentration of known impurities prepared according to the impurity limit concentration in the relevant substances), and impurity localization solution (prepared according to the impurity limit concentration in the relevant substances) were prepared according to the analytical method. Experiments were conducted according to the analytical method, and the experimental results are shown in Table 3.

[0086] Table 3. Results of Specificity Experiment

[0087]

[0088] Note: The maximum absorption wavelength of impurity BⅢ546CL is 326nm, so no peak was observed at 262nm; no peak was observed in the localized solution of impurity BⅢ820BS, and no peak was observed at the isomer peak.

[0089] Conclusion: The blank solvent and blank excipient solution did not interfere with the isomer peak positions; in the mixed solution, the resolution between the isomer and the main peak was 1.52, which is greater than 1.5. All known impurities in pramipexole hydrochloride tablets did not interfere with the isomers, which meets the requirements.

[0090] 3.2 Limit of Detection and Limit of Quantification

[0091] Prepare solutions containing the isomers so that the signal-to-noise ratio of the isomer peaks is approximately 10 (limit of quantitation solution) or 3 (limit of detection solution). Perform experiments according to the analytical method; the results are shown in Table 4.

[0092] Table 4. Experimental results for limits of detection and limits of quantitation.

[0093]

[0094] Conclusion: The limit of quantitation for the isomer was 3.7604 ng, the limit of detection was 1.5042 ng, and the RSD of the peak area of ​​the solution after six consecutive injections was 4%, which is less than 10% and meets the requirements.

[0095] 3.3 Adsorption of Filter Membrane

[0096] Take the spiked solution of the test sample, centrifuge part of the solution (5000 rpm, 10 min), filter the other part of the solution and then conduct the experiment according to the analytical method. The experimental results are shown in Table 5.

[0097] Table 5 Results of the filter membrane adsorption experiment

[0098] Solution name Peak area Ratio of peak area to centrifuged peak area (%) Spiked solution of test sample (centrifuged) 12479 —— Spiked solution for test sample (filtered, discard 1 ml). 12087 96.9

[0099] Conclusion: The ratio of the peak area of ​​the isomers after filtration to that after centrifugation was 96.9%, which is in the range of 95.0% to 105.0%, indicating that the filter membrane did not significantly adsorb the isomers.

[0100] 3.4 Solution stability

[0101] The reference solution and the spiked solution of the test sample were placed at room temperature for 1 day and 2 days, respectively. The experiments were conducted according to the analytical method, and the relative deviation between the actual concentration on day 1 or day 2 and the concentration on day 0 was calculated. The experimental results are shown in Table 6.

[0102] Table 6 Results of solution stability experiments

[0103] Solution name Actual concentration (μg / ml) Relative deviation from 0 days (%) Reference solution 0 days 0.0752 —— Reference solution 1 day 0.0740 0.8 Reference solution 2 days 0.0769 1.1 Test sample spiking solution 0 days 0.0643 —— The test sample spiking solution was prepared 1 day ago. 0.0659 1.2 2 days after the test sample was spiked 0.0693 3.7

[0104] Conclusion: The relative deviation between the actual concentration of the reference solution and the spiked solution of the test sample after 1 or 2 days at room temperature and the concentration at day 0 was 3.7%, which was less than 4.0%, indicating that the solution was stable.

[0105] At room temperature, the reference solution and the spiked test solution showed good stability after 2 days.

[0106] 3.5 Linear

[0107] The concentration of the test solution for this product is 15 μg / ml, the isomer limit is 0.5%, and the limit concentration is 0.075 μg / ml. Linear solutions were prepared at the following concentrations: equivalent to the isomer limit concentration, 50%, 75%, 100%, 150%, and 200%. Experiments were conducted according to the analytical method. A linear regression curve was plotted with concentration on the x-axis and average peak area on the y-axis. The experimental results are shown in Table 7.

[0108] Table 7. Results of linear experiments on isomers

[0109]

[0110] Conclusion: Within the concentration range of 0.0376 μg / ml to 0.1504 μg / ml, the linear equation y = ...

[0111] 206091.1859x-333.9425, r=0.994, greater than 0.990; Y-intercept is 2% of 100% response value, less than 25%; RSD of response factor is 6%, less than 10%.

[0112] This indicates that the isomers have a good linear relationship with the peak area within the specified concentration range.

[0113] 3.6 Accuracy

[0114] Prepare spiking solutions of 50%, 100%, and 150% of the isomer limit concentration using the test sample and isomer. Perform experiments according to the analytical method, injecting one syringe into each recovery solution and calculating the recovery rate. The experimental results are shown in Table 8.

[0115] Table 8. Accuracy Experiment Results

[0116]

[0117] Conclusion: The recoveries of the nine test samples were all in the range of 91.0% to 101.0%, and in the range of 90.0% to 108.0%. The RSD of the recoveries of the nine test samples was 3.3%, indicating that the analytical method has good accuracy.

[0118] 3.7 Durability

[0119] Experiments were conducted according to the analytical method under normal conditions, flow rate ±0.1 ml / min, column temperature ±5℃, mobile phase aqueous pH ±0.5, and organic phase ratio in the mobile phase ±5%. The recovery rate of isomers in the spiked solution of the test sample was calculated, and the RSD of each changed condition and the result under normal conditions were calculated. The experimental results are shown in Table 9.

[0120] Table 9 Durability Test Results

[0121]

[0122] Conclusion: When the flow rate changes (0.7 ml / min to 0.9 ml / min), column temperature changes (35℃ to 45℃), mobile phase pH changes (1.5 to 2.5), and mobile phase ratio changes (95:5 to 85:15), the maximum RSD of the results under each changing condition and the normal condition is 3.4%, which is less than 4.0%, indicating that the analytical method has good robustness.

[0123] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of this application, and all such modifications or substitutions should be considered within the scope of protection of this application.

Claims

1. A method for detecting isomer impurities in a novel dopamine agonist, wherein the novel dopamine agonist is pramipexole hydrochloride sustained-release tablets, and the isomer impurity has the following structural formula: ， characterized in that The detection method is as follows: high performance liquid chromatography (HPLC) is used, with cyclodextrin as the packing material. The chromatographic column is a Chiral CD-Ph model with dimensions of 250 mm × 4.6 mm and 5 μm. Mobile phase A is sodium perchlorate buffer, and mobile phase B is acetonitrile. The test solution is injected into the HPLC instrument for isocratic elution, and the detection wavelength is 262 nm. The pH of the mobile phase A is 1.5 to 2.5, and the volume ratio of mobile phase A to mobile phase B is 85 to 95: 15 to 5. The preparation method of the test solution is as follows: take pramipexole hydrochloride sustained-release tablets, add disintegrant to disintegrate the tablets, then dilute quantitatively with diluent, filter, and take the filtrate to obtain the test solution; The disintegrant is a mixed solution of methanol-acetonitrile-phosphoric acid at a ratio of 60~70:39~29:1; The diluent is one or a mixture of several of the following: aluminum sulfate solution, potassium chloride solution, zinc sulfate solution, and magnesium chloride solution; The test sample was pramipexole hydrochloride sustained-release tablets, batch number: X200601, manufacturer: Hunan Jiudian Pharmaceutical Co., Ltd.

2. The detection method of claim 1, wherein, The sodium perchlorate buffer solution in the mobile phase A is prepared by dissolving sodium perchlorate monohydrate in water and then adjusting the pH with perchloric acid.

3. The detection method of claim 1, wherein, The concentration of the diluent is 0.01~1g / ml.

4. The detection method as described in claim 1, characterized in that, Add a guard column in front of the chromatographic column.

5. The detection method as described in claim 4, characterized in that, The analytical conditions for liquid chromatography include one or more of the following (i) to (v): (i) The specifications of the protective post are 10mm × 4.0mm, S-5. (ii) The protective column is model Chiral CD-Ph. (iii) The column temperature is 40±5℃. (iv) The flow rate is 0.7–0.9 ml / min. (v) The injection volume is 50–100 μl.

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