Detection method of Evocamide enantiomer

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of an evocamide enantiomer, which comprises the following steps: S1, preparing a test solution; s2, preparing a contrast solution; s3, preparing a system applicable solution; s4, carrying out a system applicability test; s5, testing a test sample and a contrast solution; a chromatographic column is a chiral chromatographic column taking a polysaccharide derivative or a cellulose derivative as a filler, and a mixed solution of n-hexane, isopropanol, trifluoroacetic acid and diethylamine is taken as a mobile phase to detect the Evocadum enantiomer. The method has the characteristics of simplicity in operation, high sensitivity and high accuracy, the chromatographic peaks are high in separation degree, do not interfere with one another, the peak shape is good, the result accuracy is guaranteed, the method can be used for detecting enantiomers in the Evocamide and the Evocamide preparation, the level of the enantiomers of the Evocamide is effectively controlled, and the medication safety is guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting enantiomers of evocalcet. Background Art

[0002] Secondary hyperparathyroidism is one of the common complications of chronic kidney disease (CKD). It is mainly due to the impaired activation of vitamin D during the development of chronic kidney disease, which reduces blood calcium and then leads to excessive secretion of parathyroid hormone (PTH).

[0003] Evocalcet belongs to the third-generation oral calcium mimetics. It acts on the Ca receptor on the surface of parathyroid cells, inhibits PTH secretion, reduces the concentration of PTH in the blood, and then alleviates symptoms. In addition, evocalcet can also regulate the biosynthesis of PTH and the proliferation of parathyroid cells, and control the production of PTH.

[0004] The chemical name of evocalcet is 2-{4-[(3S)-3-{[(1R)-1-(naphthalen-1-yl)ethyl]amino}pyrrolidin-1-yl]phenyl}acetic acid, and its structural formula is as follows: It contains a chiral carbon, so enantiomers may be introduced into evocalcet, that is, 2-{4-[(3R)-3-{[(1S)-1-(naphthalen-1-yl)ethyl]amino}pyrrolidin-1-yl]phenyl}acetic acid, and its structural formula is as follows: On the one hand, according to the guiding principles for the pharmaceutical research of chiral drugs, different stereoisomers of chiral drugs may have differences in pharmacodynamics, pharmacokinetics, and toxicology. This isomer may very likely have certain toxic side effects and affect the efficacy. On the other hand, the enantiomers of evocalcet exist in the raw material drug, directly affecting the purity of evocalcet and reducing the quality of subsequent products. Therefore, it is urgent to establish a simple, accurate, rapid, reliable, and stable method for detecting the corresponding isomers of evocalcet, so as to better control and master the product quality and improve the safety of clinical medication. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting enantiomers of evocalcet.

[0006] To solve the above problems, the technical solution adopted by the present invention is: A method for detecting enantiomers of evocalcet, comprising the following steps: S1 Preparation of the test solution: Weigh an appropriate amount of evocalcet precisely, use the mobile phase as the solvent, make up the volume to prepare the test solution; Preparation of the S2 control solution: Accurately measure an appropriate amount of the test solution obtained in S1, and quantitatively dilute it with the mobile phase to obtain the control solution. Preparation of the S3 system suitability solution: Weigh appropriate amounts of evocalcet and its enantiomer accurately, dissolve them with the mobile phase, make up the volume to obtain the system suitability solution. Test of the S3 system suitability solution: Inject an appropriate amount of the system suitability solution obtained in S3 into the liquid chromatograph, and record the chromatogram. Tests of the test sample and the control solution: Inject appropriate amounts of the test solution obtained in S1 and the control solution obtained in S2 into the liquid chromatograph, record the chromatogram, and calculate the content of the enantiomer using the peak area. The chromatographic column is a chiral chromatographic column filled with a polysaccharide derivative or a cellulose derivative. The mobile phase is a mixture of n-hexane, isopropanol, trifluoroacetic acid and diethylamine, and the flow rate is 0.5 - 1.2 ml / min.

[0007] As a further improvement of the present invention, the concentration of the test solution in S1 is 0.1 - 1.0 mg / ml. The dilution factor in S2 is 100 - 1000 times. In the S3 system suitability solution, the concentration of evocalcet is 0.1 - 0.5 mg / ml, and the concentration of the evocalcet enantiomer is 0.01 - 0.5 mg / ml.

[0008] As a further improvement of the present invention, the chromatographic column is a CHIRALPAK AD-H or CHIRALPAK OD-H chiral column, with a length range of 100 - 250 mm, an inner diameter range of 2.1 - 4.6 mm, a packing particle size of 5 μm, and the column temperature of the chromatographic column is 25 - 40 °C.

[0009] As a further improvement of the present invention, the detection wavelength is 240 - 260 nm, and the injection volume is 5 - 50 μl.

[0010] As a further improvement of the present invention, the volume ratio of n-hexane, isopropanol, trifluoroacetic acid and diethylamine in the mobile phase is 70 - 80:20 - 30:0.05 - 0.15:0.05 - 0.15.

[0011] As a further improvement of the present invention, the column temperature of the chromatographic column is 25 - 35 °C.

[0012] As a further improvement of the present invention, the flow rate of the mobile phase is 0.5 - 1 ml / min.

[0013] As a further improvement of the present invention, the detection wavelength is 253 nm - 255 nm.

[0014] As a further improvement of the present invention, the chromatographic column is a CHIRALPAK AD-H or CHIRALPAK OD-H chiral column, with a length of 250 mm, an inner diameter of 4.6 mm, a packing particle size of 5 μm, and a column temperature of 30 °C.

[0015] As a further improvement of the present invention, the concentration of the test solution in S1 is 0.5 mg / ml; The dilution factor in S2 is 100 times; In the system suitability solution of S3, the concentration of evocalcet is 0.5 mg / ml, and the concentration of the evocalcet enantiomer is 0.5 mg / ml.

[0016] As a further improvement of the present invention, the detection wavelength is 254 nm, and the injection volume is 10 μl.

[0017] As a further improvement of the present invention, the volume ratio of n-hexane, isopropanol, trifluoroacetic acid, and diethylamine in the mobile phase is 75:25:0.1:0.1.

[0018] As a further improvement of the present invention, the content of the enantiomer is calculated by the external standard method or the self-control method according to the peak area.

[0019] The beneficial effects of adopting the above technical solutions are as follows: The present invention provides a method for detecting evocalcet enantiomers. By comprehensively considering the influence of factors such as the analytical column, mobile phase, flow rate, and column temperature, efficient detection of evocalcet enantiomers is achieved. The detection method in the present invention has the characteristics of simple operation, high sensitivity, and high accuracy. Moreover, the separation degree of each chromatographic peak is high, without mutual interference, the peak shape is good, the result accuracy is guaranteed, and it can be used for the detection of enantiomers in evocalcet and its preparations, effectively controlling the level of evocalcet enantiomers and ensuring the safety of its medication. Description of the Drawings

[0020] Figure 1 It is the chromatogram of the system suitability solution in Example 1.

[0021] Figure 2 It is the chromatogram of the system suitability solution in Example 2.

[0022] Figure 3 It is the chromatogram of the test solution in the method specificity test of Example 3. Detailed Embodiments

[0023] To make the purpose, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0024] Evocarb hydrochloride enantiomers were purchased from TLC Pharmaceutical Standards Ltd., item number: E-204007.

[0025] The evocarb hydrochloride used in this application was prepared by the method described in Preparation Example 1: Preparation Example 1 Step 1. Preparation of Compound I: 1.1. Preparation of Compound I(1): 100.00 g of N-Boc-(R)-pyrrolidin-3-ol, 9.19 g of methylamine hydrochloride and 400 mL of acetonitrile were added to a 2 L three-necked flask. 70.27 g of triethylamine was added with stirring, and the temperature was lowered. At 14-16 °C, a solution prepared from 130.20 g of o-nitrobenzenesulfonyl chloride and 432 mL of toluene was slowly added. After the addition was complete, the temperature was controlled at 10-20 °C and the reaction was stirred for 4 h. After monitoring the reaction by TLC to completion, 300 mL of purified water was added to the reaction solution, and a solution prepared from 20 mL of hydrochloric acid and 176 mL of purified water was added. The layers were separated. The organic phase was added with 400 mL of purified water and 400 mL of dichloromethane, stirred, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered by suction, and the filter cake was washed with dichloromethane. The filtrate was concentrated under reduced pressure to dryness to obtain a dark brown oil, which was Compound I(1), and was directly used in the next step.

[0026] 1.2. Preparation of Compound I(2): 198.9 g of Compound I(1), 90.64 g of potassium phosphate and 796 mL of acetonitrile were added to a 1 L reaction flask. 73.12 g of (R)-1-(1-naphthyl)ethylamine was added with stirring, and the temperature was raised to reflux for 12 h. After monitoring the reaction by TLC to completion, the temperature was lowered to room temperature, and the mixture was filtered. The filtrate was concentrated under reduced pressure to dryness. 800 mL of ethyl acetate and 800 mL of saturated brine were added to the concentrated residue, and the mixture was stirred for 15 min. The layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered by suction, and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to dryness to obtain a dark brown oil, which was Compound I(2), and was directly used in the next step.

[0027] 1.3. Preparation of Compound I: 546 mL of isopropanol was added to a 2 L three-necked flask and stirred. The temperature was lowered, and 251.65 g of acetyl chloride was added dropwise at 0-15 °C. After the addition was complete, the mixture was stirred at 0-15 °C for 30 min. A solution prepared from 181.84 g of Compound I(2) and 364 mL of isopropanol was added dropwise at 0-15 °C. After the addition was complete, the temperature was raised to 60-65 °C and the reaction was carried out for 4 h. After monitoring the reaction by TLC to completion, the temperature was lowered to 20-30 °C and the mixture was stirred for crystallization for 2 h. The mixture was filtered by suction and washed with isopropanol. The filter cake was dried in vacuo at 45 °C to obtain Compound I. The total yield of the three-step reaction was 65.7%.

[0028] Step 2: Preparation of Compound II Add 250 g of Compound I, 2250 mL of dichloromethane, and 200 mL of purified water to a reaction flask, stir, adjust the pH to 14 with 4 mol / L sodium hydroxide solution, separate and collect the organic phase. Extract the aqueous phase with 500 mL of dichloromethane once, combine the organic phases, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter by suction, concentrate the filtrate to dryness to obtain a yellow oily substance. Add 1250 mL of DMF to it, stir to dissolve, add 167.34 g of ethyl 4-fluoro-3-nitrobenzeneacetate (1.1 eq), protect with nitrogen, add 32.14 g of sodium hydride (1.2 eq) in portions while controlling the temperature below 25 °C, stir at room temperature for 3 h, monitor the reaction by TLC until completion. Add the reaction solution to 5 L of purified water, stir at room temperature for 1 h to crystallize, filter, wash with purified water, and dry to obtain Compound II with a yield of 87.2%.

[0029] Step 3: Preparation of Compound III Add 150 g of Compound II, 1800 mL of absolute ethanol, and 450 mL of purified water to a reaction flask, stir, add 93.59 g of iron powder and 179.28 g of ammonium chloride. After addition, heat to reflux and react for 4 h, monitor the reaction by TLC until completion, filter, concentrate the filtrate under reduced pressure to remove ethanol, extract with 450 mL of dichloromethane three times, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate the filtrate to dryness, add 600 mL of ethyl acetate, stir at room temperature for 1 h, filter, wash with ethyl acetate, and dry to obtain Compound III with a yield of 86.7%.

[0030] Step 4: Preparation of Compound IV Add 100 g of Compound III and 1000 mL of absolute ethanol to a reaction flask, add 200 mL of concentrated sulfuric acid and 200 mL of hypophosphorous acid dropwise while controlling the temperature below 20 °C, then add an aqueous sodium nitrite solution (49.57 g of sodium nitrite dissolved in 198 mL of water) dropwise while still controlling the temperature below 20 °C. After addition, stir at room temperature for 10 h, monitor the reaction by TLC until completion. Add the reaction solution to 2000 mL of purified water, adjust the pH to 8.0 - 8.5 with sodium hydroxide solution, extract with 500 mL of dichloromethane three times, combine the organic phases, dry, filter, concentrate. After concentration, add 600 mL of ethyl acetate and stir to dissolve, adjust the pH to 3.0 - 4.0 with an ethyl acetate / hydrogen chloride mixed solution. After adjustment, stir at room temperature for 1 h, filter, wash with ethyl acetate, and dry to obtain Compound IV with a yield of 85.2%.

[0031] Step 5: Preparation of Evocalcet Add 33.87 g of sodium hydroxide and 496 mL of purified water to the reaction flask. Stir and cool down the temperature to 20 - 30 °C. Add 124 g of Compound IV and 496 mL of absolute ethanol. Heat up to 60 °C and react for 45 min. Perform hot suction filtration. Transfer the filtrate to the reaction flask. Heat up to 60 °C and slowly add 10% citric acid solution until solid precipitates. Keep stirring at a constant temperature for 0.5 h. Continue to slowly add 10% citric acid solution to adjust the pH to 6 - 7. After adjustment, keep stirring at 60 °C for 1 h. Turn off the heating and cool down to 20 °C. Stir for crystallization for 2 h. Perform suction filtration, wash with 50% ethanol, and dry to obtain evocalcet, with a yield of 96.3% and a liquid phase purity of 99.934%.

[0032] Example 1 1. Chromatographic conditions Chromatographic column: CHIRALPAK AD-H chiral column, with a specification of length 250 mm, inner diameter 4.6 mm, and the particle size of the packing material being 5 μm; Detection wavelength: 254 nm; Column temperature: 30 °C; Flow rate: 0.6 ml / min; Injection volume: 10 μl; Mobile phase: n-hexane - isopropanol - trifluoroacetic acid - diethylamine (volume ratio: 75:25:0.1:0.1).

[0033] 2. Experimental method and results S1 Preparation of test solution: Weigh accurately 10 mg of evocalcet and prepare a solution with a concentration of 0.5 mg / ml using the mobile phase as the solvent, as the test solution; S2 Preparation of control solution: Accurately measure 1.0 ml of the solution in S1 and place it in a 100-ml volumetric flask. Dilute it to the mark with the mobile phase and shake well, as the control solution; S3 Preparation of system suitability solution: Weigh accurately 10 mg of evocalcet and 10 mg of evocalcet enantiomer, and prepare a solution with a concentration of 0.5 mg / ml for each using the mobile phase as the solvent, as the system suitability solution; S4 System suitability test: Inject 10 μl of the system suitability solution prepared in S3 into the liquid chromatograph and record the chromatogram. The results are as Figure 1 shown; S5 Tests for test solution and control solution: Inject 10 μl of the test solution prepared in S1 and 10 μl of the control solution prepared in S2 respectively for testing. Analyze and inject the system suitability solution into the liquid chromatograph, record the chromatogram, and calculate the enantiomer content using the peak area.

[0034] In the chromatogram of the system suitability solution, the elution order of the peaks was the eplivanserin enantiomer peak and the eplivanserin peak successively. The resolution between the two peaks was 4.682, meeting the separation conditions, and the retention times were 7.079 min and 9.328 min respectively.

[0035] Table 1: Separation Results of Eplivanserin and Its Enantiomer Example 2 1. Chromatographic Conditions Chromatographic column: CHIRALPAK OD-H chiral column, with a specification of 250 mm in length, 4.6 mm in inner diameter, and a packing particle size of 5 μm; Detection wavelength: 254 nm; Column temperature: 30 °C; Flow rate: 1 ml / min; Injection volume: 10 μl; Mobile phase: n-hexane - isopropanol - trifluoroacetic acid - diethylamine (volume ratio: 70:30:0.1:0.1).

[0036] 2. Experimental Methods and Results S1 Preparation of the test solution: Weigh 10 mg of eplivanserin precisely, and prepare a solution with a concentration of 0.5 mg / ml using the mobile phase as the solvent, serving as the test solution; S2 Preparation of the control solution: Pipette 1.0 ml of the solution in S1 precisely, place it in a 100-ml volumetric flask, dilute it to the mark with the mobile phase, and shake well to serve as the control solution; S3 Preparation of the system suitability solution: Weigh 10 mg of eplivanserin and 1 mg of eplivanserin enantiomer precisely, and prepare a solution with an eplivanserin concentration of 0.5 mg / ml and an eplivanserin enantiomer concentration of 0.05 mg / ml using the mobile phase as the solvent, serving as the system suitability solution; S4 System suitability test: Inject 10 μl of the system suitability solution prepared in S3 into the liquid chromatograph, record the chromatogram, and the results are as Figure 2 shown; S5 Tests for the test solution and the control solution: Inject 10 μl of the test solution prepared in S1 and 10 μl of the control solution prepared in S2 respectively for testing. Analyze and inject the system suitability solution into the liquid chromatograph, record the chromatogram, and calculate the enantiomer content using the peak area.

[0037] In the chromatogram of the system suitability solution, the elution order of the peaks was the eplivanserin enantiomer peak and the eplivanserin peak successively. The resolution between the two peaks was 2.328, meeting the separation conditions, and the retention times were 10.617 min and 13.972 min respectively.

[0038] Table 2: Separation Results of Evocalcet and Its Enantiomers Example 3: Methodology Investigation 1. Method Specificity Test Take appropriate amounts of evocalcet and its enantiomer, weigh accurately, and prepare a solution with about 0.5 mg of evocalcet and 0.75 μg of evocalcet enantiomer in each 1 ml with the mobile phase as the test solution. Determine according to the chromatographic conditions and method in Example 1, record the chromatogram, and the obtained chromatogram is as Figure 3 shown.

[0039] From Figure 3 the results, it can be seen that the retention times of the evocalcet enantiomer and evocalcet are 7.681 min and 10.012 min respectively, the resolution between the evocalcet enantiomer and the adjacent peaks are 4.034 and 1.741 respectively, and the resolution between evocalcet and the adjacent peaks are 2.829 and 4.262 respectively, all greater than 1.5, indicating good resolution.

[0040] 2. Accuracy Test Take appropriate amounts of evocalcet and its enantiomer, weigh accurately, dilute with the mobile phase to prepare solutions with about 0.5 mg of evocalcet and 0.375 μg, 0.75 μg, 1.125 μg of evocalcet enantiomer in each 1 ml respectively as three concentration points of 50%, 100%, and 150% as the test solutions for recovery determination. Each concentration point is in triplicate.

[0041] Take the above solutions respectively and determine according to the chromatographic conditions in Example 1, record the chromatogram, and calculate the content of the enantiomer by the self - reference method based on the peak area. Calculate the recovery according to the measured amount and the actual added amount. The test results are shown in Table 3.

[0042] Table 3 From the results in Table 3, it can be seen that the accuracy of this method is good and meets the test requirements.

[0043] 3. Limit Test Adjust the instrument sensitivity, prepare a solution of evocalcet enantiomer with a certain concentration with the mobile phase, gradually dilute and inject according to the chromatographic conditions and method in Example 1 to make the main peak height about 3 times the baseline noise, record the chromatogram, and the minimum detection limit of evocalcet enantiomer is 0.18 ng.

[0044] Adjust the sensitivity of the instrument. Prepare a solution of evocalcet enantiomers with a certain concentration using the mobile phase. After gradual dilution, inject the sample according to the chromatographic conditions and methods in Example 1, so that the height of the main peak is about 10 times that of the baseline noise. Record the chromatogram. The minimum detection limit of evocalcet enantiomers is 0.61 ng.

[0045] 4. Linear and range test Take an appropriate amount of evocalcet enantiomers, weigh accurately, dissolve with the mobile phase and dilute successively to prepare a series of standard solutions with a concentration range of 0.0307 μg / ml to 1.2293 μg / ml. Determine according to the chromatographic conditions in Example 1, record the chromatogram, take the concentration C (μg / ml) as the abscissa and the peak area A as the ordinate for linear regression, and calculate the regression equation and correlation coefficient. The results are shown in Table 4.

[0046] Table 4 The results in Table 4 show that in the range of 0.0307 μg / ml to 1.2293 μg / ml, the linear relationship of evocalcet enantiomers is good.

[0047] 5. Precision test Take an appropriate amount of evocalcet and its enantiomers, weigh accurately, dissolve with the mobile phase to prepare a solution containing about 0.5 mg of evocalcet and 0.75 μg of evocalcet enantiomers per 1 ml. According to the chromatographic conditions in Example 1, inject the sample continuously for 6 times and record the chromatogram. Statistically analyze the peak area of evocalcet enantiomers. The test results are shown in Table 5.

[0048] Table 5 As can be seen from Table 5, the precision of the instrument for this method is good and meets the test requirements.

[0049] 6. Repeatability test Take an appropriate amount of evocalcet and its enantiomers, weigh accurately, dissolve with the mobile phase to prepare a solution containing about 0.5 mg of evocalcet and 0.75 μg of evocalcet enantiomers per 1 ml. Prepare 6 portions in parallel, inject the sample according to the chromatographic conditions in Example 1, and record the chromatogram.

[0050] The test results show that the contents of enantiomers in the 6 samples are 0.131%, 0.148%, 0.133%, 0.140%, 0.134%, and 0.150% respectively. The RSD of the 6 determination results is 5.8%, proving that this method has a certain precision.

[0051] 7. Solution stability test Take appropriate amounts of evocalcet and its enantiomer, weigh accurately, dissolve in the mobile phase to prepare a solution containing about 0.5 mg of evocalcet and 0.75 μg of the evocalcet enantiomer per 1 ml. Inject samples according to Example 1 at 0, 1, 2, 4, 6, 8, and 12 hours respectively, record the chromatograms, and the test results are shown in Table 6.

[0052] Table 6 As can be seen from Table 6, the enantiomer of evocalcet has good stability within 12 hours.

[0053] 8. Robustness Test By changing the column temperature, flow rate, and detection wavelength, evaluate the tolerance of the determination results to minor changes in the determination conditions. Under each condition, take appropriate amounts of evocalcet and its enantiomer, weigh accurately, dissolve in the mobile phase to prepare a solution containing about 0.5 mg of evocalcet and 0.75 μg of the evocalcet enantiomer per 1 ml as the test solution.

[0054] Examine the usability of the detection method when the chromatographic conditions change. Based on the chromatographic conditions described in Example 1, change the chromatographic conditions, and the resolution corresponding to the system suitability solution is shown in Table 7 (the chromatographic conditions without change are the same as those in Example 1): Table 7 Note: The chromatographic conditions in the table represent that these conditions are different from those in Example 1, and the remaining conditions are the same.

[0055] As can be seen from Table 7, when the chromatographic conditions change slightly, the resolution of evocalcet and its enantiomer in the system suitability solution hardly changes, and is greater than 1.5, meeting the requirements.

[0056] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting enantiomers of evoked, characterized in that: The steps include: S1 Preparation of test solution: Accurately weigh an appropriate amount of Evocarb, use the mobile phase as solvent, make up to volume, and prepare the test solution; Preparation of S2 control solution: Accurately measure an appropriate amount of the test solution obtained in S1 and quantitatively dilute it with the mobile phase as the control solution; S3 Preparation of system suitability solution: Accurately weigh appropriate amounts of evokedil and evokedil enantiomers, use the mobile phase as solvent, adjust the volume, and prepare the system suitability solution; S4 System suitability solution test: Take an appropriate amount of the system suitability solution obtained in S3, inject it into the liquid chromatograph, and record the chromatogram; S5 Test sample and control solution test: Take appropriate amount of the test sample solution obtained in S1 and the control solution obtained in S2 and inject them into the liquid chromatograph, record the chromatogram, and calculate the enantiomer content according to the peak area; The chromatographic column is a chiral chromatographic column using a polysaccharide derivative or a cellulose derivative as filler; The mobile phase is a mixture of n-hexane, isopropanol, trifluoroacetic acid and diethylamine, and the flow rate is 0.5-1.2 ml / min.

2. The method for detecting enantiomers of evoked ivoside according to claim 1, characterized in that: The concentration of the test solution in S1 is 0.1-1.0 mg / ml; The dilution factor in S2 is 100 to 1000 times; The concentration of evokedil in the S3 system applicability solution is 0.1-0.5 mg / ml, and the concentration of evokedil enantiomer is 0.01-0.5 mg / ml.

3. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The chromatographic column is a CHIRALPAK AD-H or CHIRALPAK OD-H chiral column, with a length ranging from 100 to 250 mm, an inner diameter ranging from 2.1 to 4.6 mm, a filler particle size of 5 μm, and a chromatographic column temperature of 25 to 40° C.

4. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The detection wavelength is 240-260 nm, and the injection volume is 5-50 μl.

5. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The volume ratio of n-hexane, isopropanol, trifluoroacetic acid and diethylamine in the mobile phase is 70-80:20-30:0.05-0.15:0.05-0.

15.

6. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The concentration of the test solution in S1 is 0.5 mg / ml; The dilution factor in S2 is 100 times; The concentration of evokedil in the S3 system suitability solution is 0.5 mg / ml, and the concentration of evokedil enantiomer is 0.5 mg / ml.

7. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The chromatographic column is a CHIRALPAK AD-H or CHIRALPAK OD-H chiral column with a length of 250 mm, an inner diameter of 4.6 mm, a filler particle size of 5 μm, and a column temperature of 30° C.

8. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The detection wavelength was 254 nm, and the injection volume was 10 μl.

9. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The volume ratio of n-hexane, isopropanol, trifluoroacetic acid and diethylamine in the mobile phase was 75:25:0.1:0.

1.

10. The method for detecting enantiomers of evoked cefixime according to claim 1, characterized in that: The content of enantiomers was calculated based on the peak area using the external standard method or the self-control method.

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