Method for separating and detecting key impurities in beraprost sodium starting material

Abstract

The invention discloses a method for separating and detecting key impurities in a beraprost sodium starting material. High performance liquid chromatography is adopted. According to the method, in a reversed-phase mode, an Agilent ZORBAX SB-AQ chromatographic column is adopted, an acid system of phosphoric acid-water and methanol / acetonitrile is used as a mobile phase, and gradient elution is adopted. The method disclosed by the invention is simple, high in accuracy, good in linearity and high in sensitivity, and can be used for effectively controlling the quality of the beraprost sodium starting material.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting key impurities in the starting material of beraprost sodium. Background Art

[0002] Beraprost sodium is a drug for treating cardiovascular diseases. Its synthesis process has a long route, and the starting material, intermediate, final product, and related impurities are mostly mixtures of non-corresponding isomers, which will show multiple peaks under non-chiral chromatographic conditions, resulting in great difficulty in developing an analytical method.

[0003] SM3 is an important starting material for the synthesis of beraprost sodium, and its synthesis route is as follows:

[0004] The inventor of the present invention found that during the synthesis of the SM3 sample, the SM3-IMP1 cis-trans isomer impurities will be generated simultaneously, and large detections have been found in multiple batches of samples; and they will participate in the subsequent reactions to generate more complex derivative impurities, which not only affect the quality of the subsequent products, but also greatly increase the difficulty of establishing an analytical method for controlling related impurities in the subsequent intermediates or finished products. Therefore, it is necessary to establish a corresponding analytical method in the starting material SM3 to detect and control the impurity SM3-IMP1.

[0005] The inventor of the present invention found many difficulties in establishing this analytical method: (1) There are only minor differences in the structures of SM3 and SM3-IMP1, and they are cis-trans isomer impurities of C=C; (2) SM3 and SM3-IMP1 are respectively composed of a mixture of a pair of enantiomers. Although they are one peak under non-chiral chromatographic conditions, they will finally be derived into non-corresponding isomers in the subsequent intermediates and finished products, and multiple peaks may appear on the non-chiral chromatographic column, bringing great challenges to the establishment of the subsequent analytical method. Therefore, it is necessary to control the impurity SM3-IMP1 in its starting material SM3 at the source. Currently, no method for detecting the cis-trans isomer impurities of SM3 in the reverse phase mode has been retrieved. Therefore, it is necessary to develop an analytical method with high accuracy, good repeatability, and high sensitivity to meet the separation and detection of the cis-trans isomer impurity SM3-IMP1 in SM3 to ensure the obtainment of qualified final products. Summary of the Invention

[0006] Aiming at the deficiencies of the current existing technologies, the purpose of the present invention is to provide a method for separating and detecting the cis-trans isomer impurity SM3-IMP1 in the starting material SM3 of beraprost sodium.

[0007] The chemical structures of SM3 and SM3-IMP1 are as follows:

[0008] To achieve the above-mentioned invention object, the present invention provides the following technical solutions: In an embodiment of the technical solution, a method for separating and detecting key impurities in the starting material of beraprost sodium, the method uses high performance liquid chromatography, and is characterized in that: its chromatographic conditions include: Chromatographic column: Agilent ZORBAX SB-AQ, Mobile phase: Mobile phase A is a phosphoric acid - aqueous solution, and mobile phase B is a mixed solution of acetonitrile - methanol, Elution method: Gradient elution, the initial ratio of the mobile phase: mobile phase A: mobile phase B is 78%: 22% to 82%: 18%, Detector: Ultraviolet detector; Among them, the starting material is the compound shown by formula SM3, and the key impurity is the compound shown by formula SM3-IMP1, .

[0009] Preferably, in the detection method of the present invention above, mobile phase A is a solution with a volume ratio of phosphoric acid to water of 1:1000, and mobile phase B is a mixed solution with a volume ratio of acetonitrile to methanol = 2:1 Furthermore, in the detection method of the present invention above, for the gradient elution, from 0 to 35 minutes: mobile phase A is 78~82% - 0, mobile phase B is 22~18% - 100%, from 35 to 50 minutes, mobile phase A is 0, mobile phase B is 100%, from 50 to 51 minutes, mobile phase A is 0 - 72~82%, mobile phase B is 100% - 22~18%, and from 51 to 65 minutes, mobile phase A is 72~82%, mobile phase B is 22~18%.

[0010] Preferably, in the detection method of the present invention above, for the gradient elution, from 0 to 35 minutes: mobile phase A is 80% - 0, mobile phase B is 20% - 100%, from 35 to 50 minutes, mobile phase A is 0, mobile phase B is 100%, from 50 to 51 minutes, mobile phase A is 0 - 80%, mobile phase B is 100% - 20%, and from 51 to 65 minutes, mobile phase A is 80%, mobile phase B is 20%.

[0011] Preferably, in the detection method of the present invention above, for the gradient elution, the initial ratio of mobile phase A: mobile phase B is 80%:20%.

[0012] Furthermore, in the detection method of the present invention above, for the chromatographic conditions, the flow rate is 0.9 ml / min to 1.1 ml / min, preferably 1.0 ml / min.

[0013] Further, in the detection method of the present invention described above, for the chromatographic conditions, the column temperature is 17°C to 23°C, preferably 20°C.

[0014] Further, in the detection method of the present invention described above, for the chromatographic conditions, the detection wavelength is 240 nm.

[0015] In a specific embodiment, a method for separating and detecting key impurities in the starting material of beraprost sodium, the method uses high performance liquid chromatography, and is characterized in that: its chromatographic conditions include: Chromatographic column: Agilent ZORBAX SB-AQ (5μm, 4.6mm×250mm), Mobile phase: Mobile phase A is a phosphoric acid - water (1:1000, v / v) solution, and mobile phase B is acetonitrile - methanol (2:1, v / v). Elution mode: Gradient elution, from 0 - 35 minutes: mobile phase A is 80% - 0, mobile phase B is 20% - 100%; from 35 - 50 minutes, mobile phase A is 0, mobile phase B is 100%; from 50 - 51 minutes, mobile phase A is 0 - 80%, mobile phase B is 100% - 20%; from 51 - 65 minutes, mobile phase A is 80%, mobile phase B is 20%. Detector: UV detector; The starting material of beraprost sodium is , and the impurity is .

[0016] Further, in the detection method of the present invention described above, the chromatographic conditions further include: the flow rate is 0.9 ml / min to 1.1 ml / min, preferably 1.0 ml / min; the column temperature is 17°C to 23°C, preferably 20°C; the detection wavelength is 240 nm.

[0017] The beneficial technical effects of the present invention are as follows: Using an Agilent ZORBAX SB-AQ chromatographic column and an acidic system of phosphoric acid water - methanol / acetonitrile as the mobile phase, and gradient elution, it can effectively separate the cis-trans isomer impurity SM3-IMP1 in the starting material SM3 of beraprost sodium, and accurately quantify it. The detection limit is as low as 0.025%, the recovery rate reaches 99.5%, and the repeatability RSD% is as low as 1.9%. The method of the present invention is simple, has the advantages of high accuracy, linearity, good repeatability, high sensitivity, etc., can effectively control the quality of the starting material SM3 of beraprost sodium, and ensure the obtained final product of beraprost sodium has qualified quality. Description of the Drawings

[0018] Figure 1 It is the HPLC chromatogram of the SM3 test solution under chromatographic condition 1 in Example 1; Figure 2 It is the HPLC chromatogram of the SM3 test solution under Chromatographic Condition 2 in Example 1; Figure 3 It is the HPLC chromatogram of the SM3 test solution under Chromatographic Condition 3 in Example 1; Figure 4 It is the HPLC chromatogram of the SM3-IMP1 positioning solution and the SM3 test solution when Mobile Phase A is ultrapure water in Example 2; Figure 5 The HPLC chromatogram of the SM3-IMP1 positioning solution at a flow rate of 1.0 ml / min in Example 3; Figure 6 The HPLC chromatogram of the SM3 positioning solution at a flow rate of 1.0 ml / min in Example 3; Figure 7 The HPLC chromatogram of the specificity solution at a flow rate of 1.0 ml / min in Example 3; Figure 8 The HPLC chromatogram of the sensitivity (detection limit) solution at a flow rate of 1.0 ml / min in Example 3; Figure 9 It is the HPLC chromatogram of the specificity solution at a flow rate of 0.9 ml / min in Example 4; Figure 10 It is the HPLC chromatogram of the specificity solution at a flow rate of 1.1 ml / min in Example 4; Figure 11 It is the HPLC chromatogram of the specificity solution at a column temperature of 17 °C in Example 5; Figure 12 It is the HPLC chromatogram of the specificity solution at a column temperature of 23 °C in Example 5; Figure 13 It is the HPLC chromatogram of the specificity solution when the initial proportion of Mobile Phase A is 18% in Example 6; Figure 14 It is the HPLC chromatogram of the specificity solution when the initial proportion of Mobile Phase A is 22% in Example 6; Figure 15 It is the linear graph of SM3 in Example 8; Figure 16 It is the linear graph of SM3-IMP1 in Example 8. Detailed implementation manners

[0019] The following examples are used to further illustrate and understand the spiritual essence of the present invention, but do not limit the protection scope of the present invention in any way.

[0020] Preparation of solutions used in the following examples 1) Solvent: Acetonitrile 2) Mobile phase A (0.1% phosphoric acid aqueous solution): Precisely measure 2 ml of phosphoric acid and 2000 ml of ultrapure water, mix well and shake, sonicate for 10 min to obtain. The volume can be enlarged or reduced in proportion (phosphoric acid: water = 1:1000).

[0021] 3) Mobile phase B: Measure 1400 ml of acetonitrile and 700 ml of methanol, mix well and sonicate for 10 min to obtain. The volume can be enlarged or reduced in proportion (acetonitrile: methanol = 2:1).

[0022] 4) Test solution: Take about 25 mg of test sample SM3, accurately weigh, place in a 25-ml volumetric flask, dissolve with solvent and dilute to the mark, shake well to obtain (concentration is about 1 mg / ml).

[0023] 5) Control solution: Precisely measure 1 ml of the test solution, place in a 100-ml volumetric flask, dilute to the mark with acetonitrile, shake well (concentration is about 10 μg / ml, equivalent to 1.0% of the test sample concentration). Determination method Inject the solvent, control solution, and test solution into the liquid chromatograph and record the chromatogram. Calculation formula Except for the solvent peak, if there are impurity peaks in the chromatogram of the test solution, calculate by the main component self-control method with correction factor based on the peak area. Impurities less than LOD (0.030%) (read by area normalization method) can be ignored and not integrated. The calculation formula is as follows:

[0024] - Peak area of impurities in the test sample; - Peak area of the main peak in the self-control solution; - Impurity correction factor, the correction factor for unknown impurities is 1.0.

[0025] Example 1

[0026] Chromatographic conditions 1: Chromatographic column: Agilent SB-C18 (3.5 μm 4.6 mm × 250 mm); Mobile phase: Mobile phase A is phosphoric acid-water (1:1000 v / v) solution, and mobile phase B is acetonitrile; Elution method: Gradient elution, see Table 1 below

[0027] Detection wavelength: 240 nm; Column temperature: 20 °C; Flow rate: 1.0 ml / min; Sample volume: 10 μl; Solvent: Acetonitrile; Concentration of test solution: 1 mg / ml Detector: UV detector.

[0028] Chromatographic condition 2: Chromatographic column: Inertsil ODS-3 3 μm 4.6 mm × 150 mm Mobile phase: Mobile phase A is phosphoric acid-water (1000:1) solution, and mobile phase B is acetonitrile; Elution mode: Gradient elution, see Table 2 below

[0029] Detection wavelength: 240 nm; Column temperature: 20 °C; Flow rate: 1.0 ml / min; Sample volume: 10 μl; Solvent: Acetonitrile; Concentration of test solution: 1 mg / ml Detector: UV detector.

[0030] Chromatographic condition 3: Chromatographic column: Inertsil ODS-3 3 μm 4.6 mm × 150 mm Mobile phase: Mobile phase A is phosphoric acid-water (1:1000) solution, and mobile phase B is acetonitrile: methanol (2:1); Elution mode: Gradient elution, see Table 3 below

[0031] Detection wavelength: 240 nm; Column temperature: 20 °C; Flow rate: 1.0 ml / min; Sample volume: 10 μl; Solvent: Acetonitrile; Concentration of test solution: 1 mg / ml Detector: UV detector.

[0032] The test results show that under the above chromatographic conditions, effective separation of sodium beraprost starting material SM3 and impurity SM3-IMP1 cannot be achieved. The typical chromatogram is shown in Figures 1 to 3 .

[0033] Example 2 Chromatographic condition: Chromatographic column: Agilent ZORBAX SB-AQ (5 μm, 4.6 mm × 250 mm); Mobile phase: Mobile phase A is ultrapure water, and mobile phase B is acetonitrile: methanol (2:1); Elution mode: Gradient elution, see Table 4.

[0034] Detection wavelength: 240 nm; Column temperature: 20 °C; Flow rate: 1.0 ml / min; Injection volume: 10 μl; Solvent: Acetonitrile; Concentration of test solution: 1 mg / ml Detector: UV detector.

[0035] The test results show that under the above chromatographic conditions, although SM3 and SM3-IMP1 are well separated, both the low-concentration SM3-IMP1 reference substance, the high-concentration SM3 test substance, and some unknown impurities in the chromatogram show splitting, indicating that there are defects in the method. The theoretical analysis shows that the reason may be the keto-enol tautomerism of SM3, M3-IMP1 and similar unknown impurities, and it is necessary to optimize the diluent, mobile phase, column temperature or use derivatization methods to avoid it.

[0036] Example 3 Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-AQ (5 μm, 4.6 mm × 250 mm); Mobile phase: Mobile phase A is a phosphoric acid-water (1:1000) solution, and mobile phase B is acetonitrile: methanol (2:1); Elution mode: Gradient elution, see Table 5

[0037] Detection wavelength: 240 nm; Column temperature: 20 °C; Flow rate: 1.0 ml / min; Injection volume: 10 μl; Solvent: Acetonitrile; Concentration of test solution: 1 mg / ml Detector: UV detector.

[0038] The test results show that under the above chromatographic conditions, the separation of the starting material SM3 of beraprost sodium from the impurity SM3-IMP1 meets the requirements; the S / N of the starting material SM3 of beraprost sodium and the impurity SM3-IMP1 meets the requirements. The typical chromatogram is shown in Figures 5 to 8

[0039] Example 4 Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-AQ (5μm, 4.6mm×250mm); Mobile phase: Mobile phase A is a phosphoric acid - water (1:1000) solution, and mobile phase B is acetonitrile: methanol (2:1); Elution method: Gradient elution, see Table 6

[0040] Detection wavelength: 240nm; Column temperature: 20°C; Flow rate: 0.9ml / min, 1.0ml / min or 1.1ml / min; Injection volume: 10µl; Solvent: Acetonitrile; Concentration of test solution: 1mg / ml Detector: UV detector.

[0041] The detection results show that under the above chromatographic conditions, the separation of the starting material SM3 of beraprost sodium and the impurity SM3-IMP1 meets the requirements at different flow rates of 0.9 - 1.1ml / ml. The typical chromatogram is shown in Figures 9 to 10 .

[0042] Example 5 Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-AQ (5μm, 4.6mm×250mm); Mobile phase: Mobile phase A is a phosphoric acid - water (1:1000) solution, and mobile phase B is acetonitrile: methanol (2:1); Elution method: Gradient elution, see Table 7 below.

[0043] Detection wavelength: 240nm; Column temperature: 17°C, 20°C, 23°C; Flow rate: 1.0ml / min Injection volume: 10µl; Solvent: Acetonitrile; Concentration of test solution: 1mg / ml Detector: UV detector.

[0044] The detection results show that under the above chromatographic conditions, the separation of the starting material SM3 of beraprost sodium and the impurity SM3-IMP1 meets the requirements at different column temperatures (17°C, 20°C, 23°C). The typical chromatogram is shown in Figures 11 to 12 .

[0045] Example 6 Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-AQ (5μm, 4.6mm×250mm); Mobile phase: Mobile phase A is a phosphoric acid - water (1:1000) solution, and mobile phase B is acetonitrile: methanol (2:1); Elution method: Gradient elution, see Table 8.1 - 8.3 below.

[0046]

[0047] Detection wavelength: 240nm; Column temperature: 20°C; Flow rate: 1.0ml / min Sample injection volume: 10µl; Solvent: Acetonitrile; Concentration of test solution: 1mg / ml Detector: UV detector.

[0048] The detection results show that under the above chromatographic conditions, the separation of the starting material SM3 of beraprost sodium and the impurity SM3-IMP1 meets the requirements at different starting ratios (mobile phase A: 18% - 22%). The typical chromatogram is shown in Figures 13 to 14

[0049] The following examples were detected according to the chromatographic conditions of Example 3

[0050] Example 7 System suitability The reference solution was detected according to the chromatographic conditions of Example 3. The reference solution was injected continuously for 6 times, and the RSD of the peak area of SM3 should not be greater than 10% The detection results are shown in Table 9. The results show that the RSD of the peak area of SM3 is 0.3%, which is less than 10%. The system suitability of the related substances detection method meets the acceptance limit.

[0051]

[0052] Example 8 Specificity experiment Preparation of solutions 1) Blank solvent: Acetonitrile solvent 2) SM3-IMP1 impurity localization solution: SM3-IMP1 stock solution: Take about 30mg of SM3-IMP1 reference substance, weigh it accurately, place it in a 20ml volumetric flask, dissolve it with the solvent and dilute to the mark, and shake well. (Concentration is about 1.5mg / ml) SM3-IMP1 reference solution: Accurately measure 0.5 ml of the SM3-IMP1 stock solution, transfer it to a 25-ml volumetric flask, dilute it to the mark with the solvent, and mix well. (Concentration is approximately 30 μg / ml) 3) The test solution is the same as the SM3 test solution Determination method: Inject the above blank solvent, SM3-IMP1 impurity location solution, and SM3 test solution once each.

[0053] Calculation and acceptance criteria: 1) The blank solvent does not interfere with the determination; 2) Impurity S-2127-SM3-IMP1 does not interfere with the determination; 3) Test solution: The resolution between the main peak and the adjacent peak, and between impurity S-2127-SM3-IMP1 and the adjacent peak shall not be less than 1.5. The test results are shown in Table 10.

[0054]

[0055] Conclusion 1) The blank solvent does not interfere with the determination; 2) Impurity S-2127-SM3-IMP1 does not interfere with the determination; 3) Test solution: The resolution between the main peak and the adjacent peak, and between impurity S-2127-SM3-IMP1 and the adjacent peak are both greater than 1.5.

[0056] The specificity of the determination method for the related substances of this product meets the acceptance criteria.

[0057] Example 9 Linear experiment Preparation of solutions 1) SM3-IMP1 stock solution: The same as the SM3-IMP1 stock solution in Example 8, 2) SM3 reference stock solution: Take about 30 mg of SM3 reference substance, accurately weigh it, transfer it to a 20-ml volumetric flask, dissolve it with the solvent and dilute it to the mark, and mix well. (Concentration is approximately 1.5 mg / ml) Take the SM3-IMP1 stock solution and SM3 reference stock solution and prepare a series of concentration linear solutions by adding the solvent according to Table 11 below:

[0058] Determination method Inject each of the above linear solutions once.

[0059] Calculation and acceptance criteria Taking the peak area as the ordinate and the concentration as the abscissa, calculate the linear regression equation by the least squares method. The linear correlation coefficient should not be less than 0.990, and calculate the impurity correction factor.

[0060] The test results are shown in Table 12, Figure 15 and 16 .

[0061]

[0062] Conclusion: In the range of 0.4242 μg / ml to 141.4 μg / ml for SM3 (equivalent to 0.042% to 14.140% of the test sample concentration, equivalent to 1.41% to 471.34% of the limit), the linear equation is: y = 9879.8 + 67.4 (where y is the peak area and x is the concentration in μg / ml), and the correlation coefficient r is 1.0000.

[0063] For the impurity SM3-IMP1 in the range of 1.247 μg / ml to 124.7 μg / ml (equivalent to 0.125% to 12.473% of the test sample concentration, equivalent to 4.16% to 415.77% of the limit), the linear equation is: y = 3800.1 + 970.1 (where y is the peak area and x is the concentration in μg / ml), and the correlation coefficient r is 1.0000. The correction factor is 2.6.

[0064] The linearity of the impurity determination method of the present invention meets the acceptance limit.

[0065] Example 10 LOQ and LOD Experiments Preparation of Solutions 1) Stock solution of LOQ solution for SM3-IMP1: Prepared by referring to the method of the stock solution in Example 9, 2) Stock solution of LOQ solution for SM3: Prepared by referring to the method of the control stock solution in Example 9 3) LOD solution: Accurately measure 1 ml of the stock solutions in 1) and 2), place them in a 5-ml volumetric flask, and dilute to the mark with the solvent respectively, and shake well (about 0.30 μg / ml, relative to 0.030% of the test sample solution concentration) Determination Method Inject the LOQ solution continuously for 6 times and inject the LOD solution once.

[0066] Calculation and Acceptance Limit For the LOQ solution: The RSD of the peak areas of the impurities SM3-IMP1 and SM3 should not exceed 10%, and the S / N should not be less than 10; for the LOD solution: The S / N should not be less than 3.

[0067] The test results are shown in Tables 13 - 15.

[0068]

[0069]

[0070]

[0071] Conclusion: Results of LOQ test: For SM3 at 0.42 μg / ml (equivalent to 0.042% of the test sample concentration), the RSD of peak area was 2.6% and the S / N was 16 - 20; for impurity SM3-IMP1 at 1.247 μg / ml (equivalent to 0.125% of the test sample concentration), the RSD of peak area was 2.6% and the S / N was 12 - 22.

[0072] Results of LOD test: For SM3 at 0.2828 μg / ml (equivalent to 0.028% of the test sample concentration), the S / N was 11; for impurity SM3-IMP1 at 0.2495 μg / ml (equivalent to 0.025% of the test sample concentration), the S / N was 4.

[0073] The LOQ and LOD of the related substances determination method for this product meet the acceptance limits.

[0074] Example 11 Accuracy Experiment Preparation of solutions 1) SM3-IMP1 reference solution: Prepared in the same method as in Example 8.

[0075] 2) Blank test sample solution: Acetonitrile.

[0076] 3) Test sample solution at 100% limit concentration: Weigh approximately 25 mg of the test sample accurately, place it in a 25-ml volumetric flask, dissolve it with acetonitrile, accurately add 0.5 ml of the SM3-IMP1 stock solution, and dilute to the mark with the solvent, then shake well. (Prepare 6 parallel portions) 4) Control solution: Accurately measure 1 ml of each of the above test sample solutions, place it in a 100-ml volumetric flask, dilute to the mark with the solvent, and shake well (equivalent to 1.0% of the test sample concentration) (total 7 portions) Determination method Inject the SM3-IMP1 reference solution 3 times, inject the test sample solution and the control solution once each, and record the chromatogram.

[0077] Calculation and acceptance limit The recovery rate of impurity SM3-IMP1 in the test sample solution at 100% limit concentration level should be between 90% and 110%, and the RSD of the recovery rate should not be greater than 5.0%.

[0078] 1) Calculate the measured amount by the self-control method with correction factor

[0079] Where: A 杂质 — Peak area of the impurity peak in the chromatogram of the test solution; f — Correction factor of the impurity; A 对照 — Peak area of the main peak in the chromatogram of the control solution; m 样品 — Sampling amount of the sample, mg; Measured amount, μg.

[0080] 2) Calculate the measured amount by the external standard method of the impurity reference substance ,

[0081] Wherein: A 杂质 — Peak area of the impurity peak in the chromatogram of the test solution; f — Response factor of the impurity; C 对照 — Concentration of the reference solution, mg / ml; A 对照 — Peak area of the impurity peak in the reference solution; V— Dilution volume, ml; Measured amount, μg.

[0082] 3) Recovery rate

[0083] Wherein: is the recovery rate, %; m 总 is the actual measured amount of the impurity, μg; m 样 is the impurity content in the sample, μg; m 加 is the added amount of the impurity, μg.

[0084] The test results are shown in Table 16-19 (in the table, DZP represents the reference substance, 100% XD-GSP represents the test solution with the impurity spiked at 100% limit, and DZ represents the reference solution).

[0085]

[0086]

[0087]

[0088]

[0089] Conclusion Calculated by the external standard method, the recovery rate of impurity SM3-IMP1 was between 98.7% and 100.3%, the average recovery rate was 99.5%, the RSD of the recovery rate was 0.6%, which was less than 5.0%.

[0090] Calculated by the self-control method of the main component with a correction factor, the recovery rate of impurity SM3-IMP1 was between 105.6% and 108.9%, the average recovery rate was 107.1%, the RSD of the recovery rate was 1.4%, which was less than 5.0%.

[0091] The accuracy of the related substances determination method of the present invention meets the acceptance limit.

Claims

1. A method for separating and detecting key impurities in a beraprost sodium starting material, the method using high performance liquid chromatography, characterized in that: The chromatographic conditions include: The chromatographic column is Agilent ZORBAX SB-AQ, Mobile phase: Mobile phase A is phosphoric acid-water solution, mobile phase B is acetonitrile-methanol mixed solution, Elution mode: gradient elution, the starting ratio of mobile phase: mobile phase A: mobile phase B is 78%: 22% to 82%: 18%, Detector: UV detector; Wherein, the starting material is a compound represented by formula SM3, and the key impurity is a compound represented by formula SM3-IMP1. 。 2. The detection method according to claim 1, wherein the mobile phase A is a solution in which the volume ratio of phosphoric acid to water is 1:1000, and the mobile phase B is a mixed solution in which the volume ratio of acetonitrile to methanol is 2:

1.

3. according to the detection method described in claim 1 or 2, described gradient elution, 0-35 minutes: mobile phase A is 78~82%-0,: mobile phase B is 22~18%-100%, 35-50 minutes, mobile phase A is 0, mobile phase B is 100%, 50-51 minutes, mobile phase A is 0-72~82%, mobile phase B is 100%-22~18%, 51-65 minutes mobile phase A is 72~82%, mobile phase B is 22~18%.

4. The detection method according to claim 1, wherein the gradient elution has an initial ratio of mobile phase A:mobile phase B of 80%:20%.

5. The detection method according to claim 1, wherein the chromatographic condition has a flow rate of 0.9 ml / min to 1.1 ml / min.

6. The detection method according to claim 5, wherein the flow rate is 1.0 ml / min.

7. The detection method according to claim 1, wherein the chromatographic condition is a column temperature of 17°C to 23°C. The detection method according to claim 7 , wherein the column temperature is 20° C.

9. The detection method according to claim 1, wherein the chromatographic condition has a detection wavelength of 240 nm.

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