Method for separating and detecting impurities in beraprost sodium intermediate

Abstract

The invention provides a high performance liquid chromatography detection method for determining two key impurities in a beraprost sodium intermediate. According to the method, a Kromasil 100-5SIL chromatographic column is adopted, normal hexane and isopropanol are used as mobile phases, and isocratic elution is used. The method disclosed by the invention is simple, high in accuracy, good in repeatability and high in sensitivity, and can be used for effectively controlling the quality of the beraprost sodium intermediate.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and particularly relates to a method for separating and detecting impurities in a beraprost sodium intermediate. Background Art

[0002] Beraprost sodium is a drug for cardiovascular disease. Its process synthesis steps are long, and the starting materials, intermediates, final products and related impurities are mostly mixtures of non-corresponding isomers, which will present multiple peaks under non-chiral chromatographic conditions, making the development of analytical methods extremely difficult.

[0003] ZA4 is an important intermediate for the synthesis of beraprost sodium, and its synthesis route is as follows:

[0004] During the synthesis of the intermediate ZA4, side reactions will generate positional isomer impurities ZA4-IMP5 and over-reduction impurities ZA4-IMP4. The test results of multiple batches of samples (small test, pilot test, and scale-up batch) show that these two impurities are detected in large amounts in ZA4 and will participate in subsequent reactions to generate derivative impurities with more complex structures, which will not only affect the quality of subsequent products, but also greatly increase the difficulty of establishing relevant impurity control analysis methods in subsequent intermediates or finished products. Therefore, it is necessary to establish corresponding analysis methods in ZA4 to detect and control impurities ZA4-IMP5 and impurity ZA4-IMP4.

[0005] The inventors found many difficulties when establishing the analytical method: (1) The structures of the three substances are only slightly different: ZA4 and ZA4-IMP5 are only positional isomers with different substitution positions, and the difference between ZA4 and ZA4-IMP4 is only the difference of a C=C double bond; (2) ZA4, ZA4-IMP4 and ZA4-IMP5 are each composed of a mixture of four non-corresponding isomers, which have multiple peaks in the non-chiral chromatographic system (theoretically, they can be divided into four peaks); (3) ZA4, ZA4-IMP4 and ZA4-IMP5 are all relatively small in polarity and have good retention capacity in the reverse phase mode. For such substances, the reverse phase analysis method is usually used, but a large number of experiments have verified that under the conditions of using a variety of different types of chromatographic columns and different system mobile phases in the reverse phase mode, these three substances cannot be effectively separated. The above difficulties have greatly increased the difficulty of specificity (separation degree) and sensitivity (multiple peaks) in the establishment of conventional liquid chromatography methods. At the same time, through the reference, patents and other materials, no existing detection methods for ZA4 and its impurities were found. Therefore, it is necessary to develop an analytical method that can simultaneously detect ZA4-IMP4 and ZA4-IMP5 in ZA4 to control the impurities from the source to ensure that a qualified final product is obtained. Summary of the invention

[0006] In view of the shortcomings of the current prior art, the purpose of the present invention is to provide a method for detecting the positional isomer impurity ZA4-IMP5 and the over-reduction impurity ZA4-IMP4 in the beraprost sodium intermediate ZA4. The structures of ZA4, ZA4-IMP4 and ZA4-IMP5 are as follows:

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for detecting impurities in a beraprost sodium intermediate using high performance liquid chromatography, characterized in that it comprises the following chromatographic conditions: Column: Kromasil 100-5SIL, Mobile phase: Mobile phase A is n-hexane, mobile phase B is isopropanol, Elution method: isocratic elution, Detector: UV detector; The beraprost sodium intermediate is , the impurities are and .

[0008] Furthermore, in the above detection method, in the chromatographic conditions, the ratio of mobile phase A:mobile phase B in the mobile phase is 98.5:1.5.

[0009] Furthermore, in the above detection method, the flow rate in the chromatographic conditions is 0.9 ml / min~1.1 ml / min, preferably 1.0 ml / min.

[0010] Furthermore, in the above detection method, the column temperature in the chromatographic conditions is 20°C to 27°C, preferably 20°C.

[0011] Furthermore, in the above detection method, the detection wavelength in the chromatographic conditions is 285 nm.

[0012] The beneficial technical effect of the present invention is that, by using a Kromasil 100-5SIL chromatographic column, n-hexane and isopropanol as mobile phases and isocratic elution, the two key impurities ZA4-IMP4 and ZA4-IMP5 in the beraprost sodium intermediate ZA4 can be effectively separated and accurately quantified, and the detection limits of the two impurities are as low as 0.015% and 0.014%, respectively. The method of the present invention is simple, and has the advantages of high accuracy, good repeatability, high sensitivity, etc., and can effectively control the quality of the beraprost sodium intermediate ZA4, ensuring that a beraprost sodium final product of qualified quality is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the HPLC chromatogram of the ZA4 positioning solution when the chromatographic condition 1 is used in Example 1; Figure 2 The HPLC chromatogram of the ZA4 positioning solution and the ZA4-IMP4 positioning solution measured in Example 2 when the ratio of n-hexane to isopropanol in the mobile phase is 98:2; Figure 3 The HPLC chromatograms of the ZA4 positioning solution, ZA4-IMP4 and ZA4-IMP5 positioning solutions measured in Example 2 when the ratio of n-hexane to isopropanol in the mobile phase is 96:4; Figure 4 is the HPLC chromatogram of the ZA4 positioning solution at a flow rate of 1.0 ml / min in Example 4; Figure 5 is the HPLC chromatogram of the ZA4-IMP4 positioning solution at a flow rate of 1.0 ml / min in Example 4; Figure 6 is the HPLC chromatogram of the ZA4-IMP5 positioning solution at a flow rate of 1.0 ml / min in Example 4; Figure 7 The HPLC chromatogram of the test solution in Example 4 at a flow rate of 1.0 ml / min; Figure 8 HPLC chromatogram of the specific solution at a flow rate of 1.0 ml / min in Example 4; Fig. 9 HPLC chromatogram of the sensitivity solution at a flow rate of 1.0 ml / min in Example 4; Fig.10 This is the HPLC chromatogram of the specific solution at a flow rate of 0.9 ml / min in Example 4; Fig.11 This is the HPLC chromatogram of the specific solution at a flow rate of 1.1 ml / min in Example 4; Fig.12 This is the HPLC chromatogram of the specific solution when the column temperature is 25°C in Example 5; Fig.13 This is the HPLC chromatogram of the specific solution in Example 5 when the column temperature is 27°C. DETAILED DESCRIPTION

[0014] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way. Example 1

[0015] Chromatographic conditions 1: Chromatographic column: ACE Excel 3 C18-PFP (150mm*4.6mm); Mobile phase: Mobile phase A is 0.1% acetic acid in water, and mobile phase B is acetonitrile; Elution method: gradient elution Time (min) A（%） B（%） 0 90 10 40 20 80 45 20 80 46 90 10 60 90 10 Detection wavelength: 285nm; Column temperature: 40°C; Flow rate: 1.0ml / min; Injection volume: 3µl; Solvent: methanol; Detector: UV detector.

[0016] Chromatographic conditions 2: Chromatographic column: Agilent zorbax SB-AQ (5μm250*4.6mm) Mobile phase: Mobile phase A is 1 mmol β-cyclodextrin aqueous solution, and mobile phase B is methanol; Elution method: gradient elution Time (min) A（%） B（%） 0 90 10 40 20 80 55 20 80 56 90 10 70 90 10 Detection wavelength: 285nm; Column temperature: 40°C; Flow rate: 1.0ml / min; Injection volume: 15µl; Solvent: methanol; Detector: UV detector.

[0017] Chromatographic conditions 3: Chromatographic column: ACE Excel 3 C18-PFP (150*4.6mm); Mobile phase: Mobile phase A was ultrapure water, and mobile phase B was methanol-acetonitrile (1:1); Elution method: gradient elution Time (min) A（%） B（%） 0 90 10 40 20 80 55 20 80 56 90 10 70 90 10 Detection wavelength: 285nm; Column temperature: 40°C; Flow rate: 1.0ml / min; Injection volume: 15µl; Solvent: methanol; Detector: UV detector.

[0018] Experimental steps: (1) Preparation of ZA4-IMP4 localization solution: Weigh about 5 mg of ZA4-IMP4 into a 5 ml volumetric flask, dissolve it with methanol, dilute to the mark, shake well, then take 75 μl into a 10 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain ZA4-IMP4 localization solution (concentration of about 7.5 μg / ml). (2) Preparation of ZA4-IMP5 localization solution: Weigh about 5 mg of ZA4-IMP5 into a 5 ml volumetric flask, dissolve it with methanol, dilute to the mark, shake well, then take 75 μl into a 10 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain ZA4-IMP5 localization solution (concentration of about 7.5 μg / ml). (3) Preparation of ZA4 test solution: Weigh about 5 mg of the sample, place it in a sample vial, add 1000 μl of methanol to dissolve, and shake well to obtain the ZA4 test solution (concentration of about 5 mg / ml); (4) Determination: Take 3 µl of the above-mentioned positioning solution and the test solution and inject them into the high performance liquid chromatograph. Use the chromatographic conditions 1, 2, and 3 for determination and record the chromatograms.

[0019] The results showed that under the above chromatographic conditions, the intermediate ZA4 of beraprost sodium could not be effectively separated from the impurities ZA4-IMP4 and ZA4-IMP5. Figure 1 .

[0020] Example 2 Chromatographic conditions: Chromatographic column: Agilent ZORBAX RX-SIL (5 μm, 4.6*250 mm); Mobile phase: n-hexane-isopropanol, the ratio of n-hexane to isopropanol is as follows: Hexane: Isopropyl alcohol 1 98:2 2 97:3 3 95:5 4 96:4 Elution mode: isocratic elution; Detection wavelength: 285nm; Column temperature: 30°C; Flow rate: 1.0 ml / min; Injection volume: 15 µl; Solvent: Same as mobile phase; Detector: UV detector.

[0021] Experimental steps: (1) Preparation of ZA4 localization solution: Take an appropriate amount of ZA4 reference substance, an intermediate of beraprost sodium, and prepare a solution containing 25 μg of reference substance per 1 ml with a solvent; (2) Preparation of ZA4-IMP4 localization solution: Take an appropriate amount of ZA4-IMP4 reference substance and use solvent to prepare a solution containing 25 μg of reference substance per 1 ml; (3) Preparation of ZA4-IMP5 localization solution: Take an appropriate amount of ZA4-IMP5 reference substance and use solvent to prepare a solution containing 25 μg of reference substance per 1 ml; (4) Determination: Take the above-mentioned positioning solutions and inject them into a high performance liquid chromatograph, use mobile phases 1, 2, 3, and 4 for determination, and record the chromatograms.

[0022] The results showed that under the above chromatographic conditions, the intermediate ZA4 of beraprost sodium could not be effectively separated from the impurities ZA4-IMP4 and ZA4-IMP5. Figure 2 and Figure 3 .

[0023] Example 3 Chromatographic conditions: Chromatographic column: Kromasil 100-5SIL (5 μm, 4.6 mm × 250 mm); Mobile phase: n-hexane-isopropanol, the ratio of n-hexane to isopropanol is as follows: Hexane: Isopropyl alcohol 1 98:2 2 98.5:1.5 3 99.1 Elution mode: isocratic elution; Detection wavelength: 285nm; Column temperature: 30°C; Flow rate: 1.0 ml / min; Injection volume: 3 µl; Solvent: Same as mobile phase; Detector: UV detector.

[0024] Experimental steps: (1) Preparation of ZA4 localization solution: Take an appropriate amount of ZA4 reference substance, an intermediate of beraprost sodium, and prepare a solution containing 25 μg of reference substance per 1 ml with a solvent; (2) Preparation of ZA4-IMP4 localization solution: Take an appropriate amount of ZA4-IMP4 reference substance and use solvent to prepare a solution containing 25 μg of reference substance per 1 ml; (3) Preparation of ZA4-IMP5 localization solution: Take an appropriate amount of ZA4-IMP5 reference substance and use solvent to prepare a solution containing 25 μg of reference substance per 1 ml; (4) Preparation of specific solution: Take about 25 mg of the beraprost sodium intermediate ZA4 test sample, accurately weigh it, and place it in a 5 ml volumetric flask. Then take appropriate amounts of ZA4-IMP4 reference substance and ZA4-IMP5 reference substance, respectively, and use solvent to prepare a mixed solution containing 5 mg of beraprost sodium intermediate ZA4, 25 μg of impurity ZA4-IMP4, and 25 μg of impurity ZA4-IMP5 per 1 ml; (5) Determination: Take the above-mentioned specific solution and positioning solution and inject them into the high performance liquid chromatograph respectively, use mobile phase 1, 2, and 3 for determination respectively, and record the chromatogram.

[0025] The results showed that under the above chromatographic conditions, when mobile phase 1 was used, the separation of beraprost sodium intermediate ZA4 and impurities ZA4-IMP4 and ZA4-IMP5 was poor and effective separation could not be achieved; when mobile phase 2 was used, beraprost sodium intermediate ZA4 and impurities ZA4-IMP4 and ZA4-IMP5 were effectively separated; when mobile phase 3 was used, no peak appeared in the test for more than 1 hour. If the elution time was further extended, no peak shape would be formed and the sensitivity would not meet the requirements.

[0026] Example 4 Chromatographic conditions: Chromatographic column: Kromasil 100-5SIL (5 μm, 4.6 mm × 250 mm); Mobile phase: n-hexane-isopropanol (98.5: 1.5); Isocratic elution: isocratic elution; Detection wavelength: 285nm; Column temperature: 20°C; Flow rate: 0.9ml / min, 1.0ml / min or 1.1ml / min; Injection volume: 15µl; Needle washing solution: methanol; Sample plate temperature: 5°C; Solvent: Same as mobile phase; Detector: UV detector.

[0027] Experimental steps: (1) Preparation of ZA4 localization solution, ZA4-IMP4 localization solution, ZA4-IMP5 localization solution and specific solution: same as in Example 3; (2) Preparation of test solution: Take about 25 mg of the intermediate ZA4 of beraprost sodium, accurately weigh it, place it in a 5 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well to obtain; (3) Preparation of sensitivity solution: Take appropriate amounts of beraprost sodium intermediate ZA4 and two impurity reference substances, and prepare a mixed solution containing 1 μg of beraprost sodium intermediate ZA4, impurity ZA4-IMP4 and impurity ZA4-IMP5 per 1 ml with a solvent; (4) Determination: Inject the above-mentioned positioning solution, specific solution, sensitivity solution and test solution into a high performance liquid chromatograph for determination and record the chromatogram.

[0028] The results showed that under the above chromatographic conditions, baseline separation was achieved between the intermediate ZA4 of beraprost sodium and the impurities ZA4-IMP4 and ZA4-IMP5; the S / N of the intermediate ZA4 of beraprost sodium, the impurity ZA4-IMP4 and the impurity ZA4-IMP5 met the requirements. Typical chromatograms are shown in Figure 4~Figure 11 .

[0029] Example 5 Chromatographic conditions: Column temperature: 20℃, 25℃ or 27℃; Flow rate: 1.0ml / min; The remaining chromatographic conditions are the same as in Example 4.

[0030] Experimental steps: (1) Preparation of specific solution: same as in Example 3; (2) Determination: Take 15 µl of the above-mentioned specific solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0031] The results showed that baseline separation was achieved between the intermediate ZA4, impurity ZA4-IMP4 and impurity ZA4-IMP5 of beraprost sodium. Figure 12~Figure 13 .

[0032] Example 6 Chromatographic conditions: Chromatographic column: Kromasil 100-5SIL (5 μm, 4.6 mm × 250 mm); Mobile phase: n-hexane-isopropanol (98.5: 1.5); Isocratic elution: isocratic elution; Detection wavelength: 285nm; Column temperature: 20°C; Flow rate: 1.0ml / min; Injection volume: 15µl; Needle washing solution: methanol; Sample plate temperature: 5°C; Solvent: Same as mobile phase; Detector: UV detector.

[0033] Experimental steps: (1) Preparation of ZA4 reference substance stock solution: Take about 5 mg of ZA4 reference substance, weigh accurately, place in a 10 ml volumetric flask, add solvent to dissolve, use vortex or ultrasound to assist dissolution, dilute to scale, shake well to obtain ZA4 reference substance stock solution (about 0.5 mg / ml); (2) Preparation of ZA4-IMP4 stock solution: Take about 5 mg of ZA4-IMP4, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it, dilute it to the scale with solvent, and shake well to obtain ZA4-IMP4 stock solution (about 0.5 mg / ml); (3) Preparation of ZA4-IMP5 stock solution: Take about 5 mg of ZA4-IMP5, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it, dilute it to the scale with solvent, and shake well to obtain ZA4-IMP5 stock solution (about 0.5 mg / ml); (4) Preparation of linear solution 1: Accurately pipette 1 ml of ZA4-IMP4 stock solution and ZA4 reference stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain linear solution 3 (containing approximately 10 μg / ml of ZA4-IMP4 and ZA4, respectively); (5) Preparation of linear solution 2: Accurately pipette 1 ml of ZA4-IMP4 stock solution and ZA4 reference stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake to mix linear solution 4 (containing approximately 50 μg / ml of ZA4-IMP4 and ZA4, respectively); (6) Preparation of linear solution 3: Accurately pipette 1 ml of ZA4-IMP4 stock solution and ZA4 reference stock solution into a 5 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain linear solution 5 (containing approximately 100 μg / ml of ZA4-IMP4 and ZA4, respectively). (7) Preparation of linear solution 4: Accurately pipette 1 ml of ZA4-IMP5 stock solution and ZA4 reference stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain linear solution 4 (containing approximately 10 μg / ml of ZA4-IMP5 and ZA4, respectively). (8) Preparation of linear solution 5: Accurately pipette 1 ml of ZA4-IMP5 stock solution and ZA4 reference stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain linear solution 5 (containing approximately 50 μg / ml of ZA4-IMP5 and ZA4, respectively). (9) Preparation of linear solution 6: Accurately pipette 1 ml of ZA4-IMP5 stock solution and ZA4 reference stock solution into a 5 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain linear solution 6 (containing approximately 100 μg / ml of ZA4-IMP5 and ZA4, respectively). (10) Prepare linear solutions 1 to 6 with solvents according to the following table to prepare linear solutions of a series of concentrations:

[0034] (11) The linear solution, linear solution 2, linear solution 3, linear solution 5 and linear solution 6 of the above series of concentrations were respectively injected into a high performance liquid chromatograph and the chromatograms were recorded.

[0035] The linear regression equation was calculated by the least square method with the peak area as the ordinate and the concentration as the abscissa. The results showed that for ZA4 in the range of 1.078μg / ml~107.8μg / ml, the linear equation was: y=7878x-914.4 (y is the peak area, x is the concentration in μg / ml), the correlation coefficient r was 0.9999, and the ratio of the absolute value of the intercept to the limit concentration response value was 0.5%; for ZA4-IMP4 in the range of 0.9659μg / ml~96.59μg / ml, the linear equation was: y=7990x-1054 (y is the peak area, x is the concentration in μg / ml), the correlation coefficient r is 0.9999, the ratio of the absolute value of the intercept to the limit concentration response value is 0.5%, the correction factor is 0.99, and no correction is required for determination; for IMP5 in the range of 1.058μg / ml~105.8μg / ml, the linear equation is: y=4433x-339.4 (y is the peak area, x is the concentration in μg / ml), the correlation coefficient r is 0.9999, the ratio of the absolute value of the intercept to the limit concentration response value is 1.0%. The correction factor is 1.78. The linearity of ZA4, ZA4-IMP4 and ZA4-IMP5 meets the acceptance limit.

[0036] Example 7 Chromatographic conditions: same as Example 6.

[0037] Experimental steps: (1) Preparation of ZA4-IMP4 localization solution: same as in Example 3; (2) Preparation of ZA4-IMP5 localization solution: same as in Example 3; (3) Preparation of LOD solution of ZA4-IMP4: Accurately pipette 3 ml of ZA4-IMP4 localization solution into a 100 ml volumetric flask, dilute to scale with solvent, and shake well to obtain LOD solution (containing about 0.75 μg / ml of ZA4-IMP4); (4) Preparation of LOD solution of ZA4-IMP5: Accurately pipette 3 ml of ZA4-IMP5 localization solution into a 100 ml volumetric flask, dilute to scale with solvent, and shake well to obtain LOD solution (containing about 0.75 μg / ml of ZA4-IMP4); (5) Take the above-mentioned LOD solution of ZA4-IMP4 and the LOD solution of ZA4-IMP5 and inject them into a high performance liquid chromatograph respectively, and record the chromatograms.

[0038] The results showed that when the LOD solutions of ZA4-IMP4 and ZA4-IMP5 were determined using the above chromatographic conditions, the concentrations of ZA4-IMP4 and ZA4-IMP5 were measured to be 0.77 μg / ml (equivalent to 0.015% of the test sample concentration) and 0.71 μg / ml (equivalent to 0.014% of the test sample concentration), respectively, with S / N of 8 and 9, respectively. The LOD met the acceptance limit.

[0039] Example 8 Chromatographic conditions: same as Example 6.

[0040] Experimental steps: (1) Preparation of blank test solution: Take about 25 mg of ZA4 test sample, weigh accurately, place in a 5 ml volumetric flask, add solvent to dissolve, dilute to the scale with solvent, shake well to obtain blank test solution (containing about 5 mg / ml ZA4), prepare 2 copies in parallel; (2) Preparation of ZA4-IMP4 secondary stock solution: take about 5 mg of ZA4-IMP4, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it, dilute it to the mark with solvent, and shake well to obtain ZA4-IMP4 stock solution; accurately measure 5 ml of ZA4-IMP4 stock solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, and shake well to obtain ZA4-IMP4 secondary stock solution (containing about 125 μg / ml of ZA4-IMP4); (3) Preparation of ZA4-IMP5 secondary stock solution: take about 5 mg of ZA4-IMP5, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve it, dilute it to the mark with solvent, and shake well to obtain ZA4-IMP5 stock solution; accurately measure 1.5 ml of ZA4-IMP5 stock solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, and shake well to obtain ZA4-IMP5 secondary stock solution (containing about 37.5 μg / ml of ZA4-IMP5); (4) Preparation of 100% limit level test solution: Take about 25 mg of ZA4 test sample, accurately weigh, place in a 5 ml volumetric flask, add 1 ml of ZA4-IMP4 secondary stock solution and 1 ml of ZA4-IMP5 secondary stock solution, add solvent to dissolve, dilute to the scale with solvent, shake well to obtain 100% limit level test solution, prepare 6 copies in parallel; (5) Take the blank test solution and the 100% limit level test solution and inject them into the HPLC respectively, and record the chromatogram.

[0041] The results showed that the recovery rate of ZA4-IMP4 in the six test solutions was between 103.2% and 104.4%, with an average recovery rate of 103.5% and an RSD of 0.6%; the recovery rate of ZA4-IMP5 was between 110.8% and 115.6%, with an average recovery rate of 113.0% and an RSD of 1.6%. The recovery rate results met the requirements, that is, the chromatographic conditions had good accuracy.

[0042] Table 1

[0043] Table 2

Claims

1. A method for detecting impurities in a beraprost sodium intermediate by high performance liquid chromatography, Features The following chromatographic conditions were included: Column: Kromasil 100-5SIL, Mobile phase: Mobile phase A is n-hexane, mobile phase B is isopropanol, Elution method: isocratic elution, Detector: UV detector; The beraprost sodium intermediate is , the impurities are and .

2. The detection method according to claim 1, wherein the ratio of mobile phase A to mobile phase B is 98.5:1.

5.

3. The detection method according to claim 1, wherein the flow rate in the chromatographic conditions is 0.9 ml / min~1.1 ml / min.

4. The detection method according to claim 3, wherein the flow rate in the chromatographic conditions is 1.0 ml / min.

5. The detection method according to claim 1, wherein the column temperature in the chromatographic conditions is 20°C to 27°C. The detection method according to claim 5 , wherein the column temperature in the chromatographic condition is 20° C.

7. The detection method according to claim 1, wherein the detection wavelength in the chromatographic conditions is 285 nm.

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