HPLC (High Performance Liquid Chromatography) detection method for sulfonate impurities in calcium dobesilate

Abstract

The invention provides an HPLC (High Performance Liquid Chromatography) detection method for sulfonate impurities in calcium dobesilate, and relates to the field of medicine detection. Comprising the following steps: a chromatographic column is an octadecyl silane bonded silica gel chromatographic column; mobile phases: taking an ammonium dihydrogen phosphate solution as a mobile phase A and methanol as a mobile phase B; the elution mode is as follows: gradient elution is carried out, the adjustment range of the mobile phase A is 80-60% v / v, and the adjustment range of the mobile phase B is 20-40% v / v. By adopting the HPLC detection method disclosed by the invention, the contents of three genotoxic impurities, namely methyl 2, 5-dihydroxybenzenesulfonate, ethyl 2, 5-dihydroxybenzenesulfonate and isopropyl 2, 5-dihydroxybenzenesulfonate, in the calcium dobesilate raw material medicine are effectively determined, the sensitivity is high, the quantitation limit is low, the quality of the calcium dobesilate raw material medicine is better controlled, and the medicine safety is better guaranteed. On the premise that the detection effect is guaranteed, the detection method is simple and easy to operate, the detection cost is effectively reduced, and an impurity control scheme which is more economical, stable and easy to popularize is achieved.

Description

Technical Field

[0001] The invention relates to the field of drug detection, and in particular to an HPLC detection method for sulfonate impurities in calcium dobesilate. Background Art

[0002] Calcium dobesilate is a widely used vascular protective agent in clinical practice, and the quality control of its raw materials and preparations is directly related to the safety of medication. In the synthesis process of calcium dobesilate (code: CDH), no materials and reagents with potential genotoxicity are involved. However, since the product itself has a sulfonic acid structure, and alcohol solvents such as ethanol and isopropanol are used in the preparation process, sulfonate impurities may be produced. These sulfonate impurities have the warning structure of genotoxic impurities, and these impurities may remain in the finished product. The existing detection methods have problems such as low sensitivity and high detection cost. It is of great significance to develop and optimize the analysis method of calcium dobesilate-based toxic impurities (sulfonates). Summary of the invention

[0003] In view of this, the present invention provides an HPLC detection method for sulfonate impurities in calcium dobesilate to solve the above problems.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A HPLC detection method for sulfonate impurities in calcium dobesilate comprises the following steps:

[0006] The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; the mobile phase is ammonium dihydrogen phosphate solution as mobile phase A and methanol as mobile phase B; the elution method is gradient elution, the mobile phase A is adjusted in the range of 80% to 60% v / v, and the mobile phase B is adjusted in the range of 20% to 40% v / v.

[0007] Furthermore, the sulfonate impurities are methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate.

[0008] Further, the procedure of gradient elution is as follows:

[0009]

[0010] Furthermore, the concentration of the diammonium phosphate solution is 0.04-0.06 mol / L.

[0011] Furthermore, the chromatographic column is Welch Ultimate AQ-C18; the specification of the chromatographic column is 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance.

[0012] Furthermore, the flow rate is 0.8 ml to 1.2 ml per minute.

[0013] Furthermore, the detection wavelength is 210nm to 230nm.

[0014] Furthermore, the column temperature is 45°C to 55°C.

[0015] Furthermore, the test solution is prepared by taking the calcium dobesilate raw material, dissolving it in a solvent, diluting it to a fixed volume, and preparing a concentration of 90 to 110 mg / ml.

[0016] Further, reference solution: take 2,5-dihydroxybenzenesulfonic acid methyl ester reference substance, 2,5-dihydroxybenzenesulfonic acid ethyl ester reference substance and 2,5-dihydroxybenzenesulfonic acid isopropyl ester reference substance respectively, dissolve them in a solvent and quantitatively dilute them to prepare a mixed solution containing 0.02-0.04 μg of 2,5-dihydroxybenzenesulfonic acid methyl ester, 2,5-dihydroxybenzenesulfonic acid ethyl ester and 2,5-dihydroxybenzenesulfonic acid isopropyl ester per 1 ml.

[0017] Furthermore, the solvent is pure water, including ultrapure water or purified water.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The HPLC detection method of the present invention is used to effectively determine the contents of three genotoxic impurities, namely methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate, in the calcium dobesilate raw material drug, and has high sensitivity and low quantitative limit, so as to better control the quality of the calcium dobesilate raw material drug and better ensure the safety of the drug.

[0020] (2) The detection method of the present invention is simple and easy to operate while ensuring the detection effect, which effectively reduces the detection cost and realizes a more economical, stable and easy-to-promote impurity control scheme. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 -HPLC spectrum of the impurity CDH-H reference solution of the present invention;

[0022] Figure 2 -HPLC spectrum of the impurity CDH-I reference solution of the present invention;

[0023] Figure 3 -HPLC spectrum of the impurity CDH-J reference solution of the present invention;

[0024] Figure 4 -HPLC spectrum of the system suitability reference solution-1 of the present invention;

[0025] Figure 5 -HPLC spectrum of the system suitability reference solution-2 of the present invention;

[0026] Figure 6 -HPLC spectrum of the system suitability reference solution-3 of the present invention;

[0027] Figure 7 -HPLC spectrum of the system suitability reference solution-4 of the present invention;

[0028] Figure 8 -HPLC spectrum of the system suitability reference solution-5 of the present invention;

[0029] Fig. 9 -HPLC spectrum of the test solution of the present invention;

[0030] Fig.10 -HPLC spectrum of the test sample spiked solution-1 of the present invention;

[0031] Fig.11 -HPLC spectrum of the test sample spiked solution-2 of the present invention;

[0032] Fig.12 -HPLC spectrum of the test sample spiked solution-3 of the present invention;

[0033] Fig.13 -HPLC spectrum of the test sample spiked solution-4 of the present invention;

[0034] Fig.14 -HPLC spectrum of the test sample spiked solution-5 of the present invention;

[0035] Fig.15 -HPLC spectrum of the test sample spiked solution-6 of the present invention;

[0036] Fig.16 HPLC spectrum of quantitative limit solution-1 of the present invention;

[0037] Fig.17 HPLC spectrum of quantitative limit solution-2 of the present invention;

[0038] Fig.18 HPLC spectrum of the quantitative limit solution-3 of the present invention;

[0039] Fig.19 HPLC spectrum of quantitative limit solution-4 of the present invention;

[0040] Fig. 20 HPLC spectrum of the quantitative limit solution-5 of the present invention;

[0041] Fig.21 HPLC spectrum of quantitative limit solution-6 of the present invention;

[0042] Fig. 22The linear relationship diagram between the peak area and concentration of 2,5-dihydroxybenzenesulfonic acid methyl ester (CDH-H) of the present invention;

[0043] Fig.23 The linear relationship diagram between the peak area and concentration of 2,5-dihydroxybenzenesulfonic acid ethyl ester (CDH-I) of the present invention;

[0044] Fig.24 A linear relationship diagram between the peak area and concentration of 2,5-dihydroxybenzenesulfonic acid isopropyl ester (CDH-J) of the present invention. DETAILED DESCRIPTION

[0045] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0047] Table 1 Summary of potential genotoxic impurities in finished calcium dobesilate

[0048]

[0049] Example 1-HPLC Detection Method for Sulfonate Impurities in Calcium Dobesilate

[0050] Solvent: ultrapure water.

[0051] Test solution: Take the product, dissolve it in solvent and quantitatively dilute it to make a solution containing about 100 mg per 1 ml.

[0052] Reference solution: Take appropriate amount of impurity CDH-H reference, impurity CDH-I reference and impurity CDH-J reference respectively, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately 0.03 μg of CDH-H, CDH-I and CDH-J per 1 ml.

[0053] Test sample spiked solution: take about 5 mg each of impurity CDH-H reference substance, impurity CDH-I reference substance, and impurity CDH-J reference substance, accurately weigh, place in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain solution 1; accurately measure 1 ml of solution 1, place in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain solution 2; accurately measure 3 ml of solution 2, place in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, as the stock solution; take about 2000 mg of the test sample, accurately weigh, place in a 20 ml brown volumetric flask, add "stock solution" to dissolve and dilute to the scale, shake well.

[0054] Quantitation limit solution: Accurately measure 7 ml of the reference solution, place it in a 10 ml volumetric flask, and dilute to the mark with solvent.

[0055] Chromatographic conditions: A chromatographic column with octadecylsilane bonded silica as filler (Welch Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance), 0.05 mol / L ammonium dihydrogen phosphate solution as mobile phase A, and methanol as mobile phase B; gradient elution according to Table 2; detection wavelength of 220 nm; flow rate of 1.0 ml per minute; column temperature of 50°C; injection volume of 50 μl.

[0056] Table 2 Gradient elution program

[0057]

[0058] System suitability requires that in the chromatogram of the reference solution, CDH-H, CDH-I and CDH-J appear in sequence, and the separation between each chromatographic peak should meet the requirements (≥1.5); the signal-to-noise ratio of the three impurity peaks should not be less than 10.

[0059] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0060] Determined by high performance liquid chromatography (General Rules 0512, Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0061] Limit: In the chromatogram of the test solution, if there are chromatographic peaks with the same retention time as CDH-H, CDH-I and CDH-J, the concentrations of CDH-H, CDH-I and CDH-J shall not exceed 0.0001% when calculated by peak area using the external standard method.

[0062] Calculation formula:

[0063]

[0064]

[0065] Where:

[0066] W STD , W S are the weights of impurity reference substances (CDH-H, CDH-I, CDH-J) and test substances, mg;

[0067] V STD 、V S are the dilution multiple of the reference solution and the dilution volume of the test solution, ml;

[0068] A STD , A Sare the peak areas of impurities (CDH-H, CDH-I, CDH-J) in the reference and test solutions, respectively;

[0069] P is the content of impurity reference substances (CDH-H, CDH-I, CDH-J) (based on wet product), %;

[0070] RF is the response factor of each impurity (CDH-H, CDH-I, CDH-J) peak in the reference solution;

[0071] C S is the content of single known impurities (CDH-H, CDH-I, CDH-J) in the test solution, %.

[0072] According to the guidelines of the 2020 edition of the Chinese Pharmacopoeia, the detection method of Example 1 was methodologically verified, and the results are as follows:

[0073] 1 System suitability

[0074] 1.1 Acceptable Standards:

[0075] (1) The reference solution was injected 5 times continuously. The RSD of the peak response factors RF values ​​of impurities CDH-H, CDH-I and CDH-J should not be greater than 20%, and the RSD of the retention time should not be greater than 1.0%;

[0076] (2) In the chromatogram of the reference solution, CDH-H, CDH-I and CDH-J appear in sequence, and the separation between each chromatographic peak should be ≥1.5; the signal-to-noise ratio of the three impurity peaks should be no less than 10.

[0077] 1.2 Test results:

[0078] (1) The reference solution was injected 5 times continuously. The RSDs of the RF values ​​of the impurities CDH-H, CDH-I and CDH-J peaks were 1.81%, 3.49% and 5.62%, respectively, all less than 20%. The RSDs of the retention times were 0.08%, 0.05% and 0.01%, respectively, all less than 1.0%.

[0079] (2) In the chromatogram of the reference solution, CDH-H, CDH-I and CDH-J appeared in sequence, and the separation between the chromatographic peaks was between 20.49 and 31.89, all greater than 1.5; the signal-to-noise ratios of the three impurity peaks were not less than 10. Figures 4 to 8 .

[0080] 2 Exclusiveness

[0081] 2.1 Acceptable Standards:

[0082] (1) The blank solvent should not interfere with the determination of target impurity peaks.

[0083] (2) The retention time of the target impurity peaks (CDH-H, CDH-I, CDH-J) in the reference solution, test solution, and test spiked solution should be consistent with the impurity peaks in the single-label solution; the separation between the impurity peaks in the reference solution should be ≥1.5.

[0084] (3) The retention time drift of each target impurity peak in the test sample spiked solution shall not exceed 3%.

[0085] 2.2 Test results:

[0086] (1) The blank solvent does not interfere with the determination of each target impurity peak.

[0087] (2) The retention time drifts of the known impurity peaks in the reference solution and the impurity peaks in the single standard solution were: CDH-H 0.07%, CDH-I 0.13% and CDH-J 0.03% respectively; the separation degrees between the impurity peaks in the reference solution were between 20.49 and 31.89, all greater than 1.5, which met the requirements.

[0088] (3) The retention time shifts of the target impurity peaks in the spiked solution of the test sample were: CDH-H 0.31%, CDH-I 0.28% and CDH-J 0.10%, which met the requirements.

[0089] 3Accuracy and precision

[0090] 3.1 Acceptable standard: Among the determination results of 6 100% test sample spiked solutions, the single recovery data should be 80.0% to 120.0%, and the RSD (n=6) should not be greater than 20.0%.

[0091] 3.2 Test results:

[0092] The recoveries of impurities CDH-H, CDH-I and CDH-J in 6 100% test sample spiked solutions were between 90.4% and 107.0%, which met the requirements; see the spectra. Figures 10 to 15 .

[0093] The RSDs (n=6) were: CDH-H 1.84%, CDH-I 2.42%, CDH-J 2.26%; all met the requirements.

[0094] 4 Linearity and Range

[0095] 4.1 Acceptable criteria: Within the range of LOQ to 400% of the limit concentration, the correlation coefficient (r) of the linear regression equation of each impurity shall be ≥ 0.99, the y-axis intercept shall be within 10.0% of the 100% theoretical concentration response value, and the RSD of the impurity peak response factor of each linear solution shall be ≤ 20%.

[0096] 4.2 Test results:

[0097] like Fig. 22 As shown, in the range of 0.015 μg / ml (50%) to 0.120 μg / ml (400%), the linear equation of CDH-H is y=144,482.3442x+144.6803, the correlation coefficient (r) is 0.9988, the absolute value of the y-axis intercept is 3.31% of the 100% theoretical concentration response value, and the RSD of the linear solution impurity peak response factor is 3.61%;

[0098] like Fig.23 As shown, in the range of 0.015 μg / ml (50%) to 0.117 μg / ml (400%), the linear equation of CDH-I is y=141,315.3818x-226.0787, the correlation coefficient (r) is 0.9991, the absolute value of the y-axis intercept is 6.0% of the 100% theoretical concentration response value, and the RSD of the linear solution impurity peak response factor is 4.52%;

[0099] like Fig.24 As shown, in the range of 0.018 μg / ml (50%) to 0.147 μg / ml (400%), the linear equation of CDH-J is y=92,804.3830x+5.4294, the correlation coefficient (r) is 0.9983, the absolute value of the y-axis intercept is 0.17% of the 100% theoretical concentration response value, and the RSD of the linear solution impurity peak response factor is 6.21%.

[0100] 5 Limit of Quantitation

[0101] 5.1 Acceptable standard: The signal-to-noise ratio (S / N) of each impurity peak in the quantitative limit solution should be ≥ 10, and the quantitative limit solution should be injected 6 times continuously, and the RSD of the area of ​​each impurity peak should not be greater than 20.0%.

[0102] 5.2 Test results:

[0103] The concentration and signal-to-noise ratio S / N of each component in the quantitative limit solution are:

[0104] Table 3 Quantitation limit results

[0105] Components Concentration μg / ml S / N RSD CDH-H 0.015 22.80～27.35 5.27％ CDH-I 0.015 15.74～19.19 5.30％ CDH-J 0.019 15.11～18.93 6.18％

[0106] All meet the requirements, see the diagram Figures 16 to 21 .

[0107] 6 Solution stability

[0108] 6.1 Acceptable Standards:

[0109] (1) Reference solution: Compared with 0 hour, the ratio of the peak area of ​​each impurity at the time point to be investigated should be 0.80-1.20, which means that the reference solution is stable at room temperature during this period of time.

[0110] (2) Test sample spiked solution: Compared with 0 hour, the ratio of the peak area of ​​each impurity at the time point to be investigated should be 0.80-1.20, which means that the test sample spiked solution is stable at room temperature during this period of time.

[0111] 6.2 Test results:

[0112] (1) Reference solution: Compared with 0 hour, the ratios of the peak areas of impurities CDH-H at each time point (7h, 17h, 24h) were 0.97, 1.01 and 0.97; the ratios of the peak areas of CDH-I were 1.03, 0.96 and 1.08; the ratios of the peak areas of CDH-J were 1.00, 1.05 and 0.98; all of them were between 0.80 and 1.20, indicating that the reference solution was stable within 24 hours at room temperature.

[0113] (2) Test sample spiked solution: Compared with 0 hour, the ratios of the impurity CDH-H peak areas at each time point (7h, 17h, 24h) were 0.99, 1.03 and 0.96; the ratios of the CDH-I peak areas were 1.09, 1.04 and 1.01; the ratios of the CDH-J peak areas were 1.06, 1.00 and 1.07; all of them were between 0.80 and 1.20, indicating that the test sample spiked solution was stable within 24 hours at room temperature.

[0114] Conclusion: The system applicability, specificity, accuracy and precision, linearity and range of this method are good; the limit of quantification meets the sensitivity requirements; the reference solution and the test solution are stable at room temperature for 24 hours. This shows that the method of the present invention can better determine the content of calcium dobesilate-based toxic impurities, with high sensitivity and low limit of quantification, and can better control the quality of drugs.

[0115] Example 2

[0116] Based on the method of Example 1, the following conditions were adjusted respectively:

[0117] Mobile phase A: The concentration of ammonium dihydrogen phosphate solution is 0.05 mol / L±0.01 mol / L, the flow rate is 1 ml±0.2 ml per minute; the detection wavelength is 220 nm±10 nm; the column temperature is 50°C±5°C.

[0118] On the basis of the method of the present invention, after adjusting the concentration range of mobile phase A, flow rate, detection wavelength, column temperature and other parameters, the separation degrees between the chromatographic peaks of methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate were all greater than 1.5, the three impurities could be effectively separated, and the detection results were accurate and effective.

[0119] Comparative Example 1

[0120] Compared with Example 1, mobile phase A was replaced with 5 mM ammonium acetate aqueous solution, and the results showed that the peaks of methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate overlapped.

[0121] Comparative Example 2

[0122] Based on Example 1, the gradient elution program was adjusted, as shown in Table 4:

[0123] Table 4 Gradient elution program

[0124]

[0125] The results showed that the separation degree of each impurity peak of methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate was less than 1.5.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the scope of the present invention should be included in the protection scope of the present invention.

Claims

1. A HPLC detection method for sulfonate impurities in calcium dobesilate, characterized in that: The following steps are involved: The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; the mobile phase is ammonium dihydrogen phosphate solution as mobile phase A and methanol as mobile phase B; the elution method is gradient elution, the mobile phase A is adjusted in the range of 80% to 60% v / v, and the mobile phase B is adjusted in the range of 20% to 40% v / v.

2. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that: The sulfonate impurities are methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate.

3. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1 or 2, characterized in that, The gradient elution procedure was as follows:

4. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1 or 2, characterized in that, The concentration of the diammonium phosphate solution is 0.04-0.06 mol / L.

5. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that, The chromatographic column is Welch Ultimate AQ-C18; the specification of the chromatographic column is 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance.

6. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that: The detection wavelength is 210~230nm.

7. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that: The column temperature is 45-55°C.

8. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that, Preparation of test solution: Take calcium dobesilate raw material, dissolve it in solvent, dilute it to a constant volume, and prepare a concentration of 90-110 mg / ml.

9. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 1, characterized in that, Reference solution: Take methyl 2,5-dihydroxybenzenesulfonate reference substance, ethyl 2,5-dihydroxybenzenesulfonate reference substance and isopropyl 2,5-dihydroxybenzenesulfonate reference substance respectively, dissolve them in solvent and quantitatively dilute them to make a mixed solution containing 0.02-0.04 μg of methyl 2,5-dihydroxybenzenesulfonate, ethyl 2,5-dihydroxybenzenesulfonate and isopropyl 2,5-dihydroxybenzenesulfonate per 1 ml.

10. The HPLC detection method for sulfonate impurities in calcium dobesilate according to claim 8 or 9, characterized in that: The solvent is pure water.

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