Method for detecting impurities in sodium phosphinate injection

Abstract

The invention provides a method for detecting impurities in a sodium phosphinate injection, which adopts high performance liquid chromatography for detection under the detection condition that a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, and the detection method comprises the following steps: step S1, preparing a mobile phase A and a mobile phase B; s2, preparing a test solution, wherein the test solution comprises an air auxiliary solution, a system applicability solution, an impurity positioning solution, a test solution and a standard test solution; and S3, sequentially carrying out gradient elution on the solution for testing by using the mobile phase A and the mobile phase B so as to obtain a high performance liquid chromatogram, and analyzing the impurities in the sodium phosphinate injection on the basis of the high performance liquid chromatogram.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and particularly to a method for detecting impurities in foscarnet sodium injection. Background Art

[0002] Foscarnet sodium injection is mainly used for the treatment of cytomegalovirus retinitis in immunocompromised patients (such as AIDS patients). It can also be used for the treatment of cutaneous and mucosal herpes simplex virus infection or varicella-zoster virus infection in immunocompromised patients resistant to acyclovir (such as HIV-infected patients).

[0003] According to the synthesis route provided by the foscarnet sodium raw material manufacturer, residual starting materials, process by-products, degradation impurities, reagent residues, etc. may exist during the production of this product. According to the information disclosed in EP (European Pharmacopoeia), the known impurities that foscarnet sodium may contain include impurity A, impurity B, impurity C, and impurity D, and their structures are shown as follows:

[0004] Impurity A:

[0005]

[0006] Impurity B:

[0007]

[0008] Impurity C:

[0009]

[0010] Impurity D:

[0011]

[0012] The detection of foscarnet sodium impurities is an important part of product quality detection. However, the detection method for related substances of foscarnet sodium is not included in the Chinese Pharmacopoeia and national standards. After inquiry, the USP (United States Pharmacopoeia) 2024, EP (European Pharmacopoeia) 11.0 foscarnet sodium raw material drug standard, and BP (British Pharmacopoeia) 2024 foscarnet sodium injection standard include the detection method for related substances, and the chromatographic conditions are basically the same. By reproducing the related substance analysis method in the EP11.0 foscarnet sodium raw material drug standard, problems such as low sensitivity, interference of blank excipients in the determination, and small resolution were found. Therefore, there is an urgent need for a new detection method for related substances of foscarnet sodium, which can meet the requirements of sensitivity, with no interference from blank excipients in the determination and good resolution of impurities. Summary of the Invention

[0013] In view of the deficiencies of the prior art, the present invention provides a method for detecting impurities in foscarnet sodium injection. The method of the present invention can not only meet the requirements of detection sensitivity, but also is not interfered by blank excipients, and has a high degree of impurity resolution.

[0014] The present invention provides a method for detecting impurities in foscarnet sodium injection, which is detected by high performance liquid chromatography. The detection conditions include: a chromatographic column filled with octadecylsilane-bonded silica gel. The steps of the detection method are as follows:

[0015] Step S1: Prepare mobile phase A and mobile phase B;

[0016] Step S2: Prepare test solutions, and the test solutions include: blank excipient solution, system suitability solution, impurity location solution, test sample solution, and spiked test sample solution;

[0017] Step S3: Gradient elute the test solutions with the mobile phase A and the mobile phase B in sequence to obtain a high performance liquid chromatography (HPLC) chromatogram, and analyze the impurities in the foscarnet sodium injection based on the HPLC chromatogram.

[0018] Furthermore, the impurities in the foscarnet sodium injection include impurity A, impurity B, and impurity C.

[0019] Furthermore, the conditions for gradient elution in step S3 are as follows:

[0020] 0 min to 5 min, 90% mobile phase A and 10% mobile phase B;

[0021] 5 min to 5.1 min, linearly change from 90% mobile phase A and 10% mobile phase B to 75% mobile phase A and 20% mobile phase B;

[0022] 5.1 min to 15 min, 75% mobile phase A and 25% mobile phase B;

[0023] 15 min to 15.1 min, linearly change from 75% mobile phase A and 25% mobile phase B to 90% mobile phase A and 10% mobile phase B;

[0024] 15.1 min to 25 min, 90% mobile phase A and 10% mobile phase B.

[0025] Further, the preparation process of the mobile phase A is as follows: Dissolve 1.42 g of anhydrous sodium sulfate in water, add 3 mL of glacial acetic acid and 6 mL of an anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L, and dilute with water to 1000 mL to obtain Solution A. Dissolve 1.42 g of anhydrous sodium sulfate in water, add 6.8 g of sodium acetate and 6 mL of an anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L, and dilute with water to 1000 mL to obtain Solution B. Subsequently, mix Solution A and Solution B evenly and then add tetraethylammonium hydrogen sulfate and mix evenly to obtain the mobile phase A;

[0026] The preparation process of the mobile phase B is as follows: Take 1000 mL of 100% acetonitrile as the mobile phase B.

[0027] Further, the ratio of Solution A, Solution B and tetraethylammonium hydrogen sulfate in the mobile phase A in step S1 is: 600 - 800 ml: 400 - 200 ml: 0.2 - 0.3 g, where the total volume of Solution A and Solution B is 1000 mL.

[0028] Further, after mixing the mobile phase A and the mobile phase B, adjust the pH value of the mixed solution to 5.0.

[0029] Further, the preparation process of the test solution is as follows: Take 1 mL of foscarnet sodium injection and dilute it with the mobile phase A to a concentration of 2.0 - 2.5 mg / mL to obtain the test solution;

[0030] The preparation process of the spiked test solution includes:

[0031] Prepare the impurity mixed stock solution: Take the reference standards of impurity A, impurity B and impurity C, and quantitatively dilute them with the mobile phase A to prepare a mixed solution containing 1 mg of each of impurity A, impurity B and impurity C per milliliter of solution;

[0032] Prepare the spiked test solution: Measure 1 ml of foscarnet sodium injection, place it in a 10 ml volumetric flask, add 1 ml of the impurity mixed stock solution, dissolve with the mobile phase A and dilute to the mark, and shake well.

[0033] Further, the preparation process of the system suitability solution is as follows: Take the foscarnet sodium reference standard, the reference standards of impurity A, impurity B and impurity C, dissolve them with the mobile phase A to form the system suitability solution;

[0034] The preparation process of the impurity localization solution is as follows: Take the reference standards of impurity A, impurity B and impurity C respectively, dissolve them with the mobile phase A to form the impurity A localization solution, the impurity B localization solution and the impurity C localization solution respectively;

[0035] The preparation process of the blank auxiliary solution is as follows: Take 1 mL of the auxiliary material solution in the prescribed amount, place it in a 10-mL volumetric flask, dilute it to the calibration line with mobile phase A, and shake well to obtain the blank auxiliary solution.

[0036] Furthermore, the test solution further includes: a test sample control solution, which is prepared by the following method:

[0037] Take 1 mL of the test sample solution, place it in a 50-mL volumetric flask, dilute it to the calibration line with the mobile phase A, shake well, then take 1 mL and place it in a 10-mL volumetric flask, dilute it to the calibration line with the mobile phase A, and shake well to obtain the test sample control solution.

[0038] Furthermore, the detection conditions include: The chromatographic column used is a Hypersil C18 250×4.6 mm, 5-μm chromatographic column, and the detection wavelength is 245 nm, the flow rate is 1.2 mL / min, and the injection volume is 50 - 70 μL.

[0039] The method for detecting impurities in foscarnet sodium injection provided by the present invention adopts gradient elution to increase the separation degree of impurities, and increases the injection volume without increasing too much detection time. While improving the detection efficiency, it also improves the detection sensitivity. In addition, the method of the present invention particularly selects a Hypersil C18 250×4.6 mm, 5-μm chromatographic column. The increase in the length of the chromatographic column is beneficial to the separation of impurities. Therefore, the method provided by the present invention has high precision, low detection limit, strong stability, good repeatability, and high accuracy. Description of the Drawings

[0040] Figure 1 It is a high-performance liquid chromatography diagram of the related substances of foscarnet sodium injection detected by the method according to the present invention.

[0041] Figure 2 It is a linear diagram of the related substances method verification - foscarnet sodium linear test.

[0042] Figure 3 It is a linear diagram of the related substances method verification - impurity A linear test.

[0043] Figure 4 It is a linear diagram of the related substances method verification - impurity B linear test.

[0044] Figure 5 It is a linear diagram of the related substances method verification - impurity C linear test. Detailed Embodiments

[0045] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. The principles and features of the present invention will be described below with reference to the drawings. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The embodiments described are only used to explain the present invention and are not used to limit the scope of the present invention.

[0046] It should be noted that in the present application, the impurities in foscarnet sodium injection mentioned refer to the general term of impurity A, impurity B, and impurity C.

[0047] In addition, the reference substance mentioned in the present invention, that is, the reference article, is the reference substance for drug standards, which is used as a measurement standard and the standard content in the content determination.

[0048] Furthermore, it should be noted that, unless otherwise specified, the so-called preparation into a solution mentioned or not mentioned in the following content of the present invention refers to the preparation using mobile phase A as the solvent. For example, in the verification of the limit of quantitation and the limit of detection, the statement "Take appropriate amounts of foscarnet sodium, impurity A, impurity B, and impurity C reference substances and prepare standard substance solutions with certain concentrations respectively" means "Take appropriate amounts of foscarnet sodium, impurity A, impurity B, and impurity C reference substances and prepare standard substance solutions with certain concentrations respectively using mobile phase A as the solvent".

[0049] Example 1

[0050] A method for detecting impurities in foscarnet sodium injection is carried out by high performance liquid chromatography. The impurities in the foscarnet sodium injection detected by the present invention include impurity A, impurity B, and impurity C, and their structures are as described above and will not be elaborated here. The steps of the detection method provided by the present invention are as follows:

[0051] Step S1:

[0052] Prepare mobile phase A: Take 1.42 g of anhydrous sodium sulfate and dissolve it in water, add 3 mL of glacial acetic acid and 6 mL of an anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L, and dilute it with water to 1000 mL as solution A. Take 1.42 g of anhydrous sodium sulfate and dissolve it in water, add 6.8 g of sodium acetate and 6 mL of an anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L, and dilute it with water to 1000 mL as solution B. Subsequently, take 700 mL of solution A and 300 mL of solution B, mix them evenly, and adjust the pH value to 5.0 with sodium hydroxide. Then add 0.25 g of tetraethylammonium hydrogen sulfate and mix evenly to obtain mobile phase A.

[0053] Prepare mobile phase B: Take 1000 mL of 100% acetonitrile as mobile phase B.

[0054] Step S2. Prepare the test solution:

[0055] Prepare the test solution: Take 1 mL of foscarnet sodium injection and dilute it with mobile phase A to a concentration of 2.4 mg / mL to obtain the test solution.

[0056] Prepare the spiked test solution: Prepare the mixed stock solution of impurities: Take the reference substances of impurity A, impurity B, and impurity C respectively, and quantitatively dilute them with mobile phase A to prepare a mixed solution containing about 1 mg of each of impurity A, impurity B, and impurity C per milliliter of solution; Measure 1 mL of foscarnet sodium injection, place it in a 10 mL volumetric flask, add 1 mL of the above-mentioned mixed stock solution of impurities, dissolve it with mobile phase A and dilute it to the mark, and shake well.

[0057] Prepare the system suitability solution: Take foscarnet sodium pure product, impurity A, impurity B, and impurity C, dissolve them with mobile phase A to form the system suitability solution.

[0058] Prepare the impurity location solution: Take the reference substances of impurity A, impurity B, and impurity C respectively, dissolve them with mobile phase A to form the impurity A location solution, impurity B location solution, and impurity C location solution respectively.

[0059] Prepare the empty excipient solution: Take 1 mL of the excipient solution of foscarnet sodium injection in the prescription amount, place it in a 10 mL volumetric flask, dilute it to the mark with mobile phase A, and shake well to obtain the empty excipient solution.

[0060] Prepare the test solution control: Take 1 mL of the test solution, place it in a 50 mL volumetric flask, dilute it to the mark with mobile phase A, shake well, then take 1 mL and place it in a 10 mL volumetric flask, dilute it to the mark with mobile phase A, and shake well to obtain the test solution control. After the test solution control is prepared, it should be sealed and stored for subsequent use.

[0061] Step S3:

[0062] Perform gradient elution on the above-mentioned test solutions successively with the mobile phase A and the mobile phase B to obtain a high-performance liquid chromatography (HPLC) chromatogram, and analyze the impurities in the foscarnet sodium injection based on the HPLC chromatogram.

[0063] In addition, it should be noted that for baseline calibration, a blank solution is also required for calibration.

[0064] The gradient elution conditions are shown in Table 1 below:

[0065] Table 1: Gradient elution conditions for the detection of impurities in foscarnet sodium injection

[0066] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 5.1 75 25 15 75 25 15.1 90 10 25 90 10

[0067] The chromatographic conditions were as follows: a Hypersil C18 column with dimensions of 250×4.6 mm and a particle size of 5 μm was used; the detection wavelength was 240 nm, the flow rate was 1.0 ml / min, and the injection volume was 60 μL.

[0068] Specifically, the above test solutions were injected into the liquid chromatograph in the above-mentioned order successively, and the high-performance liquid chromatogram was recorded. The chromatogram is as Figure 1 shown. Table 2 shows the results of system suitability localization.

[0069] Table 2: Chromatographic data

[0070]

[0071]

[0072] Under the above chromatographic conditions, it can be seen from Figure 1 and Table 2 that the resolution of the excipient peak, impurity A, and foscarnet sodium is 1.965 and 2.067 respectively, both of which are greater than the resolution of 1.5 specified by the industry standard. Thus, it can be seen that the blank excipient does not interfere with the determination of each impurity, and the resolution between each impurity and the main peak is good. Therefore, it can be proved that the method of the present invention can effectively detect and separate related substances.

[0073] In order to verify the method of the present invention, in this test example, method validations were carried out on the injection precision, quantitative limit, detection limit, linearity and range, solution stability, repeatability, accuracy, and durability of the method:

[0074] 1. Verification of injection precision:

[0075] Prepare the test solution for control: Take 1 mL of the test solution prepared in Example 1 above, place it in a 50 mL volumetric flask, dilute it to the scale line with mobile phase A, shake well, then take 1 mL and place it in a 10 mL volumetric flask, dilute it to the scale line with mobile phase A, and shake well to obtain the test solution for control.

[0076] According to the detection conditions in Example 1, continuously measure 6 times. Requirements: When the control solution is continuously measured 6 times, the RSD of the retention time of the foscarnet sodium main peak should not exceed 1.0%, and the RSD of the peak area should not exceed 2.0%. The results are shown in Table 3 below:

[0077] Table 3: Method validation results

[0078]

[0079] Conclusion: When the control solution is continuously measured 6 times, the RSD of the retention time is 0.1%, which is less than 1.0%; the RSD of the peak area is 0.4%, which is less than 2.0%, meeting the requirements, indicating that the injection precision of this method is good.

[0080] 2. Verification of Quantitation Limit and Detection Limit

[0081] Weigh appropriate amounts of foscarnet sodium, impurity A, impurity B, and impurity C reference standards, and prepare standard solutions with certain concentrations respectively. After dilution, measure them. When the signal values (chromatographic peak heights) of foscarnet sodium, impurity A, impurity B, and impurity C have a ratio of approximately 10 to the baseline noise, the quantitation limit is obtained (as shown in Table 4 below), and the solution with this concentration is the quantitation limit solution. When the signal values (chromatographic peak heights) of foscarnet sodium, impurity A, impurity B, and impurity C have a ratio of approximately 3 to the baseline noise, the detection limit is obtained (as shown in Table 5 below), and the solution with this concentration is the detection limit solution.

[0082] Table 4: Quantitation Limit Results of Foscarnet Sodium, Impurity A, Impurity B, and Impurity C

[0083]

[0084] Table 5: Detection Limit Results of Foscarnet Sodium, Impurity A, Impurity B, and Impurity C

[0085]

[0086]

[0087] It can be seen from Table 4 and Table 5 above that

[0088] The quantitation limit of foscarnet sodium is 97.2 ng, the quantitation limit concentration is 1.214 μg / ml, which is equivalent to 0.051% of the test solution concentration; the detection limit is 29.15 ng, the detection limit concentration is 0.3643 μg / ml, which is equivalent to 0.015% of the test solution concentration;

[0089] The quantitation limit of impurity A is 77.63 ng, the quantitation limit concentration is 0.970 μg / ml, which is equivalent to 0.040% of the test solution concentration; the detection limit is 23.29 ng, the detection limit concentration is 0.2911 μg / ml, which is equivalent to 0.012% of the test solution concentration;

[0090] The quantitation limit of impurity B is 97.9 ng, the quantitation limit concentration is 1.224 μg / ml, which is equivalent to 0.051% of the test solution concentration; the detection limit is 29.38 ng, the detection limit concentration is 0.3672 μg / ml, which is equivalent to 0.015% of the test solution concentration;

[0091] The quantitation limit of impurity C is 90.8 ng, the quantitation limit concentration is 1.135 μg / ml, which is equivalent to 0.047% of the test solution concentration; the detection limit is 27.25 ng, the detection limit concentration is 0.3406 μg / ml, which is equivalent to 0.014% of the test solution concentration.

[0092] It can be seen that the method for detecting impurities in foscarnet sodium injection provided by the present invention has low quantitative limit of detection and detection limit, high sensitivity, and meets the detection standards.

[0093] 3. Linearity and Range

[0094] Within a certain concentration range, 5 concentration points were taken for linearity study. The linear relationship was plotted as a function of the measured response value (peak area) against the solution concentration, and linear regression was performed, and the correlation coefficient r was reported to confirm a good linear relationship.

[0095] Solution preparation:

[0096] Stock solution of impurity A standard: Take the impurity A standard, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 1 mg / ml.

[0097] Stock solution of impurity B standard: Take the impurity B standard, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 1 mg / ml.

[0098] Stock solution of impurity C standard: Take the impurity C standard, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 1 mg / ml.

[0099] Stock solution of foscarnet sodium standard: Take the foscarnet sodium standard, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 1 mg / ml.

[0100] Stock solution of linear standard: Accurately measure 2 ml of the stock solution of foscarnet sodium standard, 2 ml of each of the stock solutions of impurity A, impurity B and impurity C standards, place them in a 20-ml volumetric flask, dilute to the mark with a solvent, and shake well.

[0101] Linear 5 solution: Accurately measure 2 ml of the stock solution of linear standard, place it in a 25-ml volumetric flask, dilute to the mark with a solvent, and shake well.

[0102] Linear 4 solution: Accurately measure 1.5 ml of the stock solution of linear standard, place it in a 25-ml volumetric flask, dilute to the mark with a solvent, and shake well.

[0103] Linear 3 solution: Accurately measure 1 ml of the stock solution of linear standard, place it in a 25-ml volumetric flask, dilute to the mark with a solvent, and shake well.

[0104] Linear 2 solution: Accurately measure 0.5 ml of the stock solution of linear standard, place it in a 25-ml volumetric flask, dilute to the mark with a solvent, and shake well.

[0105] Linear 1 solution: Quantitative limit solution, that is, the quantitative limit solution in the verification of the above quantitative limit and detection limit.

[0106] Determination: Accurately measure the above linear solution, inject it into the liquid chromatograph respectively, and record the chromatogram. Tables 6 to 9 below show the results of the linearity test for the related substances method verification. Figures 2 to 5 Shows the linearity graph for the related substances method verification.

[0107] Table 6: Results of the linearity test for the related substances method verification - foscarnet sodium

[0108]

[0109] Table 7: Results of the linearity test for the related substances method verification - impurity A

[0110]

[0111] Table 8: Results of the linearity test for the related substances method verification - impurity B

[0112]

[0113] Table 9: Results of the linearity test for the related substances method verification - impurity C

[0114]

[0115] Based on the above tables and Figures 2 to 5 the data, and calculate the impurity correction factor according to the formula:

[0116]

[0117] Where: A s is the linear slope of foscarnet sodium;

[0118] A r is the linear slope of the impurity.

[0119] Table 10 below shows the results of the test for the correction factors of each impurity in the related substances method verification.

[0120] Table 10: Results of the test for the correction factors of each impurity in the related substances method verification

[0121]

[0122] In summary, it can be seen that:

[0123] When the concentration of foscarnet sodium is in the range of 1.214 μg / ml to 9.72 μg / ml, the response value (peak area) and the concentration show a good linear relationship. The linear equation is y = 3.349×10³x + 1.275×10³, the linear correlation coefficient is 0.9992, and the sum of squared residuals is 789939;

[0124] When the concentration of impurity A is in the range of 0.970 μg / ml to 7.763 μg / ml, there is a good linear relationship between the response value (peak area) and the concentration. The linear equation is y = 4.930×10³x + 3.203×10², the linear correlation coefficient is 0.9999, and the sum of squared residuals is 134079;

[0125] When the concentration of impurity B is in the range of 1.224 μg / ml to 9.79 μg / ml, there is a good linear relationship between the response value (peak area) and the concentration. The linear equation is y = 4.741×10³x - 5.214×10², the linear correlation coefficient is 0.9998, and the sum of squared residuals is 425881;

[0126] When the concentration of impurity C is in the range of 1.135 μg / ml to 9.08 μg / ml, there is a good linear relationship between the response value (peak area) and the concentration. The linear equation is y = 4.501×10³x - 2.831×10², the linear correlation coefficient is 0.9995, and the sum of squared residuals is 770067;

[0127] The correction factors of impurity A, impurity B, and impurity C are 0.68, 0.71, and 0.74 respectively. The content is calculated by the self - control method without using the correction factor.

[0128] Thus, it can be seen that the method provided by the present invention makes the response signal linearly related to the sample concentration, that is, the change of the response signal is proportional to the change of the sample concentration, ensuring the accuracy and reliability of quantitative analysis.

[0129] 4. Solution stability

[0130] Method: Prepare a spiked test solution according to the method in Example 1, and prepare a test control solution according to the method for preparing the test control solution in the above injection precision verification. Place the prepared spiked test solution and test control solution at room temperature (protected from light), and measure them according to the measurement method in Example 1 at the time points of 0 h, 10 h, 24 h, 30 h, and 51 h respectively.

[0131] Requirement: After the test control solution is placed at room temperature (protected from light) for a period of time, the RSD of the measured peak area should not exceed 2.0%. After the spiked test solution is placed at room temperature (protected from light) for a period of time, the maximum difference in the measured impurities should not be greater than 0.05%, and the maximum difference in the total impurities should not be greater than 0.1%.

[0132] Table 11 below shows the results of the stability test of the test control solution, and Table 12 shows the results of the stability test of the spiked test solution.

[0133] Table 11: Results of the stability test of the test control solution

[0134]

[0135] Table 12: Results of the stability test of the spiked test solution

[0136]

[0137]

[0138] It can be seen from Table 11 and Table 12 above that: The test solution control solution was placed at room temperature (protected from light) for 51 h, and the RSD of the peak area was measured to be 0.4%, less than 2.0%, indicating that the test solution control solution was stable within 51 h when placed at room temperature (protected from light). The spiked test solution was placed at room temperature (protected from light) for 51 h, and the maximum difference in the determination of each impurity was less than 0.05%, and the maximum difference in the total impurities was less than 0.1%, indicating that the test solution was stable within at least 51 h when placed at room temperature (protected from light).

[0139] 5. Repeatability

[0140] Method: Six spiked test solutions and test solution control solutions were prepared and detected according to the determination method in Example 1 to investigate the precision among the determination results of the method.

[0141] Requirement: The maximum difference in the impurities measured in the six spiked test solutions shall not be greater than 0.05%, and the maximum difference in the total impurities shall not be greater than 0.1%.

[0142] The test results are shown in Table 13 below:

[0143] Table 13: Results of the repeatability test

[0144]

[0145]

[0146] Conclusion: Among the six spiked test solutions, the maximum difference in the change of each impurity was less than 0.05%, and the maximum difference in the change of the total impurities was less than 0.1%, indicating that the method had good repeatability.

[0147] 6. Accuracy

[0148] Method: Standard substances of impurity A, impurity B, and impurity C were respectively added to the test solution to prepare solutions at four levels of the quantitative limit concentration, 50%, 100%, and 150% of each impurity, with 3 replicates in each group, and the accuracy of the determination results of the related substances method was characterized by the impurity recovery rate.

[0149] Solution preparation:

[0150] Stock solution of reference substances: Weigh appropriate amounts of reference substances of impurity A, impurity B, and impurity C accurately, dissolve them in the mobile phase, and prepare a solution containing about 24 μg of each of impurity A, impurity B, and impurity C per 1 ml.

[0151] Stock solution for limit of quantitation: Weigh appropriate amounts of reference substances of impurity A, impurity B, and impurity C accurately, dissolve them in the mobile phase, and prepare a solution containing about 10 μg of each of impurity A, impurity B, and impurity C per 1 ml.

[0152] Reference substance solution: Pipette 2 ml of the stock solution of reference substances accurately, transfer it to a 10-ml volumetric flask, dilute it to the mark with the mobile phase, and mix well.

[0153] Solution for recovery rate at limit of quantitation concentration: Pipette 1 ml of this product accurately, transfer it to a 10-ml volumetric flask, add 1 ml of the stock solution for limit of quantitation accurately, dilute it to the mark with the mobile phase, and mix well. (Prepare 3 parallel portions)

[0154] Solution for recovery rate at low concentration (50%): Pipette 1 ml of this product accurately, transfer it to a 10-ml volumetric flask, add 1 ml of the stock solution of reference substances accurately, dilute it to the mark with the mobile phase, and mix well. (Prepare 3 parallel portions)

[0155] Solution for recovery rate at medium concentration (100%): Pipette 1 ml of this product accurately, transfer it to a 10-ml volumetric flask, add 2 ml of the stock solution of reference substances accurately, dilute it to the mark with the mobile phase, and mix well. (Prepare 3 parallel portions)

[0156] Solution for recovery rate at high concentration (150%): Pipette 1 ml of this product accurately, transfer it to a 10-ml volumetric flask, add 3 ml of the stock solution of reference substances accurately, dilute it to the mark with the mobile phase, and mix well. (Prepare 3 parallel portions)

[0157] Requirement: The recovery rates of each impurity should be within the range of 92% - 105%; the RSD values of the recovery rates should not be greater than 5.0%.

[0158] The test results are shown in Tables 14 to 16 as follows:

[0159] Table 14: Results of recovery rate of impurity A

[0160]

[0161]

[0162] Table 15: Results of recovery rate of impurity B

[0163]

[0164]

[0165] Table 16: Results of recovery rate of impurity C

[0166]

[0167] Based on the above Tables 14 to 16: Calculated by the self-control method with a correction factor added: For the 12 recovery solutions at the quantification limit, low, medium, and high concentrations, the recovery rate of impurity A is in the range of 92.5% to 98.8%, with an average value of 96.4% and an RSD of 2.0%; the recovery rate of impurity B is in the range of 97.8% to 104.3%, with an average value of 101.9% and an RSD of 1.8%; the recovery rate of impurity C is in the range of 100.0% to 104.2%, with an average value of 102.4% and an RSD of 1.6%.

[0168] Calculated by the external standard method: For the 12 recovery solutions at the quantification limit, low, medium, and high concentrations, the recovery rate of impurity A is in the range of 94.4% to 101.1%, with an average value of 98.1% and an RSD of 2.0%; the recovery rate of impurity B is in the range of 96.8% to 101.5%, with an average value of 99.2% and an RSD of 1.7%; the recovery rate of impurity C is in the range of 93.5% to 99.4%, with an average value of 96.1% and an RSD of 2.0%.

[0169] Calculated according to the external standard method and the self-control method with a correction factor added, the recovery rates of each impurity are all in the range of 92% to 105%, and the RSDs are all less than 5.0%, all meeting the requirements, indicating that the recovery rate of the related substances method is good, the measurement result accuracy is high, and the results of the self-control method and the external standard method are consistent.

[0170] 7. Durability

[0171] Method

[0172] By changing the flow rate, column temperature, detection wavelength, and chromatographic columns of the same manufacturer and model but different batches, evaluate the tolerance degree that the measurement results are not affected when there are minor changes in the related substances determination method. The test conditions are shown in Table 17 below:

[0173]

[0174] After changing the chromatographic conditions according to the above table, take the blank solution, system suitability solution, test solution, and test control solution for determination.

[0175] Requirements: When there are minor changes in the chromatographic conditions, the system suitability should meet the requirements; compared with the normal conditions, the maximum difference in the change of each impurity in the test solution should not be greater than 0.05%, and the maximum difference in the change of the total impurities should not be greater than 0.1%.

[0176] The results are shown in Tables 18 and 19 below:

[0177] Table 18: Durability - System Suitability Results

[0178]

[0179]

[0180] Table 19: Results of the durability test

[0181]

[0182] Conclusion:

[0183] When the flow rate changes by ±0.1 ml / min, the column temperature changes by ±5 °C, the wavelength changes by ±2 nm, and different batches of chromatographic columns of the same manufacturer and model are used, the system suitability meets the requirements; compared with the normal conditions, the maximum difference in the impurities in the test sample is less than 0.05%, and the maximum difference in the total impurities is less than 0.1%; the method has good durability.

[0184] In summary, the related substance method of this product has good specificity, high sensitivity, good precision, solution stability, linearity, accuracy and durability, and can meet the detection of related substances in foscarnet sodium injection.

[0185] Test Example 1

[0186] Using the method of the present invention to detect multiple samples of different batches, 6 batches of foscarnet sodium injection produced by [manufacturer] are selected for detection in this experimental example, and the detection results are shown in the following table:

[0187] Table 20: Detection results of products of different batches

[0188]

[0189] It can be seen from the above experimental examples that when the method of the present invention is used to detect the product, the detection level is consistent, the method is stable, the detection limit is low, and the sensitivity is high.

[0190] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. The specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

Claims

1. A method for detecting impurities in sodium foscarnet injection, using high performance liquid chromatography for detection, characterized in that: The detection conditions include: using octadecylsilane bonded silica gel as a chromatographic column as a filler, and the detection method steps are as follows: Step S1, preparing mobile phase A and mobile phase B; Step S2, preparing a test solution, wherein the test solution includes: an air auxiliary solution, a system suitability solution, an impurity location solution, a test solution, and a spiked test solution; Step S3, gradient eluting the test solution with the mobile phase A and the mobile phase B in sequence to obtain a high performance liquid chromatogram, and analyzing the impurities in the sodium foscarnet injection based on the high performance liquid chromatogram.

2. The method for detecting impurities in foscarnet sodium injection according to claim 1, characterized in that: The impurities of the sodium foscarnet injection include impurity A, impurity B and impurity C.

3. The method for detecting impurities of sodium foscarnet injection according to claim 2, characterized in that: The conditions of gradient elution in step S3 are as follows: 0 min to 5 min, 90% mobile phase A and 10% mobile phase B; From 5 to 5.1 min, 90% mobile phase A and 10% mobile phase B changed linearly to 75% mobile phase A and 20% mobile phase B; 5.1min~15min, 75% mobile phase A and 25% mobile phase B; 15-15.1 min, 75% mobile phase A and 25% mobile phase B linearly changed to 90% mobile phase A and 10% mobile phase B; 15.1min~25min, 90% mobile phase A and 10% mobile phase B.

4. The method for detecting impurities in foscarnet sodium injection according to claim 3, characterized in that: The preparation process of the mobile phase A is as follows: 1.42 g of anhydrous sodium sulfate is dissolved in water, 3 mL of glacial acetic acid and 6 mL of anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L are added, and the mixture is diluted with water to 1000 mL as solution A; 1.42 g of anhydrous sodium sulfate is dissolved in water, 6.8 g of sodium acetate and 6 mL of anhydrous sodium pyrophosphate solution with a concentration of 26.61 g / L are added, and the mixture is diluted with water to 1000 mL as solution B; then, the solution A and the solution B are mixed evenly, and then tetraethylammonium hydrogen sulfate is added and mixed evenly, thereby obtaining the mobile phase A; The preparation process of the mobile phase B is as follows: 1000 mL of 100% acetonitrile is taken as the mobile phase B.

5. The method for detecting impurities in foscarnet sodium injection according to claim 4, characterized in that: The ratio of solution A, solution B and tetraethylammonium hydrogen sulfate in the mobile phase A in step S1 is: 600-800 ml: 400-200 ml: 0.2-0.3 g, wherein the total volume of solution A and solution B is 1000 mL.

6. The method for detecting impurities in foscarnet sodium injection according to claim 5, characterized in that: After the mobile phase A and the mobile phase B are mixed, the pH value of the mixed solution is adjusted to 5.

0.

7. The method for detecting impurities in foscarnet sodium injection according to claim 3, characterized in that: The preparation process of the test solution is as follows: taking 1 mL of sodium foscarnet injection and diluting it with the mobile phase A to a concentration of 2.0 to 2.5 mg / mL, thereby obtaining the test solution; The preparation process of the spiked test solution includes: Prepare impurity mixed stock solution: Take impurity A, impurity B, and impurity C standard products, and quantitatively dilute them with mobile phase A to make a mixed solution containing 1 mg of impurity A, impurity B, and impurity C per ml of solution; Prepare spiked test solution: measure 1 ml of sodium foscarnet injection, place it in a 10 ml volumetric flask, add 1 ml of the impurity mixed stock solution, add mobile phase A to dissolve and dilute to the scale, and shake well.

8. The method for detecting impurities in foscarnet sodium injection according to claim 3, characterized in that: The preparation process of the system suitability solution is as follows: taking a standard product of sodium foscarnet, a standard product of impurity A, a standard product of impurity B and a standard product of impurity C, adding them into mobile phase A to dissolve, and forming a system suitability solution; The preparation process of the impurity localization solution is as follows: respectively taking standard samples of impurity A, impurity B and impurity C, dissolving them in mobile phase A, and forming impurity A localization solution, impurity B localization solution and impurity C localization solution respectively; The preparation process of the air auxiliary solution is as follows: take 1 mL of the auxiliary solution of the prescribed amount, put it in a 10 mL volumetric bottle, dilute it to the scale line with mobile phase A, and shake it well to obtain the air auxiliary solution.

9. The method for detecting impurities in foscarnet sodium injection according to claim 7, characterized in that: The test solution also includes: a test sample control solution, and the test sample control solution is prepared by the following method: Take 1 mL of the test solution, place it in a 50 mL volumetric flask, dilute it to the scale with the mobile phase A, shake well, take 1 mL and place it in a 10 mL volumetric flask, dilute it to the scale with the mobile phase A, and shake well to obtain the test control solution.

10. The method for detecting impurities in foscarnet sodium injection according to any one of claims 1 to 8, characterized in that: The detection conditions include: the chromatographic column uses a Hypersil C18 250×4.6 mm, 5 μm chromatographic column, the detection wavelength is 245 nm, the flow rate is 1.2 ml / min, and the injection volume is 50-70 μL.

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