Method for detecting related substances in phloroglucinol injection

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for detecting related substances in phloroglucinol injection. The invention provides a method for detecting related substances in phloroglucinol injection. The related substances in the phloroglucinol injection can be accurately detected by limiting a chromatographic column, an elution mode and a specific elution procedure of high performance liquid chromatography detection. And the method has the advantages of high accuracy, high sensitivity, high precision and good specificity.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting related substances in phloroglucinol injection. Background Technology

[0002] Phloroglucinol injection is a smooth muscle relaxant developed by TEVA SANTE. It is mainly used to treat acute spasmodic pain caused by digestive system and biliary dysfunction; acute spasmodic urethral, ​​bladder, and renal colic; and gynecological spasmodic pain. It has been used clinically in France as an antispasmodic drug for nearly 30 years.

[0003] Currently, among the methods for determining phloroglucinol included in the mainstream pharmacopoeias of various countries, only the European Pharmacopoeia and the British Pharmacopoeia include quality standards for phloroglucinol and its dihydrate. A comparison shows that the methods for related substances in the European and British Pharmacopoes are consistent. However, the peak shapes for phloroglucinol and 2,4,6-trihydroxybenzoic acid in the detection chromatograms of this method are abnormal, indicating low specificity and sensitivity. Furthermore, no method for detecting related substances in phloroglucinol injection is described in any of the pharmacopoeias. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting related substances in phloroglucinol injection. The detection method provided by this invention can achieve qualitative or quantitative detection of related substances in phloroglucinol injection.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for detecting related substances in phloroglucinol injection, comprising the following steps: High performance liquid chromatography was used to detect the test sample and obtain the detection results of related substances in the test sample. The detection conditions for the high-performance liquid chromatography include: the mobile phase system comprises mobile phase A and mobile phase B; mobile phase A is a phosphate buffer-methanol system; and mobile phase B is methanol. The chromatographic column for the high-performance liquid chromatography is C18. 18 column; The flow rate of the mobile phase system is 1 mL / min; The elution method is gradient elution; The gradient elution procedure is as follows: 0~5min: The volume percentage of mobile phase A is 100%; 5~15min: The volume percentage of mobile phase A decreases uniformly from 100% to 94%; the volume percentage of mobile phase B increases uniformly from 0% to 6%. 15~45min: The volume percentage of mobile phase A decreases uniformly from 94% to 16%; the volume percentage of mobile phase B increases uniformly from 6% to 84%. 45~45.01 min: The volume percentage of mobile phase A increases uniformly from 16% to 100%; the volume percentage of mobile phase B decreases uniformly from 84% to 0%. 45.01~60min: The volume percentage of mobile phase A is 100%; The detector is an ultraviolet detector with a detection wavelength of 265 nm; The related substances include at least one of phloroglucinol, impurity D, trimethylphloroglucinol, and 2,4,6-trihydroxybenzoic acid.

[0006] Preferably, the phosphate buffer solution in the mobile phase A is an aqueous solution of potassium dihydrogen phosphate.

[0007] Preferably, the concentration of the potassium dihydrogen phosphate aqueous solution is 0.01 mol / L; and the pH value of the potassium dihydrogen phosphate aqueous solution is 2.7.

[0008] Preferably, the volume ratio of phosphate buffer to methanol in the mobile phase A is 95:5.

[0009] Preferably, the column temperature of the high-performance liquid chromatography is 35°C.

[0010] Preferably, the injection volume of the high performance liquid chromatography is 20 μL.

[0011] Preferably, the detection includes qualitative detection or quantitative detection.

[0012] Preferably, the quantitative detection includes: using high performance liquid chromatography to detect the sample to be tested, and obtaining a chromatogram of the sample to be tested; The chromatogram of the sample to be tested is compared with a predetermined standard curve, and the content of related substances in the sample to be tested is calculated using the external standard method.

[0013] This invention provides a method for detecting related substances in phloroglucinol injection, comprising the following steps: high-performance liquid chromatography (HPLC) is used to detect the sample to obtain the detection results of related substances in the sample; the detection conditions of the HPLC include: the mobile phase system includes mobile phase A and mobile phase B; mobile phase A is a phosphate buffer-methanol system; mobile phase B is methanol; the HPLC column is a C18 column; the flow rate of the mobile phase system is 1 mL / min; the elution method is gradient elution; the gradient elution program is: 0~5 min: the volume percentage of mobile phase A is 100%; 5~15 min: the volume percentage of mobile phase A decreases uniformly from 100% to 9%. 4%; the volume percentage of mobile phase B increases uniformly from 0% to 6%; 15~45 min: the volume percentage of mobile phase A decreases uniformly from 94% to 16%; the volume percentage of mobile phase B increases uniformly from 6% to 84%; 45~45.01 min: the volume percentage of mobile phase A increases uniformly from 16% to 100%; the volume percentage of mobile phase B decreases uniformly from 84% to 0%; 45.01~60 min: the volume percentage of mobile phase A is 100%; the detector is an ultraviolet detector with a detection wavelength of 265 nm; the related substances include at least one of resorcinol, impurity D, trimethylresorcinol, and 2,4,6-trihydroxybenzoic acid. This invention, by defining the chromatographic column, elution method, and specific elution procedure for high-performance liquid chromatography (HPLC), enables qualitative or quantitative detection of related substances in resorcinol injection. Furthermore, this method has the advantages of high accuracy, high sensitivity, high precision, and good specificity. Attached Figure Description

[0014] Figure 1 Line graph of phloroglucinol; Figure 2 Line graph of impurity D; Figure 3 Line graph of 2,4,6-trihydroxybenzoic acid; Figure 4 This is a line graph of trimethylphloroglucinol. Detailed Implementation

[0015] This invention provides a method for detecting related substances in phloroglucinol injection, comprising the following steps: High performance liquid chromatography was used to detect the test sample and obtain the detection results of related substances in the test sample. The detection conditions for the high-performance liquid chromatography include: the mobile phase system comprises mobile phase A and mobile phase B; mobile phase A is a phosphate buffer-methanol system; and mobile phase B is methanol. The chromatographic column for the high-performance liquid chromatography is C18.18 column; The flow rate of the mobile phase system is 1 mL / min; The elution method is gradient elution; The gradient elution procedure is as follows: 0~5min: The volume percentage of mobile phase A is 100%; 5~15min: The volume percentage of mobile phase A decreases uniformly from 100% to 94%; the volume percentage of mobile phase B increases uniformly from 0% to 6%. 15~45min: The volume percentage of mobile phase A decreases uniformly from 94% to 16%; the volume percentage of mobile phase B increases uniformly from 6% to 84%. 45~45.01 min: The volume percentage of mobile phase A increases uniformly from 16% to 100%; the volume percentage of mobile phase B decreases uniformly from 84% to 0%. 45.01~60min: The volume percentage of mobile phase A is 100%; The detector is an ultraviolet detector with a detection wavelength of 265 nm; The related substances include at least one of phloroglucinol, impurity D, trimethylphloroglucinol, and 2,4,6-trihydroxybenzoic acid.

[0016] In this invention, the phosphate buffer in the mobile phase A is preferably an aqueous solution of potassium dihydrogen phosphate; the concentration of the aqueous solution of potassium dihydrogen phosphate is preferably 0.01 mol / L; the pH value of the aqueous solution of potassium dihydrogen phosphate is preferably 2.7, and is preferably adjusted using phosphoric acid.

[0017] In this invention, the volume ratio of phosphate buffer and methanol in the mobile phase A is preferably 95:5.

[0018] In this invention, the C 18 The column is preferably a YMC-Pack ODS-AQ (4.6 mm × 250 mm, 5 µm). In this invention, the column temperature of the high-performance liquid chromatography (HPLC) column is preferably 35 °C. In this invention, the injection volume of the HPLC is preferably 20 μL.

[0019] In this invention, the detection preferably includes qualitative or quantitative detection. In this invention, the quantitative detection preferably includes: detecting the sample to be tested using high-performance liquid chromatography (HPLC) to obtain a chromatogram of the sample; comparing the chromatogram of the sample to be tested with a predetermined standard curve, and calculating the content of related substances in the sample to be tested using the external standard method. This invention does not impose any special limitation on the method for obtaining the predetermined standard curve; any method well known to those skilled in the art can be used.

[0020] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0021] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] In embodiments of the present invention, the detection conditions for high-performance liquid chromatography are as follows: The chromatographic column was a YMC-Pack ODS-AQ (4.6 mm × 250 mm, 5 µm); the column temperature was 35 °C; the injection volume was 20 μL; mobile phase A was a phosphate buffer (0.01 mol / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 2.7 with phosphoric acid)-methanol system (volume ratio 95:5); mobile phase B was methanol; the flow rate of the mobile phase system was 1 mL / min; the elution method was gradient elution; the gradient elution degree was: 0~5 min: the volume percentage of mobile phase A was 100%; 5~15 min: the volume percentage of mobile phase A decreased uniformly from 100%. The volume percentage of mobile phase B increases uniformly from 0% to 6% within 15-45 min; the volume percentage of mobile phase A decreases uniformly from 94% to 16% within 15-45 min; the volume percentage of mobile phase B increases uniformly from 6% to 84% within 45-45.01 min; the volume percentage of mobile phase A increases uniformly from 16% to 100% within 45-45.01 min; the volume percentage of mobile phase B decreases uniformly from 84% to 0% within 45.01-60 min; the volume percentage of mobile phase A is 100% within 45.01 min; the ultraviolet detection wavelength is 265 nm.

[0023] Limits: If impurity peaks are present in the chromatogram of the test solution (excluding the trimethylphloroglucinol peak), the peak area shall be calculated using the external standard method. The content of 2,4,6-trihydroxybenzoic acid shall not exceed 0.5%, impurity D shall not exceed 0.2%, and other single unknown impurities shall be calculated using the external standard method based on the phloroglucinol peak area, and shall not exceed 0.2%. The total amount of impurities shall not exceed 1.0%. Chromatographic peaks smaller than 0.1 times (0.02%) the peak area of ​​the main peak in the reference solution shall be ignored.

[0024] Example 1 Analytical Methodology Validation - Specificity Blank solvent (solvent): Mobile phase A.

[0025] Blank excipient solution: Accurately measure 2 mL of blank excipient into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0026] Phloroglucinol Stock Solution 1: Accurately weigh approximately 13 mg of phloroglucinol reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of solvent to dissolve it, dilute to the mark with solvent, and shake well. (This can also be used as a positioning solution).

[0027] Impurity D Stock Solution 1: Accurately weigh approximately 10 mg of impurity D reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of solvent to dissolve it, dilute to the mark with solvent, and shake well. (This can also be used as a positioning solution).

[0028] 2,4,6-Trihydroxybenzoic acid stock solution 1: Accurately weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of solvent to dissolve it, dilute to the mark with solvent, and shake well. (This solution can also be used as a positioning solution).

[0029] Trimethylphloroglucinol Stock Solution 1: Accurately weigh approximately 10 mg of trimethylphloroglucinol reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well. (This solution can also be used as a positioning solution).

[0030] Reference solution: Accurately measure 2 mL of phloroglucinol stock solution 1, 2 mL of impurity D stock solution 1, 5 mL of 2,4,6,-trihydroxybenzoic acid stock solution 1, and 1 mL of trimethylphloroglucinol stock solution 1 into the same 100 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution.

[0031] Acid-base blank: Accurately measure 1 mL each of 1 mol / L hydrochloric acid solution and 1 mol / L sodium hydroxide solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0032] Oxidation blank: Accurately measure 1 mL of 30% hydrogen peroxide into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the blank.

[0033] Undamaged: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the final product.

[0034] Alkali destruction: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, add 1 mL of 1 mol / L sodium hydroxide solution, let stand at room temperature for 4 h, then add 1 mL of 1 mol / L hydrochloric acid solution, dilute to the mark with solvent, and shake well to obtain the solution.

[0035] Acid destruction: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, let stand at room temperature for 8 h, then add 1 mL of 1 mol / L sodium hydroxide solution, dilute to the mark with solvent, and shake well to obtain the solution.

[0036] Oxidative degradation: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, add 1 mL of 30% hydrogen peroxide, let stand at room temperature for 8 hours, then dilute to the mark with solvent and shake well to obtain the solution.

[0037] High-temperature destruction: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, place it in a 60℃ electric heating drying oven for 8 hours, remove it, let it cool, dilute it to the mark with solvent, and shake well to obtain the final product.

[0038] Light damage: Take 2 mL of phloroglucinol injection solution that has been placed in a light chamber (5500 lx ± 500 lx) for 10 days and put it into a 10 mL volumetric flask. Dilute to the mark with solvent and shake well to obtain the solution.

[0039] Degradation of blank excipients: Undamaged: Accurately measure 2 mL of blank excipient into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the final product.

[0040] Alkali destruction: Accurately measure 2 mL of blank excipient into a 10 mL volumetric flask, add 1 mL of 1 mol / L sodium hydroxide solution, let stand at room temperature for 8 h, then add 1 mL of 1 mol / L hydrochloric acid solution, dilute to the mark with solvent, and shake well to obtain the product.

[0041] Acid destruction: Accurately measure 2 mL of blank excipient injection into a 10 mL volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, let stand at room temperature for 8 h, then add 1 mL of 1 mol / L sodium hydroxide solution, dilute to the mark with solvent, and shake well to obtain the final product.

[0042] Oxidative degradation: Accurately measure 2 mL of blank excipient injection solution into a 10 mL volumetric flask, add 1 mL of 30% hydrogen peroxide, let stand at room temperature for 8 hours, then dilute to the mark with solvent and shake well to obtain the final product.

[0043] High-temperature degradation: Accurately measure 2 mL of blank excipient injection solution into a 10 mL volumetric flask, place it in a 60℃ electric heating drying oven for 8 hours, remove it, let it cool, dilute it to the mark with solvent, and shake well to obtain the final product.

[0044] Light damage: Take 2 mL of blank excipient that has been placed in a light exposure chamber (5500 lx ± 500 lx) for 10 days and put it into a 10 mL volumetric flask. Dilute to the mark with solvent and shake well to obtain the excipient.

[0045] Accurately measure 20 μL of each solution and inject it into the liquid chromatograph, then record the chromatogram.

[0046] The specificity test results are shown in Table 1, and the degradation test results are shown in Table 2. Table 1 Results of specificity test

[0047] Table 2 Degradation test results

[0048] The experimental results show that the blank solvent and blank excipients do not interfere with the detection of various impurities. In the reference solution, phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol eluted in sequence, and the theoretical plate number was greater than 5000 for all of them. Under all forced degradation conditions, impurity D was not detected in the phloroglucinol injection. Under alkaline degradation conditions, an unknown impurity with a percentage area of ​​1.73% was found. No large impurities were found under other conditions, and the excipients did not interfere under all degradation conditions. The peak purity of phloroglucinol was >980, and the material balance was within the range of 90% to 110%. The method can effectively detect related substances that may be present in phloroglucinol injection, and the method has good specificity.

[0049] Example 2 Analytical Methodology Validation – Solution Stability For each impurity stock solution 1, please refer to the "Specificity" test item in Example 1.

[0050] Reference solution: Accurately measure 2 mL of phloroglucinol stock solution 1, 2 mL of impurity D stock solution 1, 5 mL of 2,4,6,-trihydroxybenzoic acid stock solution 1, and 1 mL of trimethylphloroglucinol stock solution 1 into the same 100 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the reference solution.

[0051] Test solution: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the test solution.

[0052] Accurately measure 20 μL of each solution and inject it into the liquid chromatograph, then record the chromatogram.

[0053] The results of the stability test of the test sample are shown in Table 3, and the results of the stability test of the reference sample are shown in Table 4. Table 3 Results of stability test on the test sample

[0054] Table 4. Results of stability tests on reference standards

[0055] The experimental results show that when the test solution was left at room temperature for 13 hours and injected at different time points, the change rate of 2,4,6-trihydroxybenzoic acid decreased to below 95% after 6 hours compared to 0 hours, indicating that the test solution was relatively stable within 6 hours at room temperature. Therefore, its use should be controlled within 6 hours. When the reference solution was left at room temperature for 12 hours and injected at different time points, the change rate of 2,4,6-trihydroxybenzoic acid decreased to 95% after 5 hours compared to 0 hours, while the change rate of phloroglucinol increased to 106.44% after 4 hours compared to 0 hours. This indicates that the reference solution was relatively stable within 4 hours at room temperature. Therefore, its use should be controlled within 4 hours.

[0056] Example 3 Analytical Methodology Validation – Injection Precision Take the reference solution from the "solution stability" test item in Example 2, inject it into the liquid chromatograph, inject it 6 times consecutively, record the chromatogram, calculate the results according to the retention time and peak area, and the test results are shown in Table 5. Table 5. Injection precision results

[0057] The reference solution was injected six times consecutively, and the retention time RSD of each impurity peak was less than 1.0%; the peak area RSD was less than 2.0%, indicating good instrument injection precision.

[0058] Example 4 Analytical Method Validation – Limit of Quantitation, Limit of Detection Limit of Quantitation (LOQ) Solution: Prepare the solution by serially diluting the stock solutions of phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol until the signal-to-noise ratio is >10. Prepare six parallel solutions.

[0059] Detection limit solution: Take stock solutions of phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol and dilute them stepwise until the signal-to-noise ratio is >3.

[0060] Accurately measure 20 μL of each solution and inject it into the liquid chromatograph, then record the chromatogram.

[0061] The results of the limit of quantitation test are shown in Table 6, and the results of the limit of detection test are shown in Table 7. Table 6 Results of Limit of Quantitation Test

[0062] Table 7 Results of the detection limit test

[0063] The experimental data show that for six consecutive injections of the limit of quantitation (LOQ) solution, the S / N values ​​of all impurities were >10, and the RSD of the peak areas of all impurities in the six LQ solutions was less than 15.0%. Specifically, the LQ concentration of phloroglucinol was 0.0829 μg / mL, equivalent to 0.0041% of the test sample concentration and 2.07% of the limit concentration; the Detection Limit (LOD) concentration was 0.0414 μg / mL, equivalent to 0.0021% of the test sample concentration and 1.04% of the limit concentration; the LQ concentration of impurity D was 0.0231 μg / mL, equivalent to 0.0012% of the test sample concentration and 0.58% of the limit concentration; the LOD concentration was 0.0069 μg / mL, equivalent to 0.0003% of the test sample concentration and 0.17% of the limit concentration. The quantitation limit (LOQ) of 2,4,6-trihydroxybenzoic acid was 0.0240 μg / mL, equivalent to 0.0012% of the sample concentration and 0.24% of the limit concentration; the detection limit (LOD) was 0.0072 μg / mL, equivalent to 0.0004% of the sample concentration and 0.07% of the limit concentration. The LOQ of trimethylphloroglucinol was 0.0813 μg / mL, equivalent to 0.0041% of the sample concentration and 4.07% of the limit concentration; the LOD was 0.0407 μg / mL, equivalent to 0.0020% of the sample concentration and 2.03% of the limit concentration. The S / N values ​​of phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol in the detection limit solution were all >3. This method has high sensitivity.

[0064] Example 5 Analytical Methodology Validation – Linearity and Range Phloroglucinol stock solution: Weigh approximately 13 mg of phloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of mobile phase, sonicate to dissolve, dilute to the mark with mobile phase, and shake well to obtain the solution.

[0065] Impurity D stock solution: Weigh approximately 10 mg of impurity D reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with the mobile phase, and shake well to obtain the solution.

[0066] 2,4,6-Trihydroxybenzoic acid stock solution: Weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with the mobile phase, and shake well to obtain the solution.

[0067] Trimethylphloroglucinol stock solution: Weigh approximately 10 mg of trimethylphloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well to obtain the solution.

[0068] 200% Linearity: Accurately measure 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution, place them in the same 50 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0069] Linear 150%: Accurately measure 15 mL of linear 200% solution, place it in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0070] 100% Linearity: Accurately measure 5 mL of the 200% linearity solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0071] Linear 50%: Accurately measure 5 mL of linear 200% solution, place it in a 20 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0072] Linear 20%: Accurately measure 5 mL of linear 200% solution, place it in a 50 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0073] Limit of Quantification Solution: Same as the Limit of Quantification and Limit of Detection solution.

[0074] Accurately measure 20 μL of each linear solution and inject it into the liquid chromatograph, then record the chromatogram.

[0075] The linear results for phloroglucinol are shown in Table 8, and the line graphs are as follows: Figure 1 As shown; the linear results for impurity D are shown in Table 9, and the line graph is as follows. Figure 2 As shown in the figure; the linear results for 2,4,6-trihydroxybenzoic acid are shown in Table 10, and the line graph is shown in the figure. Figure 3 As shown; the linear results for trimethylphloroglucinol are shown in Table 11, and the line graph is as follows. Figure 4 As shown; Table 8 Linearity results for phloroglucinol

[0076] Table 9 Linearity results for impurity D

[0077] Table 10 Linearity results for 2,4,6-trihydroxybenzoic acid

[0078] Table 11 Linearity results for trimethylphloroglucinol

[0079] The data above show that the correlation coefficients (r) of phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol are all greater than 0.990 within the concentration range from the limit of quantitation to 200%; the Y-axis intercepts should all be within 25% of the 100% response value, and each component has good linearity within its respective concentration range.

[0080] Example 6 Analytical Methodology Validation - Accuracy Phloroglucinol stock solution: Weigh approximately 13 mg of phloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0081] Impurity D stock solution: Weigh approximately 10 mg of impurity D reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0082] 2,4,6-Trihydroxybenzoic acid stock solution: Weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0083] Trimethylphloroglucinol stock solution: Weigh approximately 10 mg of trimethylphloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well to obtain the solution.

[0084] Reference stock solution 2: Prepared in the same way as reference stock solution 1.

[0085] Reference Solution 1 (Transfer of Reference Solution 1 stock solution): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute to the mark with solvent.

[0086] Reference Solution 2 (Transfer of Reference Solution 2 stock solution): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute with solvent to the mark.

[0087] 50% accuracy solution: Accurately measure 5 mL of reference solution 2 and 2 mL of phloroglucinol injection into the same 10 mL volumetric flask, dilute to the mark with the solvent, and shake well. (Prepare 3 parallel portions) 100% Accuracy Stock Solution (using Reference Stock Solution 2): Accurately measure 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 50 mL volumetric flask. Dilute the solvent to the mark and shake well.

[0088] 100% Accuracy Solution: Accurately measure 5 mL of 100% accuracy stock solution and 2 mL of phloroglucinol injection into the same 10 mL volumetric flask. Dilute the solvent to the mark and mix well. (Prepare 6 parallel portions) 150% accuracy stock solution (using reference stock solution 2): Accurately measure 6 mL of phloroglucinol stock solution, 6 mL of impurity D stock solution, 15 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 3 mL of trimethylphloroglucinol stock solution into the same 50 mL volumetric flask, and dilute the solvent to the mark.

[0089] 150% accuracy solution: Accurately measure 5 mL of 150% accuracy stock solution and 2 mL of phloroglucinol injection into the same 10 mL volumetric flask, dilute the solvent to the mark, and shake well. (Prepare 3 parallel portions).

[0090] Accurately measure 20 μL of each solution, inject it into the liquid chromatograph, and record the chromatogram.

[0091] The results of the accuracy (recovery rate) test for impurity D are shown in Table 12; the results of the accuracy (recovery rate) test for 2,4,6-trihydroxybenzoic acid are shown in Table 13; and the results of the accuracy (recovery rate) test for trimethylphloroglucinol are shown in Table 14. Table 12 Results of Accuracy (Recovery) Test for Impurity D

[0092] Table 13. Accuracy (Recovery) Test Results of 2,4,6-Trihydroxybenzoic Acid

[0093] Table 14 Results of Trimethylphloroglucinol Accuracy (Recovery Rate) Test

[0094] The experimental results show that the recovery rate was within the range of 90% to 108% and the RSD was less than 5% at different concentrations, indicating that the method has good accuracy.

[0095] Example 7 Analytical Methodology Validation - Repeatability Reference stock solution 1: Phloroglucinol stock solution: Weigh approximately 13 mg of phloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0096] Impurity D stock solution: Weigh approximately 10 mg of impurity D reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0097] 2,4,6-Trihydroxybenzoic acid stock solution: Weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0098] Trimethylphloroglucinol stock solution: Weigh approximately 10 mg of trimethylphloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well to obtain the solution.

[0099] Reference stock solution 2: Prepared in the same way as reference stock solution 1.

[0100] Reference solution 1 (transfer of reference stock solution 1): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute to the mark with solvent.

[0101] Reference solution 2 (transfer of reference stock solution 2): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute to the mark with solvent.

[0102] Repeatable solution: Accurately measure 2 mL of phloroglucinol injection into a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare 6 parallel portions.

[0103] Spiked repeatability solution: See “100% accuracy solution” under “Accuracy”.

[0104] Accurately measure 20 μL of each solution, inject it into the liquid chromatograph, and record the chromatogram.

[0105] The results of the repeatability test are shown in Table 15; the results of the spiked repeatability test are shown in Table 16. Table 15 Repeatability Test Results

[0106] Table 16 Results of Spike Repeatability Test

[0107] The experimental results show that the content of each impurity in the 6 test sample solutions did not exceed the limit, and the RSD was less than 3%. The RSD% of the impurity content in the 6 spiked test samples was less than 3%, indicating that the method has good repeatability.

[0108] Example 8 Analytical Methodology Validation - Intermediate Precision Different personnel, at different times, using different instruments, measured the test solution six times each to examine the intermediate precision of the method. The results of the first group of tests are detailed in the repeatability test section.

[0109] Blank excipient: Accurately measure 2 mL of blank excipient into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the blank excipient.

[0110] Reference stock solution 1: Phloroglucinol stock solution: Weigh approximately 13 mg of phloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0111] Impurity D stock solution: Weigh approximately 10 mg of impurity D reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0112] 2,4,6-Trihydroxybenzoic acid stock solution: Weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0113] Trimethylphloroglucinol stock solution: Weigh approximately 10 mg of trimethylphloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well to obtain the solution.

[0114] Reference stock solution 2: Prepared in the same way as reference stock solution 1.

[0115] Reference solution 1 (transfer of reference stock solution 1): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute with solvent to the mark.

[0116] Reference solution 2 (transfer of reference stock solution 2): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute with solvent to the mark.

[0117] 200% stock solution (using reference stock solution 1): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 50 mL volumetric flask, and dilute with solvent to the mark.

[0118] Spiked test solution: Accurately measure 5 mL of 200% stock solution and 2 mL of phloroglucinol injection into a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare 6 parallel portions.

[0119] Accurately measure 20 μL of each solution, inject it into the liquid chromatograph, and record the chromatogram.

[0120] The results of the precision test are shown in Table 17. Table 17 Precision Test Results

[0121] The test results show that when different personnel used different instruments to test the samples at different times, and a total of 12 samples were tested, the RSD of each impurity was <6%, which meets the requirements and indicates that the intermediate precision of this method is good.

[0122] Example 9 Analytical Methodology Validation - Robustness Fine-tuning various chromatographic parameters (including column temperature, flow rate, mobile phase pH, and different numbered columns of the same type) was conducted to investigate the effect of changes in chromatographic conditions on the analytical results.

[0123] The range of chromatographic conditions is shown in Table 18. Table 18 Range of Chromatographic Conditions

[0124] Blank excipient: Accurately measure 2 mL of blank excipient into a 10 mL volumetric flask, dilute with solvent to the mark, and shake well to obtain the blank excipient.

[0125] Reference stock solution 1: Phloroglucinol stock solution: Weigh approximately 13 mg of phloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0126] Impurity D stock solution: Weigh approximately 10 mg of impurity D reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0127] 2,4,6-Trihydroxybenzoic acid stock solution: Weigh approximately 10 mg of 2,4,6-trihydroxybenzoic acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, dilute to the mark with solvent, and shake well to obtain the solution.

[0128] Trimethylphloroglucinol stock solution: Weigh approximately 10 mg of trimethylphloroglucinol reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark with solvent, and shake well to obtain the solution.

[0129] Reference stock solution 2: Prepared in the same way as reference stock solution 1.

[0130] Reference solution 1 (transfer of reference stock solution 1): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute with solvent to the mark.

[0131] Reference Solution 2 (Transfer of Reference Stock Solution 2): Transfer 2 mL of phloroglucinol stock solution, 2 mL of impurity D stock solution, 5 mL of 2,4,6-trihydroxybenzoic acid stock solution, and 1 mL of trimethylphloroglucinol stock solution into the same 100 mL volumetric flask, and dilute to the mark with solvent.

[0132] 200% stock solution (using reference stock solution 1): Transfer 2 mL of phloroglucinol, 2 mL of impurity D, 5 mL of 2,4,6-trihydroxybenzoic acid, and 1 mL of trimethylphloroglucinol into the same 50 mL volumetric flask, and dilute with solvent to the mark.

[0133] Spiked test solution: Accurately measure 5 mL of 200% stock solution and 2 mL of phloroglucinol injection into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0134] Accurately measure 20 μL of each solution and inject it into the liquid chromatograph, then record the chromatogram.

[0135] System suitability requirements: In the chromatogram of the reference solution, phloroglucinol, impurity D, 2,4,6-trihydroxybenzoic acid, and trimethylphloroglucinol should elute in that order, and the theoretical plate number calculated based on the phloroglucinol peak should be no less than 5000.

[0136] The results of the system suitability test are shown in Table 19; the results of the durability test are shown in Table 20. Table 19 System Suitability Test Results

[0137] Table 20 Durability Test Results

[0138] Under all changing conditions, the blank solvent and blank excipient did not interfere with the detection of known impurities. Compared with the original conditions, the RSD of each impurity was <6%, indicating that the method has good robustness.

[0139] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting related substances in phloroglucinol injection, characterized in that, Includes the following steps: High performance liquid chromatography was used to detect the test sample and obtain the detection results of related substances in the test sample. The detection conditions for the high-performance liquid chromatography include: the mobile phase system comprises mobile phase A and mobile phase B; mobile phase A is a phosphate buffer-methanol system; and mobile phase B is methanol. The chromatographic column for the high-performance liquid chromatography is C18. 18 column; The flow rate of the mobile phase system is 1 mL / min; The elution method is gradient elution; The gradient elution procedure is as follows: 0~5min: The volume percentage of mobile phase A is 100%; 5~15min: The volume percentage of mobile phase A decreases uniformly from 100% to 94%; the volume percentage of mobile phase B increases uniformly from 0% to 6%. 15~45min: The volume percentage of mobile phase A decreases uniformly from 94% to 16%; the volume percentage of mobile phase B increases uniformly from 6% to 84%. 45~45.01 min: The volume percentage of mobile phase A increases uniformly from 16% to 100%; the volume percentage of mobile phase B decreases uniformly from 84% to 0%. 45.01~60min: The volume percentage of mobile phase A is 100%; The detector is an ultraviolet detector with a detection wavelength of 265 nm; The related substances include at least one of phloroglucinol, impurity D, trimethylphloroglucinol, and 2,4,6-trihydroxybenzoic acid.

2. The detection method according to claim 1, characterized in that, The phosphate buffer solution in the mobile phase A is an aqueous solution of potassium dihydrogen phosphate.

3. The detection method according to claim 2, characterized in that, The concentration of the potassium dihydrogen phosphate aqueous solution is 0.01 mol / L; the pH value of the potassium dihydrogen phosphate aqueous solution is 2.

7.

4. The detection method according to claim 1, characterized in that, The volume ratio of phosphate buffer to methanol in the mobile phase A is 95:

5.

5. The detection method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography was 35°C.

6. The detection method according to claim 1, characterized in that, The injection volume for the high-performance liquid chromatography was 20 μL.

7. The detection method according to claim 1, characterized in that, The detection includes qualitative detection or quantitative detection.

8. The detection method according to claim 7, characterized in that, The quantitative detection includes: using high performance liquid chromatography to detect the sample to be tested, and obtaining a chromatogram of the sample to be tested; The chromatogram of the sample to be tested is compared with a predetermined standard curve, and the content of related substances in the sample to be tested is calculated using the external standard method.

Images