Method for determining related substances of resorcinol

Abstract

The invention discloses a method for determining related substances of resorcinol, and belongs to the technical field of medicines. The method comprises the following steps: firstly, preparing a reference solution and a test solution for later use; a chromatographic column with octadecyl bonded silica gel as a filler is adopted, a 0.2% dipotassium phosphate buffer solution is taken as a mobile phase A, acetonitrile-methanol is taken as a mobile phase B, and gradient elution is carried out; respectively sucking the contrast solution and the test solution, injecting the contrast solution and the test solution into a liquid chromatograph, and recording chromatograms. The high performance liquid chromatography can rapidly, effectively, accurately and reliably separate and detect the impurities of resorcinol, including phenol, resorcinol, p-phenylenediamine, catechol and hydroquinone, and by controlling the quality of resorcinol, the quality of the resorcinol bulk drug can be improved, and the purity of the resorcinol is improved. And the product quality of the finished raw material medicine prepared from the raw materials can be improved, so that the medication safety of patients is further improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for determining resorcinol-related substances. Background Technology

[0002] Resorcinol, also known as 1,3-benzenediol, has the chemical formula C6H6O2 and CAS number 108-46-3. It is widely used in the preparation of rubber adhesives, wood adhesives, and ultraviolet absorbers. In the pharmaceutical field, it is often used to prepare pharmaceutical intermediates, and it can also be used as a disinfectant and preservative in the treatment of skin diseases.

[0003] The synthesis process of resorcinol uses m-phenylenediamine as a raw material, which is hydrolyzed with sulfuric acid, followed by extraction and purification. Impurities in the process may include phenol, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and hydroquinone. The quality standard for resorcinol in the 2025 edition of the Chinese Pharmacopoeia, Part II, only includes phenol and o-phenylenediamine as specific impurities in the related substances test. Hydroquinone, m-phenylenediamine, and p-phenylenediamine are not included as specific impurities in the research. In combination with the production process, adding specific impurities and developing new related substances methods are of great significance for the production and storage of active pharmaceutical ingredients and preparations.

[0004] On October 27, 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization published a preliminary list of carcinogens, which included hydroquinone, m-phenylenediamine, and p-phenylenediamine in Group 3. Therefore, there is an urgent need to develop a rapid, effective, accurate, and reliable method for detecting the levels of various impurities in resorcinol. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for determining resorcinol-related substances. This method can quickly, effectively, accurately and reliably detect various impurities in resorcinol, which is beneficial to improving the product quality of raw materials for pharmaceutical products made from this material and improving the safety of medication for patients.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for determining resorcinol-related substances, using liquid chromatography, includes the following steps:

[0008] 1) Prepare the reference solution and the test solution for later use;

[0009] 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed.

[0010] 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms;

[0011] The resorcinol-related substance is hydroquinone.

[0012] A method for determining resorcinol-related substances, using liquid chromatography, includes the following steps:

[0013] 1) Prepare the reference solution and the test solution for later use;

[0014] 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed.

[0015] 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms;

[0016] The resorcinol-related substances are hydroquinone and catechol.

[0017] A method for determining resorcinol-related substances, using liquid chromatography, includes the following steps:

[0018] 1) Prepare the reference solution and the test solution for later use;

[0019] 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed.

[0020] 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms;

[0021] The related substances of resorcinol are phenol, m-phenylenediamine, p-phenylenediamine, hydroquinone, and catechol.

[0022] Further, in step 1), the test solution is prepared as follows: dissolve resorcinol in 0.2% potassium dihydrogen phosphate buffer and dilute to a final volume to prepare a solution containing approximately 1 mg per 1 mL; the reference solution is prepared by accurately transferring an appropriate amount of the test solution and quantitatively diluting it with 0.2% potassium dihydrogen phosphate buffer to prepare a solution containing approximately 5 μg per 1 mL, which serves as the self-control solution.

[0023] Further, in step 1), the method for preparing the reference solution is as follows: accurately transfer an appropriate amount of the test solution and quantitatively dilute it with 0.2% potassium dihydrogen phosphate buffer to prepare a solution containing approximately 5 μg per 1 mL, which serves as the self-control solution.

[0024] Furthermore, in step 2), the chromatographic column is an Agilent ZORBAX SB-Aq with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0025] Further, in step 2), the column temperature is 25~35℃; the flow rate is 0.8~1.2mL / min; the detection wavelength is 275~285nm; the injection volume is 10µL; the solvent is 0.2% potassium dihydrogen phosphate buffer, and the pH is 3.9-4.1.

[0026] Further, in step 2), the gradient elution conditions are as follows: 0-15 min, the volume percentage of mobile phase A is 100%-96%, and the volume percentage of mobile phase B is 0-4%; 15-30 min, the volume percentage of mobile phase A is 96%-80%, and the volume percentage of mobile phase B is 4%-20%; 30 min-50 min, the volume percentage of mobile phase A is 80%, and the volume percentage of mobile phase B is 20%.

[0027] Further, in step 3), the self-comparison method is used to determine that the content of phenol, m-phenylenediamine, p-phenylenediamine, hydroquinone, and resorcinol is not greater than 0.1%, the content of catechol is not greater than 0.089%, other individual impurities are not greater than 0.1%, and the total impurities are not greater than 0.3%.

[0028] Furthermore, the method for determining resorcinol-related substances employs liquid chromatography and includes the following steps:

[0029] 1) Prepare the reference solution and the test solution for later use;

[0030] 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl-bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, performing gradient elution; the column was an Agilent ZORBAX. SB-Aq, specifications: 4.6mm × 250mm, 5μm; column temperature: 25~35℃; flow rate: 0.8~1.2mL / min; detection wavelength: 275~285nm; injection volume: 10µL; solvent: 0.2% potassium dihydrogen phosphate buffer, pH: 3.9-4.1; gradient elution conditions: 0~15min, mobile phase A volume percentage: 100%-96%, mobile phase B volume percentage: 0-4%; 15~30min, mobile phase A volume percentage: 96%-80%, mobile phase B volume percentage: 4%-20%; 30min~50min, mobile phase A volume percentage: 80%, mobile phase B volume percentage: 20%.

[0031] 3) Take the control solution and the test solution separately, inject them into the liquid chromatograph, and record the chromatograms.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) Compared with the prior art, the present invention uses high performance liquid chromatography to determine resorcinol-related substances. The main component and known impurities such as phenol, m-phenylenediamine, p-phenylenediamine, catechol, and hydroquinone, as well as other unknown impurities, have sufficient separation. Moreover, the blank solvent peak does not interfere with the determination of the main peak and each impurity, and the separation effect is more comprehensive and excellent.

[0034] (2) Under various destructive conditions such as acid, alkali, oxidation, high temperature, pyrolysis, and light, the degradation products generated by the test sample can be completely separated from the main peak. The degradation products do not interfere with the determination of known impurities. The reduction in the amount of main peak destruction is basically consistent with the decrease in its content. The purity of the main peak is not less than 990. Material balance is maintained, and the specificity of related substances detection is good. At the same time, when there are slight changes in chromatographic conditions such as column temperature, flow rate, mobile phase pH and chromatographic column, the impurity separation and inspection results are not affected, and the durability is good.

[0035] (3) The present invention can quickly, effectively, accurately and reliably detect a variety of related substances of resorcinol, fill the detection blind spots of existing standards, meet the quality control requirements of resorcinol raw materials and preparations, thereby improving the quality of finished products made from this raw material and ensuring the safety of patients' medication. Attached Figure Description

[0036] Figure 1 The chromatogram is the result of testing under the chromatographic conditions described in this application.

[0037] Figure 2 This application does not damage the spectrum;

[0038] Figure 3 This is the photodestruction spectrum of this application;

[0039] Figure 4 This is a high-temperature damage diagram of this application;

[0040] Figure 5 This is the pyrolysis damage diagram of this application;

[0041] Figure 6 This is the acid damage spectrum of this application;

[0042] Figure 7 This is the alkali damage spectrum of this application;

[0043] Figure 8 This is a graph of oxidation damage in this application;

[0044] Figure 9 This is a standard curve of resorcinol for this application;

[0045] Figure 10 This is a phenol standard curve diagram for this application;

[0046] Figure 11 This is a standard curve diagram of m-phenylenediamine in this application;

[0047] Figure 12 This is the standard curve diagram of p-phenylenediamine in this application;

[0048] Figure 13 This is a standard curve of catechol in this application;

[0049] Figure 14 This is the hydroquinone standard curve for this application. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] Example 1

[0052] A method for determining resorcinol-related substances includes the following steps:

[0053] 1) The instrument and samples were selected from: Agilent 1260 high performance liquid chromatograph with VWD detector; information on each raw material is shown in Table 1-2 below.

[0054] Table 1. Reference Standard Information

[0055]

[0056] Table 2 Sample Information

[0057]

[0058] 2) Setting up the liquid chromatography conditions: The column was an Agilent ZORBAX SB-Aq, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm; mobile phase A was 0.2% potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid), and mobile phase B was acetonitrile-methanol (50:50); the column temperature was 30℃; the flow rate was 1.0 mL / min; the detection wavelength was 280 nm; the injection volume was 10 µL; the gradient elution conditions were as follows: 0–15 min, mobile phase A volume percentage was 100%–96%, mobile phase B volume percentage was 0–4%; 15–30 min, mobile phase A volume percentage was 96%–80%, mobile phase B volume percentage was 4%–20%; 30–50 min, mobile phase A volume percentage was 80%, mobile phase B volume percentage was 20%.

[0059] 3) Preparation of sample solution:

[0060] Test solution: Dissolve resorcinol in 0.2% potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid) and dilute to a final volume to prepare a solution containing approximately 1 mg per mL;

[0061] Reference solution: Accurately transfer an appropriate amount of the test solution and quantitatively dilute it with 0.2% potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid) to prepare a solution containing approximately 5 μg per mL, which serves as the self-control solution;

[0062] Mixed solution: Accurately weigh an appropriate amount of the test sample into a suitable volumetric flask, then add appropriate amounts of each impurity stock solution to the same volumetric flask. Dissolve and dilute with a solvent to prepare a mixed solution containing approximately 1 mg of resorcinol, 1 μg each of phenol, m-phenylenediamine, p-phenylenediamine, and hydroquinone, and 0.89 μg of catechol per 1 mL. This solution is used as the system suitability solution. The solvent in the reference solution and the mixed solution is 0.2% potassium dihydrogen phosphate buffer (adjusted to pH 4.0 with phosphate).

[0063] 4) Sample detection method: Accurately pipette 10µL of the test solution and the reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms. The results are as follows: Figure 1 As shown.

[0064] Depend on Figure 1 It can be seen that under these chromatographic conditions, the known and unknown impurities of the main component can be well separated, and there are no small peaks interfering in the blank solvent, so this method is applicable.

[0065] Example 2

[0066] The detection method of Example 1 was validated in several aspects, including system suitability, destructive testing, limit of quantitation, limit of detection, linearity, precision, accuracy, and solution stability. These are described in detail below.

[0067] 1. System suitability test

[0068] Blank solution: 0.2% potassium dihydrogen phosphate buffer.

[0069] Impurity positioning solutions: Accurately weigh appropriate amounts of resorcinol reference standard and phenol, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and hydroquinone. Dissolve and dilute with solvent to prepare stock solutions containing approximately 1 mg of each impurity per mL. Accurately weigh an appropriate amount of catechol, dissolve and dilute with 0.2% potassium dihydrogen phosphate buffer to prepare impurity stock solutions containing approximately 0.89 mg of each impurity per mL. Transfer appropriate amounts of each impurity stock solution and add 0.2% potassium dihydrogen phosphate buffer to prepare positioning solutions containing 1 μg each of resorcinol, phenol, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and hydroquinone, and 0.89 μg of catechol per mL.

[0070] System suitability solution: Accurately weigh an appropriate amount of the test sample into a suitable volumetric flask, then add an appropriate amount of each impurity stock solution into the same volumetric flask, and dissolve and dilute with 0.2% potassium dihydrogen phosphate buffer to prepare a mixed solution containing approximately 1 mg of resorcinol, 1 μg each of phenol, m-phenylenediamine, p-phenylenediamine, and hydroquinone, and 0.89 μg of catechol per 1 mL.

[0071] 10 µL each of the blank solution, the localization solutions for each impurity, and the system suitability solution were injected into the high-performance liquid chromatograph (HPLC). The detection wavelength was 280 nm, and the chromatograms were recorded to examine the separation of each component. The results are shown in Table 3.

[0072] Table 3 Impurity Location and Separation

[0073]

[0074] As shown in Table 3, at a detection wavelength of 280 nm, the peak elution order of the mixed solution is p-phenylenediamine, m-phenylenediamine, hydroquinone, catechol, resorcinol, phenol. Resorcinol, all known impurities and other impurities can be effectively separated, and the solvent peak does not interfere with the determination of the main peak and each impurity.

[0075] 2. Destructive testing

[0076] Undamaged solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and use as the solution before destruction.

[0077] Acid destruction solution: Take about 20 mg of this product and place it in a 20 mL volumetric flask. Add 1 mL of 1 mol / L hydrochloric acid solution and destroy at room temperature for 4 hours. Add 1 mL of 1 mol / L sodium hydroxide solution to neutralize. Dissolve and dilute to the mark with solvent and shake well to obtain the acid destruction solution.

[0078] Alkali destruction solution: Take about 20 mg of this product and place it in a 20 mL volumetric flask. Add 1 mL of 1 mol / L sodium hydroxide solution and destroy at room temperature for 4 hours. Add 1 mL of 1 mol / L hydrochloric acid solution to neutralize. Dissolve and dilute to the mark with solvent and shake well to obtain the alkali destruction solution.

[0079] Oxidative destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add 1 mL of 3% hydrogen peroxide, incubate in a 60 °C water bath for 4 hours, dissolve and dilute to the mark with a solvent, and shake well to obtain the oxidative destruction solution.

[0080] Thermal decomposition solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add an appropriate amount of solvent to dissolve it, heat at 100 °C for 5 hours to decompose it, cool to room temperature, then dissolve and dilute with solvent to the mark, shake well, and use it as the thermal decomposition solution.

[0081] High-temperature destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, heat at 100 °C for 5 hours to destroy it, cool to room temperature, dissolve and dilute with solvent to the mark, shake well, and use as the high-temperature destruction solution.

[0082] Photodegradation solution: Take the undegradable solution and place it under 254nm, 365nm and visible light for 8 hours to degrade it, and use it as the photodegradation solution.

[0083] Acid-base blank: Take 1 mL of 1 mol / L hydrochloric acid and place it in a 20 mL volumetric flask. Add 1 mL of 1 mol / L sodium hydroxide to neutralize it. Dilute to the mark with solvent and shake well to obtain the acid-base blank solution.

[0084] Oxidation blank: Take 1 mL of 3% hydrogen peroxide and place it in a 20 mL volumetric flask. Incubate in a 60 °C water bath for 4 hours. Dilute to the mark with solvent and shake well to obtain the oxidation blank solution.

[0085] Inject 10 µL of each of the above test solutions into the chromatograph, record the chromatograms, and analyze the changes in each impurity. For the test solutions under various destructive conditions, a diode array was used to determine peak purity or spectral similarity. The results are shown in Table 4 and... Figure 2-8 .

[0086] Table 4. Results of Material Balance Study

[0087]

[0088] From Table 4 and Figure 2-8 It can be seen that under various destructive conditions, the degradation products generated by the test sample can be effectively separated from the main peak, the degradation products do not interfere with the determination of known impurities, the reduction in the amount of main peak destruction should be basically consistent with the decrease in its content, the purity of the main peak is not less than 990, and the material balance is maintained.

[0089] 3. Limit of Quantitation and Limit of Detection Tests

[0090] Accurately weigh appropriate amounts of each impurity reference standard of resorcinol and the resorcinol reference standard, dissolve and dilute them in solvent to prepare a mixed stock solution containing 1 μg each of resorcinol, phenol, m-phenylenediamine, p-phenylenediamine, and hydroquinone, and 0.89 μg of catechol per 1 mL. The limits of quantitation (S / N ≥ 10) and the limits of detection (S / N ≥ 3) were determined using the stepwise dilution method. The results are shown in Table 5.

[0091] Table 5 Limit of Quantification and Limit of Detection

[0092]

[0093] As shown in Table 5, under the chromatographic conditions, the limits of quantitation and detection of each impurity meet the requirements.

[0094] 4. Linear relationship

[0095] Accurately weigh appropriate amounts of resorcinol impurity reference standards and resorcinol reference standard, dissolve and dilute them in solvent to prepare a mixed solution containing 10 μg each of phenol, m-phenylenediamine, p-phenylenediamine, and hydroquinone, 8.9 μg of catechol, and 10 μg of resorcinol per 1 mL, as the linear stock solution. Dilute the above solution with solvents according to the table below to prepare linear solutions of various concentrations. Prepare linear solutions according to Table 6.

[0096] Table 6. Preparation of Linear Solutions

[0097]

[0098] Accurately measure 10 µl of the above solution and inject it into the high-performance liquid chromatograph. Record the chromatogram, measure the peak area, and perform linear regression with peak area A as the ordinate and concentration C as the abscissa. The results are shown in Tables 7-12 below. Figure 9-14 .

[0099] Table 7. Results of linearity test for phenol

[0100]

[0101] Table 8. Linearity results for m-phenylenediamine

[0102]

[0103] Table 9. Linearity results for p-phenylenediamine

[0104]

[0105] Table 10 Linearity results for catechol

[0106]

[0107] Table 11 Linearity results for hydroquinone

[0108]

[0109] Table 12 Linearity results for resorcinol

[0110]

[0111] The results of the impurity correction factor calculation are shown in Table 13:

[0112] Calculation formula:

[0113] In the formula: K 主成分 : Slope of the resorcinol standard curve;

[0114] K 杂质 : Slope of the impurity standard curve.

[0115] Table 13 Known Impurity Correction Factors

[0116]

[0117] As shown in Table 13, the relative correction factors for all known impurities are in the range of 0.2 to 5, and the content of each impurity can be calculated by the self-comparison method with correction factors.

[0118] 5. Sample injection precision

[0119] Take appropriate amounts of each impurity and resorcinol reference standard, dilute with solvent to prepare a mixed solution containing 1 μg each of resorcinol, phenol, m-phenylenediamine, p-phenylenediamine, and hydroquinone, and 0.89 μg of catechol per 1 mL, as the injection precision solution. Inject 10 µL into the liquid chromatograph, perform 6 consecutive injections, and record the peak areas. The results are shown in Table 14.

[0120] Table 14 Injection Precision Results

[0121]

[0122] As shown in Table 14, with 6 consecutive injections, the peak area RSD% is ≤2%, indicating good injection precision.

[0123] 6. Solution stability test

[0124] Solution stability tests were performed on the reference solution and the test solution, respectively.

[0125] Stability of reference solution

[0126] Samples for injection precision were injected at 0, 3, 7, 11, 16, 19, 29, 34, 37, 40, and 44 hours to examine the stability of each impurity solution. Solutions containing 5 μg resorcinol per 1 mL were injected at 0, 8, 11, 13, 16, 19, 23, and 24 hours to examine solution stability. The results are shown in Tables 15 and 16.

[0127] Table 15 Stability results of impurity reference solutions

[0128]

[0129] Table 16 Stability results of self-control solutions

[0130]

[0131] Table 15-16 shows the stability of the impurity reference solution and the self-reference solution in the solvent within 24 hours (RSD≤2.0%).

[0132] Stability of the test solution

[0133] Weigh an appropriate amount of resorcinol test sample, dissolve and dilute it with a solvent to prepare a solution containing approximately 1 mg per mL, and use this solution as the test sample. Inject the sample at 0, 2, 8, 15, 19, 24, 27, 38, and 49 hours. Calculate the impurity content using the area normalization method and examine the impurity stability. The results are shown in Table 17.

[0134] Table 17 Stability results of the test solution

[0135]

[0136] Table 17 shows the stability of the test sample in the solvent within 49 hours (absolute deviation from 0 hours ≤ 10% of the limit).

[0137] 7. Repeatability test

[0138] Take the resorcinol sample and perform the related substance determination according to the proposed method. Repeat the determination 6 times. The results are shown in Table 18.

[0139] Table 18 Repeatability Results

[0140]

[0141] As shown in Table 18, the absolute deviation of each impurity content in the test results of the 6 repeatable samples is ≤10.0% of the limit, indicating good repeatability.

[0142] 8. Accuracy test (recovery rate)

[0143] Take each impurity, dissolve and dilute it with solvent to prepare a mixed solution containing 10 μg each of phenol, m-phenylenediamine, p-phenylenediamine, hydroquinone, and catechol, and 8.9 μg of catechol per 1 mL, as the recovery stock solution.

[0144] Accurately pipette the above mixed stock solution and dilute it according to the table below to prepare 20%, 50%, 100%, and 200% solutions, with three portions prepared for each concentration. Prepare the recovery solution according to Table 17.

[0145] Inject 10 µL of each of the above samples, record the peak area of ​​each known impurity, calculate the recovery rate and RSD of each impurity, and examine the solubility recovery rate of each impurity in a solution with a 20% recovery rate. The results are shown in Tables 19-24.

[0146] Table 19 Recovery Rate Solution Preparation

[0147]

[0148] Table 20 Results of Phenol Accuracy Measurement

[0149]

[0150] Table 21 Results of accuracy determination of m-phenylenediamine

[0151]

[0152] Table 22 Results of accuracy determination for p-phenylenediamine

[0153]

[0154] Table 23 Results of Catechol Accuracy Measurement

[0155]

[0156] Table 24 Results of Hydroquinone Accuracy Measurement

[0157]

[0158] As shown in Tables 19-24, the recovery rates of each impurity concentration at 20% are all in the range of 85% to 110%, and the recovery rates of impurity concentrations at 50% to 200% are all in the range of 90% to 108%. The average recovery rate is in the range of 90% to 108%, and the RSD is less than or equal to 10.0%, indicating good recovery.

[0159] 9. Intermediate precision

[0160] Related substances tests were performed on the same batch of samples by different operators at different times using different instruments, following repeatability testing methods. The results are shown in Table 25.

[0161] Table 25 Results of intermediate precision test

[0162]

[0163] As shown in Table 25, the absolute deviation of the measurement results from the average value is ≤20.0% of the limit, indicating good intermediate precision.

[0164] 10. Durability test

[0165] The robustness of the resorcinol related substances method was verified mainly by using different flow rates, column temperatures, pH values, and chromatographic columns.

[0166] The experiment was conducted according to the conditions in Table 26, and the results are shown in Tables 27 and 28.

[0167] Table 26 Durability Conditions

[0168]

[0169] Table 27 Summary of Durability Separation Results

[0170]

[0171] Table 28 Summary of Durability Separation Results

[0172]

[0173] As shown in Tables 27 and 28, under all durability conditions, the system suitability and resolution of all impurities in the solution all meet the requirements. The absolute deviation of the results from the mean value when the column temperature, flow rate, pH and different chromatographic columns are slightly changed is ≤20.0% of the limit, indicating good durability.

[0174] 11. Testing Implementation Cases

[0175] Multiple batches of raw material samples were tested. Water was used as the negative control, and the positive control was added at the corresponding limit concentration for each target impurity, with an addition amount of 0.1% by mass. The results are shown in Table 29 below.

[0176] Table 29 Summary of test results from multiple batches

[0177]

[0178] As shown in Table 29, the content of each target impurity in the positive control sample was close to or reached the set limit, and the total impurity exceeded the standard, verifying the effectiveness of the detection method. Among the 5 batches of resorcinol raw material samples, the three carcinogens, namely p-phenylenediamine, m-phenylenediamine, and phenol, were not detected. Only 2 batches were found to contain small amounts of hydroquinone (0.060% and 0.062%), and 1 batch was found to contain trace amounts of catechol (0.002%). The content of other individual impurities ranged from 0.007% to 0.067%, and the total impurity content ranged from 0.026% to 0.117%. All indicators met the set limit requirements, indicating that the related substances control of the tested resorcinol raw material was stable and of qualified quality, which can meet the quality control requirements of subsequent raw material and preparation production and ensure the safety of patients' medication.

[0179] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining resorcinol-related substances, characterized in that: The liquid chromatography method includes the following steps: 1) Prepare the reference solution and the test solution for later use; 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed. 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms; The resorcinol-related substance is hydroquinone.

2. A method for determining resorcinol-related substances, characterized in that: The liquid chromatography method includes the following steps: 1) Prepare the reference solution and the test solution for later use; 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed. 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms; The resorcinol-related substances are hydroquinone and catechol.

3. A method for determining resorcinol-related substances, characterized in that: The liquid chromatography method includes the following steps: 1) Prepare the reference solution and the test solution for later use; 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, and gradient elution was performed. 3) Pipette the control solution and the test solution separately into the liquid chromatograph and record the chromatograms; The related substances of resorcinol are phenol, m-phenylenediamine, p-phenylenediamine, hydroquinone, and catechol.

4. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 1), the test solution is prepared as follows: dissolve resorcinol in 0.2% potassium dihydrogen phosphate buffer and dilute to a final volume to prepare a solution containing approximately 1 mg per mL; the reference solution is prepared by accurately transferring an appropriate amount of the test solution and quantitatively diluting it with 0.2% potassium dihydrogen phosphate buffer to prepare a solution containing approximately 5 μg per mL, which serves as the self-control solution.

5. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 1), the preparation method of the reference solution is as follows: accurately transfer an appropriate amount of the test solution and quantitatively dilute it with 0.2% potassium dihydrogen phosphate buffer to prepare a solution containing about 5 μg per 1 mL, which is used as the self-control solution.

6. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 2), the chromatographic column is an Agilent ZORBAX SB-Aq with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

7. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 2), the column temperature is 25~35℃; the flow rate is 0.8~1.2mL / min; the detection wavelength is 275~285nm; the injection volume is 10µL; the solvent is 0.2% potassium dihydrogen phosphate buffer, and the pH is 3.9-4.

1.

8. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 2), the gradient elution conditions are as follows: 0-15 min, the volume percentage of mobile phase A is 100%-96% and the volume percentage of mobile phase B is 0-4%; 15-30 min, the volume percentage of mobile phase A is 96%-80% and the volume percentage of mobile phase B is 4%-20%; 30 min-50 min, the volume percentage of mobile phase A is 80% and the volume percentage of mobile phase B is 20%.

9. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: In step 3), the self-comparison method was used to determine that the contents of phenol, m-phenylenediamine, p-phenylenediamine, hydroquinone, and resorcinol were not greater than 0.1%, the contents of catechol were not greater than 0.089%, the contents of other individual impurities were not greater than 0.1%, and the total impurities were not greater than 0.3%.

10. The method for determining resorcinol-related substances according to claim 1, 2, or 3, characterized in that: The liquid chromatography method includes the following steps: 1) Prepare the reference solution and the test solution for later use; 2) Setting up high-performance liquid chromatography (HPLC) detection conditions: A column packed with octadecyl-bonded silica gel was used, with 0.2% dipotassium hydrogen phosphate buffer as mobile phase A and acetonitrile-methanol as mobile phase B, performing gradient elution; the column was an Agilent ZORBAX. SB-Aq, specifications: 4.6mm × 250mm, 5μm; column temperature: 25~35℃; flow rate: 0.8~1.2mL / min; detection wavelength: 275~285nm; injection volume: 10µL; solvent: 0.2% potassium dihydrogen phosphate buffer, pH: 3.9-4.1; gradient elution conditions: 0~15min, mobile phase A volume percentage: 100%-96%, mobile phase B volume percentage: 0-4%; 15~30min, mobile phase A volume percentage: 96%-80%, mobile phase B volume percentage: 4%-20%; 30min~50min, mobile phase A volume percentage: 80%, mobile phase B volume percentage: 20%. 3) Take the control solution and the test solution separately, inject them into the liquid chromatograph, and record the chromatograms.

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