A method for detecting impurities in diphenhydramine hydrochloride bulk drug by HPLC

Abstract

The application discloses a HPLC detection method for an impurity of raw phenylamine hydrochloride, and adopts high performance liquid chromatography to detect, and the chromatographic conditions are as follows: a chromatographic column is filled with octadecylsilane bonded silica gel with a particle size of 4-5 microns, the column length is 250 mm, and the column inner diameter is 3-4.6 mm; a mobile phase is a mixture of phosphate buffer and acetonitrile, and acetonitrile is used as the mobile phase B; gradient elution is adopted; the injection amount is 10-20 microliters; the flow rate is 1.0-1.4 ml / min; the detection wavelength is 210-230 nm; and the column temperature is 28-32 DEG C. The detection method is simple in operation, high in sensitivity, good in specificity, high in accuracy, and can accurately control the content of specific impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G) and non-specific impurities in the phenylamine hydrochloride, thereby effectively controlling the quality of the phenylamine hydrochloride and realizing the quality control of the raw phenylamine hydrochloride.

Description

Technical Field

[0001] This application belongs to the field of active pharmaceutical ingredient (API) analysis technology, specifically relating to an HPLC method for detecting impurities in diphenhydramine hydrochloride API. Background Technology

[0002] Diphenhydramine hydrochloride, chemically known as N,N-dimethyl-2-(diphenylmethoxy)ethylamine hydrochloride, is a derivative of ethanolamine and can be synthesized artificially. Diphenhydramine hydrochloride is a white or off-white crystalline powder, readily soluble in water, and is an antihistamine; it has antihistamine H1 receptor activity, a strong inhibitory effect on the central nervous system, and also anticholinergic effects, relieving bronchospasm. It is also suitable for allergic diseases of the skin and mucous membranes, such as urticaria, hay fever, and allergic rhinitis, and can be used to prevent motion sickness, nausea, and vomiting.

[0003] Analyzing impurities in active pharmaceutical ingredients (APIs) not only ensures drug safety and quality but also guides process optimization and supports drug development. The structural information of diphenhydramine hydrochloride and its known impurities is shown in the table below:

[0004]

[0005] Although existing technical literature has developed methods for the determination of diphenhydramine hydrochloride impurities, these methods can only analyze or detect a very limited number of impurities, and cannot conduct a comprehensive and effective study of the known impurities mentioned above. Summary of the Invention

[0006] In view of this, the primary objective of this application is to provide an HPLC detection method for impurities in diphenhydramine hydrochloride raw material. The detection method of this application can effectively detect the content of diphenhydramine hydrochloride raw material and seven known impurities, thereby achieving effective research on impurities in diphenhydramine hydrochloride raw material.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] This application provides an HPLC method for detecting impurities in diphenhydramine hydrochloride raw material, wherein the HPLC conditions are as follows:

[0009] Chromatographic column: The packing material is octadecylsilane-bonded silica gel with a particle size of 4-5 μm; the column length is 250 mm and the inner diameter is 3-4.6 mm.

[0010] Mobile phase: Mobile phase A is a mixture of phosphate buffer and acetonitrile, and mobile phase B is acetonitrile; gradient elution; the selection of mobile phases in this application has the advantages of a longer storage period, which is not conducive to bacterial growth; at the same time, gradient operation can avoid the generation of a large number of fine bubbles, resulting in better online mixing of the mobile phase and better peak elution.

[0011] Injection volume: 10–20 μl; flow rate: 1.0–1.4 ml / min; column temperature: 28–32 °C; detection is performed using an ultraviolet detector with a detection wavelength of 210–230 nm.

[0012] In a further embodiment, the chromatographic column parameters are as follows: the packing material is octadecylsilane-bonded silica gel with a particle size of 5 μm; the column length is 250 mm and the column inner diameter is 4.6 mm.

[0013] In a further embodiment, the volume ratio of phosphate buffer to acetonitrile in the mobile phase A is (60-80):(20-40);

[0014] Preferably, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is (68-72):(28-32);

[0015] Preferably, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is 70:30.

[0016] In a further embodiment, the concentration of the phosphate buffer solution is 0.02–0.04 mol / L, and the pH is 2.8–3.2.

[0017] Preferably, the concentration of the phosphate buffer solution is 0.025–0.035 mol / L;

[0018] Preferably, the concentration of the phosphate buffer solution is 0.03 mol / L and the pH is 3.0.

[0019] In a further embodiment, the phosphate is potassium dihydrogen phosphate.

[0020] In a further embodiment, the gradient elution procedure is as follows:

[0021]

[0022]

[0023] Further proposed protocols include a flow rate of 1.2 ml / min, a column temperature of 30°C, and detection using a UV detector at a wavelength of 218–222 nm.

[0024] Preferably, the detection wavelength is 220 nm.

[0025] In a further embodiment, the HPLC detection method also includes a solution preparation step, comprising:

[0026] Test solution: Accurately weigh the active pharmaceutical ingredient diphenhydramine hydrochloride, add solvent to dissolve and dilute to a quantitative amount, shake well, and prepare a solution with a concentration of 0.7 mg / ml;

[0027] Control solution: Accurately measure the test solution, dilute it with solvent to prepare a solution with a concentration of 0.7 μg / ml;

[0028] Impurity stock solution: Accurately weigh impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, dissolve them in solvent, dilute and measure them, shake well, and prepare a solution with a concentration of 0.3 mg / ml for each impurity.

[0029] Impurity localization solution: Accurately measure the impurity stock solution, dilute it quantitatively with solvent, and prepare a solution with a concentration of 1.5 μg / ml for each impurity;

[0030] System suitability solution: Accurately weigh the reference standard diphenhydramine hydrochloride, and simultaneously accurately measure 0.1 ml of each impurity stock solution. Dilute with solvent to prepare a solution with a diphenhydramine hydrochloride concentration of 0.7 mg / ml and each impurity concentration of 1.5 μg / ml.

[0031] The solvent is prepared by mixing phosphate buffer and acetonitrile in a volume ratio of 50:50, wherein the phosphate buffer is the same as that in mobile phase A.

[0032] In a further embodiment, the formula for calculating the content of the impurities is as follows:

[0033] The formula for calculating impurity content is:

[0034]

[0035] Among them, A 单 For the impurity area, A 对照 The peak area of ​​the principal component in the 0.1% control solution is given, and F is the correction factor.

[0036] Preferably, the correction factors for known impurities A, B, C, D, E, F, and G are 1.0, 0.6, 1.0, 0.6, 1.1, 1.0, and 1.0, respectively; the correction factor for the remaining unknown impurities is calculated as 1.0.

[0037] In a further embodiment, the resolution of each impurity peak in the results of the detection method should be no less than 1.0, and the resolution between the main component peak and the preceding and following peaks should be no less than 1.5.

[0038] The beneficial effects of this application are:

[0039] This application discloses an HPLC method for detecting impurities in diphenhydramine hydrochloride raw material. This method can accurately detect diphenhydramine hydrochloride and seven known impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G). The detection method in this application is highly specific, accurate, and has good separation, providing accurate and reliable results. It allows for comprehensive analysis and research of impurities in diphenhydramine hydrochloride raw material, effectively achieving quality control of diphenhydramine hydrochloride raw material and showing promising application prospects. Attached Figure Description

[0040] Figure 1 This is a chromatogram of the system suitability solution in Example 1.

[0041] Figure 2 This is the chromatogram of the blank solvent in Example 1.

[0042] Figure 3 This is a superimposed comparison of the chromatograms of the solutions in Example 2.

[0043] Figure 4 The chromatogram is of the 0.6% limit spiked test solution in Example 6.

[0044] Figure 5 This is a comparison overlay of chromatograms of the system suitability solution after changing chromatographic conditions in the durability test of Example 7. Detailed Implementation

[0045] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0047] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0048] The reagent sample information used in the following examples is as follows:

[0049]

[0050] Example 1: HPLC System Suitability Test

[0051] 1. Solution preparation

[0052] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0053] Impurity stock solution: Accurately weigh approximately 3 mg each of reference impurities A, B, C, D, E, F, and G, and place them in separate 10 ml volumetric flasks. Add 2 ml of acetonitrile to dissolve them, then dilute to the mark with solvent and shake well.

[0054] System suitability solution: Accurately weigh approximately 14 mg of diphenhydramine hydrochloride reference standard, and simultaneously accurately measure 0.1 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution, impurity F stock solution, and impurity G stock solution, place them in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0055] 2. HPLC chromatographic conditions

[0056] High performance liquid chromatograph: Shimadzu LC20A.

[0057] Column: Agilent TC-C18, 4.6×250mm, 5μm;

[0058] Mobile phase: Mobile phase A was prepared by mixing buffer salt and acetonitrile at a volume ratio of 70:30. The buffer salt was 0.03 mmol / L potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid); Mobile phase B was acetonitrile; Gradient elution was performed, and the elution program was as follows:

[0059]

[0060] The flow rate was 1.2 ml / min, the column temperature was 30℃, the injection volume was 10 μl, and the detection wavelength of the UV detector was 220 nm.

[0061] 3. Experimental Procedure

[0062] After the system stabilizes, inject the blank solvent and system suitability solution into the high-performance liquid chromatograph separately and record the chromatograms. Figure 1 and Figure 2 The test results are shown in Table 1:

[0063] Table 1. Results of System Applicability Test

[0064]

[0065]

[0066] pass Figure 1 and Figure 2 As can be seen, in the chromatogram of the system suitability solution, the elution order is as follows: impurity A, diphenhydramine hydrochloride, impurity C, impurity F, impurity G, impurity D, impurity E, and impurity B, with each peak well separated; combined with Figure 2 It can be seen that the blank solvent does not interfere with the detection of various impurities. Combined with the results in Table 1, it can be concluded that the resolution of each impurity peak is greater than 1.0, and the resolution between the main component peak and its adjacent peaks is greater than 1.5.

[0067] Example 2 Specificity Experiment

[0068] 1. Solution preparation

[0069] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0070] Impurity stock solution: Accurately weigh approximately 3 mg each of reference impurities A, B, C, D, E, F, and G, and place them in separate 10 ml volumetric flasks. Add 2 ml of acetonitrile to dissolve them, then dilute to the mark with solvent and shake well.

[0071] System suitability solution: Accurately weigh approximately 14 mg of diphenhydramine hydrochloride reference standard, and simultaneously accurately measure 0.1 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution, impurity F stock solution, and impurity G stock solution, place them in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0072] Impurity localization solution: Accurately measure 0.1 ml of each impurity stock solution and place them in separate 20 ml volumetric flasks. Dilute to the mark with solvent and shake well.

[0073] Test solution: Accurately weigh approximately 14 mg of the active pharmaceutical ingredient diphenhydramine hydrochloride, place it in a 20 ml volumetric flask, dissolve it in solvent, dilute to the mark, and shake well.

[0074] Control solution: Accurately measure 1 ml of the test solution and place it in a 1000 ml volumetric flask. Dilute to the mark with solvent.

[0075] 2. HPLC chromatographic conditions

[0076] Same as Example 1.

[0077] 3. Experimental Procedure

[0078] Inject the reference solution into the high-performance liquid chromatograph (HPLC), adjust the detection sensitivity so that the peak height of the main component is 15% of full scale; accurately measure 10 μl each of the test solution and the reference solution, inject them separately into the HPLC, and record the chromatograms. In the chromatogram, the peak area of ​​impurity A in the test solution should not be greater than the peak area of ​​the reference solution (0.5%); the peak area of ​​impurity B multiplied by the correction factor should not be greater than 0.3 times (0.15%) of the peak area of ​​the reference solution; the peak area of ​​impurity C should not be greater than 0.3 times (0.15%) of the peak area of ​​the reference solution; the peak area of ​​impurity D multiplied by the correction factor should not be greater than 0.6 times (0.3%) of the peak area of ​​the reference solution; the peak area of ​​impurity E should not be greater than 0.6 times (0.3%) of the peak area of ​​the reference solution; the peak area of ​​impurity F should not be greater than 10 μl of the peak area of ​​the reference solution. The peak area of ​​impurity G must not exceed 0.6 times (0.3%) the peak area of ​​the control solution; the peak area of ​​other individual impurities must not exceed 0.2 times (0.10%) the peak area of ​​the control solution; the total impurity content must not exceed 1.0%; the correction factors for impurities A, B, C, D, E, F, and G are 1.0, 0.6, 1.0, 0.6, 1.1, 1.0, and 1.0, respectively; the correction factor for other unknown impurities is calculated as 1.0; record the chromatogram ( Figure 3 The test results are shown in Table 2:

[0079] Table 2 Results of Specificity Experiment

[0080]

[0081] pass Figure 3 As can be seen from the results in Table 2, the detection method of this application controls 7 known impurities, and all of them achieve effective separation. The separation degree between diphenhydramine hydrochloride and adjacent peaks is greater than 1.5, the separation degree between each known impurity peak is greater than 1.5, and the impurity peaks do not interfere with each other.

[0082] Example 3: Limit of Quantitation and Limit of Detection Experiment

[0083] 1. Solution preparation

[0084] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0085] Accurately weigh appropriate amounts of the reference standard diphenhydramine hydrochloride and impurities A, B, C, D, E, F, and G, and quantitatively dilute them with solvent to prepare solutions with concentrations of 0.2279 μg / ml, 0.0764 μg / ml, 0.1054 μg / ml, 0.1486 μg / ml, 0.0628 μg / ml, 0.1227 μg / ml, 0.1486 μg / ml, and 0.1010 μg / ml, respectively. Use an injection volume with a signal-to-noise ratio of approximately 10 as the limit of quantitation (LOQ). Take appropriate amounts of the reference standard diphenhydramine hydrochloride and impurities A, B, C, D, E, F, and G, accurately weigh them, and quantitatively dilute them with solvent to prepare solutions with concentrations of 0.0684 μg / ml, 0.0229 μg / ml, 0.0316 μg / ml, 0.0446 μg / ml, 0.0188 μg / ml, 0.0368 μg / ml, 0.0446 μg / ml, and 0.0303 μg / ml, respectively. Use an injection volume with a signal-to-noise ratio of approximately 3 as the limit of detection (LOD).

[0086] 2. Chromatographic conditions

[0087] Same as Example 1.

[0088] 3. Experimental Procedure

[0089] After the system stabilized, the above solutions were injected into the high-performance liquid chromatograph, and the detection and analysis results are shown in Table 3:

[0090] Table 3. Experimental results for limit of quantitation and limit of detection.

[0091]

[0092] The test results in Table 3 demonstrate that the detection method of Example 1 can meet the quantitative requirements for impurities A, B, C, D, E, F, G, and diphenhydramine hydrochloride at a concentration of at least 2.3 ng. The detection method provided in this application has good sensitivity and can effectively detect related substances at low concentrations, eliminating potential medication risks and ensuring the effectiveness and safety of drug quality.

[0093] Example 4: Linearity and Correction Factor Experiment

[0094] 1. Solution preparation

[0095] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0096] Accurately weigh appropriate amounts of the reference standard diphenhydramine hydrochloride and impurities A, B, C, D, E, F, and G, dissolve and dilute them in solvent, and prepare linear solutions of different concentrations, as detailed in Table 4.

[0097] Table 4. Linear solution concentrations (μg / ml) of each substance

[0098] Serial Number Linear 1 Linear 2 Linear 3 Linear 4 Linear 5 Linear 6 Linear 7 diphenhydramine hydrochloride 0.2110 0.7034 1.0551 2.1102 3.5170 7.0340 14.0679 Impurity A 0.0691 0.6912 1.0368 2.0736 3.4560 6.9119 13.8239 Impurity B 0.0970 0.6926 1.0388 2.0777 3.4628 6.9257 13.8513 Impurity C 0.1392 0.6961 1.0441 2.0883 3.4805 6.9609 13.9219 Impurity D 0.0552 0.6896 1.0344 2.0689 3.4481 6.8963 13.7925 Impurity E 0.1101 0.6884 1.0325 2.0651 3.4418 6.8835 13.7670 impurity F 0.1376 0.6881 1.0322 2.0644 3.4407 6.8813 13.7626 Impurity G 0.0864 0.6173 0.9260 1.8519 3.0866 6.1731 12.3462

[0099] 2. Chromatographic conditions

[0100] Same as Example 1.

[0101] 3. Experimental Procedure

[0102] After the system stabilized, the linear solutions prepared in Table 4 were injected into the high-performance liquid chromatograph, and the experimental results were recorded. The linearity results are shown in Table 5.

[0103] Table 5. Results of the linear experiment

[0104] compound Linear equations Correlation coefficient r Concentration range Correction factor diphenhydramine hydrochloride y = 18676.6787x - 399.2189 0.9999 0.2110~14.0679μg / ml / Impurity A y = 19490.1088x + 69.1176 1.0000 0.0691~13.8239μg / ml 1.0 Impurity B y = 31452.9197x + 286.1812 1.0000 0.0970~13.8513μg / ml 0.6 Impurity C y = 19027.6172x - 394.0606 1.0000 0.1392~13.9219μg / ml 1.0 Impurity D y = 29597.2484x - 67.7138 1.0000 0.0552~13.7925μg / ml 0.6 Impurity E y = 18153.9536x - 106.1592 1.0000 0.1101~13.7670μg / ml 1.1 impurity F y = 19293.0709x - 239.8727 1.0000 0.1376~13.7625μg / ml 1.0 Impurity G y = 17847.4304x - 138.6298 1.0000 0.0864~12.3462μg / ml 1.0

[0105] The results in Table 5 show that, using the detection method provided in this application, impurities A, B, C, D, E, F, G, and diphenhydramine hydrochloride exhibit good linearity within the limit of quantitation (LOQ) to approximately 13 μg / ml, with a wide linear range. This indicates that the detection method of this application has good linearity, can quantitatively detect impurities, and has high accuracy. The correction factors are all between 0.2 and 5.0; therefore, the content of each impurity can be calculated using the principal component self-comparison method with correction factors for related substances testing.

[0106] Specifically, the formula for calculating impurity content is:

[0107]

[0108] Among them, A 单 For the impurity area, A 对照 The peak area of ​​the main component in the 0.1% control solution is given, and F is the correction factor.

[0109] Example 5 Precision Experiment

[0110] 1. Solution preparation

[0111] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0112] Impurity stock solution: Accurately weigh approximately 7 mg each of reference impurities A, B, C, D, E, F, and G, and place them in separate 50 ml volumetric flasks. Add 2 ml of acetonitrile to dissolve them, dilute to the mark with solvent, and shake well.

[0113] Mixed impurity stock solution: Accurately measure 2.0 ml of each impurity stock solution and place it in a 20 ml volumetric flask. Dilute the solvent to the mark and shake well.

[0114] 0.2% limit spiked test solution: Accurately weigh about 14 mg of the active pharmaceutical ingredient diphenhydramine hydrochloride and place it in a 20 ml volumetric flask. At the same time, accurately measure 2 ml of the mixed impurities stock solution, add solvent to dissolve and dilute to the mark, and shake well.

[0115] Control solution: Accurately measure 1 ml of 0.2% limit standard test solution, place it in a 200 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0116] 2. Chromatographic conditions

[0117] Same as Example 1.

[0118] 3. Experimental Procedure

[0119] In this embodiment, different analysts prepared six parallel 0.2% limit-spike test solutions on different dates, following the method described in this embodiment, and measured them on a different instrument to examine the repeatability and precision of the detection method of this application. The results are shown in Table 6:

[0120] Table 6 Results of Repeatability and Precision Experiments

[0121]

[0122]

[0123] The results in Table 6 show that the RSD of each impurity in the 6 spiked test solutions was no greater than 5%, indicating good repeatability of the method; and the RSD of each impurity in the 12 spiked test solutions was less than 7.5%, indicating good intermediate precision of the method.

[0124] Example 6 Accuracy Experiment

[0125] 1. Solution preparation

[0126] Solvent: The buffer salt and acetonitrile are mixed in a volume ratio of 50:50; wherein the buffer salt is a 0.03 mol / L potassium dihydrogen phosphate solution (the pH is adjusted to 3.0 with phosphoric acid).

[0127] Impurity stock solution: Accurately weigh approximately 7 mg each of reference impurities A, B, C, D, E, F, and G, and place them in separate 50 ml volumetric flasks. Add 2 ml of acetonitrile to dissolve them, dilute to the mark with solvent, and shake well.

[0128] Mixed impurity stock solution: Accurately measure 5.0 ml of each impurity stock solution and place it in a 50 ml volumetric flask. Dilute the solvent to the mark and shake well.

[0129] Test solution: Accurately weigh about 14 mg of the active pharmaceutical ingredient diphenhydramine hydrochloride, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0130] Spiked test solution: Accurately weigh approximately 14 mg of the active pharmaceutical ingredient diphenhydramine hydrochloride, in 12 portions, and place them in separate 20 ml volumetric flasks. Dissolve the solutions in solvent, and divide each 3 portions into 4 groups. Add 1 ml, 1.5 ml, 3 ml, and 6 ml of mixed impurity stock solution to each group, and dilute to the mark with solvent. Shake well.

[0131] Reference solution: Accurately measure 1.5 ml of the mixed impurity stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0132] 2. Chromatographic conditions

[0133] Same as Example 1

[0134] 3. Experimental Procedure

[0135] After the system stabilizes, inject the prepared solutions into the high-performance liquid chromatograph and record the experimental results. The detection and analysis results are shown in Table 7.

[0136] Table 7. Accuracy Experiment Results

[0137] name Spicing situation Average recovery rate RSD Impurity A 0.1％、0.15％、0.3％、0.6％ 100.8 1.6(n＝12) Impurity B 0.1％、0.15％、0.3％、0.6％ 98.3 1.2(n＝12) Impurity C 0.1％、0.15％、0.3％、0.6％ 100.7 1.8(n＝12) Impurity D 0.1％、0.15％、0.3％、0.6％ 101.6 1.7(n＝12) Impurity E 0.1％、0.15％、0.3％、0.6％ 98.9 1.0(n＝12) impurity F 0.1％、0.15％、0.3％、0.6％ 100.8 1.2(n＝12) Impurity G 0.1％、0.15％、0.3％、0.6％ 100.9 1.2(n＝12)

[0138] The results in Table 7 show that the average recoveries of impurities A, B, C, D, E, F, and G at limit concentrations of 0.1%, 0.15%, 0.3%, and 0.6% were all within the range of 90%–108%, and the RSDs of the recoveries for all 12 samples were less than 10%, indicating that the detection method in this application has good recovery and high accuracy. Furthermore, Figure 4 The chromatogram shown is a chromatogram with a 0.6% spike concentration. It can be seen that the peak of impurity A is well separated from the peak of diphenhydramine hydrochloride. Impurities B, C, D, E, F, and G are also effectively separated from their adjacent peaks, indicating that the detection method of this application can effectively control the quality of diphenhydramine hydrochloride raw material.

[0139] Example 7 Durability Test

[0140] 1. Solution preparation

[0141] The preparation of the test solution and the system suitability solution is the same as in Example 1.

[0142] 2. Chromatographic conditions

[0143] Based on the chromatographic conditions of Example 1, different chromatographic conditions were varied to examine the robustness of the detection method of this application, as detailed in Table 8 (all varied conditions in the table are single variables, and other conditions not specified are the same as in Example 1):

[0144] Table 8 Chromatographic conditions for durability testing

[0145]

[0146]

[0147] For the chromatogram of the robustness test of the system suitability solution, please refer to [the provided text]. Figure 5 The test and analysis results are shown in Tables 9 and 10:

[0148] Table 9. Results of the robustness test - system suitability separation degree

[0149]

[0150] Table 10 Durability Test Results - Test Specimens

[0151]

[0152]

[0153] pass Figure 5 The results in Tables 9 and 10 show that, after fine-tuning the chromatographic conditions—flow rate (±0.2 ml / min), wavelength (220±2 nm), column temperature (30±2 ℃), pH of mobile phase A buffer salt (3.0±0.2), concentration of mobile phase A buffer salt (0.03±0.005 mol / L), proportion of buffer salt in mobile phase A (70%±2%), and column shifting—compared with the conditions in Example 1, the system suitability solution chromatograms showed that the resolution between impurity peaks and between the main peak and adjacent peaks was greater than 1.5. The content of each impurity and the total impurity content showed no significant difference compared to the standard conditions (all less than 0.05%), indicating that the method has good robustness.

[0154] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. An HPLC method for detecting impurities in diphenhydramine hydrochloride raw material, characterized in that, The high-performance liquid chromatography (HPLC) conditions are as follows: Chromatographic column: The packing material is octadecylsilane-bonded silica gel with a particle size of 4~5μm; the column length is 250mm and the inner diameter is 3~4.6mm; Mobile phase: Mobile phase A is a mixture of phosphate buffer and acetonitrile at a volume ratio of (60~80):(20~40), wherein the concentration of the phosphate buffer is 0.02~0.04 mol / L, the pH is 2.8~3.2, the phosphate is potassium dihydrogen phosphate, and mobile phase B is acetonitrile; gradient elution is performed, and the gradient elution program is as follows: ； Injection volume: 10-20 μl; Flow rate: 1.0-1.4 ml / min; Column temperature: 28-32℃; Detection wavelength: UV 210-230 nm; The known impurity analyzed by the HPLC detection method is impurity AG, and the molecular structure of impurity AG is shown below: 。 2. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, The column parameters are as follows: the packing material is octadecylsilane-bonded silica gel with a particle size of 5 μm; the column length is 250 mm and the column inner diameter is 4.6 mm.

3. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, In the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is (68~72):(28~32).

4. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, In the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is 70:

30.

5. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, The concentration of the phosphate buffer solution is 0.025~0.035 mol / L.

6. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 5, characterized in that, The phosphate buffer solution has a concentration of 0.03 mol / L and a pH of 3.

0.

7. The HPLC method for detecting impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, Flow rate 1.2 ml / min; column temperature 30℃; detection wavelength UV 218~222 nm.

8. The HPLC method for detecting impurities in diphenhydramine hydrochloride raw material as described in claim 7, characterized in that, The detection wavelength is 220nm.

9. The HPLC method for detecting impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, The HPLC detection method further includes a solution preparation step, including: Test solution: Accurately weigh the active pharmaceutical ingredient diphenhydramine hydrochloride, add solvent to dissolve and dilute to a quantitative amount, shake well, and prepare a solution with a concentration of 0.7 mg / ml; Control solution: Accurately measure the test solution, dilute it with solvent to prepare a solution with a concentration of 0.7 μg / ml; Impurity stock solution: Accurately weigh impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G respectively, dissolve them in solvent, dilute and measure them, shake well, and prepare a solution with a concentration of 0.3 mg / ml for each impurity. Impurity localization solution: Accurately measure the impurity stock solution, dilute it quantitatively with solvent, and prepare a solution with a concentration of 1.5 μg / ml for each impurity; System suitability solution: Accurately weigh 14 mg of diphenhydramine hydrochloride reference standard and place it in a 20 ml volumetric flask. At the same time, accurately measure 0.1 ml of each impurity stock solution, dilute with solvent, and prepare a solution with a diphenhydramine hydrochloride concentration of 0.7 mg / ml and each impurity concentration of 1.5 μg / ml. The solvent is prepared by mixing phosphate buffer and acetonitrile in a volume ratio of 50:50, and the phosphate buffer is the same as that in mobile phase A.

10. The HPLC method for detecting impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, The formula for calculating the content of the impurities is as follows: The formula for calculating impurity content is: Known impurity content = ; Other impurity content = Among them, A 单 For the impurity area, A 对照 The peak area of ​​the principal component in the 0.1% control solution is given, and F is the correction factor.

11. The HPLC method for detecting impurities in diphenhydramine hydrochloride raw material as described in claim 10, characterized in that, The correction factors for impurities A, B, C, D, E, F, and G are known to be 1.0, 0.6, 1.0, 0.6, 1.1, 1.0, and 1.0, respectively; the correction factor for the remaining unknown impurities is calculated as 1.

0.

12. The HPLC detection method for impurities in diphenhydramine hydrochloride raw material as described in claim 1, characterized in that, In the results of the detection method, the resolution of each impurity peak should be no less than 1.0, and the resolution between the main component peak and the preceding and following peaks should be no less than 1.5.

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