HPLC (High Performance Liquid Chromatography) detection method for impurities in diphenhydramine hydrochloride bulk drug

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) detection method for impurities in a diphenhydramine hydrochloride bulk drug, which adopts a high performance liquid chromatography method for detection, and the chromatographic conditions are as follows: a chromatographic column takes octadecylsilane chemically bonded silica with the particle size of 4-5 [mu] m as a filler; the column length is 250 mm, and the column inner diameter is 3-4.6 mm; mobile phases: a mobile phase A is a mixture of a phosphate buffer solution and acetonitrile, and a mobile phase B is acetonitrile; performing gradient elution; the sample size is 10-20 [mu] l; the flow velocity is 1.0 to 1.4 ml / min; the detection wavelength is 210 to 230 nm; the column temperature is 28-32 DEG C; the detection method is easy to operate, high in sensitivity, good in specificity and high in accuracy, and the content of specific impurities (the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G) and non-specific impurities in diphenhydramine hydrochloride can be accurately controlled, so that the quality of diphenhydramine hydrochloride is effectively controlled, and quality control over the diphenhydramine hydrochloride raw material is achieved.

Description

Technical Field

[0001] The present application belongs to the technical field of raw material drug analysis, and specifically relates to an HPLC detection method for impurities of diphenhydramine hydrochloride raw material drug. Background Art

[0002] Diphenhydramine hydrochloride, whose chemical name is 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, easily soluble in water, and is an antihistamine; it has an antihistamine H1 receptor effect and has a strong inhibitory effect on the central nervous system; it also has an anticholinergic effect and can relieve bronchial smooth muscle spasms; it is also suitable for allergic diseases of the skin and mucous membranes, such as urticaria, hay fever, allergic rhinitis, etc., and can also be used to prevent motion sickness, nausea, and vomiting.

[0003] Analysis of impurities in raw materials can not only ensure drug safety and quality, but also guide process optimization and provide support for drug research and development. Currently, the structural information of diphenhydramine hydrochloride and its known impurities is shown in the table below:

[0004]

[0005] Although relevant methods have been developed in the existing technical literature for the determination of impurities in diphenhydramine hydrochloride, the types of impurities analyzed or detected by these methods are very limited and it is impossible to conduct a comprehensive and effective study on the above-mentioned known impurities. Summary of the invention

[0006] In view of this, the primary purpose of the present application is to provide an HPLC detection method for impurities in diphenhydramine hydrochloride raw material. The detection method of the present application can effectively detect the contents of diphenhydramine hydrochloride raw material and 7 known impurities, thereby realizing effective research on impurities in diphenhydramine hydrochloride raw material.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] The present application provides an HPLC detection method for impurities of diphenhydramine hydrochloride raw material medicine, and its high performance liquid chromatography conditions are as follows:

[0009] Chromatographic column: The filler is octadecylsilane bonded silica gel with a particle size of 4 to 5 μm; the column length is 250 mm and the inner diameter is 3 to 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 choice of mobile phase in the present application, on the one hand, has a longer storage period, which is not conducive to the growth of bacteria; at the same time, the generation of a large number of fine bubbles can be avoided by gradient operation, the online mixing effect of the mobile phase is better, and the peak effect of the chromatographic peak is good.

[0011] Injection volume: 10-20 μl; flow rate: 1.0-1.4 ml / min; column temperature: 28-32°C; detection by ultraviolet detector, detection wavelength: 210-230 nm.

[0012] In a further embodiment, the chromatographic column parameters are as follows: the filler 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, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile 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 is 0.02-0.04 mol / L, and the pH is 2.8-3.2;

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

[0018] Preferably, the concentration of the phosphate buffer 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] .

[0024] Further scheme, flow rate 1.2ml / min; column temperature 30℃; detection by ultraviolet detector, detection wavelength 218~222nm;

[0025] Preferably, the detection wavelength is 220 nm.

[0026] In a further embodiment, the HPLC detection method further comprises the step of preparing a solution, comprising:

[0027] Test solution: accurately weigh the raw material diphenhydramine hydrochloride, add solvent to dissolve and dilute quantitatively, shake well to make a solution with a concentration of 0.7 mg / ml;

[0028] Control solution: Accurately measure the test solution and dilute it with solvent to make a solution with a concentration of 0.7 μg / ml;

[0029] Impurity stock solution: Accurately weigh impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, respectively, dissolve them in solvents, dilute them quantitatively, and shake well to prepare solutions with a concentration of 0.3 mg / ml for each impurity;

[0030] Impurity localization solution: accurately measure the impurity stock solution, dilute it with a solvent to make a solution with a concentration of 1.5 μg / ml for each impurity;

[0031] System suitability solution: Accurately weigh the reference substance diphenhydramine hydrochloride, and at the same time accurately measure 0.1 ml of each impurity stock solution, add solvent to dilute and quantify, to prepare a solution with a diphenhydramine hydrochloride concentration of 0.7 mg / ml and each impurity concentration of 1.5 μg / ml;

[0032] 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.

[0033] In a further embodiment, the impurity content is calculated as follows:

[0034] The calculation formula for impurity content is:

[0035]

[0036] Among them, A 单 is the impurity area, A 对照 is the peak area of ​​the main component of the 0.1% control solution, and F is the correction factor;

[0037] Preferably, the correction factors of 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 factors of the remaining unknown impurities are calculated as 1.0;

[0038] In a further embodiment, in the results of the detection method, the separation degree of each impurity peak should be no less than 1.0, and the separation degree of the main component peak and the preceding and following peaks should be no less than 1.5.

[0039] Beneficial effects of this application:

[0040] The present application discloses an HPLC detection method for impurities of diphenhydramine hydrochloride raw material medicine, and the present application can accurately detect diphenhydramine hydrochloride and 7 known impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G). The detection method in the present application has strong specificity, precision, good separation, accurate and reliable detection results, can comprehensively analyze and study the impurities of diphenhydramine hydrochloride raw material medicine, can well realize the quality control of diphenhydramine hydrochloride raw material medicine, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the chromatogram of the system suitability solution in Example 1.

[0042] Figure 2 It is the chromatogram of the blank solvent in Example 1.

[0043] Figure 3 The chromatograms of the solutions in Example 2 are compared and overlaid.

[0044] Figure 4 This is the chromatogram of the 0.6% limit spiked test solution in Example 6.

[0045] Figure 5 This is a comparative overlay of the system suitability solution chromatograms after changing the chromatographic conditions in the durability experiment in Example 7. DETAILED DESCRIPTION

[0046] The following will clearly and completely describe the implementation methods of the present application. The technical solutions in the implementation methods described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the implementation methods of the present application to obtain other implementation methods without creative work, and these implementation methods are also within the scope of protection of the present application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0048] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0049] The reagent sample information used in the following examples is as follows:

[0050]

[0051] Example 1 HPLC system suitability experiment

[0052] 1. Solution preparation

[0053] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0054] Impurity stock solution: Accurately weigh about 3 mg of each of the reference impurities A, B, C, D, E, F and G, place them in different 10 ml volumetric flasks, add 2 ml of acetonitrile to dissolve, then add solvent to dilute to the scale and shake well.

[0055] System suitability solution: Accurately weigh about 14 mg of the reference substance diphenhydramine hydrochloride. At the same time, accurately measure 0.1 ml each of the 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, add solvent to dilute to the scale, and shake well.

[0056] 2. HPLC chromatographic conditions

[0057] High performance liquid chromatograph: Shimadzu LC20A.

[0058] Chromatographic column: Agilent TC-C18, 4.6×250 mm, 5 μm;

[0059] Mobile phase: Mobile phase A is a mixture of buffer salt and acetonitrile in a volume ratio of 70:30, the buffer salt is 0.03mmol / L potassium dihydrogen phosphate (phosphoric acid is adjusted to pH 3.0); mobile phase B is acetonitrile; gradient elution, the elution procedure is as follows:

[0060]

[0061] The flow rate was 1.2 ml / min, the column temperature was 30 °C, the injection volume was 10 μl, and the detection wavelength of the UV detector was 220 nm.

[0062] 3. Experimental operation

[0063] After the system is stable, inject the blank solvent and system suitability solution into the HPLC, and record the chromatogram ( Figure 1 and Figure 2 ), the test results are shown in Table 1:

[0064] Table 1 System applicability experimental results

[0065]

[0066]

[0067] pass Figure 1 and Figure 2 It can be seen that in the chromatogram of the system suitability solution, the peak order is impurity A, diphenhydramine hydrochloride, impurity C, impurity F, impurity G, impurity D, impurity E and impurity B, and each peak is well separated; combined with Figure 2 It can be seen that the blank solvent has no interference with the detection of each impurity. Combining the results in Table 1, it can be concluded that the separation of each impurity peak is greater than 1.0, and the separation of the main component peak and the adjacent peaks is greater than 1.5.

[0068] Example 2 Specificity Experiment

[0069] 1. Solution preparation

[0070] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0071] Impurity stock solution: Accurately weigh about 3 mg of each of the reference impurities A, B, C, D, E, F and G, place them in different 10 ml volumetric flasks, add 2 ml of acetonitrile to dissolve, then add solvent to dilute to the scale and shake well.

[0072] System suitability solution: Accurately weigh about 14 mg of the reference substance diphenhydramine hydrochloride. At the same time, accurately measure 0.1 ml each of the 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, add solvent to dilute to the scale, and shake well.

[0073] Impurity localization solution: Accurately measure 0.1 ml of each impurity stock solution and place it in different 20 ml volumetric bottles, add solvent to dilute to the scale and shake well.

[0074] Test solution: Accurately weigh about 14 mg of the raw material diphenhydramine hydrochloride, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0075] Control solution: Accurately measure 1 ml of the test solution, place it in a 1000 ml volumetric flask, and dilute to the mark with solvent.

[0076] 2. HPLC chromatographic conditions

[0077] Same as Example 1.

[0078] 3. Experimental operation

[0079] Take the control solution and inject it into the high performance liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component peak is 15% of the full scale; accurately measure 10μl of the test solution and the control solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. In the chromatogram, the peak area of ​​impurity A in the test solution shall not be greater than the peak area of ​​the control solution (0.5%); the peak area of ​​impurity B multiplied by the correction factor shall not be greater than 0.3 times the peak area of ​​the control solution (0.15%); the peak area of ​​impurity C shall not be greater than 0.3 times the peak area of ​​the control solution (0.15%); the peak area of ​​impurity D multiplied by the correction factor shall not be greater than 0.6 times the peak area of ​​the control solution (0.3%); the peak area of ​​impurity E shall not be greater than 0.6 times the peak area of ​​the control solution (0.3%); the peak area of ​​impurity F shall not be greater than 0.6 times the peak area of ​​the control solution (0.3%). The peak area of ​​impurity G shall not be greater than 0.6 times (0.3%) of the peak area of ​​the control solution; the peak area of ​​other individual impurities shall not be greater than 0.2 times (0.10%) of the peak area of ​​the control solution; the total amount of impurities shall not be greater than 1.0%; the correction factors of 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 factors of other unknown impurities are calculated as 1.0; record the chromatogram ( Figure 3 ), the test results are shown in Table 2:

[0080] Table 2 Specificity test results

[0081]

[0082] pass Figure 3 It can be seen from the results in Table 2 that the detection method of the present application controls 7 known impurities and achieves effective separation. The separation between diphenhydramine hydrochloride and adjacent peaks is greater than 1.5, the separation between each known impurity peak is greater than 1.5, and the impurity peaks do not interfere with each other.

[0083] Example 3 Limit of Quantitation and Limit of Detection Experiment

[0084] 1. Solution preparation

[0085] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0086] Take appropriate amount of reference substance diphenhydramine hydrochloride, impurities A, B, C, D, E, F, G, accurately weigh, add solvent for quantitative dilution, and make solutions with concentrations of 0.2279μg / ml, 0.0764μg / ml, 0.1054μg / ml, 0.1486μg / ml, 0.0628μg / ml, 0.1227μg / ml, 0.1486μg / ml, 0.1010μg / ml, respectively. The injection volume with a signal-to-noise ratio of about 10 is taken as the quantification limit (LOQ ); Take appropriate amounts of reference substance diphenhydramine hydrochloride, impurities A, B, C, D, E, F, and G, accurately weigh them, add solvent and quantitatively dilute them to make solutions with concentrations of 0.0684μg / ml, 0.0229μg / ml, 0.0316μg / ml, 00446μg / ml, 0.0188μg / ml, 0.0368μg / ml, 0.0446μg / ml, and 0.0303μg / ml, respectively, and take the injection volume with a signal-to-noise ratio of about 3 as the detection limit (LOD).

[0087] 2. Chromatographic conditions

[0088] Same as Example 1.

[0089] 3. Experimental operation

[0090] After the system is stable, the above solutions are injected into the high performance liquid chromatograph respectively. The detection and analysis results are shown in Table 3:

[0091] Table 3 Results of quantitation limit and detection limit experiments

[0092]

[0093] The test results in Table 3 show that the detection method of Example 1 can meet the quantitative requirements of impurities A, B, C, D, E, F, G and diphenhydramine hydrochloride at least 2.3 ng. The detection method provided in this application has good sensitivity and can effectively detect related substances at lower contents, eliminate potential medication risks, and ensure the effectiveness and safety of drug quality.

[0094] Example 4 Linearity and correction factor experiment

[0095] 1. Solution preparation

[0096] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0097] Take appropriate amounts of the reference substance diphenhydramine hydrochloride and impurities A, B, C, D, E, F, and G, accurately weigh them, add solvent to dissolve and dilute to a certain amount, and prepare linear solutions of different concentrations, as shown in Table 4:

[0098] Table 4 Linear solution concentration of each substance (μg / ml)

[0099] 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

[0100] 2. Chromatographic conditions

[0101] Same as Example 1.

[0102] 3. Experimental operation

[0103] After the system is stable, the linear solutions prepared in Table 4 are injected into the HPLC respectively, and the experimental results are recorded. The linear results are shown in Table 5:

[0104] Table 5 Linearity test results

[0105] 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

[0106] It can be seen from the results in Table 5 that, using the detection method provided by the present application, impurities A, B, C, D, E, F, G and diphenhydramine hydrochloride have good linear relationships within the range of quantitative limit to about 13 μg / ml, and the linear range is wide, indicating that the linear relationship of the detection method of the present application is good, and the impurity content can be quantitatively detected with high accuracy. The correction factors are all between 0.2 and 5.0, so the main component self-comparison method with correction factors can be used to calculate the content of each impurity for the inspection of related substances.

[0107] Specifically, the impurity content is calculated as:

[0108]

[0109] Among them, A 单 is the impurity area, A 对照 is the peak area of ​​the main component of 0.1% control solution, and F is the correction factor.

[0110] Example 5 Precision Experiment

[0111] 1. Solution preparation

[0112] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0113] Impurity stock solution: Accurately weigh about 7 mg of each of the reference impurities A, B, C, D, E, F and G, place them in different 50 ml volumetric flasks, add 2 ml of acetonitrile to dissolve them, add solvent to dilute to the scale, and shake well.

[0114] Mixed impurity stock solution: Accurately measure 2.0 ml of each impurity stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0115] 0.2% limit spiked test solution: Accurately weigh about 14 mg of the raw material diphenhydramine hydrochloride and place it in a 20 ml volumetric flask. At the same time, accurately measure 2 ml of the mixed impurity stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0116] Control solution: Accurately measure 1 ml of the 0.2% limit spiked test solution and place it in a 200 ml volumetric flask. Add solvent to dissolve and dilute to the scale and shake well.

[0117] 2. Chromatographic conditions

[0118] Same as Example 1.

[0119] 3. Experimental operation

[0120] In this example, different analysts prepared 6 0.2% limit spiked test sample solutions in parallel on different dates according to the method in this example, and measured them on another instrument of different models to examine the repeatability and precision of the detection method of this application. The results are shown in Table 6:

[0121] Table 6 Repeatability and precision test results

[0122]

[0123]

[0124] From the results in Table 6, it can be seen that the RSD of each impurity in the 6 spiked test solutions was no more than 5%, indicating that the repeatability of this method was good; the RSD of each impurity in the 12 spiked test solutions was less than 7.5%, indicating that the intermediate precision of this method was good.

[0125] Example 6 Accuracy Experiment

[0126] 1. Solution preparation

[0127] Solvent: 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 value is adjusted to 3.0 with phosphoric acid).

[0128] Impurity stock solution: Accurately weigh about 7 mg of each of the reference impurities A, B, C, D, E, F and G, place them in different 50 ml volumetric flasks, add 2 ml of acetonitrile to dissolve them, add solvent to dilute to the scale, and shake well.

[0129] Mixed impurity stock solution: Accurately measure 5.0 ml of each impurity stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0130] Test solution: Accurately weigh about 14 mg of the raw material diphenhydramine hydrochloride, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0131] Spiked test solution: Accurately weigh about 14 mg of the raw material diphenhydramine hydrochloride, a total of 12 portions, put them into different 20 ml volumetric flasks, add solvent to dissolve them, set each 3 portions as a group, a total of 4 groups, add 1 ml, 1.5 ml, 3 ml, and 6 ml of the mixed impurity stock solution to each group, respectively, add solvent to dilute to the scale, and shake well.

[0132] Reference solution: Accurately measure 1.5 ml of the mixed impurity stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0133] 2. Chromatographic conditions

[0134] Same as Example 1

[0135] 3. Experimental operation

[0136] After the system is stable, the above prepared solutions are injected into the high performance liquid chromatograph respectively, and the experimental results are recorded. The detection and analysis results are shown in Table 7:

[0137] Table 7 Accuracy experimental results

[0138] name Spiking 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)

[0139] The results in Table 7 show that the mean recoveries of impurities A, B, C, D, E, F, and G at the limit concentrations of 0.1%, 0.15%, 0.3%, and 0.6% are all in the range of 90% to 108%, and the RSDs of the 12 recoveries are all less than 10%, indicating that the detection method in this application has a good recovery rate and high accuracy. In addition, Figure 4 The chromatogram at a spiked concentration of 0.6% is shown in the figure. It can be seen that the impurity A peak is well separated from the diphenhydramine hydrochloride peak, and impurities B, C, D, E, F, and G are effectively separated from adjacent impurity peaks, indicating that the detection method of the present application can effectively control the quality of the diphenhydramine hydrochloride raw material.

[0140] Example 7 Durability Test

[0141] 1. Solution preparation

[0142] The preparation of the test solution and system suitability solution is the same as in Example 1.

[0143] 2. Chromatographic conditions

[0144] On the basis of the chromatographic conditions in Example 1, different chromatographic conditions were changed to investigate the durability of the detection method of the present application, as shown in Table 8 (the changed conditions in the table are all single variables, and other unspecified conditions are the same as those in Example 1):

[0145] Table 8 Chromatographic conditions for durability experiments

[0146]

[0147]

[0148] The robustness test chromatograms for the system suitability solutions are shown in Figure 5 , the test and analysis results are shown in Tables 9 and 10:

[0149] Table 9 Durability test - system suitability separation results

[0150]

[0151] Table 10 Durability test - test results of test products

[0152]

[0153]

[0154] pass Figure 5 It can be seen from the results of Tables 9 and 10 that after fine-tuning the chromatographic conditions of flow rate (±0.2ml / min), wavelength (220±2nm), column temperature (30±2℃), mobile phase A buffer salt pH (3.0±0.2), mobile phase A buffer salt concentration (0.03±0.005mol / L), buffer salt ratio in mobile phase A (70%±2%) and changing the chromatographic column, compared with the conditions of Example 1, in the system suitability solution chromatogram, the separation between each impurity peak and between the main peak and the adjacent peaks is greater than 1.5, and the content of each impurity and the total impurity content are compared with those under standard conditions. There is no significant difference in the detected impurities (all less than 0.05%), indicating that this method has good durability.

[0155] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A HPLC detection method for impurities in diphenhydramine hydrochloride bulk drug, characterized in that: The HPLC conditions are as follows: Chromatographic column: The filler is octadecylsilane bonded silica gel with a particle size of 4 to 5 μm; the column length is 250 mm and the inner diameter is 3 to 4.6 mm; Mobile phase: Mobile phase A is a mixture of phosphate buffer and acetonitrile, mobile phase B is acetonitrile; gradient elution; Injection volume: 10-20 μl; flow rate: 1.0-1.4 ml / min; column temperature: 28-32°C; detection wavelength: UV: 210-230 nm.

2. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The parameters of the chromatographic column are as follows: the filler is octadecylsilane bonded silica gel, the particle size is 5 μm; the column length is 250 mm, and the column inner diameter is 4.6 mm.

3. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, is characterized in that, In the mobile phase A, the volume ratio of phosphate buffer and acetonitrile is (60-80): (20-40); Preferably, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is (68-72): (28-32); Preferably, in the mobile phase A, the volume ratio of phosphate buffer to acetonitrile is 70:

30.

4. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The concentration of the phosphate buffer is 0.02-0.04 mol / L, and the pH is 2.8-3.2; Preferably, the concentration of the phosphate buffer is 0.025 to 0.035 mol / L; Preferably, the concentration of the phosphate buffer is 0.03 mol / L and the pH is 3.

0.

5. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The phosphate is potassium dihydrogen phosphate.

6. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The gradient elution procedure is as follows:

7. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, Flow rate: 1.2 ml / min; column temperature: 30°C; detection wavelength: UV 218-222 nm; Preferably, the detection wavelength is 220 nm.

8. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The HPLC detection method also includes the step of solution preparation, including: Test solution: accurately weigh the raw material diphenhydramine hydrochloride, add solvent to dissolve and dilute quantitatively, shake well to make a solution with a concentration of 0.7 mg / ml; Control solution: Accurately measure the test solution and dilute it with solvent to make 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 solvents, dilute them quantitatively, and shake well to prepare solutions with a concentration of 0.3 mg / ml for each impurity; Impurity localization solution: accurately measure the impurity stock solution, dilute it with a solvent to make a solution with a concentration of 1.5 μg / ml for each impurity; System suitability solution: accurately weigh 14 mg of the reference substance diphenhydramine hydrochloride and place it in a 20 ml volumetric flask. At the same time, accurately measure 0.1 ml of each impurity stock solution and dilute it with solvent to make a solution with a diphenhydramine hydrochloride concentration of 0.7 mg / ml and each impurity concentration of 1.5 μg / ml. Wherein, 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.

9. The HPLC detection method of diphenhydramine hydrochloride bulk drug impurities as claimed in claim 1, characterized in that, The impurity content calculation formula is: The calculation formula for impurity content is: Among them, A 单 is the impurity area, A 对照 is the peak area of ​​the main component of the 0.1% control solution, and F is the correction factor; Preferably, the correction factors of 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 factors of the remaining unknown impurities are calculated as 1.

0.

10. The HPLC detection method of impurities of diphenhydramine hydrochloride bulk drug as claimed in claim 1, characterized in that, In the results of the detection method, the separation degree of each impurity peak should be no less than 1.0, and the separation degree between the main component peak and the preceding and following peaks should be no less than 1.5.

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