HPLC Detection Method for Related Substances in Bromhexine Hydrochloride API

Abstract

The invention belongs to the field of medical detection technology, and is specifically related to a high performance liquid chromatography detection method for related substances of bromhexine hydrochloride raw materials. The high performance liquid chromatography detection method for related substances of the bromhexine hydrochloride raw materials adopts high performance liquid chromatography, the chromatographic column uses octadecylsilane bonded silica gel as filler, the detection wavelength is 210-250nm, the column temperature is 38-42℃, the phosphate buffer solution-mobile phase B system is the mobile phase A, the water-methanol-acetonitrile system is the mobile phase B, and the gradient elution. The detection method of the present invention can effectively detect the content of related substances in bromhexine hydrochloride raw materials, the method is simple and fast, the specificity is strong, the sensitivity is high, the separation is good, the detection impurity types are many, and the drug safety is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a high performance liquid chromatography detection method for bromhexine hydrochloride raw material drug related substances. Background Art

[0002] Bromhexine hydrochloride is suitable for patients with chronic bronchitis and other respiratory diseases such as asthma, bronchiectasis, silicosis, etc. who have sticky sputum that is difficult to cough up. Patients with purulent sputum need to add antibiotics to control infection. It is also widely used in the prevention and treatment of postoperative pulmonary complications, preoperative airway clearance, and the treatment of premature infants, newborns, and infant respiratory distress syndrome (IRDS).

[0003] In the preparation process of bromhexine hydrochloride API, there are many impurities, including process impurities, degradation impurities, etc., all of which need to be strictly controlled. Therefore, it is necessary to detect the related substances of bromhexine hydrochloride API. At present, in addition to the related detection methods of bromhexine hydrochloride related substances recorded in the "Chinese Pharmacopoeia", in order to detect the related substances of bromhexine hydrochloride API more quickly and accurately, many new detection methods have been proposed in the prior art.

[0004] For example, in patent CN107703230A, a high performance liquid chromatography detection method for bromhexine hydrochloride related substances is used, and the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler, a buffered saline solution with a pH of 5 to 5.5 as mobile phase A, methanol as mobile phase B, and a chromatographic column temperature of 30-35°C for gradient elution. This detection method achieves the complete separation of bromhexine hydrochloride related substances AQ, and the separation degree is good, and baseline separation is achieved; thereby achieving the simultaneous determination of the contents of bromhexine hydrochloride and its 15 related substances. However, it does not involve specific limitations on the content of each impurity in the bromhexine hydrochloride related substances, which is likely to affect the quality of bromhexine hydrochloride and increase the risk of side effects.

[0005] Patent CN113533578A discloses a quality control method for related substances in bromhexine hydrochloride tablets, which is carried out by high performance liquid chromatography, wherein the liquid chromatography determination conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel, mobile phase A is phosphate buffer, pH value is 2.8-3.6, mobile phase B is acetonitrile, and gradient elution is used for detection. This method is suitable for qualitative and quantitative detection of related substances in bromhexine hydrochloride. However, the impurity types detected are less, and it is not suitable for detection of more impurities.

[0006] Therefore, developing a method for rapidly, simply and accurately detecting more related substances in bromhexine hydrochloride API is of great significance for the quality control of bromhexine hydrochloride API. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a high performance liquid chromatography detection method for related substances of bromhexine hydrochloride raw material medicine, which can effectively detect the content of related substances in bromhexine hydrochloride raw material medicine, the method is simple and fast, has strong specificity, high sensitivity, good separation, can detect many types of impurities, and improves the safety of medicines.

[0008] The high performance liquid chromatography detection method of bromhexine hydrochloride raw material drug related substances adopts high performance liquid chromatography, the chromatographic column uses octadecylsilane bonded silica gel as filler, the detection wavelength is 210-250nm, the column temperature is 38-42°C, the phosphate buffer solution-mobile phase B system is used as mobile phase A, the water-methanol-acetonitrile system is used as mobile phase B, and gradient elution is performed.

[0009] Preferably, the gradient elution conditions are:

[0010] The first gradient elution time was 0 min, mobile phase A accounted for 95±5%, and mobile phase B accounted for 5±5%;

[0011] The second gradient elution time was 30±3min, with mobile phase A accounting for 5±5% and mobile phase B accounting for 95±5%;

[0012] The third gradient elution time is 60±3min, the mobile phase A accounts for 5±5%, and the mobile phase B accounts for 95±5%;

[0013] The fourth gradient elution time was 65±3 min, the mobile phase A accounted for 95±5%, and the mobile phase B accounted for 5±5%;

[0014] The fifth gradient elution time was 75±3 min, the mobile phase A accounted for 95±5%, and the mobile phase B accounted for 5±5%.

[0015] Preferably, the volume ratio of water, methanol and acetonitrile in mobile phase B is 5:20:75. Under this ratio, the separation degree of related substances is better.

[0016] Preferably, the volume ratio of the phosphate buffer solution to the mobile phase B in the mobile phase A is 62:38; the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution whose pH value is adjusted to 6.8-7.2 using a 0.5 mol / L sodium hydroxide solution.

[0017] Under acidic conditions, bromhexine hydrochloride peaks at the front, but the peak shape is poor, which is inseparable from related impurities. The present invention uses 0.5 mol / L sodium hydroxide solution to adjust the mobile phase A to near neutrality. Under neutral conditions, the peak shape of each impurity chromatographic peak is improved and meets the baseline separation.

[0018] Preferably, the HPLC uses a PDA or UV detector.

[0019] Preferably, the flow rate of the mobile phase is 0.8-1.2 ml / min.

[0020] Preferably, the injection volume is 5-20 μl.

[0021] In a most preferred embodiment, the chromatographic conditions are as follows:

[0022] Chromatographic column: Xtimate C18, specification 4.6mm×250mm, 5μm, filled with octadecylsilane bonded silica gel;

[0023] Column temperature: 40°C;

[0024] Detection wavelength: 245nm;

[0025] Flow rate: 1.0ml / min;

[0026] Injection volume: 10 μl;

[0027] Mobile phase: Mobile phase B is a water-methanol-acetonitrile system with a volume ratio of 5:20:75, and mobile phase A is a phosphate buffer solution-mobile phase B system with a volume ratio of 62:38, wherein the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution with a pH value of 6.8-7.2 adjusted with 0.5 mol / L sodium hydroxide solution; gradient elution;

[0028] Gradient elution conditions:

[0029] The first gradient elution time is 0 min, mobile phase A accounts for 100%, and mobile phase B accounts for 0%;

[0030] The second gradient elution time was 30 min, with mobile phase A accounting for 0% and mobile phase B accounting for 100%;

[0031] The third gradient elution time was 60 min, the mobile phase A accounted for 0%, and the mobile phase B accounted for 100%;

[0032] The fourth gradient elution time was 65 min, the mobile phase A accounted for 100%, and the mobile phase B accounted for 0%;

[0033] The fifth gradient elution time was 75 min, the mobile phase A accounted for 100%, and the mobile phase B accounted for 0%.

[0034] Preferably, the detection method comprises the following steps:

[0035] 1) Preparation of reference solution:

[0036] Take bromhexine hydrochloride reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 2.5 μg per 1 ml as reference substance solution;

[0037] 2) Preparation of test solution:

[0038] Take bromhexine hydrochloride test sample, weigh accurately, dissolve and dilute with solvent to make a solution containing 2.5 mg per 1 ml as the test solution;

[0039] 3) System suitability solution preparation:

[0040] Take bromhexine hydrochloride reference substance and impurity A, C, F, G, N, Q, S reference substances respectively, weigh accurately, add methanol to dissolve, and then add solvent to dilute to prepare a mixed solution containing 2.5 mg of bromhexine hydrochloride, 5 μg of impurities A, C, N, Q, S, and 2.5 μg of impurities F and G per 1 ml as the system suitability solution;

[0041] 4) Detection:

[0042] Accurately measure the solvent, system suitability solution, reference solution and test solution respectively, inject them into a liquid chromatograph, record the chromatogram, and separate bromhexine hydrochloride and its related substances (impurities A, C, F, G, N, Q, S);

[0043] In the chromatogram of the test solution, if there is a chromatographic peak in the obtained test solution that is consistent with the retention time of the related substance, calculate the impurity A, impurity C, impurity F and G, impurity N, impurity Q, and impurity S according to the peak area after correction by the external standard method, that is, multiply them by the correction factors of 0.64, 1.00, 1.00, 0.72, 1.16, and 1.56, respectively. The impurity A, impurity C, impurity N, impurity Q, and impurity S shall not exceed 0.1%, and the impurities F and G shall not exceed 0.1%; other individual impurities shall not exceed 0.10%; the total amount of impurities shall not exceed 0.2%.

[0044] Preferably, in the above method, the solvent used to prepare the solution is a phosphate buffer solution-mobile phase B system with a volume ratio of 20:80, wherein the phosphate buffer solution is a potassium dihydrogen phosphate solution with a concentration of 1 g / L and a pH value of 6.8-7.2 adjusted by 0.5 mol / L sodium hydroxide solution.

[0045] The specific impurities are as follows:

[0046] Impurity A is 2-amino-3,5-dibromobenzyl alcohol, structural formula:

[0047]

[0048] Impurity C is 2-[(N-methyl-N-cyclohexylamino)methyl]aniline, structural formula:

[0049]

[0050] Impurity F is N-methyl-N-cyclohexyl-2-amino-3-chloro-5-bromobenzylamine, structural formula:

[0051]

[0052] Impurity G is N-methyl-N-cyclohexyl-2-amino-5-chloro-3-bromobenzylamine, structural formula:

[0053]

[0054] Impurity N is 2,4-dibromo-6-(ethoxymethyl)aniline, structural formula:

[0055]

[0056] Impurity Q is N-methyl-N-cyclohexyl-2-amino-3,5-dibromobenzylamine oxide, structural formula:

[0057]

[0058] Impurity S is 6,8-dibromo-3-cyclohexyl-3,4-dihydro-2(1H)-quinazolinone, structural formula:

[0059]

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The invention optimizes the best analysis conditions and the preparation method of the test solution after a large number of experiments. It is shown through multiple experimental verifications that the high performance liquid chromatography determination method of the related substances of the bromhexine hydrochloride raw material medicine provided by the invention has high sensitivity, good separation degree and strong specificity, can accurately qualitatively and quantitatively detect various impurities in the bromhexine hydrochloride raw material medicine, thereby objectively, accurately and comprehensively evaluating the quality of the bromhexine hydrochloride raw material medicine, and has important practical significance for the control of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is the HPLC chromatogram of the blank solvent of the present invention;

[0063] Figure 2 It is a high performance liquid chromatogram of the specific mixed solution of the present invention;

[0064] Figure 3 It is the high performance liquid chromatogram of the reference substance solution of the present invention;

[0065] Figure 4 It is a high performance liquid chromatogram of the test solution of the present invention;

[0066] Figure 5This is the HPLC chromatogram of the specific mixed solution of Comparative Example 1;

[0067] Figure 6 It is the HPLC chromatogram of the specific mixed solution of Comparative Example 2. DETAILED DESCRIPTION

[0068] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto, and any changes made to the technical solution of the present invention by professionals in the field should all fall within the protection scope of the present invention.

[0069] The raw materials used in the examples, unless otherwise specified, are all commercially available conventional raw materials; the process methods used in the examples, unless otherwise specified, are all conventional methods in the art.

[0070] In the following examples, the HPLC conditions were as follows:

[0071] Detector: UV detector;

[0072] Chromatographic column: Octadecylsilane bonded silica gel column (Xtimate C 18 , 4.6mm×250mm, 5μm);

[0073] Detection wavelength: 245nm;

[0074] Column temperature: 40°C;

[0075] Flow rate: 1.0ml / min;

[0076] Injection volume: 10 μl;

[0077] Mobile phase: Mobile phase B is a water-methanol-acetonitrile system with a volume ratio of 5:20:75, and mobile phase A is a phosphate buffer solution-mobile phase B system with a volume ratio of 62:38, wherein the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution with a pH value of 7.0±0.2 adjusted with 0.5 mol / L sodium hydroxide solution; gradient elution;

[0078] Gradient elution conditions:

[0079] The first gradient elution time is 0 min, mobile phase A accounts for 100%, and mobile phase B accounts for 0%;

[0080] The second gradient elution time was 30 min, with mobile phase A accounting for 0% and mobile phase B accounting for 100%;

[0081] The third gradient elution time was 60 min, the mobile phase A accounted for 0%, and the mobile phase B accounted for 100%;

[0082] The fourth gradient elution time was 65 min, the mobile phase A accounted for 100%, and the mobile phase B accounted for 0%;

[0083] The fifth gradient elution time was 75 min, the mobile phase A accounted for 100%, and the mobile phase B accounted for 0%.

[0084] Solvent: a phosphate buffer solution-mobile phase B system with a volume ratio of 20:80, wherein the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution whose pH value is adjusted to 6.8-7.2 using 0.5 mol / L sodium hydroxide solution.

[0085] Example 1

[0086] Exclusive properties:

[0087] Stock solutions of each impurity: Take 10 mg each of impurity A, impurity C, impurity F, impurity G, impurity N, impurity Q, and impurity S, place them in different 20 ml volumetric flasks, dissolve them with methanol and dilute to the scale, shake well, and obtain.

[0088] Mixed solution: Accurately weigh 50 mg of bromhexine hydrochloride and place it in a 20 ml volumetric flask. Add 0.2 ml of impurity A, C, Q, N, S stock solutions and 0.1 ml of impurity F and impurity G stock solutions respectively. Dilute to the scale with solvent and shake well to obtain.

[0089] Precisely measure the blank solvent and the specific mixed solution and inject them according to the above chromatographic conditions, record the chromatogram, and the results are shown in Table 1. Figure 1 , Figure 2 .

[0090] Table 1 Specificity test results (mixed solution)

[0091] name Retention time (min) Separation Impurity A 14.678 — Impurity Q 16.285 6.543 Impurity C 25.763 32.153 Impurity N 26.198 1.484 Impurity S 28.327 8.444 Impurities F and G 38.290 35.413 Bromhexine 39.164 3.035

[0092] Conclusion: The blank solvent does not interfere with the detection of related substances, the separation degree between each peak is greater than 1.2, and the separation effect is good.

[0093] Example 2

[0094] Forced degradation test:

[0095] Reference solution (content): Accurately weigh 25 mg of bromhexine hydrochloride reference substance, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0096] Reference solution (impurities): Accurately measure 5 ml of the above reference solution (content), place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0097] Preparation of undegraded test sample solution: Accurately weigh 125 mg of the test sample, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0098] Preparation of acid degradation test solution: Accurately weigh 125 mg of the test sample, place it in a 50 ml volumetric flask, add 5 ml of 1 mol / L hydrochloric acid solution, leave it at room temperature for 24 hours, then add 5 ml of 1 mol / L sodium hydroxide solution to neutralize it, dilute it to the scale with solvent, shake well, and obtain.

[0099] Preparation of alkaline degradation test solution: Accurately weigh 125 mg of the test sample, place it in a 50 ml volumetric flask, add 5 ml of 1 mol / L sodium hydroxide solution, leave it at room temperature for 24 hours, then add 5 ml of 1 mol / L hydrochloric acid solution to neutralize it, dilute it to the scale with solvent, shake well, and obtain.

[0100] Preparation of oxidative degradation test solution: Accurately weigh 125 mg of the test sample, place it in a 50 ml volumetric flask, add 5 ml of 3% hydrogen peroxide solution, leave it at room temperature for 1.5 hours, dilute to the scale with solvent, shake well, and obtain.

[0101] Preparation of high temperature degradation test solution: Take an appropriate amount of the test sample, place it in a 60℃ oven, leave it for 30 days, take it out, cool it to room temperature, accurately weigh 125 mg, place it in a 50ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0102] Preparation of high humidity degradation test sample solution: Take an appropriate amount of the test sample, place it at a relative humidity of 92.5% for 30 days, take it out, accurately weigh 125 mg, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0103] Preparation of light degradation test sample solution: Take an appropriate amount of the test sample, place it under light conditions (4500lx±500lx), leave it for 30 days, take it out, accurately weigh 125mg, place it in a 50ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0104] Acid and alkali degradation blank solution: Take 5 ml of 1 mol / L hydrochloric acid solution and 1 mol / L sodium hydroxide solution, place them in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and use them as the acid and alkali degradation blank solutions;

[0105] Oxidative degradation blank solution: Take 5 ml of 3% hydrogen peroxide solution, place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and use it as the oxidative degradation blank solution.

[0106] Accurately measure each of the above solutions and inject them according to the above chromatographic conditions, record the chromatogram, and calculate the content of each impurity by the external standard method. The results are shown in Table 2. Figure 4 .

[0107] Table 2 Results of forced degradation test

[0108]

[0109]

[0110] Conclusion: The solvent and blank solutions had no interference on the sample determination. Under each degradation condition, the purity similarity of the main peak was greater than the purity threshold, and the separation between the main peak and the adjacent peaks was greater than 1.5, indicating that the method had strong specificity.

[0111] Example 3

[0112] System precision:

[0113] Reference substance solution: Accurately weigh 25 mg of bromhexine hydrochloride reference substance, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain the reference substance stock solution; accurately measure 1 ml, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, shake well, and use it as the reference substance solution.

[0114] Accurately measure the reference solution and inject it according to the above chromatographic conditions, record the chromatogram, calculate the relative standard deviation (RSD) of the peak area and retention time of each peak when the reference solution is injected 6 times continuously, and the results are shown in Table 3. Figure 3 .

[0115] Table 3 System precision test results

[0116] Serial number Retention time (min) Peak area 1 39.148 36573 2 39.160 35821 3 39.151 36472 4 39.135 36213 5 39.141 35888 6 39.131 36286 average 39.144 36209 RSD(%) 0.1 0.9

[0117] Conclusion: The reference solution was injected 6 times continuously, the RSD of the bromhexine peak area was less than 10.0%, and the RSD of the retention time was less than 1.0%, indicating that the system precision was good.

[0118] Example 4

[0119] Limit of Detection / Limit of Quantitation:

[0120] Using the solvent as blank, adjust the instrument sensitivity, inject samples three times continuously, record the instrument noise level within the peak time range of the analyte, and calculate the average noise.

[0121] Detection limit: Accurately prepare the reference solution of the substance to be tested, gradually dilute it to a certain concentration and inject it, and measure it three times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 3 is the detection limit concentration, and its ratio to the theoretical sample concentration is the detection limit.

[0122] Limit of quantitation: Accurately prepare the reference solution of the substance to be measured, gradually dilute it to a certain concentration and inject it, and measure it 6 times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 10 is the limit of quantitation concentration, and its ratio to the theoretical sample concentration is the limit of quantitation.

[0123] Quantitative limit solution: Accurately measure 1 ml of the impurity stock solution under the "Specificity" item and 2 ml of the bromhexine hydrochloride reference stock solution under the "System Precision" item, place them in the same 100 ml volumetric flask, dilute to the mark with solvent, shake well, then accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, and use this as the quantitative limit solution;

[0124] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as the detection limit solution.

[0125] The solvent was accurately measured and injected continuously for 3 times according to the above chromatographic conditions, the detection limit solution was injected continuously for 3 times, and the quantification limit solution was injected continuously for 6 times. The chromatograms were recorded and the signal-to-noise ratio was calculated. The results are shown in Tables 4, 5 and 6.

[0126] Table 4 Baseline noise measurement results

[0127] name Noise 1 Noise 2 Noise 3 Average noise Impurity A 28 28 22 26 Impurity C 24 24 24 24 Impurity F 29 27 28 28 Impurity G 27 29 26 27 Impurity N 27 34 35 32 Impurity Q 17 19 19 18 Impurity S 20 19 18 19 Bromhexine 26 24 27 26

[0128] Table 5 Quantitation limit determination results

[0129]

[0130]

[0131] Table 6 Detection limit determination results

[0132] name Concentration (μg / ml) Average peak height (μV) S / N Equivalent to test concentration (%) Impurity A 0.0773 211 8.1 0.003 Impurity C 0.0788 119 5.0 0.003 Impurity F 0.0719 155 5.5 0.003 Impurity G 0.0815 154 5.7 0.003 Impurity N 0.0815 194 6.1 0.003 Impurity Q 0.0798 106 5.9 0.003 Impurity S 0.0787 93 4.9 0.003 Bromhexine 0.0774 147 5.7 0.003

[0133] Conclusion: Under this method, the sensitivity of bromhexine hydrochloride and its impurities meet the requirements.

[0134] Comparative Example 1

[0135] The HPLC conditions of this comparative example differ from those of the embodiment only in that the mobile phase A and the phosphate buffer solution in the solvent are not adjusted to a near-neutral pH value using 0.5 mol / L sodium hydroxide solution, and the mobile phase A is acidic (pH 2.5).

[0136] The HPLC chromatogram of its specific mixed solution is as follows: Figure 5 As shown in the figure, it can be seen that the bromhexine peak is located at the front, the peak shape is worse, and the separation degree from impurities is poor.

[0137] Comparative Example 2

[0138] The HPLC conditions of this comparative example are different from those of the embodiment only in that mobile phase A is a 1 g / L potassium dihydrogen phosphate solution adjusted to a pH of 7.0±0.2 with a 0.5 mol / L sodium hydroxide solution, and mobile phase B is acetonitrile.

[0139] The HPLC chromatogram of its specific mixed solution is as follows: Figure 6 As shown in the figure, it can be seen that the separation degree between impurity A and impurity Q is 1.183, and impurity D / M / H has two connected peaks with poor separation degree.

Claims

1. A high performance liquid chromatography detection method for bromhexine hydrochloride raw material drug related substances, characterized in that: High performance liquid chromatography was used, with octadecylsilane bonded silica gel as the filler of the chromatographic column, a detection wavelength of 210-250nm, a column temperature of 38-42°C, a phosphate buffer solution-mobile phase B system as mobile phase A, a water-methanol-acetonitrile system as mobile phase B, and gradient elution; The gradient elution conditions were: The first gradient elution time was 0 min, mobile phase A accounted for 95±5%, and mobile phase B accounted for 5±5%; The second gradient elution time was 30±3min, with mobile phase A accounting for 5±5% and mobile phase B accounting for 95±5%; The third gradient elution time was 60±3min, with mobile phase A accounting for 5±5% and mobile phase B accounting for 95±5%; The fourth gradient elution time was 65±3min, the mobile phase A accounted for 95±5%, and the mobile phase B accounted for 5±5%; The fifth gradient elution time was 75±3min, the mobile phase A accounted for 95±5%, and the mobile phase B accounted for 5±5%; The volume ratio of water, methanol, and acetonitrile in mobile phase B was 5:20:75; The volume ratio of phosphate buffer solution to mobile phase B in mobile phase A is 62:38; the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution whose pH value is adjusted to 6.8-7.2 using 0.5 mol / L sodium hydroxide solution; The detection method includes the following steps: 1) Preparation of reference solution: Take bromhexine hydrochloride reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 2.5µg per 1ml as reference substance solution; 2) Preparation of test solution: Take bromhexine hydrochloride test sample, weigh accurately, dissolve and dilute with solvent to make a solution containing 2.5 mg per 1 ml as the test solution; 3) System suitability solution preparation: Take bromhexine hydrochloride reference substance, impurity A, C, F, G, N, Q, S reference substances respectively, weigh accurately, add methanol to dissolve, and then add solvent to dilute to make a mixed solution containing 2.5 mg of bromhexine hydrochloride, 5 µg of impurities A, C, N, Q, S, and 2.5 µg of impurities F and G per 1 ml as the system suitability solution; 4) Detection: Accurately measure the solvent, system suitability solution, reference solution and test solution respectively, inject them into a liquid chromatograph, record the chromatogram, and separate bromhexine hydrochloride and its related substances; In the chromatogram of the test solution, if there is a chromatographic peak in the obtained test solution that is consistent with the retention time of the related substance, calculate the impurity A, impurity C, impurity F and G, impurity N, impurity Q, and impurity S according to the peak area after correction by the external standard method, that is, multiply by the correction factor 0.64, 1.00, 1.00, 0.72, 1.16, and 1.56 respectively; Impurity A is 2-amino-3,5-dibromobenzyl alcohol, structural formula: ； Impurity C is 2-[(N-methyl-N-cyclohexylamino)methyl]aniline, structural formula: ； Impurity F is N-methyl-N-cyclohexyl-2-amino-3-chloro-5-bromobenzylamine, structural formula: ； Impurity G is N-methyl-N-cyclohexyl-2-amino-5-chloro-3-bromobenzylamine, structural formula: ； Impurity N is 2,4-dibromo-6-(ethoxymethyl)aniline, structural formula: ； Impurity Q is N-methyl-N-cyclohexyl-2-amino-3,5-dibromobenzylamine oxide, structural formula: ； Impurity S is 6,8-dibromo-3-cyclohexyl-3,4-dihydro-2(1H)-quinazolinone, structural formula: 。 2. the high performance liquid chromatography detection method of bromhexine hydrochloride bulk drug related substance according to claim 1, is characterized in that: High performance liquid chromatography uses a PDA or UV detector.

3. the high performance liquid chromatography detection method of bromhexine hydrochloride bulk drug related substance according to claim 1, is characterized in that: The flow rate of the mobile phase is 0.8-1.2 ml / min.

4. the high performance liquid chromatography detection method of bromhexine hydrochloride bulk drug related substances according to claim 1, is characterized in that: The injection volume is 5-20μl.

5. according to the high performance liquid chromatography detection method of the bromhexine hydrochloride bulk drug related substance described in any one of claims 1-4, it is characterized in that: The chromatographic conditions were as follows: Chromatographic column: Xtimate C18, size 4.6mm×250mm, 5µm, filled with octadecylsilane bonded silica gel; Column temperature: 40°C; Detection wavelength: 245nm; Flow rate: 1.0ml / min; Injection volume: 10 μl; Mobile phase: Mobile phase B is a water-methanol-acetonitrile system with a volume ratio of 5:20:75, and mobile phase A is a phosphate buffer solution-mobile phase B system with a volume ratio of 62:38, wherein the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution with a pH value of 6.8-7.2 adjusted with 0.5 mol / L sodium hydroxide solution; gradient elution; Gradient elution conditions: The first gradient elution time is 0 min, mobile phase A accounts for 100%, and mobile phase B accounts for 0%; The second gradient elution time was 30 min, with mobile phase A accounting for 0% and mobile phase B accounting for 100%; The third gradient elution time is 60 min, mobile phase A accounts for 0%, and mobile phase B accounts for 100%; The fourth gradient elution time was 65 min, the mobile phase A accounted for 100%, and the mobile phase B accounted for 0%; The fifth gradient elution time was 75 min, with mobile phase A accounting for 100% and mobile phase B accounting for 0%.

6. The high performance liquid chromatography detection method of bromhexine hydrochloride bulk drug related substances according to claim 1, characterized in that: The solvent used in preparing the solution is a phosphate buffer solution-mobile phase B system with a volume ratio of 20:80, wherein the phosphate buffer solution is a 1 g / L potassium dihydrogen phosphate solution with a pH value adjusted to 6.8-7.2 using a 0.5 mol / L sodium hydroxide solution.

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