Method for separating and / or detecting bromhexine hydrochloride and related substances thereof

Abstract

The invention provides a method for separating and / or detecting bromhexine hydrochloride and related substances thereof. A chromatographic column stationary phase of the high performance liquid chromatography is octadecylsilane chemically bonded silica, a mobile phase is composed of a phase A and a phase B, the phase A is ammonium acetate, the phase B is methanol-acetonitrile, the mobile phase is gradient elution, and the detection wavelength is 250nm. The related substance is selected from any one or a combination of a related substance 1, a related substance 2, a related substance 3, a related substance 4, a related substance 5, a related substance 6, a related substance 7, a related substance 8 and a related substance 9. According to the method disclosed by the invention, efficient separation and detection of bromhexine hydrochloride and related substances thereof can be realized, and the drug quality of bromhexine hydrochloride can be effectively controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, in particular to a method for separating and / or detecting bromhexine hydrochloride and related substances thereof. Background Art

[0002] Bromhexine hydrochloride (see formula I below) is an expectorant with mucolytic effect developed by Boehringer Ingelheim KG. PMDA approved the marketing of Boehringer Ingelheim KG's bromhexine hydrochloride injection in 1976 under the trade name The dosage form is 2ml:4mg. The original formulation is not imported into China, but several domestically produced generic versions have been approved for marketing, with dosage forms including injection, tablets, oral solution, and granules.

[0003]

[0004] The presence of related substances is directly related to the quality and safety of drugs. These substances include those introduced into the drug raw materials, those generated during the process, and those related to degradation. They can directly affect the purity and quality of the drug and may even cause serious adverse reactions, affecting the effectiveness and safety of clinical medication. To improve the quality of bromhexine hydrochloride injection and ensure its safe and effective clinical use, it is necessary to fully study its related substances and develop appropriate separation and detection methods to control the related substances within safe limits, achieve controllable drug quality, and ensure the safety and effectiveness of clinical medication. Summary of the Invention

[0005] The present invention aims to provide a method for separating and / or detecting bromhexine hydrochloride and its related substances, which comprises the following features: high performance liquid chromatography is adopted, the chromatographic column stationary phase is octadecylsilane bonded silica gel, the mobile phase is composed of phase A and phase B, phase A is ammonium acetate, phase B is methanol-acetonitrile, the mobile phase is gradient elution, and the related substances are selected from any one of related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8, and related substance 9, or a combination thereof:

[0006]

[0007] In a preferred technical solution of the present invention, the column temperature of the chromatographic column is 30°C-50°C, preferably 35°C-45°C.

[0008] In a preferred technical solution of the present invention, the concentration of ammonium acetate in the mobile phase A is 0.005 mol / L-0.05 mol / L, preferably 0.005 mol / L-0.025 mol / L, and more preferably 0.01 mol / L.

[0009] In a preferred technical solution of the present invention, the pH value of the ammonium acetate aqueous solution in the mobile phase A is adjusted to 4.0-4.8, preferably to 4.2-4.6; particularly preferably, phosphoric acid is used to adjust the pH value of the ammonium acetate aqueous solution.

[0010] In a preferred technical solution of the present invention, the volume ratio of methanol to acetonitrile in the mobile phase B is 15-25:85-75, preferably 20:80.

[0011] In a preferred technical solution of the present invention, the flow rate of the mobile phase is 0.8 ml / min-1.2 ml / min, preferably 0.9 ml / min-1.1 ml / min.

[0012] In a preferred technical solution of the present invention, the injection volume of the method is 5 μl-30 μl, preferably 5 μl-20 μl, and more preferably 10 μl-15 μl.

[0013] In a preferred technical solution of the present invention, the method is equipped with an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 240nm-250nm, preferably 243nm-247nm.

[0014] In the preferred technical solution of the present invention, the octadecylsilane bonded silica gel chromatographic column is commercially available, preferably an octadecylsilane bonded silica gel column produced by any of ThermoFisher, YMC, Phenomenex, ES, Merck, Agilent, Kromasil, Agela, Techmate, and Waters.

[0015] In the preferred technical solution of the present invention, the octadecylsilane bonded silica gel chromatographic column is selected from Eclipse Plus C18, Kromasil 100-5-C18, Kromasil Eternity-5-C18, Kromasil Eternity XT-10-C18, Kromasil 100-10-C18, Kromasil 300-5-C18, YMC Pack ODS-AQ C18, YMC Pack ODS-AMC18, YMC Triart C18, Phenomenex kinetex C18, Titank C18, ES-C18, Epic C18, ZORBAX SB-C18, ZORBAX 300SB-C18, Poroshell 120EC-C18, XDB-C18, TC-C18, Extend-C18, Bonshell C18, Bonshell ASB C18, Venusil HLP C18, Venusil C18 Plus, Venusil XBPC18 (A), Venusil XBP C18 (B), Venusil MP C18, Innoval Neo XD C18, Techmate C18-ST, Techmate C18-STⅡ, Techmate CI8 UG, Welch Ultimate XS-C18, Waters Atlantis T3, Waters Xbridge C18, or any combination thereof.

[0016] In a preferred technical solution of the present invention, the octadecylsilane bonded silica gel chromatographic column is Venusil C18Plus.

[0017] In a preferred technical solution of the present invention, the octadecylsilane bonded silica gel chromatographic column is Venusil C18Plus, 4.6 mm × 250 mm, 5 μm.

[0018] In the preferred technical solution of the present invention, the gradient elution procedure is:

[0019]

[0020]

[0021] Preferably, the gradient elution procedure is:

[0022]

[0023] In the preferred technical solution of the present invention, the order of peaks is related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8, related substance 9, and bromhexine hydrochloride, wherein the separation between the peaks of bromhexine hydrochloride and related substance 9 and between the peaks of each related substance is greater than 1.5.

[0024] In the preferred technical solution of the present invention, the method comprises the following steps:

[0025] 1) Solution preparation:

[0026] Test solution: Bromhexine hydrochloride injection

[0027] Control solution: Accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well; Accurately measure 1 ml of the test solution, place it in a 10 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well

[0028] System suitability solution: Weigh approximately 2 mg of each of the related substances 1-9, accurately weigh them, place them in a 25 ml volumetric flask, add methanol to dissolve them and dilute to the mark, shake well, and use them as the related substance stock solution; weigh approximately 0.2 g of L-tartaric acid and approximately 9.5 g of glucose, place them in a beaker, add 80 ml of water to dissolve them, add approximately 0.4 g of bromhexine hydrochloride, stir to dissolve in an 80°C water bath, cool, transfer to a 100 ml volumetric flask, dilute to the mark with water, and shake well; then accurately measure 1 ml of the related substance stock solution and 10 ml of the above solution, place them in a 20 ml volumetric flask, dilute to the mark with methanol, and prepare a solution containing approximately 2 mg of bromhexine hydrochloride and approximately 4 μg of each related substance per 1 ml, which is used as the system suitability solution.

[0029] 2) Octadecylsilane bonded silica gel was used as the stationary phase, the column temperature was 38°C-42°C, the mobile phase consisted of phase A and phase B, phase A was 0.01 mol / L ammonium acetate (adjusted to pH 4.2-4.6 with phosphoric acid), phase B was methanol-acetonitrile (20:80), and the mobile phase was gradient elution. The gradient elution procedure was as follows:

[0030]

[0031] The eluted samples were injected into the liquid chromatograph with an injection volume of 10 μl, the detection wavelength of the UV detector was 243-247 nm, the flow rate was 0.9 ml-1.1 ml per minute, and the chromatogram was recorded.

[0032] In the preferred technical solution of the present invention, the method comprises the following steps:

[0033] 1) Solution preparation:

[0034] Test solution: Bromhexine hydrochloride injection

[0035] Control solution: Accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well; Accurately measure 1 ml of the test solution, place it in a 10 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well

[0036] System suitability solution: Weigh approximately 2 mg of each of the related substances 1-9, accurately weigh them, place them in a 25 ml volumetric flask, add methanol to dissolve them and dilute to the mark, shake well, and use them as the related substance stock solution; weigh approximately 0.2 g of L-tartaric acid and approximately 9.5 g of glucose, place them in a beaker, add 80 ml of water to dissolve them, add approximately 0.4 g of bromhexine hydrochloride, stir to dissolve in an 80°C water bath, cool, transfer to a 100 ml volumetric flask, dilute to the mark with water, and shake well; then accurately measure 1 ml of the related substance stock solution and 10 ml of the above solution, place them in a 20 ml volumetric flask, dilute to the mark with methanol, and prepare a solution containing approximately 2 mg of bromhexine hydrochloride and approximately 4 μg of each related substance per 1 ml, which is used as the system suitability solution.

[0037] 2) A chromatographic column, Venusil C18 Plus, 4.6 mm × 250 mm, 5 μm, was used as the stationary phase. The column temperature was 40°C. The mobile phase consisted of phase A and phase B. Phase A was 0.01 mol / L ammonium acetate (adjusted to pH 4.4 with phosphoric acid), and phase B was methanol-acetonitrile (20:80). The mobile phase was gradient elution. The gradient elution program was as follows:

[0038]

[0039]

[0040] The eluted samples were injected into the liquid chromatograph with an injection volume of 10 μl, a UV detector detection wavelength of 245 nm, a flow rate of 1.0 ml per minute, and eluted and recorded the chromatogram.

[0041] Another object of the present invention is to provide an application of the method of the present invention in separating and / or detecting bromhexine hydrochloride and its related substances.

[0042] Unless otherwise indicated, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage (ml / g); when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage (g / ml); and the rest are weight / weight percentages.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention scientifically screens and optimizes the elution conditions of high-performance liquid chromatography (HPLC), achieving effective separation and detection of 10 structurally similar, difficult-to-separate compounds, including bromhexine hydrochloride and related substances, including process impurities and degradation impurities. The separation effect is excellent, effectively controlling drug quality, reducing potential adverse reactions during clinical trials and treatments, and ensuring the effectiveness and safety of medications for patients. Furthermore, the method of the present invention can also be used to assess drug stability, ensuring stable drug quality during storage and transportation.

[0045] The method of the present invention has strong specificity, the related substances and the blank solvent do not interfere with each other, and the separation between the peaks of each component is greater than 1.5; the method of the present invention has high sensitivity in detecting related substances; when the chromatographic conditions of the method of the present invention change slightly, it does not affect the effective separation of the peaks of each component, and the method has good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the chromatogram of the mixed solution of Comparative Example 1;

[0047] Figure 2 It is the chromatogram of the mixed solution of Comparative Example 2;

[0048] Figure 3 This is the chromatogram of the mixed solution of Comparative Example 3;

[0049] Figure 4 is the mixed solution chromatogram of Example 1;

[0050] Figure 5 1 is the chromatogram of the mixed solution of Example 2. DETAILED DESCRIPTION

[0051] To make the purpose, technical solutions and advantages of the present invention more clearly understood, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the embodiments of the present invention and the features therein can be combined with each other in any manner without conflict.

[0052] Comparative Example 1 HPLC separation and detection of related substances in bromhexine hydrochloride injection

[0053] Chromatographic column: Venusil C18 Plus (4.6 mm × 250 mm, 5 μm)

[0054] Column temperature: 40°C

[0055] Mobile phase: phosphate buffer (weigh 1.0 g of potassium dihydrogen phosphate, dissolve in 900 ml of water, adjust the pH to 7.0 with 0.5 mol / L sodium hydroxide solution, and then dilute to 1000 ml with water) - acetonitrile (20:80)

[0056] Flow rate: 1.0ml / min

[0057] UV detector detection wavelength: 245nm

[0058] Injection volume: 10 μl

[0059] Mixed solution: Take about 2 mg of each of the related substances 2-9, place them in a 25 ml volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and use them as the stock solution of each related substance (80 μg / ml); take about 10 mg of the related substance 1, place it in a 100 ml volumetric flask, add water to dissolve, dilute to the mark, shake well, and use them as the stock solution of the related substance 1 (100 μg / ml); take about 20 mg of bromhexine hydrochloride, place it in a 10 ml volumetric flask, add methanol to dissolve, measure 0.5 ml of each of the stock solutions of the related substances 2-9, and 0.4 ml of the stock solution of the related substance 1, dissolve them in methanol and dilute to the mark, shake well, and use them as the mixed solution of the related substances

[0060] The chromatographic results of the mixed solution are shown in Figure 1 .

[0061] The results showed that the separation degree of the related substances was poor, effective separation could not be achieved, and product quality could not be controlled.

[0062] Comparative Example 2 HPLC Separation and Detection of Related Substances in Bromhexine Hydrochloride Injection

[0063] Chromatographic column: Venusil C18 Plus (4.6 mm × 250 mm, 5 μm)

[0064] Column temperature: 40°C

[0065] Mobile phase: Phase A is 0.01 mol / L ammonium acetate (adjusted to pH 5.0 with phosphoric acid), and phase B is methanol:acetonitrile (20:80)

[0066] Flow rate: 1.0ml / min

[0067] UV detector detection wavelength: 245nm

[0068] Injection volume: 10 μl

[0069] Preparation of each reference substance stock solution: Weigh about 2 mg of bromhexine hydrochloride reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as the bromhexine hydrochloride reference substance stock solution; weigh about 2 mg of each of the related substances 2-9 reference substances, accurately weigh it, place it in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the reference substance stock solution of each related substance; weigh about 5 mg of related substance 1, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference substance stock solution of related substance 1.

[0070] Mixed solution: Weigh about 40 mg of bromhexine hydrochloride and place it in a 20 ml volumetric flask. Accurately add 1 ml each of the reference solution of related substances 2-9 and the stock solution of related substance 1. Dissolve in mobile phase B and dilute to the mark. Shake well.

[0071] Positioning solution: Measure 1 ml of each reference stock solution and place them in 20 ml volumetric flasks, add mobile phase B to dilute to the mark, shake well, and use as each positioning solution.

[0072] The gradient elution program is as follows:

[0073]

[0074] The chromatographic results of the mixed solution are shown in Figure 2 .

[0075] The results showed that the relevant substances 2 and 3 overlapped, and could not be effectively separated, and the product quality could not be controlled.

[0076] Comparative Example 3 HPLC Separation and Detection of Related Substances of Bromhexine Hydrochloride Injection

[0077] The gradient elution program is as follows:

[0078]

[0079] The remaining conditions are the same as those in Comparative Example 2.

[0080] The chromatographic results of the mixed solution are shown in Figure 3 .

[0081] The results showed that the related substances 2 and 3 could not be effectively separated.

[0082] Example 1 HPLC separation and detection of related substances of bromhexine hydrochloride injection

[0083] Chromatographic column: Venusil C18 Plus (4.6 mm × 250 mm, 5 μm)

[0084] Column temperature: 40°C

[0085] Mobile phase: Phase A is 0.01 mol / L ammonium acetate (adjusted to pH 4.4 with phosphoric acid), and phase B is methanol:acetonitrile (20:80)

[0086] Flow rate: 1.0ml / min

[0087] UV detector detection wavelength: 245nm

[0088] Injection volume: 10 μl

[0089] Solution preparation is the same as Comparative Example 2

[0090] The gradient elution program is as follows:

[0091]

[0092] The chromatographic results of the mixed solution are shown in Figure 4 .

[0093] The results showed that the separation degrees between the related substances and between bromhexine hydrochloride and adjacent related substances were all greater than 1.5.

[0094] Example 2 Specificity of the HPLC separation and detection method of the present invention

[0095] Chromatographic column: Venusil C18 Plus (4.6 mm × 250 mm, 5 μm)

[0096] Column temperature: 40°C

[0097] Mobile phase: Phase A is 0.01 mol / L ammonium acetate (adjusted to pH 4.4 with phosphoric acid), and phase B is methanol:acetonitrile (20:80)

[0098] Flow rate: 1.0ml / min

[0099] UV detector detection wavelength: 245nm

[0100] Injection volume: 10 μl

[0101] Solution preparation is the same as Comparative Example 2

[0102] The gradient elution program is as follows:

[0103]

[0104] The chromatographic results of the mixed solution are shown in Table 1 and Figure 5 .

[0105] Table 1

[0106]

[0107] The results showed that the retention time of each related substance in the mixed solution was consistent with that of the related substance positioning solution, and the minimum resolution between adjacent peaks was 4.31, which was greater than 1.5. This method has good specificity.

[0108] Example 3 Sensitivity of the HPLC separation and detection method of the present invention

[0109] The chromatographic conditions were the same as in Example 2.

[0110] Quantitation limit solution: accurately measure each related substance and bromhexine hydrochloride respectively, and prepare a positioning solution (4μg / ml) with methanol; accurately measure 0.5ml of the positioning solution of related substance 7, 2ml of the positioning solutions of related substances 2-4, 1.25ml of the positioning solutions of related substances 5 and 6, 1ml of the positioning solution of related substance 8, and 2.5ml of the positioning solution of related substance 9, place them in the same 100ml volumetric flask, dilute to the scale with methanol, shake well, and use them as the quantitation limit solution of each related substance; accurately measure 1ml of the bromhexine hydrochloride positioning solution, place it in a 20ml volumetric flask, add methanol to dilute to the scale, shake well, and use it as the quantitation limit solution of bromhexine hydrochloride. The quantitation limit solution requires a signal-to-noise ratio S / N of not less than 10.

[0111] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution of each related substance, place it in a 10 ml volumetric flask, add methanol to dilute to the scale, shake well, and use it as the detection limit solution of each related substance. Accurately measure 2 ml of the quantitative limit solution of bromhexine hydrochloride, place it in a 10 ml volumetric flask, add methanol to dilute to the scale, shake well, and use it as the detection limit solution of bromhexine hydrochloride. The detection limit solution requires a signal-to-noise ratio S / N of not less than 3.

[0112] The results of the limit of quantification and the limit of detection are shown in Table 2 below.

[0113] Table 2

[0114]

[0115]

[0116] The results show that the method of the present invention is highly sensitive in detecting bromhexine hydrochloride and related substances, and can complete quantitative detection at a level as low as ng for each compound.

[0117] Example 4 Durability of the HPLC separation and detection method of the present invention

[0118] The chromatographic conditions were the same as in Example 2.

[0119] Stock solutions of related substance reference substances: Weigh approximately 4 mg each of the related substance 1, 3, 5, 6, 7, and 9 reference substances, accurately weigh them, and place them in 25 ml volumetric flasks respectively. Dissolve them in methanol and dilute to the mark. Shake well to prepare the stock solutions of the related substance reference substances.

[0120] Test sample stock solution: Take 80 ml of water, place it in a 250 ml beaker, heat it in a water bath to 80°C, weigh about 11.88 g of glucose and add it and stir until dissolved. After dissolution, add about 250 mg of L-tartaric acid and stir to dissolve. After dissolution, add about 500 mg of bromhexine hydrochloride and stir until dissolved. After cooling, transfer all the solution to a 100 ml volumetric flask, dilute to the scale with water, and shake well.

[0121] Spiked test solution: Accurately measure 1 ml of the reference stock solution of each relevant substance, place it in a 10 ml volumetric flask, then add 4 ml of the test sample stock solution, dissolve it in solvent and dilute it to the scale, shake well, and obtain.

[0122] The retention time (RT / min) and resolution results are shown in Tables 3 and 4 below, respectively.

[0123] Table 3

[0124]

[0125] Table 4

[0126]

[0127] The results show that after a slight adjustment to the chromatographic conditions of Example 2 of the present invention, the separation of each component still meets the requirements, and the durability of the method of the present invention is good.

[0128] Although the embodiments disclosed herein are described above, the contents are merely embodiments for facilitating understanding of the present invention and are not intended to limit the present invention. Any person skilled in the art may make any modifications and changes in the form and details of the embodiments without departing from the spirit and scope of the present invention. However, the scope of patent protection of the present invention shall still be subject to the scope defined by the appended claims.

Claims

1. A method for separating and / or detecting bromhexine hydrochloride and its related substances, comprising the following features: using high performance liquid chromatography, the chromatographic column stationary phase is octadecylsilane bonded silica gel, the mobile phase consists of phase A and phase B, phase A is ammonium acetate, phase B is methanol-acetonitrile, the mobile phase is gradient elution, and the related substances are selected from any one of related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8, and related substance 9, or a combination thereof:

2. The method according to claim 1, characterized in that The method satisfies at least one of the following conditions (1)-(6): (1) The column temperature of the chromatographic column is 30°C-50°C, preferably 35°C-45°C, (2) The concentration of ammonium acetate in the mobile phase A is 0.005 mol / L-0.05 mol / L, preferably 0.005 mol / L-0.025 mol / L, more preferably 0.01 mol / L, and the pH value of the ammonium acetate aqueous solution in the mobile phase A is adjusted to 4.0-4.8, preferably to 4.2-4.6; particularly preferably, phosphoric acid is used to adjust the pH value of the ammonium acetate aqueous solution. (3) The volume ratio of methanol to acetonitrile in the mobile phase B is 15-25:85-75, preferably 20:

80. (4) The flow rate of the mobile phase is 0.8 ml / min-1.2 ml / min, preferably 0.9 ml / min-1.1 ml / min, (5) The injection volume of the method is 5 μl-30 μl, preferably 5 μl-20 μl, more preferably 10 μl-15 μl, (6) The method is equipped with an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 240nm-250nm, preferably 243nm-247nm.

3. The method according to any one of claims 1-2, characterized in that The octadecylsilane bonded silica gel chromatographic column is Venusil C18 Plus, preferably Venusil C18 Plus, 4.6 mm × 250 mm, 5 μm.

4. The method according to any one of claims 1 to 3, characterized in that The gradient elution procedure is: Preferably, the gradient elution procedure is:

5. The method according to any one of claims 1 to 4, characterized in that The order of the peaks of bromhexine hydrochloride and related substances is related substance 1, related substance 2, related substance 3, related substance 4, related substance 5, related substance 6, related substance 7, related substance 8, related substance 9, and bromhexine hydrochloride, wherein the separation between the peaks of bromhexine hydrochloride and related substance 9 and between the peaks of each related substance is greater than 1.

5.

6. The method according to any one of claims 1 to 5, characterized in that The method comprises the following steps: 1) Solution preparation: Test solution: Bromhexine hydrochloride injection Control solution: Accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well; Accurately measure 1 ml of the test solution, place it in a 10 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well System suitability solution: Weigh approximately 2 mg of each of the related substances 1-9, accurately weigh them, place them in a 25 ml volumetric flask, add methanol to dissolve them and dilute to the mark, shake well, and use them as the related substance stock solution; weigh approximately 0.2 g of L-tartaric acid and approximately 9.5 g of glucose, place them in a beaker, add 80 ml of water to dissolve them, add approximately 0.4 g of bromhexine hydrochloride, stir to dissolve in an 80°C water bath, cool, transfer to a 100 ml volumetric flask, dilute to the mark with water, and shake well; then accurately measure 1 ml of the related substance stock solution and 10 ml of the above solution, place them in a 20 ml volumetric flask, dilute to the mark with methanol, and prepare a solution containing approximately 2 mg of bromhexine hydrochloride and approximately 4 μg of each related substance per 1 ml, which is used as the system suitability solution. 2) Octadecylsilane bonded silica gel was used as the stationary phase, the column temperature was 38°C-42°C, the mobile phase consisted of phase A and phase B, phase A was 0.01 mol / L ammonium acetate (adjusted to pH 4.2-4.6 with phosphoric acid), phase B was methanol-acetonitrile (20:80), and the mobile phase was gradient elution. The gradient elution procedure was as follows: The eluted samples were injected into the liquid chromatograph with an injection volume of 10 μl, the detection wavelength of the UV detector was 243-247 nm, the flow rate was 0.9 ml-1.1 ml per minute, and the chromatogram was recorded.

7. The method according to claim 6, characterized in that The method comprises the following steps: 1) Solution preparation: Test solution: Bromhexine hydrochloride injection Control solution: Accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well; Accurately measure 1 ml of the test solution, place it in a 10 ml volumetric flask, dilute it to the mark with [mobile phase A-mobile phase B (65:35, V / V)], and shake well System suitability solution: Weigh approximately 2 mg of each of the related substances 1-9, accurately weigh them, place them in a 25 ml volumetric flask, add methanol to dissolve them and dilute to the mark, shake well, and use them as the related substance stock solution; weigh approximately 0.2 g of L-tartaric acid and approximately 9.5 g of glucose, place them in a beaker, add 80 ml of water to dissolve them, add approximately 0.4 g of bromhexine hydrochloride, stir to dissolve in an 80°C water bath, cool, transfer to a 100 ml volumetric flask, dilute to the mark with water, and shake well; then accurately measure 1 ml of the related substance stock solution and 10 ml of the above solution, place them in a 20 ml volumetric flask, dilute to the mark with methanol, and prepare a solution containing approximately 2 mg of bromhexine hydrochloride and approximately 4 μg of each related substance per 1 ml, which is used as the system suitability solution. 2) A chromatographic column, Venusil C18 Plus, 4.6 mm × 250 mm, 5 μm, was used as the stationary phase. The column temperature was 40°C. The mobile phase consisted of phase A and phase B. Phase A was 0.01 mol / L ammonium acetate (adjusted to pH 4.4 with phosphoric acid), and phase B was methanol-acetonitrile (20:80). The mobile phase was gradient elution. The gradient elution program was as follows: The eluted samples were injected into the liquid chromatograph with an injection volume of 10 μl, a UV detector detection wavelength of 245 nm, a flow rate of 1.0 ml per minute, and eluted and recorded the chromatogram.

8. the application of the described method of any one of claim 1-7 for separating and / or detecting bromhexine hydrochloride and related substances thereof.

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