A method for detecting n-methylcyclohexylamine hydrochloride in bromhexine hydrochloride

Abstract

This invention relates to the field of pharmaceutical analysis technology, and provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The method involves dissolving N-methylcyclohexylamine in an organic solvent as a reference solution, and dissolving bromhexine hydrochloride in an organic solvent containing an acid-binding agent as the test solvent, followed by headspace gas chromatography for detection. This method is simple, efficient, specific, sensitive, repeatable, and stable, and can be used for the quality control of bromhexine hydrochloride raw material, providing technical support for its production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. Background Technology

[0002] Bromhexine hydrochloride is an expectorant developed by Boehringer Ingelheim in Germany. Acquired by Sanofi in 2015, it is marketed as Bisolvon. It is used alone or in combination with antibiotics to treat respiratory diseases associated with mucus secretion disorders. Bromhexine hydrochloride is a mucomodulator with strong mucolytic properties. It primarily acts on the tracheal and bronchial mucosa, reducing the viscosity of acidic mucopolysaccharide molecules in sputum and inhibiting their synthesis, thereby alleviating symptoms such as excessive or sticky respiratory mucus. It is particularly effective in treating cough and pneumonia in children.

[0003] In pharmaceutical manufacturing, detecting the residual levels of starting materials in the active pharmaceutical ingredient (API) is a crucial requirement of GMP and drug registration regulations. This ensures the completeness and consistency of the reaction, and can also be used to assess reaction efficiency and economic costs during production, as well as its impact on product quality. Therefore, detecting the residual levels of starting materials is an important quality control measure.

[0004] There are currently several synthetic methods for bromhexine hydrochloride, but most of them require N-methylcyclohexylamine (SM2) as a cyclohexylamine donor to participate in the reaction, such as the following synthetic route:

[0005]

[0006] During the reaction, SM2 is usually added in excess. The remaining SM2 reacts with hydrochloric acid to form SM2 hydrochloride. If this is not removed, it will affect the quality of bromhexine hydrochloride raw material. Therefore, it is necessary to detect the residual amount of SM2 hydrochloride. However, there are currently no reports on the determination of residual SM2 hydrochloride in bromhexine hydrochloride raw material. In order to more comprehensively and effectively control the overall quality of bromhexine hydrochloride raw material, it is necessary to develop a method for detecting SM2 hydrochloride in bromhexine hydrochloride raw material. Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to provide a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] A method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride involves dissolving N-methylcyclohexylamine in an organic solvent as a reference solution and dissolving bromhexine hydrochloride in an organic solvent containing an acid-binding agent as a test solvent, followed by detection using headspace gas chromatography.

[0010] Furthermore, the organic solvent is N-methylpyrrolidone (NMP), N,N-dimethylformamide, or heptane (DMF), among which DMF has the best solubility for bromhexine hydrochloride, while NMP and heptane have slightly worse solubility, resulting in slight precipitation of the sample in the headspace vial.

[0011] Furthermore, the acid-binding agent is triethylamine, cyclohexylamine, pyridine, or ethanolamine, preferably ethanolamine.

[0012] Further, in the test solution, the volume ratio of the organic solvent to the acid-binding agent is (50-95):(5-50), preferably (88-92):(8-12).

[0013] N-Methylcyclohexylamine (SM2) is a volatile amine, but its hydrochloride salt has a high boiling point, making it unsuitable for gas chromatography detection. Therefore, an acid-binding agent is needed to neutralize the hydrochloric acid and release SM2. The choice of acid-binding agent has a significant impact on the detection of SM2; it is necessary to ensure the release of SM2 without affecting its peak elution. This invention achieves the detection of SM2 residues by selecting the appropriate acid-binding agent and adjusting the ratio of solvent to acid-binding agent. Ethanolamine shows the best effect, exhibiting good sample solubility before and after injection, no precipitation, good SM2 peak specificity, and good recovery and repeatability of the determination results.

[0014] Furthermore, the gas chromatographic column is SE-30, DB-624 or CP-Volamine, preferably CP-Volamine column, which has a better SM2 peak shape.

[0015] Furthermore, the CP-Volamine column has dimensions of 30m × 0.32mm.

[0016] Furthermore, the column temperature of the gas chromatograph is programmed, with an initial temperature of 55-65°C, maintained for 2-5 minutes, increased to 140-160°C at a rate of 18-22°C per minute, maintained for 2-5 minutes, and increased to 230-250°C at a rate of 8-12°C per minute, maintained for 8-12 minutes.

[0017] Furthermore, the injection port temperature of the gas chromatograph is 240-260℃.

[0018] Furthermore, the detector of the gas chromatograph is an FID detector, and the detector temperature is 270-290℃.

[0019] Furthermore, the split ratio of the gas chromatograph is (18-22):1.

[0020] Furthermore, the carrier gas flow rate of the gas chromatograph is 2-4 mL, the headspace equilibrium temperature is 80-100℃, and the equilibrium time is 20-40 minutes.

[0021] Furthermore, the content of N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride was determined using the standard curve method.

[0022] This invention enables the detection of N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride raw material by screening solvents and acid-binding agents for the test sample solution and selecting gas chromatography conditions. The method is simple, efficient, specific, sensitive, repeatable and stable, and can be used for the quality control of bromhexine hydrochloride raw material, providing technical support for the production of bromhexine hydrochloride raw material. Attached Figure Description

[0023] Figure 1 This is the chromatogram of the blank solution in Example 1 of the present invention;

[0024] Figure 2 This is a chromatogram of the reference solution in Example 1 of the present invention.

[0025] Figure 3 This is the chromatogram of the test solution in Example 1 of the present invention;

[0026] Figure 4 This is the chromatogram of the 100% sample solution in Example 1 of the present invention;

[0027] Figure 5 The chromatograms are of DMF, blank solution and reference solution in Example 2 of the present invention, where 1: DMF, 2: reference solution, 3: blank solution;

[0028] Figure 6 The chromatograms are of the reference solution (cyclohexylamine-DMF as solvent), blank solution 2 (ethanolamine-DMF), and blank solution 3 (pyridine-DMF) in Example 3 of the present invention, where 1: reference solution, 2: blank solution 2, and 3: blank solution 3;

[0029] Figure 7 This is the chromatogram of the test solution in Example 4 of the present invention;

[0030] Figure 8 This is the chromatogram of the reference solution in Example 4 of the present invention;

[0031] Figure 9 This is the chromatogram of the 100% sample solution in Example 4 of the present invention;

[0032] Figure 10This is the chromatogram of the test solution in Example 5 of the present invention;

[0033] Figure 11 This is the chromatogram of the reference solution in Example 5 of the present invention;

[0034] Figure 12 This is the chromatogram of the 100% sample solution in Example 5 of the present invention;

[0035] Figure 13 This is the chromatogram of the test solution in Example 6 of the present invention;

[0036] Figure 14 This is a chromatogram of the test solution in Example 7 of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Example 1

[0039] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The solution preparation method is as follows:

[0040] 1. Blank solution: Take 100 mL of ethanolamine and 900 mL of DMF, mix well, and the blank solution is obtained.

[0041] 2. Reference solution: Weigh approximately 100 mg of SM2 reference standard accurately and place it in a 50 mL volumetric flask containing an appropriate amount of DMF. Dissolve and dilute to the mark with DMF, and shake well to obtain the reference standard stock solution. Accurately measure 5 mL of the reference standard stock solution into a 200 mL volumetric flask, dilute to the mark with blank solution, and shake well to obtain the reference standard solution. Accurately measure 2 mL of the reference standard solution into a headspace vial and seal it.

[0042] 3. Test solution: Weigh approximately 100 mg of bromhexine hydrochloride raw material accurately and place it in a 20 mL headspace vial. Accurately measure 2 mL of blank solution and place it in the same headspace vial. Seal the vial to obtain the test solution.

[0043] 4. 100% spiking solution: Weigh approximately 100 mg of bromhexine hydrochloride raw material accurately and place it in a 20 mL headspace vial. Accurately measure 2 mL of the reference solution and place it in the same headspace vial. Seal the vial to obtain the 100% spiking solution.

[0044] Gas chromatography was used for detection, and the chromatographic conditions were as follows:

[0045] Chromatographic column: CP-Volamine (30m × 0.32mm); initial temperature 60℃, hold for 3 minutes, increase to 150℃ at a rate of 20℃ / min, hold for 3 minutes, increase to 240℃ at a rate of 10℃ / min, hold for 10 minutes; injection port temperature 250℃; detector (FID) temperature 280℃; split ratio 20:1; carrier gas flow rate 3ml / min; headspace equilibration temperature 90℃, equilibration time 30 minutes.

[0046] Methodological Validation

[0047] 1. Specificity test

[0048] The blank solution, reference solution, test solution, and 100% spiking solution from Example 1 were respectively injected and analyzed under the chromatographic conditions of Example 1. The chromatograms are shown in the figure. Figures 1-4 Chromatographic results showed that the blank solution did not interfere with the detection of SM2, and the retention times of SM2 in the test solution and the 100% spiking solution were consistent with those in the reference solution. This indicates that the method has good specificity.

[0049] 2. Limit of Quantification and Limit of Detection

[0050] Accurately transfer 3 mL of the reference stock solution from Example 1 into a 20 mL volumetric flask, dilute to the mark with blank solution, and mix well to obtain the limit of quantitation (LOQ) stock solution. Accurately measure 1 mL of the LOQ stock solution into a 20 mL volumetric flask, dilute to the mark with blank solution, and mix well to obtain the limit of quantitation (LOQ) solution with a concentration of 14.25 mg / L. Injection analysis showed that the RSD of the SM2 peak area of ​​the LOQ solution for six consecutive injections was 4.4%, and the signal-to-noise ratio was greater than 10. Accurately transfer 3 mL of the LOQ solution into a 10 mL volumetric flask, dilute to the mark with blank solution, and mix well to obtain the limit of detection (LOD) solution with a concentration of 4.28 mg / L. Injection analysis showed that the SM2 signal-to-noise ratio was greater than 3.

[0051] 3. Linearity and Range

[0052] Accurately measure 5 mL (L3) of the limit of quantitation (LOQ) stock solution under "2. Limit of Quantitation and Limit of Detection", 5 mL (L4), 7.5 mL (L5), and 10 mL (L6) of the reference stock solution from Example 1, and place them in 20 mL volumetric flasks respectively. Dilute to the mark with blank solution and shake well to obtain a series of standard solutions. Inject the LQ solution (L1), reference solution (L2), and series of standard solutions sequentially under the chromatographic conditions of Example 1. Perform linear regression with peak area A as the ordinate and concentration ρ (mg / L) as the abscissa. The regression equation for SM2 is shown in Table 1. The results show that SM2 has a good linear relationship within the mass concentration range.

[0053] Table 1. Linearity test results of SM2

[0054] Linear equations <![CDATA[R 2 ]]> Concentration (mg / L) Equivalent to the limit % Equivalent to % of the test sample A = 0.6274ρ - 2.4846 0.999 1 14.25～949.74 28～1899 0.03～1.9

[0055] 4. Repeatability

[0056] Approximately 100 mg of bromhexine hydrochloride raw material (batch number 80351462412001) was taken in six parallel samples. The test solution was prepared according to the method in Example 1. Each sample was injected once under the chromatographic conditions of Example 1. The results showed that the average residual amount of SM2 hydrochloride was 0.024%, and the RSD was 9.1% (n=6), indicating that the method had good repeatability.

[0057] 5. Recovery rate

[0058] Take approximately 100 mg of bromhexine hydrochloride raw material (batch number 80351462412001), totaling 12 portions. For each LOQ spiking solution, accurately measure 2 mL of the LOQ solution under "2. Limit of Quantitation and Limit of Detection" (n=3); for each 100% spiking solution, accurately measure 2 mL of the reference solution from Example 1 (n=6); for each 150% spiking solution, accurately measure 2 mL of the L3 linearity solution under "3. Linearity and Range" (n=3). Inject and analyze according to the chromatographic conditions of Example 1. Using the SM2 residue under "4. Repeatability" as a blank, the recoveries and RSDs for each spiking are shown in Table 2.

[0059] Table 2. Recovery results of SM2 (n=12)

[0060]

[0061] Note: The measured amount, inherent amount, and added amount are all converted to SM2 hydrochloride (SM2 molecular weight 113.20, SM2 hydrochloride molecular weight 149.66).

[0062] 6. Solution stability

[0063] The reference solution from Example 1 was taken and placed at room temperature. It was then injected and analyzed at 0, 9.3h, 15.7h, 23.9h, 28.5h, and 34.9h according to the chromatographic conditions of Example 1. The results showed that the RSD of the SM2 peak area at different time points and the peak area at 0h was between 3.0% and 7.2%, indicating that the reference solution had good stability within 34.9h at room temperature.

[0064] 7. Sample Determination

[0065] The raw materials of bromhexine hydrochloride with batch numbers 20241016B, 20241118 and 20241119 were accurately weighed and analyzed by chromatographic conditions under section “2.3.1”. The results showed that the content of SM2 hydrochloride in the three batches of raw materials was within the limit of quantitation. The results are shown in Table 3.

[0066] Table 3 Sample test results

[0067]

[0068] As can be seen, the method for determining the SM2 hydrochloride content in bromhexine hydrochloride raw material of this invention is simple, efficient, specific, sensitive, repeatable, and stable, and can be used for the quality control of bromhexine hydrochloride raw material. It provides technical support for the production of bromhexine hydrochloride raw material.

[0069] Example 2

[0070] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The solution preparation method is as follows:

[0071] 1. Blank solution: Take 500 mL of cyclohexylamine and 500 mL of DMF, mix well, and the blank solution is ready.

[0072] 2. Reference solution: Weigh approximately 100 mg of SM2 reference standard accurately and place it in a 50 mL volumetric flask containing an appropriate amount of DMF. Dissolve and dilute to the mark with DMF, and shake well to obtain the reference standard stock solution. Accurately measure 5 mL of the reference standard stock solution into a 200 mL volumetric flask, dilute to the mark with blank solution, and shake well to obtain the reference standard solution. Accurately measure 2 mL of the reference standard solution into a headspace vial and seal it.

[0073] The DMF solvent and blank solution were separately analyzed by gas chromatography.

[0074] The chromatographic conditions were as follows: column: CP-Volamine (60m × 0.32mm); temperature: 60℃ for 3 min, ramped to 150℃ at a rate of 20℃ / min, held for 3 min, ramped to 240℃ at a rate of 10℃ / min, held for 10 min; injection port temperature: 250℃; detector (FID) temperature: 280℃; split ratio: 20:1; carrier gas flow rate: 2 ml / min; headspace equilibration temperature: 90℃; equilibration time: 30 min.

[0075] Chromatogram as shown Figure 5 As shown, cyclohexylamine interferes with the detection of the SM2 peak.

[0076] Example 3

[0077] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The solution preparation method is as follows:

[0078] 1. Blank solution 1: Take 500 mL of cyclohexylamine and 500 mL of DMF, mix well, and you will get the blank solution.

[0079] 2. Reference solution: Weigh approximately 100 mg of SM2 reference standard accurately and place it in a 50 mL volumetric flask containing an appropriate amount of DMF. Dissolve and dilute to the mark with DMF, and shake well to obtain the reference standard stock solution. Accurately measure 5 mL of the reference standard stock solution into a 200 mL volumetric flask, dilute to the mark with blank solution 1, and shake well to obtain the reference standard solution. Accurately measure 2 mL of the reference standard solution into a headspace vial and seal it.

[0080] 3. Blank solution 2: Take 500 mL of ethanolamine and 500 mL of DMF, mix well, and you will get the blank solution.

[0081] 4. Blank solution 3: Take 500 mL of pyridine and 500 mL of DMF, mix well, and you will get the blank solution.

[0082] The above reference solution, blank solution 2, and blank solution 3 were analyzed by gas chromatography.

[0083] The chromatographic conditions were the same as in Example 1.

[0084] Chromatogram as shown Figure 6 As shown, cyclohexylamine interferes with the detection of the SM2 peak, while ethanolamine-DMF and pyridine-DMF as solvents do not interfere with the detection of the SM2 peak.

[0085] Example 4

[0086] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The solution preparation method is as follows:

[0087] 1. Blank solution: Take 500 mL of ethanolamine and 500 mL of DMF, mix well, and the blank solution is obtained.

[0088] 2. Reference solution: Weigh approximately 100 mg of SM2 reference standard accurately and place it in a 50 mL volumetric flask containing an appropriate amount of DMF. Dissolve and dilute to the mark with DMF, and shake well to obtain the reference standard stock solution. Accurately measure 5 mL of the reference standard stock solution into a 200 mL volumetric flask, dilute to the mark with blank solution, and shake well to obtain the reference standard solution. Accurately measure 2 mL of the reference standard solution into a headspace vial and seal it.

[0089] 3. Test solution: Weigh approximately 100 mg of bromhexine hydrochloride accurately and place it in a 20 mL headspace vial. Accurately measure 2 mL of blank solution and place it in the same headspace vial. Seal the vial to obtain the test solution.

[0090] The chromatographic conditions were the same as in Example 1.

[0091] Chromatogram as shown Figures 7-9 As shown, the recovery rate of SM2 was (121%–123%).

[0092] Example 5

[0093] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The solution preparation method is as follows:

[0094] 1. Blank solution: Take 50 mL of ethanolamine and 950 mL of DMF, mix well, and the blank solution is ready.

[0095] 2. Reference solution: Weigh approximately 100 mg of SM2 reference standard accurately and place it in a 50 mL volumetric flask containing an appropriate amount of DMF. Dissolve and dilute to the mark with DMF, and shake well to obtain the reference standard stock solution. Accurately measure 5 mL of the reference standard stock solution into a 200 mL volumetric flask, dilute to the mark with blank solution, and shake well to obtain the reference standard solution. Accurately measure 2 mL of the reference standard solution into a headspace vial and seal it.

[0096] 3. Test solution: Weigh approximately 100 mg of bromhexine hydrochloride accurately and place it in a 20 mL headspace vial. Accurately measure 2 mL of blank solution and place it in the same headspace vial. Seal the vial to obtain the test solution.

[0097] The chromatographic conditions were the same as in Example 1.

[0098] Chromatogram as shown Figures 10-12 As shown, the recovery rate of SM2 was (73%–79%).

[0099] Example 6

[0100] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The chromatographic column is SE-30 (30m×0.32mm×0.25μm), and other conditions are the same as in Example 1.

[0101] Chromatogram as shown Figure 13 As shown, the peak shape of SM2 is poor, with a tail.

[0102] Example 7

[0103] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride. The chromatographic column is DB-624 (30m×0.32mm×1.8μm), and other conditions are the same as in Example 1.

[0104] Chromatogram as shown Figure 14 As shown, SM2 has a poor peak shape and an unstable baseline.

[0105] Example 8

[0106] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride, wherein the volume ratio of organic solvent to acid-binding agent in the test sample solvent is 88:12, and other conditions are the same as in Example 1. The detection effect is comparable to that in Example 1.

[0107] Example 9

[0108] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride, wherein the volume ratio of organic solvent to acid-binding agent in the test sample solvent is 92:8, and other conditions are the same as in Example 1. The detection effect is comparable to that in Example 1.

[0109] Example 10

[0110] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride, wherein the chromatographic conditions are as follows:

[0111] Chromatographic column: CP-Volamine (30m × 0.32mm); temperature maintained at 65℃ for 2 min, then increased to 140℃ at a rate of 18℃ / min and held for 5 min, followed by an increase to 250℃ at a rate of 12℃ / min and held for 8 min; injection port temperature: 240℃; detector (FID) temperature: 270℃; split ratio: 18:1; carrier gas flow rate: 2 ml / min; headspace equilibration temperature: 100℃; equilibration time: 20 min. Other conditions were the same as in Example 1. The detection results were comparable to those in Example 1.

[0112] Example 11

[0113] This embodiment provides a method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride, wherein the chromatographic conditions are as follows:

[0114] Chromatographic column: CP-Volamine (30m × 0.32mm); temperature maintained at 55℃ for 5 min, then increased to 160℃ at a rate of 22℃ / min, held for 2 min, then increased to 230℃ at a rate of 8℃ / min, held for 12 min; injection port temperature: 260℃; detector (FID) temperature: 290℃; split ratio: 22:1; carrier gas flow rate: 4 ml / min; headspace equilibration temperature: 80℃; equilibration time: 40 min. Other conditions were the same as in Example 1. The detection results were comparable to those in Example 1.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride, characterized in that, N-methylcyclohexylamine was dissolved in an organic solvent to serve as a reference solution; bromhexine hydrochloride was dissolved in an organic solvent containing an acid-binding agent to serve as a test solvent, and headspace gas chromatography was used for detection.

2. The method as described in claim 1, characterized in that, The organic solvent is N-methylpyrrolidone, N,N-dimethylformamide, or heptane, preferably N,N-dimethylformamide.

3. The method as described in claim 1, characterized in that, The acid-binding agent is triethylamine, cyclohexylamine, pyridine, or ethanolamine, preferably ethanolamine.

4. The method as described in claim 1, characterized in that, In the test solution, the volume ratio of the organic solvent to the acid-binding agent is (50-95):(5-50), preferably (88-92):(8-12).

5. The method according to any one of claims 1-4, characterized in that, The gas chromatograph uses an SE-30, DB-624, or CP-Volamine column, preferably CP-Volamine, with a preferred size of 30m × 0.32mm.

6. The method according to any one of claims 1-4, characterized in that, The column temperature of the gas chromatograph is programmed, starting at 55-65℃ and holding for 2-5 minutes, then increasing to 140-160℃ at a rate of 18-22℃ per minute and holding for 2-5 minutes, then increasing to 230-250℃ at a rate of 8-12℃ per minute and holding for 8-12 minutes.

7. The method according to any one of claims 1-4, characterized in that, The injection port temperature of the gas chromatograph is 240-260℃.

8. The method according to any one of claims 1-4, characterized in that, The gas chromatograph uses an FID detector with a detector temperature of 270-290℃; and / or the split ratio of the gas chromatograph is (18-22):

1.

9. The method according to any one of claims 1-4, characterized in that, The carrier gas flow rate of the gas chromatograph is 2-4 mL, the headspace equilibrium temperature is 80-100℃, and the equilibrium time is 20-40 minutes.

10. The method according to any one of claims 1-4, characterized in that, The content of N-methylcyclohexylamine hydrochloride in bromhexine hydrochloride was determined by the standard curve method.

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