Detection method for rapidly monitoring synthesis of octenidine hydrochloride
By monitoring the synthesis process of oteninidine hydrochloride using liquid chromatography, the problem of impurity control was solved, and efficient and low-cost product quality assurance was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-03-03
AI Technical Summary
Existing technologies lack effective methods for monitoring and controlling impurities during the synthesis of octinididine hydrochloride, especially the residues of N-octyl-4-pyridinium amine and monosubstituted impurities, which affect product quality.
Liquid chromatography was used with an octadecyl-bonded silica column and a UV detector to control the reaction process and reduce the residue of monosubstituted products by detecting the contents of octinidin hydrochloride and N-octyl-4-pyridinium.
It enables rapid and accurate impurity control, ensures product quality, reduces instrument operating costs, and improves the simplicity and repeatability of the testing process.
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Figure CN121595752A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical technology for novel broad-spectrum antibacterial agents, and more particularly to a liquid phase detection method for rapidly monitoring the synthesis of oxytinididine hydrochloride. Background Technology
[0002] A novel broad-spectrum antibacterial agent, chemically named N,N'-[1,10-decylidene bis-1(4H)-pyridin-4-ylidene]bis(1-octylamine) dihydrochloride, commonly known as octinidin hydrochloride, CAS number: 70775-75-6, has the following structural formula:
[0003] Otinididine hydrochloride is a novel antibacterial agent with similar effects to quaternary ammonium compounds, but it has a broader antibacterial spectrum. It is commonly used for disinfection of skin, mucous membranes, and open wounds. Otinididine hydrochloride exerts its activity by binding to cytotoxins at its site of action, exhibiting excellent antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. This antibacterial ability is unaffected by interfering substances (such as blood and mucus). Furthermore, otinididine hydrochloride has a strong residual effect on the skin surface; its antibacterial effect can still be observed 24 hours after application. Because it can directly act on the human skin surface and wounds for antibacterial purposes, its product quality directly affects human adaptability during use. Especially since it is a one-step synthesis process, impurity control in the product is particularly critical; therefore, monitoring the reaction during its synthesis is essential. The synthetic route for otinididine hydrochloride is as follows: ; Because side reactions during the reaction process can seriously affect product quality, it is crucial to develop a liquid phase detection method for process monitoring of this reaction.
[0004] The synthesis of octinibidine hydrochloride is a disubstituted reaction. N-octyl-4-pyridinamine is a key raw material in the synthesis of this product, and its amount involved in the reaction is a critical parameter, generally requiring an excess feed. The generation of monosubstituted impurities as a side reaction is closely related to the amount of this raw material fed. Simultaneously, monosubstituted impurities are difficult to remove from the product. Therefore, it is particularly necessary to develop an analytical method that can simultaneously monitor the residues of N-octyl-4-pyridinamine and monosubstituted impurities. This invention develops a rapid detection method for monitoring the reaction process of octinibidine hydrochloride, which monitors the reaction progress in real time and terminates the reaction promptly, thereby better controlling the impurity content at extremely low levels. Currently, there are no relevant detection methods for octinibidine hydrochloride in the prior art. Based on the structure of this product and its impurities, a high-performance liquid chromatography (HPLC) method has been developed. This method is simple and rapid to operate, completely avoiding the occurrence of side reactions and raw material residues. Furthermore, this method has good separation effect and high detection sensitivity, and has passed methodological validation. This method fully meets the requirements for monitoring the reaction process of octinibidine hydrochloride. Summary of the Invention
[0005] The present invention aims to develop a rapid detection method for monitoring the synthesis of octinididine hydrochloride, enabling timely monitoring of the synthesis process and effective control of related impurities. This method features high detection sensitivity and short running time, and can efficiently and accurately control impurities such as N-octyl-4-pyridinamine and monosubstituted impurities.
[0006] The technical solution of this invention is as follows: A rapid detection method for monitoring the synthesis of oxytinididine hydrochloride, wherein the synthetic route of oxytinididine hydrochloride is as follows:
[0007] During synthesis, N-octyl-4-pyridineamine is in excess. The reaction process is controlled by real-time monitoring of the contents of octinididine hydrochloride and N-octyl-4-pyridineamine to reduce the residue of the monosubstituted product 1-(10-chlorodecyl)-N-octylpyridine-4(1H)-imine. The detection was performed using liquid chromatography with an octadecyl-bonded silica column as the packing material. The column length was 150 mm and the inner diameter was 3-4.6 mm. The injection volume was 10-20 μl, the flow rate was 1.0-1.5 ml / min, the detection wavelength was 220 nm-254 nm, the column temperature was 25-35 ℃, and a VWD ultraviolet detector was used. The blank solvent, N-octyl-4-pyridinamide positioning solution, reference solution, and test solution were respectively subjected to liquid chromatography detection, and the chromatograms were recorded. The reaction process of octinidin hydrochloride was rapidly monitored based on the comparison results between the chromatogram of the test solution and the chromatogram of the N-octyl-4-pyridinamide positioning solution.
[0008] The specific testing steps are as follows: (1) Preparation of mobile phase A: Take 1.0 ml of phosphoric acid, place it in a 1000 ml volumetric flask, dilute with water to the mark, shake well, filter, and degas by sonication. (2) Mobile phase B: Acetonitrile; (3) Diluent: Acetonitrile-water, with a volume ratio of acetonitrile to water of 40:60; (4) N-octyl-4-pyridinamide positioning solution: Take an appropriate amount of raw material N-octyl-4-pyridinamide, add diluent to dissolve and dilute to the mark, shake well to obtain a positioning solution of 1.0 mg / ml; (5) Reference solution: Take oxytetracycline hydrochloride reference standard, dissolve and dilute to the mark with diluent, shake well to obtain a 1.0 mg / ml reference solution; (6) Preparation of test solution: Take 1 ml of oxytetracycline hydrochloride intermediate control reaction solution, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, and the solution is obtained; (7) Setting the parameters of the liquid chromatograph: Column: octadecyl bonded silica gel as the packing material, column length 150 mm, column inner diameter 4.6 mm, injection volume 10 μl; flow rate 1.0 ml / min; detection wavelength 230 nm; column temperature 30 ℃; mobile phase A is 0.1% phosphoric acid-water solution, mobile phase B is acetonitrile; gradient elution; Using acetonitrile-water solution as blank solvent, the blank solvent, N-octyl-4-pyridinamine positioning solution, reference solution, and test solution were subjected to liquid chromatography detection, and the chromatograms were recorded. The reaction process of octinidin hydrochloride was rapidly monitored based on the comparison results between the chromatogram of the test solution and the chromatogram of the N-octyl-4-pyridinamine positioning solution.
[0009] The steps for determining the standard curves of oxytinididine hydrochloride and N-octyl-4-pyridinium chloride include the following: S01) Weigh 10 mg each of octyl-4-pyridinium hydrochloride reference standard and N-octyl-4-pyridinium accurately into the same 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain linear mixed stock solution; S02) Accurately transfer 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the linear stock solution into 100 ml volumetric flasks, add diluent to the mark, and shake well to obtain standard curve solutions with concentrations of 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml. S03) The standard curve solution was analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: an octadecyl bonded silica column with a size of 4.6 mm × 150 mm × 5 μm was used; the mobile phase was phosphoric acid-water solution and acetonitrile; gradient elution was used; the flow rate was 1.0 ml / min; the column temperature was 30 ℃; the injection volume was 10 μl; and a VWD ultraviolet detector was used. The standard curve was plotted according to the relationship between the concentration (μg / ml) and the chromatographic peak area.
[0010] By plotting a standard curve, the range of its linear relationship can be determined. Beneficial effects
[0011] The method provided by this invention has low instrument operating costs, a simple and convenient detection process, and good repeatability, and can be used to evaluate product quality.
[0012] The present invention meets the requirements in terms of system applicability and specificity, linearity and scope.
[0013] The detection method provided by this invention can quickly and accurately control the amount of raw materials and oxidized impurities during the reaction process, effectively ensuring product quality. Attached Figure Description
[0014] Figure 1 This is the liquid chromatogram of the control sample of oxytinididine hydrochloride.
[0015] Figure 2 The liquid chromatogram for N-octyl-4-pyridinamine is shown.
[0016] Figure 3 This is the liquid chromatogram of oltinidin hydrochloride reference standard.
[0017] Figure 4 This is the hydrogen spectrum of oxytetracycline hydrochloride.
[0018] Figure 5 This is the carbon spectrum of oxytinidin hydrochloride.
[0019] Figure 6 The graph shows the linearity of oxytinididine hydrochloride.
[0020] Figure 7 The graph shows the linearity of N-octyl-4-pyridineamine. Detailed Implementation
[0021] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Example 1
[0022] A rapid detection method for monitoring the reaction process of otinibidine hydrochloride, wherein the synthetic route of otinibidine hydrochloride is as follows:
[0023] During synthesis, N-octyl-4-pyridineamine is in excess. The reaction process is controlled by real-time monitoring of the contents of octinididine hydrochloride and N-octyl-4-pyridineamine to reduce the residue of the monosubstituted product 1-(10-chlorodecyl)-N-octylpyridine-4(1H)-imine. The relevant material information is as follows:
[0024] The detection was performed using liquid chromatography with an octadecyl-bonded silica column as the packing material. The column length was 150 mm and the inner diameter was 3-4.6 mm. The injection volume was 10-20 μl, the flow rate was 1.0-1.5 ml / min, the detection wavelength was 220 nm-254 nm, the column temperature was 25-35 ℃, and a VWD ultraviolet detector was used. The blank solvent, N-octyl-4-pyridinamide positioning solution, reference solution, and test solution were respectively subjected to liquid chromatography detection, and the chromatograms were recorded. The reaction process of octinidin hydrochloride was rapidly monitored based on the comparison results between the chromatogram of the test solution and the chromatogram of the N-octyl-4-pyridinamide positioning solution.
[0025] Specifically, the following steps are included: (1) Preparation of mobile phase A: Take 1.0 ml of phosphoric acid, place it in a 1000 ml volumetric flask, dilute with water to the mark, shake well, filter, and degas by sonication. (2) Mobile phase B: Acetonitrile; (3) Diluent: Acetonitrile-water, with a volume ratio of acetonitrile to water of 40:60; (4) N-octyl-4-pyridinamide localization solution: Weigh 10 mg of N-octyl-4-pyridinamide accurately into a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain a 1.0 mg / ml N-octyl-4-pyridinamide localization solution; (5) Reference solution: Weigh 10 mg of oxytetracycline hydrochloride reference standard accurately into a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain an oxytetracycline hydrochloride reference solution with a concentration of 1.0 mg / ml; (6) Test solution: Take 1 ml of octinidin hydrochloride intermediate control reaction solution, put it into a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, and you will get the test solution.
[0026] (7) Setting the parameters of the liquid chromatograph: The liquid chromatograph is an Agilent 1260, the software is Openlab, the column is an octadecyl bonded silica column (Agilent HC C18 4.6×150mm 5μm), the injection volume is 10μl; the flow rate is 1.0ml / min; the detection wavelength is 230nm; the column temperature is 30℃; the mobile phase A is 0.1% phosphoric acid-water solution, and the mobile phase B is acetonitrile; gradient elution is performed, and the gradient program is as follows:
[0027] Using the diluent as a blank solution, the blank solution, the N-octyl-4-pyridinamide localization solution, the reference solution, and the test solution were subjected to high-performance liquid chromatography (HPLC). Chromatograms were recorded. Based on the chromatogram results of the test solution, the normalized contents of N-octyl-4-pyridinamide and monosubstituted impurities were obtained. Simultaneously, the chromatogram of the N-octyl-4-pyridinamide peak localization solution was obtained. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, the 1H and 1C spectra of oltinidin hydrochloride are detailed in [reference needed]. Figure 4 and Figure 5 .
[0028] In the test solution, the retention time of N-octyl-4-pyridinamine was 5.592 min, the retention time of the monosubstituted impurity was 9.955 min, and the retention time of oteninidine hydrochloride was 12.908 min. The minimum resolution was 2.01, which meets the requirement. Generally, a resolution greater than or equal to 1.5 is considered to indicate baseline separation between the two chromatographic peaks, meaning they are completely separated and do not interfere with each other. The minimum resolution in this example is 2.01, which is far higher than the requirement of 1.5.
[0029] The steps for determining the standard curves of oxytinididine hydrochloride and N-octyl-4-pyridinium chloride include: S01) Weigh 10 mg each of octyl-4-pyridinium hydrochloride reference standard and N-octyl-4-pyridinium accurately into the same 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain linear mixed stock solution; S02) Accurately transfer 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the linear stock solution into 100 ml volumetric flasks, add diluent to the mark, and shake well to obtain standard curve solutions with concentrations of 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml. S03) The standard curve solution was analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: an octadecyl bonded silica column with a size of 4.6×150mm×5μm was used; the mobile phase was phosphoric acid-water solution and acetonitrile; gradient elution was used; the flow rate was 1.0ml / min; the column temperature was 30℃; the injection volume was 10μl; a VWD ultraviolet detector was used; and the detection wavelength was 230nm. The standard curve was plotted according to the relationship between the concentration (μg / ml) and the chromatographic peak area.
[0030] Example 2 Specificity and System Applicability The blank solution, N-octyl-4-pyridinamide solution, octinidinidine hydrochloride reference solution, and test solution were injected according to the chromatographic conditions of Example 1 for specificity detection. The results are shown in Table 1.
[0031] Table 1: Specificity Detection Results
[0032] As shown in Table 1, the minimum resolution of the test sample solution is 2.01, and the resolution is greater than 1.5, which meets the separation requirements. Reference solution: Take an appropriate amount of oxytinididine hydrochloride reference standard, dissolve and dilute to the mark with diluent, shake well to obtain a reference solution with a concentration of 1.0 mg / ml; inject the reference solution into the liquid chromatograph and inject 6 times consecutively. The detection results are shown in Table 2.
[0033] Table 2 Detection results of reference solution
[0034] As shown in Table 2, the peak area and retention time RSD of the reference solution were both less than 2.0% after six consecutive injections.
[0035] Linearity and Range Preparation of linear stock solution: Weigh 10 mg each of N-octyl-4-pyridinamide and octinidin hydrochloride reference standards into the same 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain the solution.
[0036] Preparation of 20% linear solution: Take 0.2 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0037] Preparation of 50% linear solution: Take 0.5 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0038] Preparation of 80% linear solution: Take 0.8 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0039] Preparation of 100% linear solution: Take 1.0 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0040] Preparation of 150% linear solution: Take 1.5 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0041] Preparation of 200% linear solution: Take 2.0 ml of linear stock solution and place it in a 100 ml volumetric flask. Add diluent to the mark and shake well.
[0042] Take 10 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, plot the standard curve with concentration as the x-axis and peak area as the y-axis, and calculate the regression equation and correlation coefficient r. At the same time, calculate the correction factors for N-octyl-4-pyridinamide and octinidin hydrochloride. The linearity results of octinidin hydrochloride are shown in Table 3, and the linearity results of N-octyl-4-pyridinamide are shown in Table 4. Table 3. Linearity results of oltinidin hydrochloride
[0043] Table 4. Linearity results for N-octyl-4-pyridineamine
[0044] As shown in the table above, the correction factor F for N-octyl-4-pyridinamide is calculated as: Main peak slope / Impurity slope = 0.8205 / 1.1994 = 0.68. Therefore, the area normalization method can be used to detect N-octyl-4-pyridinamide during the monitoring of the otinidin hydrochloride reaction. Since the correction factor of 0.68 overestimates the amount of impurities, a limit of 0.1% is sufficient to meet the impurity control requirements of the intermediate and subsequent active pharmaceutical ingredient.
[0045] Example 4 A rapid liquid-phase detection method for monitoring the synthesis of oteninidine hydrochloride, specifically including the following steps in the detection of the reaction solution for producing oteninidine hydrochloride intermediates: Take 1.0 ml of the central control reaction solution into a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, mix well, and inject blank solvent, N-octyl-4-pyridinamide positioning solution and reaction solution according to the chromatographic conditions described in Example 1. Record the chromatogram, calculate the purity of octinidin hydrochloride and the content of N-octyl-4-pyridinamide and monosubstituted impurities according to the area normalization method. The results are shown in Table 5. Table 5. Test results of each batch of central control reaction solution
[0046] As shown in Table 5, in the central control reaction solution, the normalized content of N-octyl-4-pyridinamide was 6.91%, and the content of monosubstituted impurities was 0.15%. As the reaction continued, in the final product, the purity of octinididine hydrochloride was 99.94%, N-octyl-4-pyridinamide was not detected, and the normalized content of monosubstituted impurities was 0.04%. This indicates that the method can accurately monitor the reaction and meet the experimental requirements.
[0047] The above demonstrates that this method can rapidly and accurately monitor the changes in raw materials during the reaction of octinibidine hydrochloride. Methodological validation has shown that the content of N-octyl-4-pyridinamine and monosubstituted impurities in the reaction solution can be quantitatively controlled using the area normalization method, with accurate and reliable results. This method can rapidly and accurately monitor the reaction process of octinibidine hydrochloride and strictly control the amount of N-octyl-4-pyridinamine and monosubstituted impurities to provide octinibidine hydrochloride with stable quality.
Claims
1. A rapid detection method for monitoring the synthesis of oxytinididine hydrochloride, characterized in that, The synthetic route for the otinididine hydrochloride is as follows: ， During synthesis, N-octyl-4-pyridineamine was used in excess. The reaction process was controlled by real-time monitoring of the contents of octinididine hydrochloride and N-octyl-4-pyridineamine to reduce the residual monosubstituted product 1-(10-chlorodecyl)-N-octylpyridine-4(1H)-imine. The detection was performed using liquid chromatography, with an octadecyl bonded silica column as the packing material. The column length was 150 mm and the inner diameter was 3-4.6 mm. The injection volume was 10-20 μl, the flow rate was 1.0-1.5 ml / min, the detection wavelength was 220 nm-254 nm, and the column temperature was 25-35 ℃. A VWD ultraviolet detector was used. The blank solvent, N-octyl-4-pyridinamide positioning solution, reference solution, and test solution were respectively subjected to liquid chromatography detection, and the chromatograms were recorded. The reaction process of octinidin hydrochloride was rapidly monitored based on the comparison results between the chromatogram of the test solution and the chromatogram of the N-octyl-4-pyridinamide positioning solution.
2. The method for rapid monitoring of oxytinididine hydrochloride synthesis according to claim 1, characterized in that, The specific testing steps are as follows: (1) Preparation of mobile phase A: Take 1.0 ml of phosphoric acid, place it in a 1000 ml volumetric flask, dilute with water to the mark, shake well, filter, and degas by sonication. (2) Mobile phase B: Acetonitrile; (3) Diluent: Acetonitrile-water, with a volume ratio of acetonitrile to water of 40:60; (4) N-octyl-4-pyridinamide positioning solution: Take an appropriate amount of raw material N-octyl-4-pyridinamide, add diluent to dissolve and dilute to the mark, shake well to obtain a positioning solution of 1.0 mg / ml; (5) Reference solution: Take oxytetracycline hydrochloride reference standard, dissolve and dilute to the mark with diluent, shake well to obtain a 1.0 mg / ml reference solution; (6) Preparation of test solution: Take 1 ml of oxytetracycline hydrochloride intermediate control reaction solution, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, and the solution is obtained; (7) Setting the parameters of the liquid chromatograph: Column: octadecyl bonded silica gel as the packing material, column length 150 mm, column inner diameter 4.6 mm, injection volume 10 μl; flow rate 1.0 ml / min; detection wavelength 230 nm; column temperature 30 ℃; mobile phase A is 0.1% phosphoric acid-water solution, mobile phase B is acetonitrile; gradient elution; Using acetonitrile-water solution as blank solvent, the blank solvent, N-octyl-4-pyridinamine positioning solution, reference solution, and test solution were subjected to liquid chromatography detection, and the chromatograms were recorded. The reaction process of octinidin hydrochloride was rapidly monitored based on the comparison results between the chromatogram of the test solution and the chromatogram of the N-octyl-4-pyridinamine positioning solution.
3. The method for rapid monitoring of oxytinididine hydrochloride synthesis according to claim 1, characterized in that, This includes the determination of standard curves for oxytinididine hydrochloride and N-octyl-4-pyridineamine.
4. The method for rapid monitoring of oxytinididine hydrochloride synthesis according to claim 3, characterized in that, The steps for determining the standard curves of oxytinididine hydrochloride and N-octyl-4-pyridinium chloride include: S01) Weigh 10 mg each of octyl-4-pyridinium hydrochloride reference standard and N-octyl-4-pyridinium accurately into the same 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain linear mixed stock solution; S02) Accurately transfer 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the linear stock solution into 100 ml volumetric flasks, add diluent to the mark, and shake well to obtain standard curve solutions with concentrations of 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml. S03) The standard curve solution was analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: an octadecyl bonded silica column with a size of 4.6×150mm×5μm was used; the mobile phase was phosphoric acid-water solution and acetonitrile; gradient elution was used; the flow rate was 1.0ml / min; the column temperature was 30℃; the injection volume was 10μl; and a VWD ultraviolet detector was used. The standard curve was plotted according to the relationship between the concentration (μg / ml) and the chromatographic peak area.