High performance liquid chromatography method for detecting residual dimethylaminohydrochloride in dimethylaminochloroethane hydrochloride
By employing a derivatization reaction of acetonitrile, benzoylchloroacetonitrile, and sodium hydroxide solution, combined with a specific chromatographic column and gradient elution program, the problem of detecting dimethylaminohydrochloride residues in dimethylaminochloroethane hydrochloride was solved, achieving efficient and accurate detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-24
AI Technical Summary
Existing technologies are insufficient for the efficient detection of dimethylaminohydrochloride residues in dimethylaminochloroethane hydrochloride, especially due to its poor peak shape and weak ultraviolet absorption in gas chromatography columns, which makes detection difficult.
Acetonitrile was used as the solvent, benzoyl chloride acetonitrile solution as the derivatization reagent, and sodium hydroxide solution as the acid-binding agent. Dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride were converted into derivatized products with good UV response by heating reaction, and then detected by combining an octadecylsilane-bonded silica gel column with an optimized gradient elution program.
Complete separation of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride was achieved, significantly improving the detector response signal. The detection limit was as low as 0.2 μg/ml, and it exhibited good sensitivity, linearity, and repeatability, meeting the accuracy requirements for drug analysis.
Smart Images
Figure CN121721177A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a high performance liquid chromatography method for detecting dimethylamine hydrochloride residues in dimethylaminoethyl hydrochloride. BACKGROUND
[0002] Drug impurities refer to chemical components that should not exist in active pharmaceutical ingredients (API, raw materials) or drug formulations. Impurities in raw materials have a wide range of sources, mainly falling into three categories: introduced during the production process, including starting material residues, synthesis intermediates, incomplete removal of solvents and catalysts, and reaction-generated byproducts; generated during storage and compounding, such as degradation of raw materials due to their own chemical instability, or incompatibility reactions with formulation excipients; introduced by external contact, such as substances generated by chemical reactions with drug packaging materials.
[0003] Dimethylaminoethyl hydrochloride has a molecular formula of C4H 11 Cl2N and a molecular weight of 144.04, with a CAS number of 4846-46-7. It is an important fine chemical with a wide range of applications, including medicine, agriculture, and chemical industry. It can be used as a local anesthetic, pesticide, or surfactant, and also as an intermediate for various dyes and pharmaceuticals. Therefore, quality control of dimethylaminoethyl hydrochloride is particularly important, as reducing the content of possible impurities is related to the quality control of subsequent products.
[0004] Dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride are starting materials and intermediates of dimethylaminoethyl hydrochloride, which may remain in the product during the synthesis of dimethylaminoethyl hydrochloride. There have been reports on detection techniques for impurities in dimethylaminoethyl hydrochloride, generally using gas or liquid direct injection methods. However, small molecule amines have adsorptive properties, and dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride have poor peak shapes in gas chromatography columns, requiring the use of alkali-modified chromatography columns, which is costly. Additionally, dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride have weak ultraviolet absorption, making it difficult to detect low concentrations of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride using liquid DAD detectors. SUMMARY
[0005] (I) Technical problems solved: To address the shortcomings of the prior art, the present application provides a high performance liquid chromatography method for detecting dimethylamine hydrochloride residues in dimethylaminoethyl hydrochloride, which solves the problems mentioned in the background.
[0006] (II) Technical solutions: To achieve the above objectives, the present invention provides the following technical solution: a high-performance liquid chromatography (HPLC) method for detecting dimethylaminoacetonitrile residues in dimethylaminochloroethane hydrochloride, using acetonitrile as a solvent, benzoylchloroacetonitrile solution as a derivatizing reagent, and sodium hydroxide solution as an acid-binding agent. Dimethylamine hydrochloride, N,N-dimethylethanolamine hydrochloride, and benzoylchloroacetonitrile solution are reacted under heating conditions. The HPLC detection method includes the following steps: S01. Prepare the reference solution and sample solution; S02, HPLC detection: An octadecylsilane-bonded silica gel column was used as the stationary phase; acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase; a DAD detector was used; gradient elution was performed; S03. Result Calculation: Inject multiple reference solutions and dimethylaminochloroethane hydrochloride sample solution sequentially according to the chromatographic conditions in step S02, record the DAD spectrum, and plot a linear correlation curve based on the chromatographic data and concentration data of multiple reference solutions. Substitute the chromatographic data of the dimethylaminochloroethane hydrochloride sample into the curve and calculate the residual concentration of the reference standard in the sample, thereby completing the residual determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride in dimethylaminochloroethane hydrochloride.
[0007] Preferably, in step S01, the specific preparation method of the reference solution and sample solution is as follows: accurately weigh dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride reference standards, add acetonitrile solution to dilute and make up to volume, take a certain amount of solution, add 25 mg / ml benzoyl chloride acetonitrile solution, then add 10 mg / ml sodium hydroxide solution and make up to volume, heat in a water bath at 60°C for 3 hours, cool to room temperature, take the supernatant and filter to prepare multiple reference solutions with concentration gradients; Accurately weigh dimethylaminochloroethane hydrochloride, dissolve it in a certain amount of water, then add benzoylchloroacetonitrile solution and sodium hydroxide solution respectively and make up to volume. Heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant and filter to obtain the sample solution.
[0008] Preferably, the concentrations of the reference solution are, in sequence, 0.4, 1.0, 2.0, 3.0, 4.0, and 8.0 μg / ml of dimethylamine hydrochloride; and the concentrations of N,N-dimethylethanolamine hydrochloride are, in sequence, 0.4, 5.0, 10.0, 15.0, 20.0, and 40.0 μg / ml.
[0009] Preferably, in step S02, the chromatographic column is an Agilent ZORBAX SB-C18 5µm, 4.6 250mm.
[0010] Preferably, in step S02, gradient elution is performed with an initial acetonitrile ratio of 5% for 3 minutes, followed by a rise to 15% within 6 minutes for 3 minutes, a rise to 90% within 16 minutes for 1 minute, and a drop back to 5% for 6 minutes, with a flow rate of 1.0 ml / min.
[0011] Preferably, in step S02, the injection volume is 1 μl to 5 μl.
[0012] Preferably, in step S02, the detection wavelength is 210nm~230nm.
[0013] Preferably, in step S02, the column temperature is 38℃~42℃.
[0014] Preferably, in step S02, the column temperature is 40°C.
[0015] (III) Beneficial Effects: This invention provides a high-performance liquid chromatography (HPLC) method for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl chloroethane hydrochloride. Compared with existing technologies, it has the following advantages: (1) In this invention, by using benzoyl chloride as a derivatization reagent, dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride are converted into derivatized products with good UV response. Combined with an octadecylsilane-bonded silica gel column and an optimized gradient elution program, the two target impurities are completely separated. At the same time, the blank solution has no response peak in the retention time range of the target substance, and the derivatization reaction has no interfering byproducts. The interference of solvent, reagent and sample matrix is completely eliminated, and two structurally similar residual impurities can be accurately distinguished. False positive results are effectively avoided, and the specificity is far superior to the traditional direct injection method.
[0016] (2) In this invention, the technical pain point of weak ultraviolet absorption of small molecule amine hydrochloride is solved by derivatization reaction, which significantly improves the response signal of DAD detector. The detection limit of this method for dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride is as low as 0.2 μg / ml, and the quantitation limit can reach 0.4 μg / ml. Moreover, it has excellent linearity over a wide concentration range (r≥0.9995), which can accurately capture trace residues in the sample and fully meet the stringent requirements for low concentration impurity detection in the quality control of dimethylaminochloroethane hydrochloride.
[0017] (3) In this invention, continuous injection tests show that the retention time RSD of the two target substances is ≤0.1% and the peak area RSD is only 0.6%, which proves that the method has extremely strong repeatability and small operational error. In the accuracy test at concentration levels of 50%-150%, the average recovery rate is 104.7%-113.4% and the RSD is ≤1.2%, which meets the industry standards in the field of pharmaceutical analysis. This ensures that the test results can truly reflect the actual residual amount of impurities in the sample and provide scientific and reliable data support for product quality assessment.
[0018] In summary, this invention can effectively detect the residues of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride in dimethylaminochloroethane hydrochloride, with a limit of quantitation of 0.4 μg / ml for dimethylamine hydrochloride. Compared with existing direct injection methods using gas chromatography and liquid chromatography, the reagents and consumables used are all conventional, the operation is safe and simple, and the processing is convenient and fast, improving detection efficiency and reducing costs. The equipment is readily available, the chromatographic baseline is stable and does not drift, and the DAD detector has less interference, improving the precision and accuracy of the method. It has good sensitivity, linearity, precision, accuracy and stability for the determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride residues, and has important research value in the quality study of dimethylaminochloroethane hydrochloride and the analysis and control of impurities. Attached Figure Description
[0019] Figure 1 This is a specific chromatogram of dimethylamine hydrochloride in an embodiment of the present invention; Figure 2 This is a specific chromatogram of N,N-dimethylethanolamine hydrochloride in an embodiment of the present invention; Figure 3 This is a chromatogram of dimethylamine hydrochloride in the mixed reaction solution of an embodiment of the present invention; Figure 4 This is a chromatogram of the mixed reaction solution N,N-dimethylethanolamine hydrochloride in an embodiment of the present invention; Figure 5 The above are liquid chromatograms of dimethylamine hydrochloride in blank solution, reference solution and 100% accuracy injection solution in the embodiments of the present invention; Figure 6 The above are liquid chromatograms of N,N-dimethylethanolamine hydrochloride in blank solution, reference solution and 100% accuracy injection solution in the embodiments of the present invention. Figure 7 This is the working curve for the linearity test of dimethylamine hydrochloride in the embodiments of the present invention; Figure 8 This is the working curve for the linearity test of N,N-dimethylethanolamine hydrochloride in the embodiments of the present invention. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] The method for detecting dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride provided by this invention uses acetonitrile as a solvent, benzoyl chloride acetonitrile solution as a derivatizing reagent, and sodium hydroxide solution as an acid-binding agent. The inventors discovered that the reaction of dimethylamine hydrochloride, N,N-dimethylethanolamine hydrochloride, and benzoyl chloride acetonitrile solution in acetonitrile under heating conditions is mild, rapid, and complete. The solution can be directly detected after preparation, without the need for complex derivatizing reagents and extraction. This method is applicable to dimethylaminochloroethane, dimethylaminochloroethane hydrochloride, and other salt forms. This invention uses dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride as examples. Other salt forms of dimethylamine and N,N-dimethylethanolamine can also be applied to the method described in this invention, including the following steps: (1) Preparation of reference solutions and sample solutions: Accurately weigh appropriate amounts of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride reference standards, dilute with acetonitrile solution and make up to volume, take a certain amount of solution, add benzoyl chloride acetonitrile solution, add sodium hydroxide solution and make up to volume, heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant and filter to prepare multiple reference solutions with concentration gradients; accurately weigh dimethylaminochloroethane hydrochloride, dissolve in a certain amount of water, add benzoyl chloride acetonitrile solution and sodium hydroxide solution respectively and make up to volume, heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant and filter to obtain the sample solution.
[0022] (2) HPLC method conditions: The chromatographic column used was an Agilent ZORBAX SB-C18 5µm, 4.6 250mm; Gradient elution was performed with an initial acetonitrile concentration of 5% for 3 min, followed by a 6-minute increase to 15% and a 3-minute hold, then a 16-minute increase to 90% and a 1-minute hold, followed by a decrease back to 5% and a 6-minute hold. The flow rate was 1.0 ml / min, the injection volume was 2 μl, the column temperature was 40 °C, and a DAD detector was used with detection wavelengths of 210 nm and 230 nm.
[0023] (3) Calculation of results: The multiple reference solutions and sample solutions were injected sequentially under the chromatographic conditions of step (2), and the chromatograms were recorded. A linear correlation working curve was prepared based on the chromatographic data and concentration data of the multiple reference solutions. The chromatographic data of the sample were substituted into the curve to calculate and obtain the concentration of the sample solution, thus completing the determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride residues.
[0024] Example 1: Specificity study for the determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride residues in dimethylaminochloroethane hydrochloride: Accurately weigh an appropriate amount of dimethylamine hydrochloride reference standard, dilute it with acetonitrile solution and make up to volume. Take a certain amount of the solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and make up to volume to a solution containing 2.0 μg / ml dimethylamine hydrochloride per milliliter. Heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant, filter it, and label it as dimethylamine hydrochloride reaction solution.
[0025] Accurately weigh an appropriate amount of N,N-dimethylethanolamine hydrochloride reference standard, dilute with acetonitrile solution and bring to volume. Take a certain amount of the solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and bring to volume to a solution containing 10.0 g / ml of N,N-dimethylethanolamine hydrochloride per milliliter. Heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant, filter, and label it as N,N-dimethylethanolamine hydrochloride reaction solution.
[0026] Accurately weigh appropriate amounts of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride reference standards, dilute with acetonitrile solution and bring to volume. Take a certain amount of the solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and bring to volume to prepare a solution containing 2.0 μg / ml dimethylamine hydrochloride and 10.0 μg / ml N,N-dimethylethanolamine hydrochloride per milliliter. Heat in a water bath at 60℃ for 3 hours, cool to room temperature, filter the supernatant, and label it as the mixed reaction solution.
[0027] HPLC analysis was performed on the reaction solution of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride. Method conditions: The chromatographic column used was an Agilent ZORBAX SB-C18 5µm, 4.6 250mm; Gradient elution was performed with an initial acetonitrile concentration of 5% for 3 min, followed by a 6-minute increase to 15% and a 3-minute hold, then a 16-minute increase to 90% and a 1-minute hold, followed by a decrease back to 5% and a 6-minute hold. The flow rate was 1.0 ml / min, the injection volume was 2 μl, the column temperature was 40 °C, and a DAD detector was used with detection wavelengths of 210 nm and 230 nm. The DAD spectrum of the dimethylamine hydrochloride reaction solution is shown below. Figure 1 As shown, the DAD spectrum of the N,N-dimethylethanolamine hydrochloride reaction solution is as follows. Figure 2 As shown, the DAD spectrum of the mixed reaction solution is as follows: Figure 3 As shown, dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride were completely converted in the solution after adding 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution and then adding 0.2 ml of 10 mg / ml sodium hydroxide solution and heating in a water bath at 60°C for 3 hours. This derivatization reaction was mild, rapid and complete.
[0028] Depend on Figure 1 It can be seen that the derivatized dimethylamine hydrochloride presents a single, sharp chromatographic peak with symmetrical peak shape and no tailing or interference from other peaks; indicating that the benzoyl chloride derivatization reaction is highly targeted, does not produce byproducts that affect detection, and has excellent separation effect of the target analyte.
[0029] Depend on Figure 2 It can be seen that after derivatization, this impurity also produces a single pure peak with a regular shape, similar to... Figure 1 The retention times of dimethylamine hydrochloride showed a significant difference (approximately 11.3 min vs. approximately 14.8 min), laying the foundation for the subsequent separation of the two impurities in the mixed system.
[0030] Depend on Figure 3 Can Figure 4 It can be seen that the chromatographic peaks of the two impurities are completely separated, with no overlap or peak shape distortion, and the resolution meets the requirements for quantitative analysis (resolution ≥ 1.5); this proves that the method can effectively distinguish two structurally similar residual impurities, avoid mutual interference, and has extremely high specificity.
[0031] Example 2: Limits of detection, sensitivity, and linearity studies for the determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride residues in dimethylaminochloroethane hydrochloride: Accurately weigh dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride, dilute with acetonitrile solution and bring to volume. Take a certain amount of solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and bring to volume to prepare a solution containing 100.0 μg / ml dimethylamine hydrochloride and 500.0 μg / ml N,N-dimethylethanolamine hydrochloride per milliliter. Label this solution as reference stock solution 1.
[0032] Accurately weigh dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride, dilute with acetonitrile solution and bring to volume. Take a certain amount of solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and bring to volume to prepare a solution containing 100.0 μg / ml dimethylamine hydrochloride and 100.0 μg / ml N,N-dimethylethanolamine hydrochloride per milliliter. Label this solution as reference stock solution 2.
[0033] HPLC analytical method conditions: The chromatographic column used was an Agilent ZORBAX SB-C18 5µm, 4.6 250mm; Gradient elution was performed with an initial acetonitrile concentration of 5% for 3 min, followed by a 6-minute increase to 15% and a 3-minute hold, then a 16-minute increase to 90% and a 1-minute hold, followed by a decrease back to 5% and a 6-minute hold. The flow rate was 1.0 ml / min, the injection volume was 2 μl, the column temperature was 40 °C, and a DAD detector was used with detection wavelengths of 210 nm and 230 nm.
[0034] Detection limit test: Accurately transfer 0.05 ml of the reference stock solution 2 into a 25 ml volumetric flask containing a certain amount of acetonitrile, add 1.0 ml of derivatization reagent, then add 0.2 ml of sodium hydroxide solution, dilute to volume with acetonitrile, heat in a water bath at 60 °C for 3 h, cool to room temperature, take the supernatant, filter, and label as the detection limit solution (dimethylamine hydrochloride 0.2 μg / ml, N,N-dimethylethanolamine hydrochloride 0.2 μg / ml).
[0035] In the DAD chromatogram of the detection limit solution test, the signal-to-noise ratio of the target peak dimethylamine hydrochloride was 21, and the signal-to-noise ratio of N,N-dimethylethanolamine hydrochloride was 5, which fully meets the requirements of the Chinese Pharmacopoeia for the limit of quantitation (S / N≥3).
[0036] Sensitivity test: Accurately transfer 0.1 ml of the reference stock solution 2 into a 25 ml volumetric flask containing a certain amount of acetonitrile, add 1.0 ml of derivatization reagent, then add 0.2 ml of sodium hydroxide solution, dilute to volume with acetonitrile, heat in a water bath at 60 °C for 3 h, cool to room temperature, take the supernatant, filter, and label as the sensitivity solution (dimethylamine hydrochloride 0.4 μg / ml, N,N-dimethylethanolamine hydrochloride 0.4 μg / ml).
[0037] In the DAD chromatogram of the sensitivity solution test, the signal-to-noise ratio of the target peak dimethylamine hydrochloride was 85, and the signal-to-noise ratio of N,N-dimethylethanolamine hydrochloride was 11, which fully meets the requirements of the limit of quantitation (S / N≥10) in the Chinese Pharmacopoeia.
[0038] Linearity test: Accurately transfer 0.1 ml of reference stock solution 2, 0.25 ml of reference stock solution 1, 0.5 ml of reference stock solution 1, 0.75 ml of reference stock solution 1, 1.0 ml of reference stock solution 1, and 2.0 ml of reference stock solution 1 into 25 ml volumetric flasks containing a certain amount of acetonitrile. Add 1.0 ml of derivatization reagent, then add 0.2 ml of sodium hydroxide solution, and dilute to volume with acetonitrile. Heat in a water bath at 60°C for 3 hours, cool to room temperature, and filter the supernatant. The concentrations of the obtained linearity test solutions are, in order, dimethylamine hydrochloride 0.4, 1.0, 2.0, 3.0, 4.0, and 8.0 μg / ml, and N,N-dimethylethanolamine hydrochloride 0.4, 5.0, 10.0, 15.0, 20.0, and 40.0 μg / ml.
[0039] In the DAD chromatograms for linearity testing, the linear correlation coefficients for the peak areas of the five test solutions were r = 0.9999 for dimethylamine hydrochloride and r = 0.9995 for N,N-dimethylethanolamine hydrochloride. This indicates that the method exhibits good linearity for dimethylamine hydrochloride in the range of 0.4 μg / ml–18.0 μg / ml and for N,N-dimethylethanolamine hydrochloride in the range of 0.4 μg / ml–40 μg / ml. Figure 7 and Figure 8 ).
[0040] Depend on Figure 7 As can be seen, the horizontal axis represents the concentration (0.4-8.0 μg / ml), and the vertical axis represents the peak area. The data points are evenly distributed on the fitted straight line, and the linear correlation coefficient r=0.9999, which is close to the ideal value of 1.0. This indicates that within the test concentration range, the peak area and concentration of this impurity have a strict linear relationship, and the error in quantitative calculation is extremely small, which can accurately cover low, medium and high residue levels.
[0041] Depend on Figure 8 It can be seen that the linear correlation coefficient r=0.9995 for this impurity in the concentration range of 0.4-40.0 μg / ml, which also shows a very strong linear correlation. Combined with the detection limit / sensitivity data described in the technical solution, it can be confirmed that the method has high sensitivity and can accurately detect low concentration residues of 0.2 μg / ml, which meets the detection requirements of trace impurities in dimethylaminochloroethane hydrochloride.
[0042] Example 3: Precision, accuracy, and stability study of the determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride residues in dimethylaminochloroethane hydrochloride: Accurately weigh dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride, dilute with acetonitrile solution and bring to volume. Take a certain amount of solution, add 1.0 ml of 25 mg / ml benzoyl chloride acetonitrile solution, then add 0.2 ml of 10 mg / ml sodium hydroxide solution and bring to volume to prepare a solution containing 100.0 μg / ml dimethylamine hydrochloride and 500.0 μg / ml N,N-dimethylethanolamine hydrochloride per milliliter. Label this solution as reference stock solution 3.
[0043] HPLC analytical method conditions: The chromatographic column used was an Agilent ZORBAX SB-C18 5 µm, 4.6 250mm; Gradient elution was performed with an initial acetonitrile concentration of 5% for 3 min, followed by a 6-minute increase to 15% and a 3-minute hold, then a 16-minute increase to 90% and a 1-minute hold, followed by a decrease back to 5% and a 6-minute hold. The flow rate was 1.0 ml / min, the injection volume was 2 μl, the column temperature was 40 °C, and a DAD detector was used with detection wavelengths of 210 nm and 230 nm.
[0044] Precision testing: Accurately transfer 0.5 ml of the reference stock solution 3 into a 25 ml volumetric flask containing a certain amount of acetonitrile, add 1.0 ml of derivatization reagent, then add 0.2 ml of sodium hydroxide solution, dilute to volume with acetonitrile, heat in a water bath at 60 °C for 3 h, cool to room temperature, take the supernatant, filter, and label it as the reference solution (dimethylamine hydrochloride 2.0 μg / ml, N,N-dimethylethanolamine hydrochloride 10.0 μg / ml).
[0045] Six consecutive injections of the reference solution were performed. In the DAD chromatogram, the relative standard deviations (RSDs) of the retention times of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride in the six reference solutions were 0.02% and 0.1%, respectively, and the relative standard deviations (RSDs) of the peak areas of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride in the six reference solutions were both 0.6%. This demonstrates that the method exhibits good precision.
[0046] Accuracy test: Nine samples of dimethylaminochloroethane hydrochloride, each containing approximately 50 mg of known residual dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride, were accurately weighed and placed in 25 ml volumetric flasks. 1.0 ml of solvent 1 was added to dissolve the samples. 0.25 ml, 0.50 ml, and 0.75 ml of reference stock solution 3 were transferred to each flask, followed by the addition of acetonitrile, 1.0 ml of derivatization reagent, and 0.2 ml of sodium hydroxide solution. The solutions were then diluted to volume with solvent 2 and heated in a 60°C water bath for 3 hours. After cooling to room temperature, the supernatant was collected and filtered. These solutions were labeled as 50%, 100%, and 150% accuracy solutions, and three recoveries of each were prepared for testing. The overlay diagram of the reference solution and accuracy solution is shown below. Figure 5 and Figure 6 The recovery rates of the nine solutions are shown in Table 1 below. This demonstrates that the method has good accuracy.
[0047] Table 1
[0048] Table 2 As shown in Table 1, the RSD of the recovery rate of dimethylamine hydrochloride at all concentration levels is ≤1.0%, indicating that the quantitative accuracy of the method is stable under different residual concentrations, with no concentration-dependent error, and can truly reflect the actual residual amount of this impurity in the sample.
[0049] As shown in Table 2, the RSD of N,N-dimethylethanolamine hydrochloride at all levels is ≤1.2%, which proves that the method has high reliability in quantifying both impurities, is not affected by the sample matrix, and can effectively avoid false positives or quantitative deviations.
[0050] Depend on Figure 5 and Figure 6 It can be seen that the blank solution has no response peaks within the retention time range of the two target substances, indicating that acetonitrile solvent, benzoyl chloride derivatizing reagent, sodium hydroxide acid-binding agent, etc. will not interfere with the detection. The target peak positions of the reference solution and the sample accuracy solution are completely consistent, further verifying the specificity of the method, ensuring the authenticity of the residue detection results, and eliminating the risk of false positives.
[0051] Stability test: The same reference solution (dimethylamine hydrochloride 2.0 μg / ml, N,N-dimethylethanolamine hydrochloride 10.0 μg / ml) was injected and measured at 0 h, 15 h, and 25 h, respectively. The results showed that the peak areas of dimethylamine hydrochloride at 15 h and 25 h were 101% and 98% of the peak area at 0 h, respectively; the peak areas of N,N-dimethylethanolamine hydrochloride at 15 h and 25 h were 99% and 99% of the peak area at 0 h, respectively; indicating that the reference solution was stable within 25 hours.
[0052] Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.
[0053] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0054] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A high-performance liquid chromatography method for detecting dimethylaminohydrochloride residues in dimethylaminochloroethane hydrochloride, characterized in that, Using acetonitrile as a solvent, benzoyl chloride acetonitrile solution as a derivatizing reagent, and sodium hydroxide solution as an acid-binding agent, dimethylamine hydrochloride, N,N-dimethylethanolamine hydrochloride, and benzoyl chloride acetonitrile solution are reacted under heating conditions. The liquid chromatography detection method includes the following steps: S01. Prepare the reference solution and sample solution; S02, HPLC detection: An octadecylsilane-bonded silica gel column was used as the stationary phase; acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase; a DAD detector was used; gradient elution was performed; S03. Result Calculation: Inject multiple reference solutions and dimethylaminochloroethane hydrochloride sample solution sequentially according to the chromatographic conditions in step S02, record the DAD spectrum, and plot a linear correlation curve based on the chromatographic data and concentration data of multiple reference solutions. Substitute the chromatographic data of the dimethylaminochloroethane hydrochloride sample into the curve and calculate the residual concentration of the reference standard in the sample, thereby completing the residual determination of dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride in dimethylaminochloroethane hydrochloride.
2. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S01, the specific preparation method of the reference solution and sample solution is as follows: accurately weigh dimethylamine hydrochloride and N,N-dimethylethanolamine hydrochloride reference standards, add acetonitrile solution to dilute and make up to volume, take a certain amount of solution, add 25 mg / ml benzoyl chloride acetonitrile solution, then add 10 mg / ml sodium hydroxide solution and make up to volume, heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant and filter to prepare multiple reference solutions with concentration gradients; Accurately weigh dimethylaminochloroethane hydrochloride, dissolve it in a certain amount of water, then add benzoylchloroacetonitrile solution and sodium hydroxide solution respectively and make up to volume. Heat in a water bath at 60℃ for 3 hours, cool to room temperature, take the supernatant and filter to obtain the sample solution.
3. The high-performance liquid chromatography method according to claim 2 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: The concentrations of the reference solutions were, in order, 0.4, 1.0, 2.0, 3.0, 4.0, and 8.0 μg / ml of dimethylamine hydrochloride; and the concentrations of N,N-dimethylethanolamine hydrochloride were, in order, 0.4, 5.0, 10.0, 15.0, 20.0, and 40.0 μg / ml.
4. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, the chromatographic column is an Agilent ZORBAX SB-C18 5µm column, 4.6 μm in diameter. 250mm.
5. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, gradient elution is performed with an initial acetonitrile ratio of 5% for 3 minutes, followed by a rise to 15% within 6 minutes for 3 minutes, a rise to 90% within 16 minutes for 1 minute, and a drop back to 5% for 6 minutes. The flow rate is 1.0 ml / min.
6. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, the injection volume is 1 μl to 5 μl.
7. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, the detection wavelength is 210nm~230nm.
8. The high-performance liquid chromatography method according to claim 1 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, the column temperature is 38℃~42℃.
9. A high-performance liquid chromatography method according to claim 8 for detecting dimethylaminoethyl hydrochloride residues in dimethylaminoethyl hydrochloride, characterized in that: In step S02, the column temperature is 40°C.