Determination method of ethyl nicotinate related substances
The content of niacin in niacin ethyl niacin was detected by liquid chromatography, which solved the problem of difficulty in effectively detecting and controlling the quality of niacin in the prior art, and achieved the stability and safety of product quality.
Patent Information
- Application Number
- CN202510110377.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to effectively detect and control the content of niacin in ethyl niacin, affecting product quality control.
The content of impurity A (niacin) in the test sample solution was calculated by preparing appropriate mobile phase and sample solution, combined with relative correction factors.
Accurate detection of the content of niacin in ethyl niacin is achieved, ensuring the stability and safety of product quality.
Smart Images
Figure CN119619368A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining nicotinate ethyl ester-related substances. Background Art
[0002] As an important organic compound, nicotinate ethyl ester has a wide range of applications in the field of organic synthesis. It can be used as a raw material or intermediate for the synthesis of other compounds and participate in a variety of chemical reactions. At the same time, it is also used as a raw material or intermediate for drug synthesis; through specific chemical reactions, nicotinate ethyl ester can be converted into pharmacologically active compounds for the treatment or prevention of certain diseases. Nicotinic acid ethyl ester has a variety of pharmacological effects such as lowering blood lipids and anti-oxidation, and is widely used in the pharmaceutical industry. It is an ester compound generated by the reaction of nicotinic acid and ethanol. The specific reaction formula is as follows: ; Nicotinic acid and ethanol react at appropriate temperature and under the action of catalyst to produce nicotinate ethyl ester; then nicotinate ethyl ester is separated from the reactants by distillation and crystallization. During the synthesis of nicotinate ethyl ester, part of nicotinic acid (impurity A) is not reacted to form impurities, and toluene may remain in the process production. The production process and quality control of nicotinate ethyl ester are important links to ensure product quality. Therefore, the detection of related substances in nicotinate ethyl ester is crucial. Summary of the invention
[0003] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for determining related substances of nicotinate ethyl ester. The nicotinic acid content in nicotinate ethyl ester is controlled by the method of the present invention, thereby achieving quality control of nicotinate ethyl ester.
[0004] Technical solution: The present invention provides a method for determining related substances of nicotinate ethyl ester, comprising the following steps: (1) Prepare the mobile phase: acetic acid-water-acetonitrile mixture in a ratio of 1:29:70; (2) Prepare sample solution: Accurately weigh an appropriate amount of nicotinate ethyl ester sample to be tested, add mobile phase to dissolve and dilute, and shake well to obtain sample solution; (3) Prepare the test solution: accurately measure an appropriate amount of the sample solution, add the mobile phase to dilute it, and shake well to obtain the test solution; (4) Prepare the control solution: accurately measure an appropriate amount of the test solution, dilute it with the mobile phase, and shake well to obtain the control solution; (5) Determination: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram until 3 times the peak time of the main component peak; Chromatographic conditions: using octadecylsilane bonded silica gel as filler (4.0*250mm, 4μm) Detection wavelength: 261nm; Flow rate: 1.0ml / min; Injection volume: 20 μl; The related substance is nicotinic acid (impurity A).
[0005] Furthermore, the principal component self-control method with a relative correction factor was used to calculate the content of impurity A in the test solution using the peak area; the relative correction factor of impurity A was 0.77.
[0006] Furthermore, before step (5), the method further includes: accurately measuring the system suitability solution and the toluene positioning solution, injecting them into a liquid chromatograph respectively, and recording a chromatogram.
[0007] Furthermore, the system suitability solution is prepared by: accurately weighing an appropriate amount of niacin reference substance, adding a mobile phase to dissolve and dilute to obtain a niacin reference substance solution; accurately measuring an appropriate amount of niacin reference substance solution and sample solution, adding a mobile phase to dilute, and shaking to obtain a system suitability solution; The preparation method of the toluene positioning solution is: accurately weigh an appropriate amount of toluene, add the mobile phase to dissolve and dilute, and shake well to obtain the toluene positioning solution. (In the process of nicotinate ethyl ester production, there may be toluene residues in the product, so the toluene solution is positioned. The toluene is not involved in the calculation after positioning and will be removed in the later stage of the process) Furthermore, the content of nicotinic acid in the system suitability solution is 0.005 mg / ml; the content of nicotinic acid ethyl ester is 0.005 mg / ml.
[0008] Furthermore, the content of toluene in the toluene positioning solution is 0.002 mg / ml.
[0009] Furthermore, the content of nicotinic acid ethyl ester in the control solution is 0.002 mg / ml; the content of nicotinic acid ethyl ester in the test solution is 0.1 mg / ml.
[0010] Beneficial effects: Compared with the prior art, the method of the present invention can effectively detect related substances in ethyl nicotinate, and the detection method is simple, easy, convenient and fast. The present invention can ensure that the product quality is stable, safe and controllable, provide a guarantee for safe medication, and further improve the drug quality and drug safety of ethyl nicotinate. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the chromatogram of nicotinate ethyl ester reference solution; Figure 2 is the chromatogram of the test solution; Figure 3 is the system suitability solution chromatogram; Figure 4 Position the solution chromatogram for toluene; Figure 5 This is the standard curve of related substances of nicotinate ethyl ester. DETAILED DESCRIPTION
[0012] The present invention is described in detail below in conjunction with the embodiments.
[0013] Implementation 1: The determination method of related substances of nicotinate ethyl ester is as follows: 1. Chromatographic conditions Chromatographic column: Octadecylsilane bonded silica gel as filler (4.0*250mm, 4μm) Mobile phase: acetic acid: water: acetonitrile (1:29:70) Detection wavelength: 261nm Flow rate: 1.0ml / min Injection volume: 20 μl 2. Solution preparation Sample solution: Take about 10 mg of nicotinic acid ethyl ester sample, weigh accurately, place in a 10 ml volumetric flask, dissolve with mobile phase and make up to the mark, shake well.
[0014] System suitability solution: Take about 25 mg of niacin reference substance, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it with mobile phase and make up to the scale, accurately measure 0.5 ml of niacin reference solution, add 0.5 ml of sample solution, place it in a 100 ml volumetric flask, add mobile phase to make up to the scale, shake well, and set aside.
[0015] Control solution: Accurately measure 1 ml of the sample solution, place it in a 50 ml volumetric flask, dissolve it with mobile phase and make up to the mark, then accurately measure 1 ml of the solution, place it in a 10 ml volumetric flask, make up to the mark with mobile phase, shake well and set aside.
[0016] Toluene fixation solution: Take about 10 mg of toluene, weigh accurately, place in a 10 ml volumetric flask, dissolve with mobile phase and dilute to scale, shake well, and set aside. Accurately measure 1 ml, place in a 50 ml volumetric flask, dissolve with mobile phase and dilute to scale, then accurately measure 1 ml of solution, place in a 10 ml volumetric flask, dilute to scale with mobile phase, shake well, and set aside.
[0017] 3. System applicability Accurately measure 20 μl of the system suitability solution, inject it into the liquid chromatograph, and record the separation degree of the main peaks of nicotinic acid (impurity A) and nicotinate ethyl ester, which should be no less than 2. Accurately measure 20 μl of the toluene positioning solution, inject it into the liquid chromatograph, and record the chromatogram.
[0018] 4. Determination Accurately measure 20 μl of the test solution and the reference solution, and record the chromatogram to about 3 times the peak time of the main component peak. If there is impurity A in the chromatogram of the test solution, the main component self-control method with relative correction factor is used to calculate the content of impurity A in the test solution by peak area. The peak area of impurity A is not greater than the peak area of reference solution b (≤0.2%), and the relative correction factor of impurity A is 0.77.
[0019] 5. Calculation Impurity A% = (0.77×A (杂质A) / A (对照) )×1 / 50×1 / 10×100% Where: 0.77—— Relative correction factor of impurity A; A (杂质A) ——The peak area of impurity A in the test solution; A (对照) —— Peak area of the reference solution.
[0020] If there are other single impurities (except toluene), the peak area of the single impurity shall not be greater than 2.5 times (≤0.5%) the peak area of the reference solution b when calculated by the self-control method. The sum of the impurity peak areas (except toluene) shall not be greater than 5 times (≤1.0%) the peak area of the reference solution b when calculated by the self-control method.
[0021] Implementation 2: Specificity of the determination method for related substances of nicotinate ethyl ester: Blank solution: accurately measure 20 μl of blank solvent (mobile phase) and inject it into the liquid chromatograph to record the chromatogram. The results show that the blank solvent does not interfere with the determination of related substances of nicotinate ethyl ester.
[0022] System suitability solution: Take an appropriate amount of nicotinic acid reference substance 25.29mg (about 25mg), accurately weigh, place in a 25ml volumetric flask, dissolve with mobile phase and dilute to scale, accurately measure 0.5ml of nicotinic acid reference solution, add 0.5ml sample solution, place in a 100ml volumetric flask, add mobile phase to dilute to scale, shake well, and set aside. Accurately measure 20μl and inject into the liquid chromatograph, record the chromatogram. The results show that the separation degree between impurity A (nicotinic acid) and the main component (nicotinic acid ethyl ester) is 4.4, and the method system has good adaptability.
[0023] Undamaged samples: Take an appropriate amount of 10.19 mg of nicotinic acid ethyl ester sample, weigh accurately, place in a 10 ml volumetric flask, dilute to the mark with mobile phase, and shake well. Accurately measure 20 μl and inject into the liquid chromatograph to record the chromatogram. The results show that in the undamaged samples, each impurity peak and each impurity and the main component (main peak) can be effectively separated.
[0024] Light-damaged sample: Take 10.37 mg of nicotinic acid ethyl ester, weigh accurately, put it in a 10 ml volumetric bottle, place it under light conditions (5000 lx) for 48 hours, take it out, add mobile phase to the scale, shake well, set aside, accurately measure 20 μl and inject it into the liquid chromatograph, record the chromatogram. The results show that nicotinic acid ethyl ester has good stability under light-damaged conditions.
[0025] High humidity damage sample: Take 10.64 mg of nicotinic acid ethyl ester, weigh accurately, put it in a 10 ml volumetric bottle, place it in high humidity conditions (relative humidity 92.5%) for 48 hours, take it out, add mobile phase to dilute to the scale, shake well, set aside, accurately measure 20 μl and inject it into the liquid chromatograph, record the chromatogram. The results show that nicotinic acid ethyl ester has good stability under high humidity damage conditions.
[0026] High temperature destruction sample: Take 10.53 mg of nicotinic acid ethyl ester, weigh accurately, put it in a 10 ml volumetric flask, put it in a boiling water bath for 5 hours, take it out, cool it, add mobile phase to dilute it to the scale, shake it well, accurately measure 20 μl and inject it into the liquid chromatograph, and record the chromatogram. The results show that nicotinic acid ethyl ester has good stability under high temperature destruction conditions.
[0027] Acid blank: accurately measure 2ml of 2mol / L hydrochloric acid solution, place it in a 10ml volumetric flask, place it at room temperature for 30 minutes, then add 2ml of 2mol / L sodium hydroxide solution to neutralize it, add mobile phase to the scale, shake well, accurately measure 20μl and inject it into the liquid chromatograph, and record the chromatogram. The results show that the acid-destroyed blank does not interfere with the determination of related substances of nicotinate ethyl ester.
[0028] Acid destruction sample: Take 10.70mg of nicotinic acid ethyl ester, weigh accurately, place in a 10ml volumetric flask, add 2ml of 2mol / L hydrochloric acid solution, place at room temperature for 30min, add 2ml of 2mol / L sodium hydroxide solution to neutralize, add mobile phase to the scale, shake well, accurately measure 20μl and inject into the liquid chromatograph, record the chromatogram. Add 1ml of 1mol / l hydrochloric acid solution to the sample for 1min, and the sample is unstable. The results show that the impurity peaks generated can be effectively separated from each other and from each impurity peak to the main peak.
[0029] Alkali blank: accurately measure 1 ml of 0.01 mol / L sodium hydroxide solution, place it in a 10 ml volumetric flask, place it at room temperature for 10 minutes, add 1 ml of 0.01 mol / L hydrochloric acid solution to neutralize, add mobile phase to the scale, shake well, accurately measure 20 μl and inject it into the liquid chromatograph, and record the chromatogram. The results show that the alkali destruction blank does not interfere with the determination of related substances of nicotinate ethyl ester.
[0030] Alkali destruction sample: Take 10.47mg of nicotinic acid ethyl ester, accurately weigh and place in a 10ml volumetric flask, add 1ml of 0.01mol / L sodium hydroxide solution, place at room temperature for 10min, add 1ml of 0.01mol / L hydrochloric acid solution to neutralize, add mobile phase to dilute to scale, shake well, accurately measure 20μl and inject into liquid chromatograph, record chromatogram. The results show that adding 1ml of 0.01mol / l sodium hydroxide solution to the sample for 1min makes the sample unstable, and the impurity peaks generated and the impurity peaks and the main peaks can be effectively separated.
[0031] Oxidation blank: accurately measure 5 ml of hydrogen peroxide solution (30%), place in a 10 ml volumetric flask, place at room temperature for 24 hours, add mobile phase to dilute to scale, shake well, accurately measure 20 μl and inject into the liquid chromatograph, record the chromatogram. The results show that the oxidation blank is destroyed with a solvent peak at about 2 minutes, which does not interfere with the determination of related substances of nicotinate ethyl ester.
[0032] Oxidative damage sample: Take 11.02 mg of nicotinate ethyl ester, weigh accurately, place in a 10 ml volumetric flask, add 5 ml of 30% hydrogen peroxide solution, place at room temperature for 24 hours, add mobile phase to dilute to scale, shake well, accurately measure 20 μl and inject into liquid chromatograph, record chromatogram. The results show that nicotinate ethyl ester has good stability under oxidative conditions.
[0033] The results showed that the blank solvent did not interfere with the determination of related substances of nicotinate ethyl ester, nicotinate ethyl ester was stable under high temperature, high humidity, light and oxidation conditions, and unstable under acidic and alkaline conditions, and the impurity peaks produced by degradation and between the impurity peaks and the main peak could be effectively separated. The determination method of related substances of nicotinate ethyl ester had good specificity.
[0034] Implementation 3: Detection limit, quantification limit and repeatability of quantification limit for determination of related substances of nicotinate ethyl ester: 1. Detection limit of impurity A The detection limit was determined by the signal-to-noise ratio method, and the corresponding concentration was determined when the signal-to-noise ratio was 3:1. 2.5 ml of the quantitative limit solution was accurately measured into a 10 ml volumetric flask and diluted to the scale with mobile phase. The concentration was 2.7 ng / ml. The signal-to-noise ratio was 4.9, which was 0.13% of the limit, when measured according to the chromatographic conditions of the relevant substances.
[0035] 2. Impurity A Quantitative Limit The quantification limit was determined by the signal-to-noise ratio method, and the corresponding concentration was determined when the signal-to-noise ratio was 10:1. Accurately measure 0.5 ml of impurity A linear 1 solution into a 20 ml volumetric flask, add mobile phase to dilute to the scale, the concentration is 10.9 ng / ml, and the signal-to-noise ratio is 12.0 according to the chromatographic conditions of the relevant substances.
[0036] Repeatability of quantification limit of impurity A Repeat the preparation of 6 portions of the quantitative limit solution, take 20 μl of each portion and inject it into the liquid chromatograph, and record the chromatogram. According to the high performance liquid chromatography method, determine their peak areas under the conditions specified in this protocol. Calculate the ratio of the peak area to the weight of the sample solution, referred to as the F value. The RSD of the F values of the 6 results should meet the requirements (pharmacopoeia requirements).
[0037]
[0038] The results showed that the repeatability of the quantification limit of impurity A met the standard requirements.
[0039] Implementation 4: Linear Relationship and Range of Determination Method for Related Substances of Ethyl Nicotinate Impurity A linear relationship: Take about 10 mg of nicotinic acid (impurity A), accurately weigh it, put it in a 50 ml volumetric flask, dilute to the mark with mobile phase, shake well, and set aside as the reference stock solution.
[0040]
[0041] Accurately measure 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml, 1.5ml, 2.0ml of the reference stock solution, place them in 100ml volumetric flasks, dilute to scale with mobile phase, shake well, and use them as linear 1, linear 2, linear 3, linear 4, linear 5, linear 6, and linear 7 solutions. According to the chromatographic conditions of the relevant substances, measure the quantitative limit respectively, inject 20μl of the above solution into the liquid chromatograph, record the chromatogram, determine the peak area, draw the standard curve with the test sample concentration as the horizontal axis and the peak area as the vertical axis, and calculate the regression equation.
[0042]
[0043] The results showed that the impurity A had a good linear relationship in the concentration range of 0.0109μg / ml to 4.348μg / ml, and the linear equation was y=27267x-384.72, R 2 =0.9998.
[0044] Implementation 5: Solution stability of the determination method of related substances of nicotinate ethyl ester: 1. Verification method Take about 10 mg of nicotinic acid ethyl ester sample, weigh accurately, place in a 10 ml volumetric flask, dissolve with mobile phase and make up to scale, shake well. Store at room temperature, inject every 2 hours, examine the stability of the sample solution within 24 hours, and record the chromatogram.
[0045] 2. Verify the results
[0046] The results showed that the sample solution of nicotinate-related substances was stable within 27 hours at room temperature.
[0047] Implementation 6: Robustness of the determination method for related substances of nicotinate ethyl ester: Verification Method Using the standard addition method, take about 10 mg of nicotinic acid ethyl ester, accurately weigh it, and place it in a 10 ml volumetric flask. Add 5 ml of the linear 7 solution under the linear item of impurity A (implementation method 4), shake it to dissolve the nicotinic acid ethyl ester, add the mobile phase to dilute to the scale, and shake it well. Inject the sample and examine the durability under different flow rates of 1.0±0.1 ml / min, different detection wavelengths of 261±2 nm, and different chromatographic columns.
[0048]
[0049] The results showed that the analytical method for related substances of ethyl nicotinate had good durability.
[0050] Implementation 7: Precision of the determination method for related substances of nicotinate ethyl ester: 1. Instrument precision Verification method: Take 20μl of the reference solution and repeat the injection for 6 times, calculate the RSD% of the peak area of ethyl nicotinate, and examine the precision of the instrument (the instrument used in the verification process).
[0051] Verification results:
[0052] The results showed that the precision of the instruments used in the validation of the analytical method for related substances of ethyl nicotinate was good.
[0053] 2. Repeatability Verification method: Take 6 samples of nicotinate ethyl ester, about 10 mg, accurately weigh, and place in 10 ml volumetric flasks, add 5 ml of the linear 7 solution of impurity A in Implementation Method 4, shake to dissolve, add mobile phase to dilute to scale, and use as sample solution. Repeat the determination of related substances in 6 samples, and calculate the RSD% of the recovery rate of impurity A.
[0054] Verification results:
[0055] The results showed that the method for related substances of nicotinate ethyl ester had good repeatability.
[0056] 3. Intermediate precision Verification method: The solution preparation is shown in Implementation Example 8. At different times, different analysts use different instruments to measure the related substances of 6 samples respectively, and calculate the RSD of the recovery rate of impurity A.
[0057] Verification results:
[0058] The results showed that the intermediate precision of the method for related substances of ethyl nicotinate was good.
[0059] Implementation 8: Accuracy of the determination method for related substances of nicotinate ethyl ester: 1. Verification method Using the standard addition method, take 9 portions of nicotinate ethyl ester, about 10 mg, accurately weigh, and place in 9 10 ml volumetric flasks, then add 4 ml, 5 ml, and 6 ml of the linear 7 solution under the linear item of impurity A (implementation method 4) into each group of 3, respectively, shake to dissolve the nicotinate ethyl ester, add the mobile phase to dilute to the scale, shake well, and use as 80% recovery solution, 100% recovery solution, and 120% recovery solution; Take another portion of about 10 mg of nicotinic acid ethyl ester, weigh it accurately, put it in a 10 ml volumetric flask, add an appropriate amount of mobile phase, shake to dissolve the nicotinic acid ethyl ester and dilute to the scale, shake well, and use it as the blank test solution; determine it using the related substance method, deduct other impurities measured in the blank test solution accordingly, and calculate the recovery rate of impurity A.
[0060] System suitability solution: Take about 25 mg of niacin reference substance, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it with mobile phase and make up to the scale, accurately measure 0.5 ml of niacin reference solution, add 0.5 ml of sample solution, place it in a 100 ml volumetric flask, add mobile phase to make up to the scale, shake well, and set aside.
[0061] Control solution: Prepare 9 accuracy samples and one blank sample control solution respectively. The preparation method refers to 1.1.3.
[0062] 2. Verify the results
[0063] The results showed that the method for related substances of ethyl nicotinate had good accuracy.
[0064] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for determining related substances of nicotinate ethyl ester, characterized in that: The following steps are involved: (1) Prepare the mobile phase: acetic acid-water-acetonitrile mixture in a ratio of 1:29:70; (2) Prepare sample solution: Accurately weigh an appropriate amount of nicotinate ethyl ester sample to be tested, add mobile phase to dissolve and dilute, and shake well to obtain sample solution; (3) Prepare the test solution: accurately measure an appropriate amount of the sample solution, add the mobile phase to dilute it, and shake well to obtain the test solution; (4) Prepare the control solution: accurately measure an appropriate amount of the sample solution, dilute it with the mobile phase, and shake well to obtain the control solution; (5) Determination: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram until 3 times the peak time of the main component peak; Chromatographic conditions: using octadecylsilane bonded silica gel as filler (4.0*250mm, 4μm) Detection wavelength: 261nm; Flow rate: 1.0ml / min; Injection volume: 20 μl; The related substance is nicotinic acid (impurity A).
2. The method for determining the related substances of nicotinate ethyl according to claim 1, wherein: The content of impurity A in the test solution was calculated by peak area using the principal component self-reference method with relative correction factor added; the relative correction factor of impurity A was 0.
77.
3. The method for determining the related substances of nicotinate ethyl according to claim 1, wherein: Prior to step (5), the method further includes: accurately measuring the system suitability solution and the toluene positioning solution, injecting them into the liquid chromatograph respectively, and recording the chromatogram.
4. The method for determining the related substances of nicotinate ethyl according to claim 3, wherein: The preparation method of the system suitability solution is as follows: accurately weighing an appropriate amount of niacin reference substance, adding a mobile phase to dissolve and dilute to obtain a niacin reference substance solution; accurately measuring an appropriate amount of niacin reference substance solution and a sample solution, adding a mobile phase to dilute, and shaking to obtain a system suitability solution; The preparation method of the toluene positioning solution is: accurately weigh an appropriate amount of toluene, add a mobile phase to dissolve and dilute, and shake well to obtain the toluene positioning solution.
5. The method for determining the related substances of nicotinate ethyl according to claim 4, characterized in that: The content of nicotinic acid in the system suitability solution is 0.005 mg / ml; the content of nicotinic acid ethyl ester is 0.005 mg / ml.
6. The method for determining the related substances of nicotinate ethyl according to claim 4, characterized in that: The content of toluene in the toluene positioning solution is 0.002 mg / ml.
7. The method for determining the related substances of nicotinate ethyl according to claim 1, characterized in that: The content of nicotinic acid ethyl ester in the control solution is 0.002 mg / ml; the content of nicotinic acid ethyl ester in the test solution is 0.1 mg / ml.