Method for measuring content of ethyl nicotinate

Abstract

The invention relates to the technical field of drug detection, and discloses a method for determining the content of ethyl nicotinate, which comprises the following steps: preparing a mobile phase, namely an acetic acid-water-acetonitrile mixed solution; preparing a sample solution: precisely weighing a proper amount of a to-be-detected ethyl nicotinate sample, adding the mobile phase for dissolving and diluting, and uniformly shaking to obtain the sample solution; preparing a test solution: precisely measuring a proper amount of the sample solution, and adding the mobile phase for dilution to obtain the test solution; preparing a system applicable solution: precisely weighing a proper amount of a nicotinic acid reference substance, adding a mobile phase, dissolving and diluting to obtain a nicotinic acid reference substance solution; precisely measuring proper amounts of the nicotinic acid reference substance solution and the sample solution, adding a mobile phase for dilution, and uniformly shaking to obtain a system applicable solution; preparing a toluene positioning solution: precisely weighing a proper amount of toluene, adding a mobile phase for dissolving and diluting, and uniformly shaking to obtain the toluene positioning solution; and measuring: precisely measuring the test solution, and injecting the test solution into a liquid chromatograph. The detection method is high in specificity and sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the content of ethyl nicotinate. 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:

[0003] The production process and quality control of nicotinate ethyl ester are important links to ensure product quality. Impurities in drugs are the main factor affecting purity. If the drug contains impurities exceeding the limit, it may cause changes in physical and chemical constants, variations in appearance, and affect the stability of the drug; an increase in impurities will also cause a significantly lower content or reduced activity, and significantly increase toxic and side effects. Therefore, the detection of nicotinate ethyl ester purity is extremely important. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for determining the content of nicotinate ethyl ester. By determining the content of nicotinate ethyl ester by the method of the present invention, the quality control of nicotinate ethyl ester can be achieved.

[0005] Technical solution: The present invention provides a method for determining the content 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 and dilute it with the mobile phase to obtain the test solution; (4) Prepare system suitability solution: accurately weigh an appropriate amount of niacin reference substance, add mobile phase to dissolve and dilute to obtain niacin reference substance solution; accurately weigh an appropriate amount of niacin reference substance solution and sample solution, add mobile phase to dilute, and shake well to obtain system suitability solution; (5) Prepare toluene positioning solution: accurately weigh an appropriate amount of toluene, add mobile phase to dissolve and dilute, and shake well to obtain toluene positioning solution; (In the process of nicotinate ethyl ester production, there may be toluene residue in the product, so the toluene solution is positioned. Toluene is not included in the calculation after positioning and will be removed in the later stage of the process) (6) System suitability: Accurately measure the system suitability solution and toluene positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram; (7) Determination: Accurately measure the test solution, inject it into the liquid chromatograph, measure it twice in parallel, record the chromatogram, calculate it by the area normalization method, and calculate the result as the average value; Chromatographic conditions: using octadecylsilane bonded silica gel as filler (4.0*250mm, 4μm) Detection wavelength: 261nm; Flow rate: 1.0ml / min; Injection volume: 20μl.

[0006] Furthermore, the content of niacin in the system suitability solution is 0.005 mg / ml.

[0007] Furthermore, the content of nicotinate ethyl ester in the system suitability solution 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 nicotinate ethyl ester in the test solution is 0.1 mg / ml.

[0010] Beneficial effects: Compared with the prior art, the present invention can accurately and quickly measure the content of nicotinate ethyl ester, and the detection method has strong specificity and high sensitivity. The present invention can ensure that the product quality is stable, safe and controllable, provide a guarantee for safe medication, and further improve the quality and safety of nicotinate ethyl ester drugs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the chromatogram of the test solution; Figure 2 is the system suitability solution chromatogram; Figure 3 Position the solution chromatogram for toluene; Figure 4 This is the standard curve 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 nicotinate ethyl ester content is as follows: 1. Standard: Contains nicotinate ethyl ester (C 8 H 9 NO 2 ) (Except toluene) should be ≥95.0% 2. 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 3. Solution preparation Test solution: Take about 10 mg of nicotinic acid ethyl ester sample, accurately weigh, put it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, accurately measure 2 ml into a 20 ml volumetric flask, add mobile phase to dilute to the scale, shake well, and set aside.

[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] 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.

[0016] 4. System applicability Accurately measure 20 μl of the system suitability solution and inject it into the liquid chromatograph. Record the separation of the main peaks of nicotinic acid (impurity A) and nicotinate ethyl ester. The separation should be no less than 2.

[0017] Accurately measure 20 μl of the toluene positioning solution, inject it into the liquid chromatograph, and record the chromatogram.

[0018] 5. Determination Accurately take 20 μl of the test solution and inject it into the liquid chromatograph. Determine twice in parallel, record the chromatogram, calculate by the area normalization method, and calculate the result as the average value.

[0019] Implementation 2: Specificity of the method for determining the content 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 nicotinate ethyl ester content.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 nicotinate ethyl ester content.

[0025] 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.

[0026] 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 nicotinate ethyl ester content.

[0027] 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.

[0028] 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 nicotinate ethyl ester content.

[0029] 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.

[0030] The results show that the blank solvent does not interfere with the determination of nicotinate ethyl ester content. Nicotinate ethyl ester is stable under high temperature, high humidity, light and oxidation conditions, but unstable under acidic and alkaline conditions. The impurity peaks produced by degradation and the impurity peaks and the main peak can be effectively separated. Nicotinate ethyl ester The determination method of nicotinate ethyl ester content has good specificity.

[0031] Implementation 3: Limit of quantitation and repeatability of the method for determining the content of nicotinate ethyl ester: 1. Detection limit of nicotinate ethyl ester 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. Accurately measure 2.5 ml of the quantitative limit solution into a 10 ml volumetric flask, add the mobile phase to dilute to the scale, the concentration is 5.3 ng / ml, and the signal-to-noise ratio is 5.6 according to the chromatographic conditions of the relevant substances, which is 0.10% of the limit.

[0032] 2. Limit of Quantification of Ethyl Nicotinate 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. The results were verified by the chromatographic conditions of the relevant substances: an appropriate amount of linear solution 4 under the linear item of nicotinate ethyl ester was accurately measured and diluted 5000 times with the mobile phase, with a concentration of 0.02μg / ml, which was 2% of the limit, and the signal-to-noise ratio was 12.7.

[0033] 3. Repeatability of Quantification Limit of Ethyl Nicotinate 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).

[0034]

[0035] The results showed that the repeatability of the quantitative limit of nicotinate ethyl ester met the standard requirements.

[0036] Implementation 4: Linear relationship and range of the determination method of nicotinate ethyl ester content: 1. Operation steps Take about 10 mg of nicotinic acid ethyl ester, weigh accurately, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and set aside as a reference stock solution.

[0037]

[0038] Accurately measure 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml, 1.5ml and 2.0ml of the reference stock solution, respectively, and place them in 10ml volumetric flasks, dilute to the 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. Take the quantitative limit and the peak area of ​​the above solutions respectively according to the method under the content determination item, and obtain the results. See the table below.

[0039]

[0040] The results showed that nicotinate ethyl ester had a good linear relationship in the concentration range of 0.02106μg / ml to 210.6μg / ml, and the linear equation was y=20273.6228x-5955.6015, with a correlation coefficient R 2 =0.9999.

[0041] Implementation 5: Solution stability of the nicotinate ethyl ester content determination method: 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.

[0042] 2. Verify the results

[0043] The results showed that the nicotinate ethyl ester content determination solution was stable within 24 hours at room temperature.

[0044] Implementation 6: Robustness of the method for determining nicotinate ethyl ester content: 1. Verification method 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 dilute to scale, shake well. Inject, examine the durability under different flow rates 1.0±0.1 ml / min, different detection wavelengths 261±2 nm, and different chromatographic columns.

[0045] 2. Verify the results

[0046] The results showed that the analytical method for nicotinate ethyl ester content had good durability.

[0047] Implementation 7: Precision of the method for determining the content of nicotinate ethyl ester: 1. Instrument precision Verification method: Prepare 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 dilute to scale, shake well. Take 20 μl of sample solution and repeat injection 6 times, calculate the RSD% of nicotinic acid ethyl ester peak area, and examine the precision of the instrument.

[0048] Verification results:

[0049] The results showed that the instrumental precision of the method for analyzing nicotinate ethyl ester content was good.

[0050] 2. Repeatability Verification method: Repeat the preparation of 6 sample solutions (sample solution: take about 10 mg of nicotinic acid ethyl ester sample, accurately weigh, place in a 10 ml volumetric flask, dissolve with mobile phase and dilute to scale, shake well), accurately take 20 μl and inject into the liquid chromatograph, record the chromatogram. Determine the content of the 6 samples respectively, and calculate the RSD of the 6 contents.

[0051] Verification results:

[0052] The results showed that the analytical method for nicotinate ethyl ester had good repeatability.

[0053] 3. Intermediate precision Verification method: Prepare sample solution: take about 10 mg of nicotinic acid ethyl ester sample, weigh accurately, place in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to scale, shake well, accurately measure 2 ml to a 20 ml volumetric flask, add mobile phase to dilute to scale, shake well, and set aside. At different times, different analysts, using different instruments, measure the content of a sample separately, and calculate the RSD of the nicotinic acid ethyl ester content.

[0054] Verification results:

[0055] The results showed that the intermediate precision of the analytical method for nicotinate ethyl ester was good.

[0056] 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 the content of ethyl nicotinate, 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 and dilute it with the mobile phase to obtain the test solution; (4) Prepare system suitability solution: accurately weigh an appropriate amount of niacin reference substance, add mobile phase to dissolve and dilute to obtain niacin reference substance solution; accurately weigh an appropriate amount of niacin reference substance solution and sample solution, add mobile phase to dilute, and shake well to obtain system suitability solution; (5) Prepare toluene positioning solution: accurately weigh an appropriate amount of toluene, add mobile phase to dissolve and dilute, and shake well to obtain toluene positioning solution; (6) System suitability: Accurately measure the system suitability solution and toluene positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram; (7) Determination: Accurately measure the test solution, inject it into the liquid chromatograph, measure it twice in parallel, record the chromatogram, calculate it by the area normalization method, and calculate the result as the average value; Chromatographic conditions: using octadecylsilane bonded silica gel as filler (4.0*250mm, 4μm) Detection wavelength: 261nm; Flow rate: 1.0ml / min; Injection volume: 20μl.

2. The method for determining the content of ethyl nicotinate according to claim 1, wherein: The content of niacin in the system suitability solution is 0.005 mg / ml.

3. The method for determining the content of ethyl nicotinate according to claim 1, wherein: The content of nicotinate ethyl ester in the system suitability solution is 0.005 mg / ml.

4. The method for determining the content of ethyl nicotinate according to claim 1, wherein: The content of toluene in the toluene positioning solution is 0.002 mg / ml.

5. The method for determining the content of ethyl nicotinate according to claim 1, wherein: The content of nicotinate ethyl ester in the test solution is 0.1 mg / ml.

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