Method for detecting ethanolamine in Nicodil bulk drug
By using chloroformate-9-fluorenyl methyl chloroformate as a derivatization reagent in nicodil raw materials, the derivatization reaction conditions were optimized, and the pre-column derivatization high-performance liquid chromatography was established, which solved the shortcomings in the determination of ethanolamine content in the prior art, and achieved high sensitivity and accuracy measurement results.
Patent Information
- Application Number
- CN202411851867.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art lacks effective methods for determining the ethanolamine content in nicodil raw materials, making it difficult to accurately control the content during drug synthesis.
methyl chloroformate-9-fluorenyl ester was used as the derivatization reagent, and pre-column derivatization high-performance liquid chromatography was established to determine the ethanolamine content in nicodil raw materials.
The accurate determination of the ethanolamine content in nicordil raw materials is achieved. The method is highly sensitive and simple to operate, and is suitable for drug quality control.
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Figure CN119827650A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for detecting ethanolamine in Nicorandil raw material medicine. Background Art
[0002] Nicorandil, whose chemical name is N-(2-hydroxyethyl) nicotinamide nitrate, is the first ATP-sensitive potassium channel opener used clinically. It is used to treat various types of angina pectoris. Its preparations include Nicorandil tablets and Nicorandil for injection.
[0003] Ethanolamine, English name: Monoethanolamine, referred to as MEA, CAS number 141-43-5, is a colorless viscous liquid with an ammonia smell at room temperature. It is soluble in water and the solution is strongly alkaline. It is miscible with water, ethanol and acetone. It is usually used as one of the starting materials for the synthesis of Nicorandil API: .
[0004] In order to ensure the safety of the drug, the ethanolamine used in the synthesis process of Nicorandil API was studied and controlled with an identification limit of 0.10% with reference to ICH Q3A.
[0005] There are many reports on the detection methods of ethanolamine at home and abroad. The trace analysis methods in aqueous solution include ion pair chromatography analysis, and the trace analysis methods in industrial gas include derivatization gas chromatography analysis, derivatization liquid chromatography analysis, derivatization thin layer chromatography analysis, capillary electrophoresis, gas chromatography analysis, ion exclusion chromatography analysis, etc. There are also methods for determining trace monoethanolamine in cosmetics by high performance liquid chromatography-tandem mass spectrometry, and methods for detecting trace ethanolamine in high purity water in power plants by ion chromatography. The above methods are mainly aimed at the analysis of trace or trace ethanolamine, but there are few reports at home and abroad specifically for the determination of ethanolamine content in raw materials. Summary of the invention
[0006] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for detecting ethanolamine in Nicorandil raw materials. Using 9-fluorenylmethyl chloroformate as a derivatization reagent, a pre-column derivatization HPLC method is established to determine ethanolamine in Nicorandil raw materials by optimizing derivatization conditions and methodological verification. The method has mild conditions, simple operation and high sensitivity.
[0007] Technical solution: The present invention provides a method for detecting ethanolamine in Nicorandil API, comprising the following steps: (1) Using 9-fluorenylmethyl chloroformate as a derivatization reagent, the ethanolamine in the Nicorandil raw material was derivatized to obtain a sample containing an ethanolamine derivatization product; (2) preparing a test solution with the sample containing the ethanolamine derivatization product, and detecting the test solution by high performance liquid chromatography.
[0008] Furthermore, in step (1), the mass ratio of the 9-fluorenylmethyl chloroformate to the ethanolamine is 500:1-50:1.
[0009] Furthermore, in step (1), the specific process of the derivatization reaction is: adding 9-fluorenylmethyl chloroformate solution to ethanolamine solution, letting it stand for a period of time, adding boric acid buffer solution, and letting it stand for a period of time.
[0010] Preferably, in step (1), the reaction time of the derivatization reaction is 20-25 min.
[0011] Preferably, in step (1), the derivatization reaction is carried out in an aprotic polar solvent, and the aprotic polar solvent is acetonitrile.
[0012] Further, the chromatographic conditions for detection by high performance liquid chromatography include: Chromatographic column: Welch Ultimate XB-C18 column; Mobile phase: Formic acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution; Flow rate: 1.0 ml / min; Column temperature: 30 °C; Detection wavelength: 265 nm; Injection volume: 10 μl.
[0013] Furthermore, after the ethanolamine standard was derivatized with 9-fluorenylmethyl chloroformate as a derivatization reagent, the volume was fixed with acetonitrile to obtain a series of ethanolamine standard solutions with concentration gradients, which were injected for analysis; With the injection concentration as the abscissa and the peak area of the standard as the ordinate, linear regression fitting was performed to obtain the external standard method working curve for the determination of ethanolamine content.
[0014] Furthermore, the concentration of the ethanolamine standard is 0.0081 μg / ml~1.0772 μg / ml.
[0015] Beneficial effects: Compared with the prior art, the present invention has the following specific beneficial effects: In this paper, 9-fluorenylmethyl chloroformate was used as the derivatization reagent. By optimizing the derivatization reaction conditions, the conditions that can make the derivatization reaction of the reference solution, the test solution and the test spiked solution complete were obtained. A pre-column derivatization high performance liquid chromatography method was established. The method has strong specificity, high sensitivity, accuracy and rapidity, and is suitable for the determination of ethanolamine in Nicorandil raw materials. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the working curve of ethanolamine with a concentration between 0.0081 μg / ml and 1.0772 μg / ml; Figure 2 Specific blank solvent chromatogram for verification of the method for detecting ethanolamine content in Nicorandil API; Figure 3 Chromatogram of the specific reference solution for verification of the detection method of ethanolamine content in Nicorandil API; Figure 4 Chromatogram of the test sample solution for verification of the specificity of the detection method for ethanolamine content in Nicorandil API; Figure 5 Chromatogram of the spiked solution of the test sample for verification of the specificity of the detection method of ethanolamine content in Nicorandil API; Figure 6 Validation of detection limit spectrum for the detection method of ethanolamine content in Nicorandil API; Figure 7 To verify the repeatability of the blank solvent spectrum for the detection method of ethanolamine content in Nicorandil API; Figure 8 The test sample solution spectrum was used to verify the repeatability of the test method for ethanolamine content in Nicorandil API; Fig. 9 The spectrum of the reference solution for verifying the repeatability of the detection method of ethanolamine content in Nicorandil API; Fig.10 Spectrum of the spiked solution of the test sample for the verification of the repeatability of the detection method of ethanolamine content in Nicorandil API-1; Fig.11 Spectrum of the spiked solution of the test sample for the verification of the repeatability of the method for the detection of ethanolamine content in Nicorandil API-2; Fig.12 Spectrum of the spiked solution of the test sample for the verification of the repeatability of the detection method of ethanolamine content in Nicorandil API-3; Fig.13 Spectrum 4 of the spiked solution of the test sample for verifying the repeatability of the detection method for ethanolamine content in Nicorandil API. DETAILED DESCRIPTION
[0017] The present invention is described in detail below in conjunction with the embodiments.
[0018] The experimental instruments, chromatographic columns and reagents are shown in Table 1:
[0019] Experimental conditions: Use octadecylsilane bonded silica gel as filler (Welch Ultimate XB-C18, 4.6mm×150mm, 5μm or chromatographic column with equivalent performance); use 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B, and perform gradient elution according to Table 2; flow rate is 1.0ml per minute; detection wavelength is 265nm; column temperature is 30℃; injection volume is 10μl.
[0020]
[0021] Embodiment 1: Linear relationship: (1) Preparation of ethanolamine standard stock solution, derivatization reagent, and boric acid buffer solution: Ethanolamine standard stock solution: Take an appropriate amount of ethanolamine, weigh it accurately, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 0.01 mg per 1 ml.
[0022] Derivatization reagent: Weigh 50 mg of 9-fluorenylmethyl chloroformate, place in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.
[0023] Boric acid buffer solution: Weigh an appropriate amount of borax, add water to dissolve it, and prepare a solution with a pH of about 9.18.
[0024] (2) Pre-column derivatization and sample injection analysis Accurately measure 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml, and 2.0ml of the ethanolamine standard stock solution, respectively, and place them in 20ml volumetric bottles, add 1ml of the derivatization reagent, shake well, and let it stand for 10 minutes, add 1ml of the boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use them as linear 1 solution to linear 6 solution. Inject them into the liquid chromatograph, record the chromatogram, and calculate the linear regression equation. The linear determination results of ethanolamine are shown in Table 3. With the injection concentration as the horizontal axis and the peak area of the standard as the vertical axis, linear regression fitting is performed to determine the external standard method working curve (see Figure 1 ).
[0025]
[0026] The results showed that ethanolamine had a good linear relationship in the concentration range of 0.0081μg / ml~1.0772μg / ml, and the linear regression equation was: y =172196.4869x -405.0279, r = 1.0.
[0027] Actual sample testing (taking the ethanolamine content of 6 batches of Nicorandil from Jiangsu Tasly Diyi Pharmaceutical Co., Ltd. as an example): Take 10 mg of ethanolamine sample, weigh accurately, place in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve, add 1 ml of derivatization reagent, shake well, let stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let stand for 10 minutes, dilute to scale with acetonitrile, shake well. Inject into liquid chromatograph and record chromatogram. The results are shown in Table 4.
[0028]
[0029] The results showed that the ethanolamine content in 6 batches of Nicorandil was determined according to the analytical method for determining the ethanolamine content in Nicorandil, and the results were in compliance with the regulations.
[0030] Embodiment 2: Exclusive properties: (1) Blank solvent: Take 10 ml of acetonitrile and place it in a 20 ml volumetric flask. Add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile and shake well.
[0031] (2) Reference solution: Take an appropriate amount of ethanolamine standard, weigh it accurately, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 0.01 mg per 1 ml as the reference stock solution; accurately measure 1 ml and place it in a 20 ml volumetric flask, add 10 ml of acetonitrile, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of borate buffer solution, shake well, let it stand for 10 minutes, add acetonitrile to dilute to the scale and shake well.
[0032] (3) Test solution: Take 10 mg of ethanolamine sample, weigh accurately, place in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve, add 1 ml of derivatization reagent, shake well, let stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let stand for 10 minutes, dilute to the scale with acetonitrile, and shake well.
[0033] (4) Test sample spiked solution: Take 10 mg of ethanolamine sample, weigh accurately, place in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve, accurately add 1 ml of reference stock solution, add 1 ml of derivatization reagent, shake well, let stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let stand for 10 minutes, dilute to scale with acetonitrile, and shake well.
[0034] Take each of the above solutions and inject them into the liquid chromatograph respectively, and record the chromatogram. Figure 2-5 .
[0035] Compare the blank solution and the reference solution to determine the ethanolamine derivative peak. The derivatization reaction equation is as follows: .
[0036] The results show that the impurities in the blank solution and the test solution do not interfere with the determination of the ethanolamine derivative peak, the ethanolamine derivative peak is well separated from the adjacent peaks, and the analytical method for determining the ethanolamine content in Nicorandil provided by the present invention has good specificity.
[0037] Limit of detection and limit of quantification: The detection limit and quantification limit were determined by the signal-to-noise ratio method. The quantification limit was determined by the corresponding concentration when the signal-to-noise ratio was approximately 10:1, and the detection limit was determined by the corresponding concentration when the signal-to-noise ratio was approximately 3:1.
[0038] (1) Detection limit Accurately measure 0.2 ml of the reference stock solution, place it in a 20 ml volumetric flask, add acetonitrile to dilute to the scale, and shake well; accurately measure 0.5 ml again, place it in a 20 ml volumetric flask, add 1 ml of the derivatization reagent respectively, shake well, let it stand for 10 minutes, add 1 ml of the boric acid buffer solution respectively, shake well, let it stand for 10 minutes, dilute with acetonitrile to the scale, shake well, and use this as the ethanolamine detection limit solution with a concentration of 0.0027 μg / ml, which is 0.54% of the limit.
[0039] (2) Limit of quantification Accurately measure 0.2 ml of the reference stock solution, place it in a 20 ml volumetric flask, add acetonitrile to dilute to the scale, and shake well; then accurately measure 1.5 ml, place it in a 20 ml volumetric flask, add 1 ml of the derivatization reagent respectively, shake well, let it stand for 10 minutes, add 1 ml of the boric acid buffer solution respectively, shake well, let it stand for 10 minutes, dilute with acetonitrile to the scale, shake well, and use this as the ethanolamine quantitative limit solution with a concentration of 0.0081 μg / ml, which is 1.62% of the limit.
[0040] The ethanolamine quantitative limit solution was taken and injected 6 times to examine the peak area RSD. The results are shown in Table 5.
[0041]
[0042] The results showed that the quantification limit solution of ethanolamine had good precision.
[0043] Accuracy: (1) Take 10 ml of acetonitrile and place it in a 20 ml volumetric flask. Add 1 ml of derivatization reagent, shake well, and let it stand for 10 minutes. Add 1 ml of boric acid buffer solution, shake well, and let it stand for 10 minutes. Dilute to the scale with acetonitrile and shake well. This is used as the blank solution.
[0044] (2) Take about 10 mg of ethanolamine sample, weigh accurately, place in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve, add 1 ml of derivatization reagent, shake well, let stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use as the test solution.
[0045] (3) Take an appropriate amount of ethanolamine standard, weigh it accurately, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 0.01 mg per 1 ml; accurately measure 1 ml and place it in a 20 ml volumetric flask, add 10 ml of acetonitrile, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of borate buffer solution, shake well, let it stand for 10 minutes, add acetonitrile to dilute to the scale, shake well, and use as the reference solution.
[0046] (4) Take about 10 mg of ethanolamine sample, weigh it accurately, place it in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve it, accurately add 0.1 ml of reference stock solution, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use this as the 10% spike recovery solution.
[0047] (5) Take about 10 mg of ethanolamine sample, weigh it accurately, place it in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve it, accurately add 0.8 ml of reference stock solution, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use this as the 80% spike recovery solution.
[0048] (6) Take about 10 mg of ethanolamine sample, weigh it accurately, place it in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve it, accurately add 1.0 ml of reference stock solution, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use this as the 100% spike recovery solution.
[0049] (7) Take about 10 mg of ethanolamine sample, weigh it accurately, place it in a 20 ml volumetric flask, add 10 ml of acetonitrile to dissolve it, accurately add 1.2 ml of reference stock solution, add 1 ml of derivatization reagent, shake well, let it stand for 10 minutes, add 1 ml of boric acid buffer solution, shake well, let it stand for 10 minutes, dilute to the scale with acetonitrile, shake well, and use this as the 120% spike recovery solution.
[0050] (8) Take each of the above solutions and inject them into the liquid chromatograph respectively. Record the chromatogram and examine the average value and RSD (%) of the recovery rate of spiked ethanolamine. The results are shown in Table 6.
[0051]
[0052] The results showed that the analytical method for determining the ethanolamine content in Nicorandil had good accuracy.
[0053] Precision: (1) Instrument precision Take the reference solution and repeat the measurement 6 times to examine the RSD (%) of the ethanolamine peak area and retention time. The results are shown in Table 7-8.
[0054]
[0055]
[0056] The results showed that the analytical method for determining the content of ethanolamine in Nicorandil had good instrumental precision.
[0057] (2) Repeatability One portion of the test sample solution and six portions of the test sample spiked solutions were prepared (the preparation method was the same as the 100% spiked recovery solution under the accuracy item). The average value and RSD (%) of the ethanolamine content of the test sample spiked solutions were investigated according to the analytical method for the determination of ethanolamine content in Nicorandil. The results are shown in Table 9.
[0058]
[0059] The results showed that the analytical method for determining the content of ethanolamine in Nicorandil had good repeatability.
[0060] (3) Intermediate precision Different personnel were investigated to prepare 1 test sample solution and 6 test sample spiked solutions (preparation method was the same as 100% spiked recovery solution under accuracy) on different dates. The average values and RSD (%) of ethanolamine contents in 12 test sample spiked solutions (including 6 test sample spiked solutions under repeatability) were investigated using different instruments according to the analytical method for determination of ethanolamine content in nicorandil. The results are shown in Table 10.
[0061]
[0062] The results showed that the intermediate precision of the analytical method for determining the content of ethanolamine in Nicorandil was good.
[0063] Durability: The test sample solution and the test sample spiked solution were prepared (the preparation method was the same as the 100% spiked recovery solution under the accuracy item). The average value and RSD (%) of the ethanolamine content in the test sample spiked solution were investigated at different flow rates (1.0±0.1ml / min), different column temperatures (30±2℃), and different wavelengths (265±2nm). The analytical method for determining the ethanolamine content in Nicorandil was used to investigate the results. The results are shown in Table 11.
[0064]
[0065] The results showed that the analytical method for determining the ethanolamine content in Nicorandil had good durability.
[0066] Solution stability: Prepare 1 portion of test sample spiked solution (preparation method is the same as 100% spiked recovery solution under accuracy), test sample solution and reference solution, and examine the average value and RSD (%) of ethanolamine peak area of each solution within 24 hours. The results are shown in Table 12.
[0067]
[0068] The results showed that the test solution, reference solution and test spiked solution of the analytical method for the determination of ethanolamine in nicorandil had good stability within 24 hours.
[0069] The validation of analytical methodology for the determination of ethanolamine content is summarized in the following table:
[0070]
[0071] In summary, this paper used 9-fluorenylmethyl chloroformate as the derivatization reagent and optimized the derivatization reaction conditions to obtain conditions that could completely derivatize the reference solution, test solution and test spiked solution. A pre-column derivatization HPLC method was established with strong specificity, high sensitivity, accuracy and rapidity, which is suitable for the determination of ethanolamine in Nicorandil API.
[0072] 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 detecting ethanolamine in Nicorandil bulk drug, characterized in that: The following steps are involved: (1) Using 9-fluorenylmethyl chloroformate as a derivatization reagent, the ethanolamine in the Nicorandil raw material was derivatized to obtain a sample containing an ethanolamine derivatization product; (2) preparing a test solution with the sample containing the ethanolamine derivatization product, and detecting the test solution by high performance liquid chromatography.
2. The method for detecting ethanolamine in Nicorandil bulk drug according to claim 1, characterized in that: In step (1), the mass ratio of the 9-fluorenylmethyl chloroformate to the ethanolamine is 500:1-50:
1.
3. The method for detecting ethanolamine in Nicorandil bulk drug according to claim 1, characterized in that: In step (1), the specific process of the derivatization reaction is: adding 9-fluorenylmethyl chloroformate solution to ethanolamine solution, letting it stand for a period of time, adding boric acid buffer solution, and letting it stand for a period of time.
4. The method for detecting ethanolamine in Nicorandil bulk drug according to claim 1, characterized in that: In step (1), the reaction time of the derivatization reaction is 20-25 min.
5. The method for detecting ethanolamine in Nicorandil bulk drug according to claim 1, characterized in that: In step (1), the derivatization reaction is carried out in an aprotic polar solvent, and the aprotic polar solvent is acetonitrile.
6. The method for detecting ethanolamine in Nicorandil bulk drug according to claim 1, characterized in that: The chromatographic conditions for detection by HPLC include: Chromatographic column: Welch Ultimate XB-C18 column; Mobile phase: Formic acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution; Flow rate: 1.0 ml / min; Column temperature: 30 °C; Detection wavelength: 265 nm; Injection volume: 10 μl.
7. The detection method for determining the content of monoethanolamine according to claim 1, characterized in that: After the ethanolamine standard was derivatized with 9-fluorenylmethyl chloroformate as a derivatization reagent, the solution was fixed to volume with acetonitrile to obtain a series of ethanolamine standard solutions with concentration gradients, which were then injected for analysis; With the injection concentration as the abscissa and the peak area of the standard as the ordinate, linear regression fitting was performed to obtain the external standard method working curve for the determination of ethanolamine content.
8. The detection method for determining the content of monoethanolamine according to claim 7, characterized in that: The concentration of the ethanolamine standard is 0.0081 μg / ml~1.0772 μg / ml.