Detection method of cyanoacetamide related substances

Abstract

The invention relates to the technical field of chemical analysis, and particularly discloses a method for detecting cyanoacetamide related substances. SHIMADZU SH-624 (0.53 mm * 30m * 3.0 [mu] m) is adopted as a detection chromatographic column, 38-42 DEG C is set and maintained for 4-6 min, then the temperature is raised to 235-245 DEG C at the rate of 9-11 DEG C / min and maintained for 8-12 min, the temperature of a sample inlet is 245-255 DEG C, the temperature of a detector is 295-305 DEG C, and the split ratio is 3: 1-15: 1, so that simultaneous quantitative detection of various impurities in cyanoacetamide is realized, and the detection accuracy is high. The method is more beneficial to truly reflecting the quality of the cyanoacetamide, is beneficial to monitoring a cyanoacetamide synthesis process, and has a relatively high practical value.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, and in particular to a method for detecting cyanoacetamide-related substances. Background Technology

[0002] Cyanacetamide, also known as cyanoacetamide, is typically a white or pale yellow crystal with a pungent odor. It is an important organic compound that serves as a starting material for drugs such as allopurinol, triamterene, aminopterin, and pregabalin, demonstrating wide-ranging applications in chemical, pharmaceutical, and materials fields. With continuous technological advancements and increasing demand for high-performance chemicals across industries, the research and development of cyanacetamide has garnered increasing attention. In-depth research on cyanacetamide not only helps expand its application areas but also provides strong support for the development of related industries.

[0003] Currently, cyanoacetamide is generally prepared industrially by reacting ethyl cyanoacetate with ammonia. Using ethyl cyanoacetate and ammonia as starting materials, the reaction occurs under certain conditions to produce the target product cyanoacetamide and the byproduct ethanol. The reaction equation is as follows: NC-CH2-COOC2H5 + NH3·H2O → NC-CH2-CONH2 + C2H5OH. During the synthesis of cyanoacetamide, related substances such as methyl cyanoacetate, cyanoacetic acid, and malondiamide are inevitably produced. When cyanoacetamide is used as a starting material for allopurinol, its quality directly affects the purity of the final product. Therefore, the content of related substances in cyanoacetamide must be strictly controlled. Currently, there is no detection method for related substances in cyanoacetamide (methyl cyanoacetate, cyanoacetic acid, malondiamide). Therefore, this study aims to develop a detection method for related substances in cyanoacetamide to fully detect impurities. This is of great significance for effectively controlling the purity and quality safety of dihydropyridine calcium channel blocker products. Summary of the Invention

[0004] To address the lack of effective techniques for detecting related substances in cyanoacetamide in existing technologies, this invention provides a method for detecting related substances in cyanoacetamide.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] The first aspect of this invention provides a method for detecting cyanoacetamide-related substances, wherein the detection method is gas chromatography, comprising the following steps:

[0007] (1) Preparation of test solution and mixed reference solution:

[0008] Preparation of mixed reference solution: Take cyanoacetic acid, methyl cyanoacetate and malondiamide reference standards and prepare a mixed reference solution with solvent;

[0009] Preparation of test solution: Take the cyanoacetamide sample and prepare the test solution with a solvent;

[0010] (2) Take the mixed reference solution and the test solution, and perform the detection under the following gas chromatography conditions:

[0011] Detector: Hydrogen flame ionization detector;

[0012] Chromatographic column: SHIMADZU SH-624, 0.53mm × 30m × 3.0μm;

[0013] Column temperature: 38℃-42℃ for 4min-6min, then increase to 235℃-245℃ at a rate of 9℃ / min-11℃ / min and hold for 8min-12min;

[0014] Inlet temperature: 245℃-255℃;

[0015] Detector temperature: 295℃-305℃;

[0016] Split ratio: 3:1-15:1.

[0017] The method for detecting related substances in cyanoacetamide provided by this invention employs gas chromatography (GC) with a SHIMADZU SH-624 column (0.53 mm × 30 m × 3.0 μm) as the detection column. Combined with specific temperature programming and specific GC conditions such as injection port temperature, detector temperature, and split ratio, it achieves simultaneous quantitative detection of multiple known and unknown impurities in cyanoacetamide. This method is more conducive to accurately reflecting the quality of cyanoacetamide and can serve as a qualitative and quantitative detection method for multiple impurities in the cyanoacetamide synthesis process, providing a technical basis for improving production processes and determining product quality. Furthermore, the method for detecting related substances in cyanoacetamide provided by this invention has been validated through methodology, demonstrating high specificity, accuracy, sensitivity, repeatability, and stability. It is also simple to operate, enabling qualitative and quantitative analysis of multiple impurities in cyanoacetamide in a single detection, filling the gap in existing technologies that cannot effectively detect the quality of cyanoacetamide.

[0018] Preferably, the column temperature is maintained at 40°C for 5 minutes, and then increased to 240°C at a rate of 10°C / min and maintained for 10 minutes.

[0019] The optimal temperature program, combined with a specific chromatographic column, can produce good peak shapes for cyanoacetamide and various impurities, and is conducive to improving the separation between cyanoacetamide and various impurities, thereby achieving accurate quantitative analysis of multiple impurities in cyanoacetamide.

[0020] Preferably, the temperature of the injection port is 250°C.

[0021] Optimal column temperature and inlet temperature matching help ensure effective sample separation, maintain peak shapes for main components and various impurities, and improve detection accuracy and sensitivity.

[0022] Preferably, the detector temperature is 300°C.

[0023] Optimal detector temperature ensures baseline stability, reduces baseline noise, and thus improves the accuracy and sensitivity of detection results.

[0024] Preferably, the split ratio is 10:1.

[0025] Preferably, the column flow rate is 2 mL / min-4 mL / min.

[0026] Further preferred, the column flow rate is 3 mL / min.

[0027] Preferably, the injection volume is 1.0 μL.

[0028] Preferably, the concentrations of cyanoacetic acid, methyl cyanoacetate, and malondiamide in the mixed reference solution are 50 μg / mL.

[0029] Preferably, the concentration of the test solution is 10 mg / mL.

[0030] In summary, the gas chromatography detection method provided by this invention can effectively detect multiple impurities in cyanoacetamide. Methodological studies and verification have demonstrated that the method of this invention has good sensitivity, accuracy, and reproducibility, filling the gap in the existing technology for effective detection of cyanoacetamide quality. It is also beneficial for monitoring the cyanoacetamide synthesis process and has high practical value. Attached Figure Description

[0031] Figure 1 This is the gas chromatogram of the blank solvent (methanol) under the specificity item in Example 2 of this invention;

[0032] Figure 2 This is a gas chromatogram of the mixed reference solution under the specificity item in Example 2 of the present invention; wherein, peak 1 is the solvent peak, peak 2 is cyanoacetic acid, peak 3 is methyl cyanoacetate, and peak 4 is malondiamide;

[0033] Figure 3 This is a gas chromatogram of the test sample (cyanoacetamide) solution under the specificity item in Example 2 of the present invention; wherein, peak 1 is the solvent peak, peak 2 is methyl cyanoacetate, and peak 3 is cyanoacetamide;

[0034] Figure 4The above is a gas chromatogram of the spiked test solution under the specificity item in Example 2 of the present invention, wherein peak 1 is the solvent peak, peak 2 is cyanoacetic acid, peak 3 is methyl cyanoacetate, peak 4 is cyanoacetamide, and peak 5 is malondiamide.

[0035] Figure 5 This is a gas chromatogram of the spiked test solution in Comparative Example 1 of the present invention; wherein, peak 1 is cyanoacetic acid, peak 2 is the solvent peak, peak 3 is methyl cyanoacetate, peak 4 is cyanoacetamide, and peak 5 is malondiamide;

[0036] Figure 6 This is a gas chromatogram of the spiked test solution in Comparative Example 2 of the present invention; wherein, peak 1 is cyanoacetic acid, peak 2 is the solvent peak, peak 3 is methyl cyanoacetate, peak 4 is cyanoacetamide, and peak 5 is malondiamide. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Example 1

[0039] 1.1 Solution preparation:

[0040] Blank solvent: methanol.

[0041] Preparation of mixed reference stock solution: Take appropriate amounts of cyanoacetic acid, methyl cyanoacetate and malondiamide reference standards, accurately weigh them, and prepare a mixed solution containing about 500 μg of each in 1 mL with methanol, as the mixed reference stock solution.

[0042] Mixed reference solution: Take the mixed reference stock solution prepared above and dilute it quantitatively with methanol to prepare a mixed solution containing approximately 50 μg of cyanoacetic acid, 50 μg of methyl cyanoacetate and 50 μg of malondiamide per 1 mL, which is used as the mixed reference solution.

[0043] Preparation of test solution: Weigh approximately 100 mg of cyanoacetamide sample accurately and place it in a 10 mL volumetric flask. Prepare a test solution with a concentration of 10 mg / mL using methanol.

[0044] Preparation of spiked test solution: Weigh approximately 100 mg of cyanacetamide sample accurately and place it in a 10 mL volumetric flask. Add 1 mL of the mixed reference stock solution prepared above, dissolve and dilute to the mark with methanol, and shake well to obtain the spiked test solution.

[0045] 1.2 Gas Chromatography Conditions:

[0046] Detector: Hydrogen flame ionization detector;

[0047] Chromatographic column: SHIMADZU SH-624, 0.53mm × 30m × 3.0μm;

[0048] Column temperature: The column temperature was maintained at 40℃ for 5 min, and then increased to 240℃ at a rate of 10℃ / min and maintained for 10 min.

[0049] Column flow rate: 3 mL / min;

[0050] Injection volume: 1.0 μL;

[0051] Inlet temperature: 250℃;

[0052] Detector temperature: 300℃;

[0053] Split ratio: 10:1.

[0054] Example 2

[0055] Methodological validation:

[0056] 2.1 Specificity

[0057] Take the blank solvent, the above mixed reference solution, the test solution, and the spiked test solution, and perform gas chromatography under the conditions described in Example 1. Record the chromatograms, and the results are as follows: Figures 1-4 As shown, where, Figure 1 The chromatogram is for the blank solvent. Figure 2 This is the chromatogram of the mixed reference solution. Figure 3 The chromatogram of the test solution is shown. Figure 4 The chromatogram is shown in Table 1. The resolution results of each component in the spiked test solution are shown in Table 1.

[0058] Table 1. Resolution of each component in the spiked test solution

[0059] Element Retention time (min) Resolution methanol 4.455 - Cyanoacetic acid 6.081 12.44 Methyl cyanoacetate 13.551 92.97 cyanoacetamide 17.468 30.77 malondiamide 21.503 29.28

[0060] As can be seen from the table above, the blank solvent provided by this scheme does not interfere with the detection of each component, the separation degree between each component meets the requirements, and the method has good specificity.

[0061] 2.2 Limit of Detection and Limit of Quantification

[0062] Accurately weigh appropriate amounts of cyanoacetic acid, methyl cyanoacetate, malonamide, and cyanoacetamide, and prepare solutions containing approximately 25 μg of methyl cyanoacetate, 32 μg of cyanoacetic acid, 100 μg of malonamide, and 120 μg of cyanoacetamide per mL of methanol. Take appropriate amounts of each solution and perform serial dilutions for detection. The limit of detection is defined as the peak height approximately three times the baseline noise, and the limit of quantitation is defined as the peak height approximately ten times the baseline noise. Prepare six reference solutions at the limit of quantitation concentrations and perform detection under the gas chromatography conditions described in Example 1. Record the peak areas of each chromatographic peak; the results are shown in Tables 2-3.

[0063] Table 2 Results of Limit of Detection and Limit of Quantitation tests

[0064]

[0065] Table 3 Results of repeatability tests at the limit of quantitation

[0066]

[0067]

[0068] The experimental results show that the maximum RSD of the peak area of ​​each component after six repeated measurements is 4.12%, indicating that the method provided by this protocol has good repeatability at the limit of quantitation.

[0069] 2.3 Linear Range

[0070] Accurately weigh appropriate amounts of cyanoacetamide reference standard and each impurity reference standard, and prepare a series of linear solutions of different concentrations with methanol. Accurately measure 1.0 μL of each solution and inject it for detection under the gas chromatography conditions described in Example 1. Record the chromatogram, measure the peak area, and plot a linear regression curve with concentration (μg / mL) on the x-axis and peak area on the y-axis. The results are shown in Tables 4-7.

[0071] Table 4. Results of linearity test for cyanoacetic acid

[0072]

[0073] Table 5. Results of linearity test for methyl cyanoacetate

[0074]

[0075] Table 6 Results of linearity test for cyanoacetamide

[0076]

[0077]

[0078] Table 7 Results of the linearity test for malondiamide

[0079]

[0080] The above results show that the linear relationship among the components is good, indicating that the method provided in this scheme has a good linear relationship.

[0081] 2.4 Recovery rate

[0082] Accurately weigh appropriate amounts of cyanoacetic acid, methyl cyanoacetate, and malondiamide, and prepare a mixed solution containing approximately 500 μg of each per mL with methanol as a stock solution. Accurately weigh 100 mg of cyanoacetamide sample and place it in a 10 mL volumetric flask. Accurately add an appropriate volume of the mixed stock solution and dilute to the mark, then mix well. Perform triplicate for each concentration to obtain low, medium, and high recovery solutions. Perform detection according to the detection method and chromatographic conditions outlined in this protocol.

[0083] Nine recovered solutions were tested under the gas chromatography conditions described in Example 1. Chromatograms were recorded and the recovery rates were calculated. The results are shown in Tables 8-10.

[0084] Table 8 Results of Cyanoacetic Acid Recovery Rate Detection

[0085]

[0086]

[0087] Table 9 Results of Methyl Cyanate Recovery Rate Detection

[0088]

[0089] Table 10 Results of malondiamide recovery rate detection

[0090]

[0091] The experimental results show that the recovery rates of each component are between 80% and 120%, and the maximum RSD is 6.10%, indicating that the method provided by this invention has good accuracy.

[0092] 2.5 Repeatability

[0093] Six cyanoacetamide samples were accurately weighed and prepared into test solutions with a concentration of 10 mg / mL using methanol. The solutions were then tested under the gas chromatography conditions described in Example 1, and the results are shown in Table 11.

[0094] Table 11 Results of Repeatability Tests

[0095]

[0096]

[0097] The experimental results showed that the results of the six test solutions were basically consistent, indicating that the method has good repeatability.

[0098] 2.6 Solution stability

[0099] Accurately weigh appropriate amounts of cyanoacetamide and each impurity reference standard, and prepare the test solution and mixed reference solution according to the above method. Samples were taken at 0h, 3h, 8h, 16h and 22h respectively, and detected according to the gas chromatography conditions described in Example 1. The results are shown in Tables 12-13.

[0100] Table 12 Stability results of mixed reference solutions

[0101]

[0102] Table 13 Stability results of the test solution

[0103]

[0104]

[0105] The experimental results showed that after standing at room temperature for 22 hours, the RSD of the peak area of ​​each component was less than 10%; there was no significant difference in the detection results of the test solution at different time points.

[0106] 2.7 Durability

[0107] Each impurity reference standard was accurately weighed and mixed reference standard solution was prepared as described above. The initial column temperature, heating rate, injection port temperature and detector temperature were changed respectively, and the gas chromatography detection conditions described in Example 1 were used for detection. The results are shown in Tables 14-17.

[0108] Table 14 Separation Degree at Different Initial Temperatures

[0109] condition Initial temperature 40℃ Initial temperature 38℃ Initial temperature 42℃ methanol -- -- -- Cyanoacetic acid 12.63 9.35 14.06 Methyl cyanoacetate 91.85 93.78 90.61 cyanoacetamide 31.8 31.32 31.45 malondiamide 30.48 29.79 29.98

[0110] Table 15 Separation Degree at Different Heating Rates

[0111] condition Heating rate 10℃ / min Heating rate 9℃ / min Heating rate 11℃ / min methanol -- -- -- Cyanoacetic acid 12.63 11.05 13.18 Methyl cyanoacetate 91.85 95.56 87.84 cyanoacetamide 31.8 32.7 29.74 malondiamide 30.48 31.33 28.75

[0112] Table 16 Resolution at Different Inlet Temperatures

[0113] condition Inlet temperature 250℃ Inlet temperature 245℃ Inlet temperature 255℃ methanol -- -- -- Cyanoacetic acid 12.63 12.77 12.69 Methyl cyanoacetate 91.85 90.97 92.01 cyanoacetamide 31.8 31.5 31.56 malondiamide 30.48 29.9 30.21

[0114] Table 17 Temperature separation of different detectors

[0115] condition Detector 300℃ Detector 295℃ Detector 305℃ methanol -- -- -- Cyanoacetic acid 12.63 12.67 12.54 Methyl cyanoacetate 91.85 92.02 91.55 cyanoacetamide 31.8 31.4 31.18 malondiamide 30.48 30.37 29.72

[0116] As can be seen from the table above, the separation degree of each component meets the requirements even when the conditions are changed, indicating that the method provided by the present invention has good durability.

[0117] Comparative Example 1

[0118] This comparative example provides a method for detecting cyanoacetamide-related substances. The detection method is exactly the same as the gas chromatography conditions under section 1.2 of Example 1, except that the chromatographic column is HP-FFAP (30m×0.53mm×1.0μm), and 100% polyethylene glycol (or similar polarity) is used as the stationary liquid, while other conditions remain unchanged.

[0119] The spiked test solution from Example 1.1 was injected for analysis, and the chromatogram was recorded. The results are as follows: Figure 5 As shown.

[0120] The experimental results showed that cyanoacetamide and malondiamide did not produce the main peak.

[0121] Comparative Example 2

[0122] This comparative example provides a method for detecting cyanoacetamide-related substances. The detection method is exactly the same as the gas chromatography conditions under section 1.2 of Example 1, except that the chromatographic column is HP-5 (30m×0.32mm×0.25μm), and 5% phenyl-95% dimethylpolysiloxane (or similar polarity) is used as the stationary phase. All other conditions remain unchanged.

[0123] The spiked test solution from Example 1.1 was injected for analysis, and the chromatogram was recorded. The results are as follows: Figure 6 As shown.

[0124] The test results showed that methyl cyanoacetate, cyanoacetamide, and malondiamide exhibited severe tailing.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting cyanoacetamide-related substances, characterized in that, The detection method is gas chromatography, which includes the following steps: (1) Preparation of test solution and mixed reference solution: Preparation of mixed reference solution: Take cyanoacetic acid, methyl cyanoacetate and malondiamide reference standards and prepare a mixed reference solution with solvent; Preparation of test solution: Take the cyanoacetamide sample and prepare the test solution with a solvent; (2) Take the mixed reference solution and the test solution, and perform the detection under the following gas chromatography conditions: Detector: Hydrogen flame ionization detector; Chromatographic column: SHIMADZU SH-624, 0.53mm × 30m × 3.0μm; Column temperature: 38℃-42℃ for 4min-6min, then increase to 235℃-245℃ at a rate of 9℃ / min-11℃ / min and hold for 8min-12min; Inlet temperature: 245℃-255℃; Detector temperature: 295℃-305℃; Split ratio: 3:1-15:

1.

2. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The column temperature was maintained at 40°C for 5 minutes, and then increased to 240°C at a rate of 10°C / min, and maintained for 10 minutes.

3. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The injection port temperature is 250℃; and / or The detector temperature is 300℃.

4. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The split ratio is 10:

1.

5. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The column flow rate is 2 mL / min - 4 mL / min.

6. The method for detecting cyanoacetamide-related substances as described in claim 5, characterized in that, The column flow rate was 3 mL / min.

7. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The injection volume was 1.0 μL.

8. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The mixed reference solution contained 50 μg / mL cyanoacetic acid, 50 μg / mL methyl cyanoacetate, and 50 μg / mL malonamide.

9. The method for detecting cyanoacetamide-related substances as described in claim 1, characterized in that, The concentration of the test solution is 10 mg / mL.

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