CN113607837B - Method for detecting cyanoacetamide and related substances thereof - Google Patents
Method for detecting cyanoacetamide and related substances thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a method for detecting cyanoacetamide and related substances thereof, which adopts a high performance liquid chromatography method to qualitatively or/and quantitatively detect cyanoacetamide and cyanoacetic acid and methyl cyanoacetate therein, wherein the detection conditions of the liquid chromatography comprise: the chromatographic column is a C18 chromatographic column which can tolerate 100% of water phase, and the mobile phase is phosphoric acid aqueous solution. The method can effectively detect the cyanoacetamide and cyanoacetic acid and methyl cyanoacetate in the cyanoacetamide at the same time, and is favorable for carrying out stricter quality control on the cyanoacetamide.
Description
Technical Field
The invention relates to the field of detection methods, in particular to a method for detecting cyanoacetamide and related substances thereof.
Background
Cyanoacetamide is a white powdery crystal, is usually obtained by the action of methyl cyanoacetate and ammonia, is soluble in various organic solvents and water, is toxic, is an important organic synthesis intermediate, can be used for synthesizing malononitrile and electroplating solution, and is also used for synthesizing drugs of triamterene, aminopterin, milrinone and the like.
As an important synthesis starting material, the quality of the cyanoacetamide directly influences the quality of subsequent reaction intermediates and even final products, so that the quality control of the cyanoacetamide has important significance on the aspect of subsequent quality control of the final products.
At present, a quality control method for cyanoacetamide has been reported, and the cyanoacetamide has a large polarity, and under the condition of reverse phase chromatography, the retention of the cyanoacetamide on a C18 column or a C8 column is weak, and almost peaks at dead time, so that the detection of the cyanoacetamide is mostly carried out by gas phase or normal phase chromatography at first.
For example, in patent CN103926333A, a method for determining the content of cyanoacetamide is provided, which uses normal phase high performance liquid chromatography, the mobile phase is acetonitrile-methyl chloride mixed solution, and the detector is Evaporative Light Scattering Detector (ELSD); wei Yang et al also detected cyanoacetamide by normal phase high performance liquid chromatography with ethanol and n-hexane as mobile phases (normal phase high performance liquid chromatography for cyanoacetamide, modern pesticide, vol 13, no. 3, 6/2014).
However, these methods are complicated in operation and require long time for equilibration, which brings about many inconveniences to the analysis work, and the solvents used in normal phase chromatography have high toxicity and volatility, which may harm the health of operators.
In order to improve the detection method of cyanoacetamide, CN111413451a discloses a method for detecting cyanoacetamide by using reversed phase high performance liquid chromatography, wherein the mobile phase is acetonitrile-ammonium formate, the detection wavelength is 200nm, and the concentration of cyanoacetamide in a test sample is 0.5mg/mL.
Although the method realizes the reversed-phase high performance liquid chromatography detection of the cyanoacetamide, shortens the operation time of the detection process, simplifies some operation steps compared with the forward chromatography, the preparation of the mobile phase requires that the pH value of the ammonium formate solution is adjusted firstly and then mixed with acetonitrile in a certain proportion, the preparation is complicated, the problem of complex operation still exists, the uncertain factors of the experimental process are more directly caused, the reproducibility is poorer, in addition, the acetonitrile which belongs to the class II solvents with limited use is still used in the mobile phase, the environment is not friendly, and the health of operators is still damaged.
Therefore, how to realize the detection of the cyanoacetamide with high efficiency, convenience, greenness and safety by the reversed-phase high performance liquid chromatography is to be further developed, improved and perfected.
In addition, the production process of the cyanoacetamide is chemical synthesis, so that some process impurities are inevitably introduced, and partial degradation impurities are also generated in the storage process. However, there is no literature report on the types of related substances in cyanoacetamide, which also causes limitations in the quality control process of cyanoacetamide, only detection and control of cyanoacetamide can be performed, and if the related substances of cyanoacetamide can be researched and controlled, the quality of cyanoacetamide and the subsequent products thereof can be guaranteed better.
Disclosure of Invention
The invention aims to provide a method for detecting cyanoacetamide and/or related substances thereof, which can better control the quality of cyanoacetamide.
The cyanoacetamide has important practical significance as an important medical intermediate for quality control, although the cyanoacetamide is widely used at present, no research related to substances related to the cyanoacetamide exists, no research report related to detection of the substances related to the cyanoacetamide is found, and the cyanoacetamide and the substances related to the cyanoacetamide cannot be comprehensively controlled in terms of quality.
The main reasons for this situation are that the chemical structure and properties of the related substances of cyanoacetamide have certain similarity with those of cyanoacetamide, and the problems of weak retention and peak appearance of dead time on a chromatographic column are mostly existed in reverse chromatography, so that effective separation, identification and detection of the related substances are difficult to realize.
In addition, the chemical structures of cyanoacetamide and related substances determine that the cyanoacetamide has weak ultraviolet absorption, which easily causes insufficient detection sensitivity, and even if effective separation is realized, the compounds with low content of impurities are easy to miss detection and cannot be detected. Therefore, the research on the cyanoacetamide-related substance is difficult.
Through continuous research and exploration, the inventor successfully separates and identifies two related substances of cyanoacetamide synthesized by the prior art (obtained by reacting methyl cyanoacetate with ammonia): cyanoacetic acid, methyl cyanoacetate.
The structures of cyanoacetamide, cyanoacetic acid and methyl cyanoacetate are as follows:
on the basis, the inventor develops a new detection method, can effectively separate and realize simultaneous detection of cyanoacetamide, cyanoacetic acid and methyl cyanoacetate, and further can perform better quality control on cyanoacetamide.
The invention provides a method for detecting cyanoacetamide and/or related substances thereof, which adopts high performance liquid chromatography to carry out qualitative or/and quantitative detection, wherein the detection conditions of the liquid chromatography comprise:
a chromatographic column: a C18 or equivalent chromatographic column;
mobile phase: an aqueous phosphoric acid solution.
The inventor verifies through a large number of experiments that under the detection condition of the invention, the phosphoric acid aqueous solution in the mobile phase can realize effective separation between the main peak and the impurity peak within a common reasonable application range, such as when the volume fraction of phosphoric acid is 0.005-0.5%, so as to achieve the detection purpose.
When the concentration of phosphoric acid in the mobile phase is too high, damage may be caused to the column, and the performance and the service life of the column may be reduced, and therefore, the volume fraction of phosphoric acid in the aqueous phosphoric acid solution used in the art as the mobile phase is generally controlled in the range of 0.005 to 0.2%.
In a specific embodiment of the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is controlled to be not more than 0.16% for the pH value of the chromatographic column to be used, and the mobile phase is a phosphoric acid aqueous solution with a volume fraction of 0.01-0.16%, preferably a phosphoric acid aqueous solution with a volume fraction of 0.1%.
Further, the related substances at least comprise one or two of cyanoacetic acid and methyl cyanoacetate.
The phrase "at least comprising one or two of cyanoacetic acid and methyl cyanoacetate" means that the related substances may be cyanoacetic acid or methyl cyanoacetate, cyanoacetic acid and methyl cyanoacetate, cyanoacetic acid (or methyl cyanoacetate) and other impurities, cyanoacetic acid, methyl cyanoacetate and other impurities, and the like.
Further, the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i chromatographic column specification: 4.6X 250mm, 3-5 μm;
ii column temperature: 25 to 40 ℃;
iii flow rate: 0.5-1.0 ml/min;
iv detection wavelength: 200nm to 230nm.
Further, the liquid chromatography detection conditions further include one or more of the following i to iv:
i specification of chromatographic column: 4.6X 250mm,5 μm;
ii column temperature: 30 ℃;
iii flow rate: 0.5 to 0.8ml/min, preferably 0.6ml/min;
iv detection wavelength: 210 +/-2 nm;
further, the sample injection amount is 5 to 50. Mu.L.
In a specific embodiment of the present invention, the sample size is 20. Mu.L.
Further, the column may be a C18 column or equivalent column that is tolerant of 100% aqueous phase.
For the column of the present invention, the inventors tried various commercial C18 columns that can tolerate 100% aqueous phase on the market, including but not limited to waters xbridge C18, ultimate AQ-C18, YMC-Pack ODS-AQ, agilent Poroshall 120 Box-RP, agilent Poroshall 120SB-Aq, agilent Zorbax SB-Aq, polaris C18-A, zorbax Bonus-RP, zorbax SB-Aq, and Shimadzuki InSuerttain AQ-C18, all of which can achieve the technical effects of effective detection of cyanoacetamide, cyanoacetic acid, and methyl cyanoacetate. In the present invention, an embodiment using a YMC-Pack ODS-AQ column is described.
Of course, it should be understood that the alternative chromatographic columns of the present invention are not limited to the above commercial products, and the commercial products of the chromatographic columns are only exemplary of the possible technical solutions, and thus the scope of the present invention should not be limited thereby, and any chromatographic column satisfying the above limitations or equivalent to the commercial products can implement the present invention.
Furthermore, the detection method also comprises the step of preparing a test solution, which comprises the following steps: mixing the test sample with a diluent.
Further, the diluent is a phosphoric acid aqueous solution with a volume fraction of 0.005-0.5%, preferably a phosphoric acid aqueous solution with a volume fraction of 0.005-0.2%, preferably a phosphoric acid aqueous solution with a volume fraction of 0.01-0.16%, and more preferably a phosphoric acid aqueous solution with a volume fraction of 0.1%.
In practice, the diluent may be kept in line with the mobile phase for ease of handling.
Further, the concentration of the sample solution is 10mg/mL to 60mg/mL, preferably 20mg/mL to 50mg/mL, more preferably 20mg/mL to 30mg/mL, and still more preferably 25mg/mL.
The detection method can calculate the detection result by methods commonly used in the field, such as analysis by an area normalization method, a self-comparison method, an internal standard method, an external standard method and the like.
In the specific implementation mode of the invention, the contents of cyanoacetic acid and methyl cyanoacetate are calculated by adopting an external standard method, and the contents of other impurities are calculated by adopting a self-comparison method.
The invention has the beneficial effects that:
(1) The invention discovers and identifies two related substances of cyanoacetamide for the first time: cyanoacetic acid and methyl cyanoacetate are of great significance for the quality control research of cyanoacetamide.
(2) The detection method provided by the invention realizes effective separation of cyanoacetamide, cyanoacetic acid and methyl cyanoacetate, can simultaneously perform quality control on cyanoacetamide and related substances, breaks through the limitation that only cyanoacetamide can be detected for quality control in the past, more comprehensively guarantees the quality safety of cyanoacetamide, and provides a reliable method for quality control of cyanoacetamide.
(3) The detection method adopts a single water phase, does not need to adjust the pH value, is convenient to operate, has few influencing factors and has good reproducibility; the mobile phase does not contain an organic phase, so that the method is green and environment-friendly and can better ensure the health of operators; the system has good applicability and specificity and good linearity; the sensitivity is high, and related substances with very low content in the raw material of cyanoacetamide can be accurately detected (the quantitative limit of cyanoacetic acid as a process impurity is 1.9 mu g/mL, and the quantitative limit of methyl cyanoacetate is 0.98 mu g/mL); the accuracy and the durability are good, the quality control can be better carried out on related products of the cyanoacetamide, and the method is worthy of popularization and application.
Drawings
FIG. 1 is a graph of the UV absorption of cyanoacetamide;
FIG. 2 is a graph of the UV absorption of cyanoacetic acid;
FIG. 3 is a graph of the UV absorption of methyl cyanoacetate;
FIG. 4 is a detection spectrum of the chromatographic conditions of Condition 1;
FIG. 5 is a detection spectrum under the chromatographic conditions of condition 2 (concentration of the test sample: 0.5 mL/min);
FIG. 6 is a detection spectrum under the chromatographic conditions of condition 2 (concentration of the test sample: 0.6 mL/min);
FIG. 7 is a detection spectrum under the chromatographic conditions of condition 2 (concentration of the test sample: 0.8 mL/min);
FIG. 8 is a detection spectrum under the chromatographic conditions of Condition 2 (concentration of the test sample: 1.0 mL/min);
FIG. 9 is a detection spectrum of a cyanoacetic acid control substance under the chromatographic condition of condition 2 (concentration of the test substance: 0.6 mL/min);
FIG. 10 is a detection spectrum of a methyl cyanoacetate control under the chromatographic conditions of Condition 2 (concentration of the test substance: 0.6 mL/min);
FIG. 11 is a detection spectrum (column temperature: 25 ℃ C.) of the chromatographic conditions of condition 3;
FIG. 12 is a detection spectrum (column temperature: 35 ℃ C.) of the chromatographic conditions of condition 3;
FIG. 13 is a detection spectrum (column temperature: 40 ℃ C.) of the chromatographic conditions of condition 3;
FIG. 14 is a detection spectrum (mobile phase: volume fraction 0.01% phosphoric acid aqueous solution) of the chromatographic conditions of example 3;
FIG. 15 is a detection spectrum (mobile phase: volume fraction 0.16% phosphoric acid aqueous solution) of the chromatographic conditions of example 3;
FIG. 16 is a graph showing a detection spectrum under the chromatographic conditions in example 4 (concentration of the test solution: 10 mg/mL);
FIG. 17 is a chromatogram obtained by examining the chromatographic conditions in example 4 (concentration of the sample solution: 30 mg/mL);
FIG. 18 is a graph showing a detection spectrum under the chromatographic conditions in example 4 (concentration of the test solution: 40 mg/mL);
FIG. 19 is a graph showing a detection spectrum under the chromatographic conditions in example 4 (concentration of the test solution: 50 mg/mL);
FIG. 20 is a graph showing a detection spectrum under the chromatographic conditions in example 4 (concentration of the test solution: 60 mg/mL);
FIG. 21 is a line graph of cyanoacetamide;
FIG. 22 is a line graph of methyl cyanoacetate;
FIG. 23 is a line graph of cyanoacetic acid.
Detailed description of the preferred embodiment
The detection method of the present invention is further described below by way of specific embodiments and experiments.
The relevant experimental procedures in the examples are as follows:
preparation of a test solution: weighing a proper amount of cyanoacetamide sample, precisely weighing, adding a diluent to dissolve, and quantitatively diluting to prepare a solution containing about 25mg of the sample per 1 mL.
Preparation of a reference solution: taking a proper amount of a cyanoacetic acid methyl ester reference substance and a cyanoacetic acid reference substance, precisely weighing, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1.25mg of the reference substance in every 1mL, and taking the solution as a reference substance solution stock solution; precisely measuring a proper amount of the reference solution stock solution, adding a diluent to dissolve the reference solution stock solution, and quantitatively diluting the reference solution stock solution to prepare a solution containing about 125 mu g of the reference substance per 1 mL.
Preparation of self-control solution: a proper amount of the test solution is precisely measured, and a diluent is added to prepare a solution containing about 25 mu g of the test solution in each 1 mL.
System applicability solution: a proper amount of stock solutions of a test solution and a reference solution are precisely measured, and a diluent is added to dilute the stock solutions to prepare solutions containing 125 mu g of each of the test solution, the methyl cyanoacetate and the cyanoacetic acid in each 1 mL.
EXAMPLE 1 selection of detection wavelength
The ultraviolet absorption wavelengths of cyanoacetamide, methyl cyanoacetate and cyanoacetic acid are respectively detected, the results are shown in figures 1-3, the compounds have strong absorption in the range of 200 nm-230 nm, and finally 210nm is selected as the detection wavelength for carrying out subsequent experiments.
Example 2 screening of chromatographic conditions
Condition 1:
a chromatographic column: waters Symmetry C18, 4.6X 250mm,5 μm;
mobile phase: phosphoric acid aqueous solution (volume fraction 0.11%) -methanol (V/V = 80;
column temperature: 30 deg.C
Detection wavelength: 210nm;
sample introduction amount: 20 mu L of the solution;
diluent agent: a mobile phase;
concentration of the test solution: 10mg/mL;
flow rate: 0.8mL/min.
The results of the detection are shown in FIG. 4.
As shown in FIG. 4, the retention time of the main peak was 2.890min, and the impurity compounds such as cyanoacetic acid and methyl cyanoacetate could not be detected, and the chromatographic conditions were further adjusted.
Condition 2:
a chromatographic column: YMC-Pack ODS-AQ, 4.6X 250mm,5 μm;
mobile phase: phosphoric acid aqueous solution with mass fraction of 0.1%;
column temperature: 30 ℃;
detection wavelength: 210nm;
sample introduction amount: 20 mu L of the solution;
diluent agent: a mobile phase;
concentration of the test solution: 25mg/mL;
flow rate:
the flow rates were set to 0.5mL/min, 0.6mL/min, 0.8mL/min, and 1.0mL/min, respectively.
The results of the measurements are shown in Table 1 and FIGS. 5 to 8.
TABLE 1
As can be seen from the results in Table 1, the impurities cyanoacetic acid and methyl cyanoacetate can be detected well after the chromatographic column and the mobile phase are replaced under the condition, the sensitivity is high, and the effective separation of the impurities from the main peak can be realized. When the flow rate is 0.5-1.0 mL/min, the main peaks of cyanoacetamide, cyanoacetic acid and methyl cyanoacetate can be effectively separated, and the separation effects of different flow rates are not greatly different. The flow rate was selected to be 0.6mL/min, taking into account the analysis time and sample retention factors.
Condition 3:
a chromatographic column: YMC-Pack ODS-AQ, 4.6X 250mm,5 μm;
mobile phase: phosphoric acid aqueous solution with volume fraction of 0.1 percent;
column temperature:
setting the flow rates at 25 ℃, 30 ℃ (directly adopting the data in the condition 2), 35 ℃ and 40 ℃ respectively;
detection wavelength: 210nm;
sample introduction amount: 20 mu L of the solution;
diluent agent: a mobile phase;
concentration of the test solution: 25mg/mL;
flow rate: 0.6mL/min.
The results of the measurements are shown in table 2 and fig. 6 and fig. 11 to 13.
TABLE 2
As can be seen from the results in Table 2, the column temperature has a small influence on the separation, and a conventional column temperature of 30 ℃ may be selected.
The preferred chromatographic conditions ultimately determined are:
a chromatographic column: YMC-Pack ODS-AQ, 4.6X 250mm,5 μm;
mobile phase: phosphoric acid aqueous solution with volume fraction of 0.1 percent;
column temperature: 30 ℃;
detection wavelength: 210nm;
sample injection amount: 20 mu L of the solution;
diluent (b): a mobile phase;
concentration of the test solution: 25mg/mL;
flow rate: 0.6mL/min.
The results are shown in FIG. 6.
Example 3
Chromatographic conditions are as follows:
a chromatographic column: YMC-Pack ODS-AQ, 4.6X 250mm,5 μm;
mobile phase: respectively adopting phosphoric acid aqueous solution with the volume fraction of 0.01 percent and 0.16 percent;
column temperature: 30 ℃;
detection wavelength: 210nm;
sample introduction amount: 20 mu L of the solution;
diluent agent: a mobile phase;
concentration of the test solution: 25mg/mL;
flow rate: 0.6mL/min;
the detection result map is shown in FIGS. 14 and 15.
FIG. 14 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample when the mobile phase is a 0.01% volume fraction aqueous phosphoric acid solution, wherein the separation degree of cyanoacetamide (6.534 min) and cyanoacetic acid (8.483 min), which is a main peak, from the adjacent impurity is 5.87.
FIG. 15 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample when the mobile phase is a 0.16% volume fraction aqueous phosphoric acid solution, wherein the separation degree of cyanoacetamide (6.519 min) and cyanoacetic acid (8.283 min), which is a main peak, from the adjacent impurity is 5.88.
From the above results, it can be seen that effective detection can be achieved in different phosphoric acid concentration ranges of the mobile phase under the detection conditions of the present invention, and the mobile phase is only an aqueous phosphoric acid solution, but when the volume fraction of phosphoric acid is greater than 0.16%, the pH value of the mobile phase is lower than the pH value range of the AQ column, which may damage the chromatographic column, and in practical applications, an aqueous phosphoric acid solution with a volume fraction of 0.16% or less is usually used as the mobile phase.
Example 4
Chromatographic conditions are as follows:
a chromatographic column: YMC-Pack ODS-AQ, 4.6X 250mm,5 μm;
mobile phase: 0.1% by volume of phosphoric acid aqueous solution;
column temperature: 30 ℃;
detection wavelength: 210nm;
sample introduction amount: 20 mu L of the solution;
diluent agent: a mobile phase;
concentration of the test solution: 10mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL;
flow rate: 0.6mL/min.
The detection result map is shown in FIGS. 16 to 20.
FIG. 16 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample at a sample concentration of 10mg/mL, wherein the separation of cyanoacetamide (6.607 min) as a main peak from cyanoacetic acid (8.269 min) as an adjacent impurity is 6.51.
FIG. 17 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample at a sample concentration of 30mg/mL, showing that the degree of separation of cyanoacetamide (6.523 min) as a main peak from cyanoacetic acid (8.249 min), which is an adjacent impurity, is 5.50.
FIG. 18 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample at a sample concentration of 40mg/mL, showing that the degree of separation of cyanoacetamide (6.504 min) from cyanoacetic acid (8.234 min), which is an adjacent impurity, is 5.16.
FIG. 19 is a chromatogram showing the separation of cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample at a sample concentration of 50mg/mL, showing that the degree of separation of cyanoacetamide (6.521 min) from cyanoacetic acid (8.220 min), which is an adjacent impurity, is 4.81.
FIG. 20 is a chromatogram for separating cyanoacetamide from cyanoacetic acid and methyl cyanoacetate in a sample at a sample concentration of 60mg/mL, wherein the separation degree of cyanoacetamide (6.521 min) as a main peak from cyanoacetic acid (8.206 min) as an adjacent impurity is 4.54, the separation is slightly deteriorated, and the overload of a chromatographic column is severe.
Methodology verification (preferred conditions in example 2):
1. stability of cyanoacetamide solution:
TABLE 3
2. Linear equation of equations
Cyanoacetamide linearity:
TABLE 4
| Name of Linear solution | Concentration (mg/mL) | Percentage of sample solution (%) | Peak area |
| L6 | 0.00122364 | 0.005 | 5901 |
| L5 | 0.00244728 | 0.01 | 11132 |
| L4 | 0.0061182 | 0.02 | 27450 |
| L3 | 0.0152955 | 0.06 | 70079 |
| L2 | 0.030591 | 0.12 | 138949 |
| L1 | 0.061182 | 0.24 | 273910 |
The linear equation: y =4,478,458x +679,R 2 =0.9999, linear range 0.001 mg/mL-0.06 mg/mL, see fig. 21.
Linear cyanoacetic acid methyl ester:
TABLE 5
The linear equation: y =823380x-512, correlation coefficient r =0.99995, slope 823380, intercept equivalent to 4.6% of 100% concentration, linear range 0.001mg/mL to 0.057mg/mL, see fig. 22.
Linear cyanoacetic acid:
TABLE 6
| Name of Linear solution | Concentration (mg/ml) | Percentage of sample solution (%) | Peak area |
| L6 | 0.00125875 | 0.005 | 1154 |
| L5 | 0.0025175 | 0.01 | 4712 |
| L4 | 0.00629375 | 0.025 | 8898 |
| L3 | 0.015734375 | 0.06 | 15375 |
| L2 | 0.03146875 | 0.12 | 31248 |
| L1 | 0.0629375 | 0.25 | 71546 |
The linear equation: y =1104326x +30, correlation coefficient r =0.996, slope 1104326, intercept equivalent to 0.1% of 100% concentration, linear range 0.001 mg/mL-0.06 mg/mL, see fig. 23.
Methyl cyanoacetate correction factor =4478458/823380=5.44.
Cyanoacetic acid correction factor =4478458/1104326=4.06.
The correction factors of the cyanoacetic acid methyl ester and the cyanoacetic acid are both over or close to 5, so that the external standard method is used for detection.
3. Detection limit and quantification limit:
methyl cyanoacetate:
the detection limit concentration is 0.00057mg/ml, which is equivalent to 0.0023 percent of the sample test solution, the sample introduction is carried out for three times continuously, and the S/N range is 4.0 to 8.2.
The quantitative limit concentration is 0.0019mg/ml, which is equivalent to 0.0076% of the sample test solution, the sample is continuously fed for six times, the S/N range is 11.5-16.7, the peak area average value is 21.016 (Shimadzu), and the peak area RSD is 0.33%.
Cyanoacetic acid:
the detection limit concentration is 0.00029mg/ml, which is equivalent to 0.0012% of the sample test solution, the sample is continuously injected for three times, and the S/N range is 4.4-6.7.
The quantitative limit concentration is 0.00098mg/ml, which is equivalent to 0.0039 percent of the sample test solution, the sample introduction is carried out for six times, the S/N range is 16.7 to 33.5, the peak area average value 905 (Shimadzu) and the peak area RSD is 0.14 percent.
Cyanoacetamide:
detection is not performed.
Limit of quantification: the S/N ratio is not about 10. The lowest concentration of 0.00122mg/ml is achieved in a linear experiment, which is equivalent to 0.005% of the sample test solution, and the range of S/N is 162.8-168.2. Peak area average 5900 (shimadzu).
4. Durability:
TABLE 7
5. System applicability
The system suitability test is carried out for a plurality of times, and the test data of the methyl cyanoacetate and the ethyl cyanoacetate are shown in a table 8.
TABLE 8
| Name (R) | |
|
|
|
Peak area RSD/%) |
| Cyanoacetic acid methyl ester | 13184 | 13768 | 14205 | 14561 | 4.3 |
| Cyanoacetic acid | 22709 | 22798 | 22634 | 22669 | 0.3 |
6. Specificity
TABLE 9
The result shows that the method has good system applicability and specificity, good linearity, good accuracy and durability, and meets the requirements of linear range, detection limit and quantitative limit, and the method is proved to be suitable for detecting the cyanoacetamide and related substances thereof.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (11)
1. The detection method of cyanoacetamide and/or related substances thereof is characterized in that high performance liquid chromatography is adopted for qualitative or/and quantitative detection, and the detection conditions of the liquid chromatography comprise:
a chromatographic column: a C18 chromatographic column;
mobile phase: phosphoric acid aqueous solution with volume fraction of 0.01-0.16%;
the related substances are one or two of cyanoacetic acid and methyl cyanoacetate;
detection wavelength: 200nm to 230nm.
2. The detection method according to claim 1, wherein the mobile phase is a 0.1% volume fraction phosphoric acid aqueous solution.
3. The detection method according to any one of claims 1 to 2, wherein the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6X 250mm, 3-5 μm;
ii column temperature: 25 to 40 ℃;
iii flow rate: 0.5-1.0 ml/min.
4. The detection method according to claim 3, wherein the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6X 250mm,5 μm;
ii column temperature: 30 ℃;
iii flow rate: 0.5-0.8 ml/min, iv detection wavelength: 210 +/-2 nm.
5. The assay of claim 4, wherein the flow rate is 0.6ml/min.
6. The detection method according to any one of claims 1 to 3, wherein the amount of sample is 5 to 50. Mu.L.
7. The detection method according to any one of claims 1 to 3, wherein the column is a C18 column which is resistant to 100% aqueous phase.
8. The detection method according to claim 7,
the C18 column which is tolerant of 100% aqueous phase is selected from the group consisting of waters xbridge C18, ultimateAQ-C18, YMC-Pack ODS-AQ, poroshall 120 Box-RP, poroshall 120SB-Aq, zorbax SB-Aq, polaris C18-A, zorbax Box-RP, zorbax SB-Aq, inertsustain AQ-C18 columns.
9. The detection method according to any one of claims 1 to 3, further comprising preparing a test solution, comprising the steps of: mixing the test sample with a diluent;
the diluent is phosphoric acid aqueous solution with volume fraction of 0.005-0.5%.
10. The assay of claim 9, wherein the diluent is in line with the mobile phase.
11. The detection method according to claim 9, wherein the concentration of the sample solution is 10mg/mL to 60mg/mL.
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