CN111413451B - Method for detecting cyanoacetamide by reversed-phase high performance liquid chromatography - Google Patents
Method for detecting cyanoacetamide by reversed-phase high performance liquid chromatography Download PDFInfo
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Abstract
The invention relates to the technical field of detection and analysis, in particular to a method for detecting cyanoacetamide by reversed-phase high performance liquid chromatography. The following chromatographic conditions were employed: and (3) chromatographic column: agilent ZORBAX Eclipse XDB-CN liquid chromatography column (4.6 mm. times.250 mm, 5 um); mobile phase: acetonitrile-ammonium formate volatile buffer solution; flow rate: 0.5 mL/min; detection wavelength: 200 nm; column temperature: 40 ℃; sample introduction amount: 5 uL; the detector is a diode array detector. The method can better realize the quality control of the cyanoacetamide, improves the accuracy of analysis, and has high accuracy, and good detection linearity and range, recovery rate, precision and other tests.
Description
Technical Field
The invention relates to the technical field of detection and analysis, in particular to a method for detecting cyanoacetamide by using a reversed-phase high performance liquid chromatography.
Background
Cyanoacetamide is a commonly used organic synthetic raw material for the synthesis of malononitrile, electroplating solution and pesticides triamterene and aminopterin. It is used as an important synthesis intermediate, and malononitrile can be generated by dehydration; brominating to obtain 2, 2-dibromo-2-cyanoacetamide; cyclizing the compound with ethyl trifluoroacetoacetate to obtain a substituted pyridine compound, and synthesizing to obtain the insecticide flonicamid; also, cyanoacetamide is one of the important raw materials for the fungicide fludioxonil. The structural formula is as follows:
The cyanoacetamide is used as an important raw material in the chemical industry, the residue of the cyanoacetamide is generally detected by a quantitative method at present, and the like adopts normal-phase high performance liquid chromatography, an XDB-CN column is used, and a mixed solvent of ethanol and normal hexane is used as a mobile phase to separate and quantitatively analyze a cyanoacetamide sample. Patent CN103926333A provides a high performance liquid chromatography detection method of cyanoacetamide, the chromatographic conditions adopted by the method are: a chromatographic column: Polar-Silica, particle size 5 μm, length 250mm, diameter 4.6mm, pore diameter 120A; the mobile phase is a mixed system of acetonitrile and methane chloride; the detector is ELSD, and is suitable for quantitative detection of industrial coarse cyanoacetamide and intermediate. Quantitative detection can be realized by using the normal phase liquid chromatography, but the operation is complex, which brings inconvenience to analysis work, particularly, the reaction condition is not timely and directly monitored by the liquid phase, the detection period is long, time is consumed, the reaction condition can not be accurately and visually reflected, and the normal phase liquid chromatography can not effectively track the product or another raw material due to small polarity. The widenin et al adopts a high performance liquid phase reversed phase ODS column, C18 or C8 for detection, but the polarity of the cyanoacetamide is larger, so the retention on the reversed phase ODS column is weak, and the widenin et al often appears simultaneously with an inverted peak or a solvent peak and an impurity peak, and the accurate quantitative detection of the cyanoacetamide is difficult to realize. Therefore, there is a need for a reversed-phase high performance liquid chromatography method for measuring cyanoacetamide with good separation degree and high column stability.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the method for detecting the cyanoacetamide by the reversed-phase high performance liquid chromatography, which has the advantages of good separation degree, high accuracy and good column stability.
The invention provides a method for detecting cyanoacetamide by reversed-phase high-performance chromatography, which adopts the following chromatographic conditions:
and (3) chromatographic column: agilent ZORBAX Eclipse XDB-CN liquid chromatography column (4.6 mm. times.250 mm, 5 um);
mobile phase: acetonitrile-ammonium formate volatile buffer solution;
flow rate: 0.5 mL/min;
detection wavelength: 200 nm;
column temperature: 40 ℃;
sample introduction amount: 5 uL;
the detector is a diode array detector.
The volume ratio of acetonitrile to the volatile buffer solution of ammonium formate in the mobile phase is 7-15: 90. The time to peak is shortened with increasing acetonitrile ratio.
The pH of the volatile buffer solution of ammonium formate = 6.5-7.5. Maintaining this pH prevents deterioration of the cyanoacetamide and is better suited for chromatographic performance.
The mobile phase can also be acetonitrile-methanol-ammonium formate volatile buffer solution.
The volatile buffer solution of ammonium formate and the organic phase in the mobile phase do not need to be premixed (namely, the organic phase and the aqueous phase are mixed in proportion in advance and then used), and the organic phase and the volatile buffer solution of ammonium formate are directly and separately fed by a double pump; if the organic phase is a mixed phase of methanol and acetonitrile, the organic phase can be premixed and then the organic phase and the volatile buffer solution of ammonium formate can be pumped in by a double pump.
The detection wavelength is 200nm, the area of the cyanoacetamide peak is in a descending trend along with the red shift of the wavelength, the wavelength is in a blue shift, and the baseline is deteriorated.
The chromatographic conditions are preferably: the volume ratio of acetonitrile to ammonium formate buffer solution in the mobile phase is 10: 90; the flow rate is 0.5 mL/min; the detection wavelength is 200 nm; the column temperature was 40 ℃ and the injection volume was 5 uL.
A method for detecting cyanoacetamide by reversed phase high performance chromatography comprises the following steps:
(1) preparation of standard sample solution
Accurately weighing a standard sample containing 0.05g (accurate to 0.0002 g) of cyanoacetamide, placing the standard sample in a 100mL volumetric flask, adding 90mL of acetonitrile solution, dissolving by ultrasonic oscillation, cooling to room temperature, using acetonitrile to scale, and shaking uniformly for later use;
(2) preparation of sample solution
Accurately weighing a sample containing 0.05g (accurate to 0.0002 g) of cyanoacetamide, placing the sample in a 100mL volumetric flask, adding 90mL of acetonitrile solution, dissolving the sample by ultrasonic oscillation, cooling the solution to room temperature, using acetonitrile to scale, and shaking the solution uniformly for later use;
(3) measurement of
Under the operating conditions, after the baseline of the instrument is stable, continuously injecting a plurality of needles of standard sample solution until the response value of the cyanoacetamide of two adjacent needles relatively changes by less than 0.5 percent, and then measuring according to the sequence of the standard sample solution, the sample solution and the standard sample solution;
(4) Calculating out
The areas of the cyanoacetamide peaks of the two needle sample solutions and the two needle sample solutions before and after the sample are respectively averaged.
Agilent ZORBAX Eclipse XDB chromatography columns (C18, C8, phenyl and cyano) provided four bound stationary phases for optimization of method development. These columns can provide good peak shape over a wide pH range (pH = 2-9), Eclipse XDB columns can be used for process development under low pH (2-3) conditions, and the same columns can also be used for process development over a medium pH range (6-8) where residual silanol groups are more active and interactions are more likely to occur, causing peak tailing. To overcome these interactions Eclipse XDB columns were ultra-densely bonded and double-capped by proprietary processes to cover as many active silanol groups as possible. The final basic compound has an excellent peak shape in the pH =2-9 range. High speed, high separation degree, analysis and preparation amplification separation are realized.
The beneficial effect of the invention is that,
(1) acetonitrile-ammonium formate volatile buffer solution is used as a mobile phase, and the pH =6.5-7.5 of the ammonium formate volatile buffer solution can effectively avoid damage to a chromatographic column and interference of absorption of the mobile phase or a solvent under short wave on sample detection;
(2) The effective retention of the cyanoacetamide can be realized, so that the interference of solvent peaks, inverted peaks and impurity peaks is avoided, and the rapid and accurate detection can be realized;
(3) the effective separation of the cyanoacetamide and impurities corresponding to the retention time of 6.5min in the liquid chromatogram can be realized;
(4) the selected chromatographic column has good stability, the preparation of the mobile phase is simple, and the detection cost is low;
(5) the method can better realize the quality control of the cyanoacetamide, improves the accuracy of analysis, and has high accuracy, and good detection linearity and range, recovery rate, precision and other tests.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a graph of a cyanoacetamide standard curve;
FIG. 2 is a chromatogram of a sample from example 1.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for detecting cyanoacetamide by reversed phase high performance liquid chromatography comprises the following chromatographic conditions: the chromatographic column is an Agilent ZORBAX Eclipse XDB-CN liquid chromatographic column (4.6 mm multiplied by 250mm, 5 um), and the mobile phase is acetonitrile-ammonium formate volatile buffer solution; the flow rate is 0.5 mL/min; the detection wavelength is 200 nm; the column temperature was 40 ℃; the sample injection amount is 5 uL; the detector is a diode array detector.
The specific operation steps are as follows:
(1) preparation of standard sample solution
Accurately weighing a standard sample containing 0.05g (accurate to 0.0002 g) of cyanoacetamide, placing the standard sample in a 100mL volumetric flask, adding 90mL of acetonitrile solution, dissolving by ultrasonic oscillation, cooling to room temperature, adding acetonitrile to the scale, and shaking uniformly for later use.
(2) Preparation of sample solution
Accurately weighing a sample containing 0.05g (accurate to 0.0002 g) of cyanoacetamide, placing the sample in a 100mL volumetric flask, adding 90mL of acetonitrile solution, dissolving the sample by ultrasonic oscillation, cooling to room temperature, adding acetonitrile to the scale, and shaking uniformly for later use.
(3) Measurement of
Under the operating conditions, after the baseline of the instrument is stable, a plurality of needles of standard sample solution are continuously injected until the response value of the cyanoacetamide of two adjacent needles relatively changes by less than 0.5%, and then the standard sample solution, the sample solution and the standard sample solution are sequentially measured. The sample chromatogram is shown in FIG. 2.
(4) Calculating out
The areas of the cyanoacetamide peaks of the two needle sample solutions and the two needle sample solutions before and after the sample are respectively averaged. The mass fraction of the cyanoacetamide is X1(%), calculated as follows:
in the formula:
A1-average value of area of cyanoacetamide in the standard solution;
A2-average value of area of cyanoacetamide in the sample solution;
m1-mass of cyanoacetamide standard, g;
m2-mass of sample, g;
mass fraction of p-cyanoacetamide,%.
The test data are shown in the following table 1:
TABLE 1 relevant test data
The mass fraction of the sample is calculated to be 98.08%.
(5) Authentication
Accuracy analysis of samples is shown in table 1.
TABLE 1 accuracy test results of samples
Analysis of the added standard recovery experimental data:
TABLE 2 results of sample spiking recovery test data
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. A method for detecting cyanoacetamide by reversed phase high performance liquid chromatography is characterized by adopting the following chromatographic conditions:
a chromatographic column: an Agilent ZORBAX Eclipse XDB-CN liquid chromatographic column, wherein the specification of the liquid chromatographic column is as follows: 4.6mm × 250mm, 5 um;
mobile phase: acetonitrile-ammonium formate volatile buffer solution;
flow rate: 0.5 mL/min;
detection wavelength: 200 nm;
column temperature: 40 ℃;
sample introduction amount: 5 uL;
the detector is a diode array detector.
2. The method for detecting cyanoacetamide by reverse-phase high performance liquid chromatography as claimed in claim 1, wherein the volume ratio of acetonitrile to volatile buffer solution of ammonium formate in the mobile phase is 7-15: 90.
3. The method for detecting cyanoacetamide according to claim 1, wherein the volatile buffer solution of ammonium formate has a pH = 6.5-7.5.
4. The method for detecting cyanoacetamide by reverse-phase high performance liquid chromatography as claimed in claim 1, wherein the chromatographic conditions are as follows: the volume ratio of acetonitrile to ammonium formate buffer solution in the mobile phase is 10: 90; the flow rate is 0.5 mL/min; the detection wavelength is 200 nm; the column temperature was 40 ℃ and the injection volume was 5 uL.
5. The method for detecting cyanoacetamide by reverse-phase high performance liquid chromatography as claimed in claim 1, wherein the mobile phase is acetonitrile-methanol-ammonium formate volatile buffer solution.
6. The method for detecting cyanoacetamide by reverse-phase high performance liquid chromatography as claimed in claim 1, which comprises the following steps:
(1) weighing a proper amount of cyanoacetamide standard substance, and dissolving the cyanoacetamide standard substance in acetonitrile to obtain a cyanoacetamide standard substance solution;
(2) weighing a proper amount of cyanoacetamide sample, and dissolving the cyanoacetamide sample in acetonitrile to obtain a cyanoacetamide standard solution;
and (3) taking the cyanoacetamide standard solution and the sample solution, injecting the cyanoacetamide standard solution and the sample solution into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions.
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| CN114577927B (en) * | 2022-01-24 | 2023-09-01 | 河北广祥制药有限公司 | Method for detecting residual impurities in entacapone |
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| JP2009270966A (en) * | 2008-05-08 | 2009-11-19 | Denki Kagaku Kogyo Kk | Measuring method of molecular weight distribution of hyaluronic acid in biological sample of mammal origin |
| CN103926333A (en) * | 2013-01-16 | 2014-07-16 | 苏州赛分科技有限公司 | High performance liquid chromatography detection method for cyanoacetamide |
| CN110057959A (en) * | 2019-05-24 | 2019-07-26 | 山东新华制药股份有限公司 | A kind of analysis method of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance |
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| JP2009270966A (en) * | 2008-05-08 | 2009-11-19 | Denki Kagaku Kogyo Kk | Measuring method of molecular weight distribution of hyaluronic acid in biological sample of mammal origin |
| CN103926333A (en) * | 2013-01-16 | 2014-07-16 | 苏州赛分科技有限公司 | High performance liquid chromatography detection method for cyanoacetamide |
| CN110057959A (en) * | 2019-05-24 | 2019-07-26 | 山东新华制药股份有限公司 | A kind of analysis method of the high effective liquid chromatography for measuring Febustat intermediate in relation to substance |
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Address after: 256500 Boxing Economic Development Zone, Shandong, Binzhou Patentee after: Shandong Jingbo Agrochemical Technology Co.,Ltd. Address before: 256500 Boxing Economic Development Zone, Shandong, Binzhou Patentee before: JINGBO AGROCHEMICALS TECHNOLOGY Co.,Ltd. |