Method for simultaneously determining contents of glycine methyl ester hydrochloride and 2, 5-piperazine diketone

Abstract

The invention provides a method for simultaneously determining the content of glycine methyl ester hydrochloride and the content of 2, 5-piperazine diketone, and belongs to the field of chemical detection and analysis. According to the method, acetonitrile and an ammonium formate aqueous solution with the volume ratio of 3: 97 are used as a mobile phase, the concentration of ammonium formate is 1.26 g / L, formic acid is used for adjusting the pH value of the solution to 3.95, meanwhile, an electric fog detector is selected, and a simertorin Accuore TM aQ C18 chromatographic column is used for detection, so that effective separation of glycine methyl ester hydrochloride and 2, 5-piperazine diketone is realized, and the method is suitable for large-scale production of glycine methyl ester hydrochloride and 2, 5-piperazine diketone. The method can be used for rapidly and accurately detecting the content of glycine methyl ester hydrochloride and 2, 5-piperazine diketone, and can be applied to the central control monitoring of the reaction for synthesizing 2, 5-piperazine diketone by glycine methyl ester hydrochloride.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical detection and analysis, and particularly relates to a method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione. BACKGROUND

[0002] Glyphosate (C3H8NO5P, CAS registration number: 1071-83-6) is an organic phosphorus herbicide. Because it has good control effect on annual and perennial weeds, it is widely used in farmland, orchard, forest and uncultivated land, etc., and becomes one of the most widely used agricultural herbicides in the world. Glyphosate is an internal conductive type of broad-spectrum herbicide, which can penetrate into the plant body from the leaves or stems of the plant, and block the synthesis of aromatic amino acids in the plant body by inhibiting the activity of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid synthesis enzyme system of the plant body, so as to achieve the purpose of weeding.

[0003] 2,5-piperazinedione is an important intermediate for synthesizing glyphosate. Glycine methyl ester hydrochloride, as a cheap and widely used pesticide industrial raw material, can be used for the synthesis of 2,5-piperazinedione. Because glycine methyl ester hydrochloride has a wide source of raw materials, low cost and moderate reactivity, it is widely used in actual production, and is an important basis for realizing the green, efficient and low-cost synthesis of glyphosate. However, in the process of synthesizing 2,5-piperazinedione, due to the simple molecular structure of glycine methyl ester hydrochloride, the response of glycine methyl ester hydrochloride in the ultraviolet detector is greatly different from that of the product 2,5-piperazinedione, so that it is difficult to accurately determine the residual amount of the reactant under the conventional analysis means such as high performance liquid chromatography, which affects the judgment of the synthesis reaction effect, and further affects the yield and purity of 2,5-piperazinedione, and further affects the overall efficiency and product quality of the subsequent glyphosate synthesis.

[0004] Therefore, it is of great significance to establish a sensitive, accurate and specific glycine methyl ester hydrochloride detection method to effectively monitor the reaction of glycine methyl ester hydrochloride, improve the yield and quality of 2,5-piperazinedione, and effectively improve the yield of glyphosate product. SUMMARY

[0005] The main purpose of the present application is to provide a method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, which solves the problems in the background art. The method realizes the effective separation of glycine methyl ester hydrochloride and 2,5-piperazinedione by optimizing the chromatographic conditions, not only has high sensitivity, high selectivity and wide linear range, but also can complete the simultaneous detection of double components in a short time, and significantly improves the analysis efficiency.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: A method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, characterized by comprising the following steps: (1) Standard solution preparation: preparing glycine methyl ester hydrochloride and diketopiperazine standard solutions with a mobile phase as a solvent; (2) Standard curve establishment: sequentially injecting the standard solutions prepared in step (1) and analyzing them by using a high-performance liquid chromatography system to establish a standard curve of the concentration of the standard solution and the corresponding peak area; (3) Sample determination and quantitative analysis: preparing a sample solution with a mobile phase as a solvent, injecting the sample solution, and calculating the contents of each measured substance according to the standard curve of step (2).

[0007] Further, the mobile phase of step (1) uses an ammonium formate aqueous solution as an aqueous phase and acetonitrile as an organic phase.

[0008] Further, the concentration of the ammonium formate aqueous solution is 1-3 g / L, and the solution pH is adjusted to 2-5 by using phosphoric acid, acetic acid or formic acid.

[0009] Further, the concentration of the ammonium formate aqueous solution is 1.26 g / L, and the solution pH is adjusted to 3.95 by using formic acid. This condition can effectively suppress ionization interference and improve separation selectivity and detection repeatability.

[0010] Further, in step (1), the volume ratio of acetonitrile to ammonium formate aqueous solution is 1-10:90-100.

[0011] Further, in step (1), the volume ratio of acetonitrile to ammonium formate aqueous solution is 3:97. This ratio can ensure good separation of the two components while achieving a shorter analysis period.

[0012] Further, in step (2), the isocratic elution mode is used, and the volume ratio of acetonitrile to ammonium formate aqueous solution is 1-10:90-100, preferably 3:97.

[0013] Further, in step (2), the liquid chromatography conditions are as follows: the detector is an electrospray detector, the detector evaporation tube temperature is 25-55℃, the flow rate is 0.5-1.5 mL / min, the column temperature is 20-50℃, and the injection volume is 5-15 μL.

[0014] Further, in step (2), the liquid chromatography conditions are as follows: the detector is an electrospray detector, the detector evaporation tube temperature is 35℃, the flow rate is 1.0 mL / min, the column temperature is 40℃, and the injection volume is 10 μL.

[0015] Further, in step (2), the specifications of the liquid chromatography column are 250 mm x 4.6 mm with a particle size of 5 μm.

[0016] Further, the concentration of the glycine methyl ester hydrochloride standard solution in step (1) is 50-500 mg / L, and the concentration of the 2,5-piperazinedione standard solution is 50-500 mg / L.

[0017] The application provides a rapid, efficient, sensitive and accurate two-component synchronous detection method, solves the technical problem that glycine methyl ester hydrochloride and 2,5-piperazinedione have large response differences and are difficult to quantify under conventional detection means, and has important significance for improving the synthesis efficiency and product quality of key intermediates of glyphosate; the method is not only suitable for the intermediate control analysis of the synthesis reaction of 2,5-piperazinedione, but also can be applied to the quality monitoring of the synthesis process of other pesticides and medicines containing polar small molecule intermediates, and has good industrialization popularization value.

[0018] The application has the following beneficial effects: 1. The application uses acetonitrile and an aqueous ammonium formate solution as the mobile phase, adopts isocratic elution, realizes the efficient separation of glycine methyl ester hydrochloride and 2,5-piperazinedione, and can realize the quantification of the two substances, and the responses of the two substances in the electric fog detector are equivalent, which is beneficial to the control of the reaction progress in synthesis.

[0019] 2. The application uses liquid chromatography to quickly and accurately determine the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, and the test can be completed within 10 minutes, greatly shortening the detection time and realizing the control monitoring of the synthesis reaction and improving the synthesis efficiency.

[0020] 3. The method of the application is simple and easy to operate, and has high detection sensitivity. DETAILED DESCRIPTION

[0021] The application will be further described below with reference to the drawings: Figure 1 is the glycine methyl ester hydrochloride standard curve of Example 1 of the application; Figure 2 is the 2,5-piperazinedione standard curve of Example 1 of the application; Figure 3 is the HPLC spectrum of the glycine methyl ester hydrochloride and 2,5-piperazinedione standard sample in Example 1 of the application; Figure 4 is the HPLC spectrum of the reaction liquid sample in Example 1 of the application; Figure 5 is the HPLC spectrum of the A) glycine methyl ester hydrochloride and B) 2,5-piperazinedione standard sample in Example 2 of the application; Figure 6 is the HPLC spectrum of the glycine methyl ester hydrochloride standard sample when the pH of the mobile phase is adjusted to 2 in Example 3 of the application; Figure 7 is the HPLC chromatogram of the glycine methyl ester hydrochloride standard sample tested in Example 3 of the present application when the pH of the mobile phase is adjusted to 5; Figure 8 is the HPLC chromatogram of the glycine methyl ester hydrochloride standard sample tested in Example 4 of the present application when the temperature of the evaporation tube is 25°C; Figure 9 is the HPLC chromatogram of the glycine methyl ester hydrochloride standard sample tested in Example 4 of the present application when the temperature of the evaporation tube is 45°C. DETAILED DESCRIPTION

[0022] In order to make the person skilled in the art better understand the technical solutions in the present application, the solutions of the present application will be specifically and clearly described below in combination with examples. Obviously, the described examples are only some of the embodiments of the present application, but not all.

[0023] Example 1: A method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, the specific steps are as follows: (1) Preparation of the mobile phase: Ammonium formate aqueous solution: take 1.26 g of ammonium formate and dilute to 1000 mL with ultrapure water, adjust the pH to 3.95 with formic acid, shake well, and pass through a 0.45 μm mixed fiber filter membrane for standby; Mobile phase solution: mix 970 mL of ammonium formate aqueous solution with 30 mL of chromatographic acetonitrile uniformly, and standby for use for 20 min.

[0024] (2) Preparation of the standard solution: A series of mixed standard solutions of glycine methyl ester hydrochloride and 2,5-piperazinedione are prepared with the mobile phase as the diluent. The standard solution of glycine methyl ester hydrochloride has concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 350 mg / L and 500 mg / L; and the standard solution of 2,5-piperazinedione has concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 350 mg / L and 500 mg / L.

[0025] (3) Drawing of the standard curve: The standard solutions prepared in step (2) are injected in order from low concentration to high concentration, and analyzed by a high performance liquid chromatography system. The concentration of the standard solution and the corresponding peak area are taken as the abscissa and the ordinate respectively, and a standard curve is established.

[0026] As Figures 1-3The standard curve equation of glycine methyl ester hydrochloride is y = 0.0963x + 1.6799, and the correlation coefficient is R2 = 0.9999; the standard curve equation of 2,5-piperazinedione is y = 0.0975x + 0.8445, and the correlation coefficient is R2 = 0.9996; wherein, the retention time of glycine methyl ester hydrochloride is about 2.78 min, and the retention time of 2,5-piperazinedione is about 3.36 min.

[0027] (4) Liquid chromatography conditions: Chromatographic column: Thermo AccucoreTM aQ C18 chromatographic column, 5 μm, 4.6 x 250 mm; Detector: Electrospray detector; Column temperature: 40℃; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Evaporation tube temperature: 35℃; Mobile phase: 970 mL of 1.26 g / L ammonium formate aqueous solution with pH of 3.95 was mixed with 30 mL of chromatographic acetonitrile; The isocratic elution setting is:

[0028] (5) Determination method The 2,5-piperazinedione reaction solution was diluted with the mobile phase to prepare a 400 mg / L sample to be tested, and the peak area of glycine methyl ester hydrochloride was 8.969 pA·min and the peak area of 2,5-piperazinedione was 6.655 pA·min by high performance liquid chromatography-electrospray detector detection, as shown in Figure 4 .

[0029] The content of glycine methyl ester hydrochloride was accurately calculated as 18.92% by external standard method, and the content of 2,5-piperazinedione was 14.63%, and the signals of glycine methyl ester hydrochloride and 2,5-piperazinedione in the electrospray detector were equivalent, so the reaction process could be directly monitored according to the liquid chromatogram.

[0030] Example 2: The difference from Example 1 is that a UV detector is used, and the detection wavelength is 195 nm. Because the formic acid and formate system has ultraviolet absorption at 195 nm, the mobile phase is replaced with a phosphate system.

[0031] The specific chromatographic conditions are as follows: Chromatographic column: Thermo AccucoreTM aQ C18 chromatographic column, 5 μm, 4.6 x 250 mm; Detector: UV detector; Column temperature: 40℃; Flow rate: 1.0 mL / min; Detection wavelength: 195 nm; Mobile phase: 970 mL of 0.27 g / L potassium dihydrogen phosphate aqueous solution with pH of 3.95 and 30 mL of chromatography acetonitrile were uniformly mixed; Preparation of sample solution: 1 mg / mL glycine methyl ester hydrochloride standard solution and 1 mg / mL glycine anhydride standard solution were prepared with the mobile phase as diluent, wherein the injection volume of the glycine methyl ester hydrochloride standard solution was 10 μL, and the injection volume of the 2,5-piperazinedione standard solution was 1 μL.

[0032] The results are shown in Table 1. Figure 5 The retention time of glycine methyl ester hydrochloride and 2,5-piperazinedione is close, and it is difficult to achieve baseline separation; secondly, the injection volume of glycine methyl ester hydrochloride is 10 times that of 2,5-piperazinedione at the same concentration, and the ultraviolet response is close, which indicates that the response of the two to the ultraviolet detector is very different, and the method is not suitable for the monitoring of reaction liquid.

[0033] Example 3: Example 3-1: The detection method and condition are the same as those in Example 1, and only the pH of the mobile phase in the chromatographic condition is adjusted to 2.

[0034] Example 3-1: The detection method and condition are the same as those in Example 1, and only the pH of the mobile phase in the chromatographic condition is adjusted to 5.

[0035] The results are shown in Table 1. Figures 6-7 The pH of the mobile phase has a great influence on the peak shape of glycine methyl ester hydrochloride. Figure 6 When the pH of the mobile phase is 2, glycine methyl ester hydrochloride ionizes, and two chromatographic peaks are shown in the chromatogram. Figure 7 When the pH of the mobile phase is 5, the retention time of glycine methyl ester hydrochloride and 2,5-piperazinedione is coincident, and baseline separation cannot be achieved. In these two cases, it is difficult to achieve the monitoring and quantification of the two compounds.

[0036] Example 4: Example 4-1: The detection method and condition are the same as those in Example 1, and only the evaporation tube temperature in the chromatographic condition is adjusted to 25°C.

[0037] Example 4-2: The detection method and condition are the same as those in Example 1, and only the evaporation tube temperature in the chromatographic condition is adjusted to 45°C.

[0038] The results are shown in Table 1. Figures 8-9 Figure 8 When the evaporation tube temperature is 25°C, the temperature is too low, resulting in incomplete solvent evaporation, low charging efficiency, low chromatographic peak response, and poor reproducibility. Figure 9 ​When the temperature of the evaporation tube is 45℃, the temperature is too high, and glycine methyl ester hydrochloride and 2,5-piperazinedione show four chromatographic peaks, which is not suitable for the monitoring and quantification of the two compounds.

[0039] In summary, the present application uses the AccucoreTM aQ C18 column of Thermo, under the condition of the electrospray detector, with acetonitrile and pH 3.95 ammonium formate aqueous solution (1.26 g / L) as the mobile phase, the flow rate is 1.0 mL / min, the temperature of the evaporation tube of the detector is 35℃, which can effectively realize the baseline separation of glycine methyl ester hydrochloride and 2,5-piperazinedione, the peak shape is sharp and symmetrical, the retention time is stable, and the analysis cycle is controlled within 10 minutes. This method can not only be used for quantification, but also can realize the real-time monitoring of glycine methyl ester hydrochloride (raw material) and 2,5-piperazinedione (product) in the reaction process, accurately judge the reaction endpoint, and provide strong technical support for optimizing the process conditions, improving the yield of intermediates and ensuring the stability of product quality.

[0040] The above embodiments are only preferred technical solutions of the present application, and should not be regarded as a limitation of the present application. The protection scope of the present application should be based on the technical solutions recited in the claims, including equivalent replacement solutions of the technical features recited in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present application.

Claims

1. A method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, characterized in that: Includes the following steps: (1) Preparation of standard solutions: Using the mobile phase as the solvent, prepare standard solutions of glycine methyl ester hydrochloride and diketopiperazine respectively; (2) Establishment of standard curve: The standard solutions prepared in step (1) are injected sequentially and analyzed by high performance liquid chromatography system. The standard curve is established by the concentration of the standard solution and the corresponding peak area. (3) Sample determination and quantitative analysis: Prepare the sample solution to be tested using the mobile phase as solvent, and then inject the sample solution to be tested. Calculate the content of each substance to be tested according to the standard curve in step (2).

2. The method according to claim 1, characterized in that: In step (1), the mobile phase consists of an aqueous solution of ammonium formate as the aqueous phase and acetonitrile as the organic phase.

3. The method according to claim 2, characterized in that: The concentration of ammonium formate aqueous solution is 1~3g / L, and the pH of the solution is adjusted to 2~5 using phosphoric acid, acetic acid or formic acid.

4. The method according to claim 2, characterized in that: The concentration of ammonium formate aqueous solution is 1.26 g / L, and the pH of the solution is adjusted to 3.95 with formic acid.

5. The method according to claim 2, characterized in that: In step (1), the volume ratio of acetonitrile and ammonium formate aqueous solution is 1~10:90~100.

6. The method according to claim 2, characterized in that: In step (1), the volume ratio of acetonitrile to ammonium formate aqueous solution is 3:

97.

7. The method according to claim 1, characterized in that: The liquid chromatography conditions for step (2) are as follows: the detector is an electro-fogging detector, the temperature of the detector evaporation tube is 25~55℃, the flow rate is 0.5~1.5mL / min, the column temperature is 20~50℃, and the injection volume is 5~15μL.

8. The method according to claim 1, characterized in that: The liquid chromatography conditions for step (2) are as follows: the detector is an electro-fogging detector, the detector evaporation tube temperature is 35℃, the flow rate is 1.0mL / min, the column temperature is 40℃, and the injection volume is 10μL.

9. The method according to claim 1, characterized in that: The liquid chromatography column in step (2) has a size of 250 mm × 4.6 mm and a particle size of 5 μm.

10. The method according to claim 1, characterized in that: In step (1), the concentration of glycine methyl ester hydrochloride standard solution is 50-500 mg / L, and the concentration of 2,5-piperazindione standard solution is 50-500 mg / L.

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