A method for simultaneously determining the content of glycine methyl ester hydrochloride and 2,5-piperazinedione

Abstract

The application provides a method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, and belongs to the field of chemical detection and analysis. The method uses acetonitrile and ammonium formate aqueous solution with a volume ratio of 3:97 as a mobile phase, the ammonium formate concentration is 1.26 g / L, formic acid is used to adjust the pH of the solution to 3.95, an electro-spray detector is selected, and a Thermo AccucoreTM aQ C18 chromatographic column is used for detection, so that the glycine methyl ester hydrochloride and 2,5-piperazinedione are effectively separated, the contents of the glycine methyl ester hydrochloride and 2,5-piperazinedione are quickly and accurately detected, and the method can be applied to the reaction control monitoring of the synthesis of 2,5-piperazinedione from glycine methyl ester hydrochloride.

Description

Technical Field

[0001] This invention relates to the field of chemical testing and analysis, specifically to a method for simultaneously determining the content of glycine methyl ester hydrochloride and 2,5-piperazinedione. Background Technology

[0002] Glyphosate (C3H8NO5P, CAS Registry No.: 1071-83-6) is an organophosphorus herbicide. Due to its good control effect on both annual and perennial weeds, it is widely used in farmland, orchards, forests, and non-arable land, becoming one of the most widely used agricultural herbicides globally. Glyphosate is a systemic, broad-spectrum, non-selective herbicide that penetrates into the plant through the leaves or stems. It inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid synthase system, thereby blocking the synthesis of aromatic amino acids in the plant and achieving the purpose of weed control.

[0003] 2,5-Piperazinedione is an important intermediate in the synthesis of glyphosate. Glycine methyl ester hydrochloride, as an inexpensive and widely used raw material in the pesticide industry, can be used in the synthesis of 2,5-piperazinedione. Due to its wide availability, low cost, and moderate reactivity, glycine methyl ester hydrochloride is widely used in actual production and is a crucial foundation for achieving green, efficient, and low-cost glyphosate synthesis. However, in the synthesis of 2,5-piperazinedione, the simple molecular structure of glycine methyl ester hydrochloride results in a significant difference in response to the product 2,5-piperazinedione in UV detection. This makes it difficult to accurately determine the residual amount of reactants using conventional analytical methods such as high-performance liquid chromatography (HPLC), affecting the assessment of the synthesis reaction effect, and consequently impacting the yield and purity of 2,5-piperazinedione, further affecting the overall efficiency and product quality of subsequent glyphosate synthesis.

[0004] Therefore, establishing a sensitive, accurate, and specific detection method for glycine methyl ester hydrochloride is of great significance for effectively monitoring the reaction of glycine methyl ester hydrochloride, improving the yield and quality of 2,5-piperazinedione, and thus effectively increasing the yield of glyphosate products. Summary of the Invention

[0005] The main objective of this invention is to provide a method for simultaneously determining the content of glycine methyl ester hydrochloride and 2,5-piperazinedione, thereby solving the problems mentioned in the background art. This method achieves effective separation of glycine methyl ester hydrochloride and 2,5-piperazinedione by optimizing chromatographic conditions; it not only possesses high sensitivity, high selectivity, and a wide linear range, but also enables simultaneous detection of both components in a short time, significantly improving analytical efficiency.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, characterized by comprising the following steps: (1) Preparation of standard solutions: Prepare standard solutions of glycine methyl ester hydrochloride and diketopiperazine using the mobile phase as the solvent; (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).

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

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

[0009] Furthermore, the concentration of the ammonium formate aqueous solution was 1.26 g / L, and the pH of the solution was adjusted to 3.95 with formic acid. This condition effectively suppressed ionization interference and improved separation selectivity and detection repeatability.

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

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

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

[0013] Further, the liquid chromatography conditions in 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.

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

[0015] Furthermore, the liquid chromatography column in step (2) has a specification of 250 mm × 4.6 mm and a particle size of 5 μm.

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

[0017] This invention provides a rapid, efficient, sensitive, and accurate two-component simultaneous detection method, which solves the technical problem of large response differences and difficulty in quantification between glycine methyl ester hydrochloride and 2,5-piperazinedione under conventional detection methods. This method is of great significance for improving the synthesis efficiency and product quality of key glyphosate intermediates. This method is not only suitable for the mid-control analysis of the 2,5-piperazinedione synthesis reaction, but can also be applied to the quality monitoring of other pesticide and pharmaceutical synthesis processes containing polar small molecule intermediates, and has good industrialization and promotion value.

[0018] The beneficial effects of this invention are as follows: 1. This invention uses acetonitrile and ammonium formate aqueous solution as mobile phase and employs isocratic elution to achieve efficient separation of glycine methyl ester hydrochloride and 2,5-piperazine dione, and enables quantitative analysis of the two substances. Furthermore, the responses of the two substances are comparable in an electro-fogging detector, which is beneficial for controlling the reaction process during synthesis.

[0019] 2. The present invention uses liquid chromatography to rapidly and accurately determine the content of glycine methyl ester hydrochloride and 2,5-piperazine dione. The test can be completed within 10 minutes, which greatly shortens the detection time, enables the mid-control monitoring of the synthesis reaction, and improves the synthesis efficiency.

[0020] 3. The method of the present invention is simple and easy to operate, and has high detection sensitivity. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings: Figure 1 This is the standard curve of glycine methyl ester hydrochloride from Example 1 of the present invention; Figure 2 This is the 2,5-piperazinedione standard curve of Example 1 of the present invention; Figure 3 This is the HPLC chromatogram of glycine methyl ester hydrochloride and 2,5-piperazindione standard samples in Example 1 of the present invention; Figure 4 This is the HPLC spectrum of the reaction solution sample in Example 1 of the present invention; Figure 5 These are the HPLC spectra of standard samples A) glycine methyl ester hydrochloride and B) 2,5-piperazinedione in Example 2 of the present invention. Figure 6 This is the HPLC chromatogram of the glycine methyl ester hydrochloride standard sample tested when the mobile phase pH was adjusted to 2 in Example 3 of the present invention; Figure 7 This is the HPLC spectrum of standard samples A) glycine methyl ester hydrochloride and B) 2,5-piperazinedione when the mobile phase pH was adjusted to 5 in Example 3 of the present invention. Figure 8 This is the HPLC spectrum of the glycine methyl ester hydrochloride standard sample tested at an evaporation tube temperature of 25°C in Example 4 of this invention; Figure 9 This is the HPLC chromatogram of the glycine methyl ester hydrochloride standard sample tested at an evaporator temperature of 45°C in Example 4 of this invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the solutions of this invention will be described in detail and clearly below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0023] Example 1: A method for simultaneously determining the contents of glycine methyl ester hydrochloride and 2,5-piperazinedione, comprising the following steps: (1) Preparation of the mobile phase: Ammonium formate aqueous solution: Take 1.26g of ammonium formate and dilute it to 1000mL with ultrapure water. Adjust the pH to 3.95 with formic acid, shake well, and filter through a 0.45μm mixed fiber membrane for later use. Mobile phase solution: Mix 970 mL of ammonium formate aqueous solution with 30 mL of chromatographic acetonitrile, and sonicate for 20 min for later use.

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

[0025] (3) Plotting the standard curve: The standard solutions prepared in step (2) were injected sequentially from low concentration to high concentration, and analyzed using a high performance liquid chromatography system. A standard curve was established by using the concentration of the standard solution and its corresponding peak area as the abscissa and ordinate, respectively.

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

[0027] (4) Liquid chromatography conditions: Chromatographic column: Thermo Fisher Accucore™ aQ C18 column, 5μm, 4.6×250mm; Detector: Electro-fog detector; Column temperature: 40℃; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Evaporator tube temperature: 35℃; Mobile phase: Mix 970 mL of 1.26 g / L ammonium formate aqueous solution with pH 3.95 with 30 mL of chromatographic acetonitrile; Isocratic elution settings:

[0028] (5) Measurement method The 2,5-piperazinedione reaction solution was diluted with the mobile phase to prepare a 400 mg / L sample for testing. High-performance liquid chromatography-electrospray ionization (HPLC-ESI) analysis revealed a glycine methyl ester hydrochloride peak area of ​​8.969 pA·min and a 2,5-piperazinedione peak area of ​​6.655 pA·min. (See attached table). Figure 4 .

[0029] The content of glycine methyl ester hydrochloride was accurately calculated to be 18.92% and the content of 2,5-piperazinedione was 14.63% using the external standard method. At the same time, the signals of glycine methyl ester hydrochloride and 2,5-piperazinedione were comparable in the electro-fogging detector, and the reaction process could be monitored intuitively based on the liquid chromatogram.

[0030] Example 2: The difference from Example 1 is that an ultraviolet detector is used with a detection wavelength of 195nm, because the formic acid and formate system has ultraviolet absorption at 195nm, and the fluidity is changed to a phosphate system.

[0031] The specific chromatographic conditions are as follows: Chromatographic column: Thermo Fisher Accucore™ aQ C18 column, 5μm, 4.6×250mm; Detector: Ultraviolet detector; Column temperature: 40℃; Flow rate: 1.0 mL / min; Detection wavelength: 195nm; Mobile phase: Mix 970 mL of 0.27 g / L potassium dihydrogen phosphate aqueous solution (pH 3.95) with 30 mL of chromatographic acetonitrile. Sample solution preparation: Using the mobile phase as a diluent, prepare a 1 mg / mL glycine methyl ester hydrochloride standard solution and a 1 mg / mL glycine anhydride standard solution, wherein the injection volume of the glycine methyl ester hydrochloride standard solution is 10 μL and the injection volume of the 2,5-piperazindione standard solution is 1 μL.

[0032] The results are as follows Figure 5 The retention times of glycine methyl ester hydrochloride and 2,5-piperazine dione are similar, making baseline separation difficult. Secondly, at the same concentration, the injection volume of glycine methyl ester hydrochloride is 10 times that of 2,5-piperazine dione, and the corresponding UV values ​​are similar. This indicates that the responses of the two in the UV detector are very different, and the method is not suitable for the central control monitoring of the reaction solution.

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

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

[0035] The results are as follows Figure 6-7 The pH of the mobile phase has a significant impact on the peak shape of glycine methyl ester hydrochloride. Figure 6 When the mobile phase pH is 2, glycine methyl ester hydrochloride ionizes, which is represented by two chromatographic peaks. Figure 7 When the mobile phase pH is 5, the retention times of glycine methyl ester hydrochloride and 2,5-piperazinedione overlap, making baseline separation impossible. Both of these conditions make it difficult to achieve centralized monitoring and quantification of the two compounds.

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

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

[0038] The results are as follows Figure 8-9 : Figure 8 When the evaporation tube temperature is 25℃, the temperature is too low, resulting in incomplete solvent evaporation, low charging efficiency, low chromatographic peak response, and poor reproducibility. Figure 9When the evaporator temperature is 45℃, the temperature is too high, and glycine methyl ester hydrochloride and 2,5-piperazine dione show 4 chromatographic peaks, which is not suitable for the central control monitoring and quantification of the two compounds.

[0039] In summary, this invention utilizes a Thermo Fisher Accucore™ aQ C18 column under electrospray detector conditions, with acetonitrile and a pH 3.95 ammonium formate aqueous solution (1.26 g / L) as the mobile phase, a flow rate of 1.0 mL / min, and a detector evaporation tube temperature of 35°C. This method effectively achieves baseline separation of glycine methyl ester hydrochloride and 2,5-piperazinedione, producing sharp peaks with good symmetry, stable retention times, and an analysis cycle controlled within 10 minutes. This method can not only be used for quantification but also enables real-time monitoring of glycine methyl ester hydrochloride (starting material) and 2,5-piperazinedione (product) during the reaction, accurately determining the reaction endpoint. This provides strong technical support for optimizing process conditions, improving intermediate yields, and ensuring product quality stability.

[0040] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

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). In step (1), the volume ratio of acetonitrile to ammonium formate aqueous solution in the mobile phase is 1~10:90~100, the concentration of ammonium formate aqueous solution is 1~3g / L, and the pH is 3.95; The liquid chromatography conditions were as follows: Column: Thermo Fisher Accucore™ aQ C18 column, 5 μm, 4.6 × 250 mm; Detector: Electro-fog detector; Evaporator tube temperature: 35℃.

2. The method according to claim 1, characterized in that: The concentration of the ammonium formate aqueous solution was 1.26 g / L, and the pH of the solution was adjusted to 3.95 with formic acid.

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

97.

4. The method according to claim 1, characterized in that: The liquid chromatography conditions for step (2) are: flow rate of 0.5~1.5 mL / min, column temperature of 20~50℃, and injection volume of 5~15 μL.

5. The method according to claim 4, characterized in that: The liquid chromatography conditions for step (2) are: flow rate of 1.0 mL / min, column temperature of 40℃, and injection volume of 10 μL.

6. 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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