A method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC

Abstract

The invention discloses a method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC. The method uses Shimadzu LC2030C3DPlus high performance liquid chromatograph, Shim-pack GISTC18, 250 × 4.6 mm, 5 μm chromatographic column and PDA detector to carry out qualitative and quantitative analysis of 4-(4-nitrophenyl)-3-morpholinone. The method 4-(4-nitrophenyl)-3-morpholinone peaks at 8.4 min, has good peak separation, no interference, strong specificity, and detection limit and quantitative limit are 6.42 μg / L and 17.60 μg / L respectively, with high sensitivity. The method has the characteristics of being easy to operate, fast, reproducible, accurate and having high recovery rate.

Description

Technical Field

[0001] The invention belongs to the field of soil and water environment detection, and particularly relates to a method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water by utilizing HPLC. Background Art

[0002] Rivaroxaban is a highly selective, oral drug that directly inhibits factor Xa. It is used in clinical treatment to prevent and treat venous thromboembolism in adults undergoing elective hip or knee replacement surgery and to prevent stroke and systemic embolism in patients with non-valvular atrial fibrillation.

[0003] 4-(4-nitrophenyl)-3-morpholinone, with a molecular formula of C10H10N2O4 and a CAS number of 446292-04-2, is often used as an intermediate for rivaroxaban and is an important raw material for organic synthesis, with a wide range of applications in industry. However, 4-(4-nitrophenyl)-3-morpholinone is environmentally polluting. If wastewater treatment is not in place, it is likely to pollute the surrounding soil and water environments. In order to detect and determine the impact of 4-(4-nitrophenyl)-3-morpholinone on the soil and water environments and to ensure its production, application, and discharge safety, it is necessary to establish a specific, effective, and accurate detection method for it. Currently, there are few studies on 4-(4-nitrophenyl)-3-morpholinone, and no qualitative and quantitative detection methods have been reported. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide an efficient and accurate detection method for the qualitative and quantitative detection of 4-(4-nitrophenyl)-3-morpholinone in soil and water.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC comprises the following steps:

[0007] S1: Preparation of soil sample solution: Collect the soil sample to be tested, dry, grind, and sieve it in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat it for 10 to 20 minutes. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested.

[0008] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, and then add 10 mL of acetonitrile for extraction. Shake vigorously. After the acetonitrile is separated, take out the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22μm filter membrane. The filtrate is the water sample solution to be tested.

[0009] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it with acetonitrile and make up to the mark, shake well, and prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; draw an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0010] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve and calculate the regression equation and correlation coefficient.

[0011] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0012] Preferably, in step S1, the drying method is air drying or vacuum freeze drying.

[0013] Preferably, in step S1, the ultrasonic treatment time is 15 minutes.

[0014] The instrument operating conditions of HPLC were as follows: chromatographic column: Shim-pack GIST C18, 250×4.6 mm, 5 μm; injection volume: 20–40 μL; flow rate: 0.5–1.5 mL / min; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 30:70; column temperature: 35–45° C.; and detection wavelength: 290–300 nm.

[0015] Preferably, the injection volume is 25 to 35 μL.

[0016] More preferably, the injection volume is 30 μL.

[0017] Preferably, the flow rate is 0.8 to 1.2 mL / min.

[0018] More preferably, the flow rate is 1.0 mL / min.

[0019] Preferably, the column temperature is 40°C.

[0020] Preferably, the detection wavelength is 296 nm.

[0021] The present invention has the following advantages:

[0022] 1. The results of the methodological verification test of the present invention show that the injection concentration of 4-(4-nitrophenyl)-3-morpholinone is in the range of 10.0 μg / L to 400.0 μg / L and has a good linear relationship. The linear equation is y=62.33x-24.72 and the linear correlation coefficient R 2 =0.9999; the average recoveries in soil and water ranged from 98.2% to 104.9% and 80.3% to 98.4%, respectively; the target peak RSD R.T. 0.12%, RSD Area The invention has the advantages of simple and convenient operation, rapidity, good repeatability, high accuracy, high recovery rate, good separation effect, etc.

[0023] 2. The detection limit of the present invention is 6.42 μg / L, the quantification limit is 17.60 μg / L, and the sensitivity is high, which can well detect the content of 4-(4-nitrophenyl)-3-morpholinone in soil and water.

[0024] 3. The present invention overcomes the gap in the existing field of 4-(4-nitrophenyl)-3-morpholinone detection technology and provides a reliable and effective method for qualitative and quantitative detection of 4-(4-nitrophenyl)-3-morpholinone. The present invention provides a technical reference for quality control in the production and application of 4-(4-nitrophenyl)-3-morpholinone. The method of the present invention is of great significance to the protection of soil and water environment in the discharge area of ​​4-(4-nitrophenyl)-3-morpholinone. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a calibration curve diagram of 4-(4-nitrophenyl)-3-morpholinone;

[0026] Figure 2 This is a typical spectrum of 4-(4-nitrophenyl)-3-morpholinone (40.0 μg / L);

[0027] Figure 3 This is a typical reproducible spectrum of 4-(4-nitrophenyl)-3-morpholinone (80.0 μg / L);

[0028] Figure 4 This is a typical spectrum of the detection limit of 4-(4-nitrophenyl)-3-morpholinone (10.0 μg / L);

[0029] Figure 5 This is a typical spectrum of the limit of quantification of 4-(4-nitrophenyl)-3-morpholinone (20.0 μg / L);

[0030] Figure 6 This is a typical recovery graph of 4-(4-nitrophenyl)-3-morpholinone added in soil (blank control);

[0031] Figure 7 This is a typical recovery graph of 4-(4-nitrophenyl)-3-morpholinone spiked in soil (10.0 mg / L);

[0032] Figure 8 The typical recovery spectrum of 4-(4-nitrophenyl)-3-morpholinone in soil (100.0 mg / L) is shown;

[0033] Figure 9 This is a typical graph of the recovery rate of 4-(4-nitrophenyl)-3-morpholinone added in water (blank control);

[0034] Figure 10 This is a typical recovery graph of 4-(4-nitrophenyl)-3-morpholinone added to water (10.0 mg / L);

[0035] Figure 11 This is a typical recovery graph of 4-(4-nitrophenyl)-3-morpholinone in water (100.0 mg / L). DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the raw materials and equipment used in the present examples are conventional commercially available raw materials and equipment in the art.

[0037] The instruments and reagents involved in the following examples and experimental examples include but are not limited to:

[0038] 1. Main instruments and equipment

[0039] (1) High performance liquid chromatograph: Shimadzu LC2030C 3D Plus, PDA detector;

[0040] (2) Electronic balance: Mettler-Toledo (China) Co., Ltd., XSE205DU;

[0041] (3) Electronic balance: Mettler-Toledo (China) Co., Ltd., MS1602S / 01;

[0042] (4) Commonly used instruments such as ultrasonic analyzers, volumetric flasks, and pipettes.

[0043] 2. Main reagents

[0044] (1) 4-(4-nitrophenyl)-3-morpholinone standard sample, purity 99.85%;

[0045] (2) Phosphoric acid: analytical grade, Tianjin Kemeiou Chemical Reagent Co., Ltd.

[0046] (3) Acetonitrile: chromatographic grade, ANPEL Inc.;

[0047] (4) Sodium chloride: analytical grade, Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0048] (5) Formic acid: analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.

[0049] (5) Anhydrous sodium sulfate: analytical grade, Chengdu Kelong Chemical Reagent Factory.

[0050] 3. Test samples

[0051] 3.1 Soil samples to be tested

[0052] The soil used in this experiment was paddy soil T2020002 purchased from the Ministry of Agriculture and Rural Affairs in Shaoxing, Zhejiang Province. The soil was air-dried, passed through a 1.0 mm sieve, and stored at room temperature in the dark until ready for use.

[0053] The soil sample to be tested was prepared as follows: 10 g of the above-mentioned paddy soil was weighed into a 250 mL conical flask, 0.1 mL of a 10.0 mg / L 4-(4-nitrophenyl)-3-morpholinone standard working solution was added thereto, and the mixture was vortexed to obtain a 0.1 mg / kg soil sample to be tested.

[0054] 3.2 Water samples to be tested

[0055] This experiment uses UP water as a representative of environmental water bodies. UP water comes from the tap water of this laboratory and is prepared by an ultrapure water machine. The resistivity of UP water is 18.2 MΩ×cm.

[0056] The preparation steps of the water sample to be tested are as follows: 0.2 mL of 4-(4-nitrophenyl)-3-morpholinone standard working solution diluted with acetonitrile to a concentration of 10.0 mg / L is taken into a 100 mL volumetric flask, and the volume is made up with UP water to obtain a water sample to be tested with a concentration of 20 μg / L.

[0057] Example 1:

[0058] S1: Preparation of soil sample solution: Collect the soil sample to be tested, air-dry, grind, and sieve in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat for 15 min. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested;

[0059] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested;

[0060] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0061] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient;

[0062] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0063] The instrument operating conditions of HPLC are:

[0064] Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm;

[0065] Injection volume: 30 μL;

[0066] Flow rate: 1.0 mL / min;

[0067] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70;

[0068] Column temperature: 40°C;

[0069] Detection wavelength: 296nm.

[0070] Example 2:

[0071] S1: Preparation of soil sample solution: Collect the soil sample to be tested, freeze-dry it in vacuum, grind it, and sieve it in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat it for 10 minutes. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested;

[0072] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested;

[0073] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0074] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient;

[0075] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0076] The instrument operating conditions of HPLC are:

[0077] Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm;

[0078] Injection volume: 20 μL;

[0079] Flow rate: 0.5 mL / min;

[0080] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70;

[0081] Column temperature: 35°C;

[0082] Detection wavelength: 290nm.

[0083] Example 3:

[0084] S1: Preparation of soil sample solution: Collect the soil sample to be tested, air-dry, grind, and sieve in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat for 12 minutes. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested;

[0085] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested;

[0086] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0087] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient;

[0088] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0089] The instrument operating conditions of HPLC are:

[0090] Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm;

[0091] Injection volume: 25 μL;

[0092] Flow rate: 0.8 mL / min;

[0093] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70;

[0094] Column temperature: 38°C;

[0095] Detection wavelength: 294nm.

[0096] Example 4:

[0097] S1: Preparation of soil sample solution: Collect the soil sample to be tested, air-dry, grind, and sieve in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat for 18 minutes. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested;

[0098] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested;

[0099] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0100] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient;

[0101] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0102] The instrument operating conditions of HPLC are:

[0103] Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm;

[0104] Injection volume: 35 μL;

[0105] Flow rate: 1.2 mL / min;

[0106] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70;

[0107] Column temperature: 42°C;

[0108] Detection wavelength: 298nm.

[0109] Example 5:

[0110] S1: Preparation of soil sample solution: Collect the soil sample to be tested, air-dry, grind, and sieve in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat for 20 min. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested;

[0111] S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested;

[0112] S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations.

[0113] S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient;

[0114] S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method.

[0115] The instrument operating conditions of HPLC are:

[0116] Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm;

[0117] Injection volume: 40 μL;

[0118] Flow rate: 1.5 mL / min;

[0119] Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70;

[0120] Column temperature: 45°C;

[0121] Detection wavelength: 300nm.

[0122] Experimental example:

[0123] To prove the scientificity and rationality of the present invention, the inventors conducted a large number of experimental studies, some of which are excerpted as follows:

[0124] 1. Chromatographic conditions investigation test

[0125] The soil sample solutions, water sample solutions and standard working solutions prepared in Examples 1-5 were subjected to high performance liquid chromatography, and the degree to which the results were not affected was examined by examining different chromatographic conditions such as injection volume, flow rate, column temperature, wavelength, etc.

[0126] 1.1 Investigation of injection volume

[0127] The chromatographic conditions are as follows:

[0128] Chromatographic column: Shim-pack GIST C18, 250×4.6mm, 5μm; injection volume: 20μL, 25μL, 30μL, 35μL, 40μL; flow rate: 1.0mL / min; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70; column temperature: 40℃; detection wavelength: 296nm.

[0129] The results showed that the injection volume of this test method varied between 20 and 40 μL, which had little effect on the test results, and the separation degree could achieve the detection effect. When the injection volume was 30 μL, the peak shape of the chromatographic peak was the best, so it was the optimal injection volume.

[0130] 1.2 Investigation of flow rate

[0131] The chromatographic conditions are as follows:

[0132] Chromatographic column: Shim-pack GIST C18, 250×4.6mm, 5μm; injection volume: 30μL; flow rate: 0.5mL / min, 0.8mL / min, 1.0mL / min, 1.2mL / min, 1.5mL / min; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70; column temperature: 40℃; detection wavelength: 296nm.

[0133] The results showed that the flow rate of this test method varied between 0.5 and 1.5 mL / min, which had little effect on the determination results. The separation degree could achieve the detection effect. When the flow rate was 1.0 mL / min, the separation effect was the best and the peak time was short, so it was the preferred flow rate.

[0134] 1.3 Investigation of column temperature

[0135] The chromatographic conditions are as follows:

[0136] Chromatographic column: Shim-pack GIST C18, 250×4.6 mm, 5 μm; injection volume: 30 μL; flow rate: 1.0 mL / min; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70; column temperature: 35°C, 38°C, 40°C, 42°C, 45°C; detection wavelength: 296 nm.

[0137] The results showed that the change of column temperature between 35 and 45℃ had little effect on the test results, and the separation degree could achieve the detection effect. When the column temperature was 30℃, the chromatographic peak separation was the best and the peak elution time was short, so it was the optimal column temperature.

[0138] 1.4 Investigation of detection wavelength

[0139] The chromatographic conditions are as follows:

[0140] Chromatographic column: Shim-pack GIST C18, 250×4.6 mm, 5 μm; injection volume: 30 μL; flow rate: 1.0 mL / min; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70; column temperature: 40°C; detection wavelengths: 290 nm, 294 nm, 296 nm, 298 nm, 300 nm.

[0141] The results showed that the change of detection wavelength between 290 and 300 nm had little effect on the determination results, and the separation degree could achieve the detection effect. When the detection wavelength was 296 nm, the chromatographic peak shape was the best and the separation degree was the best, so it was the preferred detection wavelength.

[0142] 2. Methodology validation test

[0143] 2.1 Specificity test

[0144] The HPLC analysis method of Example 1 of the present invention was used to measure the water blank sample, the water spiked recovery sample, the soil blank sample, and the soil spiked recovery sample. Comparing the spectra of the water and soil blank samples and the spiked recovery sample, 4-(4-nitrophenyl)-3-morpholinone peaked at 8.4 min, and the blank sample had no interfering peak at this time point. The results showed that this method has good specificity for 4-(4-nitrophenyl)-3-morpholinone.

[0145] 2.1 Linearity test

[0146] The HPLC analysis method of Example 1 of the present invention was used to determine the concentrations of the standard working solutions prepared in Example 1, which were 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L. The analysis results are shown in Table 1. When the concentration of 4-(4-nitrophenyl)-3-morpholinone was in the range of 10.0 μg / L to 400.0 μg / L, there was a linear relationship between y and x, with the linear regression equation y=62.33x-24.72 and the linear correlation coefficient R 2 =0.9999, linear fitting curve see Figure 1 .

[0147] Table 1: Linearity test results

[0148]

[0149] 2.3 Repeatability test

[0150] The 80.0 μg / L 4-(4-nitrophenyl)-3-morpholinone standard working solution in Example 1 was selected for the method repeatability test and the injection was repeated 6 times. The analysis results are shown in Table 2. Figure 3 , RSD of target peak R.T. 0.12%, RSD Area is 2.54%, which is less than 5.00%. The method can be used for qualitative and quantitative analysis.

[0151] Table 2: Repeatability test results

[0152] frequency Retention time (min) Peak area ( / ) 1 8.429 4846 2 8.433 4907 3 8.440 4867 4 8.444 5154 5 8.452 4984 6 8.456 5097 average value 8.442 4976 RSD (%) 0.12 2.54

[0153] 2.4 Limit of detection and limit of quantification

[0154] The 10.0 μg / L 4-(4-nitrophenyl)-3-morpholinone standard working solution in Example 1 was selected for the detection limit test, and the 20.0 μg / L 4-(4-nitrophenyl)-3-morpholinone standard working solution was selected for the quantification limit test. The samples were injected three times respectively, and the LOD and LOQ were calculated from the S / N. The analysis results are shown in Table 3. Figure 4 、 Figure 5 The LOD and LOQ of this method for 4-(4-nitrophenyl)-3-morpholinone were 6.42 μg / L and 17.60 μg / L.

[0155] Table 3: Limits of detection and quantification

[0156] Number of measurements Analytical concentration (μg / L) S / N LOD (μg / L) LOQ (μg / L) 1 10.0 4.92 6.10 - 2 10.0 3.87 7.75 - 3 10.0 5.54 5.42 - 1 20.0 11.63 - 17.20 2 20.0 11.80 - 16.95 3 20.0 10.72 - 18.66 average value - - 6.42 17.60

[0157] 2.5 Recovery rate of soil and water sample spikes

[0158] 2.5.1 Recovery rate of soil sample spikes

[0159] 10g of paddy soil was weighed separately in two 250mL Erlenmeyer flasks. 0.1mL of a standard working solution of 4-(4-nitrophenyl)-3-morpholinone at concentrations of 10.0mg / L and 100.0mg / L was added, respectively. Vortex mixing was performed to prepare two soil recovery samples at concentrations of 0.10mg / kg and 1.0mg / kg. 49.0mL of acetonitrile and 1.0mL of formic acid were added to the Erlenmeyer flasks, ultrasonicated for 15min, and the supernatant was filtered through a 0.22μm filter membrane after standing. The filtrate was then used for subsequent use. Five replicates of each concentration sample were prepared in parallel, and a soil blank test was performed simultaneously. The 4-(4-nitrophenyl)-3-morpholinone concentration was determined under the HPLC operating conditions of the present invention.

[0160] The analysis results are shown in Table 4. Figure 6 、 Figure 7 、 Figure 8 The average recovery rates of two different concentrations of 4-(4-nitrophenyl)-3-morpholinone in soil were 104.9% and 98.2%, respectively, and the relative standard deviations of the recoveries were 3.0% and 1.1%, respectively.

[0161] Table 4: Recovery results of soil spikes

[0162]

[0163] 2.5.2 Recovery rate of water sample addition

[0164] Get 0.2mL, 1.0mL concentration respectively and be the standard working solution of 10mg / L, 100mg / L in 100mL volumetric flask, use UP water constant volume, obtain the water body recovery sample of 20 μ g / L and 1.0mg / L respectively.Get the water body recovery sample 10mL of 20 μ g / L in colorimetric tube, add enough NaCl, then add 10mL acetonitrile and extract, forcefully vibrate, after the acetonitrile layering, take out supernatant and add appropriate Na SO Remove excess moisture, leave standstill and get supernatant with 0.22 μm membrane filtration, filtrate is standby.Each concentration sample is all parallel to make 5 parts, does water body blank test simultaneously, measures 4-(4-nitrophenyl)-3-morpholinone concentration by HPLC operational condition of the present invention.

[0165] The analysis results are shown in Table 5. Figure 9 、 Figure 10 、 Figure 11 The average recoveries of 4-(4-nitrophenyl)-3-morpholinone at two different concentrations in water were 80.3% and 98.4%, respectively, and the relative standard deviations of the recoveries were 8.6% and 1.6%, respectively.

[0166] Table 5: Recovery results of water addition

[0167]

[0168] The above is only a preferred embodiment of the present invention, but the embodiment of the present invention is not limited to the above embodiments and experimental examples. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC, characterized in that: The method steps are as follows: S1: Preparation of soil sample solution: Collect the soil sample to be tested, dry it, grind it, and sieve it in sequence; weigh 10 g of the treated soil sample into a 250 mL conical flask, add 49.0 mL of acetonitrile and 1.0 mL of formic acid to the conical flask, and ultrasonically treat it for 10-20 minutes. After standing, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the soil sample solution to be tested; S2: Preparation of water sample solution: Pipette 10 mL of the water sample to be tested into a colorimetric tube, add sufficient NaCl, then add 10 mL of acetonitrile for extraction, shake vigorously, wait for the acetonitrile to separate, remove the supernatant, add appropriate amount of Na2SO4 to remove excess water, let it stand, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is the water sample solution to be tested; S3: Preparation of standard solution: Accurately weigh 0.10016 g of 4-(4-nitrophenyl)-3-morpholinone standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and bring to volume, shake well to prepare a standard stock solution of 4-(4-nitrophenyl)-3-morpholinone with a concentration of 1000.10 mg / L; pipette an appropriate amount of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions of different concentrations. S4: Drawing a standard curve: Take an appropriate amount of the standard stock solution in step S3 and dilute it with acetonitrile to a series of standard working solutions with concentrations of 10.0 μg / L, 20.0 μg / L, 40.0 μg / L, 60.0 μg / L, 80.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L for HPLC determination. Use the concentration of the series of standard working solutions as the abscissa and the peak area of ​​4-(4-nitrophenyl)-3-morpholinone as the ordinate to draw a standard curve, and calculate the regression equation and correlation coefficient; S5: Determination: The soil sample solution, water sample solution, and standard working solution are injected into a high performance liquid chromatograph for determination, and the content of 4-(4-nitrophenyl)-3-morpholinone in the sample is calculated according to the external standard method; The instrument operating conditions of the HPLC are: Column: Shim-pack GIST C18, 250 × 4.6 mm, 5 μm; Injection volume: 20-40 μL; Flow rate: 0.5-1.5 mL / min; Mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 30:70; Column temperature: 35-45°C; Detection wavelength: 290~300nm.

2. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, wherein In step S1, the drying method is air drying or vacuum freeze drying.

3. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, wherein: In step S1, the ultrasonic treatment time is 15 min.

4. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, wherein: The injection volume is 25 to 35 μL.

5. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 4, characterized in that: The injection volume was 30 μL.

6. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, wherein: The flow rate is 0.8-1.2 mL / min.

7. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 6, characterized in that: The flow rate was 1.0 mL / min.

8. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, wherein: The column temperature was 40°C.

9. The method for detecting 4-(4-nitrophenyl)-3-morpholinone in soil and water using HPLC according to claim 1, characterized in that: The detection wavelength is 296 nm.

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