LC-MS / MS-based method for the detection of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water

Abstract

The present invention discloses a method for detecting 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water based on LC-MS / MS. In this method, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine has a peak at 0.5 min, good peak separation, and strong method specificity. Target peak RSD R.T. 0.49%, RSD Area The detection limit is 0.87 ng / L, the quantitative limit is 5.98 ng / L, and the sensitivity is high. When the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample is low, the purpose of detection can still be achieved. The invention has the characteristics of simple operation, high detection efficiency, high sensitivity, good repeatability, high accuracy, good precision, etc.

Description

Technical Field

[0001] The invention belongs to the field of soil and water environment detection, and specifically relates to a method for detecting 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water based on LC-MS / MS. Background Art

[0002] Pyrrole [2,3-d] pyrimidine derivatives are effective protease inhibitors that can effectively and selectively inhibit JAK3 and block cytokine signals and cytokine-induced gene expression. They can be used for organ transplantation and the treatment of various autoimmune diseases. In addition, pyrrole [2,3-d] pyrimidine derivatives can also effectively treat rheumatoid arthritis, psoriasis, colitis and other conditions.

[0003] 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine is an important intermediate for the synthesis of pyrrolo[2,3-d]pyrimidine derivatives and is widely used in the synthesis of many pharmaceutical intermediates at home and abroad, with broad market prospects.

[0004] At present, there are many published synthesis methods for 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, and the published detection and analysis methods include infrared spectrophotometry and high performance liquid chromatography, but there are no reports on the use of LC-MS / MS detection methods. Therefore, it is very important to explore a unique and effective LC-MS / MS detection method with a low detection limit for the analysis of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine.

[0005] For example, the enterprise standard Q / 341226JTY003-2020 '4-chloropyrrolo[2,3-d]pyrimidine' discloses its high performance liquid chromatography detection method, specifically: ① Chromatographic conditions and system suitability test: use octadecylsilane bonded silica gel as filler; use 0.1% phosphoric acid solution as mobile phase A, and acetonitrile as mobile phase B; the detection wavelength is 225nm; ② Determination method: Take about 12.5g of this product, accurately weigh it, put it into a 25ml volumetric flask, add ethyl acetate solution (50:50, v / v) to dissolve and dilute to the scale, shake well, and use it as the test solution for gradient elution.

[0006] For example, the enterprise standard Q / ASN 001-2018 '4-chloropyrrolopyrimidine' discloses its high performance liquid chromatography detection method, ① operating conditions: chromatographic column: Zorbax CN PN884950-507, 4.6×250m, 5μm or equivalent chromatographic column; mobile phase preparation: mobile phase A: 100% water; mobile phase B: 100% methanol; diluent: 100% methanol; injection volume: 10.0ul; UV detector wavelength: 220nm; column temperature: 35℃; automatic sampler temperature: room temperature; flow rate: 1.2mL / min. ② Preparation of test solution: accurately weigh about 20g of sample, dilute with diluent, and prepare a test solution containing about 0.1mg of 4-chloropyrrolopyrimidine per 1ml for gradient elution.

[0007] The above-mentioned detection instruments have a long running time, large solvent consumption, low detection efficiency, poor peak separation, and detection limits at the mg / mL level, which are higher than the LOD of the present invention of 0.87ng / L and LOQ of 5.98ng / L, and have low sensitivity, and cannot achieve the purpose of detection when the concentration of the sample to be tested is low. The determination requirements of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water environments cannot be met.

[0008] Therefore, it is necessary to develop an efficient and sensitive method for the determination of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water environments. Summary of the invention

[0009] The purpose of the present invention is to overcome the problems of low detection efficiency and poor sensitivity in the prior art and to provide a method for detecting 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water based on LC-MS / MS.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] (1) Preparation of soil sample solution: Collect the soil sample containing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, air-dry, grind and sieve in sequence for later use. Weigh 10.0 g of the treated soil sample into a 250 mL conical flask, add 45.0-55.0 mL of acetonitrile and 1.0 mL of formic acid into the conical flask, sonicate for 10-20 min, let stand and cool, and filter the supernatant with a 0.22 μm filter membrane to obtain the soil sample solution.

[0012] (2) Preparation of water sample solution: Take an appropriate amount of water sample and filter it through a 0.22 μm filter membrane to obtain the water sample solution to be tested.

[0013] (3) Preparation of standard working solution: Accurately weigh 0.10031 g of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard sample into a 100-mL volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and prepare a standard stock solution with a concentration of 1000.09 mg / L of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine; take an appropriate amount of the standard stock solution and dilute it with acetonitrile.

[0014] (4) Preparation of standard curve: Take an appropriate amount of the standard stock solution of step (3), dilute it with acetonitrile to prepare a series of standard working solutions of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with gradient concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L, respectively, and perform HPLC-MS / MS determination. Draw a standard curve with the concentration of the series of gradient standard working solutions as the abscissa and the peak area of ​​4-chloro-7H-pyrrolo[2,3-d]pyrimidine as the ordinate.

[0015] (5) Determination: 5 μL of the soil sample solution, water sample solution, and standard working solution prepared in the above step were each taken for HPLC-MS / MS determination, and the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample was calculated according to the external standard method.

[0016] Preferably, the amount of acetonitrile is 49.0 mL and the ultrasonic time is 15 min.

[0017] In the HPLC-MS / MS determination, the high performance liquid chromatography operating conditions are as follows: the chromatographic column is Eclipse Plus C18RRHD, with specifications of 50×2.1 mm, 1.8 μm; acetonitrile is mobile phase A, 0.1% formic acid water is mobile phase B, and the volume ratio of mobile phase A to B is 70:30; the flow rate is 0.3-0.5 mL / min; and the column temperature is 30-35° C.

[0018] The operating conditions of the mass spectrometer were:

[0019] Ion source: AJS ESI source, positive ion mode.

[0020] Ion source parameters: Drying gas: N 2 ; Drying gas temperature: 300℃; Drying gas flow rate: 5L / min; Sheath gas: N 2 ; Sheath gas temperature: 250℃; Sheath gas flow rate: 11L / min; Nebulizer pressure: 45psi; Nozzle voltage: +500 / -500V; Capillary voltage: +3500 / -3500V.

[0021] Monitoring mode: MRM.

[0022] The MRM ion acquisition parameters are:

[0023]

[0024] * is the quantitative ion.

[0025] Preferably, in the HPLC operating conditions, the flow rate is 0.3 mL / min and the column temperature is 30°C.

[0026] The LOD and LOQ of this method for 4-chloro-7H-pyrrolo[2,3-d]pyrimidine were 0.87 ng / L and 5.98 ng / L respectively.

[0027] The present invention has the following advantages:

[0028] 1. The results of the validation test of the methodology of the present invention show that: 4-chloro-7H-pyrrolo[2,3-d]pyrimidine peaks at 0.5min, and the blank sample has no interference peak at this time point. The method has strong specificity, high detection efficiency, low solvent usage, and saves detection costs; 4-chloro-7H-pyrrolo[2,3-d]pyrimidine concentrations in the range of 1.00μg / L to 20μg / L show a good linear relationship, the linear equation y=3309.72x+313.69, and the correlation coefficient R 2 =0.9982; the average recovery rates of addition in soil and water were 92.3% to 95.4% and 94.2% to 96.4% respectively; the target peak RSD R.T. 0.49%, RSD Area The invention has the advantages of simple operation, high detection efficiency, high sensitivity, good repeatability, high accuracy, good precision, high recovery rate, good separation effect and the like.

[0029] 2. The detection limit of the present invention is 0.87 ng / L, the quantitative limit is 5.98 ng / L, and the sensitivity is high. When the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample is low, the purpose of detection can still be achieved. The content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water environment can be well detected, which is of great significance to the protection of soil and water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a calibration curve diagram of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine;

[0031] Figure 2 It is a typical spectrum of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (8.00μg / L);

[0032] Figure 3It is a typical repetitive spectrum of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (4.00μg / L);

[0033] Figure 4 This is a typical spectrum of the detection limit of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (0.01μg / L);

[0034] Figure 5 This is a typical spectrum of the quantification limit of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2.00μg / L);

[0035] Figure 6 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine added to soil (blank control);

[0036] Figure 7 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine added to soil (1.0 mg / L);

[0037] Figure 8 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil (100.0 mg / L);

[0038] Fig. 9 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine added to water (blank control);

[0039] Fig.10 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine added to water (1.0 mg / L);

[0040] Fig.11 This is a typical graph of the recovery rate of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine added to water (10.0 mg / L). DETAILED DESCRIPTION

[0041] The present invention is 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.

[0042] The instruments and reagents involved in the following embodiments and experimental examples include but are not limited to:

[0043] 1. Main instruments and equipment

[0044] (1) LC-MS / MS: Agilent 1260-6470A;

[0045] (2) Electronic balance: Mettler-Toledo (China) Co., Ltd., XSE205DU;

[0046] (3) Electronic balance: Mettler-Toledo (China) Co., Ltd., MS1602S / 01;

[0047] (4) Ultrasonic cleaner: Shanghai Kedao Ultrasonic Instrument Co., Ltd., SK250H;

[0048] (5) Commonly used glass instruments such as volumetric flasks and pipettes.

[0049] 2. Main reagents

[0050] (1) 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine, provided by the customer, purity 99.7%;

[0051] (2) Acetonitrile: chromatographic grade, ANPEL Inc.;

[0052] (3) Formic acid: analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.

[0053] 3. Test samples

[0054] 3.1 Soil samples to be tested

[0055] The soil used in this experiment was paddy soil T2020002 from Shaoxing City, Zhejiang Province, purchased from the Pesticide Testing Institute of the Ministry of Agriculture and Rural Affairs. The soil was air-dried and passed through a 1.0 mm sieve and stored at room temperature away from light.

[0056] The preparation steps of the soil sample to be tested are as follows: weigh 10 g of the above paddy soil into a 250 mL conical flask, add 0.1 mL of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution diluted with acetonitrile to a concentration of 1.0 mg / L, and vortex mix to obtain a soil sample to be tested with a concentration of 10 μg / kg.

[0057] 3.2 Water samples to be tested

[0058] 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.

[0059] The preparation steps of the water sample to be tested are as follows: aspirate 0.2 mL of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution diluted with acetonitrile to a concentration of 1.0 mg / L into a 100 mL volumetric flask, and dilute to volume with UP water to obtain a water sample to be tested with a concentration of 2 μg / L.

[0060] Embodiment 1:

[0061] (1) Preparation of soil sample solution: soil samples containing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine were collected, air-dried, ground and sieved in sequence for later use. 10.0 g of the treated soil sample was weighed into a 250 mL conical flask, 49.0 mL of acetonitrile and 1.0 mL of formic acid were added to the conical flask, and ultrasonicated for 15 min. After cooling, the supernatant was filtered through a 0.22 μm filter membrane to obtain the soil sample solution.

[0062] (2) Preparation of water sample solution: Take an appropriate amount of water sample and filter it through a 0.22 μm filter membrane to obtain the water sample solution to be tested.

[0063] (3) Preparation of standard working solution: Accurately weigh 0.10031 g of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard sample into a 100-mL volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and prepare a standard stock solution with a concentration of 1000.09 mg / L of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine; take an appropriate amount of the standard stock solution and dilute it with acetonitrile.

[0064] (4) Preparation of standard curve: Take an appropriate amount of the standard stock solution of step (3), dilute it with acetonitrile to prepare a series of standard working solutions of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with gradient concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L, respectively, and perform HPLC-MS / MS determination. Draw a standard curve with the concentration of the series of gradient standard working solutions as the abscissa and the peak area of ​​4-chloro-7H-pyrrolo[2,3-d]pyrimidine as the ordinate.

[0065] (5) Determination: 5 μL of the soil sample solution, water sample solution, and standard working solution prepared in the above step were each taken for HPLC-MS / MS determination, and the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample was calculated according to the external standard method.

[0066] The operating conditions of HPLC were as follows: the chromatographic column was Eclipse Plus C18 RRHD, with specifications of 50×2.1 mm, 1.8 μm; acetonitrile was mobile phase A, 0.1% formic acid water was mobile phase B, and the volume ratio of mobile phase A to B was 70:30; the flow rate was 0.3 mL / min; and the column temperature was 30°C.

[0067] The operating conditions of the mass spectrometer were:

[0068] Ion source: AJS ESI source, positive ion mode.

[0069] Ion source parameters: Drying gas: N 2; Drying gas temperature: 300℃; Drying gas flow rate: 5L / min; Sheath gas: N 2 ; Sheath gas temperature: 250℃; Sheath gas flow rate: 11L / min; Nebulizer pressure: 45psi; Nozzle voltage: +500 / -500V; Capillary voltage: +3500 / -3500V.

[0070] Monitoring mode: MRM.

[0071] The MRM ion acquisition parameters are:

[0072]

[0073] * is the quantitative ion.

[0074] Embodiment 2:

[0075] (1) Preparation of soil sample solution: soil samples containing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine were collected, air-dried, ground and sieved in sequence for later use. 10.0 g of the treated soil sample was weighed into a 250 mL conical flask, 45.0 mL of acetonitrile and 1.0 mL of formic acid were added to the conical flask, and ultrasonicated for 10 min. After cooling, the supernatant was filtered through a 0.22 μm filter membrane to obtain the soil sample solution.

[0076] (2) Preparation of water sample solution: Take an appropriate amount of water sample and filter it through a 0.22 μm filter membrane to obtain the water sample solution to be tested.

[0077] (3) Preparation of standard working solution: Accurately weigh 0.10031 g of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard sample into a 100-mL volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and prepare a standard stock solution with a concentration of 1000.09 mg / L of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine; take an appropriate amount of the standard stock solution and dilute it with acetonitrile.

[0078] (4) Preparation of standard curve: Take an appropriate amount of the standard stock solution of step (3), dilute it with acetonitrile to prepare a series of standard working solutions of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with gradient concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L, respectively, and perform HPLC-MS / MS determination. Draw a standard curve with the concentration of the series of gradient standard working solutions as the abscissa and the peak area of ​​4-chloro-7H-pyrrolo[2,3-d]pyrimidine as the ordinate.

[0079] (5) Determination: 5 μL of the soil sample solution, water sample solution, and standard working solution prepared in the above step were each taken for HPLC-MS / MS determination, and the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample was calculated according to the external standard method.

[0080] The operating conditions of HPLC were as follows: the chromatographic column was Eclipse Plus C18 RRHD, with specifications of 50×2.1 mm, 1.8 μm; acetonitrile was mobile phase A, 0.1% formic acid water was mobile phase B, and the volume ratio of mobile phase A to B was 70:30; the flow rate was 0.4 mL / min; and the column temperature was 32°C.

[0081] The operating conditions of the mass spectrometer were:

[0082] Ion source: AJS ESI source, positive ion mode.

[0083] Ion source parameters: Drying gas: N 2 ; Drying gas temperature: 300℃; Drying gas flow rate: 5L / min; Sheath gas: N 2 ; Sheath gas temperature: 250℃; Sheath gas flow rate: 11L / min; Nebulizer pressure: 45psi; Nozzle voltage: +500 / -500V; Capillary voltage: +3500 / -3500V.

[0084] Monitoring mode: MRM.

[0085] The MRM ion acquisition parameters are:

[0086]

[0087] * is the quantitative ion.

[0088] Embodiment 3:

[0089] (1) Preparation of soil sample solution: soil samples containing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine were collected, air-dried, ground and sieved in sequence for later use. 10.0 g of the treated soil sample was weighed into a 250 mL conical flask, 55.0 mL of acetonitrile and 1.0 mL of formic acid were added to the conical flask, and ultrasonicated for 20 min. After cooling, the supernatant was filtered through a 0.22 μm filter membrane to obtain the soil sample solution.

[0090] (2) Preparation of water sample solution: Take an appropriate amount of water sample and filter it through a 0.22 μm filter membrane to obtain the water sample solution to be tested.

[0091] (3) Preparation of standard working solution: Accurately weigh 0.10031 g of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard sample into a 100-mL volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and prepare a standard stock solution with a concentration of 1000.09 mg / L of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine; take an appropriate amount of the standard stock solution and dilute it with acetonitrile.

[0092] (4) Preparation of standard curve: Take an appropriate amount of the standard stock solution of step (3), dilute it with acetonitrile to prepare a series of standard working solutions of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with gradient concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L, respectively, and perform HPLC-MS / MS determination. Draw a standard curve with the concentration of the series of gradient standard working solutions as the abscissa and the peak area of ​​4-chloro-7H-pyrrolo[2,3-d]pyrimidine as the ordinate.

[0093] (5) Determination: 5 μL of the soil sample solution, water sample solution, and standard working solution prepared in the above step were each taken for HPLC-MS / MS determination, and the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample was calculated according to the external standard method.

[0094] The operating conditions of HPLC were as follows: the chromatographic column was Eclipse Plus C18 RRHD, with specifications of 50×2.1 mm, 1.8 μm; acetonitrile was mobile phase A, 0.1% formic acid water was mobile phase B, and the volume ratio of mobile phase A to B was 70:30; the flow rate was 0.5 mL / min; and the column temperature was 35°C.

[0095] The operating conditions of the mass spectrometer were:

[0096] Ion source: AJS ESI source, positive ion mode.

[0097] Ion source parameters: Drying gas: N 2 ; Drying gas temperature: 300℃; Drying gas flow rate: 5L / min; Sheath gas: N 2 ; Sheath gas temperature: 250℃; Sheath gas flow rate: 11L / min; Nebulizer pressure: 45psi; Nozzle voltage: +500 / -500V; Capillary voltage: +3500 / -3500V.

[0098] Monitoring mode: MRM.

[0099] The MRM ion acquisition parameters are:

[0100]

[0101] * is the quantitative ion.

[0102] Comparative Example 1:

[0103] Source of method: refer to the method of 'Q / 341226JTY003-20204-chloropyrrolo[2,3-d]pyrimidine'.

[0104] Conclusion: ① The treatment of the sample solution by this method is suitable for the detection of pure 4-chloropyrrolo[2,3-d]pyrimidine, but not for the preparation of soil and water samples; ② The instrument operation time of this method is more than 30 minutes, the instrument operation time is long, the detection efficiency is low, the solvent consumption is large, and the detection cost is high; ③ The target peak separation of this method is poor, the detection sensitivity is low, and it cannot meet the detection requirements of 4-chloropyrrolo[2,3-d]pyrimidine in soil and water environments. This method is not used.

[0105] Comparative Example 2:

[0106] Method source: Executed according to the method of 'Q / ASN 001-2018 4-Chloropyrrolopyrimidine'.

[0107] Conclusion: The instrument running time of this method is 25 minutes, the detection efficiency is low, the solvent capacity is large, the sensitivity is low, and the peak shape is poor. This method is not used.

[0108] Experimental example:

[0109] In order to prove the scientificity and rationality of the present invention, the inventors have carried out a large number of experimental studies, some of which are excerpted as follows:

[0110] 1. Chromatographic conditions investigation test

[0111] The soil sample solution, water sample solution and standard working solution prepared in Examples 1-3 were used for LC-MS / MS determination, and the degree to which the results were not affected was investigated by examining different flow rates, column temperatures and other chromatographic conditions.

[0112] 1.1 Investigation of flow rate

[0113] The chromatographic conditions were as follows:

[0114] The chromatographic column, mobile phase, column temperature, and mass spectrometry operating parameters in the LC-MS / MS instrument operating conditions were the same as those in Example 1, and the HPLC flow rates were 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min.

[0115] The results showed that the flow rate of this test method varied between 0.3 and 0.5 mL / min, which had little effect on the measurement results, and the separation degree could achieve the detection effect. When the flow rate was 0.3 mL / min, the separation effect was the best and the peak time was short, so it was the preferred flow rate.

[0116] 1.2 Investigation of column temperature

[0117] The chromatographic conditions were as follows:

[0118] The chromatographic column, mobile phase, flow rate, and mass spectrometry operating parameters in the LC-MS / MS instrument operating conditions were the same as those in Example 1, and the HPLC column temperatures were 30° C., 32° C., and 35° C.

[0119] The results showed that the change of column temperature between 30 and 45°C had little effect on the test results, and the separation degree could reach the detection effect. When the column temperature was 30°C, the chromatographic peak separation degree was the best and the peak elution time was short, so it was the optimal column temperature.

[0120] 2. Methodology validation test

[0121] 2.1 Specificity test

[0122] The LC-MS / MS analysis method established by the present invention was used to measure water blank samples, water spiked recovery samples, soil blank samples, and soil spiked recovery samples. The spectra of water and soil blank samples and spiked recovery samples were compared. 4-chloro-7H-pyrrolo[2,3-d]pyrimidine peaked at 0.5 min, and the blank sample had no interfering peak at this time point. The results showed that the method had good specificity for 4-chloro-7H-pyrrolo[2,3-d]pyrimidine.

[0123] 2.2 Linearity test

[0124] The LC-MS / MS analysis method established by the present invention was used to determine the series of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solutions with concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L prepared in the embodiment. The analysis results are shown in Table 1. When the concentration of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine is in the range of 1.00 μg / L to 20 μg / L, there is a linear relationship between y and x. The linear regression equation y=3309.72x+313.69, the linear correlation coefficient R 2 =0.9982, linear fitting curve see Figure 1 .

[0125] Table 1: Linearity test results

[0126]

[0127]

[0128] 2.3 Repeatability test

[0129] The 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution with a concentration of 4.00 μg / L in the example was selected for the method repeatability test, and the injection was repeated 6 times. The results are shown in Table 2. Figure 3 . RSD of target peak R.T. 0.49%, RSD Area为 2.57%, all less than 5.00%, indicating that this method can be used for qualitative and quantitative analysis.

[0130] Table 2: Repeatability test results

[0131] frequency Retention time (min) Peak area( / ) 1 0.498 14728 2 0.504 14092 3 0.504 13700 4 0.504 14511 5 0.504 14435 6 0.504 14479 average value 0.503 14324 RSD(%) 0.49 2.57

[0132] 2.4 Limit of detection and limit of quantification

[0133] A 0.10 μg / L 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution was selected for the detection limit test, and a 2.00 μg / L 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution was selected for the quantification limit test. The injections were repeated 3 times, and the LOD and LOQ were calculated by S / N. The average results of the 3 analyses showed that the LOD and LOQ of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine were 0.87 ng / L and 5.98 ng / L, respectively. The detailed results are shown in Table 3. Figure 4 , Figure 5 .

[0134] Table 3: Limits of detection and quantification

[0135]

[0136] 2.5 Recovery rate of soil and water sample addition

[0137] 2.5.1 Recovery rate of soil sample addition

[0138] Weigh about 10.0g of the prepared paddy soil into a 250mL conical flask, add 0.1mL of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard working solution with concentrations of 1.0 and 100.0mg / L, vortex mix, and prepare two soil recovery samples with concentrations of 10μg / kg and 1.0mg / kg, respectively. Add 49.0mL acetonitrile and 1.0mL formic acid to the conical flask, ultrasonicate for 15min, let it stand and cool, take the supernatant and filter it with a 0.22μm filter membrane (high concentration acetonitrile is diluted 20 times), and then measure the concentration of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine on the machine. Each concentration sample was prepared in parallel for 5 times, and a soil blank test was performed at the same time.

[0139] The analysis results are shown in Table 4. Figure 6 , Figure 7 , Figure 8The average recovery rates of two different concentrations of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil were 95.4% and 92.3%, respectively, and the standard deviations of the recovery rates were 5.1% and 2.5%, respectively.

[0140] Table 4: Results of soil recovery test

[0141]

[0142]

[0143] 2.5.2 Recovery rate of water sample addition

[0144] Take 0.2mL and 1mL of working solution with concentrations of 1.0mg / L and 10.0mg / L in a 100mL volumetric flask, dilute with UP water, and obtain 2μg / L and 100μg / L water recovery samples, respectively. Take the water recovery sample and filter it with a 0.22μm filter membrane (high concentration acetonitrile is diluted 10 times) and measure the concentration of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine on the machine. Each concentration sample is made in parallel for 5 times, and a water blank test is performed at the same time.

[0145] The analysis results are shown in Table 5. Fig. 9 , Fig.10 , Fig.11 The average recovery rates of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine at two different concentrations in water were 96.4% and 94.2%, respectively, and the standard deviations of the recoveries were 2.8% and 3.2%, respectively.

[0146] Table 5: Results of water addition recovery test

[0147]

[0148] The above are only preferred implementations of the present invention, but the implementations of the present invention are 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 protection scope of the present invention.

Claims

1. A method for detecting 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in soil and water based on LC-MS / MS, characterized in that: The method steps are as follows: (1) Preparation of soil sample solution: collect the soil sample to be tested containing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, air-dry, grind and sieve in sequence for later use, weigh 10.0 g of the treated soil sample into a 250 mL conical flask, add 45.0-55.0 mL of acetonitrile and 1.0 mL of formic acid into the conical flask, sonicate for 10-20 min, let stand and cool, and filter the supernatant with a 0.22 μm filter membrane to obtain the soil sample solution to be tested; (2) Preparation of water sample solution: Take an appropriate amount of water sample and filter it with a 0.22 μm filter membrane to obtain the water sample solution to be tested; (3) Preparation of standard working solution: Accurately weigh 0.10031 g of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine standard sample into a 100 mL volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and prepare a standard stock solution with a concentration of 1000.09 mg / L of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine; take an appropriate amount of the standard stock solution and dilute it with acetonitrile; (4) Drawing of a standard curve: Take an appropriate amount of the standard stock solution of step (3), dilute it with acetonitrile to prepare a series of standard working solutions of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with gradient concentrations of 1.00 μg / L, 2.00 μg / L, 4.00 μg / L, 6.00 μg / L, 8.00 μg / L, 10.0 μg / L, and 20.0 μg / L, perform HPLC-MS / MS determination, and draw a standard curve with the concentration of the series of gradient standard working solutions as the abscissa and the peak area of ​​4-chloro-7H-pyrrolo[2,3-d]pyrimidine as the ordinate; (5) Determination: The soil sample solution, water sample solution, and standard working solution prepared in the above step are subjected to HPLC-MS / MS determination, and the content of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in the sample is calculated according to the external standard method; In step (1), the amount of acetonitrile is 49.0 mL, and the ultrasonic time is 15 min; In the HPLC-MS / MS determination, the chromatographic column used for high performance liquid chromatography detection is EclipsePlus C18 RRHD, with specifications of 50×2.1 mm, 1.8 μm; In the HPLC-MS / MS determination, the high performance liquid chromatography detection uses acetonitrile as mobile phase A, 0.1% formic acid water as mobile phase B, and the volume ratio of mobile phase A to B is 70:30; the flow rate is 0.3-0.5 mL / min; and the column temperature is 30-35° C.; The injection volume for the HPLC-MS / MS determination was 5 μL; The HPLC-MS / MS mass spectrometry detection uses an AJS ESI source in positive ion mode; The ion source parameters are as follows: drying gas: N2; drying gas temperature: 300°C; drying gas flow rate: 5L / min; sheath gas: N2; sheath gas temperature: 250°C; sheath gas flow rate: 11L / min; nebulizer pressure: 45psi; nozzle voltage: +500 / -500V; capillary voltage: +3500 / -3500V; The HPLC-MS / MS mass spectrometry detection uses the MRM monitoring mode, and the MRM ion acquisition parameters are: compound name 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, parent ion 153.9, daughter ion quantitative ion 118.1, fragmentation voltage 100V, acceleration voltage 4V, polarity is positive; parent ion 153.9, daughter ion 64.1, fragmentation voltage 100V, acceleration voltage 4V, polarity is positive.

2. A method according to claim 1 for detecting 4-chloro-7H-pyrrolo[2-( ... [2,3-d]pyrimidine method, characterized in that The flow rate was 0.3 mL / min and the column temperature was 30 °C.

3. A method according to claim 1 for detecting 4-chloro-7H-pyrrolo[3-( ... [2,3-d]pyrimidine method, characterized in that The LOD of the method for 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was 0.87 ng / L and the LOQ was 5.98 ng / L.

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