Method for testing content of residual chlorothalonil and phoxim in traditional Chinese medicinal materials

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a method for testing the content of residual chlorothalonil and phoxim in traditional Chinese medicinal materials. Aiming at the problems of experimental error and recovery rate caused by adopting a single detection method in the prior art, the invention provides a detection method based on chemical properties and thermal stability of different to-be-detected substances. The method comprises the following specific steps: grinding a sample, extracting, purifying, detecting phoxim by adopting LC-MSMS (Liquid Chromatography-Mass Spectrometry Mass Spectrometry), and detecting chlorothalonil by adopting GC-MSMS. By optimizing chromatographic conditions and gradient elution procedures, matrix interference can be effectively eliminated, the detection sensitivity, accuracy and efficiency are improved, meanwhile, the analysis time is shortened, and reliable technical support is provided for precise detection of pesticide residues of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and particularly to a method for testing the contents of chlorothalonil and phoxim residues in Chinese herbal medicines. Background Art

[0002] Plant protectants play an important role in modern agricultural production, significantly increasing crop yields and quality by controlling pests and diseases. Chlorothalonil, as a broad-spectrum fungicide, is widely used to control fungal diseases of vegetables, fruits and cash crops; phoxim is an efficient organophosphorus insecticide, commonly used to control pests such as Lepidoptera and Coleoptera, especially showing excellent residual activity in soil treatment. However, their overuse or improper management may lead to excessive residues in agricultural products and environmental media. Chlorothalonil and its metabolites have potential ecological toxicity, which may interfere with the functions of soil microbial communities and can accumulate through the food chain, threatening human health; phoxim, due to its neurotoxicity, poses a significant hazard to non-target organisms and the central nervous system of humans. Therefore, there is an urgent need to establish a precise and efficient synchronous detection method for chlorothalonil and phoxim residues to ensure food safety, ecological balance and the supervision of rational pesticide use.

[0003] Currently, the residual detection techniques for chlorothalonil and phoxim mainly include gas chromatography (GC), high performance liquid chromatography (HPLC) and their hyphenated techniques with mass spectrometry (GC-MS, LC-MS). For example, Chinese invention patent CN105866272A discloses a method for detecting pesticide residues in health products, and its test steps include (1) sample preparation; (2) GC-MS detection; (3) LC-MS / MS detection. This method can detect common pesticides in health products, including: chlorothalonil, phorate, triadimefon, carbaryl, atrazine, fenthion, aldrin, buprofezin, edifenphos, quinalphos, permethrin, paclobutrazol, fenamiphos, triadimenol, amitraz, endrin, dimethomorph, edifenphos, ethoprophos, fenamiphos, imidacloprid, methamidophos, methomyl, metolcarb, monocrotophos, omethoate, paclobutrazol, phorate, phosalone, phosmet, phoxim, etc. However, in fact, for substances such as chlorothalonil and phoxim, due to the different polarities and thermal stabilities of the substances themselves, simply using the method of first detecting by GC-MS and then by LC-MS / MS will obviously lead to certain experimental errors and also affect the recovery rate of the substances to be detected.

[0004] Therefore, there is an urgent need for a new method for testing the contents of residual chlorothalonil and phoxim, which can accurately test the contents of chlorothalonil and phoxim, thereby ensuring the medicinal safety of Chinese herbal medicines such as Astragalus membranaceus, and at the same time providing reliable technical support for pesticide residue supervision. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for testing the residues of chlorothalonil and phoxim in traditional Chinese medicines, comprising the following steps:

[0006] S1. Grind and sieve the sample to be tested, place it in a stoppered centrifuge tube, add inorganic salts, shake well, add an organic phase, homogenize for 1 - 3 min, then centrifuge, let stand, and collect the precipitate and the first supernatant;

[0007] S2. Add an organic phase to the precipitate, homogenize for 1 - 3 min, centrifuge, and collect the second supernatant;

[0008] S3. Combine the first supernatant and the second supernatant, concentrate under reduced pressure to 5 - 10 mL, then cool to 20 - 30 °C, add an organic phase for dilution, shake well, to obtain a crude product solution;

[0009] S4. Purify the crude product solution using an HLB SPE solid phase extraction column, and collect the purified solution;

[0010] S5. For the phoxim component in the purified solution, measure it using the LC - MSMS method; for the chlorothalonil component in the purified solution, detect it using the GC - MSMS method.

[0011] As an implementable case, the mesh number of the grinding and sieving is 50 - 100 meshes.

[0012] Further, the mesh number of the grinding and sieving is 50 meshes.

[0013] As an implementable case, the inorganic salts include one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate, or ammonium sulfate.

[0014] Further, the inorganic salts include sodium chloride.

[0015] During the extraction process, sodium chloride increases the ionic strength of the solution, reduces the solubility of the target substances chlorothalonil and phoxim in the aqueous phase, thereby promoting their easier distribution into the organic phase and improving the extraction efficiency. At the same time, the shaking operation enables sodium chloride to come into full contact with the powder of the sample to be tested, preventing caking and ensuring the uniform progress of the salting - out effect, optimizing the release and distribution of the target substances from the complex matrix. In addition, at high concentrations, sodium chloride can also change the density of the aqueous phase, making the organic phase that is easily miscible with water easier to layer, further improving the extraction and dehydration efficiency of the components. Through these effects, not only is the distribution efficiency of the target substances enhanced, but also the high efficiency and stability of the extraction process are ensured.

[0016] As an implementable case, the organic phase includes acetonitrile.

[0017] As an implementable case, the rotation speed of the homogenization treatment is greater than 12000 rpm.

[0018] Further, the rotation speed of the homogenization treatment is 16,000 rpm.

[0019] As an implementable case, the rotation speed of the centrifugation is 3,000 - 6,000 rpm.

[0020] Further, the rotation speed of the centrifugation is 4,000 rpm.

[0021] In the present invention, an HLB SPE solid-phase extraction column is used to purify the crude solution. Due to its unique hydrophilic-lipophilic balance characteristics, the HLB SPE solid-phase extraction column can adsorb both polar and non-polar compounds simultaneously, thereby effectively removing matrix interferences such as pigments, polysaccharides, and lipids in the substance to be detected. Therefore, the testing method proposed in the present invention can significantly reduce miscellaneous interferences and improve the signal-to-noise ratio and accuracy of the detection of chlorothalonil and phoxim by liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry. In addition, the solid-phase extraction column of the present invention has good chemical stability and a wide pH application range, and can be compatible with a variety of solvents, ensuring high efficiency and reliability in a complex sample environment.

[0022] The present invention uses the LC-MSMS (liquid chromatography-tandem mass spectrometry) method to detect the phoxim component in the purified solution. Utilizing its polar and thermally unstable characteristics, through the high separation ability of liquid chromatography, matrix interferences are effectively reduced, and the sensitivity and accuracy of the detection are improved. At the same time, the GC-MSMS (gas chromatography-tandem mass spectrometry) method is used to detect the chlorothalonil component in the purified solution. Utilizing its non-polar and thermally stable characteristics, through the high separation ability of gas chromatography, matrix interferences are further reduced to ensure the reliability of the detection results.

[0023] If the GC-MSMS method is used to detect both phoxim and chlorothalonil, due to components such as pigments, polysaccharides, and lipids in the matrix may co-elute or interact with the target compounds, resulting in signal enhancement or inhibition, thus generating false positive phenomena. The present invention significantly reduces matrix interferences and ensures the accuracy and stability of the detection results by optimizing the detection method and combining the purification effect of the HLB solid-phase extraction column.

[0024] As an implementable case, in the LC-MSMS test, the liquid phase conditions are as follows:

[0025] Column temperature: 30 - 40 °C;

[0026] Mobile phase A: Ammonium formate with a formic acid mass concentration of 0.01 - 0.1 wt%;

[0027] Mobile phase B: A mixed solvent of mobile phase A and acetonitrile;

[0028] Running time: 5 - 10 min;

[0029] Elution method: gradient elution;

[0030] In the gradient elution, the volume percentage of mobile phase A is 0 - 90%, and the volume percentage of mobile phase B is 10 - 100%.

[0031] Furthermore, the parameters of the gradient elution are as described in Table 1:

[0032] Table 1

[0033]

[0034]

[0035] As an implementable case, in the LC-MSMS test, the mass spectrometry conditions are as described in Table 2:

[0036] Table 2

[0037] Serial number Test substance Q1 Q3 DP(V) CE(V) 1 Phoxim 299.16 77.06 85 43 2 Phoxim 299.16 126.01 85 19

[0038] As an implementable case, in the GC-MSMS test, the gas phase conditions are as follows: phenyl methyl polysiloxane is used as the elastic quartz capillary column, the column length is 30 m, the column inner diameter is 0.25 mm, and the film thickness is 0.254 m; the injection port temperature is 200 - 250 °C, splitless injection; the carrier gas is helium; the injection port is in constant pressure mode, and the column front pressure is 140 - 150 kPa; programmed temperature rise, the initial temperature is 50 - 60 °C, held for 1 minute, heated at a rate of 20 - 30 °C / min to 120 - 150 °C, and then heated at a rate of 5 - 10 °C / min to 290 °C, held for 2 minutes.

[0039] As an implementable case, in the GC-MSMS test, the mass spectrometry conditions are as follows: triple quadrupole tandem mass spectrometer test, the ion source is an electron impact source, the ion source temperature is 200 - 250 °C, the collision gas is nitrogen, the mass spectrometry transfer interface temperature is 200 - 250 °C, the mass spectrometry detection mode is MRM (multiple reaction monitoring), and the mass spectrometry parameters are as shown in Table 3.

[0040] Table 3

[0041] Serial number Test substance Parent ion Daughter ion CE(V) 1 Chlorothalonil 265.9 168 22 2 Chlorothalonil 265.9 230.8 14 3 Triphenyl phosphate 326 215 21 4 Triphenyl phosphate 326 233 12

[0042] Beneficial effects

[0043] (1) Based on the characteristics of different chemical properties and thermal stabilities of the analytes to be measured, the present invention adopts different test methods for chlorothalonil and phoxim. Specifically, phoxim adopts the LC-MSMS test method, and chlorothalonil adopts the GC-MSMS test method, which can effectively eliminate false positive results caused by matrix interference, thereby improving the stability of experimental test results.

[0044] (2) The method validation results show that the correlation coefficient R within the linear range is greater than 0.99, and the recovery rate is between 70 - 120%, indicating that the test method provided by the present invention has high accuracy and reliability, and can ensure the credibility of the detection results of chlorothalonil and phoxim.

[0045] (3) In the present invention, through the optimized chromatographic conditions and gradient elution program parameters, the sample analysis time can be significantly shortened, and the running time is only 8 minutes, improving the detection efficiency.

[0046] (4) The relative standard deviation RSD of the parallel determination experiments is less than 15%, indicating that the test method provided by the present invention has good precision and repeatability, and is suitable for the detection of chlorothalonil and phoxim in batch samples.

[0047] (5) The test method provided by the present invention shows good stability and durability under different equipment and conditions, can adapt to a variety of experimental environments and sample types, and has wide applicability. Description of the Drawings

[0048] Figure 1 It is the durability spectrum of phoxim on LC - MSMS.

[0049] Figure 2 It is the durability spectrum of chlorothalonil on GC - MSMS.

[0050] Figure 3 It is the durability spectrum of chlorothalonil on GC - MSMS.

[0051] Figure 4 It is the sample peak map of phoxim on GC - MSMS.

[0052] Figure 5 It is the sample peak map of phoxim on GC - MSMS.

[0053] Figure 6 It is the standard product peak map of phoxim on GC - MSMS.

[0054] Figure 7 It is the standard product peak map of phoxim on GC - MSMS.

[0055] Figure 8 It is the sample peak map of phoxim on GC - MSMS after adjusting the programmed temperature rise.

[0056] Figure 9 It is the sample peak map of phoxim on GC - MSMS after adjusting the programmed temperature rise.

[0057] Figure 10 It is the standard product peak map of phoxim on GC - MSMS after adjusting the programmed temperature rise.

[0058] Figure 11 To adjust the peak pattern of phoxim standard on GC-MSMS after programmed temperature rise.

[0059] Figure 12 Schematic diagram of the peak emergence of phoxim standard on LC-MSMS.

[0060] Figure 13 Test chart of phoxim in the sample on LC-MSMS. It can be seen that phoxim is not detected. Specific implementation mode

[0061] Comparative example 1

[0062] This example provides a method for testing the contents of chlorothalonil and phoxim residues in traditional Chinese medicinal materials, including the following steps:

[0063] S1. Grind the sample of Astragalus membranaceus traditional Chinese medicinal material to be tested, sieve it through a 50-mesh sieve, place it in a 100 mL stoppered centrifuge tube, add 1 g of sodium chloride, shake it evenly, add 50 mL of acetonitrile, homogenize it at 16000 rpm for 1 min, then centrifuge it at 4000 rpm, let it stand, and collect the precipitate and the first supernatant.

[0064] S2. Add 50 mL of acetonitrile to the precipitate, homogenize it at 16000 rpm for 1 min, centrifuge it at 4000 rpm, and collect the second supernatant.

[0065] S3. Combine the first supernatant and the second supernatant, concentrate it under reduced pressure to 5 - 6 mL, then cool it to 25 °C, add acetonitrile to dilute and make up the volume to 10 mL, shake it well to obtain a crude product solution.

[0066] S4. Purify the crude product solution using an HLB SPE solid-phase extraction column and collect the purified solution.

[0067] S5. For the phoxim and chlorothalonil components in the purified solution, both are detected by the GC-MSMS method.

[0068] 1. Test the phoxim solvent standard:

[0069] In the described GC-MSMS method, the chromatographic conditions are as follows: A phenylmethyl polysiloxane is used as the fused silica capillary column, with a column length of 30 m, an inner column diameter of 0.25 mm, and a film thickness of 0.254 m; the inlet temperature is 250 °C, and splitless injection is used; the carrier gas is helium; the inlet is in a constant pressure mode, and the column inlet pressure is 146 kPa; programmed temperature rising, the initial temperature is 50 °C, held for 1 minute, then heated at a rate of 25 °C / min to 125 °C, and then heated at a rate of 10 °C / min to 290 °C. The model of the gas chromatographic column is DB-17MS, as shown in Table 4 specifically; the mass spectrometry conditions are as follows: Tested with a triple quadrupole tandem mass spectrometer, the ion source is an electron impact source, the ion source temperature is 250 °C, the collision gas is nitrogen, the mass spectrometry transfer interface temperature is 250 °C, the mass spectrometry detection mode is multiple reaction monitoring, and the mass spectrometry parameters are shown in Table 5, and phoxim solvent standard is tested.

[0070] Table 4

[0071]

[0072] Table 5

[0073] Test substance Parent ion Daughter ion CE(V) Phoxim 130 76 25 Phoxim 130 103 10 Phoxim 103 76 15

[0074] 2. Test the solvent standard mixture (chlorothalonil, phoxim) and the matrix standard (added with internal standard - triphenyl phosphate)

[0075] The GC-MSMS method is used to test the solvent standard mixture (chlorothalonil, phoxim) and the matrix standard (added with internal standard - triphenyl phosphate). The chromatographic conditions are as follows: A phenylmethyl polysiloxane is used as the fused silica capillary column, with a column length of 30 m, an inner column diameter of 0.25 mm, and a film thickness of 0.254 m; the inlet temperature is 250 °C, and splitless injection is used; the carrier gas is helium; the inlet is in a constant pressure mode, and the column inlet pressure is 146 kPa; programmed temperature rising, the initial temperature is 50 °C, held for 1 minute, then heated at a rate of 25 °C / min to 125 °C, and then heated at a rate of 10 °C / min to 290 °C. The model of the gas chromatographic column is DB-17MS, as shown in Table 6 specifically; the mass spectrometry conditions are as follows: Tested with a triple quadrupole tandem mass spectrometer, the ion source is an electron impact source, the ion source temperature is 250 °C, the collision gas is nitrogen, the mass spectrometry transfer interface temperature is 250 °C, the mass spectrometry detection mode is multiple reaction monitoring, and the mass spectrometry parameters are shown in Table 7.

[0076] Table 6

[0077]

[0078] Table 7

[0079] Test substance Parent ion Daughter ion CE(V) Phoxim 130 76 25 Phoxim 130 103 10 Phoxim 103 76 15 Chlorothalonil 265.9 168 22 Chlorothalonil 265.9 230.8 14 Triphenyl phosphate 326 215 21 Triphenyl phosphate 326 233 12

[0080] The solvent standard shows no abnormality. For the triphenyl phosphate in the astragalus matrix standard, the collection is incomplete. Adjust the temperature programming to ensure that the temperature programming for triphenyl phosphate is adjusted as shown in Table 8.

[0081] Table 8

[0082]

[0083]

[0084] Meanwhile, when screening the astragalus blank matrix, phoxim is detected in all cases. The experimental spectra are as shown in Figure 1 、 Figure 2 It is suspected that the matrix interference of phoxim is severe, affecting the accuracy of the experimental results.

[0085] Therefore, adjust the temperature programming to eliminate matrix interference, and use the LC-MSMS method to measure the phoxim component.

[0086] The sample peak diagram of phoxim on GC-MSMS is as shown in Figure 4 shown.

[0087] The sample peak diagram of phoxim on GC-MSMS is as shown in Figure 5 shown.

[0088] The standard peak diagram of phoxim on GC-MSMS is as shown in Figure 6 shown.

[0089] The standard peak diagram of phoxim on GC-MSMS is as shown in Figure 7 shown.

[0090] The sample peak diagram of phoxim on GC-MSMS after adjusting the temperature programming is as shown in Figure 8 shown.

[0091] The sample peak diagram of phoxim on GC-MSMS after adjusting the temperature programming is as shown in Figure 9 shown.

[0092] The standard peak diagram of phoxim on GC-MSMS after adjusting the temperature programming is as shown in Figure 10 shown.

[0093] The standard peak diagram of phoxim on GC-MSMS after adjusting the temperature programming is as shown in Figure 11 shown.

[0094] The schematic diagram of the standard peak of phoxim on LC-MSMS is as shown in Figure 12 shown.

[0095] The test diagram of phoxim in the sample on LC-MSMS is as shown in Figure 13 shown, and it can be seen that phoxim is not detected.

[0096] Example 1

[0097] This example provides a method for testing the residues of chlorothalonil and phoxim in traditional Chinese medicines, including the following steps:

[0098] S1. Grind the sample of Astragalus membranaceus traditional Chinese medicine to be tested, sieve it through a 50-mesh sieve, place it in a 100 mL stoppered centrifuge tube, add 1 g of sodium chloride, shake it evenly, add 50 mL of acetonitrile, homogenize it at 16000 rpm for 1 min, then centrifuge it at 4000 rpm, let it stand, and collect the precipitate and the first supernatant;

[0099] S2. Add 50 mL of acetonitrile to the precipitate, homogenize it at 16000 rpm for 1 min, centrifuge it at 4000 rpm, and collect the second supernatant;

[0100] S3. Combine the first supernatant and the second supernatant, concentrate it under reduced pressure to 5 mL, then cool it to 25 °C, add acetonitrile to dilute and make up the volume to 10 mL, shake it well to obtain a crude solution;

[0101] S4. Purify the crude solution using an HLB SPE solid-phase extraction column and collect the purified solution;

[0102] S5. For the phoxim component in the purified solution, measure it using the LC-MSMS method; for the chlorothalonil component in the purified solution, detect it using the GC-MSMS method.

[0103] In the LC-MSMS test described above, the liquid phase conditions are as follows:

[0104] Instrument name: ACQUITY I-CALSS + AB5500 +;

[0105] Chromatographic column: Waster HSS T3;

[0106] Column temperature: 35 °C;

[0107] Mobile phase A: Ammonium formate with a mass concentration of 0.05 wt% formic acid;

[0108] Mobile phase B: A mixed solvent of mobile phase A and acetonitrile with a volume ratio of 5:95;

[0109] Running time: 8 min;

[0110] Elution mode: Gradient elution;

[0111] The parameters of the gradient elution are as described in Table 9:

[0112] Table 9

[0113]

[0114] In the LC-MSMS test described above, the mass spectrometry conditions are as described in Table 10:

[0115] Table 10

[0116] Test substance Q1 Q3 DP(V) CE(V) Phoxim 299.16 77.06 85 43 Phoxim 299.16 126.01 85 19

[0117] In the described GC-MSMS test, the gas phase conditions are as follows: phenyl methyl polysiloxane is used as the elastic quartz capillary column, with a column length of 30 m, an inner column diameter of 0.25 mm, and a film thickness of 0.254 m; the inlet temperature is 200 - 250 °C, and splitless injection is used; the carrier gas is helium; the inlet is in a constant pressure mode, and the pressure before the column is 140 - 150 kPa; programmed temperature rise, with an initial temperature of 50 - 60 °C, held for 1 minute, then heated at a rate of 20 - 30 °C / min to 120 - 150 °C, and then heated at a rate of 5 - 10 °C / min to 290 °C, held for 2 minutes; the name of the gas chromatograph is Agilent 8890 - 7000D, and the model of the chromatographic column is DB-17MS.

[0118] In the described GC-MSMS test, the mass spectrometry conditions are as follows: a triple quadrupole tandem mass spectrometer is used for testing, the ion source is an electron impact source, the ion source temperature is 250 °C, the collision gas is nitrogen, the mass spectrometry transfer interface temperature is 250 °C, the mass spectrometry detection mode is MRM, and the mass spectrometry parameters are shown in Table 11.

[0119] Table 11

[0120] Test substance Parent ion Daughter ion CE(V) Chlorothalonil 265.9 168 22 Chlorothalonil 265.9 230.8 14 Triphenyl phosphate 326 215 21 Triphenyl phosphate 326 233 12

[0121] 1. Preparation of the control solution:

[0122] Preparation of the mixed reference solution: Accurately measure 10 μL of the chlorothalonil and phoxim reference solutions, with the concentration of the reference solution being 1000 μg / mL, place them in a 10 mL volumetric flask, and dilute and make up the volume with acetonitrile.

[0123] Preparation of the internal standard solution for GC-MSMS analysis: Take triphenyl phosphate and dilute it with acetonitrile to 0.1 μg / mL.

[0124] Preparation of the blank matrix solution: Take the blank matrix sample and process it into a blank matrix solution (an astragalus sample that has been detected to not contain the analyte (chlorothalonil / phoxim)).

[0125] Preparation of the matrix mixed control solution: Measure 1.0 mL of the blank matrix solution in six portions, place them on a nitrogen blower, concentrate them to 0.4 mL in a 40 °C water bath, add 3 μL, 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of the mixed reference solution respectively, dilute to 1 mL with acetonitrile, and vortex and mix well to obtain.

[0126] For GC-MS / MS, respectively pipette 1 mL each of the matrix mixed control solution and the purified solution, add 0.3 mL of the internal standard solution, mix well, filter and take the filtrate. Respectively pipette 1 μL each of the above two solutions, inject into the gas chromatography-tandem mass spectrometer, and calculate according to the internal standard standard curve method to obtain the result.

[0127] For LC-MS / MS, respectively pipette 1 mL each of the matrix mixed control solution and the purified solution, accurately add 0.3 mL of water, mix well, filter and take the filtrate. Respectively and accurately pipette 2 μL each of the above two solutions, inject into the liquid chromatography-tandem mass spectrometer, and calculate according to the external standard standard curve method to obtain the result.

[0128] 2. Linear relationship test

[0129] Take 1 mL of the purified solution, place it on a nitrogen evaporator, concentrate it to 0.4 mL in a 40 °C water bath, respectively add 3 μL, 10 μL, 20 μL, 50 μL, 100 μL, 200 μL of the standard solution, dilute to 1 mL with acetonitrile and inject into the instrument, test the response values of each concentration, and the experimental test results are shown in Table 12.

[0130] Table 12

[0131]

[0132] In this experiment, different concentrations were injected, and the linear R was all > 0.99, indicating that the linear relationship between the concentration and the response value was significant, stable and had good repeatability within the test concentration range, and could be used for accurate quantitative analysis of the residues of chlorothalonil and phoxim in unknown samples.

[0133] 3. Detection limit test

[0134] Respectively take 1.0 mL (10 components) of the blank matrix solution, place it on a nitrogen evaporator, concentrate it to a volume of about 0.4 mL in a 40 °C water bath, add 3 μL of the mixed reference substance solution, then dilute to 1 mL with acetonitrile, vortex and mix well, detect the signal-to-noise ratio of each component, and the experimental results are shown in Tables 13 - 14.

[0135] Table 13

[0136]

[0137] Table 14

[0138]

[0139]

[0140] It can be seen from the experimental results in Tables 13 - 14 that the signal-to-noise ratio results of this experiment are all > 3, indicating that this method can reliably detect the target substances chlorothalonil and phoxim, and has good anti-interference ability and reproducibility, both meeting the requirements of the pharmacopoeia.

[0141] 4. Quantitative limit detection

[0142] Take 1.0 mL of blank matrix solution (10 components) respectively, place it on a nitrogen blower, concentrate it in a 40 °C water bath to a volume of about 0.6 mL, add 10 μL of mixed reference solution, then dilute it to 1 mL with acetonitrile, vortex and mix evenly, and detect the signal-to-noise ratio of each component. The experimental results are shown in Table 15-16.

[0143] Table 15

[0144]

[0145] Table 16

[0146]

[0147]

[0148] In the quantitative limit test, 10 groups were determined in parallel, and the signal-to-noise ratio was > 10 for all. The test results showed that the method could accurately quantify the target compounds chlorothalonil and phoxim, and the precision, anti-interference ability and robustness all met the requirements of the pharmacopoeia.

[0149] 5. Quantitative test

[0150] Take 1.0 mL of blank matrix solution (6 components) respectively, place it on a nitrogen blower, concentrate it in a 40 °C water bath to a volume of about 0.4 mL, add 10 μL of mixed reference solution, then dilute it to 1 mL with acetonitrile, vortex and mix evenly, and detect the determined concentration of the spiked samples of each component. The experimental results are shown in Table 17.

[0151] Table 17

[0152]

[0153] In this experiment, 10 μL of mixed reference solution was added to the blank matrix, and it was determined in parallel 6 times. The relative standard deviation of repeatability was ≤ 15%.

[0154] 6. Quantitative determination

[0155] Take the purified solution and add the reference substance, vortex and mix evenly, and detect the maximum recovery rate and minimum recovery rate of the spiked samples of each component. The experimental results are shown in Table 18.

[0156] Table 18

[0157]

[0158] In this experiment, 100 μL of mixed reference solution was added during the pretreatment process of the blank matrix, and it was determined in parallel 6 times. The recovery rate range of the purified solution was 91-114%, and the standard recovery rate range was 70-120%.

[0159] 7. Durability Test

[0160] Durability refers to the degree of tolerance that the measurement results are not affected when there are slight changes in the measurement conditions. In this experiment, durability tests were carried out using the same chromatographic column and mass spectrometry conditions with different equipment. The TIC ion current diagram is as Figure 1 - 3 shown, and the specific parameters are shown in Table 19.

[0161] Table 19

[0162] Test substance Quantitative ion pair Retention time Scan mode Collision voltage (V) Phoxim 299.2-＞70.1 5.195 Positive 35

[0163] The durability experiment shows that the durability experiment of this experimental test method using different equipment has results that meet the durability requirements.

Claims

1. A test method for the residues of chlorothalonil and phoxim in Chinese herbal medicines, characterized in that, It includes the following steps: S1. Grind and sieve the sample to be tested, place it in a stoppered centrifuge tube, add inorganic salts, shake well, add an organic phase, homogenize for 1 - 3 min, then centrifuge, let stand, and collect the precipitate and the first supernatant; S2. Add an organic phase to the precipitate, homogenize for 1 - 3 min, centrifuge, and collect the second supernatant; S3. Combine the first supernatant and the second supernatant, concentrate under reduced pressure to 5 - 10 mL, then cool to 20 - 30 °C, add an organic phase for dilution, shake well to obtain a crude product solution; S4. Purify the crude product solution using an HLB SPE solid-phase extraction column and collect the purified solution; S5. For the phoxim component in the purified solution, measure it by the LC-MSMS method; for the chlorothalonil component in the purified solution, detect it by the GC-MSMS method.

2. The test method for the residues of chlorothalonil and phoxim in Chinese medicinal materials according to claim 1, wherein The inorganic salts include one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate, or ammonium sulfate.

3. The method for testing the residues of chlorothalonil and phoxim in traditional Chinese medicinal materials according to claim 1, wherein The rotation speed of the homogenization treatment is greater than 12000 rpm.

4. The method for testing the residues of chlorothalonil and phoxim in Chinese medicinal materials according to claim 1, characterized in that, The rotation speed of the centrifugation treatment is 3000 - 6000 rpm.

5. The test method for the residues of chlorothalonil and phoxim in traditional Chinese medicines according to claim 1, wherein The mesh number of the grinding and sieving is 50 - 100 meshes.

6. The test method for the residues of chlorothalonil and phoxim in Chinese medicinal materials according to claim 1, characterized in that, In the LC-MSMS test, the liquid phase conditions are as follows: The column temperature is 30 - 40 °C; Mobile phase A is ammonium formate with a formic acid mass concentration of 0.01 - 0.1 wt%; Mobile phase B is a mixed solvent of mobile phase A and acetonitrile; The running time is 5 - 10 min; The elution method is gradient elution.

7. The method for testing the residues of chlorothalonil and phoxim in traditional Chinese medicines according to claim 6, characterized in that, In the gradient elution, the volume percentage of mobile phase A is 0 - 90%.

8. The test method for the residues of chlorothalonil and phoxim in Chinese medicinal materials according to claim 6, characterized in that, In the gradient elution, the volume percentage of mobile phase B is 10 - 100%.

9. The test method for the residues of chlorothalonil and phoxim in traditional Chinese medicines according to claim 1, characterized in that, In the GC-MSMS test, the gas phase conditions are as follows: Use a phenyl methyl polysiloxane as an elastic quartz capillary column; the inlet temperature is 200 - 250 °C, splitless injection; the carrier gas is helium; the inlet is in a constant pressure mode, and the column front pressure is 140 - 150 kPa; programmed temperature rise, the initial temperature is 50 - 60 °C, hold for 1 min, increase the temperature at a rate of 20 - 30 °C / min to 120 - 150 °C, and then increase the temperature at a rate of 5 - 10 °C / min to 290 °C and hold for 2 min.

10. The test method for the residual contents of chlorothalonil and phoxim in traditional Chinese medicinal materials according to claim 9, wherein In the GC-MSMS test, the mass spectrometry conditions are as follows: Test with a triple quadrupole tandem mass spectrometer, the ion source is an electron impact source, the ion source temperature is 200 - 250 °C, the collision gas is nitrogen, the mass spectrometry transfer interface temperature is 200 - 250 °C, and the mass spectrometry detection mode is multiple reaction monitoring.

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