Method for detecting various plant-derived biochemical pesticides in apples

Abstract

The invention provides a method for detecting various plant-derived biochemical pesticides in apples, and belongs to the technical field of pesticide detection. According to the method, acetonitrile is used for extracting the plant-derived biochemical pesticide, graphene / nano-ZrO2 / C18 is used for purification, and qualitative and quantitative detection and analysis are carried out through high performance liquid chromatography-ultraviolet absorption spectrum-QDa mass spectrum. The result shows that the linear relation of triptolide, thidiazuron, indolebutyric acid, 2, 4-D, paclobutrazol and uniconazole in apples is good in the concentration range of 0.1-10 mg / L, and R2 ranges from 0.9990 to 0.9999; when the addition level is 0.1-10 mg / kg, the average recovery rate in an apple matrix is 80.3-92.3%, the relative standard deviation (RSDs) is 1.1-6.4%, the method detection limit (LOD, S / N > = 3) is 10-15 [mu] g / kg, and the quantitation limit is 50 [mu] g / kg.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide detection, and in particular relates to a method for detecting multiple plant-derived biochemical pesticides in apples. Background Art

[0002] As people's living standards continue to improve, high-quality fruits rich in nutrients and vitamins have become an essential food for residents' daily lives. Apples, as a fruit with rich nutritional value and crisp taste, are very popular among the public. Therefore, its consumer market is very broad. With the continuous increase in demand for apples, the planting area and scale have also been expanding in recent years. In recent years, improper use and excessive use of plant growth regulators have occurred from time to time. The residues of plant growth regulators in agricultural products have become one of the main hidden dangers of food safety. Therefore, the control of apple quality and safety is particularly important. Food safety is an important livelihood issue. At present, residents' requirements for apple quality are constantly increasing. Initially, they only focused on the residual safety of chemical pesticides in apples, but now they have begun to pay attention to the residual safety of biochemical pesticides and other risky additives. This undoubtedly puts forward new standards and requirements for fruit quality and safety testing technology.

[0003] With the development of modern agricultural science and technology, plant-derived biochemical pesticides are widely used. They are a type of artificially synthesized plant hormones that have plant hormone activity and the same physiological effects. They can help to fully develop the potential of improved varieties and are mainly used to regulate plant growth and development. Although these plant growth regulators are not highly toxic substances, they also have certain toxicity. The toxic hazards mainly include reproductive toxicity, nephrotoxicity, hepatotoxicity, developmental toxicity, immunotoxicity, neurotoxicity, teratogenicity, carcinogenicity, mutagenicity and bone damage. In summary, it is imperative to strengthen the management and control of the use of plant growth regulators, try to avoid the abuse of plant growth regulators, and study new general plant growth regulator detection technologies.

[0004] At present, the analysis and detection technology of pesticide residues in apples mainly involves chemical pesticides. The detection methods mainly include gas chromatography, gas chromatography-mass spectrometry, liquid chromatography and liquid chromatography-mass spectrometry, etc. However, there are few reports on the detection technology of plant-derived biochemical pesticides.

[0005] Therefore, based on the current status of inspection and testing technical standards, it is urgent to develop a new detection method for multiple plant-derived biochemical pesticides in apples, taking plant-derived biochemical pesticides in apples as the implementation object. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for detecting multiple plant-derived biochemical pesticides in apples, which simultaneously screens and accurately quantitatively detects and analyzes multiple plant-derived biochemical pesticides in apple matrices by combining spectroscopy with mass spectrometry.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting plant-derived biochemical pesticides in apples, comprising the following steps:

[0009] 1) Mix apples and acetonitrile, then add sodium chloride and anhydrous magnesium sulfate for vortex extraction, and centrifuge to obtain supernatant A;

[0010] 2) Supernatant A, graphene / nano-ZrO2 / C 18 Mix with anhydrous magnesium sulfate, vortex purify, centrifuge to obtain supernatant B;

[0011] 3) Prepare the botanical biochemical pesticide standard into a standard stock solution with a concentration of 500 mg / L, and then dilute it with acetonitrile and blank matrix solution to a series of standard solutions with concentrations of 10, 5, 1, 0.5, and 0.1 mg / L;

[0012] 4) subjecting the series of standard solutions of step 3) to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection, and drawing a standard curve with the mass concentration of the plant-derived biochemical pesticide as the X-axis and the corresponding peak area as the Y-axis;

[0013] Under the same conditions, the supernatant B in step 2) is subjected to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection to determine the type of botanical biochemical pesticides based on the retention time and the relative abundance of the chromatographic peak area corresponding to the characteristic ions and the quantitative ions;

[0014] The measured peak area of ​​the plant-derived biochemical pesticide in the supernatant B is substituted into the standard curve to obtain the content of the plant-derived biochemical pesticide in the supernatant B.

[0015] Preferably, in step 2) the graphene / nano-ZrO2 / C 18 Graphene, nano-ZrO2 and C 18 The mass ratio is 2-10:5-25:5-30.

[0016] Preferably, the HPLC conditions for the HPLC-UV absorption spectroscopy-QDa mass spectrometry detection in step 4) are: C 18 The chromatographic column uses acetonitrile and water as the mobile phases, and gradient elution is used to separate the target compounds. The injection volume is 3 to 8 μL, the column temperature is 30 to 40°C, and the flow rate is 0.5 to 1.5 mL / min.

[0017] Preferably, the water in the mobile phase contains formic acid and ammonium acetate, the volume fraction of formic acid in the mobile phase is 0.03-0.1%, and the concentration of ammonium acetate in the mobile phase is 1-3 mM.

[0018] Preferably, the ultraviolet absorption spectrum conditions of the high performance liquid chromatography-ultraviolet absorption spectrum-QDa mass spectrometry detection in step 4) are: using the maximum absorption wavelength of the plant-based biochemical pesticide for detection, and simultaneously performing 3D spectrum scanning for qualitative analysis, and the range of the 3D spectrum scanning is 210 to 400 nm.

[0019] Preferably, the QDa mass spectrometry conditions of the high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection in step 4) are: electrospray ionization, positive and negative ion scanning modes, the scanning mode is selected ion monitoring, the temperature is 550-650°C, the power voltage is 0.5-1.5 kV, and the cone voltage is 5-30 V.

[0020] Preferably, in step 1), the mass volume ratio of the apple to acetonitrile is 2 g: 3-5 mL; the mass ratio of the apple to sodium chloride is 10: 1-3; and the mass ratio of the apple to anhydrous magnesium sulfate is 5: 1-3.

[0021] Preferably, in step 2), the supernatant A, graphene / nano-ZrO2 / C 18 The volume mass ratio of magnesium sulfate to anhydrous magnesium sulfate is 1.5 mL: 12-65 mg: 160-200 mg.

[0022] Preferably, the rotation speed of the vortex extraction in step 1) is 2000-3000 rpm, and the time of the vortex extraction is 3-8 min;

[0023] The centrifugal speed is 7000-9000 rpm, and the centrifugal time is 2-4 min;

[0024] Step 2) the rotation speed of the vortex purification is 2000-3000 rpm, and the time of the vortex purification is 1-3 min;

[0025] The centrifugal speed is 11000-13000 rpm, and the centrifugal time is 1-3 min.

[0026] Preferably, the plant-derived biochemical pesticide is triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and niclobutrazol.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses acetonitrile to extract plant-derived biochemical pesticides, graphene / nano-ZrO2 / C 18Purification, rapid and accurate quantitative detection and analysis by high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry, and the detection method of various plant-derived biochemical pesticides in apples was established. The six plant-derived biochemical pesticides in apples (triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and uniclobutrazol) had good linear relationships in the concentration range of 0.1-10 mg / L, and R 2 The results show that the average recoveries of 6 kinds of plant biochemical pesticides in apple matrix were between 80.3% and 92.3%, the relative standard deviations (RSDs) were 1.1-6.4%, the detection limits (LOD, S / N≥3) were 10-15μg / kg, and the quantitative limits were 50μg / kg. This method is accurate, fast and stable, and meets the needs of actual detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the chromatograms of 6 plant-derived biochemical pesticides (among which, 1 is triptolide; 2 is thidiazuron; 3 is indolebutyric acid; 4 is 2,4-D; 5 is paclobutrazol; 6 is nitrothiocyanate);

[0030] Figure 2 It is the Me of 6 plant-derived biochemical pesticides in apple matrix under different purifier combinations;

[0031] Figure 3 is the response peak area of ​​6 plant-derived biochemical pesticides under different cone voltages. DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting plant-derived biochemical pesticides in apples, comprising the following steps:

[0033] 1) Mix apples and acetonitrile, then add sodium chloride and anhydrous magnesium sulfate for vortex extraction, and centrifuge to obtain supernatant A;

[0034] 2) Supernatant A, graphene / nano-ZrO2 / C 18 Mix with anhydrous magnesium sulfate, vortex purify, centrifuge to obtain supernatant B;

[0035] 3) Prepare the botanical biochemical pesticide standard into a standard stock solution with a concentration of 500 mg / L, and then dilute it with acetonitrile and blank matrix solution to a series of standard solutions with concentrations of 10, 5, 1, 0.5, and 0.1 mg / L;

[0036] 4) subjecting the series of standard solutions of step 3) to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection, and drawing a standard curve with the mass concentration of the plant-derived biochemical pesticide as the X-axis and the corresponding peak area as the Y-axis;

[0037] Under the same conditions, the supernatant B in step 2) is subjected to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection to determine the type of botanical biochemical pesticides based on the retention time and the relative abundance of the chromatographic peak area corresponding to the characteristic ions and the quantitative ions;

[0038] The measured peak area of ​​the plant-derived biochemical pesticide in the supernatant B is substituted into the standard curve to obtain the content of the plant-derived biochemical pesticide in the supernatant B.

[0039] In the present invention, apples and acetonitrile are mixed, and then sodium chloride and anhydrous magnesium sulfate are added for vortex extraction and centrifugation to obtain supernatant A. The mass volume ratio of the apples to acetonitrile is preferably 2g:3-5mL, and more preferably 2g:3.5-4.5mL; the mass ratio of the apples to sodium chloride is preferably 10:1-3, and more preferably 10:1.5-2.5; the mass ratio of the apples to anhydrous magnesium sulfate is preferably 5:1-3, and more preferably 5:1.5-2.5; the rotation speed of the vortex extraction is preferably 2000-3000rpm, and more preferably 2200-2800rpm; the time of the vortex extraction is preferably 3-8min, and more preferably 4-7min; the rotation speed of the centrifugation is preferably 7000-9000rpm, and more preferably 7500-8500rpm; the time of the centrifugation is preferably 2-4min, and more preferably 2.5-3.5min.

[0040] In the present invention, the supernatant A, graphene / nano-ZrO2 / C 18 and anhydrous magnesium sulfate, vortex purified, and centrifuged to obtain supernatant B. 18 Graphene, nano-ZrO2 and C 18 The mass ratio of the supernatant A, graphene / nano-ZrO2 / C is preferably 2-10:5-25:5-30, and more preferably 4-8:10-20:10-25; 18 The volume mass ratio of the anhydrous magnesium sulfate is preferably 1.5mL:12-65mg:160-200mg, and more preferably 1.5mL:20-50mg:170-190mg; the rotation speed of the vortex purification is preferably 2000-3000rpm, and more preferably 2200-2800rpm; the time of the vortex purification is preferably 1-3min, and more preferably 1.5-2.5min; the rotation speed of the centrifuge is preferably 11000-13000rpm, and more preferably 11500-12500rpm; the time of the centrifugation is preferably 1-3min, and more preferably 1.5-2.5min.

[0041] In the present invention, the botanical biochemical pesticide standard is prepared into a standard mother solution with a concentration of 500 mg / L, and then diluted with acetonitrile and a blank matrix solution to a series of standard solutions with concentrations of 10, 5, 1, 0.5, and 0.1 mg / L. The botanical biochemical pesticides are triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and niclobutrazol.

[0042] In the present invention, the series of standard solutions in step 3) are subjected to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection, and a standard curve is drawn with the mass concentration of the plant-derived biochemical pesticide as the X-axis and the corresponding peak area as the Y-axis;

[0043] Under the same conditions, the supernatant B in step 2) is subjected to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection to determine the type of botanical biochemical pesticides based on the retention time and the relative abundance of the chromatographic peak area corresponding to the characteristic ions and the quantitative ions;

[0044] The measured peak area of ​​the plant-derived biochemical pesticide in the supernatant B is substituted into the standard curve to obtain the content of the plant-derived biochemical pesticide in the supernatant B.

[0045] In the present invention, the HPLC conditions for HPLC-UV absorption spectroscopy-QDa mass spectrometry detection are preferably: C 18 A chromatographic column, acetonitrile and water are used as mobile phases, the water in the mobile phase contains formic acid and ammonium acetate, the volume fraction of formic acid in the mobile phase is preferably 0.03-0.1%, and more preferably 0.04-0.09%; the concentration of ammonium acetate in the mobile phase is preferably 1-3 mM, and more preferably 1.5-2.5 mM; gradient elution is used to separate the target compounds, and the injection volume is preferably 3-8 μL, and more preferably 4-7 μL; the column temperature is preferably 30-40° C., and more preferably 32-38° C.; the flow rate is preferably 0.5-1.5 mL / min, and more preferably 0.8-1.3 mL / min.

[0046] In the present invention, the ultraviolet absorption spectrum conditions of the high performance liquid chromatography-ultraviolet absorption spectrum-QDa mass spectrometry detection are: using the maximum absorption wavelength of plant-derived biochemical pesticides for detection, and simultaneously performing 3D spectrum scanning for qualitative analysis, and the range of the 3D spectrum scanning is 210 to 400 nm.

[0047] In the present invention, the QDa mass spectrometry conditions of the high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection are: electrospray ionization, positive and negative ion scanning modes, the scanning mode is selected ion monitoring, the temperature is preferably 550-650°C, and more preferably 580-620°C; the power voltage is preferably 0.5-1.5 kV, and more preferably 0.8-1.2 kV; the cone voltage is preferably 5-30 V, and more preferably 10-25 V.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] 1. Preparation of standard solution

[0051] Accurately weigh a certain amount of triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and uniconazole standards to prepare standard stock solutions with a concentration of 500 mg / L, respectively, and then dilute them with acetonitrile and blank matrix solution to a series of standard solutions of 10, 5, 1, 0.5, and 0.1 mg / L.

[0052] 2. Sample pretreatment

[0053] Use a homogenizer to fully grind the frozen apple sample, accurately weigh 10 g of the homogenized sample into a 50 mL centrifuge tube, add 20 mL of acetonitrile and shake quickly, then add 2 g of sodium chloride and 3 g of anhydrous magnesium sulfate, vortex and oscillate at 2500 rpm for 5 min, and then centrifuge at 8000 rpm for 3 min, and the supernatant is to be purified.

[0054] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube (containing graphene / nano-ZrO2 / C18=5 / 15 / 10 mg and 180 mg anhydrous magnesium sulfate), vortex oscillate at 2500 rpm for 2 min, and then centrifuge at 12000 rpm for 2 min. The supernatant is filtered through a 0.22 μm organic filter membrane for detection.

[0055] 3. Instrumental analysis method

[0056] 3.1 Chromatographic conditions

[0057] The chromatographic column was C18 (3.5 μm*2.1 mm*50 mm), and acetonitrile and water (containing 0.05% formic acid and 2 mM ammonium acetate) were used as the mobile phases. The target compounds were separated by gradient elution. The gradient elution program was shown in Table 1. The injection volume was 5 μL and the column temperature was 35°C.

[0058] Table 1 Gradient elution program of target compounds

[0059]

[0060] 3.2UV detection parameters

[0061] Six plant-derived biochemical pesticides were tested at their maximum absorption wavelengths. The detection wavelength of each compound is shown in Table 2. In addition, 3D spectral scanning (range 210-400nm) was performed simultaneously for qualitative analysis.

[0062] 3.3QDa monitoring parameters

[0063] Electrospray ionization (ESI); positive and negative ion scanning modes, scanning mode is selected ion monitoring (SIM), temperature is 600 ° C, power voltage is 0.8 kV; compound monitoring ion parameters are shown in Table 2, chromatograms are shown in Figure 1 .

[0064] Table 26 Detection parameters of plant-derived biochemical pesticides

[0065]

[0066] Example 2

[0067] QuEChERS pretreatment and matrix effect optimization

[0068] The matrix effect (Me) refers to the phenomenon that the signal of the target compound is enhanced or weakened due to the interference of co-eluting impurities. During the analysis of complex samples, the matrix effect will affect the degree of ionization and thus affect the quantitative results of the target compound.

[0069] This study used the post-extraction addition test method to calculate the extraction and addition matrix effect, and used Me to evaluate the purification effect. The calculation formula is shown below:

[0070] Me=Se2 / Se1

[0071] Wherein, Se1 is the response intensity of the pure standard solution, and Se2 is the response intensity of the sample matrix with the same amount of analyte added. Se1 and Se2 must be added with the same amount of analyte.

[0072] When Me is between 0.8 and 1.2, it is considered that there is no obvious matrix effect, that is, the purification effect is good; when Me < 0.8, it is a matrix inhibition effect. The smaller the value, the stronger the matrix inhibition effect and the worse the purification effect; when Me > 1.2, it is a matrix enhancement effect. The larger the value, the greater the matrix enhancement effect and the worse the purification effect.

[0073] Graphene can effectively adsorb impurities such as pigments, heavy metals, inorganic non-metallic ions, aromatic hydrocarbons and antibiotics. It is a highly efficient adsorbent. However, because graphene adsorbs target compounds while adsorbing impurities, its dosage needs to be slightly optimized.

[0074] At an addition level of 200 μg / kg, the purification effect of the graphene / nano-ZrO2 / C18 purifier combination on six plant-derived biochemical pesticides in apple matrix was investigated (Table 3).

[0075] Table 3. Combination usage of purifiers in apple matrix

[0076]

[0077] Experimental results: When the dosage of Graphene / nano-ZrO2 / C18 is 5 / 20 / 10 mg, the Me of the six compounds is between 0.88 and 0.91 (e.g. Figure 2 As shown in the figure, there is no obvious matrix effect and the purification effect is the best. Therefore, this study finally selected Graphene / nano-ZrO2 / C18=5 / 15 / 10mg for the purification of apple matrix.

[0078] Example 3

[0079] Optimization of chromatography and mass spectrometry conditions

[0080] A mixed standard solution with a concentration of 20 mg / L was selected to optimize the optimal absorption wavelengths of thiamethoxam and imidacloprid.

[0081] Experimental results: Through full-band UV spectrum scanning (200-400nm), it was found that the optimal absorption peaks of triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol and uniconazole were 230, 290, 280, 290, 220 and 254nm, respectively.

[0082] A mixed standard solution with a concentration of 5 mg / L was selected to optimize the mass spectrometry parameters of 6 plant-derived chemical pesticides. First, the cone voltage was fixed at 15 V, and the scanning range was set to 100-400 m / z. The parent ion screening scans of thiazolyl and imidacloprid were performed in ESI+ and ESI- modes, respectively.

[0083] Experimental results: 2,4-D has the highest response intensity in ESI-mode, and the other compounds have the best response in ESI+ mode. The parent ions of triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and vinclozolin were obtained by screening: [M+H] + =378.25m / z, [M+H] + =221.04m / z, [M+H] + =204.09m / z, [MH]-=219.05m / z, [M+H] + = 294.15 m / z and [M+H] + =292.14m / z.

[0084] To further enhance the sensitivity of the six target compounds, based on the above optimization results, the cone voltage was set to 5, 10, 15, 20, 25 and 30 V, respectively, and the cone voltage was optimized for thiophanate-methyl, thiabendazim, indolebutyric acid, 2,4-D, paclobutrazol and thiabendazole.

[0085] Experimental results: When the cone voltage was 10V, the response intensity of 2,4-D was the highest, and the other five compounds obtained the highest response intensity when the voltage was 15V (such as Figure 3 shown).

[0086] In summary, the present invention finally determined the optimal spectral and mass spectrometry monitoring parameters for thiophanate-methyl, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and uniconazole through optimal wavelength selection, mother-child screening, and cone voltage optimization.

[0087] In order to improve the working efficiency of the method of the present invention, reduce the analysis time and prevent the co-elution of interferences and target compounds, and ensure that each compound obtains the best separation and sensitivity, the present invention is based on the above-mentioned determination of monitoring parameters, selects a C18 chromatographic column with a particle size of 3.5 μm and a length of only 50 mm, and adopts four different gradient elution procedures of (Ⅰ) acetonitrile-water solution, (Ⅱ) acetonitrile-0.05% formic acid aqueous solution, (Ⅲ) acetonitrile-0.1% formic acid aqueous solution and (Ⅳ) methanol-0.05% formic acid aqueous solution to carry out chromatographic separation of six compounds.

[0088] Experimental results: When the mobile phase system was acetonitrile-0.05% formic acid aqueous solution, the comprehensive response intensity of the six compounds was the best and the separation degree was the best. By continuously optimizing the elution procedure, the efficient separation and determination of thiophanate-methyl, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol, and thiabendazole were finally achieved within 10.5 minutes.

[0089] Example 4

[0090] Standard curve

[0091] With mass concentration as the X-axis and its corresponding peak area as the Y-axis, a matrix standard curve of the apple matrix was drawn.

[0092] Detection limit

[0093] The LODs and LOQs of the six compounds in apple matrix were obtained using 3 times the signal-to-noise ratio (S / N) as the LOD and the lowest addition level as the LOQ.

[0094] Experimental results: as shown in Table 4.

[0095] Table 4 Detection limit, quantification limit, standard curve and R of 6 kinds of plant-derived biochemical pesticides 2

[0096]

[0097] As shown in Table 4, the six plant-derived biochemical pesticides in apple matrix have a good linear relationship in the concentration range of 0.1-10 mg / L, R 2 Between 0.9990 and 0.9999.

[0098] The LODs of the six compounds in apple matrix were 10-15 μg / kg, and the LOQs were all 50 μg / kg.

[0099] Example 5

[0100] Accuracy and precision

[0101] With reference to the minimum MRLs value in GB 2763-2022, three addition levels of 0.1, 1 and 10 mg / kg were set in the blank apple matrix, and five parallel tests were conducted.

[0102] Experimental results: as shown in Table 5.

[0103] Table 5 Recovery and RSDs% of 6 plant-derived biochemical pesticides in apple matrix

[0104]

[0105] As shown in Table 5, when the addition level was 0.1-10 mg / kg, the average recoveries of the six compounds in the apple matrix were between 80.3% and 92.3%, and the relative standard deviations (RSDs) were between 1.1 and 6.4%.

[0106] Example 6

[0107] Application of analytical methods

[0108] The preferred analysis method determined by the present invention was used to analyze the residues of 6 kinds of plant-derived biochemical pesticides in apples purchased from the market, and the residues were quantitatively calculated using an apple matrix standard curve.

[0109] Experimental results: as shown in Table 6.

[0110] Table 6 Detection results of 6 kinds of plant-derived biochemical pesticide residues in apples on the market

[0111]

[0112]

[0113] It can be seen from the above examples that the method of the present invention can accurately, quickly and stably determine triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol and clobutrazol in apples, shortening the detection time and improving the detection efficiency.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting plant-derived biochemical pesticides in apples, characterized in that: The following steps are involved: 1) Mix apples and acetonitrile, then add sodium chloride and anhydrous magnesium sulfate for vortex extraction, and centrifuge to obtain supernatant A; 2) Supernatant A, graphene / nano-ZrO2 / C 18 Mix with anhydrous magnesium sulfate, vortex purify, centrifuge to obtain supernatant B; 3) Prepare the botanical biochemical pesticide standard into a standard stock solution with a concentration of 500 mg / L, and then dilute it with acetonitrile and blank matrix solution to a series of standard solutions with concentrations of 10, 5, 1, 0.5, and 0.1 mg / L; 4) subjecting the series of standard solutions of step 3) to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection, and drawing a standard curve with the mass concentration of the plant-derived biochemical pesticide as the X-axis and the corresponding peak area as the Y-axis; Under the same conditions, the supernatant B in step 2) is subjected to high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection to determine the type of botanical biochemical pesticides based on the retention time and the relative abundance of the chromatographic peak area corresponding to the characteristic ions and the quantitative ions; The measured peak area of ​​the plant-derived biochemical pesticide in the supernatant B is substituted into the standard curve to obtain the content of the plant-derived biochemical pesticide in the supernatant B.

2. The detection method according to claim 1, characterized in that: Step 2) the graphene / nano-ZrO2 / C 18 Graphene, nano-ZrO2 and C 18 The mass ratio is 2-10:5-25:5-30.

3. The detection method according to claim 1, characterized in that: Step 4) The HPLC conditions for HPLC-UV absorption spectroscopy-QDa mass spectrometry detection are: C 18 The chromatographic column uses acetonitrile and water as the mobile phases, and gradient elution is used to separate the target compounds. The injection volume is 3 to 8 μL, the column temperature is 30 to 40°C, and the flow rate is 0.5 to 1.5 mL / min.

4. The detection method according to claim 3, characterized in that: The water in the mobile phase contains formic acid and ammonium acetate. The volume fraction of formic acid in the mobile phase is 0.03-0.1%, and the concentration of ammonium acetate in the mobile phase is 1-3 mM.

5. The detection method according to claim 1, characterized in that: Step 4) The ultraviolet absorption spectrum conditions of the high performance liquid chromatography-ultraviolet absorption spectrum-QDa mass spectrometry detection are: using the maximum absorption wavelength of the plant-derived biochemical pesticide for detection, and simultaneously performing 3D spectrum scanning for qualitative analysis, and the range of the 3D spectrum scanning is 210 to 400 nm.

6. The detection method according to claim 1, characterized in that: Step 4) The QDa mass spectrometry conditions of the high performance liquid chromatography-ultraviolet absorption spectroscopy-QDa mass spectrometry detection are: electrospray ionization, positive and negative ion scanning mode, the scanning mode is selected ion monitoring, the temperature is 550-650°C, the power voltage is 0.5-1.5 kV, and the cone voltage is 5-30 V.

7. The detection method according to claim 1, characterized in that: Step 1) The mass volume ratio of the apple to acetonitrile is 2 g: 3-5 mL; the mass ratio of the apple to sodium chloride is 10: 1-3; the mass ratio of the apple to anhydrous magnesium sulfate is 5: 1-3.

8. The detection method according to claim 1, characterized in that: Step 2) the supernatant A, graphene / nano-ZrO2 / C 18 The volume mass ratio of magnesium sulfate to anhydrous magnesium sulfate is 1.5 mL: 12-65 mg: 160-200 mg.

9. The detection method according to claim 1, characterized in that: Step 1) The rotation speed of the vortex extraction is 2000-3000 rpm, and the time of the vortex extraction is 3-8 min; The centrifugal speed is 7000-9000 rpm, and the centrifugal time is 2-4 min; Step 2) the rotation speed of the vortex purification is 2000-3000 rpm, and the time of the vortex purification is 1-3 min; The centrifugal speed is 11000-13000 rpm, and the centrifugal time is 1-3 min.

10. The detection method according to claim 1, characterized in that: The plant-derived biochemical pesticides are triptolide, thidiazuron, indolebutyric acid, 2,4-D, paclobutrazol and niclobutrazol.

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