Method for detecting residues of two novel herbicides in plant-derived food by QuEChERS-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of food safety detection, and discloses a method for detecting residues of two novel herbicides in plant-derived food by QuEChERS-high performance liquid chromatography-tandem mass spectrometry, the method is simple to operate, high in sensitivity and suitable for rapid quantitative analysis of multiple matrixes in cross-border plant-derived food, and the detection accuracy is high. Technical support is provided for cross-border food safety detection; comprising the following steps: (1) respectively absorbing Tolpyralate and Cyclopyralate standard substances to prepare a 10 [mu] g / mL standard stock solution, and preserving in a dark place at-20 DEG C; (2) sucking each standard stock solution into a brown volumetric flask, preparing a mixed standard intermediate solution by using acetonitrile, and further preparing a series of standard working solutions with the mass concentration of 0.5-20.0 ng / mL for HPLC-MS / MS detection; (3) sucking a proper amount of the mixed standard intermediate solution into the purified blank matrix to prepare a series of matrix standard solutions, and drawing each matrix standard curve by taking the peak area response value of each substance as a vertical coordinate and the concentration as a horizontal coordinate, and (4) carrying out pretreatment on the sample by adopting different pretreatment methods respectively.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for detecting two new herbicide residues in plant-derived foods by using QuEChERS-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] In order to meet the needs of the growing population and increase crop yields, pesticides are widely used in agricultural production. Pesticide residues may accumulate in the food chain and pollute the environment, thus posing potential hazards to human health. The acute and chronic effects of pesticide pollution can cause various carcinogenic, tumorogenic, genotoxic and teratogenic effects on humans. Plant-derived foods represented by vegetables, fruits, and grains are necessities for people's lives, and it is crucial to strictly monitor the problem of pesticide residues in plant-derived foods. In recent years, the types of pesticides have been updated rapidly. Tolpyralate is a new herbicide developed by Ishihara Industries of Japan. By acting on the HPPD target in the plant body, it achieves a good control effect on a variety of grass weeds and broadleaf weeds in corn fields. In 2016-2017, the herbicide was registered and listed in Japan and the United States. In 2020, the Japan Institute of Food Chemistry set its limit in corn at 0.05 mg / kg. On November 24, 2023, the U.S. Environmental Protection Agency issued Notice No. 2023-25871, revising the limit of Tolpyralate in matrices such as barley and wheat from the original "no maximum limit" to "0.01 mg / kg". On January 18, 2024, Health Canada issued Notice PMRL2024-01, also revising the maximum residue limit of Tolpyralate in barley and wheat to 0.01 mg / kg. Cyclopyrimorate is a new phenoxypyridazine herbicide developed by Mitsui Chemicals of Japan, targeting homogentisate solanine transferase (HST) and can be used to control broadleaf weeds and resistant weeds in rice fields. In 2019, cyclopyrimorate was launched in Japan. In the same year, the Japan Institute of Food Chemistry stipulated that its limit in rice was 0.01 mg / kg. my country's latest version of the pesticide limit standard GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" and GB 2763.1-2022 "National Food Safety Standard Maximum Residue Limits of 112 Pesticides including 2,4-D Butyrate Sodium Salt in Food" are used in combination. However, the relevant limit standards and detection method recommendations for the above two new herbicides are not yet involved. Therefore, establishing their detection methods is innovative and forward-looking.

[0003] QuEChERS is a fast, easy, cheap, effective, rugged and safe extraction and purification technology, originally developed for the recovery of pesticide residues in fruits and vegetables. The QuEChERS method has developed rapidly and its application has exceeded its original scope, but it has always been considered one of the most convenient and quickest pretreatment methods for extracting pesticide residues. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting residues of two new herbicides in plant-derived foods by using QuEChERS-high performance liquid chromatography-tandem mass spectrometry. A detection method for Tolpyralate and Cypermethrin in nine matrices including cabbage, apple, citrus, shiitake mushroom, raisin, corn, rice, wheat and barley is established by using QuEChERS-high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS). The method is simple to operate and has high sensitivity. It is suitable for rapid quantitative analysis of multiple matrices in cross-border plant-derived foods, and provides technical support for cross-border food safety detection.

[0005] The present invention is achieved by the following technical scheme: a method for detecting residues of two new herbicides in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0006] 1) Preparation of standard solution: Accurately pipette 1 mL and 100 μL of Tolpyralate (100 μg / mL) and Cyclopyrimorate (1000 μg / mL) standard into 10 mL volumetric flasks, respectively, dissolve to volume with acetonitrile to prepare a standard stock solution with a mass concentration of 10 μg / mL, and store at –20°C in the dark for later use;

[0007] 2) Preparation of standard working solutions: Accurately pipette 1 mL of each standard stock solution into a brown volumetric flask, and prepare 10 mL of a mixed standard intermediate solution with a mass concentration of 1 μg / mL with acetonitrile, and then prepare a series of standard working solutions with a mass concentration of 0.5-20.0 ng / mL for HPLC-MS / MS detection;

[0008] 3) Preparation of matrix standard curve: Pipette an appropriate amount of the mixed standard intermediate solution into the purified blank matrix to prepare a series of matrix standard solutions with mass concentrations of 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 and 20.0 ng / mL. Draw the matrix standard curve with the peak area response value of each substance as the ordinate and the concentration as the abscissa;

[0009] 4) The samples were pre-treated using different pre-treatment methods; the cabbage, apple, citrus, and mushrooms in the samples were treated using the same pre-treatment method, the raisins were treated using one pre-treatment method, and the corn, rice, wheat, and barley were treated using the same pre-treatment method.

[0010] In order to better implement the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: in the step 2), when performing HPLC-MS / MS detection:

[0011] The chromatographic conditions are:

[0012] CAPCELL PAK C18 MGIII column (2.0*100mm, 5μm) or SHIMADZU Shim-pack GISTC18 column (2.1*150mm, 3μm) or GL Sciences InertSustain C18 chromatographic column (2.1mm*150mm, 3μm); mobile phase A is methanol or acetonitrile; mobile phase B is 0.2% formic acid water, 5mmol / L ammonium acetate or 5mmol / L ammonium acetate + 0.1% formic acid water; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B;

[0013] The mass spectrometry conditions were:

[0014] An electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500 V; ion source temperature (TEM): 450°C; nebulizer gas (GS1): 50.0 kPa; auxiliary heating gas (GS2): 50.0 kPa; curtain gas (CUR): 30.0 kPa.

[0015] In order to better implement the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: in the step 4),

[0016] 4.1) For samples of cabbage, apple, citrus and mushroom, the pretreatment method for each sample is:

[0017] 4.1.1) Weigh 10 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 4 glass homogenizer balls; add 10 mL of extraction solvent, shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0018] 4.1.2) After step 4.1.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 rpm for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading;

[0019] 4.2) The pre-treatment method of raisins is:

[0020] 4.2.1) Weigh 5 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 10 mL of extraction solvent and shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0021] 4.2.2) After step 4.2.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading;

[0022] 4.3) For corn, rice, wheat and barley samples, the pretreatment method for each sample is:

[0023] 4.3.1) Weigh 5 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 15 mL of extraction solvent; shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1.0 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0024] 4.3.2) After step 4.31.), take 1 mL of the supernatant and add 50 mg PSA, 0-75 mg C 18 (Preferably 50 mg C 18 ), 150 mg of anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading.

[0025] In order to better realize the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the extraction solvent is acetonitrile, methanol, acetonitrile: methanol (1:1), 0.1% acetic acid acetonitrile, 0.1% formic acid acetonitrile, acetone or ethyl acetate; preferably, the extraction solvent directly uses acetonitrile.

[0026] In order to better implement the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: in the step 2), when performing HPLC-MS / MS detection:

[0027] The chromatographic conditions are:

[0028] GL Sciences InertSustain C18 chromatographic column; mobile phase A is acetonitrile; mobile phase B is 5mmol / L ammonium acetate; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B;

[0029] The mass spectrometry conditions were:

[0030] An electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500 V; ion source temperature (TEM): 450°C; nebulizer gas (GS1): 50.0 kPa; auxiliary heating gas (GS2): 50.0 kPa; curtain gas (CUR): 30.0 kPa.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] The present invention adopts QuEChERS-high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) to establish a detection method for Tolpyralate and Cypermethrin in 9 matrices including cabbage, apple, citrus, shiitake mushroom, raisin, corn, rice, wheat and barley. The method is simple to operate and has high sensitivity, and is suitable for rapid quantitative analysis of multiple matrices in cross-border plant-derived foods, providing technical support for cross-border food safety detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1a The peak shape and response diagram of Cyclopyrimorate under different mobile phase combinations.

[0034] Figure 1b The peak shape and response diagram of Tolpyralate under different mobile phase combinations.

[0035] Figure 2a The peak shape and response diagram of Cyclopyrimorate under different chromatographic columns.

[0036] Figure 2b The peak shape and response diagram of Tolpyralate under different chromatographic columns.

[0037] Figure 3 This is the effect of different extraction solvents on the recovery rates of two pesticides.

[0038] Figure 4a Optimization diagram of GCB in Apple.

[0039] Figure 4b Optimization diagram of GCB in citrus.

[0040] Figure 4c Optimization diagram of GCB in shiitake mushrooms.

[0041] Figure 4d Optimization diagram of GCB in raisins.

[0042] Figure 5a Rice C 18 Optimization diagram of

[0043] Figure 5b Wheat C 18 Optimization diagram.

[0044] Figure 5c Barley C 18 Optimization diagram.

[0045] Figure 5d Corn C 18 Optimization diagram. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0047] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0048] In order to achieve better technical effects, the present invention involves the following instruments, materials and reagents:

[0049] Nexera X2 ultra-high performance liquid chromatograph (Shimadzu Corporation, Japan); AB Sciex Triple Quad 500 liquid chromatograph-mass spectrometer (ABSCIEX Corporation, USA); 3H20RI centrifuge (Huxi Corporation, China); XS205 electronic balance (Mettler-Toledo Corporation, Switzerland); XW-80A vortex mixer (HUXI Corporation, China); Turbovap LV fully automatic concentrated nitrogen blowdown apparatus (Biotage Corporation, Sweden); Milli-Q ultrapure water device (Millipore Corporation, USA); 0.22 μm PTFE filter membrane (Tianjin Aiger Co., Ltd.).

[0050] Two pesticide residue standards: Tolpyralate, purity 99.1% (Tianjin Alta Co., Ltd.); cycloheximide, purity 97.7% (DR Company). The corresponding CAS, molecular weight and structural formula information are shown in Table 1. Acetonitrile, ethyl acetate, acetone, and methanol were all chromatographically pure (Fisher Company, USA); formic acid and acetic acid were all chromatographically pure, and ammonia and ammonium acetate were all analytically pure (Chengdu Kelong Chemical Co., Ltd.); anhydrous magnesium sulfate (MgSO 4 ) and sodium chloride (NaCl) were of analytical grade (Shanghai Sinopharm Chemical Reagent Co., Ltd.); octadecyl-bonded silica gel (C 18 ), N-(n-propyl)ethylenediamine bonded silica gel (PSA), and graphitised carbon black (GCB) (Shanghai Anpu Experimental Technology Co., Ltd.).

[0051] More than 20 batches of plant samples, including cabbage, apple, citrus, mushroom, raisin, corn, rice, wheat, and barley, were purchased from the local market. The samples were homogenized and stored at -20°C for later use.

[0052] Table 1 CAS, molecular weight and structural formula of two herbicides

[0053]

[0054] Embodiment 1:

[0055] The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry comprises the following steps:

[0056] 1) Preparation of standard solution: Accurately pipette 1 mL and 100 μL of Tolpyralate (100 μg / mL) and Cyclopyrimorate (1000 μg / mL) standard into 10 mL volumetric flasks, respectively, dissolve to volume with acetonitrile to prepare a standard stock solution with a mass concentration of 10 μg / mL, and store at –20°C in the dark for later use;

[0057] 2) Preparation of standard working solutions: Accurately pipette 1 mL of each standard stock solution into a brown volumetric flask, and prepare 10 mL of a mixed standard intermediate solution with a mass concentration of 1 μg / mL with acetonitrile, and then prepare a series of standard working solutions with a mass concentration of 0.5-20.0 ng / mL for HPLC-MS / MS detection;

[0058] 3) Preparation of matrix standard curve: Pipette an appropriate amount of the mixed standard intermediate solution into the purified blank matrix to prepare a series of matrix standard solutions with mass concentrations of 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 and 20.0 ng / mL. Draw the matrix standard curve with the peak area response value of each substance as the ordinate and the concentration as the abscissa;

[0059] 4) The samples were pre-treated using different pre-treatment methods; the cabbage, apple, citrus, and mushrooms in the samples were treated using the same pre-treatment method, the raisins were treated using one pre-treatment method, and the corn, rice, wheat, and barley were treated using the same pre-treatment method.

[0060] Embodiment 2:

[0061] This embodiment is further optimized on the basis of the above embodiment, and the same points as the above technical solution are not repeated here. In order to better realize the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: In the step 2), when performing HPLC-MS / MS detection:

[0062] The chromatographic conditions are:

[0063] CAPCELL PAK C18 MGIII column (2.0*100mm, 5μm) or SHIMADZU Shim-pack GISTC18 column (2.1*150mm, 3μm) or GL Sciences InertSustain C18 chromatographic column (2.1mm*150mm, 3μm); mobile phase A is methanol or acetonitrile; mobile phase B is 0.2% formic acid water, 5mmol / L ammonium acetate or 5mmol / L ammonium acetate + 0.1% formic acid water; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B;

[0064] The mass spectrometry conditions were:

[0065] Electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500V; ion source temperature (TEM): 450℃; nebulizer gas (GS1): 50.0kPa; auxiliary heating gas (GS2): 50.0kPa; curtain gas (CUR): 30.0kPa. The LC-MS / MS characteristic selected ions and mass spectrometry parameters of the two pesticide residues are shown in Table 2.

[0066] Table 2 Retention time and mass spectrometry parameters of two pesticide residues

[0067]

[0068] Embodiment 3:

[0069] This embodiment is further optimized on the basis of any of the above embodiments, and the same points as the above technical solutions are not repeated here. In order to better realize the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: in the step 4),

[0070] 4.1) For samples of cabbage, apple, citrus and mushroom, the pretreatment method for each sample is:

[0071] 4.1.1) Weigh 10 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 4 glass homogenizer balls; add 10 mL of extraction solvent, shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0072] 4.1.2) After step 4.1.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 rpm for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading;

[0073] 4.2) The pre-treatment method of raisins is:

[0074] 4.2.1) Weigh 5 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 10 mL of extraction solvent and shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0075] 4.2.2) After step 4.2.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading;

[0076] 4.3) For corn, rice, wheat and barley samples, the pretreatment method for each sample is:

[0077] 4.3.1) Weigh 5 g of sample (accurate to 0.0001 g) and place in a 50 mL centrifuge tube with a screw cap, add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 15 mL of extraction solvent; shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1.0 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min;

[0078] 4.3.2) After step 4.31.), take 1 mL of the supernatant and add 50 mg PSA, 0-75 mg C 18 (Preferably 50 mg C 18 ), 150 mg of anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading.

[0079] In order to obtain better detection results, the invention optimizes the type and amount of purification powder during the research and development stage, and the optimization is as follows:

[0080] The QuEChERS technique uses the interaction between the adsorbent filler and the interfering matrix to achieve the purpose of impurity removal and purification. It has the advantages of simple operation, high recovery rate, accurate detection results, and a wide range of applications. At present, the purification materials used in the QuEChERS technique mainly include C18 、PSA、MgSO 4 , GCB, etc. PSA is mainly used to remove organic acids, pigments and metal ions, and is a commonly used purification powder in plant-derived foods. 18 It has a higher capacity for non-polar substances and can effectively remove long-chain lipid compounds such as oils and fat-soluble pigments. Since vegetables and fruits contain almost no fat, C was not used in the experiment when optimizing the pretreatment of cabbage, apples, citrus, mushrooms and raisins. 18 As a purification powder. GCB is mainly used to adsorb pigments and other substances. Considering that apples, citrus, mushrooms and raisins contain pigments, they may interfere with the extraction of target pesticide residues. Therefore, the experiment compared the effects of 0mg GCB and 25mg GCB on the recovery rate of the target substance for the above four matrices ( Figure 4a , Figure 4b , Figure 4c , Figure 4d ). The results showed that adding 25 mg of GCB during the purification process reduced the recovery of apple, citrus and shiitake mushroom matrices. Although the recovery rate of raisin matrix was slightly improved by adding GCB powder to the purification, good recovery effect could still be achieved without adding GCB. Therefore, GCB was not added when extracting cabbage, apple, citrus, shiitake mushroom and raisin.

[0081] Similarly, considering that rice, corn, wheat, and barley contain a certain amount of fat but almost no pigment, the experiment added C 18 powder and optimized its dosage without considering the addition of GCB. The results showed that ( Figure 5a , Figure 5b , Figure 5c , Figure 5d ), in rice, corn and wheat matrices, both pesticide residues were above 50 mgC 18 The recovery efficiency was higher under the conditions of 25-75 mg C 18 The recovery rate remained almost stable under the same conditions. In barley matrix, although cypermethrin was 18 The recovery efficiency under the conditions was slightly higher than 50 mg C 18 , but Tolpyrlate at 50mg C 18 Therefore, the experiment finally focused on the C 18 The dosage of purified powder is 50 mg.

[0082] Embodiment 4:

[0083] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the extraction solvent is acetonitrile, methanol, acetonitrile: methanol (1:1), 0.1% acetic acid acetonitrile, 0.1% formic acid acetonitrile, acetone or ethyl acetate; preferably, the extraction solvent directly uses acetonitrile.

[0084] In order to achieve better detection results, the present invention optimized the extraction solvent during the research and development stage, and the optimization is as follows: This experiment selected acetonitrile, methanol, acetonitrile: methanol (1:1), 0.1% acetic acid acetonitrile, 0.1% formic acid acetonitrile, acetone, and ethyl acetate as extraction solvents to compare the extraction effects. Figure 3 The results showed that acetonitrile and 0.1% acetic acid acetonitrile showed good extraction efficiency when extracting 9 kinds of plant-derived foods (cabbage, apple, citrus, mushroom, raisin, corn, rice, wheat, barley). Among them, in the raisin and cabbage matrix, the recovery rate of 0.1% acetic acid acetonitrile was slightly better than that of acetonitrile, but a satisfactory recovery effect could still be achieved under acetonitrile conditions. And considering that the extraction efficiency of acetonitrile in barley and wheat is significantly better than that of 0.1% acetic acid acetonitrile. Therefore, the experiment finally selected acetonitrile as the extraction solvent for the two target pesticide residues. The recovery rates of the two pesticide residues studied in this experiment in barley and wheat matrices were low, especially the recovery rate of tolpyralate in wheat was almost difficult to exceed 70%, which may be due to the fact that the flour matrix has a certain adsorption effect on the pesticide residue, resulting in extraction loss.

[0085] Embodiment 5:

[0086] This embodiment is further optimized on the basis of any of the above embodiments, and the same points as the above technical solutions are not repeated here. In order to better realize the method of detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: In the step 2), when performing HPLC-MS / MS detection:

[0087] The chromatographic conditions are:

[0088] GL Sciences InertSustain C18 chromatographic column; mobile phase A is acetonitrile; mobile phase B is 5mmol / L ammonium acetate; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B;

[0089] The mass spectrometry conditions were:

[0090] An electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500 V; ion source temperature (TEM): 450°C; nebulizer gas (GS1): 50.0 kPa; auxiliary heating gas (GS2): 50.0 kPa; curtain gas (CUR): 30.0 kPa.

[0091] In order to achieve better technical effects, the present invention optimizes the mobile phase during the research and development stage:

[0092] Methanol and acetonitrile are common organic phases in liquid mass analysis. Studies have shown that when acetonitrile is used as the organic phase, the separation, peak shape and response of the target are better than methanol. Therefore, this study uses acetonitrile as the organic phase (mobile phase A), 0.2% formic acid water, 5mmol / L ammonium acetate, and 5mmol / L ammonium acetate + 0.1% formic acid water as the mobile phase (mobile phase B), and examines the separation effect and response differences of different combinations on each target ( Figure 1a , Figure 1b ). The results showed that under the same elution procedure, when acetonitrile-5mmol / L ammonium acetate (0.1% formic acid) was used as the mobile phase, the two pesticide residues had the lowest response. Cyclopyridazine had a higher response under 0.2% formic acid water conditions than 5mmol / L ammonium acetate, but the difference in response between the two was not large. As for Tolpyrate, it had a higher response under 5mmol / L ammonium acetate conditions, and was significantly higher than 0.2% formic acid water conditions. This may be because ammonium acetate, as a buffer salt, can provide a relatively stable ionic environment in the solution, maintain a relatively stable pH of the mobile phase, and make it easier for ester herbicide molecules to accept protons to form positive ions, thereby improving the ionization efficiency. Although formic acid can also promote the formation of positive ions to a certain extent, its buffering capacity is weak, and the pH stability in the mobile phase system may not be as good as ammonium acetate, resulting in large fluctuations in ionization efficiency. Therefore, the experiment finally selected acetonitrile-5mmol / L ammonium acetate as the mobile phase.

[0093] In terms of analytical columns:

[0094] The C18 column uses octadecyl silane bonded phase as the stationary phase and has strong hydrophobicity. It is widely used in the field of pesticide residue analysis and is suitable for the separation of various pesticides such as benzimidazoles, carbamates, and organophosphorus. The experiment compared the separation effects of three columns: CAPCELL PAK C18 MGIII (2.0*100mm, 5μm), Shim-pack GIST C18 (2.1*150mm, 3μm) (SHIMADZU, Japan) and InterSuatain C18 (2.1*150mm, 3μm) (GL Science Japan) on two herbicides. See Figure 2a and Figure 2b . The results showed that under the same liquid phase conditions, the Shim-pack GIST C18 column was not suitable for the separation of the target substance, with poor peak shape, severe burrs and tailing. The separation using the InterSuatain C18 column was better than that of the MGIII, and could achieve better peak shape and response, and the retention time was suitable. This may be due to the fact that the silanol on the silica surface of the column can achieve 100% complete end-capping, reducing the secondary interaction between the active silanol groups and the alkaline impurities and cations, thereby avoiding tailing and improving the symmetry of the peak. Therefore, the InterSuatain C18 column was selected as the analytical column.

[0095] Based on the above embodiments, the present invention performs the following analyses:

[0096] 1. Matrix effect:

[0097] When pesticide residues are detected, direct quantification using solvent standard curves may cause matrix effects to seriously affect the accuracy of the results. Therefore, it is necessary to evaluate the matrix effects to determine whether they will affect the precision of the method and the accuracy of the results. Matrix effects (ME) are the effects of enhanced or suppressed response of target compounds caused by competitive ionization between impurities and target compounds in the test solution at the ion source.

[0098]

[0099] Different types of substances have different matrix effects. When ME<0, it is matrix inhibition. The larger the absolute value, the stronger the matrix effect. When |ME|<20%, it is a weak matrix effect, indicating that its matrix effect is not significant; when |ME|<50%, it is a moderate matrix effect; when |ME|>50%, it is a strong matrix effect, and the matrix effect is considered significant. The experiment uses vegetables (cabbage), fruits (citrus, apples), fruit products (raisins), and grains (corn, rice, barley, and wheat) as typical matrices to evaluate the matrix effects of the detection targets in different product categories. As shown in Table 3, the two target pesticides showed obvious matrix inhibition in all 9 matrices (ME<0). Tolpyralate showed a weak matrix effect (|ME|<20%) in apple, shiitake mushroom, and wheat matrices; cyclopyridazine showed a weak matrix effect (|ME|<20%) in apple, rice, and raisins, and a strong matrix effect (|ME|>50%) in the remaining matrices. Therefore, the present invention adopts the matrix matching standard curve external standard method for quantification to reduce the interference of the matrix effect on the actual sample determination and increase the accuracy of the method quantification.

[0100] Table 3Solvent curves, matrix curves and matrix effects of 2 herbicides in 9 plant-derived matrices

[0101]

[0102]

[0103] 2. Linear range, detection limit and quantification limit:

[0104] The linear range, repeatability, detection limit and quantification limit of the established method were evaluated according to the optimized experimental conditions. The series of matrix standard solutions of 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 and 20.0 ng / mL prepared in step 3) were used to draw the standard curves of the two target pesticides with the analyte concentration as the abscissa x and the measured peak area as the ordinate y. The results are shown in Table 4. In the range of 0.5-20.0 ng / mL, the correlation coefficient (R2) of the working curve was 0.9914-0.9999, showing a good linear relationship. The method detection limit (LOD, S / N=3) was determined to be 0.14-0.90 μg / kg by the spike recovery method, and the quantification limit (LOQ, S / N=10) was 0.47-3.00 μg / kg. By comparison, the results were significantly lower than the maximum limit requirements in the relevant matrices established in Japan, the United States and Canada.

[0105] Table 4 Linear equations, correlation coefficients (R2), method limits of detection (LODs) and limits of quantification (LOQs) for 2 pesticides

[0106]

[0107] 3. Recovery and precision of the present invention:

[0108] The experiment used blank cabbage, apple, citrus, shiitake mushroom, raisin, corn, rice, wheat and barley as sample matrices, and used the established HPLC-MS / MS method to perform an addition recovery test to evaluate the accuracy and precision of the method in the determination of two target herbicides. Because my country has not yet established the maximum residue limit requirements for the target pesticide residues measured, the experiment was designed based on GB / T27404-2008 "Laboratory Quality Control Specifications for Food Physical and Chemical Testing", and the maximum limit (MRL) established by the Japan Food Chemistry Research Institute, the US Environmental Protection Agency Announcement No. 2023-25871 and the Canadian Ministry of Health PMRL 2024-01 Notice was used as the maximum spike amount. The LOQ (3μg / kg), 2LOQ (6μg / kg), and MRL (10μg / kg) three spike levels were determined, and the experiment was repeated 6 times for each spike level, and the external standard method was used for quantification. The results are shown in Table 5. Within the three spiked concentration ranges, the recoveries of Tolpyralate and Cyclopyralid in nine matrices were 65.4-106.3% and 76.4-111.2%, respectively, with RSDs of 1.9-10.0% and 1.7-10.2%. The precision and accuracy of the method are good, in line with the requirements of GB / T 27404-2008, and can meet the requirements of accurate analysis and quantification of two new herbicides in various plant-derived foods in daily supervision and sampling.

[0109] Table 5 Average recovery and precision (RSD) of 2 herbicides, n=6

[0110]

[0111] 4. Test the actual samples:

[0112] The present invention was used to test 24 batches of plant-derived foods from the local market, including apples (4 batches), citrus (4 batches), mushrooms (4 batches), and 3 batches of other matrices. The results showed that neither of the two target substances was detected.

[0113] 5. Compared with other public technologies:

[0114] At present, the latest version of my country's pesticide limit standards GB 2763-2021 and GB 2763.1-2022 do not involve the HPLC-MS / MS detection method and limit requirements of the two herbicides, and there are few similar studies internationally. Han Sol Lee et al. used QuEChERS combined with LC-MS / MS to determine the Tolpyralate residues in five agricultural products and set the LOQ to 0.01 mg / kg. In contrast, the present invention has a lower limit of quantification and can be applied to the detection of more plant-derived matrices. Therefore, the present invention is more advantageous.

[0115] The present invention establishes a detection method for two new pesticides in plant-derived foods by QuEChRES-HPLC-MS / MS, which can achieve rapid detection in multiple matrices (cabbage, apple, citrus, mushroom, raisin, corn, rice, wheat, barley). Through the selection and optimization of mobile phase, chromatographic column, extraction solvent, purification powder and other conditions, the two target compounds have a good linear relationship in the range of 0.5-20.0 ng / mL (R 2 >0.9914), the average recovery rate was 65.4-111.2%, and RSD ≤10.2%. This method is simple, rapid, and highly sensitive, and provides reliable technical support for the extraction and determination of Tolpyralate and Cyclopyralid in multi-matrix plant-derived foods. The quantitative limit of the method can also meet the detection requirements of pesticide residues in Japan, the United States, and Canada.

[0116] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry, characterized in that: The steps include: 1) Pipette 1 mL and 100 μL of Tolpyralate and Cyclopyrimorate standards into 10 mL volumetric flasks respectively, dissolve to volume with acetonitrile to prepare a standard stock solution with a mass concentration of 10 μg / mL, and store at -20 °C in the dark for later use; 2) Pipette 1 mL of each standard stock solution into a brown volumetric flask, and prepare 10 mL of a mixed standard intermediate solution with a mass concentration of 1 μg / mL with acetonitrile, and then prepare a series of standard working solutions with a mass concentration of 0.5-20.0 ng / mL for HPLC-MS / MS detection; 3) Pipette an appropriate amount of the mixed standard intermediate solution into the purified blank matrix to prepare a series of matrix standard solutions with mass concentrations of 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 and 20.0 ng / mL, and draw the standard curve of each matrix with the peak area response value of each substance as the ordinate and the concentration as the abscissa; 4) The samples were pre-treated using different pre-treatment methods; the cabbage, apple, citrus, and mushrooms in the samples were treated using the same pre-treatment method, the raisins were treated using one pre-treatment method, and the corn, rice, wheat, and barley were treated using the same pre-treatment method.

2. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 2), when performing HPLC-MS / MS detection: The chromatographic conditions are: CAPCELL PAK C18 MGIII column or SHIMADZU Shim-pack GIST C18 column or GLSciences InertSustain C18 column; mobile phase A is methanol or acetonitrile; mobile phase B is 0.2% formic acid water, 5mmol / L ammonium acetate or 5mmol / L ammonium acetate + 0.1% formic acid water; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B; The mass spectrometry conditions were: An electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500 V; ion source temperature (TEM): 450°C; nebulizer gas (GS1): 50.0 kPa; auxiliary heating gas (GS2): 50.0 kPa; curtain gas (CUR): 30.0 kPa.

3. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 4), 4.1) For samples of cabbage, apple, citrus and mushroom, the pretreatment method for each sample is: 4.1.1) Weigh 10 g of sample into a 50 mL centrifuge tube with a screw cap and add the standard; add 4 glass homogenizer balls; add 10 mL of extraction solvent and shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min; 4.1.2) After step 4.1.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 rpm for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading; 4.2) The pre-treatment method of raisins is: 4.2.1) Weigh 5 g of sample into a 50 mL centrifuge tube with a screw cap and add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 10 mL of extraction solvent and shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min; 4.2.2) After step 4.2.1), take 1 mL of the supernatant, add 50 mg PSA and 150 mg anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading; 4.3) For corn, rice, wheat and barley samples, the pretreatment method for each sample is: 4.3.1) Weigh 5 g of sample into a 50 mL centrifuge tube with a screw cap and add the standard; add 10 mL of water and let stand for 30 min, add 4 glass homogenizer balls; add 15 mL of extraction solvent; shake the sample for 1 min; add 4 g of anhydrous magnesium sulfate and 1.0 g of sodium chloride, shake vigorously for 1 min, and centrifuge at 8000 r / min for 5 min; 4.3.2) After step 4.31.), take 1 mL of the supernatant and add 50 mg PSA, 0-75 mg C 18 , 150 mg of anhydrous magnesium sulfate, vortex mix for 1 min, centrifuge at 8000 r / min for 5 min, pass through a 0.22 μm PTFE membrane, and wait for loading.

4. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 3, characterized in that: In step 4.3.2) C 18 The dosage is 50mg.

5. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 3 or 4, characterized in that: The extraction solvent is acetonitrile, methanol, acetonitrile:methanol (1:1), 0.1% acetic acid acetonitrile, 0.1% formic acid acetonitrile, acetone or ethyl acetate.

6. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 5, characterized in that: The extraction solvent is acetonitrile.

7. The method for detecting two new herbicide residues in plant-derived foods by QuEChERS-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: In the step 2), when performing HPLC-MS / MS detection: The chromatographic conditions are: GL Sciences InertSustain C18 chromatographic column; mobile phase A is acetonitrile; mobile phase B is 5mmol / L ammonium acetate; flow rate: 0.3mL / min; injection volume: 5.0μL; column temperature is 40℃; gradient elution program of mobile phase B: 0.00~1.00min, 90%B; 1.00~2.00min, 90~40%B; 2.00~4.00min, 40-10%B; 4.00~6.00min, 10%B; 6.10~8.00min, 90%B; The mass spectrometry conditions were: An electrospray ion source (ESI+) was used; scanning mode: multiple reaction ion monitoring (SRM); ion spray voltage (IS): 5500 V; ion source temperature (TEM): 450°C; nebulizer gas (GS1): 50.0 kPa; auxiliary heating gas (GS2): 50.0 kPa; curtain gas (CUR): 30.0 kPa.

Images