A method for detecting morpholinium guanidine hydrochloride in a complex matrix of agricultural products

Abstract

The present application relates to the field of analytical chemistry, and provide a kind of method for detecting complex matrix of agricultural products in guanidine morpholino salt hydrochloride.The extraction solvent of the present application uses acetonitrile+acid+aqueous solution, different acid is selected according to the water content of sample, trifluoroacetic acid is used for high water content sample, and formic acid is used for low water content sample;In purification, pH value is adjusted according to the oil content of sample, ammonia is used to adjust pH to 8.5 for high oil content sample, and pH value can not be adjusted for low oil content sample.In the method, NaCl is not added to make organic phase and aqueous phase in extraction liquid stratified, through the way of extracting three times repeatedly, the recovery rate of the method is guaranteed;And using ultra-high phase liquid chromatography triple quadrupole mass spectrometry technology, guanidine morpholino salt hydrochloride in complex matrix of agricultural products is successfully detected, the extraction method is fast and simple, the quantitative limit of guanidine morpholino salt hydrochloride in tea is 0.1mg / kg, and the quantitative limit in other matrix is 0.05mg / kg, which is suitable for routine analysis, and can meet the quantitative detection requirements of guanidine morpholino salt hydrochloride in complex matrix of agricultural products.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products. Background Technology

[0002] Moroxydine hydrochloride is a broad-spectrum morpholine virus control agent with strong alkalinity and water solubility. When diluted and sprayed onto the plant surface, the solution enters the plant through stomata, inhibiting or disrupting the formation of nucleic acids and lipoproteins, thus preventing viral replication and controlling viruses. This drug is inexpensive and has therapeutic, attenuating, and protective effects against viral diseases such as mosaic virus, fern leaf spot, leaf streak, and leaf blight in vegetables, melons, fruit trees, and field crops. It is currently widely used in agricultural production as a broad-spectrum virus control agent. Its chemical structure is as follows:

[0003]

[0004] Morpholine guanidine hydrochloride consists of a morpholine ring and a guanidine group, contains an amino group, and belongs to organic bases. It is a water-soluble compound, requiring a polar aqueous solution for extraction. However, due to the diverse and complex matrix types in agricultural products, including those with high water content, high oil content, and low water content, conventional aqueous solutions are often insufficient for extraction and purification. Currently, there are few studies on the detection and analysis of morpholine guanidine hydrochloride in agricultural products. Zhao Lin et al. (Zhao Lin and Zhang Xiaobo et al., 2013) added 10g of brown rice and 5g of plant material to 40mL of 5% trichloroacetic acid, shook for 60min, added 0.5mL of 0.5mol / L sodium heptanesulfonate solution to the supernatant, mixed, and purified using an Oasis HLB or C18 solid-phase extraction column, followed by UPLC-MS / MS analysis. The detection limit in brown rice and plant material was 0.005mg / kg. The average recovery rate at 0.5mg / kg was 105.4%, with a relative standard deviation of 7.2%. Zheng Haixiang et al. (2012) extracted 10g of tobacco sample with trichloroacetic acid, neutralized with sodium heptanesulfonate, purified by liquid-liquid extraction, and then determined by high-performance liquid chromatography (HPLC) with a DAD detector, followed by reversed-phase HPLC analysis. The average recovery rate of the method was 79.88–90.98% and the relative standard deviation was 1.27–4.05% at concentrations of 0.05–1.0 mg / kg. Shao Hui et al. (2011) extracted 10g of tomato sample with trichloroacetic acid, purified by HLB solid-phase extraction, and analyzed by UPLC-MS / MS. The limit of quantitation was 0.005 mg / kg, and the average recovery rate at 0.5 mg / kg was 86.7% with a relative standard deviation of 6.3%. All of these techniques require relatively long extraction times and cannot meet the needs of extraction from various complex matrices.

[0005] Morpholine guanidine hydrochloride is a water-soluble substance with an early elution time, making it susceptible to interference from impurities. Hilic columns, while retaining highly polar analytes, also exhibit excellent peak shape separation for basic solutes, providing better peak shape. When using a C18 column, a 0.1% formic acid aqueous solution + acetonitrile solution can be used as the mobile phase, offering good stability. However, when using a Hilic column, a formic acid + ammonium formate buffer system is required as the mobile phase to improve the applicability of this method; this mobile phase is also suitable for C18 columns. The chemical structure of morpholine guanidine hydrochloride contains many amino groups, which readily form a buffer solution system, aiding in the equilibration of pesticides within the column and prolonging their retention time. Summary of the Invention

[0006] In view of this, this invention proposes a method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products. In pesticide residue analysis, the extraction solvent must be suitable for complex matrices with varying moisture, oil, sugar, and other substance contents to extract compounds with different polarities. In samples with low moisture content, such as grains and tea, even polar solvents cannot completely extract the compounds; a solvent containing 20-40% water must be used, or an equal amount of water must be added to the sample before extraction. Therefore, this method uses acetonitrile + water as the extraction solvent, and adds acid to directly extract the analyte from the sample. The extraction solvent is in the form of acetonitrile + acid + water. Moroline guanidine hydrochloride is readily soluble in water; this method does not add NaCl to separate the organic and aqueous phases in the extract. The recovery rate is ensured by repeating the extraction three times. Moroline guanidine hydrochloride in complex matrices of agricultural products was successfully detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The extraction method is rapid and simple, using conventional extraction solvents, and different extraction and purification conditions are selected according to the water and oil content. Morpholine guanidine hydrochloride has a limit of quantification (LOQ) of 0.1 mg / kg in tea and 0.05 mg / kg in other matrices, making it suitable for routine analysis and meeting the requirements for quantitative detection of morpholine guanidine hydrochloride in complex matrices of agricultural products.

[0007] The technical solution of this invention is as follows:

[0008] A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products includes the following steps:

[0009] a) Extraction and treatment: Weigh the complex matrix sample of the agricultural product to be tested into a centrifuge tube, add acetonitrile + acid + aqueous solution, vortex and centrifuge, pour out the extract, add acetonitrile + acid + aqueous solution to the centrifuge tube containing the remaining residue, vortex and centrifuge again, combine the extracts, make up the volume, and wait for purification.

[0010] b) Purification treatment: Take a small amount of extract, add purifying agent, vortex and centrifuge, take the supernatant and filter it through a microporous membrane to obtain the test solution;

[0011] c) The prepared test solution is separated by high performance liquid chromatography using a chromatographic column, and then analyzed and determined by mass spectrometry.

[0012] Preferred extraction process for vegetable and fruit products: Weigh 10g of sample into a 50mL centrifuge tube, add 20mL of acetonitrile-trifluoroacetic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 10mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, vortex again for 10min, centrifuge at 8000r / min for 5min, combine the extracts, and dilute to 50mL with the extract for purification.

[0013] Preferably, the volume ratio of acetonitrile-trifluoroacetic acid-aqueous solution is 49.5:1:49.5.

[0014] Preferred extraction processes for grains, oilseeds, nuts, edible fungi, vegetable oils, spices, and sugar products: Weigh 5g into a 50mL centrifuge tube. Except for vegetable oil, add water to the other products until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, remove the extract, add another 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, vortex again for 10min, centrifuge at 8000r / min for 5min, combine the extracts, and dilute to 25mL with the extract for purification.

[0015] Preferred method for tea product extraction: Weigh 2g of sample into a 50mL centrifuge tube, add water until the sample is completely wetted, let stand for 30min, add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, vortex again for 10min, centrifuge at 8000r / min for 5min, combine the extracts, and dilute to 20mL with the extract for purification.

[0016] Preferably, the volume ratio of acetonitrile-formic acid-aqueous solution is 10:1:89.

[0017] Preferably, in step b) purification: Take 5 mL of the extract. For high oil content samples such as oilseeds, nuts and vegetable oils, the pH must be adjusted to 8.5 with ammonia. For other low oil content samples, the pH value does not need to be adjusted. Add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination.

[0018] Preferably, in step c), the high-performance liquid chromatography (HPLC) uses a C18 or Hilic column, 50 mm × 2.1 mm; the mobile phase is: phase A is 0.1% formic acid + 5 mmol formic acid amine solution, and phase B is acetonitrile, with the following gradient elution conditions: 0–1.5 min, mobile phase B remains at 5%; 1.5–3.5 min, mobile phase B changes from 5% to 95%; 3.5–5 min, mobile phase B changes from 95% to 5%; column temperature: 30 °C; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0019] Preferred mass spectrometry operating conditions in step c): Scanning method: electrospray positive ion scanning; Detection method: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0020] Preferably, the complex agricultural product matrix includes fruit products such as apples, peaches, grapes and citrus; vegetable products such as cabbage, celery, tomatoes, eggplants, potatoes, carrots, green beans and leeks; grain products such as brown rice, wheat and corn; oil crops such as peanuts, nuts such as peanuts, edible fungi such as oyster mushrooms, rapeseed oil, green tea, spices such as pepper, and sugar crops such as sugarcane.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) Based on the polarity of the pesticide itself and the solubility of different agricultural product matrices in the extraction solvent, the present invention uses acetonitrile + acid + aqueous solution to repeatedly extract complex agricultural product matrices three times, which is fast and simple.

[0023] (2) The extraction solvent of this invention is acetonitrile + acid + aqueous solution. Different acids are selected according to the water content of the sample. Trifluoroacetic acid is used for samples with high water content and formic acid is used for samples with low water content. This avoids the formation of gel when trifluoroacetic acid is used in samples with low water content and high oil content, which would prevent the extraction solution from being obtained.

[0024] (3) In the purification process of this invention, the pH value is adjusted according to the oil content of the sample. For samples with high oil content, the pH value must be adjusted to 8.5 using ammonia water, while for samples with low oil content, the pH value does not need to be adjusted. This avoids the situation where the extract is turbid when extracting samples with high oil content using acetonitrile + formic acid + aqueous solution, which would prevent the extract from passing through the membrane and easily lead to instrument contamination.

[0025] (4) The content of morpholine guanidine hydrochloride was determined by ultra-high performance liquid chromatography-tandem mass spectrometry. In the concentration range of 0.01-0.2 μg / mL, the linear correlation coefficient of morpholine guanidine hydrochloride was greater than 0.999. The error of the results was within a reasonable range and met the requirements of the national food safety standard "Maximum Residue Limits of Pesticides in Food". Moreover, the limit of quantification of morpholine guanidine hydrochloride in tea was 0.1 mg / kg, and the limit of quantification of other agricultural product matrices was 0.05 mg / kg. It is suitable for accurate and rapid analysis of morpholine guanidine hydrochloride residues.

[0026] (5) The present invention preferably uses PSA as the purifying agent, which has better purification effect, higher recovery rate and lower cost. Attached Figure Description

[0027] Figure 1 This is a comparison chart showing the extraction effects of different extraction solvents on morpholine guanidine hydrochloride according to the present invention.

[0028] Figure 2 This is a comparison chart showing the purification effects of different purifying agents of the present invention on morpholine guanidine hydrochloride.

[0029] Figure 3 This image shows the gel-like phenomenon observed when the high oil content sample of this invention is extracted using a trifluoroacetic acid system.

[0030] Figure 4 The image shows turbidity when extracting samples with high oil content using a formic acid system.

[0031] Figure 5 The typical chromatograms of morpholine guanidine hydrochloride of the present invention separated by C18 and Hilic columns in 0.005 mg / kg celery matrix are shown (A is C18 column; B is Hilic column).

[0032] Figure 6 This describes the cleavage pathway of morpholine guanidine hydrochloride according to the present invention. Detailed Implementation

[0033] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0034] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0036] Example 1

[0037] A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products, comprising the following steps:

[0038] a) Extraction of vegetables and fruits: Weigh 10g (accurate to 0.01g) of grape sample into a 50mL centrifuge tube, add 20mL of acetonitrile-trifluoroacetic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 10mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 50mL with the extract for purification.

[0039] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0040] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0041] The chromatographic operating conditions were as follows: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, with gradient elution conditions as follows: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0042] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0043] Example 2

[0044] A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products, comprising the following steps:

[0045] a) Extraction of vegetable and fruit products: Weigh 10g (accurate to 0.01g) of citrus sample into a 50mL centrifuge tube, add 20mL of acetonitrile-trifluoroacetic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 10mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 50mL with the extract for purification.

[0046] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0047] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0048] The chromatographic operating conditions were as follows: UPLC column: C18, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, with gradient elution conditions as follows: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0049] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0050] Example 3

[0051] A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products, comprising the following steps:

[0052] a) Extraction of vegetable and fruit products: Weigh 10g (accurate to 0.01g) of celery sample into a 50mL centrifuge tube, add 20mL of acetonitrile-trifluoroacetic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 10mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 50mL with the extract for purification.

[0053] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0054] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0055] The chromatographic operating conditions were as follows: UPLC column: C18 or Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, with gradient elution conditions as follows: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0056] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0057] This method uses C18 and Hilic columns for injection of morpholine guanidine hydrochloride, respectively, and the chromatograms are shown below. Figure 5 Both chromatographic columns produced good peak shapes, but because morpholine guanidine hydrochloride is a water-soluble pesticide, its retention on the C18 column was poor, resulting in a higher peak appearance. The Hilic column, on the other hand, not only retained polar and highly polar analytes but also exhibited excellent peak separation for basic solutes, providing better peak shapes. Morpholine guanidine hydrochloride contains many amino groups in its chemical structure, which readily forms a buffer solution system, aiding in the pesticide's equilibration within the chromatographic column and prolonging its retention time. This method preferentially utilizes the Hilic column for the experiment of this pesticide, achieving a retention time of 1.64 min for morpholine guanidine hydrochloride under Hilic column conditions. This method can also be used to determine morpholine guanidine hydrochloride using a C18 column, with a retention time of 0.63 min.

[0058] Example 4

[0059] A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products, comprising the following steps:

[0060] a) Extraction of vegetable and fruit products: Weigh 10g (accurate to 0.01g) of carrot sample into a 50mL centrifuge tube, add 20mL of acetonitrile-trifluoroacetic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour out the extract, add 10mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 50mL with the extract for purification.

[0061] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0062] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0063] The chromatographic operating conditions were as follows: UPLC column: C18, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, with gradient elution conditions as follows: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0064] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0065] Example 5

[0066] a) Extraction process: Weigh 5g of wheat (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and collect all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract solution for purification.

[0067] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0068] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0069] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0070] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0071] Example 6

[0072] a) Extraction process: Weigh 5g of peanuts (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and take out all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract. Allow to purify.

[0073] b) Take 5 mL of the extract, adjust the pH to 8.5 with ammonia, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry analysis.

[0074] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0075] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0076] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0077] Example 7

[0078] a) Extraction process: Weigh 5g of walnuts (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and take out all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract, and then purify.

[0079] b) Take 5 mL of the extract, adjust the pH to 8.5 with ammonia, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry analysis.

[0080] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0081] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0082] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0083] Example 8

[0084] a) Extraction process: Weigh 5g of oyster mushrooms (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and take out all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract. Allow to purify.

[0085] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0086] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0087] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0088] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0089] Example 9

[0090] a) Extraction process: Weigh 5g of rapeseed oil (accurate to 0.01g) into a 50mL centrifuge tube, add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, take out all the extract, add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 25mL with the extract for purification.

[0091] b) Take 5 mL of the extract, adjust the pH to 8.5 with ammonia, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry analysis.

[0092] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0093] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0094] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0095] Example 10

[0096] a) Extraction process: Weigh 5g of pepper (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely wetted, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and take out all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract. Allow to purify.

[0097] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0098] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0099] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0100] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0101] Example 11

[0102] a) Extraction process: Weigh 5g of sugarcane (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, and take out all the extract. Add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, and repeat the above extraction process. Combine the three extracts, and make up to 25mL with the extract. Allow to purify.

[0103] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0104] c) The prepared test solution is separated by high-performance liquid chromatography (HPLC) using a chromatographic column, and then analyzed and determined by mass spectrometry (MS).

[0105] Chromatographic operating conditions: UPLC column: Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, gradient elution conditions: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 μL;

[0106] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0107] In this study, the vegetable oil extract was the lower layer, while the other matrix extracts were the upper layer. Note: When determining morpholine guanidine hydrochloride in grains, edible fungi, spices, and sugar crops, pH adjustment is not required; however, when determining the pH in oil crops and nuts, it needs to be adjusted to 8.5±0.1 with ammonia. During testing, it was found that oilseeds, nuts, and vegetable oils, due to their high oil content, exhibited a gel-like consistency after the addition of trifluoroacetic acid. This is likely because the samples contain a large amount of oil, which coagulates under acidic conditions. Even after high-speed centrifugation at 10,000 rpm, the sample and extract could not be well separated, making it difficult to collect the extract and reducing the recovery rate. After switching to a formic acid system, the sample and extract could be separated better, but the extract became turbid. Figure 4 The purification effect was still poor even after adding various purifying agents, so pH adjustment was used for treatment.

[0108] Example 12

[0109] a) Extraction of tea products: Weigh 2g of green tea (accurate to 0.01g) into a 50mL centrifuge tube, add water until the sample is completely moistened, and let stand for 30min. Add 10mL of acetonitrile-formic acid-water solution, vortex for 10min, centrifuge at 8000r / min for 5min, pour off the extract, add 5mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the three extracts, and make up to 20mL with the extract for purification;

[0110] b) Take 5 mL of the extract, add 150 mg of PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry determination;

[0111] c) The prepared test solution is separated by high performance liquid chromatography using a chromatographic column, and then analyzed and determined by mass spectrometry;

[0112] The chromatographic operating conditions were as follows: UPLC column: C18 or Hilic, 50 mm × 2.1 mm; mobile phase: Phase A: 0.1% formic acid + 5 mmol formic acid amine solution, Phase B: acetonitrile, with gradient elution conditions as follows: 0–1.5 min, mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changed from 5% to 95%; 3.5–5 min, mobile phase B changed from 95% to 5%; column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 2 μL.

[0113] Mass spectrometry operating conditions: Scanning mode: electrospray positive ion scanning; Detection mode: multiple reaction monitoring (MRM); Nebulizer gas flow rate: 600 L / h; Spray voltage: 5.50 kV; Capillary temperature: 500 °C; Collision gas type: N2.

[0114] Comparative Example 1

[0115] Comparison of extraction effects of different extractants on morpholine guanidine hydrochloride: The extraction effects of four extractants—methanol, formic acid + water, acetonitrile + trifluoroacetic acid + water (49.5 + 1 + 49.5, volume ratio), and acetonitrile + formic acid + water (10 + 1 + 89, volume ratio)—on morpholine guanidine hydrochloride were analyzed. The recovery rates of the samples were examined, and the results are as follows: Figure 1As shown, the extraction efficiency of mixed solutions of acetonitrile + trifluoroacetic acid + water and acetonitrile + formic acid + water is superior to that of the other two solvents. Specifically, the trifluoroacetic acid system is more effective than the formic acid system for vegetable and fruit samples, but for other types of samples, formic acid is more effective than the trifluoroacetic acid system. This is because strong acids are more effective for samples with high water content, but too low a concentration will result in insufficient acidity for recovery, while too high a concentration is unstable and can easily damage the instrument. Therefore, this method preferably uses acetonitrile + trifluoroacetic acid + water for the extraction of fruit and vegetable samples, while acetonitrile + formic acid + water is preferred for other samples.

[0116] Example 13

[0117] The purification effects of different purifying agents on morpholine guanidine hydrochloride were compared. Moroline guanidine hydrochloride is water-soluble; therefore, NaCl was not added in this method to separate the organic and aqueous phases in the extract, resulting in a high level of impurities in the extract. The samples were purified using three commonly used purifying agents: C18, GCB (graphite carbon), and PSA. The purification effects of the three agents were compared, and the results are shown in the figure. Figure 2 Under the same conditions, the recovery rates all meet the standards. Compared with other purifying agents, PSA has better purification effect, higher recovery rate and lower cost. Taking all factors into consideration, PSA is finally selected as the purifying agent for this method.

[0118] Example 14

[0119] The tests revealed that oilseeds and nuts, due to their high oil content, exhibited a gel-like consistency after the addition of trifluoroacetic acid, such as... Figure 3 As shown, the possible reason is that the sample contains a large amount of oil, which coagulates under acidic conditions. Even after high-speed centrifugation at 10,000 rpm, the sample and extract cannot be well separated, making it difficult to collect the extract and reducing the recovery rate. After using a formic acid system for extraction, the sample and extract can be separated better, but the extract becomes turbid. Figure 4 The purification effect remained poor even after adding various purifying agents, so pH adjustment was attempted as a treatment method. Adjusting the pH to 8.5 ± 0.1 using ammonia ensured sample separation and met the required recovery rate. Therefore, in subsequent experiments, for samples with high oil content, pH adjustment during pretreatment is necessary to ensure good separation of the sample and extract.

[0120] Example 15

[0121] Morpholine guanidine hydrochloride is composed of morpholine and hydrochloric acid, and its fragmentation behavior during ionization in mass spectrometry is crucial for its accurate quantification. This method first performs excitation in positive ion detection mode and optimizes the conditions for mass spectrometric analysis of morpholine guanidine hydrochloride using direct injection. The fragmentation fragments of morpholine guanidine hydrochloride in mass spectrometry are shown below. Figure 6As shown, in primary mass spectrometry, morpholine guanidine hydrochloride produces a stable [M+H] group. - The ion was selected as the parent ion, with an m / z of 172.10. The declustering voltage was further optimized, with an optimal DP of 30V. Secondary mass spectrometry analysis was performed on the parent ion fragments to obtain the full spectrum information of the secondary mass spectrometry fragments. Among them, the mass spectrometry fragment ions had m / z of 112.9 (qualitative ion) and m / z of 129.7 (quantitative ion).

[0122] To investigate the recovery rate of morpholine guanidine hydrochloride using this method, spiking recovery and precision experiments were conducted on 22 plant-derived food samples from 10 categories that did not contain the analyte pesticide or its metabolites. After adding the pesticide standard solution to the samples, the samples were left to stand for 30 minutes to allow for complete pesticide absorption. Then, each type of sample underwent extraction, purification, and instrument testing according to the method described herein. A total of three spiking levels (0.05, 0.1, and 1 mg / kg for others, and 0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg for tea) and five parallel experiments were performed. The recovery rate and relative standard deviation requirements for different spiking levels in the "Guideline for the Compilation of National Standards for Pesticide Residue Detection Methods" are shown in Table 1.

[0123] Table 1. Requirements for recovery rate at different spiking levels

[0124] Addition level, mg / kg scope,% Relative standard deviation, % ≤0.001 50～120 ≤35 ＞0.001≤0.01 60～120 ≤30 ＞0.01≤0.1 70～120 ≤20 ＞0.1≤1 70～120 ≤15 ＞1 70～120 ≤10

[0125] Data analysis revealed that the recovery rates of most of the different addition levels in each matrix met the requirements in the table above. Table 2 shows the recovery rates of representative matrices (brown rice, wheat, corn, peanut, cabbage, celery, tomato, eggplant, potato, radish, green bean, leek, apple, peach, grape, citrus, walnut, oyster mushroom, rapeseed oil, green tea, Sichuan pepper, and sugarcane) from different matrices.

[0126] Table 2 Recovery rates of 22 matrices in morpholine guanidine hydrochloride

[0127]

[0128]

[0129] (The addition levels of green tea were 0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg.)

[0130] The recovery data for each matrix above show that the average recoveries of the three pesticides and their metabolites measured by this method are mostly between 70% and 110%, with a small portion between 110% and 120%, and the RSDs are mostly below 10%. This indicates that the recovery rate of this method is very good and can meet the requirements for residue detection.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products, characterized in that: Includes the following steps: a) Extraction and processing: Extraction of vegetable and fruit products: Weigh the sample into a centrifuge tube, add 20 mL of acetonitrile-trifluoroacetic acid-water solution with a volume ratio of 49.5:1:49.5, vortex for 10 min, centrifuge at 8000 r / min for 5 min, pour off the extract, add 10 mL of acetonitrile-trifluoroacetic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the extracts, and make up to 50 mL with the extract for purification; Extraction of grains, oilseeds, nuts, edible fungi, vegetable oils, spices, and sugar products: Weigh the samples into centrifuge tubes. Except for vegetable oils, add water to the other products until the samples are completely moistened, and let them stand for 30 minutes. Add 10 mL of acetonitrile-formic acid-water solution with a volume ratio of 10:1:89, vortex for 10 minutes, centrifuge at 8000 r / min for 5 minutes, pour off the extract, add another 5 mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the extracts, and make up to 25 mL with the extract for purification. Extraction of tea products: Weigh the sample into a centrifuge tube, add water until the sample is completely wetted, let stand for 30 min, add 10 mL of acetonitrile-formic acid-water solution with a volume ratio of 10:1:89, vortex for 10 min, centrifuge at 8000 r / min for 5 min, pour out the extract, add 5 mL of acetonitrile-formic acid-water solution to the centrifuge tube containing the remaining residue, repeat the above extraction process, combine the 3 extracts, and make up to 20 mL with the extract, and wait for purification; b) Purification treatment: Take a small amount of extract. For oilseed, nut and vegetable oil samples with high oil content, adjust the pH to 8.5 with ammonia water and add 150 mg PSA; vortex and centrifuge, take the supernatant and filter it through a microporous membrane to obtain the test solution. c) The prepared test solution is separated by high performance liquid chromatography using a chromatographic column, and then analyzed and determined by mass spectrometry.

2. The method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products as described in claim 1, characterized in that: Step b) Purification: Take 5 mL of the extract. For oilseed, nut and vegetable oil samples with high oil content, adjust the pH to 8.5 with ammonia water, add 150 mg PSA, vortex for 2 min, centrifuge at 8000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm microporous membrane for liquid chromatography-tandem mass spectrometry analysis.

3. The method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products as described in claim 1, characterized in that: The high-performance liquid chromatography (HPLC) in step c) uses a C18 or Hilic column, 50 mm × 2.1 mm; the mobile phase is: phase A is 0.1% formic acid + 5 mmol formic acid amine solution, and phase B is acetonitrile, with gradient elution conditions of 0–1.5 min, and mobile phase B maintained at 5%; 1.5–3.5 min, mobile phase B changes from 5% to 95%; 3.5–5 min, mobile phase B changes from 95% to 5%; column temperature: 30℃, flow rate: 0.25 mL / min, injection volume: 2 µL.

4. The method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products as described in claim 1, characterized in that: The mass spectrometry operating conditions in step c) are as follows: scanning mode: electrospray positive ion scanning; detection mode: multiple reaction monitoring (MRM); nebulizer gas flow rate: 600 L / h; spray voltage: 5.50 kV; capillary temperature: 500℃; collision gas type: N2.

5. The method for detecting morpholine guanidine hydrochloride in complex matrices of agricultural products as described in claim 1, characterized in that: The complex agricultural product matrix includes fruit products such as apples, peaches, grapes and citrus; vegetable products such as cabbage, celery, tomatoes, eggplants, potatoes, carrots, green beans and leeks; grain products such as brown rice, wheat and corn; oil crops such as peanuts, nuts such as walnuts, edible fungi such as oyster mushrooms, rapeseed oil, green tea, spices such as pepper, and sugar crops such as sugarcane.

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