Determination of fungicide residues in fruit juice using MSPE system coupled with GC-MS / MS

Abstract

This paper proposes a method for detecting the content of multiple fungicides in fresh-squeezed juice using a novel magnetic solid-phase extraction system based on NH2-Fe3O4@GO coupled with a triple quadrupole tandem gas chromatography-mass spectrometer (GC-MS / MS). The specific steps are as follows: fresh-squeezed juice is centrifuged, the sample is extracted and enriched using the novel magnetic solid-phase extraction system, and then eluted twice with acetonitrile. The eluted solution is concentrated by nitrogen purging, fixed to volume with acetone, and then filtered through a membrane. The eluted solution is then analyzed by GC-MS / MS using the EI multiple reaction monitoring scanning mode and external standard quantification. The experimental results show good linear relationships for 11 fungicides. Compared with existing methods, this method has the advantages of simple operation, high sensitivity, good reproducibility, and low consumption of organic reagents, and is suitable for the qualitative and quantitative analysis of fungicides in fresh-squeezed juice.

Description

Technical Field

[0001] The present invention relates to a method for detecting fungicides in food, in particular to a method for detecting the content of multiple fungicides in freshly squeezed juice by combining a novel magnetic solid-phase extraction system based on NH2-Fe3O4@GO and a triple quadrupole tandem gas chromatography-mass spectrometer, belonging to the technical field of chemical analysis and detection. Background Art

[0002] Fungicides are widely used to prevent and control plant diseases caused by pathogenic microorganisms during the planting and post-harvest storage, transportation, and marketing of crops, vegetables, and fruits. Currently, the commonly used organic fungicides include amides, triazoles, imidazoles, strobilurins, pyrimidines, and antibiotics. Among them, strobilurins, chlorothalonil, and procymidone are widely used on fruit trees such as apples, pears, grapes, and citrus. However, the problem of excessive fungicide residues in fruits due to the improper use of pesticides is a common occurrence, seriously affecting food safety, export trade, and consumer health. In order to monitor pesticide residues in fresh agricultural products such as fruits and vegetables, my country has established residue limits for 548 pesticides in GB2763-2021. This standard has greatly expanded the scope of pesticide residues and played a positive role in regulating the use of pesticides.

[0003] Currently, the main detection methods for fungicide residues in fruits and vegetables include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC), and liquid chromatography-mass spectrometry (LC-MS / MS). Sample pretreatment techniques, including extraction, separation, cleanup, and enrichment, prior to instrumental testing are a key focus of pesticide residue detection and analysis research. These pretreatment techniques have gradually expanded from traditional solid-phase extraction (SPE) to solid-phase microextraction (SPM), QuEChERS, and the more recently developed magnetic solid-phase extraction (MSPE). SPM suffers from poor stability, and while QuEChERS is simple to operate, it suffers from low extraction efficiency. MSPE, on the other hand, is increasingly widely used in pretreatment techniques due to its unique advantages, including high adsorption, superparamagnetism, ease of modification, and magnetic separation. Furthermore, the magnetic adsorption material can be reused, offering advantages such as simplicity, time-saving speed, and the absence of centrifugal filtration. However, there is currently no relevant literature or process method in this technical field that records magnetic solid-phase extraction materials or detection standards for the detection of fungicide content in fresh-squeezed juice. Therefore, continuing to develop new magnetic solid-phase extraction adsorption materials and establishing a fast, efficient, sensitive solution that can simultaneously detect multiple fungicides is of great significance for ensuring the quality and safety of fresh-squeezed juice products. Summary of the Invention

[0004] The purpose of the present invention is to solve the above-mentioned problems existing in the detection methods of fungicide residues in fruit and vegetable products. For the first time, a newly prepared magnetic nanomaterial NH2-Fe3O4@GO is used as a magnetic adsorbent. Then, a independently designed and assembled magnetic separation device is used in combination with GC-MS / MS to detect the residues of 11 fungicides in freshly squeezed juices. A rapid, accurate and environmentally friendly analysis method is established.

[0005] The technical solution of the present invention is a method for determining fungicide residues in fruit juice using a MSPE system coupled with GC-MS / MS, which specifically comprises the following steps:

[0006] 1) Preparation of magnetic solid phase extraction rod

[0007] The magnetic solid-phase extraction rod includes a frosted glass cover, a hollow glass tube and a strong magnet. The bottom of the hollow glass tube is closed and the top is provided with an opening. The frosted glass cover is installed on the top opening of the hollow glass tube. A circular hole with the same diameter as the opening is provided in the center. The cylindrical strong magnet is inserted through the circular hole and is vertically arranged inside the hollow glass tube.

[0008] 2) Synthesis of NH2-Fe3O4@GO magnetic material powder

[0009] ① Preparation of graphene oxide dispersion: Graphene oxide (GO) was prepared by a modified Hummers method. The prepared graphene oxide was dried and then ultrasonicated in deionized water for 1 h to obtain a graphene oxide dispersion.

[0010] ② Preparation of NH2-Fe3O4 dispersion: Weigh 100 mg of Fe3O4 nanoparticles and ultrasonically disperse them in 20 mL of ethanol for 10 minutes; add 80 mL of ethanol-water (v / v, 1:1) to it and continue ultrasonicating for 20 minutes; transfer this system to a 250 mL flask, then add 1 mL of APTES and stir the reaction at 120°C for 7 hours; after the reaction, separate the reaction products by magnetic separation technology and wash them with ethanol four times; vacuum freeze-dry the thoroughly washed NH2-Fe3O4 composite nanoparticles, dissolve the NH2-Fe3O4 particles in PBS buffer solution with a pH of 9 and ultrasonicate to obtain a 10% NH2-Fe3O4 dispersion;

[0011] ③ Preparation of magnetic graphene oxide powder: 20 mL of 10% NH2-Fe3O4 dispersion prepared in step ② was added to 20 mL of graphene oxide dispersion prepared in step ①. After vigorous stirring for 5 h, a magnetic NH2-Fe3O4@GO dispersion was obtained. Solid-liquid separation was performed using a magnetic solid phase extraction rod, and the solid phase was vacuum dried at 120°C to obtain NH2-Fe3O4@GO magnetic material powder.

[0012] 3) Sample collection and processing

[0013] ① Collect fruit samples from different areas, with each sample weighing no less than 1000g. Weigh and mix 500g of the fruit sample and squeeze the juice with a juicer for later use. Store in a refrigerator at -20℃.

[0014] ② Accurately transfer 10g of juice sample to a 50mL centrifuge tube. After refrigerated centrifugation, accurately weigh 5.0g of the supernatant into a 25mL sample tube and dilute to 10mL with pure water. Add 40mg of NH2-Fe3O4@GO magnetic material powder and ultrasonically disperse for 10min. Insert a magnetic solid-phase extraction rod and stir thoroughly to adsorb the NH2-Fe3O4@GO magnetic material powder, which is the target of adsorbing the fungicide residue.

[0015] ③ Remove the stirring bar and place it in a 10 mL elution bottle containing 3 mL of acetonitrile eluent. Remove the strong magnet from the hollow glass tube of the magnetic solid phase extraction rod, cover with a frosted glass cover and sonicate for 5 minutes. Then insert the strong magnet into the hollow glass tube, transfer the eluent to a 15 mL nitrogen blowpipe, add 3 mL of eluent and repeat the above steps. Concentrate the collected eluent with nitrogen blowpipe, make up to volume with 0.5 mL of acetone, and filter through a 0.22 μm PTFE membrane for GC-MS / MS analysis.

[0016] 4) GC-MS / MS determination

[0017] Gas phase conditions: Injection port at 300°C, splitless high-pressure injection, injection time 1 minute, flow control mode: linear velocity at 40.2 cm / sec, column flow rate 1.2 mL / min; Rtx-5MS column: hold at 70°C for 1 minute, ramp to 280°C at 15°C / min, hold for 2 minutes, then ramp to 300°C at 30°C / min, hold for 10 minutes; injection volume 1 μL. Mass spectrometry conditions: EI source at 230°C, interface temperature at 280°C, solvent delay 3 minutes, detector voltage 0.6 kV relative to the tuning voltage, time-sliced multiple reaction selective monitoring mode.

[0018] 5) Calculation of results

[0019] The calculation formula for the fungicide content C in the sample is:

[0020]

[0021] Where C0 is the relevant concentration of the fungicide in the sample after concentration calculated on the standard curve (μg / L); v is the volume of the sample after concentration (mL); and m is the mass of the juice (g).

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] 1) Compared with conventional magnetic solid-phase extraction technology, the present invention has obvious advantages in terms of efficient separation of target substances after magnetic material adsorption, convenient recovery of magnetic materials, and higher recovery rate of target components. The independently designed and assembled magnetic separation device effectively reduces sample matrix interference;

[0024] 2) The present invention optimizes and analyzes magnetic solid-phase extraction and instrument conditions, and confirms the feasibility and durability of the method through tests such as method spike recovery, RSD, and material practical times;

[0025] 3) The magnetic solid-phase extraction system established in the present invention has the characteristics of convenient and rapid pretreatment, low organic solvent consumption and little interference with the matrix. It also has the advantages of high accuracy and good repeatability, providing a powerful technical means for the detection of multiple pesticide residues and is of great significance in ensuring food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Attachment Figure 1 This is a flow chart of the novel magnetic solid-phase extraction method proposed in the present invention.

[0027] Attachment Figure 2 It is a structural diagram of the magnetic solid phase extraction rod.

[0028] Attachment Figure 3 These are the MRM mass spectra of 11 fungicides.

[0029] Caption: 1-pentachloronitrobenzene, 2-chlorothalonil, 3-fucymidone, 4-picoxystrobin, 5-E-fenoxystrobin, 6-dioxastrobin, 7-pyrimidifen, 8-oxathiocarb, 9-dioxastrobin, 10-fluoxastrobin, 11-pyrazostrobin.

[0030] Figure 3a This is the SEM image of NH2-Fe3O4@GO.

[0031] Figure 3b This is the SEM image of NH2-Fe3O4.

[0032] Figure 3c This is the infrared spectrum of NH2-Fe3O4@GO.

[0033] Figure 3d This is the infrared spectrum of Fe3O4@GO.

[0034] Figure 4a This is a line chart comparing the effect of extractant dosage on the extraction efficiency of 11 fungicides.

[0035] Figure 4b It is a line graph comparing the effect of extraction solution pH on extraction efficiency.

[0036] Figure 4cIt is a line chart comparing the effect of the number of extraction agents on the extraction efficiency. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention are further described below based on examples. In this specification, the content of each example means that the specific technical features described in conjunction with it are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific technical features described can be combined in any appropriate manner in any one or more embodiments or examples.

[0038] In this example, a newly prepared magnetic nanomaterial, NH2-Fe3O4@GO, was used as a magnetic adsorbent. A magnetic separation device was then used in conjunction with GC-MS / MS to detect the residues of 11 fungicides in freshly squeezed juice. The specific instruments and operating steps used in this method are as follows:

[0039] 1. Instruments and Reagents

[0040] Instruments: TQ-8040 gas chromatography-mass spectrometer (Shimadzu, Japan); Rtx-5MS chromatographic column (30 m × 0.25 mm × 0.25 μm); fully automatic nitrogen blowdown apparatus (Horizon, USA); constant temperature ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu); refrigerated centrifuge (Thermo Scientific, USA); VORTEX1 vortex mixer (Aika, Germany); DF-101s constant temperature magnetic stirrer (Shanghai Lichen Bangxi Instrument Technology Co., Ltd., Shanghai); Million-Q ultrapure water machine (MillionPore, USA); vacuum drying oven.

[0041] Reagents: graphene (>95%, Shanghai Aladdin Biochemical Technology Co., Ltd.), Fe3O4 nanoparticles (particle size range 100-200 nm, Tianjin Bestle Chromatography Technology Development Center), APTES (3-aminopropyltriethoxysilane), methanol, ethanol, acetonitrile, acetone, dichloromethane, n-hexane and ethyl acetate (all analytical grade, purchased from Merck, Germany), phosphoric acid, potassium dihydrogen phosphate and potassium hydroxide (all analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.).

[0042] Picoxystrobin, E-methoxystrobin, kresoxim-methyl, kresoxim-methyl, pyrimidifen, trifloxystrobin, fluoxastrobin, and azoxystrobin were all prepared as standard solutions at a concentration of 1000 mg / mL. Procymidone, chlorothalonil, and pentachloronitrobenzene were all prepared at a concentration of 100 mg / mL. These standard solutions were diluted with acetone to a 1000 mg / L standard mixed stock solution, sealed, and stored in a dark refrigerator at 4°C until ready for use. A mixed standard series was prepared by serial dilution with acetone to prepare concentrations of 50, 100, 200, 500, 1000, 2000, and 4000 μg / L for chlorothalonil, fluoxastrobin, and azoxystrobin, and concentrations of 25, 50, 100, 250, 500, 1000, and 2000 μg / L for the other components.

[0043] Soaking solution: methanol-water solution in a volume ratio of 1:1.

[0044] PBS buffer: Use 0.2 mol of potassium dihydrogen phosphate, 0.2 mol of potassium dihydrogen phosphate, and 0.2 mol of phosphoric acid solution to prepare buffer solutions with pH values of 3, 5, 7, 9, and 10, respectively.

[0045] 2. Detection Method Process

[0046] 1) Preparation of magnetic solid phase extraction rod

[0047] like Figure 2 As shown, the magnetic solid-phase extraction rod consists of a frosted glass cover, a hollow glass tube, and a strong magnet. The hollow glass tube is closed at the bottom and has an opening at the top. The frosted glass cover is mounted on the top opening of the hollow glass tube. A circular hole with the same diameter as the opening is located in the center of the cover. A cylindrical strong magnet is inserted through the circular hole and positioned vertically inside the hollow glass tube. The strong magnet can be inserted and removed from the hollow glass tube, facilitating the separation of magnetic materials from solutions.

[0048] 2) Synthesis of NH2-Fe3O4@GO magnetic material powder

[0049] ① Preparation of graphene oxide dispersion: Graphene oxide (GO) was prepared by a modified Hummers method. The prepared graphene oxide was dried and then ultrasonicated in deionized water for 1 h to obtain a graphene oxide dispersion.

[0050] ② Preparation of NH2-Fe3O4 dispersion: Weigh 100 mg of Fe3O4 nanoparticles and ultrasonically disperse them in 20 mL of ethanol for 10 minutes; add 80 mL of ethanol-water (v / v, 1:1) and continue ultrasonicating for 20 minutes; transfer the system to a 250 mL flask, then add 1 mL of APTES and stir the reaction at 120°C for 7 hours; after the reaction, separate the reaction products by magnetic separation technology and wash them with ethanol four times; freeze-dry the thoroughly washed NH2-Fe3O4 composite nanoparticles in a vacuum, dissolve the NH2-Fe3O4 particles in a pH = 9 PBS buffer solution and ultrasonicate to obtain a 10% NH2-Fe3O4 dispersion;

[0051] ③ Preparation of magnetic graphene oxide powder: Add 20 mL of 10% NH2-Fe3O4 dispersion prepared in step ② to 20 mL of graphene oxide dispersion prepared in step ①. After vigorous stirring for 5 h, a magnetic NH2-Fe3O4@GO dispersion was obtained. The solid-liquid separation was performed using a strong magnet, and the solid phase was vacuum dried at 120°C to obtain NH2-Fe3O4@GO magnetic material powder.

[0052] 3) Sample collection and processing

[0053] ① Collect 30 samples of apples, strawberries, bayberries, pears, oranges, watermelons, pineapples, etc. from farmers' markets in different regions. Each sample should be no less than 1000g. 500g of the mixed fruit sample should be weighed and squeezed with a juicer for later use. The juice should be stored in a refrigerator at -20℃.

[0054] ② Accurately transfer 10g of juice sample to a 50mL centrifuge tube. After refrigerated centrifugation, accurately weigh 5.0g of the supernatant into a 25mL sample tube and dilute to 10mL with pure water. Add 40mg of NH2-Fe3O4@GO magnetic material powder and ultrasonically disperse for 10min. Insert a magnetic solid-phase extraction rod and stir thoroughly to adsorb the NH2-Fe3O4@GO magnetic material powder, which is the target of adsorbing the fungicide residue.

[0055] ③ Remove the stirring rod and place it in a 10mL elution bottle filled with 3mL acetonitrile eluent. Remove the strong magnet from the hollow glass tube of the magnetic solid phase extraction rod, cover with a frosted glass cover and sonicate for 5 minutes. Then insert the strong magnet into the hollow glass tube, transfer the eluent to a 15mL nitrogen blowpipe, add 3mL acetonitrile and repeat the above steps. Concentrate the collected eluent with nitrogen blowpipe, then dilute with 0.5mL acetone, filter through a 0.22μm PTFE membrane, and prepare for GC-MS / MS determination. The specific flow chart is as follows: Figure 1 shown.

[0056] 4) GC-MS / MS instrument conditions

[0057] Gas phase conditions: injection port 300℃, splitless high-pressure injection, injection time 1 min, flow control mode is linear velocity, linear velocity is 40.2 cm / sec, column flow rate is 1.2 mL / min; Rtx-5MS chromatographic column is maintained at 70℃ for 1 min, increased to 280℃ at 15℃ / min and maintained for 2 min, then increased to 300℃ at 30℃ / min and maintained for 10 min, injection volume 1.0 μL.

[0058] Mass spectrometry conditions: EI source temperature 230°C, interface temperature 280°C, solvent delay 3 min, detector voltage relative to tuning voltage 0.6 kV, time-sliced multiple reaction selective monitoring (MRM) mode.

[0059] 5) Calculation of results

[0060] The external standard method was used for quantification. The signal response (y) of the quantitative ion pair of each component in the standard solution and the corresponding mass concentration (x, μg / mL) were used to draw a standard curve. The concentration of the target component C0 in the sample solution was obtained from the standard curve. Then, the content C (μg / Kg) of each component in the sample was calculated according to formula (1). The calculation formula is:

[0061]

[0062] Where C0 is the relevant concentration of the fungicide in the sample after concentration calculated on the standard curve (μg / L); v is the volume of the sample after concentration (0.5 mL); and m is the mass of the juice (10 g).

[0063] Other data were analyzed using Origin statistical analysis software.

[0064] 3. Results and Discussion

[0065] 1) Optimization of gas chromatography-mass spectrometry conditions

[0066] The total ion current of 11 fungicides was scanned in the electron impact ion source EI mode. The precursor ions of the 11 compounds were found respectively. The collision energy was set at 3-45eV. The product ion scan was performed on the selected precursor ions. The appropriate quantitative ion and qualitative ion pairs were selected according to the secondary mass spectrometry. The relevant detection parameters and optimization conditions are shown in Table 1. The optimized MRM mass spectra of the 11 fungicides are shown in Figure 3 shown.

[0067]

[0068] Table 1 Mass spectrometry parameters of 11 fungicides

[0069] 2) Characterization of NH2-Fe3O4@GO nanomaterials

[0070] The synthesized NH2-Fe3O4@GO was subjected to SEM and the FI-IR characterization of NH2-Fe3O4@GO and Fe3O4@GO materials was compared. The SEM image of NH2-Fe3O4@GO is shown in Figure 2. Figure 3a As shown. NH2-Fe3O4 presents a regular spherical shape, as shown Figure 3b As shown, smooth spherical Fe3O4 nanoparticles are uniformly dispersed on the surface of GO sheets. SEM images further demonstrate the successful synthesis of NH2-Fe3O4@GO.

[0071] FI-IR spectrum of NH2-Fe3O4@GO Figure 3c As shown, the characteristic peak of Fe-O is located at 570 cm -1 Inches, the GO spectrum shows 3424 cm -1 The OH stretching vibration band and the NH stretching vibration band at 1620 cm-1 are the C=C skeleton vibration band and the NH bending vibration band at 1726 cm-1. -1 The stretching vibration band of C=O is 1391cm -1 The CO stretching vibration band is 2925cm -1 (CC stretch); through Figure 3c and Figure 3d Comparison, 3400cm -1 and 1600cm -1 The left and right infrared absorption becomes stronger, indicating the existence of -NH2, proving the successful formation of NH2-Fe3O4@GO.

[0072] 3) Optimization of solid phase extraction conditions

[0073] The present invention uses a single-factor method to optimize the relevant conditions affecting the recovery rate of magnetic solid-phase extraction. During the optimization process, the actual juice spiked with 11 fungicides is all at 1.0 μg / mL.

[0074] ①Influence of extraction agent dosage

[0075] The effect of NH2-Fe3O4@GO dosage (10-100 mg) on the extraction recovery rate was investigated. Figure 4a As shown, when the NH2-Fe3O4@GO dosage is 40 mg, the extraction recovery rate reaches the maximum. Excessive NH2-Fe3O4@GO may form permanent adsorption of the four analytes, thereby reducing the extraction recovery rate. Therefore, the dosage of NH2-Fe3O4@GO selected in this invention is 40 mg.

[0076] ②Influence of ultrasonic extraction time

[0077] Ultrasound helps to disperse NH2-Fe3O4@GO in water, increase the contact area and promote mass transfer. Figure 4b As shown, due to the significantly greater hydrophilicity of NH2-Fe3O4@GO than magnetic graphene, equilibrium was reached after only 5 minutes of ultrasonic extraction. However, increasing the ultrasonication time may cause the NH2-Fe3O4 to fall off the graphene oxide, resulting in incomplete recovery of the magnetic extractant and a decrease in the extraction recovery rate. Therefore, the ultrasonic extraction time selected in this invention is 5 minutes.

[0078] ③The influence of eluent type and dosage

[0079] The present invention investigated the elution effects of eluents such as acetonitrile, acetone, dichloromethane, n-hexane, and ethyl acetate. The experimental results showed that acetonitrile was the most effective. The effect of acetonitrile dosage on extraction recovery was then investigated. The results showed that eluting twice with 3 mL of acetonitrile initially resulted in better extraction efficiency.

[0080] ④Influence of solution pH

[0081] The pH value of the sample will affect the existence form of the target in water. This paper investigated the extraction recovery rate of 11 fungicides by NH2-Fe3O4@GO under the condition of sample solution pH=3-11. The results are as follows: Figure 4b As shown, the recovery rates of the 11 fungicides reached their maximum when the water sample had a pH of 8. At a pH of 3, some NH2-Fe3O4 hydrolyzed, preventing magnetic recovery of the graphene oxide adsorbed with the target species. Neutral substances such as esters, procymidone, and chlorothalonil maintained electrical neutrality across the entire pH range. When the pH was < 9, the π-π and hydrophobic interactions between them and NH2-Fe3O4@GO were unaffected, and the extraction recoveries remained largely unchanged. On the other hand, amines, being weakly alkaline, ionized more rapidly as the pH decreased, weakening the hydrophobic interactions between them and NH2-Fe3O4@GO, leading to a decrease in the extraction recoveries. The pH of the extraction sample solution was ultimately determined to be 9.

[0082] 4) Stability of extraction materials

[0083] In order to investigate the reuse times of the adsorbent, NH2-Fe3O4@GO was washed with soaking solution after desorption and then reused in magnetic solid phase extraction process. The experiment investigated the changes in the extraction recovery rate of 40mg NH2-Fe3O4@GO after repeated use for 5 times. Figure 4c As shown in the figure: After 5 cycles of use, although the extraction recovery rate decreased slightly, it was still above 81%. Therefore, NH2-Fe3O4@GO has certain reusability. The results show that NH2-Fe3O4@GO nanomaterials have good stability and can be used repeatedly.

[0084] 5) Methodological evaluation

[0085] ① Under the optimal extraction conditions, the linear range, detection limit, and quantification limit of this method were evaluated. The results are shown in Table 2 below: the linear range, detection limit, and quantification limit of this method were good when the concentrations of chlorothalonil, fluoxastrobin, and azoxystrobin were 50-4000 μg / L, and the concentrations of the other eight fungicides were 25-2000 μg / L. The detection limit (S / N=3) was 0.5-3.5 μg / Kg, and the quantification limit was between 3.0-10.5 μg / Kg. The detection limit and quantification limit were calculated as 3 times and 10 times the signal-to-noise ratio, respectively.

[0086]

[0087] Table 2 Regression equation, correlation coefficient, linear range, detection limit and quantification limit of 11 fungicides

[0088] ②Actual sample detection and spiked recovery

[0089] 30 samples of fruits including apples, mangoes, peaches, bayberries, pears, oranges and watermelons were collected from different farmers' markets. After the skin of the fruits was peeled and the juice was squeezed, the 30 samples were processed using the established method under the same experimental conditions. Among them, 2 samples contained chlorothalonil with concentrations of 11.6μg / Kg and 25.5μg / Kg; 2 samples contained chlorpyrifos with concentrations of 6.30μg / Kg and 12.2μg / Kg respectively; 1 sample contained fluazifop-butyl with a concentration of 12.3μg / Kg, all of which were within the limit standards.

[0090] Watermelon and orange, matrix blanks, were selected for sample spike recovery experiments. The spike concentrations were 5.0 μg / Kg, 50.0 μg / Kg, and 200.0 μg / Kg, respectively. Each concentration was measured six times in parallel. The results are shown in Table 3. The experimental results showed that the spike recovery rates of the detected samples were between 75.3% and 103.9%, and the relative standard deviations were between 1.89% and 4.78%.

[0091]

[0092] Table 3 Sample spike recovery and relative standard deviation of 11 fungicides (n=6)

[0093] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be encompassed therein. Any reference in a claim should not be construed as limiting the claim to which it relates.

[0094] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. The method for determining fungicide residues in fruit juice using the MSPE system coupled with GC-MS / MS comprises the following steps: 1) Preparation of magnetic solid phase extraction rod 2) Synthesis of NH2-Fe3O4@GO magnetic material powder; 3) Sample collection and processing; 4) GC-MS / MS determination; 5) Calculation of results; Its characteristics are: The fungicides include: pentachloronitrobenzene, chlorothalonil, procymidone, picoxystrobin, E-benzamide, kresoxim-methyl, pyrimidifen, trifloxystrobin, kresoxim-methyl, fluazifop-methyl, and azoxystrobin; The synthesis of NH2-Fe3O4@GO magnetic material powder in step 2) specifically includes the following steps: ① Preparation of graphene oxide dispersion: Graphene oxide (GO) was prepared by the improved Hummers method. The prepared graphene oxide was dried and then ultrasonicated in deionized water for 1 h to obtain a graphene oxide dispersion. ② Preparation of NH2-Fe3O4 dispersion: Weigh 100 mg of Fe3O4 nanoparticles and disperse them in 20 mL of ethanol by ultrasonication for 10 min. Add 80 mL of ethanol-water (v / v, 1:1) and continue ultrasonication for 20 min. Transfer the system to a 250 mL flask, then add 1 mL of APTES and stir at 120°C for 7 h. After the reaction, separate the reaction products by magnetic separation and wash them four times with ethanol. Freeze-dry the washed NH2-Fe3O4 composite nanoparticles in a vacuum oven and dissolve the NH2-Fe3O4 particles in a pH 9 PBS buffer solution by ultrasonication to obtain a 10% NH2-Fe3O4 dispersion. ③ Preparation of magnetic graphene oxide powder: 20 mL of 10% NH2-Fe3O4 dispersion prepared in step ② was added to 20 mL of graphene oxide dispersion prepared in step ①. After vigorous stirring for 5 h, a magnetic NH2-Fe3O4@GO dispersion was obtained. The solid-liquid separation was performed using a strong magnet, and the solid phase was vacuum dried at 120°C to obtain NH2-Fe3O4@GO magnetic material powder; The step 3) sample collection and processing specifically includes the following steps: ① Collect fruit samples from different areas, with each sample weighing no less than 1000 g. Weigh and mix 500 g of the fruit sample and squeeze the juice with a juicer for later use. Store in a refrigerator at -20°C. ② Accurately transfer 10 g of juice sample to a 50 mL centrifuge tube. After refrigerated centrifugation, accurately weigh 5.0 g of the supernatant into a 25 mL sample tube and dilute to 10 mL with pure water. Add 40 mg of NH2-Fe3O4@GO magnetic material powder and ultrasonically disperse for 10 minutes. Insert a magnetic solid-phase extraction rod and stir thoroughly to adsorb the NH2-Fe3O4@GO magnetic material powder, which is the target substance for adsorbing the fungicide residue, onto the surface of the magnetic solid-phase extraction rod. ③ Remove the stirring bar and place it in a 10 mL elution bottle containing 3 mL of acetonitrile. Remove the strong magnet from the hollow glass tube of the magnetic solid phase extraction rod, cover with a frosted glass cover and sonicate for 5 minutes. Then insert the strong magnet into the hollow glass tube. Transfer the eluate to a 15 mL nitrogen blowpipe, add 3 mL of eluate and repeat the above steps. Concentrate the collected eluate with nitrogen blowpipe, then make up to volume with 0.5 mL of acetone. Pass it through a 0.22 μm PTFE filter membrane for GC-MS / MS analysis.

2. The method for determining fungicide residues in fruit juice by combining the MSPE system according to claim 1 with GC-MS / MS, characterized in that: The magnetic solid-phase extraction rod in step 1) includes a frosted glass cover, a hollow glass tube, and a strong magnet, wherein the hollow glass tube is closed at the bottom and has an opening at the top. The frosted glass cover is mounted on the top opening of the hollow glass tube, and a circular hole with the same diameter as the opening is provided in the center thereof. A cylindrical strong magnet is inserted through the circular hole and is vertically arranged inside the hollow glass tube.

3. The method for determining fungicide residues in fruit juice by combining the MSPE system according to claim 1 with GC-MS / MS, characterized in that: The gas phase conditions in the step 4) GC-MS / MS determination are as follows: injection port 300°C, splitless high-pressure injection, injection time 1 min, flow control mode is linear velocity, linear velocity is 40.2 cm / sec, column flow rate is 1.2 mL / min; Rtx-5MS chromatographic column is maintained at 70°C for 1 min, increased to 280°C at 15°C / min and maintained for 2 min, then increased to 300°C at 30°C / min and maintained for 10 min, and the injection volume is 1 μL.

4. The method for determining fungicide residues in fruit juice by combining the MSPE system according to claim 1 with GC-MS / MS, characterized in that: The mass spectrometry conditions in the step 4) GC-MS / MS determination are specifically as follows: EI ion source 230° C., interface temperature 280° C., solvent delay 3 min, detector voltage 0.6 kV relative to the tuning voltage, and time-segmented multi-reaction selective monitoring mode.

5. The method for determining fungicide residues in fruit juice by combining the MSPE system according to claim 1 with GC-MS / MS, characterized in that: The formula for calculating the fungicide content C in the sample in the step 5) is: Where C0 is the relevant concentration of the fungicide in the sample after concentration calculated on the standard curve in μg / L; v is the volume of the sample after concentration in mL; and m is the mass of the juice in g.

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