Method for determining residual quantity of abamectin in fruits and vegetables by using MSPD-HPLC (Mass Spectrometry-High Performance Liquid Chromatography) combined

Abstract

The invention discloses a method for measuring abamectin residual quantity in fruits and vegetables by using an MSPD-HPLC (Mass Spectrometry-High Performance Liquid Chromatography) coupling technique, which comprises the following steps: (1) putting a fruit and vegetable sample to be measured into a centrifugal tube, adding acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane chemically bonded silica, uniformly mixing, and then centrifugally separating to obtain supernate; (2) blow-drying the supernate with protective gas, then adding methanol for redissolving, uniformly mixing, and passing through a membrane to prepare a detection sample; and (3) testing the detection sample by adopting a high performance liquid chromatography (HPLC) technology, and determining the residual amount of abamectin in the fruits and vegetables. According to the method, on the basis of reference to abamectin residue determination-liquid chromatography in fruits and vegetables, the pretreatment process is optimized, acetonitrile extraction and octadecylsilane chemically bonded silica are adopted as adsorbents for matrix dispersive solid-phase extraction, and through optimization of extraction conditions and combination of high performance chromatography separation analysis, the content of abamectin residues in fruits and vegetables is determined. And an efficient, green and high-sensitivity method for measuring the residual quantity of abamectin is established.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide residue detection, and in particular to a method for determining avermectin residues in fruits and vegetables using a MSPD-HPLC coupling technique. Background Art

[0002] Abamectin is a group of macrolide antibiotics produced by actinomycetes. It is a mixture of 16-membered macrolides composed of eight structurally similar homologues. It is an insecticide and acaricide widely used on crops such as vegetables, fruit trees, and cotton. It is a highly toxic pesticide. The main residue of the avermectin pesticide is B1a (structure shown in the figure below). Small amounts of pesticide residue will not cause acute poisoning in humans, but long-term consumption of foods containing excessive avermectin levels can have certain impacts on human health. The "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" (GB2763-2021) clearly stipulates the maximum residue limit of avermectin in fruits and vegetables, not exceeding 0.1 mg / kg. Traditional detection methods use sample pretreatment techniques with disadvantages such as low extraction efficiency, high organic solvent consumption, and cumbersome operation.

[0003] Summary of the Invention

[0004] To this end, the present invention provides a method for determining the amount of avermectin residues in fruits and vegetables using a MSPD-HPLC coupling technique, the steps comprising: (1) Place the fruit and vegetable sample to be tested in a centrifuge tube, add acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane bonded silica gel, mix well, and then centrifuge to obtain a supernatant; (2) drying the supernatant with protective gas, then adding methanol to dissolve it, mixing it evenly, and filtering it to obtain a test sample; (3) High performance liquid chromatography (HPLC) was used to test the test samples and determine the avermectin residues in the fruits and vegetables.

[0005] Furthermore, in step (1), the mixing ratio of the fruit and vegetable sample to be tested, acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane bonded silica gel is as follows: fruit and vegetable sample to be tested: acetonitrile: anhydrous magnesium sulfate: sodium chloride: graphitized carbon black: octadecylsilane bonded silica gel = 10 g: 10 mL: 150 mg: 3 g: 5 mg: 40-80 mg.

[0006] Furthermore, the mixing ratio of the fruit and vegetable sample to be tested, acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane bonded silica gel is: fruit and vegetable sample to be tested: acetonitrile: anhydrous magnesium sulfate: sodium chloride: graphitized carbon black: octadecylsilane bonded silica gel = 10 g: 10 mL: 150 mg: 3 g: 5 mg: 60 mg.

[0007] Furthermore, the protective gas is nitrogen; after each 2 mL of the supernatant is dried, 1 mL of the methanol is used to redissolve the dried product.

[0008] Furthermore, the chromatographic conditions are as follows: chromatographic column: InertSustain C18 column (4.6 mm × 250 mm, 5 μm); temperature: 40°C; mobile phase: methanol-water solution (90:10, v / v); flow rate: 1 mL / min; injection volume: 20 μL; detection wavelength: 245 nm.

[0009] The beneficial effects of the present invention are as follows: based on the reference to the determination of avermectin residues in fruits and vegetables - liquid chromatography (GB23200.19-2016), the present invention optimizes the pre-treatment process, and proposes to use acetonitrile extraction, octadecylsilane bonded silica gel (C 18 ) as an adsorbent for matrix-dispersed solid-phase extraction. By optimizing extraction conditions and combining them with high-performance chromatographic separation and analysis, an efficient, green, and highly sensitive method for the determination of avermectin residues has been established. Using MSPD as a sample pretreatment step for the HPLC-UV determination of avermectins in fruits and vegetables significantly shortens pretreatment time and steps, reduces extraction solvents, and saves money compared to the existing national standard liquid chromatography method. Furthermore, this method offers high recovery and excellent reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 (A) Effect of adsorbent dosage on extraction efficiency; (B) C 18 and PSA as adsorbents on the extraction effect (purple line: PSA; black line: C 18 )); Figure 2 (A) Blank matrix spectrum; (B) Blank matrix spiked spectrum; (C) Actual sample measurement spectrum; Figure 3 Comparison of chromatograms between MSPD treatment and national standard method; (blue line: result of pretreatment according to GB23200.19-2016; purple line: 5μg / mL standard solution; black line: result of pretreatment according to MSPD) DETAILED DESCRIPTION

[0011] The present invention will be further described below with reference to the embodiments.

[0012] Example A method for determining the amount of avermectin residues in fruits and vegetables using MSPD-HPLC is described. The method comprises the following steps: accurately weighing 10.00 g of fruit and vegetable sample and placing it in a 50 mL conical centrifuge tube; adding 10.00 mL of acetonitrile, 150 mg of anhydrous magnesium sulfate, 3.0 g of NaCl, 5 mg of graphitized carbon black, and 60 mg of octadecylsilane bonded silica gel C; and 18 The supernatant was collected by vortexing for 1 minute and centrifuging for 5 minutes. 2.00 mL of the supernatant was placed in a 10 mL stoppered graduated tube and purged with nitrogen until nearly dry. 1.00 mL of methanol was added for reconstitution, vortexing for 1 minute, and filtration was performed to obtain the test sample. The test sample was analyzed using high-performance liquid chromatography (HPLC) to determine the residual avermectin in fruits and vegetables. The chromatographic conditions were as follows: InertSustain C18 column (4.6 mm × 250 mm, 5 μm); temperature: 40°C; mobile phase: methanol-water solution (90:10, v / v); flow rate: 1 mL / min; injection volume: 20 μL; detection wavelength: 245 nm.

[0013] The models of the testing instruments are: LC-20AT high-performance liquid chromatograph (with SPD-M20A ultraviolet detector, Shimadzu Corporation, Japan), TG16-WS centrifuge (Hunan Xiangyi Centrifuge Instrument Co., Ltd.), mixplus vortex mixer (Hefei Ebenson Scientific Instrument Co., Ltd.), and MD-200 nitrogen blower (Hangzhou Aosheng Instrument Co., Ltd.).

[0014] The source of reagents or samples is: the concentration of avermectin standard stock solution is , purchased from Shanghai Anpu Laboratory Technology Co., Ltd. Octadecyl bonded silica gel (C 18 ), ethylenediamine-N-propylsilane silica gel (PSA), all chromatographically pure, with particle sizes of 40–60 μm, were purchased from Shanghai Anpu Laboratory Technology Co., Ltd. Graphitized carbon black (GCB) (particle sizes of 40–120 μm) was purchased from Shanghai Xinhu Laboratory Equipment Co., Ltd. Methanol and acetonitrile were chromatographically pure, and all other reagents were analytically pure and purchased from Sinopharm Group. Laboratory water was obtained from a Milli-Q water system.

[0015] The standard stock solution was diluted with methanol to obtain a series of standard solutions. Both the standard stock solution and the standard solution were stored in a refrigerator at 4°C.

[0016] Sample: Cabbage samples purchased from Dongzhiyuan Supermarket near Hunan Agricultural University.

[0017] Result analysis: 1. Optimization of matrix dispersed solid phase extraction adsorbent Matrix dispersion solid phase extraction combines conventional solid phase dispersion technology with reversed phase bonded fillers to complete homogenization, extraction and purification in the same operation. Among them, the adsorbent plays a key role in the extraction results, and its role is to adsorb impurities in the sample. Common adsorbents include C 18 and PSA. 18 It has strong hydrophobicity and mainly adsorbs non-polar compounds through hydrophobic interactions, making it suitable for extracting non-polar substances; PSA has weak anion exchange capacity and polarity characteristics, and mainly adsorbs polar compounds through ion exchange and polar interactions, which can effectively remove polar interferences in samples. This experiment investigated different dosages of 40, 50, 60, 70, and 80 mg of C 18 The effects of and PSA as adsorbents on the extraction of avermectin are shown in Figure 1 A. As can be seen from the figure, the amount of adsorbent has a certain influence on the extraction effect. Both of them reach the best adsorption effect at 60 mg, and C 18 The effect is better than PSA.

[0018] Under the best conditions, C 18 The chromatogram obtained by using PSA as adsorbent is shown in Figure 1 B. As can be seen from the figure, using C 18 As an adsorbent, the sample recovery rate is slightly higher and the purification effect is relatively better. This is because the non-polar compounds or compounds with a certain degree of hydrophobicity in the sample can produce hydrophobic interactions with the non-polar surface of the adsorbent, thereby being adsorbed to the surface of the adsorbent. Abamectin is weakly polar and will not be adsorbed, thus achieving the purification effect. Therefore, C is selected 18 as the best adsorbent.

[0019] 2. Method linear range, detection limit, recovery and precision To evaluate the feasibility of the method, a methodological investigation was conducted to assess its linear range, accuracy, precision, and limit of detection (LOD). Spike recovery was tested using Chinese cabbage as the matrix, and the limit of detection (LOD) of avermectin residues was determined using a three-fold noise threshold. A calibration curve was constructed with the concentration of the standard solution as the abscissa and the peak area of ​​the analyte after MSPD analysis of the standard solution as the ordinate. Results demonstrated good linearity for avermectin over the concentration range of 0.20-20 μg / mL, with a correlation coefficient (r²) of 0.9999 and a LOD of 0.005 mg / kg. Matrix spike recoveries ranged from 92% to 107%. The precision of the method was evaluated using a 5.0 μg / mL standard solution and was less than 3% (n=6).

[0020] 3. Actual sample analysis The developed method was applied to the determination of avermectin in Chinese cabbage samples purchased from a nearby Toshiba Source supermarket. The relevant chromatograms are shown in Figure 2. Figure 2 shown. Figure 2 A is a blank matrix image, Figure 2 B is the chromatogram of the blank matrix spiked with 5 μg / mL standard solution. Figure 2 C is the measurement result diagram of the actual sample. It can be seen from the figure that the use of MSPD as a pretreatment method achieves effective enrichment of the analyte, and no avermectin residues are detected in the sample.

[0021] 4. Comparison with national standard law This method was further compared with the determination method of GB23200.19-2016. Figure 3 The figure is a comparison of the recovery rates of MSPD treatment and the national standard method. Table 1 is a comprehensive comparison of this method and the determination method of GB23200.19-2016. Figure 3 As shown in Table 1, compared with the method in GB23200.19-2016, this method has the advantages of fewer steps, significantly shortened pretreatment time, reduced extraction solvent consumption, and good reproducibility. The results show that the proposed MSPD-HPLC method is simple, rapid, economical, and environmentally friendly, and can be successfully used for the rapid determination of avermectins in fruits and vegetables.

[0022] Table 1 5. Conclusion This study demonstrated that the use of MSPD as a sample pretreatment step for the HPLC-UV determination of avermectins in fruits and vegetables significantly shortened pretreatment time, streamlined pretreatment steps, reduced extraction solvents, and saved costs compared to the existing national standard liquid chromatography method. Furthermore, the method achieved high recovery and good reproducibility. The results demonstrate that the proposed MSPD-HPLC method is simple and rapid, making it particularly suitable for the rapid analysis of avermectins in fruits and vegetables, and can provide a reference for monitoring avermectins in fruits and vegetables.

[0023] The technical solutions provided by the present invention are described in detail above. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for determining the amount of avermectin residues in fruits and vegetables using MSPD-HPLC technology, characterized in that the steps include: (1) Place the fruit and vegetable sample to be tested in a centrifuge tube, add acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black, and octadecylsilane bonded silica gel, mix well, and then centrifuge to obtain the supernatant; (2) drying the supernatant with protective gas, then re-dissolving it in methanol, mixing it evenly, and filtering it through a membrane to obtain a test sample; (3) High performance liquid chromatography (HPLC) was used to test the test samples and determine the avermectin residues in fruits and vegetables.

2. The method for determining the residual amount of avermectin in fruits and vegetables by using a MSPD-HPLC coupling technique according to claim 1, characterized in that: In the step (1), the mixing ratio of the fruit and vegetable sample to be tested, acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane bonded silica gel is as follows: fruit and vegetable sample to be tested: acetonitrile: anhydrous magnesium sulfate: sodium chloride: graphitized carbon black: octadecylsilane bonded silica gel = 10 g: 10 mL: 150 mg: 3 g: 5 mg: 40-80 mg.

3. The method for determining the residual amount of avermectin in fruits and vegetables by using a MSPD-HPLC coupling technique according to claim 2, characterized in that: The mixing ratio of the fruit and vegetable samples to be tested, acetonitrile, anhydrous magnesium sulfate, sodium chloride, graphitized carbon black and octadecylsilane bonded silica gel is as follows: fruit and vegetable samples to be tested: acetonitrile: anhydrous magnesium sulfate: sodium chloride: graphitized carbon black: octadecylsilane bonded silica gel = 10 g: 10 mL: 150 mg: 3 g: 5 mg: 60 mg.

4. The method for determining the residual amount of avermectin in fruits and vegetables by using a MSPD-HPLC coupling technique according to claim 1, characterized in that: The protective gas is nitrogen; after each 2 mL of the supernatant is dried, 1 mL of the methanol is used to re-dissolve the dried product.

5. The method for determining the residual amount of avermectin in fruits and vegetables by using MSPD-HPLC coupling technology according to claim 1, characterized in that: The chromatographic conditions are as follows: chromatographic column: InertSustain C18 column (4.6 mm×250 mm, 5 μm); temperature: 40°C; mobile phase: methanol-water solution (90:10, v / v); flow rate: 1 mL / min; injection volume: 20 μL; detection wavelength: 245 nm.

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