Solid-phase microextraction derived fiber head as well as preparation method and application thereof

Abstract

The invention discloses a solid-phase microextraction derived fiber head and a preparation method and application thereof, the solid-phase microextraction derived fiber head comprises a solid-phase matrix and polystyrene microspheres adhered to the surface of the solid-phase matrix, and a chitosan cross-linked network layer is also cross-linked on the polystyrene microspheres; cysteine is also adsorbed on the polypropylene microsphere and chitosan cross-linked network layer. According to the solid-phase microextraction derivative fiber head, a large number of hydrophilic action sites such as amino groups and carboxyl groups exist on the surface, so that synchronous extraction and detection of the contents of three target analytes, namely cysteine-chlorothalonil association compounds, sulfydryl chlorothalonil and hydroxychlorothalonil, are realized.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis, and relates to a solid-phase microextraction-derived fiber head, its preparation method and application. Specifically, it relates to a solid-phase microextraction-derived fiber head, its preparation method and its application in detecting the content of chlorothalonil and its two metabolites (hydroxychlorothalonil and mercaptochlorothalonil) in plants. Background Technology

[0002] Pesticides play a crucial role in ensuring agricultural yields. Chlorothalonil, a broad-spectrum fungicide, is widely used both domestically and internationally. In my country, over 30 crop varieties are officially registered with chlorothalonil. However, in actual production, the misuse or improper application of chlorothalonil frequently occurs, easily leading to residues of chlorothalonil and its metabolites in food, posing a potential threat to human health. The main intermediate degradation products of chlorothalonil in vegetables are hydroxychlorothalonil and mercaptochlorothalonil. Because the toxicity of mercaptochlorothalonil is preliminarily estimated to be higher than that of its parent compound, hydroxychlorothalonil (for example, its acute toxicity to mammals is 30 times that of its parent compound) has attracted widespread attention. Developing detection methods for chlorothalonil and its metabolites in vegetables is of paramount importance for ensuring food safety for consumers. Because chlorothalonil is significantly less polar than its metabolites thiol chlorothalonil and hydroxychlorothalonil, the main detection method for chlorothalonil is gas chromatography-mass spectrometry (GC-MS). However, thiol chlorothalonil and hydroxychlorothalonil, due to their higher boiling points, are not suitable for GC and can only be detected by liquid chromatography (LC) or LC-MS. Therefore, both domestically and internationally, different instrumental methods are used to detect chlorothalonil and its metabolites separately. This seriously affects the efficiency of detection and analysis, is not conducive to the daily monitoring of large batches of samples, and separate detection and quantification can easily increase analytical errors.

[0003] Furthermore, the difficulty in detecting chlorothalonil and its metabolites lies in the instability of chlorothalonil, which readily reacts chemically with endogenous substances in vegetable matrices. For example, sulfur-rich vegetables such as cabbage, onions, leeks, and shiitake mushrooms release large amounts of sulfur-containing volatile organic compounds, including isothiocyanates and polysulfides, after homogenization. These sulfur-containing substances possess strong nucleophilic substitution and redox reactivity, and can react instantly with chlorothalonil. Traditional methods for detecting pesticides and their metabolites in vegetables include vegetable tissue homogenization, solution extraction, and purification. However, chlorothalonil readily reacts with chemically active components in vegetable homogenates during sample homogenization and solution extraction pretreatment, leading to degradation and making the chlorothalonil parent compound "inaccurately detectable or undetectable."

[0004] In vivo solid-phase microextraction (SPE) is a technique that utilizes micro-solid-phase extraction fibers to achieve non-destructive or minimally destructive sampling, avoiding the metabolic transformation of target analytes during pretreatment processes such as homogenization and extraction of in vitro samples. However, currently available commercially available SPE probes are not suitable for the simultaneous extraction of chlorothalonil and its metabolites, which have significantly different polarities. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a solid-phase microextraction-derived fiber head, which is applied to the non-destructive (no homogenization pretreatment required) extraction of chlorothalonil from plants (such as vegetables), and simultaneously extracts thiophanate-methyl and hydroxychlorothalonil. Furthermore, high-performance liquid chromatography-mass spectrometry is used to construct a method for the simultaneous detection of chlorothalonil, thiophanate-methyl, and hydroxychlorothalonil in plants.

[0006] The technical solution of the present invention is as follows:

[0007] A solid-phase microextraction-derived fiber head comprises a solid matrix and polystyrene microspheres adhered to the surface of the solid matrix, wherein the polystyrene microspheres are further cross-linked with a chitosan cross-linked network layer.

[0008] Furthermore, cysteine ​​is also adsorbed on the polypropylene microspheres and chitosan cross-linked network layer.

[0009] According to an embodiment of the present invention, the solid matrix includes at least one of stainless steel wire, fiberglass wire, glass capillary tube, etc.

[0010] According to an embodiment of the present invention, the polystyrene microspheres are physically adsorbed onto the surface of a solid matrix by an adhesive. The adhesive may be polyacrylonitrile. Preferably, the polyacrylonitrile has a weight-average molecular weight of 50,000-300,000, and more preferably 100,000-200,000.

[0011] According to an embodiment of the present invention, the polystyrene microspheres have a particle size of 50-500 nm, preferably 80-300 nm.

[0012] According to an embodiment of the present invention, the chitosan may be carboxymethyl chitosan.

[0013] According to an embodiment of the present invention, the chitosan crosslinked network layer is a crosslinked product made from carboxymethyl chitosan catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl).

[0014] According to an embodiment of the present invention, the cysteine ​​is physically adsorbed on the polypropylene microspheres and the chitosan cross-linked network layer.

[0015] This invention also provides a method for preparing the above-mentioned solid-phase microextraction-derived fiber head, the method comprising:

[0016] (1) Mix the adhesive, polystyrene microspheres and DMF to obtain a mixture;

[0017] (2) Mix carboxymethyl chitosan solution, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with the mixture in step (1) to prepare intermediate product 1;

[0018] (3) Impregnate the solid matrix in the intermediate product 1 of step (2) to obtain intermediate product 2;

[0019] (4) Immerse the intermediate product 2 from step (3) in a cysteine ​​solution to obtain the solid-phase microextraction-derived fiber head.

[0020] According to an embodiment of the present invention, in step (1), the mass ratio of the adhesive to the polystyrene microspheres is 1:1-6, preferably 1:1-4.

[0021] According to an embodiment of the present invention, in step (1), the content of DMF is not particularly limited, as long as the raw materials are partially or completely dissolved. The mixing temperature and time are not particularly limited, as long as the raw materials are partially or completely dissolved. For example, ultrasonic mixing can be used, for example, for 0.1-24 hours.

[0022] According to an embodiment of the present invention, step (1) specifically involves: first mixing the adhesive with DMF, and then adding polystyrene microspheres for mixing.

[0023] According to an embodiment of the present invention, in step (2), the carboxymethyl chitosan solution is an aqueous solution of carboxymethyl chitosan, and the concentration of the carboxymethyl chitosan solution is 5-20 mg / mL.

[0024] According to an embodiment of the present invention, in step (2), the mass ratio of carboxymethyl chitosan, catalyst and polystyrene microspheres is 1:0.01-0.2:1-6, preferably 1:0.05-0.1:1-4.

[0025] According to an embodiment of the present invention, in step (3), before impregnating the solid matrix, the surface of the solid matrix can be cleaned to remove impurities; for example, at least one of water, methanol and acetone can be used for cleaning.

[0026] According to an embodiment of the present invention, step (3) further includes a post-processing step, which involves drying the product, for example, at 40-70°C for 4-24 hours.

[0027] According to an embodiment of the present invention, in step (4), the concentration of the cysteine ​​solution is 50-200 mg / L, preferably 80-150 mg / L, for example 100.0 mg / L.

[0028] According to an embodiment of the present invention, step (4) further includes a post-processing step, in which the product is washed with water and then dried. Washing with water is used to remove unadsorbed free cysteine ​​from the product.

[0029] The present invention also provides a solid-phase microextraction device containing the above-mentioned solid-phase microextraction-derived fiber head.

[0030] The present invention also provides a method for extraction, separation, purification or detection, the method comprising using the above-described solid-phase microextraction derivatized fiber head or the solid-phase microextraction device.

[0031] According to an embodiment of the present invention, the method further includes the use of liquid chromatography or a liquid chromatography-tandem mass spectrometry device.

[0032] The present invention also provides an application of the above-mentioned solid-phase microextraction derived fiber head or the above-mentioned solid-phase microextraction device in the enrichment, extraction, separation, purification or detection of the content of chlorothalonil and its metabolites (including thiophanate-methyl and hydroxychlorothalonil) in plants.

[0033] According to an embodiment of the present invention, the plant is sampled non-destructively, i.e., no plant tissue sectioning is required.

[0034] This invention also provides a method for detecting the content of chlorothalonil and its metabolites in plants, the method comprising:

[0035] The solid-phase microextraction-derived fiber head was inserted into the plant. After extraction, the fiber head was removed and immersed in methanol. The contents of chlorothalonil and its metabolites were then detected by liquid chromatography-tandem mass spectrometry.

[0036] According to an embodiment of the present invention, the solid-phase microextraction-derived fiber head is inserted into the plant to a depth of 2-4 mm, for example, 3 mm.

[0037] According to an embodiment of the present invention, the extraction time is 15-40 min.

[0038] According to an embodiment of the present invention, the soaking time in methanol is 5-60 minutes.

[0039] As an exemplary embodiment of the present invention, the detection method is specifically as follows: the above-mentioned solid-phase microextraction-derived fiber head is inserted into plant tissue (stem or leaf) to a depth of 3 mm, extracted for 20 minutes, the solid-phase microextraction fiber head is removed, and it is immersed in a methanol solution for elution for 30 minutes. Finally, the content of chlorothalonil and its metabolites is detected by liquid chromatography-tandem mass spectrometry.

[0040] The beneficial effects of this invention are:

[0041] 1. The solid-phase microextraction derivatization fiber head of the present invention derivatizes chlorothalonil through a nucleophilic substitution reaction between cysteine ​​and chlorothalonil to prepare cysteine-chlorothalonil complex. The polarity of the derivatized product (i.e., cysteine-chlorothalonil complex) increases, which lays the foundation for subsequent simultaneous detection and analysis using high performance liquid chromatography-mass spectrometry.

[0042] 2. The solid-phase microextraction-derived fiber head of the present invention has a large number of hydrophilic interaction sites such as amino and carboxyl groups on its surface, which enables the simultaneous extraction and detection of the contents of three target analytes: cysteine-chlorothalonil complex, thiochlorothalonil, and hydroxychlorothalonil. Attached Figure Description

[0043] Figure 1 The concentrations of chlorothalonil and its metabolites were determined on the 7th day after spraying chlorothalonil onto rapeseed using the solid-phase microextraction-derived fiber head, liquid nitrogen cryogenic homogenization, and conventional room temperature homogenization methods described in Example 1. Detailed Implementation

[0044] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0045] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0046] Example 1

[0047] (1) Synthetic solid-phase microextraction-derived fiber heads

[0048] a. Cut a 3mm diameter stainless steel wire into 5cm long segments and clean them ultrasonically with pure water, methanol and acetone in sequence.

[0049] b. Dissolve 150 mg of polyacrylonitrile (PAN, weight average molecular weight of 100,000) in 2 g of anhydrous DMF and disperse by ultrasonication for 1 h to prepare a uniform yellow gel-like liquid.

[0050] c. Add 200 mg of polystyrene (PS, particle size 100 nm) microspheres to the above gel solution and sonicate for 1 hour to form a uniform slurry.

[0051] d. Dissolve 200mg of carboxymethyl chitosan in 20ml of pure water. Just before soaking, add 20mg of catalyst (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and mix well.

[0052] e. Insert the treated stainless steel wire into the above solution to a depth of 1 cm and let it stand for 1 minute. Gently remove it to allow the adsorption filler to adhere evenly to the surface of the stainless steel wire. Repeat the insertion-removal operation 3-5 times to ensure that a sufficient amount of adsorption filler adheres to the surface of the stainless steel wire. Place the stainless steel wire in an oven at 50°C for 12 hours.

[0053] Prepare a cysteine ​​solution (100.0 mg / L), immerse the cross-linked chitosan-modified solid-phase microextraction probe in the cysteine ​​solution, let it stand for 24 hours, and then immerse it in pure water for 24 hours to wash away the unadsorbed free cysteine ​​on the surface of the solid-phase microextraction probe.

[0054] f. Place the prepared solid-phase microextraction-derived fiber head into an oven and dry it overnight at 50°C. Remove it for later use.

[0055] (2) Nucleophilic substitution reaction of chlorothalonil and cysteine

[0056] A nucleophilic substitution reaction occurs between cysteine ​​and chlorothalonil to prepare chlorothalonil derivatives (i.e., cysteine-chlorothalonil complexes). The chemical reaction principle is as follows: chlorothalonil derivatization chemical reaction.

[0057]

[0058] To verify the structure of the reaction product, the derivative (cysteine-chlorothalonil compound) was characterized by high-resolution mass spectrometry. The precise molecular weight information and deviations of the parent ion and secondary fragment ions of the derivative are shown in Table 1.

[0059] (3) Quantitative detection of chlorothalonil, mercaptochlorothalonil and hydroxychlorothalonil

[0060] The instrument conditions for liquid chromatography-tandem triple quadrupole mass spectrometry were as follows: column: Agilent XDB C18 2.1×150mm, 3.5μm; injection volume: 5μL; mobile phase: A was 5mM ammonium formate aqueous solution, B was 5mM ammonium formate methanol; scanning mode: negative ion scanning; detection mode: multiple reaction monitoring; electrospray voltage: -4500V; nebulizer gas pressure, curtain gas pressure, and auxiliary heating gas pressure were 60psi, 30psi, and 60psi, respectively; ion source temperature: 550℃. The multiple reaction monitoring parameters for cysteine-chlorothalonil complex, mercaptochlorothalonil, and hydroxychlorothalonil are shown in Tables 1 and 2. Table 1 shows that the precise molecular weight deviations of the deprotonated ion and secondary fragment ions of the cysteine-chlorothalonil complex structure are all less than 2.0ppm, meeting the requirements of the Organization for the Prohibition of Chemical Weapons (OPCW) for inferring compound structures (precise molecular weight deviation less than 2.5ppm).

[0061] The solid-phase microextraction (SPE) methodology validation process references (Journal of Hazardous Materials, 2020, 399, 123013; Analytical Chemistry, 2016, 88, 5841-5848). The chlorothalonil content can be indirectly determined by measuring the content of cysteine-chlorothalonil conjugates. The derivatization detection technique employed is a known technique in the field. The quantitative analytical methodology validation results show that the linear detection ranges for chlorothalonil, thiochlorothalonil, and hydroxychlorothalonil are 20 μg / kg-500 μg / kg, 20 μg / kg-500 μg / kg, and 10 μg / kg-200 μg / kg, respectively. The limits of detection (LODs) for the three targets are: chlorothalonil 5 μg / kg, thiochlorothalonil 5 μg / kg, and hydroxychlorothalonil 2 μg / kg. The recoveries of chlorothalonil, mercaptochlorothalonil, and hydroxychlorothalonil (all at a concentration of 100 μg / L) were 70.6%, 84.6%, and 96.2%, respectively.

[0062] Table 1. Molecular formula of cysteine-chlorothalonil compound and precise molecular weight deviation of deprotonated ions and secondary fragment ions.

[0063]

[0064] Table 2. Multiple reaction monitoring parameters for the detection and analysis of cysteine-chlorothalonil compound, mercaptochlorothalonil, and hydroxychlorothalonil by high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0065]

[0066] (4) Non-destructive sampling and analysis of chlorothalonil, thiophanate-methyl, and hydroxychlorothalonil during rapeseed cultivation.

[0067] Field trials of pesticides were conducted on rapeseed grown in the open field. When each rapeseed plant had 3-4 leaves, a suspension of 75% chlorothalonil wettable powder was sprayed at a concentration of 1732 g ai / hm². 2 (Maximum recommended concentration), apply the pesticide twice, with a 7-day interval between the first and second applications. Measure the concentrations of chlorothalonil, thiophanate-methyl, and hydroxychlorothalonil in rapeseed 7 days after the second application (safety interval).

[0068] The extraction and detection methods for chlorothalonil and its main metabolites are as follows:

[0069] The solid-phase microextraction (SPME) derivatization fiber head from Example 1 was inserted into a rapeseed stem sample and allowed to stand for 30 minutes. After the derivatization and adsorption processes were completed, the SPME derivatization fiber head was removed from the vegetable tissue. 0.5 mL of methanol was added to a 1.5 mL sample vial as the elution solvent, and the SPME derivatization fiber head was immersed in the methanol for elution for 30 minutes. The analysis was then performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0070] Meanwhile, to ensure the accuracy of the solid-phase microextraction-derived fiber head test results, rapeseed stem samples were collected. In vitro sample pretreatment was performed using both liquid nitrogen cryogenic homogenization (a traditional method to avoid degradation of active ingredients during homogenization) and conventional room temperature homogenization, and control experiments were conducted. Liquid nitrogen cryogenic homogenization is a traditional method to avoid degradation of active ingredients during homogenization and is widely recognized both domestically and internationally; therefore, it was used as the reference method. For specific liquid nitrogen cryogenic homogenization or conventional room temperature homogenization methods, please refer to Analytical Biochemistry 347 (2005) 234-243.

[0071] The results of the three different sample pretreatment methods are shown in the figure. Figure 1 ,from Figure 1 As can be seen, the results of in vivo non-destructive sampling (i.e., solid-phase microextraction) and liquid nitrogen cryogenic homogenization for determining chlorothalonil and thiochlorothalonil are basically consistent, but significantly different from traditional room temperature homogenization. The traditional room temperature homogenization method shows a significantly lower chlorothalonil content and a significantly higher thiochlorothalonil content. This is because the plant tissue homogenate of rapeseed at room temperature is broken down, releasing endogenous myrosinase, which further activates glucosinolates to hydrolyze into a series of sulfur-containing substances. These substances react chemically with chlorothalonil to generate thiochlorothalonil, resulting in a decrease in chlorothalonil content and an increase in thiochlorothalonil content. The hydroxychlorothalonil content measured by the three sample pretreatment methods is basically consistent because chlorothalonil is not significantly converted to hydroxychlorothalonil during the traditional sample pretreatment process. These results indicate that the solid-phase microextraction-derived fiber head and the extraction and testing methods of this invention are significant for improving the accuracy and efficiency of the detection and analysis of chlorothalonil and its metabolites in vegetables.

[0072] The embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A solid-phase microextraction-derived fiber head, characterized in that, It comprises a solid matrix and polystyrene microspheres adhered to the surface of the solid matrix, wherein the polystyrene microspheres are also cross-linked with a chitosan cross-linked network layer; Furthermore, cysteine ​​is also adsorbed on the polypropylene microspheres and chitosan cross-linked network layer.

2. The fiber head according to claim 1, characterized in that, The solid matrix includes at least one of stainless steel wire, fiberglass wire, and glass capillary. Preferably, the polystyrene microspheres are physically adsorbed onto the surface of a solid matrix using an adhesive. Preferably, the adhesive is polyacrylonitrile.

3. The fiber head according to claim 1 or 2, characterized in that, The chitosan is carboxymethyl chitosan. Preferably, the chitosan crosslinked network layer is a crosslinked product made from carboxymethyl chitosan catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Preferably, the cysteine ​​is physically adsorbed onto the polypropylene microspheres and the chitosan cross-linked network layer.

4. The method for preparing the solid-phase microextraction-derived fiber head according to any one of claims 1-3, characterized in that, The method includes: (1) Mix the adhesive, polystyrene microspheres and DMF to obtain a mixture; (2) Mix carboxymethyl chitosan solution, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with the mixture in step (1) to prepare intermediate product 1; (3) Impregnate the solid matrix in the intermediate product 1 of step (2) to obtain intermediate product 2; (4) Immerse the intermediate product 2 from step (3) in a cysteine ​​solution to obtain the solid-phase microextraction-derived fiber head.

5. The method according to claim 4, characterized in that, In step (1), the mass ratio of the adhesive to the polystyrene microspheres is 1:1-6. Preferably, in step (2), the carboxymethyl chitosan solution is an aqueous solution of carboxymethyl chitosan, and the concentration of the carboxymethyl chitosan solution is 5-20 mg / mL. Preferably, in step (2), the mass ratio of carboxymethyl chitosan, catalyst and polystyrene microspheres is 1:0.01-0.2:1-6.

6. A solid-phase microextraction device, characterized in that, It contains the solid-phase microextraction-derived fiber head as described in any one of claims 1-3.

7. A method for extraction, separation, purification, or detection, characterized in that, The method includes using a solid-phase microextraction-derived fiber head as described in any one of claims 1-3 or a solid-phase microextraction device as described in claim 6. Preferably, the method further includes the use of liquid chromatography or liquid chromatography-tandem mass spectrometry.

8. The application of the solid-phase microextraction-derived fiber head according to any one of claims 1-3 or the solid-phase microextraction device according to claim 6 in the enrichment, extraction, separation, purification or detection of chlorothalonil and its metabolites in plants. Preferably, the metabolite includes at least one of thiophanate-methyl and hydroxychlorothalonil.

9. The application according to claim 8, characterized in that, The sampling of the plant is non-destructive, meaning that there is no need to use plant tissue sections.

10. A method for detecting the content of chlorothalonil and its metabolites in plants, characterized in that, The method is as follows: The solid-phase microextraction-derived fiber head according to any one of claims 3 is inserted into the plant. After extraction, the solid-phase microextraction-derived fiber head is removed and soaked in methanol. The content of chlorothalonil and its metabolites is then detected by liquid chromatography-tandem mass spectrometry.

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