Preparation of a β-cyclodextrin polymer solid-phase extraction material and solid-phase extraction method

Abstract

The present invention belongs to the field of solid-phase extraction of trace organic pollutants in environmental samples, and specifically relates to a preparation method of a β-cyclodextrin polymer solid-phase extraction material and a solid-phase extraction method. First, cellulose-supported β-cyclodextrin polymer is prepared by a one-step crosslinking method; then, the cellulose-supported β-cyclodextrin polymer is subjected to a reduction reaction to prepare amino-functionalized cellulose-supported β-cyclodextrin polymer. The polymer prepared above is made into a solid-phase extraction column for the extraction of various trace organic pollutants in urine, so as to determine the content of organic pollutants in urine and further evaluate the situation of human exposure to pollutants. The solid-phase extraction agent material of the present invention has both a hydrophobic cavity and a charged group, which improves the selectivity of the solid-phase extraction material. The use of cellulose as a carrier improves the loading rate, overcoming the problems of slow loading rate and low recovery rate of cyclodextrin-based solid-phase extraction agents.

Description

Technical Field

[0001] The present invention belongs to the field of solid-phase extraction of trace organic pollutants in environmental samples, and particularly relates to the preparation of a β-cyclodextrin polymer solid-phase extraction material and a solid-phase extraction method. Background Art

[0002] With the rapid improvement of the production and living capabilities of human society, a large number of chemical substances are used commercially and widely present in foods and human daily consumer goods, and then released into the environment. Humans come into contact with these chemicals through skin, oral, or inhalation routes. In recent years, emerging pollutants such as plasticizers (PAEs), per- and polyfluoroalkyl substances (PFAS), pharmaceuticals and personal care products (PPCPs), etc. have been found to be related to endocrine disruption, cytotoxicity, genotoxicity, reproductive toxicity, and neurotoxicity. Therefore, the exposure of humans to emerging pollutants is evaluated by monitoring the pollutant content in human body fluids and tissues. Since urine can be obtained in large quantities through non-invasive sampling, it is currently the most common analytical matrix.

[0003] At present, solid-phase extraction is the most important pretreatment method for separating organic pollutants in urine. However, the currently widely used solid-phase extraction materials still have the disadvantages of poor repeatability, low recovery rate, and being easily affected by complex matrices. Developing a solid-phase extractant with strong selectivity and high extraction efficiency is the key to accurately understanding the concentration levels of organic pollutants in complex matrices. β-cyclodextrin is a water-soluble cyclic oligosaccharide with 7 glucose units and can be easily obtained from natural wheat, corn, and potato starches. In terms of its three-dimensional structure, β-cyclodextrin has a hydrophobic inner cavity and a hydrophilic outer surface. The abundant primary (located at C-6) and secondary hydroxyl groups (located on the C-2 and C-3 atoms of the glucose unit) on its surface edge endow β-cyclodextrin with the ability to accept various organic guests through non-covalent interactions. Coupled with the moderate flexibility of the β-cyclodextrin macrocycle and its unique three-dimensional structure, it endows the ability to distinguish molecular sizes. Since cyclodextrin itself is soluble in water, various cyclodextrin polymers are prepared by reacting cyclodextrin with flexible cross-linking agents or rigid cross-linking agents, which have advantages such as certain acid resistance and oxidation resistance. However, so far, most of the reported β-cyclodextrin polymers in the literature are used for the adsorption and removal of pollutants. Although the regeneration performance is also mentioned, it is all for substances with strong hydrophobicity and no charge. The desorption of charged substances is still an urgent problem to be solved. At the same time, due to the strong hydrophobicity of β-cyclodextrin polymers, the loading speed is greatly limited during the pretreatment process, which is also one of the factors restricting the wide use of this material. In addition, the process is cumbersome during the material preparation stage and requires a large amount of solvents, including dichloromethane which is not environmentally friendly. Therefore, it is necessary to explore in both material synthesis and application aspects to invent a solid-phase extractant with low cost, simple synthesis, environmental friendliness, strong selectivity, and high extraction efficiency. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a cyclodextrin polymer for extracting various trace organic substances in urine, which has simple synthesis, environmental friendliness, and high extraction efficiency.

[0005] The first technical solution proposed by the present invention to solve the technical problems raised in the prior art is: A cyclodextrin polymer for solid-phase extraction of various trace organic substances in urine, and the preparation steps are as follows:

[0006] 1) Prepare β-cyclodextrin polymer loaded on cellulose by one-step cross-linking method;

[0007] 2) Prepare amino-functionalized β-cyclodextrin polymer loaded on cellulose by reduction method.

[0008] Further, step 1) specifically includes: Mix 1 part by mass of β-cyclodextrin, 0.5 - 0.7 part by mass of 2,3,5,6-tetrafluoroterephthalonitrile, 1.5 - 2 parts by mass of cellulose, and 2 - 2.5 parts by mass of anhydrous potassium carbonate in dimethyl sulfoxide solution. After stirring at 80 °C for 40 h, a dark brown gel-like solid is obtained. The solid is dispersed in tetrahydrofuran solvent, stirred well, and then filtered by suction. 1M HCl aqueous solution is added to the solid after suction filtration until no more bubbles are generated, and then it is washed with deionized water and methanol in sequence until neutral. Finally, it is dried in vacuum at 60 °C for 10 h to obtain β-cyclodextrin polymer supported on cellulose.

[0009] Further, the volume dosage of dimethyl sulfoxide is 100 mL of dimethyl sulfoxide per 5 - 10 g of β-cyclodextrin, and the volume dosage of tetrahydrofuran is 50 mL of tetrahydrofuran per 5 - 10 g of β-cyclodextrin.

[0010] Step 2) specifically includes: Disperse the β-cyclodextrin polymer solid prepared in step 1) in tetrahydrofuran solution, cool it in an ice bath, and slowly add an excessive amount of borane dimethyl sulfide complex while stirring. After stirring for 40 h, filter by suction, and wash it with 1M HCl methanol solution, 5% NaOH aqueous solution, water, and methanol in sequence until neutral. Finally, it is dried in vacuum at 60 °C for 10 h to obtain amino-functionalized β-cyclodextrin polymer supported on cellulose.

[0011] Further, the volume dosage of tetrahydrofuran is 100 mL of tetrahydrofuran per 10 - 15 g of β-cyclodextrin polymer. 6. The β-cyclodextrin polymer according to claim 1, wherein the particle size of the β-cyclodextrin polymer is 300 - 500 mesh.

[0012] The second technical solution proposed by the present invention to solve the problems of the prior art is to provide a solid-phase extraction column for extracting various trace organic pollutants. Take a clean empty solid-phase extraction column, first install a sieve plate at the bottom to block the outflow port of the extraction column, then quantitatively fill the amino-functionalized β-cyclodextrin polymer filler supported on cellulose, and then install a sieve plate above the filler to encapsulate the filler in the extraction tube. The sieve pore diameter of the sieve plate is smaller than the particle diameter of the filler, thus obtaining a solid-phase extraction column for extracting various trace organic matters in urine.

[0013] Further, the effective volume of the solid-phase extraction column of the present invention is 6 mL, and the filler dosage is 0.1 - 0.3 g.

[0014] The third technical solution proposed by the present invention to solve the problems of the prior art is to provide a method for solid-phase extraction of various trace organic matters in urine on a solid-phase extraction column. The specific solid-phase extraction method includes the following steps:

[0015] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water;

[0016] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the column, immediately add the solution to be extracted, and keep the flow rate at 4 - 5 mL / min;

[0017] (3) Drying: After loading is completed, air-dry for 10 min;

[0018] (4) Elution: Elute with 5 mL of methanol solvent;

[0019] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream, and make up the volume to 1 mL with the initial mobile phase of liquid chromatography.

[0020] Furthermore, in step (4), 0.1 - 1 M ammonium acetate and 0.01 - 0.05 M CaCl₂ solution are added to the methanol solvent according to the charged property of the extraction target substance to improve the elution efficiency.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. The solid-phase extraction agent material of the present application has both the hydrophobic cavity of β-cyclodextrin and the amino charged group, which can adsorb organic pollutants with a wide range of physical and chemical properties. At the same time, the hydrophobic cavity can also isolate macromolecular organic substances, having good selectivity and reducing the matrix effect;

[0023] 2. The amino-functionalization step of the solid-phase extraction agent material of the present application improves the hydrophobicity of the cyclodextrin polymer. At the same time, using cellulose as the carrier and the screening particle size being 300 - 500 mesh significantly improves the loading speed;

[0024] 3. The material synthesis method of the preparation method of the present application is simple. During the material washing process, a large amount of organic solvents do not need to be used, and the use of the harmful organic solvent dichloromethane is avoided. The raw material cost is low and it is environmentally friendly;

[0025] 4. The present invention can achieve the efficient extraction of various trace organic pollutants in complex matrices, generally meeting the requirements of green, safe and efficient analysis. Description of the Drawings

[0026] Table 1 shows the linear regression equations and peak emergence times of 45 organic substances on the ultra-high performance liquid chromatography tandem mass spectrometer;

[0027] Figure 1 are the spiked recoveries obtained by the solid-phase extraction method of the present invention for 7 organic substances in deionized water in Example 5 and Example 6;

[0028] Figure 2are the spiked recoveries obtained by solid-phase extraction of 7 organic compounds in deionized water by the solid-phase extraction method of the present invention in Example 6 and Example 7;

[0029] Figure 3 are the spiked recoveries obtained by solid-phase extraction of 45 organic compounds in deionized water by the solid-phase extraction method of the present invention in Example 8;

[0030] Figure 4 are the spiked recoveries obtained by solid-phase extraction of 45 organic compounds in urine by the solid-phase extraction method of the present invention in Example 9. Detailed implementation manners

[0031] The present invention will be further described below by specific examples and accompanying drawings. The examples of the present invention are for better enabling those skilled in the art to understand the present invention and do not impose any limitations on the present invention.

[0032] Example 1

[0033] A preparation method of an amino-functionalized β-cyclodextrin polymer solid-phase extraction agent supported on cellulose specifically includes the following steps:

[0034] 1) Synthesis of β-cyclodextrin polymer supported on cellulose: First, weigh 6.0 g of β-cyclodextrin solid, 3.5 g of 2,3,5,6-tetrafluoroterephthalonitrile solid, 10.0 g of cellulose powder and 15.0 g of anhydrous potassium carbonate, mix them in 100 mL of dimethyl sulfoxide solution, heat to 80 °C, cool to room temperature after reacting for 40 h. Add 50 mL of tetrahydrofuran solution to the reaction solvent, stir well, and filter. Pour the filtered solid into a 250 mL conical flask, and then add 1 M HCl aqueous solution until no more bubbles are generated. Filter, wash with deionized water and methanol in turn until neutral, and finally dry in vacuum at 60 °C for 10 h to obtain β-cyclodextrin polymer supported on cellulose.

[0035] 2) Synthesis of amino-functionalized β-cyclodextrin polymer supported on cellulose: Weigh 10.0 g of β-cyclodextrin polymer supported on cellulose, disperse it in 100 mL of tetrahydrofuran solution, cool it in an ice bath, and slowly add 50 mL of borane dimethyl sulfide complex solution (2 M in tetrahydrofuran solvent) at the same time. Keep stirring for 40 h and then filter by suction. Wash the obtained solid with 1 M HCl methanol solution, 5% NaOH aqueous solution, water and methanol in turn until neutral, and finally dry in vacuum at 60 °C for 10 h to obtain amino-functionalized β-cyclodextrin polymer supported on cellulose.

[0036] Example 2

[0037] A preparation method of an amino-functionalized β-cyclodextrin polymer solid-phase extraction agent specifically includes the following steps:

[0038] 1) Synthesis of β-cyclodextrin polymer: First, weigh 6.0 g of β-cyclodextrin solid, 3.5 g of 2,3,5,6-tetrafluoroterephthalonitrile solid, and 15.0 g of anhydrous potassium carbonate, mix them in 100 mL of dimethyl sulfoxide solution, heat to 80 °C, cool to room temperature after reacting for 40 h. Add 50 mL of tetrahydrofuran solution to the reaction solvent, stir well, and filter. Pour the filtered solid into a 250 mL conical flask, then add 1M HCl aqueous solution until no more bubbles are generated. Filter, wash with deionized water and methanol in turn until neutral, and finally dry in vacuum at 60 °C for 10 h to obtain β-cyclodextrin polymer.

[0039] 2) Synthesis of amino-functionalized β-cyclodextrin polymer: Weigh 5.0 g of β-cyclodextrin polymer supported on cellulose, disperse it in 50 mL of tetrahydrofuran solution, cool in an ice bath, and slowly add 25 mL of borane dimethyl sulfide complex solution (2M in tetrahydrofuran solvent) while stirring continuously. After stirring for 40 h, filter by suction. Wash the obtained solid with 1M HCl methanol solution, 5% NaOH aqueous solution, water and methanol in turn until neutral, and finally dry in vacuum at 60 °C for 10 h to obtain amino-functionalized β-cyclodextrin polymer.

[0040] Example 3

[0041] Use the amino-functionalized β-cyclodextrin polymer supported on cellulose prepared in Example 1 as a solid-phase extraction agent and pack it into a solid-phase extraction column: Take a clean extraction column with an effective volume of 6 mL. Before packing the extraction agent, first fit a sieve plate at the bottom to block the outlet of the extraction column, then fill 0.2 g of amino-functionalized β-cyclodextrin polymer packing supported on cellulose into the extraction column, and then fit a sieve plate above the packing to enclose the packing in the extraction tube. Finally, adjust the packing to make the packing evenly spread on the bottom sieve plate and ensure that the upper sieve plate is parallel to the bottom sieve plate.

[0042] Example 4

[0043] Use the amino-functionalized β-cyclodextrin polymer prepared in Example 2 as a solid-phase extraction agent and pack it into a solid-phase extraction column: Take a clean extraction column with an effective volume of 6 mL. Before packing the extraction agent, first fit a sieve plate at the bottom to block the outlet of the extraction column, then fill 0.2 g of amino-functionalized β-cyclodextrin polymer packing into the extraction column, and then fit a sieve plate above the packing to enclose the packing in the extraction tube. Finally, adjust the packing to make the packing evenly spread on the bottom sieve plate and ensure that the upper sieve plate is parallel to the bottom sieve plate.

[0044] Example 5

[0045] In this example, the solid-phase extraction column in Example 3 was mainly used for the solid-phase extraction of organic pollutants in the spiked water sample, and the content of organic matter was measured by high performance liquid chromatography (HPLC).

[0046] Preparation of the solution to be extracted: A mixed solution containing 7 kinds of organic substances with a concentration of 1 mg / L was prepared with deionized water. The solid-phase extraction process of the experimental water sample included the following steps:

[0047] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water;

[0048] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the small column, immediately add 5 mL of the solution to be extracted, and keep the flow rate at 4 - 5 mL / min;

[0049] (3) Drying: After loading, air dry for 10 min;

[0050] (4) Elution: Elute with 5 mL of methanol solvent;

[0051] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream, and make up the volume to 1 mL with the initial mobile phase of liquid chromatography.

[0052] (6) Filter the eluent collected in step (5) after volume makeup through a 0.22 μm filter membrane, and then inject the sample into HPLC for measurement.

[0053] The recovery rate calculated by solid-phase extraction of the water sample in this example is as Figure 1 shown

[0054] Example 6

[0055] In this example, the solid-phase extraction of the water sample with known concentration was carried out according to the method of Example 5. The difference was that the methanol elution solvent in the solid-phase extraction step (4) was replaced with 3 mL of 1 M ammonium acetate methanol solvent and 2 mL of 0.05 M CaCl2 methanol. The solid-phase extraction process of the experimental water sample included the following steps:

[0056] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water;

[0057] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the small column, immediately add 5 mL of the solution to be extracted, and keep the flow rate at 4 - 5 mL / min;

[0058] (3) Drying: After loading, air dry for 10 min;

[0059] (4) Elution: First, add 3 mL of 1 M ammonium acetate methanol solvent for elution. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 2 mL of 0.05 M CaCl2 methanol solvent for elution;

[0060] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream and make up the volume to 1 mL with the initial mobile phase of liquid chromatography.

[0061] (6) Filter the eluent collected in step (5) after volume adjustment through a 0.22 μm filter membrane, and then inject it into HPLC for measurement.

[0062] The recovery rate calculated for solid-phase extraction of the water sample in this example is as Figure 1 shown.

[0063] Example 7

[0064] In this example, solid-phase extraction of the water sample with known concentration was carried out according to the method of Example 6, except that the solid-phase extraction column was replaced with the solid-phase extraction tube column in Example 4. The process of solid-phase extraction of the experimental water sample includes the following steps:

[0065] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water;

[0066] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the small column, immediately add 5 mL of the solution to be extracted, and keep the flow rate at 4 - 5 mL / min;

[0067] (3) Drying: After loading, air dry for 10 min;

[0068] (4) Elution: First, add 3 mL of 1 M ammonium acetate methanol solvent for elution. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 2 mL of 0.05 M CaCl2 methanol solvent for elution;

[0069] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream and make up the volume to 1 mL with the initial mobile phase of liquid chromatography.

[0070] (6) Filter the eluent collected in step (5) after volume adjustment through a 0.22 μm filter membrane, and then inject it into HPLC for measurement.

[0071] The recovery rate calculated for solid-phase extraction of the water sample in this example is as Figure 2 shown.

[0072] Example 8

[0073] In this example, a solid-phase extraction column from Example 3 was mainly used for the solid-phase extraction of organic pollutants in spiked blank water samples, and the content of organic substances was measured by an ultra-high performance liquid chromatography tandem mass spectrometer (LC-MS / MS).

[0074] Preparation of the solution to be extracted: First, a mixed solution containing 45 organic substances with a concentration of 1 mg / L was prepared as the stock solution, and then the stock solution was diluted with deionized water to 2 μg / L as the solution to be extracted. The process of solid-phase extraction of the experimental water sample includes the following steps:

[0075] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water;

[0076] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the column, immediately add 5 mL of the solution to be extracted, and keep the flow rate at 4 - 5 mL / min;

[0077] (3) Drying: After loading, air dry for 10 min;

[0078] (4) Elution: First, add 3 mL of 1M ammonium acetate methanol solvent for elution. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 2 mL of 0.05M CaCl2 methanol solvent for elution;

[0079] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream, and make the volume constant to 1 mL with the initial mobile phase of liquid chromatography.

[0080] (6) Filter the collected eluent after volume constant in step (5) through a 0.22 μm filter membrane, and then inject it into the LC-MS / MS for measurement.

[0081] Table 1 shows the linear regression equations and peak elution times of 45 organic substances on the ultra-high performance liquid chromatography tandem mass spectrometer. The recovery rates calculated after solid-phase extraction of the spiked blank water sample in this example are as Figure 3 shown.

[0082] Example 9

[0083] In this example, the solid-phase extraction of urine samples was carried out according to the method of Example 8, except that the solution to be extracted was a mixture of 45 organic substances prepared with urine.

[0084] Preparation of the solution to be extracted: Take 2 mL of urine, dilute it to 5 mL with deionized water, adjust the pH to 4 - 6, add 10 μL of the mixed solution containing 45 organic substances with a concentration of 1 mg / L, and prepare a urine spiked sample with a concentration of 2 μg / L.

[0085] The process of solid-phase extraction of the spiked urine includes the following steps:

[0086] (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water.

[0087] (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the column, immediately add 5 mL of the solution to be extracted, and keep the flow rate at 4 - 5 mL / min.

[0088] (3) Drying: After loading, air-dry for 10 min.

[0089] (4) Elution: First, add 3 mL of 1 M ammonium acetate methanol solvent for elution. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 2 mL of 0.05 M CaCl2 methanol solvent for elution.

[0090] (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream, and make up the volume to 1 mL with the initial mobile phase of liquid chromatography.

[0091] (6) Filter the eluent collected in step (5) after volume making through a 0.22 μm filter membrane, and then inject the sample into LC-MS / MS for measurement.

[0092] The recovery rate calculated after solid-phase extraction of the spiked urine sample in this example is as Figure 4 shown.

[0093] Table 1 Linear regression equations and retention times of organic compounds

[0094]

[0095]

Claims

1. A preparation method of a β-cyclodextrin polymer solid-phase extraction material, characterized in that, It is prepared by the following method: 1) Prepare β-cyclodextrin polymer supported on cellulose by one-step crosslinking method; 2) Prepare amino-functionalized β-cyclodextrin polymer supported on cellulose by reduction method; Step 1) specifically includes: Mix 1 part by mass of β-cyclodextrin, 0.5 - 0.7 part by mass of 2,3,5,6-tetrafluoroterephthalonitrile, 1.5 - 2 parts by mass of cellulose, and 2 - 2.5 parts by mass of anhydrous potassium carbonate in dimethyl sulfoxide solution, stir at 80 °C for 40 h to obtain a dark brown gel-like solid. Disperse the solid in tetrahydrofuran solvent, stir well and then filter by suction. Add 1M HCl aqueous solution until no more bubbles are generated, and then wash with deionized water and methanol in turn until neutral. Finally, dry in vacuum at 60 °C for 10 h to obtain β-cyclodextrin polymer supported on cellulose; In step 1), the volume of dimethyl sulfoxide used is 100 mL of dimethyl sulfoxide per 5 - 10 g of β-cyclodextrin, and the volume of tetrahydrofuran used is 50 mL of tetrahydrofuran per 5 - 10 g of β-cyclodextrin; Step 2) specifically includes: Disperse the β-cyclodextrin polymer solid prepared in step 1) in tetrahydrofuran solution, cool in an ice bath, and slowly add an excessive amount of borane dimethyl sulfide complex while stirring. After stirring for 40 h, filter by suction, and wash with 1M HCl methanol solution, 5% NaOH aqueous solution, water and methanol in turn until neutral. Finally, dry in vacuum at 60 °C for 10 h to obtain amino-functionalized β-cyclodextrin polymer supported on cellulose; In step 2), the volume of tetrahydrofuran used is 100 mL of tetrahydrofuran per 10 - 15 g of β-cyclodextrin polymer; 2. The method according to claim 1, characterized in that, The particle size of the β-cyclodextrin polymer is 300 - 500 mesh; 3. A solid-phase extraction method for multiple trace organic compounds in urine using the solid-phase extraction material prepared by the method described in claim 1 or 2, comprising the following steps: (1) Activation: Add 5 mL of methanol to the solid-phase extraction column. When the methanol liquid level is about to reach the upper sieve plate of the solid-phase extraction column, immediately add 5 mL of deionized water; (2) Loading: When the deionized water liquid level is about to reach the upper sieve plate of the column, immediately add the solution to be extracted, and keep the flow rate at 4 - 5 mL / min; (3) Drying: After loading, dry in air for 10 min; (4) Elution: Elute with 5 mL of methanol solvent; (5) Nitrogen blowing: Blow the solution to dryness with a gentle nitrogen stream, and make up the volume to 1 mL with the initial mobile phase of liquid chromatography; 4. The method according to claim 3, wherein In step (4), the methanol solvent is added with 0.1 - 1M ammonium acetate and 0.01 - 0.05M CaCl2 solution according to the charged property of the extraction target substance to improve the elution efficiency.

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