A method for extracting and purifying organic material from a biological matrix sample

Abstract

The present application relates to the field of separation and purification, and discloses a method for extracting and purifying organic substances from a biological matrix sample, comprising the following steps: after adjusting the pH of the sample to 1-3, performing sample loading and adsorption; through magnetic force, transferring the adsorption material to perform cleaning; after cleaning, before elution, performing infrared or microwave heating on the adsorption material, aiming at evaporating residual solvent, and denaturing and coagulating co-adsorbed proteins to prevent them from being co-eluted, thereby significantly improving the purity of the final eluate; and finally, performing elution on the heated adsorption material. The present application combines special materials and innovative processes, realizes automatic sample pretreatment with high recovery rate and high purity, and can be applied to the extraction of oligonucleotides, peptides, small molecules and the like; and solves the problems of low recovery rate and poor reproducibility caused by non-specific adsorption of proteins in biological samples.

Description

Technical Field

[0001] This invention relates to the field of separation and purification, and more particularly to a method for extracting and purifying organic substances from biological matrix samples. Background Technology

[0002] Solid phase extraction (SPE) is a key technology widely used in the pretreatment of biological samples (such as plasma and serum) to enrich target analytes and remove interfering impurities. However, the abundant presence of proteins in biological samples poses a significant challenge to this technology. Proteins readily undergo non-specific adsorption on the surface of traditional SPE packing materials, leading to a series of problems: 1) physical blockage of the packing pores, affecting flow rate and operational stability; 2) occupation of active adsorption sites, significantly reducing the recovery rate of target analytes; 3) elution along with the target analytes during the elution step, generating a matrix effect that interferes with subsequent instrumental analysis (such as LC-MS); and 4) poor reproducibility of the entire extraction process.

[0003] To overcome the drawbacks of traditional column-based solid-phase extraction (SPE), such as cumbersome operation, clogging susceptibility, and difficulty in automation, magnetic solid-phase extraction (MSE) technology has emerged. This technology utilizes magnetic microspheres as a carrier, achieving solid-liquid separation through an external magnetic field, eliminating the need for liquid transfer and fundamentally avoiding physical clogging, thus enabling high-throughput automated sample processing. However, the fundamental problem of non-specific protein adsorption remains. If the surface of the magnetic beads cannot effectively resist protein adhesion, residual proteins will still severely affect the recovery rate, accuracy, and sensitivity of the analysis, limiting its application. Therefore, there is an urgent need in this field to develop a novel magnetic solid-phase extraction material and a method for extracting and purifying organic matter from biological matrix samples that can effectively resist or repel the non-specific adsorption of proteins in biological samples, thereby fundamentally improving the efficiency, reliability, and accuracy of biological sample pretreatment while ensuring the convenience of magnetic operation. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in related technologies. To this end, the present invention provides a method for extracting and purifying organic matter from biological matrix samples.

[0005] A method for extracting and purifying organic matter from biological matrix samples.

[0006] C1, employs a paramagnetic mesoporous adsorbent material, the outer surface of which has a hydrophilic organic material modification layer;

[0007] C2, the adsorbent material is mixed with a biological matrix sample containing organic matter so that the organic matter is adsorbed onto the adsorbent material to obtain an adsorbent material loaded with organic matter.

[0008] C3, the adsorbent material carrying organic matter is cleaned to remove impurities, and the cleaned adsorbent material is obtained.

[0009] C4, the cleaned adsorbent material is heated to evaporate the residual solvent and denature and coagulate the co-adsorbed proteins, thus obtaining the heated adsorbent material.

[0010] C5, the heat-treated adsorbent material is eluted to obtain an eluent containing the organic matter.

[0011] Furthermore, prior to step C2, the method includes the step of adjusting the pH of the biological matrix sample to 1-3.

[0012] Furthermore, prior to step C2, the method includes the step of adding a protein stabilizer to the biological matrix sample, wherein the protein stabilizer is selected from one or more of trifluoroacetic acid, octane sulfonic acid, and benzene sulfonic acid.

[0013] Furthermore, the heating treatment employs microwave radiation or far-infrared radiation.

[0014] Furthermore, the adsorbent material is spherical mesoporous particles, comprising:

[0015] paramagnetic core;

[0016] The mesoporous matrix encapsulating the paramagnetic core; and

[0017] A hydrophilic organic material layer modified on the outer surface of the mesoporous matrix, the hydrophilic organic material layer being used to resist non-specific adsorption of proteins.

[0018] Furthermore, the mesoporous matrix is ​​a polymer matrix or an inorganic metal oxide matrix;

[0019] The polymer matrix is ​​formed by the polymerization of monomers containing hydrophobic monomers and amphoteric monomers;

[0020] The inorganic metal oxide matrix is ​​silicon dioxide.

[0021] Furthermore, the hydrophilic organic material layer comprises one or more selected from polyethylene glycol, dextran, agarose, zwitterionic groups, carboxyl groups, or amino groups.

[0022] Further, the hydrophobic monomer is selected from one or more of divinylbenzene, styrene, and acrylate; the amphoteric monomer is selected from one or more of N-vinylimidazolium, N-vinylpyridine, N-vinylpyrrolidone, and sulfobetaine methacrylate block copolymer; the weight ratio of the hydrophobic monomer to the amphoteric monomer is 1-10:1.

[0023] Furthermore, the adsorbent material is a silica-based magnetic adsorbent material, and the preparation steps include:

[0024] D11 involves mixing magnetic porous silica filler, organic solvent, and hydrophilic material, followed by heating and reaction to obtain a magnetic silica material with a hydrophilic surface.

[0025] D12, the hydrophilic magnetic silica gel material is mixed with toluene, dehydrated, and then silane and imidazole are added. The mixture is refluxed to obtain the paramagnetic spherical mesoporous adsorbent material. The hydrophilic material is selected from one or more of polyethylene glycol, polyvinyl alcohol, dextran, and agarose. The organic solvent is selected from one or more of N,N-dimethylformamide and toluene. The silane is selected from one or more of octadecyl dimethylchlorosilane, octadecyl trichlorosilane, octyl dimethylchlorosilane, propyl dimethylchlorosilane, butyl dimethylchlorosilane, aminoethylaminopropyltrimethoxysilane, phenethyltrimethoxysilane, ureapropyltrimethoxysilane, and cyanopropyl dimethylchlorosilane.

[0026] Furthermore, the adsorbent material has an average particle size of 3–150 µm, an average pore size of 4–80 nm, and a specific surface area of ​​50–1000 m². 2 / g.

[0027] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0028] By employing paramagnetic mesoporous adsorbent materials with surfaces modified by hydrophilic organic materials, the non-specific adsorption of proteins in biological matrices is effectively suppressed, avoiding the problems of filler blockage and active site competition in traditional solid phase extraction (SPE).

[0029] The mesoporous structure of the adsorption material allows for the selective entry of small organic molecules while eliminating interference from large molecules; combined with pH adjustment and ion-pairing reagents, the adsorption selectivity of the target analyte is enhanced, improving the recovery rate and purity.

[0030] By utilizing a magnetic solid-phase extraction device, the adsorbed material is transferred via a magnetic rod, eliminating the need for liquid transfer. This overcomes the drawbacks of traditional SPE, such as cumbersome operation and easy clogging, and significantly improves sample processing efficiency and operational consistency.

[0031] After cleaning, infrared heating (60–180 seconds) evaporates residual solvent and coagulates proteins, reducing protein and magnetic material contamination in the eluent, minimizing matrix effects, and improving the accuracy and reproducibility of subsequent instrumental analyses (such as LC-MS).

[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 It is the traditional SPE process flow;

[0035] Figure 2 This is a process flow diagram of the present invention;

[0036] Figure 3 This is a schematic diagram of the structure of the magnetic solid-phase extraction material used in this invention.

[0037] Figure label:

[0038] 1. Magnetic particle core; 2. Protective layer; 3. Modification layer of hydrophilic organic material; 4. Pores; 5. Bonded functional groups. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention but should not be used to limit the scope of this invention.

[0040] Figure 1 The process of traditional solid-phase extraction is demonstrated, including activation, loading, washing, and elution steps.

[0041] Unlike traditional solid-phase extraction, the method of this invention uses... Figure 2 The specific process of the method used in this invention is clearly demonstrated.

[0042] Figure 2 The yellow pentagram represents large molecular interference, the purple triangle represents the target substance, the green circle represents impurity 1, and the red rectangle represents impurity 2. The gray circle represents magnetic material, and the gray circle represents magnetic extraction material.

[0043] The specific steps include activation, sample loading, washing, repeated washing, elution, and waste liquid treatment.

[0044] A method for extracting and purifying organic matter from a biological matrix sample includes the following steps:

[0045] Adsorbent activation—The adsorbent is activated in an organic or aqueous buffer solution;

[0046] Equilibration—Equilibrate the activated adsorbent in a buffer solution selected based on the polarity and pH stability of the target analyte.

[0047] Sample loading—Introducing the sample to allow particles to adsorb the analyte of interest.

[0048] Cleaning – Use an appropriate solution to clean and remove impurities.

[0049] Elution – Desorption of analytes using solvents or conditions that disrupt analyte-adsorbent interactions.

[0050] Unlike traditional SPE, this workflow eliminates the need to transfer liquid supernatant. Instead, it uses magnetic rods to move adsorbent particles between pores, enabling fully automated, high-throughput operation.

[0051] To improve the consistency of extraction, sample cleanliness, and analyte recovery, this method also includes the following steps:

[0052] Adsorbent particles containing both the target substance and co-adsorbed impurities can be magnetically transferred to subsequent wells for further processing; adsorbent particles containing both the target substance and some impurities can be magnetically transferred for selective cleaning; and adsorbent particles containing only the target substance can be directly transferred to the elution wells magnetically.

[0053] Depending on the properties of the target analyte and the experimental objective, one or more adsorbent types can be selected for co-extraction. For ionic analytes, ion-pairing reagents can be used, such as trifluoroacetic acid (TFA) for cationic substances or triethylamine (TEA) for anionic substances.

[0054] Before extraction, the pH of the sample is adjusted to 1-3 to reduce protein deposition on the particle surface.

[0055] Controlling the ratio of organic solvent to water solvent to below 10%, ideally below 5%, and maintaining the temperature between 15-25℃ can further reduce protein adsorption.

[0056] Add protein stabilizers, such as TFA, octane sulfonic acid, benzene sulfonic acid, or other acidic compounds with hydrophobic portions, to further inhibit protein deposition on the particle surface.

[0057] This method also includes a heating process for evaporating solvents adhering to the surface of magnetic materials, thereby enabling the use of incompatible solvents in the same extraction process.

[0058] The magnetic rod and the magnetic rod sleeve move up and down synchronously, which effectively ensures the uniform mixing and adsorption of magnetic materials and prevents residue accumulation.

[0059] One specific implementation method is to first install the magnetic rod sleeve onto the magnetic rod sleeve holder before implementing the method, and then place the well plate (pre-loaded with magnetic materials and reagents) onto the buffer plate.

[0060] Magnetic material mixing: The magnetic rod sleeve support descends, so that the bottom of the magnetic rod sleeve is in close contact with the bottom of the orifice plate, and makes a rapid vertical reciprocating motion to ensure that the magnetic material and reagents are fully mixed.

[0061] Adsorption of magnetic materials: The magnetic rod support and the magnetic rod sleeve support descend simultaneously, bringing the magnetic rod and the magnetic rod sleeve close to the bottom of the orifice plate. The magnetic force of the magnetic rod is used to adsorb the magnetic material to the bottom of the magnetic rod sleeve.

[0062] Transfer of magnetic material: Two supports rise simultaneously, lifting the adsorbed magnetic material from the solvent and moving it to the top. Then, according to the method requirements, they move horizontally above the corresponding solvent orifices in the well plate.

[0063] Release of magnetic material: The two supports descend again, bringing the magnetic rod and magnetic sleeve close to the bottom of the target hole. The magnetic rod support first rises to the top, and then the magnetic rod sleeve support moves rapidly vertically back and forth, releasing the magnetic material.

[0064] Heating is achieved using microwave or far-infrared radiation, which evaporates the solvent in the particles on the magnetic rod and in the surrounding air, thus promoting the conversion of hydrophilic solvents to hydrophobic solvents, and vice versa. When the magnetic materials are uniformly mixed in the liquid and attracted by the bottom of the magnetic rod sleeve, they accumulate in this area. Due to the surface tension of the liquid, residual solvent remains on the particle surface. Directly transferring such particles to solvents with significantly different polarities or pH values ​​would adversely affect the experimental results. The heating step effectively avoids this problem.

[0065] Another important reason for the improved effectiveness of this method is the use of a more efficient magnetic extraction material that avoids protein adsorption. The specific preparation method of this magnetic extraction material will be discussed in more detail.

[0066] The magnetic extraction material used has properties including an average pore size of 40–200 Å, allowing small organic molecules to enter while excluding larger molecules (such as proteins and nucleotides).

[0067] The particle core is paramagnetic, and its surface is coated with a polymer or inorganic metal oxide (e.g., silica). One or more organic reagents can be used for modification to adjust adsorption / desorption. Particle size: 3–100 µm; specific surface area: 50–1000 m². 2 / g.

[0068] The surface of the magnetic extraction material is modified with hydrophilic groups.

[0069] Figure 3 The structure of the anti-protein magnetic extraction material is shown.

[0070] The anti-protein magnetic extraction material has a magnetic particle core 1 at its center and a protective layer 2 on its surface. The protective layer 2 is a mesoporous matrix, which can be a polymer matrix or silica.

[0071] A hydrophilic organic material modification layer 3 is provided on the protective layer. The modification layer has bonding functional groups 5, which are located on the material surface and in the pores 4.

[0072] The bonding functional group 5 is mainly distributed in the pore 4, which reduces the adsorption of macromolecular impurities.

[0073] The preparation process of anti-protein magnetic extraction materials will be described below through specific preparation examples.

[0074] S1: Preparation of magnetic adsorption materials

[0075] S11 mixes hydrophobic monomers, amphoteric monomers, initiators, magnetic microparticles, and pore-forming agents to obtain an oil phase;

[0076] S12 dissolves the water-soluble dispersant in water to obtain an aqueous phase;

[0077] S13 adds the above oil phase to the above aqueous phase, disperses at high speed, polymerizes at high temperature, separates by vacuum filtration, and separates under a magnetic field to obtain magnetic adsorption material.

[0078] The magnetic adsorption materials include: polyimidazolium-divinylbenzene-pyrrolidone magnetic adsorption materials; polystyrene-sulfobetaine methacrylate block copolymer zwitterionic magnetic materials; polypyridine-divinylbenzene-pyrrolidone magnetic adsorption materials; and polyimidazolium-acrylic acid-styrene magnetic adsorption materials.

[0079] The hydrophobic monomer is an olefin monomer, such as one or more of divinylbenzene, chlorodivinylbenzene, styrene, chlorostyrene, acrylate, acrylic acid, etc.

[0080] Amphoteric monomers are olefin monomers that contain both hydrophilic and hydrophobic groups, such as one or more of N-vinylimidazolium, N-vinylpyridine, N-vinylpyrrolidone, and sulfobetaine methacrylate block copolymers.

[0081] The initiator is a hydrophobic initiator, which can be a hydrophobic organic peroxide initiator (e.g., di-tert-butyl peroxide DTBP), an azo initiator (e.g., azobisisobutyronitrile AIBN), etc.

[0082] The ratio of initiator to amphoteric monomer is 0.01-0.1:1.

[0083] The weight ratio of hydrophobic monomer to amphoteric monomer is 1-10:1.

[0084] The pore-forming agent is one or more of toluene, n-hexane, liquid paraffin, and isooctane.

[0085] The magnetic particles can be selected from one or more magnetic materials such as Fe3O4, Fe2O3, NiFe2O4, CuFe2O4, iron, nickel, and cobalt; the weight percentage of the magnetic particles in the anti-protein adsorption material is 0.5% to 30%.

[0086] The average pore size of the magnetic adsorption material is 4-30 nm; the average particle size is 3-100 μm; and the specific surface area ranges from 50-1000 m². 2 / g.

[0087] The preparation of S2 anti-protein magnetic extraction material also includes the functionalization of magnetic adsorption materials to achieve further inhibition and adsorption of proteins.

[0088] S21 involves dissolving the magnetic adsorbent material prepared in S1 in dichloroethane, adding chloroacetyl chloride and a catalyst, heating the mixture for a certain time, and then washing the material to prepare benzylated magnetic adsorbent material.

[0089] S22 benzylated magnetic adsorption resin was further functionalized to prepare various anti-protein magnetic extraction materials with corresponding carboxyl, amino, pyrimidine, PEG groups, and sugar surfaces.

[0090] Step S21 involves benzylic chloromethylation of the magnetic adsorption material prepared in step S1.

[0091] The ion exchange capacity of various groups in the anti-protein magnetic extraction material prepared by S22 can be 0.1 to 2.0 mmol / g, so as to obtain multiple separation effects such as hydrophobic interaction, polar dipole interaction, hydrogen bonding interaction, ion pairing interaction and ion exchange.

[0092] The catalysts are anhydrous zinc chloride, anhydrous tin tetrachloride, and anhydrous aluminum trichloride;

[0093] The heating reaction time is 1–48 hours;

[0094] The anti-protein magnetic extraction materials include carboxylated anti-protein magnetic extraction materials, primary / secondary amine anti-protein magnetic extraction materials, PEG-modified hydrophilic anti-protein magnetic extraction materials, sugar-modified hydrophilic anti-protein magnetic extraction materials, and anti-protein magnetic extraction materials with increased surface hydrophilicity due to the incorporation of pyrimidine groups.

[0095] The carboxyl-based anti-protein magnetic extraction material is a weak cation exchange group obtained by oxidizing benzyl chloride in an acidic solution;

[0096] Amine-based antiprotein magnetic adsorption materials are obtained by replacing the chlorine at the benzylic position with an amine to obtain a weak anion / strong anion exchange group (amine group).

[0097] The PEG-modified surface-hydrophilic anti-protein magnetic extraction material is obtained by substituting PEG at the benzylic chlorine position to obtain PEG-grafted surface-hydrophilic groups; the molecular weight of the PEG is 400-20000;

[0098] The sugar-modified surface hydrophilic anti-protein extraction material is obtained by substituting the chlorine at the benzylic position with sugar to obtain a sugar-grafted surface hydrophilic group; the sugar is dextran or agarose.

[0099] Preparation process 1: (Imidazole / pyrimidine modified, chloromethyl, carboxyl and amino functionalized magnetic materials)

[0100] Preparation of magnetic fillers with divinylbenzene-imidazolium-pyrrolidone groups

[0101] Experiment 1: 400 mL of pure water and 1 g of hydroxypropyl cellulose were added to a 1 L reaction flask. After heating to dissolve, the solution was cooled to below 40 °C. 1.3 g of 0.5 μm hydrophobic magnetic cores, 15 g of divinylbenzene, 10 g of vinylpyrrolidone, 0.2 g of AIBN, 20 g of toluene, and 2 g of N-vinylimidazole were added to the above aqueous solution after thorough stirring. The suspension was stirred at 500 rpm, and the temperature was gradually increased to 78 °C. Stirring and maintaining this temperature for 20 hours was then performed. The mixture was filtered, washed with ethanol, extracted with petroleum ether, vacuum dried, and then redispersed in ethanol. The magnetic material was extracted using a magnetic rod and vacuum dried to obtain 20 g of brown magnetic particles with a particle size of approximately 10-50 μm, an average pore size of approximately 6.2 nm, a pore volume of 1.26 mL / g, and a specific surface area of ​​753 μm. 2 / g (BET method test), nitrogen content 3.2%.

[0102] Preparation of chloromethyl magnetic filler

[0103] Experiment 2: 50g of the magnetic filler with divinylbenzene-imidazolium-pyrrolidone group prepared in Experiment 1 was swollen in dichloroethane for 2h, 25g of chloroacetyl chloride and 15g of anhydrous zinc chloride were added, and the reaction was carried out at 80℃ for 24h. The discharged material was washed with ethanol and acetone, extracted, and dried under vacuum at 40℃. The degree of chloromethyl substitution was 0.95mmol / g.

[0104] Carboxyl modification

[0105] Experiment 3: 50g of the chloromethyl magnetic filler prepared in Experiment 2 was reacted with 500g of 6% nitric acid pure water at 90℃ for 6h, and washed with pure water until neutral to prepare a carboxyl magnetic filler with a carboxyl substitution degree of 0.88mmol / g.

[0106] Amine modification

[0107] Experiment 4: Take 50g of the chloromethyl magnetic filler prepared in Experiment 2, 500ml of N,N dimethylformamide, swell for 2h, then add 40g of diethylenetriamine, 60ml of tetrabutylammonium hydroxide, and 0.5g of sodium hydroxide, react at 80℃ for 16h, cool down and discharge, wash with DMF and methanol, dry under vacuum at 40℃, and the degree of amino substitution is 1.25mmol / g.

[0108] Pyrimidine modification

[0109] Experiment 5: 10g of chloromethyl magnetic filler prepared in Experiment 1, 100g of toluene, and 2g of 2-amino-4,6-dihydroxypyrimidine were added to a 1-liter reaction flask and stirred for 16 hours. The mixture was filtered, washed with toluene and ethanol, extracted, and dried under vacuum at 40°C. The nitrogen content was determined to be 4.93%.

[0110] Preparation process 2: (zwitterionic magnetic materials)

[0111] Aqueous phase preparation: Add 400 mL of pure water and 1 g of hydroxypropyl cellulose to a 1 L reaction flask, heat to dissolve, and then cool to below 40 °C;

[0112] Oil phase preparation: Take 7.5g of 0.5um surface hydrophobic magnetic core, 15g of styrene, 10g of sulfobetaine methacrylate block copolymer, 0.2g of AIBN, and 10g of toluene, and stir until homogeneous;

[0113] Mixed polymerization: After mixing, add to the above aqueous solution, stir the suspension to 500 rpm, gradually increase the temperature to 78°C, and maintain the temperature while stirring for 20 hours;

[0114] Post-processing and detection: Filtration, ethanol washing, petroleum ether extraction, vacuum drying, and redisperse in ethanol. Magnetic material was extracted using a magnetic rod and vacuum dried to obtain 20 g of brown magnetic particles with a particle size of 10–50 μm, an average pore size of 5.3 nm, a pore volume of 0.95 mL / g, and a specific surface area of ​​639 m². 2 / g (BET method test).

[0115] Preparation process 3: (Polymer magnetic material on PEG surface)

[0116] 1) Add 10g of the chloromethyl magnetic filler prepared in Experiment 2, 400ml of toluene, 10g of hexadecyl ammonium bromide, 20g of PEG6000, and 300g of 33% NaOH to a 1-liter reaction flask, stir at 80℃ for 16h; filter, wash with toluene and methanol, extract, and dry under vacuum at 40℃.

[0117] 2) Add 10g of the chloromethyl magnetic filler prepared in Experiment 2, 400ml of toluene, 10g of cetyl ammonium bromide, 20g of PEG10000, and 300g of 33% NaOH to a 1-liter reaction flask, stir and react at 80℃ for 16h; filter, wash with toluene and methanol, extract, and dry under vacuum at 40℃.

[0118] Depending on the specific needs, different types of PEG can be used to prepare materials, such as amino-PEG grafted onto carboxylated magnetic particles.

[0119] 3) Add 10g of the carboxyl magnetic filler prepared in Experiment 3, 200ml of dichloromethane, 10g of monoamino PEG, 5g of triethylamine, 5g of EDCI, and 3.5g of HOBT to a 1-liter reaction flask, stir at room temperature for 16h; filter, wash with toluene and methanol, extract, and dry under vacuum at 40℃.

[0120] EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. It is a condensing agent whose core function is to activate the carboxyl group, making it more readily react with nitrogen- or oxygen-containing nucleophiles.

[0121] HOBT is 1-hydroxybenzotriazole. As an additive for condensation reactions, its core function is to be used in combination with carbodiimide condensing agents such as EDCI and DCC to solve the side reactions (such as racemization and low reaction efficiency) when the condensing agent is used alone.

[0122] Preparation process 4: (Magnetic materials on the surface of polysaccharides)

[0123] Add 10g of the chloromethyl magnetic filler prepared in Experiment 2, 400ml of toluene, 10g of hexadecyl ammonium bromide, 20g of dextran, and 300g of 33% NaOH to a 1-liter reaction flask, stir at 80℃ for 16h; filter, wash with toluene and methanol, extract, and dry under vacuum at 40℃.

[0124] Anti-protein magnetic extraction materials also include anti-protein adsorption silica magnetic extraction materials. By making the surface of hydrophobic silica magnetic adsorption materials hydrophilic, it is also possible to reduce surface protein adsorption, improve the recovery rate in extraction and purification, and solve related problems such as magnetic residue.

[0125] The preparation method of D1 anti-protein adsorption silica magnetic extraction material specifically includes:

[0126] D11 involves mixing magnetic porous silica filler, organic solvent, and hydrophilic material, heating to complete the reaction, cooling, filtering, cleaning, extracting, and drying.

[0127] D12: The hydrophilic magnetic silica material prepared in D11 is mixed with toluene, dehydrated, and then silane and imidazole are added. After the reaction is completed under reflux, the mixture is cooled, filtered, washed, and dried to obtain a silica magnetic extraction material that resists protein adsorption.

[0128] The hydrophilic material is PEG; the organic solvent is N,N dimethylformamide.

[0129] The protein-resistant silica magnetic extraction material prepared by D1 is a PEG-modified hydrophilic silica magnetic material, a sugar-modified hydrophilic silica magnetic material, or a polyvinyl alcohol-modified hydrophilic surface magnetic material.

[0130] The specific surface area of ​​magnetic porous silica gel fillers ranges from 1 to 800 m². 2 / g, with an average pore size of 1-80 nanometers and an average particle size of 1-150 micrometers.

[0131] In magnetic porous silica gel, the magnetic core accounts for 0.5% to 30% of the material by weight, and the magnetic particles are selected from one or more of Fe3O4, Fe2O3, NiFe2O4, CuFe2O4, iron, nickel, and cobalt.

[0132] The hydrophilic material is one or more of PEG, polyvinyl alcohol, dextran, and agarose;

[0133] The organic solvents are toluene, DMF, dichloromethane, and dichloroethane;

[0134] The reaction time is 1–48 hours;

[0135] The silanes are octadecyl dimethylchlorosilane, octadecyl trichlorosilane, octyl dimethylchlorosilane, octyl trichlorosilane, butyl dimethylchlorosilane, and propyl dimethylchlorosilane.

[0136] Preparation process 5: (PEG-modified silicone magnetic material)

[0137] Add 50g of silica magnetic filler, 200ml of N,N dimethylformamide, and 20g of PEG4000 to a 1L reaction flask. Heat to 90 degrees Celsius and react for 16 hours. Cool down, filter, wash three times with toluene and methanol, extract, and dry under vacuum at 40°C.

[0138] Add the magnetic packing material from the previous step, 200 ml of toluene, 30 g of octadecyl dimethyl chlorosilane, and 6 g of imidazole to a 1 L reaction flask. Reflux for 16 h, cool, filter, wash three times with toluene and methanol, extract, and dry under vacuum at 40 °C.

[0139] Protein coagulation effect example

[0140] During mSPE of biological samples, proteins can sometimes bind to magnetic materials and migrate with them through the solvent, resulting in residual protein or magnetic material in the eluent. To address this issue, controlled heating of the magnetic materials can be applied during processing. This heat treatment coagulates the proteins, effectively trapping them in the wash solution and significantly reducing protein or magnetic material contamination in the eluent.

[0141] Heating experiments were conducted on the extraction materials obtained using preparation processes 1 and 5, and the results are as follows:

[0142] Table 1 shows the magnetic extraction material obtained using preparation process 1 and then subjected to heating.

[0143]

[0144] Table 2 shows the magnetic extraction materials obtained using preparation process 5, after processing and heating.

[0145]

[0146] Examples of purifying steroid hormones

[0147] To establish a method that effectively resists interference from biological matrices and efficiently extracts and purifies organic matter from complex samples, this study used modified HLB magnetic solid-phase extraction material to extract various steroid hormones from protein-rich bovine plasma. The core advantage of this method lies in utilizing the properties of magnetic materials, effectively avoiding column clogging and low recovery rates caused by non-specific protein adsorption in traditional methods through simple magnetic separation. The specific operation is as follows: Steroid hormone mixed standards were spiked into the bovine plasma matrix, treated with magnetic solid-phase extraction, and then detected, with the spiked recovery rate calculated. Experimental results (Table 3) show that in the high-protein matrix of bovine plasma, this method exhibits excellent and stable recovery performance for most target steroid hormones. Specific data are as follows:

[0148] Table 3. Recovery rates of steroid hormones from bovine plasma using modified HLB magnetic solid-phase extraction materials

[0149]

[0150] Experimental results demonstrate that the modified HLB magnetic solid-phase extraction method employed in this study successfully extracted and purified various steroid hormones directly from bovine plasma without complex deproteinization pretreatment. As shown in Table 3, the recoveries of the target analytes were concentrated within the ideal range of 80%–120%. This result fully proves that this method can effectively resist or repel the non-specific adsorption of proteins in biological samples, avoiding the negative impact of protein blockage and co-precipitation on the recovery rate.

[0151] Examples of purifying different biological sample matrices

[0152] To further determine the versatility and effectiveness of the method described in this invention in extracting and purifying biological matrices, this embodiment uses the modified magnetic solid-phase extraction material to extract dexamethasone from five representative biological sample matrices: whole blood, plasma, serum, urine, and liver. Specifically, dexamethasone standard was added to a blank biological sample matrix, followed by extraction and purification. Simple magnetic separation replaced traditional centrifugation or negative pressure filtration. The eluent was collected and analyzed by LC-MS / MS.

[0153] The experimental results are shown in the table below. The specific data demonstrate the superior performance of the method of this invention:

[0154] Table 4. Recovery and precision results of dexamethasone in biological sample matrices using modified magnetic solid-phase extraction materials.

[0155]

[0156] As shown in Table 4, without any special protein removal pretreatment, this invention effectively extracted and purified dexamethasone from five biological samples, demonstrating excellent comprehensive performance: the recovery rates of the five representative biological sample matrices all exceeded 85%, fully meeting the requirements for biological sample analysis, which fully confirms that the magnetic solid-phase extraction material can effectively resist the non-specific adsorption of proteins in biological samples, achieving efficient capture and release of the target analyte; at the same time, the method showed good precision, with the relative standard deviation (RSD) of the five representative matrices all below 10%, indicating high reproducibility and reliable results; in addition, combined with the inherent advantages of magnetic materials—simple operation, speed, and ease of automation for high-throughput processing—further validates the significant potential and practical value of this method in the purification of complex biological matrix samples.

[0157] Based on the above embodiments, the following conclusions can be drawn:

[0158] The magnetic solid-phase extraction method provided by this invention demonstrates excellent effectiveness and versatility in the extraction and purification of steroidal substances from biological sample matrices. This method effectively overcomes the non-specific interference of proteins in biological samples, achieving satisfactory recovery rates and precision. Experimental results fully demonstrate that the method described in this invention has significant advantages such as strong anti-interference ability, simple operation, high recovery rate, and good reproducibility, providing a universal and powerful technical solution for the efficient extraction and purification of various organic substances from complex biological matrices.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting and purifying organic matter from a biological matrix sample, characterized in that, C1, employs a paramagnetic mesoporous adsorbent material, the outer surface of which has a hydrophilic organic material modification layer; C2, the adsorbent material is mixed with a biological matrix sample containing organic matter, and the organic matter is adsorbed on the adsorbent material to obtain an adsorbent material loaded with organic matter; C3, the adsorbent material carrying organic matter is cleaned to remove impurities, and the cleaned adsorbent material is obtained. C4, the cleaned adsorbent material is heated to evaporate the residual solvent and denature and coagulate the co-adsorbed proteins, thus obtaining the heated adsorbent material. C5, the heat-treated adsorbent material is eluted to obtain an eluent containing the organic matter; The heating treatment employs microwave radiation or far-infrared radiation. The adsorbent material is spherical mesoporous particles, comprising: paramagnetic core; The mesoporous matrix encapsulating the paramagnetic core; and A hydrophilic organic material layer modified on the outer surface of the mesoporous matrix, the hydrophilic organic material layer being used to resist non-specific adsorption of proteins.

2. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, Before step C2, the method further includes the step of adjusting the pH of the biological matrix sample to 1-3.

3. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, Before step C2, the method further includes the step of adding a protein stabilizer to the biological matrix sample, wherein the protein stabilizer is selected from one or more of trifluoroacetic acid, octane sulfonic acid, and benzene sulfonic acid.

4. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, The mesoporous matrix is ​​a polymer matrix or an inorganic metal oxide matrix; The polymer matrix is ​​formed by the polymerization of monomers containing hydrophobic monomers and amphoteric monomers; The inorganic metal oxide matrix is ​​silicon dioxide.

5. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, The hydrophilic organic material layer comprises one or more selected from polyethylene glycol, dextran, agarose, zwitterionic groups, carboxyl groups, or amino groups.

6. The method for extracting and purifying organic matter from a biological matrix sample according to claim 4, characterized in that, The hydrophobic monomer is selected from one or more of divinylbenzene, styrene, and acrylate; the amphoteric monomer is selected from one or more of N-vinylimidazolium, N-vinylpyridine, N-vinylpyrrolidone, and sulfobetaine methacrylate block copolymer; the weight ratio of the hydrophobic monomer to the amphoteric monomer is 1-10:

1.

7. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, The adsorbent material is a silica-based magnetic adsorbent material, and the preparation steps include: D11 involves mixing magnetic porous silica filler, organic solvent, and hydrophilic material, followed by heating and reaction to obtain a magnetic silica material with a hydrophilic surface. D12, the hydrophilic magnetic silica gel material is mixed with toluene, dehydrated, and then silane and imidazole are added. The mixture is refluxed to obtain a paramagnetic spherical mesoporous adsorbent material. The hydrophilic material is selected from one or more of polyethylene glycol, polyvinyl alcohol, dextran, and agarose. The organic solvent is selected from one or more of N,N-dimethylformamide and toluene. The silane is selected from one or more of octadecyl dimethylchlorosilane, octadecyl trichlorosilane, octyl dimethylchlorosilane, propyl dimethylchlorosilane, butyl dimethylchlorosilane, aminoethylaminopropyltrimethoxysilane, phenethyltrimethoxysilane, ureapropyltrimethoxysilane, and cyanopropyl dimethylchlorosilane.

8. The method for extracting and purifying organic matter from a biological matrix sample according to claim 1, characterized in that, The adsorbent material has an average particle size of 3–150 µm, an average pore size of 4–80 nm, and a specific surface area of ​​50–1000 m². 2 / g.

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