A core-shell structure adsorbent, a preparation method and a kit thereof, and a method for detecting the concentration of styrene metabolites in plasma

Abstract

The invention discloses a core-shell structure adsorbent and a preparation method and a kit thereof, as well as a detection method for the concentration of styrene metabolites in plasma. The core-shell structure adsorbent includes ferroferric oxide magnetic microspheres, petal-shaped titanium dioxide coated on the surface of the ferroferric oxide magnetic microspheres, and a quaternary amine-carbon 18 bifunctionalized oligomeric polymer compound modified on a partial area of ​​the surface of the petal-shaped titanium dioxide. The core of the adsorbent of the present invention is ferroferric oxide magnetic microspheres, and the adsorbent as a whole is magnetic, which can achieve rapid solid-liquid phase magnetic separation, improve the sample purification efficiency, the titanium dioxide component in the material can effectively adsorb the phospholipid components in the plasma, and the quaternary amine-carbon 18 bifunctionalized oligomeric polymer component can effectively adsorb impurities such as fat and inorganic anions in the plasma, thereby effectively reducing the matrix interference effect in the sample detection process, so as to achieve the purpose of quickly and accurately detecting the concentration of trace phenylglyoxylic acid and mandelic acid in the plasma sample.

Description

Technical Field

[0001] The present invention relates to the technical field of bioanalysis, and in particular to a core-shell structure adsorbent and a preparation method and a kit thereof, as well as a method for detecting the concentration of styrene metabolites in plasma. Background Art

[0002] Styrene is an important raw material for the plastic and rubber industry. Plastic and rubber products containing styrene are widely used in daily life and industrial production. Styrene also exists in certain plants, tobacco and automobile exhaust. Occupational exposure is the most likely to cause poisoning reactions. Styrene in the working environment mainly enters the human body in the form of gas through the respiratory tract or skin contact. Short-term exposure to high concentrations of styrene can cause respiratory irritation symptoms such as coughing and sore throat. Long-term exposure can cause damage to the lung tissue structure. This makes people more susceptible to diseases such as pneumonia and emphysema.

[0003] Styrene is reacted with the P450 enzyme system to generate styrene epoxide, which then undergoes a series of metabolic reactions in the body to form phenylglyoxylic acid and mandelic acid (chemical structure as shown in Figure 1 As shown), and excreted through urine. Therefore, there are many studies on phenylglyoxylic acid and mandelic acid in urine at present. Patent documents CN103837624A and CN103852549A respectively establish detection methods for phenylglyoxylic acid and mandelic acid in urine. The technical scheme is to directly use liquid chromatography-tandem mass spectrometry to detect urine after dilution with water. The above method does not use suitable sample pretreatment technology to remove difficult volatile inorganic salts and organic interferences in urine, which will cause irreversible damage to the detection instrument; at the same time, liquid chromatography-tandem mass spectrometry has a large matrix interference effect during urine detection, and the linear range is narrow, and it is difficult to take into account the simultaneous detection of high concentration span phenylglyoxylic acid and mandelic acid in urine. In addition, the content levels of phenylglyoxylic acid and mandelic acid in urine are affected by individual differences, water intake, diet and metabolic levels, and the content levels in different time periods fluctuate greatly, making it difficult to accurately assess the actual level of exposure in the human body.

[0004] The concentration of toxic chemicals in plasma is relatively stable, and the level of styrene exposure in the human body can be accurately assessed by measuring the content of phenylglyoxylic acid and mandelic acid in plasma. However, the plasma matrix is ​​more complex, containing fat, protein, phospholipids and inorganic salts, which brings greater challenges to phenylglyoxylic acid and mandelic acid. It is necessary to select appropriate sample pretreatment technology to achieve accurate determination. In addition, due to factors such as occupational exposure levels and individual differences, the concentration span of phenylglyoxylic acid and mandelic acid in plasma is large (changing from μg / L concentration level to mg / L concentration level). Existing technologies rely on a single liquid chromatography-ultraviolet detection method (HPLC-UV) and liquid chromatography-mass spectrometry (LC-MS) to achieve simultaneous detection of phenylglyoxylic acid and mandelic acid at different concentration levels. Summary of the invention

[0005] The problem to be solved by the present invention is how to develop an extraction and purification technology for phenylglyoxylic acid and mandelic acid, styrene metabolites in plasma, so as to achieve the purpose of quickly and accurately detecting the concentration of trace amounts of phenylglyoxylic acid and mandelic acid in plasma samples.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a core-shell structure adsorbent, including ferroferric oxide magnetic microspheres, petal-shaped titanium dioxide coated on the surface of the ferroferric oxide magnetic microspheres, and a quaternary amine-carbon 18 bifunctionalized oligomeric polymer compound modified on a partial surface area of ​​the petal-shaped titanium dioxide.

[0007] The core of the adsorbent of the present invention is ferroferric oxide magnetic microspheres. The adsorbent as a whole is magnetic and can achieve rapid solid-liquid magnetic separation, thereby improving sample purification efficiency. The titanium dioxide component in the material can effectively adsorb phospholipid components in plasma, and the modification of the titanium dioxide surface with quaternary amine-carbon 18 bifunctionalized oligomeric polymer compounds can not only effectively retain the adsorption performance of titanium dioxide, but also the quaternary amine-carbon 18 bifunctionalized oligomeric polymer components can effectively adsorb impurities such as fat and inorganic anions in plasma, thereby effectively reducing the matrix interference effect in the sample detection process, so as to achieve the purpose of quickly and accurately detecting the concentration of trace phenylglyoxylic acid and mandelic acid in plasma samples.

[0008] The second aspect of the present invention provides a method for preparing the core-shell structure adsorbent, comprising the following steps:

[0009] S1, preparing ferroferric oxide magnetic microspheres;

[0010] S2, modifying the surface of the magnetic microspheres obtained in step S1 with titanium dioxide by a solvothermal reaction and calcining at high temperature to obtain magnetic microspheres with petal-shaped titanium dioxide coated on the surface;

[0011] S3, modifying partial surface areas of the magnetic microspheres obtained in step S2 with sodium thiopropane sulfonate by directional coupling technology to achieve double-bond modification of the surface of the magnetic microspheres;

[0012] S4. Using microwave-assisted distillation precipitation polymerization reaction, 1-octadecene and dimethyldiallylammonium chloride are used as functional monomers to modify the surface of the magnetic microspheres obtained in step S3 with a quaternary amine-carbon 18 bifunctional oligomeric polymer compound to prepare core-shell structured quaternary amine-carbon 18 bifunctional magnetic titanium dioxide oligomeric polymer composite microspheres.

[0013] The preparation method of the present invention first modifies the surface of Fe3O4 magnetic microspheres with titanium dioxide that has been calcined at high temperature, which can effectively remove the phospholipid components in the plasma sample; and then continues to modify the oligomeric polymer components in a partial area of ​​the titanium dioxide surface through microwave-assisted distillation precipitation polymerization reaction, which can simultaneously exert the adsorption properties of titanium dioxide and functionalized oligomeric polymer components, thereby effectively reducing the matrix interference effect in the detection process of plasma phenylglyoxylic acid and mandelic acid.

[0014] Furthermore, the step S2 specifically includes: dispersing the ferroferric oxide magnetic microspheres in a solvent, adding tetrabutyl titanate to the dispersion, reacting the mixture at 150-250°C for 6-12 hours, washing and drying the reaction product, and then calcining at 450-600°C for 2-8 hours to obtain titanium dioxide-modified ferroferric oxide magnetic microspheres. After high-temperature calcination, the titanium dioxide component is in a thin sheet shape, and the magnetic microspheres have a flower-like structure as a whole, which is conducive to increasing the active adsorption sites of the material.

[0015] Furthermore, the step S3 specifically includes: dispersing titanium dioxide modified ferroferric oxide magnetic microspheres in a solvent, adding triethylamine to the dispersion, adding sodium thiopropane sulfonate (MPS) solution under nitrogen protection, reacting at 60-80° C. for 8-16 hours, washing and drying the reaction product, and obtaining double-bond modified titanium dioxide-based magnetic microspheres. MPS is modified on the surface of the flaky titanium dioxide component by directional coupling technology to achieve double-bond modification of a partial area of ​​the material surface.

[0016] Furthermore, the step S4 specifically includes: dispersing the double-bond modified titanium dioxide-based magnetic microspheres in a solvent, placing the container in a microwave reactor, adding 1-octadecene, dimethyldiallyl ammonium chloride, a crosslinking agent, and an initiator mixed solution to the dispersion, heating to 78-80°C, maintaining for 20-40 minutes, and then heating to 81-82°C, condensing and refluxing for 2-3 hours, washing and drying the reaction product, and obtaining a core-shell structure quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres. 18 The group can effectively improve the acid and alkali resistance of the material; the ratio between titanium dioxide on the surface of the magnetic microspheres and the quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer components can be flexibly adjusted according to the sample type and the type of matrix impurities, so as to achieve the simultaneous removal of multiple impurities and achieve the purpose of reducing the matrix interference effect in the detection process of the target analyte.

[0017] Furthermore, in step S4, the molar ratio of 1-octadecene to dimethyldiallylammonium chloride is 1:(0.5-2). When the two functional monomers are within the specified ratio range, the core-shell structure adsorbent has the best purification performance for plasma samples.

[0018] The third aspect of the present invention provides a method for detecting the concentration of styrene metabolites in plasma, using the core-shell structure adsorbent described above, the detection method comprising the following steps:

[0019] A. Transfer 500 μL of plasma sample into a centrifuge tube, add concentrated hydrochloric acid for acid hydrolysis, and then add acetonitrile for vortex extraction;

[0020] B. Add sodium chloride and anhydrous magnesium sulfate powder, vortex mix and centrifuge at high speed;

[0021] C. Take out the upper acetonitrile extract into another centrifuge tube, then add the core-shell structure adsorbent, vortex extract for 5 minutes, and then place it on a magnetic stand for magnetic separation;

[0022] D. Aspirate the supernatant, blow dry with nitrogen gas, reconstitute with methanol-water solution, and analyze by LC-MS-DAD.

[0023] Furthermore, in the step D, liquid chromatography separation is adopted, and the mass spectrometer detector and the diode array detector are connected in series, and the diode array detector is set with a detection wavelength of 254 nm for phenylglyoxylic acid and a detection wavelength of 225 nm for mandelic acid.

[0024] Furthermore, in step A, the amount of concentrated hydrochloric acid added is 40 to 50 μL.

[0025] Furthermore, in the step C, the amount of the core-shell structure adsorbent added is 20 to 50 mg.

[0026] In order to solve the problem of detecting high concentration span of phenylglyoxylic acid and mandelic acid in plasma, this paper proposes to use liquid chromatography (LC) separation, connect mass spectrometer (MS) and diode array detector (DAD) in series, and establish LC-MS-DAD to detect high concentration span of phenylglyoxylic acid and mandelic acid in plasma. This can give full play to the high qualitative ability and high sensitivity of mass spectrometer, and at the same time give full play to the accurate determination of high concentration of phenylglyoxylic acid and mandelic acid by DAD detector, so as to establish a simultaneous detection method of high concentration span of phenylglyoxylic acid and mandelic acid (0.001mg / L~500mg / L) in plasma, so as to meet the needs of phenylglyoxylic acid and mandelic acid detection in plasma.

[0027] The fourth aspect of the present invention provides a kit, comprising the above-mentioned core-shell structure adsorbent, concentrated hydrochloric acid, sodium chloride, anhydrous magnesium sulfate, an anticoagulant tube, a syringe, a standard series of working solutions and a centrifuge tube.

[0028] The kit of the present invention can efficiently and quickly purify and process plasma samples, and can realize on-site sampling, processing and storage of plasma samples in conjunction with a magnetic stand and a small vortex instrument, greatly reducing the changes and losses of styrene metabolites phenylglyoxylic acid and mandelic acid during sampling and transportation, and improving the accuracy of the test results.

[0029] Compared with the prior art, the beneficial effects of the present invention include:

[0030] (1) The core-shell structure adsorbent of the present invention can effectively extract and purify styrene metabolites phenylglyoxylic acid and mandelic acid in plasma samples, and reduce matrix interference effects. The adsorbent has a petal-shaped core-shell structure on the surface, which is beneficial to increase the specific surface area of ​​the adsorbent, thereby increasing the active adsorption sites; the surface of the magnetic microspheres is modified with titanium dioxide calcined at high temperature, which can effectively remove the phospholipid components in the plasma sample; through microwave-assisted distillation precipitation polymerization, the surface of titanium dioxide is modified with oligomeric polymer components (not completely covering titanium dioxide), which can simultaneously exert the adsorption properties of titanium dioxide and functionalized oligomeric polymer components, and can adsorb phospholipids in plasma through titanium dioxide components, and can also adsorb impurities such as fat and inorganic salts in plasma through functionalized oligomeric polymers, thereby effectively reducing the matrix interference effect in the detection process of plasma phenylglyoxylic acid and mandelic acid.

[0031] (2) The core-shell structure adsorbent of the present invention has high extraction and purification efficiency for plasma samples. The quaternary amine-carbon 18 dual functional design has a synergistic effect and can more efficiently remove impurities such as fat and inorganic salts in plasma, thereby greatly improving the purification efficiency of plasma samples; the polymerization reaction method is used to graft C 18 Functional groups, rather than traditional silanization reaction grafting, can increase the acid and alkali resistance of the material and make the material more stable.

[0032] (3) The core-shell structure adsorbent of the present invention is used to extract phenylglyoxylic acid and mandelic acid from plasma samples, which can shorten the operation time and reduce costs. Compared with the purification results of the commercial PRiME HLB solid phase extraction column, the core-shell structure adsorbent has a better ability to remove matrix interference, and can achieve accurate quantitative analysis without the use of a matrix matching working curve or an internal standard method, which reduces the tediousness of the matrix matching working curve preparation process. Not using the internal standard method for quantification can greatly reduce costs; because the magnetic dispersion solid phase extraction method can ensure that the adsorbent is in full contact with the sample matrix solution, it can be fully mixed with the target analyte to achieve the purpose of complete adsorption; at the same time, compared with the traditional dispersion solid phase extraction method, the magnetic solid phase extraction method used in the present invention can achieve rapid magnetic separation (10s) under the action of a magnetic field, eliminating time-consuming operations such as centrifugation and filtration.

[0033] (4) In order to solve the problem of detecting high concentrations of phenylglyoxylic acid and mandelic acid in plasma, the present invention adopts liquid chromatography (LC) separation, connects mass spectrometer (MS) and diode array detector (DAD) in series, and establishes LC-MS-DAD to detect the high concentrations of phenylglyoxylic acid and mandelic acid in plasma. This method can give full play to the advantages of high qualitative ability and high sensitivity of mass spectrometer, and at the same time give full play to the accurate determination of high concentrations of phenylglyoxylic acid and mandelic acid by DAD detector, thereby establishing a simultaneous detection method of high concentrations of phenylglyoxylic acid and mandelic acid (0.001 mg / L to 500.0 mg / L) in plasma, which meets the needs of phenylglyoxylic acid and mandelic acid detection in plasma.

[0034] (5) The present invention provides a rapid test kit suitable for on-site sampling. In view of the urgency of styrene poisoning in petrochemical enterprises, the test kit includes the materials and instruments required for detection. In combination with a small vortex analyzer, on-site sampling, processing and storage of plasma samples can be achieved. Subsequently, the pre-treated sample solution only needs to be brought back to the laboratory for concentration and re-dissolution for detection. This greatly reduces the changes and losses of styrene metabolites phenylglyoxylic acid and mandelic acid during sampling and transportation, improves the accuracy of the test results, and provides more accurate guidance for clinical precision treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the chemical structural formula of phenylglyoxylic acid and mandelic acid.

[0036] Figure 2 This is the TME image of the core-shell structure adsorbent in Experimental Example 1.

[0037] Figure 3 This is the TEM-mapping elemental spectrum of the core-shell structure adsorbent in Experimental Example 1.

[0038] Figure 4 This is the TG-DTG spectrum of the core-shell structure adsorbent in Experimental Example 1.

[0039] Figure 5 This is the FTIR spectrum of the core-shell structure adsorbent in Experimental Example 1.

[0040] Figure 6 This is a test result diagram of the effect of the amount of concentrated hydrochloric acid used in the acidolysis solution on the extraction effect of phenylglyoxylic acid and mandelic acid in plasma in Experimental Example 2.

[0041] Figure 7 This is a test result diagram of the effect of the amount of core-shell structure adsorbent used in Experimental Example 3 on the extraction effect of phenylglyoxylic acid and mandelic acid in plasma.

[0042] Figure 8This is a test result diagram of the effect of the ratio of bifunctional monomers in the core-shell structure adsorbent in Experimental Example 4 on the extraction effect of phenylglyoxylic acid and mandelic acid in plasma. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0044] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0047] Example 1

[0048] In this embodiment, a core-shell structure adsorbent is prepared. The adsorbent material can extract and purify phenylglyoxylic acid and mandelic acid, metabolites of styrene, in plasma.

[0049] The preparation method comprises the following steps:

[0050] Step 1: Preparation of ferroferric oxide magnetic microspheres

[0051] Using the solvothermal method, 0.4g sodium citrate and 0.75g FeCl3·6H2O were weighed in a 250mL flask, 80mL ethylene glycol was added, and mechanical stirring was performed to dissolve for 30min at room temperature, and then 5g sodium acetate was added and stirred for 30min. The mixed solution was placed in a 100mL autoclave, reacted at 200℃ for 8h, and cooled to room temperature. Then, it was washed with pure water and ethanol for 3 times, and vacuum dried at 60℃ overnight to obtain Fe3O4 magnetic microspheres.

[0052] Step 2: Preparation of titanium dioxide modified ferroferric oxide magnetic microspheres

[0053] 1.5g Fe3O4 magnetic microspheres were ultrasonically dispersed in 20mL DMF and 60mL isopropanol, and then 4mL tetrabutyl titanate (TBOT) was added dropwise under ultrasonic conditions within 10min, and the mixture was transferred to an autoclave, and then reacted at 200℃ for 10h. The reaction product was washed several times with anhydrous ethanol and dried at 60℃ overnight to obtain Fe3O4@TiO2. Fe3O4@TiO2 was calcined at 500℃ for 5h to obtain titanium dioxide-modified ferroferric oxide magnetic microspheres (Fe3O4@flower TiO2).

[0054] Step 3: Preparation of double-bond modified titanium dioxide-based magnetic microspheres

[0055] Weigh 1.5g Fe3O4@flower TiO2 microspheres, disperse them in 250mL isopropanol, add 2mL triethylamine while stirring, add 25mL MPS (6mL) ethanol solution dropwise under nitrogen protection, react at 70℃ for 12h, wash with pure water and ethanol three times to obtain Fe3O4@flower TiO2-MPS microspheres modified with double bonds.

[0056] Step 4: Preparation of core-shell structured quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres

[0057] 1.5g Fe3O4@flower TiO2-MPS microspheres were ultrasonically dispersed in 200mL isopropanol and placed in a 500mL three-necked flask. The three-necked flask was placed in a microwave reactor and mechanically stirred for 10min. 1.0g divinylbenzene, 1.0g 1-octadecene, 0.4g dimethyldiallylammonium chloride and 0.1g AIBN were dissolved in 250mL acetonitrile and added to the above dispersion. Under ultrasonic-assisted mechanical stirring, the temperature was raised to 78-80°C and maintained for 30min, then rapidly raised to 81-82°C and condensed and refluxed for 2h. The product obtained by the reaction was washed three times with pure water and ethanol to obtain a petal-shaped core-shell structure quaternary amine-C 18 Dual-functionalized magnetic titanium dioxide oligomer composite microspheres.

[0058] Example 2

[0059] The difference between this embodiment and embodiment 1 is that the amount of 1-octadecene used in step 4 is 0.5 g.

[0060] Example 3

[0061] The difference between this embodiment and embodiment 1 is that the amount of dimethyldiallylammonium chloride used in step 4 is 0.6 g.

[0062] Example 4

[0063] The difference between this embodiment and embodiment 1 is that in step 4, the amount of 1-octadecene used is 0.5 g, and the amount of dimethyldiallylammonium chloride used is 0.3 g.

[0064] Example 5

[0065] The difference between this embodiment and embodiment 1 is that in step 4, the amount of 1-octadecene used is 1.1 g, and the amount of dimethyldiallylammonium chloride used is 1.0 g.

[0066] Example 6

[0067] This embodiment provides a kit for extracting and purifying phenylglyoxylic acid and mandelic acid, metabolites of styrene in plasma, comprising a core-shell structure adsorbent, concentrated hydrochloric acid, an anticoagulant tube, a syringe, a standard series of working solutions, and a centrifuge tube. The core-shell structure adsorbent used in the kit is a petal-shaped core-shell structure quaternary amine-C prepared in any one of Embodiments 1 to 5. 18 Dual-functionalized magnetic titanium dioxide oligomer composite microspheres.

[0068] Example 7

[0069] In this embodiment, the kit of Example 6 is used to extract phenylglyoxylic acid and mandelic acid, the styrene metabolites, from the plasma sample, including the following steps: 500 μL of plasma sample is transferred to a 5 mL polypropylene centrifuge tube, 40 to 50 μL of concentrated hydrochloric acid is added for acid hydrolysis for 10 min, and then 1.0 mL of acetonitrile is added for vortex extraction for 5 min. 0.1 g of sodium chloride and 0.4 g of anhydrous magnesium sulfate powder are added, and after vortex mixing for 20 s, high-speed centrifugation at 15000 r / min is performed for 5 min. The upper acetonitrile extract is taken out and placed in another 2 mL polypropylene centrifuge tube, and then 10 to 20 mg of core-shell structure adsorbent is added, vortexed for extraction for 5 min, and then placed on a magnetic stand for magnetic separation for 10 s, 0.5 mL of supernatant is drawn, dried with nitrogen, and reconstituted with 250 μL of 10% methanol aqueous solution, and analyzed by LC-MS-DAD sampling.

[0070] LC-MS instrument conditions are as follows:

[0071] (1) Liquid phase conditions

[0072] Liquid chromatography separation was performed using an Accucore Ph / Hexyl (100 mm×2.1 mm, 2.6 μm) column, with the column temperature set at 40°C, the flow rate at 0.3 mL / min, and the injection volume at 5.0 μL. Mobile phase A: 5 mM ammonium formate-0.1% formic acid aqueous solution (v / v), mobile phase B: methanol. Gradient elution conditions: 10% B (0.00-1.50 min), 90%-80% B (1.50-5.00 min), 80% B (5.00-7.00 min), 80%-5% B (7.00-8.50 min), 5% B (8.50-9.00 min), 5%-90% B (9.00-9.10 min), 10% B (9.10-12.00 min).

[0073] (2) Mass spectrometry conditions

[0074] Electrospray ion source (ESI-), negative ion mode and full scan-data-dependent secondary mass spectrometry scanning mode (Full mass-ddMS2), electrospray voltage-3.0 kV, desolvation gas pressure 275.8 kPa, auxiliary gas rate 180 L / h, backflush gas pressure 13.8 kPa, RF prism voltage 50%, auxiliary gas heating temperature 280 ° C, ion transfer tube heating temperature 200 ° C, primary scanning resolution 70000, secondary scanning resolution 35000, automatic gain control (AGC Target) 1E5, maximum injection time (Maximum IT) 100 ms, mass-to-charge ratio isolation window (Isolation window) 2.0 m / z. Other mass spectrometry conditions are shown in Table 1.

[0075] Table 1 Mass spectrometry parameters and retention times of phenylglyoxylic acid and mandelic acid

[0076]

[0077] (2)DAD conditions

[0078] The detection wavelength of phenylglyoxylic acid was set to 254 nm, and that of mandelic acid to 225 nm.

[0079] Comparative Example 1

[0080] Referring to step 1 and step 2 of Example 1, titanium oxide modified ferroferric oxide magnetic microspheres (Fe3O4@flower TiO2) were prepared.

[0081] Comparative Example 2

[0082] Referring to steps 1 to 4 of Example 1, the difference from Example 1 is that 1-octadecene is not added in step 4.

[0083] Comparative Example 3

[0084] Referring to step 1 to step 4 of Example 1, the difference from Example 1 is that dimethyldiallylammonium chloride is not added in step 4.

[0085] Experimental Example 1 Characterization of Core-Shell Structure Adsorbent

[0086] The morphology of the core-shell structure adsorbent prepared in Example 1 was characterized by transmission electron microscopy (TEM). Figure 2 As shown. Figure 2 It can be seen that the synthesized quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomeric polymer composite microspheres present a significant petal-shaped core-shell structure.

[0087] The elemental composition of the core-shell structure adsorbent prepared in Example 1 was characterized by TEM-mapping. Figure 3 shown. Figure 3 The distribution images of Fe, O, Ti, C, and N are displayed, which verifies the rationality of the material synthesis route and shows that magnetic TiO2, C 18 The groups and quaternary amine groups have been successfully assembled on the Fe3O4 surface.

[0088] The thermal stability of the core-shell structure adsorbent prepared in Example 1 was characterized by thermogravimetry-differential thermal (TG-DTG). Figure 4 As shown. Figure 4 It can be seen that the mass loss of quaternary ammonium-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres can be divided into three temperature stages. The mass loss at 154.7℃ is caused by the quaternary ammonium functional groups on the surface; the mass loss at 332.4℃ is caused by the carbon 18 The mass loss at 631.2℃ is caused by the dehydration bonding of Ti-OH on the TiO2 surface. The overall mass loss ratio of the material is 8.74%, which is mainly due to the loss of polymer components on the surface of the material. This further shows that only a small amount of bifunctionalized oligomeric polymer components are modified on the surface of Fe3O4@flower TiO2, and the coverage of TiO2 is not thorough, which is conducive to the synergistic effect of TiO2 and quaternary amine-carbon 18 bifunctionalized oligomeric polymer components.

[0089] The functional groups of the core-shell structure adsorbent prepared in Example 1 were characterized by infrared spectroscopy (FTIR). Figure 5 shown. Figure 5 Medium, 3422cm -1 The absorption of Ti-OH on the surface of the material can be attributed to ~2900cm -1 The absorption band at can be attributed to the surface C 18 Absorption of the group, ~1400cm-1 The absorption at 580 cm-1 can be attributed to the stretching vibration of the quaternary amine group. -1 The absorption of Fe3O4 can be attributed to this, which further proves that the quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres have been successfully synthesized.

[0090] Experimental Example 2 Effect of the amount of concentrated hydrochloric acid in the acid solution on the extraction efficiency

[0091] 0μL, 10μL, 20μL, 40μL, 50μL and 100μL of concentrated hydrochloric acid solution were selected as acid hydrolysis solution, and their effects on the dissociation and extraction efficiency of styrene metabolites phenylglyoxylic acid and mandelic acid in plasma were investigated.

[0092] Accurately transfer 6 portions of 500 μL plasma spiked samples (the spiked concentrations of phenylglyoxylic acid and mandelic acid are both 200 μg / L) into 5 mL polypropylene centrifuge tubes, add different volumes of concentrated hydrochloric acid to acidify for 10 min, add 1 mL of acetonitrile and vortex extract for 5 min, then add 0.1 g of sodium chloride and 0.4 g of anhydrous magnesium sulfate powder, vortex mix for 20 s, and then centrifuge at 15000 r / min for 5 min. Take out the upper acetonitrile extract into another 2 mL polypropylene centrifuge tube, then add 20 mg of the core-shell structure adsorbent prepared in Example 1, vortex extract for 5 min, then place on a magnetic stand for magnetic separation for 10 s, draw 0.5 mL of the supernatant, blow dry with nitrogen, reconstitute with 250 μL of 10% methanol aqueous solution, and analyze by LC-MS-DAD. The results are as follows: Figure 6 shown.

[0093] Depend on Figure 6 It can be seen that with the increase of the volume of concentrated hydrochloric acid in the acidolysis solution, the peak areas of phenylglyoxylic acid and mandelic acid gradually increased, and when the volume increased to 40 μL, the peak areas of the two reached the maximum value, and the peak areas of the two remained the same when the volume of concentrated hydrochloric acid was further increased.

[0094] Experimental Example 3 Effect of the amount of core-shell structure adsorbent on the extraction efficiency

[0095] Accurately transfer 7 portions of 500 μL plasma spiked samples (the spiked concentrations of phenylglyoxylic acid and mandelic acid are both 200 μg / L) into 5 mL polypropylene centrifuge tubes, add 40 μL hydrochloric acid to acidify for 10 min, then add 1 mL acetonitrile and vortex extract for 5 min, then add 0.1 g sodium chloride and 0.4 g anhydrous magnesium sulfate powder, vortex mix for 20 s, and then centrifuge at 15000 r / min for 5 min. Take out the upper acetonitrile extract and put it into another 2 mL polypropylene centrifuge tube, add 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg and 200 mg of the core-shell structure adsorbent prepared in Example 1, vortex extract for 5 min, vortex extract for 5 min, then place it on a magnetic stand for magnetic separation for 10 s, draw 0.5 mL of the supernatant, blow dry with nitrogen, reconstitute with 250 μL 10% methanol aqueous solution, and analyze by LC-MS-DAD injection. The results are as follows Figure 7 shown.

[0096] Depend on Figure 7 It can be seen that when the amount of adsorbent is small, the purification effect is poor, and the peak areas of phenylglyoxylic acid and mandelic acid are small; when the amount of adsorbent is 20 mg, the peak areas of the two reach the maximum value, and the peak areas of the two remain basically unchanged when the amount of adsorbent is further increased.

[0097] Experimental Example 4 Effect of the ratio of bifunctional monomers in core-shell structure adsorbent on extraction efficiency

[0098] Accurately transfer 4 portions of 500 μL plasma spiked samples (the spiked concentrations of phenylglyoxylic acid and mandelic acid are both 200 μg / L) into 5 mL polypropylene centrifuge tubes, add 40 μL hydrochloric acid to acidify for 10 min, then add 1 mL acetonitrile and vortex extract for 5 min, then add 0.1 g sodium chloride and 0.4 g anhydrous magnesium sulfate powder, vortex mix for 20 s, and then centrifuge at 15000 r / min for 5 min. Take out the upper acetonitrile extract and put it into another 2 mL polypropylene centrifuge tube, add 20 mg of the adsorbent prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively, vortex extract for 5 min, then place it on a magnetic stand for magnetic separation for 10 s, draw 0.5 mL of the supernatant, blow dry with nitrogen, reconstitute with 250 μL 10% methanol aqueous solution, and analyze by LC-MS-DAD injection. The results are as follows Figure 8 shown.

[0099] The experimental results show that when the two functional monomers dimethyldiallylammonium chloride and 1-octadecene are not used in the preparation process of the adsorbent (Comparative Example 1), the purification performance of the adsorbent for phenylglyoxylic acid and mandelic acid in plasma is poor; when only one of the functional monomers is used (Comparative Examples 2 and 3), the purification performance of phenylglyoxylic acid and mandelic acid in plasma is worse than the purification performance when both functional monomers are used (Example 1), but the performance is better than Comparative Example 1. It can be seen that the addition of the two functional monomers petal-shaped core-shell structure quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres can improve the purification performance of the adsorbent for plasma samples, and when the molar ratio of the two is close, the purification performance is optimal.

[0100] Experimental Example 5 Evaluation of Matrix Effect

[0101] This experimental example compares the ability of the kit of Example 6 to resist matrix interference with the commercial Waters PRiME HLB solid phase extraction column.

[0102] Accurately pipette 500 μL of blank plasma sample into 5 mL polypropylene centrifuge tubes, add 40 μL of hydrochloric acid to acidify for 10 min, then add 1 mL of acetonitrile and vortex extract for 5 min, then add 0.1 g of sodium chloride and 0.4 g of anhydrous magnesium sulfate powder, vortex mix for 20 s, and then high-speed centrifuge at 15000 r / min for 5 min. The upper acetonitrile extract was taken out and placed in another 2 mL polypropylene centrifuge tube, and then 20 mg of the core-shell structure adsorbent prepared in Example 1 was added, and vortex extraction was performed for 5 min. Subsequently, the extract was placed on a magnetic stand for magnetic separation for 10 s, and 0.5 mL of the supernatant was drawn, dried with nitrogen, and reconstituted with 250 μL of 10% methanol aqueous solution. Then, appropriate amounts of standard stock solutions of phenylglyoxylic acid and mandelic acid were added respectively, and the purified plasma extract was used to prepare 1 μg / L, 5 μg / L, 10 μg / L, 100 μg / L, 500 μg / L and 1000 μg / L standard series solutions. At the same time, the matrix matching working curve was cleaned up with a Waters PRiME HLB solid phase extraction column and compared with a 10% methanol aqueous solution to prepare a 1 μg / L, 5 μg / L, 10 μg / L, 100 μg / L, 500 μg / L and 1000 μg / L solvent standard series solutions.

[0103] The formula [matrix effect η = (matrix matching standard curve slope K a -Solvent standard curve slope K b ) / Solvent standard curve slope K b 】The matrix effect of petal-shaped core-shell structured quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomeric polymer composite microspheres in the extraction and purification of phenylglyoxylic acid and mandelic acid in plasma was evaluated. The results are shown in Table 1.

[0104] Table 2 Comparison of the ability of the kit of the present invention and commercial solid phase extraction column to resist matrix interference

[0105]

[0106]

[0107] As shown in Table 2, the petal-shaped core-shell structure quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomer composite microspheres of the present invention can effectively reduce the matrix effect in the extraction and purification process of phenylglyoxylic acid and mandelic acid in plasma samples, and the absolute value of the matrix effect η1 is less than 10%, which is a weak matrix effect and can be ignored, that is, there is no need to prepare a matrix matching working curve or use an isotope internal standard in the quantitative process. For the commercialized Waters PRiME HLB solid phase extraction column, the absolute value of its matrix effect η2 is ~30%, which is a medium-intensity matrix effect. It is necessary to use a matrix matching working curve or an isotope internal standard for quantitative analysis, which increases the cumbersomeness of the experimental operation and increases the operating cost of the detection method. Therefore, the kit of the present invention can effectively remove interfering impurities in plasma, reduce the matrix interference effect of phenylglyoxylic acid and mandelic acid, and improve the accuracy of phenylglyoxylic acid and mandelic acid detection in plasma, with the advantages of fast and simple detection and accurate results.

[0108] Experimental Example 6 Evaluation of LC-MS-DAD Detection Method

[0109] LC-MS-DAD was used to simultaneously detect the contents of phenylglyoxylic acid and mandelic acid in plasma. The results are shown in Table 3. For LC-MS, phenylglyoxylic acid and mandelic acid showed a good linear relationship in the concentration range of 0.001 to 1.0 mg / L (correlation coefficient R 2 >0.999); for LC-DAD, phenylglyoxylic acid and mandelic acid showed a good linear relationship in the concentration range of 1.0~500.0mg / L (correlation coefficient R 2 >0.999). The LC-MS-DAD method established by the present invention can realize the accurate detection of phenylglyoxylic acid and mandelic acid in plasma within the concentration range of 0.001 to 500.0 mg / L.

[0110] Table 3 Parameters of LC-MS-DAD detection of phenylglyoxylic acid and mandelic acid in plasma

[0111]

[0112]

[0113] Experimental Example 7 Evaluation of accuracy, precision, method detection limit and quantification limit

[0114] This experimental example investigates the accuracy and precision of the kit used in Implementation 6 in extracting phenylglyoxylic acid and mandelic acid from plasma samples.

[0115] The spiked levels of phenylglyoxylic acid and mandelic acid in the plasma samples were controlled to be 0.01, 0.10, 0.80, 2.0, 100.0 and 400.0 mg / L, respectively. 500 μL of spiked plasma samples were accurately transferred to a 5 mL polypropylene centrifuge tube, 40 μL of hydrochloric acid was added for acidification for 10 min, and then 1 mL of acetonitrile was added for vortex extraction for 5 min, followed by 0.1 g of sodium chloride and 0.4 g of anhydrous magnesium sulfate powder, vortex mixing for 20 s, and then high-speed centrifugation at 15000 r / min for 5 min. The upper acetonitrile extract was taken out and placed in another 2 mL polypropylene centrifuge tube, and then 20 mg of the core-shell structure adsorbent prepared in Example 1 was added, vortexed for 5 min, and then placed on a magnetic stand for magnetic separation for 10 s, 0.5 mL of the supernatant was drawn, dried with nitrogen, and reconstituted with 250 μL of 10% methanol aqueous solution, and analyzed by LC-MS-DAD injection.

[0116] The results are shown in Table 4. The spiked recovery of phenylglyoxylic acid and mandelic acid in plasma extracted and purified by the kit method is between 88.6% and 99.2%, and the relative standard deviations (RSDs) are 1.1% to 5.5%. The detection limit and quantification limit of the method are defined by the signal-to-noise ratio S / N≥3 and S / N≥9, respectively. The detection limit and quantification limit of the method developed by the kit for phenylglyoxylic acid and mandelic acid in plasma are 0.3 to 0.4 μg / L and 0.9 to 1.2 μg / L, respectively. It can be seen that the extraction and purification method based on the kit of the present invention has the advantages of rapidity, sensitivity and accuracy.

[0117] Table 4 Method spike recovery, precision, detection limit and quantification limit (n = 6)

[0118]

[0119]

[0120] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A core-shell structure adsorbent, characterized in that: The core-shell structure adsorbent comprises ferroferric oxide magnetic microspheres, petal-shaped titanium dioxide coated on the surface of the ferroferric oxide magnetic microspheres, and a quaternary amine-carbon 18 dual-functionalized oligomeric polymer compound modified on a partial area of ​​the surface of the petal-shaped titanium dioxide. The preparation method of the core-shell structure adsorbent comprises the following steps: S1, preparing ferroferric oxide magnetic microspheres; S2, modifying the surface of the magnetic microspheres obtained in step S1 with titanium dioxide by a solvothermal reaction and calcining at high temperature to obtain magnetic microspheres with petal-shaped titanium dioxide coated on the surface; S3, modifying a part of the surface of the magnetic microspheres obtained in step S2 with sodium thiopropane sulfonate by directional coupling technology to achieve double bond modification on the surface of the magnetic microspheres; S4, using microwave-assisted distillation precipitation polymerization, using 1-octadecene and dimethyldiallylammonium chloride as functional monomers, modifying the surface of the magnetic microspheres obtained in step S3 with quaternary amine-C 18 The dual-functionalized oligomeric polymer compound is used to prepare core-shell structured quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomeric polymer composite microspheres.

2. A method for preparing a core-shell structure adsorbent as claimed in claim 1, characterized in that: The following steps are involved: S1, preparing ferroferric oxide magnetic microspheres; S2, modifying the surface of the magnetic microspheres obtained in step S1 with titanium dioxide by a solvothermal reaction and calcining at high temperature to obtain magnetic microspheres with petal-shaped titanium dioxide coated on the surface; S3, modifying a part of the surface of the magnetic microspheres obtained in step S2 with sodium thiopropane sulfonate by directional coupling technology to achieve double bond modification on the surface of the magnetic microspheres; S4, using microwave-assisted distillation precipitation polymerization, using 1-octadecene and dimethyldiallylammonium chloride as functional monomers, modifying the surface of the magnetic microspheres obtained in step S3 with quaternary amine-C 18 The dual-functionalized oligomeric polymer compound is used to prepare core-shell structured quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomeric polymer composite microspheres.

3. The preparation method according to claim 2, characterized in that: The step S4 specifically comprises: dispersing double-bond modified titanium dioxide-based magnetic microspheres in a solvent, placing the container in a microwave reactor, adding 1-octadecene, dimethyldiallylammonium chloride, a crosslinking agent, and an initiator mixed solution to the dispersion, heating the mixture to 78-80° C., maintaining the mixture for 20-40 min, and then heating the mixture to 81-82° C., condensing and refluxing for 2-3 h, washing and drying the reaction product, and obtaining core-shell structured quaternary amine-carbon 18 dual-functionalized magnetic titanium dioxide oligomeric polymer composite microspheres.

4. The preparation method according to claim 3, characterized in that: In the step S4, the molar ratio of 1-octadecene to dimethyldiallylammonium chloride is 1:(0.5-2).

5. The preparation method according to claim 2, characterized in that: The step S2 specifically includes: dispersing ferroferric oxide magnetic microspheres in a solvent, adding tetrabutyl titanate to the dispersion, reacting the mixture at 150-250° C. for 6-12 hours, washing and drying the reaction product, and then calcining at 450-600° C. for 2-8 hours to obtain titanium dioxide-modified ferroferric oxide magnetic microspheres.

6. A method for detecting the concentration of styrene metabolites in plasma, characterized in that: Using the core-shell structure adsorbent as claimed in claim 1, the detection method comprises the following steps: A. Transfer 500 μL of plasma sample into a centrifuge tube, add concentrated hydrochloric acid for acid hydrolysis, and then add acetonitrile for vortex extraction; B. Add sodium chloride and anhydrous magnesium sulfate powder, vortex mix and centrifuge at high speed; C. Take out the upper acetonitrile extract into another centrifuge tube, then add the core-shell structure adsorbent, vortex extract for 5 minutes, and then place it on a magnetic stand for magnetic separation; D. Aspirate the supernatant, blow dry with nitrogen gas, reconstitute with methanol-water solution, and analyze by LC-MS-DAD.

7. The method for detecting the concentration of styrene metabolites in plasma according to claim 6, characterized in that: In the step D, liquid chromatography separation is adopted, and a mass spectrometer detector and a diode array detector are connected in series. The diode array detector is set with a detection wavelength of 254 nm for phenylglyoxylic acid and a detection wavelength of 225 nm for mandelic acid.

8. The method for detecting the concentration of styrene metabolites in plasma according to claim 6, characterized in that: In step A, the amount of concentrated hydrochloric acid added is 40-50 μL.

9. The method for detecting the concentration of styrene metabolites in plasma according to claim 6, characterized in that: In the step C, the amount of the core-shell structure adsorbent added is 20-50 mg.

10. A kit, characterized in that: The invention comprises the core-shell structure adsorbent as claimed in claim 1, concentrated hydrochloric acid, an anticoagulant tube, a syringe, a standard series working solution and a centrifuge tube.

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