Magnetic nano material for enriching glucocorticoid and preparation method of magnetic nano material

Abstract

The invention discloses a magnetic nano material for enriching glucocorticoid and a preparation method thereof, the magnetic nano material comprises: a core formed by ferroferric oxide; the shell covers at least part of the surface of the core body, and the shell is composed of a repeating unit shown in the formula I in the specification. The magnetic nano material is high in stability and large in specific surface area, has a classical mesoporous structure and good heat resistance, has specific adsorbability to glucocorticoid, is large in adsorption capacity, is high in magnetization intensity, can be quickly dispersed in a sample solution and can quickly realize magnetic separation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular, to a magnetic nanomaterial for enriching glucocorticoids and a preparation method thereof. BACKGROUND

[0002] Glucocorticoids are a kind of steroid hormones, and the molecular structure features thereof are containing cyclopentane and polycyclic phenanthrene. Glucocorticoids have the effects of anti-inflammatory, detoxification and anti-shock, and thus are usually used in combination with other veterinary drugs. The administration of prednisolone to lactating cows to solve bovine mastitis through intramammary administration poses a great threat to dairy product production. In addition, the high tolerance of animals to these drugs requires high-dose administration, and combined with improper treatment and drug withdrawal duration, which can lead to drug residues in animal-derived foods. A large number of studies have shown that drug residues caused by improper use of glucocorticoids in livestock can cause diseases such as obesity, hypertension, osteoporosis, vasodilation, immunosuppression and diabetes in humans. And long-term use of steroid anti-inflammatory drugs can lead to the development of drug resistance and promote the spread of pathogenic drug-resistant strains. Therefore, it is crucial to monitor the level of glucocorticoids in animal-derived foods. Both China and the European Union prohibit the use of glucocorticoids as growth promoters in livestock and poultry, and the limit standards of dexamethasone and betamethasone in milk are both 0.3 mg / kg.

[0003] Therefore, in order to ensure that the glucocorticoid residues in food meet the requirements, a detection method with simple operation and high sensitivity needs to be studied. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, one object of the present application is to propose a magnetic nanomaterial for enriching glucocorticoids, which has strong stability, large specific surface area, classical mesoporous structure and good heat resistance, has specific adsorption to glucocorticoids, has large adsorption capacity, and can be used multiple times, and has high magnetization intensity, can be rapidly dispersed in a sample solution and can be rapidly magnetically separated.

[0005] According to one aspect of the present application, a magnetic nanomaterial for enriching glucocorticoids is provided. According to an embodiment of the present application, the magnetic nanomaterial includes: a core formed of ferroferric oxide; and a shell covering at least a part of a surface of the core, the shell being composed of a repeating unit represented by Formula I.

[0006]

[0007] The magnetic nanomaterial for enriching glucocorticoids has strong stability, large specific surface area, classical mesoporous structure and good heat resistance, has specific adsorption to glucocorticoids, has large adsorption capacity, and has high magnetization intensity with the ferriferrous oxide as a core body, can be rapidly dispersed in a sample solution and can be rapidly separated by magnetism, is especially suitable for adsorption and enrichment of glucocorticoids in complex samples such as food, significantly shortens the pretreatment time of the sample, has large adsorption capacity and fast adsorption speed, and can complete adsorption and desorption of glucocorticoids in a few minutes, has the advantages of simple operation, green environmental protection, high extraction efficiency and the like.

[0008] In addition, the magnetic nanomaterial for enriching glucocorticoids according to the above-mentioned embodiments of the application can have the following additional technical features:

[0009] According to the embodiments of the application, the shell has a porous network structure.

[0010] According to the embodiments of the application, the average pore size of the shell is 3-5 nm.

[0011] According to the embodiments of the application, the specific surface area of the shell is 300-350 m 2 ·g -1 .

[0012] According to the embodiments of the application, the average particle size of the core body is 300-350 nm, and the average particle size of the magnetic nanomaterial is 370-450 nm.

[0013] According to the embodiments of the application, the shell has crystal diffraction peaks at 2θ of 18.30°, 30.11°, 35.48°, 43.18°, 53.56°, 56.92° and 62.51° in X-ray powder diffraction data.

[0014] According to the embodiments of the application, the contact angle of the magnetic nanomaterial is 20.0-21.5°.

[0015] According to another aspect of the application, the application provides a method for preparing the aforementioned magnetic nanomaterial. According to the embodiments of the application, the method comprises: providing a ferriferrous oxide nanomaterial; contacting the ferriferrous oxide nanomaterial with an organic solution containing an amino monomer, ultrasonic treatment, and mixing and stirring to obtain a first mixed solution; and contacting an aldehyde monomer with the first mixed solution to perform a Schiff base reaction to obtain the magnetic covalent organic framework material.

[0016] The shell synthesized by the amino monomer and the aldehyde monomer is coated on the surface of the magnetic ferroferric oxide nanomaterial to form a magnetic covalent organic framework material, which constitutes the shell of the nanomaterial and has superparamagnetism and is easy to be adsorbed and separated. Meanwhile, the prepared shell has the advantages of high stability and large specific surface area, and has a classical mesoporous structure and good heat resistance, and has specific adsorption to glucocorticoids and large adsorption capacity. Moreover, the preparation method has simple requirements for test equipment, simple operation, effective energy saving, green environmental protection and easy popularization and application.

[0017] According to an embodiment of the present application, the aldehyde monomer is 1,3,5-trialdehyde phloroglucinol (TP), and the amino monomer is 1,3,6,8-tetra-(p-aminophenyl)-pyrene (PY).

[0018] According to an embodiment of the present application, the molar ratio of the aldehyde monomer to the amino monomer is 1-2:1, preferably 1.5:1.

[0019] According to an embodiment of the present application, the mass ratio of the ferroferric oxide nanomaterial to the amino monomer is 1:0.5-1.5, preferably 1:1.

[0020] According to an embodiment of the present application, the ultrasonic treatment time is 10-30 minutes.

[0021] According to an embodiment of the present application, the mixing and stirring is carried out at 55-65℃ and a rotation speed of 500-600 rpm for 40-80 minutes.

[0022] According to an embodiment of the present application, the Schiff base reaction is carried out at 55-65℃ for 3-5 hours.

[0023] According to an embodiment of the present application, the Schiff base reaction uses acetic acid as a catalyst.

[0024] According to another aspect of the present application, the present application provides a method for enriching glucocorticoids. According to an embodiment of the present application, the method is carried out by using the aforementioned magnetic nanomaterial for enriching glucocorticoids. Thus, the adsorption treatment by using the aforementioned magnetic nanomaterial has high specificity to glucocorticoids, good adsorption effect, fast adsorption speed, large adsorption capacity, and simple and fast operation, and is especially suitable for the extraction of glucocorticoids in complex samples. In the adsorption process, the magnetic nanomaterial greatly shortens the pretreatment time, and the adsorption and desorption of glucocorticoids can be completed in a few minutes by using a very small amount, which has the advantages of simple operation, green environmental protection, high extraction efficiency and the like. It should be noted that the magnetic nanomaterial has all the technical features and effects of the aforementioned magnetic nanomaterial, which will not be described here.

[0025] According to an embodiment of the present application, the glucocorticoid is at least one selected from dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide.

[0026] According to an embodiment of the present application, the glucocorticoid enriches the adsorption equilibrium of the magnetic nanomaterial in an amount of 80-175 mg / g.

[0027] According to an embodiment of the present application, the adsorption equilibrium time of the glucocorticoid enriching the magnetic nanomaterial is 1-5 min.

[0028] According to an embodiment of the present application, the amount of the magnetic nanomaterial is 0.8-1.2 mg based on 1 mL of the liquid sample.

[0029] According to an embodiment of the present application, the extraction time of the method is 4-8 min.

[0030] According to an embodiment of the present application, acetonitrile is used for the elution treatment.

[0031] According to an embodiment of the present application, the elution treatment time is 0.5-1.5 min, and the elution liquid volume is 4-6 mL.

[0032] According to another aspect of the present application, a method for detecting the content of glucocorticoid in a sample is provided. According to an embodiment of the present application, the method comprises: enriching the glucocorticoid in the sample by using the aforementioned method for enriching glucocorticoid, so as to obtain an extract; and analyzing and detecting the extract by using a high performance liquid chromatography-mass spectrometry system, so as to obtain the content of glucocorticoid in the sample. Thus, the aforementioned magnetic nanomaterial is used for adsorption treatment, which has high specificity for glucocorticoid, good adsorption effect, fast adsorption speed, large adsorption capacity, and simple and fast operation, and is especially suitable for the extraction of glucocorticoid in complex samples; the sample containing glucocorticoid obtained by adsorption is analyzed and detected by using a high performance liquid chromatography-mass spectrometry system, which has high accuracy and sensitivity, and is especially suitable for the quantitative detection of glucocorticoid in complex samples. It should be noted that the magnetic nanomaterial has all the technical features and effects of the aforementioned magnetic nanomaterial, which will not be described here.

[0033] According to an embodiment of the present application, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry system are as follows: a chromatographic column: Hypersil GOLD aQ column, specifications: 100*2.1mm, 1.9um; mobile phase: A phase: 5mmol / L ammonium formate solution containing 0.1% formic acid; B phase: 0.1% formic acid acetonitrile solution; flow rate of mobile phase: 0.3mL / min; injection volume: 3.0ul; gradient elution conditions: 0-1min, 27% B; 1-5min, 37% B; 5-12min, 75% B; 12-12.1min, 95% B; 12.1-15min, 95% B.

[0034] According to an embodiment of the present application, the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry system are as follows: monitoring mode: multiple reaction monitoring (MRM) mode under ESI(+) conditions; spray voltage: 5500V; ion source temperature: 500 DEG C; evaporation temperature: 5500 DEG C; curtain gas pressure: 30psi; collision gas pressure: 9psi.

[0035] According to an embodiment of the present application, the glucocorticoid is at least one selected from dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide.

[0036] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0037] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the accompanying drawings, wherein:

[0038] Figure 1 Formation of Fe3O4@PY-TP covalent bond and covalent organic framework structure according to an embodiment of the present application is shown;

[0039] Figure 2 Preparation flow chart of Fe3O4@PY-TP adsorbent according to an embodiment of the present application is shown;

[0040] Figure 3SEM images and TEM images of Fe3O4 nanomaterials and Fe3O4@PY-TP nanomaterials according to an embodiment of the present application are shown, A is the SEM image of Fe3O4, B is the SEM image of Fe3O4@PY-TP, C is the TEM image of Fe3O4, D is the TEM image of Fe3O4@PY-TP, E-H are EDS element maps of Fe3O4@PY-TP:

[0041] Figure 4 XPS spectra of 4Fe3O4 nanomaterials according to an embodiment of the present application are shown, wherein A is the total spectrum, B is the C1s spectrum, C is the N1s spectrum;

[0042] Figure 5 FI-TR spectra of magnetic nanomaterials according to an embodiment of the present application are shown, wherein TP (a), PY (b), Fe3O4 (b), Fe3O4@PY-TP (d);

[0043] Figure 6 Hysteresis curve diagrams of Fe3O4 and Fe3O4@PY-TP according to an embodiment of the present application are shown;

[0044] Figure 7 XRD results of Fe3O4 and Fe3O4@PY-TP according to an embodiment of the present application are shown;

[0045] Figure 8 TGA results of Fe3O4@PY-TP according to an embodiment of the present application are shown;

[0046] Figure 9 shows nitrogen adsorption desorption isotherms of Fe3O4@PY-TP and Fe3O4 (A), pore size distribution of Fe3O4@PY-TP (B) according to an embodiment of the present application;

[0047] Figure 10 Hydrophilicity results of Fe3O4@PY-TP according to an embodiment of the present application are shown;

[0048] Figure 11 A flow chart of MSPE according to an embodiment of the present application is shown;

[0049] Figure 12 The influence of extraction parameters on extraction recovery results according to an embodiment of the present application are shown: adsorbent dosage (A); adsorption time (B); PH value (C); salt concentration (D); elution solvent type (E); elution solvent volume (F); elution time (G);

[0050] Figure 13shows a schematic diagram of adsorption isotherms (A) and adsorption kinetics curves (B) according to one embodiment of the present application;

[0051] Figure 14 shows a schematic diagram of linear fitting curves of Langmuir model of six glucocorticoids according to one embodiment of the present application;

[0052] Figure 15 shows a schematic diagram of fitting curves of Freundlich model of six glucocorticoids according to one embodiment of the present application;

[0053] Figure 16 shows a schematic diagram of fitting results of pseudo-first-order quasi-kinetics model of six glucocorticoids according to one embodiment of the present application;

[0054] Figure 17 shows a schematic diagram of linear fitting results of pseudo-second-order quasi-kinetics model of six glucocorticoids according to one embodiment of the present application;

[0055] Figure 18 shows a schematic diagram of results of reusability study of Fe3O4@PY-TP according to one embodiment of the present application. DETAILED DESCRIPTION

[0056] Embodiments of the present application are described in detail below with reference to several drawings. The embodiments of this application described below are exemplary and are not intended to be limiting of the scope of the application, as defined by the appended claims.

[0057] It should be noted that the terms "first", "second", and the like, are used herein only to describe various elements, and do not imply or imply relative importance or a number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0058] According to an aspect of the present application, the present application provides a magnetic nanomaterial for enriching glucocorticoids. The magnetic nanomaterial for enriching glucocorticoids according to the embodiments of the present application has strong stability, large specific surface area, classical mesoporous structure and good heat resistance, has specific adsorption to glucocorticoids, has large adsorption capacity, and has high magnetization intensity with the ferriferrous oxide as the core, can be rapidly dispersed in sample solution and can be rapidly separated by magnetism, is especially suitable for adsorption and enrichment of glucocorticoids in complex samples such as food, significantly shortens the sample pretreatment time, has large adsorption capacity and fast adsorption speed, and can complete adsorption and desorption of glucocorticoids in a few minutes, has the advantages of simple operation, green environmental protection, high extraction efficiency and the like.

[0059] According to the embodiments of the present application, the magnetic nanomaterial has good thermal stability below 450°C.

[0060] According to the embodiments of the present application, the magnetic nanomaterial contains hydroxyl functional groups and has large specific surface area, and in some embodiments, the specific surface area reaches 300m 2· g -1 The above.

[0061] In order to facilitate understanding of the above-mentioned magnetic nanomaterial, the magnetic nanomaterial is explained and described herein, and according to the embodiments of the present application, the magnetic nanomaterial comprises:

[0062] (1) core

[0063] According to the embodiments of the present application, the core is formed by ferriferrous oxide.

[0064] (2) shell

[0065] According to the embodiments of the present application, the shell covers at least part of the surface of the core, and the shell is composed of the repeating units shown in formula I.

[0066] According to the embodiments of the present application, the shell has a porous network structure. Thus, the specific surface area is large and the adsorption capacity is high.

[0067] According to the embodiments of the present application, the average pore size of the shell is 3-5nm. Thus, it is proved that the covalent organic framework material of the shell has typical mesoporous structure, can form space embedding effect with target substances, and is easy to adsorb glucocorticoids.

[0068] According to the embodiments of the present application, the specific surface area of the shell is 300-350m 2 ·g -1 . Thus, the specific surface area is large, the adsorption efficiency is high, and the adsorption capacity is large.

[0069] According to the embodiment of the present application, the average particle size of the core is 300-350 nm, and the average particle size of the magnetic nanomaterial is 370-450 nm. In this way, the specific surface area of the magnetic nanomaterial is large, the shell thickness is large, and the adsorption capacity is strong.

[0070] According to the embodiment of the present application, the shell has crystal diffraction peaks at 18.30°, 30.11°, 35.48°, 43.18°, 53.56°, 56.92° and 62.51° in the X-ray powder diffraction data. In this way, the successful coating of the COF shell layer on the surface of Fe3O4 does not destroy the crystal structure of Fe3O4 itself, and the crystal structure of the shell material appears between 10-30°, indicating that the COF shell layer is successfully synthesized on the surface of the Fe3O4 nanomaterial.

[0071] According to the embodiment of the present application, the contact angle of the magnetic nanomaterial is 20.0-21.5°. In this way, the hydrophilic performance is good, and the sample solvent can be quickly dispersed in the sample solvent during the extraction process, and rapid enrichment can be achieved in a short time.

[0072] According to another aspect of the present application, a method for preparing the aforementioned magnetic nanomaterial is provided. According to the method for preparing the aforementioned magnetic nanomaterial of the present application, the shell synthesized by the amino monomer and the aldehyde monomer is coated on the surface of the magnetic magnetite nanomaterial to form a magnetic covalent organic framework material, which constitutes the shell of the nanomaterial and has superparamagnetic properties, is easy to adsorb and separate, and has the advantages of strong stability and large specific surface area, and has a classic mesoporous structure and good heat resistance, and has specific adsorption of glucocorticoids and large adsorption capacity. Moreover, the preparation method has simple requirements for test equipment, simple operation, effective energy saving, green environmental protection, and easy popularization and application.

[0073] Reference Figure 1 and 2 The method for preparing the aforementioned magnetic nanomaterial is explained and described as follows according to the embodiment of the present application, which comprises the following steps:

[0074] S100 providing a core

[0075] According to the embodiment of the present application, a magnetite nanomaterial is provided.

[0076] S200 ultrasonic stirring

[0077] According to the embodiment of the present application, the magnetite nanomaterial is contacted with an organic solution containing an amino monomer, and after ultrasonic treatment, mixing and stirring are performed to obtain a first mixed solution. In this way, the magnetite nanomaterial and the PY monomer containing an amino group are subjected to condensation reaction, so as to ensure that the organic monomer of the magnetic covalent organic framework can be successfully coated on the surface of the Fe3O4 nanoparticles.

[0078] According to the embodiment of the present application, the mass ratio of the ferroferric oxide nanomaterial to the amino monomer is 1:0.5-1.5, preferably 1:1. In this ratio, the ferroferric oxide nanomaterial and the amino monomer can be electrostatically adsorbed sufficiently, and the same monomer can be avoided from agglomeration.

[0079] According to the embodiment of the present application, the ultrasonic treatment time is 10-30 minutes. In this way, the ferroferric oxide and the amino monomer can be mixed and contacted sufficiently.

[0080] According to the embodiment of the present application, the mixing and stirring is carried out at 55-65℃, at a speed of 500-600 rpm for 40-80 minutes. In this temperature condition, the ferroferric oxide nanomaterial and the amino monomer can be electrostatically adsorbed, and suitable temperature conditions can be provided for the reaction between the two monomers.

[0081] S300 Schiff base reaction

[0082] According to the embodiment of the present application, the aldehyde monomer is contacted with the first mixed solution to carry out Schiff base reaction, so as to obtain the magnetic covalent organic framework material. In this way, Schiff base reaction occurs between the PY modified on Fe3O4 and the added TP monomer to form an imine bond, and the target covalent organic framework polymer is synthesized.

[0083] According to the embodiment of the present application, the aldehyde monomer is 1,3,5-trialdehyde phloroglucinol (TP), and the amino monomer is 1,3,6,8-tetra-(p-aminophenyl)-pyrene (PY). It should be noted that the selection of these two monomers is determined by the inventors after comparing a plurality of monomers and fully considering the spatial structure of the target adsorbate glucocorticoid.

[0084] According to the embodiment of the present application, the molar ratio of the aldehyde monomer to the amino monomer is 1-2:1, preferably 1.5:1. In this ratio, the two monomers participating in the reaction can react more completely.

[0085] According to the embodiment of the present application, the temperature of the Schiff base reaction is 55-65℃, and the time is 3-5 hours. In this way, the Schiff base reaction can be carried out efficiently and sufficiently.

[0086] According to the embodiment of the present application, the Schiff base reaction uses acetic acid as a catalyst. In this way, the Schiff base reaction can be catalyzed and carried out efficiently.

[0087] According to another aspect of the present application, the present application provides a method for enriching glucocorticoids. According to an embodiment of the present application, the method is performed by using the aforementioned magnetic nanomaterial for enriching glucocorticoids. Thus, the aforementioned magnetic nanomaterial is used for adsorption treatment, which has high specificity for glucocorticoids, good adsorption effect, fast adsorption speed, large adsorption capacity, and simple and fast operation, and is particularly suitable for extraction of glucocorticoids in complex samples. In the adsorption process, the magnetic nanomaterial greatly shortens the pretreatment time, and the adsorption and desorption of glucocorticoids can be completed in a few minutes by using a very small amount, which has the advantages of simple operation, green environmental protection, high extraction efficiency, etc.

[0088] According to an embodiment of the present application, the glucocorticoids are at least one selected from dexamethasone, cortisone, fluocinolone, prednisone, hydrocortisone and triamcinolone acetonide.

[0089] According to an embodiment of the present application, the adsorption equilibrium amount of the glucocorticoid-enriched magnetic nanomaterial is 80-175 mg / g. Thus, the magnetic nanomaterial has strong adsorption capacity and large adsorption amount, and can efficiently extract glucocorticoids in complex samples.

[0090] According to an embodiment of the present application, the adsorption equilibrium time of the glucocorticoid-enriched magnetic nanomaterial is 1-5 min. Thus, the magnetic nanomaterial has high adsorption rate and can quickly extract glucocorticoids in complex samples.

[0091] According to an embodiment of the present application, the amount of the magnetic nanomaterial is 0.8-1.2 mg based on 1 mL of liquid sample. Thus, the amount of the magnetic nanomaterial is beneficial to fully enrich low-concentration glucocorticoids, and avoids waste and reagent pollution caused by excessive magnetic nanomaterial.

[0092] According to an embodiment of the present application, the extraction time of the method is 4-8 minutes. Thus, the magnetic nanomaterial adsorption sites are fully contacted with the analyte, glucocorticoids are fully adsorbed, and the recovery rate and accuracy of detection are high.

[0093] According to an embodiment of the present application, acetonitrile is used for elution treatment. Thus, the extraction recovery rate of glucocorticoids is good and the results are similar, and the error is small.

[0094] According to an embodiment of the present application, the elution treatment time is 0.5-1.5 minutes, and the elution liquid volume is 4-6 mL. Thus, the extraction recovery rate of glucocorticoids is good and the results are similar, and the error is small.

[0095] According to another aspect of the present application, the present application provides a method for detecting the content of glucocorticoid in a sample to be tested. According to an embodiment of the present application, the method comprises: enriching glucocorticoid in the sample to be tested by using the aforementioned method for enriching glucocorticoid, so as to obtain an extract; and analyzing and detecting the extract by using a high performance liquid chromatography-mass spectrometry system, so as to obtain the content of glucocorticoid in the sample to be tested. Thus, the aforementioned magnetic nanomaterial is used for adsorption treatment, which is specific to glucocorticoid, has good adsorption effect, fast adsorption speed, large adsorption capacity, and is simple and fast to operate, and is especially suitable for extraction of glucocorticoid in a complex sample; the sample to be tested containing glucocorticoid obtained by adsorption is analyzed and detected by using the high performance liquid chromatography-mass spectrometry system, which is high in accuracy and sensitivity of detection, and is especially suitable for quantitative detection of glucocorticoid in a complex sample.

[0096] According to an embodiment of the present application, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry system are as follows: chromatographic column: Hypersil GOLD aQ column, specification: 100*2.1mm, 1.9μm; mobile phase: A phase: 5mmol / L ammonium formate solution containing 0.1% formic acid; B phase: 0.1% formic acid acetonitrile solution; flow rate of mobile phase: 0.3mL / min; injection volume: 3.0μL; gradient elution condition: 0-1min, 27% B; 1-5min, 37% B; 5-12min, 75% B; 12-12.1min, 95% B; 12.1-15min, 95% B. Thus, each glucocorticoid compound has suitable peak time and good peak shape, and each compound has good separation effect.

[0097] According to an embodiment of the present application, the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry system are as follows: monitoring mode: multiple reaction monitoring (MRM) mode under ESI(+) condition; spray voltage: 5500V; ion source temperature: 500℃; evaporation temperature: 5500℃; curtain gas pressure: 30psi; collision gas pressure: 9psi. Thus, the sensitivity and accuracy of detection of glucocorticoid are high.

[0098] According to an embodiment of the present application, the glucocorticoid is at least one selected from the group consisting of dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide.

[0099] The present application will be described below with reference to specific embodiments, and it should be noted that these embodiments are merely illustrative and should not be construed as limiting the present application.

[0100] The following will explain the schemes of the present application in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be purchased, for example, from Sigma.

[0101] Example 1

[0102] In this example, the magnetic nanomaterial is prepared according to the method of the present application, COF is synthesized by ammonia aldehyde condensation reaction as the shell, Fe3O4 nanoparticles are used as the core, and a magnetic COF material with core-shell structure is constructed, as follows:

[0103] 1. Preparation of Fe3O4 nanoparticles

[0104] FeCl3·6H2O (1.35 g) and NaAc (3.60 g) were dissolved in 50 mL of ethylene glycol, and the mixture was uniformly dispersed by continuous stirring at 60°C for 1 hour. Then the obtained yellow solution was transferred to a polytetrafluoroethylene-lined high-pressure reaction kettle, which was sealed and heated to 200°C, and reacted for 8 h. After the reaction was completed, the high-pressure reaction kettle was cooled to room temperature, and the obtained black magnetic product was washed with EtOH and ultrapure water alternately for 6 times until the supernatant was clear. Finally, the product was collected with an external magnet and dried at 60°C for standby use.

[0105] 2. Preparation of magnetic nanomaterial Fe3O4@PY-TP

[0106] The above-prepared Fe3O4 nanoparticles (100 mg) and PY (100 mg, 0.18 mM) were uniformly dispersed in a double-necked flask containing 25 mL of THF, and the system was uniformly mixed by ultrasonic for 15 min. Then the reaction system was transferred to a 60°C water bath for heating and mechanical stirring for 1 h at a stirring speed of 560 r / min. Subsequently, a solution of TP (50 mg, 0.24 mM) dissolved in THF (5 mL) was added to the system, and at the same time, 2 mL of AcOH solution as a catalyst was added dropwise into the double-necked flask. The system was mechanically stirred at 60°C water bath heating for 4 h. After the reaction was completed, the brown product was collected with an external magnet, washed with ACN and MeOH alternately until the supernatant was clear and transparent, and dried at 60°C for 12 h for further use. The preparation process of Fe3O4@PY-TP adsorbent is shown in Figure 2 .

[0107] Example 2

[0108] In this example, the magnetic nanomaterial prepared in Example 1 is characterized, as follows:

[0109] 1. SEM characterization

[0110] The surface characteristics of the synthesized Fe3O4 nanomaterials and the magnetic covalent organic framework material Fe3O4@PY-TP were analyzed using scanning electron microscopy. Figure 3 As shown in Figure A, the Fe3O4 nanomaterials exhibit a uniform spherical shape, are individually dispersed without agglomeration, and have a diameter of approximately 330 nm. Figure 3 As shown in Figure B, Fe3O4@PY-TP also appears as a uniform sphere with a diameter larger than that of Fe3O4 nanomaterials, approximately 400 nm. This indicates that the covalent organic framework (PY-TP) was successfully coated onto the surface of Fe3O4 nanomaterials.

[0111] 2. TEM characterization

[0112] Transmission electron microscopy was used to observe the morphological characteristics of the synthesized Fe3O4 nanomaterials and the magnetic covalent organic framework material Fe3O4@PY-TP. For example... Figure 3 As shown in Figure C, the surface of the bare Fe3O4 nanomaterial is smooth, and its diameter is approximately 330 nm. Figure 3 As shown in Figure D, it is easy to see that Fe3O4@PY-TP has a core-shell structure. A light-colored outer shell, approximately 44.93 nm thick, coats the surface of the Fe3O4 nanomaterial, and a magnetic covalent organic framework adsorbent was successfully synthesized on the surface. Furthermore, energy spectrum of elements (EDS) analysis more directly confirmed the presence of Fe, C, N, and O elements in Fe3O4@PY-TP, as shown in the results. Figure 3 As shown in EH, their composition and distribution are consistent with those of core-shell particles, further proving the existence of a core-shell structure.

[0113] 3. XPS Analysis

[0114] The chemical composition and structure of Fe3O4@PY-TP were characterized by XPS. Figure 4 As shown in AC, the peaks at 284, 399, and 531 eV are attributed to C1s, N1s, and O1s, respectively, confirming the presence of C, N, and O elements on the Fe3O4@PY-TP surface. Figure 4 In (B), the peaks at 284.12 and 284.97 eV correspond to CC and C=N, respectively. The π-π* satellite at 290.08 eV is allocated to extended delocalized electrons in the aromatic-ring-containing polymer, exhibiting binding energies several eV higher than the main peak. This same characteristic is also shown in the N1s spectrum. Figure 4 C). This figure shows the main peak (399.53 eV) and the π-π* satellite peak (403.68 eV).

[0115] 4. FT-IR analysis

[0116] FT-IR was used to analyze the functional groups and chemical bonds of the Fe3O4 nanomaterials and the magnetic covalent organic framework material Fe3O4@PY-TP synthesized in Example 1. The results are as follows: Figure 5 As shown, the 590 cm⁻¹ in Fe₃O₄@PY-TP (curve d) indicates... -1 The vibrational peak is attributed to the Fe-O-Fe vibration, proving the successful coating of COF on the surface of Fe3O4 nanomaterials. Furthermore, it can be seen more clearly in curve b that the peak at 3343 cm⁻¹... -1 and 3213cm -1 The vibrational peak at 1643 cm⁻¹ originates from the tensile vibration of NH₄⁺ in the PY monomer. In curve a, the peak is at 1643 cm⁻¹. -1 The vibration peak at this location originates from the C=O stretching vibration within the TP monomer. The characteristic peak of the C=N stretching vibration appears at 1619 cm⁻¹. -1 The result indicates that the Schiff base reaction of TP and PY successfully synthesized an imine bond. (3441cm) -1 The OH vibration peak at the position indicates that the modification of the hydroxyl group was successful.

[0117] 5. VSM Analysis

[0118] The hysteresis curves of the synthesized Fe3O4 nanomaterials and the magnetic covalent organic framework material Fe3O4@PY-TP in Example 1 are shown below. Figure 6 As shown, the magnetization of the bare Fe3O4 nanomaterial is 24.1 emu g. -1 The magnetization of Fe3O4@PY-TP is 66.7 emu g. -1 Although the COF shell affects the magnetism of Fe3O4 nanomaterials, they still exhibit significant magnetization. Therefore, Fe3O4@PY-TP, as a magnetic solid-phase extraction adsorbent, can meet the requirements for rapid separation of sample matrix solutions.

[0119] 7. XRD Analysis

[0120] The crystalline state of the Fe3O4 nanomaterials and the magnetic covalent organic framework Fe3O4@PY-TP synthesized in Example 1 was analyzed by XRD. Figure 7As shown, six characteristic peaks (30.08°, 35.43°, 43.08°, 53.46°, 56.94°, and 62.58°) at the same position in the 2θ angle range from 10° to 90° can be observed in Fe3O4 and Fe3O4@PY-TP. This is attributed to the crystal indices (220), (311), (400), (422), (511), and (440) of Fe3O4. This demonstrates that successfully coating the Fe3O4 surface with a COF shell does not disrupt the crystal structure of Fe3O4 itself. Furthermore, the COF material exhibits a crystal structure between 10° and 30°, indicating that a COF shell was successfully synthesized on the surface of Fe3O4 nanomaterials.

[0121] 8. TGA Analysis

[0122] The thermal stability of Fe3O4@PY-TP was studied using TGA. For example... Figure 8 As shown, with the temperature rising to approximately 450°C, there is a mass loss of about 13.2 wt%, which is attributed to the loss of residual moisture on the adsorbent. As the temperature continues to rise to 800°C, the weight will decrease by approximately 36.2 wt% due to the loss of the COF shell layer. This demonstrates that Fe3O4@PY-TP provides good thermal stability up to 450°C.

[0123] 9. Specific surface area and porosity detection of Fe3O4@PY-TP

[0124] The specific surface area of ​​Fe3O4@PY-TP is as follows Figure 9A As shown, the aperture distribution is as follows Figure 9B As shown. From Figure 9B It can be seen that bare Fe3O4 primarily exhibits a type II isotherm, indicating its non-porous structure. Fe3O4@PY-TP demonstrates a type IV isotherm in nitrogen adsorption-desorption experiments, which may be due to adsorbate condensation within the material's pores. The specific surface area of ​​Fe3O4@PY-TP is 329.7 m². 2 g -1 The specific surface area of ​​Fe3O4 is 7.97 m². 2 g -1 Furthermore, the average pore size of Fe3O4@PY-TP is 3.91 nm, demonstrating that this magnetic covalent organic framework material has a typical mesoporous structure. These results indicate that Fe3O4@PY-TP provides a large specific surface area and sufficient adsorption sites for glucocorticoids, resulting in excellent adsorption performance.

[0125] 10. Hydrophilicity analysis of Fe3O4@PY-TP

[0126] The hydrophilicity of Fe3O4@PY-TP and Fe3O4@PY-TF was analyzed, and the results are as follows:Figure 10 It can be observed that the contact angle of Fe3O4@PY-TP is 20.79°, which has excellent hydrophilic performance, can be rapidly dispersed in the sample solvent during the extraction process, and can achieve rapid enrichment in a short time.

[0127] Therefore, the structural characteristics (including morphology characteristics, chemical composition and crystal structure) and performance (hydrophilicity and thermal stability) of Fe3O4@PY-TP are characterized by various characterization techniques. The results show that the prepared magnetic covalent organic framework material Fe3O4@PY-TP has a core-shell spherical structure, the shell layer coated on the outer surface of the bare Fe3O4 nanomaterial has a thickness of about 44.9 nm, and the diameter is about 400 nm. Fe3O4@PY-TP contains hydroxyl functional groups, has a large specific surface area (329.7 m 2 g -1 ), a classic mesoporous structure (pore size of about 3.91 nm), and good heat resistance (good thermal stability below 450℃). The magnetization of Fe3O4@PY-TP is 66.7 emu g -1 , which can be rapidly dispersed in the sample solution and can be quickly separated by magnetic separation.

[0128] Example 3

[0129] The method for detecting glucocorticoids according to the embodiment of the application is used to detect glucocorticoids in commercially available honey and eggs, and the specific process is as follows:

[0130] I. Experimental method

[0131] 1. Pretreatment

[0132] Honey and eggs are purchased from the local market (Beijing, China) and stored at 4℃ before analysis. First, 5g (accurate to 0.01g) of honey is accurately weighed and placed in a 50mL centrifuge tube, 20mL of 80% acetonitrile aqueous solution (containing 0.2% formic acid) is added, vortexed for 2min, ultrasonicated for 2min at a power of 120W, and centrifuged at 9500r / min for 5min at 4℃. The supernatant is collected and evaporated under a gentle stream of nitrogen, and the residue is dissolved in 5mL of water. The resulting reconstituted solution is filtered through a 0.22μm polytetrafluoroethylene filter membrane for subsequent magnetic solid-phase extraction.

[0133] Take 2±0.05mL of milk sample in a 50mL centrifuge tube, add 20mL of ethyl acetate (EA), mix by vortex, shake on a 200r / min shaker for 15min, and centrifuge at 8000r / min for 15min. Collect the EA layer, evaporate under a gentle stream of nitrogen, and dissolve the residue in 5mL of water. The resulting reconstituted solution is filtered through a 0.22μm polytetrafluoroethylene filter membrane for subsequent magnetic solid-phase extraction.

[0134] 2. Magnetic solid phase extraction (MSPE) procedure

[0135] The main parameters of MSPE procedure for 6 glucocorticoids (dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide) using Fe3O4@PY-TP were optimized by single factor method. The main contents of optimization included adsorption time, desorption time, adsorbent dosage, eluent type, eluent volume, sample solution pH and salt concentration. Finally, the MSPE procedure was carried out under the optimized conditions.

[0136] Specifically, 5 mg of Fe3O4@PY-TP was precisely weighed (accurate to 0.01 g), dispersed into 5 mL of sample solution, and vortexed at a speed of 1800 r / min for 6 min in a multi-tube vortexer to ensure complete adsorption. The supernatant was then discarded with the help of an external magnet. Subsequently, the adsorbed glucocorticoids were eluted with ACN under ultrasonic treatment for 2 min. The eluent was concentrated to 5 mL with ACN, and then filtered through a 0.22 μm filter membrane for subsequent HPLC-MS / MS analysis. The MSPE flow chart is shown in Figure 11 .

[0137] To ensure the accuracy and repeatability of the experimental results, all the magnetic solid phase extraction optimization experiments were performed in triplicate. The recovery rate was based on the average value of the results of three parallel experiments, and the relative standard deviations (RSDs) between the three parallel experimental data were also calculated to evaluate the accuracy of the experiments. In the magnetic solid phase extraction parameter optimization chart, each data was accompanied by an error bar to represent the corresponding RSD value.

[0138] 3. HPLC-MS / MS analysis

[0139] All determinations were performed using a Shimadzu Exion LC AD HPLC system (Shimadzu, Japan) coupled to a 6500 +QqQ MS (AB SCIEX, USA) was used. The chromatographic separation of 29 glucocorticoids was performed on a Hypersil GOLD aq column (100 x 2.1 mm, 1.9 μm, Thermo) with mobile phase of 5 mmol / L ammonium formate solution (containing 0.1% formic acid) (A) and 0.1% formic acid acetonitrile solution (B). The flow rate was 0.3 mL / min and the injection volume was 3.0 μL. The optimized gradient elution conditions were as follows: 0-1 min, 27% B; 1-5 min, 37% B; 5-12 min, 75% B; 12-12.1 min, 95% B; 12.1-15 min, 95% B; 15-15.1 min, 27% B; 15.1-18 min, 27% B.

[0140] MS analysis was performed under ESI(+) conditions using multiple reaction monitoring (MRM) mode. The spray voltage was 5500 V, the ion source temperature was 500 °C, and the evaporation temperature was 5500 °C. The gas curtain pressure and the collision gas pressure were 30 psi and 9 psi, respectively. The mass spectrometry optimization parameters of 29 glucocorticoids were shown in Table 1.

[0141] 29 glucocorticoid solid standard samples were weighed and prepared into 1 mg / mL standard stock solution with ACN; the standard stock solution of 29 glucocorticoids was diluted into 10 μg / mL standard intermediate solution with 5 mmol / L ammonium formate solution (containing 0.1% formic acid) / 0.1% formic acid acetonitrile solution (7:3, v / v); the standard intermediate solution was accurately measured and diluted into 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02 and 0.01 μg / mL working solutions with 5 mmol / L ammonium formate solution (containing 0.1% formic acid) / 0.1% formic acid acetonitrile solution (7:3, v / v) in sequence, and detected by machine.

[0144] Table 1 Mass spectrometry optimization parameters of 29 glucocorticoids

[0145]

[0146] 4、Fe3O4@PY-TP adsorption behavior investigation

[0147] The adsorption behavior of Fe3O4@PY-TP for dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide was studied in this example. The single stock solution (1000 pg / mL) of six glucocorticoids was diluted with water to obtain solutions of different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 pg / mL) of six glucocorticoids. 1 mg of Fe3O4@PY-TP was weighed and dispersed in 4 mL of aqueous solution. After adsorption equilibrium was reached by vortexing for 90 min, an appropriate amount of supernatant was diluted to 1 mL of 5 mmol / L ammonium formate solution (containing 0.1% formic acid) / 0.1% formic acid acetonitrile solution (7:3, v / v) for subsequent HPLC-MS / MS analysis. The adsorption capacity equation is as follows:

[0148]

[0149] where q t is (mg / g) the adsorbed amount of glucocorticoids at time t; C0and C t ( pg / mL) are the concentrations of glucocorticoids in the supernatant at the start time and time t, respectively. V (mL) is the volume of the adsorption mixture (mL); m is the mass of the adsorbent (mg).

[0150] II. Results and discussion

[0151] 1. MSPE condition optimization

[0152] The adsorption conditions are crucial for the effect of MSPE. In order to achieve the best effect of Fe3O4@PY-TP for glucocorticoids, a series of factors affecting the adsorption effect were studied by single factor experiment method, including adsorption time, desorption time, adsorbent dosage, elution solvent type, elution solvent volume, sample solution pH and salt concentration, to achieve the most suitable extraction of six glucocorticoids (dexamethasone, cortisone, fluocinolone acetonide, prednisone, hydrocortisone and triamcinolone acetonide). The MSPE process was carried out in a solution containing 20 ng / mL of six glucocorticoids, and the recovery rate was used as an evaluation index.

[0153] (1) Fe3O4@PY-TP dosage optimization

[0154] The adsorbent dosage is a key factor of MSPE conditions. Seven different dosages of Fe3O4@PY-TP (0.5, 1, 2, 3, 4, 5 and 6 mg) were selected to extract six glucocorticoids. The recoveries of six glucocorticoids extracted by different adsorbent dosages are as follows: Figure 12The adsorbent dosage was increased from 0.5 to 3 mg, and the recovery of the six glucocorticoids increased continuously. When the dosage of the adsorbent was further increased, the recovery remained almost unchanged. Therefore, 3 mg of the adsorbent was considered to be sufficient to enrich the six glucocorticoids at a low concentration, and was selected as the optimal dosage for further procedures.

[0155] (2) Adsorption time optimization

[0156] To study the effect of the adsorption time on the recovery, nine time points of 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 min were selected for the adsorption time study, and the experimental time was as shown in Table 2. Figure 12 As shown in Fig. B, when the adsorption time was increased from 0.5 min to 5 min, the extraction recovery generally showed an upward trend. After 5 min, the extraction recovery of the six glucocorticoids did not increase significantly with the increase of the adsorption time, which may be because the analytes had been in sufficient contact with the adsorbent and reached adsorption equilibrium. This indicates that an adsorption time of 5 min is sufficient to ensure that the adsorption sites are in complete contact with the analytes. Therefore, 5 min was selected as the optimal adsorption time for further experiments.

[0157] (3) Salt concentration optimization

[0158] The addition of salt in the sample solution can increase the ion concentration in the sample solution, which can affect the extraction of glucocorticoids. Therefore, in order to investigate the effect of salt concentration on the extraction efficiency of glucocorticoids, we adjusted the ionic strength of the sample solution and investigated the effect of different concentrations of sodium chloride in the sample solution on the extraction efficiency of the six glucocorticoids. The results are shown in Fig. C. Figure 12 As can be seen from the figure, the increase of the salt concentration did not significantly increase the extraction recovery of the six glucocorticoids. This may be because, on the one hand, increasing the salt concentration can decrease the solubility of the target analytes in the aqueous phase, thereby increasing the distribution of the target analytes in the adsorbent, but at the same time, high ionic strength can increase the viscosity of the sample aqueous solution, resulting in a decrease in the mass transfer of the target analytes to the adsorbent. Therefore, the subsequent MSPE procedure was carried out without the addition of sodium chloride.

[0159] (4) PH value optimization

[0160] PH value is an important parameter that can affect the stability and existence form of some analytes, and can significantly affect the extraction recovery. 1 mol / L dilute hydrochloric acid and 1 mol / L sodium hydroxide were used as PH adjusters to adjust the PH of the sample solution in the range of 2-12, and the effect of PH value on the extraction efficiency was investigated. The experimental results are shown in Fig. D. Figure 12D. As can be seen from the figure, when the pH value of the sample solution is in the range of 2-12, the extraction recovery of the six glucocorticoids is not significantly inhibited or promoted, which may be because the glucocorticoids have fewer ionizable groups and stronger structural stability, and exist in the form of molecules under general acid-base conditions. Therefore, in order to simplify the MSPE process, the subsequent MSPE procedure does not need to adjust the pH.

[0161] (5) Optimization of elution solvent type

[0162] The elution solvent is mainly to destroy the hydrogen bonding force between the target analyte and the adsorbent, and to elute the target analyte from Fe3O4@PY-TP. Different polar elution solvents have different elution abilities, which have a great influence on the elution efficiency of the six glucocorticoids on Fe3O4@PY-TP. Therefore, five different polar organic solvents were selected as the elution solvents of Fe3O4@PY-TP, including MeOH, ACN, EtOH, EA and IPA. The elution ability of the five elution solvents is shown in Figure 12 E. As can be seen from the figure, under the same elution conditions, ACN has good extraction recovery of the six glucocorticoids and the results are similar, and the error is small, therefore, ACN is selected as the elution solvent for the subsequent MSPE procedure.

[0163] (6) Optimization of elution solvent volume

[0164] In order to ensure that the target analyte on the adsorbent can be eluted by the elution solvent to the greatest extent, ACN was used as the elution solvent to further optimize the volume of the elution solvent. The volume of the elution solvent was optimized by using 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0 and 10.0 mL of ACN as the elution solvent of Fe3O4@PY-TP, and the experimental results are shown in Figure 12 F. When the volume of ACN increases from 0.5 mL to 5 mL, the recovery reaches the maximum value. Further increasing the volume of the elution solvent has little effect on the recovery. Reducing the use of organic solvents is conducive to environmental protection. Therefore, 5 mL of elution solvent is selected for subsequent experiments.

[0165] (7) Optimization of elution solvent type

[0166] Sufficient elution time can ensure the complete separation of the target analyte and the adsorbent, so as to achieve the best elution effect. In order to obtain the most suitable elution time, the elution time was set to 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 min, respectively, for optimizing the elution time. The experimental results are shown in Figure 12G. The results showed that the recovery rates of the six glucocorticoids increased within 0.5-1 min, reached the maximum at 1 min, and did not change significantly with the increase of the elution time, indicating that the desorption equilibrium was reached. Therefore, 1 min was selected as the optimal desorption time for the subsequent experiments.

[0167] 2. Adsorption performance of Fe3O4@PY-TP for glucocorticoids

[0168] (1) Adsorption isotherm

[0169] To study the adsorption performance of Fe3O4@PY-TP, the adsorption kinetics and adsorption isotherm were investigated. The standard solutions of the six glucocorticoids were added to pure water to prepare 4 mL single standard solutions with different concentrations (10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, and 100.0 mg / L), and then 1 mg of Fe3O4@PY-TP was added. The mixture was vortexed at room temperature for 99 min to reach adsorption equilibrium. The supernatant was collected and diluted by a certain multiple, and then analyzed by high-performance liquid chromatography-tandem triple quadrupole mass spectrometry. The maximum adsorption capacity was calculated based on the data of the diluted supernatant. The results of the adsorption isotherm are shown in FIG. A. Figure 13 As shown in the figure, the adsorption capacity of the six glucocorticoids increased with the increase of the standard solution concentration from 10.0 mg / L to 80 mg / L, and reached a plateau at 90 mg / L. The calculated saturated adsorption capacities of dexamethasone, cortisone, flumethasone, prednisone, hydrocortisone, and triamcinolone acetonide were 176.53, 102.93, 176.23, 84.53, 102.80, and 156.53 mg / g, respectively. The results showed that Fe3O4@PY-TP adsorbent had good binding capacity for the target analytes and could meet the extraction and analysis of glucocorticoids in actual samples.

[0170] To better understand the adsorption process between Fe3O4@PY-TP and glucocorticoids, the Freundlich and Langmuir models were used to analyze the adsorption behavior of Fe3O4@PY-TP for the six glucocorticoids.

[0171] The Langmuir model is commonly used to describe the uniform distribution of adsorption sites on the surface of the adsorbent, and the formation of a monolayer of target molecules on the surface of the adsorbent. The linear form and characteristic parameters R L The expression is as follows:

[0172]

[0173] where Ce and C0(μg / mL) represent the supernatant concentration at adsorption equilibrium and initial, respectively, q e (mg / g) represents the adsorption capacity at equilibrium. q s represents the adsorption capacity at the theoretical saturation state, K L is the Langmuir constant. R L is the characteristic parameter of Langmuir thermodynamics, which is related to the shape of Langmuir isotherm, and is used to evaluate whether the adsorption process is beneficial. R L = 0, the adsorption process is not feasible; 0 < R L < 1, beneficial to adsorption; R L = 1, the adsorption process is linear adsorption; R L > 1, not conducive to adsorption.

[0174] The fitting parameters of the Langmuir model are shown in Table 2. The calculation range of the linear correlation constant (R 2 ) is 0.0524-0.91631, and the linear fitting curve is shown in Figure 14 . It can be seen that not all glucocorticoids are suitable for this model.

[0175] The Freundlich isotherm model is suitable for the adsorption of multilayer cover on non-uniform surface, and its expression is as follows:

[0176]

[0177] where n represents the Freundlich constant, k f represents the binding energy constant. As shown in Table 2, the Freundlich model is more suitable for describing the adsorption process of glucocorticoids on Fe3O4@PY-TP (R>0.95), and the fitting results are shown in Figure 15 . The Freundlich isotherm model is generally used to describe the adsorption process of different phase surfaces, which assumes that the distribution of adsorption sites on the surface is non-uniform, and the adsorption process often exists multiple forces, which belongs to multilayer adsorption. Therefore, the above experiments show that the adsorption of Fe3O4@PY-TP on glucocorticoids may be a multilayer adsorption of hydrogen bond force, electrostatic interaction, hydrophilic interaction and π-π interaction.

[0178] Table 2 Adsorption isotherm parameters of 6 glucocorticoids on Fe3O4@PY-TP

[0179]

[0180] (2) Adsorption kinetics

[0181] The adsorption kinetics of Fe3O4@PY-TP was studied, and the adsorption capacity of Fe3O4@PY-TP for 6 kinds of glucocorticoids in aqueous solution containing 80 μg / mL glucocorticoids was obtained under different time conditions, as shown in FIG. 2B. As can be seen from the figure, the adsorption capacity of Fe3O4@PY-TP for 6 kinds of glucocorticoids increases with time, and the adsorption rate also decreases with time. The adsorption equilibrium is reached at about 10-40 min, and the maximum adsorption capacity is between 84.53-176.53 mg / g. The adsorption kinetics data is fitted by pseudo-first-order and pseudo-second-order kinetic models, and the expressions are as follows: Figure 13

[0182]

[0183] wherein q t and q m represent the adsorption capacity of Fe3O4@PY-TP at time t and the theoretical saturation state, respectively. k1 and k2 are the adsorption rate constants of pseudo-first-order and pseudo-second-order models. The results are shown in Table 3, and compared with the pseudo-first-order kinetic model (R 2 > 0.89186), the pseudo-second-order kinetic model (R 2 > 0.98782) is more suitable for simulating the adsorption process of Fe3O4@PY-TP. The fitting equations are as follows: Figure 16 Figure 17 It can be seen from the above that the chemical adsorption between Fe3O4@PY-TP and glucocorticoids is dominated by the rate-determining step of sharing or exchanging electrons.

[0184] Table 3 Adsorption kinetics of 6 kinds of glucocorticoids on Fe3O4@PY-TP

[0185]

[0186] (3) Batch reproducibility and reusability

[0187] ​​Batch reproducibility and reusability of the adsorbent are crucial for the newly synthesized adsorbent. Therefore, in order to further evaluate the performance of Fe3O4@PY-TP, the batch reproducibility and reusability of Fe3O4@PY-TP were investigated in this section. Three batches of Fe3O4@PY-TP synthesized under the same experimental conditions were selected, and the adsorption effect of different batches of Fe3O4@PY-TP on six kinds of glucocorticoids was compared to evaluate the batch reproducibility. Each experiment was repeated three times, and the relative standard deviations (RSDs) of each parallel experiment were calculated for each batch. The experimental results are shown in Table 4. As can be seen from the table, the RSDs of different batches are 2.61-4.56%. This shows that the adsorption effect of different batches of Fe3O4@PY-TP on six kinds of glucocorticoids is basically the same, and the preparation method has high stability.

[0188] Reusability of the adsorbent is one of the important parameters for evaluating its adsorption performance. 3 mg of Fe3O4@PY-TP was taken in 200 ng / mL of 6 kinds of glucocorticoid solution to carry out MSPE program, and its recovery was detected. After completing one adsorption-desorption program, before entering the next MSPE program, the material used last time was washed with ACN for three times and dried for standby. As shown in the results Figure 18 , Fe3O4@PY-TP can still maintain a high recovery rate for 6 kinds of glucocorticoids after being used for 8 cycles in succession, which can indicate that Fe3O4@PY-TP can be reused at least seven times.

[0189] Table 4 Adsorption reproducibility of Fe3O4@PY-TP on 6 kinds of glucocorticoids

[0190]

[0191]

[0192] II. Methodology validation

[0193] Fe3O4@PY-TP as adsorbent in MSPE procedure was applied to real samples. The 29 glucocorticoids in milk and honey with different spiked concentrations were adsorbed, and the target compounds were eluted and enriched, then detected by HPLC-MS / MS. A high efficient method for trace level glucocorticoids was established. In addition, the linear range, correlation coefficient (r), limits of detection (LODs), limits of quantitation (LOQs), precision, accuracy and matrix effects (MEs) of the method were investigated, and the experimental results are shown in Tables 5 and 6.

[0194] As can be seen from the table, the linear range of the method in milk and honey was 0.003-100 μg / kg and 0.012-100 μg / kg, respectively, and the correlation coefficient was 0.9947-0.9992 and 0.9946-0.9993, respectively, indicating that the method had good linearity for the 29 glucocorticoids. At the same time, the method also had high sensitivity, and the LODs in milk and honey matrix were 0.001-0.3 μg / kg and 0.003-0.4 μg / kg, respectively, and the LOQs were 0.003-0.9 μg / kg and 0.012-0.9 μg / kg, respectively, which were lower than the limit standards of glucocorticoids in national standards and European regulations. The intra-day (n=5) and inter-day (n=5) precision of the method was studied at different spiked concentrations. The RSDs of intra-day precision were ≤12.31%, and the RSDs of inter-day precision were ≤11.91%, indicating that the method had good repeatability and reproducibility. In addition, the matrix effects of the method were also investigated by comparing the slopes of solvent calibration curves and matrix calibration curves of the 29 glucocorticoids. The matrix effects of the 29 glucocorticoids in milk and honey were -17-17.12% and -17.77-13%, respectively. The matrix effect in the range of -20%-20% was acceptable matrix effect. Therefore, the matrix effect of the method could be ignored.

[0195] In summary, the MSPE procedure with Fe3O4@PY-TP as adsorbent could effectively reduce the interference of matrix and realize the efficient enrichment of glucocorticoids. The established HPLC-MS / MS method had high practicability and reliability in real sample analysis.

[0196] Three, real sample analysis

[0197] To further investigate the practicability of the above MSPE-HPLC-MS / MS method, 29 glucocorticoids in milk and honey were detected and analyzed in this section. As shown in Table 7, no glucocorticoids were detected in milk and honey matrices. Since no positive samples were detected in real samples, the accuracy of the method was investigated by the method of standard addition recovery. The spiked concentrations of milk were 1, 5, 10 μg / kg, and the spiked concentrations of honey were 1.2, 6, 12 μg / kg. As shown in Table 6, the recoveries of spiked samples in milk matrix were 79.09 ± 1.87-108.83 ± 4.58%, and the recoveries of spiked samples in honey matrix were 74.48 ± 1.82-102.06 ± 5.20%, indicating that the method could obtain good accuracy when applied to real samples.

[0198] Table 5 Linearities, LODs, LOQs and MEs of 29 glucocorticoids in different media

[0199]

[0200] Table 5 Linearities, LODs, LOQs and MEs of 29 glucocorticoids in different media

[0201]

[0202] Table 5 Linearities, LODs, LOQs and MEs of 29 glucocorticoids in different media

[0203]

[0204] Table 6 Intra-day and inter-day precisions of 29 glucocorticoids

[0205]

[0206] Table 6 Intra-day and inter-day precisions of 29 glucocorticoids

[0207]

[0208] Table 7 Recoveries and repeatabilities of 29 glucocorticoids

[0209]

[0210] Table 7 Recoveries and repeatabilities of 29 glucocorticoids

[0211]

[0212] Four, Comparison with other methods

[0213] The advantages of the developed MSPE-HPLC-MS / MS method using Fe3O4@PY-TP as adsorbent for glucocorticoid analysis were compared with the previously reported literature, and the results are shown in Table 8. As can be seen from the table, most of the methods focus on detecting fewer types and quantities of glucocorticoids, and therefore, the potential harm caused by incomplete detection in animal-derived foods is quite serious. This work provides a highly feasible solution for the simultaneous enrichment and extraction of 29 common glucocorticoids. In terms of operational efficiency, the method only requires 3 mg of Fe3O4@PY-TP to complete adsorption within 6 min, which is superior to other types of adsorbents reported in the past, such as multi-walled carbon nanotube composites, polystyrene nanofibers, graphene / mesoporous silica composites, DVB, NVP and oleic acid modified magnetic microspheres and MOF materials. At the same time, the method provides a larger linear range, satisfactory recovery and lower LOD (0.001-0.3 μg / kg and 00.003-0.4 μg / kg), which are closely related to the excellent adsorption performance of Fe3O4@PY-TP.

[0214] Table 8 Comparison of magnetic nanomaterials with other adsorbents

[0215]

[0216] In summary, the MSPE-HPLC-MS / MS established based on the Fe3O4@PY-TP of the embodiments of the present application can effectively reduce the matrix effect of glucocorticoids in foods, obtain good linear relationship (r > 0.9946), lower detection limit (0.001-0.3 μg / kg and 00.003-0.4 μg / kg) and quantification limit (0.003-0.9 μg / kg and 0.012-0.9 μg / kg) and higher accuracy (74.48 ± 1.82-108.83 ± 4.58%, RSD ≤ 12.12%), and the RSD of intra-day and inter-day precision is ≤ 12.31%. The method has high application value in the analysis of actual samples.

[0217] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0218] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and application of the present application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the present application, which is defined by the following claims and their equivalents.

Claims

1. A magnetic nanomaterial for enriching glucocorticoids, characterized in that, The magnetic nanomaterial comprises: a core formed by ferroferric oxide; and a shell covering at least part of the surface of the core, the shell being composed of repeating units represented by Formula I.

2. The magnetic nanomaterial of claim 1, wherein, The shell has a porous network structure, optionally, the average pore size of the shell is 3-5 nm, Optionally, the specific surface area of the shell is 300-350 m 2 • g -1 .

3. The magnetic nanomaterial of claim 1, wherein, the average particle size of the core is 300-350 nm, and the average particle size of the magnetic nanomaterial is 370-450 nm.

4. The magnetic nanomaterial of claim 1, wherein, The shell has crystal diffraction peaks at 2θ of 18.30°, 30.11°, 35.48°, 43.18°, 53.56°, 56.92° and 62.51° in X-ray powder diffraction data, optionally, the contact angle of the magnetic nanomaterial is 20.0-21.5°.

5. A method for preparing the magnetic nanomaterial according to any one of claims 1-4, characterized in that, The method comprises: providing ferroferric oxide nanomaterial; contacting the ferroferric oxide nanomaterial with an organic solution containing an amino monomer, ultrasonic treatment, and then mixing and stirring to obtain a first mixed solution; and contacting an aldehyde monomer with the first mixed solution to perform a Schiff base reaction to obtain the magnetic covalent organic framework material.

6. The method of claim 5, wherein, The aldehyde monomer is 1,3,5-triformylphloroglucinol (TP), and the amino monomer is 1,3,6,8-tetra-(p-aminophenyl)-pyrene (PY), optionally, the molar ratio of the aldehyde monomer to the amino monomer is 1-2:1, preferably 1.5:1, optionally, the mass ratio of the ferroferric oxide nanomaterial to the amino monomer is 1:0.5-1.5, preferably 1:1, optionally, the ultrasonic treatment time is 10-30 minutes, optionally, the mixing and stirring is performed at 55-65°C, at a rotation speed of 500-600 rpm, and for 40-80 minutes. optionally, the temperature of the Schiff base reaction is 55-65°C, and the time is 3-5 hours, optionally, the Schiff base reaction uses acetic acid as a catalyst.

7. A method of enriching a glucocorticoid, characterized by, The method is performed using the magnetic nanomaterial for enriching glucocorticoids according to any one of claims 1-4, optionally, the glucocorticoid is at least one selected from dexamethasone, cortisone, fluocinolone, prednisone, hydrocortisone and triamcinolone acetonide, optionally, the adsorption capacity of the glucocorticoid-enriched magnetic nanomaterial is 80-175 mg / g, optionally, the adsorption equilibrium time of the glucocorticoid-enriched magnetic nanomaterial is 1-5 min, optionally, the amount of the magnetic nanomaterial is 0.8-1.2 mg based on 1 mL of liquid sample, optionally, the extraction time of the method is 4-8 minutes, optionally, acetonitrile is used for elution treatment, optionally, the elution treatment time is 0.5-1.5 minutes, and the elution liquid volume is 4-6 mL.

8. A method for detecting the content of glucocorticoid in a sample to be measured, characterized by, The method comprises: enriching glucocorticoids in the sample to be tested using the method for enriching glucocorticoids according to claim 7 to obtain an extract; and analyzing and detecting the extract using a high-performance liquid chromatography-mass spectrometry system to obtain the content of glucocorticoids in the sample to be tested, Optionally, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry system are as follows: Chromatographic column: Hypersil GOLD aQ column, specification: 100 x 2.1 mm, 1.9 μm; Mobile phase: A phase: 5 mmol / L ammonium formate solution containing 0.1% formic acid; B phase: 0.1% formic acid acetonitrile solution; Flow rate of mobile phase: 0.3 mL / min; Injection volume: 3.0 μL; Gradient elution conditions: 0-1 min, 27% B; 1-5 min, 37% B; 5-12 min, 75% B; 12-12.1 min, 95% B; 12.1-15 min, 95% B, Optionally, the mass spectrometry conditions of the high performance liquid chromatography-mass spectrometry system are as follows: Monitoring mode: multiple reaction monitoring (MRM) mode under ESI (+) condition; Spray voltage: 5500 V; Ion source temperature: 500 °C; Evaporation temperature: 5500 °C; Gas curtain pressure: 30 psi; Collision gas pressure: 9 psi.

Images