Magnetic covalent framework material and application thereof in detection of quinolones in bee products

Abstract

The invention relates to a magnetic covalent framework material and application thereof in bee product quinolone drug detection, the magnetic covalent framework material is a magnetic material taking magnetic nanoparticles as a core and loading organic monomers melamine and 2, 4, 6-triformyl benzenetriol on the surfaces of the magnetic nanoparticles, the specific surface area is 1000-1500m < 2 > / g, the pore volume ratio is 4-5cm < 3 > / g, and the magnetic covalent framework material is a covalent framework material. The solid-phase extraction adsorbent can be used as a solid-phase extraction adsorbent for extracting quinolones in bee products, the extraction time is as low as 4 minutes, and the solid-phase extraction adsorbent can be repeatedly used for more than 6 times; according to the present invention, the quinolone drug residue in the bee product is detected by combining the liquid chromatography-tandem mass spectrometry, the elution time of the chromatographic mobile phase of the method can be shortened to 14 min, the detection limit is as low as 0.04 [mu] g / kg, the quantification limit is as low as 0.12 [mu] g / kg, and compared with the existing national standard method, the method has advantages of high detection sensitivity, high accuracy and short detection time.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically to a magnetic covalent framework material and application thereof in the detection of quinolone drugs in bee products. Background Art

[0002] Honey is a natural substance collected by bees from flowers and thoroughly manufactured in the hive. It contains a variety of vitamins, minerals and amino acids. Royal jelly is a secretion from the pharyngeal glands of young worker bees (nanny bees) that cultivate larvae in the beehive, which contains high levels of protein, fat, sugar and other valuable nutrients. Due to the various precious ingredients of honey and royal jelly, these natural products have always been favored by consumers. However, bees are susceptible to several infectious and transmissible diseases. In order to maintain honey production, beekeepers generally use antibiotics to prevent and / or treat bacterial infections in bees. The use of these antibiotics can lead to the accumulation of antibiotic residues in honey and royal jelly, and the residues exceeding a certain amount may be harmful to human health.

[0003] Quinolones (QNs) are synthetic broad-spectrum antibiotics that exert their antibacterial effects by inhibiting DNA gyrase. They show strong antibacterial activity against Gram-negative bacteria and weak antibacterial activity against Gram-positive bacteria. At present, QNs are also the main therapeutic drugs for bacterial infections in bees and are widely used to prevent or treat bacterial infections in bees. However, according to GB31650.1-2022 "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food", the maximum residue limit of quinolone synthetic antibacterial drugs in honey samples is 5 μg / kg. It can be seen that even at trace concentrations in food, their overuse and environmental cumulative effects are potentially dangerous to animals and humans. After their residues enter the human body through the food chain, they may induce the spread of drug resistance, thereby affecting the clinical efficacy of QNs. Therefore, it is necessary to monitor their residues.

[0004] At present, the multi-residue of quinolones in honey is mainly determined by GB 31657.2-2021 "National Food Safety Standard for the Determination of Multi-Residue of Quinolones in Bee Products by Liquid Chromatography-Tandem Mass Spectrometry", but this method takes a long time to detect, has low detection sensitivity (detection limit of 0.5μg / kg, quantification limit of 1μg / kg), and the solid phase column cannot be reused, which is costly. Patent CN 111426767 B combines ionic liquid dispersed liquid-liquid microextraction and magnetic solid phase extraction (IL-DLLME-MSPE) for the detection of quinolone antibiotics in honey, but only three quinolones (ciprofloxacin, enrofloxacin, flumequine) are detected, and the types of residues detected are relatively small, while the number of quinolone compound drugs remaining in honey can be as high as dozens. Due to the large number of drug residues in bee products, it is generally difficult to detect quickly and accurately. Therefore, the study of a highly sensitive and rapid detection method for quinolone residues in bee products is of great significance for the precise supervision of the bee product market. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a magnetic covalent framework material and its application in the detection of quinolone drugs in bee products. The purpose is to synthesize a magnetic covalent framework material that can be used as a magnetic solid phase extractant for the extraction of quinolone drugs in bee products. The extraction time is as low as 4 minutes. The detection limit of the method is as low as 0.04 μg / kg, and the quantitative limit is as low as 0.12 μg / kg. The magnetic covalent framework material can be reused at least 6 times, thereby solving the technical problems of long detection time, low sensitivity and high cost of quinolone drugs in existing bee products.

[0006] To achieve the above object, according to one aspect of the present invention, a magnetic covalent framework material is provided, which is a magnetic material with magnetic nanoparticles as the core and the surface of the magnetic nanoparticles loaded with organic monomers melamine and 2,4,6-triformylbenzene triphenol, wherein the magnetic nanoparticles include Fe 3 O 4 or its modified product, Fe 3 O 4 NPs, NiFe 2 O 4 ,γ-Fe 2 O 3 Any one of the following; the specific surface area of ​​the magnetic material is 1000 to 1500m 2 / g, pore volume ratio is 4-5cm 3 / g.

[0007] Preferably, the magnetic covalent framework material is prepared according to the following method:

[0008] Magnetic nanoparticles and melamine are added to tetrahydrofuran and uniformly dispersed by ultrasound; 2,4,6-triformylbenzenetriol is dissolved in tetrahydrofuran and added dropwise to the reaction system, acetic acid is used as a catalyst, and after sufficient reaction, acetonitrile and ethanol are used for washing, and the magnetic covalent framework material is obtained by drying.

[0009] Preferably, the magnetic covalent framework material, wherein the magnetic nanoparticles are Fe 3 O 4 The mass ratio of melamine to 2,4,6-triformylbenzene is 3:(0.3~1.2):(0.6~2.0).

[0010] Preferably, the catalytic reaction temperature of the magnetic covalent framework material is 40-75° C., and the reaction time is 2 h.

[0011] In addition, according to another aspect of the present invention, a method for detecting quinolone residues in bee products is provided, which comprises the following steps:

[0012] (1) Extraction and purification of quinolone drugs: Using the magnetic covalent framework material of the present invention as a magnetic solid phase extractant, 20 quinolone drugs in the sample are extracted and enriched, separated, cleaned and impurities removed;

[0013] (2) Elution: vortex desorption with a desorption solvent containing formic acid to desorb and elute the 20 quinolone target compounds from the magnetic covalent framework material, collect the eluate, and filter it using a nylon or polytetrafluoroethylene microporous filter membrane;

[0014] (3) Qualitative or quantitative detection: Qualitative or quantitative detection is performed using HPLC-MS or MS.

[0015] Preferably, in the detection method, the sample is honey or royal jelly diluted to a concentration of 0.05-0.3 g / mL, and the 20 quinolone drugs are oxolinic acid, flumequine, cinoxacin, difloxacin, sarafloxacin, ofloxacin, enrofloxacin, lomefloxacin, pefloxacin, norfloxacin, ciprofloxacin, nalidixic acid, enoxacin, danofloxacin, marbofloxacin, fleroxacin, gatifloxacin, orbifloxacin, pipemidic acid and sparfloxacin.

[0016] Preferably, in the detection method, the magnetic covalent framework material is Fe 3 O 4 @MA-Tp, extraction time is 4 to 10 minutes, and the sample is honey, preferably Fe 3 O 4 @MA-Tp addition amount is 4mg, the sample is royal jelly preferred Fe 3 O 4 @The amount of MA-Tp added is 6 mg.

[0017] Preferably, in the detection method, the desorption solvent containing formic acid is a mixed solvent of formic acid and acetonitrile, methanol, ethanol or acetone, wherein the volume of formic acid accounts for 10% to 15% of the volume of the mixed solvent.

[0018] Preferably, in the detection method, the desorption solvent is 10% formic acid methanol, the addition amount is 4.0 mL to 8.0 mL, and the vortex desorption is performed for more than 2 minutes.

[0019] Preferably, the detection method adopts HPLC-MS quantitative detection, wherein the total volume of mobile phase A and mobile phase B in the liquid chromatography mobile phase gradient elution process is 100%, specifically:

[0020] 0-1min: maintain 10% B; 1-4.5min: the volume of mobile phase B increases from 10% to 45%; 4.5-7min: the volume of mobile phase B increases from 45% to 95%; 7-9.5min: maintain 95% B; 9.5-9.6min: the volume of mobile phase B decreases from 95% to 10%; 9.6-14min: maintain 10% B.

[0021] In general, compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects.

[0022] The magnetic covalent framework material provided by the present invention is a magnetic material with magnetic nanoparticles as the core and the surface of the magnetic nanoparticles loaded with organic monomers melamine and 2,4,6-triformylbenzene triphenol. The magnetic nanoparticles include Fe 3 O 4 or its modified product, Fe 3 O 4 NPs, NiFe 2 O 4 ,γ-Fe 2 O 3 Any one of the following; the specific surface area of ​​the magnetic material is 1000 to 1500m 2 / g, pore volume ratio is 4-5cm 3 / g. The magnetic material is used as a magnetic solid phase extractant to extract and enrich 20 quinolones in bee products, and the extraction time can be as low as 4 minutes. Combined with HPLC-MS or MS for qualitative or quantitative analysis, compared with the existing national standard method GB31650.1-2022, the elution time of this detection method can be shortened to 14 minutes, the detection limit is as low as 0.04μg / kg, and the quantitative limit is as low as 0.12μg / kg. The magnetic covalent framework material can be reused at least 6 times, which is conducive to reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is a magnetic covalent framework material Fe 3 O 4 @MA-Tp performance test;

[0024] Figure 2 is the total ion current diagram; Figure 2 A is blank negative honey matrix, and B is blank negative honey matrix with 5 ng / mL QNs standard added;

[0025] Figure 3 It is Fe 3 O 4 @The total ion current corresponding to the extraction after the addition of 2 mg of MA-Tp;

[0026] Figure 4 It is Fe 3 O 4 @The total ion current corresponding to the extraction after the addition of 4 mg of MA-Tp;

[0027] Figure 5 It is Fe 3 O 4 @The total ion current corresponding to the extraction after the addition of 12 mg of MA-Tp;

[0028] Figure 6 It is Fe 3 O 4 @The total ion current corresponding to the extraction after the addition of 20 mg of MA-Tp;

[0029] Figure 7 Different dosage of Fe 3 O 4 @MA-Tp corresponding recovery rates of 20 quinolones;

[0030] Figure 8 is the recovery rate of 20 quinolones corresponding to microporous filter membranes of different materials;

[0031] Fig. 9 It is Fe 3 O 4 @The recovery rates of 20 quinolones corresponding to repeated use of MA-Tp. DETAILED DESCRIPTION

[0032] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0033] Magnetic solid phase extraction (MSPE) is an important extraction and enrichment method for chromatographic detection. Magnetic solid phase extraction is a dispersed solid phase extraction technology using magnetic or magnetizable materials as adsorbents. For example, in patent CN111650320B, magnetic ferroferric oxide is used as a magnetic nano core, polydopamine is used as a transition layer, and a microporous organic polymer containing urea functional groups is used as a shell layer to synthesize magnetic nanoparticles, which are used to extract, enrich and detect drugs (enrofloxacin, ciprofloxacin, pefloxacin, sarafloxacin and lomefloxacin) in milk. F-Fe / NG@CP-d-MSPE is synthesized in CN115055163B to extract, enrich and detect drugs (sulfadiazine, sulfamethoxazole, levofloxacin, norfloxacin, enrofloxacin) in water or milk. Compared with milk, bee products have a large viscosity, complex ingredients and residual quinolone drugs up to 20 kinds. At present, there is no application of magnetic materials in the detection of quinolone drugs in bee products.

[0034] The present invention synthesizes a magnetic covalent framework material, with magnetic nanoparticles as magnetic nanocores, and the surface of the nanoparticles is loaded with organic monomers melamine and 2,4,6-triformylbenzene triphenol, and the specific surface area of ​​the material is 1000-1500m 2 / g, pore volume ratio is 4-5cm 3 / g, the magnetic nanoparticles include Fe 3 O 4 or its modified product, Fe 3 O 4 NPs, NiFe 2 O 4 ,γ-Fe 2 O 3 The magnetic material can be used as a solid phase extraction adsorbent for extracting quinolone drugs from bee products, the extraction time is as low as 4 minutes, and it can be reused for more than 6 times.

[0035] Preferably, the specific surface area of ​​the magnetic covalent framework material is 1490 m 2 / g, prepared as follows:

[0036] Add magnetic nanoparticles and melamine to tetrahydrofuran, disperse melamine and magnetic nanoparticles uniformly under ultrasound, and stir at 40-75°C; then dissolve 2,4,6-triformylbenzenetriol in tetrahydrofuran, add it dropwise to the reaction system, use acetic acid as a catalyst, and stir at 40-75°C to fully react. After the reaction, wash with acetonitrile and ethanol, and dry to obtain a magnetic covalent framework material.

[0037] The magnetic nanoparticles are Fe 3 O 4The mass ratio of melamine to 2,4,6-triformylbenzene is 3:(0.3~1.2):(0.6~2.0).

[0038] In addition, the present invention also provides an application of the magnetic covalent framework material of the present invention in the detection of quinolone drug residues in bee products.

[0039] The present invention also provides a method for detecting quinolone drug residues in bee products, which comprises the following steps:

[0040] (1) Extraction and purification of quinolone drugs: honey or royal jelly is diluted to 0.05-0.3 g / mL, and the magnetic covalent framework material of the present invention is added as a magnetic solid phase extractant for extraction and enrichment; an external magnetic field is applied for separation, impurities are washed with water, and an external magnetic field is applied for separation, and the supernatant is discarded; a desorption solvent containing formic acid is added, vortex desorption is performed to elute the target analyte, and the filtrate is separated and collected; filtration is performed using a nylon or polytetrafluoroethylene microporous filter membrane, and the filtrate is collected as a sample for the machine.

[0041] (2) Determination: Qualitative or quantitative detection is performed using high performance liquid chromatography-mass spectrometry, wherein a matrix-matched standard curve is prepared and the residual amounts of 20 quinolones in the sample are quantitatively calculated.

[0042] In the present invention, the magnetic covalent framework material can be reused at least 6 times. Preferably, the magnetic covalent framework material is Fe 3 O 4 @MA-Tp, extraction time is 4 to 10 minutes; if the sample is honey, Fe 3 O 4 @MA-Tp addition amount is 4mg, the sample is royal jelly preferred Fe 3 O 4 @The amount of MA-Tp added is 6 mg.

[0043] Preferably, the desorption solvent containing formic acid is a mixed solvent of formic acid and acetonitrile, methanol, ethanol or acetone, wherein the volume of formic acid accounts for 10% to 15% of the volume of the mixed solvent, preferably 10% of the volume of formic acid, and more preferably 10% formic acid methanol is used as the desorption solvent. In some embodiments, the amount of the desorption solvent containing formic acid is 4.0 mL-8.0 mL, and the vortex desorption is performed for more than 2 min.

[0044] Preferably, the microporous filter membrane is a 0.22 μm nylon or polytetrafluoroethylene filter membrane.

[0045] In some embodiments of the HPLC-MS detection, the liquid chromatography conditions are as follows:

[0046] Chromatographic column: ChromCore C18 column (2.1 mm × 150 mm, 1.8 μm);

[0047] Column temperature: 40°C; injection volume: 5 μL; flow rate: 0.25 mL / min; mobile phase: A is 0.1% formic acid water-5 mmol / L ammonium acetate solution, B is methanol; wherein the total volume of mobile phase A and mobile phase B during the liquid chromatography mobile phase gradient elution process is 100%, specifically:

[0048] 0-1min: maintain 10% B; 1-4.5min: the volume of mobile phase B increases from 10% to 45%; 4.5-7min: the volume of mobile phase B increases from 45% to 95%; 7-9.5min: maintain 95% B; 9.5-9.6min: the volume of mobile phase B decreases from 95% to 10%; 9.6-14min: maintain 10% B.

[0049] The mobile phase A can be prepared by adding 0.5 mL of formic acid and 0.19 g of ammonium acetate, diluting to 500 mL with water, and mixing well.

[0050] The mass spectrometry conditions were as follows:

[0051] Ion source: ESI ion source; Scan mode: positive ion scan; Detection mode: multiple reaction monitoring mode (MRM); Optimized electrospray conditions: curtain gas, 35 psi; ion source gas GS1, 55 psi; ion source gas GS2, 55 psi; ion spray voltage, 5500 V; Source temperature, 450 °C nitrogen collision gas, medium.

[0052] Compared with the national standard method GB 31657.2-2021, the extraction time of this method for detecting quinolone residues in bee products is as low as 4 minutes, the gradient elution time is shortened to 14 minutes, the detection limit is as low as 0.04 μg / kg, and the quantification limit is as low as 0.12 μg / kg. The magnetic covalent framework material can be reused at least 6 times, which is conducive to reducing costs.

[0053] The following are examples

[0054] Example 1 Synthesis of Magnetic Covalent Framework Materials

[0055] 0.3 g of magnetic nanoparticles (magnetic nanoparticles are selected from Fe 3 O 4 or its modified product, Fe 3 O 4 NPs, NiFe 2 O 4 ,γ-Fe 2 O 3Any one of the above) and 0.3-0.9 mmol of melamine (MA) were added into 20-60 mL of tetrahydrofuran, and the mixture was ultrasonicated for 30 min to uniformly disperse MA and magnetic beads in the tetrahydrofuran, and stirred at 40-75° C. for 30 min.

[0056] 0.3-0.9 mmol of 2,4,6-triformylphenol (TP) was dissolved in 10 mL of tetrahydrofuran, added dropwise to the reaction system, and 1 mL of acetic acid was added as a catalyst, and stirred at 40-75°C for 2 h. After the reaction, acetonitrile and ethanol were used for washing, and the powder of magnetic covalent framework material was obtained by drying at 60°C overnight. 3 O 4 The magnetic covalent framework material synthesized by magnetic nanoparticles is Fe 3 O 4 @MA-Tp.

[0057] According to 0.3g magnetic nanoparticles Fe 3 O 4 , 0.6mmol melamine, 0.6mmol 2,4,6-triformylbenzenetriol and synthesized magnetic covalent framework material Fe 3 O 4 @MA-Tp is used as an example for characterization. 3 O 4 Magnetic nanoparticles, monomeric melamine (MA) and 2,4,6-triformylphenol (TP) were used as references, and Fourier transform infrared spectroscopy was used to detect functional groups, as follows:

[0058] Fourier transform infrared spectroscopy is in the range of 400-4000cm -1 The range is obtained using the potassium bromide tablet method. The material to be tested and dried potassium bromide are added to a mortar at a ratio of 1:100, ground and mixed evenly, placed in a tablet mold, pressed into a tablet, and the prepared tablet is placed on a sample rack for infrared analysis. The results are as follows Figure 1 As shown in A.

[0059] Depend on Figure 1 The results of A show that Fe 3 O 4 @MA-Tp spectrum at 590cm -1 、1388cm -1 、1530cm -1 and 1604cm -1 There are characteristic peaks at 590cm -1 The characteristic Fe-O peak is shown at3 O 4 In the spectrum of 590cm -1 The Fe-O characteristic peaks shown at 3 O 4 Magnetic nanoparticles exist in Fe 3 O 4 @MA-Tp.

[0060] In the spectrum Fe 3 O 4 @MA-Tp, 1604cm -1 、1530cm -1 and 1388cm -1 The characteristic peaks at are attributed to the C=O stretching band, C=C stretching band and aromatic CN stretching band respectively. 3 O 4 @MA-Tp and the spectrum curves of monomers MA and Tp, in Fe 3 O 4 @MA-Tp spectrum at 1641cm -1 and 2895cm -1 The characteristic peak Tp at 3461cm -1 and 3412cm -1 The characteristic peak of MA (NH stretching vibration) disappears, and the characteristic peak of MA Tp is at 1255cm -1 (CN stretching vibration) appears, indicating that the two monomers are completely reacted, that is, Fe 3 O 4 @MA-Tp synthesis is completed.

[0061] Further Fe 3 O 4 Using magnetic nanoparticles as a reference, X-ray diffraction was used to gain a deeper understanding of the prepared magnetic covalent framework material Fe 3 O 4 @MA-Tp crystal structure: The copper target radiation wavelength is 0.154056nm, the scanning mode is θ / θ, the scanning speed is 2° / min, the scanning range is 5°-80°, the scanning voltage is 40kV, and the scanning current is 40mA. The results are as follows Figure 1 As shown in B.

[0062] Depend on Figure 1 The results of B show that the diffraction peaks at 2θ = 30.32°, 35.66°, 43.44°, 54.70°, 57.12° and 63.02° are attributed to the (220), (311), (400), (422), (511) and (440) reflection planes, respectively. This result is consistent with the Fe 3 O 4Cubic crystals (JCPDS card 19-0629) are consistent, indicating that the preparation of Fe 3 O 4 @MA-Tp process, Fe 3 O 4 The crystal phase is not destroyed.

[0063] The physical adsorption analyzer was used to analyze the Fe 3 O 4 @MA-Tp N2 adsorption-desorption isotherms were measured to calculate the specific surface area and pore size of the material. About 0.0449g of sample was degassed at 120℃ for 12h and then analyzed and tested. The results are as follows Figure 1 As shown in C.

[0064] Depend on Figure 1 The results of C show that Fe 3 O 4 @MA-Tp exhibits a type II isotherm with macroporous characteristics. The Brunauer-Emmett-Teller (BET) method was used to determine the Fe 3 O 4 The surface area and pore volume of @MA-Tp are 66.9152 square meters and 0.217672 cubic centimeters, respectively, which means the specific surface area is 1490m 2 / g, pore volume ratio is 4.85cm 3 / g. The Barrett–Joyner–Halenda model was used to detect Fe 3 O 4 @MA-Tp pore size distribution, the results are as follows Figure 1 As shown in D. Figure 1 The results of D determination show that Fe 3 O 4 The average pore size of @MA-Tp is 13.0118nm.

[0065] VSM refers to the vibrating sample magnetometer, which is an experimental device used to measure the magnetic properties of materials. The vibrating sample magnetometer is used to detect the magnetic size of the material: weigh an appropriate amount of sample and record the value, put it into a capsule filled with cotton for testing, and the test range is -20000 to 20000Oe. 3 O 4 For reference, the Fe of the prepared material was measured by VSM at room temperature. 3 O 4 @MA-Tp magnetic properties, the material's magnetization curve is as follows Figure 1 As shown in E.

[0066] Depend on Figure 1 The results of E show that Fe 3 O 4 The saturation magnetization is 57.67emu g -1 , Fe 3 O 4 @MA-Tp's saturation magnetization is 44.22emu g -1 , although compared to Fe 3 O 4 The magnetization intensity of Fe 3 O 4 @MA-Tp's saturation magnetization is reduced (mainly Fe 3 O 4 @MA-Tp material has a COF surface coating), but it still has good magnetic response and dispersion properties.

[0067] Magnetic nanoparticles Fe 3 O 4 As a reference, Fe 3 O 4 @MA-Tp was subjected to thermogravimetric analysis (TGA) at a heating rate of 10°C / min from 30 to 800°C in a nitrogen atmosphere to evaluate its thermal stability. Figure 1 As shown in F.

[0068] Depend on Figure 1 From the results of Fe-2+, both materials show a slight mass decay in the range of room temperature to 300 °C, which is mainly attributed to the loss of water and solvent. 3 O 4 Magnetic nanoparticles and Fe 3 O 4 The weight losses of @MA-Tp were approximately 8% and 34%, respectively, which were similar to those of Fe 3 O 4 In comparison, Fe 3 O 4 The weight loss of @MA-Tp is larger, which is mainly attributed to the mass loss of COF (i.e., MA-Tp) shell. 3 O 4 The weight loss of @MA-Tp at 450 °C was about 15%, indicating that the prepared Fe 3 O 4 @MA-Tp has good thermal stability below 450℃.

[0069] Example 2 Detection of quinolone drug residues in bee products based on magnetic covalent framework materials

[0070] In the present invention, quinolones in bee products are adsorbed and extracted using a magnetic covalent framework material as a solid phase extraction adsorbent, desorbed and eluted using a desorption solvent, and then determined by liquid chromatography-tandem mass spectrometry and quantitatively detected by an external standard method. In the following examples, negative honey that does not contain quinolones is used as a matrix, and 20 quinolone standard products specified in GB 31657.2-2021 "National Food Safety Standard for the Determination of Multi-Residue Quinolones in Bee Products by Liquid Chromatography-Tandem Mass Spectrometry" are used as standard products. These 20 quinolone standard products are added to the matrix and mixed as samples to be tested, and the magnetic covalent framework material Fe synthesized according to the method in Example 1 is added to the matrix. 3 O 4 @MA-Tp was used as a solid phase extraction adsorbent to adsorb and extract the quinolones in the sample to be tested. The target substance was desorbed and eluted by the desorption solvent and then detected by HPLC-MS / MS. The external standard method was used for quantification. The liquid chromatography conditions were as follows:

[0071] Chromatographic column: ChromCore C18 column (2.1mm×150mm, 1.8μm); column temperature: 40℃; injection volume: 5μL; flow rate: 0.25mL / min; mobile phase: A is 0.1% formic acid water-5mmol / L ammonium acetate solution, and B is methanol; mobile phase A can be prepared by adding 0.5mL formic acid and 0.19g ammonium acetate, diluting to 500mL with water and mixing well.

[0072] In the liquid chromatography mobile phase gradient elution process of this embodiment, the total volume of mobile phase A and mobile phase B is 100%, and the optimized gradient elution is as follows:

[0073] 0-1min: maintain 10% B, 90% A; 1-4.5min: the volume of mobile phase B increases from 10% to 45%, and the volume of mobile phase A decreases from 90% to 55%; 4.5-7min: the volume of mobile phase B increases from 45% to 95%, and the volume of mobile phase A decreases from 55% to 5%; 7-9.5min: maintain 95% B, 5% A; 9.5-9.6min: the volume of mobile phase B decreases from 95% to 10%, and the volume of mobile phase A increases from 5% to 90%; 9.6-14min: maintain 10% B, 90% A, with a total running time of 14min.

[0074] The mass spectrometry conditions were as follows:

[0075] Ion source: ESI ion source; Scan mode: positive ion scan; Detection mode: multiple reaction monitoring mode (MRM); Optimized electrospray conditions: curtain gas, 35 psi; ion source gas GS1, 55 psi; ion source gas GS2, 55 psi; ion spray voltage, 5500 V; source temperature, 450 ° C nitrogen collision gas, medium. The specific MRM parameters for testing these 20 quinolones are shown in Table 1.

[0076] Table 1 Detailed information on multiple reaction monitoring (MRM) parameters for 20 quinolones (QNs)

[0077]

[0078]

[0079] The negative honey refers to blank honey on the market that does not contain quinolones. GB 31657.2-2021 "National Food Safety Standard for the Determination of Quinolone Residues in Bee Products by Liquid Chromatography-Tandem Mass Spectrometry" can be used to detect and screen out negative honey as a matrix. The standard products of the 20 quinolones are added to the matrix and mixed as the sample to be tested. The main parameters affecting the efficiency of magnetic solid phase extraction (MSPE) are optimized as follows:

[0080] (1) The influence of the characteristics of honey matrix on the detection results of target analytes

[0081] The main components of honey include glucose and fructose, which account for about 65% to 80% of the total honey content, followed by water, which accounts for 16% to 25% of the total honey content. It also contains other polysaccharides, minerals, organic acids, various celluloses and proteins. During the magnetic solid phase extraction process, due to the high viscosity of the honey matrix and the fact that some components in the honey may also cover the adsorption sites on the surface of the magnetic material, forming a certain competitive relationship with the target analyte, reducing the extraction efficiency of the target analyte, we first investigated the effect of the negative honey matrix on the detection results of the target analyte, as follows:

[0082] ① Preparation of test samples: Taking blank negative honey matrix as control, weigh negative honey matrix and dilute it with water to 0.05g / mL, 0.1g / mL, 0.2g / mL, 0.3g / mL, 0.4g / mL, 0.5g / mL, 0.6g / mL. Add 20 kinds of quinolone drugs in Table 1 to each group and mix them evenly to make the concentration of each standard product 5ng / mL, and measure the pH of each group of test samples. The results are shown in the following table.

[0083] Table 2 Matrix concentration and corresponding pH of the samples to be tested

[0084]

[0085] Add 4 mg of magnetic material Fe to each group of sample solutions. 3 O 4 @MA-Tp, vortex adsorption for 4 minutes, separate by magnetic adsorption, and discard the supernatant; then add 2.0 mL of water and vortex for 2 minutes to wash away impurities, separate by magnetic adsorption, and discard the supernatant.

[0086] Then add 4.0mL 10% formic acid-methanol (take 10mL formic acid, dilute to 100mL with methanol, mix well), vortex desorption for 2min to release the target analyte adsorbed on the magnetic material, separate by magnetic adsorption, take all the supernatant, blow dry in nitrogen at 40℃ water bath. Add 0.3mL 50% acetonitrile-water to dissolve, filter through 0.22μm nylon filter membrane, and collect the filtrate as the sample for the machine.

[0087] ② High performance liquid chromatography-mass spectrometry detection: The above samples were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The results are as follows: Figure 2 As shown, it indicates that the honey matrix has no effect on the detection of target analytes.

[0088] The standard stock solution and 20 quinolones mixed standard working solutions were prepared according to the preparation methods of the standard stock solution (1 mg / mL) and mixed standard working solution (10 μg / mL) in GB 31657.2-2021 "National Food Safety Standard for the Determination of Multi-Residues of Quinolones in Bee Products by Liquid Chromatography-Tandem Mass Spectrometry". Using negative honey as the matrix, the standard was added to prepare the samples containing mixed standards of different concentrations. The same pretreatment and detection conditions were used to draw the matrix matching standard curve between the characteristic ion mass chromatographic peak area of ​​the 20 quinolones and the concentration of the standard.

[0089] According to the obtained matrix matching standard curve, the concentration of the 20 quinolones in the sample is calculated according to the mass chromatographic peak area of ​​the characteristic ions of the 20 quinolones in the total ion flow diagram of the sample, and then the recovery rate of the quinolone target is calculated according to the ratio of the measured concentration of the quinolone target in the sample to the actual standard concentration of the target, in %. The ideal recovery rate should be close to 100%. In food analysis, the accuracy and reliability of the detection method are generally evaluated at a recovery rate of 80-120%, and the preferred recovery rate is 90-110%, which is considered to be a good method with good accuracy and reliability.

[0090] The analysis results show that the recovery rates of the 20 target analytes are between 95.31% and 112.63% when the concentration of the honey matrix is ​​0.05-0.3 g / mL, and the detection is better in this concentration range, among which the best is achieved when the concentration of the honey matrix is ​​0.3 g / mL. As the concentration of the honey matrix increases from 0.3 g / mL to 0.6 g / mL, the recovery rates of the target analytes gradually decrease. It is speculated that the honey matrix gradually covers the adsorption sites of the magnetic material, and the target analytes are difficult to bind to their adsorption sites, and are lost along with the washing agent. That is, the concentration of the honey sample diluted to 0.05-0.3 g / mL is better, preferably 0.3 g / mL.

[0091] Furthermore, the effect of the volume of the sample to be tested on the test results was compared. The volume of the sample to be tested (the concentration of the negative honey matrix was 0.2 g / mL, and the concentration of the 20 standard products was 5 ng / mL) was gradually increased from 1 mL to 4 mL. 3 O 4 @MA-Tp adsorption extraction, analytical solvent elution, 50% acetonitrile-water dissolution, microporous nylon membrane filtration and then HPLC-MS detection, wherein the sample volume gradually increased from 1 mL to 4 mL. The HPLC-MS detection results showed that with the increase of the sample volume, the peak areas corresponding to the 20 target analytes did not change significantly, indicating that the magnetic material has good adsorption capacity for the 20 quinolone target analytes. In order to obtain a relatively high enrichment efficiency, the sample volume is preferably 2 mL.

[0092] (2)Fe 3 O 4 @MA-Tp dosage optimization

[0093] Weigh 0.6g of negative honey matrix and add water to make up to 2mL, add 20 kinds of quinolone drug standards in Table 1 and mix well to make the concentration of each standard product 5ng / mL, which is the sample to be tested, and add 2mg, 4mg, 6mg, 8mg, 10mg, 12mg, 14mg, 16mg, 18mg, 20mg of Fe 3 O 4 @MA-Tp magnetic material, vortex adsorption for 4 minutes, magnetic adsorption separation, and discard the supernatant.

[0094] Add 2.0 mL of water and vortex for 2 min to wash away impurities, then separate by magnetic adsorption and discard the supernatant. Add 4.0 mL of 10% formic acid and methanol, vortex for desorption for 2 min, separate by magnetic adsorption, take all the supernatant, blow dry with nitrogen in a water bath at 40°C. Add 0.3 mL of 50% acetonitrile-water (take 50 mL of acetonitrile, add 50 mL of water and mix well) to dissolve, collect the filtrate by filtering with a 0.22 μm nylon filter membrane, and use HPLC-MS or MS for detection to obtain the total ion flow diagram, among which Fe3 O 4 @MA-Tp added 2mg, 4mg, 12mg and 20mg corresponding total ion current diagram as shown Figures 3 to 6 shown.

[0095] According to the total ion current graph obtained, the peak areas corresponding to the above 20 quinolones were calculated, and the results showed that:

[0096] Using different amounts of Fe 3 O 4 @MA-Tp was used as a solid phase extraction adsorbent for extraction. The peak areas corresponding to the 20 target analytes showed no significant difference, indicating that the synthesized Fe 3 O 4 @MA-Tp magnetic material has good specific adsorption capacity for these 20 quinolone compounds.

[0097] And calculate the recovery rate of these 20 quinolones. The results are as follows: Figure 7 As shown in A, the recoveries of the 20 target analytes with different amounts of adsorbents did not vary significantly, indicating that the synthesized Fe 3 O 4 @MA-Tp material has good specific adsorption capacity for these 20 quinolones, among which the concentration of honey sample (g / mL) and Fe 3 O 4 @The mass (mg) ratio of MA-Tp is 0.3:2. Adding magnetic material can meet the extraction requirements.

[0098] (3) Optimization of adsorption time

[0099] Weigh 0.6 g of negative honey matrix, dilute with water and make up to 2.0 mL, add 20 quinolone drug standards in Table 1, make the concentration of each standard product 5 ng / mL, add 4 mg Fe 3 O 4 @MA-Tp, vortex adsorption for 2 min, 4 min, 6 min, 8 min, and 10 min respectively. Other operations were the same as in (2). HPLC-MS / MS analysis was used to calculate the recoveries of these 20 quinolones. The results are shown in Figure 7 As shown in B.

[0100] The analysis results show that as the adsorption time increases from 2 min to 4 min, the peak areas (recoveries) corresponding to the 20 quinolones gradually increase, and the corresponding peak areas (recoveries) remain basically unchanged in the range of 4 to 10 min. This may be because the adsorption equilibrium is reached at 4 min, and the adsorption of each target has been completed. Considering the saving of detection time, the preferred adsorption time is 4 min.

[0101] (4) Optimization of analytical solvent and analytical method

[0102] The desorption solvent is a key factor affecting the detection results of the target. In order to select the best desorption solvent, the effects of different organic solvents (acetonitrile, methanol, ethanol, acetone, 10% formic acid acetonitrile, 10% formic acid methanol, 10% formic acid ethanol and 10% formic acid acetone) on the detection results of the target analytes of these 20 quinolone drugs were compared. Among them, 10% formic acid acetonitrile, 10% formic acid methanol, 10% formic acid ethanol or 10% formic acid acetone means that the volume of formic acid accounts for 10% of the total volume of the desorption solvent, such as 10% formic acid acetonitrile: take 10mL formic acid, add 90mL acetonitrile and mix well; the specific experimental process is as follows:

[0103] Weigh 0.6 g of negative honey as the matrix, dilute with water and make it 2 mL, add the 20 quinolone drug standards in Table 1 to make the concentration of each standard 5 ng / mL, add 4 mg Fe 3 O 4 @MA-Tp, vortex adsorption for 4 minutes, magnetic adsorption separation, and discard the supernatant.

[0104] Then add 2.0mL of water and vortex for 2min to wash away impurities, then separate by magnetic adsorption and discard the supernatant. Then add analytical solvents (acetonitrile, methanol, ethanol, acetone, 10% formic acid acetonitrile, 10% formic acid methanol, 10% formic acid ethanol or 10% formic acid acetone) and vortex desorb for 2min, separate by magnetic adsorption, dry the supernatant with nitrogen, then re-dissolve and filter with microporous nylon filter membrane to prepare the sample for the machine, and analyze it by optimized HPLC-MS / MS. The results show that:

[0105] When acetonitrile, methanol, ethanol or acetone are used as desorption solvents, the characteristic peaks corresponding to the 20 targets are not found in the total ion current map obtained, but the characteristic peaks corresponding to the 20 targets can be accurately detected in the total ion map obtained after formic acid is added to the desorption solvent. This indicates that it is difficult to elute quinolone drugs from the adsorption material when acetonitrile, methanol, ethanol and acetone are used as the four conventional desorption solvents for desorption, but the 20 targets can be eluted from the adsorption material after formic acid is added to the desorption solvent, and the desorption ability of 10% formic acid methanol for the 20 quinolone compounds is significantly better than that of the other three acid-added desorption solvents.

[0106] The effect of formic acid content on desorption efficiency was further compared. Specifically, taking the negative honey concentration of 0.3 g / mL and the concentration of 20 quinolone drug standards of 5 ng / mL as an example, the Fe 3 O 4@MA-Tp adsorption extraction, water washing to remove impurities, magnetic adsorption separation, comparison of methanol, 1% formic acid methanol, 5% formic acid methanol, 10% formic acid methanol, 20% formic acid methanol five desorption solvents on the desorption of these 20 target substances, after re-dissolution and filtration, HPLC-MS / MS detection and analysis, and calculation of the recovery of these 20 quinolones, the results are as follows Figure 7 As shown in C.

[0107] The analysis results show that when the formic acid content is in the range of 0%-10%, as the formic acid content increases, the characteristic ion mass chromatographic peak area (recovery rate) corresponding to the 20 target substances presents an increasing trend, wherein the recovery rate corresponding to the 10% formic acid methanol group is 94.64%-109.47%, and the desorption effect is the best, and when the formic acid content increases to 20%, the recovery rates of the 20 target substances are 104.3%-136.0%, which is speculated to be due to the excessive acid content, resulting in more interfering substances being desorbed and eluted by the desorption solvent, thereby increasing the matrix enhancement effect. Therefore, the formic acid content in the preferred desorption solvent is 10%.

[0108] In addition, taking the negative honey concentration of 0.3g / mL and the concentration of 20 quinolone drugs standard products of 5ng / mL as an example, the Fe 3 O 4 @MA-Tp adsorption extraction, water washing to remove impurities, magnetic adsorption separation, adding 10% formic acid methanol for desorption, in the desorption process, vortex and ultrasonic desorption methods were compared, the desorption time was 2min, after re-dissolution and filtration, HPLC-MS / MS detection and analysis were used, and the recovery rates of 20 quinolones in the sample were calculated. The results are as follows Figure 7 As shown in D.

[0109] The analysis results show that the recovery rate of 20 quinolones in the sample using vortex desorption is basically twice that of ultrasonic desorption, indicating that vortex desorption is more effective. It may be that during ultrasonic desorption, the materials are not fully dispersed and are gathered at the bottom of the centrifuge tube, resulting in insufficient desorption and low recovery rate.

[0110] (5) Optimization of desorption solvent addition amount and desorption time

[0111] 10% formic acid in methanol was used as the desorption solvent for optimization as follows:

[0112] Weigh 0.6 g of negative honey matrix, dilute with water and make up to 2.0 mL, add 20 quinolone drug standards in Table 1, make the concentration of each standard product 5 ng / mL, add 4 mg Fe 3 O 4 @MA-Tp, vortex adsorption for 4 minutes, magnetic adsorption separation, and discard the supernatant.

[0113] Then add 2.0 mL of water and vortex for 2 min to wash away impurities, then separate by magnetic adsorption and discard the supernatant. Then add 4.0 mL of 10% formic acid in methanol, set the vortex desorption time to 2 min, 4 min, 6 min, 8 min, and 10 min, separate by magnetic adsorption, blow dry with nitrogen, re-dissolve and filter, and analyze by HPLC-MS / MS, and calculate the recovery rate of the 20 quinolones in the sample. The results are as follows Figure 7 As shown in E.

[0114] The analysis results show that with the increase of desorption time, the recovery rates of the 20 quinolones have no obvious change, indicating that these 20 quinolones can be completely desorbed and eluted by vortex desorption for 2 minutes.

[0115] The effect of desorption time on the elution effect of the 20 quinolones was further studied. Taking the sample in (5) as an example, the amount of 10% formic acid in methanol was set to 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, 6.0 mL, 7.0 mL, and 8.0 mL. The samples were vortexed for desorption for 2 min, separated by magnetic adsorption, dried with nitrogen, and re-dissolved and filtered to prepare the samples for the machine. HPLC-MS / MS was used for detection and analysis, and the recoveries of the 20 quinolones in the samples were calculated. The results are shown in Figure 2. Figure 7 As shown in F.

[0116] The analysis results show that the elution efficiency of the 20 quinolones gradually increased as the desorption solvent volume increased from 1.0 mL to 4.0 mL, especially the recovery rates of enoxacin, marbofloxacin and pipemidic acid increased significantly. When 4.0 mL of desorption solvent was added for elution, the recovery rates of the 20 quinolones reached 92.6-115.2%, while the recovery rate remained basically unchanged when the volume increased from 4.0 mL to 8.0 mL. When 4.0 mL of desorption solvent was added for elution, the recovery rates of each target analyte in the sample were as follows.

[0117] Table 3 Recovery rates of 20 target analytes in samples

[0118]

[0119] (6) Optimization of microfiltration membrane

[0120] It is very necessary to filter the sample solution before using the machine, mainly to effectively filter out large particles of impurities, reduce the interference of impurities on the target object and damage to the instrument. However, the filter membrane may also have an adsorption effect on the target analyte in the solution. This example studies whether the microporous filter membranes of three different materials have an adsorption effect on these 20 quinolones, as follows:

[0121] Weigh 0.6 g of negative honey matrix, dilute with water and make up to 2.0 mL, add 20 quinolone drug standards in Table 1, make the concentration of each standard product 5 ng / mL, add 4 mg Fe 3 O 4 @MA-Tp, vortex adsorption for 4 minutes, magnetic adsorption separation, and discard the supernatant. Add 2.0mL of water and vortex for 2 minutes to wash away impurities, and then separate by magnetic adsorption and discard the supernatant. Add 4.0mL of 10% formic acid methanol, vortex desorption for 2 minutes, magnetic adsorption separation, take all the supernatant, and blow dry with nitrogen in a water bath at 40°C. Add 0.3mL of 50% acetonitrile-water to dissolve, filter with 0.22μm nylon filter membrane, 0.22μm polytetrafluoroethylene filter membrane, and 0.22μm polyethersulfone filter membrane respectively, and then use HPLC-MS / MS to detect and analyze, and calculate the recovery rate of 20 quinolones in the sample. The results are as follows Figure 8 shown.

[0122] Depend on Figure 8 The results show that the recovery rate of most target analytes in the sample is less than 20% when the polyethersulfone filter membrane is used for filtration, indicating that the polyethersulfone filter membrane has a strong adsorption effect on most of the target substances and cannot be used as a filter membrane for the detection of quinolones. When the nylon filter membrane and polytetrafluoroethylene filter membrane are used for filtration, the recovery rates of the 20 target analytes in the sample are mostly between 80% and 110%, indicating that the nylon filter membrane and polytetrafluoroethylene filter membrane have no obvious adsorption to the 20 target analytes and can be used as filter membranes for the detection of quinolones. The recovery rate of the national standard method is 60-120%, indicating that the detection accuracy of this method is better than that of the national standard method.

[0123] In summary, this method is based on the extraction and purification of target substances in bee products by magnetic solid phase extraction agent, and then high performance liquid chromatography-mass spectrometry is used for detection and analysis. The optimized pretreatment is to add 4 mg of Fe 3 O 4 @MA-Tp, vortex adsorption for 4 minutes, magnetic adsorption separation, and discard the supernatant. Add 2.0mL of water and vortex for 2 minutes to wash away impurities, and then separate by magnetic adsorption and discard the supernatant. Add 4.0mL of 10% formic acid methanol, vortex desorption for 2 minutes, magnetic adsorption separation, take all the supernatant, blow dry in a water bath with nitrogen at 40°C, add 0.3mL of 50% acetonitrile-water to dissolve, filter through a 0.22μm nylon or polytetrafluoroethylene filter; use high performance liquid chromatography-mass spectrometry for detection and external standard quantification.

[0124] Example 3Fe 3 O 4 @MA-Tp material reusability

[0125] In order to study Fe 3 O4 @MA-Tp material as an adsorbent, a cyclic adsorption and desorption experiment was conducted. The sample to be tested (honey or royal jelly) was diluted to a concentration of 0.3 g / mL, and the test was repeated according to the optimized test method. 3 O 4 The MA-Tp material was washed with 10% formic acid and methanol until no quinolone residue was detected. It was then used as an adsorbent for the next MSPE. After the test, the Fe 3 O 4 @MA-Tp, wash until no quinolone residue is detected, and reuse. The test results of repeated use are as follows Fig. 9 .

[0126] Depend on Fig. 9 The results show that the recovery rates of the 20 quinolones did not change significantly after repeated use for 6 times compared with the first use. 3 O 4 @MA-Tp magnetic material can be reused as a magnetic solid phase adsorbent, which is beneficial to reduce the detection cost of quinolone residues in bee products.

[0127] Example 4 Evaluation of the detection method

[0128] In order to verify the applicability of this method for the determination of NQ, the linear range, correlation coefficient (r), matrix effect (ME), limit of detection (LOD), limit of quantification (LOQ), recovery and precision of this method were evaluated under the optimized detection conditions. The results are shown in Tables 4 and 5.

[0129] Table 4 Matrix-matched and solvent-matched standard curves, median error, detection limit and quantification limit of the method

[0130]

[0131]

[0132]

[0133] The matrix matching standard curve in Table 4 is based on negative honey or negative royal jelly as the matrix, and each standard product is added to the matrix separately to prepare a standard solution, wherein the concentration of the standard product is 0.5μg / kg, 1μg / kg, 5μg / kg, 10μg / kg, 25μg / kg, 50μg / kg; the solvent matching standard curve is based on water as the solvent, and each standard product is added separately to prepare a standard solution of the same concentration. The treatment was carried out under the optimized pretreatment conditions, and then the high performance liquid chromatography-mass spectrometry was used for detection. The matrix matching standard curve was drawn with the mass chromatographic peak area of ​​each drug characteristic ion as the ordinate and the concentration of the standard solution as the abscissa, and the regression equation and correlation coefficient r were calculated.

[0134] For the 20 QNs, honey samples were in the range of 0.5–50 μg kg -1 In the concentration range of royal jelly samples, the concentration range is 1.0-50 μg / kg -1 A satisfactory linear relationship was obtained over the concentration range, with a correlation coefficient of more than 0.9980 for 20QNs. The calculated detection limit was between 0.04 and 0.21 μg kg at a signal-to-noise ratio of 3. -1 The LOQ was calculated at a S / N ratio of 10 and was between 0.12 and 0.70 μg / kg. -1 The matrix effect (ME) was calculated based on the slope of the matrix matching curve and the standard solvent calibration curve. The ME of most QNs ranged from 0.80 to 1.20, which was negligible.

[0135] Table 5 Recovery and precision of fluoroquinolones added to blank honey and blank royal jelly samples

[0136]

[0137] In Table 5, the concentrations of the 20 quinolones in the sample were calculated based on the matrix-matched standard curve obtained, and the recovery rate was calculated according to the ratio of the measured concentration of the quinolone target in the sample to the actual standard concentration of the target, in %.

[0138] Example 5 Practical Application

[0139] 90 batches of honey samples and 10 batches of royal jelly were randomly selected from local supermarkets and measured according to the optimized method. The results showed that norfloxacin was detected in 10 batches of honey samples (the detection value of norfloxacin was 2.05-14.2μg / kg), and ofloxacin was also detected in 1 batch (the detection value of ofloxacin was 1.45μg / kg). According to GB 31650.1-2022 "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food", the maximum residue limits of norfloxacin and ofloxacin in honey samples are both 5μg / kg. Among the positive samples, 4 honey samples failed, and the total sample failure rate was 4.0%. At the same time, GB31657.2-2021 "National Food Safety Standard Determination of Multi-Residues of Quinolone Drugs in Bee Products" was used to verify the data of these 10 batches of positive samples, and the results are shown in Table 6.

[0140] Table 6 Comparison of the results of this method and the national standard method

[0141]

[0142] From the verification results in the table above, it can be seen that the detection results of quinolone compounds in bee products by this method are not significantly different from those by the national standard method, and can be used for the determination of quinolone drug residues in bee products. The national standard method takes 37.6 minutes to detect quinolone drug residues in honey, with a detection limit of 0.5μg / kg and a quantitative limit of 1μg / kg. The organic solvent consumption is high and the solid phase column cannot be reused, resulting in high detection costs.

[0143] The detection time of this method can be as low as 14 minutes, and the detection limit is as low as 0.04μg / kg, the quantitative limit is as low as 0.12μg / kg, and the magnetic covalent framework material can be reused at least 6 times. Compared with the existing national standard method, this method is used to determine quinolone compounds in bee products, which takes less time, is more efficient, has higher detection sensitivity and accuracy, and is low in cost.

[0144] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A magnetic covalent framework material, characterized in that: The invention relates to a magnetic material with magnetic nanoparticles as the core and organic monomers melamine and 2,4,6-triformylbenzene triphenol loaded on the surface of the magnetic nanoparticles, wherein the magnetic nanoparticles include any one of Fe3O4 or its modified product, Fe3O4 NPs, NiFe2O4 and γ-Fe2O3; the specific surface area of ​​the magnetic material is 1000-1500m 2 / g, pore volume ratio is 4-5cm 3 / g.

2. The magnetic covalent framework material according to claim 1, characterized in that: Prepared as follows: Magnetic nanoparticles and melamine are added to tetrahydrofuran and uniformly dispersed by ultrasound; 2,4,6-triformylbenzenetriol is dissolved in tetrahydrofuran and added dropwise to the reaction system, acetic acid is used as a catalyst, and after sufficient reaction, acetonitrile and ethanol are used for washing, and the magnetic covalent framework material is obtained by drying.

3. The magnetic covalent framework material according to claim 2, characterized in that: The magnetic nanoparticles are Fe3O4, and the mass ratio of Fe3O4 to melamine and 2,4,6-triformylbenzene is 3:(0.3-1.2):(0.6-2.0).

4. The magnetic covalent framework material according to claim 3, characterized in that: The catalytic reaction temperature is 40-75°C and the reaction time is 2h.

5. A method for detecting quinolone residues in bee products, characterized in that: The following steps are involved: (1) Extraction and purification of quinolone drugs: using the magnetic covalent framework material as claimed in any one of claims 1 to 4 as a magnetic solid phase extractant to extract and enrich, separate, clean and remove impurities from 20 quinolone drugs in the sample; (2) Elution: vortex desorption with a desorption solvent containing formic acid to desorb and elute the 20 quinolone target compounds from the magnetic covalent framework material, collect the eluate, and filter it using a nylon or polytetrafluoroethylene microporous filter membrane; (3) Qualitative or quantitative detection: HPLC-MS is used for qualitative or quantitative detection.

6. The detection method according to claim 5, characterized in that The sample is honey or royal jelly diluted to a concentration of 0.05-0.3 g / mL, and the 20 quinolone drugs are oxolinic acid, flumequine, cinoxacin, difloxacin, sarafloxacin, ofloxacin, enrofloxacin, lomefloxacin, pefloxacin, norfloxacin, ciprofloxacin, nalidixic acid, enoxacin, danofloxacin, marbofloxacin, fleroxacin, gatifloxacin, orbifloxacin, pipemidic acid and sparfloxacin.

7. The detection method according to claim 6, characterized in that The magnetic covalent framework material is Fe3O4@MA-Tp, and the extraction time is 4 to 10 minutes. If the sample is honey, the Fe3O4@MA-Tp addition amount is preferably 4 mg, and if the sample is royal jelly, the Fe3O4@MA-Tp addition amount is preferably 6 mg.

8. The detection method according to claim 7, characterized in that The desorption solvent containing formic acid is a mixed solvent of formic acid and acetonitrile, methanol, ethanol or acetone, wherein the volume of formic acid accounts for 10% to 15% of the volume of the mixed solvent.

9. The detection method according to claim 8, characterized in that: The desorption solvent is 10% formic acid methanol, the addition amount is 4.0mL-8.0mL, and the desorption is vortexed for more than 2 minutes.

10. The detection method according to claim 9, characterized in that: HPLC-MS quantitative detection was adopted, wherein the total volume of mobile phase A and mobile phase B in the liquid chromatography mobile phase gradient elution process was 100%, specifically: 0-1min: maintain 10% B; 1-4.5min: the volume of mobile phase B increases from 10% to 45%; 4.5-7min: the volume of mobile phase B increases from 45% to 95%; 7-9.5min: maintain 95% B; 9.5-9.6min: the volume of mobile phase B decreases from 95% to 10%; 9.6~14min: Maintain 10% B.

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