Fe3O4 / GO / CS magnetic nanocomposite and application thereof in detecting content of danofloxacin in pig urine

Abstract

The application provides a Fe3O4 / GO / CS magnetic nanocomposite and application thereof in detection of the content of darifloxacin in pig urine, and belongs to the technical field of antibiotic detection. The Fe3O4 / GO / CS magnetic nanocomposite is prepared from Fe3O4, graphene oxide and chitosan. The detection method comprises dispersing the Fe3O4 / GO / CS into a sample solution to be detected, obtaining a sample to be analyzed through magnetic solid phase extraction, and detecting the content of darifloxacin in the sample to be analyzed through HPLC-FLD. The Fe3O4 / GO / CS magnetic nanocomposite simplifies the sample pretreatment process, improves the sample extraction rate, and the detection method has high sensitivity and accuracy, and is suitable for detection of darifloxacin in pig urine samples.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibiotic detection, and particularly relates to a Fe3O4 / GO / CS magnetic nanocomposite and application thereof in detecting the content of danofloxacin in pig urine. BACKGROUND

[0002] Danofloxacin is an animal-specific quinolone antibiotic with the advantages of wide antibacterial spectrum and strong antibacterial activity, and is therefore widely used in the treatment of respiratory diseases of livestock and poultry in veterinary clinics in China. Although the use is allowed by the relevant national standards, excessive use or improper use can cause drug residues in animal products, which may pose potential harm to the health of consumers and lead to bacterial resistance and other problems. The maximum residue limit of danofloxacin in pork is 100 μg / kg, the maximum residue limit in beef, mutton and poultry meat is 200 μg / kg, and the maximum residue limit in cow's milk and goat's milk is 30 μg / kg according to GB31650-2019. The maximum residue limit of danofloxacin in poultry eggs is 10 μg / kg according to GB31650.1-2022. Therefore, it is of great significance to establish an efficient, sensitive and reliable detection method for monitoring the residue of danofloxacin in food.

[0003] At present, the commonly used methods for detecting danofloxacin include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS). Among them, the HPLC method is widely used in antibiotic residue detection due to its simple operation, low cost and high sensitivity. However, the traditional HPLC method often faces serious matrix interference and complicated sample pretreatment when dealing with complex matrix samples such as pig urine, which affects the accuracy and reliability of the detection results.

[0004] In order to overcome the above problems, in recent years, solid phase extraction (SPE) technology has been widely used in sample pretreatment to improve the selectivity and sensitivity of the detection method. However, the traditional SPE technology has the disadvantages of limited adsorbent capacity, complex operation steps and long time-consuming. Therefore, developing an efficient and simple sample pretreatment technology has become a research hotspot.

[0005] Magnetic solid phase extraction (MSPE) is a new sample pretreatment technology that combines the traditional SPE technology and the unique properties of magnetic materials. MSPE uses magnetic materials as adsorbents to separate the adsorbents from the sample quickly by applying a magnetic field, greatly simplifying the operation steps and shortening the processing time. In addition, magnetic materials have a large specific surface area and good adsorption performance, which can effectively improve the enrichment efficiency of target analytes.

[0006] High performance liquid chromatography-fluorescence detector (HPLC-FLD) is a high-sensitivity detection technology, especially suitable for the detection of trace analytes. The fluorescence detector has high detection sensitivity and selectivity, which can effectively reduce matrix interference and improve the accuracy of detection results. The combination of MSPE and HPLC-FLD can fully exert the advantages of both and realize the efficient and sensitive detection of enrofloxacin in pig urine.

[0007] Although MSPE combined with HPLC-FLD shows great potential in antibiotic residue detection, there are still some challenges in practical application. First, how to choose the appropriate magnetic material and surface modification method to improve the adsorption efficiency and selectivity of the target analyte is a key problem. Second, the matrix effect and removal of interfering substances during sample pretreatment are also difficult. In addition, how to optimize the detection conditions to improve the sensitivity and reproducibility of the detection method is also a problem to be solved.

[0008] Therefore, there is an urgent need for a method to efficiently and sensitively detect the content of enrofloxacin in pig urine to overcome the shortcomings of traditional detection methods. SUMMARY

[0009] The purpose of the present application is to provide a Fe3O4 / GO / CS magnetic nanocomposite material, and to use the composite material as an adsorbent combined with HPLC-FLD detection technology for a method of efficiently and sensitively detecting the content of enrofloxacin in pig urine. This method not only effectively overcomes the shortcomings of traditional detection methods, but also significantly improves the accuracy and reliability of detection, providing strong technical support for food safety and animal health.

[0010] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0011] The present application provides a Fe3O4 / GO / CS magnetic nanocomposite material in the first aspect, which is prepared from Fe3O4, graphene oxide and chitosan.

[0012] Specifically, the preparation method of the Fe3O4 / GO / CS magnetic nanocomposite material comprises the following steps:

[0013] (i) Add ferrous sulfate and ferric chloride to water, stir and mix well, add polyethylene glycol aqueous solution, and time for 30 min when the temperature reaches 50℃. After 30 min, vacuum pump air, introduce N2, add ammonia water, and increase the temperature to 75-85℃. Water bath reaction for 20-40 min;

[0014] (ii) The product obtained in step (i) is washed with distilled water and ethanol in turn, and Fe3O4 is obtained after vacuum drying;

[0015] (iii) mixing and grinding Fe3O4, graphene oxide and chitosan, rinsing with ultrapure water and ethanol after grinding, and vacuum drying to obtain Fe3O4 / GO / CS magnetic nanocomposite.

[0016] Further, in step (i), the mass ratio of the ferrous sulfate, ferric chloride and polyethylene glycol is (2-3):(5-6):(3-4); the mass concentration of the mixture obtained by adding the ferrous sulfate and ferric chloride into water is 0.08-0.2 g / mL; the mass concentration of the polyethylene glycol aqueous solution is 0.08-0.2 g / mL; the mass concentration of the ammonia water is 25-28%, and the volume ratio of the ammonia water to the polyethylene glycol aqueous solution is 1:(0.8-1.2).

[0017] Further, in step (ii), the drying temperature is 50-60℃, and the drying time is 10-20 h.

[0018] Further, in step (iii), the mass ratio of the Fe3O4, graphene oxide and chitosan is 1:(1-1.5):(1-1.5); the grinding time is 20-40 min; the drying temperature is 50-60℃, and the drying time is 10-20 h.

[0019] In the Fe3O4 / GO / CS magnetic nanocomposite of the application, Fe3O4 is a magnetic material with superparamagnetism, which can be quickly separated under the action of an external magnetic field, and this feature enables the composite material to be quickly and effectively separated from a complex matrix through a simple magnetic separation step during sample processing, simplifying the operation steps and improving the processing efficiency. Graphene oxide (GO) has a two-dimensional nanostructure and a large specific surface area, and can provide a large number of adsorption sites to enhance the adsorption capacity of the target analyte. Chitosan (CS) is a porous natural polymer material with abundant pore structure, which can further increase the specific surface area of the composite material and improve the adsorption capacity. Fe3O4 and chitosan have good chemical stability and can maintain the integrity of the structure in acidic, alkaline or organic solvents, and are suitable for various chemical environments. The synthesis of Fe3O4 uses a solvothermal method or a co-precipitation method, which is simple, mild and easy to control, and can be mass-produced; by precisely controlling the proportion of raw materials and reaction conditions, high-purity Fe3O4 nanoparticles are prepared, reducing the introduction of impurities. The mixing and grinding process of Fe3O4, graphene oxide and chitosan is simple, and uniform dispersion and close combination can be achieved through mechanochemical action without complex chemical reactions; during the mixing and grinding process, Fe3O4 nanoparticles can be uniformly dispersed in graphene oxide and chitosan, avoiding agglomeration and improving the dispersibility and stability of the composite material.

[0020] The second aspect of the application provides application of the Fe3O4 / GO / CS magnetic nanocomposite in detecting the content of darifloxacin in pig urine.

[0021] Specifically, the detection method comprises the following steps:

[0022] (1) Magnetic solid phase extraction:

[0023] Fe3O4 / GO / CS is dispersed into a sample solution to be detected, oscillation extraction is performed, Fe3O4 / GO / CS is separated by an external magnet, the separated Fe3O4 / GO / CS is cleaned with ultrapure water, and then an eluent is added for desorption, Fe3O4 / GO / CS and the eluent are separated, and the separated eluent is filtered with a 0.22 μm organic filter membrane to obtain a sample to be analyzed;

[0024] (2) HPLC-FLD detection:

[0025] The content of darifloxacin in the sample to be analyzed is detected by HPLC-FLD.

[0026] Further, the preparation method of the sample solution to be detected in step (1) is as follows: according to the expected concentration of darifloxacin in pig urine, the collected pig urine sample is diluted 1-10 times with ultrapure water, and the pH value is adjusted to 4-8.

[0027] Further, the mass-volume ratio of Fe3O4 / GO / CS to the sample solution to be detected in step (1) is 1 mg:(1-4) mL.

[0028] Further, the oscillation extraction time in step (1) is 10-30 min.

[0029] Further, the eluent in step (1) is a mixture of ethanol and 3 mol / L NaOH aqueous solution with a mass ratio of 3:(0.8-1.2);

[0030] Further, the mass-volume ratio of Fe3O4 / GO / CS to the eluent in step (1) is 10 mg:(1.5-2.5) mL.

[0031] Further, the HPLC chromatographic conditions in step (2) are as follows:

[0032] Mobile phase: A phase is 0.03-0.08 mol / L phosphoric acid / triethylamine solution, B phase is acetonitrile, and the ratio of the mobile phase is: A:B=(80-90):(10-20);

[0033] Detection wavelength: excitation wavelength is 280 nm, and emission wavelength is 450 nm;

[0034] The injection amount is 20 μL;

[0035] The flow rate is 0.5-1.0 mL / min.

[0036] In the detection method of the present application, the magnetic solid phase extraction technology simplifies the sample pretreatment process, quickly separates the sample, improves the work efficiency, the composite material has a high specific surface area and a porous structure, and can efficiently adsorb the target analyte; the HPLC-FLD technology has high sensitivity and selectivity, can effectively reduce the matrix interference, and improves the accuracy of the detection result; and the system-optimized experimental conditions ensure the stability and reproducibility of the method.

[0037] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0038] 1. Simplify the sample pretreatment process: the present application uses the magnetic nano composite material Fe3O4 / GO / CS as the adsorbent, and through the magnetic solid phase extraction technology, the sample is quickly separated through an external magnetic field, greatly simplifying the sample pretreatment process, avoiding the complicated operations such as centrifugation or filtration in the traditional solid phase extraction, and saving time and labor cost.

[0039] 2. Improve the sample extraction rate: the Fe3O4 / GO / CS composite material has a large specific surface area and good adsorption performance, can efficiently adsorb the danofloxacin in pig urine, improve the sample extraction rate, and through the optimization of the extraction time, adsorbent dosage, sample solution volume and other conditions, ensure the highest recovery rate and extraction efficiency.

[0040] 3. Enhance the sensitivity and accuracy of the detection method: the present application uses high performance liquid chromatography-fluorescence detector (HPLC-FLD) for detection, and the fluorescence detector has high detection sensitivity and selectivity, can effectively reduce the matrix interference, and improve the accuracy of the detection result. In the detection method of the present application, the danofloxacin shows a good linear relationship in the concentration range of 0.001 to 0.1 mg / L, the linear equation is Y=15573X-5.1604 (R 2 =0.9990), the detection limit is 1.0 μg / L, and the quantification limit is 3.0 μg / L, which is suitable for the detection of samples with different concentration ranges.

[0041] 4. Improve the reliability and reproducibility of the method: through the system optimization of the pH value, adsorbent dosage, extraction time, sample solution volume and other conditions, the reliability and reproducibility of the method are ensured.

[0042] 5. Suitable for various sample types: the present application is not only suitable for the detection of danofloxacin in pig urine samples, but also has wide applicability, and can be extended to the detection of antibiotics in other animal urine and biological samples. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1HPLC chromatogram of daflloxacin with the spiked amount of 0.05 mg / L in Example 10. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0045] The instruments and buffer solutions used in the examples are as follows:

[0046] Agilent 1260 high performance liquid chromatograph equipped with Agilent-G1321B fluorescence detector, Waters C18 chromatographic column (3.9x150 mm, 4 μm);

[0047] Britton-Robinson (B-R) buffer solution, acid solution: 0.6183 g of boric acid, 0.58 mL of phosphoric acid and 0.572 mL of acetic acid were added into a conical flask and diluted to 250 mL; base solution: 2 g of NaOH was added into a conical flask and diluted to 250 mL, and the acid solution and the base solution were mixed to prepare the B-R buffer solution.

[0048] The raw materials used in the examples are all commercially available products unless otherwise specified, and the following are exemplary descriptions:

[0049] Polyethylene glycol was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., PEG-400;

[0050] Graphene oxide was purchased from Nanjing Xianfeng Nanometer Material Technology Co., Ltd., GO-100;

[0051] Chitosan was purchased from Qingdao Boyite Biological Material Co., Ltd., CS-200;

[0052] Amino dextran was purchased from Shandong Lu Kang Pharmaceutical Co., Ltd., DS-200;

[0053] Polyethyleneimine was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., PEI-200;

[0054] Activated carbon was purchased from Jiangsu Huachang Chemical Co., Ltd., 200 mesh;

[0055] Palygorskite was purchased from Anhui Xuan Cheng Huiluo Group Co., Ltd., 200 mesh.

[0056] (I) Preparation of magnetic nanocomposite

[0057] Example 1

[0058] The embodiment provides a Fe3O4 / GO / CS magnetic nanocomposite, and a preparation method thereof.

[0059] (i) adding ferrous sulfate and iron chloride into water, stirring and uniformly mixing, adding a polyethylene glycol aqueous solution, timing for 30 min when the temperature reaches 50 DEG C, vacuum pumping after 30 min, introducing N2, adding ammonia water, increasing the temperature to 80 DEG C, and performing water bath reaction for 30 min;

[0060] The mass ratio of the ferrous sulfate, the iron chloride and the polyethylene glycol is 2.7:5.4:3.12; the total mass concentration of the mixture obtained by adding the ferrous sulfate and the iron chloride into water is 0.12 g / mL; the mass concentration of the polyethylene glycol aqueous solution is 0.1 g / mL; the mass concentration of the ammonia water is 26%, and the volume ratio of the ammonia water to the polyethylene glycol aqueous solution is 1:1.

[0061] (ii) sequentially washing the product obtained in step (i) with distilled water and ethanol until the product is clear, and vacuum drying at 55 DEG C for 12 h to obtain Fe3O4;

[0062] (iii) mixing Fe3O4, graphene oxide and chitosan according to a mass ratio of 1:1:1, grinding in a marcasite grinding bowl for 30 min, rinsing with ultrapure water and ethanol for 3 times after grinding, vacuum drying at 60 DEG C for 12 h to obtain a Fe3O4 / GO / CS magnetic nanocomposite.

[0063] Comparative Example 1

[0064] The comparative example provides a magnetic nanocomposite, and the difference from the embodiment 1 is that the chitosan is replaced by aminodextran.

[0065] Comparative Example 2

[0066] The comparative example provides a magnetic nanocomposite, and the difference from the embodiment 1 is that the chitosan is replaced by polyethyleneimine.

[0067] Comparative Example 3

[0068] The comparative example provides a magnetic nanocomposite, and the difference from the embodiment 1 is that the graphene oxide is replaced by activated carbon.

[0069] Comparative Example 4

[0070] The comparative example provides a magnetic nanocomposite, and the difference from the embodiment 1 is that the graphene oxide is replaced by attapulgite.

[0071] (ii) Magnetic solid phase extraction condition optimization

[0072] In order to optimize the extraction conditions of Fe3O4 / GO / CS magnetic nanocomposites for danofloxacin, examples 2-9 were carried out to test the magnetic solid phase extraction with danofloxacin standard solution as sample solution.

[0073] Example 2

[0074] The present example provides a method for detecting the content of danofloxacin using the Fe3O4 / GO / CS magnetic nanocomposites prepared in example 1, which comprises the following steps:

[0075] (1) Preparation of standard solution: accurately weigh danofloxacin standard, dissolve in deionized water, prepare a standard solution with a concentration of 100 mg / L, pH = 7;

[0076] (2) accurately weigh 10 mg of Fe3O4 / GO / CS and add it to a conical flask containing 30 mL of standard solution, shake for 10 min, then collect Fe3O4 / GO / CS with an external magnet, transfer the separated Fe3O4 / GO / CS to a 10 mL glass centrifuge tube, rinse with ultrapure water for 2-3 times, add 1.5 mL of eluent (mass ratio of 3:1 ethanol-3 moL / L NaOH) for desorption, separate Fe3O4 / GO / CS and eluent, and filter the separated eluent with a 0.22 μm organic filter membrane to obtain the sample to be analyzed;

[0077] (3) detect the content of danofloxacin in the sample to be analyzed by HPLC-FLD, and calculate the recovery rate, the HPLC chromatographic conditions are as follows:

[0078] Mobile phase: A phase is 0.05 mol / L phosphoric acid / triethylamine solution, B phase is acetonitrile, and the proportion of mobile phase is: A:B = 83:17;

[0079] Detection wavelength: excitation wavelength is 280 nm, and emission wavelength is 450 nm;

[0080] The injection volume is 20 μL;

[0081] The flow rate is 0.8 mL / min.

[0082] Example 3

[0083] According to the detection method of example 2, the difference is that by adding B-R buffer solution with different acidity to the standard solution, the influence of Fe3O4 / GO / CS on the adsorption performance of danofloxacin is investigated in the range of pH 4.0-8.0, and the danofloxacin recovery rate under different pH is shown in table 1, when pH = 7, the maximum recovery rate of danofloxacin is 95.76%, so pH = 7 is selected for subsequent research.

[0084] Table 1 Influence of sample solution pH on recovery rate

[0085]

[0086]

[0087] Example 4

[0088] The detection method of Example 2 was followed, except that the amount of Fe304 / GO / CS was changed (1 mg to 20 mg) to investigate the effect on the recovery rate of daflloxacin. The results, as shown in Table 2, showed that the recovery rate gradually increased as the amount of Fe304 / GO / CS increased. When the amount of Fe304 / GO / CS reached 10 mg, the recovery rate reached a maximum of 95.76%, and then remained stable (recovery rate ≥ 95%). Therefore, the amount of Fe304 / GO / CS was selected as 10 mg for subsequent studies.

[0089] Table 2 Effect of Fe304 / GO / CS amount on recovery rate

[0090]

[0091] Example 5

[0092] The detection method of Example 2 was followed, except that the extraction time was changed to 5 to 25 min to investigate the effect of Fe304 / GO / CS on the adsorption performance of daflloxacin. The results, as shown in Table 3, showed that the recovery rate of daflloxacin remained stable (recovery rate ≥ 98%) after 20 min of extraction time. Therefore, 20 min was the optimal extraction time.

[0093] Table 3 Effect of extraction time on recovery rate

[0094] Extraction time (min) Recovery (%) 5 92.58 10 97.75 15 97.98 20 98.88 25 98.43

[0095] Example 6

[0096] The detection method of Example 2 was followed, except that the sample solution volume was changed (10 to 40 mL) to investigate the effect of the sample solution volume on the recovery rate of daflloxacin. The results, as shown in Table 4, showed that the recovery rate gradually decreased as the sample solution volume increased, and decreased to 83.60% at 35 mL. Therefore, the maximum sample solution volume was selected as 35 mL.

[0097] Table 4 Effect of sample solution volume on recovery rate

[0098]

[0099] Example 7

[0100] The detection method of Example 2 was followed, except that the concentration of the sample solution was changed, and the effect of different concentrations of the sample solution on the adsorption capacity was investigated. The results are shown in Table 5. The adsorption capacity of the Fe3O4 / GO / CS magnetic nanocomposite was up to 44.79 mg / g, and 10 mg of Fe3O4 / GO / CS was close to saturation when the adsorption capacity was 90 mg / L.

[0101] Table 5. Effect of different concentrations of sample solution on adsorption capacity

[0102]

[0103]

[0104] Example 8

[0105] The detection method of Example 2 was followed, except that the eluent was changed, and the effect of ethanol, methanol, acetonitrile, and ethanol-3 moL / L NaOH (prepared according to a mass ratio of 3:1) as the eluent on the recovery rate of daflloxacin was investigated. The results are shown in Table 6. Among the four eluents, the elution effect of ethanol-3 moL / L NaOH was the best (recovery rate > 85%), so ethanol-3 moL / L NaOH was selected as the eluent for the experiment.

[0106] Table 6. Effect of different eluents on the recovery rate of daflloxacin

[0107] Eluent Recovery (%) Ethanol 10.47 Methanol 5.30 Acetonitrile 12.4 Ethanol-3 mol / L NaOH 87.12

[0108] Example 9

[0109] The detection method of Example 2 was followed, except that the amount of ethanol-3 moL / L NaOH eluent was changed, and the effect of the amount of eluent on the recovery rate was investigated. The results are shown in Table 7. The recovery rate increased with the increase of the volume of the eluent, and when the volume of the eluent was greater than 1.5 mL, the recovery rate remained stable (recovery rate > 85%), so the volume of the eluent was selected as 1.5 mL for subsequent research.

[0110] Table 7. Effect of eluent volume on recovery rate

[0111]

[0112]

[0113] Comparative Example 5

[0114] The detection method of Example 2 was followed, except that the Fe3O4 / GO / CS was replaced with the magnetic nanocomposite prepared in Comparative Example 1. The sample recovery rate was 71.08% after detection.

[0115] Comparative Example 6

[0116] The comparative example refers to the method of Example 2, except that Fe3O4 / GO / CS is replaced by the magnetic nanocomposite prepared in Comparative Example 2. The sample recovery rate is 76.35% as detected

[0117] Comparative Example 7

[0118] The comparative example refers to the method of Example 2, except that Fe3O4 / GO / CS is replaced by the magnetic nanocomposite prepared in Comparative Example 3. The sample recovery rate is 60.52% as detected

[0119] Comparative Example 8

[0120] The comparative example refers to the method of Example 2, except that Fe3O4 / GO / CS is replaced by the magnetic nanocomposite prepared in Comparative Example 4. The sample recovery rate is 68.14% as detected

[0121] (III) Method evaluation

[0122] Example 10

[0123] To prove the application of the Fe3O4 / GO / CS magnetic nanocomposite provided by the present application in detecting danofloxacin in pig urine, based on the above-mentioned (II) optimized magnetic solid-phase extraction conditions, pig urine sample solutions with different spiked concentrations (0.001-0.1 mg / L) were prepared for methodological verification.

[0124] The preparation method of pig urine sample solutions with different spiked concentrations (0.001-0.1 mg / L) is as follows: 10 mg of danofloxacin standard was weighed into a 100 mL volumetric flask, dissolved with ultrapure water and diluted to the mark to obtain a 100 mg / L standard stock solution, which was diluted with collected negative pig urine not containing the test substance to obtain a sample solution.

[0125] The working curve was plotted with the peak area (Y) against the mass concentration of the sample solution (X, μg / mL). The test results showed that the mass concentration of danofloxacin was in good linear relationship within the range of 0.001-0.1 mg / L, the linear equation was Y=15573X-5.1604 (R 2 =0.9990), the detection limit of danofloxacin was 1.0 μg / L (S / N=3, the lowest detection limit measured when the signal-to-noise ratio was 3), and the quantification limit of danofloxacin was 3.0 μg / L according to three times the detection limit. Taking the mass concentration of danofloxacin as an example, the measured HPLC chromatogram is shown in Figure 1

[0126] ​When the spiked concentration was 0.001-0.1 mg / L, the recovery was 82.1%-90.5%, and the RSD was 0.2%-4.8% (Table 8). The experimental results showed that the method had good accuracy and precision, and could be used for analysis of actual samples.

[0127] Table 8 Accuracy and precision

[0128] Spiked concentration (mg / L) Recovery (%) RSD (%) 0.001 90.4 4.7 0.05 82.1 0.2 0.1 90.5 4.8

[0129] The above are preferred embodiments of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. Application of Fe3O4 / GO / CS magnetic nanocomposite in detecting the content of danofloxacin in pig urine, the method comprising the following steps: (1) dispersing Fe3O4 / GO / CS into the sample solution to be detected, oscillating extraction, separating Fe3O4 / GO / CS with external magnet, washing the separated Fe3O4 / GO / CS with ultrapure water, then adding eluent for desorption, separating Fe3O4 / GO / CS and eluent, filtering the separated eluent with 0.22 μm organic filter membrane to obtain sample to be analyzed; (2) detecting the content of danofloxacin in the sample to be analyzed by HPLC-FLD; The Fe3O4 / GO / CS magnetic nanocomposite in step (1) is prepared from Fe3O4, graphene oxide and chitosan by the following method: (i) adding ferrous sulfate and ferric chloride into water, stirring and mixing, adding polyethylene glycol aqueous solution, timing for 30 min when the temperature reaches 50℃, vacuum pumping after 30 min, introducing N2, adding ammonia, increasing the temperature to 75-85℃, water bath reaction for 20-40 min; (ii) washing the product obtained in step (i) with distilled water and ethanol in sequence, vacuum drying to obtain Fe3O4; (iii) mixing, grinding and washing Fe3O4, graphene oxide and chitosan with ultrapure water and ethanol, vacuum drying to obtain Fe3O4 / GO / CS magnetic nanocomposite; The mass ratio of ferrous sulfate, ferric chloride and polyethylene glycol in step (i) is (2-3):(5-6):(3-4); The mass concentration of the mixture obtained by adding ferrous sulfate and ferric chloride into water is 0.08-0.2 g / mL; The mass concentration of the polyethylene glycol aqueous solution is 0.08-0.2 g / mL; The mass concentration of the ammonia is 25-28%, and the volume ratio of ammonia to polyethylene glycol aqueous solution is 1:(0.8-1.2); The mass ratio of Fe3O4, graphene oxide and chitosan in step (iii) is 1:(1-1.5):(1-1.5); The HPLC chromatographic conditions in step (2) are as follows: Mobile phase: A phase is 0.03-0.08 mol / L phosphoric acid / triethylamine solution, B phase is acetonitrile, and the ratio of mobile phase is A:B=(80-90):(10-20); Detection wavelength: excitation wavelength is 280 nm, and emission wavelength is 450 nm; Injection volume is 20 μL; Flow rate is 0.5-1.0 mL / min; In the method for detecting, the good linear relationship of the daflloxacin is in the concentration range of 0.001 to 0.1 mg / L, the linear equation is Y=15573X-5.1604, R 2 =0.9990, X is the mass concentration of sample solution μg / mL, Y is the unit peak area, the detection limit is 1.0 μg / L, and the quantification limit is 3.0 μg / L.

2. Use according to claim 1, characterized in that, The drying temperature in step (ii) is 50-60℃, and the drying time is 10-20 h.

3. Use according to claim 1, characterized in that, The preparation method of the sample solution to be detected in step (1) is as follows: diluting the collected pig urine sample with ultrapure water by 1-10 times, and adjusting the pH value to 4-8; The mass / volume ratio of Fe3O4 / GO / CS to the sample solution to be detected is 1 mg:(1-4) mL.

4. Use according to claim 1, characterized in that, The oscillation extraction time in step (1) is 10-30 min. The eluent is a mixture of ethanol and 3 mol / L NaOH aqueous solution with a mass ratio of 3:(0.8-1.2); The mass-volume ratio of the Fe3O4 / GO / CS and the eluent is 10 mg:(1.5-2.5) mL.

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