Fe3O4 / GO / CS magnetic nano composite material and application thereof in detecting content of danofloxacin in swine urine

Abstract

The invention provides a Fe3O4 / GO / CS magnetic nano composite material and application thereof in detecting the content of danofloxacin in swine urine, and belongs to the technical field of antibiotic detection. The Fe3O4 / GO / CS magnetic nano composite material is prepared from Fe3O4, graphene oxide and chitosan, and the Fe3O4 / GO / CS magnetic nano composite material is prepared from Fe3O4, graphene oxide and chitosan. The detection method comprises the following steps: dispersing Fe3O4 / GO / CS into a to-be-detected sample solution, carrying out magnetic solid-phase extraction to obtain a to-be-analyzed sample, and detecting the content of danofloxacin in the to-be-analyzed sample by adopting HPLC-FLD. According to the Fe3O4 / GO / CS magnetic nano composite material provided by the invention, the pretreatment process of a sample is simplified, the sample extraction rate is improved, and the detection method has relatively high sensitivity and accuracy and is suitable for detecting danofloxacin in a pig urine sample.

Description

Technical Field

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

[0002] Danofloxacin is a quinolone antibiotic for animals. It has the advantages of a broad antibacterial spectrum and strong antibacterial activity. Therefore, it is widely used in the treatment of respiratory diseases of livestock and poultry in veterinary clinics in my country. Although the relevant national standards allow its use, excessive or improper use will cause drug residues in animal products, which may cause potential harm to consumers' health after human ingestion and lead to problems such as bacterial resistance. GB31650-2019 stipulates that the maximum residue limit of danofloxacin in pork is 100μg / kg, the maximum residue limit in beef, mutton and poultry is 200μg / kg, and the maximum residue limit in milk and goat milk is 30μg / kg. GB31650.1-2022 stipulates that the maximum residue limit of danofloxacin in poultry eggs is 10μg / kg. 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, commonly used methods for detecting danofloxacin include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), etc. Among them, HPLC is widely used in antibiotic residue detection due to its simple operation, low cost and high sensitivity. However, when dealing with complex matrix samples, such as pig urine, traditional HPLC often faces problems such as severe matrix interference and cumbersome sample pretreatment, which affects the accuracy and reliability of the test results.

[0004] In order to overcome the above problems, solid phase extraction (SPE) technology has been widely used in sample pretreatment in recent years 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 consumption. Therefore, the development of 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 traditional SPE technology with the unique properties of magnetic materials. MSPE uses magnetic materials as adsorbents and quickly separates the adsorbent from the sample through an external 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 properties, which can effectively improve the enrichment efficiency of target analytes.

[0006] High performance liquid chromatography-fluorescence detector (HPLC-FLD) is a highly sensitive detection technology, especially suitable for the detection of trace analytes. Fluorescence detector has high detection sensitivity and selectivity, which can effectively reduce matrix interference and improve the accuracy of detection results. Combining MSPE with HPLC-FLD can give full play to the advantages of both and achieve efficient and sensitive detection of danofloxacin in pig urine.

[0007] Although MSPE combined with HPLC-FLD shows great potential in the detection of antibiotic residues, it still faces some challenges in practical application. First, how to choose suitable magnetic materials and surface modification methods to improve the adsorption efficiency and selectivity of target analytes is a key issue. Secondly, the matrix effect and removal of interfering substances during sample pretreatment are also difficult points. In addition, how to optimize the detection conditions and improve the sensitivity and reproducibility of the detection method are also problems that need to be solved.

[0008] Therefore, there is an urgent need for an efficient and sensitive method for detecting the content of danofloxacin in pig urine to overcome the shortcomings of traditional detection methods. Summary of the invention

[0009] The purpose of the present invention 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, to efficiently and sensitively detect the content of danofloxacin in pig urine. The method can not only effectively overcome the shortcomings of traditional detection methods, but also significantly improve the accuracy and reliability of detection, providing strong technical support for food safety and animal health.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] A first aspect of the present invention provides a Fe3O4 / GO / CS magnetic nanocomposite material, 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, add polyethylene glycol aqueous solution, and when the temperature reaches 50°C, time for 30 minutes. After 30 minutes, evacuate with a vacuum pump, introduce N2, add ammonia water, raise the temperature to 75-85°C, and react in a water bath for 20-40 minutes;

[0014] (ii) washing the product obtained in step (i) with distilled water and ethanol in turn, and drying in vacuo to obtain Fe3O4;

[0015] (iii) Fe3O4, graphene oxide and chitosan are mixed and ground, rinsed with ultrapure water and ethanol after grinding, and vacuum dried to obtain Fe3O4 / GO / CS magnetic nanocomposites.

[0016] Furthermore, in step (i), the mass ratio of ferrous sulfate, ferric chloride and polyethylene glycol 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 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] Furthermore, in step (ii), the drying temperature is 50° C. to 60° C., and the drying time is 10-20 hours.

[0018] Furthermore, in step (iii), the mass ratio of 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°C to 60°C, and the drying time is 10-20 h.

[0019] In the Fe3O4 / GO / CS magnetic nanocomposite material of the present invention, Fe3O4 is a magnetic material with superparamagnetism, which can be quickly separated under the action of an external magnetic field. This characteristic enables the composite material to be quickly and effectively separated from a complex matrix through a simple magnetic separation step during sample processing, thereby 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 target analytes. Chitosan (CS) is a porous natural polymer material with a rich 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, can maintain structural integrity in acidic, alkaline or organic solvents, and are suitable for a variety of chemical environments. The synthesis of Fe3O4 adopts a solvothermal method or a coprecipitation method, which is simple to operate, mild in conditions, easy to control, and can be mass-produced; by accurately controlling the proportion of raw materials and reaction conditions, high-purity Fe3O4 nanoparticles are prepared, reducing the introduction of impurities. The mixed grinding process of Fe3O4, graphene oxide and chitosan is simple, and can achieve uniform dispersion and tight bonding through mechanical chemical action without the need for complex chemical reactions; during the mixed grinding process, Fe3O4 nanoparticles can be evenly dispersed in graphene oxide and chitosan, avoiding agglomeration and improving the dispersibility and stability of the composite material.

[0020] The second aspect of the present invention provides the use of the Fe3O4 / GO / CS magnetic nanocomposite material in detecting the content of danofloxacin in pig urine.

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

[0022] (1) Magnetic solid phase extraction:

[0023] Disperse Fe3O4 / GO / CS into the sample solution to be tested, extract by oscillation, separate Fe3O4 / GO / CS by using an external magnet, wash the separated Fe3O4 / GO / CS by ultrapure water, add eluent to desorb, separate Fe3O4 / GO / CS and eluent, filter the separated eluent by using a 0.22 μm organic filter membrane to obtain the sample to be analyzed;

[0024] (2) HPLC-FLD detection:

[0025] HPLC-FLD was used to detect the content of danofloxacin in the samples to be analyzed.

[0026] Furthermore, the preparation method of the test sample solution in step (1) is as follows: according to the expected concentration of danofloxacin 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] Furthermore, in step (1), the mass volume ratio of Fe3O4 / GO / CS to the sample solution to be tested is 1 mg:(1-4) mL.

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

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

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

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

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

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

[0034] The injection volume was 20 μL;

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

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

[0037] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0038] 1. Simplified sample pretreatment process: The present invention adopts magnetic nanocomposite material Fe3O4 / GO / CS as adsorbent, and uses magnetic solid phase extraction technology to quickly separate samples through an external magnetic field, which greatly simplifies the sample pretreatment process, avoids the cumbersome operations such as centrifugation or filtration required in traditional solid phase extraction, and saves time and labor costs.

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

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

[0041] 4. Improved reliability and reproducibility of the method: The reliability and reproducibility of the method are ensured by systematic optimization of conditions such as pH value, adsorbent dosage, extraction time, and sample solution volume.

[0042] 5. Applicable to various sample types: The present invention is not only applicable to 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 THE DRAWINGS

[0043] Figure 1This is the HPLC chromatogram of danofloxacin spiked at 0.05 mg / L in Example 10. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[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 column (3.9×150 mm, 4 μm);

[0047] Britton-Robinson (BR) buffer solution, acid: 0.6183g boric acid, 0.58mL phosphoric acid and 0.572mL acetic acid are added to a conical flask and the volume is adjusted to 250mL; alkali: 2g NaOH is added to a conical flask and the volume is adjusted to 250mL, and the acid and alkali are mixed to prepare a BR buffer solution.

[0048] Unless otherwise specified, the raw materials used in the examples are all commercially available products, and the following is an exemplary description:

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

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

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

[0052] Glycosaminoglycan was purchased from Shandong Lukang Pharmaceutical Co., Ltd., DS-200;

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

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

[0055] Attapulgite was purchased from Anhui Xuancheng Conch Group Co., Ltd., 200 mesh.

[0056] (I) Preparation of magnetic nanocomposites

[0057] Example 1

[0058] This embodiment provides a Fe3O4 / GO / CS magnetic nanocomposite material, and the preparation method thereof comprises the following steps:

[0059] (i) Add ferrous sulfate and ferric chloride to water, stir and mix, add polyethylene glycol aqueous solution, and when the temperature reaches 50°C, time for 30 minutes. After 30 minutes, evacuate with a vacuum pump, introduce N2, add ammonia water, raise the temperature to 80°C, and react in a water bath for 30 minutes;

[0060] The mass ratio of the ferrous sulfate, ferric chloride and polyethylene glycol is 2.7:5.4:3.12; the total mass concentration of the mixture obtained by adding the ferrous sulfate and ferric 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) washing the product obtained in step (i) with distilled water and ethanol in sequence until it is clear, and drying it in vacuum at 55° C. for 12 h to obtain Fe3O4;

[0062] (iii) Fe3O4, graphene oxide and chitosan were mixed in a mass ratio of 1:1:1 and ground in an agate mortar for 30 min. After grinding, the mixture was rinsed with ultrapure water and ethanol for 3 times and dried in vacuum at 60°C for 12 h to obtain Fe3O4 / GO / CS magnetic nanocomposite materials.

[0063] Comparative Example 1

[0064] This comparative example provides a magnetic nanocomposite material, which is different from Example 1 in that chitosan is replaced by aminodextran.

[0065] Comparative Example 2

[0066] This comparative example provides a magnetic nanocomposite material, which is different from Example 1 in that chitosan is replaced by polyethyleneimine.

[0067] Comparative Example 3

[0068] This comparative example provides a magnetic nanocomposite material, which is different from Example 1 in that graphene oxide is replaced by activated carbon.

[0069] Comparative Example 4

[0070] This comparative example provides a magnetic nanocomposite material, which is different from Example 1 in that graphene oxide is replaced by attapulgite.

[0071] (II) Optimization of magnetic solid phase extraction conditions

[0072] In order to optimize the extraction conditions of danofloxacin by Fe3O4 / GO / CS magnetic nanocomposite materials, magnetic solid phase extraction experiments were carried out using danofloxacin standard solution as the sample solution in Example 2-9.

[0073] Example 2

[0074] This embodiment provides a method for detecting the content of danofloxacin using the Fe3O4 / GO / CS magnetic nanocomposite prepared in Example 1, comprising the following steps:

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

[0076] (2) Accurately weigh 10 mg of Fe3O4 / GO / CS and add it to a conical flask containing 30 mL of standard solution. After oscillation extraction for 10 min, Fe3O4 / GO / CS was collected with an external magnet. The separated Fe3O4 / GO / CS was transferred to a 10 mL glass centrifuge tube, rinsed with ultrapure water 2 to 3 times, and 1.5 mL of eluent (ethanol-3 mol / L NaOH with a mass ratio of 3:1) was added for desorption to separate Fe3O4 / GO / CS and the eluent. The separated eluent was filtered with a 0.22 μm organic filter membrane to obtain the sample to be analyzed;

[0077] (3) HPLC-FLD was used to detect the danofloxacin content in the sample to be analyzed and the recovery rate was calculated. The HPLC chromatographic conditions were set as follows:

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

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

[0080] The injection volume was 20 μL;

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

[0082] Example 3

[0083] The detection method of Example 2 was used, except that BR buffer solutions of different acidities were added to the standard solution to investigate the effect of Fe3O4 / GO / CS on the adsorption performance of danofloxacin in the pH range of 4.0 to 8.0. The recovery rates of danofloxacin at different pH values ​​are shown in Table 1. When pH = 7, the maximum recovery rate of danofloxacin was 95.76%, so pH = 7 was selected for subsequent research.

[0084] Table 1 Effect 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 Fe3O4 / GO / CS was changed (1 mg to 20 mg) to investigate the effect on the recovery of danofloxacin. The results are shown in Table 2. As the amount of Fe3O4 / GO / CS increased, the recovery gradually increased. When the amount of Fe3O4 / GO / CS reached 10 mg, the recovery reached a maximum value of 95.76%, and then remained stable (recovery ≥ 95%). Therefore, the amount of Fe3O4 / GO / CS of 10 mg was selected for subsequent research.

[0089] Table 2 Effect of Fe3O4 / GO / CS dosage on recovery rate

[0090]

[0091] Example 5

[0092] According to the detection method of Example 2, the difference is that the extraction time is changed to 5-25 minutes, and the effect of Fe3O4 / GO / CS on the adsorption performance of danofloxacin is investigated. The results are shown in Table 3. After the extraction time is 20 minutes, the recovery rate of danofloxacin remains basically stable (recovery rate ≥ 98%). Therefore, 20 minutes is the optimal extraction time.

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

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

[0095] Example 6

[0096] According to the detection method of Example 2, the difference is that the sample solution volume (10-40mL) is changed to investigate the influence of the sample solution volume on the recovery of danofloxacin. The results are shown in Table 4. As the sample solution volume increases, the recovery gradually decreases. When reaching 35mL, the recovery is reduced to 83.60%. Therefore, the maximum sample solution volume is selected to be 35mL.

[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 danofloxacin in the sample solution was changed, and the effect of different danofloxacin concentrations on the adsorption capacity was investigated. The results are shown in Table 5. When 10 mg Fe3O4 / GO / CS adsorbed 90 mg / L, it was close to saturation, and the adsorption capacity of the Fe3O4 / GO / CS magnetic nanocomposite material could reach up to 44.79 mg / g.

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

[0102]

[0103]

[0104] Example 8

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

[0106] Table 6 Effect of different eluents on the recovery of danofloxacin

[0107] Eluent Recovery rate (%) Ethanol 10.47 Methanol 5.30 Acetonitrile 12.4 Ethanol-3moL / LNaOH 87.12

[0108] Example 9

[0109] According to the detection method of Example 2, the difference is that the amount of ethanol-3moL / LNaOH eluent is changed, and the effect of the amount of eluent on the recovery rate is investigated. The results are shown in Table 7. The recovery rate increases with the increase of the eluent volume. When the eluent volume is greater than 1.5mL, the recovery rate remains stable (recovery rate>85%), so the eluent volume of 1.5mL is selected for subsequent research.

[0110] Table 7 Effect of eluent volume on recovery

[0111]

[0112]

[0113] Comparative Example 5

[0114] This comparative example refers to the method of Example 2, except that Fe3O4 / GO / CS is replaced by the magnetic nanocomposite material prepared in Comparative Example 1. After testing, the sample recovery rate is 71.08%

[0115] Comparative Example 6

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

[0117] Comparative Example 7

[0118] This 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. After testing, the sample recovery rate is 60.52%

[0119] Comparative Example 8

[0120] This 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. After testing, the sample recovery rate is 68.14%

[0121] 3. Method evaluation

[0122] Example 10

[0123] In order to demonstrate the application of the Fe3O4 / GO / CS magnetic nanocomposite material provided by the present invention in the detection of danofloxacin in pig urine, based on the above (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: weigh 10 mg of danofloxacin standard into a 100 mL volumetric flask, dissolve and dilute with ultrapure water to obtain a 100 mg / L standard stock solution, and dilute the standard stock solution with the collected negative pig urine that does not contain the substance to be tested to obtain the sample solution.

[0125] The working curve was drawn by comparing the peak area (Y) with the mass concentration (X, μg / mL) of the sample solution. The test results showed that the mass concentration of danofloxacin was in the range of 0.001 to 0.1 mg / L and the linear equation was Y=15573X-5.1604(R 2 =0.9990), the detection limit of danofloxacin is 1.0μg / L (S / N=3, the lowest detection limit measured when the signal-to-noise ratio is 3), and the quantitative limit of danofloxacin is 3.0μg / L according to the three times detection limit. Taking the mass concentration of danofloxacin as 0.05mg / L as an example, the measured HPLC chromatogram is as follows Figure 1 shown.

[0126] When the spike 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 actual sample analysis.

[0127] Table 8 Accuracy and precision

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

[0129] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Fe3O4 / GO / CS magnetic nanocomposite material, characterized in that: It is prepared from Fe3O4, graphene oxide and chitosan.

2. The Fe3O4 / GO / CS magnetic nanocomposite material according to claim 1, characterized in that: Prepared by the following method: (i) Add ferrous sulfate and ferric chloride to water, stir and mix, add polyethylene glycol aqueous solution, and when the temperature reaches 50°C, time for 30 minutes. After 30 minutes, evacuate with a vacuum pump, introduce N2, add ammonia water, raise the temperature to 75-85°C, and react in a water bath for 20-40 minutes; (ii) washing the product obtained in step (i) with distilled water and ethanol in turn, and drying in vacuo to obtain Fe3O4; (iii) Fe3O4, graphene oxide and chitosan are mixed and ground, rinsed with ultrapure water and ethanol after grinding, and vacuum dried to obtain Fe3O4 / GO / CS magnetic nanocomposites.

3. The Fe3O4 / GO / CS magnetic nanocomposite material according to claim 2, characterized in that: 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 water is 25-28%, and the volume ratio of the ammonia water to the polyethylene glycol aqueous solution is 1:(0.8-1.2).

4. The Fe3O4 / GO / CS magnetic nanocomposite material according to claim 2, characterized in that: The drying temperature in step (ii) is 50° C. to 60° C., and the drying time is 10-20 hours.

5. The Fe3O4 / GO / CS magnetic nanocomposite material according to claim 2, characterized in that: The mass ratio of Fe3O4, graphene oxide and chitosan in step (iii) is 1:(1-1.5):(1-1.5); The grinding time is 20-40min; The drying temperature is 50° C. to 60° C., and the drying time is 10-20 hours.

6. Use of the Fe3O4 / GO / CS magnetic nanocomposite material according to any one of claims 1 to 5 in detecting the content of danofloxacin in pig urine.

7. The use according to claim 6, characterized in that: The detection method comprises the following steps: (1) dispersing Fe3O4 / GO / CS into a sample solution to be tested, extracting by oscillation, separating Fe3O4 / GO / CS by using an external magnet, washing the separated Fe3O4 / GO / CS with ultrapure water, adding an eluent to desorb, separating Fe3O4 / GO / CS and the eluent, filtering the separated eluent with a 0.22 μm organic filter membrane to obtain a sample to be analyzed; (2) HPLC-FLD was used to detect the content of danofloxacin in the samples to be analyzed.

8. The use according to claim 7, characterized in that: The preparation method of the test sample solution in step (1) is as follows: diluting the collected pig urine sample by 1-10 times with ultrapure water and adjusting the pH value to 4-8; The mass volume ratio of Fe3O4 / GO / CS to the sample solution to be tested is 1 mg:(1-4) mL.

9. The use according to claim 7, 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 in 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.

10. The use according to claim 7, characterized in that: In step (2), the HPLC chromatographic conditions are set as follows: Mobile phase: Phase A is 0.03-0.08 mol / L phosphoric acid / triethylamine solution, phase B is acetonitrile, and the mobile phase ratio is: A:B=(80-90):(10-20); Detection wavelength: excitation wavelength is 280nm, emission wavelength is 450nm; The injection volume was 20 μL; The flow rate is 0.5-1.0 mL / min.

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