A Fe3O4@C-NFs composite material and a method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS with the use of this composite material.

Abstract

This invention discloses a Fe3O4@C-NFs composite material, synthesized using a simple one-step hydrothermal method, which exhibits excellent selective adsorption and elution performance for four typical tetracyclines. It can be used as an adsorbent for magnetic solid-phase extraction (MSPE) pretreatment of four typical tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in aquatic products, solving problems such as slow column throughput and column clogging in conventional tetracycline antibiotic pretreatment. This invention also discloses a method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS with the Fe3O4@C-NFs composite material, comprising the following steps: (1) sample preparation, (2) magnetic solid-phase extraction, and (3) loading the solution onto the UPLC-MS / MS instrument for scanning detection of the four tetracycline antibiotics. This method is simple, economical, and sensitive, meeting the requirements for the detection of trace tetracycline antibiotics in aquatic products.

Description

Technical Field

[0001] This invention relates to the field of tetracycline antibiotic detection technology, and in particular to a Fe3O4@C-NFs composite material and a method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS with the composite material. Background Technology

[0002] Tetracycline antibiotics, as typical representatives of hydrophilic antibiotics, are widely used in animal husbandry and aquaculture due to their broad antibacterial spectrum, good water solubility, and low cost. They play an important role in preventing and treating bacterial infections and promoting growth [3]. Toxicological studies of tetracycline antibiotics have shown that these antibiotics have quite serious side effects, such as abdominal discomfort, upper abdominal pain, nausea, vomiting and anorexia, tooth discoloration and inhibition of bone growth in children. In addition, tetracycline antibiotics may induce and spread drug-resistant bacteria after entering the environment, posing a long-term potential threat to human health and the ecological environment.

[0003] The determination of tetracycline antibiotics in food commonly utilizes liquid chromatography coupled with UV, fluorescence, and mass spectrometry (MS). Among these methods, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) offers significant advantages in quantitative analysis due to its short analysis time, high sensitivity, and good separation efficiency. However, considering the trace levels of the target analyte and the high levels of matrix interference, sample pretreatment is also a crucial step for accurate determination.

[0004] While solid-phase extraction (SPE) offers superior extraction and enrichment compared to traditional methods, it also suffers from drawbacks such as high cost and column clogging, particularly for tetracyclines. Tetracycline antibiotics contain hydrophilic hydroxyl groups, making them readily soluble in polar solvents like aqueous solutions but less soluble in nonpolar organic solvents. Furthermore, tetracyclines exhibit strong protein-binding abilities, necessitating the use of acidic deproteinizing agents for extraction from biological samples. Therefore, most studies on the extraction of tetracyclines from biological matrices, including aquatic products, utilize weakly acidic Na₂EDTA-Mcllvaine buffer solutions. The addition of Na₂EDTA effectively reduces the chelation of tetracyclines with metal ions, improving analyte extraction efficiency. However, Na₂EDTA-Mcllvaine buffer solutions are prone to protein precipitation, resulting in turbid extracts with high levels of impurities such as fats, hindering purification. The turbid extract requires low-temperature, high-speed centrifugation and filtration before loading onto the SPE column, significantly extending pretreatment time.

[0005] In recent years, magnetic solid-phase extraction (MSPE) has attracted widespread attention as a promising extraction method. MSPE can adsorb and desorb analytes onto magnetic adsorbents using an external magnetic field, and can avoid the column packing problems encountered in traditional solid-phase extraction (SPE). However, current research on the determination of tetracycline antibiotics using MSPE has certain limitations: First, most synthetic processes are complex, with harsh conditions, and use expensive and toxic materials and solvents, causing environmental pollution; second, MSPE is mainly used to extract or enrich tetracycline antibiotics from water or milk, and rarely involves aquatic products. The complex substrates of aquatic products make the successful application of MSPE in aquatic products more challenging, and there are currently no reports of MSPE being used to determine tetracycline antibiotic residues in aquatic products. Summary of the Invention

[0006] The purpose of this invention is to provide a Fe3O4@C-NFs composite material that has low preparation cost, large specific surface area and uniform pore radius distribution, and good selectivity, adsorption and elution performance for tetracycline antibiotics.

[0007] Another objective of this invention is to provide a method for the trace determination of four tetracycline antibiotics in aquatic products using Fe3O4@C-NFs composite material and UPLC-MS / MS. This method is simple, economical, and sensitive, and can meet the detection requirements for trace tetracycline antibiotics in aquatic products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A Fe3O4@C-NFs composite material of the present invention is prepared by the following method: Ferric chloride hexahydrate and sodium acetate are dispersed in a mixture of ethylene glycol and water; carbon nanofibers are dispersed in the mixture of ethylene glycol and water to obtain a dispersion; the dispersion is placed in a stainless steel autoclave for heating and reaction; after the reaction, it is cooled to room temperature; the precipitate is recovered using a magnet; the precipitate is washed several times with deionized water and ethanol; and then vacuum dried to obtain the Fe3O4@C-NFs composite material.

[0009] Preferably, the amount of ferric chloride hexahydrate added is 2.5g, the amount of sodium acetate added is 4.0g, the volume of deionized water in the mixture of ethylene glycol and water is 25mL, the volume of ethylene glycol is 50mL, and the amount of carbon nanofiber added is 0.5g; the reaction is heated to 180℃ for 10h; and then dried under vacuum at 70℃.

[0010] Preferably, the carbon nanofibers have a diameter of 50–200 nm and a length of 1–15 μm.

[0011] A method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS includes the following steps:

[0012] (1) Sample preparation

[0013] Take the muscle or edible part of aquatic products, cut it into pieces, and homogenize it thoroughly. Weigh 2.00±0.01g of the homogenized sample into a 50mL polypropylene centrifuge tube, then add 10mL of glacial acetic acid-acetonitrile-water mixture and 0.1g of disodium ethylenediaminetetraacetate. Vortex and sonicate to homogenize, centrifuge, collect the supernatant, and re-extract the residue with 5mL of glacial acetic acid-acetonitrile-water mixture. Combine the supernatants, concentrate to 2.5mL, dilute with water to 15mL, filter, and collect the filtrate to obtain the sample solution. Selecting a suitable extraction solution is key to applying the MSPE method to extract four tetracycline antibiotics from aquatic products. A suitable extraction solution must have both high extraction efficiency for the target analytes and ensure high adsorption capacity of the magnetic material for the target analytes. Due to the presence of hydroxyl groups in their structure, tetracycline antibiotics exhibit high stability and good solubility in acidic aqueous solutions. A weakly acidic Na2EDTA-Mcllvaine buffer solution is typically used to extract tetracycline antibiotics from aquatic products. The inventors tested the adsorption of four tetracycline antibiotics by the Fe3O4@C-NFs composite material in pure water, Na2EDTA-Mcllvaine buffer solution, and a glacial acetic acid-acetonitrile-water mixed solution (1:84:15, v / v / v). The results showed that the magnetic material achieved an adsorption rate of over 90% for all four tetracycline drugs in pure water and the glacial acetic acid-acetonitrile-water mixed solution, while the adsorption rate in the Na2EDTA-Mcllvaine buffer solution was only about 50%. Therefore, this invention uses a glacial acetic acid-acetonitrile-water mixed solution (1:84:15, v / v / v) as the extraction solvent to extract tetracycline antibiotics from the samples.

[0014] (2) Magnetic solid phase extraction

[0015] 10g of Fe3O4@C-NFs composite material activated with methanol and water was added to the sample solution and shaken for adsorption. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside of the tube. The liquid in the polypropylene centrifuge tube was poured out, and 4mL of elution buffer was added. The mixture was shaken for elution. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside of the tube. The liquid was poured out and filtered to obtain the solution for use in the centrifuge. The dosage of the Fe3O4@C-NFs composite adsorbent is a crucial parameter affecting the adsorption of target analytes. Given that mass spectrometry can detect tetracycline antibiotics at trace levels with high sensitivity, and that the synthesized Fe3O4@C-NFs composite possesses excellent specific surface area and adsorption performance, the dosage of Fe3O4@C-NFs composite does not need to be too high. The inventors investigated the effect of the dosage of Fe3O4@C-NFs composite on the extraction rate of tetracycline antibiotics. By adjusting the dosage from 5 to 20 mg (5, 10, 15, and 20 mg), they optimized the dosage of Fe3O4@C-NFs composite. They found that increasing the mass of Fe3O4@C-NFs composite from 5.0 mg to 10.0 mg improved the extraction rate of tetracycline antibiotics. However, when the amount of tetracycline antibiotics added exceeds 10 mg, the extraction rate of tetracycline antibiotics begins to decrease. Therefore, in this invention, the amount of Fe3O4@C-NFs composite material added is 10.0 mg, which can ensure a good adsorption effect. The inventors investigated the effect of the volume of the eluent (1.0-5.0 mL) on the recovery rate. When the volume of the eluent is 4.0 mL, a satisfactory recovery rate can be obtained. Unlike the traditional solid phase extraction method, the use of Fe3O4@C-NFs composite material as an adsorbent for adsorption extraction does not require activation, sample loading, rinsing and other steps. The sample pretreatment steps are more convenient, which helps to greatly improve the detection efficiency. With the assistance of ultra-high performance liquid chromatography (UPLC), the separation of four tetracycline antibiotics can be achieved in less than 5 minutes.

[0016] (3) The sample solution was loaded onto the UPLC-MS / MS instrument, and the four tetracycline antibiotics were scanned and detected using UPLC-MS / MS. The peak areas of the quantitative ions of the four tetracycline antibiotics were compared with the standard curve, and the actual concentration of the tetracycline antibiotics in the sample could be obtained by conversion. The four tetracycline antibiotics were oxytetracycline, tetracycline, chlortetracycline, and doxycycline. Plotting the standard curves of the four tetracycline antibiotics is a conventional technique in this field, and therefore will not be described in detail here.

[0017] Preferably, in step (1), the volume ratio of glacial acetic acid, acetonitrile, and water in the glacial acetic acid-acetonitrile-water mixture is 1:84:15; the mixture is centrifuged at 8000 rpm for 5 min at 4°C; and filtered through a 0.45 μm membrane filter.

[0018] Preferably, in step (2), the mixture is shaken for 20 minutes; the eluent is a mixture of acetonitrile and oxalic acid solution, with a volume ratio of acetonitrile to oxalic acid solution of 1:8, and the oxalic acid concentration in the oxalic acid solution is 0.02 mol / L; the liquid is filtered through a 0.22 μm organic microporous membrane. Adsorption time is one of the decisive factors affecting the extraction rate, and effective contact with the sample solution containing the target analyte is an important condition for obtaining a high extraction rate. The inventors investigated the effect of different shaking times within the range of 5–40 min on the extraction rate. The results showed that the extraction rate gradually increased with the extension of time, and the change in extraction rate was not significant after 20 min, indicating that a rapid distribution equilibrium between tetracycline antibiotics and Fe3O4@C-NFs composite material was established within 20 min. Therefore, the shaking adsorption time was selected as 20 min. One of the most critical steps in MSPE is the complete elution of the analyte adsorbed on the Fe3O4@C-NFs composite material. A suitable eluent should first be able to completely elute the analyte from the Fe3O4@C-NFs composite material to obtain reliable and reproducible analytical results, and secondly, it should dissolve a high percentage of the analyte. To elute tetracycline antibiotics from Fe3O4@C-NFs composite materials and apply them to subsequent mass spectrometry analysis, the inventors tested several commonly used eluents, including acetonitrile (ACN), methanol (MeOH), and formic acid solutions containing methanol (2%, 5%, 10%, V / V). They found that acetonitrile and methanol could not elute tetracycline antibiotics from the Fe3O4@C-NFs composite material, while the formic acid solutions containing methanol (2%, 5%, 10%, V / V) achieved elution rates of 2.7%–7.4%. The inventors ultimately discovered that a mixed solution of acetonitrile and oxalic acid (with a volume ratio of 1:8 and an oxalic acid concentration of 0.02 mol / L) had a good elution effect on tetracycline antibiotics. Therefore, this invention selected a mixed solution of acetonitrile and oxalic acid (with a volume ratio of 1:8 and an oxalic acid concentration of 0.02 mol / L) as the eluent.

[0019] Preferably, in step (3), Waters Acquity UPLC is used. TM The system and chromatographic conditions were as follows: chromatographic separation was performed using Waters BEHC. 18The chromatographic column was 2.1 × 100 mm with a particle size of 1.7 μm. 0.1% formic acid solution was used as mobile phase A, and acetonitrile as mobile phase B. A binary gradient mobile phase consisting of mobile phases A and B was used. The flow rate was 0.3 mL / min, and the gradient was designed as follows: 0–1.0 min, 90% A; 1.0–2.0 min, 90%–70% A; 2.0–3.5 min, 70%–40% A; 3.5–5.0 min, 40% A; 5.0–5.5 min, 40%–90% A; 5.5–7.0 min, 90% A. The column temperature was 35℃, and the autosampler temperature was 10℃. The injection volume was 10 μL. MassLynx software 4.1 was used for instrument control and data acquisition.

[0020] Preferably, in step (3), a Quattro Premier XE Micromass triple quadrupole mass spectrometer is used. The mass spectrometry conditions are as follows: electrospray ionization is performed in positive ion mode, and multiple reaction monitoring mode is used for mass spectrometry analysis. The MS / MS parameters are as follows: cone and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.9999%; source temperature 150℃; desolvation gas temperature: 380℃; cone gas is high-purity nitrogen, flow rate: 50L / h; desolvation gas is high-purity nitrogen, flow rate: 600L / h.

[0021] Therefore, the present invention has the following beneficial effects:

[0022] (1) A Fe3O4@C-NFs composite material is provided, which is synthesized by a simple one-step hydrothermal method. The synthesis method is simple and environmentally friendly, requires few reagents, and has low safety and toxicity. The Fe3O4@C-NFs composite material has the characteristics of large specific surface area, highly interwoven and branched mesoporous structure and excellent magnetic properties. It has excellent selective adsorption and elution performance for four typical tetracyclines. It can be used as an adsorbent for the pretreatment of four typical tetracyclines (oxytetracycline, tetracycline, chlortetracycline and doxycycline) in aquatic products by magnetic solid phase extraction (MSPE) to solve the problems of slow column passage and column blockage in conventional pretreatment of tetracycline antibiotics.

[0023] (2) A method for the trace determination of four tetracycline antibiotics in aquatic products using Fe3O4@C-NFs composite material by UPLC-MS / MS is provided. This method is simple, economical and sensitive, and can meet the detection requirements of trace tetracycline antibiotics in aquatic products. Unlike the traditional solid phase extraction method, this method does not require complex steps such as activation, sample loading, rinsing and elution, and the adsorbent has the advantage of being recyclable. Attached Figure Description

[0024] Figure 1This is a SEM image (1.00 μm) of the Fe3O4@C-NFs composite material.

[0025] Figure 2 This is a SEM image (500 nm) of the Fe3O4@C-NFs composite material.

[0026] Figure 3 This is the FT-IR spectrum of the Fe3O4@C-NFs composite material.

[0027] Figure 4 This is the X-ray diffraction pattern of the Fe3O4@C-NFs composite material.

[0028] Figure 5 This is the nitrogen adsorption / desorption isotherm diagram of the Fe3O4@C-NFs composite material. Detailed Implementation

[0029] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0030] In the following examples: carbon nanofibers were purchased from Jiangsu Pioneer Nanomaterials Technology Co., Ltd. (Nanjing); ferric chloride hexahydrate, sodium acetate, ethylene glycol, and oxalic acid dehydration were all analytical grade and purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China; methanol, acetonitrile, acetic acid, and formic acid were high performance liquid chromatography grade (99.9%) and purchased from Merck AG (Darmstadt), Germany; tetracycline standards, including oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DC) in hydrochloride form, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0031] In the following examples, the Fe3O4@C-NFs composite material was prepared by the following method: 2.5 g of ferric chloride hexahydrate and 4.0 g of sodium acetate were dispersed in a mixture of 50 mL ethylene glycol and 25 mL water. 0.5 g of carbon nanofibers were ultrasonically dispersed in the same mixture to obtain a dispersion. The dispersion was placed in a stainless steel autoclave and heated to 180 °C for 10 h. After the reaction, the mixture was cooled to room temperature, and the precipitate was recovered using a magnet. The precipitate was washed several times with deionized water and ethanol, and then vacuum dried at 70 °C to obtain the Fe3O4@C-NFs composite material. The SEM image of the obtained Fe3O4@C-NFs composite material is shown below. Figure 1 , Figure 2 As shown. From Figure 1 and Figure 2 It can be seen that the Fe3O4@C-NFs composite material has a large surface area and many adsorption sites, and the Fe3O4 particles are uniformly distributed on the fiber rods of c-nanofibers.

[0032] Fourier transform infrared spectroscopy (FT-IR) was performed on the Fe3O4@C-NFs composite material, and the obtained FT-IR spectrum is shown below. Figure 3 As shown. From Figure 3 It can be seen that at 3437cm -1 1624cm -1 and 578cm -1 The characteristic peaks at 3425 cm⁻¹ belong to the tensile vibrations of OH, HOH, and Fe-O, respectively. The spectrum of the Fe₃O₄@C-NFs nanocomposite is consistent with that of Fe₃O₄ particles, showing no peak shift. However, in the nanocomposite, the peak at 3425 cm⁻¹ of Fe₃O₄@C-NFs... -1 2918cm -1 The peaks shifted to 3437 cm. -1 2922cm -1 The slight shift in the peak can be attributed to the introduction of Fe3O4 particles into the C-NFs. FTIR analysis results indicate that the Fe3O4@C-NFs nanocomposite was successfully synthesized.

[0033] The crystal structure of the Fe3O4@C-NFs composite material was determined by X-ray diffraction (XRD), and the obtained X-ray diffraction pattern is shown below. Figure 4 As shown. From Figure 4 It can be seen that the Fe3O4 microspheres in the Fe3O4@C-NFs composite material have a set of diffraction peaks near 30.2° (220), 35.5° (311), 53.7° (422), 57.0° (511), 62.7° (440) and 74.3° (533), among which the diffraction peak observed at 2θ: 43.3 (400) is a specific peak of C-NFs.

[0034] The specific surface area and pore size of the Fe3O4@C-NFs composite material were calculated using N2 adsorption-desorption isotherms. The average pore size (4V / A) for BJH adsorption was 10.7 nm. The nitrogen adsorption / desorption isotherms of the Fe3O4@C-NFs composite material were calculated using the Barrett-Joyner-Halenda (BJH) method, as shown below. Figure 5 As shown. From Figure 5 It can be seen that the BET specific surface area of ​​the Fe3O4@C-NFs composite material is 30.7 cm². 2 / g, total pore volume is 0.30cm³ 3 / g. The results confirmed that the Fe3O4@C-NFs composite material has a large specific surface area and a uniform pore radius distribution.

[0035] Method verification:

[0036] Accurately weigh 5.00 mg of OTC, TC, CTC, and DC standards, dissolve them in methanol, and dilute to 50 mL. Calculate the mass concentration of the standard stock solution to 100 μg / mL according to their respective hydrochloride salts and purities. Store at 4°C protected from light. Before use, dilute the standard stock solution with deionized water to prepare a mixed standard working solution.

[0037] 1. Linear range

[0038] Appropriate amounts of a mixed standard working solution of four tetracycline antibiotics were transferred and diluted to 1.0 mL with ultrapure water to prepare a series of standard working solutions with mass concentrations of 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0 ng / mL. These solutions were then analyzed by UPLC-MS / MS. A standard curve was plotted by performing a linear regression analysis on the peak area of ​​the quantitative ion against the mass concentration. The linear regression equations, linear ranges, and correlation coefficients for the four tetracycline antibiotics are shown in Table 1.

[0039] Table 1. Linear regression equations, linear ranges, and correlation coefficients for four tetracycline antibiotics.

[0040] Analytes Linear regression equation Linear range (ng / mL) <![CDATA[Correlation coefficient (r 2 )]]> OTC y = 6515.65x + 596.784 1.0～200 0.9991 TC y = 7958.82x + 674.114 1.0～200 0.9992 CTC y = 4449.41x - 533.554 1.0～200 0.9996 DC y = 16990.5x + 5282.66 1.0～200 0.9997

[0041] As shown in Table 1, within the range of 1.0–200 ng / mL, the four tetracycline antibiotics exhibited a good linear relationship, with correlation coefficients (r) of [missing value]. 2 The value is ≥0.995, which meets the requirements of instrumental analysis.

[0042] 2. Limit of detection

[0043] Grass carp, Litopenaeus vannamei, and swimming crab samples that were confirmed to be free of analytes were collected and processed according to the methods described in steps (1) to (2) above. A certain concentration of a mixed standard working solution of four tetracycline antibiotics was added, and the samples were tested according to step (3) above. The limits of detection (LOD) for CTC, OTC, TC, and DC were determined with a signal-to-noise ratio (S / N) ≥ 3, and the limits of quantitation (LOQ) for CTC, OTC, TC, and DC were determined with an S / N ≥ 10. The test results are shown in Table 2.

[0044] Table 2. Limits of detection and limits of quantitation for four tetracycline antibiotics.

[0045]

[0046]

[0047] According to Table 2, the limits of detection (LOD) for CTC, OTC, TC, and DC are 0.7 μg / kg, and the limits of quantitation (LOQ) for CTC, OTC, TC, and DC are 2.0 μg / kg.

[0048] 3. Recovery and Precision of the Method: Spiking recovery experiments were conducted using negative samples of grass carp, Litopenaeus vannamei, and Swimming crab as blank matrices. 2.0 g of each of the three types of samples was weighed into centrifuge tubes, and a certain volume of tetracycline antibiotic mixed standard solution was added to each, resulting in three concentration levels of tetracycline antibiotics in the samples after addition: 2.0 μg / kg, 10.0 μg / kg, and 50.0 μg / kg, respectively. After addition, the samples were analyzed using the methods described in steps (1) to (3) above, and compared with the standard curve. The final concentrations of the four tetracycline antibiotics in the grass carp, Litopenaeus vannamei, and Swimming crab samples were obtained through conversion. Each sample was measured in parallel six times, and each spiking level was measured repeatedly six times according to this method. The measurements were conducted continuously for 5 days, and the spiked recovery rate, intra-day precision, and inter-day precision were calculated. The recovery rate was calculated using the following formula:

[0049]

[0050] Where: R - recovery rate, %; Cs - the determined concentration of tetracycline antibiotics in the spiked sample, μg / kg; C0 - the concentration of tetracycline antibiotics in the actual sample, μg / kg; C - the theoretical spiking concentration of tetracycline antibiotics in the spiked sample, μg / kg.

[0051] The measurement results are shown in Table 3.

[0052] Table 3. Recovery and precision results of spiked samples.

[0053]

[0054] As shown in Table 3, the average recoveries of tetracycline antibiotics under three spiking levels and in three sample matrices ranged from 80.7% to 97.7%. The intra-day and inter-day precisions were 3.7%–8.2% (n=6) and 4.2%–9.7% (n=5), respectively, which met the recovery requirements of the analytical method.

[0055] 4. Recyclability of Fe3O4@C-NFs composite materials

[0056] When the Fe3O4@C-NFs composite material of this invention is applied to the method of this invention, the results of the experiment show that there is no significant difference in extraction efficiency between the recovered Fe3O4@C-NFs composite material and the newly prepared Fe3O4@C-NFs composite material. The Fe3O4@C-NFs composite material can be reused at least 10 times after recovery, with a loss of 5% in extraction recovery rate.

[0057] Example 1

[0058] (1) Sample preparation

[0059] The aquatic product selected was large yellow croaker purchased from a seafood market in Zhoushan. The muscle of the large yellow croaker was taken, cut into pieces, and thoroughly homogenized. 2.00g of the homogenized sample was weighed into a 50mL polypropylene centrifuge tube, and then 10mL of glacial acetic acid-acetonitrile-water mixture and 0.1g of disodium ethylenediaminetetraacetate were added. After vortexing, the mixture was sonicated and homogenized. It was centrifuged at 8000rpm for 5min at 4℃. The supernatant was collected, and the residue was re-extracted with 5mL of glacial acetic acid-acetonitrile-water mixture. The supernatants were combined and concentrated to 2.5mL. The mixture was diluted with water to 15mL and filtered through a 0.45μm membrane filter. The filtrate was collected to obtain the sample solution. In the glacial acetic acid-acetonitrile-water mixture, the volume ratio of glacial acetic acid, acetonitrile, and water was 1:84:15.

[0060] (2) Magnetic solid phase extraction

[0061] 10g of Fe3O4@C-NFs composite material activated with methanol and water was added to the sample solution and shaken for 20min. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside of the tube. The liquid in the polypropylene centrifuge tube was discarded, and 4mL of eluent was added. The mixture was shaken and eluted. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside of the tube. The liquid was then filtered through a 0.22μm organic microporous membrane and used as the loading solution. The eluent was a mixture of acetonitrile and oxalic acid solutions with a volume ratio of 1:8. The oxalic acid concentration in the oxalic acid solution was 0.02mol / L.

[0062] (3) The solution was loaded onto the UPLC-MS / MS instrument, and the four tetracycline antibiotics were scanned and detected using UPLC-MS / MS. The peak areas of the quantitative ions of the four tetracycline antibiotics were compared with the standard curve, and the actual concentration of the tetracycline antibiotics in the sample could be obtained by conversion. The four tetracycline antibiotics were oxytetracycline, tetracycline, chlortetracycline, and doxycycline. The chromatographic conditions were: Waters Acquity UPLC. TM The system, chromatographic separation was performed using Waters BEH C 18The chromatographic column was 2.1 × 100 mm with a particle size of 1.7 μm. 0.1% formic acid solution was used as mobile phase A, and acetonitrile as mobile phase B. A binary gradient mobile phase consisting of mobile phases A and B was used at a flow rate of 0.3 mL / min. The gradient was designed as follows: 0–1.0 min, 90% A; 1.0–2.0 min, 90%–70% A; 2.0–3.5 min, 70%–40% A; 3.5–5.0 min, 40% A; 5.0–5.5 min, 40%–90% A; 5.5–7.0 min, 90% A. The column temperature was 35℃, and the autosampler temperature was 10℃. The injection volume was 10 μL. MassLynx software 4.1 was used for instrument control and data acquisition. The mass spectrometry conditions were: QuattroPremier XE. The Micromass triple quadrupole mass spectrometer operates in positive ion mode using electrospray ionization. Mass spectrometry analysis employs multiple reaction monitoring (MRM). MS / MS parameters are as follows: cone and desolvation gas: nitrogen, 99.9% purity; collision gas: argon, 99.9999% purity; source temperature: 150℃; desolvation gas temperature: 380℃; cone gas: high-purity nitrogen, flow rate: 50 L / h; desolvation gas: high-purity nitrogen, flow rate: 600 L / h. The MRM conditions for four tetracycline antibiotics are shown in Table 4.

[0063] Table 4. Multiple reaction monitoring conditions for four tetracycline antibiotics

[0064] Analytes Parent ion (m / z) Daughter ions (m / z) Tapered hole voltage (V) Collision energy (eV) TC 445.1 <![CDATA[410 * ,427]]> 25 20,14 OTC 461.1 <![CDATA[426 * ,442.9]]> 20 20,15 CTC 479.1 <![CDATA[443.9 * ,462]]> 30 20,18 DC 445.2 <![CDATA[428 * ,154.1]]> 30 18,24

[0065] No tetracycline antibiotics were detected.

[0066] Example 2

[0067] In this embodiment, the aquatic products selected were wild-caught swimming crabs purchased from a seafood market in Zhoushan. The muscle portion of the swimming crabs was selected, and the detection method was the same as in Example 1. No four tetracycline antibiotics were detected.

[0068] Example 3

[0069] In this embodiment, the aquatic product selected was Litopenaeus vannamei from a certain aquaculture farm in Zhoushan. The tail muscle of the Litopenaeus vannamei was selected, and the detection method was the same as in Example 1. No four tetracycline antibiotics were detected.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS, characterized in that, Includes the following steps: (1) Sample preparation Take the muscle or edible part of aquatic products, cut them into pieces, and homogenize them thoroughly. Weigh 2.00±0.01 g of the homogenized sample into a 50 mL polypropylene centrifuge tube, then add 10 mL of glacial acetic acid-acetonitrile-water mixture and 0.1 g of disodium ethylenediaminetetraacetate. Vortex and sonicate to homogenize, centrifuge, collect the supernatant, and re-extract the residue with 5 mL of glacial acetic acid-acetonitrile-water mixture. Combine the supernatants, concentrate to 2.5 mL, dilute with water to 15 mL, filter, and collect the filtrate to obtain the sample solution. In the glacial acetic acid-acetonitrile-water mixture, the volume ratio of glacial acetic acid, acetonitrile, and water is 1:84:

15. (2) Magnetic solid phase extraction 10g of Fe3O4@C-NFs composite material activated with methanol and water was added to the sample solution and shaken for adsorption. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside. The liquid in the polypropylene centrifuge tube was discarded, and 4mL of eluent was added. The mixture was shaken and eluted. The Fe3O4@C-NFs composite material was magnetically fixed inside the polypropylene centrifuge tube using a magnet on the outside. The liquid was then discarded, filtered, and used as the loading solution. The eluent was a mixture of acetonitrile and oxalic acid solution. The volume ratio of oxalic acid to the oxalic acid solution is 1:8, and the concentration of oxalic acid in the oxalic acid solution is 0.02 mol / L. The Fe3O4@C-NFs composite material is prepared by the following method: ferric chloride hexahydrate and sodium acetate are dispersed in a mixture of ethylene glycol and water, and carbon nanofibers are dispersed in the mixture of ethylene glycol and water to obtain a dispersion. The dispersion is placed in a stainless steel autoclave for heating and reaction. After the reaction, it is cooled to room temperature, and the precipitate is recovered using a magnet. The precipitate is washed several times with deionized water and ethanol, and then vacuum dried to obtain the Fe3O4@C-NFs composite material. (3) The solution was loaded onto the instrument and the four tetracycline antibiotics were scanned and detected by UPLC-MS / MS using the Waters Acquity UPLC™ system. The peak areas of the quantitative ions of the four tetracycline antibiotics were compared with the standard curve, and the actual concentration of the tetracycline antibiotics in the sample could be obtained by conversion. The four tetracycline antibiotics were oxytetracycline, tetracycline, chlortetracycline and doxycycline. The chromatographic conditions were as follows: the chromatographic separation used a Waters BEH C18 column with a column specification of 2.1×100 mm and a particle size of 1.7 μm. 0.1% formic acid solution was used as mobile phase A and acetonitrile was used as mobile phase B. The binary gradient mobile phase composed of mobile phase A and mobile phase B was used with a flow rate of 0.3 mL / min and a gradient design of 0~1.0 min, 90% A. 1.0~2.0 min, 90%~70% A; 2.0~3.5 min, 70%~40%A; 3.5~5.0 min, 40% A; 5.0~5.5 min, 40%~90% A; 5.5–7.0 min, 90% A; column temperature 35℃, autosampler temperature 10℃; injection volume 10 μL; instrument control and data acquisition were performed using MassLynx software 4.1; a Quattro Premier XE Micromass triple quadrupole mass spectrometer was used, with the following mass spectrometry conditions: electrospray ionization in positive ion mode, multiple reaction monitoring (MRM) mode for mass spectrometry analysis, and the following MS / MS parameters: cone and desolvation gas: nitrogen, purity 99.9%; collision gas: argon, purity 99.9999%; source temperature 150℃; desolvation gas temperature: 380℃; cone gas: high-purity nitrogen, flow rate: 50 L / h; desolvation gas: high-purity nitrogen, flow rate: 600 L / h.

2. The method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS according to claim 1, characterized in that, In step (1), centrifuge at 8000 rpm for 5 min at 4℃; filter through a 0.45 μm membrane filter.

3. The method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS according to claim 1, characterized in that, In step (2), the liquid is shaken for 20 minutes and then filtered through a 0.22 μm organic microporous membrane.

4. The method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS according to claim 1, characterized in that, In step (2), 2.5g of ferric chloride hexahydrate and 4.0g of sodium acetate are added. In the mixture of ethylene glycol and water, the volume of deionized water is 25mL, the volume of ethylene glycol is 50mL, and the amount of carbon nanofibers added is 0.5g. The mixture is heated to 180℃ and reacted for 10h. The mixture is then dried under vacuum at 70℃.

5. A method for trace determination of four tetracycline antibiotics in aquatic products using UPLC-MS / MS according to claim 1 or 4, characterized in that, In step (2), the carbon nanofibers have a diameter of 50~200nm and a length of 1~15μm.

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