Magnetic shrimp shell biochar as well as preparation method and application thereof

Abstract

The invention discloses magnetic shrimp shell biochar as well as a preparation method and application thereof. The preparation method comprises the following steps: 1) pretreating shrimp shells; 2) preparing shrimp shell biochar; and 3) preparing the magnetic shrimp shell biochar. According to the invention, the shrimp shell is used as a carbon source, a high-temperature pyrolysis method is adopted for magnetizing and modifying to prepare the shrimp shell biochar magnetic material, the green and recyclable biochar magnetic material is developed, waste is turned into wealth, and the application range of the biochar material in pretreatment analysis of typical hydrophilic organic matters of aquatic products is expanded; according to the application, the pretreatment process of UPLC-MS / MS detection of tetracycline drugs is effectively simplified, the experimental analysis efficiency is improved, a novel green and efficient method can be provided for detection of TCs in aquatic products, technical support is provided for effective monitoring of tetracycline drugs in aquatic products, and application of shrimp shell biochar materials in the fields of food safety and analytical chemistry is promoted.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and specifically relates to a magnetic shrimp shell biochar, a preparation method and an application of the biochar in combination with MSPE-UPLC-MS / MS in the determination of trace tetracycline and its metabolites in aquatic products. Background Art

[0002] . Since their discovery, antibiotics have been widely used in medical care, aquaculture and other fields. However, due to human abuse and the limited absorption capacity of humans and animals for antibiotics, the problem of antibiotic pollution has become increasingly serious. Among the routinely monitored antibiotics, chloramphenicol, nitrofuran metabolites, pentachlorophenol, etc. can be directly extracted with organic solvents, and the operation steps are relatively simple, while some water-soluble antibiotics need to be extracted with aqueous solutions, and then solid phase extraction and other methods are needed to convert the extract into an organic phase for concentration and enrichment, which is more complicated. Tetracyclines (TCs) are typical representatives of hydrophilic antibiotics. They are widely used in the aquaculture industry because of their broad antibacterial spectrum, good water solubility and low price. They cannot be metabolized and absorbed by humans and animals, and will be excreted and accumulated in the environment with feces and urine, affecting plant growth. They are also transported and enriched through the food chain, which will also endanger human health. With dodecahydronaphthacene as the basic skeleton, the structure contains two groups, hydroxyl and dimethylamino, and has acid-base properties. Common TCs include tetracycline (TC), oxytetracycline (OTC), chorltetracycline (CTC) and doxycycline (DC). The molecular structure of TCs contains multiple dissociable functional groups, which are chemically unstable and prone to degradation reactions under acidic and alkaline conditions. They can form corresponding metabolites through diastereoisomerism, dehydration and diastereoisomerization. TCs can cause damage to the digestive tract and liver and kidneys, trigger allergic and poisoning reactions, and affect the development of teeth and bones. In addition, TCs entering the environment can cause drug-resistant bacteria to be induced and spread, posing a long-term potential threat to human health and the ecological environment. Therefore, the efficient and accurate detection of trace TCs in aquatic products is an important technical support to ensure the effective monitoring of pesticide and veterinary drug residues and conduct risk assessment research.

[0003] It is very important to achieve efficient enrichment and purification of TCs in complex samples before LC-MS / MS analysis. Currently, most pretreatment methods have problems such as cumbersome operation, time-consuming, low efficiency, and high consumption of organic solvents, and cannot effectively reduce impurity interference and matrix effects.

[0004] Magnetic solid-phase extraction (MSPE) is a type of sample pretreatment technology that has flourished in recent years. It can avoid the use of solid phase extraction columns in the pretreatment of food TCs, overcome the above-mentioned pretreatment problems, effectively shorten the pretreatment time, reduce the amount of organic solvents used, and can be recycled. It has the advantages of being green, efficient, fast, and easy to operate. At present, the research on the detection of TCs using MSPE technology has certain limitations: First, although there are many types of magnetic functional materials, most of them have complicated synthesis processes and harsh synthesis conditions, and often require expensive, toxic, and harmful materials and solvents, causing certain environmental pollution; second, it is mainly used in water or liquid food, and relatively few aquatic product matrices are involved; third, it only targets a few common TCs, and relatively few metabolites such as diastereomers and dehydrated products are simultaneously enriched, purified, and detected.

[0005] Magnetic functional materials, as adsorbents for MSPE, are the core of MSPE technology. Finding new, efficient and highly specific adsorbents is the key to the role of magnetic solid phase extraction technology. With the deepening of the concept of green sustainable chemistry, reducing the introduction of organic solvents and secondary pollution in processes such as material synthesis and mixture separation is also an important part of green separation chemistry. Therefore, developing low-cost, simple and green material synthesis methods is a development trend. The preparation of biochar magnetic materials generally has few steps, simple processes and is easy to synthesize. Biochar magnetic materials combine the high adsorption properties of biochar and the easy recycling of magnetic media. Compared with other magnetic materials, biochar has a wide source, low price, large quantity and easy availability. It can synthesize magnetic materials in large quantities, making up for the shortcomings of high synthesis cost and weak practical application of magnetic materials. It has unique advantages in the preparation of magnetic materials; at the same time, the preparation of biochar is also an important way to efficiently and greenly utilize waste biomass. At present, magnetic biochar has been widely used in the adsorption and removal of organic pollutants and heavy metals such as antibiotics. At present, the main methods for removing antibiotics are photolysis, ozone oxidation, Fenton method, photocatalysis, microbial treatment, electrochemical method, adsorption method, etc. Compared with other methods, the engineering operation of the adsorption method is simpler, and the adsorption reactor can be easily connected in series or in parallel to increase the processing capacity; and the adsorbent can be prepared from different wastes, and the raw materials are cheap and the cost is low. It is precisely because biochar comes from biomass pyrolysis, is cheap and easy to obtain, and has the potential for energy and solid waste resource utilization that many researchers have used biochar to study the adsorption and removal of antibiotics. However, although the biochar currently prepared can adsorb antibiotics, the adsorption effect and removal efficiency are difficult to meet the demand. Shrimp shell-derived magnetic biochar uses marine waste shrimp shells as biomass, which can not only achieve efficient utilization of aquatic processing by-products, but also the rich calcium carbonate and chitin contained in shrimp shells can provide pore templates and activated gases for the production of biochar. The protein and chitin contained in the shrimp shells can increase the additional nitrogen content of the biochar, so that the shrimp shell-derived magnetic biochar has the advantages of excellent mechanical strength, strong hydrophilicity, and strong regeneration under extreme conditions. At present, shrimp shell-derived magnetic biochar is mostly used to adsorb petroleum in seawater, heavy metal ions such as Cu(II), Pb(II), Se(IV) in water, and organic pollutants such as 2,4-dichlorophenol, tetracycline and rhodamine B in water. There are few studies on its application in the pretreatment of TCs detection in animal-derived solid foods such as aquatic products, as well as the desorption of pollutants and repeated regeneration of adsorbents. The iron oxide groups, oxygen-containing functional groups and aromatic rings rich in the surface of shrimp shell-derived magnetic biochar can react with the benzene ring, amino group, carboxyl group and acid-base groups in the tetracycline molecular structure to form metal ion complexation, cation-π interaction, hydrogen bonding and π-π conjugation, thereby efficiently adsorbing TCs. Then, by destroying these interactions with appropriate reagents, TCs can be resolved.

[0006] The invention uses discarded shrimp shells in aquatic product processing as a carbon source, adopts a high-temperature pyrolysis method and magnetic modification to prepare shrimp shell biochar magnetic materials, selects the optimal biochar magnetic solid phase extraction material as an MSPE adsorbent through morphological characterization and performance evaluation comparative analysis; takes factors such as extractant type, extraction time, amount of magnetic functional material, adsorption and analytical conditions as investigation indicators, establishes a green, simple and specific MSPE pretreatment method for TCs in aquatic products; and then, with the advantages of high sensitivity and rapid analysis of ultra-high performance liquid chromatography tandem mass spectrometry, establishes a MSPE-UPLC-MS / MS green and efficient analytical determination technology for tetracycline precursors and their metabolites in aquatic products. The implementation of the project can develop a green and recyclable biochar magnetic material, turning waste into treasure and expanding the application scope of biochar materials in the pretreatment analysis of typical hydrophilic organic matter in aquatic products; the establishment of the project analysis method will effectively simplify the pretreatment process of UPLC-MS / MS detection of tetracycline drugs, overcome the problems of column blockage and slow column speed in solid phase extraction, shorten the experimental time and analysis time, and improve the efficiency of experimental analysis. It can provide a new green and efficient method for the detection of TCs in aquatic products, provide technical support for the effective monitoring of tetracycline drugs in aquatic products, and promote the application of shrimp shell biochar materials in the fields of food safety and analytical chemistry. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a magnetic shrimp shell biochar, a preparation method and a technical solution for its application in the determination of trace tetracycline and its metabolites in aquatic products by coupling with MSPE-UPLC-MS / MS, which is specifically achieved by the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing magnetic shrimp shell biochar, the method comprising the following steps:

[0009] 1) Shrimp shell pretreatment: Sorting shrimp shells to remove impurities, washing, drying at 60°C, and crushing through a 100-mesh sieve to obtain shrimp shell powder;

[0010] 2) Preparation of shrimp shell biochar: The shrimp shell powder was heated to 600°C in a muffle furnace at a heating rate of 10°C / L under nitrogen protection and maintained for 2 h, and then cooled to obtain shrimp shell biochar;

[0011] 3) Preparation of magnetic shrimp shell biochar: The shrimp shell biochar obtained in step 2) is added into distilled water to prepare a biochar suspension, and ferric chloride and ferrous sulfate are dissolved in distilled water to obtain Fe 3+ and Fe 2+ The mixed solution was dripped into the biochar suspension, and then the NaOH solution was slowly added and stirred to adjust the pH to 10. 2After being stirred and boiled in air, the mixture was aged, washed with distilled water until neutral, and then dried in an oven to obtain magnetic shrimp shell biochar.

[0012] Further, in step 3), the amount of shrimp shell biochar and distilled water is 3 g of biochar added to 200 mL of distilled water; Fe 3+ and Fe 2+ Fe in mixed solution 3+ / Fe 2+ The molar ratio is 1 / 1.

[0013] Further, in step 3), N 2 The mixture was stirred at 100 r / min for 1 h in an atmosphere, and then the suspension was boiled at 100°C for 1 h and aged at room temperature for 12 h. It was washed with distilled water until neutral and dried in an oven at 105°C for 12 h.

[0014] A second aspect of the present invention provides magnetic shrimp shell biochar prepared by any of the above preparation methods.

[0015] The third aspect of the present invention provides the magnetic shrimp shell biochar and

[0016] Application of MSPE-UPLC-MS / MS in the determination of trace tetracycline and its metabolites in aquatic products.

[0017] Further, the application comprises the following steps:

[0018] (1) After the aquatic product to be tested is processed, it is cut into small pieces no larger than 0.5 cm × 0.5 cm × 0.5 cm, mixed and homogenized, and stored in a freezer below -18°C for later use;

[0019] (2) Take the homogenized sample into a polypropylene centrifuge tube, then add the glacial acetic acid-acetonitrile-water mixed extract and Na 2 EDTA, vortex homogenization for 1 min, ultrasonic homogenization for 15 min, centrifugation at 8000 rpm for 5 min at 4 °C, collect the supernatant, re-extract the sample residue with glacial acetic acid-acetonitrile-water mixed extraction solution, collect the supernatant, and heat at 40 °C N 2 L was concentrated in the flow, then diluted with water and filtered through a 0.45 μm membrane filter for magnetic solid phase extraction;

[0020] (3) The magnetic shrimp shell biochar was activated with methanol and water respectively, and added to the diluted supernatant as an adsorbent to extract the residual TCs in the product, and the TCs were adsorbed by parallel oscillation on a constant temperature air bath vibrator. The adsorbent remained in the tube, and the supernatant was discarded with the help of an external magnet, and the eluent was added to elute the tetracycline in the adsorbent by parallel oscillation; then the eluent was separated from the adsorbent with the help of an external magnet;

[0021] (4) The eluate was filtered through a 0.22 μm organic microporous filter membrane and then the tetracycline in the eluate was determined using UPLC-MS / MS.

[0022] Furthermore, in the application, the volume ratio of the glacial acetic acid-acetonitrile-water mixed extract in step (2) is 1:84:15.

[0023] Further, the extraction conditions in step (3) of the application are: pH = 4-7, adsorbent dosage of 5 mg, adsorption time of 30 min, and eluent of methanol / acetonitrile / 0.04 mol·L -1 The volume ratio of oxalic acid aqueous solution was 1:2:7, the elution volume was 4 mL, and the desorption time was 15 min.

[0024] Further, the TCs described in this application include tetracycline, doxycycline, oxytetracycline, chlortetracycline, methacycline, demeclocycline, meclocycline, minocycline, anhydrotetracycline, 4-different chlortetracycline, 4-different tetracycline, and 4-different oxytetracycline.

[0025] The invention uses discarded shrimp shells in aquatic product processing as a carbon source, adopts a high-temperature pyrolysis method and magnetic modification to prepare shrimp shell biochar magnetic materials, selects the best biochar magnetic solid phase extraction material as an adsorbent for MSPE (magnetic solid phase extraction) through morphological characterization and performance evaluation comparative analysis; takes factors such as extractant type, extraction time, amount of magnetic functional material, adsorption and analytical conditions as investigation indicators, establishes a green, simple and specific MSPE pretreatment method for TCs in aquatic products; and then, with the advantages of high sensitivity and rapid analysis of ultra-high performance liquid chromatography tandem mass spectrometry, establishes a green and efficient MSPE-UPLC-MS / MS analysis and determination technology for tetracycline precursors and their metabolites in aquatic products.

[0026] The present invention develops a green and recyclable biochar magnetic material, which turns waste into treasure and expands the application scope of biochar materials in the pretreatment analysis of typical hydrophilic organic matter in aquatic products; this application will effectively simplify the pretreatment process of UPLC-MS / MS detection of tetracycline drugs, overcome the problems of column clogging and slow column flow in solid phase extraction, shorten the experimental time and analysis time, and improve the experimental analysis efficiency. It can provide a new green and efficient method for the detection of TCs in aquatic products, provide technical support for the effective monitoring of tetracycline drugs in aquatic products, and promote the application of shrimp shell biochar materials in the fields of food safety and analytical chemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the adsorption equilibrium time of 12 tetracyclines on MBC-600, MBC-800, MHBC-600, and MHBC-800 (condition: TCs concentration in 50 mL diluted sample solution is 10 ng mL -1, adsorbent dosage 30 mg, pH = 5, sample solution at 5, 10, 20, 30, 40, 50, 60 min was taken for evaluation of adsorption efficiency);

[0028] Figure 2 is the hysteresis regression curve of shrimp shell-derived magnetic biochar;

[0029] Figure 3 The elution effects of different elution solutions on 12 tetracyclines on MBC-600, MBC-800, MHBC-600, and MHBC-800 (condition: TCs concentration in 50 mL of diluted sample solution is 10 ng mL -1 , adsorbent dosage 30 mg, pH = 5, adsorption time 60 min, elution volume 5 mL and desorption time 20 min, four solutions of methanol, acetonitrile, 20% formic acid in methanol, methanol / acetonitrile / 0.04 mol / L oxalic acid (1:2:7, v / v) were used to adsorb the four materials, and the elution efficiency was evaluated);

[0030] Figure 4 Adsorption kinetics curves of TCs on MBC-600 ((a) pseudo-first-order kinetic model; (b) pseudo-second-order kinetic model);

[0031] Figure 5 The adsorption isotherm curves of TCs on MBC-600 ((a) Langmuir isotherm model, (b) Freundlich isotherm model);

[0032] Figure 6 This is the FTIR spectrum of shrimp shell-derived magnetic biochar;

[0033] Figure 7 This is the XRD pattern of shrimp shell-derived magnetic biochar;

[0034] Figure 8 SEM and SEM-mapping images of shrimp shell-derived magnetic biochar;

[0035] Fig. 9 The nitrogen adsorption-desorption curve and pore size distribution diagram of shrimp shell-derived magnetic biochar;

[0036] Fig.10 This is the thermogravimetric analysis diagram of shrimp shell-derived magnetic biochar;

[0037] Fig.11 This is the XPS spectrum of shrimp shell-derived magnetic biochar;

[0038] Fig.12 MRM diagram of a mixed standard solution of 12 tetracycline drugs at a concentration of 20 ng / mL

[0039] Fig.13 The effect of sample solution on MSPE efficiency and the Zeta potential diagram of MBC-600 ((a) is the effect of sample solution pH on MSPE efficiency; (b) is the Zeta potential diagram of MBC-600);

[0040] Fig.14 is the effect of adsorbent dosage on MSPE efficiency;

[0041] Fig.15 is the effect of adsorption time on MSPE efficiency;

[0042] Fig.16 The effect of elution solvent type on MSPE efficiency;

[0043] Fig.17 The effect of eluent volume and desorption time on MSPE efficiency;

[0044] Fig.18 For the reusability of MBC-600. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0046] Example 1: Preparation of shrimp shell-derived biochar magnetic material

[0047] The shrimp shells used in the experiment are Chinese tube shrimp shells.

[0048] 1. Shrimp shell pretreatment: sort the shrimp shells to remove impurities, wash, dry, crush and sieve to obtain shrimp shell powder. Collect the shrimp shells, soak them in tap water, scrub them to remove impurities, then wash them three times with ultrapure water, put them in an oven to dry at 60°C, and after they are completely dried, crush them with a grinder, and then pass them through a 100-mesh sieve to finally obtain 100-mesh shrimp shell powder, which is transferred to a sealed container for later use.

[0049] 2. Preparation of shrimp shell biochar:

[0050] (1) Preparation of BC: The shrimp shells were heated to 600 °C in a muffle furnace at a heating rate of 10 °C / L under nitrogen protection and maintained for 2 h, then cooled to obtain biochar for later use;

[0051] (2) Preparation of HBC: The biochar obtained after cooling was placed in an excess of 5% hydrochloric acid (solid-liquid ratio = 1 g: 40 mL), stirred with a magnetic stirrer for 12 h, then repeatedly washed with deionized water, dried in an oven at 80 °C to constant weight, ground, passed through a 100-mesh sieve, and placed in a sealed container for later use;

[0052] 3. Preparation of magnetic shrimp shell biochar: 3.0 g of the biochar obtained above was added to 200 mL of distilled water to prepare a biochar suspension. 2.7 g of ferric chloride (FeCl 3 6H 2 O) and 2.78 g ferrous sulfate (FeSO 4 7H 2 O) was dissolved in 30 mL of distilled water to obtain Fe 3+ and Fe 2+ Mixed solution (molar ratio Fe 3+ / Fe 2+ =1 / 1) was added to the biochar suspension, and the NaOH solution was slowly added and stirred to adjust the pH to 10. 2 The mixture was stirred at a speed of 100 r / min for 1 hour in the atmosphere. The suspension was then boiled at 100°C for 1 hour and aged at room temperature for 12 hours. The suspension was washed with distilled water until neutral and then dried in an oven at 105°C for 12 hours to obtain the shrimp shell source magnetic biochar.

[0053] In this embodiment, a variety of biochars were prepared simultaneously, including placing shrimp shell powder in a tube furnace and pyrolyzing it at 600 and 800 ° C in a nitrogen atmosphere for 2 hours to obtain BC600 and BC800. It was placed in a 1.5 mol / L HCl solution and continuously magnetically stirred for 24 hours. After filtering, washing and drying, hierarchical shrimp shell biochar HBC600 and HBC800 were obtained. Appropriate amounts of BC600, BC800, HBC600 and HBC800 were weighed and placed in a pre-mixed FeCl 2 and FeCl 3 The aqueous solution was stirred magnetically for 10 min, and the NaOH solution was slowly added and stirred. After cooling to room temperature, the material was separated by a magnet, and the material was repeatedly rinsed with deionized water and anhydrous ethanol until neutral, and dried in an oven at 50°C to obtain magnetic biochar MBC-600, MBC-800, MHBC-600 and MHBC-800.

[0054] Different from the simple adsorption of target objects, the goal of the present invention is to detect tetracycline drugs contained in aquatic matrices, so the prepared materials are required to have not only good adsorption performance, but also easy analysis of the target objects. Therefore, the above four synthesized materials were compared from the two aspects of adsorption and analysis. Figure 1As shown, the order of the adsorption equilibrium time of 12 tetracyclines on the four materials from long to short is: MBC-600>MBC-800>MHBC-600=MHBC-800. The adsorption equilibrium time of MHBC-600 and MHBC-800 after acidification is significantly shorter than that of non-acidified MBC-600 and MBC-800, indicating that the adsorption rate of the acidified materials is significantly improved. The increase in specific surface area and total pore volume after acidification of the material represents the increase in adsorption sites, which can effectively increase the adsorption rate. The results calculated by the Barrett-Joyner-Halenda (BJH) method are shown in Table 1. It can be seen that the specific surface area and total pore volume of MBC-600, MBC-800, MHBC-600 and MHBC-800 increase from small to large, which corresponds to the adsorption rate of the four. However, from the results of hysteresis curve characterization Figure 2 It is known that the magnetic saturation strength of biochar after acidification is low, and it is difficult to achieve the purpose of rapid separation of solids and liquids. The performance of four magnetic materials, MBC-600, MBC-800, MHBC-600 and MHBC800, in different elution solutions is shown in Figure 2. Figure 3 As shown in the figure, MBC-800 and MHBC-800 prepared at 800°C have obvious difficulty in target resolution, and the target resolution rate is below 35.98%. At 600°C, the target resolution rate of MHBC-600 prepared after acidification (10.43%-47.45%) is lower than that of MBC-600 without acidification (80.15%-86.79%). Due to the high sensitivity of liquid-mass spectrometry, the magnetic biochar MBC-600 prepared at 600°C was selected in consideration of energy saving, elution effect and magnetic strength. Its adsorption capacity can fully meet the requirements of trace analysis of target objects.

[0055] Table 1: Specific surface area of ​​various biochars

[0056]

[0057]

[0058] Example 2: Adsorption performance experimental method

[0059] Formula: Qt=(C 0 -C t )V / m

[0060] In the formula, C 0 and C t represent the concentration of TC before and after adsorption (mg / L), V represents the volume of the solution (mL), and m represents the mass of the polymer (mg).

[0061] Adsorption kinetics experiment: Accurately weigh 10.0 mg of magnetic biochar material, activate it, add 10 mL of 10 ug / mL TCs mixed standard solution, oscillate at 220 r / min at 25 ° C for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 60 min, 120 min, then dilute through a membrane for detection.

[0062] Using pure water as the adsorption solution, the relationship between reaction time and the adsorption capacity of MBC-600 was studied at 25°C when the initial concentration of TCs was 40 mg / L. The adsorption rate of MBC-600 reached adsorption equilibrium at 30 min, and the rapid adsorption rate of TCs can be attributed to the abundant adsorption sites on the surface of the adsorbent. The first-order and second-order kinetic models were used to simulate the adsorption kinetics of TCs on the MBC-600 surface, and the data were linearly fitted. The linear model can be expressed as:

[0063] Pseudo-first-order kinetic model:

[0064]

[0065] Pseudo-second-order kinetic model:

[0066]

[0067] In the formula, qe(mg·g -1 )、qt(mg·g -1 ) represent the adsorption amount at the equilibrium moment and at a specific moment in the adsorption process, and t (min) represents the adsorption time. K 1 (min -1 ) and K 2 (g·mg -1 ·min -1 ) represent the rate constants of pseudo-first-order and pseudo-second-order kinetic models, respectively.

[0068] Figure 4 (a) shows the relationship between log(qe-qt) and t / qt versus time, indicating the external mass transfer of TC from the aqueous solution to the adsorbent. Figure 4 (b) It can be seen that the adsorption behavior between MBC-600 and TCs conforms to the pseudo-second-order kinetic model and belongs to chemical adsorption behavior. The relevant parameters of the fitting results are shown in Table 2. The determination coefficient (R 2 >0.988) was higher than the pseudo-first-order kinetic model (R 2>0.445), so the chemical adsorption process is the main rate-determining step of the interaction between MBC-600 and TCs, which should be attributed to electrostatic and coordination interactions. At the same time, the high specific surface area of ​​MBC-600 provides more active sites, which is also one of the reasons for accelerating the binding of the adsorbent with the target analyte.

[0069] Table 2 Adsorption kinetic parameters of TCs on MBC-600

[0070]

[0071] Static adsorption experiment: In order to investigate the binding ability of the prepared magnetic biochar to TCs, 10.0 mg of magnetic material was accurately weighed. After activation, 10 mL of TCs mixed standard solution with concentrations of 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, and 260 ug / mL was added respectively. The mixture was oscillated at a constant temperature of 220 r / min at 25 °C for 30 min, then stopped, diluted through a membrane, and tested.

[0072] The adsorption isotherm can describe the surface properties and affinity of the adsorbent. Therefore, this experiment used two different isotherm models, Langmuir and Freundlich, to simulate the experimental data to evaluate the adsorption performance of MBC-600. The adsorption isotherm of TCs (40-260 mg·L -1 ) isotherm at 25°C. The Langmuir model states that the same adsorption sites are evenly distributed on the adsorbent material, and each adsorption site only adsorbs one unit of the adsorbate. Therefore, the adsorbate is adsorbed on the adsorbent material in the form of a monolayer, which is called the single molecule adsorption theory, and mainly describes chemical adsorption. The Freundlich model is an empirical model that describes the surface of the adsorbent material as a heterogeneous surface, and explains the physical adsorption and chemical adsorption processes between the adsorbent material and the target compound. The calculation formulas of the two models are:

[0073] Langmuir isotherm model:

[0074] Freundlich isotherm model:

[0075] In the formula, qe(mg·g -1 )、qm(mg·g -1 ) represent the adsorption amount and maximum adsorption capacity at equilibrium, KL (L·mg -1 ) represents the adsorption equilibrium constant of the Langmuir isotherm model and is related to the adsorption rate. The larger the value, the stronger the adsorption performance of the adsorbent. -1)1 / n) represents the affinity coefficient of the Freundlich isotherm model and is related to the adsorption capacity. Ce (mg·L -1 ) represents the concentration of the target substance at the adsorption equilibrium moment, and 1 / n represents the empirical constant of Freundlich, which indicates the adsorption strength.

[0076] The nonlinear fitting diagram of Langmuir and Freundlich isotherm models is: Figure 5 (a), (b), the nonlinear fitting correlation coefficients are shown in Table 3. The results show that the overall linear regression coefficient of the Langmuir isotherm is (0.988 <R 2 <0.999) value is higher than Freundlich (0.914 <R 2 <0.998), indicating that the adsorption between MBC-600 and TCs was monolayer, and the adsorption of TCs on the surface of MBC-600 was uniform. The maximum adsorption amounts qm,cal calculated by Langmuir equation were 92.08, 113.90, 109.41, 110.62, 132.10, 141.44, 138.31, 109.53, 109.29, 116.14, 131.06 and 109.89 mg g -1 From the Freundlich model, it was found that the KF values ​​of the 12 TCs were all greater than 9.98, and 1 / n<0.5, indicating that MBC-600 has a strong adsorption capacity for TCs.

[0077] Table 3: Static adsorption of TCs on MBC-600

[0078]

[0079] Example 3: Characterization Methods

[0080] The surface morphology of biochar was observed using a scanning electron microscope (SEM) (Hitachi Regulus8100, Japan); the types of surface functional groups of biochar were analyzed using a Fourier transform infrared spectrometer (FT-IR) (Thermo Scientific Nicolet iS20, USA); the surface area, average pore size and pore volume of biochar were measured at 200°C using a surface area and porosity analyzer (ASAP 2460, USA); the crystalline structure of biochar was analyzed using an X-ray diffractometer (XRD) (X'PertPro MPD, Nalytical, the Netherlands); and the magnetic strength of biochar was analyzed using a hysteresis regression tester (Model 8604, LakeShore, USA).

[0081] Infrared spectroscopy analysis: During the pyrolysis process, atoms such as C, H, and O on the surface and in the pores of biochar will form various functional groups such as hydroxyl, carboxyl, and carbonyl. FTIR is used to identify various functional groups in biochar, such as Figure 6 As shown, 3414.64cm -1 The absorption peaks can be attributed to the stretching vibration of -OH (alcohols and phenols); at 2700-3000cm -1 Band 2930.64cm -1 The weaker peak at 3 and -CH 2 The CH stretching vibration indicates that with the decomposition of chitin and other organic matter in shrimp shells, the number of alkyl groups decreases and the degree of aromatization of biochar increases; 1620.14cm -1 The absorption peak at 1417.00 cm is attributed to the stretching vibration of the aromatic ring or C=C, which further indicates the carbonization of the shrimp shell; -1 The characteristic peaks are attributed to the characteristic peaks of C=O or CO; 1040.22cm -1 The characteristic peak at 868.66cm is attributed to the stretching vibration of CO; -1 The characteristic peak at 564.12cm is attributed to the unsaturated absorption bond of biochar or the CH bending vibration on the carbon ring; -1 The characteristic peak at belongs to the stretching vibration of Fe-O. The FTIR analysis results show that the shrimp shell biochar magnetic material was successfully synthesized, and there are a large number of oxygen-containing functional groups such as -OH, -COOH and -CHO on the surface.

[0082] XRD analysis: X-ray diffractometer (XRD) was used to determine the crystal structure of the shrimp shell-derived magnetic biochar material. Figure 7 As shown, the diffraction angles of 29.7°(220), 36.1°(311), 57.3°(511), and 61.0°(440) in the composite material are Fe 3 O 4 The characteristic diffraction peaks of Fe 3 O 4 The XRD standard card (PDF#97-015-9967) of the company is consistent. The diffraction angle of 23.2° corresponds to the (002) carbon crystal plane, which is a typical characteristic peak of carbonaceous materials, indicating the existence of parallel stacked and interconnected graphene layers; the diffraction peak observed at a diffraction angle of 43.3° (400) corresponds to the (100) carbon crystal plane.

[0083] SEM analysis: Figure 8 (ab) are SEM images of shrimp shell biochar before and after magnetization. Figure 8 (a) It can be observed that the surface of BC-600 is irregular, with a large number of impurity particles and no obvious pores. Figure 8 (b) Fe 3 O 4 As can be seen from the figure, MBC-600 shows obvious surface changes. It can be easily observed that a large number of obvious pore structures appear on the surface. These pore structures may originate from impurity sites, indicating that they have been successfully removed. The increase in pores will expand the specific surface area of ​​the carbon material and increase the active sites, which is conducive to the adsorption of TCs into the interior of MBC-600 through pore filling. Figure 8 The SEM-mapping image of (ch) shows the distribution of elements on the surface of the material. The analysis results show that the material contains C, N, O, P, S and Fe elements, indicating that Fe 3 O 4 Successfully attached to biochar.

[0084] BET analysis: using N 2 The adsorption-desorption isotherm was used to calculate the specific surface area, pore size and pore volume of the material. Fig. 9 As shown in Table 1, the characteristic hysteresis curves of BC-600 and MBC-600 belong to the type IV isotherm. Although the pore size distribution of BC-600 involves both the mesopore and macropore regions, it is mainly concentrated in the mesopore position. The pore size distribution of MBC-600 is concentrated in the mesopore region, indicating that BC-600 and MBC-600 are mesoporous materials, and the adsorption sites are more easily exposed. As shown in Table 1, the average pore size (4V / A) of BC-600 is 31.53nm, and the BET specific surface area is 5.57m 2 g -1 , the total pore volume is 0.044 cm 3 g -1 The average pore size (4V / A) of MBC-600 is 9.07nm, and the BET specific surface area is 71.30m 2 g -1 , with a total pore volume of 0.16 cm 3 g -1 . Compared with BC-600, MBC-600 has a higher total pore volume and a smaller pore size value, and the results are consistent with the SEM scanning results. Studies have shown that when the average pore size of the adsorbent is 1.7-3 times larger than the pore size of the adsorbate, the adsorbent is more likely to adsorb the adsorbate. The structural size of TC is about 2nm, which is more suitable as an adsorbent material for adsorbing residual TCs in water products than BC-600 (31.53nm).

[0085] Hysteresis loop: The magnetic properties of MBC were determined by VSM method. The magnetization curves of MBC-600 after initial preparation and 5 cycles are shown in Figure 2. Figure 2The VSM test curves of the two are typical S-shaped curves. With the increase of magnetic field strength, the magnetization intensity gradually increases and reaches saturation. The saturation magnetization intensity of the two is 7.04emu·g -1 and 4.84emu·g -1 , no obvious hysteresis, remanence and coercivity, showing paramagnetism, further proving that Fe 3 O 4 The loading on biochar was successful. Although the magnetization intensity of MBC-600 and MBC-600 after 5 cycles was lower than that of other magnetic materials, it still had the ability to quickly separate the adsorbent from the solution within 10 seconds during the experiment.

[0086] Thermogravimetric analysis: In order to investigate the thermal stability of MBC-600, TGA test analysis was performed on it. The results are as follows Fig.10 As shown in Figure 2, it decreased by 3.64% from 30℃ to 200℃, and the decreased part can be considered to be the evaporation of water molecules in the sample; it decreased by 12.99% from 200℃ to 700℃. The thermogravimetric differential curve (DTG) shows that the weight loss of MBC-600 is the largest at 658.2℃, which may be attributed to the thermal decomposition of the carbon phase and the evaporation of carbon and Fe 3 O 4 The total mass loss of MBC-600 during the whole heating process is 16.63%, and it has good thermal stability in a wide temperature range.

[0087] XPS: Select XPS to analyze the surface composition and valence state changes of MBC-600. The results are as follows Fig.11 (a) As shown in the figure, the Fe 2p XPS spectrum of MBC-600 shows that six components, Fe, O, N, Ca, C, and P, are detected. Fig.11 (b)) Two characteristic peaks at 710.77eV and 724.21eV were observed, corresponding to Fe 2p3 / 2 and Fe 2p1 / 2, respectively, proving that Fe 3 O 4 The XPS analysis results further confirmed that Fe 3 O 4 The presence of α-hydroxy-1-nitropropene is consistent with the results of FT-IR analysis.

[0088] Example 4: Standards and Standard Solutions

[0089] 12 kinds of standard products: tetracycline (TC) purity ≥99%, doxycycline (DC) purity ≥98%, oxytetracycline (OTC) purity ≥95%, chlortetracycline (CTC) purity ≥99%, methacycline (MTC) purity ≥95.19%, demeclocycline (DMC) purity ≥90.4%, meclocycline (MCC) purity ≥97%, minocycline (MNC) purity ≥98.8%, anhydrotetracycline (ATC) purity ≥98.0%, 4-epi chlortetracycline (ECTC) purity ≥84.9%, 4-epi tetracycline (ETC) purity ≥84.9%. Tetracycline) with a purity of ≥86.3% and 4-epi oxytetracycline (EOTC) with a purity of ≥80.7% were purchased from Dr. Ehrenstorfer, Germany.

[0090] Accurately weigh 5.00 mg of each of the 12 standards, dissolve in methanol and dilute to 50 mL, then calculate the mass concentration of the standard stock solution of about 100 μg / mL according to the hydrochloride and purity of each. Store at 4°C in the dark. Before use, dilute the single standard stock solution with deionized water to prepare the 12 TCs mixed standard working solutions.

[0091] Example 5: Chromatographic and mass spectrometric conditions

[0092] Chromatography using WatersAcquity UPLC TM System (Waters, Milford, MA, USA) and QuattroPremier XE Micromass triple quadrupole mass spectrometer (Waters, Manchester, UK). Chromatographic separation was performed on a Waters BEH C18 column (2.1 × 100 mm, particle size 1.7 μm) with a binary gradient mobile phase consisting of 0.1% formic acid solution (a) and acetonitrile (b) at a flow rate of 0.3 mL min -1. Gradient program: 0-6.0 min, 90%-80% A; 6.0-6.5 min, 80%-78% A; 6.5-8.5 min, 78%-60% A; 8.5-8.7 min, 60%-5.0% A; 8.7-9.7 min, 5% A; 9.7-10.0 min, 5%-90% A; 10.0-13.0 min, 90% A. Column temperature 35 °C, autosampler temperature 10 °C. Injection volume 10 μL. MassLynx software 4.1 was used for instrument control and data acquisition.

[0093] The mass spectrometer (MS) was operated in positive ion mode using electrospray ionization (ESI). The mass spectrometry analysis was performed in multiple reaction monitoring (MRM) mode. The optimized MS / MS parameters are: cone and desolvation gas: nitrogen (purity 99.9%), collision gas: argon (purity 99.9999%), source temperature 150°C, desolvation gas temperature: 380°C; cone gas is high-purity nitrogen, flow rate: 50L / h; desolvation gas is high-purity nitrogen, flow rate: 600L / h. The mass spectrometry multiple reaction monitoring conditions of TCs are shown in Table 4. The MRM spectra of the 12 analytes are shown in Fig.12 .

[0094] Table 4 Multi-reaction monitoring conditions of TCs

[0095]

[0096] *quantitative ion

[0097] Example 6: Sample preparation

[0098] The samples collected from the market were processed according to the provisions of SC / T 3016-2004 "Aquatic Product Sampling Method" and sealed and stored at -18℃. Fish meat, skin, shrimp shells were removed and thawed before use. Fish, scales and skin were removed, and muscles were taken along the spine; shrimp, head, shells and intestinal glands were removed, and muscles were taken; turtles, crabs and shellfish, edible parts were taken, cut into small pieces no larger than 0.5cm×0.5cm×0.5cm, mixed, fully homogenized, and frozen below -18℃ for later use.

[0099] 2.00 ± 0.01 g of the homogenized sample was weighed into a 50 mL polypropylene centrifuge tube, and then 10 mL of glacial acetic acid-acetonitrile-water mixed extract (1:84:15, v / v) and 0.1 g Na 2 EDTA. Vortex homogenize for 1 min, ultrasonic homogenize for 15 min, and centrifuge at 8000 rpm for 5 min at 4°C. Collect the supernatant and re-extract the sample residue with 5 mL of glacial acetic acid-acetonitrile-water mixed extraction solution (1:84:15, v / v). Collect the supernatant and heat at 40°C N 2The solution was concentrated to approximately 2.5 mL, then diluted with water to 50 mL, and filtered through a 0.45 μm membrane filter for magnetic solid phase extraction.

[0100] Example 7: Magnetic Solid Phase Extraction

[0101] The synthesized biochar magnetic material was activated with 5.0 mL of methanol and 5.0 mL of water, respectively. 15 mg of activated carbon nanofiber magnetic material was directly added to the diluted supernatant and oscillated in parallel on a constant temperature air bath oscillator to adsorb TCs for 20 min. The adsorbent remained in the tube and the supernatant was discarded with the help of an external magnet. 4 mL of acetonitrile and 0.02 mol L -1 The eluent, which consisted of an oxalic acid solution (1:8, v / v), was used to elute tetracycline from the adsorbent by parallel oscillation. Subsequently, the eluent was separated from the adsorbent with the aid of an external magnet and filtered through a 0.22 μm organic microporous filter membrane for UPLC-MS / MS analysis. The reusability of the material was evaluated by repeated extraction and desorption.

[0102] In order to obtain the best adsorption effect of 12 TCs in the pretreatment of aquatic products, the synthesized MBC-600 was used as the adsorbent of MSPE, and UPLC-MS / MS was used to perform trace analysis of drug residues in aquatic products. The main parameters affecting the performance of MSPE, including the pH of the sample solution, the amount of adsorbent, the extraction time, the selection of elution solvent, the volume of the elution solution, and the desorption time, were optimized. The desorption efficiency value was used as an evaluation index for extraction and desorption. Considering the influence of the sample matrix, the optimization of MSPE conditions was carried out in the blank sample matrix.

[0103] Effect of extraction solvent type: Different from water or liquid samples, choosing a suitable extraction solvent is important and key to the application of MSPE method in aquatic products. The appropriate extraction liquid should have a high extraction efficiency for the target analyte and ensure that the magnetic material has a high adsorption capacity for the target analyte. Due to the presence of hydroxyl groups in the structure, TCs has high stability and good solubility in acidic aqueous solutions. Weakly acidic Na 2 EDTA-Mcllvaine buffer solution was used to extract TCs from aquatic products. According to the QuEChERS pretreatment method, acidified acetonitrile aqueous solution and appropriate amount of Na 2 The present invention firstly tests the magnetic material in pure water, Na+ and different pH values. 2 The adsorption of TCs in EDTA-Mcllvaine buffer solution and acetic acid-acetonitrile-water mixed solution (1:84:15, v / v). The results show that the adsorption rate can reach more than 90% in pure water and acetic acid-acetonitrile-water mixed solution, while in Na 2In the EDTA-Mcllvaine buffer solution, the adsorption rate is only about 50%, which may be due to the complexing ions in citric acid inhibiting adsorption. Therefore, a mixed solution of glacial acetic acid-acetonitrile-water (1:84:15, v / v / v) was used to extract TCs from fortified samples, and then adsorbed with magnetic materials. The results showed that the adsorption rate was approximately zero. The results showed that when the adsorbent was directly placed in the extraction solution, the adsorption process could not proceed. It was necessary to dilute the solution and convert it to pure water or concentrate and then dissolve it, and then adsorb again.

[0104] pH of the sample solution: The pH of the adsorption solution can affect the existence form of TCs molecules and the charge properties of the adsorbent surface, thus having a significant impact on the extraction efficiency. The experimental results are as Fig.13 (a) shown. It was observed that as the pH increased, the recovery efficiency also gradually increased, reaching a maximum at pH = 4. When the pH value increased from 4 to 7, the recovery rate gradually leveled off, and when pH > 7, the adsorption efficiency showed a downward trend. The above changes indicated that the pH value had an important impact on the adsorption of 12 TCs on the MBC-600 adsorbent material, and the maximum recovery rate was reached in the range of 4 < pH < 7.

[0105] The above phenomenon can be explained by the pH PZC value of MBC-600 revealed by Zeta potential ( Fig.13 (b)) and the PKa value of tetracycline. The pH PZC value of MBC-600 is 4.64. TC has multiple ionizable functional groups. When pH < 3.30, TCs exist in the form of TCH 3+ ; when 3.32 < pH < 7.69, TCs exist in the form of TCH 2 ; when 7.69 < pH < 9.69, TCs exist in the form of TCH - ; when pH > 9.69, TCs exist in the form of TC 2- . Therefore, when the pH value of the solution is lower than 3.30, MBC-600 is positively charged, and the repulsive force of the same charge will repel the adsorption of TCs. When the solution is between 3.32 < pH < 7.69, TCs mainly exist in the form of TCH 2 , and in this range, the surface negative charge of MBC-600 dominates, so MBC-600 can generate electrostatic attraction with TCs that lose a proton and present the TCH 2 form. It was shown that the surface complexation of functional groups between antibiotics and biochar was accompanied by the release of H + , and the electrostatic interaction and hydrogen bond enhanced the adsorption of TC molecules on the adsorbent. When the recovery rate showed a downward trend at pH > 7.69, this may be because as the pH increased, the proportion of -OH in the solution increased, and the active sites on the adsorbent surface were occupied by -OH. At the same time, under alkaline conditions, TCs exist in the form of TC 2-The positive charge on the adsorbent surface gradually decreases with the increase of pH, and the two produce electrostatic repulsion, resulting in a decrease in recovery rate. Therefore, the optimal pH for MBC-600 to adsorb TCs is between 4 and 7, and the extraction efficiency is maintained above 90.18% within this range. There is no need to adjust the pH of the sample solution during the experiment.

[0106] Effect of adsorbent dosage: The dosage of adsorbent is an important parameter affecting the adsorption of target analytes. Given that TCs can be detected at trace levels using the high sensitivity of mass spectrometry and that the synthesized adsorbent has good specific surface area and adsorption performance, the dosage of adsorbent does not need to be too large. The dosage of MBC-600 was optimized in the range of 3.0-18.0 mg. Fig.14 As shown in the figure, when the adsorbent mass increased from 3.0 mg to 15.0 mg, the extraction rate of TCs gradually increased, reached a maximum value at 15 mg, and then reached equilibrium. This phenomenon shows that the increase in the amount of adsorbent is beneficial to increase the specific surface area and the number of active adsorption centers. From the perspective of effective use of resources, the amount of adsorbent material should be reduced, and too much adsorbent will lead to more stringent elution conditions. Therefore, 15.0 mg was selected as the optimal adsorbent dosage for subsequent experiments.

[0107] Effect of adsorption time: In MSPE, the effective contact time between the adsorbent and the target compound in the sample solution is an important criterion for obtaining high extraction efficiency. The effects of different extraction times (5, 10, 15, 20, 25, 30, 35 and 40 min) on MSPE efficiency were studied. Fig.15 It shows that the extraction efficiency increases continuously from 5 min. At 30 min, the extraction efficiency of 12 TCs reaches the highest value and basically maintains a balance within 30-40 min. This indicates that within 30 min, a rapid distribution equilibrium between TCs and MBC-600 has been established. Therefore, the optimal adsorption time is set to 30 min.

[0108] Effect of eluent type: In MSPE, one of the key steps is to thoroughly elute the target compounds adsorbed on the magnetic material. A suitable eluent should firstly be able to thoroughly elute the analytes from the adsorbent to obtain reliable and reproducible analytical results, and secondly dissolve a high percentage of the target compounds and be suitable for subsequent mass spectrometry analysis of the sample. First, acetonitrile and methanol were tested as eluents. The results are shown in Fig.16 As shown in the figure: 100% acetonitrile and methanol as elution solutions can only elute a very small amount of target from the adsorbent, and the elution efficiency is less than 44.17%, which cannot effectively leach TCs from MBC-600. Therefore, several eluents composed of acetonitrile, methanol, oxalic acid and formic acid in different proportions were studied as elution solutions for desorbing TCs from magnetic microspheres. The results are shown in the figure. Fig.16As shown, methanol / acetonitrile / 0.04mol·L -1 Oxalic acid aqueous solution (1:2:7, v / v / v) can release TCs more effectively as an elution solution, and the elution efficiency of 12 TCs is above 90.84%. The results show that the presence of a certain amount of oxalic acid is beneficial to the elution of TCs. This may be because oxalic acid can form a complex in aqueous solution as a ligand of metal cations to compete with TCs, reduce the complexation of TCs with metal ions, and thus change the morphology of TCs. At the same time, TCs contain carboxylic acids and C 3 -OH, C 12 -OH, organic acid can effectively prevent the interaction between Fe(II) and TCs. Therefore, methanol / acetonitrile / 0.04 mol·L -1 Aqueous oxalic acid solution (1:2:7, v / v / v) is the best eluent.

[0109] The influence of eluent volume and desorption time: The volume of the eluent will directly affect the enrichment factor and elution efficiency of the target. Too large a volume will cause the target to be over-diluted, reducing the enrichment effect; too small a volume may not fully elute the target, affecting the recovery rate. Therefore, choosing the appropriate eluent volume is a key step to ensure the experimental effect and data accuracy. The effect of eluent volumes of 1, 2, 3, 4, 5 and 6 mL on the elution efficiency was investigated. The results are as follows: Fig.17 As shown in (a), within the range of 1-4 mL, the elution efficiency shows an upward trend as the volume increases, and the elution efficiency tends to be balanced after 4 mL. In order to reduce the amount of organic solvent used, 4 mL was selected as the optimal eluent volume. At the same time, in order to ensure that the target reaches desorption equilibrium from the adsorbent, the desorption time of 5, 10, 15, 20, 25 and 30 min was investigated, and other conditions were determined. The results are shown in Fig.17 As shown in (b), the recovery rate of TCs gradually increased with the increase of elution time. When the desorption time reached 15 min, the recovery rate of 12 TCs reached the maximum value, and the elution efficiency remained basically balanced within 15 to 30 min. Considering the extraction amount and extraction time, the elution time of 15 min was sufficient to elute most TCs.

[0110] Method validation

[0111] Under the optimized experimental conditions, the linear range, accuracy, precision, sensitivity and matrix effect of the method were studied. The method was validated using fish, crab and shrimp samples that were confirmed to be free of target analytes. -1 , 10.0 μg kg -1 and 50.0μg kg -1 ), and each spiked amount was repeated 6 times for method validation.

[0112] Linear range: Pipette appropriate amount of 12 kinds of TCs mixed standard working solution, dilute to 1.0mL with ultrapure water, and prepare a series of standard solutions with mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0ng / mL for UPLC-MS / MS determination. The internal standard method was used for quantification, and the standard curve was drawn with the peak area ratio of the measured component to the internal standard as the ordinate and the concentration of the standard solution as the abscissa. The results are shown in Table 5. In the range of 0.5 to 200ng / mL, the 12 kinds of TCs showed a good linear relationship, and the correlation coefficient (R 2 )≥0.959, meeting the needs of instrumental analysis.

[0113] Detection limit and quantification limit: The grass carp and white shrimp samples that were confirmed to be free of analytes were pre-treated according to the test method and then added with a certain concentration of 12 TCs mixed standard working solutions for testing. The results are shown in Table 5. The detection limit (LOD) of the 12 TCs was determined to be 0.15-0.49μg / kg with a signal-to-noise ratio (S / N) ≥ 3, and the quantification limit (LOQ) of the 12 TCs was determined to be 0.50-1.63μg / kg with S / N ≥ 10. The test results are in compliance with the provisions of GB 31650-2019 "Maximum Residue Limits of Veterinary Drugs in Food". It shows that this method has good accuracy in the determination of TCs in aquatic products and can meet the needs of TCs residue analysis of actual samples.

[0114] Table 5: Linear range, detection limit and quantification limit

[0115]

[0116]

[0117] Recovery rate and precision of the method: In order to investigate the accuracy and precision of the method, grass carp samples that were confirmed to be free of analytes were used as blank matrix for the recovery experiment. 2.0 g of the two types of samples were weighed into centrifuge tubes, and a certain volume of 12 TCs mixed standard working solutions were added to each of them, so that the three concentration levels of TCs in the added samples were 2.0 μg / kg -1 , 10.0 μg kg -1 and 50.0μg kg -1 After addition, the samples were pre-treated according to the test method and then measured by UPLC-MS / MS. The samples were compared with the standard curve and the concentrations of 12 TCs in the samples were finally obtained by conversion. Each sample was measured 6 times in parallel, and each addition level was measured 6 times according to this method. The measurement was continued for 5 days, and the spike recovery, intra-day precision and inter-day precision were calculated. The recovery was calculated according to the following formula:

[0118]

[0119] Where: R is the recovery rate, %; Cs is the measured concentration of TCs in the spiked sample, μg kg -1 ; C0 is the concentration of TCs in the actual sample, μg kg -1 ; C is the theoretical spike concentration of TCs in the spiked sample, μg kg -1 The results showed that the average recoveries of TCs under three addition levels and in two sample matrices were 86.60% to 98.90%. The intra-day precision and inter-day precision were 1.62% to 8.80% (n=6) and 2.41% to 11.63% (n=5), respectively. See Table 6.

[0120] Table 6: Recovery and precision of 12 tetracycline drugs

[0121]

[0122] Matrix effect: Aquatic products contain complex matrices. When analyzed by UPLC-MS / MS, the matrix can cause ion suppression or enhancement, affecting the accuracy of detection. Therefore, in order to reduce the matrix effect, improve the signal intensity, and obtain more accurate results, it is necessary to choose a suitable sample pretreatment method. In order to investigate the matrix effect of MBC-600 as a magnetic adsorbent in MSPE, blank samples were selected for experiments under optimal conditions. Matrix effect = (slope of matrix matching standard curve / slope of reagent standard curve-1) × 100%.

[0123] The results are shown in Table 7. The ME of DMC, MCC, OTC, TC, CTC, and DC are all acceptable within ±15%. The reason for the low matrix effect may be that the adsorbent in MSPE acts as a barrier to block the highly polar matrix components outside the adsorbent while adsorbing the target. The ME of the remaining six TCs is within ±20%, MTC and EOTC have matrix inhibition effects, and MNC, ETC, ECTC, and ATC have matrix enhancement effects. The results show that the MBC-600 has good enrichment ability, can significantly reduce and narrow the matrix effect to an acceptable range, and can reduce some interfering components and improve detection sensitivity.

[0124] Table 7: Matrix effects of 12 tetracycline drugs in shrimp and crab

[0125]

[0126] Reproducibility and recyclability of MBC-600: Regeneration ability is a key indicator for evaluating the adsorption performance of magnetic materials. In this experiment, MBC-600 adsorbed TCs and then used methanol / acetonitrile / 0.04 mol·L -1 After elution with oxalic acid aqueous solution (1:2:7, v / v / v), the solid was collected and vortexed with methanol and water (1:1) for 1 min, washed twice, the waste liquid was discarded, dried in an oven at 60°C for 5 h, and collected for the next adsorption experiment. In each cycle, the cycle experiment was repeated 5 times with the optimal MSPE conditions to verify the material reuse performance. The results are shown in Fig.18 As shown, after 5 recycling cycles (TCs: 20 ng mL -1 , blank sample: 10mL), the overall recovery rate is above 72.15%, and the loss is less than 20.28%. The adsorbent after 5 cycles of recovery was analyzed by VSM. The results are as follows Figure 2 As shown in the figure, after five cycles, although the magnetic properties of the adsorbent material were low, it could still meet the requirements of solid-liquid separation. Therefore, MBC-600 can be used as an adsorbent to remove residual TCs in aquatic products.

Claims

1. A method for preparing magnetic shrimp shell biochar, characterized in that: The following steps are involved: 1) Shrimp shell pretreatment: Sorting shrimp shells to remove impurities, washing, drying at 60°C, and crushing through a 100-mesh sieve to obtain shrimp shell powder; 2) Preparation of shrimp shell biochar: The shrimp shell powder was heated to 600°C in a muffle furnace at a heating rate of 10°C / L under nitrogen protection and maintained for 2 h, and then cooled to obtain shrimp shell biochar; 3) Preparation of magnetic shrimp shell biochar: The shrimp shell biochar obtained in step 2) is added into distilled water to prepare a biochar suspension, and ferric chloride and ferrous sulfate are dissolved in distilled water to obtain Fe 3+ and Fe 2+ The mixed solution was dripped into the biochar suspension, and then the NaOH solution was slowly added and stirred, and the pH was adjusted to 10. The mixture was stirred and boiled in a N2 atmosphere at room temperature and then aged. The mixture was washed with distilled water until neutral and then dried in an oven to obtain the magnetic shrimp shell biochar.

2. The method for preparing magnetic shrimp shell biochar according to claim 1, characterized in that: In step 3), the amount of shrimp shell biochar and distilled water is 3 g biochar added to 200 mL distilled water; Fe 3+ and Fe 2+ Fe in mixed solution 3+ / Fe 2+ The molar ratio is 1 / 1.

3. The method for preparing magnetic shrimp shell biochar according to claim 1, characterized in that: In step 3), the mixture is stirred at 100 r / min in a N2 atmosphere at room temperature for 1 hour, and then the suspension is boiled at 100° C. for 1 hour and aged at room temperature for 12 hours. The suspension is washed with distilled water until neutral and then dried in an oven at 105° C. for 12 hours.

4. The magnetic shrimp shell biochar prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the magnetic shrimp shell biochar as claimed in claim 4 in combination with MSPE-UPLC-MS / MS in the determination of trace tetracycline and its metabolites in aquatic products.

6. The use according to claim 5, characterized in that The application includes the following steps: (1) After the aquatic product to be tested is processed, it is cut into small pieces no larger than 0.5 cm × 0.5 cm × 0.5 cm, mixed and homogenized, and stored in a freezer below -18°C for later use; (2) The homogenized sample was placed in a polypropylene centrifuge tube, and then glacial acetic acid-acetonitrile-water mixed extract and Na2EDTA were added, vortexed and homogenized for 1 min, ultrasonically homogenized for 15 min, centrifuged at 4°C, 8000 rpm for 5 min, and the supernatant was collected. The sample residue was re-extracted with glacial acetic acid-acetonitrile-water mixed extract, and the supernatant was collected. L was concentrated in a 40°C N2 flow, and then L was diluted with water and filtered using a 0.45 μm membrane filter for magnetic solid phase extraction; (3) The magnetic shrimp shell biochar was activated with methanol and water respectively, and added to the diluted supernatant as an adsorbent to extract the residual TCs in the product, and the TCs were adsorbed by parallel oscillation on a constant temperature air bath vibrator. The adsorbent remained in the tube, and the supernatant was discarded with the help of an external magnet, and the eluent was added to elute the tetracycline in the adsorbent by parallel oscillation; then the eluent was separated from the adsorbent with the help of an external magnet; (4) The eluate was filtered through a 0.22 μm organic microporous filter membrane and then the tetracycline in the eluate was determined using UPLC-MS / MS.

7. The use according to claim 6, characterized in that The volume ratio of the glacial acetic acid-acetonitrile-water mixed extract in step (2) is 1:84:

15.

8. The use according to claim 6, characterized in that The extraction conditions in step (3) are: pH = 4-7, adsorbent dosage 5 mg, adsorption time 30 min, eluent methanol / acetonitrile / 0.04 mol·L -1 The volume ratio of oxalic acid aqueous solution was 1:2:7, the elution volume was 4 mL, and the desorption time was 15 min.

9. The use according to claim 6, characterized in that: The TCs include tetracycline, doxycycline, oxytetracycline, aureomycin, methacycline, demeclocycline, meclocycline, minocycline, anhydrotetracycline, 4-different aureomycin, 4-different tetracycline, and 4-different oxytetracycline.

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