Bifunctionalized mesoporous silica material and method of preparation and use

Abstract

The application discloses a bifunctional mesoporous silica material and a preparation method and application thereof, and the bifunctional mesoporous silica material is MCM-41@COOH@C 18 mesoporous microspheres; the MCM-41@COOH@C 18 The mesoporous microspheres are obtained by modifying MCM-41 through 3-aminopropyl triethoxysilane, succinyl and octadecyl trimethoxysilane. The application optimizes solid phase extraction parameters, uses an amide chromatographic column for separation, and establishes an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) detection method for six kinds of aminoglycoside drugs (AGs) residues in milk. The material is low in cost, simple in production, and high in operability as a SPE filler, and can be applied to detection of AGs in other food or agricultural product samples in the future.

Description

Technical Field

[0001] The present invention relates to a silicon dioxide material, a preparation method and an application, and in particular to a bifunctionalized mesoporous silicon dioxide material, a preparation method and an application, belonging to the technical field of chemical materials. Background Art

[0002] Aminoglycosides are a class of broad-spectrum antibiotics that have played an important role in treating major bacterial infections and tuberculosis since their discovery in 1943. However, toxicological studies have shown that aminoglycosides exhibit significant side effects, such as ototoxicity, nephrotoxicity, and allergic reactions. Milk is a common food in our daily diet, containing a variety of nutrients such as fat, peptides, protein, and minerals. Aminoglycosides possess broad-spectrum antimicrobial properties and can treat conditions such as mastitis and tuberculosis in dairy cows. They can also act as growth promoters to improve feed conversion rates, and therefore are used directly or indirectly in the production, processing, and storage of milk and dairy products. Human consumption of animal products containing these drugs also poses a risk, and can also contribute to the development of bacterial resistance, complicating disease treatment. GB 31650-2019, the National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food, stipulates maximum residue limits (MRLs) for aminoglycosides in various animal tissues. These limits stipulate that gentamicin, kanamycin, and neomycin should not exceed 200 μg / kg, 150 μg / kg, and 1500 μg / kg, respectively, in milk. To regulate the use of aminoglycosides, the government must invest significant manpower and resources annually to ensure agricultural product safety. Therefore, establishing highly accurate and sensitive detection methods for aminoglycosides is crucial.

[0003] Solid phase extraction (SPE) plays a very important role in the pretreatment of veterinary drugs. It can not only enrich the target compound, but also purify the sample matrix. The most commonly used aminoglycoside drug in milk is C 18Solid phase extraction or MCX, WCX ion exchange mode solid phase extraction. Compared with single mode adsorbent, mixed mode adsorbent has better adsorption selectivity by multiple interactions, and impurities can be easily removed by controlling the washing conditions. In addition to the commonly used solid phase extraction column, many researchers use molecular imprinting or magnetic beads, graphene and other materials to bond boronic acid groups, hydroxyl groups and carboxyl groups to develop a series of adsorbents for aminoglycoside drugs. The skeleton of mesoporous material MCM-41 type material is composed of hexagonal arranged cylindrical mesopores, has high surface area and narrow pore size distribution, excellent pore structure, high thermal stability and chemical stability, and the surface has a large number of silicon hydroxyl groups, which can be modified by functional groups to increase the function, which has caused great research interest in the field of adsorption chemistry. At present, there are literatures reported that the surface modified mesoporous material is applied in lead, anionic toxic azo dye, biological toxin and the like, but it is less applied in aminoglycoside drug enrichment.

[0004] Since aminoglycoside drugs do not have volatility, do not have chromophores or fluorophores, and are not suitable for direct ultraviolet or fluorescence detection, they need to be derivatized, which increases the complexity of the pretreatment. With the emergence of mass spectrometry technology, liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology has become the simplest and most accurate detection method. In chromatographic separation, aminoglycosides are compounds with high polarity, and their retention on ordinary reversed-phase chromatographic columns is quite weak. The most common solution is to add ion-pair reagents to the mobile phase. However, these additives can cause severe contamination of the chromatographic column and ion suppression of electrospray ionization. SUMMARY

[0005] The present application aims to provide a bifunctional mesoporous silica material which can be used as a SPE filler and has strong operability; another object of the present application is to provide a preparation method of the bifunctional mesoporous silica material; another object of the present application is to provide an application of the bifunctional mesoporous silica material in aminoglycoside drug detection.

[0006] Technical scheme: the bifunctional mesoporous silica material of the present application is MCM-41@COOH@C 18 mesoporous microspheres; the MCM-41@COOH@C 18 The MCM-41 is modified by 3-aminopropyl triethoxysilane, succinyl and octadecyl trimethoxysilane to obtain the mesoporous microspheres.

[0007] On the other hand, the present application provides a preparation method of the above-mentioned bifunctional mesoporous silica material, which comprises the following steps:

[0008] (1) preparing MCM-41@NH2@C 18Materials: By C 18 / NH2 functionalized mesoporous MCM-41 material to obtain MCM-41@NH2@C 18 Material;

[0009] (2) Preparation of MCM-41@COOH@C 18 Mesoporous microspheres: through C 18 / COOH functionalized MCM-41@NH2@C 18 Materials, prepared MCM-41@COOH@C 18 Mesoporous microspheres.

[0010] As a further improvement of the above scheme, in step (1), MCM-41@NH2@C 18 The material specifically includes the following steps:

[0011] MCM-41 was dispersed in anhydrous toluene and stirred evenly, and then 3-aminopropyltriethoxysilane (APTES) and octadecyltrimethoxysilane (C 18 TMS), stirred vigorously in an oil bath and refluxed for a period of time, the resulting white solid product was collected, washed and dried to obtain MCM-41@NH2@C 18 Material.

[0012] Preferably, the oil bath temperature is 110-120° C., and the reflux time is 15-16 h; and / or, the drying is vacuum drying, and the drying conditions are: vacuum degree -0.08-0.10 MPa, 65-70° C., and 12-14 h.

[0013] As a further improvement of the above scheme, in step (2), MCM-41@COOH@C 18 The mesoporous microspheres specifically include the following steps:

[0014] MCM-41@NH2@C 18 The materials were dispersed in DMF and stirred for a period of time to obtain a mixed solution;

[0015] SAA was dissolved in DMF and slowly added dropwise to the above mixed solution. After the addition, stirring was continued for a period of time. After the reaction was completed, the white powder was collected, washed and dried to obtain MCM-41@COOH@C 18 Mesoporous microspheres.

[0016] Preferably, SAA solution and MCM-41@NH2@C 18 The reaction temperature of the solution is room temperature, and the reaction time is 24 to 25 hours.

[0017] In another aspect, the present invention provides a method for detecting aminoglycoside drugs, comprising:

[0018] (1) Prepare the sample to be tested into a test solution;

[0019] (2) Through solid phase extraction of the test solution, MCM-41@COOH@C 18 Mesoporous microspheres are packed in solid phase extraction columns as adsorbents in a reverse phase / cation exchange mixed mode;

[0020] (3) The purified test solution is tested to determine the content of aminoglycoside drugs therein.

[0021] Preferably, in step (1), 5-10 mL of 5-10% trichloroacetic acid and 5-10 mL of 0.05-0.1 mol / L dipotassium hydrogen phosphate are used as the extraction solution of the sample to be tested; and / or the pH value of the final test solution is controlled at 6-7.

[0022] As a further improvement of the above scheme, the step (2) specifically includes the following purification steps:

[0023] MCM-41@COOH@C 18 The mesoporous microspheres are loaded into a solid phase extraction column with frits at both ends; the filler is activated with methanol and water before use;

[0024] The test solution was passed through the SPE cartridge, then rinsed with water and methanol in sequence and dried;

[0025] Elute with eluent and collect the eluate.

[0026] Preferably, in step (2), MCM-41@COOH@C 18 As a filler, the amount used is 25-30 mg; and / or, water / acetonitrile / formic acid in the eluent = 93:4-5:2-3 (v:v:v).

[0027] The present invention uses 3-aminopropyltriethoxysilane, succinate glucoside and octadecyltrimethoxysilane to modify MCM-41 and synthesizes a new reverse phase / cation exchange mixed mode mesoporous silica adsorbent as a solid phase extraction (SPE) filler. The solid phase extraction parameters were optimized, and an amino (Amide) chromatographic column was used for separation to establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) detection method for the residues of 6 aminoglycoside drugs (AGs) in milk. The synthetic material was structurally characterized, and the results of the characterization showed that the synthetic material has a large specific surface area, uniform pore size and functional group modification, and exhibits strong adsorption capacity and selectivity for AGs. Compared with the material that is only carboxylated, the dual-functionalized synthetic material significantly improves the matrix interference of milk samples.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: MCM-41@COOH@C is synthesized by a two-step reaction method. 18 A mesoporous material was used as a SPE adsorbent for the analysis of aminoglycoside residues in milk. The analysis revealed good linearity (0.9927–0.9995) for six aminoglycosides within a concentration range of 5–500 ng / mL, with limits of detection (LODs) ranging from 0.30 to 2.50 ng / mL, average recoveries of 74.3%–107%, and relative standard deviations of 2.6%–15.7%. The material is low-cost, simple to prepare, and highly adaptable as an SPE filler. Future applications include the detection of aminoglycosides in other food or agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 MCM-41@COOH@C 18 Schematic diagram of synthesis and detection flow chart.

[0030] Figure 2 MCM-41@COOH@C prepared in Example 1 of the present invention 18 Electron microscopy images of MCM-41@COOH@C 18 Scanning electron microscopy images, scale 1 μm (interpolated scale is 100 nm); (b) MCM-41@COOH@C 18 Transmission electron microscopy images

[0031] Figure 3 N2 adsorption-desorption isotherms and pore size distribution curves in Example 1 of the present invention, (a) MCM-41; (b) MCM-41@COOH@C 18

[0032] Figure 4MCM-41, MCM-41@COOH@C in Example 1 of the present invention 18 Fourier transform infrared spectrum of

[0033] Figure 5 Figures 1 and 2 show the results of optimized SPE conditions in Example 3 of the present invention; (a) shows the effect of different sample pH values ​​on enrichment efficiency; (b) shows the effect of different AGs concentrations using 30 mg of filler; (c) shows the effect of different FA concentrations in the elution solvent on recovery; (d) shows the effect of different elution solvent volumes on recovery (n=3);

[0034] Figure 6 This is the extracted ion chromatogram of six aminoglycoside drugs in Example 4 of the present invention, wherein the spiked concentration in milk is 100 ng / mL;

[0035] Figure 7 Graph showing the influence of different SPE fillers on matrix effect in Example 6 of the present invention; DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] The embodiment of the present invention discloses a new reverse phase / cation exchange mixed mode adsorbent based on the mesoporous material MCM-41, with six aminoglycoside drugs, including apramycin (APC), gentamicin (GTC), kanamycin (KNM), neomycin (NMC), amikacin (AKH) and tobramycin (TBC) as target analytes. The synthesized bifunctionalized adsorbent contains abundant carboxyl groups and can dissociate at a suitable pH value. It is expected that AGs can be effectively extracted through cation exchange. At the same time, the synthetic material and AGs have a certain hydrophobic interaction, and impurities can be washed away by adjusting the eluent to further achieve purification. The developed adsorbent has good adsorption capacity and good selectivity for the target, and its improvement effect on the matrix effect in LC-MS / MS analysis is further evaluated.

[0038] Example 1 Preparation of bifunctionalized mesoporous silica material

[0039] like Figure 1 As shown, this embodiment provides a method for preparing a bifunctionalized mesoporous silica material, the method comprising the following steps:

[0040] (1)C 18 Preparation of / NH2 functionalized mesoporous MCM-41 materials

[0041] 1 g of MCM-41 was dispersed in 100 mL of anhydrous toluene and mechanically stirred at 60 °C for 20 min, followed by the dropwise addition of APTES and C18 1.2 mL of each TMS was stirred vigorously in an oil bath at 110°C for 15 h, and the resulting white solid product was collected and washed three times with anhydrous ethanol, and dried in a vacuum oven (vacuum degree -0.08 MPa, 65°C, 12 h) to obtain C 18 / NH2 functionalized mesoporous MCM-41 materials (MCM-41@NH2@C 18 ).

[0042] (2)C 18 Preparation of MCM-41 / COOH functionalized mesoporous materials

[0043] Weigh C 18 0.8 g of / NH2 functionalized mesoporous MCM-41 material was dispersed in 100 mL of DMF and stirred at room temperature for 20 min. 11.5 g of SAA was weighed and dissolved in 50 mL of DMF and slowly added dropwise to the stirred reaction system. After the addition was complete, stirring was continued at room temperature for 24 h. After the reaction was completed, the white powder was collected and washed three times with anhydrous ethanol and dried in a vacuum oven (65 ° C, 12 h) to obtain the final reaction product (MCM-41@COOH@C 18 ).

[0044] The MCM-41@COOH@C prepared by the above method 18 Conduct performance testing.

[0045] Characterization of adsorption materials: Since MCM-41 mesoporous material has a long-range ordered pore structure with uniform pore size and extremely high specific surface area, it is very suitable as an adsorption material. 18 Without destroying the original pore structure, Figure 2 In a), worm-like particles can be clearly observed, growing in clusters and arranged densely, with a minimum particle size of about 200 nm. Figure 2 As shown in b, the TEM micrograph demonstrates a highly ordered mesoporous structure, showing the presence of ordered channels of approximately 4 nm in width.

[0046] Through nitrogen adsorption and desorption technology, MCM-41 and MCM-41@COOH@C 18 The adsorption-desorption curves and pore size distributions of the two materials are shown in Figure 2. Figure 3 As shown. According to the IUPAC classification, these two curves are type IV. This isotherm is a typical characteristic curve of mesoporous materials. The BET specific surface area of ​​MCM-41 is 1049.9 m 2 / g, the average pore size is 3.6nm. Figure 3As can be seen in a, when P / P0 < 0.3, the amount of nitrogen adsorption increases with the increase of P / P0. This is because monolayer adsorption occurs in the initial stage. When P / P0 is 0.3-0.4, the amount of nitrogen adsorption increases sharply due to capillary aggregation of nitrogen in the mesoporous material. When P / P0 is 0.4-1, the amount of nitrogen adsorption increases slowly, indicating that the material has a low external specific surface area and a small number of pores. 18 and -COOH groups, the sample exhibits less sharp steps, and MCM-41@COOH@C 18 The pore size shows a bimodal pore distribution. The specific surface area and pore size of the material after double functional group modification are reduced to a certain extent. The specific surface area is 505.8m 2 / g, and the average pore size is 2.6 nm, which is attributed to the modified groups occupying the structural channels.

[0047] In order to verify the effective groups of the synthesized adsorbent, Figure 4 Shows the MCM-41 raw material and the synthesized MCM-41@COOH@C 18 FTIR spectra of MCM-41 and MCM-41@COOH@C 18 At 808cm -1 , 978cm -1 and 1088cm -1 The typical characteristic peaks of silicon dioxide are shown at 3445cm, corresponding to the vibration of Si-O-Si symmetric bond, Si-OH stretching bond, and Si-O-Si asymmetric bond. -1 and 3550cm -1 and 1632cm -1 , 1651cm -1 The adsorption band at is the -OH stretching of the silanol group. 18 and carboxyl group modification, the synthesized MCM-41@COOH@C 18 At 2855cm -1 and 2928cm -1 Two new peaks appear at 18 CH vibration of -CH2- and -CH3 on the chain. 1724cm -1 The new peak is the C=O peak of the carboxyl group. These results confirm the successful surface modification of MCM-41.

[0048] The above results prove that the mesoporous material was successfully synthesized and the prepared MCM-41@COOH@C 18 The material has good water dispersibility and can be evenly dispersed in aqueous solution without any aggregation.

[0049] Comparative Example 1

[0050] This comparative example provides a method for preparing a mesoporous silica material, which comprises the following steps:

[0051] (1) Preparation of NH2-functionalized mesoporous MCM-41 materials

[0052] 1 g of MCM-41 was dispersed in 100 mL of anhydrous toluene and mechanically stirred at 60 °C for 20 min. Then 1.2 mL of APTES was added dropwise and vigorously stirred and refluxed in an oil bath at 110 °C for 15 h. The resulting white solid product was collected and washed three times with anhydrous ethanol. It was then dried in a vacuum oven (vacuum degree -0.08 MPa, 65 °C, 12 h) to obtain NH2-functionalized mesoporous MCM-41 material (MCM-41@NH2).

[0053] (2) Preparation of COOH-functionalized mesoporous MCM-41 materials

[0054] 0.8 g of NH2-functionalized mesoporous MCM-41 was dispersed in 100 mL of DMF and stirred at room temperature for 20 minutes. 11.5 g of SAA was dissolved in 50 mL of DMF and slowly added dropwise to the stirred reaction system. Stirring was continued at room temperature for 24 hours. After the reaction, the white powder was collected, washed three times with anhydrous ethanol, and dried in a vacuum oven (65°C for 12 hours) to obtain the final product (MCM-41@COOH).

[0055] Example 2 Preparation of bifunctionalized mesoporous silica material

[0056] This embodiment provides a method for preparing a bifunctionalized mesoporous silica material. The difference between this embodiment and Example 1 lies in that the oil bath temperature and reflux time in step (1), the reaction time in step (2), and the drying conditions in steps (1) and (2) are changed. Specifically, the method comprises the following steps:

[0057] (1)C 18 Preparation of / NH2 functionalized mesoporous MCM-41 materials

[0058] 1 g of MCM-41 was dispersed in 100 mL of anhydrous toluene and mechanically stirred at 60 °C for 20 min, followed by the dropwise addition of APTES and C 18 1.2 mL of each TMS was stirred vigorously in an oil bath at 120°C for 16 h, and the resulting white solid product was collected and washed three times with anhydrous ethanol, and dried in a vacuum oven (vacuum degree -0.10 MPa, 70°C, 14 h) to obtain C 18 / NH2 functionalized mesoporous MCM-41 materials (MCM-41@NH2@C 18 ).

[0059] (2)C 18 Preparation of MCM-41 / COOH functionalized mesoporous materials

[0060] Weigh C 18 0.8 g of / NH2 functionalized mesoporous MCM-41 material was dispersed in 100 mL of DMF and stirred at room temperature for 20 min. 11.5 g of SAA was weighed and dissolved in 50 mL of DMF and slowly added dropwise to the stirred reaction system. After the addition was complete, stirring was continued at room temperature for 25 h. After the reaction was completed, the white powder was collected and washed three times with anhydrous ethanol. It was then dried in a vacuum oven (70 ° C, 14 h) to obtain the final reaction product (MCM-41@COOH@C 18 ).

[0061] Example 3 Optimization of SPE conditions

[0062] The enrichment mechanism is determined by both reverse phase and ion exchange modes. 18 Contains abundant carboxyl groups, which can dissociate under appropriate pH conditions and can effectively enrich AGs through cation exchange. 18 There is a certain hydrophobic interaction with AGs, and the choice of eluent can be used to further separate the target and impurities. 18 The applicability of mesoporous materials in enriching aminoglycosides was further optimized by optimizing the parameters that may affect the extraction, such as the type of extraction solution, pH value during extraction (3.0–7.0), amount of adsorbent, type of elution solvent, and volume of elution solvent.

[0063] (1) Selection of extract type

[0064] Commonly used extraction solvents for aminoglycosides include phosphate buffer, trichloroacetic acid, and ethylenediaminetetraacetic acid disodium salt. A comparison of the extraction performance of these three solutions revealed that the sample extracts treated with phosphate buffer and ethylenediaminetetraacetic acid disodium salt were turbid, affecting subsequent SPE column cleanup and recovery experiments. After trichloroacetic acid extraction, the centrifuged solution was relatively clear and free of flocculent impurities. This suggests that trichloroacetic acid easily precipitates proteins in milk, and the addition of phosphate buffer helps adjust the pH. Therefore, 10 mL of 5% trichloroacetic acid and 5 mL of 0.1 mol / L potassium dihydrogen phosphate were ultimately selected as the extraction solution.

[0065] (2) Optimization of pH value

[0066] The prepared MCM-41@COOH@C 18It has a large specific surface area, uniform pore size, and good bifunctional group modification. During solid-phase extraction, the pH of the reserve solution affects the ionization of the target. The synthesized bifunctional adsorbent contains abundant carboxyl groups, which can dissociate at appropriate pH values ​​and undergo ion exchange with the amino groups on aminoglycoside drugs. Therefore, the retention of the target on the SPE column was compared at reserve solution pH values ​​of 3, 4, 5, 5.5, 6, 6.5, and 7. The AGs content in the milk before enrichment was 100 ng / mL. The SPE column was filled with the same 30 mg volume. Sample solutions at different pH values ​​were collected after passing through the SPE column and tested using GB / T 22969-2008 (preliminary experimental verification shows that this method has a recovery range of 86.9%-98.5%) to determine the AGs content in the reserve solution before and after enrichment. Figure 5 The results in Figure a show that at solution pH values ​​of 3 and 4, the enrichment of each target drug on the synthesized adsorption medium did not exceed 50%. At pH values ​​of 5 and 5.5, the enrichment of AKH and KNM did not exceed 80%. At pH values ​​of 6-7, the enrichment of aminoglycosides in the solution on the SPE column was higher. This indicates that sample pH is a key factor influencing ion exchange mode. Taking all factors into consideration, a sample solution pH of 6 was selected, resulting in target enrichment of 84.0%-97.6%.

[0067] (3) Selection of SPE column filler dosage

[0068] Because the adsorbent has a limited capacity to carry the target, sufficient adsorbent is essential to achieve the maximum enrichment effect. The SPE column filling volume was selected to be 30 mg, and the recovery rates of aminoglycoside solutions with different concentrations (40, 80, 400 and 2000 ng / mL) were compared ( Figure 5 b) It was found that for each aminoglycoside drug, the corresponding target compound at different concentrations achieved relatively consistent and stable recoveries, and the recovery rate ranged from 70% to 110%, meeting the requirements of the analytical method. This indicates that a filler content of 30 mg will not cause column overloading. Therefore, the SPE filler weight of 30 mg was selected.

[0069] (4) Selection of eluent

[0070] During the complete SPE process, the eluent washes away co-extractables accumulated on the SPE medium, effectively separating the target compound from interfering components. The ideal eluent should not lose the target compound, while also washing away impurities and reducing matrix interference. Two different solvents, 2 mL of water and 2 mL of methanol, were selected as eluents. After collection and LC-MS / MS analysis, no target compound was found in either solution, demonstrating that the eluent does not cause target compound loss.

[0071] (5) Selection of elution solvent

[0072] Aminoglycoside drug molecules have abundant amino groups, which can react with the synthesized MCM-41@COOH@C 18 The carboxyl groups in the solution produce a cation exchange effect, and adding acid to the elution solvent will help weaken the cation exchange interaction. Aminoglycoside drugs are a class of drugs with higher polarity, so when choosing an elution solvent, a mixture of acetonitrile, water and formic acid is selected as the elution solvent. Among them, the proportion of acetonitrile should not be too high. Excessive acetonitrile content will make the hydrophilic interaction between the target drug and the adsorbent difficult to destroy. The concentration of acetonitrile in the entire elution system is set to 5% (v:v). The choice of acid solution can provide a certain acidity and must be suitable for ionization of the mass spectrometry ion source, so the volatile acid formic acid is selected. The pH of the extract is selected to be 6, the amount of SPE filler is 30 mg, water and methanol are used as eluents, and the formic acid concentration is compared from 0% to 5% to select the optimal elution solvent. From Figure 5 As can be seen in Figure c, when the formic acid concentration is 2%, the absorption of all six aminoglycosides exceeds 70%. Therefore, the eluent ratio was selected to be water / acetonitrile / formic acid = 93:5:2 (v:v:v).

[0073] (6) Optimization of eluent volume

[0074] In addition to the type of elution solvent, the volume of the elution solvent also affects the recovery rate of the entire SPE. The effects of different volumes of water / acetonitrile / formic acid = 93:5:2 (v:v:v) 1mL and 2mL on the recovery rate of the target compound were compared, while other parameters remained the same. Figure 5 As shown in Figure d, the two elution volumes did not significantly differ in the recovery of the target compound. The recovery rate exceeded 70% with 1 mL of eluent, indicating that 1 mL of elution volume is sufficient for eluting aminoglycosides. Increasing the elution volume results in dilution of the target compound, requiring subsequent concentration steps and increasing experimental complexity. Therefore, a 1 mL eluent volume was selected.

[0075] Example 4 Optimization of Chromatographic and Mass Spectrometric Conditions

[0076] In chromatographic separation, AGs are highly polar compounds and exhibit relatively poor retention on conventional reversed-phase columns. To achieve better retention on reversed-phase columns, ion-pairing agents such as trifluoroacetic acid are often added to the mobile phase. However, ion-pairing agents can cause matrix inhibition and reduce column life, making the separation of AGs a challenge for analytical personnel. In recent years, amino columns have also been widely used for the analysis of AGs. These columns utilize amide groups bonded to high-purity ethylene-bridged silica particles, offering greater stability than HILIC columns. This experiment utilized a Waters ACQUITY BEH Amide column, which is suitable for separating highly polar compounds. The separation of AGs using pure water, formic acid in water, acetic acid in water, ammonium acetate buffer as the aqueous phase, and acetonitrile as the organic phase was investigated. The peak shape and sensitivity of 2 mmol / L ammonium acetate in water were found to be optimal. Adding 1% formic acid to the mobile phase enhances ionization and improves sensitivity. The addition of 2 mmol / L ammonium acetate to the organic phase creates a stable acidic environment, enhancing elution of the target compound and reducing tailing. Figure 6 The chromatogram of the milk matrix spiked with 100 ng / mL aminoglycosides shows no obvious interference near the target peaks, and the peak shape and response are good, indicating that the LC conditions can separate the six aminoglycosides.

[0077] Aminoglycosides contain aminocyclyl, hydroxyl, and primary or secondary amine groups in their structures, exhibiting strong polarity and weak basicity, making them suitable for electrospray ionization in positive ion scanning mode. A 100 ng / mL mixed standard solution was continuously injected into the mass spectrometer ion source inlet. The optimized mass spectrometry conditions are shown in Table 1.

[0078] Table 1 Mass spectrometry optimization parameters for aminoglycosides in MRM mode

[0079]

[0080] Note: * represents quantitative ion

[0081] Example 5

[0082] This example provides a method for detecting aminoglycosides. The detection method uses the optimal conditions determined in Examples 3 and 4, and uses six aminoglycosides, namely apramycin (APC), gentamicin (GTC), kanamycin (KNM), neomycin (NMC), amikacin (AKH), ​​and tobramycin (TBC), as target analytes. The method specifically includes the following steps:

[0083] (1) Sample preparation

[0084] Accurately pipette 1.0 mL of milk sample, add 10 mL of 5% trichloroacetic acid and 5 mL of 0.1 mol / L potassium dihydrogen phosphate, and mix in a 50 mL plastic centrifuge tube. Vortex for 30 seconds and shake for 5 minutes. Centrifuge at 10,000 rpm for 2 minutes at 4°C. Transfer the supernatant to another 50 mL centrifuge tube, adjust the pH to 6.0 with 10 mol / L sodium hydroxide, and set aside.

[0085] (2) Preparation of standard stock solution: Accurately weigh an appropriate amount of standard sample into a 10 mL polypropylene volumetric flask, dissolve it in water and dilute to volume to prepare a 0.5 mg / mL standard stock solution. Preparation of mixed standard intermediate solution: Pipette 5 mL of standard stock solution into a 50 mL polypropylene volumetric flask, dilute to volume with water / acetonitrile / formic acid = 93:5:2 (v:v:v) to prepare a 50 μg / mL intermediate solution. Preparation of mixed standard working solution: For the solution standard curve, dilute the mixed standard intermediate solution to a series of concentrations of 5.0, 10.0, 20.0, 50.0, 100 and 500.0 ng / mL with water / acetonitrile / formic acid = 93:5:2 (v:v:v). Dilute the matrix-matched standard solution to the same concentration with the matrix blank solution.

[0086] (3) Solid phase extraction step

[0087] The MCM-41@COOH@C prepared in Example 1 18 Mesoporous microspheres (30 mg) were loaded into a solid phase extraction cartridge (3 mL) with frits at both ends. The filler was pre-activated with 3 mL of methanol and 3 mL of water before use. The extract solution was passed through the SPE cartridge, followed by 2 mL of water and 2 mL of methanol, and then drained. The extract was eluted with 1 mL of water / acetonitrile / formic acid = 93:5:2 (v:v:v), and the eluate was collected and filtered using a 0.22 μm filter membrane before UPLC-MS / MS analysis. The SPE process diagram is shown in the figure. Figure 1 .

[0088] Intra-day recovery experiment: Add three different concentrations of AGs standards, 5 ng / mL, 50 ng / mL, and 200 ng / mL, to blank milk samples. AGs standards include tobramycin (TBC), kanamycin (KNM), gentamicin (GTC), neomycin (NMC), apramycin (APC), and amikacin (AKH). Repeat 6 times for each concentration. Perform the experiment according to the established method, and calculate the recovery and precision. Inter-day recovery: Add three different concentrations of AGs standards, 5 ng / mL, 50 ng / mL, and 200 ng / mL, to blank milk samples, and repeat 6 times for each concentration. Test for 3 days according to the established method, and calculate the recovery and precision.

[0089] (4) UPLC-MS / MS detection

[0090] Chromatographic separation was performed on a Waters ACQUITY BEH Amide column (2.1 mm × 100 mm ID, 1.7 μm). Mobile phase A consisted of 2 mmol / L ammonium acetate in 1% formic acid, and mobile phase B consisted of 2 mmol / L ammonium acetate in acetonitrile in 1% formic acid. The linear gradient was as follows: 0.0 → 2.5 min, 20.0% A; 2.5 → 3.5 min, 20.0 → 65.0% A; 3.5 → 5.5 min, 65.0% → 90.0% A; 5.5 → 6.5 min, 90.0 → 90.0% A; 6.5 → 6.6 min, 90.0% → 20.0% A; 6.6 → 9 min, 20.0 → 20.0% A. The mobile phase flow rate was 0.3 mL / min. The column temperature was set at 40°C, and the injection volume was 2 μL.

[0091] The ESI was optimized by continuously infusing the AGs mixed standard solution (100.0 ng / mL) at a flow rate of 10.0 μL / min using a syringe pump. + The MS parameters in the ESI mode are as follows: + The ion source voltage was 5.5 kV, the nebulizer temperature was 550°C, the gas flow rate (GAS1) was 50°C, the gas flow rate (GAS2) was 55°C, and the curtain gas (CUR) was 45.0. Acquisition was performed in MRM scan mode. The protonated molecular ion was selected as the parent ion, and two daughter ions were used for quantitative and qualitative analysis. The parent ion (Q1), daughter ion (Q3), declustering voltage (DP) (V), and collision voltage (CE) (V) for MRM are shown in Table 1 in Example 4.

[0092] Test results:

[0093] Table 2 Linear equations, correlation coefficients, detection limits, and quantification limits of six aminoglycosides

[0094]

[0095] Matrix-matched standard curves were used for quantification. The standard curve concentrations were 5.0, 10.0, 20.0, 50.0, 100, and 500.0 ng / mL. The linear equations are shown in Table 2 , and the correlation coefficients (r) were 0.9927–0.9995.

[0096] Table 3 Recovery and repeatability of aminoglycosides added to milk at three different concentrations

[0097]

[0098] Based on the signal-to-noise ratio S / N = 3, the LODs of aminoglycosides in milk were 0.30-2.50 ng / mL, and based on the signal-to-noise ratio S / N = 10, the LOQs of aminoglycosides in milk were 1.00-10.0 ng / mL, which fully met the requirements for the detection of aminoglycosides in milk. The recovery rate was studied using low (LQC), medium (MQC), and high (HQC) three different concentrations (5 ng / mL, 50 ng / mL, 200 ng / mL) of blank samples (Table 3). Each concentration level was repeated 6 times, the average intra-day recovery rate of the 6 aminoglycosides ranged from 74.3% to 107%, and the RSD value ranged from 3.0% to 13.3%; the inter-day recovery rate ranged from 77.2% to 106%, and the RSD value ranged from 2.6% to 15.7%. These results showed that the synthesized MCM-41@COOH@C 18 The accuracy and precision of the filler applied to the detection of aminoglycosides in milk can meet the requirements of routine monitoring.

[0099] Table 4 Comparison with other reported aminoglycoside LC-MS / MS detection methods

[0100]

[0101] Note: dSPE (dispersive solid phase extraction), MDSPE (magnetic dispersive solid phase extraction), streptomycin (SPM), boronic acid functionalization (APB), dihydrostreptomycin (DHS), paromomycin sulfate (PRM), polyvinyl alcohol (PVA), pectin (SPC).

[0102] In the above table, the detection method used in Comparative Example 2 is the same as that described in “FENG Jianan, SHE Xiaojian, HE Xinying, et al. Synthesis of magnetic graphene / mesoporous silica composites with boronic acid-functionalized pore-walls for selective and efficient residue analysis of aminoglycosides in milk [J]. Food Chemistry, 2018, 239: 612-621. DOI: 10.1016 / j.foodchem.2017.06.052.”.

[0103] The detection method used in Comparative Example 3 is the same as that described in “ZHANG Zheng, CAO Xiaolin, ZHANG Ziping, et al. Synthesis of dummy-template molecularly imprinted polymer adsorbents for solid phase extraction of aminoglycosides antibiotics from environmental water samples [J]. Talanta, 2020, 208: 1-8. DOI: 10.1016 / j.talanta.2019.120385.”

[0104] The detection method used in Comparative Example 4 is the same as that described in “HAMIDI H, ZARRINEH M, ES-HAGHI A, et al. Rapid and sensitive determination of neomycin and kanamycin in measles, mumps, and rubella vaccine via high-performance liquid chromatography-tandem mass spectrometry using modified super-paramagnetic Fe3O4 nanospheres [J]. Journal of Chromatography A, 2020, 1625: 1-12. DOI: 10.1016 / j.chroma.2020.461343.”

[0105] The detection method used in Comparative Example 5 is the same as that described in “LI Duo, LI Tengfei, WANG Lei, et al. A polyvinylalcohol-coated core-shell magnetic nanoparticle for the extraction of aminoglycoside antibiotics residues from honey samples [J]. Journal of Chromatography A, 2018, 1581: 1-7. DOI: 10.1016 / j.chroma.2018.10.048.”

[0106] The detection method used in Comparative Example 6 is the same as that described in “CAO Xiaolin, ZHANG Zheng, LIU Guangyang, et al. Preparation of magnetic dummy template molecularly imprinted polymers for the determination of aminoglycosides antibiotics in milk[J]. Food Analytical Methods, 2021, 14(10): 2111-2120. DOI: 10.1007 / s12161-021-02042-z.”

[0107] The detection method used in Comparative Example 7 is the same as that described in “CONTIN MD, QUINSAAT J, MARTIN NR, et al. Development of carbohydrate functionalized magnetic nanoparticles for aminoglycosides magnetic solid phase extraction[J]. Analytica Chimica Acta, 2019, 1082: 37-48. DOI: 10.1016 / j.aca.2019.07.038.”

[0108] The established method was compared with other methods for aminoglycoside drugs in different matrix samples recorded in Comparative Examples 2-7 (Table 4). Comparative Examples 2-7 are mainly based on the coupling of SPE, magnetic dispersed solid phase extraction (MDSPE) and MS / MS detection system. Regardless of the type of solid phase extraction used, the adsorbents in the comparative examples are based on molecular imprinting materials, reverse phase or ion exchange, and are only for single adsorption mode. In contrast, the reverse phase / cation exchange adsorbent of the present invention matches the structure of aminoglycoside drugs, so it can selectively adsorb the target through electrostatic groups, and effectively separate the target and impurities by reverse phase action. In terms of material preparation, the raw material cost is low and the synthesis method is simple; in terms of target detection, the established method covers many types and can effectively determine 6 kinds of aminoglycoside drugs at the same time, indicating that the synthesized MCM-41@COOH@C 18 It has good adsorption and elution properties for aminoglycosides. In terms of method performance, the recoveries of AKH, TBC, KNM, GTC, NMC, and APC were all between 74.3% and 107%, and the LODs of the method were also better than other reported methods in the detection of similar samples.

[0109] Example 6 Matrix Effect (ME) Evaluation

[0110] In this embodiment, the UPLC-MS / MS detection method described in Example 5 was used to detect an unpurified milk sample, a milk sample after solid phase extraction using the filler prepared in Example 1, and a milk sample after solid phase extraction using the filler prepared in Comparative Example 1 to evaluate the matrix effect.

[0111] In LC-MS / MS, signal enhancement or suppression effects are often observed, resulting in higher or lower recoveries. Milk contains some endogenous substances such as proteins, lipids, amino acids and other complex components. ME can be evaluated by matrix-matched standard curves and solution standard curves. ME is expressed as a percentage (%) and is calculated as the ratio of the peak area (A) of the standard substance in the blank matrix to the peak area (B) of the standard substance in the solvent. When the ME is in the range of 80-120%, the matrix effect is considered to be negligible. ME (%) greater than 120% indicates signal enhancement, while ME (%) less than 80% indicates signal suppression.

[0112] Without any purification method, direct liquid chromatography-tandem mass spectrometry detection of the reserve solution will result in very low ME values ​​(42.0-62.0%) ( Figure 7 ), indicating signal suppression. In contrast, the ME values ​​of some drugs were improved when the samples were purified using MCM-41@COOH material modified with only carboxyl groups. For example, the ME values ​​of AKH and KNM were within the reasonable range of 80%-120%, but the ME value of GTC was as high as 229.9%. 18 The ME values ​​of the target products ranged from 78.0% to 120.0%, indicating that the difunctionalized MCM-41@COOH@C 18 The material has more advantages in matrix purification. 18 The adsorbent was compared with the commercial product WCX (3 mL / 60 mg, Waters). Figure 7 As shown in the figure, since the WCX SPE column is a cation exchange column, its ME value is 72.0-114%, mainly because it can selectively adsorb alkaline target compounds, while washing with water and methanol can eliminate neutral and lipid compounds. Therefore, the mixed-mode SPE of the present invention is comparable to the commercial WCX SPE solid phase extraction column in reducing matrix effects, indicating that the synthesized MCM-41@COOH@C 18 Theoretically, the ability of mixed-mode adsorbents to reduce matrix effects is related to the presence of a large number of hydrophobic and neutral impurities, which can be removed by washing with water and methanol.

[0113] The present invention synthesizes MCM-41@COOH@C by a two-step reaction method. 18 Mesoporous materials are prepared and used as SPE adsorbent fillers for the residual analysis of aminoglycoside drugs in milk. Based on the large surface area and mesoporous channels of the mesoporous material, it is modified with carboxyl and alkyl functional groups. Due to the ion exchange interaction between the carboxyl and amino groups on the material, the sample matrix is ​​purified by adjusting the eluent, showing the great ability of the synthetic material to selectively enrich and purify aminoglycoside drugs in complex matrices. The method of the present invention is simple, economical, sensitive, and highly operational, and has been successfully applied to the extraction of 6 aminoglycoside drugs in milk samples. In the future, the MCM-41@COOH@C 18 Mesoporous materials are used as adsorbents in other food matrices.

Claims

1. A method for detecting aminoglycoside drugs, characterized in that: The detection method comprises: (1) Prepare the sample to be tested into a test solution; (2) The solid phase extraction of the test solution was performed, and MCM-41@COOH@C 18 Mesoporous microspheres were filled in solid phase extraction columns as adsorbents in a reverse phase / cation exchange mixed mode; among them, MCM-41@COOH@C 18 The preparation method is as follows: MCM-41 is dispersed in anhydrous toluene and stirred evenly, and then 3-aminopropyltriethoxysilane and octadecyltrimethoxysilane are added dropwise, and the mixture is vigorously stirred and refluxed in an oil bath for a period of time. The resulting white solid product is collected, washed and dried to obtain MCM-41@NH2@C 18 Materials: MCM-41@NH2@C 18 The materials were dispersed in DMF and stirred for a period of time to obtain a mixed solution; succinoside was dissolved in DMF and slowly added dropwise to the mixed solution. After the addition was completed, stirring was continued for a period of time. After the reaction was completed, white powder was collected, washed and dried to obtain MCM-41@COOH@C 18 Mesoporous microspheres; (3) The purified test solution was tested to determine the content of aminoglycoside drugs therein.

2. The method for detecting aminoglycosides according to claim 1, wherein In the step (1), 5-10 mL of 5-10% trichloroacetic acid and 5-10 mL of 0.05-0.1 mol / L dipotassium hydrogen phosphate are used as the extraction solution of the sample to be tested.

3. The method for detecting aminoglycosides according to claim 1, wherein In the step (1), the pH value of the final test solution is controlled at 6-7.

4. The method for detecting aminoglycosides according to claim 1, wherein The step (2) specifically includes the following purification steps: MCM-41@COOH@C 18 The mesoporous microspheres are loaded into a solid phase extraction column with frits at both ends; the filler is activated with methanol and water before use; The test solution was passed through the SPE cartridge, then rinsed with water and methanol in sequence and dried; Elute with eluent and collect the eluate.

5. The method for detecting aminoglycosides according to claim 1, wherein In step (2), MCM-41@COOH@C 18 As a filler, the dosage is 25~30 mg.

6. The method for detecting aminoglycosides according to claim 1, wherein: In step (2), the eluent contains water / acetonitrile / formic acid = 93:4~5:2~3 (v:v:v).

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