A natural steroidal estrogen extraction and detection method based on multi-mechanism synergistic adsorption

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a natural steroidal estrogen extraction and detection method based on multi-mechanism synergistic adsorption; the method provided by the present application comprises steps of sample extraction, dispersion adsorption, magnetic separation, elution, elution and chromatographic detection; in the sample purification process, the adsorption material is dispersed in the sample solution, so that the adsorption material is fully contacted with the target, the synergistic enrichment of different steroidal estrogens is realized through multiple action mechanisms, and the influence of interfering substances in the complex matrix is reduced in the adsorption process; after the adsorption is completed, the rapid separation is realized through the external magnetic field, and after elution, the sample enters the chromatographic analysis stage; the present application can realize the synchronous extraction and detection of multiple natural steroidal estrogens, and has the advantages of good selectivity, strong anti-matrix interference ability and high detection stability.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a method for the extraction and detection of natural steroidal estrogens based on multi-mechanism synergistic adsorption. Background Technology

[0002] Steroid estrogens are a class of endocrine-active substances with a typical steroidal skeleton structure, mainly including estriol, estradiol, and estrone. These substances are not only widely present in animals but also exist naturally in plants and have been detected in agricultural products such as vegetables. Due to their strong biological activity, steroid estrogens can interfere with the endocrine system even at low concentrations. Therefore, accurate and sensitive detection of natural steroid estrogens in vegetables and other foods is of great significance.

[0003] Currently, the detection of steroidal estrogens in vegetables is mostly carried out using analytical methods such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In actual detection processes, vegetable samples usually contain pigments, polysaccharides, proteins, and other complex organic components. These matrix components are prone to co-extraction with the target analyte and enter the detection system, thus interfering with chromatographic separation and detection results. Therefore, extraction and purification treatment is usually required before detection.

[0004] In existing technologies, solid-phase extraction columns are commonly used for sample purification. However, these methods typically rely on a single adsorption mechanism, making it difficult to achieve simultaneous and efficient enrichment of multiple steroid estrogens with significant structural differences. Furthermore, large molecules or polar interfering substances in complex matrices can compete with the adsorbent, affecting the recovery rate and detection stability of the target analyte. In addition, traditional column-based solid-phase extraction methods generally require multiple steps such as activation, sample loading, rinsing, and elution, making the process cumbersome and limiting its efficiency, thus restricting its application in the detection of complex samples.

[0005] Therefore, there is an urgent need to provide a method for the extraction and detection of natural steroidal estrogens that is applicable to complex matrix samples, has high selectivity and enrichment efficiency, and simplifies the operation process, so as to improve the accuracy and reliability of detection. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for the extraction and detection of natural steroidal estrogens based on multi-mechanism synergistic adsorption, as detailed below.

[0007] S1. Sample extraction: S1-1. The freeze-dried vegetable samples are divided into aboveground parts and roots. Weigh 0.49-0.51 g of aboveground part sample and 0.049-0.051 g of root sample respectively, and place them in a container with a volume of not less than 40 mL. S1-2, After adding the extraction agent and extracting, the supernatant is obtained; S1-3. Take the supernatant and filter it through a 0.22 μm organic phase filter membrane to collect the extract; repeat the extraction once and combine the two extracts. S1-4. The combined extract was concentrated to 1.0-1.2 mL by nitrogen blowing in a 40℃ water bath, 1 mL of acetonitrile was added to redissolve the extract, and the volume was adjusted to 40 mL with ultrapure water to obtain the sample solution to be purified. S2. Pretreatment of adsorbent material: S2-1. Weigh 20-60 mg of magnetic composite adsorption material; S2-2, Add 2 mL of methanol and 2 mL of ultrapure water sequentially to wet and disperse, and vortex for 30 s each time; S2-3. Apply an external magnetic field to perform magnetic separation, discard the supernatant, and obtain the pre-activated magnetic composite adsorbent material; S3, Dispersed Adsorption: Add the pre-activated magnetic composite adsorbent material in S2-3 to the sample solution to be purified in S1-4; vortex for 30 s, then shake at 200-250 rpm for 8-10 min to adsorb the natural steroidal estrogens in the vegetable sample onto the surface of the magnetic composite adsorbent material. S4, Magnetic Separation: Apply an external magnetic field to the system after S3 treatment to perform magnetic separation for 1-2 min, and discard the supernatant; S5. Eluting: Add 5 mL of ultrapure water to the magnetic composite adsorbent material treated in S4, vortex for 15-20 s and then magnetically separate, discarding the eluent; then add 2 mL of 5% (v / v) methanol aqueous solution, vortex for 10-15 s and then magnetically separate again, discarding the eluent. S6, Elution: The magnetic composite adsorbent material treated by S5 is eluted to obtain the eluent; S7. Concentration, Reconstitution, and Detection: S7-1. The eluent obtained in S6 is concentrated by nitrogen blowing under a 40℃ water bath until the residual volume is 0.1-0.3 mL. 2 mL of methanol is added to redissolve and vortex to mix. After filtration through a 0.22 μm organic phase filter membrane, it is transferred to a sample vial. S7-2. High-performance liquid chromatography-fluorescence detector was used for analysis to complete the extraction and detection of natural steroidal estrogens.

[0008] Description: This invention presents a method for the extraction and detection of natural steroidal estrogens based on multi-mechanism synergistic adsorption. This method combines sample extraction, dispersion adsorption using magnetic composite adsorbents, magnetic separation, elution, and chromatographic detection to construct a dispersed magnetic solid-phase extraction and detection system distinct from traditional column-based solid-phase extraction. This method utilizes the highly efficient dispersion characteristics of magnetic composite adsorbents in solution to improve the contact efficiency between the adsorbent and the target analyte. Furthermore, magnetic separation replaces traditional centrifugation or column separation, significantly simplifying the operation process and shortening processing time. Simultaneously, the combination of multi-mechanism synergistic adsorption enables the simultaneous and efficient enrichment of steroidal estrogens with different structures and effectively reduces interference from complex matrices, thereby improving the selectivity, sensitivity, and stability of the detection method.

[0009] Further, the extraction and processing steps in S1-2 are as follows: add 10 mL of extraction agent, then vortex for 30-40 s, ultrasonically extract for 20-40 min, shake for 20-40 min, and centrifuge at 8000-12000 r / min for 8-15 min to obtain supernatant; The extractant is prepared by mixing acetonitrile and water in a volume ratio of 1:1.

[0010] Description: This invention employs a mixed extraction system of acetonitrile and water, combined with a synergistic extraction method of vortexing, ultrasound, and oscillation, to fully release natural steroidal estrogens of different polarities in the sample, thereby improving extraction efficiency. Simultaneously, by performing multiple extractions and combining the extracts, the recovery rate of the target analyte is further improved, thus providing stable and sufficient sample pretreatment conditions for subsequent adsorption and enrichment.

[0011] Furthermore, the magnetic composite adsorbent in S3 adsorbs estrone and / or estradiol from vegetable samples through hydrophobic interactions, adsorbs estriol through polar interactions, and reduces the interference of large-molecule polar interfering substances in the vegetable sample matrix on the adsorption process through molecular size exclusion.

[0012] Description: The magnetic composite adsorption material of this invention achieves selective adsorption of steroidal estrogens with different structures through a synergistic mechanism of hydrophobic and polar interactions. The hydrophobic interaction is conducive to the enrichment of estrone and / or estradiol, while the polar interaction is conducive to the enrichment of estriol. At the same time, through the molecular size exclusion effect, it effectively prevents large polar interfering molecules from entering the adsorption interface, reduces matrix competitive adsorption, and thus significantly improves the selective enrichment ability and detection accuracy of the target analyte.

[0013] Further, the elution process in S6 is as follows: 10 mL of eluent is added to the magnetic composite adsorbent material after S5 treatment, and then the mixture is vortexed for 30-40 s, sonicated for 3-8 min, and oscillated for 3-8 min before magnetic separation is performed. The eluent is collected. Then, 5 mL of eluent is added to repeat the elution once and the two eluents are combined. The eluent was prepared by mixing ethyl acetate, methanol and glacial acetic acid in a volume ratio of 50:50:1.

[0014] Note: This invention employs a composite elution system composed of ethyl acetate, methanol, and glacial acetic acid, which effectively disrupts the interaction between the target analyte and the magnetic composite adsorption material, achieving thorough elution of natural steroidal estrogens. By using a two-stage elution process and combining the eluents, the recovery rate of the target analyte and the repeatability of the method are further improved, thereby ensuring the accuracy and stability of the detection results.

[0015] Furthermore, the settings for the high-performance liquid chromatography-fluorescence detector in S7-2 are as follows: The chromatographic column is a C18 column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; The mobile phase was prepared by mixing acetonitrile and water at a volume ratio of 40:60, the flow rate was 0.8 mL / min, the column temperature was 40 ℃, and the injection volume was 20 μL. The fluorescence detector has an excitation wavelength of 280 nm, an emission wavelength of 310 nm, and a single detection time of 20 min.

[0016] Description: This invention optimizes high-performance liquid chromatography-fluorescence detection conditions to achieve good separation and high-sensitivity response of different steroid estrogens. The matching of chromatographic conditions and fluorescence detection parameters can effectively improve the detection signal intensity and reduce background interference, thereby achieving accurate detection of natural steroid estrogens in complex samples.

[0017] As another aspect of the present invention, a method for preparing the above-mentioned magnetic composite adsorbent material is also provided, denoted as the multiplier coefficient of the amount of each component added in the following method, and is a positive number that can satisfy the operation system of this method, as follows.

[0018] SA1. Preparation and modification of the magnetic core: SA1-1, Preparation of the magnetic core: First, under a nitrogen atmosphere, 100 mL of deionized water, 5.2–5.6 g of FeCl3·6H2O, and 2.0–2.2 g of FeCl2·4H2O were added sequentially to the reactor and stirred until homogeneous. Then, the system was heated to 80 °C and 10–11 mL of 25 wt% ammonia water was added, and the mixture was stirred at 800–1000 rpm for 30–40 min. Finally, the product was collected and washed with deionized water and anhydrous ethanol sequentially to obtain Fe3O4 nanoparticles (particle size 500–800 μm), i.e., magnetic cores. SA1-2, Preparation of silanol-modified sites: 0.5–1.0 g of Fe3O4 nanoparticles from SA1-1 were added to a mixed solution of 160 mL ethanol and 40 mL deionized water and ultrasonically dispersed for 10–15 min. Then, 6–8 mL of 25 wt% ammonia solution was added and stirred until homogeneous. Next, 2–3 mL of TEOS (tetraethyl orthosilicate) was added dropwise over 10–15 min, and stirring was maintained for 6–7 h. The magnetically separated product was then washed with anhydrous ethanol and deionized water in sequence, and finally dried to obtain Fe3O4@SiO2 particles. SA2, Preparation of emulsion: Maintaining a nitrogen-protected environment, 0.5–0.7 g of Fe3O4@SiO2 particles from SA1 were added to a mixed solution of 40 mL ethanol and 40 mL deionized water and ultrasonically dispersed for 3–5 min. Then, 20 mL of anhydrous toluene was added and stirred until homogeneous. Next, the mixture was emulsified at 8000 rpm for 1–2 min using a homogenizer to obtain an emulsion in which Fe3O4@SiO2 particles were distributed at the oil / water phase interface. The side of the Fe3O4@SiO2 particles located in the oil phase was defined as the first reaction zone, and the side located in the water phase was defined as the second reaction zone. SA3, Preparation of hydrophobic adsorption layer: First, maintain the emulsion state in SA2 and keep the stirring speed at 200~300 rpm; then add 0.6~0.8 mL of ODTMS (octadecyltrimethoxysilane) to the oil phase side, and then react at 55~60℃ for 2~4 h. After the reaction is completed, cool the system to room temperature to obtain the first functional zone. SA4. Preparation of polar adsorption layer: First, maintain the emulsion state in SA3, then add 1.0~1.5 mL NVP (N-vinylpyrrolidone), 20~30 mg APS (ammonium persulfate), and 20~30 μL TEMED (N,N,N',N'-tetramethylethylenediamine) sequentially to the aqueous phase side, and then react at 20~25℃ for 30~40 min to form a polar adsorption layer on the second reaction zone; SA5. Preparation of a semi-permeable shielding shell: Keep the emulsion in SA4, then add 0.2~0.3 mL of PEGDA (polyethylene glycol diacrylate), and react at 30~35℃ for 1~1.5 h to form a semi-permeable shielding shell on the polar adsorption layer, thus obtaining the complete second functional region; SA6, Demulsification - Washing: Add 100 mL of anhydrous ethanol to the SA5 system, stir at 400-500 rpm for 1-3 min, magnetically absorb the product, and wash it clean with anhydrous ethanol, deionized water and anhydrous ethanol in sequence, and dry it at 50-60℃ for 10-12 h to obtain the composite adsorbent material.

[0019] Description: In terms of preparation, this invention introduces silanol-modified sites on the surface of Fe3O4 nanoparticles to form Fe3O4@SiO2 particles, enabling subsequent functional layers to grow stably on the particle surface and improving the structural stability of the material. Subsequently, by constructing an oil-water interface system where oil and water phases coexist, the Fe3O4@SiO2 particles are positioned at the interface between the oil and water phases, thus forming a spatially distinguishable first and second reaction zone. In the first reaction zone, ODTMS is introduced to form a hydrophobic adsorption layer, thereby constructing a hydrophobic interface with a C18 alkyl chain structure on one side of the particle. In the second reaction zone, a polar adsorption layer is formed by NVP under the action of an APS and TEMED initiation system, resulting in a polymer structure containing polar functional groups on the other side of the particle. Subsequently, a semi-permeable shielding shell with a cross-linked polymer network structure is further formed outside the polar adsorption layer by PEGDA, thereby constructing a protective layer with size exclusion function. Through the above interface orientation construction process, the first and second functional zones form a spatially asymmetric distribution structure on the same particle surface, thereby achieving the synergistic effect of different adsorption mechanisms. As another aspect of the present invention, the structure of the above-mentioned magnetic composite adsorption material is also provided, which includes a magnetic core and a functional layer disposed on the outer surface of the magnetic core from the inside out. The magnetic core consists of magnetic nanoparticles composed of Fe3O4 nanoparticles. The functional layer consists of a first functional area and a second functional area, which are spatially asymmetrically distributed around the magnetic core. The first functional region is a hydrophobic adsorption layer, which includes a C18 alkyl chain and adsorbs estrone and / or estradiol from steroid estrogens through hydrophobic interactions. The second functional area consists of a polar adsorption layer and a semi-permeable shielding shell from the inside out. The polar adsorption layer includes one or more of hydroxyl, amide, carboxyl, or amino groups, which adsorb estriol from steroid hormones through hydrogen bonding. The semi-permeable shielding shell has a cross-linked polymer network structure, which forms a molecular-scale exclusion structure to block large polar interfering molecules from entering the polar adsorption layer while allowing estriol to pass through.

[0020] Description: The magnetic composite adsorbent material designed in this invention constructs a bifunctional adsorption system with a spatially asymmetric structure, enabling differentiated recognition and synergistic enrichment of steroid estrogens with different structural characteristics on the same material. The magnetic core consists of magnetic nanoparticles, which can achieve rapid magnetic separation under an external magnetic field. This makes the dispersion and subsequent separation and recovery of the adsorbent material in the sample solution more efficient and stable, while avoiding the clogging problems that may occur with traditional solid-phase extraction columns in complex sample systems. A functional layer is disposed on the outer surface of the magnetic core, and through the spatially asymmetric distribution of the first and second functional regions, the material simultaneously possesses hydrophobic and polar adsorption properties on the same particle surface, thereby achieving selective recognition and adsorption of multiple steroid estrogens molecules. The first functional region is a hydrophobic adsorption layer containing a C18 alkyl chain structure, which can interact with... Stable hydrophobic interactions are formed between estrone and / or estradiol molecules in steroidal estrogens, thereby improving the enrichment efficiency of the aforementioned target substances. The second functional region consists of a polar adsorption layer and a semi-permeable shielding shell. The polar adsorption layer contains one or more polar functional groups, such as hydroxyl, amide, carboxyl, or amine groups. These functional groups can form hydrogen bonds with estriol molecules in steroidal hormones, enabling effective capture of estriol in complex sample systems. Simultaneously, a semi-permeable shielding shell is further constructed outside the polar adsorption layer. This semi-permeable shielding shell is a cross-linked polymer network structure. By adjusting the cross-linking density, a stable molecular size exclusion structure is formed, making it difficult for proteins, polysaccharides, and other large molecular polar interfering substances in the sample system to enter the polar adsorption layer region. However, smaller estriol molecules can pass through smoothly and be adsorbed by the polar adsorption layer, thereby effectively reducing matrix interference and improving detection selectivity.

[0021] Compared with existing methods for extracting and detecting natural steroidal estrogens, the advantages of this invention are: (1) This invention transforms the traditional column solid phase extraction method into a dispersed adsorption process in a solution system by constructing an integrated processing flow of “sample extraction-dispersion adsorption-magnetic separation-rinsing-elution-detection”, so that the adsorption medium can be fully dispersed in the sample solution and come into efficient contact with the target analyte, thereby significantly improving the enrichment efficiency of natural steroid estrogens; at the same time, by introducing magnetic separation to replace the traditional column separation or centrifugation separation steps, the operation process is simplified, the processing time is shortened, and the operation convenience and repeatability of the method are improved.

[0022] (2) The present invention introduces a multi-mechanism synergistic effect in the adsorption process, enabling steroid estrogens with different structures to be simultaneously enriched in the same processing step, and effectively reducing the influence of interfering substances in complex matrices during the enrichment process, thereby improving the selectivity and stability of the detection method.

[0023] (3) By optimizing the rinsing and elution steps, the present invention achieves effective removal of non-target impurities and full desorption of target substances, thereby further improving the recovery rate and detection accuracy of target substances; combined with optimized chromatographic detection conditions, different steroid estrogens can achieve good separation effect and detection response, thus meeting the detection needs of trace natural steroid estrogens in complex vegetable samples.

[0024] (4) The method of the present invention has the advantages of high extraction efficiency, good selectivity, simple operation, strong repeatability and applicability to complex matrix samples, and has good application prospects in the field of detection of natural steroidal estrogens. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the present invention.

[0026] Figure 2 This is a graph showing the average recovery rate of the detection method extracted in Experimental Example A1; Figure 3 This is the enrichment factor test chart of the detection method extracted in Experiment Example A2; Figure 4 This is the matrix interference test diagram of the detection method in Experiment Example A3; Figure 5 This is a graph showing the effect of different structures on the extraction and detection of natural steroidal estrogens in Experimental Example B1; Figure 6 This is a graph showing the effect of different purification methods on the extraction and detection of natural steroidal estrogens in Experiment B2. Detailed Implementation

[0027] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0028] Example 1: This example aims to provide a method for the extraction and detection of natural steroidal estrogens under certain parameters.

[0029] Weigh 0.50 g of freeze-dried aerial part of vegetables and 0.050 g of root sample, and place them in a 40 mL centrifuge tube. Add 10 mL of an extraction solvent with an acetonitrile-to-water ratio of 1:1, and perform vortexing for 35 s, ultrasonic extraction at 2500 rpm, 40 kHz, and 300 W for 30 min, shaking at 220 rpm for 30 min, and centrifugation at 10000 r / min for 10 min to obtain the supernatant. Filter the supernatant through a 0.22 μm organic phase filter membrane, repeat the extraction once, and combine the two extracts. Concentrate the combined extract to 1.1 mL under nitrogen blowing in a 40 ℃ water bath, add 1 mL of acetonitrile to redissolve, and then make up to 40 mL with ultrapure water to obtain the sample solution to be purified. Weigh 40 mg of magnetic composite adsorbent material, add 2 mL of methanol and 2 mL of ultrapure water to wet and disperse, and vortex for 30 s each; apply an external magnetic field of 0.4 T for magnetic separation for 45 s, discard the supernatant to obtain the pre-activated magnetic composite adsorbent material; The pre-activated magnetic composite adsorption material was added to the above sample solution to be purified. After vortexing for 30 s, it was shaken at 220 rpm for 8 min to adsorb natural steroidal estrogens. An external magnetic field of 0.4 T was applied for magnetic separation for 1.5 min, and the supernatant was discarded. Add 5 mL of ultrapure water, vortex for 15 s, and then magnetically separate the mixture, discarding the eluent; then add 2 mL of 5% (v / v) methanol aqueous solution, vortex for 10 s, and then magnetically separate the mixture again, discarding the eluent. Add 10 mL of eluent in the ratio of ethyl acetate:methanol:glacial acetic acid = 50:50:1 to the magnetic composite adsorbent material, vortex for 35 s, sonicate for 5 min at 2500 rpm, 40 kHz, and 250 W, then oscillate at 230 rpm for 5 min and perform magnetic separation. Collect the eluent; add another 5 mL of eluent and repeat the elution once, then combine the two eluents. The eluent was concentrated to 0.2 mL by nitrogen blowing under a 40 ℃ water bath, redissolved in 2 mL of methanol, filtered through a 0.22 μm filter membrane, and then injected. It was analyzed by high performance liquid chromatography-fluorescence detector.

[0030] Examples 2-4: Variable amount of adsorbent material.

[0031] Example 2: The amount of adsorbent material used was 20 mg, and the rest was the same as in Example 1.

[0032] Example 3: The amount of adsorbent material used was 40 mg, and the rest was the same as in Example 1.

[0033] Example 4: The amount of adsorbent material used was 60 mg, and the rest was the same as in Example 1.

[0034] Examples 5-7: Adsorption time variable.

[0035] Example 5: The oscillation adsorption time was 5 min, and the rest was the same as in Example 1.

[0036] Example 6: The oscillation adsorption time was 8 min, and the rest was the same as in Example 1.

[0037] Example 7: The oscillation adsorption time was 15 min, and the rest was the same as in Example 1.

[0038] Examples 8-10: Rinsing conditional variables.

[0039] Example 8: Only ultrapure water was used for rinsing, and the rest was the same as in Example 1.

[0040] Example 9: Washing was performed with water and 5% methanol, and the rest was the same as in Example 1.

[0041] Example 10: Washing was performed with water and 10% methanol, and the rest was the same as in Example 1.

[0042] Examples 11-13: Elution of conditional variables.

[0043] Example 11: Single elution with 10 mL, the rest is the same as in Example 1.

[0044] Example 12: Two washes (10 mL + 5 mL), the rest is the same as in Example 1.

[0045] Example 13: Single elution of 15 mL, the rest is the same as in Example 1.

[0046] Example 14: Comparison with traditional methods.

[0047] A C18 solid-phase extraction column was used instead of the magnetic composite adsorption material, and the remaining steps were the same as in Example 1.

[0048] Example 15: Non-dispersive adsorption control.

[0049] The adsorbent material is allowed to stand and adsorb without being shaken or dispersed; otherwise, it is the same as in Example 1.

[0050] Example 16: All parameters are taken as the lower limit of the claims, and the rest are the same as in Example 1, and are not included in the experimental comparison.

[0051] Weigh 0.49 g of freeze-dried aerial part of vegetables and 0.049 g of root sample, and place them in a 40 mL centrifuge tube. Add 10 mL of acetonitrile to water extractant at a volume ratio of 1:1, and perform vortexing for 30 s, ultrasonic extraction at 2000 rpm, 38 kHz, and 280 W for 30 min, shaking at 200 rpm for 30 min, and centrifugation at 8000 r / min for 8 min to obtain the supernatant. Filter the supernatant through a 0.22 μm organic phase filter membrane, repeat the extraction once, and combine the two extracts. Concentrate the combined extract to 1.0 mL under nitrogen blowing in a 40 ℃ water bath, add 1 mL of acetonitrile to redissolve, and make up to 40 mL with ultrapure water to obtain the sample solution to be purified.

[0052] Weigh 20 mg of magnetic composite adsorbent material, add 2 mL of methanol and 2 mL of ultrapure water to wet and disperse, and vortex for 30 s each; apply an external magnetic field of 0.3 T for magnetic separation for 40 s, discard the supernatant to obtain the pre-activated magnetic composite adsorbent material.

[0053] The pre-activated magnetic composite adsorbent material was added to the above sample solution to be purified. After vortexing for 30 s, it was shaken at 200 rpm for 5 min to adsorb the natural steroidal estrogen. An external magnetic field of 0.3 T was applied for magnetic separation for 1 min, and the supernatant was discarded.

[0054] Add 5 mL of ultrapure water, vortex for 15 s, and then magnetically separate the mixture, discarding the eluent. Add 2 mL of 5% methanol aqueous solution, vortex for 10 s, and then magnetically separate the mixture again, discarding the eluent.

[0055] Add 10 mL of eluent in the ratio of ethyl acetate:methanol:glacial acetic acid = 50:50:1 to the magnetic composite adsorption material, vortex for 30 s, sonicate for 5 min at 2000 rpm, 35 kHz, and 200 W, then oscillate at 200 rpm for 3 min and magnetically separate, collect the eluent; add another 5 mL of eluent to repeat the elution once and combine the two eluents.

[0056] The eluent was concentrated to 0.1 mL by nitrogen blowing in a 40°C water bath, then reconstituted with 2 mL of methanol, filtered through a 0.22 μm filter membrane, and then injected for analysis.

[0057] Example 17: All parameters are taken at the upper limit of the claims, and the rest are the same as in Example 1, and are not included in the experimental comparison.

[0058] Weigh 0.51 g of freeze-dried aerial part of vegetables and 0.051 g of root sample, and place them in a 40 mL centrifuge tube. Add 10 mL of an extraction solvent with an acetonitrile-to-water ratio of 1:1, and perform vortexing for 40 s, ultrasonic extraction at 3000 rpm, 45 kHz, and 320 W for 30 min, shaking at 220 rpm for 30 min, and centrifugation at 12000 r / min for 15 min to obtain the supernatant. Filter the supernatant through a 0.22 μm organic phase filter membrane, repeat the extraction once, and combine the two extracts. Concentrate the combined extract to 1.2 mL under nitrogen blowing in a 40 ℃ water bath, add 1 mL of acetonitrile to redissolve, and make up to 40 mL with ultrapure water to obtain the sample solution to be purified.

[0059] Weigh 60 mg of magnetic composite adsorbent material, add 2 mL of methanol and 2 mL of ultrapure water to wet and disperse, and vortex for 30 s each; apply an external magnetic field of 0.5 T for magnetic separation for 60 s, discard the supernatant to obtain the pre-activated magnetic composite adsorbent material.

[0060] The pre-activated magnetic composite adsorbent material was added to the above sample solution to be purified. After vortexing for 30 s, it was shaken at 250 rpm for 15 min to adsorb natural steroidal estrogens. An external magnetic field of 0.5 T was applied for magnetic separation for 2 min, and the supernatant was discarded.

[0061] Add 5 mL of ultrapure water, vortex for 20 s, and then magnetically separate the mixture, discarding the eluent. Add 2 mL of 5% methanol aqueous solution, vortex for 15 s, and then magnetically separate the mixture again, discarding the eluent.

[0062] Add 10 mL of eluent in the ratio of ethyl acetate:methanol:glacial acetic acid = 50:50:1 to the magnetic composite adsorption material, vortex for 40 s, sonicate for 5 min at 3000 rpm, 45 kHz, and 300 W, then oscillate at 250 rpm for 8 min and magnetically separate, collect the eluent; add another 5 mL of eluent to repeat the elution once and combine the two eluents.

[0063] The eluent was concentrated to 0.3 mL by nitrogen blowing in a 40°C water bath, then reconstituted with 2 mL of methanol, filtered through a 0.22 μm filter membrane, and then injected for analysis.

[0064] Experimental Example 1: Recovery Rate Test.

[0065] Steroidal estrogen standards were added to vegetable samples at concentrations of 10 μg / kg, 50 μg / kg, and 100 μg / kg, respectively. The steroidal estrogens included a mixed standard solution of estrone, estradiol, and estriol. After spiking, the samples were allowed to stand for 12 h to allow for full equilibration between the target analyte and the matrix.

[0066] Weigh the spiked samples and process them according to the methods in Examples 1-7 and 11-13, respectively. Three parallel samples were set up for each sample. After processing, the samples were detected by high performance liquid chromatography-fluorescence detector. The recovery rate was calculated based on the measured concentration and the spiked concentration. The result was the average of three parallel determinations.

[0067] Combining the above table with Figure 2 As can be seen, the method of the present invention exhibits high recovery rates at all concentration levels, indicating that the method can effectively achieve the extraction and enrichment of natural steroidal estrogens. Further analysis of the data trends reveals that: when the amount of adsorbent material increases from 20 mg to 40 mg, the recovery rate significantly improves, while the improvement is limited when further increased to 60 mg, indicating the existence of an optimal adsorbent dosage; simultaneously, the recovery rate significantly improves when the adsorption time increases from 5 min to 8 min, while the improvement is not significant when further extended to 15 min, indicating that the adsorption process reaches equilibrium in a relatively short time. In addition, the use of two elutions (Example 12) has a higher recovery rate than single elutions (Examples 11, 13), indicating that multiple elutions are beneficial for the complete desorption of the target analyte.

[0068] Experimental Example 2: Enrichment Factor Test.

[0069] Vegetable samples were taken and steroidal estrogen standard solution was added to make the initial concentration 50 μg / kg; after spiking, the samples were allowed to stand for 12 hours. The samples were then processed according to the methods of Examples 1, 11-13 and Example 14, with 3 parallel samples set up for each group.

[0070] Record the target analyte concentration C0 in the sample before extraction and the target analyte concentration C1 in the final detection solution, and calculate the enrichment factor according to the following formula: Enrichment factor = C1 / C0

[0071] See the table above and Figure 3The enrichment factor of the method of this invention is significantly higher than that of the traditional C18 solid-phase extraction method. Further analysis shows that the two-stage elution method (Example 12) maintains a high enrichment factor while ensuring the recovery rate, while the single-stage elution method (Example 11), although simple to operate, results in a decrease in enrichment capacity. In addition, compared with the traditional SPE method, this invention significantly improves the contact efficiency between the target analyte and the adsorption medium through dispersion adsorption, thereby achieving a higher enrichment effect and demonstrating the advantages of the method in the pretreatment stage.

[0072] Experimental Example 3: Evaluation of matrix interference.

[0073] Vegetable samples were taken and steroidal estrogen standard solution was added to make the spiking concentration 50 μg / kg; after spiking, the samples were allowed to stand for 12 hours. The samples were then treated according to the methods in Examples 8-10, 14, and 15, with 3 parallel samples in each group.

[0074] The matrix effect is calculated as follows: Let A1 be the detection response value of the solution obtained after sample processing, and A2 be the response value of the standard solution of the same concentration in pure solvent. The matrix effect is then calculated using the following formula: Matrix effect (%) = A1 / A2 × 100%

[0075] Combining the above table with Figure 4 It can be seen that the method of the present invention has a significant advantage in suppressing matrix interference. Further analysis of the data trends shows that matrix interference is relatively large when only water washing is used (Example 8), while adding a low concentration of organic solvent for rinsing (Example 9) can significantly reduce the matrix effect; when the concentration of organic solvent is further increased (Example 10), the matrix interference slightly rebounds, indicating that there are optimal rinsing conditions. In addition, compared with the conventional SPE method (Example 14), the method of the present invention can more effectively reduce matrix interference, while the control group without the dispersion adsorption method (Example 15) shows the highest matrix effect, indicating that the dispersion adsorption process plays an important role in reducing non-specific adsorption.

[0076] Example 18: This example aims to illustrate the provided magnetic composite adsorption material and its preparation method.

[0077] The provided magnetic composite adsorption material comprises, from the inside out, a magnetic core and a functional layer disposed on the outer surface of the magnetic core; the magnetic core is Fe3O4 nanoparticles; the functional layer consists of a first functional region and a second functional region, which are spatially asymmetrically distributed with the magnetic core as the center; the first functional region is a hydrophobic adsorption layer, and the second functional region comprises, from the inside out, a polar adsorption layer and a semi-permeable shielding shell.

[0078] The specific preparation steps of the above-mentioned magnetic composite adsorption material are as follows: SA1. Preparation and modification of magnetic cores: Under nitrogen atmosphere, 100 mL of deionized water was added to the reactor, followed by 5.4 g of FeCl3·6H2O and 2.1 g of FeCl2·4H2O. After stirring evenly, the system was heated to 80℃. 10.5 mL of 25 wt% ammonia was added, and the mixture was stirred at 900 rpm for 35 min. The product was collected by magnetic separation and washed with deionized water and anhydrous ethanol to obtain Fe3O4 nanoparticles. 0.8 g of the obtained Fe3O4 nanoparticles were added to a mixed solution of 160 mL of ethanol and 40 mL of deionized water and ultrasonically dispersed for 12 min. 7 mL of 25 wt% ammonia was added, and after stirring evenly, 2.5 mL of TEOS was added dropwise over 12 min, and the reaction was continued for 6.5 h. The mixture was then magnetically separated and washed with anhydrous ethanol and deionized water, and dried to obtain Fe3O4@SiO2 particles.

[0079] SA2. Preparation of the oil-water interface: Under nitrogen protection, 0.60 g of Fe3O4@SiO2 particles were added to a mixed solution of 40 mL ethanol and 40 mL deionized water and ultrasonically dispersed for 4 min; 20 mL of anhydrous toluene was added and stirred evenly; emulsification was carried out at 8000 rpm for 2 min using a homogenizer to obtain an emulsion in which Fe3O4@SiO2 particles were located at the oil / water phase interface; the side of the Fe3O4@SiO2 particles located in the oil phase was defined as the first reaction zone, and the side of the Fe3O4@SiO2 particles located in the water phase was defined as the second reaction zone.

[0080] SA3. Prepare a hydrophobic adsorption layer on the first reaction zone: Maintain the emulsion state in S2 and keep the stirring rate at 250 rpm; add 0.70 mL of ODTMS to the oil phase side and react at 58℃ for 3 h. After the reaction is completed, cool to room temperature to obtain the first functional zone.

[0081] SA4. Preparation of a polar adsorption layer on the second reaction zone: While maintaining the emulsion state in S3, add 1.20 mL NVP, 25 mg APS and 25 μL TEMED sequentially to the aqueous phase side, and react at 23 °C for 35 min to form a polar adsorption layer on the second reaction zone.

[0082] SA5. Preparation of a semi-permeable shielding shell on the second reaction zone: While maintaining the emulsion state in S4, add 0.25 mL of PEGDA and react at 32℃ for 1.2 h to form a semi-permeable shielding shell on the polar adsorption layer, thus obtaining a complete second functional zone.

[0083] SA6, Demulsification-washing: Add 100 mL of anhydrous ethanol to the system in S5, stir at 450 rpm for 2 min, magnetically absorb and collect the product, and wash with anhydrous ethanol, deionized water and anhydrous ethanol in sequence, and dry at 55℃ for 11 h to obtain the magnetic composite adsorbent material.

[0084] When the magnetic composite adsorption material is applied to the extraction and detection of natural steroidal estrogens, it is processed according to the detection method described in Example A1.

[0085] Example 19: Compared with Example 18, only the amount of ODTMS added in S3 was changed to 0.60 mL, and the other steps and conditions were the same as in Example 18.

[0086] Example 20: Compared with Example 18, only the amount of NVP added in S4 was changed to 1.00 mL, and the remaining steps and conditions were the same as in Example B1.

[0087] Example 21: Compared with Example 18, step S5 is omitted, i.e., a semi-transparent shielding shell is not formed. The remaining steps and conditions are the same as in Example B1.

[0088] Example 22: In this example, a commercially available C18 solid-phase extraction column was used as a control; sample extraction and detection were performed according to the detection method described in Example 1, wherein the purification step was completed using a commercially available C18 solid-phase extraction column, and the other conditions were the same as in Example 1.

[0089] Example 23: Compared with Example 18, the external magnetic field was not used for separation during the extraction and detection process. Instead, the supernatant was poured out after centrifugation at 10000 r / min for 10 min for solid-liquid separation. The remaining steps and conditions were the same as in Example 18.

[0090] Example 24: In steps S2-S5, the lower limits of each parameter were used, namely 0.50 g of Fe3O4@SiO2 particles, 0.60 mL of ODTMS, 1.00 mL of NVP, 20 mg of APS, 20 μL of TEMED, and 0.20 mL of PEGDA; the reaction conditions were 55℃ for 2 h; 20℃ for 30 min; and 30℃ for 1 h, respectively; the remaining steps and conditions were the same as in Example 18. This example is only used to support the lower limit of the scope of the claims and is not used for performance comparison of experimental examples.

[0091] Example 25: In steps S2-S5, the upper limits of each parameter were used, namely 0.70 g of Fe3O4@SiO2 particles, 0.80 mL of ODTMS, 1.50 mL of NVP, 30 mg of APS, 30 μL of TEMED, and 0.30 mL of PEGDA; the reaction conditions were 60℃ for 4 h; 25℃ for 40 min; and 35℃ for 1.5 h, respectively; the remaining steps and conditions were the same as in Example 18. This example is only used to support the upper limit of the claims and is not included in the performance comparison of the experimental examples.

[0092] Experimental Example 4: The effect of synergistic structure on the detection effect of natural steroidal estrogens.

[0093] Examples 18-22 were used as test objects; according to the detection method described in Example 1, mixed spiked recovery tests of 50 μg / kg, 500 μg / kg and 1000 μg / kg were carried out on blank Shanghai bok choy aerial samples, with 6 parallel tests for each group; the average value of the three levels was taken as the comprehensive recovery rate, and the purification time of a single sample was recorded.

[0094] See above and Figure 5 Example 18 showed high and relatively balanced average recoveries for all three natural steroidal estrogens, indicating that the synergistic structure of the first functional region, the second functional region, and the semi-permeable shielding shell in the magnetic composite adsorbent material is beneficial for the simultaneous extraction and detection of natural steroidal estrogens. In Example 19, as the hydrophobic adsorption layer effect of the first functional region weakened, the recoveries of estrone and estradiol decreased significantly, indicating that the first functional region plays an important role in the enrichment of these two target substances. In Example 20, after the polar adsorption layer effect weakened, the recovery of estriol decreased significantly, indicating that the second functional region makes an important contribution to the selective adsorption of estriol. In Example 21, when the semi-permeable shielding shell was not formed, the recovery of estriol decreased, indicating that the semi-permeable shielding shell can effectively reduce matrix interference. In Example 22, when a commercially available C18 solid-phase extraction column was used, the simultaneous enrichment capacity and purification efficiency of the three target substances were lower than those in Example 18. The above results indicate that magnetic composite adsorbent materials with complete synergistic structures are more suitable for application in the extraction and detection methods of natural steroidal estrogens.

[0095] Experimental Example 5: The effect of different purification methods on the application effect of the method.

[0096] Examples 18, 22, and 23 were used as test subjects. Following the detection method described in Example 1, a 50 μg / kg mixed spike test was performed on blank Shanghai bok choy aerial samples, with six replicates per group. The enrichment fold, limit of detection, and single-sample purification time were investigated. The enrichment fold was calculated based on the ratio of the target analyte concentration in the treated detection solution to the target analyte concentration in the initial sample; the limit of detection was calculated with a signal-to-noise ratio of 3.

[0097] See the table above and Figure 6 Example 18 showed the best performance in terms of enrichment fold, detection limit, and single-sample purification time. Compared with Example 22, Example 18 showed a significantly improved enrichment fold, a significantly lower detection limit for the three natural steroidal estrogens, and a significantly shorter single-sample purification time, indicating that the magnetic composite adsorption material described above has better application performance than the traditional commercially available C18 solid-phase extraction column when used for the extraction and detection of natural steroidal estrogens. Compared with Example 23, Example 18 showed slight improvement in enrichment fold and detection limit, and a significantly shorter single-sample purification time, indicating that the magnetic separation step can further improve purification efficiency and reduce operational losses. The above results show that when the magnetic composite adsorption material described in this invention is used in conjunction with the magnetic separation step, it is more beneficial to improve the sensitivity, efficiency, and practicality of the natural steroidal estrogens extraction and detection method.

Claims

1. A method for extracting and detecting natural steroidal estrogens based on multi-mechanism synergistic adsorption, characterized in that, Includes the following steps: S1. Sample extraction: S1-1. The freeze-dried vegetable samples are divided into aboveground parts and roots. Weigh 0.49-0.51 g of aboveground part sample and 0.049-0.051 g of root sample respectively, and place them in a container with a volume of not less than 40 mL. S1-2, After adding the extraction agent and extracting, the supernatant is obtained; S1-3. Filter the supernatant through a 0.22 μm organic phase filter membrane and collect the extract; repeat the extraction once and combine the two extracts. S1-4. The combined extract was concentrated to 1.0-1.2 mL by nitrogen blowing in a 40℃ water bath, 1 mL of acetonitrile was added to redissolve the extract, and the volume was adjusted to 40 mL with ultrapure water to obtain the sample solution to be purified. S2. Pretreatment of adsorbent material: S2-1. Weigh 20-60 mg of magnetic composite adsorption material; S2-2, Add 2 mL of methanol and 2 mL of ultrapure water sequentially to wet and disperse, and vortex for 30 s each time; S2-3. Apply an external magnetic field to perform magnetic separation, discard the supernatant, and obtain the pre-activated magnetic composite adsorbent material; S3, Dispersed Adsorption: Add the pre-activated magnetic composite adsorbent material described in S2-3 to the sample solution to be purified described in S1-4; vortex for 30 seconds, then shake at 200-250 rpm for 8-10 minutes to adsorb the natural steroidal estrogens in the vegetable sample onto the surface of the magnetic composite adsorbent material. S4, Magnetic Separation: Apply an external magnetic field to the system after S3 treatment to perform magnetic separation for 1-2 min, and discard the supernatant; S5. Eluting: Add 5 mL of ultrapure water to the magnetic composite adsorbent material treated in S4, vortex for 15-20 s and then magnetically separate, discarding the eluent; then add 2 mL of 5% (v / v) methanol aqueous solution, vortex for 10-15 s and then magnetically separate again, discarding the eluent. S6, Elution: The magnetic composite adsorbent material treated by S5 is eluted to obtain the eluent; S7. Concentration, Reconstitution, and Detection: S7-1. The eluent obtained in S6 is concentrated by nitrogen blowing under a 40℃ water bath until the residual volume is 0.1-0.3 mL. 2 mL of methanol is added to redissolve and vortex to mix. After filtration through a 0.22 μm organic phase filter membrane, it is transferred to a sample vial. S7-2. High-performance liquid chromatography-fluorescence detector was used for analysis to complete the extraction and detection of natural steroidal estrogens.

2. The method for extracting and detecting natural steroidal estrogens as described in claim 1, characterized in that, The extraction process described in S1-2 is as follows: add 10 mL of extraction agent, then vortex for 30-40 s, ultrasonically extract for 20-40 min, shake for 20-40 min, and centrifuge at 8000-12000 r / min for 8-15 min to obtain supernatant; The extractant is prepared by mixing acetonitrile and water in a volume ratio of 1:

1.

3. The method for extracting and detecting natural steroidal estrogens as described in claim 1, characterized in that, The elution process described in S6 is as follows: 10 mL of eluent is added to the magnetic composite adsorption material after S5 treatment, and then the mixture is vortexed for 30-40 seconds, sonicated for 3-8 minutes, and oscillated for 3-8 minutes before magnetic separation. The eluent is collected. Then, 5 mL of eluent is added to repeat the elution once and the two eluents are combined. The eluent is prepared by mixing ethyl acetate, methanol and glacial acetic acid in a volume ratio of 50:50:

1.

4. The method for extracting and detecting natural steroidal estrogens as described in claim 1, characterized in that, The setting parameters for the high-performance liquid chromatography-fluorescence detector described in S7-2 are as follows: The chromatographic column is a C18 column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; The mobile phase was prepared by mixing acetonitrile and water at a volume ratio of 40:60, the flow rate was 0.8 mL / min, the column temperature was 40 ℃, and the injection volume was 20 μL. The fluorescence detector has an excitation wavelength of 280 nm, an emission wavelength of 310 nm, and a single detection time of 20 min.

5. The method for extracting and detecting natural steroidal estrogens as described in claim 1, characterized in that, The preparation method of the magnetic composite adsorption material is as follows: SA1. Preparation and modification of the magnetic core: Fe3O4 nanoparticles were prepared and silanol-modified sites were prepared on their surface to obtain Fe3O4@SiO2 particles; SA2, Preparation of emulsion: Maintaining a nitrogen-protected environment, 0.5-0.7 g of the Fe3O4@SiO2 particles were added to a mixed solution of 40 mL ethanol and 40 mL deionized water and ultrasonically dispersed for 3-5 min. Then, 20 mL of anhydrous toluene was added and stirred until homogeneous. Next, the mixture was emulsified at 8000 rpm for 1-2 min using a homogenizer to obtain an emulsion in which the Fe3O4@SiO2 particles were distributed at the oil / water phase interface. The side of the Fe3O4@SiO2 particles located in the oil phase was defined as the first reaction zone, and the side located in the water phase was defined as the second reaction zone. SA3, Preparation of hydrophobic adsorption layer: First, maintain the emulsion state in SA2 and keep the stirring rate at 200~300 rpm; then add 0.6~0.8 mL of ODTMS to the oil phase side, and then react at 55~60℃ for 2~4 h. After the reaction is completed, cool the system to room temperature to obtain the first functional zone. SA4. Preparation of polar adsorption layer: While maintaining the emulsion state in SA3, 1.0-1.5 mL NVP, 20-30 mg APS and 20-30 μL TEMED were added sequentially to the aqueous phase side. The mixture was then reacted at 20-25 °C for 30-40 min to form a polar adsorption layer on the second reaction zone. SA5. Preparation of a semi-permeable shielding shell: Keep the emulsion in SA4, then add 0.2~0.3 mL of PEGDA and react at 30~35℃ for 1~1.5 h to form a semi-permeable shielding shell on the polar adsorption layer, thus obtaining the second functional region; SA6, Demulsification - Washing: Add 100 mL of anhydrous ethanol to the SA5 system, stir at 400-500 rpm for 1-3 min, magnetically absorb and collect the product, and wash it clean with anhydrous ethanol, deionized water and anhydrous ethanol in sequence, and dry it at 50-60℃ for 10-12 h to obtain the magnetic composite adsorption material.

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