Mass spectrum ionization substrate capable of specifically adsorbing estrogen as well as preparation method and application of mass spectrum ionization substrate

Abstract

The invention discloses a mass spectrum ionization substrate capable of specifically adsorbing estrogen and a preparation method and application thereof, and the mass spectrum ionization substrate comprises a conductive substrate which is made of stainless steel; the adsorption layer covers at least part of the surface of the substrate, the adsorption layer is formed by covalent organic polymerization nanoparticles, and the covalent organic polymerization nanoparticles are formed by repeated multi-element ring units shown in the formula I in the specification. The mass spectrum ionization substrate is uniform in coating, good in estrogen enrichment specificity and high in adsorption capacity, eluent of the mass spectrum ionization substrate can be used for detection based on a liquid chromatography method and can also serve as a mass spectrum ion source fixing substrate, mass spectrum detection is conducted through ionization and ionization, the detection steps are simple, and the background noise of detection is low.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, in particular to a mass spectrometry ionization substrate for estrogen detection and a preparation method and application thereof. Background Art

[0002] Endocrine disrupting compounds (EDCs) in food can interfere with the human hormone system and may lead to adverse health effects such as endometrial cancer, decreased male fertility, epigenetic changes and abnormal sexual maturation. As an endocrine disruptor, phytoestrogens exhibit weak estrogen agonist or antagonist activity and have similar molecular and cellular characteristics to synthetic EDCs such as bisphenol A, thus attracting much attention from the scientific community and legislative bodies. Therefore, the development of rapid and effective estrogen detection technology is crucial to prevent the health risks of endocrine disruptors in food.

[0003] In recent years, although a variety of quantitative analysis techniques for phytoestrogens have been developed, such as immunoassay, capillary electrophoresis (CE), and liquid chromatography (LC) coupled with mass spectrometry (MS) or ultraviolet (UV) detectors, the application of these methods in rapid detection is still limited by potential compound cross-reactions, highly technically dependent operational requirements, complex sample pretreatment procedures, and time-consuming column separation steps.

[0004] As an innovative mass spectrometry technology based on atmospheric pressure ionization technology, open mass spectrometry (AMS) can directly analyze samples at room temperature and pressure, and usually only requires simple sample pretreatment. Compared with traditional electron bombardment ionization plasma sources, this technology has soft ionization characteristics, high ionization efficiency, high abundance of quasi-molecular ion peaks, and few fragment ions. Compared with atmospheric pressure ionization sources such as electrospray ionization, it shows significant advantages in tolerating matrix interference, reducing solvent loss and improving sensitivity. However, given the complexity of the sample matrix and the low content of the target component, it is still essential to develop an efficient and selective mass spectrometry ionization substrate.

[0005] Therefore, research on highly selective adsorption materials and mass spectrometry detection methods for estrogen needs to be advanced urgently. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide a mass spectrometry ionization substrate for estrogen, which can efficiently enrich phytoestrogens, and the mass spectrometry ionization substrate is suitable for detection by liquid chromatography, and is also suitable for detection by analytical means such as open solid substrate electrospray mass spectrometry, and has broad application prospects.

[0007] Therefore, according to one aspect of the present invention, the present invention provides a mass spectrometry ionization substrate for estrogen. According to an embodiment of the present invention, the mass spectrometry ionization substrate comprises: a conductive substrate, the substrate is formed of stainless steel; an adsorption layer, the adsorption layer is coated on at least a portion of the surface of the substrate, the adsorption layer is formed of covalent organic polymer nanoparticles, and the covalent organic polymer nanoparticles are composed of repeated polycyclic units shown in Formula I.

[0008]

[0009] According to the mass spectrometry ionization substrate of the embodiment of the present invention, the coating is uniform, the specificity for phytoestrogen enrichment is good, the adsorption capacity is strong, and the eluent of the mass spectrometry ionization substrate can be used for detection based on liquid chromatography, and can also be used as a fixed substrate for mass spectrometry detection through ionization. The detection steps are simple, and the detection background noise is low, and the sensitivity and accuracy are high.

[0010] In addition, the mass spectrometry ionization substrate according to the above embodiment of the present invention may also have the following additional technical features:

[0011] According to an embodiment of the present invention, the particle size of the covalent organic polymer nanoparticles is 80-150 nm.

[0012] According to an embodiment of the present invention, the thickness of the adsorption layer is 10-20 μm.

[0013] According to an embodiment of the present invention, the base has a tapered tip, preferably an isosceles triangle.

[0014] According to an embodiment of the present invention, the base of the base is no greater than 2 cm, and the height of the base is no greater than 3 cm. Preferably, the base is an isosceles triangle with a base of 1 cm and a height of 2 cm.

[0015] According to an embodiment of the present invention, the hydrophilic contact angle of the adsorption layer is 23-25°.

[0016] According to an embodiment of the present invention, the estrogen is at least one selected from the group consisting of daidzein (DAID), genistein (GEN), formononetin (FORM) and coumestrol (COUM).

[0017] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned mass spectrometry ionization substrate for estrogen. According to an embodiment of the present invention, the method comprises: subjecting a stainless steel plate to an acidification treatment to obtain an acidified substrate; subjecting the acidified substrate to a first contact with an aldehyde monomer, and subjecting the substrate to an ultrasonic and oscillating treatment to obtain an ultrasonically oscillated mixture; and subjecting the ultrasonically oscillating mixture to a second contact with an amino monomer, and subjecting the mixture to a Schiff base reaction to obtain the mass spectrometry ionization substrate.

[0018] According to the preparation method of the embodiment of the present invention, the mass spectrometry ionization substrate coating prepared by the method is uniform, has good specificity for phytoestrogen enrichment, and has strong adsorption capacity. In addition, the preparation method has mild conditions, simple steps, and is convenient for industrial production.

[0019] According to an embodiment of the present invention, the first contacting is performed in tetrahydrofuran.

[0020] According to an embodiment of the present invention, the ratio of the aldehyde monomer to tetrahydrofuran is 5-6 mg:1 mL.

[0021] According to an embodiment of the present invention, the solvent of the amino monomer is tetrahydrofuran, and the ratio is 50-60 mg:3 mL.

[0022] According to an embodiment of the present invention, the molar ratio of the aldehyde monomer to the amino monomer is 0.8-1.2:1.

[0023] According to an embodiment of the present invention, the aldehyde monomer is 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and the amino monomer is 4-(4-aminophenyl)-2,6-di(4-aminophenyl)pyridine.

[0024] According to an embodiment of the present invention, the ultrasonic treatment time is 20-40 minutes.

[0025] According to an embodiment of the present invention, the temperature of the shaking treatment is 60-65° C., the time is 0.5-1.5 hours, and the shaking frequency is 150-250 rpm / min.

[0026] According to an embodiment of the present invention, the temperature of the Schiff base is 60-65° C., and the time is 3-6 hours, preferably 4 hours.

[0027] According to an embodiment of the present invention, the catalyst of the Schiff base reaction is acetic acid.

[0028] According to another aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometry device. According to an embodiment of the present invention, the device includes: the aforementioned mass spectrometry ionization substrate for estrogen; an open mass spectrometry detector, the open mass spectrometry detector includes an injection port, the injection port is arranged relative to the tip of the mass spectrometry ionization substrate; a high-voltage power supply, the high-voltage power supply is connected to the mass spectrometry ionization substrate. Thus, the aforementioned mass spectrometry ionization substrate for estrogen is directly connected to the high-voltage power supply, and under the action of high voltage electricity, the target on the mass spectrometry ionization element is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometer through the mass spectrometer inlet to obtain a collection signal, the detection step is simple, and the background noise of the detection is low, which is particularly suitable for the enrichment and detection of estrogen in food samples.

[0029] According to another aspect of the present invention, the present invention provides a method for enriching and purifying estrogen. According to an embodiment of the present invention, the method includes: filtering the sample to be tested to obtain a test liquid containing estrogen; and subjecting the test liquid to an oscillating contact treatment with the aforementioned mass spectrometry ionization substrate for estrogen to obtain a mass spectrometry ionization substrate with the estrogen adsorbed on the surface. As a result, the purification method has good specificity for estrogen and strong adsorption capacity, which is conducive to fully enriching complex matrices, such as estrogen in food. In addition, the enrichment and purification method of the embodiment of the present invention is simple to operate, does not require a complex sample pretreatment process, and has high sample extraction efficiency.

[0030] According to an embodiment of the present invention, the rotation speed of the oscillating contact treatment is 1500-2000 revolutions, and the time is 25-35 minutes.

[0031] According to another aspect of the present invention, the present invention provides a method for qualitative / quantitative detection of estrogen. According to an embodiment of the present invention, the method includes: using the aforementioned method for enriching and purifying estrogen to enrich the estrogen in the sample to be tested, so as to obtain a mass spectrometry ionization substrate with surface enriched estrogen; using the aforementioned separation ionization integrated mass spectrometry device to detect the mass spectrometry ionization substrate with surface enriched estrogen, so as to perform qualitative / quantitative detection of the estrogen. Thus, the aforementioned mass spectrometry ionization substrate has good specificity for estrogen in food, strong adsorption, and is conducive to fully enriching estrogen in complex matrices, and the mass spectrometry ionization substrate enriched with estrogen is directly connected to the high-voltage power supply, and under the action of high voltage electricity, the target on the mass spectrometry ionization substrate is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometry through the mass spectrometry detector inlet to obtain the acquisition signal, the detection step is simple, the background noise of the detection is low, and the detection sensitivity is high, which is particularly suitable for the rapid and accurate analysis of trace estrogen.

[0032] According to an embodiment of the present invention, the detection conditions of the separation and ionization integrated mass spectrometer are: high voltage power supply voltage: -3.0 kV; ionization elution solvent: methanol.

[0033] According to an embodiment of the present invention, the detection conditions of the mass spectrometer detector are: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 20psi; ion source temperature: 550°C; dwell time: 100ms.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0036] Figure 1 An experimental device according to an embodiment of the present invention and a mass spectrum actually obtained are shown, wherein A is a diagram of the experimental device and B is a mass spectrum.

[0037] Figure 2 A schematic diagram of a mass spectrometry ionization substrate scanning electron microscope according to an embodiment of the present invention is shown, wherein A is a scanning electron microscope image of an acid-treated solid substrate; B is a scanning electron microscope image of TCOPs-S; and C is a scanning electron microscope image of a cross section of TCOPs-S.

[0038] Figure 3 The material characterization diagram of the mass spectrometry ionization substrate according to one embodiment of the present invention is shown, wherein A is an infrared spectrum diagram; B is a thermogravimetric analysis curve; C is the contact angle of the solid substrate; and D is the contact angle of TCOPs-S.

[0039] Figure 4 A schematic diagram showing the selectivity and enrichment ability of the mass spectrometry ionization substrate prepared in Example 1 for four phytoestrogens according to one embodiment of the present invention is shown, wherein A is a schematic diagram of the selective extraction ability and B is a schematic diagram of the enrichment ability. DETAILED DESCRIPTION

[0040] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0041] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0042] According to one aspect of the present invention, the present invention provides a mass spectrometry ionization substrate for estrogen. The mass spectrometry ionization substrate according to the embodiment of the present invention has a uniform coating, good specificity for enrichment of phytoestrogens, strong adsorption, and the eluent of the mass spectrometry ionization substrate can be used for detection based on a liquid chromatography method, and can also be used as a fixed substrate for mass spectrometry detection through ionization, the detection steps are simple, and the detection background noise is low, and the sensitivity and accuracy are high.

[0043] In order to facilitate understanding of the mass spectrometry ionization substrate, the mass spectrometry ionization substrate is explained here. According to an embodiment of the present invention, the mass spectrometry ionization substrate includes:

[0044] Conductive substrate 100

[0045] According to an embodiment of the present invention, the substrate is formed of stainless steel. The inventors have found that when a paper base or a wood base is used as a solid substrate, when used for open solid substrate electrospray mass spectrometry, due to the poor conductivity of the paper base or the wood base, the electrospray formed after applying high voltage thereon has high background interference, and the generated mass spectrometry signal is unstable. However, due to the good conductivity of the stainless steel substrate, high voltage can be directly applied to the substrate, the ionization efficiency is high, and the experimental operation can be further simplified.

[0046] According to an embodiment of the present invention, the substrate is formed of 304 stainless steel. Therefore, the substrate formed of 304 stainless steel has good processing performance, is convenient for cutting, and has good electrical conductivity and heat resistance.

[0047] According to an embodiment of the present invention, the substrate has a conical tip, that is, there is a tip shape on the top view of the substrate. Thus, it is convenient to carry out open solid substrate electrospray mass spectrometry, the ionization element is fixed at the horizontal front end position of the mass spectrometer injection port, and the estrogen adsorbed on the substrate surface can be eluted by applying a spray solvent, and through the effect of a high-voltage power supply, it is ionized at the tip and forms a Taylor cone spray, thereby directly entering the mass spectrometry detection. According to an embodiment of the present invention, the substrate is an isosceles triangle. According to an embodiment of the present invention, the base of the substrate is not more than 2cm, and the height of the substrate is not more than 3cm, preferably, the base is 1cm, and the height is an isosceles triangle of 2cm. Thus, a spray Taylor cone is formed at the triangular tip, and the angle is too large, and the elution solvent will be hindered from spraying by surface tension. If the angle is too small, the substrate area is small, which affects the adsorption capacity of the substrate. Under this size, the elution solvent forms a spray with higher efficiency.

[0048] Adsorption layer 200

[0049] According to an embodiment of the present invention, the adsorption layer covers at least a portion of the surface of the substrate, and the adsorption layer is formed by covalent organic polymer nanoparticles, and the covalent organic polymer nanoparticles are composed of repeated polycyclic units shown in Formula I.

[0050] According to an embodiment of the present invention, the particle size of the covalent organic polymer nanoparticles is 80-150 nm. Therefore, the adsorption layer formed by the nanometer-scale particle size in the above particle size range has a large specific surface area.

[0051] According to an embodiment of the present invention, the thickness of the adsorption layer is 10-20 μm. Therefore, the thickness of the adsorption layer shortens the diffusion path between the functional particles or ligands on the membrane porous matrix and the liquid flow, accelerates mass transfer, significantly shortens the separation time of membrane adsorption, and improves separation efficiency.

[0052] According to an embodiment of the present invention, the hydrophilic contact angle of the adsorption layer is 23°-25°. Therefore, the adsorption layer has good hydrophilicity, which is conducive to the adsorption of the target in the aqueous solution.

[0053] According to an embodiment of the present invention, the estrogen is at least one selected from the group consisting of daidzein (DAID), genistein (GEN), formononetin (FORM) and coumestrol (COUM).

[0054] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned mass spectrometry ionization substrate for estrogen. According to the preparation method of the embodiment of the present invention, the mass spectrometry ionization substrate prepared by the method has a uniform coating, good specificity for enrichment of phytoestrogens, strong adsorption capacity, and the preparation method has mild conditions, simple steps, and is convenient for industrial production.

[0055] In order to facilitate understanding of the method for preparing the aforementioned mass spectrometry ionization substrate for estrogen, the method is explained here. According to an embodiment of the present invention, the method includes:

[0056] S100 acid treatment

[0057] According to an embodiment of the present invention, the stainless steel plate is subjected to an acidification treatment to obtain an acidified substrate, thereby eliminating the interference of stains or other ions on the stainless steel surface, so that the subsequent reaction has a better synthesis effect.

[0058] S200 Ultrasonic Oscillation

[0059] According to an embodiment of the present invention, the acidified substrate is first contacted with an aldehyde monomer and subjected to ultrasound and vibration treatment to obtain an ultrasound-vibrated mixture.

[0060] According to an embodiment of the present invention, the first contact is carried out in tetrahydrofuran, that is, the acidified substrate is contacted with a tetrahydrofuran solution in which the aldehyde monomer is dissolved.

[0061] According to an embodiment of the present invention, the ratio of the aldehyde monomer to tetrahydrofuran is 5-6 mg:1 mL. That is, in the tetrahydrofuran solution in which the aldehyde monomer is dissolved, the ratio of the aldehyde monomer to tetrahydrofuran is 5-6 mg:1 mL.

[0062] According to an embodiment of the present invention, the ultrasonic time is 20-40 minutes, which is conducive to the full dissolution of the aldehyde monomer in tetrahydrofuran.

[0063] According to an embodiment of the present invention, the temperature of the shaking treatment is 60-65° C., the time is 0.5-1.5 hours, and the shaking frequency is 150-250 rpm / min. This is conducive to the reactants being fully contacted for reaction.

[0064] According to an embodiment of the present invention, the aldehyde monomer is 2,4,6-tris(4-formaldehyde phenyl)-1,3,5-triazine, and the amino monomer is 4-(4-aminophenyl)-2,6-bis(4-aminophenyl)pyridine. Thus, 2,4,6-tris(4-formaldehyde phenyl)-1,3,5-triazine is used as the aldehyde monomer, the nitrogen content and porosity are high, the stability is good, it is easy to synthesize polymer materials, and the ability to resist matrix interference is strong; 4-(4-aminophenyl)-2,6-bis(4-aminophenyl)pyridine is used as the amino monomer, the nitrogen content and porosity are high, the stability is good, it is easy to form a polymer with the above-mentioned aldehyde monomer, and the specific surface area of ​​the polymer is large.

[0065] S300 Schiff base reaction

[0066] According to an embodiment of the present invention, the ultrasonically vibrated mixture is secondly contacted with an amino monomer to perform a Schiff base reaction to obtain the mass spectrometry ionization substrate. Thus, through the Schiff base reaction, the amino monomer polymerizes with the aldehyde monomer on the substrate to form an organic polymer (COP).

[0067] According to an embodiment of the present invention, the solvent of the amino monomer is tetrahydrofuran, and the ratio is 50-60 mg:3 mL. Thus, this ratio can ensure that the amino monomer is completely dissolved quickly.

[0068] According to an embodiment of the present invention, the molar ratio of the aldehyde monomer to the amino monomer is 0.8-1.2, preferably 1: 1. Thus, this ratio is conducive to keeping the number of amino groups and aldehyde groups in the amino monomer close to that in the aldehyde monomer.

[0069] According to an embodiment of the present invention, the temperature of the Schiff base should be 60-65° C., and the time should be 3-6 hours, preferably 4 hours. This is conducive to efficient and sufficient reaction and high product yield.

[0070] According to an embodiment of the present invention, the catalyst of the Schiff base reaction is acetic acid, thereby achieving good catalytic effect and high reaction efficiency.

[0071] According to another aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometry device. According to an embodiment of the present invention, the device includes: the aforementioned mass spectrometry ionization substrate for estrogen; an open mass spectrometry detector, the open mass spectrometry detector includes an injection port, the injection port is arranged relative to the tip of the mass spectrometry ionization substrate; a high-voltage power supply, the high-voltage power supply is connected to the mass spectrometry ionization substrate. Thus, the aforementioned mass spectrometry ionization substrate for estrogen is directly connected to the high-voltage power supply, and under the action of high voltage electricity, the target on the mass spectrometry ionization substrate is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometer through the mass spectrometer inlet to obtain a collection signal, the detection step is simple, and the background noise of the detection is low, which is particularly suitable for the enrichment and detection of estrogen in food samples.

[0072] According to another aspect of the present invention, the present invention provides a method for enriching and purifying estrogen. According to an embodiment of the present invention, the method includes: filtering the sample to be tested to obtain a liquid to be tested; and subjecting the liquid to be tested to an oscillating contact treatment with the aforementioned mass spectrometry ionization substrate for estrogen, so as to obtain a mass spectrometry ionization substrate with the estrogen adsorbed on the surface. As a result, the purification method has good specificity for estrogen and strong adsorption capacity, which is conducive to fully enriching complex matrices, such as estrogen in food. In addition, the enrichment and purification method of the embodiment of the present invention is simple to operate, does not require a complex sample pretreatment process, and has high sample extraction efficiency.

[0073] According to an embodiment of the present invention, the rotation speed of the oscillating contact treatment is 1500-2000 revolutions and the time is 25-35 minutes, which is conducive to the mass spectrometry ionization substrate fully absorbing the estrogen in the test solution.

[0074] According to another aspect of the present invention, the present invention provides a method for qualitative / quantitative detection of estrogen. According to an embodiment of the present invention, the method includes: using the aforementioned method for enriching and purifying estrogen to enrich the estrogen in the sample to be tested, so as to obtain a mass spectrometry ionization substrate with surface enriched estrogen; using the aforementioned separation ionization integrated mass spectrometry device to detect the mass spectrometry ionization substrate with surface enriched estrogen, so as to perform qualitative / quantitative detection of the estrogen. Thus, the aforementioned mass spectrometry ionization substrate has good specificity for estrogen in food, strong adsorption, and is conducive to fully enriching estrogen in complex matrices, and the mass spectrometry ionization substrate enriched with estrogen is directly connected to the high-voltage power supply, and under the action of high voltage electricity, the target on the mass spectrometry ionization substrate is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometry through the mass spectrometry detector inlet to obtain the acquisition signal, the detection step is simple, the background noise of the detection is low, and the detection sensitivity is high, which is particularly suitable for the rapid and accurate analysis of trace estrogen.

[0075] According to an embodiment of the present invention, the detection conditions of the separation and ionization integrated mass spectrometer are: high voltage power supply voltage: -3.0kV; ionization elution solvent: methanol. The inventor optimized the detection voltage and elution solvent, and found that within the voltage range of -1 to -5kV, the signal response is stronger when the -3kV voltage is selected; for the elution solvent, the inventor found that among ethyl acetate, acetonitrile, ethanol, isopropanol, and methanol, the signal response is higher when methanol is used as the elution solvent. Therefore, the selection of the above voltage and elution solvent is conducive to improving the detection signal of the object to be detected, making the detection sensitivity and accuracy higher.

[0076] According to an embodiment of the present invention, the detection conditions of the mass spectrometer detector are: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 20psi; ion source temperature: 550°C; dwell time: 100ms. Therefore, under the above conditions, estrogen is detected, the target response value is high, the background noise of the detection is low, the detection sensitivity and accuracy are high, and it is particularly suitable for the rapid and accurate analysis of trace phytoestrogens.

[0077] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0078] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unrepresented specific techniques or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially, for example, can be purchased from Sigma company.

[0079] Example 1

[0080] In this embodiment, the method for preparing a mass spectrometry ionization substrate for estrogen according to the embodiment of the present invention is used, 2,4,6-tris(4-aldehyde phenyl)-1,3,5-triazine is used as an aldehyde monomer, and 4-(4-aminophenyl)-2,6-bis(4-aminophenyl)pyridine is used as an amino monomer to prepare a mass spectrometry ionization substrate. The specific method is as follows:

[0081] (1) The acidified solid substrate (7 pieces) and 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (59 mg, 0.15 mmol) in a conical flask (200 mL) were dispersed or dissolved in 10 mL of tetrahydrofuran and ultrasonicated for 30 min.

[0082] (2) The mixture was then transferred to a water bath shaker at 60°C and shaken at 200 rpm / min for 1 h. 4-(4-aminophenyl)-2,6-di(4-aminophenyl)pyridine (53 mg, 0.15 mmol) was dissolved in tetrahydrofuran (3 mL) and slowly added to the above system.

[0083] (3) Acetic acid (200 μL) was added dropwise into the conical flask as a catalyst to accelerate the reaction. The mixture was mechanically stirred at 200 rpm at 60°C for 4 h.

[0084] (4) The obtained mass spectrometry ionization substrate is washed alternately with methanol and acetonitrile until the supernatant is clear. Finally, the material is stored in acetonitrile, and the reaction formula is as follows:

[0085]

[0086] Example 2

[0087] A detailed characterization experiment was conducted on the mass spectrometry ionization substrate (TCOPs-S) prepared in Example 1 to demonstrate the successful preparation of the mass spectrometry ionization substrate and its excellent physical and chemical properties. The specific contents are as follows:

[0088] 1. The surface morphologies of solid substrates, acid-treated solid substrates, and TCOPs-S were characterized, providing evidence for the successful synthesis of TCOPs on the surface of solid substrates. Figure 1 Show, Figure 2As shown in Figure A, after sulfuric acid treatment, the surface of the stainless steel solid substrate presents a rough and uneven texture, which is beneficial to increase the specific surface area. After coating the solid substrate with TCOPs coating, uniformly distributed small spherical particles are observed on the surface, each with a diameter of about 100 nm and a coating thickness of about 10 μm ( Figure 2 B and C).

[0089] 2. FT-IR spectroscopy further confirmed the successful synthesis of TCOPs ( Figure 3 A). Compared with aldehyde monomers and amino monomers, -CHO has a higher peak at 2730 and 2810 cm -1 The CH stretching band at 3200 and 3300 cm -1 The NH stretching band at 1710 cm -1 A new peak was observed at , which can be attributed to the formation of C=N bond in TCOPs. The results showed that TCOPs were successfully synthesized via Schiff base reaction.

[0090] 3. The thermal stability of TCOPs in the range of 40-800℃ was studied by thermogravimetric analysis (TGA) and differential thermal analysis (DTA). Figure 3 B). The 36.25% weight loss at 92.93°C is due to the evaporation of the solvent. When the temperature reaches 555.93°C, the weight of the material decreases by 23.82%, corresponding to the exothermic peak of the DTA curve. The observed phenomenon is believed to be caused by the degradation and carbonization of the TCOPs material. These results confirm the good thermal stability of TCOPs.

[0091] 4. The hydrophobic properties of the solid substrate and TCOPs-S surface were studied by contact angle measurement. The water contact angle of the solid substrate was 95.41°( Figure 3 C), indicating that it is hydrophobic. This can be attributed to the presence of an antioxidant oil film on the surface. After coating the TCOPs layer, the contact angle was measured to be 24.46° ( Figure 3 D), indicating that TCOPs-S has good hydrophilicity and can adsorb phytoestrogens in aqueous solution.

[0092] Example 3

[0093] In this example, the selectivity and enrichment ability of the mass spectrometry ionization substrate prepared in Example 1 were investigated. In order to study the anti-interference ability of TCOPs-S in the selective adsorption of phytoestrogens, coumarin compounds and alkaloid compounds (20 ng / mL) were used as interfering agents. Figure 4As shown in Figure A, the adsorption rate of coumarin compounds (including coumarin, 7-methylcoumarin, and 7-methoxycoumarin) on this material ranges from 5.50±0.50% to 13.67±1.53%. The adsorption rate of alkaloids (including matrine, oxymatrine, and oxysophorophyline) ranges from 33.10±4.81% to 47.67±0.21%, while the adsorption rate of phytoestrogens can be as high as 82.13±2.32% to 98.14±0.13%. These results show that the mass spectrometry ionization substrate has significant anti-interference ability and is suitable for enriching target compounds in complex samples.

[0094] In order to study the enrichment ability of the materials, the adsorption efficiency of four phytoestrogens on the acid-treated solid substrate and TCOPs-S was compared. Figure 4 As shown in B, when the acid-treated solid substrate was used for extraction, the peak area range of the four phytoestrogens (30 ng / mL) was 2.14x10 5 ~2.66x10 6 , while when TCOPs-S was used for extraction, their peak areas were 4.53x10 7 ~2.02x10 8 The adsorption efficiency of TCOPs-S for phytoestrogens was 53.2 to 212.0 times that of the acid-treated solid substrate, demonstrating the enrichment ability of the material.

[0095] Example 4

[0096] In this example, the mass spectrometry ionization substrate prepared in Example 1 was used to enrich estrogen in the milk sample. The specific method is as follows:

[0097] 1. Sample preparation

[0098] Take 7.5mL of milk sample, add 5mL of acetate buffer (pH 5), and add 50μL of β-glucuronidase. Then place the mixture in a water bath at 37°C overnight. Take 2g of milk sample and transfer it to a 50mL centrifuge tube, add ACN / water / acetic acid (90:9:1; v:v:v) until the total volume reaches 10mL. Vortex for 1min, sonicate for 20min, centrifuge at 4°C and 8000rpm for 5min, take 5mL of supernatant and evaporate to almost dryness under a gentle nitrogen flow, then dissolve in deionized water to a final volume of 5mL for further extraction.

[0099] During the extraction process, 4 mL of the above solution was transferred to a 30 mL glass bottle. Subsequently, one piece of TCOPs was added to the mixture and vigorously shaken for 30 min using a multi-tube vortexer (BV1010, Thomas Scientific, USA). TCOPs-S was then dried for further analysis.

[0100] For spiked milk samples, 2 g of blank milk sample without phytoestrogen was taken and mixed standard of corresponding concentration was added, followed by the same treatment as above to obtain extracted TCOPs-S for further analysis. 2.5. AMS analysis

[0101] TCOPs-S was mounted on a three-dimensional adjustable platform using a copper clip to ensure that its tip was 3-5 mm away from the MS inlet. The experimental setup is shown in the figure. Figure 1 As shown in A. The other end of the copper clip is connected to the negative electrode of the high-voltage power supply. The mounted TCOPs-S is then treated with 20 μL of MeOH solution and carefully dispensed on its surface with a micropipette to promote the desorption of the analyte. A high voltage of -3.0 kV is applied to TCOPs-S to generate electrospray at its tip, followed by mass spectrometry analysis. Mass spectrometry detection conditions: Detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 20 psi; ion source temperature: 550 ° C; dwell time: 100 ms.

[0102] AMS analysis was performed using a TRIPLE QUADTM 5500MS instrument (AB SCIEX Corp.). Multiple reaction monitoring (MRM) mode was used to quantify phytoestrogens. The specific parameters are shown in Table 1. The peak area of ​​the analyte quantitative ion was used for calculation. The Analyst software provided by AB SCIEX was used for data acquisition and analysis. The actual spectrum is shown in Figure 1 As shown in B.

[0103] Table 1 Range of phytoestrogens detected by MS

[0104]

[0105] *Quantitative ion

[0106] 2. Comparison of adsorption performance between acid-treated stainless steel solid substrate and TCOPs

[0107] The purpose of this example is to demonstrate that the adsorption mainly occurs in the TCOPs layer of TCOPs-S, which is composed of a stainless steel substrate and a TCOPs coating. To achieve this goal, the adsorption efficiency of the four phytoestrogens on the acid-treated stainless steel substrate and TCOPs-S was compared. Figure 4 As shown in B, when acid-treated stainless steel substrate (Acid-treated S) was used for extraction, the peak area range of the four phytoestrogens (30 ng / mL) was 2.14x10 5 Up to 2.66x10 6 , while when TCOPs (TCOPs-S) was used for extraction, their peak areas were 4.53x10 7Up to 2.02x10 8 The adsorption efficiency of TCOPs-S for phytoestrogens was 53.2-212.0 times that of the acid-treated stainless steel substrate, indicating that the main adsorption sites were located within the TCOPs layer.

[0108] 3 Method validation

[0109] Before the actual sample analysis, the established TCOPs-SS-MS method was validated according to the optimized experimental conditions. The detection limit (LOD) and quantification limit (LOQ) of each compound in milk samples were determined using a signal-to-noise ratio (S / N) of 3 and 10, respectively. The LOD values ​​of soyizin (DAID), genistein (GEN), formononetin (FORM) and coumestrol (COUM) were determined to be 0.50-0.75 μg / kg, and the LOQ values ​​were 2.00-3.00 μg / kg. The calibration curve was established by adding different concentrations of phytoestrogens to blank milk. The data in Table 2 show a robust linear correlation, R 2 The value was ≥0.9934. The method had good repeatability, with intra-day relative standard deviation (RSD) ranging from 2.87 to 8.22% and intra-day precision (RSD) ranging from 1.57 to 9.97%.

[0110] Table 2 Performance parameters of the established method

[0111]

[0112]

[0113] Application of 4TCOPs-SS-MS in real samples

[0114] To further verify the accuracy of the developed method, DAID, FORM, COUM and GEN with known concentrations (5, 25 and 100 ng / g) were added to the treated milk samples. The average recovery of the method was 84.08-109.15%, and the RSD range was 0.81%-14.14% (see Table 3). The results showed that TCOPs-S combined with AMS is a reliable method for the analysis of phytoestrogens in milk samples.

[0115] Table 3 Spiked recovery method of phytoestrogens in actual samples

[0116]

[0117] ND=Not Detected.

[0118] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0119] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A mass spectrometry ionization substrate that specifically adsorbs estrogen, characterized in that: include: a conductive substrate formed of stainless steel; as well as The adsorption layer covers at least a portion of the surface of the substrate, and the adsorption layer is formed by covalent organic polymer nanoparticles, and the covalent organic polymer nanoparticles are composed of repeated polycyclic units shown in Formula I.

2. The mass spectrometry ionization substrate according to claim 1, characterized in that The particle size of the covalent organic polymer nanoparticles is 80-150 nm. Optionally, the adsorption layer has a thickness of 10-20 μm.

3. The mass spectrometry ionization substrate according to claim 1, characterized in that: The base has a conical tip, preferably an isosceles triangle; Optionally, the base of the base is not greater than 2 cm, and the height of the base is not greater than 3 cm. Preferably, the base is an isosceles triangle with a base of 1 cm and a height of 2 cm.

4. The mass spectrometry ionization substrate according to claim 1, characterized in that: The hydrophilic contact angle of the adsorption layer is 23-25°. Optionally, the estrogen is at least one selected from the group consisting of daidzein (DAID), genistein (GEN), formononetin (FORM) and coumestrol (COUM).

5. A method for preparing a mass spectrometry ionization substrate for specific adsorption of estrogen according to any one of claims 1 to 4, characterized in that: include: Acidizing the stainless steel sheet to obtain an acidified substrate; The acidified substrate is first contacted with the aldehyde monomer, and subjected to ultrasonic and vibration treatment to obtain an ultrasonically vibrated mixture; as well as The mixture after ultrasonic vibration is brought into contact with an amino monomer for a second time to perform a Schiff base reaction, so as to obtain the mass spectrometry ionization substrate.

6. The method according to claim 5, characterized in that The first contacting is carried out in tetrahydrofuran, Optionally, the ratio of the aldehyde monomer to tetrahydrofuran is 5-6 mg:1 mL, Optionally, the solvent of the amino monomer is tetrahydrofuran, with a ratio of 50-60 mg: 3 mL, Optionally, the molar ratio of the aldehyde monomer to the amino monomer is 0.8-1.2:1, Optionally, the aldehyde monomer is 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and the amino monomer is 4-(4-aminophenyl)-2,6-di(4-aminophenyl)pyridine.

7. The method according to claim 5, characterized in that The ultrasound time is 20-40 minutes. Optionally, the shaking treatment temperature is 60-65°C, the time is 0.5-1.5 hours, and the shaking frequency is 150-250 rpm / min. Optionally, the temperature of the Schiff base is 60-65° C., and the time is 3-6 hours, preferably 4 hours. Optionally, the catalyst for the Schiff base reaction is acetic acid.

8. A separation and ionization integrated mass spectrometer, characterized in that: include: The mass spectrometry ionization substrate for specific adsorption of estrogen according to any one of claims 1 to 4; An open mass spectrometer detector, the open mass spectrometer detector comprising an injection port, the injection port being arranged opposite to the tip of the mass spectrometer ionization substrate; as well as A high voltage power supply is connected to the mass spectrometry ionization substrate.

9. A method for enriching and purifying estrogen, characterized in that: include: Pre-treating the sample to be tested to obtain a test solution; as well as The test solution is subjected to an oscillating contact treatment with the mass spectrometry ionization substrate for specific adsorption of estrogen according to any one of claims 1 to 4, so as to obtain the mass spectrometry ionization substrate with the estrogen adsorbed on the surface, Optionally, the rotation speed of the oscillating contact treatment is 1500-2000 rpm and the time is 25-35 minutes.

10. A method for qualitative / quantitative detection of estrogen, characterized in that: include: The method for enriching and purifying estrogen according to claim 9 is used to enrich the estrogen in the sample to be tested, so as to obtain a mass spectrometry ionization substrate with surface enriched estrogen; as well as The separation and ionization integrated mass spectrometer according to claim 8 is used to detect the mass spectrometry ionization substrate of the surface-enriched estrogen, so as to perform qualitative / quantitative detection of the estrogen, Optionally, the detection conditions of the separation and ionization integrated mass spectrometry device are: High voltage power supply voltage: -3.0kV; Ionization elution solvent: methanol, Optionally, the detection conditions of the mass spectrometer detector are: Detection method: multiple reaction monitoring (MRM); Atomizing gas pressure: 55psi; Auxiliary gas pressure: 50psi; Air curtain air pressure: 20psi; Ion source temperature: 550°C; Dwell time: 100ms.

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