A method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances

Abstract

The present invention relates to a method for detecting bisphenols and perfluorinated compounds in an orthodontic transparent appliance, and belongs to the field of detection technology. The method comprises the following steps: S1: sample preparation: adding an orthodontic transparent appliance sample to artificial saliva, incubating in a rotary shaker at 37°C ± 1°C for 2 weeks, and filtering to obtain a sample solution; S2: sample pretreatment: adding a magnetic composite material Fe3O4@OH‑COF to the sample solution, and vortexing; separating the magnetic composite material Fe3O4@OH‑COF from the solution; eluting the separated magnetic composite material Fe3O4@OH‑COF with an acetonitrile solution; separating to obtain an eluent; S3: detection and analysis: using a high performance liquid chromatography-tandem mass spectrometry detection method to detect and analyze the eluent. The present invention establishes a method of magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS), which can simultaneously detect the amount of bisphenol compounds and perfluorinated compounds released by an orthodontic transparent appliance, with high sensitivity, low detection limit, and short analysis time.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances. Background Art

[0002] Bisphenols (BPs) are important organic chemical raw materials. Because they significantly improve the heat resistance, moisture resistance, and insulation properties of materials, they are commonly used as modifiers, stabilizers, photoinitiators in polymer materials, rubber antioxidants and plasticizers, and reactive flame retardants for epoxy resins, polycarbonate resins, and phenolic resins in printed circuit boards. In recent years, there has been growing concern about the safety of BPs in humans. BPs are typical endocrine disruptors (EDs), binding to multiple nuclear receptors in the human body and disrupting the endocrine systems of both animals and humans even at low concentrations. With ongoing research, the toxicological effects of BPs, including endocrine disruption, cytotoxicity, reproductive toxicity, neurotoxicity, and genotoxicity, have been increasingly confirmed. BPs are associated with the development and progression of diabetes, heart disease, obesity, immune system disorders, and reproductive system diseases.

[0003] Perfluoroalkyl and polyfluoroalkyl substances (PFASs), also known as perfluorinated compounds, are a class of synthetic organic compounds. Due to their high surface tension and excellent chemical and thermal stability, they are widely used in consumer products such as non-stick cookware, stain-resistant textiles, and food packaging. However, these consumer products continuously release PFASs into the environment during production and use. Numerous studies have demonstrated that PFASs exhibit multiple biological toxicities, including neurotoxicity, immunotoxicity, hepatotoxicity, reproductive toxicity, and genotoxicity. PFASs have been detected in various environmental media and in plants and animals.

[0004] Orthodontic clear aligners are essential instruments in orthodontic treatment. Users often need to wear them in their mouths for 24 hours, and the orthodontic treatment cycle is long. In clinical practice, orthodontic clear aligners need to be worn in the user's mouth for 2 weeks before replacing them with a new pair of orthodontic clear aligners. The basic material components of orthodontic clear aligners are polymers, such as polyethylene (PE), polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyurethane (PU) and polypropylene (PP). These polymers contain various additives to improve performance, stability, elasticity, etc. Bisphenol compounds (BPs) and perfluorinated compounds (PFASs) are the most abundant of these additives and are used to harden plastics and increase their transparency.

[0005] These two types of additives in orthodontic clear aligners are easily released into the human mouth during the two-week wear period. When accumulated to a certain concentration in the human body, they can easily cause harm to the body. Due to the harmful effects and difficult degradation of these two types of pollutants, the demand for measuring the content of different BPs and different PFASs in orthodontic clear aligners is becoming increasingly urgent. In particular, it is necessary to understand the amount of BPs and PFASs that will be released into the human mouth after the patient wears the orthodontic clear aligners.

[0006] Since bisphenol compounds and perfluorinated compounds often exist in trace amounts in samples (orthodontic clear aligners), accurate quantitative determination cannot be achieved using sophisticated analytical instruments. In addition, the sample (orthodontic clear aligners) matrix is ​​complex and there is matrix interference, which also causes difficulties in detection. Sample pretreatment, enrichment and purification must be carried out before detection.

[0007] Existing methods generally target bisphenols or perfluorinated compounds, using liquid-liquid extraction, freeze-drying and other methods for sample pretreatment. These methods are not only cumbersome and time-consuming, but also fail to meet the requirements of rapid analysis. In addition, due to the large differences in the physical and chemical properties of bisphenols (BPs) and perfluorinated compounds (PFASs), simultaneous extraction and enrichment cannot be achieved. These two types of pollutants need to be extracted separately, which increases the workload and analysis costs. Moreover, the simultaneous detection of these two types of components also poses a challenge to the optimization of instrument detection parameters. In order to quickly and accurately detect BPs and PFASs in orthodontic clear aligners, a simultaneous extraction and elution strategy is an effective method. This strategy achieves the simultaneous extraction of these two types of pollutants in the sample through a single sample processing process, which not only simplifies the sample pretreatment steps and improves analysis efficiency, but also reduces the amount of reagents used and reduces analysis costs.

[0008] Currently, the detection of bisphenols or perfluorinated compounds is mostly concentrated on surface water, sediments, sewage effluent, etc., while there is little research on orthodontic clear appliance samples. This is mainly because the matrix of orthodontic clear appliance is complex and the interference is serious. The pre-analysis treatment is much more difficult than the detection of bisphenols or perfluorinated compounds in water. In addition, the current technology only targets one type of component. How to efficiently extract bisphenols or perfluorinated compounds from orthodontic clear appliance samples has become a bottleneck in the analysis of trace pollutants.

[0009] Among the commonly used extraction methods for bisphenol compounds or perfluorinated compounds in clear aligners, rapid solvent extraction or ultrasonic extraction alone have the advantages of short analysis time, low use of toxic solvents, and high extraction efficiency. However, due to their broad selectivity, the extract matrix background is complex, greatly increasing the difficulty of subsequent enrichment and purification. Among the enrichment and purification methods, the most mature and widely used is solid phase extraction (SPE), which has the advantages of simple operation, good reproducibility, and easy automation. However, for orthodontic clear aligner samples with complex matrices, it is difficult to minimize background interference with only a single cleanup step, and the separation time is relatively long.

[0010] Magnetic solid-phase extraction (MSPE) combines the advantages of dispersive solid-phase extraction (DSPE) and magnetic separation. It separates the adsorbent from the solution using an external magnetic field, avoiding tedious centrifugation steps and shortening pretreatment time. Currently, polystyrene-divinylbenzene fillers and silica gel are the primary adsorbents used to enrich bisphenols and perfluorinated compounds. However, these adsorbents suffer from poor selectivity and low adsorption capacity. Complex matrices can limit the extraction efficiency of these adsorbents. Therefore, developing novel adsorbents with high selectivity and high adsorption capacity is crucial for extracting bisphenols and perfluorinated compounds from orthodontic clear aligners while minimizing matrix interference. Covalent organic frameworks (COFs) have great potential for application in sample pretreatment and chromatographic separations due to their large surface area, tunable pore size, and ease of functionalization.

[0011] In recent years, instrumental detection technologies have greatly improved. Methods such as gas chromatography-mass spectrometry (GC / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), liquid chromatography-mass spectrometry (LC / MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS) offer high sensitivity and accuracy, primarily for quantitative determination using external standard methods. Currently, these methods are widely used for trace analysis in various complex matrices, including food, biological samples, inorganic samples, and the environment. Therefore, establishing analytical methods with high recovery, good reproducibility, accuracy, and precision for the simultaneous detection of trace amounts of bisphenols and perfluorinated compounds in orthodontic clear aligners has become a key research priority. Summary of the Invention

[0012] Based on this, the purpose of the present invention is to provide a method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances. By using a high-performance liquid chromatography-tandem mass spectrometry detection method combined with a specific sample pretreatment technology, the bisphenol compounds and perfluorinated compounds in the orthodontic clear appliances can be qualitatively detected simultaneously, and the amount of bisphenol compounds (BPs) and perfluorinated compounds (PFASs) in the orthodontic clear appliances released into the human oral cavity after the user wears the orthodontic clear appliances for 2 weeks can be determined. The method established by the present invention has high sensitivity, low detection limit and short analysis time.

[0013] To achieve the above object, the present invention is implemented through the following technical solutions:

[0014] A method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances comprises the following steps:

[0015] S1: Sample preparation:

[0016] Orthodontic clear aligner samples were added to artificial saliva, incubated in a rotary shaker at 37°C ± 1°C for 2 weeks, and filtered to obtain a sample solution;

[0017] S2: Sample pretreatment:

[0018] The magnetic composite material Fe3O4@OH-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@OH-COF is separated from the solution; the separated magnetic composite material Fe3O4@OH-COF is eluted with an acetonitrile solution; and an eluate is obtained by separation;

[0019] S3: Detection and analysis:

[0020] The eluate was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0021] The method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances of the present invention comprises placing an orthodontic clear appliance sample in artificial saliva in step S1 and incubating the sample at 37°C±1°C for 2 weeks, thereby releasing the bisphenols and perfluorinated compounds in the orthodontic clear appliance sample into the sample solution. Step S1 simulates the environment in which the orthodontic clear appliance is worn in the human oral cavity, and simulates the release of the bisphenols and perfluorinated compounds in the orthodontic clear appliance sample into the human oral cavity. Then, in step S2, the magnetic composite material Fe3O4@OH-COF is used as a magnetic solid phase extraction adsorbent to efficiently enrich the bisphenol compounds and perfluorinated compounds in the sample solution; wherein the magnetic composite material Fe3O4@OH-COF contains a triazine ring and OH, the interaction between the triazine ring in the magnetic composite material Fe3O4@OH-COF and the perfluorinated compounds is a hydrophobic adsorption effect, and the interaction between OH in the magnetic composite material Fe3O4@OH-COF and the bisphenol compounds is a hydrogen bond effect, so the magnetic composite material Fe3O4@OH-COF can simultaneously adsorb bisphenol compounds and perfluorinated compounds; then, a desorption treatment is performed to elute the bisphenol compounds and perfluorinated compounds adsorbed on the magnetic composite material Fe3O4@OH-COF, so that the bisphenol compounds and perfluorinated compounds are transferred to the eluent. Finally, in step S3, the eluate is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry detection method, and the bisphenol compounds and perfluorinated compounds are qualitatively detected, and the content of bisphenol compounds and perfluorinated compounds in the sample solution is determined, so as to further obtain the amount of bisphenol compounds and perfluorinated compounds released into the artificial saliva by the orthodontic clear appliance sample, thereby simulating the amount released into the human oral cavity after the user wears the orthodontic clear appliance for 2 weeks in clinical practice.

[0022] The present invention utilizes a high-performance liquid chromatography-tandem mass spectrometry detection method in combination with a specific sample pretreatment technology to establish a magnetic solid phase extraction-high-performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method, which can simultaneously detect bisphenol compounds and perfluorinated compounds present in trace amounts in orthodontic clear appliances, and the established method has high sensitivity, a low detection limit, and a short analysis time. The method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances of the present invention can solve the problems of existing detection methods, namely, that bisphenol compounds and perfluorinated compounds are present in trace amounts in orthodontic clear appliances and cannot be detected simultaneously, resulting in a complicated and time-consuming detection process; and that the amount of bisphenol compounds and perfluorinated compounds released into the human oral cavity after a user wears the orthodontic clear appliances for two weeks in clinical practice cannot be determined.

[0023] Furthermore, in step S1, the amount of the orthodontic clear appliance sample added is preferably: 0.1 g of the orthodontic clear appliance sample is added to 1 mL of artificial saliva. That is, the mass-to-volume ratio of the orthodontic clear appliance sample to the artificial saliva is 0.1 g / mL, simulating the human oral cavity. This allows the bisphenol compounds and perfluorinated compounds present in trace amounts in the orthodontic clear appliance sample to be released into the sample solution without saturation after incubation in a rotary shaker at 37°C ± 1°C for 2 weeks, thereby improving the accuracy of subsequent testing.

[0024] Furthermore, in step S1, the orthodontic clear appliance sample is obtained by: cutting the orthodontic clear appliance into small discs using a special orthodontic punching pliers, wherein the small discs preferably have a diameter of 5 mm and a thickness of 0.75 mm; after washing and disinfecting, the orthodontic clear appliance sample is obtained. By cutting the orthodontic clear appliance into small discs, the contact area between the orthodontic clear appliance sample and artificial saliva is increased, thereby facilitating the transfer of bisphenol compounds and perfluorinated compounds in the orthodontic clear appliance sample into the artificial saliva to form a sample solution.

[0025] Furthermore, in step S2, the preparation method of the magnetic composite material Fe3O4@OH-COF includes the following steps:

[0026] Preparation of Fe3O4 magnetic nanoparticles: Add ferric chloride hexahydrate, sodium acetate, and ethylene glycol to a hydrothermal reactor, stir to obtain a yellow suspension, and react at 180-220°C for 6-10 hours. After the reaction, separate the precipitate, wash, and dry to obtain Fe3O4 magnetic nanoparticles.

[0027] Amino modification of Fe3O4 magnetic nanoparticles: The prepared Fe3O4 magnetic nanoparticles are dispersed in a mixed solution containing ethanol, water, and ammonia, and ethyl orthosilicate is added dropwise with stirring. The mixture is reacted at room temperature for 8-12 hours. After the reaction, the precipitate is separated, washed, and dried to obtain Fe3O4@SiO2. The Fe3O4@SiO2 is ultrasonically dispersed in anhydrous toluene, 3-aminopropyltriethoxysilane is added dropwise, and the mixture is refluxed at 100-140°C for 4-8 hours. After the reaction, the precipitate is separated, washed, and dried to obtain amino-modified Fe3O4 magnetic nanoparticles, i.e., Fe3O4@NH2.

[0028] Preparation of magnetic composite material Fe3O4@OH-COF: Fe3O4@NH2, 2,5-dihydroxyterephthalaldehyde, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane / toluene mixed solution and acetic acid solution were added to a thick-walled reaction bottle, ultrasonically treated for 4-6 minutes, and reacted at 100-140°C for 60-80 hours; after the reaction, the precipitate was separated, washed, and dried to obtain the magnetic composite material Fe3O4@OH-COF.

[0029] In the preparation method of the magnetic composite material Fe3O4@OH-COF of the present invention, in the step of preparing the magnetic composite material Fe3O4@OH-COF, Fe3O4@NH2, 2,5-dihydroxyterephthalaldehyde, and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine are reacted together (Schiff base reaction), so that the prepared magnetic composite material Fe3O4@OH-COF has a triazine ring and OH, and can simultaneously adsorb bisphenol compounds and perfluorinated compounds by utilizing its structural characteristics, wherein the triazine ring in the magnetic composite material Fe3O4@OH-COF can form a hydrophobic adsorption effect with the perfluorinated compound, and the OH in the magnetic composite material Fe3O4@OH-COF forms a hydrogen bond with the bisphenol compound, so the magnetic composite material Fe3O4@OH-COF can simultaneously adsorb bisphenol compounds and perfluorinated compounds, and can simultaneously and efficiently enrich the bisphenol compounds and perfluorinated compounds in the sample solution. The magnetic composite material Fe3O4@OH-COF introduces Fe3O4 magnetic nanoparticles, which saves time compared to traditional dispersed extraction and solid phase extraction, while reducing the use of organic solvents and improving economic efficiency.

[0030] Furthermore, in the step of preparing Fe3O4 magnetic nanoparticles, the mass volume ratio of ferric chloride hexahydrate, sodium acetate and ethylene glycol is 2-3:4-5:35-40, g / g / mL.

[0031] Furthermore, in the step of amino modification of Fe3O4 magnetic nanoparticles, the volume ratio of ethanol, water and ammonia in the mixed solution of ethanol, water and ammonia is 50:10:1.2; in the step of preparing the magnetic composite material Fe3O4@OH-COF, the volume ratio of 1,4-dioxane and toluene in the 1,4-dioxane / toluene mixed solution is 4:1.

[0032] Furthermore, in both the steps of preparing Fe3O4 magnetic nanoparticles and the step of amino-modifying the Fe3O4 magnetic nanoparticles, washing was performed alternately with water and ethanol, and drying was performed at 80°C. In the step of preparing the magnetic composite material Fe3O4@OH-COF, washing was performed alternately with methanol and water. The washing and drying operations removed any unreacted reagents from the corresponding precipitates.

[0033] Furthermore, in step S3, in the high performance liquid chromatography-tandem mass spectrometry detection method:

[0034] Chromatographic conditions were: a Waters C18 column, 50 × 2.1 mm, 1.9 μm; mobile phase: A: 0.1% formic acid in water, B: methanol; gradient elution: 0-1 min, 3% B; 1-3 min, 3-20% B; 3-5 min, 20-97% B; 5-5.5 min, 97% B; 5.5-9 min, 97-3% B; flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 5 μL. Mass spectrometry conditions were: ionization mode: H-ESI+; spray voltage: 3500 V; ion source temperature: 350°C; ion transfer tube temperature: 320°C; sheath gas: 35 Arb; auxiliary gas: 10 Arb; sweep gas: 0 Arb.

[0035] Furthermore, the bisphenol compounds include bisphenol A (BPA), bisphenol B (BPB), and bisphenol F (BPF); the perfluorinated compounds include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), and perfluorotridecanoic acid (PFTriDA). The method of the present invention can be used to simultaneously detect BPA, BPB, BPF, PFBA, PFPeA, PFHxA, PFHpA, and PFTriDA in orthodontic clear aligners. It should be noted that the components listed above do not represent a limitation of this method.

[0036] Furthermore, in step S3, after the eluate is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry, the content of bisphenols and perfluorinated compounds in the sample solution is determined, thereby determining the amount of bisphenols and perfluorinated compounds released by the orthodontic clear aligner sample. Wherein: the step of determining the content of bisphenols and perfluorinated compounds in the sample solution can adopt a conventional analysis method: for example, under the same conditions, a series of standard solutions of bisphenols and perfluorinated compounds with concentration gradients are prepared respectively, added to artificial saliva, and incubated in a rotary shaker at 37°C ± 1°C for 2 weeks to obtain a standard sample solution; and the same sample pretreatment and detection and analysis steps are performed, a standard curve of the concentration-peak area of ​​the standard sample solution is drawn and the corresponding linear regression equation is obtained; the peak area value obtained by the sample detection and analysis is substituted into the corresponding linear regression equation to obtain the content of bisphenols and perfluorinated compounds in the sample solution. The above are all operations that can be performed by those skilled in the art and will not be repeated here.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances. The method simulates the human oral environment to release the bisphenols and perfluorinated compounds in the orthodontic clear appliance into a sample solution. The method utilizes the property of the magnetic composite material Fe3O4@OH-COF that can simultaneously adsorb bisphenols and perfluorinated compounds to efficiently enrich the bisphenols and perfluorinated compounds in the sample solution. A magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method is established to simultaneously and qualitatively detect the bisphenols and perfluorinated compounds in the orthodontic clear appliance and determine the content of the bisphenols and perfluorinated compounds in the sample solution. The amount of bisphenols and perfluorinated compounds released into artificial saliva from the orthodontic clear appliance sample is then determined, thereby simulating the amount released into the human oral cavity after a user wears the orthodontic clear appliance for two weeks in clinical practice. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances of the present invention has high sensitivity, low detection limit and short analysis time.

[0039] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The BPA related spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention are shown;

[0041] Figure 2The BPB correlation spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention are shown;

[0042] Figure 3 The BPF spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention are shown;

[0043] Figure 4 PFBA correlation spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention;

[0044] Figure 5 PFPeA correlation spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention;

[0045] Figure 6 PFHxA correlation spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention;

[0046] Figure 7 PFHpA correlation spectra obtained after HPLC-MS / MS testing of four different samples in Example 1 of the present invention;

[0047] Figure 8 PFTriDA correlation spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is described in detail below by specific embodiments and accompanying drawings. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0049] Example 1

[0050] Since bisphenol compounds and perfluorinated compounds exist in trace amounts in orthodontic clear aligners, it is difficult to detect them using sensitive analytical instruments, so sample pretreatment technology is needed.

[0051] This embodiment provides a method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances, which specifically includes the following steps:

[0052] S1 sample preparation:

[0053] (1) Use a special orthodontic punching pliers to cut the orthodontic clear appliance into small discs for later use; the diameter of the small discs is 5 mm and the thickness is 0.75 mm;

[0054] (2) Weigh 1 g of the small disc, wash it with ultrapure water, and sterilize it with 115V ultraviolet light in a biosafety cabinet for 15 minutes;

[0055] (3) The small disc was then turned over with sterile tweezers and sterilized in a container for another 15 minutes to obtain an orthodontic clear aligner sample;

[0056] (4) Place 1 g of orthodontic clear aligner sample in 10 mL of artificial saliva at a ratio of 0.1 g / mL, incubate in a rotary shaker (230 rpm) at 37 °C for 2 weeks, and filter to obtain the sample solution;

[0057] S2 sample pretreatment:

[0058] S21 Preparation of magnetic composite material Fe3O4@OH-COF:

[0059] ① Preparation of Fe3O4 magnetic nanoparticles (MNPs):

[0060] Ferric chloride hexahydrate (2.8 g), sodium acetate (4.9 g), and ethylene glycol (40 mL) were added to a hydrothermal reactor and stirred to obtain a yellow suspension. The suspension was then reacted at 200°C for 8 hours. After the reaction, the precipitate was collected using a magnet and washed alternately with ultrapure water and ethanol to remove unreacted reagents. The washed precipitate was then vacuum-dried at 80°C to obtain Fe3O4 magnetic nanoparticles (MNPs).

[0061] ②Amino modification of Fe3O4 magnetic nanoparticles (MNPs):

[0062] The prepared Fe3O4 magnetic nanoparticles (MNPs) were dispersed in a mixture of 50 mL of ethanol, 10 mL of ultrapure water, and 1.2 mL of ammonia. 0.4 mL of tetraethyl orthosilicate was slowly added dropwise under mechanical stirring, and the reaction was allowed to proceed at room temperature for 8 hours. After the reaction, the precipitate was collected by filtration or magnetism and washed alternately with ultrapure water and ethanol to remove unreacted reagents. The washed precipitate was vacuum-dried at 80°C to obtain Fe3O4@SiO2. Subsequently, 0.2 g of Fe3O4@SiO2 was ultrasonically dispersed in 130 mL of anhydrous toluene, and 17 mL of 3-aminopropyltriethoxysilane was added dropwise. The reaction was refluxed at 120°C for 6 hours. After the reaction, the precipitate was collected by magnetism and washed alternately with ultrapure water and ethanol to remove unreacted reagents. The washed precipitate was vacuum-dried at 80°C to obtain amino-modified Fe3O4 magnetic nanoparticles (Fe3O4@NH2).

[0063] ③ Preparation of magnetic composite material Fe3O4@OH-COF:

[0064] Fe₃O₄@NH₂ (50 mg), 2,5-dihydroxyterephthalaldehyde (DHPA) (18.9 mg), 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) (26.4 mg), a 1,4-dioxane / toluene mixture (6 mL, 4:1, v / v), and acetic acid (0.6 mL, 3 mol / L) were added to a thick-walled reaction flask. The mixture was sonicated for 5 minutes and reacted at 120°C for 60 hours. After completion of the reaction, the product was collected with a magnet and washed alternately with methanol and ultrapure water. It was then dried in vacuum for 12 hours to obtain the magnetic composite Fe₃O₄@OH-COF.

[0065] (Before preparing the magnetic composite material Fe3O4@OH-COF, in order to facilitate the use of the optimized OH-COF synthesis conditions to prepare the magnetic composite material Fe3O4@OH-COF, this embodiment also selected the optimized OH-COF synthesis conditions: 2,5-dihydroxyterephthalaldehyde (2,5-Dihydroxyterephthalaldehyde, DHPA) (18.9 mg), 2,4,6-tris (4-aminophenyl) -1,3,5-triazine (4,4',4''-(1,3,5-Triazine-2,4,6-triyl)trianiline, TAPT) (26.4 mg), anhydrous ethanol / 1,2-dichlorobenzene mixed solution (2 mL, 1:1, v / v) and acetic acid solution (0.2 mL, 12 mol / L) was added to a centrifuge tube, sonicated for 10 minutes, and then reacted at 125°C for 72 hours. After the reaction, the precipitate was collected by centrifugation and washed alternately with 1,2-dichlorobenzene and methanol to remove unreacted reagents. The washed precipitate was vacuum-dried at 60°C for 12 hours to obtain OH-COF. Research has found that OH-COF can be stably and efficiently synthesized under the above-mentioned synthesis conditions. Therefore, this example makes corresponding adjustments based on this, designing the above-mentioned step "③ Preparation of the Magnetic Composite Material Fe3O4@OH-COF". Using the optimized OH-COF synthesis conditions, Fe3O4@NH2, 2,5-dihydroxyterephthalaldehyde, and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were used as monomers and subjected to a Schiff base reaction to obtain the magnetic composite material Fe3O4@OH-COF.

[0066] S22 Magnetic Solid Phase Extraction:

[0067] 10 mg of the Fe3O4@OH-COF magnetic composite was added to 15 mL of the sample solution. After vortexing for 5 minutes, the Fe3O4@OH-COF was collected from the solution using a magnet and the supernatant removed. Next, 1.0 mL of acetonitrile was added to the centrifuge tube and vortexed for 1 minute to elute the bisphenols and perfluorinated compounds adsorbed on the Fe3O4@OH-COF. Finally, the filtered Fe3O4@OH-COF was washed alternately with 2 mL of acetonitrile and ultrapure water, then dried for subsequent use.

[0068] S3 detection analysis:

[0069] A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection and analysis method was established, and the eluate was analyzed instrumentally to simultaneously detect bisphenols and perfluorinated compounds. The contents of bisphenols and perfluorinated compounds in the sample solution and the amounts of bisphenols and perfluorinated compounds released from orthodontic clear aligner samples were determined.

[0070] Since the instruments used in the high performance liquid chromatography-tandem mass spectrometry method can be selected from existing conventional high performance liquid chromatography equipment, the operating methods of high performance liquid chromatography-tandem mass spectrometry analysis are all conventional methods. Therefore, except for the chromatographic conditions and mass spectrometry conditions in this embodiment, they will not be described in detail in the present invention specification.

[0071] Chromatographic conditions:

[0072] Column: Waters C18 (50×2.1 mm, 1.9 μm)

[0073] The mobile phase composition and gradient elution conditions are shown in Table 1

[0074] Table 1 Mobile phase composition and gradient elution

[0075]

[0076] Mass spectrometry conditions:

[0077] Mass spectrometry parameters are shown in Table 2

[0078] Table 2 Mass spectrometry parameter settings

[0079]

[0080] The method for detecting bisphenols and perfluorinated compounds in orthodontic clear aligners of this embodiment includes bisphenol compounds detected including bisphenol A (BPA), bisphenol B (BPB), and bisphenol F (BPF); the perfluorinated compounds detected include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), and perfluorotridecanoic acid (PFTriDA).

[0081] In this example, the above method was used to establish detection methods for three bisphenol compounds (BPA, BPB, and BPF) and five perfluorinated compounds (PFBA, PFPeA, PFHxA, PFHpA, and PFTriDA). Figures 1-8Four different samples were tested by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) under the above-mentioned chromatographic and mass spectrometric conditions: 2 ng / g standard of each substance, eluate obtained by pre-treatment after adding 2 ng / g standard of each substance to the sample solution, eluate obtained after step S2 (actual sample after pre-treatment), and sample solution obtained after step S1 (actual sample before pre-treatment), and the spectra related to each substance were obtained. Figure 1-8 It can be seen that the chromatographic peak shapes of the three bisphenol compounds and the five perfluorinated compounds are good. Under the current chromatographic conditions and mass spectrometry conditions, the three bisphenol compounds and the five perfluorinated compounds are well separated.

[0082] Example 2

[0083] Methodological evaluation:

[0084] In order to evaluate the analytical performance of the magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method established in the present invention, this example also prepared mother solutions with a concentration of 20 ng / g for the three bisphenol compounds (BPA, BPB, BPF) and the five perfluorinated compounds (PFBA, PFPeA, PFHxA, PFHpA, PFTriDA), and successively diluted them with methanol to a series of standard working concentrations of 0.1 ng / g, 0.6 ng / g, 0.8 ng / g, 2 ng / g, 5 ng / g, and 10 ng / g, respectively. The same sample pretreatment method as in Example 1 was used for magnetic solid phase extraction, and the corresponding eluates were detected by high performance liquid chromatography-tandem mass spectrometry using the same detection and analysis method under the same chromatographic and mass spectrometric conditions. The linear range, detection limit, quantification limit, and relative standard deviation of the method were investigated. The results are shown in Table 3, where x represents the concentration of the analyte and y represents the peak area of ​​the analyte. The results showed that the method showed good linearity in the range of 0.10-20 ng / g (R 2 ≥0.9982), LODs and LOQs were between 0.009-0.025 ng / g and 0.032-0.085 ng / g, respectively. The intra-day precision and inter-day precision were less than 10%, indicating that this method has a low detection limit.

[0085] Table 3 MSPE-HPLC-MS / MS method parameters

[0086]

[0087] Example 3

[0088] Accuracy of the method:

[0089] The target compounds (three bisphenols and five perfluorinated compounds) were spiked into blank samples (untreated sample solutions) at three concentration levels (0.5, 2.0, and 10.0 ng / g), and their spiked recoveries were determined. The results are shown in Table 4. The spiked recoveries of the bisphenols and perfluorinated compounds in the samples ranged from 87.6% to 103.2%, with RSDs ≤ 6.3%. This method demonstrates that it can accurately determine the bisphenols and perfluorinated compounds in orthodontic clear aligner sample solutions and, consequently, accurately determine the amounts of bisphenols and perfluorinated compounds released from the orthodontic clear aligner samples.

[0090] Table 4. Recovery rate of spiked samples from MSPE-HPLC-MS / MS experiments

[0091]

[0092] Example 4

[0093] Actual sample test results:

[0094] The results of the actual sample detection in Example 1 are shown in Table 5. These results indicate that the established magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method can be used to detect bisphenol compounds and perfluorinated compounds in orthodontic clear aligners. The actual results are the amount of bisphenol compounds and perfluorinated compounds released into artificial saliva in ng of the actual sample of orthodontic clear aligners.

[0095] Table 5 Test results of actual samples

[0096]

[0097] The present invention provides a method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances. By simulating the human oral environment, the bisphenols and perfluorinated compounds in the orthodontic clear appliances are released into a sample solution. The magnetic composite material Fe3O4@OH-COF is capable of simultaneously adsorbing these compounds, effectively enriching them in the sample solution. A magnetic solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method is developed to simultaneously and qualitatively detect the bisphenols and perfluorinated compounds in the orthodontic clear appliances. The content of these compounds in the sample solution is then determined, and the amount of bisphenols and perfluorinated compounds released from the orthodontic clear appliance sample into artificial saliva is further determined. This method can then be used to simulate the amount released into the human oral cavity after a user wears the orthodontic clear appliance for two weeks. The present invention provides a method for detecting bisphenols and perfluorinated compounds in orthodontic clear appliances, with high sensitivity, a low detection limit, and a short analysis time.

[0098] The above-described embodiments merely represent several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, and the present invention is intended to encompass such modifications and variations.

Claims

1. A method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances, characterized in that: The bisphenol compounds include bisphenol A, bisphenol B, and bisphenol F; the perfluorinated compounds include perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorotridecanoic acid; and the detection method includes the following steps: S1: Sample preparation: The orthodontic clear appliance sample was added to artificial saliva, incubated in a rotary shaker at 37°C ± 1°C for 2 weeks, and filtered to obtain a sample solution; S2: Sample preparation: The magnetic composite material Fe3O4@OH-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@OH-COF is separated from the solution; the separated magnetic composite material Fe3O4@OH-COF is eluted with an acetonitrile solution; and an eluent is obtained by separation; In step S2, the method for preparing the magnetic composite material Fe3O4@OH-COF comprises the following steps: Preparation of Fe3O4 magnetic nanoparticles: adding ferric chloride hexahydrate, sodium acetate and ethylene glycol into a hydrothermal reactor, stirring to obtain a yellow suspension, and reacting at 180-220°C for 6-10 hours; after the reaction, separating the precipitate, washing, and drying to obtain Fe3O4 magnetic nanoparticles; Amino modification of Fe3O4 magnetic nanoparticles: the prepared Fe3O4 magnetic nanoparticles are dispersed in a mixed solution containing ethanol, water and ammonia water, and ethyl orthosilicate is added dropwise under stirring, and the mixture is reacted at room temperature for 8-12 hours; after the reaction, the precipitate is separated, washed, and dried to obtain Fe3O4@SiO2; Fe3O4@SiO2 is ultrasonically dispersed in anhydrous toluene, 3-aminopropyltriethoxysilane is added dropwise, and the mixture is refluxed at 100-140°C for 4-8 hours. After the reaction, the precipitate is separated, washed, and dried to obtain amino-modified Fe3O4 magnetic nanoparticles, namely Fe3O4@NH2; Preparation of magnetic composite material Fe3O4@OH-COF: Fe3O4@NH2, 2,5-dihydroxyterephthalaldehyde, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane / toluene mixed solution and acetic acid solution are added into a thick-walled reaction bottle, ultrasonically treated for 4-6 minutes, and reacted at 100-140°C for 60-80 hours; after the reaction, the precipitate is separated, washed and dried to obtain the magnetic composite material Fe3O4@OH-COF; S3: Detection and analysis: The eluate was detected and analyzed using high performance liquid chromatography-tandem mass spectrometry; In step S3, in the high performance liquid chromatography-tandem mass spectrometry detection method: The chromatographic conditions were as follows: Waters C18 column, 50×2.1 mm, 1.9 μm; mobile phase: A was 0.1% formic acid aqueous solution, B was methanol solution; gradient elution: 0-1 min, 3% B; 1-3 min, 3-20% B; 3-5 min, 20-97% B; 5-5.5 min, 97% B; 5.5-9 min, 97-3% B; flow rate was 0.2 mL / min; column temperature was 40°C; injection volume was 5 μL.

2. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In step S1, the amount of the orthodontic transparent appliance sample added is: 0.1 g of the orthodontic transparent appliance sample is added to every 1 mL of artificial saliva.

3. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In step S1, the orthodontic transparent appliance sample is obtained by the following method: the orthodontic transparent appliance is cut into small discs with a special orthodontic punching pliers, the diameter of the small disc is 5 mm, and the thickness is 0.75 mm; after washing and disinfection, the orthodontic transparent appliance sample is obtained.

4. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In the step of preparing Fe3O4 magnetic nanoparticles, the mass volume ratio of ferric chloride hexahydrate, sodium acetate and ethylene glycol is 2-3:4-5:35-40, g / g / mL.

5. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In the step of amino modification of Fe3O4 magnetic nanoparticles, the volume ratio of ethanol, water and ammonia water in the mixed solution of ethanol, water and ammonia water is 50:10:1.2; in the step of preparing the magnetic composite material Fe3O4@OH-COF, the volume ratio of 1,4-dioxane and toluene in the 1,4-dioxane / toluene mixed solution is 4:

1.

6. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In the steps of preparing Fe3O4 magnetic nanoparticles and in the step of amino modification of Fe3O4 magnetic nanoparticles, washing is performed alternately using water and ethanol, and drying is performed at 80°C; in the step of preparing the magnetic composite material Fe3O4@OH-COF, washing is performed alternately using methanol and water.

7. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In step S3, in the high performance liquid chromatography-tandem mass spectrometry detection method: The mass spectrometry conditions were as follows: ionization mode H-ESI+; spray voltage 3500 V; ion source temperature 350°C; ion transfer tube temperature 320°C; sheath gas 35 Arb; auxiliary gas 10 Arb; and sweep gas 0 Arb.

8. The method for detecting bisphenols and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In step S3, after the eluent is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry, the content of bisphenol compounds and perfluorinated compounds in the sample solution is determined, thereby determining the amount of bisphenol compounds and perfluorinated compounds released by the orthodontic clear appliance sample.

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