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

Abstract

The present invention relates to a method for detecting perfluorinated and hexabromocyclododecane 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@F‑COF to the sample solution, and vortexing; separating the magnetic composite material Fe3O4@F‑COF from the solution; eluting the separated magnetic composite material Fe3O4@F‑COF with a methanol 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 perfluorinated and hexabromocyclododecane compounds released by an orthodontic transparent appliance, with high sensitivity, low detection limit, and short analysis time.

Description

Technical Field

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

[0002] Perfluoroalkyl and polyfluoroalkyl substances (PFASs), also known as perfluorinated compounds, are a class of synthetic organic compounds. They are widely used in consumer products such as non-stick cookware, stain-resistant textiles, and food packaging materials due to their high surface tension and excellent chemical and thermal stability. However, these consumer products will continuously release PFASs into the environment during the production and use process. A large number of studies have shown that PFASs have multiple biological toxicities such as neurotoxicity, immunotoxicity, hepatotoxicity, reproductive toxicity, and genotoxicity. At present, PFASs have been detected in various environmental media and in animals and plants.

[0003] Hexabromocyclododecane (HBCD) is a type of brominated flame retardant. HBCDs refers to a mixture of isomers of hexabromocyclododecane, also known as hexabromocyclododecane compounds. Hexabromocyclododecane compounds are widely used in polystyrene foam, electronic products and acrylonitrile-styrene-butadiene polymer (ABS) and other fields. Due to its excellent cost-effectiveness, it occupies an important position in the field of flame retardancy. So far, it is difficult to find its substitute in many fields. In recent years, researchers have detected brominated flame retardants in human breast milk, crayfish, fish feed and children's toys. Due to its stability and lipophilicity, HBCDs can accumulate in organisms and produce a biomagnification effect. Therefore, even a trace amount of HBCDs entering the environment can cause harm to organisms at the end of the food chain. The human body can ingest HBCDs through air, dust and food, and then transmit it to the whole body through blood circulation and accumulate in fat. Current studies have found that HBCDs have toxic effects on organisms, including neurotoxicity, hepatotoxicity, reproductive and developmental toxicity, and endocrine disruption. They are potentially persistent and bioaccumulative compounds and are receiving increasing attention from researchers.

[0004] With the rapid advancement of biomaterials, computer-aided design (CAD) and manufacturing (CAM) technologies, clear aligner treatment has become a promising alternative to traditional fixed braces in orthodontics. The global orthodontic clear aligner market size is expected to grow from $3.1 billion in 2021 to $11.6 billion in 2027, at a compound annual growth rate (CAGR) of 13%. Orthodontic clear aligners typically consist of a series of clear plastic trays that fit snugly over the teeth and are used by patients at all times except for eating and brushing, and are typically replaced every one to two weeks to complete the planned orthodontic tooth movement. Although there are a variety of commercial orthodontic clear aligner systems available worldwide today, the use of clear thermoformed plastic materials to manufacture orthodontic clear aligners remains a common feature. The most commonly used polymers (alone or in blends) used to manufacture commercial orthodontic clear aligners include polyesters, polyurethanes or copolyesters, polypropylene, polycarbonate, ethylene vinyl acetate, and polyvinyl chloride, among others. Although clear aligner materials are marketed as intraoral appliance materials as being free of hazardous substances, not expected to leach any potential toxins that could produce adverse local or systemic reactions, not carcinogenic, and not likely to produce any developmental defects, these polymers contain various additives to enhance specific properties such as performance, stability, elasticity, etc. Furthermore, the paucity of evidence-based literature due to the paucity of controlled trials on clear aligners and conflicting results reported in the published literature continues to fuel the debate regarding the potential toxic effects of using clear aligners. Furthermore, with the continued influx of multiple commercial aligner systems, there is a need to continue testing the cytotoxicity of orthodontic clear aligners produced by different manufacturers.

[0005] Orthodontic transparent appliances are essential instruments in oral 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 transparent appliances need to be worn in the user's mouth for 2 weeks before replacing the next pair of new orthodontic transparent appliances. The basic components of the materials of orthodontic transparent appliances are polymers, such as polyethylene (PE), polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyurethane (PU) and polypropylene (PP). These polymers contain various additives, and PFASs and HBCDs may be one of these additives, which are used to enhance the comprehensive performance of the materials. These two types of additives in orthodontic transparent appliances are easily released into the human mouth during the two-week clinical wearing process of the user. When a certain concentration accumulates in the human body, it is easy to cause harm to the human body. Due to the hazards and difficulty in degradation of these two types of pollutants, the demand for the determination of the content of different PFASs and HBCDs in orthodontic transparent appliances is becoming more and more urgent. In particular, when patients wear orthodontic clear aligners, it is necessary to understand how much PFASs and HBCD will be released into the human mouth.

[0006] Since PFASs and HBCDs are often present 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 will also cause difficulties in detection. Sample pretreatment, enrichment and purification must be performed before detection. Existing methods generally use liquid-liquid extraction, freeze-drying and other methods for sample pretreatment for perfluorinated compounds or hexabromocyclododecane compounds. The operation is not only cumbersome, but also time-consuming and cannot meet the requirements of rapid analysis. In addition, due to the large differences in the physical and chemical properties of PFASs and HBCDs, simultaneous extraction and enrichment cannot be achieved. These two types of pollutants need to be extracted separately, which increases the workload and analysis cost. 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 PFASs and HBCDs 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 treatment process, which can not only simplify the sample pretreatment steps and improve the analysis efficiency, but also reduce the use of reagents and reduce the analysis cost.

[0007] At present, the detection of PFASs and HBCDs is mostly concentrated on surface water, sediments, sewage effluent, etc., while there are few studies on orthodontic clear aligner samples. This is mainly because the matrix of orthodontic clear aligners is complex and the interference is serious. The difficulty of pre-analysis treatment is much greater than the detection of PFASs and HBCDs in water bodies. In addition, the current technology only targets one type of components. How to efficiently extract PFASs and HBCDs from orthodontic clear aligner samples has become a bottleneck in the analysis of trace pollutants.

[0008] Among the commonly used extraction methods for PFASs and HBCDs in transparent aligners, the use of rapid solvent extraction or ultrasonic extraction alone has the advantages of short analysis time, less use of toxic solvents, and high extraction efficiency. However, due to the wide selectivity, the background of the extract matrix is ​​complex, which greatly increases 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 is a method that uses the special interaction between the target compound and the functional groups of the SPE adsorbent to achieve sample purification. It has the advantages of simple operation, good repeatability, and easy automation. However, for orthodontic transparent aligner samples with complex matrices, it is difficult to minimize background interference by only one-step purification, and the separation time is relatively long. In order to improve the selectivity and purification effect of the adsorbent, new materials such as carbon nanotubes, titanium dioxide nanotubes, molecularly imprinted polymers, graphene, and metal-organic frameworks have been used as SPE adsorbents.

[0009] Magnetic solid-phase extraction (MSPE) combines the advantages of dispersed solid-phase extraction and magnetic separation. It separates the adsorbent from the solution by applying an external magnetic field, avoiding the tedious centrifugation step and shortening the pretreatment time. At present, the main adsorbents used for enriching PFASs and HBCDs are polystyrene-divinylbenzene fillers and silica gel, but they have disadvantages such as poor selectivity and low adsorption capacity. When faced with complex matrices, the extraction efficiency of these adsorbents will be limited. Therefore, in order to extract PFASs and HBCDs in orthodontic clear aligners and reduce the interference of the matrix, it is very important to develop new adsorbents with high selectivity and high adsorption capacity. Covalent organic frameworks (COFs) have great application potential in sample pretreatment and chromatographic separation due to their large specific surface area, adjustable pore size, and easy functionalization.

[0010] In recent years, instrument detection technology has been greatly improved, such as gas chromatography-mass spectrometry (GC / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), liquid chromatography-mass spectrometry (LC / MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc., which have high sensitivity and accuracy, and are mainly used for quantitative determination by external standard method. At present, it has been widely used in trace analysis of various complex matrices such as food, biological samples, inorganic samples, and environment. Therefore, it is the key to establish an analytical method with high recovery rate, good reproducibility, high accuracy and precision for the simultaneous detection of trace PFASs and HBCDs in orthodontic clear aligners. Summary of the invention

[0011] Based on this, the purpose of the present invention is to provide a method for detecting perfluorinated compounds and hexabromocyclododecane compounds in orthodontic transparent appliances. By using a high performance liquid chromatography-tandem mass spectrometry detection method combined with a specific sample pretreatment technology, the perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliances can be qualitatively detected simultaneously, and the amount of fluorinated compounds (PFASs) and hexabromocyclododecane compounds (HBCDs) in the orthodontic transparent appliances released into the human oral cavity after the user wears the orthodontic transparent appliances for 2 weeks can be determined; the method established by the present invention has high sensitivity, low detection limit and short analysis time.

[0012] To achieve the above object, the present invention is implemented through the following technical solutions:

[0013] A method for detecting perfluorinated and hexabromocyclododecane compounds in orthodontic transparent appliances comprises the following steps:

[0014] S1: Sample preparation:

[0015] 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;

[0016] S2: Sample preparation:

[0017] The magnetic composite material Fe3O4@F-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@F-COF is separated from the solution; the separated magnetic composite material Fe3O4@F-COF is eluted with a methanol solution; and an eluent is obtained by separation;

[0018] S3: Detection and analysis:

[0019] The eluate was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0020] The method for detecting perfluorinated and hexabromocyclododecane compounds in an orthodontic transparent appliance of the present invention comprises the following steps: placing an orthodontic transparent appliance sample in artificial saliva and incubating the sample in an environment of 37°C±1°C for 2 weeks in step S1, so that the perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance sample are released into the sample solution; thereby simulating the environment in which the orthodontic transparent appliance is worn in the human oral cavity through S1, and simulating the release of the perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance sample into the human oral cavity. Then, in step S2, the magnetic composite material Fe3O4@F-COF is used as a magnetic solid phase extraction adsorbent to efficiently enrich the perfluorinated compounds and hexabromocyclododecane compounds in the sample solution; wherein the magnetic composite material Fe3O4@F-COF contains a triazine ring and F, the interaction between the triazine ring in the magnetic composite material Fe3O4@F-COF and the hexabromocyclododecane compounds is a hydrophobic adsorption effect, and the interaction between F in the magnetic composite material Fe3O4@F-COF and the perfluorinated compounds is an FF adsorption effect, so the magnetic composite material Fe3O4@F-COF can adsorb perfluorinated compounds and hexabromocyclododecane compounds at the same time; then a desorption treatment is performed to elute the perfluorinated compounds and hexabromocyclododecane compounds adsorbed on the magnetic composite material Fe3O4@F-COF, so that the perfluorinated compounds and hexabromocyclododecane compounds are transferred to the eluent. Finally, in step S3, the eluate is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry, and the perfluorinated compounds and hexabromocyclododecane compounds are qualitatively identified, and the contents of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution are determined, so as to further obtain the amount of perfluorinated compounds and hexabromocyclododecane compounds released into the artificial saliva by the orthodontic transparent appliance sample, thereby simulating the amount released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice.

[0021] The present invention uses 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 perfluorinated compounds and hexabromocyclododecane compounds that exist in trace amounts in orthodontic transparent appliances, and the established method has high sensitivity, low detection limit, and short analysis time; the detection method of perfluorinated compounds and hexabromocyclododecane compounds in orthodontic transparent appliances of the present invention can solve the problems in the existing detection method that perfluorinated compounds and hexabromocyclododecane compounds exist in trace amounts in orthodontic transparent appliances and cannot be detected simultaneously, resulting in a complicated detection process and a long time consumption; and the problem that the amount of the orthodontic transparent appliance released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice cannot be determined.

[0022] Furthermore, in step S1, the amount of the orthodontic transparent appliance sample added is preferably: 0.1 g of the orthodontic transparent appliance sample is placed in every 1 mL of artificial saliva. That is, the mass volume ratio of the orthodontic transparent appliance sample to the artificial saliva is placed at a ratio of 0.1 g / mL, simulating the human oral cavity, so that the perfluorinated compounds and hexabromocyclododecane compounds present in trace amounts in the orthodontic transparent appliance sample are 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 detection.

[0023] Furthermore, in step S1, the orthodontic transparent appliance sample is obtained by the following method: the orthodontic transparent appliance is cut into small discs using a special orthodontic punching pliers, the diameter of the small disc is preferably 5 mm, and the thickness is preferably 0.75 mm; after washing and disinfection, the orthodontic transparent appliance sample is obtained. By cutting the orthodontic transparent appliance into small discs, the contact area between the orthodontic transparent appliance sample and the artificial saliva is increased, so that the perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance sample are easily transferred to the artificial saliva to form a sample solution.

[0024] Further, in step S2, the method for preparing the magnetic composite material Fe3O4@F-COF comprises the following steps:

[0025] 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;

[0026] 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;

[0027] Preparation of magnetic composite material Fe3O4@F-COF: Fe3O4@NH2, 2,3,5,6-tetrafluorobenzaldehyde, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane / o-dichlorobenzene mixed solution and acetic acid solution were added to a reaction bottle, ultrasonically treated for 8-12 minutes, and reacted at room temperature for 60-80 hours; after the reaction, the precipitate was separated, washed, and dried to obtain the magnetic composite material Fe3O4@F-COF.

[0028] In the method for preparing the magnetic composite material Fe3O4@F-COF of the present invention, in the step of preparing the magnetic composite material Fe3O4@F-COF, Fe3O4@NH2, 2,3,5,6-tetrafluorobenzaldehyde 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@F-COF has a triazine ring and F, and can simultaneously adsorb perfluorinated compounds and hexabromocyclododecane compounds by utilizing its structural characteristics, wherein the triazine ring in the magnetic composite material Fe3O4@F-COF can form a hydrophobic effect with the hexabromocyclododecane compound, and the F in the magnetic composite material Fe3O4@F-COF forms an FF effect with the perfluorinated compound, so the magnetic composite material Fe3O4@F-COF can simultaneously adsorb perfluorinated compounds and hexabromocyclododecane compounds, and can simultaneously and efficiently enrich the perfluorinated compounds and hexabromocyclododecane compounds in the sample solution. The magnetic composite material Fe3O4@F-COF introduces Fe3O4 magnetic nanoparticles, which saves time compared to traditional dispersed extraction and solid phase extraction, reduces the use of organic solvents, and improves economic efficiency.

[0029] 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.

[0030] Furthermore, 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 20:10:1.2; in the step of preparing the magnetic composite material Fe3O4@F-COF, the volume ratio of 1,4-dioxane and o-dichlorobenzene in the 1,4-dioxane / o-dichlorobenzene mixed solution is 2:1.

[0031] Furthermore, in the steps of preparing Fe3O4 magnetic nanoparticles and the step of amino modification of Fe3O4 magnetic nanoparticles, washing is performed by alternating water and ethanol, and drying is performed at 80°C; in the step of preparing the magnetic composite material Fe3O4@F-COF, washing is performed by alternating methanol and water. The unreacted reagents on the corresponding precipitates are removed by the washing and drying operations.

[0032] Further, in step S3, in the high performance liquid chromatography-tandem mass spectrometry detection method:

[0033] The chromatographic conditions were as follows: Hypersil GOLD C18 column, size 50×2.1 mm, 1.9 μm; mobile phase: A was 10 mmol ammonium acetate aqueous solution, B was methanol solution; gradient elution: 0-1 min, 5% B; 1-3 min, 5-20% B; 3-5 min, 20-95% B; 5-5.5 min, 95% B; 5.5-9 min, 95-5% B; flow rate was 0.3 mL / min; column temperature was 40 °C; injection volume was 5 μL; mass spectrometry conditions were as follows: ionization mode H-ESI-; spray voltage 3000 V; ion source temperature 370 °C; ion transfer tube temperature 320 °C; sheath gas 35 Arb; auxiliary gas 10 Arb; purge gas 0 Arb.

[0034] Furthermore, the perfluorinated compounds include perfluoroundecanoic acid (PFUdA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), and perfluorooctadecanoic acid (PFODA); the hexabromocyclododecane compounds include α-hexabromocyclododecane (α-HBCD). The method of the present invention can be used to simultaneously detect PFUdA, PFDoA, PFTrDA, PFTeDA, PFHxDA, PFODA, and α-HBCD in orthodontic transparent aligners. It should be noted that the components listed above do not represent a limitation on this method.

[0035] Further, in step S3, after the eluent is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry, the content of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution is determined, thereby determining the amount of perfluorinated compounds and hexabromocyclododecane compounds released by the orthodontic transparent appliance sample. Wherein: the step of determining the content of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution can be performed by conventional analysis methods: for example, under the same conditions, a series of standard solutions of perfluorinated compounds and hexabromocyclododecane compounds with concentration gradients are respectively prepared, 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, and 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 detection and analysis of the sample is substituted into the corresponding linear regression equation, and the content of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution can be obtained. The above are all capable of being operated by those skilled in the art, and will not be repeated here.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The invention discloses a method for detecting perfluorinated compounds and hexabromocyclododecane compounds in an orthodontic transparent appliance. The method simulates the human oral environment to release perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance into a sample solution. The magnetic composite material Fe3O4@F-COF is capable of simultaneously adsorbing hexabromocyclododecane compounds and perfluorinated compounds, and simultaneously and efficiently enriching perfluorinated compounds and hexabromocyclododecane compounds in the sample solution. A magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method is established. The method can simultaneously and qualitatively detect perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance, and determine the contents of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution, thereby further obtaining the amounts of perfluorinated compounds and hexabromocyclododecane compounds released from the orthodontic transparent appliance sample into artificial saliva, thereby simulating and judging the amounts released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice. The method for detecting perfluorinated and hexabromocyclododecane compounds in orthodontic transparent appliances of the present invention has high sensitivity, low detection limit and short analysis time.

[0038] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The relevant spectra of PFUdA obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention;

[0040] Figure 2 The PFDoA related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0041] Figure 3 The relevant spectra of PFTrDA obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention;

[0042] Figure 4 The PFTeDA related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0043] Figure 5 The relevant spectra of PFHxDA obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention;

[0044] Figure 6 The relevant spectra of PFODA obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention;

[0045] Figure 7 1 is a related spectrum of α-HBCD obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention. DETAILED DESCRIPTION

[0046] 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 known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications of the material composition and dosage in these embodiments also belong to the protection scope of the present invention.

[0047] Example 1

[0048] Since hexabromocyclododecane compounds and perfluorinated compounds exist in trace amounts in orthodontic transparent appliances, it is difficult to detect them using sensitive analytical instruments, so it is necessary to combine them with sample pretreatment technology.

[0049] This embodiment provides a method for detecting perfluorinated compounds and hexabromocyclododecane compounds in orthodontic transparent appliances, which specifically includes the following steps:

[0050] S1 Sample Preparation:

[0051] (1) Use a special orthodontic punching pliers to cut the orthodontic transparent appliance into small discs for later use; the diameter of the small disc is 5 mm and the thickness is 0.75 mm;

[0052] (2) Take 1 g of the small disc, wash it with ultrapure water, and sterilize it with 115V ultraviolet light in a biological safety cabinet for 15 minutes;

[0053] (3) The small disc was then turned over with sterile tweezers and sterilized in the container for another 15 minutes to obtain an orthodontic transparent aligner sample;

[0054] (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 a sample solution;

[0055] S2 sample preparation:

[0056] S21 Preparation of magnetic composite material Fe3O4@F-COF:

[0057] ① Preparation of Fe3O4 magnetic nanoparticles (MNPs):

[0058] Ferric chloride hexahydrate (2.8 g), sodium acetate (4.9 g) and ethylene glycol (40 mL) were added to a hydrothermal reactor, and a yellow suspension was obtained by stirring, and the reaction was carried out at 200°C for 8 hours. After the reaction, the precipitate was collected by a magnet, and the precipitate was washed alternately with ultrapure water and ethanol to remove the unreacted reagents; the washed precipitate was vacuum dried at 80°C to obtain Fe3O4 magnetic nanoparticles (MNPs).

[0059] ②Amino modification of Fe3O4 magnetic nanoparticles (MNPs):

[0060] The prepared Fe3O4 magnetic nanoparticles (MNPs) were dispersed in a mixed solution of 50 mL ethanol, 10 mL ultrapure water and 1.2 mL ammonia water, and 0.4 mL of tetraethyl orthosilicate was slowly added under mechanical stirring, and the reaction was carried out at room temperature for 8 hours. After the reaction, the precipitate was filtered or collected by a magnet, and the precipitate was washed alternately with ultrapure water and ethanol to remove the unreacted reagent; the washed precipitate was vacuum dried at 80 ° C to obtain Fe3O4@SiO2. Then, 0.2 g of Fe3O4@SiO2 was ultrasonically dispersed in 130 mL of anhydrous toluene, 17 mL of 3-aminopropyltriethoxysilane was added, and the reaction was refluxed at 120 ° C for 6 hours. After the reaction, the precipitate was collected by a magnet, and the precipitate was washed alternately with ultrapure water and ethanol to remove the unreacted reagent; the washed precipitate was vacuum dried at 80 ° C to obtain amino-modified Fe3O4 magnetic nanoparticles (Fe3O4@NH2).

[0061] ③ Preparation of magnetic composite material Fe3O4@F-COF:

[0062] Fe3O4@NH2 (40 mg), 2,3,5,6-tetrafluorobenzaldehyde (26.8 mg), 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (4,4',4''-(1,3,5-Triazine-2,4,6-triyl)trianiline, TAPT) (30.8 mg), 1,4-dioxane / o-dichlorobenzene mixed solution (12 mL, 2:1, v / v) and acetic acid solution (0.6 mL, 6 mol / L) were added to the reaction bottle, ultrasonically treated for 10 minutes, and reacted at room temperature for 60 hours. After the reaction, the product was collected with a magnet and washed alternately with methanol and ultrapure water, and vacuum dried for 12 hours to obtain the magnetic composite material Fe3O4@F-COF.

[0063] S22 Magnetic Solid Phase Extraction:

[0064] 10 mg of the magnetic composite Fe3O4@F-COF was added to 10 mL of the sample solution. After vortexing for 5 minutes, the magnetic composite Fe3O4@F-COF was collected from the solution with a magnet and the supernatant was removed. Then, 3.0 mL of methanol solution was added to the centrifuge tube and vortexed for 1 minute to elute the perfluorinated compounds and hexabromocyclododecane compounds adsorbed on the magnetic composite Fe3O4@F-COF. Finally, the filtered magnetic composite Fe3O4@F-COF was washed alternately with 2 mL of methanol solution and ultrapure water and dried for next use.

[0065] S3 detection analysis:

[0066] A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection and analysis method was established, and the eluent was analyzed instrumentally. The perfluorinated compounds and hexabromocyclododecanes in the orthodontic clear appliance samples were detected simultaneously, the contents of perfluorinated compounds and hexabromocyclododecanes in the sample solution were determined, and the amount of perfluorinated compounds and hexabromocyclododecanes released from the orthodontic clear appliance samples was determined.

[0067] Since the instruments used in the HPLC-MS / MS method can be selected from existing conventional HPLC equipment, the operating methods of HPLC-MS / MS 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 specification of the present invention.

[0068] Chromatographic conditions:

[0069] Chromatographic column: Hypersil GOLD C18 (100×2.1 mm, 1.9μm)

[0070] The mobile phase composition and gradient elution conditions are shown in Table 1

[0071] Table 1 Mobile phase composition and gradient elution

[0072]

[0073] The specific chromatographic conditions are as follows: Hypersil GOLD C18 chromatographic column, specifications are 100×2.1 mm, 1.9 μm; mobile phase: A is 10 mmol ammonium acetate aqueous solution, B is methanol solution; gradient elution: 0-1 min, 5% B; 1-3 min, 5-20% B; 3-5 min, 20-95% B; 5-5.5 min, 95% B; 5.5-9 min, 95-5% B; flow rate is 0.3 mL / min; column temperature is 40°C; injection volume is 5 μL.

[0074] Mass spectrometry parameters are shown in Table 2

[0075] Table 2 Mass spectrometry parameter settings

[0076]

[0077] The mass spectrometry conditions were as follows: ionization mode H-ESI-; spray voltage 3000 V; ion source temperature 370°C; ion transfer tube temperature 320°C; sheath gas 35 Arb; auxiliary gas 10 Arb; and sweep gas 0 Arb.

[0078] In the method for detecting perfluorinated and hexabromocyclododecane compounds in orthodontic transparent aligners of the present embodiment, the detected perfluorinated compounds include perfluoroundecanoic acid (PFUdA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), and perfluorooctadecanoic acid (PFODA); the detected hexabromocyclododecane compounds include α-hexabromocyclododecane (α-HBCD).

[0079] In this example, the above method was used to establish detection methods for six perfluorinated compounds (PFUdA, PFDoA, PFTrDA, PFTeDA, PFHxDA, PFODA) and one hexabromocyclododecane compound (α-HBCD). Figure 1-Figure 7 Four different samples were subjected to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection under the above-mentioned chromatographic conditions and mass spectrometry conditions: 2 ng / g of each substance standard, the eluate obtained after pretreatment of the sample solution with 2 ng / g of each substance standard, the eluate obtained after step S2 (the actual sample after pretreatment), and the sample solution obtained after step S1 (the actual sample before pretreatment), and the spectra related to each substance were obtained. Figure 1-7 It can be seen that the chromatographic peaks of the six perfluorinated compounds and one hexabromocyclododecane compound have good peak shapes. Under the current chromatographic conditions and mass spectrometry conditions, the six perfluorinated compounds and one hexabromocyclododecane compound are well separated.

[0080] Example 2

[0081] Methodological evaluation:

[0082] 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 by the present invention, the present embodiment also prepares mother liquors with a concentration of 40 ng / g for the above-mentioned 6 perfluorinated compounds (PFUdA, PFDoA, PFTrDA, PFTeDA, PFHxDA, PFODA) and 1 hexabromocyclododecane compound (α-HBCD), and dilutes them step by step with methanol to a series of standard working concentrations of 0.2 ng / g, 0.4 ng / g, 2 ng / g, 5 ng / g, 10 ng / g, and 20 ng / g; and the same sample pretreatment method is carried out for magnetic solid phase extraction with reference to the method of Example 1, and the same detection and analysis method is used, and under the same chromatographic conditions and mass spectrometry conditions, the corresponding eluents are respectively detected by high performance liquid chromatography-tandem mass spectrometry system, and the linear range, detection limit, quantification limit and relative standard deviation of the method are respectively investigated. The results are shown in Table 3, wherein x represents the concentration of the analyte and y represents the peak area of ​​the analyte. The results showed that the method exhibited good linearity in the range of 0.20-20 ng / g (R 2 ≥0.9985), LODs and LOQs were between 0.014-0.036 ng / g and 0.046-0.120 ng / g, respectively, and the intra-day precision and inter-day precision were less than 10%, indicating that this method has a low detection limit.

[0083] Table 3 MSPE-HPLC-MS / MS method parameters

[0084]

[0085] Example 3

[0086] Accuracy of the method:

[0087] The target substances (six perfluorinated compounds and one hexabromocyclododecane compound) at three concentration levels (0.4, 2.0, and 10.0 ng / g) were added to the blank sample (sample solution without pretreatment), and their spike recoveries were determined. The results are shown in Table 4. The spike recoveries of perfluorinated compounds and hexabromocyclododecane compounds in the samples were 84.4-102.7%, with RSDs ≤ 8.9%. This indicates that this method can accurately determine the perfluorinated compounds and hexabromocyclododecane compounds in the sample solution of orthodontic clear aligners, and then accurately determine the amount of perfluorinated compounds and hexabromocyclododecane compounds released from orthodontic clear aligner samples.

[0088] Table 4. Recovery rate experiment of spiked MSPE-HPLC-MS / MS

[0089]

[0090] Example 4

[0091] Actual sample test results:

[0092] 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 perfluorinated compounds and hexabromocyclododecane compounds in orthodontic clear appliances. The actual results are the number of ng of perfluorinated compounds and hexabromocyclododecane compounds released into artificial saliva per 1g of the actual sample of orthodontic clear appliances.

[0093] Table 5 Test results of actual samples

[0094]

[0095] The invention discloses a method for detecting perfluorinated compounds and hexabromocyclododecane compounds in an orthodontic transparent appliance. The method simulates the human oral environment to release perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance into a sample solution. The magnetic composite material Fe3O4@F-COF is capable of simultaneously adsorbing perfluorinated compounds and hexabromocyclododecane compounds, and simultaneously and efficiently enriching perfluorinated compounds and hexabromocyclododecane compounds in the sample solution. A magnetic solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (MSPE-HPLC-MS / MS) method is established. The perfluorinated compounds and hexabromocyclododecane compounds in the orthodontic transparent appliance can be qualitatively detected simultaneously, and the contents of perfluorinated compounds and hexabromocyclododecane compounds in the sample solution can be determined, thereby further obtaining the amounts of perfluorinated compounds and hexabromocyclododecane compounds released from the orthodontic transparent appliance sample into artificial saliva, thereby simulating and judging the amounts released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice. The method for detecting perfluorinated and hexabromocyclododecane compounds in orthodontic transparent appliances of the present invention has high sensitivity, low detection limit and short analysis time.

[0096] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A method for detecting perfluorinated and hexabromocyclododecane compounds in orthodontic transparent appliances, characterized in that: The perfluorinated compounds include perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, and perfluorooctadecanoic acid; the hexabromocyclododecane compounds include α-hexabromocyclododecane; 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@F-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@F-COF is separated from the solution; the separated magnetic composite material Fe3O4@F-COF is eluted with a methanol solution; and an eluent is obtained by separation; In step S2, the method for preparing the magnetic composite material Fe3O4@F-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@F-COF: Fe3O4@NH2, 2,3,5,6-tetrafluorobenzaldehyde, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, 1,4-dioxane / o-dichlorobenzene mixed solution and acetic acid solution were added to a reaction bottle, ultrasonically treated for 8-12 minutes, and reacted at room temperature for 60-80 hours; after the reaction, the precipitate was separated, washed, and dried to obtain the magnetic composite material Fe3O4@F-COF; S3: Detection and analysis: The eluate was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry.

2. The method for detecting perfluorinated and hexabromocyclododecane 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 perfluorinated and hexabromocyclododecane 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 perfluorinated and hexabromocyclododecane 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 perfluorinated and hexabromocyclododecane 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, ultrapure water and ammonia water is 50:10:1.2; in the step of preparing the magnetic composite material Fe3O4@F-COF, the volume ratio of 1,4-dioxane and o-dichlorobenzene in the 1,4-dioxane / o-dichlorobenzene mixed solution is 2:

1.

6. The method for detecting perfluorinated and hexabromocyclododecane 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@F-COF, washing is performed alternately using methanol and water.

7. The method for detecting perfluorinated and hexabromocyclododecane 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: Chromatographic conditions were: Hypersil GOLD C18 column, 50 × 2.1 mm, 1.9 μm; Mobile phase: A is 10mmol ammonium acetate aqueous solution, B is methanol solution; gradient elution: 0-1min, 5%B; 1-3min, 5-20%B; 3-5min, 20-95%B; 5-5.5min, 95%B; 5.5-9min, 95-5%B; flow rate is 0.3mL / min; column temperature is 40℃; injection volume is 5μL; The mass spectrometry conditions were as follows: ionization mode H-ESI-; spray voltage 3000 V; ion source temperature 370°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 perfluorinated and hexabromocyclododecane 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 perfluorinated compounds and hexabromocyclododecane compounds in the sample solution is determined, thereby determining the amount of perfluorinated compounds and hexabromocyclododecane compounds released by the orthodontic transparent appliance sample.

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