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

Abstract

The present invention relates to a method for detecting hexachlorobutadiene 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@Cl‑COF to the sample solution, and vortexing; separating the magnetic composite material Fe3O4@Cl‑COF from the solution; eluting the separated magnetic composite material Fe3O4@Cl‑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 and a high performance gas chromatography-tandem mass spectrometry detection method to detect and analyze the eluent respectively. The detection method of the present invention can simultaneously detect the amount of hexachlorobutadiene and perfluorinated 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, in particular to a method for detecting hexachlorobutadiene and perfluorinated compounds in an orthodontic transparent appliance. Background Art

[0002] 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 their teeth, 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 clear aligners remains a common feature. The most commonly used polymers (alone or in blends) used to manufacture commercial orthodontic clear plastic orthodontic aligners include polyesters, polyurethanes or copolyesters, polypropylene, polycarbonate, ethylene vinyl acetate, and polyvinyl chloride, among others. As intraoral appliance materials, although orthodontic clear aligner materials are advertised as being free of harmful 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 that enhance specific properties such as performance, stability, elasticity, etc., which may be toxic to humans. Furthermore, the scarcity of evidence-based literature due to the paucity of controlled trials on orthodontic clear aligners and the conflicting results mentioned in the published literature continues to fuel the debate on the possible toxic effects of using clear aligners. In addition, with the continued influx of multiple commercial aligner systems, there is a need to continue testing the toxicity of orthodontic clear aligners produced by different manufacturers.

[0003] 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.

[0004] Hexachlorobutadiene (HCBD) is an aliphatic halogenated hydrocarbon that was added to the Stockholm Convention control list in 2017 and is a new type of persistent organic pollutants (POPs). HCBD has a low boiling point, is volatile, and has stable chemical properties. It is often used as a solvent for rubber and other polymers. HCBD has tritoxicity and potential reproductive toxicity and is listed as a human carcinogen in Group C. As a new type of persistent organic pollutant, HCBD is difficult to degrade and has bioaccumulation. It can be absorbed by organisms through breathing, ingestion, and skin contact. HCBD has a strong irritating effect on human eyes, skin, and respiratory tract. Inhalation can cause respiratory damage, arterial hypotension, etc. Long-term contact can cause liver and kidney lesions, central nervous system depression, cyanosis, chromosome aberrations, etc. It can also be passed to infants through breast milk. At present, domestic research on HCBD is very limited. Its occurrence in orthodontic transparent aligner materials and its effects on the human body, as well as its impact on the environment, are still unclear, which is not conducive to the pollution control of this POPs.

[0005] Orthodontic transparent appliances are essential devices 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 material components of orthodontic transparent appliances are polymers, such as polyethylene (PE), polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyurethane (PU) and polypropylene (PP). PFASs and HCBD may be one of these additives used to enhance the comprehensive performance of the material. 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 different PFASs and HCBD 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 HCBD will be released into the human mouth.

[0006] Since hexachlorobutadiene and perfluorinated compounds often exist in trace amounts in samples (orthodontic transparent aligners), accurate quantitative determination cannot be achieved using sophisticated analytical instruments. In addition, the sample (orthodontic transparent 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. Sample pretreatment (including pretreatment) can not only extract and enrich the target and remove interferences, but also convert the sample into a state suitable for preservation or a form suitable for instrumental analysis. These processes can directly affect the reliability and sensitivity of the entire analytical method, especially for the detection of trace substances PFASs and HCBD, which is an indispensable and important link. Existing methods generally use liquid-liquid extraction, freeze-drying and other methods for sample pretreatment for PFASs and HCBD, which is not only cumbersome to operate, 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 hexachlorobutadiene (HCBD) 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 PFASs and HCBD in orthodontic clear aligners, the 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 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 hexachlorobutadiene and perfluorinated compounds is mostly concentrated on surface water, sediments, sewage effluent, etc., while there are few studies on orthodontic transparent aligner samples. This is mainly because the matrix of orthodontic transparent aligners is complex and the interference is serious. The difficulty of pre-analysis treatment is much greater than the detection of hexachlorobutadiene or perfluorinated compounds in water bodies. In addition, the current technology only targets one type of components. How to efficiently extract hexachlorobutadiene and perfluorinated compounds from orthodontic transparent aligner samples has become a bottleneck in the analysis of trace pollutants. Among the commonly used extraction methods for hexachlorobutadiene or perfluorinated compounds in orthodontic 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 technology (SPE), which has the advantages of simple operation, good repeatability, and easy automation. However, for orthodontic clear aligner samples with complex matrices, it is difficult to minimize background interference through only one-step purification, and the separation time is relatively long.

[0008] 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 to enrich perfluorinated compounds or hexachlorobutadiene 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 hexachlorobutadiene or perfluorinated compounds in orthodontic transparent 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.

[0009] 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) and other detection methods, which have high sensitivity and accuracy, and are mainly quantitatively determined 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 detecting trace amounts of hexachlorobutadiene and perfluorinated compounds in orthodontic clear aligners. Summary of the invention

[0010] Based on this, the purpose of the present invention is to provide a method for detecting hexachlorobutadiene and perfluorinated compounds in orthodontic transparent appliances. By using high performance liquid chromatography-tandem mass spectrometry detection method and gas chromatography-tandem mass spectrometry detection method combined with specific sample pretreatment technology, hexachlorobutadiene and perfluorinated compounds in orthodontic transparent appliances can be qualitatively detected, and the amount of hexachlorobutadiene (HCBD) and perfluorinated compounds (PFASs) in the orthodontic transparent appliance released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks can be determined; the method established by the present invention has high sensitivity, low detection limit and short analysis time.

[0011] To achieve the above object, the present invention is implemented through the following technical solutions:

[0012] A method for detecting hexachlorobutadiene and perfluorinated compounds in an orthodontic transparent appliance comprises the following steps:

[0013] S1: Sample preparation:

[0014] 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;

[0015] S2: Sample preparation:

[0016] The magnetic composite material Fe3O4@Cl-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@Cl-COF is separated from the solution; the separated magnetic composite material Fe3O4@Cl-COF is eluted with a methanol solution; and an eluent is obtained by separation;

[0017] S3: Detection and analysis:

[0018] The eluent was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry, respectively.

[0019] The method for detecting hexachlorobutadiene and perfluorinated 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 hexachlorobutadiene and perfluorinated 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 hexachlorobutadiene and perfluorinated compounds in the orthodontic transparent appliance sample into the human oral cavity. Then, in step S2, the magnetic composite material Fe3O4@Cl-COF is used as a magnetic solid phase extraction adsorbent to efficiently enrich hexachlorobutadiene and perfluorinated compounds in the sample solution; wherein the magnetic composite material Fe3O4@Cl-COF has a benzene ring and Cl, the interaction between Cl in the magnetic composite material Fe3O4@Cl-COF and hexachlorobutadiene is a Cl-Cl mutual adsorption, and the interaction between the benzene ring in the magnetic composite material Fe3O4@Cl-COF and the perfluorinated compounds is a hydrophobic adsorption, so the magnetic composite material Fe3O4@Cl-COF can adsorb hexachlorobutadiene and perfluorinated compounds at the same time; then a desorption treatment is performed to elute the hexachlorobutadiene and perfluorinated compounds adsorbed on the magnetic composite material Fe3O4@Cl-COF, so that the hexachlorobutadiene and perfluorinated compounds are transferred to the eluent. Finally, in step S3, the eluent is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry, respectively, to qualitatively detect hexachlorobutadiene and perfluorinated compounds, and determine the content of hexachlorobutadiene and perfluorinated compounds in the sample solution, thereby further obtaining the amount of hexachlorobutadiene and perfluorinated compounds released into the artificial saliva by the orthodontic transparent appliance sample, thereby simulating the content released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice.

[0020] The invention uses a high performance liquid chromatography-tandem mass spectrometry detection method and a gas 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 / gas chromatography-tandem mass spectrometry (MSPE-HPLC / GC-MS / MS) method, which can detect hexachlorobutadiene and perfluorinated compounds present in trace amounts in an orthodontic transparent appliance, and the established method has high sensitivity, low detection limit and short analysis time. The detection method of hexachlorobutadiene and perfluorinated compounds in an orthodontic transparent appliance of the invention can solve the problems in the existing detection method that hexachlorobutadiene and perfluorinated compounds are present in trace amounts in the orthodontic transparent appliance and cannot be detected at the same time, resulting in a complicated detection process and a long time consumption; and the problem that the amount of hexachlorobutadiene and perfluorinated compounds released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice cannot be determined.

[0021] 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 hexachlorobutadiene and perfluorinated 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.

[0022] 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 hexachlorobutadiene and perfluorinated compounds in the orthodontic transparent appliance sample are transferred to the artificial saliva to form a sample solution.

[0023] Further, in step S2, the preparation method of the magnetic composite material Fe3O4@Cl-COF comprises the following steps:

[0024] 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;

[0025] Amino modification of Fe3O4 magnetic nanoparticles: the prepared Fe3O4 magnetic nanoparticles are dispersed in a mixed solution containing ethanol, ultrapure 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;

[0026] Preparation of magnetic composite material Fe3O4@Cl-COF: Add Fe3O4@NH2, 2,5-dichloroterephthalaldehyde and tetrakis(4-aminophenyl)ethylene, acetonitrile and acetic acid solution into a reaction bottle, ultrasonically treat for 10-15 minutes, and react at room temperature for 65-80 hours; after the reaction, separate the precipitate, wash it, and dry it to obtain the magnetic composite material Fe3O4@Cl-COF.

[0027] In the preparation method of the magnetic composite material Fe3O4@Cl-COF of the present invention, Fe3O4@NH2, 2,5-dichloroterephthalaldehyde and tetra(4-aminophenyl)ethylene are reacted together (Schiff base reaction), so that the prepared magnetic composite material Fe3O4@F-COF has a benzene ring and Cl, and can simultaneously adsorb hexachlorobutadiene and perfluoro compounds by utilizing its structural characteristics, wherein the benzene ring in the magnetic composite material Fe3O4@Cl-COF can form a hydrophobic adsorption effect with the perfluoro compound, and Cl-Cl mutual adsorption is formed between Cl and hexachlorobutadiene in the magnetic composite material Fe3O4@Cl-COF, so the magnetic composite material Fe3O4@Cl-COF can simultaneously adsorb hexachlorobutadiene and perfluoro compounds, and can simultaneously and efficiently enrich hexachlorobutadiene and perfluoro compounds in a sample solution. In the magnetic composite material Fe3O4@Cl-COF, Fe3O4 magnetic nanoparticles are introduced, which saves more time than traditional dispersed extraction and solid phase extraction, and reduces the use of organic solvents, thereby improving economic efficiency.

[0028] 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.

[0029] Furthermore, in the step of amino modification of Fe3O4 magnetic nanoparticles, in the mixed solution of ethanol, water and ammonia water, the volume ratio of ethanol, water and ammonia water is 50:10:1.2.

[0030] Furthermore, in the steps of preparing Fe3O4 magnetic nanoparticles and the step of amino modification of Fe3O4 magnetic nanoparticles, washing is performed by alternately using water and ethanol, and drying is performed at 80°C; in the step of preparing the magnetic composite material Fe3O4@Cl-COF, washing is performed by alternately using acetonitrile and water. The unreacted reagents on the corresponding precipitates are removed by the washing and drying operations.

[0031] Further, in step S3, in the high performance liquid chromatography-tandem mass spectrometry detection method, the chromatographic conditions are: Hypersil GOLD C18 chromatographic column, specification is 50×2.1 mm, 1.8 μm; mobile phase: A is 5 mmol ammonium acetate aqueous solution, B is methanol solution; gradient elution: 0-1min, 3%B; 1-3min, 3-15%B; 3-5min, 15-90%B; 5-5.5min, 90%B; 5.5-10min, 90-3%B; flow rate is 0.3mL / min; column temperature is 35°C; injection volume is 5μL; mass spectrometry conditions are: ionization mode H-ESI-; spray voltage is 3500V; ion source temperature is 380°C; ion transfer tube temperature is 330°C; sheath gas is 40Arb; auxiliary gas is 20Arb; purge gas is 0Arb.

[0032] Further, in step S3, in the gas chromatography-tandem mass spectrometry detection method, the chromatographic conditions are: Agilent gas chromatography column, specifications are 30 m×0.25 mm, 0.25 μm; inlet temperature: 280°C, column temperature: 130°C maintained for 1 min, increased to 220°C at 15°C / min, maintained for 2 min; the carrier gas is high-purity helium (99.999%), with a flow rate of 0.8 ml / min; the mass spectrometry conditions are: ion source, electron bombardment energy: 80 eV; mass scanning range: 40-500 amu; ion source temperature: 220°C; solvent delay time: 8 min.

[0033] Furthermore, the perfluorinated compounds include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPe A), perfluorobutyl sulfonic acid (PFBS), perfluorooctane sulfonic acid (PFOS), and perfluorodecyl sulfonic acid (PFDS). The method of the present invention can be used to detect HCBD in orthodontic transparent aligners and the above-mentioned PFBA, PFPe A, PFBS, PFOS, PFDS, and HCBD. It should be noted that the above-mentioned components do not represent a limitation to the present method.

[0034] Further, in step S3, after the eluent is detected and analyzed by high performance liquid chromatography-tandem mass spectrometry, the content of perfluorinated compounds in the sample solution is determined; after the eluent is detected and analyzed by gas chromatography-tandem mass spectrometry, the content of hexachlorobutadiene in the sample solution is determined; thereby determining the amount of hexachlorobutadiene and perfluorinated compounds released by the orthodontic transparent appliance sample. Wherein: the step of determining the content of hexachlorobutadiene 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 hexachlorobutadiene or perfluorinated compounds with a concentration gradient 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, 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 to obtain the content of hexachlorobutadiene and perfluorinated compounds in the sample solution. The above operations are all capable of being performed by those skilled in the art and will not be described in detail here.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The invention discloses a method for detecting hexachlorobutadiene and perfluoro compounds in an orthodontic transparent appliance. The method simulates the human oral environment to release hexachlorobutadiene and perfluoro compounds in the orthodontic transparent appliance into a sample solution. The magnetic composite material Fe3O4@Cl-COF is capable of simultaneously adsorbing hexachlorobutadiene and perfluoro compounds, and simultaneously and efficiently enriching hexachlorobutadiene and perfluoro compounds in the sample solution. A magnetic solid phase extraction-high performance liquid chromatography / gas chromatography-tandem mass spectrometry (MSPE-HPLC / GC-MS / MS) method is established. The hexachlorobutadiene and perfluoro compounds in the orthodontic transparent appliance can be qualitatively detected simultaneously, and the contents of hexachlorobutadiene and perfluoro compounds in the sample solution can be determined, thereby further obtaining the amount of hexachlorobutadiene and perfluoro compounds released from the orthodontic transparent appliance sample into artificial saliva, thereby simulating and judging the amount released into the human oral cavity after the user wears the orthodontic transparent appliance for 2 weeks in clinical practice. The method for detecting hexachlorobutadiene and perfluorinated compounds in an orthodontic transparent appliance of the present invention has high sensitivity, low detection limit and short analysis time.

[0037] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The PFBA related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0039] Figure 2 The relevant spectra of PFPeA obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention;

[0040] Figure 3 The PFBS related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0041] Figure 4 The PFOS related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0042] Figure 5 The PFDS related spectra obtained after HPLC-MS / MS detection of four different samples in Example 1 of the present invention are shown;

[0043] Figure 6 The GC-MS spectra of HCBD obtained by respectively performing GC-MS tests on four different samples in Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0044] 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.

[0045] Example 1

[0046] Since hexachlorobutadiene and perfluorinated compounds exist in trace amounts in orthodontic transparent aligners, it is difficult to detect them using sensitive analytical instruments, so it is necessary to combine them with sample pretreatment technology.

[0047] This embodiment provides a method for detecting hexachlorobutadiene (HCBD) and perfluorinated compounds in an orthodontic transparent appliance, which specifically includes the following steps:

[0048] S1 Sample Preparation:

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] S2 sample preparation:

[0054] S21 Preparation of magnetic composite material Fe3O4@Cl-COF:

[0055] ① Preparation of Fe3O4 magnetic nanoparticles (MNPs):

[0056] 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).

[0057] ②Amino modification of Fe3O4 magnetic nanoparticles (MNPs):

[0058] 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 reagents; 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 reagents; the washed precipitate was vacuum dried at 80 ° C to obtain amino-modified Fe3O4 magnetic nanoparticles (Fe3O4@NH2).

[0059] ③ Preparation of magnetic composite material Fe3O4@Cl-COF:

[0060] Fe3O4@NH2 (50 mg), 2,5-dichloroterephthalaldehyde (38.7 mg) and tetrakis(4-aminophenyl)ethylene (43.6 mg), 12 mL of acetonitrile and acetic acid solution (0.6 mL, 12 mol / L) were added to a reaction bottle, ultrasonically treated for 15 minutes, and reacted at room temperature for 65 hours. After the reaction, the product was collected with a magnet and washed alternately with acetonitrile and ultrapure water, and vacuum dried for 12 hours to obtain the magnetic composite material Fe3O4@Cl-COF.

[0061] S2 Magnetic Solid Phase Extraction:

[0062] 10 mg of the magnetic composite Fe3O4@Cl-COF was added to 10 mL of the sample solution. After vortexing for 5 minutes, the magnetic composite Fe3O4@Cl-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 hexachlorobutadiene and perfluorinated compounds adsorbed on the magnetic composite Fe3O4@Cl-COF. Finally, the filtered magnetic composite Fe3O4@Cl-COF was washed alternately with 2 mL of methanol solution and ultrapure water and dried for next use.

[0063] S3 detection analysis:

[0064] High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and gas chromatography-tandem mass spectrometry (GC-MS) detection and analysis methods were established, and the eluents were instrumentally analyzed. Among them, the content of perfluorinated compounds in the sample solution was determined after the eluent was detected and analyzed by the high performance liquid chromatography-tandem mass spectrometry detection method; the content of hexachlorobutadiene in the sample solution was determined after the eluent was detected and analyzed by the gas chromatography-tandem mass spectrometry detection method; thus, the amount of hexachlorobutadiene and perfluorinated compounds released by the orthodontic clear aligner samples was determined, and the amount of hexachlorobutadiene (HCBD) and perfluorinated compounds released by the orthodontic clear aligner samples was determined.

[0065] Since the instruments used in the high performance liquid chromatography-tandem mass spectrometry detection method and the gas chromatography-tandem mass spectrometry detection method can be selected from existing conventional high performance liquid and gas chromatography equipment, the operating methods of the high performance liquid chromatography-tandem mass spectrometry detection method and the gas chromatography-tandem mass spectrometry detection method are conventional methods. Therefore, except for the corresponding chromatographic conditions and mass spectrometry conditions in this embodiment, they will not be repeated in the specification of the present invention.

[0066] In the liquid chromatography-tandem mass spectrometry detection method:

[0067] Chromatographic conditions:

[0068] Chromatographic column: Hypersil GOLD C18 (50×2.1 mm, 1.8μm)

[0069] The mobile phase composition and gradient elution conditions are shown in Table 1

[0070] Table 1 Mobile phase composition and gradient elution

[0071]

[0072] Mass spectrometry parameters are shown in Table 2

[0073] Table 2 Mass spectrometry parameter settings

[0074]

[0075] In the gas chromatography-tandem mass spectrometry detection method:

[0076] Chromatographic conditions were as follows: Agilent gas chromatography column (30 m×0.25 mm, 0.25 μm); injection port temperature: 280°C, column temperature: 130°C for 1 min, then increased to 220°C at 15°C / min, and maintained for 2 min. The carrier gas was high-purity helium (99.999%) at a flow rate of 0.8 ml / min.

[0077] The mass spectrometry conditions were as follows: ion source, electron bombardment energy: 80 eV; mass scanning range: 40-500 amu; ion source temperature: 220°C; solvent delay time: 8 min.

[0078] The detection method of hexachlorobutadiene (HCBD) and perfluorinated compounds in orthodontic transparent aligners of this embodiment, the detected perfluorinated compounds include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPe A), perfluorobutyl sulfonic acid (PFBS), perfluorooctane sulfonic acid (PFOS), and perfluorodecane sulfonic acid (PFDS).

[0079] In this example, the above method was used to establish a detection method for hexachlorobutadiene (HCBD) and five perfluorinated compounds (PFBA, PFPeA, PFBS, PFOS, and PFDS). Figure 1-Figure 5 For the five perfluorinated compounds (PFBA, PFPe A, PFBS, PFOS, PFDS), four different samples: 2 ng / g standard of each substance, the eluate obtained by pretreatment after adding 2 ng / g standard of each substance to the sample solution, the eluate obtained after step S2 (the actual sample after pretreatment), and the sample solution obtained after step S1 (the actual sample before pretreatment) were subjected to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) under the above-mentioned chromatographic conditions and mass spectrometry conditions, and spectra related to each substance were obtained. Figure 6For hexachlorobutadiene, four different samples were tested by gas chromatography-tandem mass spectrometry (GC-MS / MS) under the above-mentioned chromatographic conditions and mass spectrometry conditions: hexachlorobutadiene standard 2ng / g, sample solution plus hexachlorobutadiene standard 2ng / g and then pre-treated eluate, 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 hexachlorobutadiene were obtained. Figure 1-6 It can be seen that the chromatographic peaks of hexachlorobutadiene and five perfluorinated compounds have good peak shapes. Under the current chromatographic conditions and mass spectrometry conditions, hexachlorobutadiene and five perfluorinated compounds can be 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 / gas chromatography-tandem mass spectrometry (MSPE-HPLC / GC-MS / MS) method established in the present invention, the present embodiment also prepares mother solutions with a concentration of 20 ng / g for the above five perfluorinated compounds (PFBA, PFPeA, PFBS, PFOS, PFDS) and hexachlorobutadiene (HCBD), and dilutes them step by step with methanol to a series of standard solutions of 0.1 ng / g, 0.5 ng / g, 1 ng / g, 2 ng / g, 5 ng / g, 10 ng / g, and 20 ng / g. The same sample pretreatment method was used for magnetic solid phase extraction according to the method in Example 1, and the same detection and analysis method was used. Under the same chromatographic conditions and mass spectrometry conditions, the corresponding eluents were respectively detected by high performance liquid chromatography-tandem mass spectrometry detection method (MSPE-HPLC-MS / MS) and gas chromatography-tandem mass spectrometry detection method (MSPE-GC-MS). The linear range, detection limit, quantitative limit and relative standard deviation of the method were investigated respectively. The results are shown in Tables 3 and 4, where x represents concentration and y represents peak area of ​​the analyte. The results show that the method exhibits good linearity (R 2 ≥0.9982), the LODs and LOQs of perfluorinated compounds were between 0.011-0.023 ng / g and 0.043-0.076 ng / g, respectively; the LODs and LOQs of hexachlorobutadiene were 0.065 ng / g and 0.216 ng / g, respectively. The intra-day precision and inter-day precision were less than 10%, indicating that the method had a low detection limit.

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

[0084]

[0085] Table 4 MSPE-GC-MS method parameters

[0086]

[0087] Example 3

[0088] Accuracy of the method:

[0089] The target substances (five perfluorinated compounds and hexachlorobutadiene) at three concentration levels (0.2, 2.0, and 10.0 ng / g) were added to the blank sample (sample solution without pretreatment), and their spike recovery rates were determined. The results are shown in Table 5. The spike recovery rates of perfluorinated compounds and hexachlorobutadiene in the samples were 82.7-104.5%, and RSDs ≤ 9.2%. This shows that this method can accurately determine the perfluorinated compounds and hexachlorobutadiene in the sample solution of orthodontic clear aligners, and then accurately determine the amount of perfluorinated compounds and hexachlorobutadiene released from the orthodontic clear aligner samples.

[0090] Table 5. Spike recovery experiment of MSPE-HPLC / GC-MS / MS

[0091]

[0092] Example 4

[0093] Actual sample testing:

[0094] The results of the actual sample detection in Example 1 are shown in Table 6. These results indicate that the established magnetic solid phase extraction-high performance liquid chromatography / gas chromatography-tandem mass spectrometry (MSPE-HPLC / GC-MS / MS) method can be used to detect hexachlorobutadiene and perfluorinated compounds in orthodontic clear aligners. The actual results are the number of ng of perfluorinated compounds contained in each 1g of the actual sample of orthodontic clear aligners and the content of hexachlorobutadiene released into artificial saliva.

[0095] Table 6 Test results of actual samples

[0096]

[0097] The invention discloses a method for detecting perfluorinated compounds and hexachlorobutadiene in an orthodontic transparent appliance. The method simulates the human oral environment to release perfluorinated compounds and hexachlorobutadiene in the orthodontic transparent appliance into a sample solution. The magnetic composite material Fe3O4@Cl-COF is capable of simultaneously adsorbing perfluorinated compounds and hexachlorobutadiene, and simultaneously and efficiently enriching perfluorinated compounds and hexachlorobutadiene in the sample solution. A magnetic solid phase extraction-high performance liquid chromatography / gas chromatography-tandem mass spectrometry (MSPE-HPLC / GC-MS / MS) method is established. The method can simultaneously and qualitatively detect perfluorinated compounds and hexachlorobutadiene in the orthodontic transparent appliance, and determine the contents of hexachlorobutadiene and perfluorinated compounds in the sample solution, thereby further obtaining the amounts of hexachlorobutadiene and perfluorinated 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 detection method of perhexachlorobutadiene and perfluorinated compounds in an orthodontic transparent appliance of the present invention has high sensitivity, low detection limit and short analysis time.

[0098] 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 hexachlorobutadiene and perfluorinated compounds in orthodontic transparent appliances, characterized in that: The perfluorinated compounds include perfluorobutyric acid, perfluoropentanoic acid, perfluorobutylsulfonic acid, perfluorooctanesulfonic acid, and perfluorodecanesulfonic acid; 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@Cl-COF is added to the sample solution and vortexed; the magnetic composite material Fe3O4@Cl-COF is separated from the solution; the separated magnetic composite material Fe3O4@Cl-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@Cl-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@Cl-COF: Fe3O4@NH2, 2,5-dichloroterephthalaldehyde and tetrakis(4-aminophenyl)ethylene, acetonitrile and acetic acid solution are added to a reaction bottle, ultrasonically treated for 10-15 minutes, and reacted at room temperature for 65-80 hours; after the reaction is completed, the precipitate is separated, washed, and dried to obtain the magnetic composite material Fe3O4@Cl-COF; S3: Detection and analysis: The eluent was detected and analyzed by high performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry, respectively.

2. The method for detecting hexachlorobutadiene 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 hexachlorobutadiene 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 hexachlorobutadiene 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 hexachlorobutadiene and perfluorinated compounds in orthodontic transparent appliances according to claim 1, characterized in that: In the step of amino modification of Fe3O4 magnetic nanoparticles, in the mixed solution of ethanol, water and ammonia water, the volume ratio of ethanol, water and ammonia water is 50:10:1.

2.

6. The method for detecting hexachlorobutadiene 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@Cl-COF, washing is performed alternately using acetonitrile and water.

7. The method for detecting hexachlorobutadiene 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 chromatographic conditions are: Hypersil GOLDC18 chromatographic column, specification is 50×2.1 mm, 1.8 μm; Mobile phase: A is 5mmol ammonium acetate aqueous solution, B is methanol solution; gradient elution: 0-1min, 3%B; 1-3min, 3-15%B; 3-5min, 15-90%B; 5-5.5min, 90%B; 5.5-10min, 90-3%B; flow rate 0.3mL / min; column temperature 35℃; injection volume 5μL; mass spectrometry conditions: ionization mode H-ESI-; spray voltage 3500V; ion source temperature 380℃; ion transfer tube temperature 330℃; sheath gas 40Arb; auxiliary gas 20Arb; Purge gas 0Arb; In step S3, in the gas chromatography-tandem mass spectrometry detection method, the chromatographic conditions are: Agilent gas chromatography column, specifications are 30 × 0.25 mm, 0.25 μm; injection port temperature: 280°C, column temperature: 130°C for 1 min, increased to 220°C at 15°C / min, and maintained for 2 min; the carrier gas is high-purity helium with a flow rate of 0.8 ml / min; the mass spectrometry conditions are: ion source, electron bombardment energy: 80 eV; mass scanning range: 40-500 amu; ion source temperature: 220°C; solvent delay time: 8 min.

8. The method for detecting hexachlorobutadiene 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 perfluorinated compounds in the sample solution is determined; after the eluent is detected and analyzed by gas chromatography-tandem mass spectrometry, the content of hexachlorobutadiene in the sample solution is determined; thereby determining the amount of hexachlorobutadiene and perfluorinated compounds released by the orthodontic transparent appliance sample.

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