High-throughput analysis method for phthalate plasticizers based on thin film microextraction technology

Abstract

The present invention discloses a high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology, belonging to the technical field of physical and chemical inspection of phthalate plasticizers. The high-throughput analysis method is as follows: after surfactants, urea, tetraethyl orthosilicate, isopropanol, and cyclohexane are added to water and react, they are calcined to obtain nano-mesoporous silica; octadecyltrichlorosilane is used as a modifier to react with the activated mesoporous silica to obtain C18-modified mesoporous silica; then trimethylchlorosilane is used as a capping reagent for capping to obtain C18-modified nano-mesoporous silica; the C18-modified nano-mesoporous silica and polyacrylonitrile are respectively dispersed in DMF, and after mixing, they are sprayed and cured to obtain a microextraction thin film; it is installed in a high-throughput oscillating extraction device, and after the sample undergoes the thin-film microextraction analysis process, reverse-phase liquid chromatography is used for analysis to calculate the content. This method can meet the high-throughput, efficient, and rapid analysis of phthalate esters in a large number of samples.

Description

Technical Field

[0001] The present invention relates to the technical field of physical and chemical inspection of phthalate plasticizers, and more particularly to a high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology. Background Art

[0002] Phthalate (PAEs) compounds are the main components of plasticizers and are one of the synthetic compounds with the largest production volume and the widest application in the world. PAEs are endocrine disruptors with obvious reproductive toxicity, causing problems including fertility, child development, and cancer. PAEs are mainly used in the production of food plastic packaging materials and can migrate and dissolve through contact with food, resulting in food contamination and posing risks to human health when ingested. Therefore, highly sensitive and high-throughput PAEs detection technologies are very important.

[0003] The detection of PAEs includes two parts: sample pretreatment and instrumental analysis and detection. Among them, the separation and extraction of PAEs in complex samples are extremely crucial for improving detection sensitivity and accuracy and accelerating the analysis speed. Currently, PAEs sample pretreatment technologies include Soxhlet extraction, liquid-phase microextraction, solid-phase extraction, solid-phase microextraction, accelerated solvent extraction, QuEChERS method, etc. Although each method has its own unique advantages, there is still a lack of a high-throughput analysis method for PAEs that can meet the processing of large batches of samples and has the characteristics of high sensitivity, fast analysis speed, and low cost. Summary of the Invention

[0004] In view of the above problems, the present invention provides a high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology. This method has the advantages of high sample processing throughput, fast speed, low cost, high sensitivity, good precision, and good reproducibility between coating batches, and can meet the high-throughput, efficient, and rapid analysis of PAEs in large batches of samples.

[0005] The object of the present invention is to provide a high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology, which is carried out according to the following steps:

[0006] Step 1: Dissolve a surfactant and urea in water, add tetraethyl orthosilicate, isopropanol, and cyclohexane, stir evenly at room temperature, and raise the temperature to 65 - 75 °C for hydrolysis and polycondensation reaction to obtain a product, and calcine the product to obtain nano-porous silicon.

[0007] Step 2: Using absolute ethanol as a solvent, triethanolamine as a catalyst, and octadecyltrichlorosilane as a modifier, add activated mesoporous silica, and obtain C18-modified mesoporous silica through a surface covalent bonding reaction; then, using anhydrous toluene as a solvent and trimethylchlorosilane as a capping reagent, add C18-modified mesoporous silica, and perform capping through a silane coupling reaction to obtain C18-modified nano-mesoporous silica;

[0008] Step 3: Disperse C18-modified nano-mesoporous silica in N,N-dimethylformamide A to obtain solution A; disperse polyacrylonitrile in N,N-dimethylformamide B to obtain solution B, mix solution A and solution B to obtain a homogeneous slurry, spray the homogeneous slurry on the surface of the acid-etched substrate, and cure to obtain a microextraction film;

[0009] Step 4: Install the microextraction film in a high-throughput oscillating extraction device, prepare a standard mixed solution of plasticizers, and use ultrapure water to gradually dilute the concentration of the mixed standard solution to 20 ng / mL for extraction analysis;

[0010] Step 5: Use reverse-phase liquid chromatography to analyze phthalate plasticizers in the standard mixed solution of plasticizers and the sample solution, and calculate the content.

[0011] Preferably, in Step 1, the reaction time of the hydrolysis polycondensation reaction is 20 - 24 h; among them, the ratio of surfactant, urea, and water is 1 - 6 g: 1.8 g: 90 mL; the volume ratio of water, tetraethyl orthosilicate, isopropanol, and cyclohexane is 90: 5 - 10: 1 - 5: 90; the calcination temperature is 500 - 600 °C, and the calcination time is 6 - 12 h.

[0012] Preferably, in Step 2, the reaction temperature of the surface covalent bonding reaction is 60 - 90 °C, and the reaction time is 12 - 24 h; among them, the addition ratio of absolute ethanol, triethanolamine, octadecyltrichlorosilane, and activated mesoporous silica is 150 mL: 1 - 3 mL: 2.5 - 10 mL: 5 g;

[0013] The reaction temperature of the silane coupling reaction is 110 °C, and the reaction time is 12 - 24 h; the addition ratio of anhydrous toluene, trimethylchlorosilane, and C18-modified mesoporous silica is 60 mL: 1 - 3 mL: 2 g.

[0014] Preferably, in Step 2, the preparation method of the activated mesoporous silica is: disperse nano-mesoporous silica into a hydrochloric acid solution, and perform acidification treatment to obtain activated mesoporous silica; the ratio of nano-mesoporous silica and hydrochloric acid solution is 10 g: 300 ml, the hydrochloric acid concentration is 6.0 - 7.0 mol / L, the acidification treatment temperature is 115 - 125 °C, and the reaction time is 5 - 6 h.

[0015] Preferably, in step 3, the ratio of C18-modified nano-mesoporous silica to N,N-dimethylformamide A is 0.2 - 2.0 g:8 ml; the mass concentration of polyacrylonitrile in N,N-dimethylformamide B is 6% - 10%; the volume ratio of solution A to solution B is 8:6.

[0016] Preferably, in step 3, the matrix treatment method for acid etching is as follows: using a 12-tooth stainless steel sheet that supports the extraction coating as the matrix, adding hydrochloric acid, and performing ultrasonic etching at 250 W for 15 min to obtain the acid-etched matrix.

[0017] Preferably, in step 3, measure 15 mm from the bottom end of each single tooth of the 12-tooth stainless steel sheet and mark it with tape;

[0018] During spraying, pour the homogenized slurry into the liquid storage tank of an air pump spray pen, and adjust the flow rate to 0.02 - 0.12 mL / s. Align the spray nozzle with the surface of the single tooth, keep a distance of about 10 cm between the spray nozzle and the surface of the single tooth, and move the spray nozzle back and forth once between the bottom end and the tape end of the single tooth at a speed of 1 - 5 mm / s. Under the action of gas, evenly spray the homogenized slurry onto the surface of the matrix; perform the spraying and curing processes on both sides of each tooth respectively. The curing temperature is 60 - 100 °C, and the curing time is 2 - 5 min. Repeat the spraying-curing step 7 - 8 times to prepare a microextraction film with a length of 15 mm, a width of 2 mm, and a thickness of about 150 μm.

[0019] Preferably, in step 4, install a 96-well deep well plate in a high-throughput oscillating extraction device, and insert the microextraction films into each deep well for extraction analysis; the extraction analysis is carried out according to the following steps:

[0020] Step 4-1, Activation: Add 1 mL of a mixed solution of methanol and water to each deep well, and oscillate at 25 °C for 30 min to wet and activate the coating; in the mixed solution, the volume ratio of methanol to water is 1:1;

[0021] Step 4-2, Sample extraction: Add 1 mL of sample solution to each deep well and oscillate at 25 °C for 50 min for extraction;

[0022] Step 4-3, Cleaning: Immerse each coating in 1 mL of cleaning solution and let it stand at 25 °C for 15 s;

[0023] Step 4-4, Elution: Pipette 1 mL of eluent into each deep well respectively, and oscillate at 25 °C for 5 - 50 min to achieve elution. The eluent is methanol or a mixed solution of methanol and acetonitrile.

[0024] Preferably, in step 4, the concentration of the plasticizer standard mixed solution is 20 ng / mL, and the plasticizers are dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibenzyl phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, diallyl phthalate, and bis(2-ethylhexyl) phthalate, wherein the concentration of each plasticizer is 20 ng / mL.

[0025] Preferably, in step 5, the chromatographic column is SHIMADZU VP-ODS, with a specification of 150×4.6 mm, 5 μm; the flow rate is 1.0 mL / min, the column temperature is 30 °C; the detection wavelength is 226 nm;

[0026] Mobile phase A is water; mobile phase B is acetonitrile;

[0027] The gradient elution program is as follows:

[0028] 0 - 10 min, 75% - 100% B,

[0029] 10 - 15 min, 100% B,

[0030] 15 - 17 min, 75% B.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a high-throughput analysis method for plasticizers (phthalates) based on thin-film microextraction technology. This method uses self-made C18-modified dendritic mesoporous silica (C18-mesosilica) as the coating material, mixes it with polyacrylonitrile (PAN), and then uses the self-developed spraying technology to prepare a microextraction film with high extraction performance on the surface of a stainless-steel extraction wafer. The microextraction film is integrated into a high-throughput extraction device, with C18-mesosilica-PAN as the extraction phase, combined with a 96-well deep-well plate, and a high-performance liquid chromatograph as the detection device, to develop a new high-throughput analysis method for phthalate plasticizers, which can achieve quantitative analysis of PAEs in food. This method has the advantages of high sample processing throughput (simultaneously processing 96 samples at a time), fast speed (the processing time for a single sample < 1.5 min), low cost (a single coating wafer can be reused more than 10 times), high sensitivity (detection limit 0.09 - 0.25 ng / mL), good precision (RSD < 12.8%), and good reproducibility between coating batches (RSD < 11.3%), and can meet the high-throughput, efficient, and rapid analysis of PAEs in a large number of samples. Description of the Drawings

[0033] Figure 1 SEM (A) and TEM (B) diagrams of the dendritic mesoporous silica prepared in Example 1;

[0034] Figure 2 Schematic diagram (A) of the shape, size and coating size of a 12-tooth stainless steel thin sheet and the physical diagram (B) of the 96-blade high-throughput extraction device with a C18-mesosilica thin film coating on its surface;

[0035] Figure 3 Liquid chromatography separation diagram of 8 kinds of PAEs;

[0036] Figure 4 Reusability test of C18-mesosilica thin film coating. Specific implementation mode

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] Embodiment 1

[0039] A high-throughput analysis method for phthalate plasticizers based on thin film microextraction technology, comprising the following steps:

[0040] (1) Preparation of dendritic nano-mesoporous silica (mesosilica)

[0041] First, dissolve 3 g of cetyltrimethylammonium bromide (CTAB) and 1.8 g of urea in 90 mL of distilled water, add 7.5 mL of tetraethyl orthosilicate, stir and mix for 30 min, then add 2.76 mL of isopropanol and 90 mL of cyclohexane and continue stirring for 50 min; secondly, heat up to 70 °C and react under magnetic stirring at 150 rpm for 24 h. After the reaction is completed, centrifuge at 11000 rpm for 12 min to obtain a white solid, wash it twice with water, twice with absolute ethanol, and dry it at 60 °C; finally, place the product in a muffle furnace and calcine it at 550 °C for 10 h (heating for 5 h and maintaining for 5 h) to remove the template agent CTAB to obtain nano-mesoporous silica.

[0042] (2) C18 modification of the mesoporous silica surface

[0043] ① Weigh 10 g of nano-mesoporous silica microspheres, disperse them in 300 mL of hydrochloric acid solution with a concentration of 6 mol L -1 Reflux at 120 °C for 6 h under magnetic stirring to activate the surface of the mesoporous silica. After the reaction is completed, centrifuge at 11000 rpm for 12 min, and the product is washed with 2 mol L - 1Wash with NaOH solution until neutral, then wash with water and anhydrous ethanol three times in sequence, and dry at 60°C to obtain activated mesoporous silica;

[0044] ② Disperse 5g activated mesoporous silica, 5mL octadecyltrichlorosilane and 1mL triethanolamine in 150mL anhydrous ethanol, and reflux at 70℃ for 24h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with anhydrous ethanol three times, and vacuum dry at 60℃ to obtain C18 modified mesoporous silica;

[0045] ③ Weigh 2.0g C18-modified mesoporous silica, disperse it in 60mL anhydrous toluene, add 2mL trimethylsilyl chloride, reflux at 110℃ for 24h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with toluene and anhydrous ethanol twice, and dry it in vacuum at 60℃ to finally obtain C18-modified nano-mesoporous silica (C18-mesosilica).

[0046] (3) Preparation of microextraction film:

[0047] ① Stainless steel substrate treatment: 12-tooth stainless steel sheet ( Figure 2 ) The substrate was immersed in deionized water and anhydrous ethanol in turn, ultrasonically cleaned at 250W for 10 minutes, and then placed in an oven at 60°C for drying. Then it was immersed in concentrated hydrochloric acid (36-38%), ultrasonically etched at 250W for 15 minutes, and the thin slice was taken out and quickly wiped off the surface attachments with clean absorbent paper, and repeatedly rinsed with deionized water, and then placed in an oven at 60°C for drying. Finally, each single tooth was accurately measured from the bottom end with a ruler for 15mm in length, and marked with tape (see Figure 2 ).

[0048] ② Preparation of C18-mesosilica homogenate: First, weigh 1.6 g of polyacrylonitrile (PAN, Mw 15000), evenly disperse it in 18.6 g of DMF, and stir overnight to form a light yellow transparent PAN-DMF solution (8% w / w). Secondly, disperse 1.5 g of C18-mesosilica in 8 mL of DMF, add 6 mL of 8% (w / w) PAN-DMF solution, and stir magnetically for 24 hours to obtain a homogenate for coating preparation;

[0049] ③ Coating preparation: Pour the homogenized slurry into the liquid storage tank of an air pump spray pen, and adjust the flow rate to 0.1 mL / s. Align the spray nozzle with the surface of the single tooth, keeping a distance of about 10 cm between them. Move the spray nozzle reciprocally once between the bottom end and the tape end of the single tooth at a speed of 3 mm / s. Under the action of gas, evenly spray the homogenized slurry onto the surface of the stainless steel substrate. Repeat the spraying process on both sides of each tooth, and then place the stainless steel sheet in an 80 °C oven for curing for 3 min. Repeat the above spraying-curing steps 7 - 8 times to finally prepare a C18-mesosilica microextraction film coating with a length of 15 mm, a width of 2 mm, and a thickness of about 150 μm. According to the above self-developed spraying method, prepare 8 stainless steel sheets each containing a 12-tooth film coating.

[0050] (4) High-throughput extraction process: First, install 8 12-tooth stainless steel sheets in a high-throughput oscillating extraction device. Then, prepare a plasticizer standard mixed solution with a concentration of 1 mg / mL, which contains 8 plasticizers, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dibenzyl phthalate (BBP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DnOP), diallyl phthalate (DAP), and bis(2-ethylhexyl) phthalate (DEHP). Then, gradually dilute the concentration of the mixed standard solution to 20 ng / mL with ultrapure water for extraction analysis. The extraction steps include:

[0051] ① Coating activation: Add 1 mL of methanol / water (1:1 v / v) solution to each well of a 96-well plate, place it on a high-throughput oscillator, and then adjust the height of the extraction device so that the film coating is inserted into the activation solution. Set the oscillation speed to 500 rpm and oscillate for activation for 30 min.

[0052] ② Extraction: Pipette 1 mL of the plasticizer standard mixed solution or the sample solution to be tested (pH 4) into a 96-well plate, insert the film coating, and oscillate for extraction at 500 rpm at 25 °C for 50 min. During the extraction process, the pH of the solution varies within the range of 2 - 8, and the extraction time varies within the range of 5 - 100 min.

[0053] ③ Cleaning: Immerse the film coating in 1 mL of water for 15 s (static) to remove impurities attached to the surface of the coating.

[0054] ④ Elution: Add 1 mL of eluent (acetonitrile / methanol 3:1 v / v) to a 96-well plate, then immerse the thin film coating into the eluent, and shake and elute at 500 rpm at 25 °C for 40 min. Collect the eluent. During the elution process, the types of eluents include acetonitrile, acetonitrile / methanol (3:1 v / v), acetonitrile / methanol (2:1 v / v), acetonitrile / methanol (1:1 v / v), acetonitrile / methanol (1:2 v / v), and methanol. After optimizing the five eluents, it was found that the eluent with the best elution effect for PAEs was acetonitrile / methanol (3:1 v / v), and the residual amount of PAEs on the coating after elution was the least (<1 ng). The elution time was varied within 5 - 50 min, and the desorption equilibrium of PAEs was reached at about 40 min. Select 40 min as the elution time.

[0055] (5) High-performance liquid chromatography analysis: Analyze 8 kinds of PAEs in the standard solution and sample solution by reverse-phase chromatography. The liquid chromatography conditions include: chromatographic column: SHIMADZU VP-ODS (150×4.6 mm 5 μm), mobile phase: A - water, B - acetonitrile, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 226 nm;

[0056] Gradient program: 0 - 10 min 75% - 100% B, 10 - 15 min, 100% B, 15 - 17 min 75% B.

[0057] Before performing liquid chromatography analysis, first place the eluent in a nitrogen blowing instrument to evaporate the solvent to dryness at 40 °C, then add 0.1 mL of HPLC-grade acetonitrile for reconstitution, filter through a 0.22 μm filter membrane, and transfer it to a liquid chromatography vial with an inner insert tube.

[0058] Example 2

[0059] A high-throughput analysis method for phthalate plasticizers based on thin film microextraction technology, comprising the following steps:

[0060] (1) Preparation of dendritic mesoporous silica (mesosilica)

[0061] First, dissolve 1 g of cetyltrimethylammonium bromide (CTAB) and 1.8 g of urea in 90 mL of distilled water, add 5 mL of tetraethyl orthosilicate, stir and mix for 30 min, then add 1 mL of isopropanol and 90 mL of cyclohexane and continue stirring for 50 min; secondly, heat up to 75 °C and magnetically stir and react at 150 rpm for 20 h. After the reaction is completed, centrifuge at 11000 rpm for 12 min to obtain a white solid, wash it twice with water, twice with absolute ethanol, and dry it at 60 °C; finally, place the product in a muffle furnace and calcine it at 600 °C for 6 h (heating for 3 h, maintaining for 3 h) to remove the template agent CTAB to obtain mesoporous silica.

[0062] (2) C18 modification of mesoporous silica surface

[0063] ① Weigh 10g of nano-mesoporous silica microspheres and disperse them in 300mL of 7mol L -1 The surface of the mesoporous silicon was activated by refluxing at 115°C for 5 h under magnetic stirring in a hydrochloric acid solution. After the reaction, the product was centrifuged at 11000 rpm for 12 min and the product was purified by 2 mol L - 1 Wash with NaOH solution until neutral, then wash with water and anhydrous ethanol three times in sequence, and dry at 60°C to obtain activated mesoporous silica;

[0064] ② Disperse 5g activated mesoporous silica, 2.5mL octadecyltrichlorosilane and 2mL triethanolamine in 150mL anhydrous ethanol, and reflux at 60℃ for 20h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with anhydrous ethanol 3 times, and dry it in vacuum at 60℃ to obtain C18 modified mesoporous silica;

[0065] ③ Weigh 2.0g C18-modified mesoporous silica, disperse it in 60mL anhydrous toluene, add 1mL trimethylsilyl chloride, reflux at 110℃ for 12h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with toluene and anhydrous ethanol twice, and dry it in vacuum at 60℃ to finally obtain C18-modified nano-mesoporous silica (C18-mesosilica).

[0066] (3) Preparation of microextraction film:

[0067] ① Stainless steel substrate treatment: 12-tooth stainless steel sheet ( Figure 2 ) The substrate was immersed in deionized water and anhydrous ethanol in turn, ultrasonically cleaned at 250W for 10 minutes, and then placed in an oven at 60°C for drying. Then it was immersed in concentrated hydrochloric acid (36-38%), ultrasonically etched at 250W for 15 minutes, and the thin slice was taken out and quickly wiped off the surface attachments with clean absorbent paper, and repeatedly rinsed with deionized water, and then placed in an oven at 60°C for drying. Finally, each single tooth was accurately measured from the bottom end with a ruler for 15mm in length, and marked with tape (see Figure 2 ).

[0068] ② Preparation of C18-mesosilica homogenate: First, weigh 1.1 g of polyacrylonitrile (PAN, Mw 15000) and disperse it evenly in 18.6 g of DMF. Stir overnight to form a light yellow transparent PAN-DMF solution (6% w / w). Second, disperse 2.0 g of C18-mesosilica in 8 mL of DMF, add 6 mL of 6% (w / w) PAN-DMF solution, and stir magnetically for 24 h to obtain a homogenate for coating preparation;

[0069] ③ Coating preparation: Pour the homogenate into the liquid storage tank of an air pump spray pen and adjust the flow rate to 0.02 mL / s. Align the spray nozzle with the surface of the single tooth, keep a distance of about 10 cm between them, and move the spray nozzle reciprocally once between the bottom end and the tape end of the single tooth at a speed of 1 mm / s. Under the action of gas, evenly spray the homogenate onto the surface of the stainless steel substrate. Repeat the spraying process on both sides of each tooth, and then place the stainless steel sheet in an oven at 100 °C for curing for 2 min. Repeat the above spraying-curing steps 7-8 times to finally prepare a C18-mesosilica microextraction thin film coating with a length of 15 mm, a width of 2 mm, and a thickness of about 150 μm. According to the above self-made spraying method, 8 stainless steel sheets with 12-tooth thin film coatings are prepared respectively.

[0070] (4) High-throughput extraction process: First, install 8 stainless steel sheets with 12 teeth in a high-throughput oscillating extraction device. Then, prepare a plasticizer standard mixed solution with a concentration of 1 mg / mL, which contains 8 plasticizers, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dibenzyl phthalate (BBP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DnOP), diallyl phthalate (DAP), and bis(2-ethylhexyl) phthalate (DEHP). Then, gradually dilute the concentration of the mixed standard solution to 20 ng / mL with ultrapure water for extraction analysis. The extraction steps include:

[0071] ① Coating activation: Add 1 mL of methanol / water (1:1 v / v) solution to each well of a 96-well plate, place it on a high-throughput oscillator, and then adjust the height of the extraction device so that the thin film coating is inserted into the activation solution. Set the oscillation speed to 500 rpm and oscillate for activation for 30 min.

[0072] ② Extraction: Pipette 1 mL of the plasticizer standard mixed solution or the sample solution to be tested (pH 4) into a 96-well plate, insert the thin film coating, and oscillate for extraction at 500 rpm at 25 °C for 50 min. During the extraction process, the pH of the solution varies within the range of 2-8, and the extraction time varies within the range of 5-100 min.

[0073] ③ Cleaning: Immerse the thin film coating in 1 mL of water for 15 s (static) to remove impurities adhering to the coating surface.

[0074] ④ Elution: Add 1 mL of eluent (acetonitrile / methanol 3:1 v / v) to a 96-well plate, then immerse the thin film coating in the eluent, and shake and elute at 500 rpm for 50 min at 25 °C. Collect the eluate.

[0075] (6) High-performance liquid chromatography analysis: Analyze 8 PAEs in the standard solution and the sample solution by reversed-phase chromatography. The liquid chromatography conditions include: chromatographic column: SHIMADZU VP-ODS (150×4.6 mm 5 μm), mobile phase: A - water, B - acetonitrile, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 226 nm;

[0076] Gradient program: 0 - 10 min 75% - 100% B, 10 - 15 min, 100% B, 15 - 17 min 75% B.

[0077] Before performing liquid chromatography analysis, first place the eluate in a nitrogen blower to evaporate the solvent to dryness at 40 °C, then add 0.1 mL of HPLC-grade acetonitrile for reconstitution, filter through a 0.22 μm filter membrane, and transfer to a liquid chromatography vial with an inner insert tube.

[0078] Example 3

[0079] A high-throughput analysis method for phthalate plasticizers based on thin film microextraction technology, comprising the following steps:

[0080] (1) Preparation of dendritic mesoporous silica

[0081] First, dissolve 6 g of cetyltrimethylammonium bromide (CTAB) and 1.8 g of urea in 90 mL of distilled water, add 10 mL of tetraethyl orthosilicate, stir and mix for 30 min, then add 5 mL of isopropanol and 90 mL of cyclohexane and continue stirring for 50 min; secondly, heat up to 65 °C and react with magnetic stirring at 150 rpm for 22 h. After the reaction is completed, centrifuge at 11000 rpm for 12 min to obtain a white solid, wash it twice with water and twice with absolute ethanol, and dry it at 60 °C; finally, place the product in a muffle furnace and calcine it at 500 °C for 12 h (heating for 6 h and maintaining for 6 h) to remove the template agent CTAB to obtain mesoporous silica.

[0082] (2) C18 modification on the surface of mesoporous silica

[0083] ① Weigh 10 g of mesoporous silica microspheres and disperse them in 300 mL of a solution with a concentration of 6.5 mol L -1In hydrochloric acid solution, reflux at 125℃ for 5.5h under magnetic stirring to activate the surface of mesoporous silicon. After the reaction, centrifuge at 11000rpm for 12min, and the product is washed with 2molL -1 Wash with NaOH solution until neutral, then wash with water and anhydrous ethanol three times in sequence, and dry at 60°C to obtain activated mesoporous silica;

[0084] ② Disperse 5g activated mesoporous silica, 10mL octadecyltrichlorosilane and 3mL triethanolamine in 150mL anhydrous ethanol, and reflux at 90℃ for 12h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with anhydrous ethanol 3 times, and vacuum dry at 60℃ to obtain C18 modified mesoporous silica;

[0085] ③ Weigh 2.0g C18-modified mesoporous silica, disperse it in 60mL anhydrous toluene, add 3mL trimethylsilyl chloride, reflux at 110℃ for 20h under mechanical stirring at 170rpm. After the reaction, centrifuge at 11000rpm for 10min, wash the product with toluene and anhydrous ethanol twice, and dry it in vacuum at 60℃ to finally obtain C18-modified nano-mesoporous silica (C18-mesosilica).

[0086] (3) Preparation of microextraction film:

[0087] ① Stainless steel substrate treatment: 12-tooth stainless steel sheet ( Figure 2 ) The substrate was immersed in deionized water and anhydrous ethanol in turn, ultrasonically cleaned at 250W for 10 minutes, and then placed in an oven at 60°C for drying. Then it was immersed in concentrated hydrochloric acid (36-38%), ultrasonically etched at 250W for 15 minutes, and the thin slice was taken out and quickly wiped off the surface attachments with clean absorbent paper, and repeatedly rinsed with deionized water, and then placed in an oven at 60°C for drying. Finally, each single tooth was accurately measured from the bottom end with a ruler for 15mm in length, and marked with tape (see Figure 2 ).

[0088] ② Preparation of C18-mesosilica homogenate: First, weigh 1.9 g of polyacrylonitrile (PAN, Mw 15000), evenly disperse it in 18.6 g of DMF, and stir overnight to form a light yellow transparent PAN-DMF solution (10% w / w). Secondly, disperse 0.2 g of C18-mesosilica in 8 mL of DMF, add 6 mL of 10% (w / w) PAN-DMF solution, and stir magnetically for 24 hours to obtain a homogenate for coating preparation;

[0089] ③ Coating preparation: Pour the homogenized slurry into the liquid storage tank of an air pump spray pen, and adjust the flow rate to 0.12 mL / s. Align the spray nozzle with the surface of a single tooth, keeping a distance of about 10 cm between them. Move the spray nozzle reciprocally once between the bottom end and the tape end of the single tooth at a speed of 5 mm / s. Under the action of gas, evenly spray the homogenized slurry onto the surface of the stainless-steel substrate. Repeat the spraying process on both sides of each tooth, and then place the stainless-steel sheet in an oven at 60 °C for curing for 2 min. Repeat the above spraying-curing steps 7 - 8 times, and finally prepare a C18-mesosilica microextraction thin-film coating with a length of 15 mm, a width of 2 mm, and a thickness of about 150 μm. According to the above self-made spraying method, respectively prepare 8 stainless-steel sheets with 12-tooth thin-film coatings.

[0090] (4) High-throughput extraction process: First, install 8 stainless-steel sheets with 12 teeth in a high-throughput oscillating extraction device. Then, prepare a plasticizer standard mixed solution with a concentration of 1 mg / mL, which contains 8 plasticizers, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dibenzyl phthalate (BBP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DnOP), diallyl phthalate (DAP), and bis(2-ethylhexyl) phthalate (DEHP). Then, gradually dilute the concentration of the mixed standard solution to 20 ng / mL with ultrapure water for extraction analysis. The extraction steps include:

[0091] ① Coating activation: Add 1 mL of methanol / water (1:1 v / v) solution to each well of a 96-well plate, place it on a high-throughput oscillator, and then adjust the height of the extraction device so that the thin-film coating is inserted into the activation solution. Set the oscillation speed to 500 rpm and oscillate for activation for 30 min.

[0092] ② Extraction: Pipette 1 mL of the plasticizer standard mixed solution or the sample solution to be tested (pH 4) into a 96-well plate, insert the thin-film coating, and oscillate for extraction at 500 rpm at 25 °C for 50 min. During the extraction process, the pH of the solution varies within the range of 2 - 8, and the extraction time varies within the range of 5 - 100 min.

[0093] ③ Cleaning: Immerse the thin-film coating in 1 mL of water for 15 s (static) to remove impurities attached to the surface of the coating.

[0094] ④ Elution: Add 1 mL of eluent (acetonitrile / methanol 3:1 v / v) to a 96-well plate, then immerse the thin-film coating in the eluent, and oscillate for elution at 500 rpm at 25 °C for 50 min, and collect the eluent.

[0095] (7) High Performance Liquid Chromatography Analysis: The reverse phase chromatography method was used to analyze 8 kinds of PAEs in the standard solution and the sample solution. The liquid chromatography conditions were as follows: chromatographic column: SHIMADZU VP-ODS (150×4.6 mm 5 μm), mobile phase: A - water, B - acetonitrile, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 226 nm;

[0096] Gradient program: 0 - 10 min 75% - 100% B, 10 - 15 min, 100% B, 15 - 17 min 75% B.

[0097] Before performing liquid chromatography analysis, the eluent was placed in a nitrogen blowing instrument to evaporate the solvent to dryness at 40 °C, then 0.1 mL of HPLC-grade acetonitrile was added for reconstitution, and after filtration through a 0.22 μm filter membrane, it was transferred to a liquid chromatography vial with an inner insert tube.

[0098] Taking Example 1 as an example for analysis, according to the high-throughput extraction steps in Example 1, the total time required to complete a complete sample pretreatment step was 120 min, the sample size processed each time was 96, and the average processing time for a single sample was about 1.3 min.

[0099] The morphology and structure of the dendritic nano-mesoporous silica are as Figure 1 shown. The mesoporous silica has a porous spherical morphology with a uniform particle size distribution, and the size is about in the range of 300 - 400 nm. It has a central radially oriented mesoporous structure inside, and the pore channels are regular and uniform, showing a radial fiber state.

[0100] The size and shape of the 12-tooth stainless steel thin sheet and its surface covered with a C18-mesosilica thin film coating are as Figure 2 shown. Using the spraying preparation method independently developed by the present invention, the C18-mesosilica coating prepared on the surface of the stainless steel thin sheet has a smooth and flat surface, uniform thickness up and down, and good stability. A high-throughput extraction head containing 96 C18-mesosilica coatings can be prepared.

[0101] The liquid chromatography separation diagram of 8 kinds of PAEs is as Figure 3 shown. Under the chromatographic conditions of this method, the 8 kinds of PAEs achieved baseline separation within 15 min.

[0102] (I) Analysis Performance Verification

[0103] 1. Linear range, detection limit (LOD) and quantification limit (LOQ) of the method

[0104] Determine a series of standard solutions of 8 PAEs compounds according to the method provided in Example 1, and continuously analyze with a blank solution for 10 times. Calculate the detection limit and quantification limit based on the standard deviation of the 10 results. The results of the method's linear range, detection limit, and quantification limit are shown in Table 1.

[0105] Table 1 Linear relationship of standard curves, detection limits, and quantification limits of 8 PAEs compounds

[0106]

[0107] 2. Method accuracy, precision, and reproducibility between thin film coating batches

[0108] According to the method provided in Example 1, analyze standard samples at three concentration levels of 5 ng / mL, 20 ng / mL, and 50 ng / mL respectively, and investigate the accuracy and precision of the method within-day and between-day. The results are shown in Table 2 and Table 3. The results show that the within-day accuracy of the method at the three concentration levels of 5, 20, and 50 ng / mL is 91% - 109%, 94% - 107%, 92 - 108% respectively, and the precision RSD is 2.0% - 9.3%, 1.9 - 8.0%, 0.7% - 8.0% respectively; the between-day accuracy is 93% - 111%, 93% - 108%, 94% - 105% respectively, and the precision RSD is 3.1% - 12.8%, 3.0% - 6.0%, 3.7 - 8.4% respectively. It shows that the method has good accuracy and precision.

[0109] Table 2 Within-day and between-day accuracy and precision of this method

[0110]

[0111] Compare the reproducibility of the preparation between thin film coating batches according to the method of Example 1. Measure the results between different batches of coatings at three concentration levels of 5 ng / mL, 20 ng / mL, and 50 ng / mL respectively, and calculate the relative standard deviation RSD. The results are shown in Table 3. The results show that the RSD values of the thin film coatings prepared by this method at the three concentration levels are between 1.1% - 11.3%, 1.1% - 6.5%, and 0.7% - 6.5% respectively, showing good reproducibility between batches.

[0112] Table 3 Reproducibility between thin film coating batches

[0113]

[0114]

[0115] 3. Performance of repeated use of the coating

[0116] According to the method of Example 1, at a concentration level of 20 ng / mL, the process was cycled 10 times using the same wafer with 12 extraction coatings, and 12 samples were analyzed each time. The content of PAEs in the samples was determined, and the relative standard deviation RSD was calculated. The results are as Figure 4 shown. The results show that after analyzing 120 samples, the extraction rate of the coating can be maintained above 87%, and the RSD of the sample measurement results is between 2.3% and 8.4%, indicating that the thin film coating has good stability and reusability.

[0117] (II) Determination of actual samples

[0118] This method was applied to the analysis and determination of plasticizers in fruit tea - flavored beverages, and the accuracy of the method was verified through a standard addition recovery experiment (adding 20 ng / mL). Before the treatment steps and analysis and detection according to this method, the beverage samples were only subjected to ultrasonic degassing and centrifugation. The results show that no PAEs were detected in the beverages, and the standard addition recovery rate was between 89% and 107%. This indicates that this method can be applied to the determination of phthalate plasticizers in foods.

[0119] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology, characterized in that, Proceed as follows: Step 1: Dissolve the surfactant and urea in water, add tetraethyl orthosilicate, isopropanol and cyclohexane, stir evenly at room temperature, heat up to 65 - 75 °C to carry out hydrolysis and polycondensation reaction to obtain a product, and calcine the product to obtain nano - mesoporous silicon; the surfactant is cetyltrimethylammonium bromide; Step 2: Acidify the nano - mesoporous silicon to obtain activated mesoporous silicon; use absolute ethanol as the solvent, triethanolamine as the catalyst, octadecyltrichlorosilane as the modifier, add the activated mesoporous silicon, and obtain C18 - modified mesoporous silicon through surface covalent bonding reaction; then use anhydrous toluene as the solvent, trimethylchlorosilane as the capping reagent, add the C18 - modified mesoporous silicon, and carry out capping through silane coupling reaction to obtain C18 - modified nano - mesoporous silicon; Step 3: Disperse the C18 - modified nano - mesoporous silicon in N,N - dimethylformamide A to obtain solution A; Disperse polyacrylonitrile in N,N - dimethylformamide B to obtain solution B, mix solution A and solution B to obtain a homogeneous slurry, spray the homogeneous slurry on the surface of the acid - etched substrate, and cure to obtain a micro - extraction film; Step 4: Install the micro - extraction film in a high - throughput oscillating extraction device, prepare a standard mixed solution of plasticizers, and use ultrapure water to gradually dilute the concentration of the mixed standard solution to 20 ng / mL for extraction analysis; Step 5: Use reverse - phase liquid chromatography to analyze the phthalate plasticizers in the standard mixed solution of plasticizers and the sample solution, and calculate the content.

2. The high-throughput analysis method for phthalate plasticizers based on thin film microextraction technology according to claim 1, characterized in that, In step 1, the reaction time of the hydrolysis and polycondensation reaction is 20 - 24 h; among them, the ratio of the surfactant, urea, and water is 1 - 6 g:1.8 g:90 mL; the volume ratio of water, tetraethyl orthosilicate, isopropanol, and cyclohexane is 90:5 - 10:1 - 5:90; the calcination temperature is 500 - 600 °C, and the calcination time is 6 - 12 h.

3. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, characterized in that, In step 2, the reaction temperature of the surface covalent bonding reaction is 60 - 90 °C, and the reaction time is 12 - 24 h; among them, the addition ratio of absolute ethanol, triethanolamine, octadecyltrichlorosilane, and activated mesoporous silicon is 150 mL:1 - 3 mL:2.5 - 10 mL:5 g; The reaction temperature of the silane coupling reaction is 110 °C, and the reaction time is 12 - 24 h; the addition ratio of anhydrous toluene, trimethylchlorosilane, and C18 - modified mesoporous silicon is 60 mL:1 - 3 mL:2 g.

4. The high-throughput analysis method for phthalate plasticizers based on thin film microextraction technology according to claim 1, characterized in that In step 2, the preparation method of the activated mesoporous silicon is: disperse the nano - mesoporous silicon into a hydrochloric acid solution, and carry out acidification treatment to obtain activated mesoporous silicon; the ratio of the nano - mesoporous silicon and the hydrochloric acid solution is 10 g:300 ml, the hydrochloric acid concentration is 6.0 - 7.0 mol / L, the acidification treatment temperature is 115 - 125 °C, and the reaction time is 5 - 6 h.

5. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, characterized in that, In step 3, the ratio of the C18 - modified nano - mesoporous silicon to N,N - dimethylformamide A is 0.2 - 2.0 g:8 ml; the mass concentration of polyacrylonitrile in N,N - dimethylformamide B is 6% - 10%; the volume ratio of solution A to solution B is 8:

6.

6. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, characterized in that, In Step 3, the matrix treatment method of acid etching is as follows: using a 12-tooth stainless steel sheet that supports the extraction coating as the matrix, adding hydrochloric acid, and performing ultrasonic etching at 250 W for 15 min to obtain the acid-etched matrix.

7. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 6, wherein, In Step 3, measure 15 mm from the bottom end of each single tooth of the 12-tooth stainless steel sheet and mark it with tape; During spraying, pour the homogenate into the liquid storage tank of an air pump spray pen, adjust the flow rate to 0.02 - 0.12 mL / s, align the spray nozzle with the surface of the single tooth, keep a distance of 10 cm between the spray nozzle and the surface of the single tooth, move the spray nozzle reciprocally once between the bottom end and the tape end of the single tooth at a speed of 1 - 5 mm / s, and evenly spray the homogenate onto the surface of the matrix under the action of gas; perform the spraying and curing processes on both sides of each tooth respectively. The curing temperature is 60 - 100 °C, and the curing time is 2 - 5 min. The spraying-curing step is repeated 7 - 8 times to prepare a microextraction film with a length of 15 mm, a width of 2 mm, and a thickness of 150 μm.

8. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, characterized in that, In Step 4, in a high-throughput oscillating extraction device, install a 96-well deep-well plate, and insert the microextraction films into each deep well for extraction analysis; the extraction analysis is carried out according to the following steps: Step 4-1, Activation: Add 1 mL of a mixed solution of methanol and water to each deep well, and oscillate at 25 °C for 30 min to soak and activate the coating; in the mixed solution, the volume ratio of methanol to water is 1:1; Step 4-2, Sample Extraction: Add 1 mL of sample solution to each deep well and oscillate at 25 °C for 50 min for extraction; Step 4-3, Cleaning: Immerse each coating in 1 mL of cleaning solution and let it stand at 25 °C for 15 s; Step 4-4, Elution: Pipette 1 mL of eluent into each deep well respectively, and oscillate at 25 °C for 5 - 50 min to achieve elution. The eluent is methanol or a mixed solution of methanol and acetonitrile.

9. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, wherein In Step 4, the concentration of the phthalate standard mixed solution is 20 ng / mL, and the phthalates are dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibenzyl phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, diallyl phthalate, and bis(2-ethylhexyl) phthalate. Among them, the concentration of each phthalate is 20 ng / mL.

10. The high-throughput analysis method for phthalate plasticizers based on thin-film microextraction technology according to claim 1, wherein In Step 5, the chromatographic column is SHIMADZU VP-ODS, with a specification of 150×4.6 mm, 5 μm; flow rate: 1.0 mL / min, column temperature: 30 °C; detection wavelength is 226 nm; Mobile phase A is water; mobile phase B is acetonitrile; The gradient elution program is: 0 - 10 min 75% - 100%B, 10 - 15 min, 100%B, 15 - 17 min 75%B.

Images