Pre-treatment method for detecting organic phosphate pollutants in aquatic products and method for detecting organic phosphate pollutants

Abstract

The application provides a pretreatment method for detecting organic phosphate pollutants in aquatic products and a detection method for the organic phosphate pollutants, and belongs to the technical field of analysis and detection. The pretreatment method for detecting organic phosphate pollutants in aquatic products provided by the application comprises the following steps: mixing a to-be-detected aquatic product sample with an extraction solution to perform extraction, to obtain an extraction liquid; mixing the extraction liquid, an adsorption material and a salting-out-water-removing material to perform purification, and performing magnetic separation to obtain a purified liquid; the adsorption material is amphiphilic functional magnetic material and C18, the amphiphilic functional magnetic material is Fe3O4@SiO2 particles modified by N-vinylpyrrolidone and divinylbenzene; and filtering the purified liquid to obtain a to-be-detected sample solution. The method provided by the application is simple in operation, high in processing efficiency, and makes it possible to analyze high-throughput samples.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a pretreatment method and a detection method for organophosphate contaminants in aquatic products. Background Technology

[0002] Organophosphates (OPEs) are a new type of flame retardant, gradually replacing brominated flame retardants and widely used in building materials, textiles, chemicals, and electronics industries. Large amounts of released OPEs ultimately enter the aquatic environment, contaminating aquatic products and endangering human health through inhalation, skin contact, and bioaccumulation. Therefore, to ensure food safety and protect consumer health, research on OPE residue analysis in aquatic products is urgently needed. Current domestic and international research on contaminant residue analysis in aquatic products typically employs organic solvent extraction of samples. The extract is then purified using solid-phase extraction (SPE) or dispersive solid-phase extraction (d-SPE) methods, followed by accurate quantitative analysis using chromatography and mass spectrometry. However, both SPE and dispersive solid-phase extraction methods require centrifugation for phase separation, which limits the speed of contaminant residue analysis in batches of samples. How to further improve the efficiency of methods for batch sample analysis has become a pressing issue for researchers. Summary of the Invention

[0003] The purpose of this invention is to provide a pretreatment method and a detection method for organophosphate (OPE) contaminants in aquatic products. The method provided by this invention solves the limitations of the processing speed and time of organophosphate (OPE) contaminants in aquatic product sample pretreatment technology, and can achieve efficient and rapid analysis of OPE residues in aquatic products while ensuring the accuracy of recovery rate.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a pretreatment method for detecting organophosphate contaminants in aquatic products, comprising the following steps:

[0006] The aquatic product sample to be tested is mixed with the extraction solution and extracted to obtain the extraction liquid.

[0007] The extraction solution, adsorbent material, and salting-out dehydration material are mixed for purification, and the purified solution is obtained by magnetic separation. The adsorbent material is an amphiphilic functional magnetic material and C18. The amphiphilic functional magnetic material is Fe3O4@SiO2 particles modified with N-vinylpyrrolidone and divinylbenzene.

[0008] The purified solution was filtered to obtain the sample solution to be tested.

[0009] Preferably, the aquatic sample to be tested includes samples prepared from shrimp and / or fish.

[0010] Preferably, the extraction solution is acetonitrile, acetic acid-acetonitrile solution, or acetic acid-acetonitrile-aqueous solution; the volume fraction of acetic acid in the acetic acid-acetonitrile solution is 0.5-2%; the volume fraction of acetic acid in the acetic acid-acetonitrile-aqueous solution is 0.5-2%, and the volume ratio of the total volume of acetic acid and acetonitrile to the volume of water is (5-8):(2-4); the volume ratio of the aquatic product sample to be tested to the extraction solution is 2g:(2.5-5)mL.

[0011] Preferably, the extraction is carried out under vortex conditions; the extraction time is 0.5 to 3 minutes.

[0012] Preferably, the mass ratio of the amphiphilic functional magnetic material to C18 in the adsorbent material is (10-70):(10-50); the mass ratio of the aquatic product sample to be tested to the adsorbent material is 2:(0.02-0.12).

[0013] Preferably, the salting-out material is MgSO4 and NaCl, and the mass ratio of MgSO4 to NaCl is (3-5):1; the mass ratio of the aquatic product sample to be tested to the salting-out material is 2:(1-4).

[0014] Preferably, the purification is carried out under vortex conditions; the purification time is 0.5 to 3 minutes.

[0015] Preferably, the organophosphate contaminant includes at least one of 2-ethylhexyl diphenyl phosphate, tricresyl phosphate, thylmethyl phosphate, tri(butoxyethyl) phosphate, tributyl phosphate, triisobutyl phosphate, cresol diphenyl phosphate, tri(1,3-dichloroisopropyl) phosphate, tri(2-chloropropyl) phosphate, tripropyl phosphate, p-toluyl phosphate, triphenylphosphine oxide, tri(2-chloroethyl) phosphate, triethyl phosphate, trimethyl phosphate, and triphenyl phosphate.

[0016] This invention provides a method for detecting organophosphate contaminants in aquatic products, comprising the following steps:

[0017] The aquatic product sample to be tested is processed according to the method described in the above technical solution to obtain the sample solution to be tested.

[0018] The sample to be tested was analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0019] Preferably, the high-performance liquid chromatography (HPLC) conditions for the HPLC-tandem mass spectrometry analysis include:

[0020] The mobile phase system includes phase A and phase B; phase A is a 0.1% (v / v) aqueous solution of formic acid, and phase B is methanol; a gradient elution program is used, with the following gradient conditions: 0–1 min, phase B volume fraction maintained at 20%; 2–12 min, phase B volume fraction linearly increases from 20% to 90%; 12–14 min, phase B volume fraction maintained at 90%; 14–14.2 min, phase B volume fraction linearly decreases from 90% to 20%; 14.2–20 min, phase B volume fraction maintained at 20%.

[0021] This invention provides a pretreatment method for detecting organophosphate contaminants in aquatic products, comprising the following steps: mixing the aquatic product sample to be tested with an extraction solution for extraction to obtain an extraction liquid; mixing the extraction liquid, an adsorbent material, and a salting-out dehydration material for purification, and obtaining a purified liquid through magnetic separation; wherein the adsorbent material is an amphiphilic functional magnetic material and C18, and the amphiphilic functional magnetic material is obtained by modifying Fe3O4@SiO2 particles with N-vinylpyrrolidone (NVP) and divinylbenzene (DVB); and filtering the purified liquid to obtain the sample solution to be tested. In this invention, the amphiphilic functional magnetic material (Fe3O4@SiO2@NVP-DVB) can achieve ideal removal of matrix interferences such as fats, proteins, and fatty acids in aquatic products through the synergistic effects of hydrophobic-hydrophilic, hydrogen bonds, and π-π bonds. C18 can also remove non-polar interfering impurities such as lipids. Compared with non-magnetic purification materials such as N-propylethylenediamine-bonded silica gel (PSA) and graphitized carbon black (GCB), C18 has the best purification effect. The salting-out and dehydration material mainly plays the role of dehydration and salting out. The method provided by this invention solves the limitations of processing speed and time for organophosphate (OPE) contaminants in aquatic product sample pretreatment technology. After extraction, the obtained extract solution is directly purified without the need for other transfer or separation operations. The extraction and purification of OPEs in aquatic product samples can be achieved in the same container, effectively separating the analyte from matrix impurities. This improves the operation speed, reduces system losses and deviations caused by experimental transfer, and enables efficient and rapid analysis of OPE residues in aquatic products while ensuring the accuracy of recovery rate. It greatly improves the sample pretreatment speed, increases work efficiency, makes high-throughput sample analysis possible, and reduces the cost of experimental instruments (mainly centrifuges). Detailed Implementation

[0022] This invention provides a pretreatment method for detecting organophosphate contaminants in aquatic products, comprising the following steps:

[0023] The aquatic product sample to be tested is mixed with the extraction solution and extracted to obtain the extraction liquid.

[0024] The extraction solution, adsorbent material, and salting-out dehydration material are mixed for purification, and the purified solution is obtained by magnetic separation. The adsorbent material is an amphiphilic functional magnetic material and C18. The amphiphilic functional magnetic material is Fe3O4@SiO2 particles modified with N-vinylpyrrolidone and divinylbenzene.

[0025] The purified solution was filtered to obtain the sample solution to be tested.

[0026] In this invention, unless otherwise specified, all raw materials used are commercially available products known to those skilled in the art or prepared using methods known to those skilled in the art.

[0027] This invention involves mixing the aquatic product sample to be tested with an extraction solution for extraction to obtain an extract liquid. In this invention, the aquatic product sample to be tested preferably includes samples prepared from shrimp and / or fish. The shrimp is preferably Litopenaeus vannamei or Litopenaeus halophilus, and the fish preferably includes one or more of large yellow croaker, grass carp, silver carp, and common carp. Preferably, the shrimp and / or fish have their heads, bones, shells, or other inedible parts removed, retaining only the edible parts, and then are minced and homogenized to obtain the aquatic product sample to be tested. In this invention, the extraction solution is preferably acetonitrile, an acetic acid-acetonitrile solution, or an acetic acid-acetonitrile-aqueous solution; the volume fraction of acetic acid in the acetic acid-acetonitrile solution is preferably 0.5-2%, more preferably 0.5-1.5%, and even more preferably 0.5-1%; the volume fraction of acetic acid in the acetic acid-acetonitrile-aqueous solution is 0.5-2%, more preferably 0.5-1.5%, and even more preferably 0.5-1%; the volume ratio of the total volume of acetic acid and acetonitrile to water is preferably (5-8):(2-4), more preferably (6-7):(3-4), even more preferably 7:3 or 6:4, and even more preferably 7:3; the volume ratio of the aquatic product sample to be tested to the extraction solution is preferably 2g:(2.5-5)mL, more preferably 2g:(3-4)mL, and even more preferably 2g:4mL. In this invention, the extraction is preferably carried out under vortex conditions; the extraction time is preferably 0.5-3 min, more preferably 1-2 min. In this invention, the extraction is preferably carried out in a centrifuge tube.

[0028] After extraction, the present invention requires no further processing. The obtained extract is directly mixed with the adsorbent material and the salting-out dehydration material for purification, and the purified liquid is obtained by magnetic separation. Preferably, the adsorbent material and the salting-out dehydration material are added to the extract for purification. In the present invention, the mass ratio of the amphiphilic functional magnetic material to C18 in the adsorbent material is preferably (10-70):(10-50), more preferably (35-45):(25-35), and even more preferably 40:30; the mass ratio of the aquatic product sample to the adsorbent material is preferably 2:(0.02-0.12), more preferably 2:(0.05-0.10), and even more preferably 2:0.07. In this invention, the salting-out and dehydration materials are preferably MgSO4 and NaCl, and the mass ratio of MgSO4 to NaCl is preferably (3-5):1, more preferably 4:1; the mass ratio of the aquatic product sample to be tested to the salting-out and dehydration materials is preferably 2:(1-4), more preferably 2:(2-3), and even more preferably 1:1. This invention uses MgSO4 and NaCl as salting-out and dehydration materials. NaCl facilitates the stratification of the organic and aqueous phases, promoting the distribution of the target compound more extensively in the organic phase. Anhydrous MgSO4 has a strong water absorption capacity, and its use can remove water from the extraction solution. Therefore, mixing MgSO4 and NaCl can improve the selectivity of the extraction / distribution process, thereby increasing the recovery rate of the analyte. In this invention, the purification is preferably carried out under vortex conditions; the purification time is preferably 0.5-3 min, more preferably 0.5-1 min.

[0029] After obtaining the purified solution, the present invention filters the purified solution to obtain the sample solution to be tested. In the present invention, the filter used for filtration is preferably a 0.22μm microporous membrane filter.

[0030] In this invention, the organophosphate contaminants preferably include at least one of 2-ethylhexyl diphenyl phosphate, tricresyl phosphate, tricresyl phosphate, tri(butoxyethyl) phosphate, tributyl phosphate, triisobutyl phosphate, cresol diphenyl phosphate, tri(1,3-dichloroisopropyl) phosphate, tri(2-chloropropyl) phosphate, tripropyl phosphate, p-toluyl phosphate, triphenylphosphine oxide, tri(2-chloroethyl) phosphate, triethyl phosphate, trimethyl phosphate, and triphenyl phosphate.

[0031] This invention provides a method for detecting organophosphate contaminants in aquatic products, comprising the following steps:

[0032] The aquatic product sample to be tested is processed according to the method described in the above technical solution to obtain the sample solution to be tested.

[0033] The sample to be tested was analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0034] In this invention, the equipment used for LC-MS / MS analysis preferably includes a Shimadzu 8050 triple quadrupole mass spectrometer and a NexeraX2 ultra-high performance liquid chromatograph, and is equipped with a communication bus module (CBM-20A), two binary pumps (LC-30AD), an autosampler (SIL-30AC), and a column oven (CTO-20AC).

[0035] In this invention, the preferred high-performance liquid chromatography (HPLC) conditions for LC-MS / MS analysis include: a mobile phase system comprising phase A and phase B; phase A being a 0.1% (v / v) aqueous solution of formic acid, and phase B being methanol; a gradient elution program is used, with the following gradient conditions: 0–1 min, phase B volume fraction maintained at 20%; 2–12 min, phase B volume fraction linearly increasing from 20% to 90%; 12–14 min, phase B volume fraction maintained at 90%; 14–14.2 min, phase B volume fraction linearly decreasing from 90% to 20%; 14.2–20 min, phase B volume fraction maintained at 20%. In this invention, the preferred HPLC conditions also include: a Luna Omega C18 column (100 mm × 2.1 mm, 1.6 μm), an oven temperature of 35 °C; a mobile phase flow rate of 0.3 mL / min, and an injection volume of 1 μL.

[0036] In this invention, the preferred mass spectrometry conditions for the LC-MS / MS analysis include: an electrospray ionization (ESI) source with a capillary voltage of 4000V, an ESI capillary temperature of 300℃, a heating block temperature of 400℃, and a DL temperature of 250℃; the flow rates of the drying gas (nitrogen), heating gas (air), and nebulizing gas (nitrogen) are set to 10L / min, 10L / min, and 3L / min, respectively; nitrogen (N2) is provided by a nitrogen generator (Shimadzu, Kyoto, Japan); the collision gas is argon; the tandem mass spectrometry is operated in multiple reaction monitoring (MRM) mode; the compound information of 16 OPEs and their mass spectrometry analysis parameters are shown in Table 1 (see Example 1 for details).

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] The C18 used in the following examples was purchased from Tianjin Bona Ager Technology Co., Ltd., and the product model is: CleanertODS C18 (Endcapped), 40-60μm.

[0039] The preparation method of Fe3O4@SiO2@NVP-DVB used in the following examples is as follows:

[0040] Fe3O4 particles were prepared by a solvothermal method, and Fe3O4@SiO2 particles were prepared by coating the surface of the Fe3O4 particles with silica. 3g of Fe3O4@SiO2 particles were added to 400mL of 75% (v / v) ethanol aqueous solution, and then 8mL of 25-28wt% ammonia solution was added at room temperature (25℃) under nitrogen protection. Solution A (obtained by adding 8mL of 3-(methacryloyloxy)propyltrimethoxysilane (MPS) to 10mL of anhydrous ethanol) was added and the mixture was vigorously stirred until homogeneous. The resulting mixture was heated to 60℃ and stirred vigorously for 12h. Magnetic material was collected by magnetic separation, washed multiple times with water and ethanol, and then vacuum dried at 60℃ for 12h to obtain Fe3O4@MPS particles. 3g of Fe3O4@MPS particles were added to 500mL of acetonitrile and sonicated for 15min. 0.1g of azobisisobutyronitrile (AIBN) was added, followed by 7.5g of DVB and 6.3g of... NVP was stirred and mixed at 300 rpm for 30 min under nitrogen protection. The resulting mixture was heated to 75 °C and reacted for 16 h. The magnetic material was separated and collected using a magnet. Then, it was washed multiple times with acetone and ethanol and dried under vacuum at 60 °C for 12 h to obtain Fe3O4@SiO2@NVP-DVB particles.

[0041] Example 1

[0042] Accurately weigh 2g (±0.01g) of shrimp sample (specifically, the edible parts of *Litopenaeus vannamei* are removed, including the head and shell, and then minced and homogenized) into a 50mL centrifuge tube. Add 3mL of 1% acetic acid acetonitrile (i.e., acetic acid volume fraction of 1%) and 1mL of water, vortex for 1min, then add 40mg of Fe3O4@SiO2@NVP-DVB, 30mg of C18, and 2g of salting-out material (a mixture of MgSO4 and NaCl in a mass ratio of 4:1). Vortex for 30s, then magnetically separate for 30s. Filter the supernatant through a 0.22μm filter membrane and perform high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. The LC-MS / MS analysis conditions include:

[0043] The LC-MS / MS system consists of a Shimadzu 8050 triple quadrupole mass spectrometer and a NexeraX2 ultra-high performance liquid chromatograph, and is equipped with a communication bus module (CBM-20A), two binary pumps (LC-30AD), an autosampler (SIL-30AC), and a column oven (CTO-20AC).

[0044] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was a Luna Omega C18 column (100 mm × 2.1 mm, 1.6 μm), and the column oven temperature was 35 °C; the mobile phase system consisted of phase A and phase B, where phase A was a 0.1% (v / v) aqueous solution of formic acid, and phase B was methanol; a gradient elution program was used, with the following gradient conditions: 0–1 min, phase B volume fraction maintained at 20%; 2–12 min, phase B volume fraction linearly increased from 20% to 90%; 12–14 min, phase B volume fraction maintained at 90%; 14–14.2 min, phase B volume fraction linearly decreased from 90% to 20%; 14.2–20 min, phase B volume fraction maintained at 20%; the total run time was 20 min; the flow rate was 0.3 mL / min, and the injection volume was 1 μL.

[0045] The mass spectrometry conditions were as follows: electrospray ionization (ESI) source, capillary voltage 4000V, ESI capillary temperature 300℃, heating block temperature 400℃, DL temperature 250℃; flow rates of drying gas (nitrogen), heating gas (air), and nebulizing gas (nitrogen) were set to 10L / min, 10L / min, and 3L / min, respectively; nitrogen (N2) was provided by a nitrogen generator (Shimadzu, Kyoto, Japan); argon was used as the collision gas; tandem mass spectrometry was run in multiple reaction monitoring (MRM) mode; the compound information of 16 OPEs and their mass spectrometry analysis parameters are shown in Table 1. The residence time of each ion pair was 2ms. In the product ion column, the values ​​on the left are quantitative ions, and the values ​​on the right are qualitative ions.

[0046] Table 1. Compound information and mass spectrometry parameters of 16 OPEs

[0047]

[0048]

[0049] Example 2: Extraction Solvent Optimization

[0050] This example investigated the effects of pure acetonitrile, 1% acetonitrile acetate (i.e., 1% acetic acid by volume), and 2% acetonitrile acetate (i.e., 2% acetic acid by volume) on the recovery of 16 OPEs in shrimp samples. Specifically, 2g (±0.01g) of shrimp sample was accurately weighed into a 50mL centrifuge tube, 4mL of extraction solvent (pure acetonitrile, 1% acetonitrile acetate, or 2% acetonitrile acetate) and 2mL of water were added, and the mixture was vortexed for 1min. Then, 40mg of Fe3O4@SiO2@NVP-DVB and 3g of salting-out material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1) were added, vortexed for 1min, and then magnetically separated for 30s. The supernatant was filtered through a 0.22μm filter membrane and injected into the sample. The specific results are shown in Table 2. The results in Table 2 show that when the extraction solvent is 1% acetonitrile acetate, the recovery rate ranges from 63.3% to 112.0%, the RSD is <20%, the matrix effect is in the range of 0.8 to 1.2, and the recovery rate is good. Therefore, 1% acetic acid acetonitrile was used as the extraction solvent.

[0051] Table 2. Effect of extraction solvent type on the recovery rate and RSD value of OPEs

[0052]

[0053]

[0054] Example 3: Optimization of the ratio of extraction solvent to water

[0055] This embodiment investigated the effect of the extraction solvent to water ratio on the recovery of 16 OPEs from shrimp samples. Specifically, 2g (±0.01g) of shrimp sample was accurately weighed into a 50mL centrifuge tube, and 6mL of 1% acetonitrile + water in different volume ratios (1% acetonitrile to water volume ratio of 10:0, 7:3, or 6:4) was added. The mixture was vortexed for 1min, followed by the addition of 40mg Fe3O4@SiO2@NVP-DVB and 3g salting-out material (a mixture of MgSO4 and NaCl in a mass ratio of 4:1). After vortexing for 1min, magnetic separation was performed for 30s. The supernatant was filtered through a 0.22μm filter membrane and then injected. The specific results are shown in Table 3. Table 3 shows that when pure solvent was used as the extraction solvent, the recovery rate of OPEs ranged from 76.4% to 117.6%; when the volume ratio of 1% acetonitrile acetate to water was 7:3, the recovery rate of OPEs ranged from 85.6% to 116.1%; and when the volume ratio of 1% acetonitrile acetate to water was 6:4, the recovery rate of OPEs ranged from 70.3% to 105.5%. In all experiments, the volume ratio of 1% acetonitrile acetate to water was 7:3, resulting in the best recovery rate of OPEs, with most OPEs having an RSD of <20%. Therefore, a volume ratio of 7:3 for the extraction solvent and water was selected.

[0056] Table 3. Effect of the ratio of extraction solvent to water on the recovery rate and RSD value of OPEs.

[0057]

[0058]

[0059] Example 4: Optimization of Extraction Solution Amount

[0060] This example investigated the effect of different amounts (3 mL, 4 mL, 5 mL) of the extraction solution of 1% acetonitrile + water (7:3, v / v) on the recovery of 16 OPEs in shrimp samples. Specifically, 2 g (±0.01 g) of shrimp sample was accurately weighed into a 50 mL centrifuge tube, and different volumes of 1% acetonitrile + water (7:3, v / v) were added and vortexed for 1 min. Then, 40 mg of Fe3O4@SiO2@NVP-DVB and 3 g of salting-out material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1) were added, vortexed for 1 min, and then magnetically separated for 30 s. The supernatant was then filtered through a 0.22 μm filter membrane and injected. The specific results are shown in Table 4. Table 4 shows that the recovery rates of OPEs were between 67.0% and 108.0% when the extraction solution volume was 3 mL; between 74.1% and 127.4% when the extraction solution volume was 4 mL; and between 62.4% and 89.9% when the extraction solution volume was 5 mL. Therefore, a volume of 4 mL of 1% acetonitrile acetate + water (1% acetonitrile acetate to water volume ratio of 7:3) was selected as the optimal extraction solution volume.

[0061] Table 4. Effect of extraction solution volume on the recovery rate and RSD value of OPEs

[0062]

[0063]

[0064] Example 5: Optimization of Salting-out and Water Removal Material Dosage

[0065] This embodiment investigated the effect of the dosage of the salting-out-dehydration material NaCl + anhydrous MgSO4 (1:4, w / w) on the recovery of 16 OPEs in shrimp samples. Specifically, 2 g (±0.01 g) of shrimp sample was accurately weighed into a 50 mL centrifuge tube, 4 mL of 1% acetonitrile + water (7:3, v / v) was added, and the mixture was vortexed for 1 min. Then, 40 mg of Fe3O4@SiO2@NVP-DVB and different dosages (1 g, 2 g, or 3 g) of the salting-out-dehydration material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1) were added, vortexed for 1 min, and then magnetically separated for 30 s. The supernatant was filtered through a 0.22 μm filter membrane and then injected. The specific results are shown in Table 5. Table 5 shows that when the amount of salting-out material used for dehydration was 1 g, the recovery rate of OPEs ranged from 57.9% to 105.5%; when the amount of salting-out material used for dehydration was 2 g, the recovery rate ranged from 83.1% to 114.9%; and when the amount of salting-out material used for dehydration was 3 g, the recovery rate of OPEs ranged from 94.8% to 133.7%. In all experiments, the recovery rate of OPEs was poor when the amount of salting-out material used for dehydration was 1 g, while the recovery rates were better and the differences were small when the amount of salting-out material used for dehydration was 2 g and 3 g. However, when the amount of salting-out material used for dehydration was 3 g, the amount of supernatant obtained was small, while when the amount of salting-out material used for dehydration was 2 g, sufficient supernatant was obtained and there was no water residue. Therefore, a mixture of MgSO4 and NaCl in a mass ratio of 4:1 was selected as the salting-out material, and the amount of salting-out material added was 2 g.

[0066] Table 5. Effect of salting-out-water removal material dosage on OPE recovery rate and RSD value.

[0067]

[0068]

[0069] Example 6: Optimization of Fe3O4@SiO2@NVP-DVB dosage

[0070] This example investigated the effect of the dosage of the magnetic purification material Fe3O4@SiO2@NVP-DVB on the recovery of 16 OPEs in shrimp samples. Specifically, 2 g (±0.01 g) of shrimp sample was accurately weighed into a 50 mL centrifuge tube, 4 mL of 1% acetonitrile + water (7:3, v / v) was added, and the mixture was vortexed for 1 min. Then, different dosages (10 mg, 40 mg, or 70 mg) of Fe3O4@SiO2@NVP-DVB and 2 g of salting-out material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1) were added, vortexed for 1 min, and then magnetically separated for 30 s. The supernatant was then filtered through a 0.22 μm filter membrane and injected into the sample. The specific results are shown in Table 6. The results in Table 6 show that when the dosage of Fe3O4@SiO2@NVP-DVB was 40 mg, the recovery rate of OPEs ranged from 71.7% to 101.8%, and the RSD of all OPEs was less than 10%. Furthermore, based on actual operational observations, a dosage of 40 mg of Fe3O4@SiO2@NVP-DVB resulted in good solid magnetic adsorption, and the purified sample showed no obvious impurities. Therefore, a dosage of 40 mg of Fe3O4@SiO2@NVP-DVB was selected.

[0071] Table 6. Effect of Fe3O4@SiO2@NVP-DVB dosage on OPEs recovery rate and RSD value.

[0072]

[0073]

[0074] Example 7: Selection of Non-Magnetic Purification Materials

[0075] This example investigated the effects of adding C18, PSA, and GCB to the recovery of 16 OPEs in shrimp samples. Specifically, 2 g (±0.01 g) of shrimp sample was accurately weighed into a 50 mL centrifuge tube, 4 mL of 1% acetonitrile acetate + water (7:3, v / v) was added, and the mixture was vortexed for 1 min. Then, 40 mg of Fe3O4@SiO2@NVP-DVB, 2 g of salting-out material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1) and 10 mg of non-magnetic purification material (C18, PSA, or GCB) were added, vortexed for 1 min, and then magnetically separated for 30 s. The supernatant was filtered through a 0.22 μm filter membrane and then injected. The specific results are shown in Table 7. The results in Table 7 show that the recoveries of OPEs by C18, PSA, and GCB were all in the range of 70%–120%. However, when C18 was used as the non-magnetic purification material, the recoveries of most samples were in the range of 90%–110%, and the RSD was <12.1%. Therefore, considering both the recovery rate and precision results of the method, C18 was selected as the non-magnetic purification material.

[0076] Table 7. Effects of Nonmagnetic Material Type on OPE Recovery Rate and RSD Value

[0077]

[0078]

[0079] Example 8: C18 Dosage Optimization

[0080] This example investigated the effect of different dosages of C18 on the recovery of 16 OPEs in shrimp samples. Specifically, 2 g (±0.01 g) of shrimp sample was accurately weighed into a 50 mL centrifuge tube, 4 mL of 1% acetonitrile + water (7:3, v / v) was added, and the mixture was vortexed for 1 min. Then, 40 mg of Fe3O4@SiO2@NVP-DVB, 2 g of salting-out material (a mixture of MgSO4 and NaCl at a mass ratio of 4:1), and different dosages (10 mg, 30 mg, or 50 mg) of C18 were added. After vortexing for 1 min, magnetic separation was performed for 30 s. The supernatant was filtered through a 0.22 μm filter membrane and then injected. The specific results are shown in Table 8. The results in Table 8 show that when the dosage of C18 was 10 mg, 30 mg, and 50 mg, the recovery rate of OPEs was between 70.0% and 120.0%. However, with increasing dosage, the recovery rates of some target compounds (such as tributyl phosphate, triisobutyl phosphate, and tripropyl phosphate) decreased significantly. Therefore, considering both the accuracy and precision of the method, 30 mg C18 was selected.

[0081] Table 8. Effect of C18 dosage on the recovery rate and RSD value of OPEs

[0082]

[0083]

[0084] Example 9 Method Verification

[0085] Take an appropriate amount of mixed standard working solution and prepare a series of standard solutions with mass concentrations of 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, and 250 μg / L using methanol and the extracted and purified blank shrimp sample matrix solution, respectively. Plot the analyte concentration (x) on the x-axis and the chromatographic peak area (y) on the y-axis, and fit the curve, as shown in Table 9. Table 9 shows that the analytes exhibit good linearity within their respective linear ranges, with correlation coefficients all >0.99. The limits of detection (LOD) and quantitation (LOQ) were calculated based on a signal-to-noise ratio of 3 and 10, respectively. The LOD range was 0.005–1.76 μg / kg, and the LOQ range was 0.02–5.86 μg / kg.

[0086] Matrix effect is evaluated by the ratio of the slope of the linear equation of the matrix standard curve to the slope of the linear equation of the solvent standard curve. A slope ratio of 1 indicates no matrix effect; a slope ratio less than 1 indicates a matrix inhibition effect; and a slope ratio greater than 1 indicates a matrix enhancement effect. Table 9 shows that the slope ratios of the 13 OPEs are in the range of 0.8–1.2, indicating that the matrix effect is not significant; however, tris(1,3-dichloroisopropyl) phosphate (slope ratio 1.40), triisobutyl phosphate (slope ratio 1.53), and tributyl phosphate (slope ratio 1.41) exhibit matrix enhancement effects. To achieve accurate quantification of all analytes, matrix standard curves were used for calibration.

[0087] In addition, recovery experiments were conducted using blank shrimp sample matrix at spiking levels of 10 μg / kg, 100 μg / kg, and 250 μg / kg (each spiking level was measured in triplicate). The results are shown in Table 9. Table 9 shows that the average recoveries of the analyte at different spiking levels ranged from 73% to 113%, with RSDs ranging from 0.1% to 19%. The accuracy and precision of the method meet the requirements for residue analysis.

[0088] Table 9. Linear equations, matrix effects, LOD, LOQ, and recoveries at different spiking concentrations for OPEs (n=3)

[0089]

[0090]

[0091] Note: 1) represents the linear equation of the solvent standard calibration curve.

[0092] 2) The linear equation representing the matrix matching calibration curve.

[0093] As can be seen from the above embodiments, the method provided by the present invention completes the extraction and purification of the shrimp sample in the same container without the need for transfer. This is beneficial to improve the operation speed, reduce system losses and deviations caused by experimental transfer, and is faster and more convenient while ensuring the accuracy of recovery rate. It greatly improves the sample pretreatment speed, increases work efficiency, makes high-throughput sample analysis possible, and reduces the cost of experimental instruments (mainly centrifuges). At the same time, the present invention uses a smaller sample volume, and the extraction solution, adsorption material, and salting-out dehydration material are all reduced accordingly, thus reducing experimental costs. In particular, the reduction of organic solvents makes this method more green and environmentally friendly.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pretreatment method for detecting organophosphate contaminants in aquatic products, comprising the following steps: The aquatic product sample to be tested is mixed with the extraction solution for extraction to obtain the extraction solution; the extraction solution is an acetic acid-acetonitrile-water solution, wherein the volume fraction of acetic acid in the acetic acid-acetonitrile-water solution is 1~2%, and the volume ratio of the total volume of acetic acid and acetonitrile to water is (5~8):(2~4); the volume ratio of the aquatic product sample to be tested to the extraction solution is 2g:(2.5~4)mL; The extraction solution, adsorbent material, and salting-out dehydration material are mixed for purification, and the purified solution is obtained by magnetic separation. The adsorbent material is an amphiphilic functional magnetic material and C18. The amphiphilic functional magnetic material is obtained by modifying Fe3O4@SiO2 particles with N-vinylpyrrolidone and divinylbenzene. The mass ratio of the amphiphilic functional magnetic material to C18 in the adsorbent material is (35~45):(25~35). The mass ratio of the aquatic product sample to be tested to the adsorbent material is 2:(0.02~0.07). The purified solution was filtered to obtain the sample solution to be tested; The organophosphate contaminants are 2-ethylhexyl diphenyl phosphate, tricresyl phosphate, tricresyl phosphate, tri(butoxyethyl) phosphate, tributyl phosphate, triisobutyl phosphate, cresol diphenyl phosphate, tri(1,3-dichloroisopropyl) phosphate, tri(2-chloropropyl) phosphate, tripropyl phosphate, p-toluyl phosphate, triphenylphosphine oxide, tri(2-chloroethyl) phosphate, triethyl phosphate, trimethyl phosphate, and triphenyl phosphate.

2. The pretreatment method according to claim 1, characterized in that, The aquatic samples to be tested include samples prepared from shrimp or fish.

3. The pretreatment method according to claim 1, characterized in that, The extraction is carried out under vortex conditions; the extraction time is 0.5~3 min.

4. The pretreatment method according to claim 1, characterized in that, The salting-out and dehydration materials are MgSO4 and NaCl, and the mass ratio of MgSO4 to NaCl is (3~5):1; the mass ratio of the aquatic product sample to be tested to the salting-out and dehydration materials is 2:(1~4).

5. The pretreatment method according to claim 1 or 4, characterized in that, The purification is carried out under vortex conditions; the purification time is 0.5~3 minutes.

6. A method for detecting organophosphate contaminants in aquatic products, comprising the following steps: The aquatic product sample to be tested is processed according to the method of any one of claims 1 to 5 to obtain the sample solution to be tested. The sample solution to be tested was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), wherein the HPLC conditions for the HPLC-MS / MS analysis included: The mobile phase system includes phase A and phase B; phase A is a 0.1% (v / v) aqueous solution of formic acid, and phase B is methanol; a gradient elution program is used. The mass spectrometry conditions for the high performance liquid chromatography-tandem mass spectrometry analysis include: operating in multiple reaction monitoring mode.

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