Simultaneous determination of 14 organophosphorus flame retardants in aquatic products by impurity delayed combination UPLC-MS / MS and its application

Abstract

The application provides a method for simultaneously determining 14 kinds of organophosphate flame retardants in aquatic products by impurity delayed combination UPLC-MS / MS and application, and belongs to the technical field of aquatic product pollution detection; the determination method provided by the application mainly comprises the following steps: using 1% formic acid acetonitrile-ketone as an extraction solvent to extract aquatic products, purifying by using a hydrophilic lipophilic solid phase extraction column or a magnesium silicate solid phase extraction column, collecting effluent and adding d-SPE purifying agent for purification; preparing a matrix standard working curve, and determining by using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry; the detection method provided by the application can realize rapid qualitative and quantitative detection of up to 14 kinds of OPFRs in aquatic products in a short time.

Description

Technical Field

[0001] This invention belongs to the field of aquatic product pollution detection technology, specifically relating to a method and application for the simultaneous determination of 14 organophosphate flame retardants in aquatic products using delayed-release UPLC-MS / MS. Background Technology

[0002] Organophosphate flame retardants (OPFRs), as alternatives to traditional brominated flame retardants, have seen their production and use increase year by year, and are now widely present in various environmental media and organisms. OPFRs have multi-target toxic effects, interfering with the neural development of organisms, disrupting endocrine balance, inducing oxidative stress, and leading to reproductive and developmental abnormalities, posing multiple potential threats to ecosystems and human health. Of particular concern is that OPFRs can accumulate in aquatic organisms through the food chain, and their metabolites still possess similar or even higher biotoxicity than their parent compounds.

[0003] The pollution problem in aquaculture is becoming increasingly serious, and OPFR residues in aquatic products have become a global food safety issue. A growing body of research shows that the detection rate and content of OPFRs in aquatic products are both at high levels. Their significant bioaccumulation capacity and complex metabolic transformation pathways make related safety risk assessment and control a current research hotspot and challenge. Against this backdrop, establishing highly sensitive and accurate analytical detection methods is a prerequisite for achieving risk assessment and effective regulation of OPFRs in aquatic products, and is also a focus of common attention in the international research community.

[0004] However, current detection technologies for OPFRs differ significantly between domestic and international sources in terms of method design, limit indicators, and applicable scope. Furthermore, most standards focus only on a few significantly hazardous OPFR monomers and specific matrix types, leaving a need for improvement in detection technologies related to aquatic products. Existing methods largely rely on individual sample testing, resulting in complex, time-consuming, and highly susceptible to system interference, failing to meet the demands for high-throughput, rapid, and accurate analysis in practical applications. Simultaneously, the significant differences in the physicochemical properties of OPFR homologues create technical bottlenecks in simultaneous extraction, leading to cumbersome purification steps, severe matrix interference, and low purification efficiency in the simultaneous extraction of some homologues, resulting in high detection limits. Moreover, the widespread presence of OPFRs in the environment means that liquid phase flow systems (such as pipelines, seals, and solvents) may introduce background interference, affecting the accuracy of low-concentration sample detection. These uncertainties limit their widespread application in real-world samples.

[0005] Furthermore, aquatic products are diverse, with significant differences in their matrix (for example, sea cucumbers are rich in polysaccharides, shellfish are rich in protein, and fish are rich in fat). Existing technologies often only target a single or a few types of aquatic products and cannot simultaneously meet the testing needs of highly differentiated aquatic products such as fish, shellfish, sea cucumbers, shrimp, and crabs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method and its application for the simultaneous determination of 14 organophosphate flame retardants in aquatic products using delayed-binding UPLC-MS / MS.

[0007] The technical solution of the present invention is as follows: A method for determining organophosphate flame retardants in aquatic products includes the following steps: (1) Sample extraction: Accurately weigh 1.00~500g of homogenized sample, add 10~20 ng of mixed internal standard, 5~10 mL of 1% formic acid acetonitrile-acetone (the volume ratio of 1% formic acid acetonitrile to acetone is 1:1), and 2~5 g of anhydrous sodium sulfate. Vortex for 3~5 min, then place in a water bath for ultrasonic-assisted extraction for 5~10 min at a water bath temperature of 35~40℃. Centrifuge at 3,800~5,000 rpm for 5~10 min and collect the supernatant. Add the above 1% formic acid acetonitrile-acetone to the precipitate and repeat the extraction more than once. Combine the supernatants for purification. (2) Purification steps: The supernatant obtained in step (1) was placed in a water bath at 35-40℃ and purged with nitrogen until nearly dry. It was then reconstituted with 5-10 mL of acetonitrile-water solution (the volume ratio of acetonitrile to water was 5:95) or 5-10 mL of n-hexane and loaded onto a hydrophilic-lipophilic solid-phase extraction column or a magnesium silicate solid-phase extraction column for purification. The solution was washed and eluted, and the effluent was collected. It was then purged with nitrogen until nearly dry in a water bath at 35-40℃. 1.00-2.00 mL of methanol was added and vortexed to dissolve the effluent. d-SPE purifying agent was added, and the mixture was vortexed for 1-2 min. After centrifugation at 8,000-12,000 rpm for 10-15 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the test solution. (3) Matrix standard working curve: After homogenization, different concentrations of OPFR standard substance mixed solutions and mixed internal standards were added to the samples. The test solutions were obtained according to the corresponding extraction and purification steps in steps (1) and (2). The matrix standard working curve was prepared with a concentration range of 0.1~100 ng / mL. -1 between; (4) Sample testing: OPFRs in the test solution obtained in steps (2) and (3) were determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0008] Preferably, in step (2), the hydrophilic and lipophilic solid phase extraction column includes an Oasis PRiME HLB solid phase extraction column; the magnesium silicate solid phase extraction column includes a Florisil solid phase extraction column.

[0009] Preferably, in step (2), after redissolving in 5-10 mL of n-hexane, the solution is loaded onto a Florisil solid-phase extraction column for purification. The solid-phase extraction column has a specification of 1 g / 6 mL, and the flow rate is maintained at 1-5 mL / min. -1 Wash 2-3 times with 4-10 mL of n-hexane solution, elute 2-3 times with 4-10 mL of ethyl acetate solution, and collect the effluent. Alternatively, in step (2), after redissolving in 5-10 mL of acetonitrile-water solution (acetonitrile to water volume ratio 5:95), the solution is loaded onto an Oasis PRiME HLB solid-phase extraction column for purification. The solid-phase extraction column has a specification of 200 mg / 6 mL, and the flow rate is maintained at 1-5 mL / min. -1 Wash 2-3 times with 4-10 mL of the acetonitrile-water solution, and elute 2-3 times with 4-10 mL of acetonitrile-methanol solution, wherein the volume ratio of acetonitrile to methanol is 9:1, and collect the effluent.

[0010] Preferably, in step (2), the d-SPE purifier comprises 50-100 mg PSA and 50-100 mg C18.

[0011] Preferably, in step (3), 1.00~5.00 g of homogenized sample is taken and 100 μL of different concentrations are added: 1, 5, 10, 20, 50, 100, 200, 500, 1000 ng·ml. -1 The OPFRs standard substance mixed solution and 10~20 ng mixed internal standard were used to obtain the test solution according to the corresponding extraction and purification steps in steps (1) and (2). The matrix standard working curve was prepared with a concentration range of 0.1~100 ng / mL. -1 between.

[0012] Preferably, in step (3) or step (4), the OPFRs include one or more of TMP, TEP, TCEP, TPRP, TCIPP, TDCIPP, TPHP, TIBP, CDP, TNBP, TBOEP, TMCP, TOCP, and TMPP.

[0013] Preferably, in step (1) or step (3), the mixed internal standard includes at least one of TNBP-d27, TCEP-d12, and TPHP-d15.

[0014] Preferably, in step (1) or step (3), the sample is an edible part of aquatic products.

[0015] Preferably, the aquatic products include at least one of fish, shrimp, sea cucumber, and shellfish.

[0016] Preferably, in step (4), the chromatographic conditions for determination are: the chromatographic column is C10 ... 18 The chromatographic column is 2.1 mm × 100 mm, 1.7 µm; a Phenomenex Kinetex Biphenyl column is 2.1 mm × 100 mm, 2.6 μm, or other columns with equivalent performance; the delay column is a C18 column, 2.1 mm × 50 mm, 5 μm, or a Waters Isolator column, 2.1 mm × 50 mm, 5 μm, installed between the liquid chromatography system mixer and the injector; the column temperature is 35℃, and the mobile phase A is 5 mmol / L. -1 Ammonium acetate aqueous solution, mobile phase B: methanol, gradient elution program as follows: 0~1 min, A:B=90:10; 1~4 min, gradient increase to A:B=10:90; 4~8 min, A:B=10:90; 8~8.1 min, A:B=90:10; 8.1~10 min, A:B=90:10; injection volume 5~10 μL, flow rate 0.2~0.4 mL / min -1 ; Mass spectrometry conditions: Ion source: electrospray ionization (ESI), positive ion mode; Scanning mode: multiple reaction monitoring (MRM) mode; Nebulizer temperature: 275℃; Capillary voltage: 4100V; Ion transfer tube temperature: 325℃; Sheath gas: 35 arb; Auxiliary gas: 5 arb.

[0017] The above method is applied to the determination of OPFRs in aquatic products.

[0018] Preferably, in the application, OPFRs include one or more of TMP, TEP, TCEP, TPRP, TCIPP, TDCIPP, TPHP, TIBP, CDP, TNBP, TBOEP, TMCP, TOCP, and TMPP.

[0019] The beneficial effects of this invention are as follows: 1. This invention employs an improved QuEChERS method for pretreatment of aquatic products. On one hand, it uses 1% formic acid acetonitrile-acetone as the base extraction solvent, combining SPE and d-SPE to effectively remove interfering impurities of different properties from aquatic products, significantly improving the method's sensitivity. On the other hand, it uses less solvent, is environmentally friendly, simple to operate, rapid, and has good reproducibility, making it suitable for high-throughput rapid analysis of OPFR residues in aquatic products. The average recovery rate is between 72.3% and 110%, with a relative standard deviation of 1.5% to 15%. 2. This invention employs ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) for qualitative and quantitative analysis of target analytes. It can simultaneously and rapidly perform qualitative and quantitative analysis of up to 14 OPFRs and their internal standards within just 10 minutes. This significantly shortens the analysis time and is suitable for the practical needs of high-throughput aquatic product safety monitoring.

[0020] 3. Aquatic products are diverse, with significant differences in their substrates (e.g., sea cucumbers are rich in polysaccharides, shellfish are rich in protein, and fish are rich in fat). Existing technologies often only address a single or a few types of aquatic products. This invention can simultaneously meet the testing needs of highly differentiated aquatic products such as fish, shellfish, sea cucumbers, shrimp, and crabs, possessing strong universality and industrial application value.

[0021] 4. Given the widespread background presence of OPFRs in the environment and biological matrices, this invention employs a matrix subtraction method in the quantitative analysis stage. By pre-measuring the background response values ​​of each target analyte in the blank matrix extract and subtracting them from the matrix-matched standard curve, the influence of endogenous background interference on quantitative accuracy is effectively eliminated. Combined with the isotope dilution internal standard method, losses from the pretreatment process and complex matrix effects are further corrected, ensuring high sensitivity and high accuracy in trace detection at the ng / g level. Attached Figure Description

[0022] Figure 1 50 ng / mL of carp muscle sample in Example 1 -1 Total ion chromatogram of 14 OPFRs measured in matrix standard solution.

[0023] Figure 2 50 ng / mL of carp muscle sample in Example 1 -1 Total ion chromatogram of the three OPFRs internal standards measured in the matrix standard solution. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] All details not described in the embodiments are based on existing technology in the field.

[0026] Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0027] Instruments and reagents (1) Instruments Ultra-high performance liquid chromatography (Ultimate 3000, Thermo Fisher Scientific, USA), tandem triple quadrupole mass spectrometer (TSQ Endura), equipped with electrospray ionization (ESI) source; XW-80A vortex mixer (Shanghai Medical University Instrument Factory); HimacCR 22G II high-speed centrifuge (Hitachi, Japan); N-EVAP 112 nitrogen evaporator (Organomation, USA); Milli-Q water purification system (Millipore, USA); CP225D (0.01 mg) electronic balance (Sartorius, Germany).

[0028] (2) Reagents Formic acid and ammonium acetate, LC-MS / MS grade, were purchased from Merck (Darmstadt, Germany); acetonitrile (HPLC grade), acetone (HPLC grade), methanol (LC-MS / MS grade), C18 packing material (40-63 μm), and PSA packing material (40-63 μm) were purchased from Shanghai ANPEL Technology Co., Ltd. Waters Oasis PRiME HLB (200 mg / 6 mL) was purchased from Waters Corporation, USA. Cleanert Florisil (1 g / 6 mL) was purchased from Tianjin Bona Agilent Technologies Co., Ltd. Other reagents were purchased from Shanghai Guoyao Chemical Reagent Co., Ltd.

[0029] Fourteen OPFRs standard solutions and three deuterated OPFRs (TNBP-d27, TCEP-d12, and TPHP-d15) internal standards were purchased from Tianjin Alta Scientific Co., Ltd. and stored at -20℃. Methanol was used for dilution when preparing standard stock solutions and mixed standard working solutions.

[0030] 1% Formic Acid Acetonitrile-Acetone (1:1, v / v): Take 10 mL of formic acid, dilute it with acetonitrile to 1000 mL, mix well to obtain 1% Formic Acid Acetonitrile; then mix 1% Formic Acid Acetonitrile with acetone at a volume ratio of 1:1.

[0031] Example 1 Spiked recovery rate tests were conducted on carp as the test subject to examine the precision and accuracy of the established detection method.

[0032] Sample preparation: Accurately weigh 2.00 g of homogenate sample into a 50 mL centrifuge tube, and add 200 μL of different concentrations (20, 200, 500 ng·ml) respectively. -1 OPFRs standard mixed solution and 100 μL internal standard mixed solution (100 ng·ml) -1After mixing, add 8 mL of 1% formic acid acetonitrile-acetone (1:1, v / v) and 4.00 g of anhydrous sodium sulfate. Vortex for 3 min, then place in a 40℃ water bath for ultrasonic-assisted extraction for 5 min. Centrifuge at 3,800 rpm for 5 min and transfer the supernatant to a new tube. Add the above 8 mL of 1% formic acid acetonitrile-acetone to the precipitate and repeat the extraction once. Combine the supernatants in a new tube and blow with nitrogen to near dryness in a 40℃ water bath. Redissolve in 5 mL of n-hexane and load onto a pre-activated (activated sequentially with 4 mL of dichloromethane-n-hexane (1:9, v / v) and 4 mL of n-hexane) Florisil (Bona Ager, 1 g / 6 mL) solid-phase extraction column for purification, maintaining a flow rate of approximately 2 mL / min. -1 Wash twice with 4 mL of n-hexane solution and elute twice with 4 mL of ethyl acetate solution. Collect the effluent and blow it to near dryness with nitrogen under a 40°C water bath. Add 1 mL of methanol and vortex to dissolve the residue. Add d-SPE purification agent (50 mg PSA, 50 mg C18), vortex for 1 min, centrifuge at 12,000 rpm for 10 min, and take the supernatant. Filter the supernatant through a 0.22 μm organic nylon filter membrane to obtain the test solution.

[0033] Blank sample preparation: Accurately weigh 2.00 g of homogenate sample into a 50 mL centrifuge tube, except for the addition of 200 μL of different concentrations (20, 200, 500 ng·ml). -1 Except for the mixed solution of OPFRs standard substances, the rest are the same as the sample preparation steps.

[0034] Preparation of matrix marking samples: Accurately weigh 2.00 g of homogenate sample into a 50 mL centrifuge tube, and add 100 μL of different concentrations (1, 5, 10, 20, 50, 100, 200, 500, 1000 ng·ml). -1 OPFRs standard mixed solution and 100 μL internal standard mixed solution (100 ng·ml) -1 The remaining steps are the same as those for sample preparation.

[0035] Sample detection method: The above sample (test solution) was determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0036] The chromatographic conditions were as follows: column temperature 35℃, mobile phase A: 5 mmol / L. -1 Ammonium acetate aqueous solution, mobile phase B: methanol, gradient elution program as follows: 0~1 min, A:B=90:10; 1~4 min, gradient increase to A:B=10:90; 4~8 min, A:B=10:90; 8~8.1 min, A:B=90:10; 8.1~10 min, A:B=90:10. Injection volume 5 μL, flow rate 0.3 mL / min.-1 The chromatographic column was a Phenomenex Kinetex Biphenyl column (2.1 mm × 100 mm, 2.6 μm), and the delay column was a Waters Isolator column (2.1 mm × 50 mm, 5 μm). The column was installed between the liquid chromatography system mixer and the injector. It can effectively adsorb and delay the background interference signal of OPFRs introduced by the liquid chromatography system itself, so as to separate the chromatographic peak of the target analyte in time, thereby significantly reducing the system background noise and improving the signal-to-noise ratio.

[0037] Mass spectrometry conditions: Ion source: electrospray ionization (ESI), positive ion mode; Scanning mode: multiple reaction monitoring (MRM) mode; Nebulizer temperature: 275℃; Capillary voltage: 4100V; Ion transfer tube temperature: 325℃; Sheath gas: 35 arb; Auxiliary gas: 5 arb.

[0038] The quantitative and qualitative ion pairs of the compounds are shown in Table 1.

[0039] The detection data obtained above were used to plot a standard curve with the peak area ratio of each target compound to its corresponding internal standard quantitative ion pair as the ordinate and the target compound concentration as the abscissa. The curve equations, correlation coefficients, limits of detection, and limits of quantitation for each OPFR in the examples are shown in Table 2. The 14 OPFRs were tested at concentrations ranging from 0.1 to 100 ng / mL. -1 Linear correlation coefficient (R) within the range 2 The concentrations were between 0.995 and 0.999, showing good linear correlation. The detection limits were 0.03–0.2 ng / g. -1 Limit of quantitation: 0.1–0.6 ng g -1 This indicates that the method has high sensitivity.

[0040] Table 1. Quantitative and qualitative ion pairs of compounds

[0041] * indicates quantitative ions Table 2. Calibration curve equation, correlation coefficient, limit of detection, and limit of quantitation

[0042] Table 3. Spike recoveries and relative standard deviations of 14 OPFRs in carp muscle (n=6)

[0043] The experimental results are shown in Table 3: the recoveries of 14 OPFRs in carp ranged from 72.3% to 110%, with RSDs all less than 15%. The method recovery and precision both met the detection requirements.

[0044] Figure 1 50 ng / mL of carp muscle sample in Example 1 -1 Total ion chromatogram of 14 OPFRs measured in matrix standard solution.

[0045] Figure 2 50 ng / mL of carp muscle sample in Example 1 -1 Total ion chromatogram of the three OPFRs internal standards measured in the matrix standard solution.

[0046] Example 2 The recovery rates of spiked samples were tested using sea cucumber, prawn, and green mussel as matrices to examine the applicability of the established detection method.

[0047] Sample preparation: Accurately weigh 1.00 g of homogenate sample into a 15 mL centrifuge tube, and add 100 μL of different concentrations (20, 200, 500 ng·ml) respectively. -1 OPFRs standard mixed solution and 100 μL internal standard mixed solution (100 ng·ml) -1 After mixing, add 5 mL of 1% formic acid acetonitrile-acetone (1:1, v / v) and 2.00 g of anhydrous sodium sulfate. Vortex for 3 min, then sonicate for 5 min in a 40°C water bath. Centrifuge at 3,800 rpm for 5 min and transfer the supernatant to a new tube. Add the same 5 mL of 1% formic acid acetonitrile-acetone to the precipitate and repeat the extraction once. Combine the supernatants for purification. Purge the supernatant to near dryness with nitrogen in a 40°C water bath. Reconstitute with 5 mL of acetonitrile-water solution (5:95, v / v) and load it onto a Waters Oasis PRiME HLB (200 mg / 6 mL) column for purification, maintaining a flow rate of approximately 2 mL / min. -1 Wash twice with 4 mL of acetonitrile-water solution (5:95, v / v) and elute twice with 4 mL of acetonitrile-methanol solution (9:1, v / v). Collect the effluent and blow it to near dryness with nitrogen in a 40°C water bath. Add 1 mL of methanol and vortex to dissolve the residue. Add d-SPE purification agent (50 mg PSA, 50 mg C18), vortex for 1 min, centrifuge at 12,000 rpm for 10 min, and collect the supernatant. Filter the supernatant through a 0.22 μm organic nylon membrane to obtain the test solution.

[0048] Blank sample preparation: Accurately weigh 1.00 g of homogenate sample into a 15 mL centrifuge tube, except for the addition of 100 μL of different concentrations (20, 200, 500 ng·ml). -1 Except for the mixed solution of OPFRs standard substances, the rest are the same as the sample preparation steps.

[0049] Preparation of matrix marking samples: Accurately weigh 1.00 g of homogenate sample into a 15 mL centrifuge tube, and add 100 μL of different concentrations (1, 5, 10, 20, 50, 100, 200, 500, 1000 ng·ml). -1 OPFRs standard mixed solution and 100 μL internal standard mixed solution (100 ng·ml) -1 The remaining steps are the same as those for sample preparation.

[0050] Sample detection method: The above sample (test solution) was determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0051] The chromatographic conditions were as follows: column temperature 35℃, mobile phase A: 5 mmol / L. -1 Ammonium acetate aqueous solution, mobile phase B: methanol, gradient elution program as follows: 0~1 min, A:B=90:10; 1~4 min, gradient increase to A:B=10:90; 4~8 min, A:B=10:90; 8~8.1 min, A:B=90:10; 8.1~10 min, A:B=90:10. Injection volume 10 μL, flow rate 0.3 mL / min. -1 Chromatographic column: Phenomenex Kinetex Biphenyl column (2.1 mm × 100 mm, 2.6 μm), delay column: Waters Isolator column (2.1 mm × 50 mm, 5 μm).

[0052] Mass spectrometry conditions: Ion source: electrospray ionization (ESI), positive ion mode; Scanning mode: multiple reaction monitoring (MRM) mode; Nebulizer temperature: 275℃; Capillary voltage: 4100V; Ion transfer tube temperature: 325℃; Sheath gas: 35 arb; Auxiliary gas: 5 arb.

[0053] The quantitative and qualitative ion pairs of the compounds are shown in Table 1. The detection results for sea cucumber, shrimp, and green mussel are shown in Tables 4, 5, and 6, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) of each OPFR in sea cucumber, shrimp, and green mussel are shown in Table 7.

[0054] Table 4. Spike recoveries and relative standard deviations of 14 OPFRs in sea cucumber (n=6)

[0055] Table 5. Recovery rates and relative standard deviations of 14 OPFRs spiked in shrimp (n=6)

[0056] Table 6. Spiked recoveries and relative standard deviations of 14 OPFRs in *Echinochloa crus-galli* (n=6)

[0057] Table 7. LOD and LOQ of various OPFRs in sea cucumber, prawn, and green mussel samples (unit: μg kg) -1 )

[0058] Experimental results showed that the recoveries of 14 OPFRs in sea cucumber ranged from 70.8% to 119%, with RSDs all less than 14%. In shrimp, the recoveries ranged from 70.6% to 113.8%, with RSDs all less than 10%. In mussels, the recoveries ranged from 78.1% to 109%, with RSDs all less than 11%. The established method is applicable to the detection of OPFRs in the above matrices.

[0059] Experimental Example 1 The inventors used a single solvent, acetone, ethyl acetate-acetone (volume ratio 1:1), and 1% formic acid acetonitrile-acetone (volume ratio 1:1), to extract carp samples. The extraction method was the same as in Example 1 above, but SPE (solid phase extraction column purification) was not performed. The other steps were the same.

[0060] The detection method is the same as in Example 1, and the detection results are shown in Table 8.

[0061] Table 8 Comparison of OPFRs spiked with three different extraction solvents (dosage: 20 ng / g) -1 Recovery rate and relative standard deviation (n=3, carp substrate).

[0062] Experimental results show that compared with the 1% formic acid acetonitrile-acetone (volume ratio 1:1) used in this invention, the recovery rates of single extraction solvent (acetone) and some mixed solvents (ethyl acetate + acetone) are generally lower, and the RSD values ​​are significantly higher than those of this invention. Some RSDs are too high to detect these 14 OPFRs simultaneously.

[0063] As can be seen from the experimental data of the 1% formic acid acetonitrile-acetone group in Table 8 and the experimental data of Table 3 in Example 1, using only dispersive solid-phase extraction (d-SPE) without SPE (solid-phase extraction column purification) will result in a lower recovery rate of some OPFRs.

[0064] Experiment Example 2 Using the method provided in the above embodiments, the inventors determined 14 OPFRs in three aquatic products collected from production areas and markets: mussels (28 samples, approximately 500 g each), shrimp (39 samples, approximately 500 g each), and carp (29 samples, 3 fish per sample). After pretreatment and instrumental analysis, the content of each compound on a wet weight basis (unit: μg kg) was obtained. - 1 ww).

[0065] Table 9. Detection results of actual samples of mussels, shrimp, and carp (Note: ND means not detected, i.e., the content is below the detection limit).

[0066] The experimental results are shown in Table 9. Except for TMP, the detection rates of the other 13 OPFRs in the three types of aquatic products were all above 90%. Data analysis showed significant differences in the contamination levels of the same compounds among different aquatic products. For example, TCIPP had the highest mean value in shrimp (6.03 ± 5.62 μg kg). -1 ww), while the lowest was found in carp (0.766±0.667 μg kg). -1 Overall, the mean and median values ​​of most compounds in mussels and shrimp were higher than those in carp, reflecting differences in bioaccumulation characteristics among species and potential environmental exposure variations. The results of the assay method provided in this invention offer fundamental data for assessing the OPFR contamination characteristics of different aquatic products.

Claims

1. A method for determining organophosphate flame retardants in aquatic products, characterized in that, Includes the following steps: (1) Sample extraction: Accurately weigh 1.00~500g of homogenized sample, add 10~20 ng of mixed internal standard, 5~10 mL of 1% formic acid acetonitrile-acetone, where the volume ratio of 1% formic acid acetonitrile to acetone is 1:1, 2~5 g of anhydrous sodium sulfate, vortex for 3~5 min, then place in a water bath for ultrasonic-assisted extraction for 5~10 min at a water bath temperature of 35~40℃, centrifuge at 3,800~5,000 rpm for 5~10 min and take the supernatant. Add the above 1% formic acid acetonitrile-acetone to the precipitate and repeat the extraction more than once. Combine the supernatants for purification. (2) Purification steps: Place the supernatant obtained in step (1) under nitrogen blowing in a water bath at 35~40℃ until it is nearly dry, and then reconstitute it with 5~10 mL of acetonitrile-water solution, wherein the volume ratio of acetonitrile to water is 5:95, or 5~10 mL of n-hexane. Then, load it onto a hydrophilic-lipophilic solid phase extraction column or a magnesium silicate solid phase extraction column for purification. Wash and elute with the solution, collect the effluent, and blow it under nitrogen blowing in a water bath at 35~40℃ until it is nearly dry. Add 1.00~2.00 mL of methanol and vortex to dissolve it. Then add d-SPE purification agent, vortex for 1~2 min, centrifuge at 8,000~12,000 rpm for 10~15 min, and take the supernatant. Filter it through a 0.22 μm filter membrane to obtain the test solution. (3) Matrix standard working curve: After homogenization, different concentrations of OPFRs standard substance mixed solution and mixed internal standard were added to the sample respectively. The test solution was obtained according to the corresponding extraction and purification steps in steps (1) and (2). The matrix standard working curve was prepared with the concentration range of 0.1~100 ng / mL. -1 between; (4) Sample detection: OPFRs in the test solution obtained in steps (2) and (3) were determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

2. The method as described in claim 1, characterized in that, In step (2), the hydrophilic and lipophilic solid phase extraction column includes the OasisPRiME HLB solid phase extraction column; the magnesium silicate solid phase extraction column includes the Florisil solid phase extraction column.

3. The method as described in claim 1, characterized in that, In step (2), after redissolving in 5-10 mL of n-hexane, the solution is loaded onto a Florisil solid-phase extraction column for purification. The solid-phase extraction column has a specification of 1 g / 6 mL, and the flow rate is maintained at 1-5 mL / min. -1 Wash 2-3 times with 4-10 mL of n-hexane solution, elute 2-3 times with 4-10 mL of ethyl acetate solution, and collect the effluent. Alternatively, in step (2), after redissolving in 5-10 mL of acetonitrile-water solution (acetonitrile to water volume ratio 5:95), the solution is loaded onto an Oasis PRiME HLB solid-phase extraction column for purification. The solid-phase extraction column has a specification of 200 mg / 6 mL, and the flow rate is maintained at 1-5 mL / min. -1 Wash 2-3 times with 4-10 mL of the acetonitrile-water solution, and elute 2-3 times with 4-10 mL of acetonitrile-methanol solution, wherein the volume ratio of acetonitrile to methanol is 9:1, and collect the effluent.

4. The method as described in claim 1, characterized in that, In step (2), the d-SPE purifier includes 50-100 mg PSA and 50-100 mg C18.

5. The method as described in claim 1, characterized in that, In step (3), take 1.00~5.00 g of homogenized sample and add 100 μL of different concentrations: 1, 5, 10, 20, 50, 100, 200, 500, 1000 ng·ml. -1 The standard substance mixture solution and 10-20 ng of mixed internal standard were used to obtain the test solution according to the corresponding extraction and purification steps in steps (1) and (2). The matrix standard working curve was prepared with a concentration range of 0.1-100 ng / mL. -1 between.

6. The method as described in claim 1, characterized in that, In step (3) or step (4), the OPFRs include one or more of TMP, TEP, TCEP, TPRP, TCIPP, TDCIPP, TPHP, TIBP, CDP, TNBP, TBOEP, TMCP, TOCP, and TMPP. Preferably, in step (1) or step (3), the mixed internal standard includes at least one of TNBP-d27, TCEP-d12, and TPHP-d15.

7. The method as described in claim 1, characterized in that, In step (1) or step (3), the sample is an edible part of aquatic products.

8. The method as described in claim 7, characterized in that, The aquatic products include at least one of fish, shrimp, sea cucumber, and shellfish.

9. The method as described in claim 1, characterized in that, In step (4), the chromatographic conditions for determination are as follows: the chromatographic column is C10. 18 The chromatographic column is 2.1 mm × 100 mm, 1.7 µm; a Phenomenex Kinetex Biphenyl column is 2.1 mm × 100 mm, 2.6 μm, or other columns with equivalent performance; the delay column is a C18 column, 2.1 mm × 50 mm, 5 μm, or a Waters Isolator column, 2.1 mm × 50 mm, 5 μm, installed between the liquid chromatography system mixer and the injector; the column temperature is 35℃, and the mobile phase A is 5 mmol / L. -1 Ammonium acetate aqueous solution, mobile phase B: methanol, gradient elution program as follows: 0~1 min, A:B=90:10; 1~4 min, gradient increase to A:B=10:90; 4~8 min, A:B=10:90; 8~8.1 min, A:B=90:10; 8.1~10 min, A:B=90:10; injection volume 5~10 μL, flow rate 0.2~0.4 mL / min -1 ; Mass spectrometry conditions: Ion source: electrospray ionization (ESI), positive ion mode; Scanning mode: multiple reaction monitoring (MRM) mode; Nebulizer temperature: 275℃; Capillary voltage: 4100V; Ion transfer tube temperature: 325℃; Sheath gas: 35 arb; Auxiliary gas: 5 arb.

10. The application of the method according to any one of claims 1-9 in the determination of OPFRs in aquatic products; Preferably, the OPFRs include one or more of TMP, TEP, TCEP, TPRP, TCIPP, TDCIPP, TPHP, TIBP, CDP, TNBP, TBOEP, TMCP, TOCP, and TMPP.

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