A method for simultaneously detecting the content of 10 organophosphates in herbivore feces

Abstract

This invention discloses a method for simultaneously detecting the content of 10 organophosphates (OPEs) in herbivore feces, comprising the following steps: S1: The mixed calibration solution contains EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP, and TEP, with each target OPE having a concentration of 100 μg / L; S2: The deuterated mixed internal standard is diluted with methanol to prepare an internal standard solution with a concentration of 1000 ng / mL; S3: The mixed calibration solution is prepared into a series of concentration standard solutions, and the internal standard solution is added. After detection, a calibration curve is plotted; S4: The herbivore feces sample is pretreated to obtain the test solution; S5: The test solution is detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) and quantitative analysis is performed based on the calibration curve. This invention enables the simultaneous and accurate detection of multiple target OPEs, providing technical support for research on pollution exposure in wild herbivores, ecological environment monitoring, and risk assessment.

Description

Technical Field

[0001] This invention belongs to the field of analysis and detection technology of organic pollutants in environmental samples, specifically relating to a method for simultaneously detecting the content of 10 organic phosphate esters in the feces of herbivores. Background Technology

[0002] Organophosphate esters (OPEs) are a class of flame retardants and plasticizers widely used in plastic products, electronics, building materials, textiles, furniture, vehicle interiors, and polyurethane foams. With some traditional brominated flame retardants facing limitations due to issues such as persistence, bioaccumulation, and toxicity, OPEs have gained widespread use due to their superior flame-retardant properties and broad application range, gradually becoming a new type of pollutant that has attracted significant attention in recent years. Because OPEs are mostly added to materials physically and do not form stable chemical bonds with the matrix, they easily enter the environmental media through volatilization, abrasion, leaching, and particulate release during their entire life cycle, including production, processing, transportation, use, and disposal. With the continuous expansion of their production and use, OPEs have been widely detected in atmospheric, soil, water, sediment, vegetation, and biological samples. Existing research has shown that these compounds not only exist in cities, industrial areas, and areas with heavy traffic, but can also be transported long distances through the atmosphere to high-altitude and remote areas far from direct emission sources, where they can migrate, accumulate, and redistribute in terrestrial ecosystems, thus causing environmental damage.

[0003] Herbivores, as primary consumers in terrestrial ecosystems, are exposed to organic pollutants (OPEs) in the environment through continuous grazing on plants. For wild herbivores, the application of traditional tissue sampling methods in practical research is limited due to difficulties in capturing individuals, limited access to biological samples, and significant sampling interference. In contrast, fecal samples offer advantages such as non-invasiveness, convenient sampling, and the ability to reflect animal feeding exposure and excretion processes, making them an important medium for wildlife pollution monitoring and ecological risk research. Therefore, establishing a pretreatment and analytical method suitable for detecting OPEs in herbivore feces is crucial for revealing the exposure levels, excretion characteristics, and environmental re-release processes of OPEs in wild herbivores. However, herbivore feces are typical complex biological matrix samples, making their pretreatment significantly more challenging than that of conventional environmental media such as soil and plants. Feces typically contain a large amount of plant-derived pigments, lipid residues, digestive products, and insoluble particles, which can easily co-enter the extract with the target analytes during extraction, leading to increased matrix interference, greater purification difficulty, and unstable instrument response. If the pretreatment methods for OPEs in water, soil, sediment or vegetation samples are directly applied, it is often difficult to balance the extraction efficiency of the target compound, the effect of impurity removal and the detection sensitivity, which in turn affects the accuracy and repeatability of the analytical results.

[0004] While existing technologies have provided some foundation for the detection of OPEs in environmental media, pretreatment and detection methods for samples with high pigment, high lipid content, and complex matrices, such as herbivore feces, still require improvement. Specifically, current methods are inadequate in the simultaneous extraction of multiple OPEs, lipid removal, pigment purification, and control of interference from complex matrices. This results in poor stability of the pretreatment process, unsatisfactory purification effects, and difficulty in meeting the accurate analysis requirements of batch samples.

[0005] Therefore, how to solve the technical problems of unstable extraction efficiency, insufficient purification effect and limited detection accuracy in complex fecal matrix samples, and provide a method for the simultaneous detection of multiple organophosphates in herbivore feces with stable pretreatment process, good purification effect, high sensitivity and suitable for batch sample analysis, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to address the problems in the prior art by providing a method for simultaneously detecting the content of 10 organophosphates in the feces of herbivores.

[0007] Therefore, the above-mentioned objectives of the present invention are achieved through the following technical solutions: A method for simultaneously detecting the content of 10 organophosphates in herbivore feces includes the following steps: S1: Preparation of mixed calibration solution: The mixed calibration solution contains EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP and TEP, and the concentration of each target organophosphate is 100 μg / L. S2: Preparation of internal standard solution: Dilute the deuterated mixed internal standard with methanol to prepare an internal standard solution with a concentration of 1000 ng / mL; S3: Prepare a series of standard solutions of different concentrations from the mixed calibration solution, add the internal standard solution, and plot the calibration curve after testing; S4: Pretreatment of herbivore fecal samples to obtain the test solution: S5: Detection and Quantitative Analysis: The test solution was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitative analysis was performed according to the calibration curve. The preprocessing described in step S4 includes: S41: Freeze-dry, pulverize and homogenize the herbivore fecal sample; S42: Weigh the homogenized fecal sample, add sodium chloride and anhydrous magnesium sulfate, mix well, then add the deuterated mixed internal standard solution and extraction solvent, and shake to mix well; S43: The sample is subjected to oscillation aging followed by ultrasonic extraction, and the extract is obtained by centrifugation. S44: Repeat the extraction and combine the extracts, concentrate them and add ethylenediamine-N-propylsilane adsorbent to remove lipid interference; S45: The supernatant after degreasing is purified sequentially through a Florid solid-phase extraction column and a graphitized carbon black solid-phase extraction column, and the purified liquid is collected. S46: Concentrate the purified liquid and bring it to a fixed volume to obtain the test solution.

[0008] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions: As a preferred embodiment of the present invention, the organophosphates include: 2-ethylhexyl diphenyl phosphate, triphenyl phosphate; tris(1,3-dichloroisopropyl) phosphate, tris(2-chloropropyl) phosphate, tris(2-chloroethyl) phosphate; triethyl phosphate, tripropyl phosphate, tri-n-butyl phosphate, tris(butoxyethyl) phosphate, and tris(2-ethylhexyl) phosphate.

[0009] As a preferred technical solution of the present invention, the preparation method of the mixed calibration solution in step S1 is as follows: Take the single standard solutions of EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP and TEP respectively, and dilute them with methanol until the concentration of each single standard solution is 1000 ng / L; take equal volumes of the above 10 single standard solutions, mix them, and then dilute them with methanol to obtain a mixed calibration solution with the concentration of each target organic phosphate ester being 100 μg / L.

[0010] As a preferred technical solution of the present invention: the deuterated mixed internal standard in step S2 includes M6TBEP, D 15 -TDCPP, D 12 -TCEP、D 21 -TPRP, D 15 -TEP, D 15 -TPHP and D 27 -TNBP.

[0011] As a preferred technical solution of the present invention: the concentrations of the series of calibration solutions in step S3 are 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively; the internal standard solution is added to each calibration solution to make the final concentration of the internal standard 20 ng / mL; the calibration working curve is established by linear regression fitting with the concentration of the target compound as the abscissa and the peak area of ​​the corresponding quantitative ion pair as the ordinate.

[0012] As a preferred technical solution of the present invention: the mass of the fecal sample weighed in step S42 is 0.3 g, the mass of the added sodium chloride and anhydrous magnesium sulfate desiccant is 0.4 g, and the extraction solvent is a mixed solvent of acetone and n-hexane.

[0013] As a preferred technical solution of the present invention: the oscillation aging time in step S43 is 15 min, the ultrasonic extraction time is 30 min, and the centrifugation speed is 4000 r / min and the time is 10 min; the extraction in step S44 is repeated 3 times, and the combined extract is concentrated to 1-2 mL by rotary evaporation, and then 0.5 g of ethylenediamine-N-propylsilane adsorbent is added to remove lipid interference; the purification in step S45 is carried out in series with a Floric clay solid phase extraction column and a graphitized carbon black solid phase extraction column, wherein the graphitized carbon black solid phase extraction column is used to remove pigment interference.

[0014] As a preferred technical solution of the present invention: the activation solvent of the Florid solid phase extraction column in step S45 is a mixed solvent of acetone and dichloromethane with a volume ratio of 1:1, the equilibrium solvent is n-hexane, and the elution solvent is a mixed solvent of acetone and dichloromethane with a volume ratio of 1:1; in step S46, the eluent is concentrated to near dryness by gentle nitrogen blowing and then brought to a final volume of 500 μL with methanol.

[0015] As a preferred technical solution of the present invention: the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technique in step S5 adopts positive ion mode electrospray ionization; The liquid chromatography conditions included: a Shim-pack XR-ODS column (100 mm × 2 mm, 2.2 μm), a Shim-pack GIST-HP C18 guard column, mobile phase A (5 mmol / L ammonium acetate aqueous solution), mobile phase B (methanol), a flow rate of 0.3 mL / min, a column temperature of 40 ℃, an injection volume of 1 μL, and a gradient elution program of: 50% mobile phase B at 0 min, 90% mobile phase B at 5 min, 100% mobile phase B at 7 min, 100% mobile phase B at 9 min, 50% mobile phase B at 10 min, and 50% mobile phase B at 10.5 min.

[0016] Compared with the prior art, the method of the present invention for simultaneously detecting the content of 10 organophosphates in herbivore feces has the following beneficial effects: First, this invention addresses the complex matrix characteristics of herbivore feces, which are high in pigments, high in lipids, and contain insoluble impurities from the intestines. It establishes a pretreatment system that includes salt dispersion, oscillation aging, ultrasonic extraction, lipid removal with adsorbents, and purification using two solid-phase extraction columns in series. This system effectively solves the technical problems of unstable extraction efficiency, severe matrix interference from lipids and pigments, and insufficient purification effect in existing technologies. It effectively reduces matrix interference, improves the extraction efficiency and purification effect of target OPEs, and enables the simultaneous detection and analysis of multiple OPEs in herbivore feces.

[0017] Secondly, this invention employs ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) to detect the target compound, and combines this with a deuterated internal standard to monitor the entire process of extraction, purification, and instrumental analysis. This effectively solves the technical problems of insufficient detection sensitivity and limited quantitative accuracy in complex matrix samples, achieving the technical effect of improving method stability and accuracy. Method validation results show that the average recovery rate of the target OPEs is 60.5%–90.2%, and the linear correlation coefficient R of the standard curve is [value missing]. 2The value >0.99 indicates that the pretreatment process of the present invention is stable, highly applicable, and highly sensitive, and has great application prospects in the fields of OPEs pollution exposure research, ecological environment monitoring, and risk assessment research in herbivore feces.

[0018] This invention discloses a method for simultaneously detecting the content of 10 OPEs in herbivore feces. By optimizing the sample pretreatment process, the method employs salt dispersion, oscillation aging, ultrasonic extraction, ethylenediamine-N-propylsilane adsorbent degreasing, and purification using a Floric clay solid-phase extraction column and a graphitized carbon black solid-phase extraction column in series. This effectively reduces matrix interference caused by high pigments, high lipids, and insoluble impurities in the intestines of herbivore feces. Combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, the method achieves simultaneous and accurate detection of multiple target OPEs, providing technical support for research on pollution exposure in wild herbivores, ecological environment monitoring, and risk assessment. Attached Figure Description

[0019] Figure 1 This is a schematic flowchart of the method for pretreatment and detection of OPEs in herbivore feces according to the present invention. Figure 2 Multiple reaction monitoring chromatogram of OPE mixed standard solution. Detailed Implementation

[0020] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] The reagents involved in this invention are described below: In this invention, EHDPP is 2-ethylhexyl diphenyl phosphate, TPHP is triphenyl phosphate, TDCIPP is tris(1,3-dichloroisopropyl) phosphate, TCEP is tris(2-chloroethyl) phosphate, TCIPP is tris(2-chloropropyl) phosphate, TBOEP is tris(2-butoxyethyl) phosphate, TEHP is tris(2-ethylhexyl) phosphate, TNBP is tri-n-butyl phosphate, TPRP is tripropyl phosphate, and TEP is triethyl phosphate.

[0022] In this invention, the deuterated mixed internal standards include M6TBEP, D15-TDCPP, D12-TCEP, D21-TPRP, D15-TEP, D15-TPHP, and D27-TNBP. All 10 OPEs standards and 7 deuterated internal standards were purchased from Wellington Laboratories.

[0023] M6TBEP refers to tris(2-butoxyethyl) hexadeuterated phosphate.

[0024] In this invention, dichloromethane and n-hexane (pesticide residue grade) were both purchased from JT Baker; acetone (pesticide residue grade) and methanol (chromatographic grade) were both purchased from Fisher Scientific. Sodium chloride and anhydrous magnesium sulfate were purchased from Macklin; the ethylenediamine-N-propylsilane adsorbent used was Supelclean PSA; the graphitized carbon black solid-phase extraction column used was an Anavo CARB Glass SPE column; and the Floric clay solid-phase extraction column used was an ANPEL Floric clay SPE column.

[0025] The present invention discloses a method for simultaneously detecting the content of 10 OPEs in the feces of herbivores, the specific steps of which are as follows: S1: Preparation of Mixed Calibration Solution: Dilute the OPEs mixed standard solution with methanol to prepare a mixed calibration solution with a concentration of 1000 ng / mL. Then, use methanol to serially dilute the solution to prepare a series of standard solutions with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 ng / mL, designated as CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS9, and CS10, respectively.

[0026] S2: Preparation of internal standard solution: Dilute the 7 deuterated mixed internal standard solutions with methanol to prepare an internal standard solution with a concentration of 1000 ng / mL.

[0027] S3: Plotting standard working curves: Take the standard solutions of the CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS9 and CS10 series respectively, add the internal standard solution to each concentration standard solution to make the final concentration of the internal standard in each standard solution 20 ng / mL. After instrument detection, fit the relationship between the concentration of the target compound in each standard solution and the peak area response of the corresponding quantitative ion pair to establish a standard working curve.

[0028] S4: Sample Pretreatment: Herbivore fecal samples were freeze-dried, then pulverized and homogenized thoroughly using a stainless steel blender. 0.3 g of fecal sample was accurately weighed into a glass test tube, and 0.4 g each of sodium chloride and anhydrous magnesium sulfate were added. After thorough mixing, 10 ng of internal standard and 8 mL of a 1:1 mixture of acetone and n-hexane were added as extraction solvent. The mixture was vortexed until homogeneous, aged on a shaker at 25℃ for 15 min, followed by ultrasonic extraction for 30 min, and centrifuged at 4000 r / min for 10 min. The supernatant was transferred to a pouch using a Pasteur pipette. This extraction and transfer process was repeated three times. The combined supernatants were concentrated to 1-2 mL using a rotary evaporator. Then, 0.5 g of ethylenediamine-N-propylsilane adsorbent was added to the pouch, shaken thoroughly, and the supernatant was purified sequentially through a Florid solid-phase extraction column and a graphitized carbon black solid-phase extraction column. The Floric clay solid-phase extraction column was pre-activated with 6 mL of a 1:1 mixture of acetone and dichloromethane, and then equilibrated with 6 mL of n-hexane. After loading the sample, the target analyte was eluted with 6 mL of a 1:1 mixture of acetone and dichloromethane. The activation and elution conditions for the graphitized carbon black solid-phase extraction column were the same as those for the Floric clay solid-phase extraction column. After collecting the eluent, it was dried to near dryness under mild nitrogen blowing conditions, and then diluted to volume with 500 μL of methanol. The solution was then vortexed to obtain the test solution.

[0029] S5: Detection and Result Analysis: The analyte was qualitatively and quantitatively analyzed using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS). Detection was performed using positive ion mode electrospray ionization at an interface temperature of 300 °C. The chromatographic column was a Shim-pack XR-ODS column (100 mm × 2 mm, 2.2 μm), and the guard column was a Shim-pack GIST-HPC18 guard column. Mobile phase A was 5 mmol / L ammonium acetate aqueous solution, and mobile phase B was methanol at a flow rate of 0.3 mL / min. The gradient elution program was as follows: 50% B phase at 0 min, 90% B phase at 5 min, 100% B phase at 7 min, 100% B phase at 9 min, 50% B phase at 10 min, and 50% B phase at 10.5 min. The column temperature was 40 °C, and the injection volume was 1 μL. Quantitative analysis of the target OPEs was performed based on the standard working curve.

[0030] Ten OPEs were detected simultaneously, including EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP, and TEP.

[0031] The deuterated mixed internal standards in S2 include M6TBEP, D15-TDCPP, D12-TCEP, D21-TPRP, D15-TEP, D15-TPHP, and D27-TNBP.

[0032] The solvent used in S4 is a mixture of acetone and n-hexane in a volume ratio of 1:1.

[0033] In step S4, accurately weigh 0.3 g of herbivore feces sample that has been freeze-dried, pulverized and homogenized, add 0.4 g each of NaCl and anhydrous NaSO4, and add the internal standard solution.

[0034] After the sample in S4 is vortexed and mixed, it is first aged on a shaker at a constant temperature of 25℃ for 15 min, then extracted by ultrasound for 30 min, and centrifuged at 4000 r / min for 10 min; the extraction and centrifugation are repeated 3 times, and the supernatant is combined and concentrated to 1-2 mL.

[0035] 0.5 g of ethylenediamine-N-propylsilane adsorbent is added to S4 to remove lipid interference.

[0036] In step S4, purification is performed sequentially using a Florid solid-phase extraction column and a graphitized carbon black solid-phase extraction column.

[0037] The Florite solid-phase extraction column in S4 was activated with a 1:1 mixture of acetone and dichloromethane and then equilibrated with 6 mL of n-hexane. After loading the sample, it was eluted with a 1:1 mixture of acetone and dichloromethane. The activation and elution conditions of the graphitized carbon black solid-phase extraction column were the same as those of the Florite solid-phase extraction column.

[0038] The eluent in S4 was concentrated to near dryness by gentle nitrogen blowing, then diluted to volume with 500 μL of methanol, and vortexed to obtain the test solution.

[0039] The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) technique in S5 was performed using a Shimadzu LCMS-8050 UHPLC-MS / MS system.

[0040] The liquid chromatography conditions in S5 are as follows: the column is a Shim-pack XR-ODS column with dimensions of 100 mm × 2 mm × 2.2 μm, the guard column is a Shim-pack GIST-HP C18 guard column with dimensions of 2.1 mm × 10 mm × 2 μm; mobile phase A is 5 mmol / L ammonium acetate aqueous solution, and mobile phase B is methanol; the flow rate is 0.3 mL / min; the column temperature is 40 ℃; and the injection volume is 1 μL.

[0041] The liquid chromatography gradient elution conditions in S5 are as follows: 50% B phase at 0 min, 90% B phase at 5 min, 100% B phase at 7 min, 100% B phase at 9 min, 50% B phase at 10 min, and 50% B phase at 10.5 min.

[0042] The mass spectrometry conditions in S5 are as follows: a positive ion mode electrospray ionization source is used, and the interface temperature is 300 ℃; quantitative ion pairs, qualitative ion pairs and corresponding collision energies are set according to the target analyte to perform qualitative and quantitative analysis of the test solution.

[0043] This invention discloses a method for simultaneously detecting the content of 10 organophosphates in herbivore feces. The core of this method lies in solving the pretreatment problem of this complex matrix. The method includes: preparing a mixed calibration solution of organophosphates and a deuterated mixed internal standard solution to establish a standard working curve; pretreating the herbivore feces sample, which includes freeze-drying, homogenization, weighing the sample, adding sodium chloride and anhydrous magnesium sulfate, adding internal standard and extraction solvent, shaking and aging, ultrasonic extraction, centrifugation, concentrating the extract, adding ethylenediamine-N-propylsilane adsorbent to remove lipid interference, sequentially purifying with Floritica solid-phase extraction column and graphitized carbon black solid-phase extraction column, concentrating and adjusting the volume to obtain the test solution; and using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection and analysis. This invention addresses the complex matrix characteristics of herbivore feces, which are characterized by high pigments, high lipids, and intestinal insoluble impurities. By optimizing the pretreatment process, it can effectively reduce matrix interference and improve the extraction efficiency and detection accuracy of target compounds. It has the advantages of stable pretreatment process, strong applicability, and high sensitivity, and can be used for routine detection and ecological monitoring of various organophosphates in herbivore feces.

[0044] Example 1 1. Liquid Chromatography Conditions The chromatographic column was a Shim-pack XR-ODS column (100 mm × 2 mm, 2.2 μm, Shimadzu Corporation, Japan), and the guard column was a Shim-pack GIST-HP C18 guard column (2.1 mm × 10 mm, 2 μm). The mobile phase A was 5 mmol / L ammonium acetate aqueous solution, and the mobile phase B was methanol, with a flow rate of 0.3 mL / min. The gradient elution program was as follows: 50% of phase B at 0 min, 90% at 5 min, 100% at 7 min, 100% at 9 min, 50% at 10 min, and 50% at 10.5 min. The column temperature was 40 ℃, and the injection volume was 1 μL.

[0045] 2. Mass spectrometry conditions Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS / MS-8050, Shimadzu Corporation, Japan) was used for detection. A positive ion mode electrospray ionization (ESI) source was employed, with nitrogen, argon, and compressed air as carrier gases, and an interface temperature of 300 °C. Appropriate mass spectrometry monitoring parameters were set for each target analyte for qualitative and quantitative analysis. Other mass spectrometry parameters are shown in Table 1.

[0046] Table 1. Retention times, ion pair parameters, and collision energies of 10 OPEs and internal standards The above-mentioned OPEs mixed standard solution was analyzed and determined under the above conditions, and the results are as follows: Figure 2 As shown, the 10 OPEs mixed solutions showed good separation within 11 min, with symmetrical peak shapes.

[0047] Example 2 1. Linear range of the working curve, regression equation, and method detection limit. Within the linear range of 0.1–100 ng / g, the regression equations for all OPEs showed good linearity. The method detection limit was calculated based on the standard deviation of seven parallel blank spiked determinations throughout the entire process, as detailed in Table 2.

[0048] Table 2. Regression equations and R values ​​of 10 OPEs in herbivore feces using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. 2 And the method detection limit. Using herbivore feces as a background, a matrix spike recovery experiment was conducted by adding a mixed standard solution of 10 OPEs at a concentration of 20 ng. The experiment was repeated in triplicate. The results of matrix spike recovery of the 10 OPEs and recovery of the 7 internal standards are shown in Table 3. The matrix spike recovery of this method was 88.8%–129.6%, and the internal standard recovery was 60.5%–90.2%, indicating that the method has good accuracy and stability.

[0049] Table 3. Matrix spiked recoveries and internal standard recoveries of 10 OPEs in herbivore feces Example 3 Determination of actual samples: The method established in this study was used to analyze the feces of wild herbivores on the Qinghai-Tibet Plateau. The results are shown in Table 4. Except for TPRP, all other nine OPEs were detected in the feces of herbivores. The detection rates of TEHP and TEP were 73% and 64%, respectively, while the detection rates of the remaining monomers ranged from 81% to 96%. The concentration range of ΣOPEs was 0.60-143.87 ng / g dw. Table 4. Detection results of OPEs in feces of wild herbivores on the Qinghai-Tibet Plateau (ng / g dw) Note: ND indicates not detected.

[0050] As described above, this invention establishes a method for simultaneously detecting the content of 10 OPEs in herbivore feces. The core of this method lies in solving the pretreatment problem of this complex matrix. Targeting the complex matrix characteristics of herbivore feces—high pigment, high lipid, and containing insoluble impurities from the intestines—this method employs a two-stage purification pretreatment process: salt dispersion, oscillation aging, ultrasonic extraction, and adsorbent degreasing. This effectively reduces matrix interference and improves the extraction efficiency and detection accuracy of the target compounds. Method validation results show that when using a mixture of seven deuterated internal standards to monitor the entire process of extraction, purification, and instrumental analysis, the average recovery rate of the target OPEs is 60.5%–90.2%, indicating that the pretreatment process of this invention is stable, has good sensitivity, and is highly practical. It can be used for pollution exposure studies, ecological environment monitoring, and risk assessment studies of 10 OPEs in herbivore feces.

[0051] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces, characterized by, Includes the following steps: S1: Preparation of mixed calibration solution: The mixed calibration solution contains EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP and TEP, and the concentration of each target organophosphate is 100 μg / L. S2: Preparation of internal standard solution: Dilute the deuterated mixed internal standard with methanol to prepare an internal standard solution with a concentration of 1000 ng / mL; S3: Prepare a series of standard solutions of different concentrations from the mixed calibration solution, add the internal standard solution, and plot the calibration curve after testing; S4: Pretreatment of herbivore fecal samples to obtain the test solution: S5: Detection and Quantitative Analysis: The test solution was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and quantitative analysis was performed according to the calibration curve. The preprocessing described in step S4 includes: S41: Freeze-dry, pulverize and homogenize the herbivore fecal sample; S42: Weigh the homogenized fecal sample, add sodium chloride and anhydrous magnesium sulfate, mix well, then add the deuterated mixed internal standard solution and extraction solvent, and shake to mix well; S43: The sample is subjected to oscillation aging followed by ultrasonic extraction, and the extract is obtained by centrifugation. S44: Repeat the extraction and combine the extracts, concentrate them and add ethylenediamine-N-propylsilane adsorbent to remove lipid interference; S45: The supernatant after degreasing is purified sequentially through a Florid solid-phase extraction column and a graphitized carbon black solid-phase extraction column, and the purified liquid is collected. S46: Concentrate the purified liquid and bring it to a fixed volume to obtain the test solution.

2. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, The organophosphates include: 2-ethylhexyl diphenyl phosphate, triphenyl phosphate; tris(1,3-dichloroisopropyl) phosphate, tris(2-chloropropyl) phosphate, tris(2-chloroethyl) phosphate; triethyl phosphate, tripropyl phosphate, tri-n-butyl phosphate, tris(butoxyethyl) phosphate, and tris(2-ethylhexyl) phosphate.

3. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, The preparation method of the mixed calibration solution in step S1 is as follows: Take the single standard solutions of EHDPP, TPHP, TDCIPP, TCEP, TCIPP, TBOEP, TEHP, TNBP, TPRP, and TEP respectively, and dilute them with methanol until the concentration of each single standard solution is 1000 ng / L; take equal volumes of the above 10 single standard solutions, mix them, and then dilute them with methanol to obtain a mixed calibration solution with the concentration of each target organophosphate ester being 100 μg / L.

4. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, The deuterated mixed internal standards mentioned in step S2 include M6TBEP and D 15 -TDCPP, D 12 -TCEP、D 21 -TPRP, D 15 -TEP, D 15 -TPHP and D 27 -TNBP.

5. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, The concentrations of the series of calibration solutions mentioned in step S3 are 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. The internal standard solution is added to each calibration solution to make the final concentration of the internal standard 20 ng / mL. The calibration working curve is established by linear regression fitting with the concentration of the target compound as the abscissa and the peak area of ​​the corresponding quantitative ion pair as the ordinate.

6. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, In step S42, the mass of the fecal sample weighed is 0.3 g, the mass of the added sodium chloride and anhydrous magnesium sulfate desiccant is 0.4 g each, and the extraction solvent is a mixture of acetone and n-hexane.

7. The method for simultaneous detection of the contents of 10 organophosphate esters in herbivore feces according to claim 1, characterized in that, In step S43, the oscillation aging time is 15 min, the ultrasonic extraction time is 30 min, and the centrifugation speed is 4000 r / min for 10 min. In step S44, the extraction is repeated 3 times, and the combined extracts are concentrated to 1-2 mL by rotary evaporation. Then, 0.5 g of ethylenediamine-N-propylsilane adsorbent is added to remove lipid interference. In step S45, the purification is carried out in series with a Floric clay solid-phase extraction column and a graphitized carbon black solid-phase extraction column, wherein the graphitized carbon black solid-phase extraction column is used to remove pigment interference.

8. The method for simultaneously detecting the content of 10 organophosphates in herbivore feces as described in claim 1, characterized in that, In step S45, the activation solvent of the Florid solid-phase extraction column is a mixture of acetone and dichloromethane in a volume ratio of 1:1, the equilibration solvent is n-hexane, and the elution solvent is a mixture of acetone and dichloromethane in a volume ratio of 1:

1. In step S46, the eluent is concentrated to near dryness by gentle nitrogen blowing and then brought to a final volume of 500 μL with methanol.

9. The method for simultaneously detecting the content of 10 organophosphates in herbivore feces as described in claim 1, characterized in that, The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technique described in step S5 employs positive ion mode electrospray ionization. The liquid chromatography conditions included: a Shim-pack XR-ODS column (100 mm × 2 mm, 2.2 μm), a Shim-pack GIST-HP C18 guard column, mobile phase A (5 mmol / L ammonium acetate aqueous solution), mobile phase B (methanol), a flow rate of 0.3 mL / min, a column temperature of 40 ℃, an injection volume of 1 μL, and a gradient elution program of: 50% mobile phase B at 0 min, 90% mobile phase B at 5 min, 100% mobile phase B at 7 min, 100% mobile phase B at 9 min, 50% mobile phase B at 10 min, and 50% mobile phase B at 10.5 min.

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