A method for detecting 99 kinds of non-chiral / chiral endocrine disruptors in urine based on double column combination

Abstract

This invention discloses a method for detecting 99 achiral / chiral endocrine disruptors in urine based on a dual-column coupled analysis system, belonging to the field of pollutant monitoring and analysis technology. The method involves a single pretreatment of the urine sample, including formic acid-acetonitrile extraction and ethylenediamine-N-propyl silica gel purification. A dual-column coupled analysis mode is then used, with a C18 column simultaneously quantifying the total concentration of 93 achiral endocrine disruptors (EDCs) and 6 chiral pesticides. The chiral column yields the R / S enantiomer signal intensity ratio, and the content of each enantiomer is calculated using a "total-proportional conversion" model. This invention achieves, for the first time, a unified detection system for achiral and chiral substances, eliminating the need for a single enantiomer standard; accurate quantification can be achieved using only racemic mixtures, effectively avoiding the direct quantitative bias of chiral columns. The method has a limit of quantification as low as 0.25 μg / L, is simple to operate, low in cost, and has good reproducibility, providing an efficient and reliable analytical tool for population exposure monitoring and chiral selective metabolism research.

Description

Technical Field

[0001] This invention belongs to the field of pollutant monitoring and analysis technology, specifically relating to a method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column combined detection. Background Technology

[0002] Endocrine disrupting chemicals (EDCs) are a class of exogenous chemical substances that can interfere with the synthesis, secretion, transport, and metabolism of hormones in organisms. They are widely found in pesticides, industrial chemicals, personal care products, and environmental pollutants. Long-term exposure to EDCs is closely associated with various health effects, including reproductive and developmental abnormalities, metabolic disorders, neurotoxicity, and cancer. Population biosurveillance (such as urine analysis) has become an important technical support for environmental and health research as a direct means of assessing exposure load to EDCs.

[0003] However, end-use chemical compounds (EDCs) are diverse, encompassing pesticides, phenols, phthalate metabolites, bisphenols and their substitutes, UV absorbers, preservatives, and perfluorinated compounds, among others. Their physicochemical properties vary greatly, and in actual human exposure, they often exhibit typical characteristics of low-dose, multi-component, and combined exposure. Furthermore, many chiral substances exist within EDCs. Chiral pesticides have attracted significant attention due to their unique stereochemical structure. Chiral pesticides, represented by novel neonicotinoids such as acetamiprid, fipronil, and flonicamid, and typical organophosphorus pesticides such as fenpropathrin, fumonisin, and profenofos, are widely used in agricultural production, and the risk of human exposure persists. Although different enantiomers of chiral pesticides have highly similar physicochemical properties, they often exhibit significant differences in target binding capacity, metabolic transformation rate, tissue distribution, and toxic effects in organisms. Therefore, simply measuring the total amount of enantiomers (racemics) is insufficient to accurately assess the true risk of exposure. Differentiating and quantifying R / S enantiomers and calculating the enantiomer ratio have become key requirements for refined exposure assessment and chiral selective metabolism studies.

[0004] Current methods for detecting achiral pesticides (EDCs) mostly focus on the determination of total achiral amounts, while the detection of chiral pesticides is often limited to the enantiomeric separation of a few single compounds. These two methodologies are fragmented, making it difficult to simultaneously perform broad-spectrum screening of achiral EDCs and precise quantification of chiral pesticide enantiomeric compounds within the same analytical workflow in real-world human biosample analysis. Among existing methods for chiral pesticide enantiomeric quantification, liquid chromatography... Tandem triple quadrupole mass spectrometry (LC) MS / MS combined with chiral columns is the most mainstream technical approach. However, the quantitative accuracy of chiral columns is poor. On the one hand, the difference in the interaction forces between the chiral stationary phase and different enantiomers leads to significant differences in the mass spectrometric response of the R-type and S-type enantiomers of the same pesticide. Even at the same concentration, their peak areas or signal intensities are not directly equivalent. On the other hand, co-extracted residues in complex biological matrices (such as urine) have a more significant impact on the separation efficiency and ionization efficiency of chiral columns. The matrix effect often presents asymmetric interference between different enantiomers, further exacerbating quantitative deviations. The traditional solution relies on preparing or purchasing high-purity single R-type and S-type enantiomer standards and establishing standard curves for calibration. However, this method has significant drawbacks: first, single enantiomer standards for multiple chiral pesticides are expensive and have limited supply channels, making them difficult to popularize in routine monitoring; second, there are still differences in matrix matching between the standards and actual biological samples, making it difficult to completely correct for asymmetric interference caused by complex matrices. More importantly, this approach makes the quantification of chiral enantiomers heavily reliant on separate method development, separate sample injection, and separate calibration, resulting in long method development cycles, low analytical throughput, and poor data comparability, which contradicts the technological development direction of high-throughput EDCs screening.

[0005] In summary, there is an urgent need to establish a combined detection method capable of simultaneously covering 93 EDCs and 6 chiral pesticide enantiomers, and to overcome the technical bottleneck of inaccurate direct quantification using chiral chromatographic columns, achieving efficient, accurate, and economical quantification of achiral substances and chiral enantiomers within the same analytical framework. This method is of significant practical importance for accurately characterizing combined exposure loads in populations, identifying major exposure sources, and conducting mixed exposure risk assessments. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention discloses a method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling, so as to solve the technical problems in the prior art such as the independence of non-chiral and chiral detection systems, the poor accuracy of direct quantification by chiral chromatographic columns, and the dependence of chiral detection on a single enantiomeric standard.

[0007] To achieve the above objectives, the technical solution adopted by this invention is as follows: A method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling, wherein the non-chiral endocrine disruptors include organophosphates, phthalates, bisphenols, parabens, perfluorinated compounds, neonicotinoid pesticides, and organophosphorus pesticides; the detection method includes the following steps: S1: Adjust the pH of the urine sample to 4.5-5.5, then add β-D. A complex enzyme solution of glucuronidase and arylsulfatase is used for enzymatic hydrolysis. S2: After the enzymatic hydrolysis reaction is complete, formic acid is added to the enzymatic hydrolysis system of step S1. After mixing the acetonitrile mixture, add salt and mix again. Centrifuge to obtain supernatant A (this step is based on the QuEChERS method principle, using salting-out induced phase separation to achieve liquid-liquid extraction of the target analyte); then react supernatant A with ethylenediamine. N The propyl silica gel was mixed and centrifuged to obtain supernatant B (this step uses dispersion solid phase extraction purification method to effectively remove polar interfering impurities in the urine matrix). S3: Dry the supernatant B, dissolve it in the solvent, and filter it to obtain the test solution; S4: Liquid chromatography was employed. The test solution was detected by tandem triple quadrupole mass spectrometry, wherein: the first detection channel used an octadecylsilane-bonded silica gel column for simultaneous quantitative detection of the concentrations of 93 endocrine disruptors and the total concentration of chiral pesticides in the test solution; the second detection channel used a polysaccharide derivative-coated reversed-phase chiral column for separating and detecting the R / S enantiomeric signal intensity ratio of chiral pesticides in the test solution; the chiral pesticides were oxychloride, dinotefuran, flonicamid, fenpropathrin, fenpropathrin, and profenofos. The content of R-type and S-type enantiomers of each chiral pesticide is obtained by multiplying the total concentration of chiral pesticide measured by the first detection channel by the corresponding R / S enantiomer signal intensity ratio measured by the second detection channel.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, acetic acid is used in step S1. Sodium acetate buffer solution for pH adjustment; β The activity concentration of glucuronidase was 4.5 IU / mL, and the activity concentration of arylsulfatase was 14 IU / mL; urine samples were mixed with acetic acid. The ratio of sodium acetate buffer to complex enzyme solution is 2 mL: 0.7 mL: 10 μL.

[0010] Furthermore, in step S1, the enzymatic hydrolysis temperature is 35~37℃, and the enzymatic hydrolysis time is 11~13h.

[0011] Furthermore, in step S2, formic acid The volume ratio of formic acid to acetonitrile in the acetonitrile mixture is 1:200; the salt is a mixture of anhydrous sodium acetate and anhydrous magnesium sulfate in a mass ratio of 1:4; the enzymatic hydrolysis products and formic acid... The ratio of acetonitrile mixture to salt is 2 mL: 4 mL: 1.5 g.

[0012] Furthermore, in step S2, ethylenediamine N The ratio of propyl silica gel to urine sample was 100 mg: 2 mL.

[0013] Furthermore, in step S3, the solvent is methanol, and the volume ratio of methanol to urine sample is 400 μL: 2 mL.

[0014] Furthermore, the chromatographic conditions for the octadecylsilane-bonded silica column in step S4 are as follows: column temperature 35℃, flow rate 0.3 mL / min, column pressure ≤1300 bar, and injection volume 5 μL; the elution program uses gradient elution, with mobile phase A being methanol and mobile phase B being 1 mmol / L ammonium fluoride aqueous solution, and the elution gradient is set as follows: .

[0016] Furthermore, the chromatographic conditions of the polysaccharide derivative coated reversed-phase chiral column in step S4 are as follows: column temperature 25℃, flow rate 0.2mL / min, column pressure ≤200bar, injection volume 5μL; the elution program is isocratic elution, and the mobile phase is a methanol-water mixture containing 0.1% formic acid, wherein the volume ratio of methanol to water is 9:1.

[0017] Furthermore, the mass spectrometry analysis conditions in step S4 are as follows: the ion source is an electrospray ionization source, the nebulizer gas is N2, the ionization mode is positive ion mode, the scanning method is multiple reaction monitoring, the drying gas temperature is 300℃, the drying gas flow rate is 10L / min, the nebulizer pressure is 35psi, the capillary voltage is 4000V, the sheath gas temperature is 250℃, the sheath gas flow rate is 11L / min, and the nozzle voltage is 1500V.

[0018] The beneficial effects of this invention are as follows: (1) A highly efficient and unified pretreatment system based on the QuEChERS principle. This invention adopts a salting-out induced phase separation and dispersion solid phase extraction purification strategy, extracts the target analytes efficiently through a formic acid-acetonitrile system, and combines ethylenediamine-N-propyl silica gel to remove polar interference impurities in the urine matrix, effectively reducing the matrix effect. This pretreatment process is simple to operate, time-consuming, and consumes little reagent. A single treatment can meet the subsequent dual-channel detection requirements, significantly improving processing efficiency and batch consistency. (2) Unification of achiral and chiral detection systems. This invention achieves the first simultaneous detection and accurate quantification of 93 achiral EDCs and 6 chiral pesticide R / S enantiomers through a combined analysis mode of single pretreatment and dual-column separation, solving the problems of cumbersome pretreatment process, large sample consumption, and poor data comparability caused by separate analytical methods for achiral substances and chiral pesticides. (3) A precise quantitative model for chiral enantiomers based on "total amount-proportional conversion". This invention utilizes the advantages of high quantitative accuracy and wide linear range of C18 chromatographic columns. First, the total concentration of chiral pesticides is determined, and then the signal intensity ratio of R / S enantiomers is obtained through chiral chromatographic columns. The content of each enantiomer is obtained by multiplying the two. This strategy avoids the quantitative deviation caused by enantiomer response differences and matrix interference in chiral chromatographic columns, which is an important innovation in chiral enantiomer quantitative methodology. (4) No need for single enantiomer standard quantification. Traditional chiral enantiomer quantitative methods require the use of high-purity single R-type and S-type standards to establish calibration curves, which is costly. This invention only requires conventional racemic standards to achieve accurate enantiomer quantification, which greatly reduces costs and significantly improves the universality and scalability of the method. (5) It has both high sensitivity and high detection throughput. This invention achieves trace detection of 99 target substances through sample enrichment and concentration and multi-level mixed standard curve design, with a quantification limit as low as 0.25 μg / L, which meets the detection needs of low-level human exposure. Simultaneously, it can output high-dimensional data such as EDC concentration, chiral pesticide enantiomeric ratio, and excess value, adapting to high-throughput detection needs. (6) The method has a high degree of standardization and broad prospects for promotion and application. The reagents and consumables used in this invention are all conventional commercially available products; the pretreatment process is based on the mature QuEChERS system, and the dual-column combination strategy only requires a conventional C18 column and a chiral column. This method has excellent reproducibility and can provide an efficient and reliable analytical tool for environmental exposure monitoring, population health risk assessment, and chiral selective metabolism research. Attached Figure Description

[0019] Figure 1 The total ion flow chromatogram of the 99 EDCs in Example 1; Figure 2 This is a diagram of the multiple reaction monitoring mode of non-chiral BPs in Example 1; Figure 3 This is a diagram of the multiple reaction monitoring mode of non-chiral mPAEs in Example 1; Figure 4 This is a diagram of the multiple reaction monitoring mode of non-chiral NNIs in Example 1; Figure 5 This is a diagram of the multiple reaction monitoring mode of non-chiral OPEs in Example 1; Figure 6 This is a diagram of the multiple reaction monitoring mode for non-chiral OPs in Example 1; Figure 7 This is a diagram of the multiple reaction monitoring mode of non-chiral PFASs in Example 1; Figure 8 This is a diagram of the multiple reaction monitoring mode of non-chiral PHBAs in Example 1; Figure 9 This is a diagram of the multiple reaction monitoring mode of other non-chiral EDCs in Example 1; Figure 10 This is a diagram showing the R / S chiral enantiomeric multiple reaction monitoring (MRM) pattern of the six chiral pesticides in Example 1. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below with reference to the examples. The sources of the specifications and models of the instruments and raw materials used in the examples are as follows: The following instruments were used: a triple quadrupole liquid chromatography-mass spectrometry system (1290 Infinity II-G6475A, Agilent Technologies, USA); a nitrogen evaporator (UGC-24M); a centrifuge (KH19A, Keda); a vortex mixer (UMV-2, Yousheng); a chromatographic column (Daicel, CHIRALPAK AD-RH); acetonitrile (HPLC, Aladdin); formic acid (HPLC, Aladdin); 15 mL polypropylene centrifuge tubes (White Shark); anhydrous magnesium sulfate (Maclin); sodium acetate (Maclin); PLA (Century Bond); 93 non-chiral EDC standards (100 μg / mL, Alta) and 6 chiral pesticide standards (100 μg / mL, Alta) are detailed in Table 1.

[0021] Table 1. Compound information of 99 EDCs

[0022] Example 1: Determination of the standard curve for R / S type enantiomer pesticides (1) Preparation of standard solutions Ninety-nine standard stock solutions, each with a concentration of 100 μg / mL, were prepared using chromatographic grade methanol as the solvent. All standard stock solutions were stored in a sealed container at -20°C, protected from light. Before use, the stock solution of each standard was accurately transferred, and then the two types of mixed standard intermediate solutions were serially diluted with methanol to prepare a series of working standard solutions. The concentration points for the standard curve were set at 0.01 ng / mL, 0.025 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL.

[0023] (2) Analytical standard working solution ① The above series of non-chiral EDC standard working solutions were analyzed by liquid chromatography-tandem triple quadrupole mass spectrometry.

[0024] Chromatographic separation was performed using an octadecylsilane-bonded silica column (C18 column). The liquid chromatography conditions were set as follows: column temperature 35℃, flow rate 0.3 mL / min, maximum pressure 1300 bar, and injection volume 5 μL. Mobile phase A was methanol, and mobile phase B was 1 mmol / L ammonium fluoride aqueous solution. A gradient elution program was used, with specific settings shown in Table 2 below. Table 2

[0025] Mass spectrometry detection was performed using an electrospray ionization source with positive / negative ion switching mode and multiple reaction monitoring (MRM). The main parameters of the ion source were: drying gas temperature 300℃, drying gas flow rate 10 L / min, nebulizing gas pressure 35 psi, sheath gas temperature 250℃, sheath gas flow rate 11 L / min, capillary voltage 4000 V (positive ion mode) / 3500 V (negative ion mode), and nozzle voltage 1500 V (positive ion mode) / 1000 V (negative ion mode). The mass spectrometry acquisition parameters (parent ion, daughter ion, fragmentation voltage, and collision energy) for 93 non-chiral EDCs are detailed in Table 3.

[0026] Table 3. Mass spectrometry acquisition parameters for 93 non-chiral EDCs

[0027] ② Six chiral pesticide R / S enantiomer standard working solutions of various concentrations were analyzed by liquid chromatography-tandem triple quadrupole mass spectrometry. For chiral separation, a polysaccharide derivative coated reversed-phase chiral column (CHIRALPAK AD-RH) with specifications of 150mm×4.6mm and a packing particle size of 5μm was used. The chromatographic conditions were set as follows: column temperature of 25℃, flow rate of 0.2mL / min, upper limit of pressure of 200bar, and injection volume of 5μL. The mobile phase was a methanol-water mixture containing 0.1% formic acid (methanol:water = 9:1, v / v), and isocratic elution was used. Mass spectrometry detection was performed in positive ion electrospray ionization mode with multiple reaction monitoring scanning. The ion source parameters were the same as those in positive ion mode in (2). The mass spectrometry acquisition parameters of the six chiral pesticides are shown in Table 4.

[0028] Table 4. Mass spectrometry acquisition of six chiral pesticides

[0029] (3) Establishing a standard curve Based on the results in Tables 3 and 4, total ion current chromatograms of 99 EDCs were constructed. Figure 1 )as well as Figure 2-10 The diagram shows the multiple reaction monitoring (MRM) pattern for achiral / chiral EDCs. Each EDC was quantified based on a standard curve fitted to 13 concentration levels, where the x-axis represents concentration and the y-axis represents absolute response intensity. For chiral pesticides, based on the properties of the CHIRALPAKAD-RH chiral column, the first peak on the left in the MRM diagram represents the S-enantiomer of the amino acid, and the second peak represents the R-enantiomer. The standard curve was fitted using the weighted least squares method, with the peak area of ​​each target analyte as the y-axis and the corresponding concentration as the x-axis (ng / mL). The results showed that the 93 achiral EDCs exhibited good linearity in the concentration range of 0.01–100 ng / mL, with correlation coefficients (R²) all greater than 0.98 (as shown in Table 5).

[0030] Table 5. Linearity results of standard curves for 99 EDCs

[0031] Example 2 Spiked Recovery Test To verify the extraction efficiency, matrix adaptability, and quantitative accuracy of the detection method established in this invention for 93 achiral EDCs and 6 chiral pesticide R / S enantiomers, a spiked recovery experimental group was set up to test the recovery rate. Considering that EDCs in the human body often exist in a complex form in actual exposure scenarios, and that this invention aims to establish a broad-spectrum, high-throughput screening detection method for chiral and achiral EDCs, this experimental group adopted a mixed standard spiked method for method validation. The specific setup is as follows: A mixed standard of 93 achiral EDCs and a mixed standard of 6 chiral pesticides were simultaneously added to a standard artificial urine matrix, with a spiked concentration of 10 ng / mL (total amount of achiral EDCs and chiral pesticides). Three parallel samples were set up in the experimental group, and an unspiked blank control group was set as a background subtraction baseline. The spiked recovery rate was calculated using the following formula: Spiked recovery rate = (C 测定加标浓度 -C 空白 ) / C 实际加标浓度 ×100% The sample pretreatment and testing process is as follows: Take 2 mL of the urine sample to be tested, add 0.7 mL of 1 mol / L acetate-sodium acetate buffer to adjust the pH of the sample to 5, then add 10 μL of a compound enzyme solution of β-glucuronidase and arylsulfatase (enzyme activity concentrations of 4.5 IU / mL and 14 IU / mL, respectively), and incubate at 37℃ for 12 h to fully release the bound target substance into the free state. After the enzymatic digestion is completed, add 4 mL of formic acid-acetonitrile mixture (formic acid:acetonitrile = 1:200, v / v) to the enzymatic digestion system, vortex for 1 min, then add 1.5 g of extraction salt (anhydrous sodium acetate to anhydrous magnesium sulfate mass ratio of 1:4), vortex again for 1 min, centrifuge at 8000 r / min for 5 min, and collect the supernatant A. The obtained supernatant A was transferred to a centrifuge tube containing 100 mg of ethylenediamine-N-propyl silica gel, vortexed for 1 min, centrifuged, and the supernatant B was taken and dried under a nitrogen stream. The residue was redissolved in 400 μL of methanol and filtered through a 0.22 μm PTFE membrane to obtain the test solution.

[0032] The above-mentioned test solutions were analyzed using liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS). The first detection channel used a C18 column with methanol as mobile phase A and 1 mmol / L ammonium fluoride aqueous solution as mobile phase B, separating and detecting the total concentrations of 93 non-chiral EDCs and 6 chiral pesticides using a gradient elution program. The second detection channel used a CHIRALPAK AD-RH column with a methanol-water mixture (9:1, v / v) containing 0.1% formic acid as mobile phase, isocratic elution, separating and detecting the signal intensity ratios of the R / S enantiomers of the 6 chiral pesticides. The total concentration of chiral pesticides measured in the first channel was multiplied by the enantiomer signal intensity ratio measured in the second channel to calculate the precise content of each R / S enantiomer. The spiked recovery results are shown in Table 6, with a limit of quantitation of 0.05 ng / mL.

[0033] Table 6 Spiked recovery results of 99 EDCs

[0034] As shown in Table 6, after removing the background values ​​in the blank control, the spiked recovery rate at a spiked concentration of 10 μg / L was between 60% and 110%, with a relative deviation of less than 10%, which meets the generally accepted good recovery standard. This confirms that the method of the present invention can accurately achieve the simultaneous extraction, separation, and qualitative and quantitative detection of 93 non-chiral EDCs and 6 chiral pesticides.

[0035] Example 3: Broad-spectrum detection of non-chiral / chiral EDCs in actual human urine samples Using the LC-MS / MS detection method described in Example 1, 93 non-chiral EDCs and 6 chiral pesticide enantiomers in 50 actual human urine samples were simultaneously detected and analyzed.

[0036] Urine pretreatment process: S1: Add 0.7 mL of 1 mol / L acetic acid to a 2 mL urine sample. Sodium acetate buffer was used to adjust the sample pH to 5, and then 10 μL of β-propanediol was added. The complex enzyme solution of glucuronidase and aryl sulfatase (β-glucuronidase activity concentration of 4.5 IU / mL and aryl sulfatase activity concentration of 14 IU / mL) was then subjected to enzymatic hydrolysis at a constant temperature of 37℃ for 12 h to fully release the bound target substance into the free state. S2: Add 4 mL of formic acid to the enzymatic hydrolysis reaction system in step S1. Mix the acetonitrile mixture (formic acid to acetonitrile volume ratio 1:200) by vortexing for 1 min, then add 0.3 g of anhydrous sodium acetate and 1.2 g of anhydrous magnesium sulfate, and vortex again for 1 min. After the reaction is complete, centrifuge to extract the supernatant A; add 100 mg of ethylenediamine to supernatant A. N After mixing with propyl silica gel, vortex for 1 min. After the reaction is complete, centrifuge and collect the supernatant B. S3: Dry the supernatant B with nitrogen, then dissolve the residue in 400 μL of methanol, and then filter it through a 0.22 μm PTFE membrane to obtain the test solution.

[0037] The analytical method for the test solution is the same as in Example 1. The total amount of non-chiral EDCs and chiral pesticides is quantitatively analyzed using a C18 chromatographic column. The R / S enantiomer signal intensity ratio of the six chiral pesticides is separated and determined using a chiral chromatographic column. The enantiomer concentration is calculated according to the "total amount - proportional conversion" model.

[0038] Based on the standard curve established in Example 1, 99 detectable EDCs in human urine samples were quantitatively calculated, and the concentrations of R-type and S-type enantiomers of 6 chiral pesticides were calculated using a conversion model. The detection of various target substances in 50 human urine samples is shown in Table 7.

[0039] Table 7. Measured concentrations and detection rates of 99 EDCs

[0040] As shown in Table 7, the detection method of this invention can stably detect all 93 achiral EDCs and 6 chiral pesticide enantiomers in complex urine matrices, demonstrating high sensitivity and selectivity. The detection rates of various achiral EDCs ranged from 80% to 100%, with average concentrations ranging from 0.01 to 115.94 ± 98.40 ng / mL. The R / S enantiomers of the 6 chiral pesticides all exhibited good chromatographic peak shapes and clear separation in urine samples, with no significant interference peaks or baseline drift. The R and S enantiomers of each chiral pesticide achieved a 100% detection rate.

[0041] The detection method of this invention enables the simultaneous, stable, and accurate detection and quantification of 93 achiral EDCs and 6 chiral pesticide R / S enantiomers in a complex urine matrix. Compared to methods that only detect the total amount or a single configuration of EDCs, this invention provides more refined enantiomer-level information and a broad spectrum of EDC exposure profiles. This facilitates the analysis of the correlation between achiral and chiral substances under combined exposure backgrounds, in vivo chiral selective metabolism studies, and enantiomer toxicity difference assessments, providing higher-dimensional data support for refined health risk assessment of environmental pollutants.

[0042] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling, characterized in that, Non-chiral endocrine disruptors include organophosphates, phthalates, bisphenols, parabens, perfluorinated compounds, neonicotinoid pesticides, and organophosphate pesticides; The detection method includes the following steps: S1: Adjust the pH of the urine sample to 4.5-5.5, then add β-D. A complex enzyme solution of glucuronidase and arylsulfatase is used for enzymatic hydrolysis. S2: After the enzymatic hydrolysis reaction is complete, formic acid is added to the enzymatic hydrolysis system of step S1. After mixing the acetonitrile mixture, add salt and mix well. Centrifuge to obtain supernatant A. Then, react supernatant A with ethylenediamine. N Mix the propyl silica gel thoroughly, centrifuge, and separate to obtain supernatant B; S3: Dry the supernatant B, dissolve it in solvent, and filter it to obtain the test solution; S4: Liquid chromatography was employed. The test solution was detected by tandem triple quadrupole mass spectrometry, wherein: the first detection channel used an octadecylsilane-bonded silica gel column for simultaneous quantitative detection of the concentrations of 93 endocrine disruptors and the total concentration of chiral pesticides in the test solution; the second detection channel used a polysaccharide derivative-coated reversed-phase chiral column for separating and detecting the R / S enantiomer signal intensity ratio of chiral pesticides in the test solution; the chiral pesticides were oxychloride, dinotefuran, flonicamid, fenpropathrin, fenpropathrin, and profenofos; the content of the R-type and S-type enantiomers of each chiral pesticide was obtained by multiplying the total concentration of chiral pesticides measured by the first detection channel by the corresponding R / S enantiomer signal intensity ratio measured by the second detection channel.

2. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: Acetic acid is used in step S1 Sodium acetate buffer solution for pH adjustment; β The activity concentration of glucuronidase was 4.5 IU / mL, and the activity concentration of arylsulfatase was 14 IU / mL; urine samples, acetic acid The ratio of sodium acetate buffer to complex enzyme solution is 2 mL: 0.7 mL: 10 μL.

3. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: In step S1, the enzymatic hydrolysis temperature is 35~37℃, and the enzymatic hydrolysis time is 11~13h.

4. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: Formic acid in step S2 The volume ratio of formic acid to acetonitrile in the acetonitrile mixture is 1:200; the salt is a mixture of anhydrous sodium acetate and anhydrous magnesium sulfate in a mass ratio of 1:4; the enzymatic hydrolysis products and formic acid... The ratio of acetonitrile mixture to salt is 2 mL: 4 mL: 1.5 g.

5. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: Ethylenediamine in step S2 N The ratio of propyl silica gel to urine sample was 100 mg: 2 mL.

6. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: The solvent mentioned in step S3 is methanol, and the volume ratio of methanol to urine sample is 400 μL: 2 mL.

7. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: The chromatographic conditions for the octadecylsilane-bonded silica column in step S4 were as follows: column temperature 35℃, flow rate 0.3 mL / min, column pressure ≤1300 bar, and injection volume 5 μL. The elution program used gradient elution, with mobile phase A being methanol and mobile phase B being 1 mmol / L ammonium fluoride aqueous solution. The elution gradient was set as follows: 。 8. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: The chromatographic conditions for the polysaccharide derivative coated reversed-phase chiral column in step S4 are as follows: column temperature 25℃, flow rate 0.2mL / min, column pressure ≤200bar, injection volume 5μL; isocratic elution program is used, and the mobile phase is a methanol-water mixture containing 0.1% formic acid, wherein the volume ratio of methanol to water is 9:

1.

9. The method for detecting 99 non-chiral / chiral endocrine disruptors in urine based on dual-column coupling according to claim 1, characterized in that: The mass spectrometry analysis conditions in step S4 are as follows: the ion source is an electrospray ionization source, the nebulizer gas is N2, the ionization mode is positive ion mode, the scanning mode is multiple reaction monitoring, the drying gas temperature is 300℃, the drying gas flow rate is 10L / min, the nebulizer pressure is 35psi, the capillary voltage is 4000V, the sheath gas temperature is 250℃, the sheath gas flow rate is 11L / min, and the nozzle voltage is 1500V.

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