Method for detecting steroid endocrine disrupter based on GC-MS / MS

Abstract

The invention discloses a method for detecting steroid endocrine disruptors based on GC-MS / MS. The method comprises the following steps: carrying out derivatization treatment on a sample to be detected by using a derivatization reagent, diluting a derivatization product by using pyridine to achieve a constant volume, and carrying out detection analysis on the derivatization product by using a gas chromatography-mass spectrometry / mass spectrometry instrument, in the detection, derivatization groups are used as daughter ions to carry out quantitative analysis on different steroid derivatization products; the detection limit and the precision of the detection method are equivalent to those of an LC-MS / MS method, but the instrument cost is far lower than that of the LC-MS / MS method, the detection method has higher universality and can meet the detection requirements of most laboratories, the detection limit of steroid substances in a liquid sample is 1.80-2.69 ng / L, the detection limit of steroid substances in a solid sample is 0.174-0.282 ng / g, the precision is 1.05-3.67%, and the detection limit of the steroid substances in the liquid sample is 1.80-2.69 ng / L; the method is safer, more environmentally friendly, high in stability and good in detection accuracy.

Description

Technical Field

[0001] The invention discloses a gas chromatography-tandem mass spectrometry detection method for steroid endocrine disruptors, belonging to the technical field of detection and analysis. Background Art

[0002] Steroids are a class of bioactive substances with multi-ring structures, belonging to environmental endocrine disruptors (EDCs). These substances have stable chemical properties, wide sources, strong bioaccumulation, good concealment, persistent pollution, and pose great risks to the ecological environment and human health. Typical examples include estrone (Estrone, E1), 17β-estradiol (17β-Estradiol, E2), estriol (Estriol, E3), and 17α-ethynylestradiol (17α-ethynylestradiol, EE2). EE2 is a synthetic hormone that affects the health of organisms, such as embryotoxicity to zebrafish. E1 affects the quality and quantity of sperm. In addition, E2 in the environment also has varying degrees of impact on organisms. It can be seen that these substances have a significant impact on the ecological environment and human health. With the continuous advancement of detection technology, monitoring of this type of new pollutants has become particularly important. Therefore, it is of great significance to study accurate and stable detection methods that are generally applicable and can meet the conditions of most laboratories.

[0003] At present, the main detection methods for steroids in China include gas chromatography-mass spectrometry (Luo Qing et al. Determination of steroid estrogens in water by solid phase extraction-derivation-gas chromatography-mass spectrometry. Physical and Chemical Testing (Chemical Section). 2015, 51(10)), liquid chromatography-tandem mass spectrometry (Zhou Yongshun et al. Simultaneous determination of 24 corticosteroid hormones in sediments by ultrasonic extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry [J]. Chromatography, 2022, 40(02): 165-174.) and other detection methods. Although liquid chromatography-mass spectrometry has high sensitivity, the instrument cost is high, the experimental personnel have high operating requirements, and the system error is large. Many laboratories lack the corresponding conditions. In contrast, the single ion detection scanning (SIM) mode of gas chromatography-mass spectrometry is relatively low in sensitivity, has poor anti-interference ability for complex matrices, and is prone to false positive results. The multiple reaction monitoring (MRM) mode of gas chromatography-triple quadrupole mass spectrometry has better anti-interference ability and high sensitivity. Compared with high-performance liquid chromatography-mass spectrometry, it has lower cost, lower operating requirements for experimenters, relatively small system errors, and is more universally applicable.

[0004] Currently, estrogen derivatization mainly relies on two reagents: N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA). Researchers have used MSTFA to analyze estrogens in water and biological tissues (HMKuch, K.Ballschmiter, Determination of endogenous and exogenous estrogens in effluents from sewage treatment plants at the ng / L-level, Fresenius J Anal Chem 366(4)(2000)392-5.). BSTFA is used to detect steroid hormones in wastewater (Y.Zhou, J.Zha, Z.Wang, Occurrence and fate of steroid estrogens in the largest wastewater treatment plant in Beijing, China, Environ Monit Assess 184(11)(2012)6799-813.).

[0005] When the existing derivatization method is used to detect estrogen derivatives (including E1 or EE2), after the derivatization reaction is completed, dichloromethane, n-hexane or a mixed solvent thereof is usually used as an injection solvent to dilute the sample to a fixed volume; this will cause the derivatization product of EE2 to be converted into the derivatization product of E1, thereby affecting the accuracy of the detection result. Summary of the invention

[0006] In view of the shortcomings of existing detection methods, the present invention provides an analytical detection method for the derivatization of steroid environmental endocrine disruptors based on the gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) multiple reaction monitoring (MRM) mode with derivatized groups as daughter ions. At the same time, a new detection idea for derivatized compounds under tandem mass spectrometry is proposed, that is, the target is quantified by obtaining the structurally intact derivatized groups (TMS) from the benzene ring on the characteristic ion fragments through secondary collisions. A detection method for steroid endocrine disruptors with low cost, strong anti-interference ability, high sensitivity, good correlation and general applicability is provided for the majority of laboratories.

[0007] The present invention discloses a method for detecting steroid environmental endocrine disruptors using a derivatization group as a daughter ion. The method comprises the following steps: a sample to be detected is subjected to derivatization treatment using a derivatization agent, the derivatization product is diluted with pyridine to a fixed volume, and then a gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) instrument is used to detect and analyze the derivatization product. In the detection, the derivatization group is used as a daughter ion to perform quantitative analysis on different steroid derivatization products.

[0008] The steroid environmental endocrine disruptors are estrone (E1), 17β-estradiol (E2), estriol (E3) or 17α-ethynyl estradiol (EE2).

[0009] The samples to be tested were prepared as follows:

[0010] 1. The liquid sample is first filtered using a 0.45 μm filter membrane, and the filtrate is used to enrich the steroid environmental endocrine disruptors using an activated solid phase extraction column PLS. The solid phase extraction column is eluted with a methanol-acetone mixture, and the eluate is collected, dried, and then a derivatization reagent is added. The reaction is shaken at room temperature, and the reaction product is diluted with pyridine to obtain the sample to be tested;

[0011] 2. The solid sample is freeze-dried and mixed with diatomaceous earth. The accelerated solvent extraction method is used to extract the sample using a methanol-acetone mixture. The extract is concentrated to about 3 mL, purified using a solid phase extraction column PLS, and blown dry. A derivatization reagent is added and the sample is shaken at room temperature. The reaction product is diluted with pyridine to a fixed volume to obtain a sample to be tested, wherein the mass ratio of the freeze-dried product to the diatomaceous earth is 1-2:1.

[0012] The methanol-acetone mixed liquid is prepared by mixing methanol and acetone in a volume ratio of 1:1-3; the derivatization reagent is N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) or N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA); when the derivatization reagent is N,O-bis(trimethylsilyl)trifluoroacetamide, the reaction is carried out under shaking at room temperature; when the derivatization reagent is N-methyl-N-(trimethylsilyl)trifluoroacetamide, the reaction is carried out at 55-60°C.

[0013] The present invention uses SCAN mode scanning, collision energy optimization, and detector voltage optimization to finally establish the quantitative ion pairs of four steroid environmental endocrine disruptors as TMS-E1 257m / z>73m / z; di-TMS-E2 285m / z>73m / z; di-TMS-EE2 285m / z>73m / z; tri-TMS-E3 345m / z>73m / z; with trimethylsilyl ion group (TMS) as the daughter ion, and its mass-to-charge ratio is 73.

[0014] The GC-MS / MS instrument analysis conditions are as follows: CBX-5MS column, helium carrier gas, argon CID gas, flow rate 1 mL / min, splitless injection, injection port temperature 280°C, injection volume 1 μL; chromatographic column box: starting temperature 50°C, hold for 2 min, increase to 240°C at 20°C / min, hold for 2 min, increase to 260°C at 12°C / min, hold for 3 min, increase to 280°C at 3°C / min, hold for 4 min. Interface temperature 300°C, EI ion source temperature 250°C, electron bombardment energy 70 eV, solvent delay 12 min, mass spectrometry scan start time 14.7 min, end time 28 min.

[0015] Advantages and technical effects of the present invention:

[0016] The present invention simplifies the solvent coordination problem of derivatization, and can directly use the derivatization reagent to carry out the derivatization reaction on the extracted sample, thereby ensuring the singleness and stability of the derivatization product, and avoiding the problem that the derivatization product of EE2 is converted into the derivatization product of E1 during the analysis process; at the same time, the present invention adopts the derivatization group for quantification, and completes the determination of the detection limit and precision of the method according to the requirements of HJ168-2020. The results show that the present invention has high correlation and low detection limit, and good precision, high correlation, and a determination coefficient R 2 The detection limit of the method for liquid samples is 1.80-2.69 ng / L, the detection limit of the method for solid samples is 0.174-0.282 ng / g, and the precision is 1.05-3.67%. The method of the invention is safer, more environmentally friendly, has high stability, and good detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the spectrum analysis diagram of the derivatization product TMS-E1;

[0018] Figure 2 It is the spectrum analysis diagram of the derivatization product di-TMS-EE2;

[0019] Figure 3 It is the spectrum analysis diagram of the derivatization product di-TMS-E2;

[0020] Figure 4 This is the spectrum analysis diagram of the derivatization product tri-TMS-E3.

[0021] Figure 5 is the standard curve of compound E1 derivatives;

[0022] Figure 6 is the standard curve of compound E2 derivatives;

[0023] Figure 7is the standard curve of compound EE2 derivative;

[0024] Figure 8 is the standard curve of compound E3 derivatives. DETAILED DESCRIPTION

[0025] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified;

[0026] Chemical substances used in the following examples include estrone (solid, CAS: 53-16-7, Beijing, China), 17β-estradiol (solid, CAS: 50-28-2, Beijing, China), estriol (solid, CAS: 50-27-1, Shanghai, China), androstane (solid, CAS: 438-22-2, R. Enrenstorfer, Germany), estrone-d4 (solid, CAS: 53866-34-5, TMRM), N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) (5 mL, 98%, 10% TMCS, Sigma-aldrich, USA, CAS: 25561-30-2), N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, 95%, Shanghai, China, CAS: 24589-78-4), ammonium iodide (NH 4 I, solid, 98%, CNW, Germany, CAS: 12027-06-4), dithiothreitol (DTT, solid, 98%, West Standard (Shanghai) Technology Co., Ltd., CAS: 3483-12-3), methanol, n-hexane, ethyl acetate, acetonitrile (HPLC grade, Dikma, USA), dichloromethane (HPLC grade, CNW, Germany), pyridine (ACS grade, Hach, USA);

[0027] Example 1: Experiment on solvent effect of steroid environmental endocrine disruptors treated by different derivatization methods and then diluted with solvents

[0028] 1. Preparation of test samples

[0029] Prepare several 5 mL volumetric flasks for standby use; dissolve compounds E1, E2, EE2, E3, and E1-d4 (substitute) in methanol to prepare a solution with a concentration of 10 mg / L, and dissolve androstane (internal standard) in pyridine to prepare a solution with a concentration of 10 mg / L, and store at -20°C for standby use;

[0030] 2. Derivatization of samples and solvent effects of solvent dilution

[0031] (1) 100 μL of 10 mg / L E1-d4, E1, and EE2 were added to a chromatographic injection vial, blown dry, and then 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added. After shaking and reacting at room temperature (20°C) for 20 min, the reaction product was diluted to 1 mL with dichloromethane, n-hexane, ethyl acetate, pyridine, and acetonitrile, respectively, and the diluted sample was detected and analyzed using a gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) instrument;

[0032] GC-MS / MS instrument analysis conditions are as follows: CBX-5MS column, helium carrier gas, argon CID gas, flow rate 1 mL / min, splitless injection, injection port temperature 280°C, injection volume 1 μL; chromatographic column box: starting temperature 50°C, hold for 2 min, increase to 240°C at 20°C / min, hold for 2 min, increase to 260°C at 12°C / min, hold for 3 min, increase to 280°C at 3°C / min, hold for 4 min. Interface temperature 300°C, EI ion source temperature 250°C, electron bombardment energy 70 eV, solvent delay 12 min, mass spectrometry scan start time 14.7 min, end time 28 min;

[0033] The mass spectrometry conditions were set as follows: ion source temperature 240°C, interface temperature 280°C, detector voltage relative tuning voltage + 0.5 v, solvent delay 10 min, channel 1 (target / quantitative ion pair) 257 m / z>73 m / z, collision energy 15 eV, start time 22.00 min, scan end time 23.00 min, interval time 0.150 s; channel 1 (target / quantitative ion pair) 285 m / z>73 m / z, collision energy 23 eV, start time 23.00 mi n, scan end time 25.00min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 285m / z>73m / z, collision energy is 25eV, start time 25.00min, scan end time 26.65min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 345m / z>73m / z, collision energy is 29eV, start time 26.65min, scan end time 27.86min, interval time 0.300s;

[0034] The test results are shown in Table 1. It can be seen from the table that after the derivatization treatment, the derivatization products of E1-d4, E1, and EE2 were all single products after dilution with pyridine, while the derivatization products of EE2 diluted with other solvents contained the derivatization products of E1;

[0035] Table 1

[0036]

[0037]

[0038] (2) Take 100 μL of 10 mg / L E1-d4, E1, and EE2 and add them to the chromatographic injection vial respectively. After drying, add 150 μL of N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 50 μL of pyridine solvent. After reacting at 60 °C for 30 min (M. Ljoncheva, E. Heath, D. Heath, S. Dzeroski, T. Kosjek, Contaminants of emerging concern: Silylation procedures, evaluation of the stability of silyl derivatives and associated measurement uncertainty, Sci Total Environ 899(2023)165669.), the reaction products were diluted to 1 mL with dichloromethane, n-hexane, ethyl acetate, pyridine, and acetonitrile, respectively, and the diluted samples were detected and analyzed using a gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) instrument. The analysis conditions were the same as those in step (1). The results are shown in Table 2. It can be seen from the table that although studies have shown that BSTFA combined with pyridine can make the product of EE2 a single product after reaction at 60°C, too high a reaction temperature will still cause the derivatization product of EE2 to be partially converted into the derivatization product of E1 during the reaction;

[0039] Table 2

[0040]

[0041]

[0042] (3) Prepare the derivatization reagent according to the method in the literature (A. Temerdashev, P. Nesterenko, E. Dmitrieva, K. Zhurkina, Y. Q. Feng, GC-MS / MS Determination of Steroid Hormones in Urine Using Solid-Phase Derivatization as an Alternative to Conventional Methods, Molecules 27(18)(2022).): Mix 5 mL of MSTFA, 12 mg of NH4I, and 7.5 mg of DTT in a brown reagent bottle to prepare the derivatization reagent MSTFA / NH 4 I / DTT;

[0043] Take 100 μL of 10 mg / L E1-d4, E1, and EE2 and add them to the chromatographic injection vial. After drying, add the derivatization reagent MSTFA / NH 4 I / DTT 150μL, after reacting at 60℃ for 40min, the reaction product was diluted with dichloromethane, n-hexane, ethyl acetate, pyridine, and acetonitrile to 1mL, and the diluted sample was detected and analyzed by gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS). The analysis conditions were the same as those in step (1). The results are shown in Table 3. It can be seen from the table that after derivatization treatment, the derivatization products of E1-d4, E1, and EE2 were diluted with pyridine to a single product, and there was no interference in the determination.

[0044] Table 3

[0045]

[0046]

[0047] (4) Solvent effect experiments under different reaction conditions

[0048] A. Take 100 μL of 10 mg / L EE2 and add it to a chromatographic injection vial. After drying, add 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). At room temperature (20°C), shake and react for 20 minutes. After drying the reaction product, dilute it with pyridine to 1 mL.

[0049] B. Take 100 μL of 10 mg / L EE2 and add it to a chromatographic injection vial. After drying, add 150 μL of N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). After shaking and reacting for 20 min at room temperature (20°C), the reaction product is dried and then 150 μL of N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) is added again. After shaking and reacting for 20 min at room temperature (20°C), the volume is diluted to 1 mL with pyridine.

[0050] C. Take 100 μL of 10 mg / L EE2 and add it to a chromatographic injection vial. After drying, add 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 50 μL of pyridine solvent. At room temperature (20°C), shake and react for 20 minutes. Then, dilute the reaction product with dichloromethane and n-hexane to a fixed volume of 1 mL.

[0051] D. Take 100 μL of 10 mg / L EE2 and add it to a chromatographic injection vial. After drying, add 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 50 μL of pyridine solvent. At room temperature (20°C), shake and react for 20 minutes. After drying, the reaction product is diluted with dichloromethane, n-hexane, and pyridine to a fixed volume of 1 mL.

[0052] The diluted samples were tested and analyzed using a gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) instrument. The analysis conditions were the same as in step (1). The results are shown in Table 4.

[0053]

[0054]

[0055] It can be seen from the results in the table that the derivatization reaction of EE2 is a reaction with a certain reversibility, and its product is not a stable product as previously believed. Although the derivatization reaction with the derivatization reagent and pyridine can obtain a stable and single EE2 derivatization product, as long as the derivatization product is fixed to volume with solvents such as dichloromethane or n-hexane, TMS-E1 product will be added to the derivatization product, thereby affecting the accuracy of the detection result; if the sample is directly injected after the reaction with pyridine mixed with the derivatization reagent, there is a lack of dilution, and the instrument equipment may be contaminated due to the high sample concentration. The use of pyridine for dilution and volume will increase the use of pyridine, a carcinogen, and repeated use will pose a safety hazard to the tester; the present invention proposes a method in which the sample to be tested is directly derivatized with a derivatization reagent and then diluted and fixed to volume with pyridine, which not only ensures the stability of the reaction and the singleness of the derivatization product, but also reduces the use of carcinogenic organic solvents, reduces the cost and risk of use, and improves the safety of the experiment.

[0056] Example 2: Experiments for determining sensitivity or detection limit

[0057] 1. Preparation of calibration curves for compounds E1, E2, EE2, and E3

[0058] Compound E1, E2, EE2, and E3 standards were used to prepare compound solutions with a concentration range of 5-500 μg / L, added to a chromatographic injection vial, blown dry, and then 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added. After shaking and reacting at room temperature (20°C) for 20 min, 50 μL of internal standard solution (androstane 10 mg / L) was added to the reaction product, and pyridine was added to dilute the volume to 1 mL;

[0059] The diluted samples were detected and analyzed by gas chromatography-mass spectrometry (GC-MS / MS). The analysis conditions of the GC-MS / MS instrument were as follows: the chromatographic column was CBX-5MS, the carrier gas was helium, the CID gas was argon, the flow rate was 1 mL / min, the injection was in splitless mode, the injection port temperature was 280°C, and the injection volume was 1 μL; the chromatographic column box: the starting temperature was 50°C, maintained for 2 min, heated to 240°C at 20°C / min, maintained for 2 min, heated to 260°C at 12°C / min, maintained for 3 min, heated to 280°C at 3°C / min, maintained for 4 min. The interface temperature was 300°C, the EI ion source temperature was 250°C, the electron bombardment energy was 70 eV, the solvent delay was 12 min, the mass spectrometry scan start time was 14.7 min, and the end time was 28 min;

[0060] The mass spectrometry conditions were set as follows: ion source temperature 240°C, interface temperature 280°C, detector voltage relative tuning voltage + 0.5 v, solvent delay 10 min, channel 1 (target / quantitative ion pair) 257 m / z>73 m / z, collision energy 15 eV, start time 22.00 min, scan end time 23.00 min, interval time 0.150 s; channel 2 (target / quantitative ion pair) 285 m / z>73 m / z, collision energy 23 eV, start time 23.00 min, scan end time 25.00 min, interval time 0.3 00s; channel 1 (target / quantitative ion pair) is 285m / z>73m / z, collision energy is 25eV, start time is 25.00min, scan end time is 26.65min, and interval time is 0.300s; channel 1 (target / quantitative ion pair) is 345m / z>73m / z, collision energy is 29eV, start time is 26.65min, scan end time is 27.86min, and interval time is 0.300s; mass spectrometry analysis of derivative products TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3 see Figure 1-4 ;

[0061] The standard curve is drawn with the ratio of compound concentration to internal standard concentration as the horizontal axis and the ratio of compound peak area to internal standard peak area as the vertical axis. Figure 5-8 ;

[0062] 2. Detection limit determination experiment for compounds E1, E2, EE2, and E3

[0063] A. Detection limit of liquid samples

[0064] (1) Add the standard samples of compounds E1, E2, EE2, and E3 to 1 L of ultrapure water to prepare aqueous solutions of the compounds with a concentration of 10 ng / L;

[0065] (2) Activate the solid phase extraction column PLS column using 6 mL of methanol and water;

[0066] (3) using the PLS column activated in step (2) to enrich steroid environmental endocrine disruptors;

[0067] (4) eluting the solid phase extraction column enriched in step (3) with a methanol-acetone mixture (volume ratio 1:1) (6 mL × 3 times);

[0068] (5) Collect the eluate, blow dry it with a nitrogen blower, and then reconstitute it with 2 mL of a methanol-acetone mixture (volume ratio 1:1);

[0069] (6) Transfer the reconstituted solution from step (5) into a concentrator tube with a chromatographic injection vial and blow dry using a quantitative concentrator;

[0070] (7) Add 150 μL of BSTFA reagent to the chromatographic injection vial of step (6), shake and react at room temperature (20° C.) for 20 min, add 50 μL of internal standard solution (androstane 10 mg / L) to the reaction product, and then dilute to 1 mL with pyridine;

[0071] (8) The chromatographic and mass spectrometric conditions used in the preparation of the calibration curves of compounds E1, E2, EE2, and E3 were used for detection and analysis.

[0072] B. Detection limit of solid samples

[0073] (1) Compound E1, E2, EE2, and E3 standards were added to 10 g of quartz sand to prepare sediment samples with a concentration of 1 ng / g of the compound;

[0074] (2) adding the sediment sample from step (1) into the extraction pool, using an accelerated solvent extractor, using a methanol-acetone mixed solvent (1:1) as the extraction solvent, and the extraction conditions of the extractor are: temperature 80° C., pressure 1500 psi, heating equilibrium 5 min, cycle number 2, elution volume 50%, and purge time 80 s;

[0075] (3) drying the extract obtained in step (2) with a nitrogen blower, and re-dissolving it with a methanol-acetone mixed solvent (1:1);

[0076] (4) washing the PLS column with methanol, and purifying the reconstituted solution in step (3) through the washed PLS column;

[0077] (5) transferring the purified solution in step (4) into a concentrator tube with a chromatographic injection vial and drying it using a quantitative concentrator;

[0078] (6) Add 150 μL of BSTFA reagent to the chromatographic injection vial in step (5), shake and react at room temperature (20° C.) for 20 min, add 50 μL of internal standard solution (androstane 10 mg / L) to the reaction product, and then dilute to 1 mL with pyridine;

[0079] (7) The chromatographic and mass spectrometric conditions used in the preparation of the calibration curves of compounds E1, E2, EE2, and E3 were used for detection and analysis.

[0080] Table 5

[0081]

[0082] 3. Precision determination experiments of compounds E1, E2, EE2, and E3

[0083] (1) Compound E1, E2, EE2, and E3 standards were used to prepare compound solutions with concentrations of 10, 100, and 500 μg / L, respectively, and added to a chromatographic injection vial. After drying, 150 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added. After shaking and reacting at room temperature (20°C) for 20 min, 50 μL of internal standard solution (androstane 10 mg / L) was added to the reaction product, and pyridine was added to dilute the volume to 1 mL;

[0084] (2) The chromatographic and mass spectrometric conditions used in the preparation of the calibration curves of compounds E1, E2, EE2, and E3 were used for detection and analysis.

[0085] Table 6

[0086]

[0087]

[0088] The detection limit and precision tests of the method were completed in accordance with the calculation formula and requirements of HJ168-2020.

[0089] It can be seen from Table 5 and Table 6 that the method of the present invention has a good effect, the detection limit of the liquid sample is 1.80-2.69 ng / L; the detection limit of the method in the sediment is 0.174-0.282 ng / g, and the precision is 1.05-3.67%.

[0090] Example 3: Detection method of steroid endocrine disruptors in the surface water of Caohai Lake in Dianchi Lake

[0091] 1. Preparation of test samples

[0092] (1) Use 6 mL of methanol and 6 mL of water to activate the solid phase extraction column PLS column;

[0093] (2) 1 L of surface water samples were collected from different locations in Caohai Lake of Dianchi Lake, and filtered with a 0.45 μm filter membrane. The samples were divided into surface water samples and surface water spiked samples. The surface water spiked samples were prepared by adding 20 μL of a 10 mg / L standard solution (E1, E2, EE2, E3) to the filtered surface water. The samples were subjected to a solid phase extraction instrument and the PLS column activated in step (1) was used to enrich steroid environmental endocrine disruptors.

[0094] (3) eluting the solid phase extraction column enriched in step (2) with a methanol-acetone mixture (volume ratio 1:1) (6 mL × 3 times);

[0095] (4) Collect the eluate, blow dry it with a nitrogen blower, and then reconstitute it with 2 mL of a methanol-acetone mixture (volume ratio 1:1);

[0096] (5) transferring the reconstituted solution from step (4) into a concentrator tube with a chromatographic injection vial and drying it using a quantitative concentrator;

[0097] (6) Add 150 μL of BSTFA reagent to the chromatographic injection vial of step (5), shake and react at room temperature (20° C.) for 20 min, add 50 μL of internal standard solution (androstane 10 mg / L) to the reaction product, and then dilute to 1 mL with pyridine;

[0098] 2. Use gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) to detect and analyze the diluted samples. The analysis conditions of the GC-MS / MS instrument are as follows: the chromatographic column is CBX-5MS, the carrier gas is helium, the CID gas is argon, the flow rate is 1mL / min, the injection is in splitless mode, the injection port temperature is 280℃, and the injection volume is 1μL; the chromatographic column box: the starting temperature is 50℃, maintained for 2min, the temperature is increased to 240℃ at 20℃ / min, maintained for 2min, the temperature is increased to 260℃ at 12℃ / min, maintained for 3min, the temperature is increased to 280℃ at 3℃ / min, and maintained for 4min. The interface temperature is 300℃, the EI ion source temperature is 250℃, the electron bombardment energy is 70eV, the solvent delay is 12min, the mass spectrometry scan start time is 14.7min, and the end time is 28min;

[0099] The mass spectrometry conditions were set as follows: ion source temperature 240°C, interface temperature 280°C, detector voltage relative tuning voltage + 0.5 v, solvent delay 10 min, channel 1 (target / quantitative ion pair) 257 m / z>73 m / z, collision energy 15 eV, start time 22.00 min, scan end time 23.00 min, interval time 0.150 s; channel 1 (target / quantitative ion pair) 285 m / z>73 m / z, collision energy 23 eV, start time 23.00 mi n, scan end time 25.00min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 285m / z>73m / z, collision energy is 25eV, start time 25.00min, scan end time 26.65min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 345m / z>73m / z, collision energy is 29eV, start time 26.65min, scan end time 27.86min, interval time 0.300s;

[0100] The results are shown in Table 7, where the Caohai Center mean is the average of the parallel samples of Caohai Center-1 and Caohai Center-2, and the Broken Bridge mean is the average of the parallel samples of Broken Bridge-1 and Broken Bridge-2. The results show that estrone was detected in Caohai Water, and the recovery rate of spiked water samples was 67.0-102%;

[0101] Table 7

[0102]

[0103]

[0104] Example 4: Detection method of steroid environmental endocrine disruptors in sediments of Lake Caohai, Dianchi

[0105] 1. Preparation of test samples

[0106] (1) Sediments were collected from different locations in Caohai Lake of Dianchi Lake. After freeze-drying, 5-7 g of freeze-dried samples were taken and 5 g of diatomaceous earth was added and mixed. The samples were divided into sediment samples and sediment spiked samples. The sediment spiked samples were prepared by adding 20 μL of a 10 mg / L standard solution (E1, E2, EE2, E3) to the sediments.

[0107] (2) adding the mixture of step (1) into an extraction cell, using an accelerated solvent extractor, using a methanol-acetone mixed solvent (1:1) as the extraction solvent, and the extraction conditions of the extractor are: temperature 80° C., pressure 1500 psi, heating equilibrium 5 min, cycle number 2, elution volume 50%, and purge time 80 s;

[0108] (3) drying the extract obtained in step (2) with a nitrogen blower, and re-dissolving it with a methanol-acetone mixed solvent (1:1);

[0109] (4) washing the PLS column with methanol, and purifying the reconstituted solution in step (3) through the washed PLS column;

[0110] (5) transferring the purified solution in step (4) into a concentrator tube with a chromatographic injection vial and drying it using a quantitative concentrator;

[0111] (6) Add 150 μL of BSTFA reagent to the chromatographic injection vial in step (5), shake and react at room temperature (20° C.) for 20 min, add 50 μL of internal standard solution (androstane 10 mg / L) to the reaction product, and then dilute to 1 mL with pyridine;

[0112] 2. Use gas chromatography-mass spectrometry / mass spectrometry instrument for detection. The analysis conditions of GC-MS / MS instrument are as follows: chromatographic column is CBX-5MS, carrier gas is helium, CID gas is argon, flow rate is 1mL / min, injection is in splitless mode, injection port temperature is 280℃, injection volume is 1μL; chromatographic column box: starting temperature is 50℃, keep for 2min, increase the temperature to 240℃ at 20℃ / min, keep for 2min, increase the temperature to 260℃ at 12℃ / min, keep for 3min, increase the temperature to 280℃ at 3℃ / min, keep for 4min. Interface temperature is 300℃, EI ion source temperature is 250℃, electron bombardment energy is 70eV, solvent delay is 12min, mass spectrometry scan start time is 14.7min, and end time is 28min;

[0113] The mass spectrometry conditions were set as follows: ion source temperature 240°C, interface temperature 280°C, detector voltage relative tuning voltage + 0.5 v, solvent delay 10 min, channel 1 (target / quantitative ion pair) 257 m / z>73 m / z, collision energy 15 eV, start time 22.00 min, scan end time 23.00 min, interval time 0.150 s; channel 1 (target / quantitative ion pair) 285 m / z>73 m / z, collision energy 23 eV, start time 23.00 mi n, scan end time 25.00min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 285m / z>73m / z, collision energy is 25eV, start time 25.00min, scan end time 26.65min, interval time 0.300s; channel 1 (target / quantitative ion pair) is 345m / z>73m / z, collision energy is 29eV, start time 26.65min, scan end time 27.86min, interval time 0.300s;

[0114] The results are shown in Table 8, where the Caohai Center mean is the average of the parallel samples of Caohai Center-1 and Caohai Center-2, and the Broken Bridge mean is the average of the parallel samples of Broken Bridge-1 and Broken Bridge-2. The results show that only estrone was detected in the Caohai sediments, and the matrix spike recovery rate of the target compound in the sediments reached 48.8-91.3%.

[0115] Table 8

[0116]

Claims

1. A method for detecting steroid endocrine disruptors based on GC-MS / MS, characterized in that: The sample to be tested is derivatized with a derivatization reagent, and the derivatization product is diluted with pyridine to a fixed volume, and then detected and analyzed by a gas chromatography-mass spectrometry / mass spectrometry instrument. In the detection, the derivatization group is used as a daughter ion to quantitatively analyze different steroid derivatization products.

2. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 1, characterized in that: The steroidal environmental endocrine disruptors are estrone, 17β-estradiol, estriol or 17α-ethynyl estradiol.

3. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 1, characterized in that: The liquid sample is first filtered using a 0.45 μm filter membrane, and the filtrate is used to enrich the steroid environmental endocrine disruptors using an activated solid phase extraction column. The solid phase extraction column is eluted with a methanol-acetone mixture, and the eluate is collected and dried to obtain the sample to be tested.

4. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 1, characterized in that: The solid sample is freeze-dried and then mixed with diatomaceous earth, and extracted using a methanol-acetone mixture using an accelerated solvent extraction method. The extract is concentrated, purified, and blown dry to obtain a sample to be tested, wherein the mass ratio of the freeze-dried product to the diatomaceous earth is 1-2:

1.

5. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 1, characterized in that: The derivatization reagent is N-methyl-N-(trimethylsilyl)trifluoroacetamide or N,O-bis(trimethylsilyl)trifluoroacetamide. When the derivatization reagent is N,O-bis(trimethylsilyl)trifluoroacetamide, the reaction is carried out under shaking at room temperature; when the derivatization reagent is N-methyl-N-(trimethylsilyl)trifluoroacetamide, the reaction is carried out at 55-60°C.

6. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 2, characterized in that: The derivatization group is a trimethylsilane ion group with a mass-to-charge ratio of 73. The quantitative ion pairs for four steroid environmental endocrine disruptors are TMS-E1 257m / z>73m / z; di-TMS-E2 285m / z>73m / z; di-TMS-EE2285m / z>73m / z; tri-TMS-E3 345m / z>73m / z.

7. The method for detecting steroid endocrine disruptors based on GC-MS / MS according to claim 3 or 4, characterized in that: The methanol-acetone mixed solution is prepared by mixing methanol and acetone in a volume ratio of 1:1-3.

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