Solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water body or sludge

Abstract

The invention relates to the technical field of detection, in particular to a solid-phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in a water body or sludge, which is characterized by comprising enrichment purification, pretreatment and solid-phase extraction and detection, and ultra-high performance liquid chromatography-tandem mass spectrometry. The method specifically comprises the following steps: 1, collecting a sample; 2, treating a water body sample; step 3, sludge sample treatment; step 4, solid-phase extraction; 5, preparing a target object standard curve solution; 6, preparing an internal standard substance mixed standard solution; 7, preparing a sample solution added with an internal standard substance and a mixed standard curve solution; step 8, liquid chromatography-mass spectrometry on-machine detection; and 9, quantifying by an internal standard method. The method is suitable for simultaneous enrichment, purification and detection of 43 steroid hormones of multiple environmental media.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge. Background Art

[0002] Pharmaceuticals and personal care products (PPCPs) are a significant category of emerging pollutants, with steroid hormones being a key category within them. These substances, classified as pharmaceuticals, include glucocorticoids, androgens, and progestins. In addition to being commonly used orally or by injection, they are also frequently applied directly to the skin, occupying a significant share of the topical medication market and representing a common medication category in daily life. However, these substances have been shown to pose potential environmental and ecological risks.

[0003] Steroid hormones in the environment may cause potentially significant negative impacts and ecological risks to fish, algae, microbial communities, etc. In daily life, through bathing and excretion, steroid hormones can enter the urban sewage system with domestic sewage. Since steroid hormones are not a direct carbon source for microorganisms, traditional sewage treatment processes have limited removal of them. Therefore, their presence can often be detected in the effluent of sewage treatment plants. In addition, steroid hormones are generally lipophilic and may accumulate and exist in large quantities in sludge. Once the effluent and sludge enter the environment, the steroid hormones in them will induce ecological risks. Compared with other types of emerging pollutants (such as antibiotics and microplastics), steroid hormones have received relatively less attention, but their potential environmental risks cannot be ignored.

[0004] Therefore, it is necessary to develop a method for the simultaneous enrichment, purification and detection of typical steroid hormones (glucocorticoids, androgens, progestins) that is suitable for multiple environmental media (tap water, river water, seawater, sewage, sludge). Summary of the Invention

[0005] The purpose of the present invention is to provide a solid phase extraction-liquid chromatography-mass spectrometry detection method for simultaneously detecting 43 steroid hormones (40 glucocorticoids, 2 androgens, and 1 progesterone, quantified by internal standard method) in multiple environmental media such as water or sludge with high efficiency, rapidity, and good reproducibility, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge, comprising two parts: enrichment and purification: pretreatment + solid phase extraction and detection: ultra-high performance liquid chromatography-tandem mass spectrometry;

[0007] The specific steps include:

[0008] Step 1: Sample collection;

[0009] Step 2: water sample processing;

[0010] Step 3: Sludge sample processing;

[0011] Step 4: solid phase extraction;

[0012] Step 5: Prepare the target standard curve solution;

[0013] Step 6: Prepare internal standard mixed solution;

[0014] Step 7: Prepare the sample solution and mixed standard curve solution with added internal standard;

[0015] Step 8: Liquid chromatography-mass spectrometry (LC-MS) testing;

[0016] Step 9: Quantification by internal standard method.

[0017] Preferably, in the step 1, sample collection, the collected water samples include tap water, river water, sea water and sewage, all of which are placed in clean brown glass bottles, and the sludge is collected in clean sampling bags. The samples are stored at 4°C and quickly transported back to the laboratory. For water samples, the processing is completed within 24 hours after collection. As for sludge samples, they need to be freeze-dried immediately after delivery to the laboratory, and the corresponding processing work must be completed within 24 hours after freeze-drying is completed.

[0018] Preferably, in the step 2, water sample processing, for various water samples, use a 0.22μm water system microporous filter membrane to fully filter; for sludge samples, after freeze-drying for 48 hours, grind them to make them fully crushed, and for various water samples after filtration, use 1mol / L hydrochloric acid solution and 1mol / L sodium hydroxide solution to adjust their pH to 3.0. Thereafter, 200mL to 500mL of sewage samples are taken depending on the location of the sampling point, 500mL of river water samples are taken, 1000mL of seawater samples are taken, and 2000mL of tap water samples are taken to prepare for solid phase extraction.

[0019] Preferably, in the step three, sludge sample treatment, for the dried sludge sample, accurately weigh 0.100 g into a 50 mL centrifuge tube, add 5.00 mL of methanol: acetonitrile = 1: 1, LC-MS grade, and centrifuge for 10 minutes, collect the supernatant in a clean brown glass bottle, and perform the above oscillation, ultrasound, and centrifugation processes three times in total. A total of 15 mL of supernatant is collected, and ultrapure water is added to a volume of 800 mL. The supernatant is then fully filtered using a 0.22 μm water-based microporous filter membrane, and then adjusted to a pH of 3.0 using 1 mol / L hydrochloric acid solution and 1 mol / L sodium hydroxide solution to prepare for solid phase extraction.

[0020] Preferably, in step 4, solid phase extraction, the solid phase extraction column is inserted into the extraction channel of the solid phase extractor, the large-capacity sampler is connected, and the counterweight is placed at the bottom of the sample bottle, 4 mL of HPLC grade methanol, 4 mL of ultrapure water, and 2 mL of ultrapure water with pH = 3 are used to activate the solid phase extraction column, and the sample is loaded, and the loading flow rate is maintained at 5 mL / min. After the loading is completed, the large-capacity sampler is removed, and the solid phase column filler is rinsed with 4 mL of ultrapure water. The flow rate adopts the natural gravity flow rate. After the elution is completed, a vacuum pump is used to air dry the solid phase column filler for 30 minutes. After the drying is completed, 8 mL of eluent is used, i.e., methanol: acetonitrile = 1:1, LC-MS level, Elute the solid phase column filler at a natural gravity flow rate, collect the eluate in a clean graduated glass centrifuge tube, place the glass centrifuge tube containing the eluted sample in a nitrogen blower, and slowly blow dry with nitrogen until the volume is less than 1 mL. The nitrogen purity should be >99.99%. Use a 1:1 solution of methanol:acetonitrile, LC-MS grade, and accurately dilute to 1.0 mL. Filter the sample using a 0.22 μm syringe filter. After filtration, transfer the sample to a clean brown injection bottle and store at -20°C in the dark.

[0021] Preferably, in step five, a target standard curve solution is prepared, and LC-MS grade methanol is used to prepare 1 mg / mL stock solutions of 43 target substances, respectively. The stock solutions of 43 target substances and LC-MS grade methanol are used to prepare standard curve gradient dilution solutions with concentrations of 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

[0022] Preferably, in step six, an internal standard mixed standard solution is prepared using LC-MS grade methanol to prepare 1 mg / mL hydrocortisone-D2 internal standard stock solution, 1 mg / mL clobetasol propionate-D5 internal standard stock solution, 1 mg / mL fluticasone propionate-D5 internal standard stock solution, and 1 mg / mL testosterone-D3 internal standard stock solution. Four internal standard stock solutions and LC-MS grade methanol are used to prepare an internal standard mixed standard solution with a concentration of 2 μg / mL.

[0023] Preferably, in the step seven, a sample solution and a mixed standard curve solution with added internal standard are configured. Before liquid chromatography-mass spectrometry detection, 0.500 mL of the standard curve gradient dilution solution and the sample to be tested are accurately measured into a new, clean brown injection bottle, and 10 μL of a 2 μg / mL internal standard mixed standard solution are added to make the concentrations of both internal standards 40 ng / mL. The standard curve gradient dilution solution with added internal standard and the sample to be tested with added internal standard are prepared for machine detection.

[0024] In step eight, the liquid chromatography-mass spectrometry (LC-MS) instrument is an ultra-high performance liquid chromatography-tandem mass spectrometer (AB SCIEX Triple Quad). TM 3500LC-MS / MS;

[0025] Set up the chromatography method:

[0026] Mobile phase A: 0.1% formic acid in water (formic acid: LC-MS grade; water: ultrapure water);

[0027] Mobile phase B: methanol:acetonitrile 1:1 solution (LC-MS grade).

[0028] The mobile phase gradient program was as follows: 0 min: A5%; 0→3 min: 5%→40%; 3→18 min: A40%→65%; 18→20 min: A65%→95%; 20→22 min: A95%; 22→22.1 min: A95%→5%; 22.1→24 min: A5%.

[0029] Flow rate: 0.300 mL / min.

[0030] Separation column: WATERS ACQUITY UPLC BEH C18 Column, 1.7μm,2.1mm×100mm.

[0031] Sample detection time: 24 min; injection volume: 10 μL;

[0032] Set the detection parameters for the target substance and internal standard;

[0033] The step nine, internal standard method quantification, after the detection is completed, the internal standard method is used for quantification and target concentration calculation. Hydrocortisone-D2 quantification: prednisolone, prednisone, hydrocortisone, cortisone, methylprednisolone, betamethasone, dexamethasone; testosterone-D3 quantification: progesterone, methyltestosterone, testosterone; clobetasol propionate-D5 quantification: fluorometholone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetonide, diflorasone acetate, betamethasone valerate, prednicarbate, halcinonide, amcinonide, alclometasone dipropionate, clobetasol propionate; fluticasone acetate Flumethasone propionate-D5 quantitative: flumethasone, beclomethasone, triamcinolone acetonide, fludrocortisone, triamcinolone diacetate, prednisolone acetate, fluorometholone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluticasone propionate, mometasone furoate, betamethasone dipropionate, beclomethasone propionate, clobetasone butyrate;

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. A method for the simultaneous enrichment, purification, and detection of 43 steroid hormones applicable to multiple environmental media;

[0036] 2. Excellent target instrument detection limit (LOD) and quantification limit (LOQ);

[0037] 3. Excellent target recovery rate (taking the spiked recovery rate of tap water, river water, sea water, sewage treatment plant secondary sedimentation tank effluent, and residual sludge as examples), most of the recovery rates meet the general requirements (70% to 130%). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 This is a flow chart of the solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to the present invention. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] The existing technology for detecting new pollutants does not yet have a method suitable for the simultaneous enrichment, purification and detection of 40 glucocorticoids, 2 androgens and 1 progestin.

[0042] Existing solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) technology often requires the development of corresponding methods based on the different environmental media (tap water, river water, seawater, sewage, sludge). If there are many substances to be detected, it may also be necessary to develop methods based on their categories, which is time-consuming and labor-intensive. There is no simultaneous SPE-LC-MS method suitable for multiple environmental media.

[0043] Existing quantitative methods often use the external standard method (standard curve method) for quantification. For multi-environmental media, the matrix background is complex and easily interferes with the instrumental quantification of the target. There is no LC / MS method that uses deuterated isotope internal standards (hydrocortisone-D2 (CAS: 79037-25-5), clobetasol propionate-D5 (CAS: 2280940-18-1), fluticasone propionate-D5 (CAS: 1093258-28-6), testosterone-D3 (CAS: 77546-39-5)) to simultaneously detect 40 glucocorticoids, two androgens, and one progestogen.

[0044] See also Figure 1 , the present invention provides a technical solution:

[0045] A solid-phase extraction-liquid chromatography-mass spectrometry method for the simultaneous detection of 43 steroid hormones in water or sludge, including pretreatment + solid-phase extraction and detection and ultra-performance liquid chromatography-tandem mass spectrometry;

[0046] The specific steps include:

[0047] Step 1: Sample collection. The collected water samples include tap water, river water, sea water and sewage, all of which are placed in clean brown glass bottles, while the sludge is collected in clean sampling bags. The samples are stored at 4°C and quickly transported back to the laboratory. For water samples, processing is completed within 24 hours after collection. As for sludge samples, they need to be freeze-dried immediately after delivery to the laboratory, and the corresponding processing work must be completed within 24 hours after freeze-drying is completed;

[0048] Step 2: Water sample processing: For all water samples, use a 0.22μm water-based microporous filter membrane to fully filter; for sludge samples, freeze-dry for 48 hours and then grind them to be fully crushed. For all filtered water samples, use 1mol / L hydrochloric acid solution and 1mol / L sodium hydroxide solution to adjust their pH to 3.0. Then, depending on the location of the sampling point, 200mL to 500mL of sewage samples, 500mL of river water samples, 1000mL of seawater samples, and 2000mL of tap water samples are taken to prepare for solid phase extraction;

[0049] Step 3: Sludge sample treatment: For the dried sludge sample, accurately weigh 0.100 g into a 50 mL centrifuge tube, add 5.00 mL of methanol: acetonitrile = 1:1, LC-MS grade, and centrifuge for 10 minutes. Collect the supernatant in a clean brown glass bottle. Perform the above oscillation, ultrasonication, and centrifugation processes three times. Collect a total of 15 mL of supernatant, add ultrapure water to a volume of 800 mL, and then filter thoroughly with a 0.22 μm water-based microporous filter membrane. Then use 1 mol / L hydrochloric acid solution and 1 mol / L sodium hydroxide solution to adjust its pH to 3.0 and prepare for solid phase extraction.

[0050] Step 4, solid phase extraction, insert the solid phase extraction column into the extraction channel of the solid phase extractor, connect the large-capacity sampler, and place the counterweight at the bottom of the sample bottle. Use 4mL HPLC grade methanol, 4mL ultrapure water, and 2mL pH=3 ultrapure water to activate the solid phase extraction column, load the sample, and maintain the loading flow rate at 5mL / min. After the loading is completed, remove the large-capacity sampler and use 4mL ultrapure water to elute the solid phase column filler. The flow rate adopts the natural gravity flow rate. After the elution is completed, use a vacuum pump to air dry the solid phase column filler for 30min. After drying, use 8mL eluent, i.e. methanol: acetonitrile = 1:1, LC-MS level, to elute the solid phase column filler. Use natural gravity flow rate to collect the eluate in a clean glass centrifuge tube with a scale. For eluted sample processing, place the glass centrifuge tube containing the eluted sample in a nitrogen blower and slowly blow dry with nitrogen until the volume is less than 1 mL. The nitrogen purity should be greater than 99.99%. Use methanol:acetonitrile = 1:1 solution, LC-MS grade, and accurately dilute to 1.0 mL. Use a 0.22 μm syringe filter to filter the sample. After filtration, transfer the sample to a clean brown injection bottle and store at -20°C in the dark.

[0051] Step 5: Prepare target standard curve solutions. Use LC-MS grade methanol to prepare 1 mg / mL stock solutions of each of the 43 targets. Use the 43 stock solutions and LC-MS grade methanol to prepare gradient dilution solutions of the standard curve with concentrations of 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

[0052] Step 6. Prepare the internal standard mixed standard solution. Use LC-MS grade methanol to prepare 1 mg / mL hydrocortisone-D2 internal standard stock solution, 1 mg / mL clobetasol propionate-D5 internal standard stock solution, 1 mg / mL fluticasone propionate-D5 internal standard stock solution, and 1 mg / mL testosterone-D3 internal standard stock solution. Use these four internal standard stock solutions and LC-MS grade methanol to prepare an internal standard mixed standard solution with a concentration of 2 μg / mL.

[0053] Step 7. Prepare the sample solution with added internal standard. Before LC-MS detection, accurately measure 0.500mL of the standard curve gradient dilution solution and the sample to be tested into a new clean brown injection bottle, and add 10μL of the internal standard mixed standard solution with a concentration of 2μg / mL to each of them so that the concentration of both internal standards is 40ng / mL. Add the standard curve gradient dilution solution with internal standard and the sample to be tested with internal standard and prepare for the machine detection.

[0054] Step 8: Liquid chromatography-mass spectrometry (LC-MS) testing. The instrument used is an ultra-high performance liquid chromatography-tandem mass spectrometer (ABSCIEX Triple Quad). TM 3500LC-MS / MS.

[0055] Set up the chromatography method:

[0056] Mobile phase A: 0.1% formic acid in water (formic acid: LC-MS grade; water: ultrapure water);

[0057] Mobile phase B: methanol:acetonitrile 1:1 solution (LC-MS grade).

[0058] The mobile phase gradient was as follows: 0 min: A5%; 0–3 min: 5%–40%; 3–18 min: A40%–65%; 18–20 min: A65%–95%; 20–22 min: A95%; 22–22.1 min: A95%–5%; 22.1–24 min: A5%. Flow rate: 0.300 mL / min. Separation column: WATERS ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 100 mm. Sample detection time: 24 min; injection volume: 10 μL.

[0059] Set the mass spectrometry parameters: use positive ion source mode (ESI+); curtain gas (Curtain Gas): 30.0Psi; collision gas (Collision Gas): 9Psi; spray voltage (IonSpray Voltage) 5500V; nebulization temperature (Temperature): 550℃; nebulization gas (Ion Source Gas1) 55.0Psi; auxiliary gas (Ion Source Gas2) 60Psi.

[0060] Set the detection parameters for the target and internal standard:

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Step 9: Quantification by internal standard method. After the test is completed, the internal standard method is used for quantitative determination and concentration calculation of the target substance. Hydrocortisone-D2 quantitative determination: prednisolone, prednisone, hydrocortisone, cortisone, methylprednisolone, betamethasone, dexamethasone; Testosterone-D3 quantitative determination: progesterone, methyltestosterone, testosterone; Clobetasol propionate-D5 quantitative determination: fluorometholone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetonide, diflorasone acetate, betamethasone valerate, prednicarbate, halcinonide, amcinonide, alclometasone dipropionate, clobetasol propionate; Fluticasone Betamethasone propionate-D5 quantitative: flumethasone, beclomethasone, triamcinolone acetonide, fludrocortisone, triamcinolone diacetate, prednisolone acetate, fluorometholone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluticasone propionate, mometasone furoate, betamethasone dipropionate, beclomethasone propionate, clobetasone butyrate.

[0068] 1. The solid-phase extraction cartridge used in this method is a Waters Oasis HLB cartridge, 400 mg / 3 cc. This method may also be implemented using solid-phase extraction cartridges of other brands and specifications.

[0069] 2. The detection instrument for this method is AB SCIEX Triple Quad TM This method can also be performed on 3500 LC-MS / MS instruments.

[0070] 3. Separation column: WATERS ACQUITY UPLC BEH C18 Column, 1.7μm, 2.1mm×100mm. This method may also be achieved using other chromatographic columns.

[0071] 4. This method selects two sets of Q1 / Q3 ion pairs for each target compound and internal standard. Other ion pairs of target compounds and internal standards may also be used to implement this method.

[0072] 5. The mobile phase of this method is:

[0073] A: 0.1% formic acid aqueous solution (formic acid: LC-MS grade; water: ultrapure water);

[0074] B: Methanol: acetonitrile 1:1 solution (LC-MS grade).

[0075] The mobile phase gradient program was as follows: 0 min: A 5%; 0–3 min: 5%–40%; 3–18 min: A 40%–65%; 18–20 min: A 65%–95%; 20–22 min: A 95%; 22–22.1 min: A 95%–5%; 22.1–24 min: A 5%. Flow rate: 0.300 mL / min. Other mobile phases or mobile phase gradients are also possible.

[0076] This method uses hydrocortisone-D2 (CAS: 79037-25-5), clobetasol propionate-D5 (CAS: 2280940-18-1), fluticasone propionate-D5 (CAS: 1093258-28-6), and testosterone-D3 (CAS: 77546-39-5) as internal standards. If other deuterated derivatives of the 43 target substances are used as internal standards, this method can also be implemented. Excellent instrument detection limits (LODs) and quantification limits (LOQs) for the target substances are:

[0077]

[0078]

[0079]

[0080] Excellent target recovery rate (taking the spiked recovery rate of tap water, river water, seawater, sewage treatment plant secondary sedimentation tank effluent, and excess sludge as examples), most of the recovery rates meet the general requirements (70% to 130%);

[0081]

[0082]

[0083]

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge, characterized in that: It includes two parts: enrichment and purification: pretreatment + solid phase extraction and detection: ultra-high performance liquid chromatography-tandem mass spectrometry; The specific steps include: Step 1: Sample collection; Step 2: water sample processing; Step 3: Sludge sample processing; Step 4: solid phase extraction; Step 5: Prepare the target standard curve solution; Step 6: Prepare internal standard mixed solution; Step 7: Prepare the sample solution and mixed standard curve solution with added internal standard; Step 8: Liquid chromatography-mass spectrometry (LC-MS) testing; Step 9: Quantification by internal standard method.

2. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: The first step is sample collection. The collected water samples include tap water, river water, sea water and sewage, which are all placed in clean brown glass bottles, and the sludge is collected in clean sampling bags. The samples are stored at 4°C and quickly transported back to the laboratory. For water samples, the processing is completed within 24 hours after collection. As for sludge samples, they need to be freeze-dried immediately after delivery to the laboratory, and the corresponding processing work must be completed within 24 hours after the freeze-drying is completed.

3. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In the second step, water sample processing, for various water samples, use a 0.22 μm water system microporous filter membrane to fully filter; for sludge samples, after freeze-drying for 48 hours, grind them to be fully crushed, and for various filtered water samples, use 1 mol / L hydrochloric acid solution and 1 mol / L sodium hydroxide solution to adjust their pH to 3.

0. Thereafter, 200 mL to 500 mL of sewage samples, 500 mL of river water samples, 1000 mL of seawater samples, and 2000 mL of tap water samples are taken depending on the location of the sampling point to prepare for solid phase extraction.

4. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In the third step, sludge sample treatment, for the dried sludge sample, accurately weigh 0.100 g into a 50 mL centrifuge tube, add 5.00 mL of a methanol: acetonitrile = 1:1 solution, LC-MS grade, centrifuge for 10 minutes, collect the supernatant into a clean brown glass bottle, and perform the above oscillation, ultrasonication, and centrifugation processes three times in total. A total of 15 mL of supernatant is collected, ultrapure water is added to a volume of 800 mL, and then the solution is fully filtered using a 0.22 μm water-based microporous filter membrane, and then the pH is adjusted to 3.0 using a 1 mol / L hydrochloric acid solution and a 1 mol / L sodium hydroxide solution to prepare for solid phase extraction.

5. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In step 4, solid phase extraction, the solid phase extraction column is inserted into the extraction channel of the solid phase extraction instrument, connected to the large-capacity sampler, and the counterweight is placed at the bottom of the sample bottle. 4 mL of HPLC grade methanol, 4 mL of ultrapure water, and 2 mL of The solid phase extraction cartridge was activated with ultrapure water at pH 3 and the sample was loaded at a flow rate of 5 mL / min. After loading, the large-capacity sampler was removed and the solid phase cartridge packing was eluted with 4 mL of ultrapure water at a flow rate of natural gravity. After elution, the solid phase cartridge packing was air-dried for 30 min using a vacuum pump. After drying, the solid phase cartridge packing was eluted with 8 mL of eluent (methanol:acetonitrile = 1:1, LC-MS grade) at a flow rate of natural gravity. The eluate was collected in a clean, graduated glass centrifuge tube. The glass centrifuge tube containing the eluted sample was placed in a nitrogen blowdown apparatus and slowly blown dry with nitrogen until the volume was less than 1 mL (nitrogen purity should be >99.99%). The volume was accurately determined to 1.0 mL using a methanol:acetonitrile = 1:1 solution (LC-MS grade). The sample was filtered using a 0.22 μm syringe filter. After filtration, the sample was transferred to a clean brown injection bottle and stored at -20°C in the dark.

6. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In step 5, standard curve solutions of the target substances were prepared. LC-MS grade methanol was used to prepare 1 mg / mL stock solutions of 43 target substances. Gradient dilution solutions of the standard curve were prepared using the 43 stock solutions of the target substances and LC-MS grade methanol with concentrations of 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

7. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In step six, a mixed internal standard solution was prepared using LC-MS grade methanol to prepare 1 mg / mL hydrocortisone-D2 internal standard stock solution, 1 mg / mL clobetasol propionate-D5 internal standard stock solution, 1 mg / mL fluticasone propionate-D5 internal standard stock solution, and 1 mg / mL testosterone-D3 internal standard stock solution. The four internal standard stock solutions and LC-MS grade methanol were used to prepare a mixed internal standard solution with a concentration of 2 μg / mL.

8. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In the step seven, a sample solution and a mixed standard curve solution with added internal standard are prepared. Before liquid chromatography-mass spectrometry detection, 0.500 mL of the standard curve gradient dilution solution and the sample to be tested are accurately measured into a new, clean brown injection bottle, and 10 μL of the internal standard mixed standard solution with a concentration of 2 μg / mL are added to each of them so that the concentrations of the two internal standards are both 40 ng / mL. The standard curve gradient dilution solution with added internal standard and the sample to be tested with added internal standard are prepared for detection on the machine.

9. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: In step eight, the liquid chromatography-mass spectrometry (LC-MS) instrument is an ultra-high performance liquid chromatography-tandem mass spectrometer (AB SCIEX Triple Quad). TM 3500LC-MS / MS; Set up the chromatography method: Mobile phase A: 0.1% formic acid in water (formic acid: LC-MS grade; Water: ultrapure water); Mobile phase B: methanol: acetonitrile 1:1 solution (LC-MS grade); The mobile phase gradient program was as follows: 0 min: A5%; 0→3 min: 5%→40%; 3→18 min: A40%→65%; 18→20 min: A65%→95%; 20→22 min: A95%; 22→22.1 min: A95%→5%; 22.1→24 min: A5%; Flow rate: 0.300 mL / min; Separation column: WATERS ACQUITY UPLC BEH C18 Column, 1.7μm, 2.1mm×100mm; Sample detection time: 24 min; injection volume: 10 μL; Set the mass spectrometry parameters: positive ion source mode (ESI+); Curtain Gas: 30.0 Psi; Collision Gas: 9 Psi; Ion Spray Voltage: 5500 V; Temperature: 550°C; Ion Source Gas 1: 55.0 Psi; Auxiliary Gas: 60 Psi. Set the detection parameters for the target compound and internal standard.

10. The solid phase extraction-liquid chromatography-mass spectrometry method for simultaneously detecting 43 steroid hormones in water or sludge according to claim 1, characterized in that: The step nine, internal standard method quantification, after the detection is completed, the internal standard method is used for quantification and the concentration of the target is calculated, hydrocortisone-D2 quantification: prednisolone, prednisone, hydrocortisone, cortisone, methylprednisolone, betamethasone, dexamethasone; testosterone-D3 quantification: progesterone, methyltestosterone, testosterone; clobetasol propionate-D5 quantification: fluorometholone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetonide, diflorasone acetate, betamethasone valerate, prednicarbate, halcinonide, amcinonide, a Clomethasone dipropionate, clobetasol propionate; Fluticasone propionate-D5 quantitative: flumethasone, beclomethasone, triamcinolone acetonide, fludrocortisone, triamcinolone diacetate, prednisolone acetate, fluorometholone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluticasone propionate, mometasone furoate, betamethasone dipropionate, beclomethasone propionate, clobetasone butyrate.

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