阅读说明：本技术 一种测定高有机质固体样品中甾体激素的检测方法 (Detection method for measuring steroid hormone in high organic matter solid sample ) 是由 张云 邹华 钟琴 冯慧 周涛 顾帆 付善飞 于 2021-09-23 设计创作，主要内容包括：本发明公开了一种测定高有机质固体样品中甾体激素的检测方法,包括下列步骤：(1)将固体样品冷冻干燥,研磨后过100目筛；(2)将步骤(1)得到的样品置于容器内；(3)向步骤(2)中的容器加入萃取剂,震荡15s-30s,超声处理后离心取上清液,萃取三次；萃取三次使用的萃取剂为甲醇、乙腈；(4)将步骤(3)得到的三次萃取的上清液混合后在氮气下吹干,加入甲醇复溶后,加入PSA吸附剂进行清洗；吸附剂的投加量为400mg；(5)离心将吸附剂分离后,将清洗后的样品过膜用LC-MS/MS进行检测。采用本发明方法适用范围广,操作简便,能实现多种甾体激素回收率均达到75％以上,平均回收率达到94.65％。(The invention discloses a detection method for measuring steroid hormones in a high organic matter solid sample, which comprises the following steps: (1) freeze-drying the solid sample, grinding, and sieving with a 100-mesh sieve; (2) placing the sample obtained in the step (1) into a container; (3) adding an extracting agent into the container in the step (2), shaking for 15-30 s, centrifuging after ultrasonic treatment, taking supernate, and extracting for three times; the extracting agent used for the third extraction is methanol and acetonitrile; (4) mixing the supernatants obtained in the step (3) after the three extractions, drying the mixed supernatants under nitrogen, adding methanol for redissolution, and adding a PSA adsorbent for cleaning; the dosage of the adsorbent is 400 mg; (5) after centrifugation to separate the adsorbent, the washed sample was passed through a membrane and examined by LC-MS/MS. The method has wide application range and simple and convenient operation, and can realize that the recovery rates of various steroid hormones all reach over 75 percent and the average recovery rate reaches 94.65 percent.)

1. A pretreatment method for detecting trace steroid hormones in a high organic matter solid sample is characterized in that the pretreatment method comprises the steps of extracting the trace steroid hormones in the high organic matter solid sample by adopting an ultrasonic-assisted extraction method to obtain an extraction liquid, and then cleaning the extraction liquid by adopting N-propyl ethylenediamine as an adsorbent; the extraction is divided into three times of extraction, and methanol is adopted for the first time of extraction; extracting with acetonitrile for the second and third times; the addition amount of the N-propylethylenediamine is 200-800mg/g of the high organic solid sample, namely the addition amount of the N-propylethylenediamine is 20-80% of the mass of the high organic solid sample, and g/g.

2. The method of claim 1, wherein the sample of high organic matter solids is freeze-dried, ground and sieved.

3. The method according to claim 1 or 2, wherein the three extractions are performed under ultrasonic conditions, and each extraction time is 10-20 min.

4. The method of any one of claims 1 to 3, wherein the high organic solids sample comprises sewage plant activated sludge and excess sludge, livestock manure, high organic soil or sediments.

5. The method of any one of claims 1-4, wherein said steroid hormone comprises one or more of estrone, estradiol, estriol, ethinyl estradiol, epiandrosterone, androsterone, androstenedione, testosterone, methyltestosterone, nandrolone, progesterone, levonorgestrel, medroxyprogesterone acetate, megestrol acetate, hydroxyprogesterone, cortisone, hydrocortisone, corticosterone, prednisone, prednisolone, methylprednisolone, and dexamethasone.

6. Use of the pretreatment method according to any one of claims 1 to 5 for detecting steroid hormones in a high organic matter solid sample.

7. A method for detecting the content of micro steroid hormones in a high organic matter solid sample, which is characterized in that the method comprises the steps of treating the solid sample by the pretreatment method according to any one of claims 1 to 5, and then analyzing by high performance liquid chromatography-tandem mass spectrometry.

8. The method according to claim 7, wherein ammonia and acetonitrile are used as mobile phases when analyzing estrogens; when analyzing androgens, progestins and glucocorticoids, methanol solution containing 0.1% formic acid and acetonitrile are used as mobile phases.

9. Method according to claim 7 or 8, characterized in that it comprises the following steps:

(1) pretreatment: putting the solid sample into a culture dish, freezing the solid sample in a-80 ℃ ultra-low temperature refrigerator overnight, taking out the solid sample, immediately putting the solid sample into a freeze dryer, freeze-drying the solid sample at-45 ℃, grinding the solid sample in a mortar, and sieving the ground solid sample with a 100-mesh sieve;

(2) and (3) extraction: accurately weighing 0.5g of the solid sample obtained in the step (1), putting the solid sample into a 30mL polytetrafluoroethylene centrifugal tube, adding 5mL of methanol, screwing a screw cap, oscillating for 30s by using a vortex oscillator, then carrying out ultrasonic extraction for 15min, centrifuging for 15min at 4000rpm, carrying out solid-liquid separation, and sucking out supernatant liquid by using a glass rubber head dropper to put the supernatant liquid into the polytetrafluoroethylene centrifugal tube; repeating the steps for 2 times, but replacing the extraction solvent with 5mL of acetonitrile, collecting the extract liquid after three times of centrifugation, centrifuging for 30min again, taking the supernatant liquid to be contained in a glass nitrogen blowing tube, blowing the supernatant liquid on a nitrogen blowing instrument by using stable nitrogen flow, and accurately adding 1.0mL of methanol for redissolution;

(3) cleaning: weighing 400mg of PSA powder adsorbent, adding into the centrifugal tube containing the concentrated extract liquid in the step (2), carrying out vortex oscillation for 30s, centrifuging at 4000rpm for 20min, finally passing the supernatant through a 0.22 mu m PTFE membrane by using a needle filter, injecting into a 250 mu L inner insert tube, and storing in a brown chromatographic bottle to be tested;

(4) high performance liquid chromatography tandem mass spectrometry: the liquid chromatographic conditions for estrogen analysis were: the mobile phase A is 0.05% ammonia water, the mobile phase B is acetonitrile, the flow rate is 0.4mL/min, the sample injection amount is 5 mu L, and the column temperature is 40 ℃; the elution conditions were: 0-0.25 min mobile phase is 70% phase A + 30% phase B; gradually reducing phase A to 10% and gradually increasing phase B to 90% in 0.25-2 min; maintaining for 1min, recovering to 70% A phase + 30% B phase of initial mobile phase, and maintaining for 1 min; the mass spectrometry conditions were: the mass spectrum mode is an electrospray negative ion source; the mass spectrum scanning mode is multi-reaction ion monitoring; the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision airflow is 0.16 mL/min;

the chromatographic conditions for the analysis of androgens, progestins and glucocorticoids are: the mobile phase A is a methanol solution containing 0.1% formic acid, the mobile phase B is acetonitrile, the flow rate is 0.4mL/min, the sample injection amount is 5 mu L, and the column temperature is 40 ℃; the elution conditions were: the mobile phase is 90% phase A + 10% phase B in 0-0.25 min; gradually increasing phase B to 70% in 0.25-1 min; 1-3 min, increasing B to 95% successively, and maintaining for 1 min; then immediately returning to the initial mobile phase 90% A phase + 10% B phase for 1 min; the mass spectrometry conditions were: when detecting androgen and glucocorticoid, the mass spectrum mode is an electrospray positive ion source; when the glucocorticoid is detected, the mass spectrum mode is an electrospray negative ion source, and the mass spectrum scanning mode is multi-reaction ion monitoring; the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision gas flow was 0.16 mL/min.

10. A method for cleaning extraction liquid after extracting steroid hormones in a high organic solid sample is characterized in that N-propyl ethylenediamine powder is used as an organic adsorbent, so that the interference of organic matters is reduced, and the loss of target detection substances is avoided; the addition amount of the N-propyl ethylenediamine is 20-80 percent of the mass of the high organic solid sample, and g/g.

Technical Field

The invention relates to a detection method for measuring steroid hormones in a high organic matter solid sample, belonging to the field of detection and analysis of novel trace pollutants in a complex environment sample.

Background

Steroid hormones are also called steroid hormones, are the most potent endocrine disruptors, can also cause significant endocrine disrupting effects on aquatic organisms at environmentally relevant concentration levels, and have higher ecological risks. The method has the advantages that steroid hormones in different media of the environment are monitored and accurately reported, and the method has important significance for preventing and controlling ecological risks of the steroid hormones. Steroid hormones often exist in solid media such as livestock and poultry manure, soil around a farm, sludge of a sewage treatment plant, water body sediments and the like, the solid samples usually contain a large amount of organic matters such as humic acid, protein, lignocellulose and the like, and the accurate quantification of the steroid hormones is challenged by adsorbing and complexing the steroid hormones, interfering the response of target objects and the like. In the past, the traditional solid phase extraction method is commonly used as a cleaning step after extraction and concentration to remove the influence of organic matters, but the sample recovery rate with extremely high organic matters such as excrement, sludge and the like is still not ideal, the traditional solid phase extraction method has limited removal of the organic matters, the loss of target detection substances is increased, the operation steps are complicated, and more time and labor are consumed. Through retrieval, a steroid hormone detection method aiming at a high organic matter solid sample does not exist at present, so that a detection method which is simple and convenient to operate and good in recovery rate is urgently needed to be developed.

Disclosure of Invention

In order to solve the problems, the invention provides a method for detecting 22 steroid hormones in a high organic solid sample, which reduces organic interference in an extract and simultaneously retains a target detection object as much as possible by screening a proper organic adsorbent. The method is simple to operate, less in interference and wide in application range, and can effectively improve the recovery rate and detection accuracy of steroid hormones in the solid sample in the complex environment.

The first purpose of the invention is to provide a pretreatment method for detecting the micro steroid hormone in the high organic matter solid sample, wherein the pretreatment method comprises the steps of extracting the micro steroid hormone in the high organic matter solid sample by adopting a solid phase extraction method to obtain an extraction liquid, and then cleaning the extraction liquid by adopting N-propyl ethylenediamine as an adsorbent; the extraction is divided into three times of extraction, and methanol is adopted for the first time of extraction; extracting with acetonitrile for the second and third times; the addition amount of the N-propylethylenediamine is 200-800mg/g of the high organic solid sample, namely the addition amount of the N-propylethylenediamine is 20-80% of the mass of the high organic solid sample, and g/g.

In one embodiment of the present invention, the high organic matter solid sample is freeze-dried, ground and sieved.

In one embodiment of the present invention, the three extractions are performed under ultrasonic conditions, and each extraction time is 10-20 min.

In one embodiment of the invention, the high organic solids sample comprises sewage plant activated sludge and excess sludge, livestock manure, high organic soil or sediments.

In one embodiment of the invention, the steroid hormone comprises one or more of estrone, estradiol, estriol, ethinyl estradiol, epiandrosterone, androsterone, androstenedione, testosterone, methyltestosterone, nandrolone, progesterone, levonorgestrel, medroxyprogesterone acetate, megestrol acetate, hydroxyprogesterone, cortisone, hydrocortisone, corticosterone, prednisone, prednisolone, methylprednisolone, and dexamethasone.

The second purpose of the invention is to provide an application of the method in detecting steroid hormones in a high organic matter solid sample.

The third purpose of the invention is to provide a method for detecting the content of the micro steroid hormones in the high organic matter solid sample, wherein the method comprises the steps of firstly treating the solid sample by adopting the pretreatment method and then analyzing by adopting a high performance liquid chromatography-tandem mass spectrometry.

In one embodiment of the present invention, ammonia and acetonitrile are used as mobile phases when analyzing estrogen; when analyzing androgens, progestins and glucocorticoids, methanol solution containing 0.1% formic acid and acetonitrile are used as mobile phases.

In one embodiment of the invention, the method comprises the steps of:

(1) pretreatment: the solid sample is firstly placed in a culture dish and frozen overnight in an ultra-low temperature refrigerator at minus 80 ℃, and then taken out and immediately put in a freeze dryer to be freeze-dried at minus 45 ℃, and then ground in a mortar and sieved by a 100-mesh sieve.

(2) And (3) extraction: accurately weighing 0.5g of the solid sample obtained in the step (1), putting the solid sample into a 30mL polytetrafluoroethylene centrifugal tube, adding 5mL of methanol, screwing a screw cap, oscillating for 30s by using a vortex oscillator, then carrying out ultrasonic extraction for 15min, centrifuging for 15min at 4000rpm, carrying out solid-liquid separation, and sucking out supernatant liquid in the polytetrafluoroethylene centrifugal tube by using a glass rubber head dropper. Repeating the steps for 2 times, but replacing the extraction solvent with 5mL of acetonitrile, collecting the extract liquid after three times of centrifugation, centrifuging for 30min again, taking the supernatant liquid to be contained in a glass nitrogen blowing tube, blowing the supernatant liquid on a nitrogen blowing instrument by using stable nitrogen flow, and accurately adding 1.0mL of methanol for redissolution.

(3) Cleaning: and (3) weighing 400mg of PSA powder adsorbent, adding into the centrifugal tube containing the concentrated extract liquid in the step (2), carrying out vortex oscillation for 30s, then centrifuging at 4000rpm for 20min, finally passing the supernatant through a 0.22 mu m PTFE membrane by using a needle filter, injecting into a 250 mu L inner insert tube, and storing in a brown chromatographic bottle for testing.

(4) High performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis: the liquid chromatographic conditions for estrogen analysis were: the mobile phase A is 0.05% ammonia water, the mobile phase B is acetonitrile, the flow rate is 0.4mL/min, the sample injection amount is 5 mu L, and the column temperature is 40 ℃. The elution conditions were: 0-0.25 min mobile phase is 70% phase A + 30% phase B; gradually reducing phase A to 10% and gradually increasing phase B to 90% in 0.25-2 min; immediately after maintaining for 1min, the flow was restored to the initial flowPhases 70% A + 30% B, and maintaining for another 1 min. The mass spectrometry conditions were: the mass spectrum mode is an electrospray negative ion source (ESI)^-) (ii) a The mass spectrum scanning mode is multi-reactive ion monitoring (MRM); the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision gas flow was 0.16 mL/min. The chromatographic conditions for the analysis of androgens, progestins and glucocorticoids are: the mobile phase A was a methanol solution containing 0.1% formic acid (water: methanol 98:2, v/v), the mobile phase B was acetonitrile, the flow rate was 0.4mL/min, the amount of sample was 5. mu.L, and the column temperature was 40 ℃. The elution conditions were: the mobile phase is 90% phase A + 10% phase B in 0-0.25 min; gradually increasing phase B to 70% in 0.25-1 min; 1-3 min, increasing B to 95% successively, and maintaining for 1 min; then immediately return to the initial mobile phase 90% a phase + 10% B phase for 1 min. The mass spectrometry conditions were: the mass spectrum mode for detecting androgen and glucocorticoid is electrospray ionization (ESI)⁺) (ii) a The mass spectrum mode is electrospray negative ion source (ESI) in the detection of glucocorticoid^-) The mass spectrum scanning mode is multi-reactive ion monitoring (MRM); the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision gas flow was 0.16 mL/min.

In one embodiment of the present invention, the steroid hormone comprises estrone, estradiol, estriol, ethinyl estradiol, epiandrosterone, androsterone, androstenedione, testosterone, methyltestosterone, nandrolone, progesterone, levonorgestrel, medroxyprogesterone acetate, megestrol acetate, hydroxyprogesterone, cortisone, hydrocortisone, corticosterone, prednisone, prednisolone, methylprednisolone, dexamethasone.

The fourth purpose of the invention is to provide a method for cleaning the extract liquor after extracting steroid hormones from a high organic solid sample, wherein N-propyl ethylenediamine Powder (PSA) is used as an organic adsorbent, so that the interference of organic matters is reduced, and the loss of target detection substances is avoided.

The invention has the beneficial effects that:

1. the method can realize the determination of 22 steroid hormones, the recovery rate is 63.50-154.10%, and the average recovery rate can reach 94.65%.

2. The method utilizes PSA as an organic adsorbent to clean the extraction concentrated solution of the solid sample, only needs operations such as vortex, centrifugation and the like, and has the advantages of simple and convenient steps, low time and labor consumption, low requirements on instruments and equipment and low maintenance cost compared with the traditional solid phase extraction as a cleaning method.

3. The invention utilizes PSA as a cleaning agent, effectively controls the interference of organic matters relative to other adsorbents such as graphite carbon powder, silica gel and the like, reduces the loss of target detection objects and achieves the aim of detecting trace steroid hormones in complex samples.

Drawings

Figure 1 is a flow diagram of a method for detecting trace amounts of steroid hormones from a high organic matter solids sample.

FIG. 2 is a graph comparing the recovery of estrogen with different PSA dosages, wherein E1 is Estrone (Estrone), E2 is Estradiol (17 β -Estradiol), and EE2 is ethinyl Estradiol (Ethinylestradiol).

FIG. 3 is a graph showing the comparison of the recovery rate of androgen with different PSA dosages, wherein NDL is nandrolone, ADD is Androstenedione, TTR is Testosterone, EADR is Epiandrosterone, ADR is Androsterone, and MTTR is Methyltestosterone (17 alpha-methylistosterone).

FIG. 4 is a graph showing the comparison of progestin recovery for different dosages of PSA, where LNG is Levonorgestrel (Levonorgestrel), PGT is Progesterone (Progesterone), HPT is Hydroxyprogesterone (Hydroxyprogesterone), MTA is megestrol acetate (Megestrolacetate), and MPA is Medroxyprogesterone acetate (Medyxyprogesterone acetate).

FIG. 5 is a graph of the recovery of glucocorticoids at different PSA dosing doses, where CORT is Corticosterone (Cortisterone), PREL is Prednisolone (Prednisonone), CRN is Cortisone (Cortisone), CRL is hydrocortisone (Cortisol), MPREL is methylprednisone, and DEX is Dexamethasone (Dexamethasone).

Figure 6 is a graph of recovery of steroid hormones from various examples and comparative examples.

Detailed Description

The following describes embodiments of the present invention with reference to specific examples.

The invention provides a detection method for measuring multiple micro steroid hormones in a high organic matter solid sample, which can be used for extracting and quantitatively analyzing 22 steroid hormones such as estrogen, androgen, progestogen, glucocorticoid and the like in residual sludge, activated sludge, human and animal manure of a sewage plant and high organic matter soil or sediment, and adopts the following instruments and reagents:

the instrument comprises the following steps: ultrasonic instrument (Ningbo Xinzhi Biotechnology GmbH, SB-500 DTY); a freeze dryer (laboco); centrifuge (Eppendorf, Centrifuge 5804R); vortex oscillators (Shanghai West Analyzer Co., Ltd., WH-1 micro vortex mixer); a dry nitrogen blower (Shanghai Ougo electronics Co., Ltd., AG-DCY-12G); high performance liquid chromatography tandem mass spectrometry (Waters, ACQUITY UPLC Xevo TQ), Waters ACQUITY UPLC R BEH C18(100 mm. times.2.1 mm. times.1.7 μm).

Reagent: the extraction solvent comprises methanol and acetonitrile, which are both German Merck chromatographic pure and ultrapure water.

Adsorbent: n-propylethylenediamine powder (primarysone, PSA for short); graphite carbon powder (Supelco ENVI Carb, Carb for short); silica gel powder (Silica for short).

Example 1: establishment of a Standard Curve

(1) Accurately preparing each steroid hormone standard substance into a single-standard stock solution with the concentration of 1g/L by using methanol, then taking 10 mu L of single standard to a 10mL volumetric flask for constant volume, preparing a mixed standard of 1mg/L for LC-MS/MS analysis, and determining respective peak-out time and qualitative and quantitative ion pairs;

the preparation method of the LC-MS/MS mobile phase comprises the following steps of determining that the mobile phase A of estrogen is 0.05% ammonia water obtained by diluting 20% concentrated ammonia water by 400 times, determining that the mobile phase A of other hormones is obtained by mixing water and methanol according to the volume ratio of 98:2, and adding formic acid until the concentration of formic acid is 0.1%;

the chromatographic conditions were a Waters ACQUITY UPLC R BEH C18 column (100 mm. times.2.1 mm. times.1.7 μm), an ion source temperature and a desolvation temperature of 150 ℃ and 350 ℃ respectively, and elution was carried out using the above-prepared mobile phase A and acetonitrile as the mobile phase B at a flow rate of 0.4mL/min and a sample introduction amount of 5 μ L.

(2) Taking 100 mu L of 1g/L steroid hormone single standard to a 10mL volumetric flask, using methanol to fix the volume to prepare a mixed standard of 10mg/L, diluting the mixed standard into mixed standard solutions with the concentrations of 1, 5, 10, 50, 100 and 200 mu g/L respectively, and finally taking the sample injection concentration as a horizontal coordinate X (mu g/L) and the peak area as a vertical coordinate Y to obtain a standard curve and a detection limit of the corresponding steroid hormone. The linear range of the obtained hormone standard curve is 1-200 mug/L, and the detailed results are as follows:

TABLE 1 Standard curves and limits of detection and quantitation of steroid hormones

Example 2: detection method for detecting steroid hormones in excess sludge of sewage plant

As shown in figure 1, the solid sample is firstly placed in a culture dish and frozen in an ultra-low temperature refrigerator at-80 ℃ overnight, then is taken out and immediately placed in a freeze dryer for freeze drying at-45 ℃, and is ground in a mortar and sieved by a 100-mesh sieve for extraction. Samples which could not be extracted immediately were wrapped with aluminum foil, sealed in sealed bags and stored in a refrigerator at-20 ℃.

Accurately weighing 0.5g of freeze-dried and ground sample, putting the sample into a 30mL polytetrafluoroethylene centrifugal tube, adding 5mL of methanol, screwing a screw cap, oscillating the sample for 30s by using an eddy oscillator, then carrying out ultrasonic extraction for 15min, centrifuging the sample for 15min at 4000rpm, carrying out solid-liquid separation, and sucking out supernatant liquid in the polytetrafluoroethylene centrifugal tube by using a glass rubber-tipped dropper. Repeating the steps for 2 times, but replacing the extraction solvent with 5mL of acetonitrile, collecting the extract liquid after three times of centrifugation, centrifuging for 30min again, taking the supernatant liquid to be contained in a glass nitrogen blowing tube, blowing the supernatant liquid on a nitrogen blowing instrument by using stable nitrogen flow, and accurately adding 1.0mL of methanol for redissolution. Weighing 400mg of PSA powder adsorbent, adding into a centrifuge tube, performing vortex oscillation for 30s, centrifuging at 4000rpm for 20min, finally passing the supernatant through a 0.22 μm PTFE membrane by using a needle filter, injecting into a 250 μ L inner insert tube, and storing in a brown chromatographic bottle for testing.

Analyzing the extracted sample by using a high performance liquid chromatography tandem mass spectrum (LC-MS/MS), wherein the conditions of the liquid chromatography when the estrogen is analyzed are as follows: the mobile phase A is 0.05% ammonia water, the mobile phase B is acetonitrile, the flow rate is 0.4mL/min, the sample injection amount is 5 mu L, and the column temperature is 40 ℃. The elution conditions were: 0-0.25 min mobile phase is 70% phase A + 30% phase B; gradually reducing phase A to 10% and gradually increasing phase B to 90% in 0.25-2 min; after maintaining for 1min, the solution is immediately recovered to the initial mobile phase of 70% A phase + 30% B phase, and then maintained for 1 min. The mass spectrometry conditions were: the mass spectrum mode is an electrospray negative ion source (ESI)^-) (ii) a The mass spectrum scanning mode is multi-reactive ion monitoring (MRM); the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision gas flow was 0.16 mL/min. The chromatographic conditions for the analysis of androgens, progestins and glucocorticoids are: the mobile phase A was a methanol solution containing 0.1% formic acid (water: methanol 98:2, v/v), the mobile phase B was acetonitrile, the flow rate was 0.4mL/min, the amount of sample was 5. mu.L, and the column temperature was 40 ℃. The elution conditions were: the mobile phase is 90% phase A + 10% phase B in 0-0.25 min; gradually increasing phase B to 70% in 0.25-1 min; 1-3 min, increasing B to 95% successively, and maintaining for 1 min; then immediately return to the initial mobile phase 90% a phase + 10% B phase for 1 min. The mass spectrometry conditions were: the mass spectrum mode for detecting androgen and glucocorticoid is electrospray ionization (ESI)⁺) (ii) a The mass spectrum mode is electrospray negative ion source (ESI) in the detection of glucocorticoid^-) The mass spectrum scanning mode is multi-reactive ion monitoring (MRM); the capillary voltage is 3.5 kV; the taper hole voltage and the extracted taper hole voltage are respectively 30.0V and 3.0V; the ion source temperature and the desolventizing temperature are respectively 150 ℃ and 500 ℃; the taper hole airflow and the desolventizing airflow are respectively 50L/hr and 900L/hr; the collision gas flow was 0.16 mL/min.

The method is subjected to standard addition recovery rate detection, 200ng of steroid hormone mixed standard is added into 0.5g of residual sludge of a sewage plant, namely the standard addition amount is 400 mug/kg, meanwhile, blank samples without standard addition are prepared, 2-3 parallel samples are prepared for each experimental group, extraction and detection are carried out after standing for 2 hours, and the recovery rate is calculated according to the following formula:

wherein, R is recovery rate,%; m is the detected amount of the labeled sample, ng; m₀Ng is the detected amount of the blank sample.

As shown in figure 2, the recovery rate of each steroid hormone in example 1 is 63.5-154.1% (average 94.7%), the method is used for measuring the steroid hormone in the residual sludge of a certain municipal sewage treatment plant, and the detected estrogens E1, E2, E3 and EE2 are respectively 0.50-2.60 mug/kg, 0.90 mug/kg, 4.40 mug/kg and 2.30 mug/kg, the detected androgens EADR, ADR and ADD are respectively 14.70-381.12 mug/kg, 32.8 mug/kg and 0.40-4.70 mug/kg, and the detected progestational hormone PGT is 0.50-6.60 mug/kg.

Example 3: detection method for detecting steroid hormones in activated sludge of sewage plant

The detection steps are the same as those in example 2, the detection sample is changed into activated sludge in an oxidation ditch, an anaerobic pool, an anoxic pool and an aerobic pool of a certain municipal sewage plant, and the detection results are shown in table 2:

TABLE 2 detection of steroid hormones in activated sludge of different process stages of sewage plants

"-" is not detected.

Example 4: optimization of adsorbent PSA addition

When PSA is used as an organic adsorbent in the cleaning step and the adding amount of the extraction concentrate of 1mL of the sample is 0, 50, 100, 200, 300 and 400mg respectively, the recovery rate of each steroid hormone is compared, and the results are shown in figure 2, figure 3, figure 4 and figure 5, wherein the recovery rate is increased along with the increase of the adding amount, the recovery rate of 400mg in the test adding amount range is optimal, and if the adding amount is continuously increased, the sample amount collected after centrifugal separation is not enough for LC-MS/MS measurement, so 400mg is selected as the final adding amount.

Comparative example 1: silica gel powder as adsorbent for cleaning step

And in the cleaning step, namely blow-drying by using stable nitrogen flow on a nitrogen blowing instrument, accurately adding 1.0mL of methanol for redissolution, weighing 0, 50, 100, 200, 300 or 400mg of silica gel powder, adding the silica gel powder into a centrifuge tube, carrying out vortex oscillation for 30s, centrifuging at 4000rpm for 20min, finally passing the supernatant through a 0.22 mu m PTFE membrane by using a needle filter, injecting into a 250 mu L inner insert tube, and storing in a brown chromatographic bottle for detection. The remaining steps and parameters were in accordance with example 2. The results show that the adsorption removal effect of silica gel on organic matters is not obvious, the sample is still yellowish brown after being cleaned, the risk of damaging the liquid chromatographic column is still present, the recovery rate does not trend to rise remarkably with the increase of the adding amount, the recovery rate of each hormone under the condition of the highest adding amount of 400mg is only 12.65% -98.25% (average 62.47%), and the detection effect is not as good as that of taking PSA as a cleaning agent (example 2).

Comparative example 2: graphite carbon powder as adsorbent for cleaning step

In the cleaning step, namely blow-drying by using stable nitrogen flow on a nitrogen blowing instrument, accurately adding 1.0mL of methanol for redissolution, weighing 150mg of graphite carbon powder, adding the graphite carbon powder into a centrifuge tube, carrying out vortex oscillation for 30s, then centrifuging at 4000rpm for 20min, finally passing the supernatant through a 0.22 mu m PTFE membrane by using a needle filter, injecting the supernatant into a 250 mu L inner insert tube, and storing the supernatant in a brown chromatographic bottle for testing. The remaining steps and parameters were in accordance with example 2. As a result, the graphite carbon powder has a very good effect of removing organic matters, the extract liquid before cleaning is yellow brown or even dark brown, and the extract liquid after cleaning is transparent faint yellow, but simultaneously, the graphite carbon has a very obvious effect of removing a target object, and the recovery rate of each hormone is only 0.63% -77.55% (average 26.42%), and the detection purpose cannot be achieved.

Comparative example 3: without additional cleaning steps

Collecting the extract liquid after three times of centrifugation without adding a cleaning step, centrifuging for 30min, taking the supernatant liquid, placing the supernatant liquid in a glass nitrogen blowing tube, blowing the supernatant liquid on a nitrogen blowing instrument by using stable nitrogen flow, accurately adding 1.0mL of methanol for redissolution, and finally directly passing a sample through a 0.22 mu m PTFE membrane by using a needle filter and storing the sample in a brown chromatographic bottle to be detected. The sample without the cleaning step is yellow brown or even dark brown, so that the risk of damaging instruments is caused, the recovery rate of each hormone is 27.98-78.83% (average 43.11%), the detection effect is not good as that of the example 2, and flocculent precipitates appear after the sample is frozen and stored, so that the requirement on a computer cannot be met.

Comparative example 4: adopts the traditional solid phase extraction method as the cleaning step

The method comprises the following steps of taking a traditional SPE as a cleaning step, namely blow-drying on a nitrogen blowing instrument, adding 1.0mL of methanol for redissolution, then fixing the volume to 500mL by using ultrapure water, installing a Waters oasis HLB solid-phase extraction column (200mg, 6cc) on a solid-phase extraction device, connecting a vacuum pump, a suck-back prevention device and a sample inlet pipe, sequentially activating the solid-phase extraction column by 5mL of methanol and 5mL of ultrapure water, then completely passing 500mL of samples through the solid-phase extraction column under vacuum, controlling the flow rate at 10mL/min, and then introducing 10mL of 10% methanol aqueous solution for cleaning. And continuously performing vacuum pumping for more than 30min to blow the solid phase extraction column, eluting the extracted hormone to a glass nitrogen blow pipe by 5mL/min sequentially through 7mL of methanol and 7mL of methanol/acetone (1:1, v/v), then concentrating to 1mL under a stable nitrogen flow, passing through a 0.22 mu m PTFE membrane, and storing in a brown chromatographic bottle for detection. The color of the sample obtained by the method is slightly lighter than that of the sample without the washing step, but is not very obvious, so that the removal of organic matters is limited, and the recovery rate result shows that the method also causes certain loss on target hormone, the recovery rate is 17.80-118.60%, the average recovery rate is 53.96%, is higher than that without the washing step, but is obviously lower than that of the example 2.

In conclusion, the invention uses methanol and acetonitrile to extract 22 steroid hormones in a high organic matter solid sample, uses PSA as an adsorbent to clean and purify concentrated extract liquor, then uses LC-MS/MS to carry out quantification, and after dosage optimization, the recovery rate of the steroid hormones in 22 reaches more than 63.5 percent, the average recovery rate is 94.7 percent, the detection limit is low, the operation is simple and convenient, and the requirement of quantitative detection of trace organic pollutants is met.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.