CN107884490B - Method for simultaneously detecting multiple progestogens in environmental water body - Google Patents

Abstract

The invention discloses a method for simultaneously detecting multiple progestogens in environmental water, belonging to the field of organic matter analysis in the environment, including water sample pretreatment, novel solid-phase extraction adsorbent Oasis PRiME HLB enrichment and concentration, UPLC-MS/MS determination The five steps of progesterone concentration, standard curve drawing and on-board determination, using the new solid phase extraction sorbent Oasis PRiME HLB as the pretreatment technology of environmental water samples, not only improves the selectivity of the analyte, but also greatly increases the richness. The multiplexing and matrix effects are reduced, and 11 different progesterones in the water environment are simultaneously measured by using the ultra-high liquid phase-mass spectrometer as the detection and quantitative tool. It is an effective combination of selectivity and high sensitivity, and has the advantages of fast analysis speed and wide application range.

Description

A method for simultaneous detection of multiple progestogens in environmental water

technical field

The invention belongs to the technical field of organic matter analysis in environmental water bodies, and particularly relates to a method for simultaneously detecting multiple progestogens in environmental water bodies.

Background technique

In the past half century, progesterone has caused serious harm to the natural environment on which humans and wild animals depend. As a common endocrine disruptor, progesterone can accumulate in the environment and human body for a long time, and its metabolism is slow, and it can be transferred to various environmental media such as atmosphere, soil, and water sediments through diffusion and convection. Once it enters the body, it can not only disrupt the body's normal synthesis and metabolism of endocrine hormones, affect growth and gender differentiation, but also cause neurological, endocrine, immune and other multi-system developmental abnormalities and reproductive disorders, and even cause teratogenicity and carcinogenesis. Therefore, it is very important to establish a fast, efficient, accurate and sensitive method for the analysis and detection of progesterone in environmental water.

Due to the diverse morphology, complex composition and low progesterone content of environmental samples, it is difficult to directly analyze and detect them. In order to reduce the interference of the sample substrate and improve the analytical detection sensitivity, appropriate sample pretreatment must be carried out before the sample analysis can be carried out for various instrumental analysis. The traditional methods of environmental hormone pretreatment technology in environmental samples include ultrasonic extraction, Soxhlet extraction, microwave extraction and so on. However, the ultrasonic extraction solvent consumption is large, the Soxhlet extraction time is long, and reflux operation is required; after microwave extraction, filtration is required, which is difficult to connect with the instrument. At present, with the development of science and technology, many new sample pretreatment technologies have emerged, such as SPE and SPME. As a new type of sample pretreatment technology, SPME technology continues to expand with the continuous expansion of its application field, but some limitations of its extraction fiber itself lead to the emergence of new problems, and the recovery rate and repeatability cannot be guaranteed. SPE can be used as a new technology for environmental hormone pretreatment, with high recovery rate and stable reproducibility. higher requirement. Oasis PRiME HLB is a novel reversed-phase solid-phase extraction sorbent that simplifies and accelerates SPE processes, providing cleaner samples in less time, making LC and LC/MS systems more efficient easy. Oasis PRiME HLB columns can also be combined with multiwell plates to simplify and speed up extraction, ensuring consistent and reproducible LC and LC/MS results. Analytical laboratories can now process samples faster than other extraction techniques, up to 40% faster, with up to 70% improved sample cleanliness and less matrix effects by LC/MS. At the same time, cleaner extracts can be obtained than other sample preparation methods. Oasis PRiME HLB is a very successful next-generation reversed-phase SPE product, which can obtain cleaner extracts through simpler protocols and shorter processing times, and can effectively enrich progesterone in concentrated environmental water. As a pretreatment method for the treatment of progesterone in environmental water.

SUMMARY OF THE INVENTION

In view of the above technical problems, the present invention provides a method for simultaneously detecting multiple progestogens in environmental water, which can effectively remove impurities in environmental water and greatly reduce the matrix effect.

The technical scheme of the present invention is as follows: a method for simultaneously detecting a variety of progesterone in environmental water, the sample to be tested is pretreated by a novel solid phase extraction adsorbent Oasis PRiME HLB, and then detected by UPLC-MS/MS, including the following step:

(1) Water sample pretreatment

The collected water samples are filtered with three different pore size membranes for tertiary filtration, accurately measure 0.2-2L of the filtered water samples, and adjust the pH to 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide. 7.0-7.2;

(2) New solid phase extraction adsorbent Oasis PRiME HLB enrichment and concentration

The water sample processed in step (1) was directly enriched through the Oasis PRiME HLB solid phase extraction cartridge, and the flow rate was controlled at 7-9 mL/min. After the enrichment was completed, the solid phase was washed twice with ultrapure water. Phase extraction cartridge, and then dry the rinsed solid phase extraction cartridge in a vacuum drying oven for 1-3 hours at a drying temperature of 25-35 °C, and wash the dried solid phase extraction cartridge three times with ethyl acetate. to obtain an eluent containing the target compound, move the eluent to a nitrogen blow dryer, and purge with nitrogen with a flow rate of 1.5-2 m/s for 30-60 min to obtain a residue for later use;

(3) Determination of progesterone concentration by UPLC-MS/MS

Preparation of standard solution: Accurately weigh 1 mg of the tested progesterone standard, respectively, and dilute to a 100-mL volumetric flask with methanol. After mixing, a 10 mg/L mixed standard stock solution is obtained, which is stored in a -20°C refrigerator;

Preparation of working solution: Precisely measure 500 μL of methanol solution to dissolve the residue in step (2), after vortexing, dilute to 1 mL with a glass dropper and filter with a 0.22 μm filter membrane, and then dilute with methanol solution to obtain different concentrations of working solution;

Chromatographic conditions: column ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; mobile phase A is 0.1% formic acid, mobile phase B is methanol, gradient elution procedure is used; flow rate is 0.3 mL/min; column temperature is 40 °C , the injection volume is 10 μL; the gradient elution procedure is as follows:

time (min) A(%) B(%) 0 10 90 0.5 60 40 3 65 35 6 70 30 6.5 100 0 8 10 90

Mass spectrometry conditions: use multiple reaction detection mode, ion source is electrospray ionization source (ESI source), source temperature is 120 ° C, cone voltage: 30 V, extraction ion voltage is 2.5 kV, capillary voltage is 3 kV, desolvation gas temperature is 450℃, the flow rate of desolvation gas is 900L/hr; the scanning time is 0.1s;

(4) Drawing of standard curve

Taking the mixed standard stock solution concentration (mg/L) described in the step (3) as the abscissa, the response value is the standard curve of the progesterone drawn on the ordinate;

(5) On-board measurement

Detect the working solution described in step (3) using UPLC-MS/MS instrument to obtain a typical chromatogram of the actual sample, and compare the typical chromatogram of the actual sample with the standard curve for qualitative detection; then use The tuning function of the UPLC-MS/MS instrument determines the precursor ion, product ion, collision voltage and capillary voltage of each substance, and uses the above parameters to quantitatively detect progesterone in the working solution.

Further, the progestogens are respectively mifepristone, clodesterone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dehydroprogesterone, levonorgestrel, norethisterone , progesterone-d9, norethisterone-d6.

Further, the environmental water bodies are the influent water, the effluent water and the river water of the sewage treatment plant, and the two water sources have a high human contact rate and are more representative.

Further, in the step (1), the collection amount is 200-500mL when the water sample is the sewage treatment plant influent water sample, and the collection amount is 300-500mL when the water sample is the sewage treatment plant effluent water sample, When the water sample is river water, the collection volume is 1-2L.

Further, the Oasis PRiME HLB solid phase extraction small column described in the step (2) contains composite adsorption fillers, and the composite adsorption fillers are poly-N-vinylpyrrolidone-divinylbenzene with a mass ratio of 2:1:1. Porous resins, triolein fiber spheres, core-shell glucose polymer-silica spheres, the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin increases the The retention of polar substances provides good water wettability; the triolein fiber spheres have strong lipophilicity, and have strong adsorption force and stable performance for progesterone, a lipid compound; the core-shell glucose The polymer-silica spheres are highly hydrophilic and have good enrichment and separation effects.

Further, the Oasis PRiME HLB solid phase extraction cartridge does not need to be activated and equilibrated, which greatly saves operation time and solvent.

Further, the pore sizes of the filter membranes in step (1) are respectively 10 μm, 1 μm and 0.45 μm, and gradient filtration can minimize the influence of impurities on the analysis results.

Further, the consumption of the ultrapure water and ethyl acetate in step (2) is 1-2 times the volume of the solid phase extraction cartridge.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention adopts the novel solid phase extraction adsorbent Oasis PRiME HLB as the pretreatment technology of environmental water samples, which can effectively remove impurities in environmental water and greatly reduce the matrix effect. Combined with UPLC-MS/MS as a detector, it can quickly and accurately detect the content of progesterone in environmental water, and the detection results are accurate and reliable.

(2) The present invention adopts the novel solid-phase extraction adsorbent Oasis PRiME HLB, which not only improves the selectivity of the analyte, but also greatly increases the enrichment multiple and reduces the matrix effect, which provides a new idea for the development and application of pretreatment technology, It also adds new methods for the extraction, concentration and separation of trace drug residues in the environment.

(3) The present invention uses an ultra-high liquid phase-mass spectrometer as a detection and quantitative tool, and simultaneously measures 11 different progesterones in the water environment. Compared with ordinary detection methods, high selectivity and high progesterone are realized It is an effective combination of sensitivity, and has the advantages of fast analysis speed and wide application range.

Description of drawings

Fig. 1 is the influence of the solid phase extraction cartridge type of embodiment 2 on 9 kinds of progesterone recovery rates;

Fig. 2 is the influence of different pH of embodiment 2 on the recovery rate of 9 kinds of progestogens;

Fig. 3 is the influence of different eluents of embodiment 2 on 9 kinds of progesterone;

Fig. 4 is the influence of different eluents of embodiment 2 on the recovery rate of 9 kinds of progesterone;

Figures 5.1-5.3 are ion chromatograms extracted from 11 kinds of progestogens in Example 2.

Detailed ways

Example 1

A method for simultaneously detecting multiple progestins in an environmental water body, wherein the progestins are respectively mifepristone, clodesterone, medroxyprogesterone, drospirenone, progesterone, norethindrone, and Hydroprogesterone, levonorgestrel, norethisterone, progesterone-d9, norethisterone-d6. The samples to be tested were pretreated with a new solid-phase extraction adsorbent, Oasis PRiME HLB, and then detected by UPLC-MS/MS, including the following steps:

(1) Water sample pretreatment

The influent water, effluent water and river water of the sewage treatment plant were selected as water samples. The two water sources had high human contact rates and were more representative. The collected water samples were filtered with three different pore sizes of 10 μm, 1 μm and 0.45 μm for three-stage filtration, and the filtered water samples were accurately measured. The sample is 300mL, the river water collection volume is 1L, and the pH is adjusted to 7.0 with the concentration of 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide;

(2) New solid phase extraction adsorbent Oasis PRiME HLB enrichment and concentration

The Oasis PRiME HLB solid phase extraction cartridge does not need to be activated and equilibrated, which greatly saves operation time and solvent. The water sample treated in step (1) is directly enriched through the Oasis PRiME HLB solid phase extraction cartridge, and the flow rate is controlled at 7mL /min, after the enrichment is completed, the solid phase extraction cartridge is rinsed twice with ultrapure water, and then the rinsed solid phase extraction cartridge is dried in a vacuum drying oven for 1 hour at a drying temperature of 25°C. The dried solid-phase extraction cartridge was eluted three times with ethyl acetate, wherein the amounts of the ultrapure water and ethyl acetate were 1 times the volume of the solid-phase extraction cartridge to obtain the target compound containing the target compound. Eluent, move the eluent to a nitrogen blower, and purge it with nitrogen with a flow rate of 1.5 m/s for 30 min to obtain a residue for later use; wherein, the Oasis PRiME HLB solid phase extraction cartridge contains composite adsorption filler, the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, triolein fiber ball, core-shell glucose polymer-silica ball with a mass ratio of 2:1:1, The poly-N-vinylpyrrolidone-divinylbenzene macroporous resin increases the retention of polar substances through a "special polar capture group" to provide good water wettability; the triolein fiber The sphere has strong lipophilicity, strong adsorption force for progesterone, a lipid compound, and stable performance; the core-shell type glucose polymer-silica sphere has high hydrophilicity and good enrichment and separation effect.

(3) Determination of progesterone concentration by UPLC-MS/MS

Preparation of standard solution: Accurately weigh 1 mg of the tested progesterone standard, respectively, and dilute to a 100-mL volumetric flask with methanol. After mixing, a 10 mg/L mixed standard stock solution is obtained, which is stored in a -20°C refrigerator;

Preparation of working solution: Precisely measure 500 μL of methanol solution to dissolve the residue in step (2), after vortexing, dilute to 1 mL with a glass dropper and filter with a 0.22 μm filter membrane, and then dilute with methanol solution to obtain different concentrations of working solution;

Chromatographic conditions: column ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; mobile phase A is 0.1% formic acid, mobile phase B is methanol, gradient elution procedure is used; flow rate is 0.3 mL/min; column temperature is 40 °C , the injection volume is 10 μL; the gradient elution procedure is as follows:

time (min) A(%) B(%) 0 10 90 0.5 60 40 3 65 35 6 70 30 6.5 100 0 8 10 90

Mass spectrometry conditions: use multiple reaction detection mode, ion source is electrospray ionization source (ESI source), source temperature is 120 ° C, cone voltage: 30 V, extraction ion voltage is 2.5 kV, capillary voltage is 3 kV, desolvation gas temperature is 450℃, the flow rate of desolvation gas is 900L/hr; the scanning time is 0.1s;

(4) Drawing of standard curve

Taking the mixed standard stock solution concentration (mg/L) described in the step (3) as the abscissa, the response value is the standard curve of the progesterone drawn on the ordinate;

(5) On-board measurement

Detect the working solution described in step (3) using UPLC-MS/MS instrument to obtain a typical chromatogram of the actual sample, and compare the typical chromatogram of the actual sample with the standard curve for qualitative detection; then use The tuning function of the UPLC-MS/MS instrument determines the precursor ion, product ion, collision voltage and capillary voltage of each substance, and uses the above parameters to quantitatively detect progesterone in the working solution.

Example 2

A method for simultaneously detecting multiple progestins in an environmental water body, wherein the progestins are respectively mifepristone, clodesterone, medroxyprogesterone, drospirenone, progesterone, norethindrone, and Hydroprogesterone, levonorgestrel, norethisterone, progesterone-d9, norethisterone-d6. The samples to be tested were pretreated with a new solid-phase extraction adsorbent, Oasis PRiME HLB, and then detected by UPLC-MS/MS, including the following steps:

(1) Water sample pretreatment

The influent water, effluent water and river water of the sewage treatment plant were selected as water samples. The two water sources had high human contact rates and were more representative. The collected water samples were filtered with three different pore sizes of 10 μm, 1 μm and 0.45 μm for three-stage filtration, and the filtered water samples were accurately measured. The sample is 400mL, the river water collection volume is 1.5L, and the pH is adjusted to 7.1 with the concentration of 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide;

(2) New solid phase extraction adsorbent Oasis PRiME HLB enrichment and concentration

The Oasis PRiME HLB solid phase extraction cartridge does not need to be activated and equilibrated, which greatly saves operation time and solvent. The water sample treated in step (1) is directly enriched through the Oasis PRiME HLB solid phase extraction cartridge, and the flow rate is controlled at 8mL /min, after the enrichment is over, use ultrapure water to rinse the solid phase extraction cartridge twice, and then dry the rinsed solid phase extraction cartridge in a vacuum drying oven for 2 hours at a drying temperature of 30°C. The dried solid-phase extraction cartridge was eluted three times with ethyl acetate, wherein the amounts of the ultrapure water and ethyl acetate were both 1.5 times the volume of the solid-phase extraction cartridge to obtain the target compound containing the target compound. Eluent, move the eluent to a nitrogen blower, and purge with nitrogen with a flow rate of 1.8 m/s for 45 min to obtain a residue for later use; wherein, the Oasis PRiME HLB solid phase extraction cartridge contains composite adsorption filler, the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, triolein fiber ball, core-shell glucose polymer-silica ball with a mass ratio of 2:1:1, The poly-N-vinylpyrrolidone-divinylbenzene macroporous resin increases the retention of polar substances through a "special polar capture group" to provide good water wettability; the triolein fiber The sphere has strong lipophilicity, strong adsorption force for progesterone, a lipid compound, and stable performance; the core-shell type glucose polymer-silica sphere has high hydrophilicity and good enrichment and separation effect.

(3) Determination of progesterone concentration by UPLC-MS/MS

Preparation of standard solution: Accurately weigh 1 mg of the tested progesterone standard, respectively, and dilute to a 100-mL volumetric flask with methanol. After mixing, a 10 mg/L mixed standard stock solution is obtained, which is stored in a -20°C refrigerator;

Preparation of working solution: Precisely measure 500 μL of methanol solution to dissolve the residue in step (2), after vortexing, dilute to 1 mL with a glass dropper and filter with a 0.22 μm filter membrane, and then dilute with methanol solution to obtain different concentrations of working solution;

Chromatographic conditions: column ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; mobile phase A is 0.1% formic acid, mobile phase B is methanol, gradient elution procedure is used; flow rate is 0.3 mL/min; column temperature is 40 °C , the injection volume is 10 μL; the gradient elution procedure is as follows:

time (min) A(%) B(%) 0 10 90 0.5 60 40 3 65 35 6 70 30 6.5 100 0 8 10 90

Mass spectrometry conditions: use multiple reaction detection mode, ion source is electrospray ionization source (ESI source), source temperature is 120 ° C, cone voltage: 30 V, extraction ion voltage is 2.5 kV, capillary voltage is 3 kV, desolvation gas temperature is 450℃, the flow rate of desolvation gas is 900L/hr; the scanning time is 0.1s;

(4) Drawing of standard curve

Taking the mixed standard stock solution concentration (mg/L) described in the step (3) as the abscissa, the response value is the standard curve of the progesterone drawn on the ordinate;

(5) On-board measurement

Detect the working solution described in step (3) using UPLC-MS/MS instrument to obtain a typical chromatogram of the actual sample, and compare the typical chromatogram of the actual sample with the standard curve for qualitative detection; then use The tuning function of the UPLC-MS/MS instrument determines the precursor ion, product ion, collision voltage and capillary voltage of each substance, and uses the above parameters to quantitatively detect progesterone in the working solution.

Example 3

A method for simultaneously detecting multiple progestins in an environmental water body, wherein the progestins are respectively mifepristone, clodesterone, medroxyprogesterone, drospirenone, progesterone, norethindrone, and Hydroprogesterone, levonorgestrel, norethisterone, progesterone-d9, norethisterone-d6. The samples to be tested were pretreated with a new solid-phase extraction adsorbent, Oasis PRiME HLB, and then detected by UPLC-MS/MS, including the following steps:

(1) Water sample pretreatment

The influent water, effluent water and river water of the sewage treatment plant were selected as water samples. The two water sources had high human contact rates and were more representative. The collected water samples were filtered with three different pore sizes of 10 μm, 1 μm and 0.45 μm for three-stage filtration, and the filtered water samples were accurately measured. The sample is 500mL, the river water collection volume is 2L, and the pH is adjusted to 7.2 with the concentration of 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide;

(2) New solid phase extraction adsorbent Oasis PRiME HLB enrichment and concentration

The Oasis PRiME HLB solid phase extraction cartridge does not need to be activated and equilibrated, which greatly saves operation time and solvent. The water sample treated in step (1) is directly enriched through the Oasis PRiME HLB solid phase extraction cartridge, and the flow rate is controlled at 9mL /min, after the enrichment is over, use ultrapure water to rinse the solid phase extraction cartridge twice, and then dry the rinsed solid phase extraction cartridge in a vacuum drying oven for 3 hours at a drying temperature of 35°C, The dried solid-phase extraction cartridge was eluted three times with ethyl acetate, wherein the amounts of the ultrapure water and ethyl acetate were both twice the volume of the solid-phase extraction cartridge to obtain the target compound-containing Eluent, move the eluent to a nitrogen blower, and purge with nitrogen with a flow rate of 2 m/s for 60 min to obtain a residue for later use; wherein, the Oasis PRiME HLB solid phase extraction cartridge contains composite adsorption Filler, the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin with mass ratio of 2:1:1, triolein fiber ball, core-shell glucose polymer-silica ball, so The poly-N-vinylpyrrolidone-divinylbenzene macroporous resin provides good water wettability through a "special polar capture group" to increase the retention of polar substances; the triolein fiber balls It has strong lipophilicity, strong adsorption force for progesterone, a lipid compound, and stable performance; the core-shell glucose polymer-silica ball has high hydrophilicity and good enrichment and separation effect.

(3) Determination of progesterone concentration by UPLC-MS/MS

Preparation of standard solution: Accurately weigh 1 mg of the tested progesterone standard, respectively, and dilute to a 100-mL volumetric flask with methanol. After mixing, a 10 mg/L mixed standard stock solution is obtained, which is stored in a -20°C refrigerator;

Preparation of working solution: Precisely measure 500 μL of methanol solution to dissolve the residue in step (2), after vortexing, dilute to 1 mL with a glass dropper and filter with a 0.22 μm filter membrane, and then dilute with methanol solution to obtain different concentrations of working solution;

Chromatographic conditions: column ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; mobile phase A is 0.1% formic acid, mobile phase B is methanol, gradient elution procedure is used; flow rate is 0.3 mL/min; column temperature is 40 °C , the injection volume is 10 μL; the gradient elution procedure is as follows:

time (min) A(%) B(%) 0 10 90 0.5 60 40 3 65 35 6 70 30 6.5 100 0 8 10 90

Mass spectrometry conditions: use multiple reaction detection mode, ion source is electrospray ionization source (ESI source), source temperature is 120 ° C, cone voltage: 30 V, extraction ion voltage is 2.5 kV, capillary voltage is 3 kV, desolvation gas temperature is 450℃, the flow rate of desolvation gas is 900L/hr; the scanning time is 0.1s;

(4) Drawing of standard curve

Taking the mixed standard stock solution concentration (mg/L) described in the step (3) as the abscissa, the response value is the standard curve of the progesterone drawn on the ordinate;

(5) On-board measurement

Detect the working solution described in step (3) using UPLC-MS/MS instrument to obtain a typical chromatogram of the actual sample, and compare the typical chromatogram of the actual sample with the standard curve for qualitative detection; then use The tuning function of the UPLC-MS/MS instrument determines the precursor ion, product ion, collision voltage and capillary voltage of each substance, and uses the above parameters to quantitatively detect progesterone in the working solution.

Result analysis

The following is the analysis of the measurement results on the machine of Example 3:

(1) Qualitative detection

Among them, the detection limit of the method is 3 times of the signal-to-noise ratio, the detection limit is 0.1-0.8ng/L, and the sensitivity is high; and the linear correlation coefficients are all above 0.99, and the linear relationship is good. The results of the standard curve range, correlation coefficient and detection limit of each working solution are shown in Table 1:

Table 1 Methodological test results

(2) Quantitative detection

Then use the tuning function of the UPLC-MS/MS instrument to determine the precursor ion, product ion, collision voltage and capillary voltage of each substance, and use the above parameters to quantitatively detect progesterone in the working solution. Among them, 11 kinds of progesterone quantitative analysis mass spectrometry parameters are shown in Table 2:

Table 2 Mass spectrometry conditions for 11 progestins

(3) Calculation of matrix effect

A: The internal standard and standard solution are added to the water sample processed by the above-mentioned steps (1), and the above-mentioned progesterone is extracted according to the step (2) and detected according to the step (3), and the result is defined as C _m ;

B: The same water sample, without adding internal standard and standard solution, extract the above progestogen according to step (2), take 100 μL from the extract, dry it with nitrogen, and then add 100 μL of mixed standard solution of progesterone (50 μg/L ), the obtained result is defined as C _m+100 ;

C: The standard solution of the mixed standard solution of progesterone (50 μg/L) is analyzed according to step (3), and the result is defined as C ₁₀₀ ; then the calculation result of the matrix effect (ME%) is ME%=[(C _m+10 0 -C _m )/C ₁₀₀ ]×100%, the results are shown in Table 3:

Table 3 Progestogen matrix effect (ME%)

(4) Calculation of standard addition recovery rate

D: Take the influent and effluent water samples of the sewage treatment plant and the river water, add standard solutions of each analyte of a certain concentration to the water samples processed according to the above step (1), and after enriching and concentrating according to the above step (2), press Above-mentioned step (3) measures sample, obtains corresponding result and is defined as C _a ;

E: The standard solution of each test substance of a certain concentration, without treatment, is measured according to the above step (3) to obtain the corresponding result C _b ; then the calculation formula of the recovery rate (RE%) is: RE%=C _a / C _b × 100%

The present invention examines the recovery rates of three concentrations, and the results are shown in Table 4. The recovery rates of the substances to be tested are relatively high and meet the analysis requirements.

Table 4 The recovery rate of each test object in the sewage treatment plant inflow and outflow water and river water

(5) Content analysis

Take the influent and effluent water samples of the sewage treatment plant and the river water, add a certain concentration of the internal standard solution of each analyte to the water sample processed according to the above step (1), and after enrichment and concentration according to the above step (2), according to the above Step (3) measures sample, obtains progesterone content (ng/L) such as table 5;

Table 5 Progesterone content in sewage treatment plant and river water (ng/L)

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be The technical solutions described in the foregoing embodiments are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions in the embodiments of the present invention.

Claims (7)

1. a method for simultaneously detecting multiple progestogens in environmental water, is characterized in that, after the pretreatment of novel solid phase extraction adsorbent Oasis PRiME HLB, the sample to be tested is detected with UPLC-MS/MS, comprising the following steps : (1) Water sample pretreatment The collected water samples are filtered with three different pore size membranes for tertiary filtration, accurately measure 0.2-2L of the filtered water samples, and adjust the pH to 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide. 7.0-7.2; (2) New solid phase extraction adsorbent Oasis PRiME HLB enrichment and concentration The water sample processed in step (1) was directly enriched through the Oasis PRiME HLB solid phase extraction cartridge, and the flow rate was controlled at 7-9 mL/min. After the enrichment was completed, the solid phase was washed twice with ultrapure water. Phase extraction cartridge, and then dry the rinsed solid phase extraction cartridge in a vacuum drying oven for 1-3 hours at a drying temperature of 25-35 °C, and wash the dried solid phase extraction cartridge three times with ethyl acetate. to obtain an eluent containing the target compound, move the eluent to a nitrogen blow dryer, and purge it with nitrogen with a flow rate of 1.5-2 m/s for 30-60 min to obtain a residue for later use; wherein, the Oasis The PRiME HLB solid phase extraction cartridge contains composite adsorption fillers, and the composite adsorption fillers are poly-N-vinylpyrrolidone-divinylbenzene macroporous resin with a mass ratio of 2:1:1, triolein fiber balls, Core-shell glucose polymer-silica spheres, the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin provides excellent water infiltration through a "special polar capture group" to increase the retention of polar substances The triolein fiber ball has strong lipophilicity, and has strong adsorption force and stable performance for progesterone, a lipid compound; the core-shell glucose polymer-silica ball has high hydrophilicity, The enrichment and separation effect is good; (3) Determination of progesterone concentration by UPLC-MS/MS Preparation of standard solution: Accurately weigh 1 mg of the tested progesterone standard, respectively, and dilute to a 100-mL volumetric flask with methanol. After mixing, a 10 mg/L mixed standard stock solution is obtained, which is stored in a -20°C refrigerator; Preparation of working solution: Precisely measure 500 μL of methanol solution to dissolve the residue in step (2), after vortexing, dilute to 1 mL with a glass dropper and filter with a 0.22 μm filter membrane, and then dilute with methanol solution to obtain different concentrations of working solution; Chromatographic conditions: column ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; mobile phase A is 0.1% formic acid, mobile phase B is methanol, gradient elution procedure is used; flow rate is 0.3 mL/min; column temperature is 40 °C , the injection volume is 10 μL; the gradient elution procedure is as follows:

Mass spectrometry conditions: use multiple reaction detection mode, ion source is electrospray ionization source (ESI source), source temperature is 120 ° C, cone voltage: 30 V, extraction ion voltage is 2.5 kV, capillary voltage is 3 kV, desolvation gas temperature is 450°C, the desolvation gas flow rate is 900L/hr; the scanning time is 0.1s; (4) Drawing of standard curve Taking the mixed standard stock solution concentration mg/L described in the step (3) as the abscissa, the response value is the standard curve of the progesterone drawn on the ordinate; (5) On-board measurement Detect the working solution described in step (3) using UPLC-MS/MS instrument to obtain a typical chromatogram of the actual sample, and compare the typical chromatogram of the actual sample with the standard curve for qualitative detection; then use The tuning function of the UPLC-MS/MS instrument determines the precursor ion, product ion, collision voltage and capillary voltage of each substance, and uses the above parameters to quantitatively detect progesterone in the working solution. 2. a kind of method for simultaneously detecting multiple progestogens in environmental water body as claimed in claim 1, is characterized in that, described progesterone is respectively mifepristone, clodesterone, medroxyprogesterone, drospirenone ketone, progesterone, norethisterone, dehydroprogesterone, levonorgestrel, norethisterone, progesterone-d9, norethisterone-d6. 3 . The method for simultaneously detecting multiple progestogens in an environmental water body as claimed in claim 1 , wherein the environmental water body is the influent, effluent and river water of a sewage treatment plant. 4 . 4. a kind of method for detecting multiple progesterone in environmental water body simultaneously as claimed in claim 1, is characterized in that, in described step (1), when described water sample is sewage treatment field influent water sample, the amount of collection It is 200-500mL, when the water sample is effluent from the sewage treatment plant, the collection volume is 300-500mL, and when the water sample is river water, the collection volume is 1-2L. 5. The method for simultaneously detecting multiple progestogens in environmental water as claimed in claim 1, wherein the Oasis PRiME HLB solid phase extraction cartridge does not need to be activated and equilibrated. 6 . The method for simultaneously detecting multiple progestogens in environmental water according to claim 1 , wherein the pore sizes of the filter membranes in step (1) are 10 μm, 1 μm and 0.45 μm, respectively. 7 . 7. a kind of method of detecting multiple progestogens in environmental water body simultaneously as claimed in claim 1, is characterized in that, the consumption of ultrapure water and ethyl acetate described in step (2) is described solid phase extraction 1-2 times the volume of the cartridge.

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