CN107884490B - Method for simultaneously detecting multiple progestogens in environmental water body - Google Patents
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Abstract
The invention discloses a method for simultaneously detecting multiple progestogens in an environmental water body, which belongs to the field of organic matter analysis in the environment and comprises five steps of water sample pretreatment, novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration, UPLC-MS/MS progestogen concentration determination, drawing of a standard curve and on-machine determination.
Description
Technical Field
The invention belongs to the technical field of analysis of organic matters in an environmental water body, and particularly relates to a method for simultaneously detecting multiple progestagens in the environmental water body.
Background
In the past half century, progestagens have caused serious harm to the natural environment in which human beings and wild animals live, have become an environmental pollution problem to be solved together in urgent need in the world, and have attracted extensive attention. The progestogen as a common endocrine disrupter can be accumulated in the environment and human body for a long time, is slowly metabolized, and can be transferred to various environmental media such as atmosphere, soil, water area sediments and the like through diffusion and convection. Once entering the body, it can not only disturb the synthesis and metabolism of the normal endocrine hormone of the body, influence the growth and the sexual differentiation, but also cause the abnormal development and the reproductive disorder of multiple systems such as nerves, incretion, immunity and the like, even cause teratogenesis, carcinogenesis and the like. Therefore, it is very important to establish a rapid, efficient, accurate and sensitive method for analyzing and detecting the progestogen in the environmental water body.
Due to the characteristics of various forms, complex components and low progestogen content of the environmental sample, the environmental sample is difficult to directly analyze and detect. In order to reduce interference with the sample substrate and to improve assay detection sensitivity, appropriate sample pretreatment must be performed before sample analysis can be performed for various instrument analyses. The traditional methods of the environmental hormone pretreatment technology in the environmental sample comprise ultrasonic extraction, Soxhlet extraction, microwave extraction and the like. However, the ultrasonic extraction solvent consumption is large, the Soxhlet extraction time is long, and the reflux operation is needed; filtration is required after microwave extraction and is difficult to connect to the instrument. At present, with the development of science and technology, a plurality of novel sample pretreatment technologies, such as SPE and SPME, come into force. As a novel sample pretreatment technology, the SPME technology is continuously expanded in the application field, but some limitations of extraction fibers of the SPME technology cause new problems to continuously appear, and the recovery rate and the repeatability cannot be guaranteed. SPE can be used as a novel technology for environmental hormone pretreatment, the recovery rate is high, the reproducibility is stable, and higher requirements are provided for the SPE technology due to the characteristics of various environmental sample forms, complex components and low content of a substance to be detected. As a novel reversed-phase solid-phase extraction adsorbent, the Oasis PRIME HLB can simplify and accelerate SPE process, can provide cleaner samples in shorter time, and makes the analysis of liquid chromatography and liquid chromatography-mass spectrometry system easier. The Oasis PRiME HLB column can also be combined with a multi-well plate to simplify the extraction process and speed up the extraction, ensuring consistent and reproducible results of liquid phase and LC-MS analyses. Compared to the use of other extraction techniques for sample preparation, analytical laboratories are now able to process samples more rapidly, with up to 40% speed improvements, and up to 70% improvement in sample cleanliness, and with less matrix effects in LC-MS. Meanwhile, compared with other sample pretreatment methods, the method can obtain cleaner extract. The Oasis PRIME HLB is a very successful new-generation reversed-phase SPE product, can obtain a cleaner extract through a simpler scheme and shorter treatment time, can effectively enrich and concentrate the progestogen in the environmental water body, and can be used as a pretreatment method for treating the progestogen in the environmental water body.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for simultaneously detecting multiple progestogens in an environmental water body, which can effectively remove impurities in the environmental water body and greatly reduce matrix effect.
The technical scheme of the invention is as follows: a method for simultaneously detecting multiple progestogens in environmental water body is characterized in that a sample to be detected is pretreated by a novel solid phase extraction adsorbent Oasis PRIME HLB and then is detected by UPLC-MS/MS, and the method comprises the following steps:
(1) water sample pretreatment
Performing three-stage filtration on an acquired water sample by adopting three filter membranes with different apertures, accurately measuring 0.2-2L of the filtered water sample, and adjusting the pH to 7.0-7.2 by using hydrochloric acid with the concentration of 0.1mol/L and sodium hydroxide with the concentration of 0.1 mol/L;
(2) novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration
Directly passing the water sample treated in the step (1) through an Oasis PRIME HLB solid phase extraction column for enrichment, controlling the flow rate to be 7-9mL/min, leaching the solid phase extraction column twice by using ultrapure water after the enrichment is finished, then drying the leached solid phase extraction column for 1-3h in a vacuum drying oven at the drying temperature of 25-35 ℃, eluting the dried solid phase extraction column for three times by using ethyl acetate to obtain an eluent containing a target compound, transferring the eluent into a nitrogen blow-drying instrument, and blowing for 30-60min by using nitrogen with the flow rate of 1.5-2m/s to obtain residues for later use;
(3) UPLC-MS/MS determination of progestogen concentration
Preparing a standard solution: accurately weighing 1mg of standard progestogen to be detected respectively, and respectively using methanol to be dissolved in a volumetric flask of 100mL, mixing to obtain a mixed standard stock solution of 10mg/L, and storing in a refrigerator at the temperature of minus 20 ℃;
preparing a working solution: precisely measuring 500 mu L of methanol solution to dissolve the residues in the step (2), after vortex, fixing the volume to 1mL by using a glass dropper, filtering by using a 0.22 mu m filter membrane, and diluting by using the methanol solution to obtain working solutions with different concentrations;
chromatographic conditions are as follows: column ACQUITY UPLC BEH C18, 2.1X 100mm, 1.7 μm; the mobile phase A is 0.1 percent of formic acid, the mobile phase B is methanol, and a gradient elution procedure is adopted; the flow rate is 0.3 mL/min; the column temperature is 40 ℃, and the sample injection amount is 10 mu L; wherein 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 spectrum conditions: a multi-reaction detection mode is adopted, an ion source is an electrospray ionization source (ESI source), the source temperature is 120 ℃, the cone hole voltage: 30V, the voltage of extracted ions is 2.5kV, the voltage of a capillary tube is 3kV, the temperature of desolventizing gas is 450 ℃, and the flow rate of the desolventizing gas is 900L/hr; the scanning time is 0.1 s;
(4) drawing of standard curve
Drawing a standard curve of the progestogen by taking the concentration (mg/L) of the mixed standard stock solution in the step (3) as an abscissa and taking the response value as an ordinate;
(5) determination on machine
Detecting the working solution in the step (3) by using a UPLC-MS/MS instrument to obtain a typical chromatogram of an actual sample, and comparing the typical chromatogram of the actual sample with the standard curve to perform qualitative detection; and determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters.
Further, the progestogens are mifepristone, chlormadinone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dydrogesterone, levonorgestrel, norethindrone, progesterone-d 9, norethindrone-d 6, respectively.
Furthermore, the environmental water bodies are water inlet, water outlet and river water of a sewage treatment plant, and the two water sources have high human body contact rate and stronger representativeness.
Furthermore, in the step (1), the collection amount is 500mL when the water sample is the inlet water sample of the sewage treatment plant, 500mL when the water sample is the outlet water sample of the sewage treatment plant, and 1-2L when the water sample is river water.
Further, in the step (2), the interior of the Oasis PRIME HLB solid-phase extraction column contains a composite adsorption filler, the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, glyceryl trioleate fiber spheres and core-shell glucose polymer-silica gel spheres with the mass ratio of 2:1:1, and the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin is used for increasing retention of polar substances by a special polarity capturing group to provide good water wettability; the triolein fiber ball has stronger lipophilicity, strong adsorption capacity on lipid compounds such as progestogen and stable performance; the core-shell glucose polymer-silica gel sphere has high hydrophilicity and good enrichment and separation effects.
Furthermore, the Oasis PRiME HLB solid phase extraction cartridge does not require activation and equilibration, greatly saving operating time and solvent.
Further, the pore diameters of the filter membranes in the step (1) are respectively 10 μm, 1 μm and 0.45 μm, and the gradient filtration can maximally reduce the influence of impurities on the analysis result.
Further, the dosage of the ultrapure water and the ethyl acetate in the step (2) is 1-2 times of the volume of the solid phase extraction cartridge.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention adopts a novel solid phase extraction adsorbent Oasis PRIME HLB as a pretreatment technology of an environmental water sample, can effectively remove impurities in the environmental water body, and greatly reduces the matrix effect. And the UPLC-MS/MS is combined as a detector, so that the progestogen content of the environmental water body can be detected quickly and accurately, and the detection result is accurate and reliable.
(2) The invention adopts a novel solid phase extraction adsorbent Oasis PRIME HLB, which not only improves the selectivity to the object to be detected, but also greatly increases the enrichment multiple and reduces the matrix effect, provides a new idea for the development and application of the pretreatment technology, and also increases a new method for the extraction, concentration and separation of trace drug residues in the environment.
(3) The invention uses the ultra-high liquid phase-mass spectrometer as a detection quantitative tool, simultaneously measures 11 different progestogens in the water environment, realizes the effective combination of high selectivity and high sensitivity of the progestogens compared with the common detection mode, and has the advantages of high analysis speed, wide application range and the like.
Drawings
FIG. 1 is a graph showing the effect of the solid phase extraction cartridge of example 2 on the recovery of 9 progestogens;
FIG. 2 is a graph of the effect of different pH's on recovery of 9 progestogens from example 2;
FIG. 3 is a graph showing the effect of different leachates of example 2 on 9 progestogens;
FIG. 4 is a graph of the effect of different eluents of example 2 on recovery of 9 progestogens;
fig. 5.1-5.3 are ion flow diagrams of the 11 progestin extractions of example 2.
Detailed Description
Example 1
A method for simultaneously detecting multiple progestogens in an environmental water body, wherein the progestogens are mifepristone, chlormadinone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dydrogesterone, levonorgestrel, norethindrone, progesterone-d 9 and norethindrone-d 6. After a sample to be detected is pretreated by a novel solid phase extraction adsorbent Oasis PRIME HLB, the sample is detected by UPLC-MS/MS, and the method comprises the following steps:
(1) water sample pretreatment
The water inlet, the water outlet and the river water of the sewage treatment plant are selected as water samples, and the two water sources have high human body contact rate and stronger representativeness. Performing three-stage filtration on an acquired water sample by using three filter membranes with different apertures of 10 microns, 1 micron and 0.45 micron, and accurately measuring the filtered water sample, wherein the volume of the water sample of the sewage treatment plant is 200mL, the volume of the water sample of the sewage treatment plant is 300mL, the volume of the river water is 1L, and the pH value is adjusted to 7.0 by using hydrochloric acid with the concentration of 0.1mol/L and sodium hydroxide with the concentration of 0.1 mol/L;
(2) novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration
Directly passing the water sample treated in the step (1) through an Oasis PRIME HLB solid-phase extraction cartridge for enrichment, controlling the flow rate to be 7mL/min, leaching the solid-phase extraction cartridge twice by using ultrapure water after the enrichment is finished, drying the leached solid-phase extraction cartridge in a vacuum drying oven for 1h at the drying temperature of 25 ℃, and eluting the dried solid-phase extraction cartridge for three times by using ethyl acetate, wherein the using amounts of the ultrapure water and the ethyl acetate are 1 time of the volume of the solid-phase extraction cartridge to obtain an eluent containing a target compound, transferring the eluent into a nitrogen blow-drying instrument, and purging for 30min by using nitrogen with the flow rate of 1.5m/s to obtain a residue for later use; the Oasis PRIME HLB solid-phase extraction column comprises a composite adsorption filler, wherein the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, glycerol trioleate fiber spheres and core-shell glucose polymer-silica gel spheres with the mass ratio of 2:1:1, and the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin is used for increasing retention of polar substances by virtue of a special polar capture group so as to provide good water wettability; the triolein fiber ball has stronger lipophilicity, strong adsorption capacity on lipid compounds such as progestogen and stable performance; the core-shell glucose polymer-silica gel sphere has high hydrophilicity and good enrichment and separation effects.
(3) UPLC-MS/MS determination of progestogen concentration
Preparing a standard solution: accurately weighing 1mg of standard progestogen to be detected respectively, and respectively using methanol to be dissolved in a volumetric flask of 100mL, mixing to obtain a mixed standard stock solution of 10mg/L, and storing in a refrigerator at the temperature of minus 20 ℃;
preparing a working solution: precisely measuring 500 mu L of methanol solution to dissolve the residues in the step (2), after vortex, fixing the volume to 1mL by using a glass dropper, filtering by using a 0.22 mu m filter membrane, and diluting by using the methanol solution to obtain working solutions with different concentrations;
chromatographic conditions are as follows: column ACQUITY UPLC BEH C18, 2.1X 100mm, 1.7 μm; the mobile phase A is 0.1 percent of formic acid, the mobile phase B is methanol, and a gradient elution procedure is adopted; the flow rate is 0.3 mL/min; the column temperature is 40 ℃, and the sample injection amount is 10 mu L; wherein 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 spectrum conditions: a multi-reaction detection mode is adopted, an ion source is an electrospray ionization source (ESI source), the source temperature is 120 ℃, the cone hole voltage: 30V, the voltage of extracted ions is 2.5kV, the voltage of a capillary tube is 3kV, the temperature of desolventizing gas is 450 ℃, and the flow rate of the desolventizing gas is 900L/hr; the scanning time is 0.1 s;
(4) drawing of standard curve
Drawing a standard curve of the progestogen by taking the concentration (mg/L) of the mixed standard stock solution in the step (3) as an abscissa and taking the response value as an ordinate;
(5) determination on machine
Detecting the working solution in the step (3) by using a UPLC-MS/MS instrument to obtain a typical chromatogram of an actual sample, and comparing the typical chromatogram of the actual sample with the standard curve to perform qualitative detection; and determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters.
Example 2
A method for simultaneously detecting multiple progestogens in an environmental water body, wherein the progestogens are mifepristone, chlormadinone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dydrogesterone, levonorgestrel, norethindrone, progesterone-d 9 and norethindrone-d 6. After a sample to be detected is pretreated by a novel solid phase extraction adsorbent Oasis PRIME HLB, the sample is detected by UPLC-MS/MS, and the method comprises the following steps:
(1) water sample pretreatment
The water inlet, the water outlet and the river water of the sewage treatment plant are selected as water samples, and the two water sources have high human body contact rate and stronger representativeness. Performing three-stage filtration on an acquired water sample by adopting three filter membranes with different apertures of 10 microns, 1 micron and 0.45 micron, and accurately measuring the filtered water sample, wherein the water inlet sample of a sewage treatment plant is 350mL, the water outlet sample of the sewage treatment plant is 400mL, the river water collection amount is 1.5L, and the pH value is adjusted to 7.1 by using hydrochloric acid with the concentration of 0.1mol/L and sodium hydroxide with the concentration of 0.1 mol/L;
(2) novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration
Directly passing the water sample treated in the step (1) through an Oasis PRIME HLB solid-phase extraction cartridge for enrichment, controlling the flow rate to be 8mL/min, leaching the solid-phase extraction cartridge twice by using ultrapure water after the enrichment is finished, drying the leached solid-phase extraction cartridge in a vacuum drying oven for 2 hours at the drying temperature of 30 ℃, and eluting the dried solid-phase extraction cartridge for three times by using ethyl acetate, wherein the using amounts of the ultrapure water and the ethyl acetate are 1.5 times of the volume of the solid-phase extraction cartridge to obtain an eluent containing a target compound, transferring the eluent into a nitrogen blow-drying instrument, and purging for 45min by using nitrogen with the flow rate of 1.8m/s to obtain a residue for later use; the Oasis PRIME HLB solid-phase extraction column comprises a composite adsorption filler, wherein the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, glycerol trioleate fiber spheres and core-shell glucose polymer-silica gel spheres with the mass ratio of 2:1:1, and the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin is used for increasing retention of polar substances by virtue of a special polar capture group so as to provide good water wettability; the triolein fiber ball has stronger lipophilicity, strong adsorption capacity on lipid compounds such as progestogen and stable performance; the core-shell glucose polymer-silica gel sphere has high hydrophilicity and good enrichment and separation effects.
(3) UPLC-MS/MS determination of progestogen concentration
Preparing a standard solution: accurately weighing 1mg of standard progestogen to be detected respectively, and respectively using methanol to be dissolved in a volumetric flask of 100mL, mixing to obtain a mixed standard stock solution of 10mg/L, and storing in a refrigerator at the temperature of minus 20 ℃;
preparing a working solution: precisely measuring 500 mu L of methanol solution to dissolve the residues in the step (2), after vortex, fixing the volume to 1mL by using a glass dropper, filtering by using a 0.22 mu m filter membrane, and diluting by using the methanol solution to obtain working solutions with different concentrations;
chromatographic conditions are as follows: column ACQUITY UPLC BEH C18, 2.1X 100mm, 1.7 μm; the mobile phase A is 0.1 percent of formic acid, the mobile phase B is methanol, and a gradient elution procedure is adopted; the flow rate is 0.3 mL/min; the column temperature is 40 ℃, and the sample injection amount is 10 mu L; wherein 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 spectrum conditions: a multi-reaction detection mode is adopted, an ion source is an electrospray ionization source (ESI source), the source temperature is 120 ℃, the cone hole voltage: 30V, the voltage of extracted ions is 2.5kV, the voltage of a capillary tube is 3kV, the temperature of desolventizing gas is 450 ℃, and the flow rate of the desolventizing gas is 900L/hr; the scanning time is 0.1 s;
(4) drawing of standard curve
Drawing a standard curve of the progestogen by taking the concentration (mg/L) of the mixed standard stock solution in the step (3) as an abscissa and taking the response value as an ordinate;
(5) determination on machine
Detecting the working solution in the step (3) by using a UPLC-MS/MS instrument to obtain a typical chromatogram of an actual sample, and comparing the typical chromatogram of the actual sample with the standard curve to perform qualitative detection; and determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters.
Example 3
A method for simultaneously detecting multiple progestogens in an environmental water body, wherein the progestogens are mifepristone, chlormadinone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dydrogesterone, levonorgestrel, norethindrone, progesterone-d 9 and norethindrone-d 6. After a sample to be detected is pretreated by a novel solid phase extraction adsorbent Oasis PRIME HLB, the sample is detected by UPLC-MS/MS, and the method comprises the following steps:
(1) water sample pretreatment
The water inlet, the water outlet and the river water of the sewage treatment plant are selected as water samples, and the two water sources have high human body contact rate and stronger representativeness. Performing three-stage filtration on an acquired water sample by adopting three filter membranes with different apertures of 10 microns, 1 micron and 0.45 micron, accurately measuring the filtered water sample, wherein the water inlet sample of a sewage treatment plant is 500mL, the water outlet sample of the sewage treatment plant is 500mL, the river water collection amount is 2L, and adjusting the pH to 7.2 by using hydrochloric acid with the concentration of 0.1mol/L and sodium hydroxide with the concentration of 0.1 mol/L;
(2) novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration
Directly passing the water sample treated in the step (1) through an Oasis PRIME HLB solid-phase extraction cartridge for enrichment, controlling the flow rate to be 9mL/min, leaching the solid-phase extraction cartridge twice by using ultrapure water after the enrichment is finished, drying the leached solid-phase extraction cartridge in a vacuum drying oven for 3 hours at the drying temperature of 35 ℃, and eluting the dried solid-phase extraction cartridge for three times by using ethyl acetate, wherein the using amounts of the ultrapure water and the ethyl acetate are 2 times of the volume of the solid-phase extraction cartridge to obtain an eluent containing a target compound, transferring the eluent into a nitrogen blow-drying instrument, and purging for 60 minutes by using nitrogen with the flow rate of 2m/s to obtain a residue for later use; the Oasis PRIME HLB solid-phase extraction column comprises a composite adsorption filler, wherein the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, glycerol trioleate fiber spheres and core-shell glucose polymer-silica gel spheres with the mass ratio of 2:1:1, and the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin is used for increasing retention of polar substances by virtue of a special polar capture group so as to provide good water wettability; the triolein fiber ball has stronger lipophilicity, strong adsorption capacity on lipid compounds such as progestogen and stable performance; the core-shell glucose polymer-silica gel sphere has high hydrophilicity and good enrichment and separation effects.
(3) UPLC-MS/MS determination of progestogen concentration
Preparing a standard solution: accurately weighing 1mg of standard progestogen to be detected respectively, and respectively using methanol to be dissolved in a volumetric flask of 100mL, mixing to obtain a mixed standard stock solution of 10mg/L, and storing in a refrigerator at the temperature of minus 20 ℃;
preparing a working solution: precisely measuring 500 mu L of methanol solution to dissolve the residues in the step (2), after vortex, fixing the volume to 1mL by using a glass dropper, filtering by using a 0.22 mu m filter membrane, and diluting by using the methanol solution to obtain working solutions with different concentrations;
chromatographic conditions are as follows: column ACQUITY UPLC BEH C18, 2.1X 100mm, 1.7 μm; the mobile phase A is 0.1 percent of formic acid, the mobile phase B is methanol, and a gradient elution procedure is adopted; the flow rate is 0.3 mL/min; the column temperature is 40 ℃, and the sample injection amount is 10 mu L; wherein 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 spectrum conditions: a multi-reaction detection mode is adopted, an ion source is an electrospray ionization source (ESI source), the source temperature is 120 ℃, the cone hole voltage: 30V, the voltage of extracted ions is 2.5kV, the voltage of a capillary tube is 3kV, the temperature of desolventizing gas is 450 ℃, and the flow rate of the desolventizing gas is 900L/hr; the scanning time is 0.1 s;
(4) drawing of standard curve
Drawing a standard curve of the progestogen by taking the concentration (mg/L) of the mixed standard stock solution in the step (3) as an abscissa and taking the response value as an ordinate;
(5) determination on machine
Detecting the working solution in the step (3) by using a UPLC-MS/MS instrument to obtain a typical chromatogram of an actual sample, and comparing the typical chromatogram of the actual sample with the standard curve to perform qualitative detection; and determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters.
Analysis of results
The following is an analysis of the results of the on-machine assay of example 3:
(I) qualitative detection
Wherein, the detection limit of the method is 3 times of the signal-to-noise ratio, and the detection limit is 0.1-0.8ng/L, so the sensitivity is high; and the linear correlation coefficients are all above 0.99, and the linear relation is good. The results of the standard curve range, correlation coefficient, detection limit, etc. of each working solution are shown in table 1:
TABLE 1 methodological test results
(II) quantitative detection
And determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters. Wherein, the mass spectrum parameters of the quantitative analysis of the 11 progestogens are shown in the following table 2:
mass spectrometric conditions for the progestogens in Table 211
(III) calculation of substrate Effect
A: adding an internal standard and a standard solution into the water sample treated according to the step (1), extracting the progestogen according to the step (2) and detecting according to the step (3), wherein the result is defined as Cm；
B: extracting the progestogen according to the step (2) with the same water sample without adding the internal standard and the standard solution, taking 100. mu.L of the extract, drying the extract with nitrogen, adding 100. mu.L of the progestogen mixed standard solution (50. mu.g/L), and obtaining a result defined as Cm+100；
C: standard solutions of a Mixed Standard solution of progestogen (50. mu.g/L) according to the procedure(3) Analysis, the result is defined as C100(ii) a The calculation result of the matrix effect (ME%) is ME% ([ (C)m+100-Cm)/C100]X 100%, results are shown in table 3:
TABLE 3 progestogen matrix effect (ME%)
(IV) calculation of recovery rate by adding standard
D: taking water samples of inlet water and outlet water of a sewage treatment plant and river water, adding standard solutions of various objects to be detected with certain concentrations into the water samples treated according to the step (1), enriching and concentrating according to the step (2), and measuring samples according to the step (3) to obtain corresponding results, wherein the corresponding results are defined as Ca；
E: the standard solution of each substance to be measured with a certain concentration is measured according to the step (3) without being processed to obtain a corresponding result Cb(ii) a The recovery (RE%) is calculated by the formula: RE% ═ Ca/Cb×100％
The invention investigates the recovery rate of 3 concentrations, the result is shown in table 4, the recovery rate of each substance to be tested is high, and the analysis requirement is met.
TABLE 4 recovery rates of the influent and effluent and river water of the wastewater treatment plant
(V) analysis of content
Taking water samples of inlet water and outlet water of a sewage treatment plant and river water, adding internal standard solutions of various objects to be detected with certain concentrations into the water samples treated according to the step (1), enriching and concentrating according to the step (2), and measuring samples according to the step (3) to obtain the progestogen content (ng/L) shown in the table 5;
TABLE 5 progestogen content (ng/L) in sewage treatment plants and river waters
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for simultaneously detecting multiple progestogens in environmental water is characterized in that a sample to be detected is pretreated by a novel solid phase extraction adsorbent Oasis PRIME HLB and then is detected by UPLC-MS/MS, and comprises the following steps:
(1) water sample pretreatment
Performing three-stage filtration on an acquired water sample by adopting three filter membranes with different apertures, accurately measuring 0.2-2L of the filtered water sample, and adjusting the pH to 7.0-7.2 by using hydrochloric acid with the concentration of 0.1mol/L and sodium hydroxide with the concentration of 0.1 mol/L;
(2) novel solid phase extraction adsorbent Oasis PRIME HLB enrichment concentration
Directly passing the water sample treated in the step (1) through an Oasis PRIME HLB solid phase extraction column for enrichment, controlling the flow rate to be 7-9mL/min, leaching the solid phase extraction column twice by using ultrapure water after the enrichment is finished, then drying the leached solid phase extraction column for 1-3h in a vacuum drying oven at the drying temperature of 25-35 ℃, eluting the dried solid phase extraction column for three times by using ethyl acetate to obtain an eluent containing a target compound, transferring the eluent into a nitrogen blow-drying instrument, and blowing for 30-60min by using nitrogen with the flow rate of 1.5-2m/s to obtain residues for later use; the Oasis PRIME HLB solid-phase extraction column comprises a composite adsorption filler, wherein the composite adsorption filler is poly-N-vinylpyrrolidone-divinylbenzene macroporous resin, glycerol trioleate fiber spheres and core-shell glucose polymer-silica gel spheres with the mass ratio of 2:1:1, and the poly-N-vinylpyrrolidone-divinylbenzene macroporous resin is used for increasing retention of polar substances by virtue of a special polar capture group so as to provide good water wettability; the triolein fiber ball has stronger lipophilicity, strong adsorption capacity on lipid compounds such as progestogen and stable performance; the core-shell glucose polymer-silica gel sphere has high hydrophilicity and good enrichment and separation effects;
(3) UPLC-MS/MS determination of progestogen concentration
Preparing a standard solution: accurately weighing 1mg of standard progestogen to be detected respectively, and respectively using methanol to be dissolved in a volumetric flask of 100mL, mixing to obtain a mixed standard stock solution of 10mg/L, and storing in a refrigerator at the temperature of minus 20 ℃;
preparing a working solution: precisely measuring 500 mu L of methanol solution to dissolve the residues in the step (2), after vortex, fixing the volume to 1mL by using a glass dropper, filtering by using a 0.22 mu m filter membrane, and diluting by using the methanol solution to obtain working solutions with different concentrations;
chromatographic conditions are as follows: column ACQUITY UPLC BEH C18, 2.1X 100mm, 1.7 μm; the mobile phase A is 0.1 percent of formic acid, the mobile phase B is methanol, and a gradient elution procedure is adopted; the flow rate is 0.3 mL/min; the column temperature is 40 ℃, and the sample injection amount is 10 mu L; wherein the gradient elution procedure is as follows:
mass spectrum conditions: a multi-reaction detection mode is adopted, an ion source is an electrospray ionization source (ESI source), the source temperature is 120 ℃, the cone hole voltage: 30V, the voltage of extracted ions is 2.5kV, the voltage of a capillary tube is 3kV, the temperature of desolventizing gas is 450 ℃, and the flow rate of the desolventizing gas is 900L/hr; the scanning time is 0.1 s;
(4) drawing of standard curve
Drawing a standard curve of the progestogen by taking the concentration mg/L of the mixed standard stock solution in the step (3) as an abscissa and taking the response value as an ordinate;
(5) determination on machine
Detecting the working solution in the step (3) by using a UPLC-MS/MS instrument to obtain a typical chromatogram of an actual sample, and comparing the typical chromatogram of the actual sample with the standard curve to perform qualitative detection; and determining parent ions, ionic ions, collision voltage and capillary voltage of each substance by using the tuning function of the UPLC-MS/MS instrument, and quantitatively detecting the progestogen in the working solution by using the parameters.
2. The method of claim 1, wherein the progestogens are mifepristone, chlormadinone, medroxyprogesterone, drospirenone, progesterone, norethindrone, dydrogesterone, levonorgestrel, norethindrone, progesterone-d 9, norethindrone-d 6, respectively.
3. The method for simultaneously detecting multiple progestagens in an environmental water as claimed in claim 1, wherein the environmental water is sewage treatment plant influent water, effluent water and river water.
4. The method for simultaneously detecting multiple progestogens in an environmental water body as claimed in claim 1, wherein the collecting amount in step (1) is 200-.
5. The method of claim 1, wherein the Oasis PRiME HLB solid phase extraction cartridge does not require activation and equilibration.
6. The method for simultaneously detecting multiple progestagens in an environmental water body as claimed in claim 1, wherein the pore size of the filter membrane in step (1) is 10 μm, 1 μm and 0.45 μm, respectively.
7. The method for simultaneously detecting multiple progestogens in an environmental water body as claimed in claim 1, wherein the amount of the ultrapure water and the ethyl acetate used in step (2) is 1-2 times of the volume of the solid phase extraction cartridge.
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| CN105334282A (en) * | 2015-10-27 | 2016-02-17 | 哈尔滨工业大学 | Co-detecting method for environmental estrogens in surface water body |
| CN107121518A (en) * | 2017-06-30 | 2017-09-01 | 华东理工大学 | A kind of method of phenols, estrogens and androgens incretion interferent in Sync enrichment detection drinking water |
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