Rapid detection method for 27 steroid hormones in mangrove plant

Abstract

The invention discloses a rapid detection method for 27 steroid hormones in mangrove plants. The method comprises the following steps: S1, putting a mangrove plant sample subjected to freeze drying, grinding and sieving into a centrifugal tube A, and then adding an isotope internal standard for balancing; adding acetonitrile, carrying out vortex, ultrasonic extraction and centrifugation, and taking supernate; s2, methanol and acetonitrile are sequentially added, vortex, ultrasonic extraction and centrifugation are performed, and supernate obtained after acetonitrile extraction in the S1, supernate obtained after methanol extraction in the S2 and supernate obtained after acetonitrile extraction in the S2 are combined; s3, transferring the supernate into a centrifugal tube B containing filler, carrying out vortex centrifugation, and putting the supernate into a glass test tube; s4, extracting the filler in S3 again by using acetonitrile, carrying out vortex centrifugation, and combining the two supernatants in a glass test tube; s5, carrying out nitrogen blowing concentration on the solution in the glass test tube, and fixing the volume in methanol; s6, determining the steroid hormones and the metabolites thereof by using high performance liquid chromatography-tandem mass spectrometry, and obtaining the content level of the steroid hormones and the metabolites thereof in the plants through data analysis and quantitative calculation.

Description

Technical Field

[0001] The invention belongs to the field of organic pollutant detection, and specifically relates to a rapid detection method for 27 steroid hormones and their metabolites in mangrove plants. Background Art

[0002] Steroid hormones are derivatives of cholesterol produced by anabolism in different tissues (including the brain, adrenal glands, gonads, etc.). Steroid hormones and their metabolites can enter the freshwater environment and even the marine environment through a variety of discharge pathways such as sewage treatment plants, livestock and poultry and aquaculture farms, pharmaceutical factories and hospitals. Although the concentrations of steroid hormones and their metabolites detected in the water environment are almost always between a few nanograms per liter and a few micrograms per liter, these pollutants have strong endocrine disrupting effects and bioaccumulation, so long-term exposure can reach harmful concentrations, such as affecting the normal metabolic function of the organism, interfering with the normal growth, development and reproduction of the organism, and even causing death, carcinogenesis and teratogenesis.

[0003] With the deepening of understanding of steroid hormones and their metabolites found in environmental media and the improvement of awareness of their ecological hazards, researchers have widely carried out research on the accumulation and biological effects of these substances in animals. Mangrove wetlands purify polluted seawater that flows into the sea with various land runoffs through their soil-plant-microorganism complex system to filter and transform pollutants and capture sediments. However, there are still many difficulties in the current research on the migration and transformation of steroid hormones in mangrove plants, such as trace concentrations of steroid hormones in mangrove plants, interference of high pigment content in mangrove plants on quantitative analysis, and single extraction and analysis methods. Most analytical methods are limited to the analysis of a certain type of hormone. Therefore, establishing an integrated analytical method for multiple steroid hormones in different mangrove plant tissues is of great significance for understanding the pollution status of steroid hormones in mangrove plants, actively preventing and controlling offshore pollution of steroid hormones, and protecting the safety of offshore aquatic ecosystems. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a rapid detection method for 27 types of steroid hormones and their metabolites in mangrove plants. The detection method optimizes pretreatment and purification operations so that it can be applied to the detection of different types of mangrove plants and their different tissues. The method has strong compatibility and high detection accuracy.

[0005] The specific scheme adopted by the present invention is:

[0006] A rapid detection method for 27 steroid hormones in mangrove plants, characterized in that it comprises the following steps:

[0007] S1. Place the freeze-dried, ground and sieved mangrove plant sample in centrifuge tube A, and then add isotope internal standard balance; add acetonitrile, vortex, ultrasonically extract, centrifuge, and take the supernatant;

[0008] S2. methanol and acetonitrile were added successively, vortexed, ultrasonically extracted and centrifuged, and the supernatants of acetonitrile extraction of S1, methanol extraction of S2 and acetonitrile extraction were combined;

[0009] S3. Transfer the supernatant to centrifuge tube B containing filler, vortex centrifuge, and place the supernatant in a glass test tube;

[0010] S4. Extract the filler in S3 again with acetonitrile, vortex centrifuge, and combine the two supernatants in a glass test tube;

[0011] S5. Concentrate the solution in the glass test tube by nitrogen blowing and then adjust the volume to methanol;

[0012] S6. Detecting steroid hormones and their metabolites by high performance liquid chromatography-tandem mass spectrometry, and obtaining the content levels of steroid hormones and their metabolites in plants by data analysis and quantitative calculation;

[0013] The 27 steroid hormones are 4-Androstene-3,17-dione, Testosterone, 17β-Boldenone, 17α-Trenbolone, 17β-Trenbolone, 19-nortestosterone, Stanozolo l, 17α-methyltestosterone, Cortisol, Tetrahydrocortisol, 11-Deoxycorticosterone, Prednisolone, Progesterone, 20α-Dihydroprogester one, 17α,20α-Dihydroxyprogesterone, 17α,20β-Dihydroxyprogesterone, 17α-Hydroxyprogesterone, 17α-Hydroxyprogesterone acetate, 11-Ketoprogesterone, 1-Dehydroprogesterone, 16-Dehy droprogesterone, Medroxyprogesterone acetate,Norvinisterone,Dydrogesterone,Cyproterone acetate,Megestrol acetate, Norethindrone.

[0014] The 27 steroid hormones and the liquid chromatography-tandem mass spectrometry parameters for their analysis are as follows:

[0015]

[0016]

[0017]

[0018] Preferably, the test conditions of the high performance liquid chromatography-tandem mass spectrometry in step S6 are as follows: the liquid chromatography mobile phase used is phase A: ultrapure water containing 5 mM ammonium acetate and 0.05% formic acid by volume and phase B: methanol, the flow rate is 0.2 mL / min, the column temperature is 40°C, the gradient elution program starts with 60% methanol for 8 minutes, and is reduced to 50% methanol within 12 minutes. The post-run time is set to 3 minutes for column balance before the next injection. An Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm) reverse phase column is used to separate the target compounds, the ion source is AJS ESI positive mode, the nitrogen temperature in the ion source is 350°C, the gas flow rate is 3 L / min, and the nebulizer pressure is 50 psi; the sheath gas temperature is 350°C, the flow rate is 12 L / min; the capillary voltage is 3500V.

[0019] Preferably, in step S1, the ratio of the mangrove plant sample to acetonitrile is (0.1-0.2) g: 2 mL;

[0020] Preferably, the isotope internal standard is P-d9, CRL-d2, or T-d3. Further preferably, the equilibration time is 10 to 30 minutes.

[0021] Preferably, the filler is C18, GCB and anhydrous MgSO4, and the mass ratio of the filler is (0.15-0.2):(0.15-0.2):(0.5-0.6) g. More preferably, the mass ratio of C18, GCB and anhydrous MgSO4 is 3:3:10.

[0022] Preferably, in S5, the solution in the glass test tube is concentrated by nitrogen blowing, fixed to volume in methanol, and then filtered into a brown injection vial using a 0.22 micron organic phase filter.

[0023] Preferably, in steps S1 and S2, the ultrasonic time is 10 min, the centrifugal speed is 3500 rpm, and the time is 10 min.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] 1. The present invention simplifies the sample pretreatment steps. Compared with the traditional solid phase extraction method, the present method omits the cumbersome steps of activation, elution, and the like required for the enrichment column in solid phase extraction, greatly reduces the amount of organic solvent used and the pretreatment time, and reduces the cost at the same time, which is conducive to the large-scale processing of samples.

[0026] 2. Traditional methods mostly process a certain type of hormone, while the present invention can be used to process multiple types of hormones at the same time, significantly improving the analysis efficiency of samples. Compared with other methods, it can better reflect the concentration of 27 types of steroid hormones in mangrove plant tissues, ensure stable peaks without impurities, and can also be well separated. It can provide effective methods and means for further ecological risk assessment of steroid hormones and research on pollution status in mangrove plants.

[0027] 3. This method can be used for sample testing of different types of mangrove plants and different plant tissues. The method has strong compatibility and does not require a specific method for a specific plant or tissue. It can effectively remove chlorophyll from leaves and stems. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the total ion chromatogram of the steroid hormones and their metabolites of the standard solution and matrix spiked samples in the examples by liquid chromatography-tandem mass spectrometry.

[0029] Figure 2 This is a comparison chart of matrix spike recoveries of steroid hormones and their metabolites in quality control experiments. DETAILED DESCRIPTION

[0030] The content of the present invention is further illustrated below in conjunction with specific implementation cases, but this should not be construed as limiting the present invention.

[0031] Embodiment 1:

[0032] The mangrove plant samples in this example were collected from the coastal area of ​​Guangdong Province. All samples were packed in sealed bags and stored in a -20℃ refrigerator.

[0033] 1. Freeze-dry various tissue parts of mangrove plants, crush them with a high-speed multifunctional crusher (model Q-300B), and then pass them through a 200-mesh sieve.

[0034] 2. Take 0.1g of plant sample in a centrifuge tube, add isotope internal standards P-d9, CRL-d2, and T-d3, and balance for 15 minutes. Then add 2mL of acetonitrile, vortex for 1 minute, ultrasonically extract for 10 minutes, centrifuge at 3500rpm for 10 minutes, take the supernatant, and then extract the sample twice with 2mL of methanol and 2mL of acetonitrile respectively.

[0035] 3. Combine the three supernatants in a centrifuge tube filled with 150 mg C18, 150 mg GCB and 500 mg anhydrous MgSO4, vortex for 3 min, centrifuge at 3500 rpm for 5 min, take the supernatant, and repeat the extraction with 2 mL acetonitrile.

[0036] 4. Combine the two supernatants in a nitrogen blowpipe, blow nitrogen to a fixed volume in methanol, and filter into a brown injection vial with a 0.22μm organic phase filter. The model of the high performance liquid chromatography-tandem mass spectrometry (HPLC-MSMS) is Agilent 1260-6460 series, and the liquid chromatography mobile phase used is (phase A) ultrapure water containing 5mM ammonium acetate and 0.05% formic acid (volume fraction) and (phase B) methanol, the flow rate is 0.2mL / min, the column temperature is 40℃, the gradient elution program starts with 60% methanol for 8min, and drops to 50% methanol within 12min, and the retention time is set to 3min, which is used for column balance before the next injection. The target compounds were separated using an Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm) reverse phase column, the ion source was AJS ESI positive mode, the nitrogen temperature in the ion source was 350 ° C, the gas flow rate was 3 L / min, the nebulizer pressure was 50 psi; the sheath gas temperature was 350 ° C, the flow rate was 12 L / min; the capillary voltage was 3500 V. Other mass spectrometry parameters, such as ion pairs, collision energy and retention time, are shown in Table 1.

[0037] 5. Attachment Figure 1 The total ion chromatogram of steroid hormones and their metabolites in standard solution and matrix spiked samples was obtained by liquid chromatography-tandem mass spectrometry. Figure 1 It can be seen that the 27 steroid hormones and their corresponding isotope internal standards have the same elution time as the standard solution and the peak shape is not tailed. Steroid hormones with similar elution time can also be well separated.

[0038] 6. Attachment Figure 2 The matrix spike recoveries of 27 steroid hormones in quality control experiments were demonstrated, and the results showed that the recoveries were almost stable at around 85%.

[0039] Table 1 HPLC-MS / MS parameters for the analysis of 27 steroid hormones and their corresponding 3 isotope internal standards in mangrove plants

[0040]

[0041]

[0042]

[0043] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A rapid detection method for 27 steroid hormones in mangrove plants, characterized in that: The following steps are involved: S1. Take the freeze-dried, ground and sieved mangrove plant sample into centrifuge tube A, and then add isotope internal standard balance; Add acetonitrile, vortex, ultrasonically extract, centrifuge, and take the supernatant; S2. methanol and acetonitrile were added successively, vortexed, ultrasonically extracted and centrifuged, and the supernatants after acetonitrile extraction of S1, methanol extraction of S2 and acetonitrile extraction were combined; S3. Transfer the supernatant to centrifuge tube B containing filler, vortex centrifuge, and place the supernatant in a glass test tube; S4. Extract the filler in S3 again with acetonitrile, vortex centrifuge, and combine the two supernatants in a glass test tube; S5. The solution in the glass test tube was concentrated by nitrogen blowing and then fixed to volume in methanol; S6. Detecting steroid hormones and their metabolites by high performance liquid chromatography-tandem mass spectrometry, and obtaining the content levels of steroid hormones and their metabolites in plants by data analysis and quantitative calculation; The 27 steroid hormones are 4-Androstene-3,17-dione, Testosterone, 17β-Boldenone, 17α-Trenbolone, 17β-Trenbolone, 19-nortestosterone, Stanozolo l, 17α-methyltestosterone, Cortisol, Tetrahydrocortisol, 11-Deoxycorticosterone, Prednisolone, Progesterone, 20α-Dihydroprogester one, 17α,20α-Dihydroxyprogesterone, 17α,20β-Dihydroxyprogesterone, 17α-Hydroxyprogesterone, 17α-Hydroxyprogesterone acetate, 11-Ketoprogesterone, 1-Dehydroprogesterone, 16-Dehy droprogesterone, Medroxyprogesterone acetate,Norvinisterone,Dydrogesterone,Cyproterone acetate,Megestrol acetate, Norethindrone.

2. The rapid detection method according to claim 1, characterized in that: The 27 steroid hormones and the liquid chromatography-tandem mass spectrometry parameters for their analysis are as follows:

3. The rapid detection method according to claim 1, characterized in that The test conditions of the high performance liquid chromatography-tandem mass spectrometry described in step S6 are as follows: the liquid chromatography mobile phase used is phase A: ultrapure water containing 5 mM ammonium acetate and 0.05% formic acid by volume fraction and phase B: methanol, the flow rate is 0.2 mL / min, the column temperature is 40°C, the gradient elution program starts with 60% methanol for 8 minutes, and is reduced to 50% methanol within 12 minutes. The post-run time is set to 3 minutes for column balance before the next injection. An Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm) reverse phase column is used to separate the target compounds, the ion source is AJS ESI positive mode, the nitrogen temperature in the ion source is 350°C, the gas flow rate is 3 L / min, and the nebulizer pressure is 50 psi; the sheath gas temperature is 350°C, the flow rate is 12 L / min; the capillary voltage is 3500V.

4. The rapid detection method according to claim 1, characterized in that: In the step S1, the ratio of the mangrove plant sample to acetonitrile is (0.1-0.2) g: 2 mL.

5. The rapid detection method according to claim 1, characterized in that: The isotope internal standard is P-d9, CRL-d2, T-d3. More preferably, the equilibration time is 10 to 30 minutes.

6. The rapid detection method according to claim 1, characterized in that: The fillers are C18, GCB and anhydrous MgSO4, and the mass ratio of the fillers is (0.15-0.2):(0.15-0.2):(0.5-0.6) g. The mass ratio of C18, GCB and anhydrous MgSO4 is further preferably 3:3:

10.

7. The rapid detection method according to claim 1, characterized in that: The solution in the S5 glass tube was concentrated by nitrogen blowing, fixed to volume in methanol, and then filtered into a brown injection vial using a 0.22 micron organic phase filter.

8. The rapid detection method according to claim 1, characterized in that: In steps S1 and S2, the ultrasonic time is 10 min, and the centrifugal speed is 3500 rpm for 10 min.

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