A method for selectively separating bisphenol compounds in serum

Abstract

The application discloses a method for selectively separating bisphenol compounds in serum, and belongs to the technical field of extraction and purification of organic pollutants. The method is suitable for analyzing bisphenol compounds such as BPA, BPS, BPF, BPE, BPB, BPAF, BPAP, BPZ and TBBPA in serum. Zeolite molecular sieves are filled into a solid-phase extraction empty tube, and the filled solid-phase extraction column is activated by using an organic solvent to remove possible impurities. Then, serum extract is loaded onto the solid-phase extraction column. A certain volume of dichloromethane is used for elution to remove part of interfering substances. Then, a mixed solvent of methanol / dichloromethane with a proper ratio is used for elution to collect target substances. According to the application, some lipid macromolecules in serum can be removed through zeolite molecular sieve solid-phase extraction, which greatly reduces the matrix effect of serum, guarantees the recovery rate of bisphenol compounds, saves cost and is more environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and purification of organic pollutants in biological samples, and specifically relates to a method for selectively separating bisphenol compounds such as BPA, BPS, BPF, BPE, BPB, BPAF, BPAP, BPZ and TBBPA in serum based on zeolite molecular sieves as solid phase extraction adsorbents. Background Art

[0002] Bisphenols (BPs) are a class of chemical substances containing two phenolic hydroxyl groups and similar structures. Due to their stability, excellent heat resistance and ductility, relatively simple production process, and low cost, they are commonly used in the production of many important chemical raw materials, such as polycarbonate, epoxy resin and other polymers. They are commonly used in daily products such as water bottles, baby bottles, container coatings, and building materials. BPs, a type of non-natural new organic pollutant, are easily enriched and difficult to degrade. Long-term low-level exposure can affect human health and cause a range of diseases such as obesity, cancer, and cardiovascular disease. Currently, the production and use of bisphenols are increasing globally. Due to their structural similarity to sex hormones, bisphenols have negative effects on the human body, such as endocrine disruption, reproductive toxicity, and neurotoxicity, and are classified as endocrine disruptors. Currently, the Canadian government has identified and is subjecting 34 bisphenols, including BPS, BPF, BPAF, BPAP, and BPZ, to further risk management.

[0003] The content of bisphenol compounds in serum is mostly at the ng / g concentration level. Due to the lack of sensitive trace detection methods, the research data on bisphenol compounds is seriously insufficient. 18 Solid phase extraction columns are used as pretreatment methods for bisphenol compounds, but they have problems such as high cost and obvious matrix effect.

[0004] Zeolite molecular sieves are materials with selective adsorption properties, capable of separating compounds based on their molecular shape and size. Hundreds of framework types exist. Zeolites have been reported to selectively adsorb polychlorinated dibenzo-p-dioxins and dibenzofurans through a molecular sieving effect, with this selectivity determined by the number of chlorine atoms and the chlorine substitution pattern (regioisomers). Research has shown that zeolites with appropriate pore sizes fall within the size range of the target molecule, and that size and shape effects play a significant role in the selective separation of pollutants. 13X molecular sieves are porous aluminosilicate materials with a complete framework structure and belong to the low-silicon-to-aluminum ratio zeolite class. They are easy to synthesize, have a large surface area, and exhibit excellent hydrophilicity, resulting in excellent ion exchange and adsorption properties. They are widely used in drying and separation applications. 13X molecular sieves are primarily used for selective adsorption based on the size of their crystalline pores. They are commonly used in the drying and purification of industrial gases, the adsorption of heavy metal ions from industrial wastewater, and the desulfurization of liquid hydrocarbons and natural gas. However, the application of 13X molecular sieves in the analysis of bisphenol compounds has not yet been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for selectively separating bisphenol compounds in serum.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for selectively separating bisphenol compounds in serum mainly comprises the following steps:

[0008] (1) Liquid-liquid extraction and concentration: The bisphenol compounds in the serum are extracted and concentrated by liquid-liquid extraction, and then replaced with dichloromethane or a solvent with similar properties to obtain an extract;

[0009] (2) Filling the solid phase extraction column: Select a solid phase extraction column and fill it with zeolite molecular sieve;

[0010] (3) Activation: Use dichloromethane or a solution of similar properties to activate the filled solid phase extraction column;

[0011] (4) Sampling: The extract obtained in step (1) is loaded onto the solid phase extraction column of step (3), and the effluent is discarded;

[0012] (5) Eluent: Use dichloromethane or a solution of similar properties as the eluent to remove interfering substances and discard the effluent;

[0013] (6) Elution: Use a methanol-dichloromethane mixed solution with a volume ratio of 1:5 to 1:1 as the eluent, and collect the eluate in a glass centrifuge tube.

[0014] Furthermore, the bisphenol compound in step (1) includes compounds with similar structures such as BPA, BPS, BPF, BPE, BPB, BPAF, BPAP, BPZ and TBBPA.

[0015] Furthermore, the extraction solvent in step (1) is a single solvent such as ethyl acetate, dichloromethane, ether, toluene, n-hexane, methyl tert-butyl ether, or a mixed solvent of two or more.

[0016] Furthermore, the concentration method in step (1) includes rotary evaporation and nitrogen purging.

[0017] Furthermore, the specific process of step (2) is: selecting a glass solid phase extraction column, installing the fiber sieve plate into the glass solid phase extraction column, filling the zeolite molecular sieve and then compacting it with the fiber sieve plate.

[0018] Furthermore, the zeolite molecular sieve includes 13X molecular sieve.

[0019] Furthermore, in step (3), the amount of dichloromethane or a solution with similar properties is more than 1 times the volume of the solid phase extraction column.

[0020] Furthermore, in step (4), the volume ratio of the extract to the solid phase extraction column is not higher than 6:1.

[0021] Furthermore, in step (5), the ratio of the amount of the eluent to the amount of the zeolite molecular sieve is not greater than 20 mL:200 mg.

[0022] Furthermore, in step (6), the ratio of the eluent to the zeolite molecular sieve is not less than 10 mL:200 mg.

[0023] Furthermore, the eluate collected in step (6) is purged with nitrogen to dryness, redissolved with dichloromethane or a solution of similar properties, and analyzed and detected, or treated by a derivatization method and then analyzed and detected.

[0024] The present invention has the following beneficial effects compared to the prior art:

[0025] The present invention uses zeolite molecular sieves as solid phase extraction adsorbents to achieve quantitative analysis of bisphenol compounds in serum, effectively reduces the matrix effect of serum, ensures the accuracy of bisphenol compound determination, saves costs, and is more environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0027] Figure 1 Bovine serum was filtered through different adsorbents (13X molecular sieve, C 18Full scan (m / z 100-1025) after HPLC (HLB, PEP);

[0028] Figure 2 Bovine serum was filtered through different adsorbents (13X molecular sieve, C 18 Total ion current in SIM mode after ionization (HLB, PEP);

[0029] Figure 3 Comparison of matrix effects of bovine serum before and after purification with 13X molecular sieve;

[0030] Figure 4 Bovine serum was filtered through four different adsorbents (13X molecular sieve, C 18 , HLB, PEP) matrix effect comparison chart;

[0031] Figure 5 Recovery rate of bisphenol compounds after serum extract was purified by 13X molecular sieve. DETAILED DESCRIPTION

[0032] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0033] The nine bisphenols include BPA, BPS, BPF, BPE, BPB, BPAF, BPAP, BPZ, and TBBPA;

[0034] Six isotope labels include 13 C-BPA, 13 C-BPS, 13 C-BPF, 13 C-BPB, 13 C-BPAF, 13 C-TBBPA.

[0035] Example 1: 13X molecular sieve and three common commercial adsorbents (HLB, C 18 Purification effect of PEP on serum extract

[0036] (1) Filling the solid phase extraction column: weigh 200 mg of 13X molecular sieve, 200 mg of C 18 , 100 mg HLB and 100 mg PEP adsorbent were filled into a 3 mL glass solid phase extraction column, and both the upper and lower ends were compacted with fiber frits;

[0037] (2) Activation: Use 3 mL of dichloromethane to activate the 13X molecular sieve solid phase extraction column, 3 mL of methanol and 4 mL of 1% formic acid to activate C 18 Solid phase extraction cartridges, HLB solid phase extraction cartridges activated with 3 mL of dichloromethane, 3 mL of methanol, and 3 mL of water, and PEP solid phase extraction cartridges activated with 3 mL of methanol and 3 mL of water;

[0038] (3) Liquid-liquid extraction and nitrogen blowing: 0.5 mL of bovine serum was subjected to liquid-liquid extraction using 3*3 mL of ethyl acetate. The extracts were combined into a glass centrifuge tube and dried by nitrogen blowing;

[0039] (4) Sample loading: Add 2 mL of dichloromethane to each glass centrifuge tube in step (3) for re-dissolution, then load the sample onto a 13X molecular sieve solid phase extraction column. Wash the centrifuge tube with 0.5 mL of dichloromethane and then combine and load the sample. Add 2 mL of deionized water to each of the other three equal amounts of bovine serum (0.5 mL) and load them onto the PEP, C 18 and HLB solid phase extraction column, wash the centrifuge tubes with 0.5 mL of deionized water respectively and then combine and load the sample; discard the effluent;

[0040] (5) Washing: Use 5 mL of dichloromethane to wash the 13X molecular sieve solid phase extraction column, 2 mL of 0.5% formic acid and 4 mL of 25% methanol to wash the C 18 For solid-phase extraction (SPE) columns, rinse the HLB SPE column with 3 mL of deionized water, and the PEP SPE column with 1 mL of ultrapure water and 2 mL of 5% methanol; discard the eluents; except for the 13X molecular sieve SPE column, dry all other SPE columns using a mechanical pump for 20–30 min.

[0041] (6) Elution: 10 mL of a methanol-dichloromethane mixed solution with a volume ratio of 1:4 was used to elute the 13X molecular sieve solid phase extraction column, and 3 mL of a 5% ammonia methanol solution was used to elute the C 18 The solid phase extraction column was eluted with 5 mL of a methanol-dichloromethane mixed solution with a volume ratio of 1:1 for eluting the HLB solid phase extraction column and 5 mL of methanol for eluting the PEP solid phase extraction column; the eluates were collected respectively;

[0042] (7) Nitrogen blowing and volume adjustment: The eluate was blown dry with nitrogen, and an appropriate amount of sodium bicarbonate buffer solution (100 mM, pH 10.5) and dansyl chloride derivatization reagent were added. The reaction was heated in a water bath at 60°C for at least 1 h. After the derivatization was completed, the mixture was extracted with n-hexane for at least three times. The supernatants were combined and concentrated to dryness, and then the volume was adjusted with 250 μL of acetonitrile.

[0043] (8) Analysis: Liquid chromatography-mass spectrometry was used for analysis. 18The analysis and detection were carried out on a reverse phase chromatography column (250 mm × 4.6 mm, 5 μm), with a flow rate of 1 mL / min and a column temperature of 25°C. Gradient elution was performed using mobile phases A (acetonitrile) and B (water) as follows: 0 min, A:B = 50:50; 20 min, A:B = 80:20; 20.1 min, A:B = 100:0; 28 min, A:B = 100:0; 28.1 min, A:B = 50:50; 35 min, A:B = 50:50.

[0044] From the full scan (m / z: 100~1025) ( Figure 1 ) It can be seen that 13X molecular sieve, C 18 , HLB and PEP solid phase extraction columns have similar purification effects on serum; and from the total ion current diagram under SIM mode ( Figure 2 ) It can be seen that the purification effect of 13X molecular sieve is better than that of C 18 , HLB and PEP adsorbents.

[0045] Example 2:

[0046] (1) Weigh 200 mg of 13X molecular sieve in parallel, load each into a 3 mL solid phase extraction glass column, and compact the upper and lower ends of the 13X molecular sieve with fiber sieve plates;

[0047] (2) Activation: Activate the solid phase extraction column using 3 mL of dichloromethane;

[0048] (3) Liquid-liquid extraction and nitrogen blowdown: 0.5 mL of bovine serum was subjected to liquid-liquid extraction using 3*3 mL of ethyl acetate. The extracts were combined into glass centrifuge tubes. This operation was repeated four times in parallel. The extracts were labeled #1, #2, #3, and #4, respectively. The extracts were blown dry with nitrogen.

[0049] (4) Loading: Add 2 mL of dichloromethane to each of the glass centrifuge tubes containing extracts #1 and #2 to reconstitute the solution. Load the solution onto a 13X molecular sieve solid phase extraction column, wash with 0.5 mL of dichloromethane, and then combine and load the solution. Discard the flow-through.

[0050] (5) Washing: Use 5 mL of dichloromethane to wash the 13X molecular sieve solid phase extraction column and discard the eluent;

[0051] (6) Elution: Elute the 13X molecular sieve solid phase extraction column with 10 mL of 1:4 (v / v) methanol-dichloromethane and collect the eluate;

[0052] (7) Nitrogen blowing and spike addition: The eluents of extracts #1 and #2 were blown dry with nitrogen; 5 ng of bisphenol mixed standard was added to extracts #1, #3 and blank centrifuge tube #0 respectively, and the mixture was blown dry with nitrogen, wherein blank centrifuge tube #0 was used as a standard control sample;

[0053] (8) Derivatization reaction: 200 μL of sodium bicarbonate buffer solution (100 mM, pH 10.5) and 200 μL of dansyl chloride derivatization reagent (1 mg / mL, acetone) were added to extracts #1, #2, #3, #4, and centrifuge tube #0, respectively. The mixture was vortexed for 1 min and heated in a water bath at 60°C for 1 h. After derivatization, 1 mL of deionized water was added, and the mixture was extracted with 3 × 1 mL of n-hexane. The supernatants were combined.

[0054] (9) Nitrogen purging and reconstitution: The extract was purged with nitrogen until dry and then diluted to volume with 250 μL acetonitrile;

[0055] (10) Analysis: Liquid chromatography-mass spectrometry was used for detection and analysis, and the matrix effect was calculated. The calculation formula for the matrix effect is: M E (%)=(BC) / A*100, wherein A is the peak area of ​​#0, B is the peak area of ​​#1 or #3, and C is the peak area of ​​#2 or #4.

[0056] Depend on Figure 3 It can be seen that compared with liquid-liquid extraction, the matrix effect of bovine serum was significantly improved after purification by 13X molecular sieve.

[0057] Example 3:

[0058] (1) Filling the solid phase extraction column: the same as step (1) of Example 1;

[0059] (2) Activation: the same as step (2) of Example 1;

[0060] (3) Liquid-liquid extraction and nitrogen blowdown: 0.5 mL of bovine serum was subjected to liquid-liquid extraction using 3*3 mL of ethyl acetate. The extracts were combined into a glass centrifuge tube. This operation was repeated twice in parallel, and the extracts were labeled #5 and #6, respectively. The extracts were blown dry with nitrogen blowdown.

[0061] (4) Sample loading: Add 2 mL of dichloromethane to the glass centrifuge tubes of the above extracts #5 and #6 respectively to reconstitute, then load the samples onto a 13X molecular sieve solid phase extraction column, and wash the centrifuge tubes with 0.5 mL of dichloromethane before combining and loading; prepare six equal amounts of bovine serum (#7, #8, #9, #10, #11, #12), add 2 mL of deionized water to each, and load samples #7 and #8 onto the PEP solid phase extraction column; load samples #9 and #10 onto the C 18 Solid phase extraction column; Samples #11 and #12 were loaded onto HLB solid phase extraction column separately; Samples #7 to #12 were washed with 0.5 mL of deionized water in centrifuge tubes and then combined and loaded; the effluent was discarded;

[0062] (5) rinsing: the same as (5) in Example 1;

[0063] (6) Elution: the same as (6) in Example 1;

[0064] (7) Nitrogen blowing and spiking: The eluents of samples #5 to #12 were blown dry with nitrogen; 5 ng of bisphenol mixed standard was added to extracts #5, #7, #9, #11 and blank centrifuge tube #13, respectively, and blown dry with nitrogen. Blank centrifuge tube #13 was used as a standard control sample;

[0065] (8) Derivatization reaction: 200 μL of sodium bicarbonate buffer solution (100 mM, pH 10.5) and 200 μL of dansyl chloride derivatization reagent (1 mg / mL, acetone) were added to extracts #5 to #12 and centrifuge tube #13, respectively. The mixture was vortexed for 1 min and heated in a water bath at 60°C for 1 h. After derivatization, 1 mL of deionized water was added, and the mixture was extracted with 3 × 1 mL of n-hexane. The supernatants were combined.

[0066] (9) Nitrogen purging and re-dissolution: The extract was purged with nitrogen until dry, and the volume was adjusted to 250 μL acetonitrile;

[0067] (10) Analysis: Liquid chromatography-mass spectrometry was used for analysis and matrix effects were calculated.

[0068] Depend on Figure 4 It can be seen that 13X molecular sieve, C 18 The matrix effects of HLB and PEP solid phase extraction columns for nine bisphenol compounds were 61.05%-121.28%, 3.54%-107.10%, 43.26%-279.63% and 8.92%-142.18%, respectively. It can be seen that 13X molecular sieve has a better matrix effect than the other three commercial adsorbents.

[0069] Example 4: Analysis of the recovery rate of bisphenol compounds in serum extracts using a 13X molecular sieve solid phase extraction column

[0070] (1) Filling the solid phase extraction column: Weigh 200 mg of 13X molecular sieve and fill it into a 3 mL glass solid phase extraction column. Press the upper and lower ends with fiber sieve plates.

[0071] (2) Activation: Use 3 mL of dichloromethane to activate the 13X molecular sieve solid phase extraction column;

[0072] (3) Liquid-liquid extraction and nitrogen blowdown: 0.5 mL of bovine serum was subjected to liquid-liquid extraction using 3*3 mL of ethyl acetate. The extracts were combined into a glass centrifuge tube. This operation was performed twice in parallel, and the extracts were labeled #14 and #15 respectively. The extracts were blown dry with nitrogen blowdown.

[0073] (4) loading: 5 ng of bisphenol mixed standard and 5 ng of isotope-labeled mixed standard were added into the glass centrifuge tube of extract #14, and only 5 ng of isotope-labeled mixed standard was added into the glass centrifuge tube of extract #15; after nitrogen blowing to dryness, 2 mL of dichloromethane was added for loading into the 13X molecular sieve solid phase extraction column, and the centrifuge tube was washed with 0.5 mL of dichloromethane and then combined; the effluent was discarded;

[0074] (5) elution: 5 mL of dichloromethane was used to elute the 13X molecular sieve solid phase extraction column, and the eluent was discarded;

[0075] (6) elution: 10 mL of 1:4 (v / v) methanol-dichloromethane was used to elute the 13X molecular sieve solid phase extraction column, and the eluent was collected;

[0076] (7) nitrogen blowing: the eluent was blown dry by nitrogen;

[0077] (8) preparation of standard control sample: 5 ng of bisphenol mixed standard and 5 ng of isotope-labeled mixed standard were added into a blank centrifuge tube, and nitrogen blowing was performed to dryness;

[0078] (9) derivatization reaction: 200 μL of sodium bicarbonate buffer solution (100 mM, pH 10.5) and 200 μL of dansyl chloride derivatization reagent (1 mg / mL, acetone) were added into samples #14, #15 and the standard control sample, respectively, vortexed for 1 min, heated in a water bath at 60°C for 1 h; after the derivatization was completed, 1 mL of deionized water was added, liquid-liquid extraction was performed with 3*1 mL of n-hexane, and the supernatant was combined;

[0079] (10) nitrogen blowing and constant volume: the extract was blown dry by nitrogen, and 250 μL of acetonitrile was used for constant volume;

[0080] (11) analysis: liquid chromatography-mass spectrometry was used for detection and analysis.

[0081] According to the internal standard method and after deducting the method blank, the recovery rate of the bisphenol compound was calculated, and it could be known from the following formula that the recovery rate of the nine bisphenol compounds in serum analyzed by the 13X molecular sieve solid phase extraction column could reach 63.91% to 135.07%. Figure 5

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. A method for selectively separating bisphenol compounds in serum, characterized in that: The steps include: (1) Extracting and concentrating bisphenol compounds in serum, and replacing them with dichloromethane to obtain an extract; (2) Select a solid phase extraction column and fill it with zeolite molecular sieve; (3) Activate the solid phase extraction column using dichloromethane; (4) Loading the extract obtained in step (1) onto the solid phase extraction column in step (3), and discarding the effluent; (5) Use dichloromethane as the eluent to elute and discard the effluent; (6) Using a methanol-dichloromethane mixed solution with a volume ratio of 1:5 to 1:1 as the eluent, perform elution and collect the eluate; The zeolite molecular sieve is 13X molecular sieve.

2. The method according to claim 1, wherein: The bisphenol compounds in step (1) include BPA, BPS, BPF, BPE, BPB, BPAF, BPAP, BPZ and TBBPA.

3. The method according to claim 1, wherein: The extraction solvent in step (1) is a single solvent selected from ethyl acetate, dichloromethane, ether, toluene, n-hexane, and methyl tert-butyl ether, or a mixture of two or more solvents; the concentration method includes rotary evaporation and nitrogen purging.

4. The method according to claim 1, wherein: The specific process of step (2) is: select a glass solid phase extraction column, install the fiber sieve plate into the glass solid phase extraction column, fill the zeolite molecular sieve and then use the fiber sieve plate to compact it.

5. The method according to claim 1, wherein: The amount of dichloromethane used in step (3) is more than 1 times the volume of the solid phase extraction column.

6. The method according to claim 1, wherein: The volume ratio of the extract to the solid phase extraction column in step (4) is not higher than 6:

1.

7. The method according to claim 1, wherein: In step (5), the ratio of the eluent to the zeolite molecular sieve is not greater than 20 mL:200 mg.

8. The method according to claim 1, wherein: In step (6), the ratio of the eluent to the zeolite molecular sieve is not less than 10 mL: 200 mg.

9. The method according to claim 1, wherein: The eluate collected in step (6) is purged to dryness with nitrogen, redissolved with dichloromethane, and analyzed or treated by a derivatization method before analysis.

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