Pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum

Abstract

A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum involves denaturing and precipitating proteins in a mixed solution of acetone and n-hexane using acetone, followed by shaking extraction with a mixed solution of dichloromethane and n-hexane, and then using Supelclean ENVI-Carb. TM The solid-phase extraction column was used for purification and concentration, and high-performance liquid chromatography-tandem mass spectrometry was used for detection. The method of this application can effectively extract TBBPA-BDBPE from serum and quantify its content. It requires a small amount of serum, is simple to operate, and has a high recovery rate, providing a basic method for environmental and health research on tetrabromobisphenol A bis(2,3-dibromopropyl ether).

Description

Technical Field

[0001] This invention relates to the field of environmental pollutant detection, specifically to a pretreatment method for analyzing a novel pollutant, tetrabromobisphenol A bis(2,3-dibromopropyl ether), in serum. Background Technology

[0002] Tetrabromobisphenol A bis(2,3-dibromopropyl ether) (TBBPA-bis(2,3-dibromopropyl ether), TBBPA-BDBPE, also known as "octabromoether") is a substitute for the brominated flame retardant tetrabromobisphenol A and is widely used in the production of polymers such as polypropylene, high-density polyethylene, low-density polyethylene, and high-impact polystyrene. As an additive flame retardant, TBBPA-BDBPE is easily released from the polymer into the environment. Currently, TBBPA-BDBPE has been detected globally and in various environmental matrices, including marine mollusks, indicating its widespread environmental presence and significant risk of human exposure.

[0003] While ensuring the body's metabolism, blood circulation also transports organic pollutants that enter the body for distribution or metabolism throughout the body. Therefore, blood is an important biological sample for understanding the level of organic pollutant pollution in humans or animals. However, the analysis of organic pollutants in blood is quite challenging due to the complexity and high concentration of organic compounds, strong matrix effects, and limitations in sampling volume.

[0004] TBBPA-BDBPE has become a novel pollutant of great concern. Studying its concentration and distribution patterns in serum is crucial for elucidating the bioavailability of TBBPA-BDBPE and its impact on human health. Therefore, it is necessary to establish a pretreatment method for analyzing TBBPA-BDBPE in serum. No serum analysis of TBBPA-BDBPE has been reported in the prior art; this application represents the inventors' first related research in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a pretreatment method that addresses the problem of complex serum sample matrices by requiring small sample volumes, achieving high recovery rates, good impurity removal, and rapid processing speed, thereby enabling the quantitative analysis of TBBPA-BDBPE in serum samples. This invention employs a protein precipitation followed by shaking extraction to extract TBBPA-BDBPE from serum, using a Supelclean ENVI-Carb... TM Solid-phase extraction columns remove the matrix and concentrate TBBPA-BDBPE from serum, ensuring extraction efficiency while reducing the loss of TBBPA-BDBPE during pretreatment.

[0006] To achieve the above objectives, a pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum was established, comprising the following steps:

[0007] (1) Stable isotopes 13 C-labeled dimethylated tetrabromobisphenol A ( 13 C 12 Mix TBBPA-BME with serum, add small steel beads, and vortex to mix thoroughly.

[0008] (2) Add a mixed solution of n-hexane and acetone, vortex for 30 seconds, and then extract using a tissue homogenizer;

[0009] (3) After centrifugation, the supernatant was collected in advance, and the extraction and centrifugation were repeated three times;

[0010] (4) Continue to add the mixed solution of n-hexane and dichloromethane, use a tissue homogenizer to extract by shaking, centrifuge, add the supernatant in advance, and repeat the extraction and centrifugation three times;

[0011] (5) Collect the supernatant after six centrifugations, blow it with nitrogen until it is nearly dry using a nitrogen blower, and redissolve it in a mixed solution of n-hexane and dichloromethane;

[0012] (6) After activating the solid phase extraction column with hexane and dichloromethane respectively, the mixed solution obtained in step (5) is loaded onto the sample and then purified and eluted with the mixed solution of hexane and dichloromethane.

[0013] (7) After collecting the purified solution and blowing it with nitrogen until it is nearly dry, replace the solvent with methanol and transfer it to a sample vial;

[0014] (8) After reconstitution, add stabilizer 13 C isotope labeled tetrabromobisphenol A ( 13 C 12 The concentration of TBBPA-BDBPE was detected using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) as an internal standard.

[0015] Furthermore, the extraction method employed is oscillatory extraction, in which small steel balls with a diameter of 4 mm are used.

[0016] Furthermore, the ratio of the hexane and acetone mixture in the extract is 8:2 by volume; the ratio of the hexane and dichloromethane mixture is 1:1 by volume.

[0017] Furthermore, the tissue homogenizer used in the oscillation extraction had an oscillation frequency of 30 Hz and an extraction time of 4 min.

[0018] Further, the supernatant was extracted by centrifugation at 9000 rpm for 10 min.

[0019] Furthermore, the solutions used to activate the solid-phase extraction column were 6 mL of dichloromethane and 6 mL of n-hexane, and the solution used to elute the solid-phase extraction column was a 12 mL mixture of n-hexane and dichloromethane in a volume ratio of 1:1.

[0020] Furthermore, the internal standard added in the preprocessing is 13 C 12 -TBBPA-BME (58.4 ng); This is an internal standard added to reduce errors caused by instrument fluctuations. 13 C 12 -TBBPA (55.6ng).

[0021] Compared with existing technologies, the advantages and positive effects of this method are that the serum sample undergoes protein denaturation and precipitation under the action of acetone in a mixed solution of acetone and n-hexane, followed by shaking extraction using a mixed solution of dichloromethane and n-hexane, while using Supelclean ENVI-Carb. TM Solid-phase extraction columns remove the matrix, reducing matrix effect interference. The pretreatment method is simple to operate, uses less organic solvent, and has a high recovery rate. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Appendix Figure 1 This is a flowchart of the preprocessing method for this application;

[0024] Appendix Figure 2 This is the standard curve of the pretreatment method TBBPA-BDBPE used in this application;

[0025] Appendix Figure 3 This is a comparison of the recovery rates of different extraction methods in this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] like Figure 1As shown, this application provides a pretreatment method for analyzing the novel pollutant tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum. The method mainly involves mixing the serum sample and internal standard compound thoroughly, followed by vortexing extraction, centrifugation to extract the supernatant, and then solid-phase extraction and concentration of the supernatant to obtain the sample extract to be tested. The specific steps are as follows:

[0028] (1) Stable isotopes 13 C-labeled dimethylated tetrabromobisphenol A ( 13 C 12 Mix TBBPA-BME with serum, add small steel beads, and vortex to mix thoroughly.

[0029] (2) Add a mixed solution of n-hexane and acetone, vortex for 30 seconds, and then extract using a tissue homogenizer;

[0030] (3) After centrifugation, the supernatant was collected in advance, and the extraction and centrifugation were repeated three times;

[0031] (4) Continue to add the mixed solution of n-hexane and dichloromethane, use a tissue homogenizer to extract by shaking, centrifuge, add the supernatant in advance, and repeat the extraction and centrifugation three times;

[0032] (5) Collect the supernatant after six centrifugations, blow it with nitrogen until it is nearly dry using a nitrogen blower, and redissolve it in a mixed solution of n-hexane and dichloromethane;

[0033] (6) After activating the solid phase extraction column with hexane and dichloromethane respectively, the mixed solution obtained in step (5) is loaded onto the sample and then purified and eluted with the mixed solution of hexane and dichloromethane.

[0034] (7) After collecting the purified solution and blowing it with nitrogen until it is nearly dry, replace the solvent with methanol and transfer it to a sample vial;

[0035] (8) After reconstitution, add stabilizer 13 C isotope labeled tetrabromobisphenol A ( 13 C 12 The concentration of TBBPA-BDBPE was detected using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) as an internal standard.

[0036] The spiked recoveries of the pretreatment method in this application are shown in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] The present application will be further described below with reference to specific embodiments:

[0041] (1) Serum sample pretreatment

[0042] Accurately transfer 200 μL of serum and add 58.4 ng. 13 C 12 Add a 4mm diameter steel ball to TBBPA-BME solution and vortex to mix thoroughly. Add 1mL of a mixture of acetone and n-hexane (8:2, v / v), vortex for 30 seconds, and then extract using a tissue homogenizer. Centrifuge at 9000rpm for 10 minutes, extract the supernatant, and repeat the extraction twice. Combine the supernatants from the three extractions. Continue extraction using a mixture of dichloromethane and n-hexane (1:1, v / v), centrifuge, and extract the supernatant. Repeat the extraction three times. Combine all supernatants to obtain the initial extract, evaporate to near dryness using a nitrogen evaporator, and then redissolve in 1mL of a mixture of dichloromethane and n-hexane (1:1, v / v). Use Supelclean ENVI-Carb... TM The TBBPA-BDBPE was purified and concentrated using a solid-phase extraction (SPE) column. The SPE column was activated with 6 mL of dichloromethane and 6 mL of n-hexane, respectively. The reconstituted solution was loaded onto the SPE column, and then eluted with a 1:1 mixture of n-hexane and dichloromethane (v / v). The eluent was collected, purged to near dryness with nitrogen, and then reconstituted with 200 μL of methanol in a vial.

[0043] (2) The concentration of TBBPA-BDBPE was analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0044] Add 55.6ng 13 C 12 TBBPA was used as an internal standard for injection. The concentration of TBBPA-BDBPE was detected using high-performance liquid chromatography-tandem mass spectrometry.

[0045] (3) Plotting the standard curve

[0046] Take a well-mixed blank serum sample and process it according to the pretreatment method described in step (1). After processing, use the obtained blank serum sample to prepare a series of concentration gradients of TBBPA-BDBPE and... 13 C 12 -TBBPA-BME standard curve, with 55.6ng added. 13 C 12 -TBBPA was used as an internal standard for injection, with serial concentrations of TBBPA-BDBPE and... 13 C 12 The peak areas of -TBBPA-BME are respectively with 13 C 12The peak area ratio of TBBPA was used to construct TBBPA-BDBPE quantitative standard curves and recovery standard curves to calculate the content of TBBPA-BDBPE in the sample. The TBBPA-BDBPE concentration gradients were 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 ng·g. -1 . 13 C 12 -TBBPA-BME concentration gradients of 10, 20, 50, 100, and 200 ng·g -1 The linear correlation coefficients of the obtained standard curves are all greater than 0.99.

[0047] (4) Recovery rate determination

[0048] Take the same blank serum sample as in (3), add a known mass of TBBPA-BDBPE, and follow the pretreatment method described in step (1) but without adding any additional TBBPA-BDBPE. 13 C 12 Sample pretreatment was performed using TBBPA-BME. After pretreatment, 55.6 ng of TBBPA-BME was added. 13 C 12 -TBBPA is used as an internal standard for injection.

[0049] The mass of TBBPA-BDBPE in the pretreated sample was calculated based on the standard curve plotted in (3), and the recovery rate was calculated by comparing it with the known mass added. The method recovery rate was calculated as follows:

[0050] R = (A / A0) × 100%

[0051] A - The mass of TBBPA-BDBPE obtained from the standard curve;

[0052] A0 - The mass of the known mass of TBBPA-BDBPE added;

[0053] R - Recovery rate (%).

[0054] The spiked recoveries of this pretreatment method and the spiked recoveries of different pretreatment methods are provided in the appendix to the instruction manual. Figure 3 The demonstration was conducted in triplicate, with precision expressed as relative standard deviation (RSD). Different pretreatment methods refer to the different extraction solutions used; other procedures are the same as this pretreatment method. Method 1 uses a mixture of acetone and n-hexane (9:1, v / v), Method 2 is this method, and Method 3 uses a mixture of acetone and n-hexane (6:4, v / v); the concentration used is 500 ng / mL. -1 .

[0055] (5) Determination of TBBPA-BDBPE concentration in serum samples

[0056] Pregnant female mice in their late stages were purchased from Vital River and exposed to TBBPA-BDBPE (at a concentration of 15 or 3000 ng / mL) via drinking water starting on day 0 after delivery. -1 The mice were exposed for 21 days (after weaning), and blood was collected from them. After standing for 1 hour, the blood was centrifuged at 4000 rpm for 20 minutes to collect serum. This invention was used to quantitatively study the TBBPA-BDBPE content in the serum. A process blank was added during sample processing; no TBBPA-BDBPE was detected in the process blank.

[0057] TBBPA-BDBPE was detected in serum samples from both exposure groups. (15 ng / mL) -1 In the exposure group, the serum TBBPA-BDBPE concentration was 3.97 ng / mL. -1 ; at 3000 ng·mL -1 In the exposure group, the serum TBBPA-BDBPE concentration was 7.91 ng / mL. -1 .

[0058] The embodiments of this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum, characterized in that, Includes the following steps: (1) 13 C 12 - Mix TBBPA-BME with serum, add small steel beads, and vortex to mix thoroughly; (2) Add a mixed solution of n-hexane and acetone, vortex for 30 s, and then extract using a tissue homogenizer. The volume ratio of n-hexane to acetone in the mixed solution of n-hexane and acetone is 8:

2. (3) After centrifugation, extract the supernatant and repeat the extraction and centrifugation three times; (4) Continue to add the mixed solution of n-hexane and dichloromethane, use a tissue homogenizer to extract by shaking, centrifuge, extract the supernatant, and repeat the extraction and centrifugation three times; the volume ratio of the mixed solution of n-hexane and dichloromethane is 1:

1. (5) Collect the supernatant after six centrifugations, blow it with nitrogen until it is nearly dry using a nitrogen blower, and redissolve it in a mixed solution of n-hexane and dichloromethane; (6) Supelclean ENVI-Carb was activated with n-hexane and dichloromethane respectively. TM Solid phase extraction column: The reconstituted mixed solution obtained in step (5) is loaded onto the solid phase extraction column, and then purified and eluted using a mixed solution of n-hexane and dichloromethane; (7) After collecting the purified solution and blowing it to near dryness with nitrogen, replace the solvent with methanol and transfer it to a vial; (8) After reconstitution, add 13 C 12 -TBBPA was used as an internal standard for injection, and the concentration of tetrabromobisphenol A bis(2,3-dibromopropyl ether) was detected by high performance liquid chromatography-tandem mass spectrometry.

2. The pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum as described in claim 1, characterized in that: The extraction method used was oscillatory extraction, in which small steel balls with a diameter of 4 mm were used.

3. A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum as described in claim 1 or 2, characterized in that: The tissue homogenizer used in the oscillation extraction had an oscillation frequency of 30 Hz and an extraction time of 4 min.

4. A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum as described in claim 1 or 2, characterized in that: Centrifuge to extract the supernatant at 9000 rpm for 10 min.

5. A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum as described in claim 1 or 2, characterized in that: The solutions used to activate the solid-phase extraction column were 6 mL of dichloromethane and 6 mL of n-hexane, and the solution used to elute the solid-phase extraction column was a 12 mL mixture of n-hexane and dichloromethane in a 1:1 volume ratio.

6. A pretreatment method for analyzing tetrabromobisphenol A bis(2,3-dibromopropyl ether) in serum as described in claim 1 or 2, characterized in that: The internal standard added during pretreatment was 58.4 ng. 13 C 12 -TBBPA-BME; 55.6 ng of internal standard was added to reduce errors caused by instrument fluctuations. 13 C 12 -TBBPA.

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