Simultaneous pre-treatment and detection method of bisphenol s and bisphenol af in biological tissues

Abstract

This invention relates to the fields of environmental and bioanalytical technology, and particularly to a method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues. The technical solution includes a framework process for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues. This invention establishes a dual-solvent secondary extraction system composed of n-hexane and methyl tert-butyl ether, achieving differentiated adaptation to different tissue matrices. By combining the advantages of non-polar and moderately polar solvents, it can simultaneously handle the relatively highly polar bisphenol S and the relatively non-polar bisphenol AF in a single extraction process. Furthermore, this method sets key physical parameters during the extraction process according to the histological characteristics of each organ. For heart and lung tissues, specific ultrasonic power is used to ensure thorough cell disruption; while for liver and spleen with high lipid content, different power and time are used to ensure the full release of lipid-bound target substances.

Description

Technical Field

[0001] This invention relates to the fields of environmental and biological analysis technology, and in particular to a method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues. Background Technology

[0002] Bisphenol S and bisphenol AF, as major alternatives to bisphenol A, have seen a continuous increase in their detection rates in environmental media and in vivo in recent years. Patent document CN202310119051.5 discloses a pretreatment method for tetrabromobisphenol A and its derivatives in sludge, including freeze drying, oscillating grinding extraction, and solid phase extraction purification. However, this method is designed for environmental sludge matrix, with sample amounts up to 0.5g, while the sample amount for a single tissue in animal toxicology experiments is usually only 25-50mg. Direct application of this method would lead to insufficient detection sensitivity.

[0003] Existing technologies for treating bisphenol compounds in different tissues of rats, such as the heart, liver, spleen, lungs, and kidneys, often rely on pretreatment methods based on a single matrix or specific sample types. This fails to effectively address the inherent tissue heterogeneity within biological samples. Heart tissue is rich in myofibrillar proteins, liver tissue has a high lipid content, and the spleen contains a large number of immune cells and nucleic acids. These different biological matrices have varying tolerances and release efficiencies to the same extraction solvent. However, traditional homogenization-liquid-liquid extraction methods typically use a fixed solvent system, which can lead to incomplete extraction and low recovery rates in lipid-rich liver tissue due to lipid encapsulation. In protein-rich heart tissue, protein denaturation and precipitation can encapsulate the target compound, causing fluctuations in recovery rates. This results in a lack of comparability of toxicokinetic data from different organs, hindering in-depth research on the cardiotoxicity mechanisms of bisphenol S and bisphenol AF. Summary of the Invention

[0004] To overcome the problem that existing technologies often rely on a single matrix or a specific type of sample for pretreatment when processing bisphenol compounds in different tissues such as rat heart, liver, spleen, lung, and kidney, which fails to effectively address the inherent tissue heterogeneity within biological samples.

[0005] The technical solution of this invention is: a method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues, comprising the following steps: S11: After euthanizing the exposed rats, the five organs of heart, liver, spleen, lung and kidney were separated. After rinsing with physiological saline and drying with filter paper, 30±5mg of tissue was weighed and aliquoted into cryovials. After being flash-frozen in liquid nitrogen, the tissues were transferred to a freezer at -80℃ for long-term storage. S12: Place the frozen tissue in a freeze dryer at -50℃ and 10Pa for 48 hours until constant weight. After equilibration in the desiccator, weigh and calculate the water content. Seal the tissue for later use when the water content is less than 5%. S13: Weigh 25 mg of dried tissue into a glass centrifuge tube, add 300 μL of methanol-ammonium acetate mixed solution and zirconium oxide grinding beads, grind at 50 Hz for 120 seconds at 4 °C and repeat twice. S14: Add a mixed solvent of n-hexane and methyl tert-butyl ether and an isotope internal standard to the homogenate. After vortex mixing, ultrasonic extraction and centrifugation, collect the upper organic phase. S15: Add n-hexane and methyl tert-butyl ether to the remaining aqueous phase after the first extraction, repeat the vortexing, sonication and centrifugation operations, and combine the upper organic phases obtained from the two extractions. S16: The combined organic phase was concentrated to 200 μL by nitrogen blowing, and PSA powder was added and vortexed for dispersion solid-phase extraction. After filtration through a filter membrane, it was bleached to near dryness again by nitrogen blowing. S17: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness with nitrogen to reconstitute the solution, vortex mix, transfer to a sample vial, seal and label, and store at 4°C until analysis is performed; S18: Set up a C18 column, a mobile phase gradient containing ammonium acetate, and ESI negative ion mode. Optimize MRM parameters. Inject 2 μL of sample and run for 8 minutes to complete chromatographic separation and mass spectrometry detection. S19: Perform peak area integration on the detection results, calculate the concentrations of BPS and BPAF based on the standard curve, and ensure that the recovery rate and precision meet the requirements through blank control, spiked recovery and quality control samples, and statistically analyze the organ distribution data.

[0006] Preferably, the collection and preservation of rat tissue samples includes the following steps: S21: SPF-grade SD rats were selected. After completing the bisphenol S and bisphenol AF exposure experiments, the rats were euthanized by CO2 asphyxiation. The heart, liver, spleen, lungs and kidneys were immediately dissected and separated. S22: Rinse the tissue surface three times repeatedly with pre-cooled physiological saline at 4℃ to remove blood and hair impurities, and then blot dry the tissue surface with sterile filter paper. S23: Weigh 30±5mg of each organ tissue and record the actual mass using a precision electronic balance; S24: Transfer the weighed tissue sample to a pre-labeled 2mL sterile cryovial, seal it, and immediately place it in liquid nitrogen for rapid freezing. S25: Transfer the quick-frozen tissue samples to an ultra-low temperature freezer at -80℃ for storage, and the storage time shall not exceed three months.

[0007] Preferably, the freeze-drying process for tissue samples includes the following steps: S31: Remove the tissue sample from the cryopreservation tube from the -80°C freezer and immediately place it in a freeze dryer pre-cooled to -50°C; S32: Set the vacuum degree of the freeze dryer to 10Pa and freeze dry continuously for 48 hours until the tissue sample mass no longer changes and reaches a constant weight state; S33: After drying, remove the sample and place it in a desiccator for 30 minutes to equilibrate and return to room temperature; S34: Weigh the dried tissue sample using an analytical balance and calculate the moisture content; S35: Seal and store the dried tissue sample in a desiccator for later use.

[0008] Preferably, the grinding and homogenization of tissue samples includes the following steps: S41: Weigh 25mg of dried tissue sample and transfer it to a 2mL glass centrifuge tube; S42: Add 300 μL of methanol-ammonium acetate mixed solution to the centrifuge tube. The volume ratio of methanol to 0.1M ammonium acetate is 3:7. Adjust the pH value to 4.75 with formic acid. S43: Add a 3mm diameter zirconium oxide grinding bead to the centrifuge tube; S44: Place the centrifuge tube in a fully automatic sample grinder, set the frequency to 50Hz, grind for 120 seconds at 4℃, and repeat twice. S45: After grinding, place the centrifuge tubes on ice and let them stand for 2 minutes.

[0009] Preferably, the first liquid-liquid extraction process includes the following steps: S51: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the centrifuge tube after homogenization to form a dual solvent extraction system; S52: Add 10 μL of mixed internal standard solution containing BPS- to the centrifuge tube. 13 C 12 and BPAF- 13 C 12 The final concentrations were 250 ng / mL. S53: Place the centrifuge tube on a vortex mixer and vortex at 2500 rpm for 1 minute; S54: Place the centrifuge tube in the ultrasonic extractor, set the ultrasonic power to 100W or 150W, and the ultrasonic time to 5 minutes or 8 minutes. S55: After sonication, centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, remove the upper organic phase and transfer it to a new centrifuge tube.

[0010] Preferably, the second liquid-liquid extraction process includes the following steps: S61: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the aqueous phase remaining after the first extraction; S62: Repeat the vortex mixing operation, vortexing at 2500 rpm for 1 minute; S63: Perform ultrasonic extraction again, with the power and time settings the same as the first time; S64: Centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, collect the upper organic phase, and combine it with the organic phase obtained from the first extraction; S65: The combined organic phases are temporarily stored on ice.

[0011] Preferably, the concentration and purification of the organic phase includes the following steps: S71: Transfer the combined organic phase to a nitrogen blower, set the water bath temperature to 40℃, and the nitrogen flow rate to a moderate level, and concentrate it to 200μL. S72: Add 40 mg of PSA powder to the concentrated solution as a dispersible solid-phase extraction adsorbent to remove lipids and proteins; S73: Place the centrifuge tube on a vortex mixer and vortex for 2 minutes; S74: Filter the mixture through a 0.22μm organic filter membrane to remove PSA powder and insoluble impurities; S75: Collect the filtrate in a new centrifuge tube and dry it again in a nitrogen blower until it is nearly dry.

[0012] Preferably, the sample reconstitution and transfer process includes the following steps: S81: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness by nitrogen as a reconstitution solvent; S82: Place the centrifuge tube on a vortex mixer and vortex for 30 seconds; S83: Transfer the reconstituted solution to a vial with an inner tube to avoid the generation of air bubbles; S84: Tightly cap the sample vial and mark the sample number and date information; S85: Store the sample vial at 4°C until it is ready for instrument testing. The storage time should not exceed 24 hours.

[0013] Preferably, the setting and operation of UPLC-MS / MS detection conditions include the following steps: S91: The ultra-high performance liquid chromatography column was set to Phenomenex Luna PS C18, the column temperature was 40℃, and the flow rate was 0.4 mL / min; S92: Set mobile phase A to ultrapure water containing 0.2 mM ammonium acetate and mobile phase B to methanol. Run according to the gradient program: 0-1 min, 30% B; 1-4 min, 30 → 95% B; 4-6 min, 95% B; 6-6.1 min, 95 → 30% B; 6.1-8 min, 30% B. S93: Set the mass spectrometer to ESI negative ion mode, ion source temperature 550℃, spray voltage -4600V, curtain gas 35psi, collision gas 9psi; S94: Set MRM parameters: BPS precursor ion 249.0, quantitative ion 108.1; BPAF precursor ion 335.1, quantitative ion 200.0; synchronous detection of internal standard corresponding ion pairs; S95: The injection volume is 2 μL, and the running time for each sample is 8 minutes.

[0014] Preferably, the data processing and quality control analysis includes the following steps: S101: Use analysis software to integrate the peak area of ​​the detection results and record the peak area ratio of the target analyte to the internal standard; S102: Calculate the concentrations of BPS and BPAF in the sample based on the standard curve equation. The standard curve range is 0.05-500 ng / mL, and the linear correlation coefficient R is... 2 ≥0.99; S103: Each batch of samples shall include a blank control, a spiked recovery sample, and a quality control sample; S104: Calculate the spiked recovery and precision, requiring a recovery rate between 85-110% and an RSD of less than 10%; S105: Statistically analyze the concentration distribution of BPS and BPAF in various organs and tissues, and draw organ distribution maps.

[0015] The beneficial effects of this invention are: Existing technologies for treating bisphenol compounds from different tissues such as rat heart, liver, spleen, lung, and kidney often rely on pretreatment protocols based on a single matrix or specific sample types. This fails to effectively address the inherent tissue heterogeneity within biological samples. Heart tissue is rich in myofibrillar proteins, liver tissue has a high lipid content, and the spleen contains numerous immune cells and nucleic acids. These different biological matrices exhibit varying tolerances and release efficiencies to the same extraction solvent. However, traditional homogenization-liquid-liquid extraction methods typically use a fixed solvent system. This leads to incomplete extraction and low recovery rates in lipid-rich liver tissue due to lipid encapsulation. Conversely, in protein-rich heart tissue, protein denaturation and precipitation may encapsulate the target compound, causing fluctuations in recovery rates. Consequently, toxicokinetic data from different organs lack comparability, impacting the effectiveness of toxicokinetics analysis. This study delves into the cardiotoxicity mechanisms of bisphenol S (BPS) and bisphenol AF (BAF). A novel two-stage extraction system using hexane and methyl tert-butyl ether (MTBE) was developed, achieving differentiated adaptation to various tissue matrices. By combining the advantages of non-polar and moderately polar solvents, this method can simultaneously extract both the relatively polar BPS and the relatively non-polar BAF in a single extraction process. Furthermore, the method sets key physical parameters during extraction based on the histological characteristics of each organ. Specific ultrasonic power is used for the heart and lung tissues to ensure thorough cell disruption; while different power and time are employed for the liver and spleen, which have high lipid content, to ensure the full release of lipid-bound target compounds. This approach achieves stable and efficient extraction of BPS and BAF in five different organ tissues, significantly reducing the differences in recovery rates between tissues. Attached Figure Description

[0016] Figure 1 The diagram shows the framework flow of the method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to the present invention. Figure 2 The diagram shows the collection and preservation process of rat tissue samples for the simultaneous pretreatment and detection method of bisphenol S and bisphenol AF in biological tissues according to the present invention. Figure 3 The diagram shows a freeze-drying process of tissue samples for the simultaneous pretreatment and detection method of bisphenol S and bisphenol AF in biological tissues according to the present invention. Figure 4 The diagram shows the process flow of grinding and homogenizing tissue samples for the simultaneous pretreatment and detection method of bisphenol S and bisphenol AF in biological tissues according to the present invention. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Please see Figure 1-4This invention provides an embodiment of a method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues, comprising the following steps: S11: After euthanizing the exposed rats, the five organs of heart, liver, spleen, lung and kidney were separated. After rinsing with physiological saline and drying with filter paper, 30±5mg of tissue was weighed and aliquoted into cryovials. After being flash-frozen in liquid nitrogen, the tissues were transferred to a freezer at -80℃ for long-term storage. S12: Place the frozen tissue in a freeze dryer at -50℃ and 10Pa for 48 hours until constant weight. After equilibration in the desiccator, weigh and calculate the water content. Seal the tissue for later use when the water content is less than 5%. S13: Weigh 25 mg of dried tissue into a glass centrifuge tube, add 300 μL of methanol-ammonium acetate mixed solution and zirconium oxide grinding beads, grind at 50 Hz for 120 seconds at 4 °C and repeat twice. S14: Add a mixed solvent of n-hexane and methyl tert-butyl ether and an isotope internal standard to the homogenate. After vortex mixing, ultrasonic extraction and centrifugation, collect the upper organic phase. S15: Add n-hexane and methyl tert-butyl ether to the remaining aqueous phase after the first extraction, repeat the vortexing, sonication and centrifugation operations, and combine the upper organic phases obtained from the two extractions. S16: The combined organic phase was concentrated to 200 μL by nitrogen blowing, and PSA powder was added and vortexed for dispersion solid-phase extraction. After filtration through a filter membrane, it was bleached to near dryness again by nitrogen blowing. S17: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness with nitrogen to reconstitute the solution, vortex mix, transfer to a sample vial, seal and label, and store at 4°C until analysis is performed; S18: Set up a C18 column, a mobile phase gradient containing ammonium acetate, and ESI negative ion mode. Optimize MRM parameters. Inject 2 μL of sample and run for 8 minutes to complete chromatographic separation and mass spectrometry detection. S19: Perform peak area integration on the detection results, calculate the concentrations of BPS and BPAF based on the standard curve, and ensure that the recovery rate and precision meet the requirements through blank control, spiked recovery and quality control samples, and statistically analyze the organ distribution data.

[0019] Preferably, the collection and preservation of rat tissue samples includes the following steps: S21: SPF-grade SD rats were selected. After completing the bisphenol S and bisphenol AF exposure experiments, the rats were euthanized by CO2 asphyxiation. The heart, liver, spleen, lungs and kidneys were immediately dissected and separated. S22: Rinse the tissue surface three times repeatedly with pre-cooled physiological saline at 4℃ to remove blood and hair impurities, and then blot dry the tissue surface with sterile filter paper. S23: Weigh 30±5mg of each organ tissue and record the actual mass using a precision electronic balance; S24: Transfer the weighed tissue sample to a pre-labeled 2mL sterile cryovial, seal it, and immediately place it in liquid nitrogen for rapid freezing. S25: Transfer the quick-frozen tissue samples to an ultra-low temperature freezer at -80℃ for storage, and the storage time shall not exceed three months.

[0020] Preferably, the freeze-drying process for tissue samples includes the following steps: S31: Remove the tissue sample from the cryopreservation tube from the -80°C freezer and immediately place it in a freeze dryer pre-cooled to -50°C; S32: Set the vacuum degree of the freeze dryer to 10Pa and freeze dry continuously for 48 hours until the tissue sample mass no longer changes and reaches a constant weight state; S33: After drying, remove the sample and place it in a desiccator for 30 minutes to equilibrate and return to room temperature; S34: Weigh the dried tissue sample using an analytical balance and calculate the moisture content; S35: Seal and store the dried tissue sample in a desiccator for later use.

[0021] Preferably, the grinding and homogenization of tissue samples includes the following steps: S41: Weigh 25mg of dried tissue sample and transfer it to a 2mL glass centrifuge tube; S42: Add 300 μL of methanol-ammonium acetate mixed solution to the centrifuge tube. The volume ratio of methanol to 0.1M ammonium acetate is 3:7. Adjust the pH value to 4.75 with formic acid. S43: Add a 3mm diameter zirconium oxide grinding bead to the centrifuge tube; S44: Place the centrifuge tube in a fully automatic sample grinder, set the frequency to 50Hz, grind for 120 seconds at 4℃, and repeat twice. S45: After grinding, place the centrifuge tubes on ice and let them stand for 2 minutes.

[0022] Preferably, the first liquid-liquid extraction process includes the following steps: S51: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the centrifuge tube after homogenization to form a dual solvent extraction system; S52: Add 10 μL of mixed internal standard solution containing BPS- to the centrifuge tube. 13 C 12 and BPAF- 13 C 12 The final concentrations were 250 ng / mL. S53: Place the centrifuge tube on a vortex mixer and vortex at 2500 rpm for 1 minute; S54: Place the centrifuge tube in the ultrasonic extractor, set the ultrasonic power to 100W or 150W, and the ultrasonic time to 5 minutes or 8 minutes. S55: After sonication, centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, remove the upper organic phase and transfer it to a new centrifuge tube.

[0023] Preferably, the second liquid-liquid extraction process includes the following steps: S61: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the aqueous phase remaining after the first extraction; S62: Repeat the vortex mixing operation, vortexing at 2500 rpm for 1 minute; S63: Perform ultrasonic extraction again, with the power and time settings the same as the first time; S64: Centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, collect the upper organic phase, and combine it with the organic phase obtained from the first extraction; S65: The combined organic phases are temporarily stored on ice.

[0024] Preferably, the concentration and purification of the organic phase includes the following steps: S71: Transfer the combined organic phase to a nitrogen blower, set the water bath temperature to 40℃, and the nitrogen flow rate to a moderate level, and concentrate it to 200μL. S72: Add 40 mg of PSA powder to the concentrated solution as a dispersible solid-phase extraction adsorbent to remove lipids and proteins; S73: Place the centrifuge tube on a vortex mixer and vortex for 2 minutes; S74: Filter the mixture through a 0.22μm organic filter membrane to remove PSA powder and insoluble impurities; S75: Collect the filtrate in a new centrifuge tube and dry it again in a nitrogen blower until it is nearly dry.

[0025] Preferably, the sample reconstitution and transfer process includes the following steps: S81: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness by nitrogen as a reconstitution solvent; S82: Place the centrifuge tube on a vortex mixer and vortex for 30 seconds; S83: Transfer the reconstituted solution to a vial with an inner tube to avoid the generation of air bubbles; S84: Tightly cap the sample vial and mark the sample number and date information; S85: Store the sample vial at 4°C until it is ready for instrument testing. The storage time should not exceed 24 hours.

[0026] Preferably, the setting and operation of UPLC-MS / MS detection conditions include the following steps: S91: The ultra-high performance liquid chromatography column was set to Phenomenex Luna PS C18, the column temperature was 40℃, and the flow rate was 0.4 mL / min; S92: Set mobile phase A to ultrapure water containing 0.2 mM ammonium acetate and mobile phase B to methanol. Run according to the gradient program: 0-1 min, 30% B; 1-4 min, 30 → 95% B; 4-6 min, 95% B; 6-6.1 min, 95 → 30% B; 6.1-8 min, 30% B. S93: Set the mass spectrometer to ESI negative ion mode, ion source temperature 550℃, spray voltage -4600V, curtain gas 35psi, collision gas 9psi; S94: Set MRM parameters: BPS precursor ion 249.0, quantitative ion 108.1; BPAF precursor ion 335.1, quantitative ion 200.0; synchronous detection of internal standard corresponding ion pairs; S95: The injection volume is 2 μL, and the running time for each sample is 8 minutes.

[0027] Preferably, the data processing and quality control analysis includes the following steps: S101: Use analysis software to integrate the peak area of ​​the detection results and record the peak area ratio of the target analyte to the internal standard; S102: Calculate the concentrations of BPS and BPAF in the sample based on the standard curve equation. The standard curve range is 0.05-500 ng / mL, and the linear correlation coefficient R is... 2 ≥0.99; S103: Each batch of samples shall include a blank control, a spiked recovery sample, and a quality control sample; S104: Calculate the spiked recovery and precision, requiring a recovery rate between 85-110% and an RSD of less than 10%; S105: Statistically analyze the concentration distribution of BPS and BPAF in various organs and tissues, and draw organ distribution maps.

[0028] Example 1 SPF-grade healthy adult male Sprague Dawley rats were purchased from a medical experimental animal center to study the tissue distribution patterns of bisphenol S and bisphenol AF after oral exposure. Forty-two rats were randomly divided into seven groups of six each, including a control group, a low-dose BPS group, a high-dose BPS group, a low-dose BPAF group, a high-dose BPAF group, a low-dose BPS+BPAF combined group, and a high-dose BPS+BPAF combined group. Each group was given the corresponding dose of the exposure substance by gavage at 9:00 AM every day for 31 consecutive days. During the experiment, the rats' diet, water intake, and behavioral status were observed.

[0029] Twenty-four hours after the last exposure, rats were euthanized by CO2 asphyxiation. The heart, liver, spleen, lungs, and kidneys were immediately dissected and separated. The tissue surfaces were rinsed three times with pre-cooled physiological saline at 4°C to remove blood, hair, and other impurities. The surface moisture was blotted dry with sterile filter paper. 30±5mg of each organ tissue was weighed, and the actual mass was recorded using a precision electronic balance. The samples were transferred to pre-labeled 2mL sterile cryovials, sealed, and immediately flash-frozen in liquid nitrogen. They were then transferred to an ultra-low temperature freezer at -80°C for storage for no more than three months.

[0030] Remove the tissue sample from the cryopreservation tube from the -80℃ freezer and immediately place it in a freeze dryer pre-cooled to -50℃. Set the vacuum degree to 10Pa and freeze dry for 48 hours until the tissue sample mass no longer changes and reaches a constant weight state. After drying, remove the sample and place it in a desiccator for 30 minutes to equilibrate to room temperature. Use an analytical balance to weigh the dried tissue sample and calculate the water content to ensure that the tissue water content is less than 5%. Then, seal the dried tissue sample and store it in a desiccator for later use.

[0031] Accurately weigh 25 mg of dried tissue sample, accurate to 0.0001 g, and transfer it to a 2 mL glass centrifuge tube. Add 300 μL of methanol-ammonium acetate mixture to the centrifuge tube, with a methanol to 0.1 M ammonium acetate volume ratio of 3:7. Adjust the pH to 4.75 with formic acid. Add a 3 mm diameter zirconia grinding bead to the centrifuge tube. Place the centrifuge tube in an automated sample grinder, set the frequency to 50 Hz, and grind for 120 seconds at 4 °C. Repeat twice to ensure complete tissue disruption. After grinding, place the centrifuge tube on ice for 2 minutes to prevent sample overheating and degradation of the target analyte.

[0032] Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the homogenized centrifuge tube to form a dual-solvent extraction system. Add 10 μL of a mixed internal standard solution containing BPS- to the centrifuge tube. 13 C 12 and BPAF- 13 C 12The final concentrations were 250 ng / mL. The centrifuge tubes were placed on a vortex mixer and vortexed at 2500 rpm for 1 minute. Then, they were placed in an ultrasonic extractor with an ultrasonic power of 100 W or 150 W and an ultrasonic time of 5 or 8 minutes. After ultrasonication, the centrifuge tubes were centrifuged at 3000 rpm for 5 minutes at 4°C. The upper organic phase was then transferred to a new centrifuge tube.

[0033] Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the aqueous phase remaining after the first extraction, repeat the vortex mixing operation at 2500 rpm for 1 minute, and perform ultrasonic extraction again with the same power and time settings as the first time. Centrifuge the tube at 3000 rpm for 5 minutes at 4°C, collect the upper organic phase and combine it with the organic phase obtained from the first extraction, and temporarily store the combined organic phase on ice.

[0034] The combined organic phase was transferred to a nitrogen evaporator, the water bath temperature was set to 40℃ and the nitrogen flow rate was moderate, and the solution was concentrated to 200μL. 40mg of PSA powder was added to the concentrated solution as a dispersing solid-phase extraction adsorbent to remove lipids and proteins. The centrifuge tube was placed on a vortex mixer and vortexed for 2 minutes. The mixture was then filtered through a 0.22μm organic filter membrane to remove PSA powder and insoluble impurities. The filtrate was collected in a new centrifuge tube and dried again in a nitrogen evaporator until nearly dry.

[0035] Add 50 μL of chromatographically pure methanol as a reconstitution solvent to a centrifuge tube that has been nearly dried by nitrogen. Place the centrifuge tube on a vortex mixer and vortex for 30 seconds to ensure that the residue is completely dissolved. Transfer the reconstituted solution to a sample vial with an inner tube to avoid the generation of air bubbles. Tightly cap the sample vial and mark it with the sample number and date information. Store the sample vial at 4°C for later use in the instrument. The storage time should not exceed 24 hours.

[0036] The ultra-high performance liquid chromatography (UHPLC) column was a Phenomenex Luna PS C18, with a column temperature of 40℃ and a flow rate of 0.4 mL / min. Mobile phase A was ultrapure water containing 0.2 mM ammonium acetate, and mobile phase B was methanol. A gradient program was used: 0-1 min 30% B, 1-4 min 30% to 95% B, 4-6 min 95% B, 6-6.1 min 95% to 30% B, and 6.1-8 min 30% B. The mass spectrometer was set to ESI negative ion mode, with an ion source temperature of 550℃, a spray voltage of -4600V, a curtain gas pressure of 35 psi, and a collision gas pressure of 9 psi. MRM parameters were set as follows: BPS precursor ion 249.0, quantitative ion 108.1; BPAF precursor ion 335.1, quantitative ion 200.0. Internal standard ion pairs were detected simultaneously. The injection volume was 2 μL, and the run time for each sample was 8 minutes.

[0037] The peak area of ​​the detection results was integrated using analytical software, and the peak area ratio of the target analyte to the internal standard was recorded. The concentrations of BPS and BPAF in the sample were calculated based on the standard curve equation. The standard curve range was 0.05-500 ng / mL, and the linear correlation coefficient R0 was calculated. 2 For samples with a concentration ≥0.99, blank control, spiked recovery sample and quality control sample are set up in each batch of samples. The spiked recovery rate and precision are calculated. The recovery rate is required to be between 85-110% and the RSD is less than 10%. The concentration distribution of BPS and BPAF in each organ tissue is statistically analyzed and organ distribution map is drawn.

[0038] This method was used to perform multi-organ assays on 36 SD rats orally exposed to environmental doses of BPS and BPAF. The results showed that BPS accumulated most significantly in the spleen (89.24±7.15 ng / g), followed by the lungs (51.37±5.82 ng / g), liver (42.16±6.33 ng / g), kidneys (2.18±0.35 ng / g), and heart (1.29±0.22 ng / g). BPAF accumulated significantly in the heart (2.01±0.15 ng / g) and in the spleen. The mean recoveries of the method in 36 actual sample batches were 1.84±0.21 ng / g in the middle, 1.92±0.18 ng / g in the lungs, 1.15±0.14 ng / g in the liver, and 0.67±0.09 ng / g in the kidneys. The average recoveries of the method were 92.3%±4.7% for BPS and 90.8%±5.1% for BPAF. The matrix effect was within the range of -12% to +8% for all tissue samples. The intra-day precision RSD was less than 7.2%, and the inter-day precision RSD was less than 9.5%.

[0039] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues; characterized in that: It includes the following steps: S11: After euthanizing the exposed rats, the five organs of heart, liver, spleen, lung and kidney were separated. After rinsing with physiological saline and drying with filter paper, 30±5mg of tissue was weighed and aliquoted into cryovials. After being flash-frozen in liquid nitrogen, the tissues were transferred to a freezer at -80℃ for long-term storage. S12: Place the frozen tissue in a freeze dryer at -50℃ and 10Pa for 48 hours until constant weight. After equilibration in the desiccator, weigh and calculate the water content. Seal the tissue for later use when the water content is less than 5%. S13: Weigh 25 mg of dried tissue into a glass centrifuge tube, add 300 μL of methanol-ammonium acetate mixed solution and zirconium oxide grinding beads, grind at 50 Hz for 120 seconds at 4 °C and repeat twice. S14: Add a mixed solvent of n-hexane and methyl tert-butyl ether and an isotope internal standard to the homogenate. After vortex mixing, ultrasonic extraction and centrifugation, collect the upper organic phase. S15: Add n-hexane and methyl tert-butyl ether to the remaining aqueous phase after the first extraction, repeat the vortexing, sonication and centrifugation operations, and combine the upper organic phases obtained from the two extractions. S16: The combined organic phase was concentrated to 200 μL by nitrogen blowing, and PSA powder was added and vortexed for dispersion solid-phase extraction. After filtration through a filter membrane, it was bleached to near dryness again by nitrogen blowing. S17: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness with nitrogen to reconstitute the solution, vortex mix, transfer to a sample vial, seal and label, and store at 4°C until analysis is performed; S18: Set up a C18 column, a mobile phase gradient containing ammonium acetate, and ESI negative ion mode. Optimize MRM parameters. Inject 2 μL of sample and run for 8 minutes to complete chromatographic separation and mass spectrometry detection. S19: Perform peak area integration on the detection results, calculate the concentrations of BPS and BPAF based on the standard curve, and ensure that the recovery rate and precision meet the requirements through blank control, spiked recovery and quality control samples, and statistically analyze the organ distribution data.

2. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The following steps are included in the collection and preservation of rat tissue samples: S21: SPF-grade SD rats were selected. After completing the bisphenol S and bisphenol AF exposure experiments, the rats were euthanized by CO2 asphyxiation. The heart, liver, spleen, lungs and kidneys were immediately dissected and separated. S22: Rinse the tissue surface three times repeatedly with pre-cooled physiological saline at 4℃ to remove blood and hair impurities, and then blot dry the tissue surface with sterile filter paper. S23: Weigh 30±5mg of each organ tissue and record the actual mass using a precision electronic balance; S24: Transfer the weighed tissue sample to a pre-labeled 2mL sterile cryovial, seal it, and immediately place it in liquid nitrogen for rapid freezing. S25: Transfer the quick-frozen tissue samples to an ultra-low temperature freezer at -80℃ for storage, and the storage time shall not exceed three months.

3. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The freeze-drying process for tissue samples includes the following steps: S31: Remove the tissue sample from the cryopreservation tube from the -80°C freezer and immediately place it in a freeze dryer pre-cooled to -50°C; S32: Set the vacuum degree of the freeze dryer to 10Pa and freeze dry continuously for 48 hours until the tissue sample mass no longer changes and reaches a constant weight state; S33: After drying, remove the sample and place it in a desiccator for 30 minutes to equilibrate and return to room temperature; S34: Weigh the dried tissue sample using an analytical balance and calculate the moisture content; S35: Seal and store the dried tissue sample in a desiccator for later use.

4. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The following steps are included in the grinding and homogenization of tissue samples: S41: Weigh 25mg of dried tissue sample and transfer it to a 2mL glass centrifuge tube; S42: Add 300 μL of methanol-ammonium acetate mixed solution to the centrifuge tube. The volume ratio of methanol to 0.1M ammonium acetate is 3:

7. Adjust the pH value to 4.75 with formic acid. S43: Add a 3mm diameter zirconium oxide grinding bead to the centrifuge tube; S44: Place the centrifuge tube in a fully automatic sample grinder, set the frequency to 50Hz, grind for 120 seconds at 4℃, and repeat twice. S45: After grinding, place the centrifuge tubes on ice and let them stand for 2 minutes.

5. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The first liquid-liquid extraction process includes the following steps: S51: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the centrifuge tube after homogenization to form a dual solvent extraction system; S52: Add 10 μL of mixed internal standard solution containing BPS- to the centrifuge tube. 13 C 12 and BPAF- 13 C 12 The final concentrations were 250 ng / mL. S53: Place the centrifuge tube on a vortex mixer and vortex at 2500 rpm for 1 minute; S54: Place the centrifuge tube in the ultrasonic extractor, set the ultrasonic power to 100W or 150W, and the ultrasonic time to 5 minutes or 8 minutes. S55: After sonication, centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, remove the upper organic phase and transfer it to a new centrifuge tube.

6. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The second liquid-liquid extraction process includes the following steps: S61: Add 200 μL of n-hexane and 200 μL of methyl tert-butyl ether to the aqueous phase remaining after the first extraction; S62: Repeat the vortex mixing operation, vortexing at 2500 rpm for 1 minute; S63: Perform ultrasonic extraction again, with the power and time settings the same as the first time; S64: Centrifuge the centrifuge tube at 3000 rpm for 5 minutes at 4°C, collect the upper organic phase, and combine it with the organic phase obtained from the first extraction; S65: The combined organic phases are temporarily stored on ice.

7. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The concentration and purification of the organic phase includes the following steps: S71: Transfer the combined organic phase to a nitrogen blower, set the water bath temperature to 40℃, and the nitrogen flow rate to a moderate level, and concentrate it to 200μL. S72: Add 40 mg of PSA powder to the concentrated solution as a dispersible solid-phase extraction adsorbent to remove lipids and proteins; S73: Place the centrifuge tube on a vortex mixer and vortex for 2 minutes; S74: Filter the mixture through a 0.22μm organic filter membrane to remove PSA powder and insoluble impurities; S75: Collect the filtrate in a new centrifuge tube and dry it again in a nitrogen blower until it is nearly dry.

8. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The process of reconstitution and transfer of samples includes the following steps: S81: Add 50 μL of chromatographically pure methanol to a centrifuge tube that has been blown to near dryness by nitrogen as a reconstitution solvent; S82: Place the centrifuge tube on a vortex mixer and vortex for 30 seconds; S83: Transfer the reconstituted solution to a vial with an inner tube to avoid the generation of air bubbles; S84: Tightly cap the sample vial and mark the sample number and date information; S85: Store the sample vial at 4°C until it is ready for instrument testing. The storage time should not exceed 24 hours.

9. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The steps involved in setting and running UPLC-MS / MS detection conditions are as follows: S91: The ultra-high performance liquid chromatography column was set to Phenomenex Luna PS C18, the column temperature was 40℃, and the flow rate was 0.4 mL / min; S92: Set mobile phase A to ultrapure water containing 0.2 mM ammonium acetate and mobile phase B to methanol. Run according to the gradient program: 0-1 min, 30% B; 1-4 min, 30 → 95% B; 4-6 min, 95% B; 6-6.1 min, 95 → 30% B; 6.1-8 min, 30% B. S93: Set the mass spectrometer to ESI negative ion mode, ion source temperature 550℃, spray voltage -4600V, curtain gas 35psi, collision gas 9psi; S94: Set MRM parameters: BPS precursor ion 249.0, quantitative ion 108.1; BPAF precursor ion 335.1, quantitative ion 200.0; synchronous detection of internal standard corresponding ion pairs; S95: The injection volume is 2 μL, and the running time for each sample is 8 minutes.

10. The method for simultaneous pretreatment and detection of bisphenol S and bisphenol AF in biological tissues according to claim 1, characterized in that: The following steps are included in data processing and quality control analysis: S101: Use analysis software to integrate the peak area of ​​the detection results and record the peak area ratio of the target analyte to the internal standard; S102: Calculate the concentrations of BPS and BPAF in the sample based on the standard curve equation. The standard curve range is 0.05-500 ng / mL, and the linear correlation coefficient R is... 2 ≥0.99; S103: Each batch of samples shall include a blank control, a spiked recovery sample, and a quality control sample; S104: Calculate the spiked recovery and precision, requiring a recovery rate between 85-110% and an RSD of less than 10%; S105: Statistically analyze the concentration distribution of BPS and BPAF in various organs and tissues, and draw organ distribution maps.

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