Method for extracting 17 bisphenol compounds and application

Abstract

The invention discloses a method for extracting 17 bisphenol compounds and application, and the method comprises the following steps: fully mixing a buffer solution and a body fluid sample, fully extracting with a mixed reagent, and collecting an organic phase to obtain an extract of the 17 bisphenol compounds. According to the method, liquid-liquid extraction which is simple and efficient to operate, low in cost and good in repeatability is adopted as a body fluid sample pretreatment method and is combined with an isotope label method and an ultra-high performance liquid chromatography-tandem mass spectrometry technology, qualitative and quantitative detection and separation of 17 bisphenol compounds in different body fluid samples are achieved, the adding standard recovery rate is 70%-120%, and the detection sensitivity is high. Meanwhile, the method has the advantages of high sensitivity, small quantity of isotope internal standards and small solvent loss, meets actual detection requirements, and has higher simplicity and wider detection limit.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for extracting 17 bisphenol compounds and an application thereof. Background Art

[0002] Bisphenol A (BPA) is an environmental endocrine disruptor that can enter the human body through multiple pathways (such as breathing, eating, and skin contact), and has negative effects on the endocrine system and reproductive health. At the same time, some existing clinical data show that bisphenol compounds with similar structures to BPA may not be safe, including carcinogenicity, neurotoxicity, developmental and reproductive toxicity, etc.

[0003] At present, for bisphenol compounds in the human body, usually only one or a class of bisphenol compounds are detected, mainly focusing on several typical bisphenol compounds such as BPA, bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF), and most of the monitoring fields only detect bisphenol compounds in blood or urine. However, the monitoring of other bisphenol compounds and the monitoring projects of multiple bisphenol compounds in other body fluids such as semen have not been widely studied and applied. Therefore, in order to be able to detect multiple bisphenol compounds at the same time and expand the scope of detection projects, a simple and convenient method for detecting multiple bisphenol compounds in body fluids is urgently needed. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method for extracting 17 bisphenol compounds and application thereof.

[0005] The first object of the present invention is to provide a method for extracting 17 bisphenol compounds.

[0006] The second object of the present invention is to provide a method for obtaining the extracts of the 17 bisphenol compounds.

[0007] The third object of the present invention is to provide an application of the extracts of the 17 bisphenol compounds obtained by the method described in preparing a detection product for bisphenol compounds.

[0008] The fourth object of the present invention is to provide application of the method in detecting bisphenol compounds.

[0009] A fifth object of the present invention is to provide a method for detecting 17 bisphenol compounds in a body fluid sample.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] The present invention obtains a test sample for detecting 17 bisphenol compounds through a liquid-liquid extraction sample pretreatment extraction method, and then combines ultra-high performance liquid chromatography tandem mass spectrometry technology to successfully detect and separate the 17 bisphenol compounds in the body fluid sample.

[0012] A method for extracting 17 kinds of bisphenol compounds, wherein the bisphenol compounds are bisphenol A, bisphenol S, bisphenol AF, bisphenol AP, bisphenol BP, 2,4'-dihydroxydiphenyl sulfone, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-hydroxy-4'-benzyloxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, bisphenol TMC, bisphenol Z, bisphenol M, bisphenol P, 2,4-di(phenyl) The method comprises the following steps: fully mixing a buffer solution and a body fluid sample to obtain a mixture; then fully extracting the mixture with a mixed reagent, collecting an organic phase, and obtaining an extract of the 17 bisphenol compounds; the buffer solution is an ammonium acetate buffer containing a hydrolase; and the mixed reagent comprises methyl tert-butyl ether and ethyl acetate.

[0013] Preferably, the hydrolase is β-glucuronidase.

[0014] More preferably, the concentration of the β-glucuronidase is 72 to 74 units / mL.

[0015] Further preferably, the concentration of the β-glucuronidase is 73 units / mL.

[0016] Preferably, the concentration of the ammonium acetate buffer is 0.5-1 mM.

[0017] More preferably, the concentration of the ammonium acetate buffer is 1 mM.

[0018] Preferably, the body fluid sample is urine and / or semen.

[0019] More preferably, the body fluid sample is semen.

[0020] Preferably, the volume ratio of the buffer solution to the body fluid sample is (2-1):1.

[0021] More preferably, the volume ratio of the buffer solution to the body fluid sample is 1:1.

[0022] Preferably, the method for fully mixing the buffer solution and the body fluid sample comprises: first mixing the buffer solution and the body fluid sample, and then fully shaking them.

[0023] More preferably, the sufficient shaking condition includes shaking at 36-37° C. for 11-12 hours.

[0024] More preferably, the sufficient shaking condition includes shaking at 37° C. for 12 hours.

[0025] Preferably, in the mixed reagent, the volume ratio of methyl tert-butyl ether to ethyl acetate is (4-5):1.

[0026] More preferably, in the mixed reagent, the volume ratio of methyl tert-butyl ether to ethyl acetate is 5:1.

[0027] Preferably, the method for fully extracting the mixture with a mixed reagent includes: S1. After mixing the mixed solvent and the mixture in a volume ratio of 2:(0.4~1), vortexing and ultrasonic treatment are performed, centrifugation is performed to obtain stratified organic phase and aqueous phase, and the organic phase is collected; S2. After mixing the new mixed solvent and the aqueous phase obtained in step S1, vortexing and ultrasonic treatment are performed according to the method of step S1, centrifugation is performed to obtain stratified organic phase and aqueous phase, the organic phase is collected, and this step is repeated 1 to 3 times; S3. The organic phases obtained in step 1 and step S2 are fully mixed.

[0028] More preferably, in step S2, this step is repeated twice.

[0029] More preferably, in step S1, the mixed solvent and the mixture are mixed in a volume ratio of 2:0.6.

[0030] More preferably, the method further comprises the following steps: after purifying and drying the organic phase, fully dissolving the obtained solid with a 30-50 v / v% methanol aqueous solution, separating the solid from the liquid, and obtaining the obtained liquid as the extract.

[0031] Further preferably, the purification method comprises: washing and purifying with ultrapure water.

[0032] Further preferably, the drying method comprises: blowing with nitrogen until nearly dry.

[0033] More preferably, the obtained solid is fully dissolved in a 50 v / v% methanol aqueous solution.

[0034] Further preferably, the solid-liquid separation method comprises filtration.

[0035] Further preferably, the filter membrane used for the filtration is a 0.22 μm filter membrane.

[0036] The extracts of 17 bisphenol compounds obtained by any of the above methods should also be within the protection scope of the present invention.

[0037] The use of the extracts of 17 bisphenol compounds obtained by any of the above methods in the preparation of bisphenol compound detection products should also be within the protection scope of the present invention.

[0038] The application of any of the above methods in detecting bisphenol compounds should also be within the protection scope of the present invention.

[0039] A method for detecting 17 bisphenol compounds in a body fluid sample, comprising the following steps: extracting the body fluid sample using the method described in claim 1 to obtain an extract of the 17 bisphenol compounds, and then detecting the extract of the 17 bisphenol compounds using ultra-high performance liquid chromatography.

[0040] Preferably, the method further comprises the following steps: fully mixing the internal standard, the buffer solution and the body fluid sample to obtain a spiked mixture; then fully extracting the spiked mixture with the mixed reagent, collecting the organic phase to obtain a test sample; and then detecting the test sample with ultra-high performance liquid chromatography tandem mass spectrometry.

[0041] More preferably, the internal standard is isotope-labeled bisphenol A, isotope-labeled bisphenol S and isotope-labeled bisphenol AF.

[0042] Further preferably, the isotope is a stable isotope.

[0043] More preferably, the stable isotope is carbon-13.

[0044] Still further preferably, the internal standard is 13 C 12 -Bisphenol A, 13 C 12 -Bisphenol S and 13 C 12 -Bisphenol AF.

[0045] Said 13 C 12 -Bisphenol A is the corresponding internal standard of bisphenol A.

[0046] Said 13 C 12 -Bisphenol S is the corresponding internal standard for bisphenol S, 2,4'-dihydroxydiphenyl sulfone, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-hydroxy-4'-benzyloxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, bisphenol Z, 2,4-di(phenylsulfonyl)phenol, 4'-methylenebis(oxyethylenethio)diphenol and bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer.

[0047] Said 13 C 12-Bisphenol AF is the corresponding internal standard for Bisphenol AF, Bisphenol AP, Bisphenol TMC, Bisphenol M, Bisphenol BP and Bisphenol P.

[0048] More preferably, the mass volume ratio of the internal standard to the body fluid sample is (2-4) ng: (2-4) ng: (2-4) ng: (270-300) μL.

[0049] Further preferably, the mass volume ratio of the isotope-labeled bisphenol A, the isotope-labeled bisphenol S and the isotope-labeled bisphenol AF to the body fluid sample is 3ng:3ng:3ng:300μL.

[0050] More preferably, the chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using water as mobile phase A and methanol as mobile phase B, and in the gradient elution program, the volume percentage of mobile phase B changes as follows: 0-1 min, mobile phase B is 30%; 1-2 min, mobile phase B is 30%-60%; 2-4 min, mobile phase B is 60%; 4-5 min, mobile phase B is 60%-70%; 5-6 min, mobile phase B is 70%-80%; 6-8 min, mobile phase B is 80%-95%; 8-9 min, mobile phase B is 95%; 9-10 min, mobile phase B is 95%-30%; 10-12 min, mobile phase B is 30%.

[0051] Further preferably, the C18 chromatographic column has a column temperature of 38-40° C., a length of 90-100 mm, an inner diameter of 1.8-2.1 mm, and a filler particle diameter of 3-3.5 μm.

[0052] More preferably, the C18 chromatographic column has a column temperature of 40° C., a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 3.5 μm.

[0053] Further preferably, the chromatographic conditions of the ultra-high performance liquid chromatography also include: an injection volume of 3 to 5 μL; a flow rate of 0.30 to 0.35 mL / min.

[0054] More preferably, the injection volume is 5 μL and the flow rate is 0.35 mL / min.

[0055] More preferably, the detection conditions of the mass spectrometry include: drying gas temperature 300°C, drying gas flow rate 5.0L / min, nebulizing gas pressure 35psi, sheath gas temperature 350°C, sheath gas flow rate 11L / min, capillary voltage 3500V, and nozzle voltage 800V.

[0056] Further preferably, the method further comprises the following steps: thoroughly mixing a 45-50 v / v% methanol aqueous solution with the standard of each bisphenol compound to obtain a mixed standard mother solution, wherein the final concentration of the standard of each bisphenol compound in the mixed standard mother solution is 0.5-1 μg / mL.

[0057] More preferably, a 50 v / v% methanol aqueous solution is fully mixed with the standard of each bisphenol compound to obtain a mixed standard mother solution, wherein the final concentration of the standard of each bisphenol compound in the mixed standard mother solution is 0.5-1 μg / mL.

[0058] Still more preferably, in the mixed standard stock solution, the final concentration of each standard substance of the bisphenol compound is 1 μg / mL.

[0059] Specifically, 50 v / v% methanol is fully mixed with the mixed standard mother solution to obtain a standard working solution, in which the final concentration of the standard of each bisphenol compound in the standard working solution is 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL or 200 ng / mL.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The present invention discloses a method for extracting 17 bisphenol compounds and its application, the method comprising: fully mixing a buffer solution and a body fluid sample, then fully extracting with a mixed reagent, collecting an organic phase, and obtaining extracts of the 17 bisphenol compounds. The method of the present invention adopts liquid-liquid extraction with simple and efficient operation, low cost and good repeatability as a body fluid sample pretreatment method, and combines with isotope labeling method and ultra-high performance liquid chromatography tandem mass spectrometry technology to achieve qualitative and quantitative detection and separation of 17 bisphenol compounds in different body fluid samples, with a spike recovery rate of 70% to 120%. At the same time, it has the advantages of high sensitivity, small number of isotope internal standards and small solvent loss, meets actual detection requirements, and has higher simplicity and wider detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 TIC diagrams of 17 bisphenol compounds when 0.1 v / v% ammonium acetate aqueous solution and methanol were used as mobile phases, respectively.

[0063] Figure 2 The TIC diagrams of 17 bisphenol compounds when pure water and acetonitrile were used as mobile phases, respectively.

[0064] Figure 3TIC diagrams of 17 bisphenol compounds when pure water and methanol were used as mobile phases, respectively.

[0065] Figure 4 This is the TIC diagram of 17 bisphenol compounds at a flow rate of 0.30 min / L.

[0066] Figure 5 This is the TIC diagram of 17 bisphenol compounds at a flow rate of 0.35 min / L.

[0067] Figure 6 This is the TIC diagram of 17 bisphenol compounds at a flow rate of 0.40 min / L.

[0068] Figure 7 This is the TIC diagram of mobile phase gradient condition 1 in Example 4.

[0069] Figure 8 This is the TIC diagram of mobile phase gradient condition 2 in Example 4.

[0070] Fig. 9 This is the TIC diagram of mobile phase gradient condition 3 in Example 4.

[0071] Fig.10 This is the TIC diagram of mobile phase gradient condition 4 in Example 4. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0073] Example 1 Method for detecting 17 bisphenol compounds in semen

[0074] 1. The names, corresponding abbreviations and CAS numbers of the 17 bisphenol compounds are as follows:

[0075] Bisphenol A (BPA, CAS: 80-05-7), Bisphenol S (BPS, CAS: 80-09-1), Bisphenol AF (BPAF, CAS: 1478-61-1), Bisphenol AP (BPAP, CAS: 1571-75-1), Bisphenol BP (BPBP, CAS: 1844-01-5), 2,4'-dihydroxydiphenyl sulfone (2,4-BPS, CAS: 5397-34-2), 4,4'-sulfonylbis[2-(2-propenyl)]phenol (TGSA, CAS: 41481-66-7), 4-hydroxy-4'-isopropoxydiphenyl sulfone (BPSIP, CAS: 95235-30-6), 4-hydroxy-4'-benzyloxydiphenyl sulfone (BPS-MPE, CAS: 63134-33-8) , 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol (BPS-MAE, CAS: 97042-18-7), bisphenol TMC (BP-TMC, CAS: 129188-99-4), bisphenol Z (BPZ, CAS: 843-55-0), bisphenol M (BPM, CAS: 13595-25-0), bisphenol P (BPP, CAS: 2167-51-3), 2,4-di(phenylsulfonyl)phenol (DBSP, CAS: 177325-75-6), 4-4'-methylenebis(oxyethylenethio)diphenol (DD-70, CAS: 93589-69-6) and bis(2-chloroethyl)ether-4,4'-dihydroxydiphenylsulfone copolymer (D-90, CAS: 191680-83-8).

[0076] 2. The names and corresponding abbreviations of the three isotope-labeled internal standards are as follows:

[0077] 13 C 12 -Bisphenol A ( 13 C 12 -BPA), 13 C 12 -Bisphenol S( 13 C 12 -BPS), 13 C 12 -Bisphenol AF( 13 C 12 -BPAF).

[0078] 3. Standard solution configuration

[0079] A mixed standard stock solution containing 17 bisphenol compounds was prepared using 50 v / v% methanol: the concentration of the standard of each bisphenol compound was 1 μg / mL, and then the final concentration of the standard of each bisphenol compound was gradiently diluted with 50 v / v% methanol to 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL or 200 ng / mL to obtain a mixed standard working solution of the corresponding diluted concentration.

[0080] 4. Treatment reagent configuration

[0081] Buffer solution: 1 mM ammonium acetate buffer, containing 73 units / mL of β-glucuronidase. The volume ratio of the buffer solution to the semen sample is 1:1.

[0082] Mixed solvent: methyl tert-butyl ether and ethyl acetate in a volume ratio of 5:1.

[0083] Reconstitution reagent: 50 v / v% methanol in water.

[0084] 5. Sample pretreatment

[0085] Take 300 μL of semen sample with 17 kinds of bisphenol compound content to be tested, add 3 ng of three isotope-labeled internal standards, mix with 300 μL buffer solution, shake at 37 ° C for 12 hours to ensure full balance, and obtain balanced solution. Add 2 mL of mixed solvent to the balanced solution, vortex and 80KHZ ultrasonic treatment for ten minutes, then centrifuge at 4300 rpm for ten minutes at room temperature (25 ° C) to separate the organic reagent and the aqueous phase, collect the upper organic phase; add 2 mL of mixed solvent to the remaining lower aqueous phase, extract according to the same method, and repeat the extraction of the lower aqueous phase twice. After the upper organic phases extracted three times are combined, they are washed and purified with 1 mL of ultrapure water, blown to near dryness with nitrogen, and finally 300 μL of 50 v / v% methanol aqueous solution is added for redissolution, filtered through a 0.22 μm filter membrane, and the sample to be tested is obtained for standby use.

[0086] 6. Detection

[0087] The detection conditions of ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry are as follows:

[0088] The liquid chromatography instrument was an Agilent 1290 Infnity II ultra-high performance liquid chromatography system, the mass spectrometer was an Agilent G6470B electrospray triple quadrupole mass spectrometer, and the chromatographic column was a Waters The C18 high performance liquid chromatography column has a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 3.5 μm.

[0089] Gradient elution was performed with pure water (mobile phase A) and methanol (mobile phase B), the column temperature was 40°C, the injection volume was 5 μL, the flow rate was 0.35 mL / min, and the gradient elution program was shown in Table 1:

[0090] Table 1 Gradient elution program

[0091] Time (min) Water (v / v%) Methanol (v / v%) Flow rate (mL / min) 1 70 30 0.35 2 40 60 0.35 4 40 60 0.35 5 30 70 0.35 6 20 80 0.35 8 5 95 0.35 9 5 95 0.35 10 70 30 0.35 12 70 30 0.35

[0092] Data were collected in negative ion mode, and the tandem mass spectrometry conditions used an AJS electrospray ion source with the following ion source parameters: drying gas temperature 300°C, drying gas flow rate 5.0 L / min, nebulizing gas pressure 35 psi, sheath gas temperature 350°C, sheath gas flow rate 11 L / min, capillary voltage 3500 V, and nozzle voltage 800 V. The specific multiple reaction mass spectrometry parameters are shown in Table 2:

[0093] Table 2 Specific parameters of multiple reaction mass spectrometry

[0094]

[0095] *Quantitative ion

[0096] 7. Results Analysis

[0097] A standard curve is established according to the peak area and concentration of the standard solution of 17 bisphenol compounds, and then the content of the 17 bisphenol compounds contained in the semen sample to be tested is calculated according to the peak area of ​​the semen sample to be tested and the standard curve.

[0098] Example 2: Spiked recovery

[0099] 1. Experimental Methods

[0100] 1. Preparation of spiked samples

[0101] According to the steps of Example 1, 3 internal standards and 17 bisphenol compounds were added to 300 μL of semen to verify their recovery. The spike concentrations of the 17 bisphenol compounds were set to 1 ng / mL, 10 ng / mL and 20 ng / mL, respectively. Three parallel experiments were performed for each concentration, and an unspecified blank control was set. In each gradient concentration, the spike amount of each bisphenol compound was the same, that is, the same spike amount was added to each bisphenol compound.

[0102] 2. Detection

[0103] The configuration of the standard solution, the configuration of the reagents in the sample pre-treatment process and the detection method are the same as those in Example 1.

[0104] II. Experimental Results

[0105] (1) Standard curves were plotted based on the peak areas and concentrations of 17 bisphenol compound standard working solutions. The abscissa of the standard curve was the relative concentration, and the ordinate was the relative response value. Each bisphenol compound was quantified using standard curves with more than ten concentration levels. As shown in Table 3, the linear relationships of the 17 bisphenol compounds in the concentration range of 0.01 - 50 ng / mL were good, and the correlation coefficients (R 2 ) were all greater than 0.99, which could be used for subsequent calculation of the contents of the 17 bisphenol compounds.

[0106] Table 3 Method Linear Results for the Detection of 17 Bisphenol Compounds

[0107]

[0108]

[0109] (2) The detection limits and spiked recovery results for the detection of 17 bisphenol compounds are shown in Table 4. In the blank control, except for BPA having a background value, the background values of the other bisphenol compounds were lower than the limit of quantification (<LOQ). The spiked recoveries at three spiked concentration levels were between 78.2% and 119.0%, and the relative deviation was less than 10%. The recoveries were between 75% and 120%, meeting the generally recognized recovery standard (70% - 120%), and the simultaneous extraction and determination of 17 bisphenol compounds could be accurately achieved.

[0110] Table 4 Quantification Limits and Spiked Recovery Results for the Detection of 17 Bisphenol Compounds

[0111]

[0112] Example 3 Detection of Bisphenol Compounds in Actual Semen Samples

[0113] I. Experimental Method

[0114] Collect 27 semen samples from donors, and detect the contents of 17 bisphenol compounds in the semen samples using the method of Example 1.

[0115] II. Experimental Results

[0116] As shown in Table 5, the detection and recovery situations of 17 bisphenol compounds in the semen samples are presented. Due to instrument limitations in detection and the fact that not all 17 bisphenol compounds may be present in the semen samples, 10 bisphenol compounds were actually detected in the semen samples in Example 1 of the present invention. Among them, the detection rate of BPS was the highest, at 74.1%, followed by BPS-MPE and 2,4-BPS, with corresponding detection rates of 70.4% and 63% respectively. This indicates that the method of Example 1 of the present invention can detect the presence and content of bisphenol compounds in semen samples.

[0117] Table 5 Detection of 17 bisphenol compounds in 27 semen samples

[0118] Compound Detection rate (%) LOQ Minimum Median Maximum Arithmetic mean BPA 37.0 0.01 <LOQ <LOQ 2.11 0.166 BPS 74.1 0.01 <LOQ 0.014 0.122 0.027 BPAF 48.1 0.01 <LOQ <LOQ 0.114 0.022 BPBP 55.6 0.01 <LOQ 0.017 0.135 0.034 BPS-MPE 70.4 0.01 <LOQ 0.028 0.164 0.040 2,4-BPS 63.0 0.01 <LOQ 0.016 0.072 0.024 BPAP 40.7 0.01 <LOQ <LOQ 0.029 0.011 BPP 0 0.50 <LOQ <LOQ <LOQ <LOQ BPZ 0 0.50 <LOQ <LOQ <LOQ <LOQ BPSIP 3.7 0.01 <LOQ <LOQ 0.015 <LOQ BP-TMC 0 0.10 <LOQ <LOQ <LOQ <LOQ BPM 14.8 0.01 <LOQ <LOQ 0.031 <LOQ DBSP 0 0.05 <LOQ <LOQ <LOQ <LOQ TGSA 11.1 0.01 <LOQ <LOQ 0.034 <LOQ BPS-MAE 0 0.01 <LOQ <LOQ <LOQ <LOQ DD-90 0 0.02 <LOQ <LOQ <LOQ <LOQ D-70 0 0.02 <LOQ <LOQ <LOQ <LOQ

[0119] Example 4 Effect of different test conditions on the results

[0120] 1. Comparison of the effects of different extraction reagents on the results

[0121] 1. Experimental methods

[0122] In order to explore the effect of the extraction reagent on the results, a standard working solution of 1 ng / mL was used, and the method of Example 1 was used for detection, except that the mixed solvent was replaced with acetonitrile, ether, n-hexane, ethyl acetate or methyl tert-butyl ether.

[0123] 2. Experimental results

[0124] As shown in Table 6, when using single reagents (acetonitrile, ether, n-hexane, ethyl acetate and methyl tert-butyl ether) for extraction, methyl tert-butyl ether has the best extraction effect among single reagents, and 8 bisphenol compounds were successfully extracted. In contrast, the mixed reagent (methyl tert-butyl ether and ethyl acetate with a volume ratio of 5:1) combines the good extraction effect of methyl tert-butyl ether with the volatile and relatively low toxicity of ethyl acetate, and successfully extracts 17 bisphenol compounds, improving the extraction efficiency and covering a wider range of compounds.

[0125] Therefore, within the recovery range of 70% to 120%, the mixed reagent (methyl tert-butyl ether and ethyl acetate in a volume ratio of 5:1) was confirmed as the best extraction reagent, which has wider applicability and higher extraction efficiency.

[0126] Table 6 Comparison of the recovery rates of 17 bisphenol compounds extracted by mixed reagents and 5 single reagents

[0127]

[0128]

[0129] 2. Comparison of the effects of different extraction times on the results

[0130] 1. Experimental methods

[0131] In order to explore the effect of the number of extractions on the results, a 1 ng / mL standard working solution was used and the method of Example 1 was used for detection, except that the number of extractions was changed to 2 or 4 times.

[0132] 2. Experimental results

[0133] As shown in Table 7, when 2 mL of mixed reagent (methyl tert-butyl ether and ethyl acetate with a volume ratio of 5:1) was used for extraction twice, the recoveries of BPBP, BPZ, BP-TMC and BPM were all lower than 70%, and the recovery of TGSA was higher than 120%; when the extraction was performed four times, the recovery of BPZ was lower than 70%, and the recovery of BPS-MPE, BPSIP, BP-TMC, DBSP and TGSA was higher than 120%; therefore, extraction twice and extraction three times could not reach the qualified spike recovery standard; while when the extraction was performed three times, the recovery rates of 17 bisphenol compounds all met the recovery standards (70% to 120%). Therefore, extraction three times can ensure a good recovery rate while taking into account the economy and feasibility of the operation. Therefore, considering the recovery rate, time consumption and cost, extraction three times is the best extraction number.

[0134] Table 7 Comparison of the recovery rates of 17 bisphenol compounds extracted at different extraction times

[0135]

[0136]

[0137] 3. Comparison of the effects of different centrifugation conditions on the results

[0138] 1. Experimental methods

[0139] In order to explore the influence of centrifugation conditions on the results, a standard working solution of 1 ng / mL was used, and the method of Example 1 was used for detection, except that the centrifugation conditions were changed to: ① 25°C, 4500 rpm, 5 min; ② 25°C, 5000 rpm, 5 min; ③ 4°C, 5000 rpm, 5 min for the experiment.

[0140] 2. Experimental results

[0141] The recovery results of the 17 bisphenol compounds are shown in Table 8. When the centrifugation conditions are 25℃, 4300rmp, 10min, the recovery of the 17 bisphenol compounds is between 75% and 120%. Under other centrifugation conditions, at 25℃, 4500rmp, 5min, the recovery rate of BPBP is less than 70%, and the recovery rates of BPS-MPE, 2,4-BPS, BPZ, BP-TMC, BPM and TGSA are all higher than 120%; at 25℃, 5000rmp, 5min, the recovery rates of BPS-MPE, 2,4-BPS, BPZ, BP-TMC and TGSA are all higher than 120%; at 4℃, 5000rmp, 5min, the recovery rates of BPA, BPS-MPE, 2,4-BPS, BPP, BPZ, BPM, DBSP, TGSA, BPS-MAE and DD-90 are all higher than 120%; all of them cannot meet the qualified spike recovery standard. Therefore, taking into account the recovery rate of most compounds and the convenience of practical operation, the centrifugation condition of 25°C and 4300 rpm for 10 minutes was selected as the most suitable treatment condition.

[0142] Table 8 Comparison of the recovery rates of 17 bisphenol compounds extracted under different centrifugal conditions

[0143]

[0144]

[0145] 4. Comparison of the impact of different flows on results

[0146] 1. Experimental methods

[0147] In order to explore the influence of mobile phase on the results, a 1 ng / mL standard working solution was used and the method of Example 1 was used for detection, except that the mobile phase was changed to: ① 0.1 v / v% ammonium acetate aqueous solution (phase A) and methanol (phase B) or ② water (phase A) and acetonitrile (phase B).

[0148] 2. Experimental results

[0149] like Figure 1 As shown in the figure, using 0.1 v / v% ammonium acetate aqueous solution and methanol as the mobile phase, the peak area of ​​17 bisphenol compounds decreased, the response decreased, and the separation effect was also worse. Figure 2 As shown in Figure 2, when acetonitrile and water are used as mobile phases, the peak shape is poor and the response is reduced; Figure 3 As shown in the figure, when methanol and water solution are used as mobile phases, the peak shape is better, the response is better, and the material separation is better. In general, the water-methanol mobile phase system makes the response and separation effect of each analyte higher than the response results of other mobile phase systems.

[0150] 5. Comparison of the effects of different flow rates on the results

[0151] 1. Experimental methods

[0152] In order to explore the effect of flow rate on the results, a 1 ng / mL standard working solution was used and the method of Example 1 was used for detection, except that the flow rate was changed to 0.30 mL / min or 0.40 mL / min.

[0153] 2. Experimental results

[0154] like Figure 4 and Figure 6 As shown in Figure 2, when the flow rate is 0.30 mL / min or 0.40 mL / min, although the separation effect of bisphenol compounds is good, the response is low; Figure 5 As shown, when the flow rate is 0.35 mL / min, the separation effect of the substances is good and the response is higher. Therefore, 0.35 mL / min is selected as the detection flow rate of this method to achieve the best separation effect and response of 17 bisphenol compounds.

[0155] VI. Comparison of the effects of different elution gradients on results

[0156] 1. Experimental methods

[0157] In order to explore the effect of the elution gradient on the results, a 1 ng / mL mixed standard working solution was used and the method of Example 1 was used for detection, except that the elution gradient was changed to different elution gradient conditions as shown in Table 9.

[0158] 2. Experimental results

[0159] like Figures 7-10 As shown, compared with the elution gradient of condition 1 (Example 1), when conditions 2 to 3 are used, the separation of 17 bisphenol compounds is insufficient, the separation effect is poor, and the response is also reduced. According to the separation effect and response of the TIC diagram of bisphenol compounds, condition 1 of Example 1 is the best elution gradient and can obtain the best chromatographic analysis results.

[0160] Table 9 Different elution gradient conditions

[0161]

[0162]

[0163] Example 5 Applicability of Detecting Bisphenol Compounds in Urine Matrix

[0164] 1. Experimental Methods

[0165] 1. Preparation of spiked samples

[0166] According to the method of Example 2, spiked samples were prepared using urine samples and semen samples respectively.

[0167] 2. Detection

[0168] The contents of 17 bisphenol compounds in spiked urine samples and spiked semen samples were detected by the method of Example 1, and the spiked recovery rates of the spiked concentrations were calculated by the method of Example 2.

[0169] 2. Experimental Results

[0170] As shown in Table 10, the test results show that compared with the semen samples, except for BPP and BPZ whose recovery rates were lower than 70%, the recovery rates of most compounds in urine samples were in the range of 70% to 120%. This indicates that the detection method provided by the present invention is also applicable to the determination of the contents of 17 bisphenol compounds in urine matrix in addition to semen.

[0171] Table 10 Comparison of the recovery rates of 17 bisphenol compounds detected in urine and semen by the present invention

[0172]

[0173] Comparison of Example 6 with existing bisphenol compound detection methods

[0174] 1. Urine sample testing experiment

[0175] (1) Experimental methods

[0176] According to the method of Example 2, a spiked sample was prepared using a urine sample.

[0177] The urine spiked sample was treated with reference to the prior art "Occurrence of multiple bisphenol S analogues in children" (Pan Y, Zhu J, Zhu Z, et al. Occurrence of multiple bisphenol Sanalogues in children from Shantou, China. Environ Int. 2023; 174: 107926.), and the specific steps were as follows: 600 μL of spiked sample was taken, and 6 ng of three isotope-labeled internal standards (2 ng for each internal standard) in Example 2 were added and mixed with an equal volume of 1 mM ammonium acetate buffer solution. After oscillation at 37 ° C for 12 hours to ensure full equilibrium, 4 mL of ethyl acetate extraction solvent was added to the urine containing the internal standard. Vortex and 80KHZ ultrasonic treatment for ten minutes were performed, followed by centrifugation at 4300 rpm for five minutes to separate the organic reagent and the aqueous phase, take the upper organic phase, add 4 mL of ethyl acetate extraction solvent to the lower aqueous phase, and repeat the extraction of the lower aqueous phase twice according to the same method. The upper organic phases of the three extractions were combined, washed and purified with 1 mL of ultrapure water, blown to near dryness with nitrogen, and finally re-dissolved with 200 μL of 70 v / v% methanol water, filtered through a 0.22 μm filter membrane, and set aside.

[0178] The method of Example 1 was used for detection, and the spiked recovery rate of the spiked concentration was calculated using the method of Example 2.

[0179] (2) Experimental results

[0180] As shown in Table 11, the recovery rate of the prior art method at two spiked concentrations ranges from 70% to 120%, which is almost the same as the recovery rate of 70% to 120% in Example 2, which indicates that both detection methods can well determine the content of 17 bisphenol compounds in urine matrix.

[0181] Table 11 Comparison of the recovery rates of 17 bisphenol compounds in urine detected by the present invention and the existing detection method

[0182]

[0183]

[0184] 2. Semen sample testing experiment

[0185] (1) Experimental methods

[0186] According to the method of Example 2, a spiked sample was prepared using a semen sample.

[0187] The semen spiked sample was treated with reference to the prior art "Occurrence of multiple bisphenol S analogues in children" (Pan Y, Zhu J, Zhu Z, et al. Occurrence of multiple bisphenol Sanalogues in children from Shantou, China. Environ Int. 2023; 174: 107926.), and the specific steps were as follows: 600 μL of spiked sample was taken, and 6 ng of three isotope-labeled internal standards in Example 2 (2 ng for each internal standard) were added and mixed with an equal volume of 1 mM ammonium acetate buffer solution. After oscillation at 37 ° C for 12 hours to ensure full equilibrium, 4 mL of ethyl acetate extraction solvent was added to the urine containing the internal standard. Vortex and 80KHZ ultrasonic treatment were performed continuously for ten minutes, followed by centrifugation at 4300 rpm for five minutes to separate the organic reagent and the aqueous phase, take the upper organic phase, add 4 mL of ethyl acetate extraction solvent to the lower aqueous phase, and repeat the extraction of the lower aqueous phase twice according to the same method. The upper organic phases of the three extractions were combined, washed and purified with 1 mL of ultrapure water, blown to near dryness with nitrogen, and finally re-dissolved with 200 μL of 70 v / v% methanol water, filtered through a 0.22 μm filter membrane, and set aside.

[0188] The method of Example 1 was adopted, and the spiked recovery rate of the spiked concentration was calculated according to the method of Example 2.

[0189] (2) Experimental results

[0190] As shown in Table 12, when the prior art method is used to detect 17 bisphenol compounds in semen, the spike recovery rate ranges from 40% to 120%, and the recovery rate of 7 bisphenol compounds is less than 70%. Compared with the detection method provided by the present invention, the prior art method requires more sample volume, and the recovery rate of the detected bisphenol compounds is less than the standard. Therefore, this method is not suitable for the detection of the content of 17 bisphenol compounds in semen matrix.

[0191] Table 12 Comparison of the recovery rates of 17 bisphenol compounds in semen detected by the present invention and the existing detection method

[0192]

[0193] In summary, the detection method provided by the present invention realizes the qualitative and quantitative determination of 17 bisphenol compounds, and has the advantages of high accuracy, strong sensitivity, a small number of isotope internal standards and low solvent loss, which meets the actual detection requirements and has higher simplicity and wider detection limit than the existing technology.

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for extracting 17 bisphenol compounds, characterized in that: The bisphenol compound is bisphenol A, bisphenol S, bisphenol AF, bisphenol AP, bisphenol BP, 2,4'-dihydroxydiphenyl sulfone, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-hydroxy-4'-benzyloxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, bisphenol TMC, bisphenol Z, bisphenol M, bisphenol P, 2,4-di(benzenesulfonyl)phenol, 4-4'-methylenebis(oxyethylenethio)diphenol and bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer; The method comprises the following steps: fully mixing a buffer solution and a body fluid sample to obtain a mixture; then fully extracting the mixture with a mixed reagent, collecting an organic phase, and obtaining an extract of the 17 bisphenol compounds; The buffer solution is an ammonium acetate buffer containing a hydrolase; The mixed reagent includes methyl tert-butyl ether and ethyl acetate.

2. The method according to claim 1, characterized in that The body fluid sample is urine and / or semen.

3. The method according to claim 1, characterized in that The hydrolase is β-glucuronidase.

4. The extract of the 17 bisphenol compounds is obtained according to the method according to any one of claims 1 to 3.

5. Use of the extracts of 17 bisphenol compounds according to claim 4 in preparing a detection product for bisphenol compounds.

6. Use of the method according to any one of claims 1 to 3 in detecting bisphenol compounds.

7. A method for detecting 17 bisphenol compounds in a body fluid sample, characterized in that: The method comprises the following steps: extracting the body fluid sample by the method of claim 1 to obtain extracts of the 17 bisphenol compounds, and then detecting the extracts of the 17 bisphenol compounds by ultra-high performance liquid chromatography.

8. The method according to claim 7, characterized in that The method further comprises the following steps: The internal standard, the buffer solution and the body fluid sample are fully mixed to obtain a spiked mixture; the spiked mixture is then fully extracted with the mixed reagent, and the organic phase is collected to obtain a test sample; and the test sample is then detected by ultra-high performance liquid chromatography tandem mass spectrometry.

9. The method according to claim 7 or 8, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using water as mobile phase A and methanol as mobile phase B; in a gradient elution program, the volume percentage of mobile phase B changes as follows: 0-1min, mobile phase B is 30%; 1-2min, mobile phase B is 30%-60%; 2-4min, mobile phase B is 60%; 4-5min, mobile phase B is 60%-70%; 5-6min, mobile phase B is 70%-80%; 6-8min, mobile phase B is 80%-95%; 8-9min, mobile phase B is 95%; 9-10min, mobile phase B is 95%-30%; 10-12min, mobile phase B is 30%.

10. The method according to claim 9, characterized in that: The C18 chromatographic column has a column temperature of 38-40° C., a length of 90-100 mm, an inner diameter of 1.8-2.1 mm, and a filler particle diameter of 3-3.5 μm.

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