Method for extracting bisphenol A and / or related compounds thereof in powder food and application of bisphenol A and / or related compounds thereof

Abstract

The invention discloses a method for extracting bisphenol A and / or related compounds thereof in powder food and application, and the method comprises the following steps: fully and uniformly mixing the powder food, a salting-out reagent and an extraction solvent, carrying out solid-liquid separation, and collecting a solution to obtain the bisphenol A and / or related compounds thereof. According to the method disclosed by the invention, liquid-liquid extraction which is simple and efficient to operate, low in cost and good in repeatability is adopted as a sample pretreatment method, and is combined with an isotope label method and an ultra-high performance liquid chromatography-tandem mass spectrometry technology, so that qualitative and quantitative detection and separation of 20 bisphenol A related compounds in different powder foods are realized; 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 present invention relates to the technical field of analysis and detection, and in particular to a method for extracting bisphenol A and / or its related compounds from powdered food and applications thereof. Background Art

[0002] Bisphenol A (BPA) is one of the most widely used and highest-volume industrial chemicals among bisphenol compounds. It is widely used as a plasticizer, flame retardant, and heat stabilizer in various industrial, commercial, and personal applications. Due to the various toxic effects of BPA, BPA analogs are being widely used as BPA alternatives. Bisphenol A analogues primarily refer to compounds with structures similar to BPA, particularly bisphenol S (BPS), bisphenol AF (BPAF), and bisphenol F (BPF). BPS has become a major substitute for BPA and is widely present in the environment. Numerous toxicological studies have shown that BPS exhibits toxic effects similar to those of BPA, and its continued large-scale production and use will harm the ecological environment and human health. Therefore, the development and use of various new BPS derivatives to replace BPS is still in need. These include 4-hydroxy-4'-isopropoxydiphenyl sulfone (BPSIP or D-8), 2,4'-dihydroxydiphenyl sulfone (2,4-BPS), and 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol (BPS-MAE), which are potential BPS alternatives. These new compounds are often used as colorants in thermal paper. However, existing toxicological data indicate that these compounds exhibit the same toxic effects as BPA, or even more potent reproductive toxicity.

[0003] In the infant food sector, milk powder and rice flour, as key complementary foods, have drawn significant attention for their safety. Bisphenol compounds (such as bisphenol A, bisphenol F, and bisphenol S), widely used in food packaging materials, can easily migrate into milk powder and rice flour. Bisphenol compounds have been shown to have endocrine disrupting properties, potentially causing adverse effects on the reproductive, nervous, and metabolic systems of infants and young children. Long-term, low-dose exposure can even pose a potential carcinogenic risk, posing a serious threat to infant health. Therefore, detecting bisphenol compounds in milk powder and rice flour is crucial for ensuring infant food safety.

[0004] To accurately assess the actual exposure levels of bisphenols and their novel derivatives in infant formula and rice cereal, it is often necessary to measure the content of these compounds. Currently, various methods are available for detecting bisphenols: liquid chromatography-mass spectrometry offers high sensitivity and good accuracy, but is costly and complex. Spectrophotometry is simple and rapid, but lacks sensitivity and selectivity, making it difficult to detect low concentrations of bisphenols. Emerging biosensor technologies, such as molecular imprinting sensors, offer advantages such as speed, portability, and low cost, but face challenges in stability, reproducibility, and selectivity. High-performance liquid chromatography (HPLC) offers low equipment cost and relatively simple operation, but is less sensitive than mass spectrometry and is susceptible to matrix interference. Therefore, the development of efficient, sensitive, accurate, and low-cost detection technologies is crucial for the detection and control of bisphenols in milk powder and rice cereal. Summary of the Invention

[0005] In order to solve the problem in the prior art of lacking a method for quickly, accurately and simply detecting the exposure levels of various bisphenol A analogues and their novel derivatives in milk powder and rice flour, the present invention provides a method for extracting bisphenol A and / or its related compounds in milk powder and / or rice flour and its application.

[0006] The first object of the present invention is to provide a method for extracting bisphenol A and / or its related compounds from powdered food.

[0007] The second object of the present invention is to provide the application of the method in detecting bisphenol A and / or its related compounds.

[0008] The third object of the present invention is to provide a method for detecting bisphenol A and / or its related compounds.

[0009] In order to achieve the above object, the present invention is implemented through the following scheme:

[0010] The present invention obtains a test sample for detecting bisphenol A and / or its related compounds through a liquid-liquid extraction sample pretreatment method, and then combines it with ultra-performance liquid chromatography tandem mass spectrometry technology to successfully achieve quantitative detection of 20 bisphenol compounds in powdered food.

[0011] A method for extracting bisphenol A and / or its related compounds from powdered food, characterized by comprising the following steps: thoroughly mixing the powdered food, a salting-out reagent, and an extraction solvent, separating the solid from the liquid, and collecting the solution;

[0012] The bisphenol A-related compound includes at least one of bisphenol S, 2,4'-dihydroxydiphenyl sulfone, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 2,4-bis(phenylsulfonyl)phenol, bisphenol TMC, bisphenol M, bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer, 3-chlorobisphenol A, dichlorobisphenol S, dichlorobisphenol S isomers, trichlorobisphenol S, 4-hydroxy-4'-benzyloxydiphenyl sulfone, hexafluorobisphenol A, bisphenol F, bisphenol Z, bisphenol AP and 4,4'-dibenzylidene bisphenol;

[0013] The extraction solvent includes acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is (1-2):1.

[0014] Preferably, the powdered food comprises milk powder and / or rice flour.

[0015] More preferably, the milk powder is infant milk powder.

[0016] More preferably, the rice flour is infant rice flour.

[0017] Preferably, the salting-out reagent comprises NaCl.

[0018] Preferably, the extraction solvent comprises acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is 1:1.

[0019] Preferably, the extraction solvent is a mixed solvent of acetonitrile and dichloromethane.

[0020] More preferably, the extraction solvent is obtained by mixing acetonitrile and dichloromethane in a volume ratio of 1:1.

[0021] Preferably, the mass-to-volume ratio of the powdered food, the salting-out reagent, and the extraction solvent is (0.5-1) g: (0.5-1) g: (5-10) mL.

[0022] More preferably, the mass-to-volume ratio of the powdered food, the salting-out reagent, and the extraction solvent is 1 g:1 g:1 mL.

[0023] Preferably, the method of sufficiently mixing comprises ultrasound.

[0024] More preferably, the ultrasonic conditions include: frequency 80-100 KHz, time 10-15 min.

[0025] Further preferably, the ultrasonic conditions include: frequency 100KHz, time 15min.

[0026] Preferably, the solid-liquid separation method comprises centrifugation.

[0027] More preferably, the centrifugal conditions include: a rotation speed of 4000-4500 rpm and a time of 5-10 min.

[0028] Further preferably, the centrifugal conditions include: rotation speed 4500 rpm, time 10 min.

[0029] Preferably, the method comprises the following steps: thoroughly mixing the powdered food, salting-out reagent, and extraction solvent for a first extraction, performing solid-liquid separation, and collecting the solution to obtain supernatant 1; then thoroughly mixing the resulting solid with the extraction solvent for a second extraction, performing solid-liquid separation, and collecting the solution to obtain supernatant 2; and combining supernatant 1 and supernatant 2. In this solution, a total of two extractions are performed.

[0030] More preferably, the volume ratio of the extraction solvent used in the first extraction to the extraction solvent used in the second extraction is 4 to 5:4.

[0031] Further preferably, the volume ratio of the extraction solvent used in the first extraction to the extraction solvent used in the second extraction is 5:4.

[0032] More preferably, the volume ratio of the supernatant 1 to the supernatant 2 is (4-5):4.

[0033] Further preferably, the volume ratio of the supernatant 1 to the supernatant 2 is 5:4.

[0034] The application of any of the methods in detecting bisphenol A and / or its related compounds should also be within the scope of protection of the present invention.

[0035] A method for detecting bisphenol A and / or its related compounds comprises the following steps: extracting a powdered food to be tested using any of the methods described above to obtain an extract; removing the solvent from the extract to obtain a precipitate, dissolving the precipitate with acetonitrile to obtain a test sample; and then detecting the test sample using ultra-high performance liquid chromatography.

[0036] Preferably, the internal standard is fully mixed with the powdered food to be tested, the salting-out reagent and the extraction solvent, the solid and liquid are separated, and the solution is collected to obtain an extract.

[0037] More preferably, the internal standard comprises isotope-labeled bisphenol A, isotope-labeled bisphenol S, isotope-labeled hexafluorobisphenol A and isotope-labeled bisphenol F.

[0038] Further preferably, the internal standard comprises bisphenol A labeled with isotope 1, bisphenol S labeled with isotope 2, hexafluorobisphenol A labeled with isotope 3, and bisphenol F labeled with isotope 4, wherein the bisphenol A labeled with isotope 1 is the internal standard of bisphenol A and 3-chlorobisphenol A, and the bisphenol S labeled with isotope 2 is bisphenol S, bisphenol Z, 2,4'-dihydroxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-hydroxy-4'- Benzyloxydiphenyl sulfone, 4,4'-dihydroxydiphenyl sulfide, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer, 2,4-bis(phenylsulfonyl)phenol, dichlorobisphenol S, dichlorobisphenol S isomers, and trichlorobisphenol S are internal standards; the isotope 3-labeled hexafluorobisphenol A is the internal standard for hexafluorobisphenol A, bisphenol AP, bisphenol M, and 4,4'-dibenzylidene bisphenol; the isotope 4-labeled bisphenol F is the internal standard for bisphenol F.

[0039] The isotope 1, the isotope 2, the isotope 3 and the isotope 4 include but are not limited to carbon isotopes, nitrogen isotopes, oxygen isotopes and hydrogen isotopes.

[0040] More preferably, the isotope 1, the isotope 2, the isotope 3 and the isotope 4 are the same.

[0041] Still further preferably, the isotope 1, the isotope 2, the isotope 3 and the isotope 4 are all carbon isotopes.

[0042] Most preferably, the isotope 1, the isotope 2, the isotope 3 and the isotope 4 are 13 C 12 .

[0043] Preferably, the method of removing the solvent from the extract comprises blowing the extract to near dryness with nitrogen.

[0044] Preferably, the precipitate is dissolved in acetonitrile to obtain a reconstituted solution; the reconstituted solution is cooled to separate the solid and the liquid, and the obtained liquid is collected to obtain the test sample.

[0045] More preferably, the reconstituted solution is cold-treated at -20 to 4°C for 1 to 4 hours.

[0046] More preferably, the reconstituted solution is cold-treated at -20°C for 4 hours.

[0047] Further preferably, the solid-liquid separation method includes filtration.

[0048] More preferably, the filter membrane used for the filtration is a 0.22 μm filter membrane.

[0049] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatography include:

[0050] A C18 chromatographic column was used; water was used as mobile phase A and methanol was used as mobile phase B. In the gradient elution program, the volume percentage of mobile phase A changed as follows: from 0 to 1 minute, mobile phase A was 70%; from 1 to 2 minutes, mobile phase A decreased from 70% to 40%; from 2 to 4 minutes, mobile phase A was 40%; from 4 to 5 minutes, mobile phase A decreased from 40% to 30%; from 5 to 6 minutes, mobile phase A decreased from 30% to 20%; from 6 to 8 minutes, mobile phase A decreased from 20% to 5%; from 8 to 9 minutes, mobile phase A was 5%; from 9 to 10 minutes, mobile phase A increased from 5% to 70%; from 10 to 12 minutes, mobile phase A was 70%.

[0051] More preferably, ultrapure water is used as mobile phase A.

[0052] More preferably, the column temperature of the C18 chromatographic column is 38-40° C., the length is 90-100 mm, the inner diameter is 1.8-2.1 mm, and the filler particle diameter is 3-3.5 μm.

[0053] Further preferably, the column temperature of the C18 chromatographic column is 40° C., the length is 100 mm, the inner diameter is 2.1 mm, and the filler particle diameter is 3.5 μm.

[0054] More preferably, the chromatographic conditions of the ultra-high performance liquid chromatography further include: an injection volume of 3 to 5 μL; and a flow rate of 0.18 to 0.22 mL / min.

[0055] More preferably, the injection volume is 5 μL and the flow rate is 0.2 mL / min.

[0056] More preferably, the test sample is detected by ultra performance liquid chromatography triple quadrupole tandem mass spectrometry, and the detection conditions of the mass spectrometry include: using an electrospray ionization source, the scanning mode is positive ion mode and negative ion mode, the detection mode is multiple ion reaction detection mode, the drying gas temperature is 300 ° C, the drying gas flow rate is 5.0 L / min, the nebulizing gas pressure is 35 psi, the sheath gas temperature is 350 ° C, the sheath gas flow rate is 11 L / min, the capillary voltage is 3500 V, and the nozzle voltage is 800 V.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention establishes a detection method for 20 types of bisphenol A and its analogues, which can achieve the simultaneous extraction of all target compounds in powdered food and qualitative and quantitative detection. It is simple to operate, low in cost, has good detection accuracy, high sensitivity, strong specificity, a small number of isotope internal standards, and low solvent loss. It complies with recognized standards and meets actual detection needs, and has higher simplicity and a wider detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the TIC diagram of the 20 target compounds in Example 4. Mobile phase A is ultrapure water, mobile phase B is methanol, the gradient elution program is Program 2, and the flow rate of the mobile phase is 0.2 ml / min.

[0060] Figure 2 This is the TIC chart of the 20 target compounds in Example 4. Mobile phase A is 0.1 v / v% formic acid aqueous solution, and mobile phase B is methanol.

[0061] Figure 3 This is the TIC diagram of the 20 target compounds in Example 4. Mobile phase A is 1 mmol / L ammonium acetate aqueous solution, and mobile phase B is methanol.

[0062] Figure 4 This is the TIC diagram of the gradient elution program 1 in Example 4.

[0063] Figure 5 This is the TIC diagram of the gradient elution program 3 in Example 4.

[0064] Figure 6 This is the TIC diagram of the mobile phase in Example 4 with a flow rate of 0.3 ml / min.

[0065] Figure 7 This is the TIC diagram of the mobile phase in Example 4 with a flow rate of 0.35 ml / min. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0067] Example 1 Method for detecting bisphenol A and its related compounds in a sample

[0068] 1. Target compound

[0069] The present invention provides a detection method for bisphenol A analogs and novel derivatives thereof, taking 20 compounds as shown in Table 1 as target compounds, specifically including: bisphenol A, bisphenol S, 2,4'-dihydroxydiphenyl sulfone, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 2,4-bis(phenylsulfonyl)phenol, bisphenol TMC, bisphenol M, bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer, 3-chlorobisphenol A, dichlorobisphenol S, dichlorobisphenol S isomers, trichlorobisphenol S, 4-hydroxy-4'-benzyloxydiphenyl sulfone, hexafluorobisphenol A, bisphenol F, bisphenol Z, bisphenol AP and 4,4'-dibenzylidene bisphenol.

[0070] Table 1 Basic information of 20 bisphenol A analogues and their novel derivatives

[0071]

[0072]

[0073] 2. Internal standard

[0074] Since most of the derivatives of bisphenol A are new compounds, there are currently only four isotope-labeled internal standards. Among the 20 target compounds mentioned above, four existing isotope-labeled compounds are used as internal standards, specifically: 13 C 12 -Bisphenol A ( 13 C 12 -BPA, CAS: 80-05-7), 13 C 12 -Bisphenol S( 13 C 12 -BPS, CAS: 80-09-1), 13 C 12 -Hexafluorobisphenol A ( 13 C 12 -BPAF, CAS: 1478-61-1) and 13 C 12 -Bisphenol F( 13 C 12 -BPF, CAS: 620-92-8). 13 C 12 -BPA is the internal standard for BPA and BPA-Cl, 13 C 12 -BPS is the internal standard for BPS, BPZ, 2,4-BPS, BPS-MAE, BPS-MPE, BPSIP, TGSA, D-90, DBSP, Cl2-BPS-1, Cl2-BPS-2 and Cl3-BPS.13 C 12 -BPAF is the internal standard for BPAF, BPAP, BPM, BPBP and BP-TMC. 13 C 12 -BPF is the internal standard of BPF.

[0075] 3. Pretreatment of the sample to be tested

[0076] (1) Add 0.5g of the sample to be tested, 0.5g of NaCl and four internal standards ( 13 C 12 -BPA, 13 C 12 -BPS, 13 C 12 -BPAF and 13 C 12 -BPF) 5ng each.

[0077] (2) First extraction: 5 mL of acetonitrile-dichloromethane mixed solution (obtained by mixing acetonitrile and dichloromethane in a volume ratio of 1:1) was added to the tube for ultrasonic treatment at a frequency of 100 kHz for 15 min, followed by centrifugation at 4500 rpm for 10 min.

[0078] (3) Take the supernatant after centrifugation and transfer it to another clean glass tube, which is recorded as supernatant 1.

[0079] (4) Second extraction: 4 mL of acetonitrile-dichloromethane mixed solution was added to the tube again and mixed with the precipitate. Ultrasonic extraction was performed again at 100 kHz for 10 min. After ultrasonication, the mixture was centrifuged at 4500 rpm for 10 min. The supernatant after centrifugation was collected and recorded as supernatant 2.

[0080] (5) Combine 4 mL of supernatant 2 with 5 mL of supernatant 1, slowly blow them to dryness with nitrogen, and then redissolve them in 500 μL of acetonitrile to obtain a reconstituted solution.

[0081] (6) The reconstituted solution obtained in (5) was placed in a -20°C refrigerator for 4 h to remove fat.

[0082] (7) After filtering through a 0.22 μm filter membrane, collect the filtrate to obtain the test sample for later use.

[0083] 4. Preparation of standard solution

[0084] A methanol solution containing 20 target compounds was used as a mixed standard stock solution, wherein the concentration of each compound was the same, 1 μg / mL.

[0085] The mixed standard stock solution was gradiently diluted with pure methanol solvent into 12 standard solutions with different concentrations (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 and 50 ng / mL).

[0086] 5. Ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry

[0087] (1) Instruments and equipment

[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] (2) Ultra-high performance liquid chromatography detection conditions

[0090] Ultrapure water (mobile phase A) and methanol (mobile phase B) were used for gradient elution. The column temperature was 40°C, the injection volume was 5 μL, the flow rate was 0.20 mL / min, and the gradient elution program was shown in Table 2.

[0091] Table 2 Mobile phase elution gradient

[0092]

[0093] (3) Mass spectrometry detection conditions

[0094] Data were collected in negative ion mode using an AJS electrospray ion source. Tandem mass spectrometry parameters included: drying gas temperature of 300°C, drying gas flow rate of 5.0 L / min, nebulizer gas pressure of 35 psi, sheath gas temperature of 350°C, sheath gas flow rate of 11 L / min, capillary voltage of 3500 V, and nozzle voltage of 800 V. Specific multiple reaction mass spectrometry parameters are shown in Table 3.

[0095] Table 3 Specific parameters of multiple reaction mass spectrometry

[0096]

[0097]

[0098] Note: * indicates quantitative ion.

[0099] 6. Results Analysis

[0100] A linear regression analysis was performed based on the peak areas and concentrations of the 12 different concentrations of standard solutions, with relative concentrations plotted on the horizontal axis and relative responses plotted on the vertical axis. A standard curve was then generated. The content of the 20 target compounds in the test sample was then calculated based on the peak areas of the test sample and the resulting standard curve.

[0101] Example 2: Recovery of Bisphenol A and Its Related Compounds in Actual Infant Milk Powder

[0102] 1. Preparation of spiked samples

[0103] Recoveries were determined by spiking 0.5 g of infant formula powder with four internal standards and the standards of the 20 compounds listed in Table 1. The spiked concentrations of the 20 compounds were set at 1 ng / mL, 2 ng / mL, and 5 ng / mL, with three replicates performed at each concentration. An unspiked sample of infant formula powder was used as a blank control. The same amount of each compound was spiked throughout the concentration gradient.

[0104] 2. Detection

[0105] The spiked sample prepared in this example was used as the sample to be tested, and the content of each compound therein was detected according to the method of Example 1.

[0106] 3. Results Analysis

[0107] The standard curves of the 20 compounds are shown in Table 4. It can be seen that the 20 target analytes have good linear relationships in the concentration range of 0.01 to 50 ng / mL, with correlation coefficients (all greater than 0.99), which can be used for the subsequent calculation of the contents of the 20 compounds in the spiked samples.

[0108] Table 4 Standard curve equations for 20 bisphenol A analogs and their novel derivatives

[0109] Compound Concentration range (ng / mL) Regression equation <![CDATA[Coefficient of correlation R 2 > BPA 0.01～50 y=25436.9×x-167.8 0.9993 BPS 0.01～50 y=22208.6×x-220.0 0.9991 2,4-BPS 0.01～50 y=17208.7×x-80.6 0.9989 BPSIP 0.01～50 y=153469.8×x-870.5 0.9995 BPS-MAE 0.01～50 y=370093.8×x-2396.5 0.9995 TGSA 0.01～50 y=53055.6×x-374.1 0.9994 DBSP 0.01～50 y=60704.5×x-45.2 0.9992 BP-TMC 0.01～50 y=971.0×x-50.0 0.9994 BPM 0.01～50 y=5804.3×x-45.6 0.9997 D-90 (n=1) 0.01～50 y=12044.5×x-166.1 0.9988 BPA-Cl 0.01～50 y=5219.0×x-90.8 0.9980 <![CDATA[Cl2-BPS-1]]> 0.01～50 y=4107.5×x-5.50 0.9995 <![CDATA[Cl2-BPS-2]]> 0.01～50 y=45330.3×x-184.5 0.9992 <![CDATA[Cl3-BPS]]> 0.01～50 y=55756.8×x-892.0 0.9997 BPS-MPE 0.01～50 y=186759.6×x-1085.7 0.9994 BPAF 0.01～50 y=11064.1×x+19.1 0.9985 BPZ 0.01～50 y=373.9×x-54.7 0.9999 BPF 0.01～50 y=352.0×x+7.1 0.9997 BPAP 0.01～50 y=386396.8×x-4817.5 0.9998 BPBP 0.01～50 y=986.3×x+28.9 0.9997

[0110] The quantification limits and spiked recoveries of 20 target analytes in infant formula are shown in Table 5. The spiked recoveries of the 20 target analytes at three spiked concentrations ranged from 70% to 119%, with a relative deviation of no more than 16%, which meets the recognized recovery standard (70% to 120%) and can accurately achieve the simultaneous extraction and determination of 20 bisphenol A and / or its related compounds.

[0111] Table 5 Recovery results of bisphenol A and / or its related compounds in formula milk powder

[0112]

[0113]

[0114] The above results show that the detection method of Example 1 of the present invention can achieve the simultaneous extraction and determination of 20 target compounds in infant formula milk powder, is simple to operate, low in cost, has good detection accuracy, high sensitivity, and strong specificity, and complies with recognized standard regulations.

[0115] Example 3: Recovery and Detection of Bisphenol A and Its Related Compounds in Actual Infant Rice Cereal

[0116] 1. Preparation of spiked samples

[0117] Referring to the method of Example 2, spiked samples were prepared using infant rice cereal (under 1 year old) purchased from the market as the matrix, and unspiked infant rice cereal was set as a blank control.

[0118] 2. Detection

[0119] The spiked sample prepared in this example was used as the sample to be tested. The content of each compound therein was detected according to the method of Example 1, and the content of each target compound was calculated in combination with the standard curve equation in Example 2.

[0120] 3. Results

[0121] The limits of quantitation and spiked recoveries for the 20 target analytes in infant rice cereal are shown in Table 6. Three replicates were performed at each concentration. The recoveries for the 20 target analytes ranged from 72% to 117% at the three spiked concentrations, with relative deviations of no more than 15%, meeting the generally accepted recovery standard of 70% to 120%. This method enables the accurate simultaneous extraction and determination of 20 bisphenol A and related compounds in rice cereal.

[0122] Table 6 Recovery results of 20 target analytes in infant rice cereal

[0123]

[0124] Example 4 Effects of different conditions on test results

[0125] 1. Influence of extraction reagents on test results

[0126] Standard solutions of 20 target compounds, each with a final concentration of 5 ng / mL, were used as test samples. Testing was performed according to the method of Example 1, with three replicates for each method. The only difference was that in the "Pretreatment of Test Samples" step, combinations 1 to 9, as shown in Table 7, were used for the first and second extractions to prepare the test samples.

[0127] Table 7 Different extraction reagent combinations for 20 target analytes

[0128]

[0129]

[0130] Note: Combination 2 is the extraction reagent combination in Example 1. / indicates no second extraction

[0131] As shown in Table 8, when using different extraction solvent combinations for extraction, combination 2 achieved the best extraction results, with recoveries of 71% to 114% for the 20 target compounds, generally meeting the generally accepted recovery standard (70% to 120%). This indicates that the dichloromethane and acetonitrile mixture effectively extracts both polar and non-polar compounds. The synergistic effect of the two solvents further enhances the simultaneous extraction of the 20 target compounds. Furthermore, compared to other extraction solvent combinations, combination 2 is easier to remove by evaporation after extraction, reducing residues.

[0132] Table 8 Spiked recovery (%) ± relative standard deviation (RSD,%) of different extraction reagent combinations

[0133]

[0134] 2. The impact of different extraction times on test results

[0135] A mixed standard working solution of 20 target compound standards with a final concentration of 5 ng / mL was used as the test sample, and detection was performed according to the method of Example 1, except that in the step "pretreatment of the test sample", extraction was performed once (i.e., only the first extraction was performed), twice (i.e., the first extraction and the second extraction were performed), and three times (i.e., the first extraction and the second extraction were performed, and the second extraction step was repeated once).

[0136] The results are shown in Table 9. The best result was obtained when the extraction was repeated twice. The recovery rates of the 20 target compounds were between 72% and 110%, which were in line with the generally accepted recovery standards (70% to 120%).

[0137] Table 9 Spiked recovery (%) ± relative standard deviation (RSD,%) of different extraction reagent combinations

[0138]

[0139] 3. The influence of different mobile phases on test results

[0140] Standard solutions of 20 target compounds, each with a final concentration of 10 ng / mL, were used as test samples. Detection was then performed according to the method of Example 1, with the following differences: In the "ultra-performance liquid chromatography triple quadrupole tandem mass spectrometry detection" step, the mobile phases were changed to: ① 0.1 v / v% formic acid aqueous solution (mobile phase A) and methanol (mobile phase B); ② 1 mmol / L ammonium acetate aqueous solution (mobile phase A) and methanol (mobile phase B). A TIC (Total Ion Chromatogram) plot was plotted based on the test results.

[0141] The TIC diagram when using ultrapure water (mobile phase A) and methanol (mobile phase B) for detection in Example 1 is as follows: Figure 1 As shown in the figure, the peak shapes of the 20 target compounds are good, the responses are good, and the substances are clearly separated. Figure 2 As shown in Figure 1, when 0.1 v / v% formic acid aqueous solution was used instead of ultrapure water as mobile phase A, the peak areas of the 20 target compounds were significantly reduced, the response was reduced, and the separation effect was also worse. Figure 3 As shown, when 1 mmol / L ammonium acetate aqueous solution is used instead of ultrapure water as mobile phase A, the peak shapes of the 20 target compounds are relatively poor, the response is reduced, and miscellaneous peaks appear. Therefore, the ultrapure water-methanol mobile phase system of Example 1 has the best response and separation effect for detecting each compound.

[0142] 4. Effect of different elution gradients on test results

[0143] Standard solutions of the 20 target compounds, each with a final concentration of 10 ng / mL, were used as test samples. Testing was performed according to the method of Example 1, with the only difference being that in step "ultra-performance liquid chromatography triple quadrupole tandem mass spectrometry detection," gradient elution was performed according to procedures 1 to 3 shown in Table 10. TIC plots were plotted based on the test results.

[0144] Table 10 Different gradient elution programs

[0145]

[0146] Note: Procedure 2 is the elution gradient condition in Example 1.

[0147] The TIC diagrams of programs 1 to 3 are as follows: Figure 4 、 Figure 1 and Figure 5 As shown, the response of target compounds decreased and the separation effect became worse when gradient elution was performed using procedures 1 and 3 compared with procedure 2. Therefore, the response and separation effect of each compound detected by the elution gradient of Example 1 were the best.

[0148] 5. The impact of different flow rates on test results

[0149] Standard solutions of 20 target compounds, each with a final concentration of 10 ng / mL, were used as test samples. Testing was performed according to the method of Example 1, with the only difference being that in the "ultra-performance liquid chromatography triple quadrupole tandem mass spectrometry detection" step, the mobile phase flow rates were 0.2 ml / min, 0.3 ml / min, and 0.35 ml / min, respectively. TIC plots were drawn based on the test results.

[0150] The TIC diagram when the flow rate of the mobile phase is 0.3 ml / min is as follows Figure 6 As shown, the TIC diagram when the flow rate of the mobile phase is 0.2 ml / min is as follows Figure 1 As shown, the TIC diagram when the flow rate of the mobile phase is 0.35ml / min is as follows Figure 7 As shown, it can be seen that when the flow rate is 0.2 ml / min, the responses of the 20 target compounds are relatively the best, while when the flow rates are 0.3 ml / min and 0.35 ml / min, the responses of all compounds are reduced.

[0151] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for extracting bisphenol A and / or its related compounds from powdered food, characterized in that: The method comprises the following steps: fully mixing the powdered food, the salting-out reagent and the extraction solvent, separating the solid and the liquid, and collecting the solution to obtain the product; The bisphenol A and / or its related compounds include at least one of bisphenol A, bisphenol S, 2,4'-dihydroxydiphenyl sulfone, 4-hydroxy-4'-isopropoxydiphenyl sulfone, 4-[[4-(2-allyloxy)phenyl]sulfonyl]phenol, 4,4'-sulfonylbis[2-(2-propenyl)]phenol, 2,4-bis(phenylsulfonyl)phenol, bisphenol TMC, bisphenol M, bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone copolymer, 3-chlorobisphenol A, dichlorobisphenol S, dichlorobisphenol S isomers, trichlorobisphenol S, 4-hydroxy-4'-benzyloxydiphenyl sulfone, hexafluorobisphenol A, bisphenol F, bisphenol Z, bisphenol AP and 4,4'-dibenzylidene bisphenol; The extraction solvent includes acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is (1-2):

1.

2. The method according to claim 1, characterized in that The powdered food includes milk powder and / or rice flour.

3. The method according to claim 1, characterized in that The salting-out reagent includes NaCl.

4. The method according to claim 1, wherein The mass-to-volume ratio of the powdered food, the salting-out reagent, and the extraction solvent is (0.5-1) g: (0.5-1) g: (5-10) mL.

5. The method according to claim 1, wherein The method comprises the following steps: fully mixing powdered food, salting-out reagent and extraction solvent, separating solid and liquid, collecting the solution to obtain supernatant 1; then fully mixing the obtained solid with the extraction solvent, separating solid and liquid, collecting the solution to obtain supernatant 2; and combining supernatant 1 and supernatant 2 to obtain the product.

6. Use of the method according to any one of claims 1 to 5 in detecting bisphenol A and / or its related compounds.

7. A method for detecting bisphenol A and / or its related compounds, characterized in that: The following steps are involved: The powdered food to be tested is extracted using the method according to claim 1 to obtain an extract; the solvent of the extract is removed to obtain a precipitate, and the precipitate is dissolved in acetonitrile to obtain a test sample; and then the test sample is detected by ultra-high performance liquid chromatography.

8. The detection method according to claim 7, characterized in that In the method of claim 1, the internal standard is thoroughly mixed with the powdered food to be tested, the salting-out reagent and the extraction solvent, the solid-liquid separation is performed, and the solution is collected to obtain an extract.

9. The detection method according to claim 8, characterized in that The internal standard substances include isotope-labeled bisphenol A, isotope-labeled bisphenol S, isotope-labeled hexafluorobisphenol A and isotope-labeled bisphenol F.

10. The detection method according to claim 7, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography include: A C18 chromatographic column was used, with water as mobile phase A and methanol as mobile phase B. In the gradient elution program, the volume percentage of mobile phase A changed as follows: 0-1 min, mobile phase A was 70%; 1-2 min, mobile phase A decreased from 70% to 40%; 2-4 min, mobile phase A was 40%; 4-5 min, mobile phase A decreased from 40% to 30%; 5-6 min, mobile phase A decreased from 30% to 20%; 6-8 min, mobile phase A decreased from 20% to 5%; 8-9 min, mobile phase A was 5%; 9-10 min, mobile phase A increased from 5% to 70%; 10-12 min, mobile phase A was 70%.

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