CN112684018A - Method for detecting content of bisphenol S and bisphenol F in animal-derived food and application thereof - Google Patents
Method for detecting content of bisphenol S and bisphenol F in animal-derived food and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of analysis and detection, and discloses a method for detecting the contents of bisphenol S and bisphenol F in animal-derived food by high performance liquid chromatography-mass spectrometry, wherein the method is used for detecting the concentrations of bisphenol S and bisphenol F in pork, and subtracting the measured values of reagent blank and sample blank to obtain the actual contents of bisphenol S and bisphenol F in a sample; the standard curve is obtained by measuring bisphenol S and bisphenol F in the range of standard addition concentration of 2-100ng/mL, and the linear correlation coefficient R of BPS20.9995 with detection limit of 0.010 μ g/kg; BPF linear correlation coefficient R20.9997, detection limit of 0.126 mug/kg, standard recovery rate of 84.6-102% for different concentrations, and precision of less than 5.49%, and the product is prepared by the methodThe method has simple operation, high precision and detection limit reaching ppb level; the method is suitable for accurately, rapidly, qualitatively and quantitatively detecting the bisphenol S and the bisphenol F in the animal-derived food.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting the content of bisphenol S and bisphenol F in animal-derived food and application thereof.
Background
Bisphenol S (BPS), formula C12H10O4S (4, 4' -dihydroxy diphenyl sulfone), as bisphenol A substitute, is widely used for manufacturing epoxy resin, baby feeding bottle and heat-sensitive paper, BPS is detected in various foods (materials), the BPS has acute toxicity and estrogenic activity similar to bisphenol A, and after the animals are exposed in early stage, estrogen interference effect and neuroendocrine system development toxicity are generated, and even genotoxicity is generated. Bisphenol F (BisphenolF, BPF), formula C13H12O2(4,4 dihydroxy diphenylmethane), also used mainly in the manufacture of epoxy resins and polycarbonate plastics and products such as varnishes, coatings, gaskets, adhesives, etc. BPF homologues BPA have been shown to have estrogenic effects, and the mode of human exposure to BPF in daily life is often dominated by low-level, long-term exposure, and BPF has now been shown to be present in the environment as a food contaminant. The cytotoxicity and genotoxicity of BPF and its partial metabolites have been characterized, with BPF showing moderate cytotoxicity. At present, the content of bisphenol compounds is concentrated on bisphenol A and the detection objects are mainly water environment and food contact materials, but the research reports of BPS and BPF on animal-derived foods are less. Furthermore, the regulations for the limits of BPS and BPF in animal derived foods are not yet specified and are still in the blank phase.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect that the prior art lacks a BPF and BPS detection method and specification in animal derived food; the first purpose of the invention is to provide a sample pretreatment method for detecting bisphenol S and bisphenol F in animal derived food.
The second purpose of the invention is to provide a method for detecting the content of bisphenol S and bisphenol F in animal-derived food, which is characterized by high precision, high sensitivity and low detection limit, and can overcome the defects of complex operation and large background interference of the above technology.
The third purpose of the invention is to provide the application of the method for detecting the bisphenol S and the bisphenol F in the animal-derived food by the high performance liquid chromatography-mass spectrometry.
The purpose of the invention is realized by the following technical scheme:
a sample pretreatment method for detecting bisphenol S and bisphenol F in animal derived food comprises the following steps:
s1, homogenizing the animal-derived food to be detected, adding an extracting agent, oscillating, and carrying out ultrasonic treatment under an ice bath condition;
s2, centrifuging for the first time to obtain supernatant and precipitate;
s3, adding the extracting agent into the precipitate again, and repeating the steps to obtain supernatant liquid of secondary extraction;
and S4, combining the supernatants after the two centrifugations, and purifying the supernatant by using a QuEChERS buffer salt reagent and a PRIME HLB small column in a combined mode to obtain a target sample.
When bisphenol F and bisphenol S are detected by a traditional method, a sample is combined with an extracting agent, and the supernatant is purified after direct centrifugation, for example, a GCB column or other types of columns are used for purifying a sample matrix, so that the sample matrix can be purified, but the experimental recovery rate of BPF and BPS is not high, the background interference is large, and finally the sensitivity of the method is low. The invention optimizes the sample pretreatment, for example, before obtaining the supernatant, the extraction process needs to be carried out under the ice bath condition, the ice bath can reduce the dissolution rate of the background of the centrifugal tube, and the background effect of the experimental vessel is reduced; the purification adopts a QuEChERS buffer salt reagent bag to purify lipid in a sample, and then further purifies the lipid by using a PRIME HLB column, and the combined technology can more completely reduce matrix interference and can also consider the experimental recovery rate and sensitivity of BPF and BPS.
Preferably, in the sample pretreatment method, in step S1, ultrasonic extraction is performed for 10 to 20min under an ice bath condition. The extractant is ethyl acetate or acetonitrile.
Preferably, in the step S2, the rotation speed of the centrifugation is 8000-12000 r/min, and the centrifugation is carried out for 10-20 min each time.
Preferably, in step S4, the purified liquid is blown with nitrogen, redissolved, and passed through a membrane for further use.
The invention also provides a method for detecting the bisphenol S and the bisphenol F in the animal derived food, namely, an external standard substance is added into a sample to carry out quantitative analysis on the bisphenol S and the bisphenol F, and an internal standard substance is used for correcting the possible matrix interference effect and compensating the loss of the target compound in the pretreatment process. In the detection process, the detection values of the sample blank and the reagent blank are used as background effect detection values, and the actual content of the bisphenol S and the bisphenol F in the animal-derived food is obtained by subtracting the background effect detection values from the content of the bisphenol S and the bisphenol F obtained by detection. The method specifically comprises the following steps:
s1, obtaining a target sample solution according to the pretreatment method;
s2, preparing a standard solution, a sample blank and a reagent blank;
s3, adding an internal standard solution into the target sample solution obtained in the step S1 and the standard solution, the sample blank and the reagent blank of the step S2, and respectively detecting through a high performance liquid chromatography-mass spectrometer;
s4, taking the ratio of the quantitative ion peak area of the target compound to the internal standard peak area as a vertical coordinate, taking the mass concentration of the compound to be detected as a horizontal coordinate, drawing a quantitative standard curve, and calculating the content of bisphenol S and bisphenol F in the target sample according to the standard curve and the peak area detected by a high performance liquid chromatography-mass spectrometer;
and S5, quantifying according to a standard curve, and deducting the concentrations of corresponding reagent blanks and sample blanks to finally obtain the actual contents of bisphenol S and bisphenol F in the target sample solution to be detected.
In the detection process, three reagent blank and sample blank experiments are carried out each time when the sample is analyzed, and the average value is takenAnd as a blank correction value (namely an experimental background value) of the measurement result of the same batch of samples, the interference of the background effect is reduced to a certain extent. Meanwhile, in order to compensate the loss of the target compound in the pretreatment process, an isotope internal standard method is adopted in the quantitative method. With internal standard BPA-D of similar chemical nature16As an alternative internal standard, the detected amounts of BPS and BPF were corrected. Preparing a series of mixed standard solutions with different high and low concentrations, adding an isotope internal standard with the same concentration, taking the ratio of the quantitative ion peak area of the target compound to the internal standard peak area as a vertical coordinate, taking the mass concentration of the compound to be detected as a horizontal coordinate, and drawing a quantitative standard curve.
Specifically, the actual contents of bisphenol S and bisphenol F in the sample to be measured are calculated and obtained through the following formula:
X＝(ρ-ρ0-ρb)×V/m。
wherein X is the content of bisphenol S or bisphenol F in the sample, and is mu g/kg; v is the volume of concentrated solution of the sample with constant volume, mL; rho is the content of bisphenol S or bisphenol F in the sample solution read by the instrument, ng/mL; p0The content of bisphenol S or bisphenol F in the reagent blank is ng/mL; rhobThe content of bisphenol S or bisphenol F in the sample blank is ng/mL; m is sample weighing amount, g.
Performing three parallel determinations on each reagent blank and each sample blank, taking the arithmetic mean value of the reagent blanks, and recording the reagent blank as 0 when the reagent blank concentration is greater than the sample blank; the samples were measured in duplicate each time, and the arithmetic mean of the samples was used as a result, with three significant figures being retained.
Preferably, the chromatographic conditions of the high performance liquid phase are as follows:
a chromatographic column: ACQUITY UPLC C18, Specification: 1.7 μm, 3.0X 100 mm; sample introduction amount: 3 mu L of the solution; column temperature: 40 ℃; flow rate: 0.3 mL/min; mobile phase: the mobile phase A is pure water solution, and the mobile phase B is methanol;
the high performance liquid chromatography adopts gradient elution, and the conditions of the gradient elution are as follows: when 0-0.5min, the mobile phase is 90% A + 10% B; when the time is 0.5-1.5min, the mobile phase is changed from 90% A to 65% A; when the time is 1.5-3.5min, the mobile phase is changed from 65% A to 35% A; when the time is 3.5-6.5min, the mobile phase is changed from 35% A to 5% A; keeping the mobile phase at 5% A + 95% B within 6.5-7.5 min; when 7.5-7.6min, the mobile phase is changed from 5% A to 90% A; and when 7.6-9.00min, keeping the mobile phase at 90% A + 10% B.
Preferably, the conditions of the mass spectrum are as follows:
an ion source: electrospray ionization source (ESI); scanning mode: multiple Reaction Monitoring (MRM); positive and negative ion modes: a negative ion mode; mode temperature: 550 ℃; time: 9min, delay 0, cycle 1S; CXP voltage: 13V; air curtain gas (CUR): 35 psi; collision gas (CAD): 9; ionization voltage (IS): -4500V; temperature (TEM): 550 ℃; nebulization (GS 1): 55 psi; auxiliary heating gas (GS 2): 55 psi. The atomizing gas, the air curtain gas, the auxiliary gas and the collision gas used in the mass spectrum detection are all high-purity nitrogen. The bisphenol S takes 249/108.2 as a quantitative ion pair; DP Voltage: 150V; CE voltage: 37.2V. The bisphenol F takes 199/105.2 as a quantitative ion pair; DP Voltage: 135V; CE voltage: 28.9V. Internal standard bisphenol A-D16 had a quantitation ion of 241.2/222, DP voltage: 49V; CE voltage: 45V.
The invention also provides application of the method for detecting the bisphenol S and the bisphenol F in the animal-derived food by the high performance liquid chromatography-mass spectrometry.
In the above application, the animal-derived food includes, but is not limited to, eggs, livestock meat, and poultry meat.
Specifically, the food of animal origin comprises pork, chicken, egg, etc.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a sample pretreatment method for detecting bisphenol S and bisphenol F in animal derived food, and establishes a method for detecting the content of bisphenol S and bisphenol F in animal derived food by high performance liquid chromatography-mass spectrometry, the sample pretreatment used in the method is simple and rapid, and an internal standard substance is added at the beginning of the sample pretreatment, so that the effective quality control is performed on the whole operation process including the pretreatment process; detecting the concentrations of bisphenol S and bisphenol F in pork by the method, and finally obtaining bisphenol S and bisphenol F in the sample by deducting the measured values of reagent blank and sample blankActual content of bisphenol F; the standard curve is obtained by measuring bisphenol S and bisphenol F in the range of standard addition concentration of 2-100ng/mL, and the linear correlation coefficient R of BPS20.9995 with detection limit of 0.010 μ g/kg; BPF linear correlation coefficient R2The detection limit is 0.9997, the recovery rate of the spiked standard with different concentrations is 84.6-102%, and the precision is less than 5.49%, and the method has simple operation, high precision and low detection limit (reaching ppb level); the detection method is suitable for accurately, rapidly, qualitatively and quantitatively detecting the bisphenol S and the bisphenol F in the animal-derived food.
Drawings
FIG. 1 shows quantitative ion chromatograms of bisphenol S extracted with different solvents (A is methanol, B is acetonitrile, and C is ethyl acetate);
FIG. 2 is a quantitative ion chromatogram of bisphenol F extracted with different solvents (A is methanol, B is acetonitrile, and C is ethyl acetate);
FIG. 3 is a standard curve of bisphenol S and bisphenol F over a range of spiking concentrations from 2 to 100 ng/mL;
FIG. 4 shows bisphenol S and bisphenol F and internal standard bisphenol A-D16Ion chromatogram of Standard solution (A)1、B1Ion chromatogram map of bisphenol F quantitative characteristic and qualitative characteristic2、B2The ion chromatogram map of the quantitative characteristic and the qualitative characteristic of bisphenol S is shown, and C is internal standard bisphenol A-D16Ion chromatograms);
FIG. 5 shows an ion chromatogram of a 2.00ng/mL sample at the time of standard addition (A)1、B1Ion chromatogram map of bisphenol F quantitative characteristic and qualitative characteristic2、B2Ion chromatogram for quantitative characteristics and qualitative characteristics of bisphenol S).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following examples and experimental examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available reagents and materials; the equipment used, unless otherwise specified, is conventional laboratory equipment, and reference to "solutions" is not specifically intended to be aqueous.
Example 1 optimization of sample extractants
Pork was used as a study, and the extraction efficiency of 10mL of methanol, acetonitrile and ethyl acetate was examined.
Taking 2.00g of pork as a research object, adding 5.0 mu g/kg of mixed standard solution and 20 mu g/kg of internal standard solution, respectively adding the extracting solution, fully whirling, performing ultrasonic extraction, centrifuging, taking supernatant liquid, blowing nitrogen to be dry, re-dissolving with 50% methanol water, and performing on-machine detection to finally determine the optimal extraction solvent of the sample. The results are shown in table 1 and fig. 1-2, and show that the extraction efficiency of methanol is low in 3 kinds of extract solutions, the extraction efficiency of acetonitrile and ethyl acetate is between 96.5% and 100.5%, and the difference is not large, but the ethyl acetate is selected as the extraction solvent in the present study because the response values of BFS and BPF chromatographic peaks extracted by ethyl acetate are good, the background interference is small, and the boiling point of ethyl acetate is relatively low, so that the concentration time of nitrogen blowing can be shortened.
Table 1 comparison of extraction efficiency for different solvents (n ═ 3)
EXAMPLE 2 optimization of sample purification reagents
The sample pretreatment process comprises the following steps:
s1, homogenizing the animal-derived food to be detected, adding an extracting agent, oscillating, and carrying out ultrasonic treatment under an ice bath condition;
s2, centrifuging for the first time to obtain supernatant and precipitate;
s3, adding the extracting agent into the precipitate again, and repeating the steps to obtain supernatant liquid of secondary extraction;
and S4, combining the supernatants after the two centrifugations, and purifying the supernatants to obtain the target sample.
For some animal-derived foods with complex matrixes and high fat content, the sensitivity of the method can be reduced while removing interfering substances by traditional purification, the invention compares the purification effect of a sample extracting solution by comparing 3 different purification methods of QuEChERS buffer salt, Oasis HLB and QuEChERS buffer salt and PRIME HLB, and determines the most appropriate purification method according to the recovery rate of bisphenol F and bisphenol S of different purification methods and the time length of a pretreatment process.
Taking 2.00g of pork as a research object, obtaining centrifuged supernatant according to the pretreatment process, and carrying out the following specific steps by 3 different purification means:
1. and (3) passing the obtained supernatant through a QuEChERS buffer salt reagent bag, adding 50uL formic acid, fully whirling, centrifuging, sucking the supernatant, concentrating, re-dissolving, and measuring on a machine.
2. And (3) loading the obtained supernatant through an Oasis HLB column (sequentially activating by 6mL of methanol and 6mL of water before use), discarding the filtrate, then rinsing by using a 6mL methanol-water solution (v: v ═ 1:1), blowing the liquid in the column bed by using an aurilave after the rinsing liquid completely passes through the column bed, eluting by using 6mL of methanol, concentrating the eluent, and performing redissolution on the machine for determination.
3. Passing the obtained supernatant through a QuEChERS buffer salt reagent bag, fully whirling, centrifuging, taking the supernatant, passing through a rapid quantitative filter paper, further purifying the obtained filtrate, directly passing through PRIME HLB (without activation), concentrating the purified filtrate, re-dissolving, and measuring by using a machine.
The specific results using different means of purification are shown in table 2. The results show that recovery of BPS after QuEChERS buffer salt decontamination is higher than 80%, but recovery of BPF is lower than 70%; the recovery rates of BPF and BPS after purification by an Oasis HLB column are both around 80%; the recovery rates of BPF and BPS after the QuEChERS buffer salt is cooperated with the PRIME HLB column for purification are both higher than 80%; presumably, due to incomplete purification of the matrix of the BPF by QuEChERS buffer salt purification, the background of the purified matrix inhibits the BPF, and then the purified matrix is further purified by a PRIME HLB column, and due to the characteristic that the filler can selectively retain the matrix and can reduce the interference of matrix effect, the synergistic effect of the two purification means is better. Considering that the Oasis HLB column purification operation is complex, the column purification includes multiple steps of activation, elution, and the time consumption is long, while the whole purification process of the QuEChERS buffer salt and PRIME HLB column is simple to operate and short in time consumption, so the QuEChERS buffer salt and PRIME HLB column are selected as the purification mode of the experiment.
TABLE 2 comparison of recovery rates for different purification means (n. 3)
Example 3 establishment of method for detecting bisphenol S and bisphenol F in animal derived food by high performance liquid chromatography-mass spectrometry
(1) Pretreatment
Weighing 2.00g of pork sample, and placing the pork sample in a 50mL centrifuge tube;
adding ethyl acetate 5mL, shaking for 10min, performing ultrasonic extraction for 20min under ice bath, centrifuging for 7min after extraction, and collecting supernatant;
adding 5mL of ethyl acetate into the precipitate, repeating the steps, collecting the supernatant obtained by the second extraction, and combining the supernatants;
adding a reagent bag added with QuEChERS buffer salt, fully mixing for 1min by vortex, centrifuging for 10min, taking supernate and quickly quantifying filter paper, and keeping the obtained filtrate for later use.
Transferring 4.0mL of the filtrate, eluting with a PRIME HLB solid phase extraction column, accurately transferring the eluent into a 2.5-15 mL centrifugal tube, drying the eluent with nitrogen in a water bath at 40 ℃, metering the volume to 1.00mL with methanol-water solution (volume ratio is 1:1), filtering with a 0.22 mu m filter membrane, and measuring with a machine for use.
(2) Liquid chromatography and mass spectrometry conditions
Chromatographic conditions
An ACQUITY UPLC C181.7 μm 3.0X 100mm column; the column temperature is 40 ℃; the sample injection volume is 3 mu L; the mobile phase A is pure water solution, and the mobile phase B is methanol; the flow rate is: 0.3mL/min, and gradient elution conditions are shown in Table 3.
TABLE 3 gradient elution conditions
Conditions of Mass Spectrometry
An ion source: electrospray ionization source (ESI); scanning mode: multiple Reaction Monitoring (MRM); positive and negative ion modes: a negative ion mode; mode temperature: 550 ℃; time: 9min, delay 0, cycle 1S; CXP voltage: 13V; air curtain gas (CUR): 35 psi; collision gas (CAD): 9; ionization voltage (IS): -4500V; temperature (TEM): 550 ℃; nebulization (GS 1): 55 psi; auxiliary heating gas (GS 2): 55 psi. The atomizing gas, the air curtain gas, the auxiliary gas and the collision gas used in the mass spectrum detection are all high-purity nitrogen. Bisphenol S and bisphenol F and internal standard bisphenol A-D16The mass spectral parameters of (a) are as follows (table 4):
TABLE 4 bisphenol S, bisphenol F and bisphenol A-D16Mass spectrum number of
(3) Establishment of a Standard Curve
Performing chromatographic-mass spectrometric analysis on a pork sample according to the methods (1) and (2), adding standard, measuring, adding external scalar quantities (bisphenol S and bisphenol F) of 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL, wherein the content of the internal standard is 20 mu g/kg, taking the ratio of the quantitative ion peak area of the target compound to the internal standard peak area as a vertical coordinate, and taking the mass concentration of the compound to be measured as a horizontal coordinate, and drawing a quantitative standard curve. And obtaining a linear regression equation and a correlation coefficient. The results are shown in Table 5 and FIG. 3.
TABLE 5 Standard Curve regression equations and correlation coefficients for bisphenol S and bisphenol F
| Compound (I) | Regression equation | Coefficient of correlation R2 | Linear range (ng/mL) |
| BPS | y＝1.0042x-0.2935 | 0.9991 | 2-100 |
| BPF | y＝0.9995x+0.0386 | 0.9995 | 2-100 |
As shown in Table 4 and FIG. 3, the regression equation for bisphenol S in the linear range of 2-100ng/mL normalized concentration is Y-1.0042 x-0.2935, correlation R20.9991; the regression equation of bisphenol F in the linear range of the normalized concentration of 2-100ng/mL is that Y is 0.9995x +0.0386 and the correlation R20.9995, the linear relationship is good.
(4) Detection Limit (Limit of Detection, LOD)
According to the methods (1) and (2), pork is taken as a substrate, seven parallel addition experiments are carried out, the addition concentration of bisphenol S and bisphenol F is 2 mu g/kg, and internal standard bisphenol A-D is added16The concentration is 20 mug/kg, three reagent blanks and sample blanks are made as experiment background values, pretreatment is carried out according to the method in (1), the concentration of bisphenol S and bisphenol F of each sample is measured by a computer by adopting an internal standard method, and the final actual concentration of bisphenol S of each sample after deducting the background values is calculated as follows: 1.98. mu.g/kg, 1.99. mu.g/kg, 1.79. mu.g/kg, 1.78. mu.g/kg, 1.95. mu.g/kg, 1.79. mu.g/kg, detectedThe limit is 0.010 mu g/kg; the bisphenol F concentrations were: 2.02. mu.g/kg, 1.97. mu.g/kg, 2.00. mu.g/kg, 1.95. mu.g/kg, 1.98. mu.g/kg, 1.89. mu.g/kg, 1.96. mu.g/kg, the detection limit is: 0.126. mu.g/kg. The calculation formula is as follows:
limit of detection (LOD) ═ KxSb×C/X
In the formula: k is confidence factor, and 3 is taken; standard deviation of Sb-parallel test sample content; c-adding standard concentration; x-average of the contents of the replicates.
(5) Recovery (%) and precision (RSD%): the pork sample was analyzed according to the detection methods (1) and (2), and the amounts of bisphenol S and bisphenol F added were 2.00ng/mL, 5.0ng/mL and 50.0ng/mL, respectively, and the internal standard bisphenol A-D was added16The contents were all 20. mu.g/kg, and the concentrations determined for the three reagent blanks and the sample blank were set as experimental background values, and 6 replicates were performed for each concentration level, and the precision (RSD%) and recovery (%) of each group were calculated simultaneously. The results are shown in tables 6 to 11.
TABLE 62.00 ng/mL sample plus scalar BPF recovery and precision profiles
TABLE 72.00 ng/mL sample plus scalar BPS recovery and precision profiles
TABLE 85.0 ng/mL sample plus scalar BPF recovery and precision profiles
TABLE 95.0 ng/mL sample plus scalar BPS recovery and precision profiles
TABLE 1050.0 ng/mL sample plus scalar BPF recovery and precision profiles
TABLE 1150.0 ng/mL sample plus scalar BPS recovery and precision profiles
Note: in the table, LOD of bisphenol F is 0.126. mu.g/kg; LOD of bisphenol S is 0.010 mu g/kg;
as can be seen from tables 6 to 11 and fig. 4 and 5, the recovery rate of the results obtained by performing the liquid chromatography-mass spectrometry detection on the samples with different addition concentrations is 84.6% to 102%, and the precision is less than 5.49%, which indicates that the detection method of the present invention has high precision and recovery rate.
Example 4 detection of bisphenol S and bisphenol F in food of animal origin Using the detection method of the present invention
3 parts of animal-derived food randomly purchased from Shenzhen farmer market were subjected to liquid chromatography-mass spectrometry detection by the experimental method of example 3, wherein 2 parts of pork (numbered Y1 and Y2), 2 parts of chicken (numbered Y3 and Y4) and 2 parts of egg (numbered Y5 and Y6) were subjected to double parallel determination, and the average concentration was determined as the determination concentration of bisphenol S and bisphenol F in the sample. The final actual bisphenol S and bisphenol F concentrations are the average concentration of the measured sample concentration minus the reagent blank and sample blank. The measurement results are shown in Table 12.
As shown by the results of the experimental measurement in Table 12, bisphenol S and bisphenol F were not detected in all the samples.
TABLE 126 contents of bisphenol S and bisphenol F in animal-derived foods
Note: in the table, LOD of bisphenol F is 0.126. mu.g/kg; LOD of bisphenol S is 0.010 mu g/kg; "ND" means not detected
According to the invention, ethyl acetate is used as an extraction reagent and combined with ultrasonic in an ice bath, so that bisphenol F and bisphenol S in a sample are fully extracted, then the sample is purified by a QuEChERS buffer salt reagent bag in cooperation with a PRIME HLB column, most of matrix interference in the sample is removed, and finally the high performance liquid chromatography-mass spectrometry combined measurement is carried out. Because the plastic product container is inevitably used in the experimental process, background effect exists, in order to eliminate the influence of the factors on the experiment, the background effect detection is added while the sample is analyzed each time, the effect value is measured on a computer, and the actual content of the bisphenol S and the bisphenol F in the sample is obtained by deduction calculation. Thus, a method for measuring the contents of bisphenol S and bisphenol F in animal-derived food by high performance liquid chromatography-mass spectrometry is established, a standard curve is obtained by measuring the contents of bisphenol S and bisphenol F in the concentration range of 2-100ng/mL, and the linear correlation coefficient R of BPS20.9991, detection limit of 0.010 μ g/kg, linear correlation coefficient R of BPF20.9995 with a detection limit of 0.126 mug/kg; the standard adding recovery rate of the bisphenol S and the bisphenol F is between 84.6 and 102 percent and the precision is less than 5.49 percent when the standard adding concentration is between 2 and 50 ng/mL; the method has the advantages of low detection limit, high precision, economy, practicality and good sample extraction stability, and the detection method is suitable for rapid, accurate, qualitative and quantitative detection of the bisphenol S and the bisphenol F in the animal derived food.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (8)
1. A sample pretreatment method for detecting bisphenol S and bisphenol F in animal derived food is characterized by comprising the following steps:
s1, homogenizing the animal-derived food to be detected, adding an extracting agent, oscillating, and carrying out ultrasonic treatment under an ice bath condition;
s2, centrifuging for the first time to obtain supernatant and precipitate;
s3, adding the extracting agent into the precipitate again, and repeating the steps to obtain supernatant liquid of secondary extraction;
and S4, combining the supernatants after the two centrifugations, and purifying the supernatant by using a QuEChERS buffer salt reagent and a PRIME HLB small column in a combined mode to obtain a target sample.
2. The sample pretreatment method for detecting bisphenol S and bisphenol F in animal derived food according to claim 1, wherein in step S1, ultrasonic extraction is performed for 10-20 min under ice bath condition; the extractant is ethyl acetate or acetonitrile.
3. A method for detecting bisphenol S and bisphenol F in animal derived food by high performance liquid chromatography-mass spectrometry is characterized in that the bisphenol S and bisphenol F are quantitatively analyzed by using an external standard, and the internal standard is used for correcting matrix interference effect and compensating loss of a target compound in a pretreatment process; in the detection process, the detection values of the sample blank and the reagent blank are used as background effect detection values, and the actual content of the bisphenol S and the bisphenol F in the animal-derived food is obtained by subtracting the background effect detection values from the content of the bisphenol S and the bisphenol F obtained by detection.
4. The method for detecting bisphenol S and bisphenol F in food of animal origin by high performance liquid chromatography-mass spectrometry as claimed in claim 3, comprising the steps of:
s1, obtaining a target sample solution according to the pretreatment method of claim 1 or 2;
s2, preparing a standard solution, a sample blank and a reagent blank;
s3, adding an internal standard solution into the target sample solution obtained in the step S1 and the standard solution, the sample blank and the reagent blank of the step S2, and respectively detecting through a high performance liquid chromatography-mass spectrometer;
s4, taking the ratio of the quantitative ion peak area of the target compound to the internal standard peak area as a vertical coordinate, taking the mass concentration of the compound to be detected as a horizontal coordinate, drawing a quantitative standard curve, and calculating the content of bisphenol S and bisphenol F in the target sample according to the standard curve and the peak area detected by a high performance liquid chromatography-mass spectrometer;
and S5, quantifying according to a standard curve, and deducting the concentrations of corresponding reagent blanks and sample blanks to finally obtain the actual contents of bisphenol S and bisphenol F in the target sample solution to be detected.
5. The method for detecting bisphenol S and bisphenol F in animal derived food by high performance liquid chromatography-mass spectrometry as claimed in claim 4, wherein the internal standard is BPA-D16。
6. The method for detecting bisphenol S and bisphenol F in food of animal origin according to claim 5, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: ACQUITY UPLC C18, Specification: 1.7 μm, 3.0X 100 mm; sample introduction amount: 3 mu L of the solution; column temperature: 40 ℃; flow rate: 0.3 mL/min; mobile phase: the mobile phase A is pure water solution, and the mobile phase B is methanol;
the high performance liquid chromatography adopts gradient elution, and the conditions of the gradient elution are as follows: when 0-0.5min, the mobile phase is 90% A + 10% B; when the time is 0.5-1.5min, the mobile phase is changed from 90% A to 65% A; when the time is 1.5-3.5min, the mobile phase is changed from 65% A to 35% A; when the time is 3.5-6.5min, the mobile phase is changed from 35% A to 5% A; keeping the mobile phase at 5% A + 95% B within 6.5-7.5 min; when 7.5-7.6min, the mobile phase is changed from 5% A to 90% A; and when 7.6-9.00min, keeping the mobile phase at 90% A + 10% B.
7. The method for detecting bisphenol S and bisphenol F in food of animal origin according to claim 6, wherein the conditions of mass spectrometry are as follows:
an ion source: electrospray ionization source ESI; scanning mode: multiple reaction monitoring, MRM; positive and negative ion modes: a negative ion mode; mode temperature: 550 ℃; time: 9min, delay 0, cycle 1S; CXP voltage: 13V; air curtain air: 35 psi; collision gas: 9; ionization voltage: -4500V; temperature: 550 ℃; spraying mist: 55 psi; auxiliary heating gas: 55 psi; the bisphenol S takes 249/108.2 as a quantitative ion pair; DP Voltage: 150V; CE voltage: 37.2V; the bisphenol F takes 199/105.2 as a quantitative ion pair; DP Voltage: 135V; CE voltage: 28.9V; internal standard bisphenol A-D16 had a quantitation ion of 241.2/222, DP voltage: 49V; CE voltage: 45V.
8. Use of the method for detecting bisphenol S and bisphenol F in food of animal origin according to any of claims 3 to 7, wherein said food of animal origin includes but is not limited to eggs, livestock meat, poultry meat.
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