CN114720570B - Method for detecting 8 estrogens in fish meat - Google Patents
Method for detecting 8 estrogens in fish meat Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention provides a method for detecting 8 kinds of estrogens in fish meat, which comprises the following steps: sequentially adding an internal standard substance, an acetonitrile solution and potassium hydrogen phosphate into a fish sample to form a double-aqueous phase, taking supernatant, measuring the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, determining 8 kinds of estrogens in the sample solution according to the retention time, and quantifying by adopting an internal standard curve method to determine the content of the 8 kinds of estrogens in the sample solution. The method for detecting 8 kinds of estrogens in fish provided by the invention can be used for rapidly, efficiently, sensitively and accurately detecting 8 kinds of estrogens in fish at the same time, greatly improves the detection efficiency, and has the advantages of high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection result and the like.
Description
Technical Field
The invention belongs to the technical field of organic pollutant residue detection, relates to a method for detecting 8 kinds of estrogens in fish, and in particular relates to a method for simultaneously detecting 8 kinds of estrogens in fish by combining aqueous two-phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
Background
In recent years, the aquaculture industry in China develops rapidly, and the cultivation scale is larger and larger. In order to reduce the cultivation cost, part of farmers can adopt chicken manure as feed to raise catfish, grass carp, silver carp and the like. As the chicken manure contains more estrogen substances, the substances possibly enter the fish body through the ingestion of fish and enter the human body through a food chain, so that the residual detection of the estrogen substances in the fish body has important significance for the evaluation of the dietary exposure risk and the ecological risk of the human body.
The fish body contains abundant proteins and fats, and has great influence on extraction and purification of substances such as estrogen and the like in the body, which forms a great challenge for accurately and efficiently measuring the content of the estrogen in the fish body. Therefore, the establishment of an effective separation, purification and enrichment method is particularly important for accurately measuring the content of estrogen in fish bodies. The extraction method commonly used in the pretreatment of the sample mainly comprises a Soxhlet extraction method, an accelerated solvent extraction method, an ultrasonic extraction method, a microwave extraction method, a double water phase extraction method and the like. The aqueous two-phase extraction is based on the selective partitioning of the material between the two phases, but the nature of the extraction system is different. When the substance enters the aqueous two-phase system, the concentration of the substance in the upper phase and the lower phase is different due to the surface property, the charge effect, the existence of various forces (such as hydrophobic bond, hydrogen bond, ionic bond and the like) and the influence of environmental factors. The partition coefficient K is equal to the concentration ratio of substances in two phases, and the substances can be separated by using a double water phase extraction system due to different K values of various substances. The method has been used for separating various proteins and enzymes in organisms. The detection methods commonly used at present mainly comprise High Performance Liquid Chromatography (HPLC) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The HPLC-MS/MS has strong separation capability of HPLC, high sensitivity of MS and extremely strong qualitative identification capability, is one of the most effective means for detecting trace pollutant residues of complex matrixes in the environment at present, and has the advantages of wide analysis range, strong separation capability, reliable qualitative analysis result, low detection limit, quick analysis time, high automation degree and the like. The components of the complex mixture can be accurately, qualitatively and quantitatively determined.
At present, a complete and reliable analysis method for separating, enriching and co-detecting various estrogens in fish bodies does not exist, and the existing method generally has the problems of instability, more interference, limited types and quantity of detected estrogens, limited recovery rate and the like. Therefore, a need exists for a complete and reliable assay including separation enrichment and co-detection that rapidly, efficiently, sensitively, and accurately detects as much of the estrogen residue in an aqueous product as possible to meet increasingly stringent residue limit requirements.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for detecting 8 kinds of estrogens in fish, which can simultaneously detect 8 kinds of estrogens in fish, and has the advantages of stable detection result, small interference, rapidness, high efficiency, high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection result, etc.
To achieve the above and other related objects, the present invention provides a method for detecting 8 kinds of estrogens in fish meat, comprising: sequentially adding internal standard, acetonitrile solution and potassium hydrogen phosphate (K) into fish sample 2 HPO 4 ) Forming a double aqueous phase, taking supernatant, measuring the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, determining 8 kinds of estrogens in the sample solution according to the retention time, quantifying by adopting an internal standard curve method, and determining the content of the 8 kinds of estrogens in the sample solution.
Preferably, the 8 estrogens include 17β -estradiol (E2, CAS No. 50-28-2), estrone (E1, CAS No. 53-16-7), estriol (E3, CAS No. 50-27-1), 17α -ethinyl estradiol (EE 2, CAS No. 57-63-6), diethylstilbestrol (DES, CAS No. 56-53-1), octylphenol (OP, CAS No. 27193-28-8), nonylphenol (NP, CAS No. 25154-52-3), bisphenol A (BPA, CAS No. 80-05-7).
Preferably, the fish sample is homogenized. The homogenization is to clean the collected fish body sample, absorb the surface moisture with gauze, split the muscles on the back and the abdomen of the fish body, separate the fish skin, and homogenate the fish body sample after adding water with the same mass. The homogenized fish sample was rapidly frozen at-20 ℃.
Preferably, the internal standard comprises nonylphenol-D 8 (NP-D 8 CAS number 25154-52-3), bisphenol A-D 16 (BPA-D 16 CAS number 96210-87-6), ethinyl estradiol-D 4 (EE2-D 4 The CAS number is 350820-06-3), diethylstilbestrol-D 8 (DES-D 8 91318-10-4 CAS number), estradiol-D 3 (E2-D 3 CAS number 79037-37-9).
More preferably, in the internal standard, nonylphenol-D 8 (NP-D 8 ) Bisphenol A-D as an internal standard for Nonylphenols (NP) and Octylphenols (OP) 16 (BPA-D 16 ) Ethinyl estradiol-D as an internal standard for bisphenol a (BPA) 4 (EE2-D 4 ) diethylstilbestrol-D as an internal standard for 17α -ethinyl estradiol ((EE 2) 8 (DES-D 8 ) estradiol-D as an internal standard for Diethylstilbestrol (DES) 3 (E2-D 3 ) As internal standard for 17 beta-estradiol (E2), estrone (E1) and estriol (E3).
Given the large differences in structure and properties of the different estrogens, the addition of internal standards avoids the possible losses during pretreatment.
Preferably, the ratio of the mass of the fish sample to the mass of the addition of 5 internal standards is 3.00+/-0.02: 0.1:0.1:0.1:0.1:0.1.
preferably, the fish sample is mixed uniformly after the internal standard is added.
Preferably, the ratio of the mass g of the fish sample added to the volume mL of acetonitrile solution added is 3.00+/-0.02: 8-12, preferably 3.00±0.02:10.
preferably, the acetonitrile solution is an acetonitrile aqueous solution with the volume percentage concentration of 70-80%. More preferably, the acetonitrile solution is an aqueous acetonitrile solution with a concentration of 75% by volume.
Preferably, the acetonitrile is added followed by vortexing and standing to separate the solutions to form a single aqueous phase.
More preferably, the vortex mixing is for a period of time of 1.5 to 2.5 minutes, preferably 2 minutes.
More preferably, the time of the standing is 40 to 50min, preferably 45min.
Preferably, the mass ratio of the fish sample to the potassium hydrogen phosphate is 3.00+/-0.02: 1-3, preferably 3.00±0.02:2.
preferably, the potassium hydrogen phosphate is added and then vortexed and allowed to stand to separate the solution into two aqueous phases.
More preferably, the vortex mixing is for a period of time of 1.5 to 2.5 minutes, preferably 2 minutes.
More preferably, the time of the standing is 1.5 to 3.0 hours, preferably 2.5 hours.
Preferably, the supernatant is filtered using a glass fiber filter membrane. When forming the two aqueous phases, the target is enriched in the supernatant. The target substance matrix in the supernatant is clean, and the instrument test can be directly carried out after filtration.
More preferably, the glass fiber filter membrane has a pore size of 0.2-0.3 μm, preferably 0.22 μm.
Preferably, the sample solution is measured by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), comprising the following steps:
1) Preparing a standard solution: 8 kinds of estrogen standard substances are taken, and an internal standard substance and a methanol solution are added to prepare a standard solution;
2) Sample detection: and (2) respectively detecting the sample solution and the standard solution in the step (1) by adopting an anion mode of an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), comparing the obtained liquid chromatogram of the sample solution with the liquid chromatogram of the standard solution, identifying the characterization of the common characteristic peak according to the relative retention time, quantifying according to the chromatographic peak area of the common characteristic peak by an internal standard curve method, and determining the concentration of 8 estrogens in the sample solution.
More preferably, in step 1), the concentration of each of the 5 internal standards in the standard solution is 100.0. Mu.g.L -1 。
More preferably, in step 1), the methanol solution is an aqueous methanol solution with a volume percentage concentration of 65-75%, and the volume percentage concentration is preferably 70%.
More preferably, in step 1), the concentration of 8 estrogens in the standard solution is in the range of 1.37-333.33. Mu.g.L -1 。
More preferably, in step 2), in the ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, the negative ion mode is detected by using an shimadzu 30A ultra performance liquid chromatography tandem AB 5500Q-trap mass spectrometer (AB company in the united states).
More preferably, in step 2), when the negative ion mode is detected, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: a C18 column; column temperature: 35-45 ℃; sample injection amount: 4-6. Mu.L; flow rate: 0.2-0.4mL/min; the mobile phase A is aqueous solution of ammonia water with the volume percentage concentration of 0.05-0.15%; the mobile phase B is acetonitrile; the analysis time is 6.1min; gradient elution.
Further preferably, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1X column length 100mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 5. Mu.L; flow rate: 0.3mL/min; the mobile phase A is aqueous solution of ammonia water with the volume percentage concentration of 0.1%; the mobile phase B is acetonitrile; the analysis time is 6.1min; gradient elution.
Further preferably, the specific procedure of the gradient elution is:
0-2min, phase A: the volume ratio of the phase B is 90:10-90:10;
2-2.2min, phase A: the volume ratio of the phase B is 90:10-40:60;
2.2-3.5min, phase A: the volume ratio of the phase B is 40:60-40:60;
3.5-3.8min, phase A: the volume ratio of the phase B is 40:60-3:97;
3.8-6min, phase A: the volume ratio of the phase B is 3:97-3:97;
6-6.1min, phase A: the volume ratio of the phase B is 3:97-90:10.
more preferably, in step 2), the measurement conditions of the mass spectrum (MS/MS) when the negative ion mode is detected are:
ionization mode: electrospray ion source (ESI), negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, the auxiliary heating gas GS2 was 50psi, the collision gas CAD was Medium, and the collision gas was high purity nitrogen.
Further preferably, the quantitative ion, collision energy, declustering voltage (DP, V) are as shown in table 1.
TABLE 1
Note that: * Representing quantitative ions; * Represents the collision energy corresponding to the quantitative ion.
Preferably, in step 2), the internal standard curve method includes the following steps:
a1 Respectively carrying out negative ion mode analysis of a high performance liquid chromatography-mass spectrometry method on 8 kinds of standard solutions containing a series of different concentrations of estrogens to obtain a linear relation between the ratio of the chromatographic peak area of 8 kinds of estrogen components under different concentrations to the chromatographic peak area of an internal standard and the ratio of the different concentrations of the corresponding components to the internal standard, drawing a corresponding standard curve, and carrying out regression operation by using a weighted least square method to obtain a regression equation of the standard curve of the 8 kinds of estrogen components;
a2 Analyzing the sample solution by using a high performance liquid chromatography-mass spectrometry method, substituting the obtained ratio of the chromatographic peak areas of 8 estrogen components under different concentrations to the chromatographic peak areas of the internal standard into the regression equation of the standard working curve of the corresponding components in the step A1), and calculating the concentration of 8 estrogen components in the sample solution according to the known concentration of the internal standard.
More preferably, in step A1) or A2), the ratio of the chromatographic peak area of 8 estrogen components at different concentrations to the chromatographic peak area of the internal standard is taken as an ordinate (Y-axis), and the ratio of the different concentrations of the corresponding components to the concentration of the internal standard is taken as an abscissa (X-axis).
The water used in the present invention is ultrapure water.
As described above, the method for detecting 8 kinds of estrogens in fish provided by the invention adopts a double aqueous phase extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology, optimizes the extraction method, the purification condition and the liquid quality detection condition, and simultaneously selects a proper internal standard, so that the residual condition of 8 kinds of estrogens in fish can be detected at one time, and the method has the following beneficial effects:
(1) According to the method for detecting 8 kinds of estrogens in fish, provided by the invention, the acetonitrile water solution with specific concentration is used as the extraction liquid, so that the protein can be slowly denatured, and the estrogens in the fish can be extracted.
(2) According to the method for detecting 8 kinds of estrogens in fish, acetonitrile-water-salt extraction is adopted to form a double-water phase, impurities are effectively removed from a lower salt phase, proteins are effectively removed from an upper organic water phase, and meanwhile, fat-soluble substances are reduced to enter an extract liquid, so that a matrix effect is small.
(3) The method for detecting 8 kinds of estrogens in fish provided by the invention has the advantages of very simple extraction steps, very low analysis cost and good recovery rate in the detection process of an actual sample.
(4) According to the method for detecting 8 kinds of estrogens in fish, provided by the invention, the internal standard mark method is adopted to quantify the estrogens, the linear relation of the measurement result is good, the relative deviation is small, and the analysis precision is improved.
(5) The method for detecting 8 kinds of estrogens in fish provided by the invention adopts the ultra-high performance liquid chromatography-tandem mass spectrometry for quantitative analysis and detection, has high sensitivity, and the instrument detection limit of 8 kinds of estrogens is lower than 0.28 mug.L -1 Can meet the detection requirement of trace estrogen in fish flesh. In addition, the tandem triple quadrupole mass spectrometer performs qualitative analysis according to characteristic fragment ions generated by collision of corresponding parent ions, has strong selectivity, and eliminates interference of other impurity signals in the sample.
(6) According to the method for detecting 8 kinds of estrogens in fish, the internal standard substance indicating the recovery rate is added before the extracting solution is added, so that the loss of the target estrogens in the whole pretreatment process can be more accurately represented, meanwhile, the recovery rates of different estrogens are calibrated by adopting various internal standard substances in consideration of the fact that different physical and chemical properties of different estrogens have different degrees of loss in the pretreatment process. And the internal standard is properly selected, so that the final detection result is more real and reliable. In the conventional trace estrogen detection method, most of the trace estrogens are marked with only one internal standard substance, and thus the detected result has deviation.
(7) The method for detecting 8 kinds of estrogens in fish provided by the invention has the advantages of small using amount of organic reagent and environmental friendliness. 8 kinds of estrogens in fish meat can be detected by one sample injection, the detection process is less in time consumption, and the cost of manpower and material resources is saved.
(8) The method for detecting 8 kinds of estrogens in fish provided by the invention can be used for rapidly, efficiently, sensitively and accurately detecting 8 kinds of estrogens in fish simultaneously, has the advantages of high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection result and the like, greatly improves the detection efficiency, and has stable detection result and small interference.
Drawings
FIG. 1 is a graph showing the labeled recovery rate of target estrogen in the present invention using different extraction solvents.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The reagents or apparatus used in the examples below are conventional products available commercially without the manufacturer's knowledge.
Example 1
1. Sample pretreatment
Preferably, the fish sample is homogenized. The homogenization is to clean the collected fish body sample, absorb the surface moisture with gauze, split the muscles on the back and the abdomen of the fish body, separate the fish skin, and homogenate the fish body sample after adding water with the same mass. The treated sample is rapidly frozen at-20 ℃ and is further extracted.
3.00g of homogenized fish sample were weighed into 30mL glass centrifuge tubes, and 100ng of nonylphenol-D was added, respectively 8 Bisphenol A-D 16 Ethynyl estradiol-D 4 diethylstilbestrol-D 8 estradiol-D 3 After the 5 internal standards are fully and evenly mixed, 10mL of acetonitrile water solution with the volume percentage concentration of 75% is added, vortex mixing is carried out for 2min, and standing is carried out for 45min, so that the solution is layered to form a single water phase. 2.0. 2.0g K is added 2 HPO 4 Vortex mixing for 2min, and standing for 2.5h to separate the solution into two aqueous phases. 1mL of supernatant is filtered by a 0.22 mu m glass fiber filter membrane to obtain a sample solution to be measured.
2. Standard solution
8 kinds of estrogen standard substances are taken, 5 kinds of internal standard substances and 70% methanol aqueous solution by volume percent concentration are added to prepare a series of standard solutions with different concentrations. Among the 8 estrogens, 17 beta-estradiol (E2), estrone (E1), estriol (E3), 17 alpha-ethynyl estradiol (EE 2), diethylstilbestrol (DES), octylphenol (OP), nonylphenol (NP), bisphenol a (BPA). The concentration of 5 internal standard substances in the standard solution is 100.0 mug.L -1 . The concentration of 8 kinds of estrogens in a series of standard solutions with different concentrations is 1.37, 4.12, 12.35, 37.04, 111.11, 333.33 mug.L respectively -1 。
3. Detection of
The negative ion mode of an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method is adopted to respectively detect a sample solution and a standard solution, the obtained liquid chromatogram of the sample solution is compared with the liquid chromatogram of the standard solution, the common characteristic peak is identified according to the relative retention time to be qualitative, and then the concentration of 8 estrogens in the sample solution is determined by quantifying according to the chromatographic peak area of the common characteristic peak through an internal standard curve method.
Specifically, a series of 8 kinds of standard solutions containing different concentrations of estrogens are respectively subjected to negative ion mode analysis by a high performance liquid chromatography-mass spectrometry method, the linear relation between the ratio of the chromatographic peak area of 8 kinds of estrogen components under different concentrations to the chromatographic peak area of an internal standard and the ratio of the different concentrations of the corresponding components to the internal standard is obtained, the corresponding standard curve is drawn, and regression operation is performed by a weighted least square method, so that a regression equation of the standard curve of the 8 kinds of estrogen components is obtained. And analyzing the sample solution by using a high performance liquid chromatography-mass spectrometry method, substituting the obtained ratio of the chromatographic peak areas of 8 estrogen components under different concentrations to the chromatographic peak areas of the internal standard into a regression equation of a standard working curve of the corresponding components, and calculating the concentration of the 8 estrogen components in the sample solution according to the known concentration of the internal standard.
Wherein, the negative ion mode is detected by using an Shimadzu 30A ultra-high performance liquid chromatography tandem AB 5500Q-trap mass spectrometer (AB company in the United states).
The measurement conditions of the ultra high performance liquid chromatography (UPLC) are as follows: chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1X column length 100mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 5. Mu.L; flow rate: 0.3mL/min; the mobile phase A is aqueous solution of ammonia water with the volume percentage concentration of 0.1%; the mobile phase B is acetonitrile; the analysis time is 6.1min; gradient elution.
The specific procedure of gradient elution is:
0-2min, phase A: the volume ratio of the phase B is 90:10-90:10;
2-2.2min, phase A: the volume ratio of the phase B is 90:10-40:60;
2.2-3.5min, phase A: the volume ratio of the phase B is 40:60-40:60;
3.5-3.8min, phase A: the volume ratio of the phase B is 40:60-3:97;
3.8-6min, phase A: the volume ratio of the phase B is 3:97-3:97;
6-6.1min, phase A: the volume ratio of the phase B is 3:97-90:10.
the measurement conditions of mass spectrometry (MS/MS) were: ionization mode: electrospray ion source (ESI), negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, the auxiliary heating gas GS2 was 50psi, the collision gas CAD was Medium, and the collision gas was high purity nitrogen. The quantitative ion, collision energy, declustering voltage (DP), retention Time (RT) are shown in table 2.
TABLE 2
Note that: * Representing quantitative ions; * Represents the collision energy corresponding to the quantitative ion.
Example 2
The fish sample has complex matrix and contains protein, fat, saccharide, inorganic salt, water and other compounds. The sample matrixes can influence the extraction efficiency of the to-be-detected object, so that the detection result is error, and meanwhile, the instrument is polluted, so that the detection efficiency and the accuracy are directly influenced. Therefore, in veterinary drug residue analysis, the pretreatment technology of the sample is very critical, and the main purpose is to separate the target compound from the sample matrix and remove the interfering impurities. Meanwhile, the target antibiotics are various in types and large in physical and chemical property difference, and in order to maximize the extraction efficiency of various antibiotics in fish meat, the invention researches the extraction effect of 3 different extracting solutions on the target object.
Two sets of fish samples were prepared for comparison experiments, one set being a standard sample set and the other set being a blank sample set, each set being 3 replicates, as in step 1 of example 1. Because different extraction solvents may have different extraction efficiencies for target estrogens, the matrix removal capacities are different, and 3 extraction solvents with different proportions are adopted, namely (1) acetonitrile: water=70:30 (v: v); (2) acetonitrile: water=75:25 (v: v); (3) acetonitrile: water=85:15 (v: v), treated as in step 1 of example 1, and tested as in step 3 of example 1, and the labeled recovery of 8 estrogens in fish meat was measured, and the results are shown in fig. 1. Acetonitrile was used: water=75:25 as extraction solvent, the recovery rate was highest, RSD was lower and the effect was best. Therefore, in the actual detection of fish meat, acetonitrile is selected as: water=75:25 as extraction solution.
Example 3
The 8 estrogens mixed standard solutions were diluted with 70% methanol-water solution as in step 2 of example 1 to a series of concentration gradients (1.37, 4.12, 12.35, 37.04, 111.11, 333.33. Mu.g.L -1 ) The internal standard is 100.0 mug.L -1 Is added to a standard substance, and 6-point standard curves are drawn by an internal standard method with the concentration ratio of the component to the internal standard substance as an abscissa and the response value (response value=target peak area/internal standard substance peak area) as an ordinate, so as to obtain a standard curve and a correlation coefficient (R) 2 ). Meanwhile, the detection limit and the quantitative limit of the instrument are determined by adopting a signal-to-noise ratio method (S/N), the detection Limit (LOD) is estimated by using a signal-to-noise ratio of 3 times, and the quantitative Limit (LOQ) is estimated by using a signal-to-noise ratio of 10 times. The standard curves, correlation coefficients, detection limits and quantification limits for the 8 estrogens are shown in table 3. As is clear from Table 3, 18 kinds of estrogens have good linear relationship, and the correlation coefficient (R 2 ) Are all greater than 0.999.
TABLE 3 Table 3
Example 4
The fish samples were subjected to the standard recovery test as in steps 1 and 3 of example 1. Setting 2 groups of standard adding concentrations of 100ng g and 200ng g respectively -1 3 replicates were run for each set of samples and the normalized Recovery (RE) and Relative Standard Deviation (RSD) were calculated.
The calculation formula of the addition standard recovery rate (RE%) is as follows:
wherein C is 0 Is mixed withConcentration of the mixed standard solution, μg.L -1 ；C 1 To detect the concentration of the sample without adding the mixed standard solution, mug.L -1 ；C 2 To add the detection concentration of the mixed standard solution sample, μg.L -1 ；V 0 The volume of the mixed standard solution is L; v (V) 1 The volume is fixed before the machine is started to carry out the operation of adding no mixed standard solution sample, and L; v (V) 2 And (3) fixing the volume and L before loading the mixed standard solution sample.
The method examines the standard recovery rate of 8 kinds of estrogens with 2 concentrations in fish meat, and the result is shown in a table 4. The standard deviation of the labeled recovery rate of each estrogen is lower than 12%, and the labeled recovery rate is different, which indicates that different estrogens have different matrix interferences; the influence of matrix interference can be reduced by adding an internal standard to calculate the recovery rate of the addition standard and correcting the test result by using the recovery rate of the addition standard.
TABLE 4 labelling recovery and relative standard deviation of estrogens at different labelling concentrations in fish samples
Example 5
Collecting black carp and catfish in a certain aquaculture area of Shanghai, extracting and purifying samples according to the step 1 of the embodiment 1, and detecting and analyzing the actual concentration of target compounds in the samples according to the step 3 of the embodiment 1 to examine the applicability of the method to different fish samples. The actual measured 8 estrogen levels in muscle samples of black carp and catfish are shown in table 5. Experimental results show that the method can be applied to the determination of various estrogen contents in fish meat, and has good applicability.
TABLE 5 content of 8 estrogens in muscle samples of herring and catfish
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (5)
1. A method for detecting 8 estrogens in fish flesh, comprising: sequentially adding an internal standard substance, an acetonitrile solution and potassium hydrogen phosphate into a fish sample to form a double-aqueous phase, taking a supernatant, measuring the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, determining 8 kinds of estrogens in the sample solution according to the retention time, quantifying by adopting an internal standard curve method, and determining the content of the 8 kinds of estrogens in the sample solution;
the 8 kinds of estrogens comprise 17 beta-estradiol, estrone, estriol, 17 alpha-ethynyl estradiol, diethylstilbestrol, octylphenol, nonylphenol and bisphenol A;
the internal standard comprises nonylphenol-D 8 Bisphenol A-D 16 Ethynyl estradiol-D 4 diethylstilbestrol-D 8 estradiol-D 3 The method comprises the steps of carrying out a first treatment on the surface of the In the internal standard, nonylphenol-D 8 Bisphenol A-D as an internal standard for nonylphenols and octylphenols 16 Ethinyl estradiol-D as an internal standard for bisphenol a 4 diethylstilbestrol-D as an internal standard for 17α -ethinyl estradiol 8 estradiol-D as an internal standard for diethylstilbestrol 3 As an internal standard for 17 beta-estradiol, estrone and estriol;
the sample solution is measured by adopting an ultra-high performance liquid chromatography tandem mass spectrometry method, and the method comprises the following steps of:
1) Preparing a standard solution: 8 kinds of estrogen standard substances are taken, and an internal standard substance and a methanol solution are added to prepare a standard solution;
2) Sample detection: respectively detecting the sample solution and the standard solution in the step 1) by adopting an anion mode of an ultra-high performance liquid chromatography tandem mass spectrometry, comparing the obtained liquid chromatogram of the sample solution with the liquid chromatogram of the standard solution, identifying the qualitative characteristic peak according to the relative retention time, quantifying according to the chromatographic peak area of the common characteristic peak by an internal standard curve method, and determining the concentration of 8 estrogens in the sample solution;
in the step 2), the measurement conditions of the ultra performance liquid chromatography are as follows: chromatographic column: a C18 column; column temperature: 35-45 ℃; sample injection amount: 4-6. Mu.L; flow rate: 0.2-0.4mL/min; the mobile phase A is aqueous solution of ammonia water with the volume percentage concentration of 0.05-0.15%; the mobile phase B is acetonitrile; the analysis time is 6.1min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-2min, phase A: the volume ratio of the phase B is 90:10-90:10;2-2.2min, phase A: the volume ratio of the phase B is 90:10-40:60;2.2-3.5min, phase A: the volume ratio of the phase B is 40:60-40:60;3.5-3.8min, phase A: the volume ratio of the phase B is 40:60-3:97;3.8-6min, phase A: the volume ratio of the phase B is 3:97-3:97;6-6.1min, phase A: the volume ratio of the phase B is 3:97-90:10.
2. a method for detecting 8 estrogens in fish according to claim 1, wherein the obtaining of said sample solution comprises any one or more of the following conditions:
a1 The mass ratio of the fish sample to the addition of 5 internal standard substances is 2.98-3.02:0.1:0.1:0.1:0.1:0.1;
a2 The ratio of the mass of the fish sample added to the volume of acetonitrile solution added was 3.00.+ -. 0.02:8-12 g/mL;
a3 The acetonitrile solution is acetonitrile water solution with the volume percentage concentration of 70-80 percent;
a4 Vortex mixing and standing after the acetonitrile is added so as to separate the solution into a single water phase;
a5 The mass ratio of the fish sample to the potassium hydrogen phosphate is 2.98-3.02:1-3;
a6 After the potassium hydrogen phosphate is added, vortex mixing and standing are carried out to separate the solution to form a double water phase.
3. A method for detecting 8 estrogens in fish according to claim 1, characterized in that in step 1) it comprises any one or more of the following conditions:
b1 Concentrations of 5 internal standard substances in the standard solution are 100.0 mug.L -1 ；
B2 The methanol solution is 65-75% methanol aqueous solution by volume percent:
b3 The concentration range of 8 kinds of estrogens in the standard solution is 1.37-333.33 mug.L -1 。
4. The method for detecting 8 kinds of estrogens in fish according to claim 1, wherein in step 2), in the ultra-high performance liquid chromatography tandem mass spectrometry, an anion mode is detected by using an island jin 30A ultra-high performance liquid chromatography tandem AB 5500Q-trap mass spectrometer.
5. The method for detecting 8 kinds of estrogens in fish according to claim 1, wherein in step 2), the mass spectrum is measured under the following conditions: ionization mode: electrospray ion source ESI, negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, the auxiliary heating gas GS2 was 50psi, the collision gas CAD was Medium, and the collision gas was high purity nitrogen.
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