Method for detecting beauvericin and pesticide residues in eggs

Abstract

The application relates to the technical field of chemical detection, and specifically discloses a detection method for beauvericin and pesticide residues in eggs. 18 The yolk is treated by a purifying agent and a PSA purifying agent, and the egg white is treated by a PSA purifying agent; a standard curve is established; and the pesticide residues and the beauvericin residues are determined. The detection method is high in sensitivity, good in stability, high in recovery rate and accurate in quantification.

Description

Technical Field

[0001] This invention relates to the technical field of chemical detection, and more specifically, to a method for detecting beauveria bassiana and pesticide residues in eggs. Background Technology

[0002] Currently, the use of various pesticides and veterinary drugs in production processes makes it easy for food to contain pesticide, antibiotic, and biotoxin residues. Among these, pesticide residues have the greatest impact on human health, the longest duration of impact, and the most diverse pathways of contamination. Long-term consumption of poultry eggs with pesticide residues may harm human health.

[0003] In addition, beauveria (BEA), a biotoxin, easily contaminates crops and their products, such as corn and wheat products. When poultry consumes contaminated crops or products, beauveria residues can be found in their eggs. However, current research on pesticide and beauveria residues in poultry eggs is limited. Therefore, there is an urgent need to provide a detection method for the efficient detection of beauveria and pesticide residues in poultry eggs. Summary of the Invention

[0004] To efficiently detect beauveria bassiana residues and pesticide residues in poultry eggs, this application provides a method for detecting beauveria bassiana residues and pesticide residues in eggs.

[0005] In a first aspect, this application provides a method for detecting beauvericin and pesticide residues in eggs, employing the following technical solution:

[0006] A method for detecting beauveria bassiana and pesticide residues in eggs, comprising the following steps:

[0007] S1: Preparation of standard solution;

[0008] Each target compound standard was dissolved in acetonitrile to prepare standard solutions with a concentration of 0.9-1.2 mg / mL. The target compounds include pesticides and beauveria bassiana. The pesticides include one or more of the following: fipronil sulfone, fipronil, fipronil sulfide, flufenoxuron, emamectin benzoate, difenoconazole, triadimefon, thiamethoxam, fenitrothion, imidacloprid, methamidophos, isoprocarb, cyromazine, tebufenozide, parathion, pendimethalin, acetamiprid, and omethoate.

[0009] S2: Preprocessing;

[0010] Take the eggs, remove the shells, separate the egg whites and yolks, and stir the egg whites and yolks separately until they are evenly mixed;

[0011] Add x1 g of well-mixed egg yolk to x1 mL of water, let stand, then add 2x1 mL of 1wt% formic acid-ethyl acetate mixture, vortex mix for 1-3 min, then add 0.05x1 g of a 1:3 mixture of polyacrylamide and polyethylene glycol, vortex mix for 1-3 min, then add 0.5x1-3x1 g of sodium chloride, vortex mix for 1-3 min, shake for 20-30 min, centrifuge for 5-10 min, and take the supernatant to obtain mixture I, where x1 is a positive number;

[0012] Add x2 g of well-mixed egg white to x2 mL of water, let stand, then add 2x2 mL of 1wt% formic acid-acetonitrile mixture, vortex mix for 1-3 min, then add 0.5x2-3x2 g of sodium chloride, vortex mix for 1-3 min, shake for 20-30 min, centrifuge for 5-10 min, take the supernatant to obtain mixture II, where x2 is a positive number;

[0013] Take 1 mL of the mixture and add 80 mg of C. 18 Purifying agent, 100y1 mg PSA purifying agent, vortex for 1-3 min, centrifuge for 5-10 min, take the supernatant to obtain supernatant I, where y1 is a positive number;

[0014] Take y2 mL of mixture II, add 60y2 mg of PSA purifying agent, vortex for 1-3 min, centrifuge for 5-10 min, take the supernatant to obtain supernatant II, where y2 is a positive number;

[0015] Supernatant I and Supernatant II are mixed evenly to obtain Supernatant III;

[0016] Take z μL of supernatant III and add it to z / 4 μL of 0.1wt% formic acid aqueous solution. Mix well, filter, and obtain the test solution, where z is a positive number.

[0017] S3: Establishing the standard curve;

[0018] Using methanol as a solvent, mixed standard working solutions of b μg / kg, 2b μg / kg, 4b μg / kg, 20b μg / kg, 40b μg / kg, and 200b μg / kg were prepared from the standard solution, where b is a positive number.

[0019] The retention time and peak area of ​​each pesticide component and beauveria bassiana in the mixed standard working solution were determined by liquid chromatography-tandem mass spectrometry. A standard curve was plotted with the mass concentration of the mixed standard working solution as the abscissa and the peak area as the ordinate.

[0020] S4: Determination of pesticide residues and beauveria bassiana residues;

[0021] The retention times and peak areas of each pesticide component and beauveria bassiana in the test solution were determined using liquid chromatography-tandem mass spectrometry. The peak areas of each pesticide component and beauveria bassiana in the test solution were compared with the standard curve to obtain the measured values ​​of each pesticide component and beauveria bassiana in the test solution. The measured values ​​were then substituted into the quantitative calculation formula to finally obtain the pesticide residue and beauveria bassiana residue in poultry eggs.

[0022] The quantitative calculation formula is: ω=(ρ×v×f) / m, where ω is the pesticide residue or beauveria bassiana residue in poultry eggs, in mg / kg, ρ is the measured value, in mg / L, m is the amount of sample weighed, in g, v is the final volume, in mL, and f is the dilution factor.

[0023] This application presents a method for the detection of beauveria bassiana and pesticide residues in eggs, establishing an analytical method involving dispersive solid-phase extraction purification followed by ultra-high performance liquid chromatography-tandem mass spectrometry. Both beauveria bassiana and pesticides exhibit good linearity, with correlation coefficients ≥0.99; the limits of detection (S / N=3) range from 0.5 to 2 μg / kg; the limits of quantitation (S / N=10) range from 2.5 to 5 μg / kg; the recoveries of each target analyte are between 70.1% and 102.9%, and the relative standard deviations are between 0.7% and 9.7%. This detection method, through the synergistic effect of each step, can efficiently and accurately quantify beauveria bassiana and pesticide residues in eggs.

[0024] In this application, egg white and yolk are separated. Formic acid-ethyl acetate is used to extract pesticides and beauveria bassiana from the yolk, while formic acid-acetonitrile is used to extract pesticides and beauveria bassiana from the egg white. Because the yolk has a higher lipid content, while the egg white has higher protein and water content, acetonitrile, with its higher polarity, is used for extraction of the egg white. This method removes fewer co-soluble impurities, and most pesticides are stable in this medium, effectively precipitating proteins. For the yolk, ethyl acetate, with slightly lower polarity, is used for extraction, followed by further treatment with a mixture of polyacrylamide and polyethylene glycol. This mixture effectively precipitates proteins and has strong hydrophobicity. The high lipid content in the yolk facilitates the separation of polyacrylamide, polyethylene glycol, proteins, and lipids from the extracted target substances, reducing interference with detection.

[0025] In addition, different purifying agents were used to purify the egg white and the protein separately. For the yolk, the pigments mainly come from chlorophyll degradation products and other carotenoids, while the fats in the yolk include triglycerides, cholesterol, and phospholipids. 18Due to its hydrophobic properties, PSA exhibits excellent adsorption effects on non-polar to moderately polar fats, and shows better removal effects on more polar and organic acid pigments. Therefore, C... 18 Purifying agents and PSA purifying agents are used to treat egg yolks; for egg whites, which have lower pigment and fat content, only PSA purifying agents are used to treat the more polar impurities, reducing impurities and helping to improve C content. 18 The addition of less [agent] increases the recovery rate of each target analyte.

[0026] Optionally, the liquid chromatography conditions in S4 and S5 are as follows:

[0027] Column: Acquity BEH C 18 The chromatographic column has dimensions of 2.1 mm × 100 mm.

[0028] Mobile phase: 0.1% formic acid aqueous solution A, acetonitrile B;

[0029] Flow rate: 0.4 mL / min;

[0030] Column temperature: 35℃;

[0031] Injection volume: 10 μL;

[0032] Gradient elution program: 0-0.5 min, 85% A, 0.5-1.5 min, 5% A, 1.5-2.5 min, 5% A, 2.5-2.6 min, 85% A, 2.6-4.0 min, 85% A.

[0033] Optionally, the mass spectrometry conditions in S4 and S5 are as follows:

[0034] Ion source type: Electrospray ionization source;

[0035] Scanning method: positive ion scan, multiple reaction monitoring mode;

[0036] Collision gas: Argon;

[0037] Capillary voltage: 1.000kV;

[0038] Ion source temperature: 120℃;

[0039] Conical orifice air flow rate: 150L / h;

[0040] Desolvation gas temperature: 500℃;

[0041] Desolvation gas flow rate: 800L / h.

[0042] Optionally, in S2, the amount of sodium chloride added in the egg yolk pretreatment is x1 g, and the amount of sodium chloride added in the egg white pretreatment is 1.2 x 2 g.

[0043] Optionally, S2 is performed at 4-6°C.

[0044] Optionally, filtration in S2 is performed via a 0.22μm organic microporous membrane.

[0045] Optionally, before obtaining mixture I in S2, the centrifugation speed is 10000 r / min.

[0046] Optionally, before obtaining mixture II in S2, the centrifugation speed is 10000 r / min.

[0047] Optionally, before obtaining supernatant I in S2, the centrifugation speed is 8000 r / min.

[0048] Optionally, the centrifugation speed is 8000 r / min before obtaining supernatant II in S2.

[0049] In summary, this application has at least the following beneficial effects:

[0050] 1. The detection method of this application separates the egg white and yolk of an egg, extracts the target analytes using different extraction solvents, and purifies the impurities in the egg white and yolk using different purification agents, which enables efficient detection of pesticide and beauveria bassiana residues in eggs and results in a high recovery rate of each target analyte. Attached Figure Description

[0051] Figure 1 The chromatogram of the mixed standard working solution provided in the embodiments of this application. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the embodiments.

[0053] raw material

[0054] Acetonitrile and formic acid were of mass spectrometry grade and purchased from Fisher Scientific, USA; sodium chloride was of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; PSA and C18 adsorbent were purchased from Agilent Technologies, USA; egg samples were purchased from a local supermarket; fipronil sulfone, fipronil, fipronil sulfide, flufenoxuron, emamectin benzoate, difenoconazole, triadimefon, thiamethoxam, fenitrothion, imidacloprid, methamidophos, isoprocarb, cyromazine, tebufenozide, parathion, pendimethalin, acetamiprid, omethoate, and beauveria bassiana standards, with a concentration of 100 μg / mL and a purity of 95.0%, were purchased from Dr. Ehrenstorfer GmbH, Germany; Symmetry C 18The chromatographic column, 5μm 4.6*250mm, was purchased from Waters Corporation, USA; the organic microporous filter membrane, 0.2μm, was purchased from Pull Corporation, USA; the water used was ultrapure water; the polyacrylamide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S31321; the polyethylene glycol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30184-500.

[0055] instrument

[0056] Waters TQ-S high-performance liquid chromatography-tandem mass spectrometry system from Waters Corporation, USA; BSA6202S electronic balance from Mettler Corporation, Switzerland; MS2 vortex mixer horizontal shaker from IKA Corporation, Germany; CR30NX high-speed refrigerated centrifuge from HITACHI Corporation, Japan; and Milli-Q academic ultrapure water system from Millipore Corporation, USA.

[0057] Example

[0058] 1. Instrument operating conditions:

[0059] Liquid chromatography conditions:

[0060] An Acquity BEHC18 1.7 μm, 2.1 mm * 100 mm column was used; the mobile phase was 0.1% formic acid aqueous solution A and acetonitrile B; the flow rate was 0.4 mL / min; the column temperature was 35°C; the injection volume was 10 μL; and the gradient elution program was: 0–0.5 min, 85% A; 0.5–1.5 min, 5% A; 1.5–2.5 min, 5% A; 2.5–2.6 min, 85% A; 2.6–4.0 min, 85% A.

[0061] Mass spectrometry conditions:

[0062] Ion source type: electrospray ionization source; Scanning mode: positive ion scan (ESI+); multiple reaction monitoring mode (MRM); Collision gas: argon (Ar); Capillary voltage: 1.00 kV; Ion source temperature: 120°C; Cone gas flow rate: 150 L / h; Desolvation gas temperature: 500°C; Desolvation gas flow rate: 800 L / h. Mass spectrometry parameters for each pesticide and beauveria bassiana are shown in Table 1.

[0063] Table 1. Mass spectrometry parameters for various pesticides and beauveria bassiana.

[0064]

[0065] Where * represents quantitative ions.

[0066] 2. Preparation of standard solutions

[0067] Take 10 mg of each target standard and dissolve and dilute it in 10 mL volumetric flasks. Prepare standard solutions with a concentration of 1 mg / mL by adding acetonitrile. The target substances include pesticides and beauveria bassiana. The pesticides include: fipronil sulfone, fipronil, fipronil sulfide, flufenoxuron, emamectin benzoate, difenoconazole, triadimefon, thiamethoxam, fenitrothion, imidacloprid, methamidophos, isoprocarb, cyromazine, tebufenozide, parathion, pendimethalin, acetamiprid, and omethoate.

[0068] 3. Pretreatment

[0069] Take one egg, remove the shell, separate the egg white and yolk, and use a homogenizer to stir the egg white and yolk separately until they are evenly mixed. Store the mixture at -20℃ for later use.

[0070] Add 16 g of well-mixed egg yolk to 16 mL of pure water and let stand for 1 min. Then add 32 mL of 1 wt% formic acid-ethyl acetate mixture and vortex for 1 min. Next, add 0.8 g of a mixture of polyacrylamide and polyethylene glycol in a mass ratio of 1:3, and then add 16 g of sodium chloride. Vortex for 1 min and extract at high speed for 20 min. Centrifuge at 10000 r / min for 5 min at 4 °C and collect the supernatant to obtain 36 mL of mixture I.

[0071] Add 28 g of well-mixed egg white to 28 mL of water and let stand. Then add 56 mL of 1 wt% formic acid-acetonitrile mixture, vortex for 1-3 min, vortex for 1 min, add 33 g of sodium chloride, vortex for 1 min, extract by high-speed shaking for 20 min, and centrifuge at 10000 r / min for 5 min at 4 °C to obtain 63 mL of mixture II.

[0072] Add 2880 mg C to 36 ml of mixture I 18 The purifying agent, 3600 mg PSA purifying agent, was vortexed for 1 min and centrifuged at 8000 r / min for 5 min at 4℃ to obtain supernatant I.

[0073] Add 3780 mg of PSA purifying agent to 63 ml of mixture II, vortex for 1 min, and centrifuge at 8000 r / min for 5 min at 4 °C to obtain supernatant II.

[0074] Supernatant I and Supernatant II are mixed evenly to obtain Supernatant III.

[0075] Take 400 μL of supernatant III and add it to 100 μL of 0.1wt% formic acid aqueous solution. Mix well and filter to obtain the test solution.

[0076] 4. Establishment of the standard curve

[0077] Using methanol as a solvent, mixed standard working solutions of 2.5 μg / kg, 5 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, and 500 μg / kg were prepared using standard solutions of different target analytes.

[0078] The retention times and peak areas of each pesticide component and beauveria bassiana in the mixed standard working solution were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). A standard curve was plotted with the mass concentration of the mixed standard working solution on the x-axis and the peak area on the y-axis, and the regression equation and correlation coefficient were calculated. The results showed that 19 target compounds exhibited good linearity in the concentration range of 2.5–500 μg / kg, with a correlation coefficient R0. 2 ≥0.99, the standard curve linear regression equation and correlation coefficient are shown in Table 2.

[0079] Furthermore, based on the concentration where the signal-to-noise ratio (S / N) of the characteristic ion mass chromatographic peak is ≥3, the limit of detection (LOD) of the method is defined as the concentration where the S / N is ≥10, and the limit of quantitation (LOQ) of the method is defined as the concentration where the S / N is ≥10. The LOD range for the 19 target compounds in eggs was determined to be 0.5–2.0 μg / kg, and the LOQ range was determined to be 2.5–5.0 μg / kg. The LOD and LOQ are shown in Table 2. The chromatogram of the mixed standard working solution at 100 μg / kg is shown in the figure. Figure 1 As shown. In Figure 1 The following are listed from top to bottom: fipronil sulfone, fipronil, fipronil sulfide, flufenoxuron, emamectin benzoate, difenoconazole, beauveria bassiana, parathion, pendimethalin, acetamiprid, omethoate, triadimefon, thiamethoxam, fenitrothion, imidacloprid, methamidophos, isoprocarb, cyromazine, and tebufenozide.

[0080] Table 2. Standard Curve, Linear Regression Equation, Correlation Coefficient, Limit of Detection, and Limit of Quantification

[0081]

[0082] 5. Test Results

[0083] (1) Accuracy and precision determination

[0084] Three mixed standard working solutions with different concentrations of 10 μg / kg, 50 μg / kg, and 100 μg / kg were selected and analyzed according to the pretreatment steps and instrument operating conditions described above. Each concentration of the mixed standard working solution was measured three times, and the recovery and relative standard deviation of each target analyte were calculated. The recovery rates and relative standard deviations of the 19 target analytes are shown in Table 3. The recovery rates ranged from 70.1% to 102.9%, and the relative standard deviations (RSDs) ranged from 0.7% to 9.7%.

[0085] Table 3 Standard Curve, Linear Regression Equation, Correlation Coefficient, Limit of Detection, Limit of Quantification

[0086]

[0087] (2) Selection of mass spectrometry conditions

[0088] Nineteen mixed standard working solutions (concentration 200 μg / kg) were injected into the column for full scan to determine the characteristic ions of each target analyte. Results showed that fipronil sulfone, fipronil, fipronil sulfide, and flufenoxuron showed higher signal responses in negative ion mode, while other target compounds showed higher signal responses in positive ion mode. The 19 target analytes were scanned using both positive and negative ion modes, and the quasi-molecular ion peak with the highest response was selected as the precursor ion. Secondary mass spectrometry scans were performed on the precursor ions, selecting two daughter ions; the one with the stronger response was used for quantitative analysis, and the one with the slightly lower response value was used for qualitative analysis. Parameters such as ion source temperature, desolvation gas temperature, capillary voltage, and cone voltage were optimized in multiple reaction monitoring (MRM) mode to achieve the best overall response signal intensity for the target analytes. The optimized mass spectrometry parameters are shown in Table 1.

[0089] (3) Selection of chromatographic conditions

[0090] When separating target analytes using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the separation efficiency mainly depends on the selection of the chromatographic column and the mobile phase. For the column selection, a widely applicable reverse-phase C25 column was chosen. 18 The column was tested. During the test, C 18 The column achieved good separation and high sensitivity. For the mobile phase, acetonitrile, a highly polar and widely used organic phase, was chosen. Adding certain amounts of formic acid, ammonium acetate, and ammonium formate to the aqueous phase increased the ionization state of the target analytes, improved ion efficiency, and enhanced peak shape, thereby improving resolution. Therefore, under gradient elution conditions, four mobile phases were selected: water-acetonitrile, 0.1% formic acid-water-acetonitrile, 5 mmol / L ammonium formate-acetonitrile, and 0.1% formic acid-5 mmol / L ammonium formate-acetonitrile. The effects of different mobile phases on the chromatographic resolution, peak shape, and mass spectrometry sensitivity of the target analytes were investigated. The results showed that 0.1% formic acid-water-acetonitrile was the best mobile phase for the 19 target analytes, exhibiting better peak shapes, higher response signals, and lower interference from impurities. Therefore, 0.1% formic acid-water-acetonitrile solution was ultimately selected as the mobile phase.

[0091] (4) Selection of egg white extraction solution in pretreatment

[0092] For pesticide residue extraction, organic solvents such as methanol, acetonitrile, ethyl acetate, and acetone are commonly considered. While acetone is volatile and toxic, it is not the first choice. Ethyl acetate has strong dissolving power but tends to extract a large number of impurities simultaneously, resulting in a matrix effect. Methanol can precipitate proteins, but extraction from eggs produces turbidity, affecting detection accuracy. Acetonitrile is the best choice because it is suitable for extracting targets with broad polarity, removes few co-soluble impurities, most pesticides are stable in its medium, and it effectively precipitates proteins. Under weakly acidic conditions, using acetonitrile to extract residues is the most thorough and gentle method and has wide applications in the detection of multiple pesticide residues.

[0093] This application compared the extraction effects of three extraction solvents: 10 mL acetonitrile, 10 mL 1 wt% formic acid-acetonitrile, and 10 mL 1 wt% acetic acid-acetonitrile. When using pure acetonitrile, the egg white was clear, but the color was dark, and there was significant interference from impurity peaks; the recoveries of emamectin benzoate, cyromazine, and isoprocarb were below 40%. Compared to 1 wt% acetic acid-acetonitrile, 1 wt% formic acid-acetonitrile had a lighter color, fewer impurity peaks, and the recoveries of all target compounds were increased to over 65.1%, meeting the detection requirements. Therefore, 1 wt% formic acid-acetonitrile was used as the extraction solvent for egg white.

[0094] (5) Selection of egg yolk extract solution in pretreatment

[0095] This application compared the extraction effects of three extraction solvents: 10 mL of 1 wt% acetic acid-acetonitrile, 10 mL of 1 wt% formic acid-acetonitrile, and 10 mL of 1 wt% formic acid-ethyl acetate. Compared with 1 wt% formic acid-acetonitrile and 1 wt% acetic acid-acetonitrile, the recovery rate of all target compounds was improved in 1 wt% formic acid-ethyl acetate; however, there were more interference from impurity peaks. By adding a mixture of polyacrylamide and polyethylene glycol, the impurity peaks were reduced, meeting the detection requirements. Therefore, 1 wt% formic acid-acetonitrile was used as the extraction solution for egg yolk, and a mixture of polyacrylamide and polyethylene glycol was further added for treatment.

[0096] (6) Optimization of egg yolk dilution

[0097] The translucent, viscous colloidal nature of egg samples leads to significant differences in viscosity between samples. When extracting target compounds from egg yolks using a 1 wt% formic acid-ethyl acetate solution, high-viscosity samples often form clumps, affecting uniform dispersion during vortexing. Adding an appropriate amount of water to the egg yolks can reduce their viscosity, thus helping the extract (1 wt% formic acid-ethyl acetate solution) to fully integrate with the yolks, ensuring uniform matrix dispersion and enhancing the extraction efficiency of the target compounds. This study compared the extraction effects of diluting samples with 0 mL, 16 mL, 32 mL, and 64 mL of water before adding the extract. The results showed that as the amount of water increased, the viscosity of the egg yolks gradually decreased, improving the dispersion problem. Protein began to precipitate, forming white particles after thorough vortexing. When 64 mL of water was added, the high-speed centrifugation time was longer, but a large number of protein particles still floated on the surface of the extract, increasing the difficulty of subsequent purification. Therefore, to balance the viscosity of the egg yolks and the protein precipitation effect, 16 mL of water was added to dilute the egg yolks.

[0098] (7) Optimization of egg white dilution

[0099] The translucent, viscous colloidal nature of egg samples leads to significant differences in viscosity between samples. When extracting the target substance from egg white using a 1 wt% formic acid-acetonitrile solution, high-viscosity samples often form clumps, affecting uniform dispersion during vortexing. This study compared the extraction effects of diluting samples with 0 mL, 28 mL, 56 mL, and 112 mL of water before adding the extraction solution. The results showed that as the water volume increased, the viscosity of the egg white gradually decreased, improving the dispersion, and protein began to precipitate, forming white particles after thorough vortexing. When 112 mL of water was added, the high-speed centrifugation time was longer, but a large number of protein particles still floated on the surface of the extract, increasing the difficulty of subsequent purification. Therefore, to balance the viscosity of egg white with the protein precipitation effect, 28 mL of water was added to dilute the egg white.

[0100] (8) Selection of the amount of inorganic salts added to egg yolk

[0101] During extraction, egg emulsification may occur when the sample is shaken and mixed, resulting in the formation of a white emulsion layer. This phenomenon blurs the boundary between the aqueous and organic phases, making it difficult to recover suspended solids and thus reducing the recovery rate of the target analyte. To eliminate emulsification and achieve better separation of the aqueous and organic phases, inorganic salts are typically added to the extract. The "salting-out effect" increases the concentration of total ions in the solution, altering the polar environment and reducing the interaction between water molecules and the organic solvent. This lowers the probability of emulsification and achieves a higher recovery rate of the target analyte. In this application, 8g, 16g, 32g, and 64g of sodium chloride were added to the solution after egg yolk extraction, respectively. The results showed that when the amount of sodium chloride added reached 16g, the emulsification phenomenon was significantly suppressed, the interface between the aqueous phase and the organic phase was clear, the layering effect was significant, and the average recovery rate of the target compound reached 70.2%. As the amount of sodium chloride added continued to increase, the layering effect and the recovery rate of the compound showed a tendency to stabilize. This indicates that after reaching a certain critical concentration, further increasing the concentration of sodium chloride can no longer further promote the extraction effect. Therefore, the amount of sodium chloride added was 16g.

[0102] (9) Selection of the amount of inorganic salts added to egg white

[0103] Considering that this study used blank eggs as the subject, 16g, 33g, 50g, and 70g of sodium chloride were added to the solution after egg white extraction, respectively. The results showed that when the amount of sodium chloride added reached 33g, the emulsification phenomenon was significantly inhibited, the interface between the aqueous phase and the organic phase was clear, and the layering effect was significant. As the amount of sodium chloride added continued to increase, the layering effect and the recovery rate of compounds showed a tendency to stabilize. This indicates that after reaching a certain critical concentration, further increasing the concentration of sodium chloride can no longer further promote the extraction effect. Therefore, the amount of sodium chloride added was chosen to be 33g.

[0104] (10) Selection of purification solution for egg yolk

[0105] The presence of trace impurities such as fats and pigments in the extract can cause matrix effects, leading to deviations from expected analytical responses and contaminating the chromatographic column and analytical instrument. The determination revealed excessively high recoveries (128–135%) of fipronil sulfone, fipronil, fipronil sulfide, and flufenoxuron, indicating significant matrix effects. Eliminating matrix effects is crucial to ensuring analytical accuracy and maintaining instrument health. Pigments and fats in egg samples are mainly distributed in the yolk. Pigments in the yolk primarily originate from chlorophyll degradation products and other carotenoids, while fats include triglycerides, cholesterol, and phospholipids. 18Based on its hydrophobic properties, it exhibits excellent adsorption effects on nonpolar to moderately polar fats, and shows good removal efficiency for more polar and organic acid pigments. This application employs the dSPE purification method to remove PSA and / or C... 18 The results of the purification treatment were compared. Samples were extracted following the pretreatment steps, and 1 mL of mixture I was taken, followed by the addition of 100 mg PSA and 100 mg C. 18 and 100 mg PSA + 100 mg C 18 100 mg PSA + 80 mg C 18 80 mg PSA + 100 mg C 18 The mixture was purified. Experimental results showed that using only C... 18 The egg yolks were darker during purification, indicating that PSA was effective at adsorbing pigments from eggs; when using 100 mg PSA + 80 mg C... 18 When the mixture underwent purification, the egg yolk color became significantly lighter, and the recoveries of fipronil sulfone, fipronil, fipronil sulfide, and flufenoxuron were high, meeting the detection requirements. Further addition of C... 18 Recovery rates decreased to varying degrees with different dosages, therefore 100 mg PSA and 80 mg C were selected. 18 Mixed as a purifying agent for egg yolks.

[0106] (11) Selection of purification solution for egg white

[0107] Egg whites are high in protein and low in fat. 18 Due to its hydrophobic properties, it exhibits excellent adsorption effects on lipids. This application employs the dSPE purification method to remove PSA and / or C. 18 The results of the purification treatment were compared. Samples were extracted following the pretreatment steps. 2 mL of mixture II was taken, and 120 mg PSA and 100 mg C were added respectively. 18 120 mg PSA + 100 mg C 18 120 mg PSA + 80 mg C 18 And 120 mg PSA + 60 mg C 18 The mixture was purified. Experimental results showed that the purifying agent for egg white included C. 18 Under these conditions, the recovery rates of fipronil sulfone, fipronil, fipronil sulfide, and flufenoxuron decreased to varying degrees. Therefore, 120 mg PSA was selected as the purifying agent for egg white.

[0108] (12) Determination of pesticide residues and beauveria bassiana residues

[0109] The retention times and peak areas of the chromatographic peaks of each pesticide component and beauveria bassiana in the test solution were determined by liquid chromatography-tandem mass spectrometry. The peak areas of each pesticide component and beauveria bassiana in the test solution were compared with the standard curve to obtain the measured values ​​of each pesticide component and beauveria bassiana in the test solution. The measured values ​​were then substituted into the quantitative calculation formula to finally obtain the pesticide residue and beauveria bassiana residue in poultry eggs.

[0110] The quantitative calculation formula is: ω=(ρ×v×f) / m, where ω is the pesticide residue or beauveria bassiana residue in poultry eggs, in mg / kg, ρ is the measured value, in mg / L, m is the amount of sample weighed, in g, v is the volume of the final volume, in mL, and f is the dilution factor.

[0111] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for detecting beauvericin and pesticide residues in egg, characterized by, Comprising the following steps: S1: standard solution preparation; Respectively dissolve the standard of each target in acetonitrile, and respectively prepare the standard solution with a concentration of 0.9-1.2 mg / mL, the targets including pesticides and Beauveria bassiana, the pesticides including one or more of the following: fipronil sulfone, fipronil, fipronil sulfide, flumethrin, emamectin benzoate, difenoconazole, triflumuron, thiamethoxam, phoxim, imidacloprid, isocarbophos, isoprocarb, cyromazine, parathion, pendimethalin, acetamiprid, omethoate; S2: pretreatment; Take the poultry eggs, remove the shells, separate the egg white and the egg yolk, and respectively stir the egg white and the egg yolk until they are mixed evenly; And add x1 g of the mixed and evenly-mixed egg yolk into x1 mL of water, stand still, then add 2x1 mL of 1wt% formic acid-ethyl acetate mixture, vortex mix for 1-3 min, then add 0.05x1 g of a mixture of polyacrylamide and polyethylene glycol with a mass ratio of 1:3, vortex mix for 1-3 min, then add 0.5x1-3x1 g of sodium chloride, vortex mix for 1-3 min, shake for 20-30 min, centrifuge for 5-10 min, take the supernatant, and obtain mixture I, x1 being a positive number; And add x2 g of the mixed and evenly-mixed egg white into x2 mL of water, stand still, then add 2x2 mL of 1wt% formic acid-acetonitrile mixture, vortex mix for 1-3 min, then add 0.5x2-3x2 g of sodium chloride, vortex mix for 1-3 min, shake for 20-30 min, centrifuge for 5-10 min, take the supernatant, and obtain mixture II, x2 being a positive number; Take mixed solution I y1 mL, add 80y1 mg C 18 Purification agent, 100y1 mg PSA purification agent, vortex 1-3min, centrifugal 5-10min, take supernatant, get supernatant I, y1 is positive; Take y2 mL of mixture II, add 60y2 mg of PSA purifier, vortex for 1-3 min, centrifuge for 5-10 min, take the supernatant, and obtain supernatant II, y2 being a positive number; Mix the supernatant I and the supernatant II evenly, and obtain supernatant III; Take z μL of supernatant III, add to z / 4 μL of 0.1wt% formic acid aqueous solution, mix evenly, filter, and obtain the sample solution, z being a positive number; S3: establishment of standard curve; Use methanol as the solvent, and respectively prepare b μg / kg, 2b μg / kg, 4b μg / kg, 20b μg / kg, 40b μg / kg, 200b μg / kg of mixed standard working solution using the standard solution, b being a positive number; Use the liquid chromatography-tandem mass spectrometer to determine the chromatographic peak retention time and the chromatographic peak area of each pesticide component and Beauveria bassiana in the mixed standard working solution, take the mass concentration of the mixed standard working solution as the abscissa, and take the chromatographic peak area as the ordinate, and draw the standard curve; S4: determination of pesticide residue and Beauveria bassiana residue; The retention time and peak area of each pesticide component and Beauveria bassiana in the sample are determined by liquid chromatography-mass spectrometry, and the peak area of each pesticide component and Beauveria bassiana in the sample is compared with the standard curve to obtain the determination value of each pesticide component and Beauveria bassiana in the sample; then the determination value is brought into the quantitative calculation formula to finally obtain the pesticide residue and Beauveria bassiana residue in the egg; The quantitative calculation formula is: ω=(ρ×v×f) / m, wherein ω is the pesticide residue or Beauveria bassiana residue in the egg, the unit is mg / kg, ρ is the determination value, the unit is mg / L, m is the sample amount, the unit is g, v is the constant volume, the unit is mL, and f is the dilution multiple; The liquid chromatography conditions in S3 and S4 are as follows: Chromatographic column: Acquity BEH C 18 Chromatographic column, 2.1 mm x 100 mm in size; Mobile phase: 0.1% formic acid aqueous solution A and acetonitrile B; Flow rate: 0.4 mL / min; Column temperature: 35℃; Injection volume: 10 μL; Gradient elution; The mass spectrometry conditions in S3 and S4 are as follows: Ion source type: electrospray ion source; Scanning mode: positive ion scanning, multiple reaction monitoring mode; Collision gas: argon; Capillary voltage: 1.000 kV; Ion source temperature: 120℃; Taper hole gas flow: 150 L / h; Desolvation gas temperature: 500℃; Desolvation gas flow: 800 L / h.

2. The method according to claim 1, wherein the method is characterized by, In S2, the addition amount of sodium chloride in the yolk pretreatment is x1 g, and the addition amount of sodium chloride in the egg white pretreatment is 1.2x2 g.

3. The method according to claim 1, wherein the method is characterized by, S2 is carried out at 4-6℃.

4. The method according to claim 1, wherein the method is characterized by, In S2, the filtration is carried out through a 0.22 μm organic microporous filter membrane.

5. The method according to claim 1, wherein the method is characterized by, In S2, before obtaining the mixed solution I, the centrifugation speed is 10000 r / min.

6. The method according to claim 1, wherein the method is characterized by, In S2, before obtaining the mixed solution II, the centrifugation speed is 10000 r / min.

7. The method according to claim 1, wherein the method is characterized by, In S2, before obtaining the supernatant I, the centrifugation speed is 8000 r / min.

8. The method according to claim 1, wherein the method is characterized by, In S2, before obtaining the supernatant II, the centrifugation speed is 8000 r / min.

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