Establishment of a new method for detecting fungicide residues in animal-derived foods based on GC-MSMS

Abstract

The present invention relates to the technical field of fungicide residue detection, and discloses a novel fungicide residue detection method for animal-derived food based on GC-MSMS, a standard stock solution, and a standard stock solution diluted with a corresponding solvent to a 100 μg / mL standard stock solution, which is stored in a refrigerator at ‑18°C. By optimizing the extraction method, a stable, practical and sensitive analysis method is developed, which can simultaneously detect the contamination of 13 new fungicides in animal food; after optimizing the ratio of QuEChERS purification powder, the method optimizes the extraction solvent in animal food, and obtains a good detection limit and quantitative limit. The spiked recoveries of the 13 fungicides in chicken and beef are within 79.51%-111.39% and 83.35%-103.98%, and the RSD range is within 7.49%, which proves that the repeatability and reproducibility of the detection method are acceptable.

Description

Technical Field

[0001] The present invention relates to the technical field of fungicide residue detection, and in particular to a novel fungicide residue detection method for animal-derived food based on GC-MSMS. Background Art

[0002] Animal food plays an important role in daily life in most of the world and is considered to be a food with high nutritional value. Meat and meat products are considered to be a good source of trace elements Zn, heme iron, B vitamins and essential amino acids. In addition to the possibility of pesticides in plant foods, animals may also eat water and plants contaminated by pesticides, which may also cause pesticide residues in animal foods.

[0003] Since its development, pesticides have been considered the most important component of agricultural production. In agriculture, they have the ability to protect and prevent crops from being damaged, and increase crop yields. Although pesticides occupy such an important position in agricultural production, the widespread use of pesticides has also caused certain pollution to the environment and water. And with the transmission of the food chain, they will be enriched in animal food and even in the human body, and cause certain harm to human and animal health. Fungicides are mainly aimed at fungal diseases of crops in the production process. Fungicides occupy a very important position in agriculture. Although the role of fungicides in agriculture is not as obvious as that of herbicides and insecticides, the attention paid to fungicides cannot be ignored.

[0004] Fungicides are mainly used in agricultural production to protect crops from microbial invasion and are widely used throughout the world. Many countries are currently developing new and highly effective fungicides for crop production.

[0005] The present invention compares the limits of new fungicides set by the European Union and identifies fungicides for which limits and detection methods have not yet been established in animal foods in my country. Summary of the invention

[0006] The present invention provides a novel method for detecting fungicide residues in animal-derived foods based on GC-MSMS, which solves the problems raised by the above-mentioned background technology.

[0007] The present invention provides the following technical solution: a new method for detecting fungicide residues in animal-derived food is established based on GC-MSMS, comprising the following steps:

[0008] Step S1, preparation of standard solution:

[0009] (1) Standard stock solution: dilute the standard solution with the corresponding solvent to a 100 μg / mL standard stock solution and store it in a refrigerator at -18°C;

[0010] (2) Standard working solution intermediate solution: dilute the above standard stock solution into an intermediate working solution of 10 μg / mL and store it in a -18°C refrigerator;

[0011] (3) blank matrix solution, using the blank solution treated and purified in step (2) to prepare a series of gradient standard solutions;

[0012] Mixed standard working solution: ready for use;

[0013] Step S2, extraction, accurately weigh 5.00 g of the homogenized sample into a 50 mL centrifuge tube, add 10 mL of the extraction solution, vortex on a vortex machine for 10 min, ultrasonicate for 30 min, add 4 g of anhydrous MgSO4 and 1 g of NaCl to remove water and make it uniform, add the extraction powder, shake it thoroughly for 10 min, and centrifuge it at 10000 r / min for 5 min;

[0014] Step S3, taking 2.0 mL of the upper organic solution for purification;

[0015] Step S4, purification: add 0.2 g of purification powder to 2.0 mL of the organic solution to be purified, vortex and oscillate for 3 min, and then centrifuge the sample at 4000 r / min for 3 min;

[0016] Step S5, taking the supernatant and passing it through a 0.22 μm membrane, putting it into a sample injection vial, and putting it on the machine for testing;

[0017] Step S6, setting GC-MS / MS conditions.

[0018] Preferably, step S6 specifically includes:

[0019] GC conditions, column oven temperature: 60°C;

[0020] Equilibration time: 1.0 min;

[0021] The initial temperature was 60°C, maintained for 1 min, then raised to 130°C at a rate of 30°C / min, then raised to 250°C at a rate of 5°C / min, and then raised to 300°C at a rate of 10°C / min and maintained for 5 min, with a total time of 37.33 min;

[0022] MS conditions, ion source temperature: 230°C, interface temperature: 280°C, solvent delay time: 3.5 min;

[0023] Detector voltage (relative to tuning result): 0.3kV;

[0024] Injector: Number of solvent flushes (before injection): 2;

[0025] Number of solvent washes (after injection): 5;

[0026] Number of sample rinses: 2;

[0027] Injection volume: 1 μL.

[0028] Preferably, step S2 specifically includes that the mass error of 5.00 g of the homogenized sample is ±0.02 g.

[0029] Preferably, the preparation ratio of the purification powder in step S4 is MgSO4:PSA:C18=6:1:1.

[0030] Preferably, step S2 specifically includes preparing the extraction solution in a ratio of acetone:acetonitrile=1:1.

[0031] The present invention has the following beneficial effects:

[0032] 1. This GC-MSMS-based method for detecting new fungicide residues in animal-derived foods was developed by optimizing the extraction method to develop a stable, practical and sensitive analytical method that can simultaneously detect the contamination of 13 new fungicides in animal foods. After optimizing the ratio of QuEChERS purification powder, this method optimized the extraction solvent in animal foods and obtained good detection limits and quantification limits. The spiked recoveries of the 13 fungicides in chicken and beef were within 79.51%-111.39% and 83.35%-103.98%, and the RSD range was within 7.49%, proving that the repeatability and reproducibility of the detection method were acceptable.

[0033] 2. The new method for detecting fungicide residues in animal-derived food based on GC-MSMS is established. Acetonitrile:acetone=1:1 is used as the extraction solvent, and the fungicide in animal food is purified with purification powder after optimized ratio. Chicken is purified using MgSO4:C18=6:1, and beef is purified using MgSO4:PSA:C18=6:1:1. The recoveries of 13 fungicides in chicken and beef are 85.28-102.24% and 78.2%-106.78%, respectively. The detection limits of the 13 fungicides in chicken and beef are between 0.58mg / kg-54.44mg / kg and between 0.52mg / kg-54.44mg / kg, and the quantification limits are between 1.62mg / kg-174.82mg / kg and between 1.08mg / kg-207.68mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of retention time and ion fragments of 13 standard substances of the present invention;

[0035] Figure 2Schematic diagram of GC-MS / MS standard MRM spectra of 13 fungicides of the present invention;

[0036] Figure 3 Schematic diagram of optimization of solvents for extracting 13 fungicides from beef and chicken of the present invention;

[0037] Figure 4 It is a schematic diagram of the optimization of 13 kinds of bactericide purification powders in beef and chicken of the present invention;

[0038] Figure 5 It is a schematic diagram of the beef solvent curve, matrix curve and matrix effect of the present invention;

[0039] Figure 6 It is a schematic diagram of the chicken solvent curve, matrix curve and matrix effect of the present invention;

[0040] Figure 7 Schematic diagram of linear curves, detection limits and quantification limits of 13 fungicides in beef of the present invention;

[0041] Figure 8 Schematic diagram of linear curves, detection limits and quantification limits of 13 fungicides in chicken of the present invention;

[0042] Fig. 9 Schematic diagram of the spike recovery experiment of 13 fungicides in beef and chicken of the present invention;

[0043] Fig.10 This is a schematic diagram of the intra-day precision of 13 fungicides of the present invention in beef and chicken;

[0044] Fig.11 This is a schematic diagram of the daytime precision of 13 fungicides of the present invention in beef and chicken.

[0045] Figure 1 In: * indicates quantitative ion;

[0046] Figure 2 Chinese: Note: 1. Tebufloquin; 2. Fluopyram; 3. Thiofuran; 4. Fluopyram; 5. Penthiopyrad; 6. Fluopyram, 7. Fluopyram; 8. Inpyrflnxam; 9. Mefenoxam; 10. Fluopyram; 11. Azopyram; 12. Pydifumetofenen; 13. Bifenthion. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Materials and Reagents

[0049] The materials and reagents used in this example are as follows:

[0050] Tebufloquin, fluopyram, trifloxystrobin, inpyrflnxam, mefenoxam, and clofopyralid were purchased from Alta Company, China;

[0051] Thifuranil, penthiopyrad, fluopyram, fluopyram, pyraclostrobin, and bixafen were purchased from Bepure;

[0052] Acetonitrile (chromatographic grade), acetone (chromatographic grade), and methanol (chromatographic grade) were purchased from Fisher, USA;

[0053] Ethyl acetate and acetic acid were purchased from Sigma, USA;

[0054] Anhydrous MgSO4 and NaCl were purchased from Kelun Reagent Co., Ltd.;

[0055] PSAQuEChERS special ultra-clean filler, C18: 40-63 μm, filter membrane, and filter head (0.22 μm) were purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0056] Beef and chicken samples: all are from samples that have been tested by Chengdu Customs Technical Center.

[0057] Instruments and Equipment

[0058] The types of instruments and equipment used in this embodiment are as follows:

[0059] GCMS-TQ8040 gas chromatograph-triple quadrupole mass spectrometer (Shimadzu, Japan);

[0060] Equipped with Rtx-5 (Rtx-5MS) mass spectrometer column (30m×0.25mm×0.25μm);

[0061] KQ-800DE CNC ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd., Shanghai);

[0062] XS205 electronic balance (MettlerToledo, Switzerland);

[0063] 3H2RI desktop refrigerated centrifuge (Hexi Company, China);

[0064] MS3 vortex mixer (IKA);

[0065] GM200 knife type mixer grinder (Retsch Company).

[0066] See also Figure 1-11 , a new method for detecting fungicide residues in animal-derived foods was established based on GC-MSMS, including the following steps:

[0067] Step S1, preparation of standard solution:

[0068] (1) Standard stock solution: dilute the standard solution with the corresponding solvent to a 100 μg / mL standard stock solution and store it in a refrigerator at -18°C;

[0069] (2) Standard working solution intermediate solution: dilute the above standard stock solution into an intermediate working solution of 10 μg / mL and store it in a -18°C refrigerator;

[0070] (3) blank matrix solution, using the blank solution treated and purified in step (2) to prepare a series of gradient standard solutions;

[0071] Mixed standard working solution: ready for use;

[0072] Step S2, extraction, accurately weigh 5.00 g of the homogenized sample into a 50 mL centrifuge tube, add 10 mL of the extraction solution, vortex on a vortex machine for 10 min, ultrasonicate for 30 min, add 4 g of anhydrous MgSO4 and 1 g of NaCl to remove water and make it uniform, add the extraction powder, shake it thoroughly for 10 min, and centrifuge it at 10000 r / min for 5 min;

[0073] Step S3, taking 2.0 mL of the upper organic solution for purification;

[0074] Step S4, purification: add 0.2 g of purification powder to 2.0 mL of the organic solution to be purified, vortex and oscillate for 3 min, and then centrifuge the sample at 4000 r / min for 3 min;

[0075] Step S5, taking the supernatant and passing it through a 0.22 μm membrane, putting it into a sample injection vial, and putting it on the machine for testing;

[0076] Step S6, setting GC-MS / MS conditions.

[0077] Preferably, step S6 specifically includes:

[0078] GC conditions, column oven temperature: 60°C;

[0079] Equilibration time: 1.0 min;

[0080] The initial temperature was 60°C, maintained for 1 min, then raised to 130°C at a rate of 30°C / min, then raised to 250°C at a rate of 5°C / min, and then raised to 300°C at a rate of 10°C / min and maintained for 5 min, with a total time of 37.33 min;

[0081] MS conditions, ion source temperature: 230°C, interface temperature: 280°C, solvent delay time: 3.5 min;

[0082] Detector voltage (relative to tuning result): 0.3kV;

[0083] Injector: Number of solvent flushes (before injection): 2;

[0084] Number of solvent washes (after injection): 5;

[0085] Number of sample rinses: 2;

[0086] Injection volume: 1 μL.

[0087] Preferably, step S2 specifically includes that the mass error of 5.00 g of the homogenized sample is ±0.02 g.

[0088] Preferably, the preparation ratio of the purification powder in step S4 is MgSO4:PSA:C18=6:1:1.

[0089] Preferably, step S2 specifically includes preparing the extraction solution in a ratio of acetone:acetonitrile=1:1.

[0090] The data were analyzed using Shimadzu Labsolutions4.30 software and graphed using Origin2023b software;

[0091] The established method was validated using SANTE / 11945 / 2015. To validate the method, the limit of detection (LOD), limit of quantification (LOQ), linear range, accuracy (recovery), precision (inter-day precision and intra-day precision) and matrix effect of the method were verified.

[0092] The calibration curve was constructed with calibration concentration as the independent variable and peak area as the dependent variable. The linearity of the method was verified by preparing the corresponding concentration ranges of 13 standards with 1 ng / mL as the starting point of the linear range.

[0093] The limit of detection (LOD) and limit of quantification (LOQ) were calculated using 3 times the signal-to-noise ratio and 10 times the signal-to-noise ratio.

[0094] Three levels, low, medium and high, were selected within the linear range to carry out spike recovery experiments on chemical pollutants. Each group of samples was measured three times in parallel, and the spike recovery rate was calculated.

[0095] The intra-day and inter-day precision were measured respectively, and each sample was measured 6 times in parallel. The RSD was used to evaluate the precision of the method. matrix / S solvent -1)×100, where S matrix and S solvent Represent the matrix standard curve and solvent standard curve, respectively.

[0096] The 13 fungicide standards were diluted into a single standard of 5 ppm using acetonitrile, and the Scan mode of GC-MS / MS was used to search for relevant information of the new fungicide under the conditions of GC-MS / MS in step S6 without applying additional voltage, and to determine the basic information such as the ion fragments and retention time of the standard substances. After finding the basic information such as the retention time and ion fragments of the related substances, the voltage of each standard substance was optimized, and the voltage of the standard substance was increased from 3 V to 45 V to find the most suitable voltage for each standard substance. The basic information such as the retention time and ion fragments of the 13 standard substances was as follows: Figure 1 As shown. Based on the retention time, voltage, ion fragmentation and other information of the fungicides, the MRM method of 13 fungicides was established. The 13 fungicides were finally confirmed according to the established MRM method. The GC-MS / MS spectra of the 13 fungicides are shown in Figure 2 shown.

[0097] This study compared the extraction efficiency of 1% acetic acid in acetonitrile, acetonitrile, 1:1 acetonitrile in acetone, and 5% ethyl acetate in acetonitrile for chicken and beef. Figure 3 shown.

[0098] The fungicides in beef and chicken were extracted using 1% acetic acid and acetonitrile. Among them, the extraction efficiency of beef and chicken exceeded 80% for 11 and 10 fungicides, respectively.

[0099] When acetonitrile was used to extract 13 fungicides from chicken and beef, the extraction efficiency of 11 substances exceeded 80%;

[0100] When extracting fungicides from beef and chicken using 1:1 acetonitrile / acetone, 12 substances had an extraction efficiency of more than 80%;

[0101] The extraction efficiency of 11 fungicides in beef and chicken by 5% acetic acid and acetonitrile exceeded 80%.

[0102] In comparison, the extraction efficiency of 1:1 acetonitrile / acetone was the most suitable for the extraction of fungicides in chicken and beef.

[0103] In addition, acetone has a good extraction effect on oily substances. There are many fat-soluble substances in animal food. The combination of acetone and acetonitrile can achieve a certain extraction effect on the extraction of fungicides in animal food.

[0104] After optimizing the extraction solvent, the purification powder is optimized. The purification effect of the purification powder is crucial for the detection of pesticide residues. It is also the most important step in the QuEChERS extraction method. The types of purification powders currently on the market are C18, PSA, GCB and other common purification powders. C18 mainly adsorbs fats, pigments, vitamins and non-polar substances; PSA adsorbs sugars, pigments and organic acids; GCB mainly adsorbs and removes pigments in samples.

[0105] The ratios of purification powders in different combinations of MgSO4:PSA=6:1, MgSO4:PSA:C18=6:1:1, MgSO4:PSA:GCB=6:1:1, and MgSO4:C18=6:1 were optimized.

[0106] The optimization results are as follows Figure 4 As shown, for substances with a recovery rate of more than 80%, MgSO4:PSA:C18=6:1:1 is greater than MgSO4:C18=6:1 in beef, except for mefenamic acid and bixafen, which have a slightly lower recovery rate than MgSO4:C18=6:1. In chicken, MgSO4:C18=6:1 is greater than MgSO4:PSA:C18=6:1 in chicken, except for mefenamic acid, which has a slightly lower recovery rate than MgSO4:PSA:C18=6:1:1. MgSO4:PSA:C18=6:1:1 is more suitable for the extraction of fungicides in beef, and MgSO4:C18=6:1 is more suitable for the extraction of fungicides in chicken.

[0107] Matrix effect is the most common factor affecting the accuracy of test results in pesticide residue detection. Matrix effect ME = (slope of matrix matching standard curve / slope of solvent standard curve - 1) × 100% Different types of substances have different matrix effects. The matrix effect results of beef and chicken are as follows: Figure 5 and Figure 6 When ME>0, it is a matrix enhancement effect; when ME<0, it is a matrix inhibition effect; when |ME|<20%, it is a weak matrix effect, and the matrix effect is not significant; when |ME|<50%, it is a moderate matrix effect; when |ME|>50%, it is a strong matrix effect, and the matrix effect is considered significant.

[0108] Results of matrix effects of 13 fungicides in chicken and beef Figure 5 and Figure 6As shown in the figure, 13 fungicides showed strong matrix effects in chicken and beef (|ME|>50%). The strong matrix effect of pesticide residue determination using GC-MS / MS is due to the fact that the matrix components blocked the active sites in the capillary column or the injector lining, thereby improving the transmission of the target analyte. Especially for animal food, due to the complex composition of animal food itself, the pesticide residues in animal food can effectively protect the detected pesticides when entering the detector port, reducing the possibility of their volatilization. After the non-volatile sample components and chemical pollutant ions are co-eluted on the chromatographic column, they will compete with the vaporized ions, significantly increasing or significantly reducing the generation efficiency and ion intensity of the target ions.

[0109] The slope ratio of the matrix-matched calibration curve to the solvent standard curve was used to evaluate the influence of the matrix effect on the quantitative results and eliminate or compensate for the mechanism effect. The matrix standard curve was established for all 13 fungicides to eliminate the influence of the matrix effect.

[0110] The matrix-matched standard curve external standard method was used for quantitative analysis. Figure 7 and Figure 8 As shown. Within the linear range of -ng / ml, 13 fungicides have good linear correlation, and the correlation coefficient (R 2 ) were all greater than 0.999, and LOD and LOQ represented the limit of detection and limit of quantification of the samples, respectively, which were represented by the concentrations corresponding to signal-to-noise ratios of 3:1 and 10:1, respectively.

[0111] In the linear range, three levels of spiked amounts, low (LOQ), medium (5LOQ), and high (10LOQ), were selected to conduct spiked recovery experiments on fungicides. Each group of samples was tested three times in parallel, and the spiked recovery rate was calculated. The results are shown in Figure 2. Fig. 9 The recoveries of 13 fungicides in chicken and beef were within 79.51%-111.39% and 83.35%-103.98%, and the RSDs of the recoveries of 13 fungicides in chicken and beef were within 7.49%. The established simultaneous detection method of 13 fungicides can meet the basic detection requirements.

[0112] The precision of the established method was determined. The intra-day and inter-day compactness were measured separately, and each sample was measured 6 times in parallel. The RSD was used to evaluate the precision of the method. The results are shown in the figure. Fig.10 and Fig.11As shown. The intra-day precision of chicken and beef was less than 10.92% and 10.83%, respectively, and the inter-day precision was less than 12.99% and 12.24%, respectively. According to the AOAC guidelines, when the concentration is between 0.001 and 0.01 ppm, the intra-day and inter-day precision RSDs should be less than 30% and 32%, respectively; when the analyte concentration is between 0.01 and 0.10 ppm, the intra-day and inter-day RSDs should be less than 20% and 22%, respectively, meeting the conditions.

[0113] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A new method for detecting fungicide residues in animal-derived foods based on GC-MSMS is established, characterized in that: The following steps are involved: Step S1, preparation of standard solution: (1) Standard stock solution: dilute the standard solution with the corresponding solvent to a 100 μg / mL standard stock solution and store it in a refrigerator at -18°C; (2) Standard working solution intermediate solution: dilute the above standard stock solution into an intermediate working solution of 10 μg / mL and store it in a -18°C refrigerator; (3) blank matrix solution, using the blank solution treated and purified in step (2) to prepare a series of gradient standard solutions; Mixed standard working solution: ready for use; Step S2, extraction, accurately weigh 5.00 g of the homogenized sample into a 50 mL centrifuge tube, add 10 mL of the extraction solution, vortex on a vortex machine for 10 min, ultrasonicate for 30 min, add 4 g of anhydrous MgSO4 and 1 g of NaCl to remove water and make it uniform, add the extraction powder, shake it thoroughly for 10 min, and centrifuge it at 10000 r / min for 5 min; Step S3, taking 2.0 mL of the upper organic solution for purification; Step S4, purification: add 0.2 g of purification powder to 2.0 mL of the organic solution to be purified, vortex and oscillate for 3 min, and then centrifuge the sample at 4000 r / min for 3 min; Step S5, taking the supernatant and passing it through a 0.22 μm membrane, putting it into a sample injection vial, and putting it on the machine for testing; The animal-derived food is chicken and beef, the extraction solution is acetonitrile:acetone=1:1, when the animal-derived food is chicken, the purification powder is MgSO4:C18=6:1, when the animal-derived food is beef, the purification powder is MgSO4:PSA:C18=6:1:1; Dilute the fungicide standard with acetonitrile to a single standard of 5 ppm, use the Scan mode of GC-MS / MS, set the conditions of GC-MS / MS, and search for relevant information of the new fungicide without applying additional voltage. Determine the basic information of the ion fragments and retention time of the standard substance, optimize the voltage of each standard substance, increase the voltage of the standard substance from 3V to 45V, find the most suitable voltage for each standard substance, and establish the MRM method of the fungicide based on the retention time, voltage, and ion fragment information of the fungicide; GC conditions, column oven temperature: 60°C; Equilibration time: 1.0 min; The initial temperature was 60°C, maintained for 1 min, then raised to 130°C at a rate of 30°C / min, then raised to 250°C at a rate of 5°C / min, and then raised to 300°C at a rate of 10°C / min and maintained for 5 min, with a total time of 37.33 min; MS conditions, ion source temperature: 230°C, interface temperature: 280°C, solvent delay time: 3.5 min; Detector voltage (relative to tuning result): 0.3kV; Injector: Number of solvent flushes (before injection): 2; Number of solvent washes (after injection): 5; Number of sample rinses: 2; Injection volume: 1 μL; The retention time, ion fragmentation, and voltage of each new fungicide are as follows; 2. The novel method for detecting fungicide residues in animal-derived food based on GC-MSMS according to claim 1, characterized in that: Step S2 specifically includes that the mass error of 5.00 g of the homogenized sample is ±0.02 g.

Images