A method for detecting penthiopyrad and its metabolites in animal-derived foods

Abstract

The present invention discloses a detection method for penthiopyrad and its metabolites in animal-derived foods, comprising the following steps: S1, extraction: mixing the animal-derived food sample with an extraction solvent, then adding a dehydrating agent and a buffer salt to mix evenly, and then performing solid-liquid separation to obtain an extract; S2, purification: taking the extract of step S1 and mixing it with a purifying agent and a dehydrating agent, and then performing solid-liquid separation to obtain a purified liquid; S3, detection: using a liquid chromatography-triple quadrupole tandem mass spectrometer to detect and analyze the purified liquid obtained in step S2; the animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, liver, eggs, raw milk or casings. The present invention expands the scope of application of the detection method for penthiopyrad and its metabolites to animal-derived foods of different matrices such as poultry and livestock meat, aquatic products, fat, viscera, eggs, milk and casings for the first time, and has the advantages of being fast, easy to operate, low cost, accurate, reliable, effective and highly sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a method for detecting penthiopyrad and its metabolites in animal-derived food. Background Art

[0002] Penthiopyrad is a novel amide fungicide developed by Mitsui Chemicals, Japan. It has a different fungicide spectrum from existing carboxamide fungicides. In addition to being effective against basidiomycetes like existing carboxamide fungicides, it is also effective against ascomycetes and imperfect fungi, and is now recommended for the prevention and control of gray mold and powdery mildew that are resistant to other fungicides. According to relevant reports, penthiopyrad was confirmed in 1998 to be effective against many diseases of many crops, such as gray mold and powdery mildew of fruit trees and vegetable crops, and black spot and red spot of fruit trees. Its mechanism of action is to affect the electron transfer of the mitochondrial respiratory chain of the target bacteria by inhibiting succinate dehydrogenase, thereby destroying its activity. Studies have shown that penthiopyrad is acutely toxic to zebrafish in the early stage and induces a series of deformities during its development.

[0003] The main metabolite of penthiopyrad after field application is 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (PAM), which is more toxic than the parent. GB2763-2021 stipulates that the monitoring of penthiopyrad is defined as penthiopyrad, where the residues: plant-derived foods are penthiopyrad; animal-derived foods are the sum of penthiopyrad and its metabolite 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide, expressed as penthiopyrad. JMPR (2012) also stipulates that the evaluation of penthiopyrad is defined as the sum of penthiopyrad and its metabolite PAM, expressed as penthiopyrad. Drug detection in animal-derived foods requires the detection of components with extremely low concentrations in a complex matrix, and in liquid chromatography-tandem mass spectrometry, the matrix effect will affect the sensitivity and reproducibility of the instrument. Therefore, pretreatment is required to remove the interference of impurities on the analyte as much as possible, and the matrix preparation curve is required. At the same time, highly sensitive and specific detection methods are required to quantitatively detect the components. However, there are currently no standards available for reference regarding the detection of penthiopyrad and its metabolites in animal-derived foods, so it is necessary to explore detection methods for penthiopyrad and its metabolites.

[0004] At present, the main detection methods for penthiopyrad residues include gas chromatography (GC), solid phase extraction technology or QuEChERS combined with high performance liquid chromatography (HPLC), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), etc. However, most of the studies are limited to the detection of penthiopyrad parent. For example, some scholars have established the detection methods of penthiopyrad and trifloxystrobin in matrices such as honey, apple flowers, bees, and human serum using GC. Yang Huan et al. used the improved QuEChERS method to quickly determine 8 new succinate dehydrogenase inhibitor fungicides in fruits and vegetables. Lu Jing et al. used high performance liquid chromatography-tandem mass spectrometry to determine penthiopyrad residues in vegetable oils, but these studies are limited to the detection of the residual amount of penthiopyrad parent. Only two literatures were found reporting the simultaneous detection of penthiopyrad and its metabolite PAM on perilla leaves or tomatoes. For example, Li Ruotong et al. used QuEChES-HPLC-MS / MS to detect the residues of penthiopyrad, trifloxystrobin and their metabolites in tomatoes and their long-term dietary risk assessment. The samples were extracted with acetic acid-acetonitrile, purified with anhydrous magnesium sulfate, N-propylethylenediamine (PSA) and graphitized carbon black (GCB), and detected with HPLC-MS / MS. The limit of quantification (LOQ) was 0.05 mg / kg. The LOQ of this method could not meet the requirements for animal-derived foods in GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food". Sun Jiong et al. also studied the method for the residual analysis of penthiopyrad and its metabolites in tomatoes. The samples were extracted with 0.2% formic acid-acetonitrile, purified with N-propylethylenediamine (PSA) and anhydrous magnesium sulfate, and detected with UPLC-MS / MS. The minimum detection amount of penthiopyrad and PAM in tomatoes was 3×10-13g, and the limit of quantification was 0.002mg / kg. However, the above methods are all for the study of plant-derived foods such as tomatoes, which are different from animal-derived foods. In addition, the matrix of animal-derived foods is complex. In addition to considering the removal of pigments, the pretreatment should also consider the removal of fat and protein. Therefore, the above methods cannot be copied and applied to the detection of penthiopyrad and its metabolites in animal-derived foods.

[0005] Although, a large number of detection experts have studied the detection of penthiopyrad in animal-derived foods. For example, patent CN105699568A discloses a GC-MS / MS rapid determination method for the residual amount of penthiopyrad, which is a method for the residual content of penthiopyrad in animal-derived foods with high fat content. The residual penthiopyrad in the sample is extracted with acetonitrile homogenization, and the extract is purified by matrix dispersion with an enhanced lipid removal product (Enhanced Matrix Removal, EMR), concentrated by stripping tube extraction, and then detected by gas chromatography tandem mass spectrometry (GC-MS / MS), with a detection limit of less than 0.085μg / kg. For example, patent CN105699525A discloses a GC-EI-MS determination method for the residual amount of penthiopyrad, which comprises adding acetonitrile or an acetonitrile solution containing 1% acetic acid for homogenization or oscillating ultrasonic extraction, then adding sodium chloride or sodium acetate and anhydrous magnesium sulfate for extraction; concentrating, purifying with a C18 / PSA solid phase extraction column, eluting with acetonitrile, collecting the eluate, concentrating to dryness, dissolving with a mixed solvent of acetone / n-hexane with a volume ratio of 1 / 1, passing through a membrane, and then detecting with gas chromatography-electron bombardment ion source-mass spectrometry (GC-EI-MS); the detection limit is less than 1.68 μg / kg; in addition, patent CN1123051 31 discloses a GC-Orbitrap-MS screening and analysis method for pesticide residues in animal-derived foods. The animal-derived foods in the patent include at least one of chicken, pork, beef, mutton, duck, goose, chicken liver, pork liver and sheep liver; the extraction solvent is a 1:1 mixture of acetonitrile containing 1% acetic acid and acetone, and detection is performed using an Orbitrap-MS with an EI ion source. However, none of the above patents involve research on the detection of penthiopyrad metabolites in animal-derived foods, and the matrix range of the animal-derived products in the above patents is small, and the detection speed is slow, which is not conducive to the rapid detection of animal-derived foods with different matrices.

[0006] Therefore, obtaining a method that can quickly detect penthiopyrad and its metabolites in animal-derived foods in a variety of matrices has become one of the research directions in the field of analytical testing. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for detecting penthiopyrad and its metabolites in animal-derived foods, which realizes the detection of penthiopyrad and its metabolites in animal-derived foods in different matrices, and has the advantages of being rapid, easy to operate, low cost, accurate, reliable and highly sensitive.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for detecting penthiopyrad and its metabolites in animal-derived foods, comprising the following steps:

[0010] S1. Extraction: Evenly mix the animal-derived food sample with the extraction solvent, then add a dehydrating agent and a buffer salt and mix evenly, and then perform solid-liquid separation to obtain an extract;

[0011] S2, purification: take the extract of step S1 and mix it evenly with the purifier and the water removal agent, and then perform solid-liquid separation to obtain a purified liquid;

[0012] S3, detection: using liquid chromatography-triple quadrupole tandem mass spectrometry to detect and analyze the purified liquid obtained in step S2;

[0013] The animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, liver, eggs, raw milk or casings.

[0014] The present invention expands the applicable scope of determination of penthiopyrad and its metabolites (PAM) to animal-derived foods of different matrices such as poultry meat, aquatic products, fat, liver, eggs, raw milk and casings for the first time, mixes animal-derived food samples treated by different methods with extraction solvents for purification pre-treatment, the supernatant obtained in step S1 is purified, and a liquid chromatography-mass spectrometry is used to detect the residual amount of benzophenone in the animal food. The statistical results show that the overall recovery rate of the method ranges from 77.0% to 117.0%, and the relative standard deviation of the method is from 0.6% to 8.9%, which meets the detection needs. The verification results meet the requirements of the relevant provisions of SN / T 0001-2016 "Basic Provisions for the Preparation of Physical and Chemical Determination Methods for Exported Foods and Cosmetics", and has the advantages of being fast, easy to operate, low cost, accurate, reliable, effective and highly sensitive.

[0015] Furthermore, the purifier in step S2 includes at least one of N-propylethylenediamine (PSA), silica-bonded carbon 18 (C18), neutral alumina, graphitized carbon black (GCB) and Florisil (60-100MESH), preferably a composite of silica-bonded carbon 18 and Florisil, and the mass ratio of C18 to Florisil is 1:1.

[0016] In the present invention, the purifier is preferably a compound of C18 and florisil in a mass ratio of 1:1. The use of the purifier can further remove impurity interference, is simple and convenient to operate, saves reagent costs and time costs, and is suitable for large-scale sample testing.

[0017] Furthermore, the extraction solvent is an aqueous solution of acetic acid and acetonitrile with a mass fraction of 1%.

[0018] In the present invention, although the simplest acetic acid acetonitrile aqueous solution with a mass fraction of 1% is used as the extraction solvent, this extraction method can effectively precipitate impurities.

[0019] The method for preparing the animal-derived food sample in the present invention is:

[0020] (1) Randomly sample 500g of poultry meat, fat and viscera, crush them with a wall-breaking machine, mix them thoroughly, and put them into polyethylene bottles or bags. Store the samples according to test and reserve, and store them at -18℃ or below. Take out the samples in advance to restore them to room temperature before testing.

[0021] (2) Randomly sample 500g of aquatic products, take the edible parts, remove the bones and scales, randomly sample 500g, crush with a wall-breaking machine, mix thoroughly, and put into a polyethylene bottle or bag. Store the samples according to test and reserve, and store at -18℃ or below. Take out the samples in advance to restore to room temperature when testing.

[0022] (3) Randomly sample 500 g of raw milk (e.g., milk) or eggs, stir evenly, put into a polyethylene bottle, and refrigerate. Store the samples separately for testing and for backup.

[0023] Furthermore, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, etc., the specific steps of extraction in S1 are: weigh the sample and place it in a centrifuge tube A, add a 1% acetic acid acetonitrile solution by mass fraction, and then place it in a homogenizer for homogenization, then take another centrifuge tube B and add 1% acetic acid acetonitrile solution thereto, clean the homogenizer blade, and then merge the washing liquid in the centrifuge tube B into the centrifuge tube A, mix evenly, add a dehydrating agent and a buffer salt, mix evenly, oscillate and extract, and then centrifuge to obtain a supernatant, i.e., an extract.

[0024] Preferably, when the animal-derived food is one or more of poultry and livestock meat and aquatic products, the specific steps of extraction in S1 are: weigh 5.0g of the sample and place it in a 50mL centrifuge tube A, add 10mL of 1% acetic acid and acetonitrile by mass and homogenize under a homogenizer for 20 to 30s, then take another centrifuge tube B and add 10mL of 1% acetic acid and acetonitrile solution by mass to it to clean the residue on the blade of the homogenizer, then combine the washing liquid in centrifuge tube B into centrifuge tube A, mix evenly for 20s, then add 6.0g of anhydrous magnesium sulfate and 1.5g of sodium acetate, mix evenly, oscillate and extract for 10min, centrifuge at 4000r / min for 5min, and obtain the supernatant, i.e., the extract.

[0025] Furthermore, when the animal-derived food is one or more of liver, eggs, raw milk, fat or casings, the specific steps of extraction in S1 are: weighing the sample and placing it in a centrifuge tube, adding a 1% by mass acetic acid acetonitrile solution, mixing evenly, adding a dehydrating agent and a buffer salt, mixing evenly, oscillating and extracting, and then centrifuging to obtain a supernatant, i.e., an extract.

[0026] Preferably, 5.0 g of the sample is weighed and placed in a 50 mL centrifuge tube, 10 mL of 1% acetic acid acetonitrile solution is added, and after mixing evenly for 20 seconds, 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate are added, and after mixing evenly, the mixture is shaken and extracted for 10 minutes, and centrifuged at 4000 r / min for 5 minutes to obtain a supernatant;

[0027] Furthermore, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, raw milk or casings, the specific steps of purification in S2 are: 1.00 mL of the extract of step S1 is absorbed into a centrifuge tube containing 25-150 mg of a purifier and a dehydrating agent, oscillated for 15-20 seconds, centrifuged at 11000-13000 r / min for 5-8 minutes, and the supernatant after centrifugation is passed through an organic filter membrane to obtain a purified liquid.

[0028] Preferably, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, raw milk or casings, the specific steps of purification in S2 are: 1.00 mL of the extract of step S1 is absorbed into a centrifuge tube containing 100 mg of a purifier and 150 mg of anhydrous magnesium sulfate, oscillated for 20 seconds, centrifuged at 12000 r / min for 5 minutes, and the supernatant after centrifugation is filtered through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0029] Furthermore, when the animal-derived food is fat, the specific steps of purification in S2 are: the extract in step S1 must be frozen for 1.5-2.5 hours, and then 1.00 mL of the frozen extract is absorbed into a centrifuge tube containing 25-150 mg of a purifier and a dehydrating agent, shaken for 15-20 seconds, and centrifuged at 11000-13000 r / min for 5-8 minutes. The supernatant after centrifugation is passed through an organic filter membrane to obtain a purified liquid.

[0030] Preferably, when the animal-derived food is fat, the specific steps of purification in S2 are: first, the extract in step S1 needs to be frozen for 2 hours, then 1.00 mL of the frozen extract is absorbed into a centrifuge tube containing 100 mg of a purifier and 150 mg of anhydrous magnesium sulfate, oscillated for 20 seconds, centrifuged at 12000 r / min for 5 minutes, and the supernatant after centrifugation is filtered through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0031] Furthermore, when the animal-derived food is at least one of eggs and liver, the specific steps of purification in S2 are: 1.00 mL of the extract of step S1 is taken into a centrifuge tube containing 25-150 mg of a purifier and a dehydrating agent, oscillated for 15-20 seconds, centrifuged at 11000-13000 r / min for 5-8 minutes, 0.5-1 ml of the supernatant after centrifugation is taken and placed in a test tube, 0.5-1 ml of deionized water is added to the test tube, and the liquid in the test tube is passed through an organic filter membrane to obtain a purified liquid.

[0032] Preferably, when the animal-derived food is at least one of eggs and liver, the specific steps of purification in S2 are: 1.00 mL of the supernatant of step S1 is absorbed into a centrifuge tube containing 100 mg of purifier and 150 mg of anhydrous magnesium sulfate, mixed evenly for 20 seconds, centrifuged at 12000 r / min for 5 minutes, 0.5 ml of the supernatant after centrifugation is absorbed and placed in a test tube, and then 0.5-1 ml of deionized water and 0.22 μm organic phase type microporous filter membrane are added to the test tube to obtain a purified liquid.

[0033] Furthermore, the detection conditions of the liquid chromatography-triple quadrupole mass spectrometer are:

[0034] The chromatographic conditions of the liquid chromatography are as follows: chromatographic column: Accucore aQ (150 mm×2.1 mm 2.6 μm), injection volume 2 μL, column temperature 40° C., mobile phase A is methanol, mobile phase B is an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate for gradient elution.

[0035] The mass spectrometry conditions are: electrospray positive ion mode, multiple reaction monitoring mode acquisition, ion source temperature of 350°C, and evaporation temperature of 300°C.

[0036] Compared with the prior art, the present invention has achieved beneficial technical effects:

[0037] (1) The present invention is the first to expand the applicable scope of PAM determination of penthiopyrad and its metabolites to different matrices of animal-derived foods such as poultry and livestock meat, aquatic products, fat, viscera, eggs, milk, and casings.

[0038] (2) The present invention is the first to use a compound of C18 and floral silica as a purifier for the determination of penthiopyrad and its metabolites in animal-derived foods; the use of this purifier can further remove impurity interference, is simple and convenient to operate, saves reagent costs and time costs, and is suitable for large-scale sample testing.

[0039] (3) The method for detecting penthiopyrad and its metabolites in animal-derived foods provided by the present invention can simultaneously perform qualitative identification and quantitative detection of penthiopyrad and its metabolite PAM.

[0040] (4) The method for detecting penthiopyrad and its metabolites in animal-derived foods provided by the present invention has the advantages of being rapid, easy to operate, low cost, accurate, reliable, effective and highly sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The positive ion secondary mass spectrum of penthiopyrad, wherein the mobile phase ratio is 90% methanol-10% 5mmol / L ammonium formate (0.1% vol formic acid);

[0042] Figure 2 This is the positive ion secondary mass spectrum of the metabolite PAM of penthiopyrad, wherein the mobile phase ratio is 90% methanol-10% 5mmol / L ammonium formate (0.1% vol formic acid);

[0043] Figure 3 The effect of different solvents on the recovery of penthiopyrad in samples;

[0044] Figure 4 To study the effects of different extraction methods on the recovery of penthiopyrad and its metabolites in tilapia;

[0045] Figure 5 The response and signal-to-noise ratio of penthiopyrad when the extraction method of extraction number 3 is adopted in Example 10;

[0046] Figure 6 The response and signal-to-noise ratio of penthiopyrad when the extraction method of extraction number 4 is adopted in Example 10;

[0047] Figure 7 To investigate the effects of extraction volume and number of extractions on the recovery of penthiopyrad and its metabolites in milk;

[0048] Figure 8 To investigate the effects of extraction volume and number of extractions on the recovery of penthiopyrad and its metabolites in tilapia;

[0049] Fig. 9 The adsorption of different purifiers on pentiopyrad;

[0050] Fig.10 The adsorption of penthiopyrad metabolites by different purifiers;

[0051] Fig.11 The effect of the amount of purifier on the recovery of penthiopyrad in tilapia;

[0052] Fig.12 Effect of the amount of purifier on the recovery of penthiopyrad metabolites in tilapia

[0053] Fig.13 The effects of different compound purifiers on the recovery of penthiopyrad and its metabolites in tilapia were studied.

[0054] Fig.14 The purifying effect of 100mg of Florisil;

[0055] Fig.15 The purification effect of 100mgPSA;

[0056] Fig.16 The purification effect of 100mgC18;

[0057] Fig.17 The purification effect is 50mgPSA+50mgflorisil;

[0058] Fig.18 The purification effect is 50mgC18+50mgflorisil;

[0059] Fig.19 The effect of different ratios of C18 and Florisil on the recovery of penthiopyrad and its metabolites in tilapia.

[0060] Fig. 20 The freezing effect of pig fat at different time lengths. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. However, the scope of protection claimed in the present invention is not limited to the following specific embodiments, and the reagents or instruments used in the detection method of the present invention can be purchased from the market.

[0062] Among them, the preparation method of the animal-derived food sample in this embodiment is:

[0063] (1) Randomly sample 500g of poultry meat, fat and viscera, crush with a wall-breaking machine, mix thoroughly, and put into a polyethylene bottle or bag. Store the samples separately according to test and reserve. Store at -18℃ and above. Take out the test samples in advance and restore them to room temperature.

[0064] (2) Randomly sample 500g of aquatic products, take the edible parts, remove the bones and scales, randomly sample 500g, crush with a wall-breaking machine, mix thoroughly, and put into a polyethylene bottle or bag. Store the samples according to test and reserve. Store at -18℃ and above. Take out the test samples in advance and restore them to room temperature.

[0065] (3) Randomly sample 500g of raw milk (e.g., milk) or eggs, stir well, and place in a polyethylene bottle. Refrigerate and store the samples separately for testing and reserve.

[0066] Example 1

[0067] This embodiment provides a method for detecting penthiopyrad and its metabolites in animal-derived food (the animal-derived food in this embodiment is beef), and the detection method comprises the following steps:

[0068] S1. Extraction: Weigh 5 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube A, add 10 mL of 1% acetic acid acetonitrile solution, and homogenize it in a homogenizer at 10000 r / min for 20 s. Take another 50 mL centrifuge tube B, add 10 mL of 1% acetic acid acetonitrile solution, clean the homogenizer blade for 10 s, then combine the washing liquid in centrifuge tube B into centrifuge tube A, mix and extract on a vortex mixer for 20 s, add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate, mix well, shake and extract for 10 min, centrifuge at 4000 r / min for 5 min, and obtain the supernatant;

[0069] S2, purification: take 1.00 mL of the supernatant obtained in step S1, put it into a centrifuge tube containing 50 mg C18, 50 mg Florisil and 150 mg anhydrous magnesium sulfate, vortex and shake for 20 seconds, centrifuge at 12000 r / min for 5 minutes, take the supernatant after centrifugation and pass it through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0070] S3. Sample detection: liquid chromatography-triple quadrupole tandem mass spectrometry is used to detect and analyze the purified liquid obtained in step S2.

[0071] Example 2

[0072] The detection method of this embodiment is the same as that of Embodiment 1, except that the animal-derived food sample to be detected is replaced with chicken.

[0073] Example 3

[0074] The detection method of this embodiment is the same as that of Embodiment 1, except that the animal-derived food sample to be detected is replaced by tilapia.

[0075] Example 4

[0076] A method for detecting penthiopyrad and its metabolites in animal-derived food (in this embodiment, the animal-derived food is chicken liver), the detection method comprising the following steps:

[0077] S1. Extraction: Weigh 5.0 g of sample (accurate to 0.01 g) and place in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid acetonitrile solution, mix and extract on a vortex mixer for 20 seconds, add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate, mix well, shake and extract for 10 minutes, centrifuge at 4000 r / min for 5 minutes to obtain the supernatant;

[0078] S2, purification: 1.00 mL of the supernatant obtained in step S1 was taken and placed in a centrifuge tube C containing 50 mg C18, 50 mg Florisil and 150 mg anhydrous magnesium sulfate. After vortexing for 20 seconds, the mixture was centrifuged at 12000 r / min for 5 minutes. 0.5 ml of the supernatant after centrifugation was taken into a test tube, and 0.5 ml of deionized water was added to the test tube. The liquid in the test tube was filtered through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0079] S3. Sample detection: liquid chromatography-triple quadrupole tandem mass spectrometry is used to detect and analyze the purified liquid obtained in step S2.

[0080] Example 5

[0081] The detection method of this embodiment is the same as that of Embodiment 4, but different from that of Embodiment 4, the animal-derived food in this embodiment is eggs.

[0082] Example 6

[0083] A method for detecting penthiopyrad and its metabolites in animal-derived food (in this embodiment, the animal-derived food is milk), the detection method comprising the following steps:

[0084] S1. Extraction: Weigh 5.0 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid and acetonitrile, mix and extract on a vortex mixer for 20 seconds, add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium acetate, mix well, oscillate and extract for 10 minutes, centrifuge at 4000 r / min for 5 minutes to obtain the supernatant.

[0085] S2, purification: take 1.00 mL of the supernatant obtained in step S1, put it into a centrifuge tube containing 50 mg C18, 50 mg Florisil and 150 mg anhydrous magnesium sulfate, vortex and shake for 20 seconds, centrifuge at 12000 r / min for 5 minutes, take the supernatant after centrifugation and pass it through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0086] S3. Sample detection: liquid chromatography-triple quadrupole tandem mass spectrometry is used to detect and analyze the purified liquid obtained in step S2.

[0087] Example 7

[0088] The detection method of this embodiment is the same as that of Embodiment 1, and is different from that of Embodiment 6 in that the animal-derived food in this embodiment is sausage casing.

[0089] Example 8

[0090] A method for detecting penthiopyrad and its metabolites in animal-derived food (in this embodiment, the animal-derived food is fat), the detection method comprising the following steps:

[0091] S1. Extraction: Weigh 5.0 g sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL 1% acetic acid and acetonitrile, mix well for 20 seconds, then add 6.0 g anhydrous magnesium sulfate and 1.5 g sodium acetate, mix well, oscillate and extract for 10 minutes, centrifuge at 4000 r / min for 5 minutes to obtain the supernatant.

[0092] S2, purification: freeze the supernatant obtained in step S1 for 2 h, take 1.00 mL of the frozen supernatant, put it into a centrifuge tube containing 50 mg C18, 50 mg Florisil and 150 mg anhydrous magnesium sulfate, vortex and shake for 20 s, centrifuge at 12000 r / min for 5 min, take the supernatant after centrifugation and pass it through a 0.22 μm organic phase microporous filter membrane to obtain a purified solution.

[0093] S3. Sample detection: liquid chromatography-triple quadrupole tandem mass spectrometry is used to detect and analyze the purified liquid obtained in step S2.

[0094] The following examples investigate the effects of different factors on the detection of penthiopyrad and its metabolite PAM in animal-derived foods.

[0095] The standard working solution of penthiopyrad and its metabolite PAM is prepared as follows:

[0096] (1) Preparation of penthiopyrad standard stock solution (1000 mg / L): Accurately weigh about 10 mg (accurate to 0.1 mg) of penthiopyrad standard, dissolve it in acetonitrile and make up to 10 mL, and store it at -18°C or below in a dark place.

[0097] Preparation of standard stock solution (1000 mg / L) of pentiopyrad metabolite PAM: Accurately weigh about 10 mg (accurate to 0.1 mg) of PAM, dissolve it in acetonitrile and make up to 10 mL, and store it at -18°C or below in the dark.

[0098] (2) Preparation of mixed standard stock solution (100 mg / L): Pipette a certain amount of penthiopyrad and PAM standard stock solution into a 10 mL volumetric flask and dilute to the mark with acetonitrile. Store at -18°C or below in a dark place.

[0099] (3) Preparation of mixed standard solution (10 mg / L): Pipette a certain amount of mixed standard stock solution into a 10 mL volumetric flask, dilute to the mark with acetonitrile, and store at -18°C or below in a dark place.

[0100] (4) Working curve of mixed standard solution: Accurately measure the standard mixed standard solution and dilute it with a blank matrix (the liquid of the animal-derived food sample without penthiopyrad and its metabolite PAM after being treated in Examples S1 and S2) to prepare a series of standard working solutions of concentrations of 0.000 μg / L, 1.00 μg / L, 2.00 μg / L, 5.00 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, and 100.0 μg / L, which are prepared and used immediately.

[0101] Example 9

[0102] This example is about the optimization of liquid chromatography and mass spectrometry conditions.

[0103] 1. Optimization of liquid phase conditions

[0104] 1.1 Choice of mobile phase

[0105] The chromatographic column was Accucore aQ (150 mm × 2.1 mm 2.6 μm), the injection volume was 2 μL, and chromatographic analysis was performed at a column temperature of 40°C using two mobile phase ratios. The peak areas were compared. The specific results are shown in Table 1.

[0106] Table 1 Comparison of mobile phase selection

[0107]

[0108] As shown in Table 1, by comparing the response peak areas of 2.00 μg / L, 5.00 μg / L, 10.0 μg / L, and 20.0 μg / L standard solutions in different mobile phases, it is found that the peak areas of the corresponding concentrations of benzovindiflupyr (positive ion) in the mobile phase A of methanol and the mobile phase B of an aqueous solution containing 5 mmol / L ammonium formate (0.1% vol formic acid) are larger. Therefore, the mobile phase A of methanol and the mobile phase B of an aqueous solution containing 5 mmol / L ammonium formate (0.1% vol formic acid) are selected as the mobile phases for chromatographic analysis.

[0109] 1.2 Selection of chromatographic columns

[0110] Comparison was made between Accucore aQ C18 (150 mm × 2.1 mm 2.6 μm) and Agilent XDB C18 (150 mm × 2.1 mm 2.6 μm) columns. Under the conditions of injection volume of 2 μL and column temperature of 40°C, mobile phase A was methanol and mobile phase B was an aqueous solution containing 5 mmol / L ammonium formate (0.1% vol formic acid), and chromatographic analysis was performed. The results are shown in Table 2.

[0111] Table 2 Selection of chromatographic columns

[0112]

[0113]

[0114] As shown in Table 2, by comparing the response signal-to-noise ratios of 1.00 μg / L, 5.00 μg / L, 10.0 μg / L, 50.0 μg / L and 100.0 μg / L standard solutions, the data analysis revealed that under the same gradient conditions, the concentration responses of each standard solution of penthiopyrad were AgilentXDB C18>Accucore aQ C18, and the signal-to-noise ratio of Accucore aQ C18 was about 3 times that of AgilentXDB C18; the PAM response and signal-to-noise ratio of AgilentXDB C18 were slightly higher than those of Accucore aQ C18. After comprehensive consideration, Accucore aQC18 (150 mm×2.1 mm 2.6 μm) was selected as the chromatographic column of the present invention.

[0115] 1.3 Optimization of mobile phase ratio

[0116] In liquid chromatography, under the condition of fixed chromatographic column, the separation of the tested compound and other components in the sample is completed by mobile phase. The composition and proportion of mobile phase have an important influence on the chromatographic peak shape and separation degree of the separated components. The composition of mobile phase is related to the chromatographic column and the separated components. The elution ability of mobile phase is determined by its polarity. The mobile phase with strong polarity has a strong ability to occupy the adsorption center and has a strong elution ability, which makes the k value of the component small and the retention time short. In actual operation, a mixed solvent with more than two components is often used as the mobile phase. Because the strength of the mixed solvent changes continuously with its composition, a mobile phase with suitable solvent strength can be found, and the low viscosity of the solvent can be maintained to reduce the column pressure and improve the column efficiency, and the selectivity of separation can also be improved. An aqueous solution containing 5mmol / L ammonium formate (0.1% vol formic acid) was selected as the aqueous phase of the mobile phase for chromatographic analysis. The results are shown in Table 3.

[0117] Table 3 Selection of mobile phase ratio

[0118]

[0119]

[0120] As can be seen from Table 3, penthiopyrad elutes when the mobile phase ratio of the length gradient of 15min and 10min is 90% methanol, and PAM elutes when the mobile phase ratio of the length gradient of 15min and 10min is 55% methanol. The retention times are not much different, and the peak area of ​​15min is better than that of 10min. Taking all factors into consideration, the present invention adopts a 15min length gradient mobile phase ratio.

[0121] 1.4 Optimization of column temperature

[0122] Take 10 μg / L penthiopyrad and PAM standard solution and enter the instrument for analysis according to the above chromatographic conditions.

[0123] Table 4 Column temperature selection

[0124]

[0125] As can be seen from Table 4, the effects of the commonly used 30°C, 35°C, 40°C and 45°C on chromatographic separation were compared. The results showed that the RSD values ​​of penthiopyrad and PAM were smaller under the column temperature condition of 40°C, indicating that the responses were more stable at this column temperature. Taking comprehensive considerations, the present invention selects 40°C as the column temperature of the liquid chromatography-mass spectrometry method.

[0126] 2. Optimization of mass spectrometry conditions

[0127] 2.1 Selection of mass spectrometry parameters

[0128] A peristaltic pump was used to continuously inject 1.0 mg / L of penthiopyrad and PAM standard solutions into the ESI ion source at a flow rate of 10 μL / min. The primary mass spectrometry analysis (Q1 scan) of penthiopyrad and PAM was performed in the positive ion mode to obtain the quasi-molecular ion peaks (M-1) and (M+1), and the declustering voltage was optimized. The secondary mass spectrometry analysis (daughter ion scan) of the quasi-molecular ion peaks was performed to obtain the fragment ion information, and the collision energy was optimized. The secondary mass spectra of penthiopyrad and PAM are shown in Figure 1-2 The ion pairs with high response value and low baseline noise were selected as monitoring ion pairs (qualitative ion pairs), and the ion pairs with the strongest signal were selected as quantitative ion pairs. In addition, the parameters such as ion source temperature, sheath gas, and auxiliary gas were optimized. The results are shown in Table 5. The MRM mode was used to collect data, and the multiple reaction monitoring conditions for each ion pair were optimized as shown in Table 6.

[0129] Table 5 Optimization parameters of penthiopyrad and PAM

[0130]

[0131] Table 6 Multiple reaction monitoring conditions of penthiopyrad and PAM

[0132]

[0133] Example 10

[0134] This example investigates the effects of the extraction reagent volume and the number of extractions on the recovery of penthiopyrad and its metabolites.

[0135] 1.1 Effect of extraction solvent on the recovery of penthiopyrad and its metabolites

[0136] 0.05 ml of 1000 μg / L penthiopyrad standard solution was added to tilapia and blank milk samples, respectively, and acetonitrile, 1% acetic acid acetonitrile and 0.2% formic acid-80% acetonitrile water were used as extraction solvents, respectively. After treatment with S1 and S2 in Reference Example 3 and Example 6, liquid chromatography-triple quadrupole tandem mass spectrometry was used for detection and analysis, and the recovery rates of penthiopyrad were calculated, respectively. Figure 3 shown.

[0137] Depend on Figure 3 The results show that the recovery rate of penthiopyrad in 0.2% formic acid-80% acetonitrile water is the lowest; acetonitrile and 1% acidic acetonitrile extraction are equivalent and can be used as extraction agents. However, since protein is easy to coagulate under acidic conditions, for example, milk can be obviously flocculated when acidic acetonitrile is selected, the present invention selects 1% acetic acid acetonitrile solution, which is more conducive to the extraction of penthiopyrad from samples with high protein content.

[0138] 1.2 Effect of buffer salt and dehydrating agent selection on the recovery of penthiopyrad and its metabolites

[0139] 0.05 ml of 1000 μg / L mixed standard solution was added to tilapia, and four different extraction methods were used, as shown in Table 7. After treatment with S1 and S2 in Reference Example 3, liquid chromatography-triple quadrupole tandem mass spectrometry was used for detection and analysis, and the recovery rates of penthiopyrad and its metabolites were calculated, as shown in Table 7. Figure 4 shown.

[0140] Depend on Figure 4 It can be seen that when the extraction solvent is 1% acetic acid acetonitrile, the recovery rate of penthiopyrad metabolite PAM is higher; different buffer salt and dehydrating agent ratios have no significant effect on the recovery of the target. By comparing the response value and signal-to-noise ratio of penthiopyrad, it is found that when the extraction solvent is 1% acetic acid acetonitrile, the dehydrating agent is 6.0g anhydrous magnesium sulfate, and the buffer salt is 1.5g sodium acetate, the response of penthiopyrad is higher and the signal-to-noise ratio is better. Figure 5-6 Therefore, the extraction method in the technical solution selects 1% acetic acid acetonitrile as solvent, 6.0g anhydrous magnesium sulfate as dehydrating agent, and 1.5g sodium acetate as buffer salt.

[0141] Table 7 Extraction method

[0142]

[0143] 1.3 Selection of extraction solution volume and extraction times

[0144] 0.25 ml of 1000 μg / L mixed standard solution was added to milk respectively, and 3 samples were weighed. Sample 1 was extracted with 5 ml of 1% acetic acid acetonitrile solution at one time; Sample 2 was first extracted with 5 ml of 1% acetic acid acetonitrile solvent and centrifuged, and then 5 ml of 1% acetic acid acetonitrile solvent was added to the remaining residue for extraction and centrifugation, and the extracts were combined; Sample 3 was extracted with 10 ml of 1% acetic acid acetonitrile solution at one time. After treatment with S1 and S2 in Reference Example 6, liquid chromatography-triple quadrupole tandem mass spectrometry was used for detection and analysis, and the recovery rates of penthiopyrad and its metabolites were calculated respectively, as shown in detail. Figure 7 shown.

[0145] Depend on Figure 7 It can be seen that when the milk sample is extracted with 1% acetic acid and acetonitrile once, sample 3 is better than sample 1 (i.e., the effect of one extraction with 10 ml of 1% acetic acid and acetonitrile is better than one extraction with 5 ml). From the test results of sample 1 and sample 2, it can be seen that when 10 ml of extraction is used, there is no significant difference in the recovery effect when the number of extractions is 1 or 2. In order to save steps, reduce time cost and operation difficulty, it is better to use 10 ml of solvent for one-time extraction when determining penthiopyrad and its metabolites in milk.

[0146] 0.25 ml of the 1000 μg / L mixed standard solution was added to the tilapia, and one sample was weighed as sample 4. Sample 4 was first extracted with 10 ml of solvent and then centrifuged. Then 10 ml of solvent was added to the remaining residue and extracted and centrifuged. The extracts were combined. Sample 5 was extracted with 20 ml of solution at one time. After treatment with S1 and S2 in Reference Example 3, liquid chromatography-triple quadrupole tandem mass spectrometry was used for detection and analysis, and the recovery rates of penthiopyrad and its metabolites were calculated respectively. See Figure 8 shown.

[0147] Depend on Figure 8 It can be seen that when the tilapia samples used the same 20 ml extraction solvent volume, there was no significant difference in the recovery effect when the number of extractions was 1 or 2. In order to save steps, reduce time costs and operational difficulties, the use of 20 ml solvent for one-time extraction was the better choice when determining penthiopyrad and its metabolites in tilapia.

[0148] Embodiment 11

[0149] This embodiment is an optimization of the purifier and the purification method.

[0150] 1.1. Selection of purifier type

[0151] In this example, the QuEChERS purification method was used to investigate the adsorption of penthiopyrad and its metabolite PAM by five dispersed solid phase extraction purifiers: N-propylethylenediamine (PSA, 50 μm), silica-bonded carbon 18 (C18, 50 μm), neutral alumina, graphitized carbon black (GCB) and Florisil (60-100 MESH).

[0152] Add 25, 50, 100, and 150 mg of the 5 purifiers to the 100 μg / L standard solution, mix well, filter, and bottle. Then measure the concentration of the target substance on the machine. The results are as follows: Fig. 9 , 10 shown.

[0153] Depend on Fig. 9 , 10 The results show that GCB and neutral alumina have obvious adsorption effects on penthiopyrad metabolites and cannot be used for the determination of penthiopyrad and its metabolites in animal-derived foods. PSA, C18 and Florisil have no obvious adsorption effects on penthiopyrad and its metabolites, and there is no obvious change with the increase of the amount of purification powder. Therefore, PSA, C18 and Florisil can be considered for the detection of penthiopyrad and its metabolites in animal-derived foods.

[0154] 1.2. Investigating the effect of the amount of purifier on the recovery rate of penthiopyrad and its metabolites

[0155] 0.05 mL of a 1000 μg / L standard solution of penthiopyrad and its metabolites was added to a blank tilapia sample (blank sample refers to a sample without penthiopyrad), and PSA, C18, and Florisil were used as purifiers (the mass of each purifier was 25 mg, 50 mg, 10 mg, and 150 mg), respectively. After being treated with S1 and S2 in Reference Example 3, the samples were detected by high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, and the recovery of penthiopyrad and its metabolites was calculated, as shown in detail. Fig.11 and Fig.12 shown.

[0156] Depend on Fig.11 and 12 It can be seen that the recovery rate of 25-150mg PSA, C18, and Florisil for the determination of penthiopyrad and its metabolites in tilapia ranges from 75 to 130%. Among them, when the dosage of the purified powder is 100mg, the recovery rate of penthiopyrad and its metabolites in tilapia is 85-100%, which is relatively stable. Considering comprehensively, the experimental dosage of a single purified powder is selected as 100mg.

[0157] 1.3. Recovery of penthiopyrad and its metabolites by single purifier and mixed purifier

[0158] 0.05 mL of 1000 μg / L standard solution of penthiopyrad and its metabolites was added to a blank tilapia sample, and three composite purifiers, 50 mg PSA + 50 mg C18, 50 mg PSA + 50 mg Florisil, and 50 mg C18 + 50 mg Florisil, were used respectively. After treatment with S1 and S2 in Reference Example 3, the sample was detected by high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, and the recovery of penthiopyrad and its metabolites was calculated. Fig.13 As shown by Fig.13 The results show that the recovery rate is between 85% and 100% when the composite purifier is used.

[0159] Figure 14-18 The 5.00 μg / L standard points were prepared from tilapia matrices prepared by different purification methods. The experiment found that when 50 mg C18 and 50 mg Florisil were used as purification powder, the response, signal-to-noise ratio and linearity of the standard curve of penthiopyrad were better. Therefore, in the determination of penthiopyrad and its metabolites in animal-derived foods in this technical solution, a composite powder of 50 mg C18 + 50 mg Florisil was selected as a purifier.

[0160] 1.4. Recovery of penthiopyrad and its metabolites using different ratios of C18 and Florisil composite purifiers

[0161] 0.05 mL of 1000 μg / L penthiopyrad standard solution was added to a blank tilapia sample, and the effects of three different ratios of purification powders, 25 mg C18 and 75 mg Florisil, 50 mg C18 and 50 mg Florisil, and 75 mg C18 and 25 mg Florisil, on the determination of penthiopyrad and its metabolites in tilapia samples were respectively prepared with a composite purifier. After treatment with S1 and S2 in Reference Example 3, the samples were detected by high pressure liquid chromatography-triple quadrupole tandem mass spectrometry, and the recovery of penthiopyrad and its metabolites was calculated. The results are shown in FIG. Fig.19 As shown in the figure, the recovery rates of different ratios of purifying powder for the determination of penthiopyrad and its metabolites in tilapia samples were all in the range of 70% to 120%, but it was found that when 50mgC18 and 50mgflorisil, that is, C18 and 50mgflorisil were in a 1:1 ratio, the added recovery rate of the determination of penthiopyrad metabolites in tilapia samples was better than that of the other two ratio groups of purifiers. After comprehensive consideration, 50mgC18 and 50mgflorisil were selected as purifiers.

[0162] 1.5. Determination of grease removal method

[0163] For samples with high fat content, this technical solution uses the different freezing points of fat and solvent to achieve more effective separation. Taking pig fat as the research object, the samples were frozen for different lengths of time, and the same volume of extraction solution was nitrogen-blown until the mass (m1) remained unchanged. The appropriate freezing time was selected by comparing the percentage of reduction in m1. Fig. 20 As shown in the figure, when the freezing time is greater than 2 hours, there is no significant change in the reduction of m1. Therefore, in this technical solution, 2 hours of freezing purification is used to treat samples with high oil content.

[0164] Example 12

[0165] This embodiment investigates the influence of matrix effect, and uses eight kinds of blank matrices and acetonitrile to prepare standard curves respectively, and obtains the slope k of various matrix and water configuration curves. The specific process is: accurately measure an appropriate amount of 1000μg / ml standard solution, dilute it with blank matrix or acetonitrile into a series of matrix standard working solutions of 1μg / L, 2μg / L, 5μg / L, and 10μg / L, and prepare it for use. The standard working solution is tested and analyzed in sequence according to the above-mentioned measurement conditions using a liquid chromatography-tandem mass spectrometer according to the concentration of the standard working solution from low to high to obtain chromatographic data, and then the chromatographic data obtained by TraceFinder 3.2.368.22 data processing software is plotted with the peak area of ​​the quantitative ion chromatographic peak of penthiopyrad and metabolite PAM as the ordinate and the corresponding standard solution concentration as the abscissa to draw a standard working curve. The calculation of matrix effect ME is as follows:

[0166]

[0167] In the present invention, blank samples of tilapia, beef, chicken, chicken liver, eggs, milk, fat, and casings without penthiopyrad and its metabolite PAM were selected to investigate the matrix effect, wherein a series of working solutions with a blank matrix solution and acetonitrile were used to dilute the standard intermediate solution, and then the matrix curve and solvent curve were obtained by detection and analysis. 基质 and the slope k of the solvent curve 溶剂 Substitute into the K value calculation formula: (k 基质 / k 溶剂 -1)×100% for calculation (where K 溶剂 Refers to the slope of the standard curve of acetonitrile or water solvent prepared without matrix), and the matrix effect ME, i.e. K value, is obtained. The K value is used as the investigation index:

[0168] If K is between -10% and 10%, it means that the matrix effect is weak and not obvious;

[0169] K<-10%, it shows a strong negative effect;

[0170] When K>10%, a stronger positive effect is shown.

[0171] The K value results in different animal-derived matrices are shown in Table 8 below.

[0172] Table 8 Matrix effects in different animal-derived food matrices

[0173]

[0174]

[0175] As shown in Table 8, it can be seen that penthiopyrad and its metabolites in 8 different matrices all show different degrees of matrix inhibition effects, among which PAM in tilapia, penthiopyrad and PAM in chicken liver, PAM in eggs, PAM in fat and PAM in casings all show strong negative effects, and the matrix negative effect intensity of fat and casings exceeds 20%. Therefore, considering comprehensively, the present invention uses matrix curves for calculation and quantification.

[0176] Embodiment 13

[0177] 1. Linear range and regression equation

[0178] In this example, acetonitrile and 8 blank matrix extracts (blank matrix extracts are animal-derived food samples that do not contain penthiopyrad and its metabolites, respectively, and are treated with reference to steps S1 and S2 in Examples 1-8) are used to prepare 1 mg / L standard stock solutions into solutions with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, and 10.0 μg / L.

[0179] The method for drawing the standard curve of penthiopyrad and its metabolites: the solution concentration (1.0μg / L, 2.0μg / L, 5.0μg / L, 10.0μg / L) is used as the horizontal axis, and the ion peak area corresponding to penthiopyrad and its metabolites is used as the vertical axis to draw the standard curve. The results show that within the corresponding mass concentration range of penthiopyrad and its metabolites, the peak area of ​​the quantitative ion and the concentration show a good linear relationship, and the correlation coefficient r 2 It can reach above 0.999, which can meet the experimental analysis requirements. See Table 9 for details.

[0180] Table 9 Regression equations and correlation coefficients of penthiopyrad and metabolite standard working solutions

[0181]

[0182]

[0183] 2. Limit of detection and limit of quantification

[0184] According to the above operation, when the spiked concentration of penthiopyrad and penthiopyrad metabolites was 2.0 μg / kg with a blank beef matrix, the signal-to-noise ratio of the method was greater than 3, and the detection limit of the method was 2.0 μg / kg. Similarly, the confirmation of the quantification limit was also verified by adding certified reference materials to blank beef matrix samples. When the added concentration level was 5.0 μg / kg, the corresponding signal-to-noise ratio (S / N) was greater than 10, and the quantification limit confirmed by this method was 5.0 μg / kg.

[0185] 3. Verification of the precision and recovery rate of the detection method

[0186] Referring to the preparation and detection method of the blank matrix spiked sample solution, 5, 10, and 100 μg / kg levels of penthiopyrad and its metabolite PAM standard solutions were added to 8 sample matrices of beef, chicken, tilapia, chicken liver, pig fat, milk, eggs, and casings, respectively. The recoveries (%) and relative standard deviations (RSDs) of the three spiked concentration levels and the results obtained by testing are shown in Tables 9 and 10.

[0187] Table 10 Recovery rates and relative standard deviations of 8 blank matrix samples spiked with penthiopyrad

[0188]

[0189] Table 11 Recovery and relative standard deviation of 8 blank matrix samples spiked with penthiopyrad metabolites (PAM)

[0190]

[0191] It can be seen from Tables 10 and 11 that the overall average recovery rate of the detection method of the present invention is in the range of 77.0%-117.0%, the relative standard deviation of the method is 0.6%-8.9%, and the relative standard deviation is less than 10%. The verification results of the method meet the requirements of the relevant provisions of SN / T0001-2016 "Basic Provisions for the Preparation of Physical and Chemical Determination Methods for Export Food and Cosmetics".

[0192] Test Example 1

[0193] The detection methods of Examples 1-8 were used to determine the residual amounts of penthiopyrad and penthiopyrad metabolites in 72 samples of chicken, tilapia, beef, chicken liver, pig fat, milk, eggs, pig casings, etc. purchased from supermarkets and markets in Guangzhou. The results are shown in Table 12.

[0194] Table 12 Detection results of penthiopyrad and its metabolites in actual samples

[0195]

[0196]

[0197]

[0198] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the invention.

Claims

1. A method for detecting penthiopyrad and its metabolite PAM in animal-derived food, characterized in that: The following steps are involved: S1. Extraction: Evenly mix the animal-derived food sample with the extraction solvent, then add a dehydrating agent and a buffer salt and mix evenly, and then perform solid-liquid separation to obtain an extract; S2, purification: take the extract of step S1 and mix it evenly with the purifier and the water removal agent, and then perform solid-liquid separation to obtain a purified liquid; S3, detection: using liquid chromatography-triple quadrupole tandem mass spectrometry to detect and analyze the purified liquid obtained in step S2; The animal-derived food is one or more of aquatic products, fat, liver, or casings; the purifier is a compound of C18 and Florisil in a mass ratio of 1:1; When the animal-derived food is aquatic products, the specific steps of extraction in S1 are: weigh the sample and place it in a centrifuge tube A, add 1% by mass acetic acid acetonitrile solution and place it in a homogenizer for homogenization, then take another centrifuge tube B and add 1% by mass acetic acid acetonitrile solution thereto, clean the blade of the homogenizer, then combine the washing liquid in the centrifuge tube B into the centrifuge tube A, mix well, then add the dehydrating agent anhydrous magnesium sulfate and the buffer salt sodium acetate, mix well, shake and extract, and then centrifuge to obtain the supernatant, which is the extract. When the animal-derived food is one or more of liver, fat or casing, the specific steps of extraction in S1 are: weighing the sample and placing it in a centrifuge tube, adding 1% by mass acetic acid acetonitrile solution, mixing evenly, then adding a dehydrating agent and a buffer salt, mixing evenly, oscillating and extracting, and then centrifuging to obtain a supernatant, which is the extract. When the animal-derived food is aquatic products or casings, the specific steps of purification in S2 are: 1.00 mL of the extract of step S1 is taken into a centrifuge tube containing 25-150 mg of a purifier and anhydrous magnesium sulfate as a dehydrating agent, shaken for 15-20 seconds, centrifuged at 11000-13000 r / min for 5-8 minutes, and the supernatant after centrifugation is passed through an organic filter membrane to obtain a purified solution. When the animal-derived food is fat, the specific steps of purification in S2 are as follows: the extract in step S1 needs to be frozen for 1.5-2 hours, and then 1.00 mL of the frozen extract is taken into a centrifuge tube containing 25-150 mg of a purifier and a dehydrating agent, shaken for 15-20 seconds, centrifuged at 11000-13000 r / min for 5-8 minutes, and the supernatant after centrifugation is passed through an organic filter membrane to obtain a purified solution. When the animal-derived food is liver, the specific steps of purification in S2 are: 1.00 mL of the extract of step S1 is taken into a centrifuge tube C containing 25-150 mg of a purifier and a dehydrating agent, shaken for 15-20 seconds, centrifuged at 11000-13000 r / min for 5-8 minutes, 0.5-1 ml of the supernatant after centrifugation is taken and placed in a test tube, 0.5-1 ml of deionized water is added to the test tube, and then the liquid in the test tube is filtered through an organic filter membrane to obtain a purified liquid. The detection conditions of the liquid chromatography-triple quadrupole tandem mass spectrometer are: The chromatographic conditions of the liquid chromatography were as follows: column: Accucore aQ 150 mm × 2.1 mm 2.6 μm, injection volume 2 μL, column temperature 40 °C, mobile phase A was methanol, mobile phase B was an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate for gradient elution; The mass spectrometry conditions were as follows: electrospray positive ion mode, multiple reaction monitoring mode acquisition, ion source temperature of 350 °C, and evaporation temperature of 300 °C.

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