Method for detecting imidazolone compounds in animal-derived food

Abstract

The invention provides a method for detecting imidazolone compounds in animal-derived food, and relates to the technical field of analysis and detection. The QuEChERS method is combined for extraction, the acetic acid-acetonitrile solution serves as an extracting agent, the sodium citrate dihydrate, the disodium citrate sesquihydrate, the anhydrous magnesium sulfate and the sodium chloride serve as QuEChERS extracting salt, and the extraction rate of imazamox, imazapic, imazapic, imazalic acid and imazethapyr is high. The extracting solution is purified by using a PPR phospholipid-removing solid-phase extraction small column, and the extracting solution of a high-fat food sample is frozen, so that the matrix effect is greatly reduced. High performance liquid chromatography-tandem mass spectrometry is adopted for detection, and the sensitivity of the detection method is high. The detection method provided by the invention is simple to operate and low in pretreatment cost.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting imidazolinone compounds in animal-derived foods. Background Art

[0002] Currently, the detection and analysis of imidazolinone pesticide residues primarily focuses on plant-derived matrices, soil, and water. Analysis of residues in animal-derived foods is relatively limited, primarily involving beef and milk, and the primary imidazolinone compound detected is imazapyr. Developing methods for the simultaneous analysis of multiple imidazolinone pesticides in animal-derived foods is of great significance. Summary of the Invention

[0003] In view of this, the present invention aims to provide a method for detecting imidazolinone compounds in animal-derived foods. The detection method provided by the present invention can achieve efficient simultaneous extraction of up to five imidazolinone compounds in animal-derived foods, and uses high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) for detection, with high sensitivity.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for detecting imidazolinone compounds in animal-derived foods, comprising the following steps:

[0006] The animal-derived food sample to be tested, an extractant, and a QuEChERS extraction salt are mixed and extracted to obtain an extract; the extractant is an acetic acid-acetonitrile solution; the QuEChERS extraction salt includes sodium citrate dihydrate, disodium citrate sesquihydrate, anhydrous magnesium sulfate, and sodium chloride;

[0007] The animal-derived food samples to be tested include high-fat food samples and non-high-fat food samples, and the high-fat food samples include one or more of meat and aquatic products;

[0008] When the animal-derived food sample to be tested is a high-fat food sample, the mixed extraction further comprises: performing solid-liquid separation on the extraction system obtained by the mixed extraction and then freezing the system, wherein the supernatant is the extraction solution;

[0009] Purifying the extract using a PPR phospholipid-free solid phase extraction cartridge to obtain a sample solution to be tested;

[0010] The sample solution to be tested is subjected to high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of the imidazolinone compound;

[0011] The imidazolinone compound includes at least two of methoxam, imazapic acid, imazapic acid, imidazoquinoline acid and imazethapyr;

[0012] The high performance liquid chromatography-tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection, the chromatographic column for the high performance liquid chromatography separation includes a C18 chromatographic column; the mobile phase A is ammonium formate and formic acid aqueous solution; the mobile phase B is methanol; the elution method is gradient elution, and the gradient elution procedure is as follows:

[0013] From 0 to 0.5 min, the volume fraction of mobile phase A is 0.5%; from 0.5 to 2 min, the volume fraction of mobile phase A increases at a uniform rate from 0.5% to 40%; from 2 to 20 min, the volume fraction of mobile phase A increases at a uniform rate from 40% to 95%; from 20 to 22 min, the volume fraction of mobile phase A is 95%; from 22 to 22.1 min, the volume fraction of mobile phase A decreases at a uniform rate from 95% to 5%; from 22.1 to 27 min, the volume fraction of mobile phase A is 0.5%.

[0014] Preferably, the non-high-fat food samples include offal, eggs, milk and sausage casings.

[0015] Preferably, the volume fraction of acetic acid in the acetic acid-acetonitrile solution is 1%.

[0016] Preferably, the solid-liquid ratio of the animal-derived food sample to be tested and the extractant is 1 g: 2-4 mL.

[0017] Preferably, the mass ratio of the sodium citrate dihydrate to the disodium citrate sesquihydrate is 1:0.45-0.55;

[0018] The mass ratio of the animal-derived food sample to be tested to sodium citrate dihydrate is 1:0.15-0.25.

[0019] Preferably, the mass ratio of the animal-derived food sample to be tested to anhydrous magnesium sulfate is 1:0.75-0.85;

[0020] The mass ratio of the animal-derived food sample to be tested to sodium chloride is 1:0.15-0.25.

[0021] Preferably, the mixed extraction comprises: performing a first mixed extraction on the animal-derived food sample to be tested and the extractant, and then adding QuEChERS extraction salt to perform a second mixed extraction;

[0022] The first mixed extraction includes vortex extraction, wherein the temperature of the vortex extraction is 18 to 25° C., the rotation speed is 2000 to 3000 r / min, and the time is 15 to 25 s;

[0023] When the food sample to be tested is a high-fat food sample, the vortex extraction further includes a homogenization extraction, and the homogenization extraction temperature is 18-25° C., the rotation speed is 10000-13500 r / min, and the time is 20-30 s;

[0024] The second mixed extraction includes shaking extraction, and the shaking extraction temperature is 18-25° C., the rotation speed is 300-400 r / min, and the time is 8-12 min.

[0025] Preferably, the freezing treatment is carried out at a temperature of -16 to -20°C and for a time of 2 to 3 hours.

[0026] Preferably, the concentration of ammonium formate in the ammonium formate aqueous solution is 5 mmol / L, and the concentration of formic acid is 0.1 v / v%;

[0027] The column temperature of the HPLC separation was 40° C., the mobile phase flow rate was 0.3 mL / min, and the injection volume was 2 μL.

[0028] Preferably, the mass spectrometry detection conditions include: the ion source mode is H-ESI; the scanning mode is positive ion scanning, multiple reaction monitoring mode; the sheath gas is nitrogen, the value is 50Arb; the auxiliary gas is nitrogen, the value is 20Arb; the sweep gas is nitrogen, the value is 1Arb; the spray voltage is 1kV; the vaporization chamber temperature is 415°C; the ion transfer tube temperature is 300°C; the parent ion of the imazapic acid is 276, the daughter ions are 163 and 216, and the collision energies are 27V and 26V respectively; the imazapic acid The parent ion of imipenem is 262, the daughter ions are 217 and 220, and the collision energies are 20V and 18V, respectively; the parent ion of imidazoquinoline is 312, the daughter ions are 252 and 266, and the collision energies are 21V and 32V, respectively; the parent ion of imidazoethylnicotinic acid is 290, the daughter ions are 177 and 248, and the collision energies are 31V and 21V, respectively; the parent ion of methoxazole is 306, the daughter ions are 69 and 86, and the collision energies are 28V and 28V, respectively.

[0029] The present invention combines the QuEChERS method for extraction, wherein the extractant is an acetic acid-acetonitrile solution, and the QuEChERS extraction salts include sodium citrate dihydrate, disodium citrate sesquihydrate, anhydrous magnesium sulfate, and sodium chloride. This method can extract at least two of imazamox, imazapic, imazapyr, imazaquin, and imazethapyr in animal-derived foods. The sodium citrate dihydrate and disodium citrate sesquihydrate together form a buffer system that stabilizes the pH value of the animal-derived food sample matrix within a weakly acidic range, thereby preventing the decomposition of some acid-sensitive and alkaline-sensitive pesticides. The weakly acidic environment can also inhibit the activity and solubility of certain matrix components (such as enzymes and pigments) in the sample, reducing interference with subsequent analysis. The present invention uses a combination of the two citrates mentioned above, which not only ensures the stability of the buffering capacity but also avoids the pH fluctuations that may be caused by a single salt. It is particularly suitable for the analysis of imidazolinone compounds in animal-derived food samples with high fat content. The present invention utilizes the dehydration and salting-out effects of anhydrous magnesium sulfate, as well as its heat release property when in contact with water, to achieve a small temperature increase in the extraction system, significantly improving the extraction rate of imidazolinone compounds and reducing matrix interference. The present invention promotes the separation of acetonitrile and water by adding sodium chloride, thereby improving the extraction rate of imidazolinone compounds and reducing matrix interference. The extraction method adopted by the present invention has a high extraction rate of imidazolinone compounds and a low matrix effect. The present invention uses a PPR phospholipid-free solid-phase extraction cartridge to purify the extract, which can achieve efficient removal of phospholipid impurities and greatly reduce the matrix effect. Moreover, the present invention freezes the extract of high-fat food samples, which can cause free saturated fat impurities to solidify and precipitate under freezing conditions, thereby reducing the fat content in the supernatant and more effectively removing fat interference in the extract, achieving a good purification effect and improving the sensitivity of the detection method. The present invention adopts the above steps to perform sample pretreatment, which can improve the sensitivity of the detection method, is simple to operate, and has low sample pretreatment cost.

[0030] The present invention adopts a C18 chromatographic column and uses ammonium formate formic acid aqueous solution-methanol as the mobile phase system, which can effectively inhibit the dissociation of the sample liquid to be tested, increase the retention time and response value of the imidazolinone compounds in the stationary phase, and improve the separation degree of each imidazolinone compound. The detection method provided by the present invention can achieve efficient, accurate and highly sensitive detection of various imidazolinone compounds in animal-derived foods. Moreover, the detection method provided by the present invention is applicable to various animal-derived matrices and is suitable for large-scale detection. The methodological validation results show that the imidazolinone compounds have a good linear relationship in the linear concentration range of 1 to 100 μg / L (correlation coefficient r 2The spiking test was performed in different animal-derived foods at three concentration levels (0.005 mg / kg, 0.010 mg / kg, and 0.100 mg / kg). The recoveries ranged from 67.8 to 120.1%, the relative standard deviations ranged from 1.0 to 9.9%, the limits of detection ranged from 0.045 to 0.332 μg / kg, and the limit of quantification ranged from 5 μg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the effects of different extraction methods on the extraction of imazapyr in beef samples in Example 1;

[0032] Figure 2 This is a graph showing the mass change of soluble matter in the beef sample extract before and after freezing in Example 1;

[0033] Figure 3 Graph showing the change in signal-to-noise ratio of imidazoquinolinic acid in the extract before and after freezing in Example 1;

[0034] Figure 4 This is a diagram showing the effect of different dispersed solid phase extraction purifications on the determination of imazapyr in Example 1;

[0035] Figure 5 This is a graph showing the effect of different solid phase extraction cleanup cartridges on the determination of imidazolinone pesticides in Example 1;

[0036] Figure 6 This is a diagram showing the matrix effect results of five imidazolinone compounds in Example 2;

[0037] Figure 7 This is a peak height ratio graph of the mixed standard solution in the ammonium formate aqueous solution (5 mmol / L ammonium formate, 0.1 v / v% formic acid)-methanol mobile phase system in Example 3;

[0038] Figure 8 is the standard curve of imazapyr in Example 4;

[0039] Figure 9 is the standard curve of imazamox in Example 4;

[0040] Figure 10 is the standard curve of imazapic in Example 4;

[0041] Figure 11 is the standard curve of imazethapyr in Example 4;

[0042] Figure 12 It is the standard curve of imidazoquinolinic acid in Example 4. DETAILED DESCRIPTION

[0043] The present invention provides a method for detecting imidazolinone compounds in animal-derived foods, comprising the following steps:

[0044] The animal-derived food sample to be tested, an extractant, and a QuEChERS extraction salt are mixed and extracted to obtain an extract; the extractant is an acetic acid-acetonitrile solution; the QuEChERS extraction salt includes sodium citrate dihydrate, disodium citrate sesquihydrate, anhydrous magnesium sulfate, and sodium chloride;

[0045] The animal-derived food samples to be tested include high-fat food samples and non-high-fat food samples, and the high-fat food samples include one or more of meat and aquatic products;

[0046] When the animal-derived food sample to be tested is a high-fat food sample, the mixed extraction further comprises: performing solid-liquid separation on the extraction system obtained by the mixed extraction and then freezing the system, wherein the supernatant is the extraction solution;

[0047] Purifying the extract using a PPR phospholipid-free solid phase extraction cartridge to obtain a sample solution to be tested;

[0048] The sample solution to be tested is subjected to high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of the imidazolinone compound;

[0049] The imidazolinone compound includes at least two of methoxam, imazapic acid, imazapic acid, imidazoquinoline acid and imazethapyr;

[0050] The high performance liquid chromatography-tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection, the chromatographic column for the high performance liquid chromatography separation includes a C18 chromatographic column; the mobile phase A is ammonium formate and formic acid aqueous solution; the mobile phase B is methanol; the elution method is gradient elution, and the gradient elution procedure is as follows:

[0051] From 0 to 0.5 min, the volume fraction of mobile phase A is 0.5%; from 0.5 to 2 min, the volume fraction of mobile phase A increases at a uniform rate from 0.5% to 40%; from 2 to 20 min, the volume fraction of mobile phase A increases at a uniform rate from 40% to 95%; from 20 to 22 min, the volume fraction of mobile phase A is 95%; from 22 to 22.1 min, the volume fraction of mobile phase A decreases at a uniform rate from 95% to 5%; from 22.1 to 27 min, the volume fraction of mobile phase A is 0.5%.

[0052] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0053] The present invention performs mixed extraction on a to-be-tested animal-derived food sample, an extractant and a QuEChERS extraction salt to obtain an extract.

[0054] In the present invention, the animal-derived food samples to be tested include high-fat food samples and non-high-fat food samples. The high-fat food samples include one or more of meat and aquatic products. The meat preferably includes red meat and / or white meat, and in specific embodiments, may be one or more of beef, pork, lamb, chicken, and duck. The aquatic products preferably include one or more of fish, shrimp, crab, and shellfish. In the present invention, the non-high-fat food samples preferably include one or more of offal, eggs, milk, and casings. The offal preferably includes liver and / or kidney, and the milk preferably includes raw milk and / or cow's milk.

[0055] In the present invention, when the animal-derived food samples to be tested are high-fat food samples, offal and eggs, the animal-derived food samples to be tested are preferably pretreated before use, and the pretreatment preferably includes: crushing the edible part of the animal-derived food samples to be tested and mixing them evenly; the crushing is preferably performed using a wall breaker. In the present invention, the casings are preferably pretreated before use, and the pretreatment preferably includes, in sequence: washing the casings, cutting them to a length of less than 5 mm and drying them until no water drips. In the present invention, the storage temperature of the pretreated animal-derived food samples to be tested is preferably -18°C, and they are preferably stored in polyethylene containers, and the polyethylene containers preferably include polyethylene bottles or polyethylene bags.

[0056] In the present invention, the extractant is an acetic acid-acetonitrile solution, and the volume fraction of acetic acid in the acetic acid-acetonitrile solution is preferably 1%.

[0057] In the present invention, the solid-liquid ratio of the animal-derived food sample to be tested and the extractant is preferably 1g:2-4mL, and in specific embodiments it can be 1g:2mL, 1g:2.5mL, 1g:3mL, 1g:3.5mL or 1g:4mL.

[0058] In the present invention, the QuEChERS extraction salts include sodium citrate dihydrate, disodium citrate sesquihydrate, anhydrous magnesium sulfate, and sodium chloride. In the present invention, the mass ratio of sodium citrate dihydrate to disodium citrate sesquihydrate is preferably 1:0.45 to 0.55, and in specific embodiments, it can be 1:0.45, 1:0.48, 1:0.5, 1:0.52, or 1:0.55. In the present invention, the mass ratio of the animal-derived food sample to be tested to sodium citrate dihydrate is preferably 1:0.15 to 0.25, and in specific embodiments, it can be 1:0.15, 1:0.18, 1:0.2, 1:0.22, or 1:0.25. In the present invention, the mass ratio of the animal-derived food sample to be tested to anhydrous magnesium sulfate is preferably 1:0.75-0.85, and in specific embodiments, it can be 1:0.75, 1:0.78, 1:0.8, 1:0.82, or 1:0.85. In the present invention, the mass ratio of the animal-derived food sample to be tested to sodium chloride is preferably 1:0.15-0.25, and in specific embodiments, it can be 1:0.15, 1:0.18, 1:0.2, 1:0.22, or 1:0.25.

[0059] In the QuEChERS extraction salt used in the present invention, sodium citrate dihydrate and disodium citrate sesquihydrate together constitute a buffer system, which stabilizes the pH value of the animal-derived food sample matrix within the weak acid range (pH = 5 to 5.5), and can avoid the decomposition of some acid-sensitive pesticides and alkaline-sensitive pesticides. At the same time, the weakly acidic environment can also inhibit the activity and solubility of certain matrix components (such as enzymes and pigments) in the sample, reducing interference with subsequent analysis. The present invention adopts a combination of the above two citrates, which not only ensures the stability of the buffering capacity, but also avoids the pH fluctuations that may be caused by a single salt. It is particularly suitable for the qualitative and quantitative analysis of imidazolinone compounds in animal-derived food samples with high fat content. The present invention utilizes the dehydration and salting-out effect of anhydrous magnesium sulfate, as well as the heat release property of water, to achieve a small temperature rise in the extraction system, significantly improving the extraction rate of imidazolinone compounds and reducing matrix interference. The present invention promotes acetonitrile and water demixing by adding sodium chloride, thereby improving the extraction rate of imidazolinone compounds and reducing matrix interference.

[0060] In the present invention, the mixed extraction preferably includes: performing a first mixed extraction on the animal-derived food sample to be tested and the extractant, and then adding QuEChERS extraction salt to perform a second mixed extraction. In the present invention, the temperature of the mixed extraction is preferably 18 to 25°C, and in specific embodiments, it can be 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C. In the present invention, the first mixed extraction preferably includes vortex extraction, and the rotation speed of the vortex extraction is preferably 2000 to 3000 r / min, and in specific embodiments, it can be 2000 r / min, 2200 r / min, 2500 r / min, 2800 r / min or 3000 r / min; the vortex extraction time is preferably 15 to 25 s, and in specific embodiments, it can be 15 s, 18 s, 20 s, 22 s or 25 s.

[0061] In the present invention, when the food sample to be tested is a high-fat food sample, the vortex extraction preferably also includes homogenization extraction. The temperature of the homogenization extraction is preferably 18-25°C, and in specific embodiments it can be 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C; the rotation speed of the homogenization extraction is preferably 10000-13500 r / min, and in specific embodiments it can be 10000 r / min, 11000 r / min, 12000 r / min, 13000 r / min or 13500 r / min; the time of the homogenization extraction is preferably 20-30s, and in specific embodiments it can be 20s, 22s, 25s, 28s or 30s.

[0062] In the present invention, the second mixed extraction preferably includes shaking extraction, and the rotation speed of the shaking extraction is preferably 300-400 r / min, and in specific embodiments it can be 300 r / min, 320 r / min, 350 r / min, 380 r / min or 400 r / min; the time of the shaking extraction is preferably 8-12 min, and in specific embodiments it can be 8 min, 9 min, 10 min, 11 min or 12 min; the shaking extraction is preferably carried out on a horizontal reciprocating shaker.

[0063] When the animal-derived food sample to be tested is a non-high-fat food sample, after the mixed extraction, the present invention preferably further comprises: subjecting the extraction system obtained by the mixed extraction to solid-liquid separation, and the resulting liquid component is the extract. In the present invention, the solid-liquid separation preferably comprises centrifugal separation, and the rotation speed of the centrifugal separation is preferably 3000-5000 r / min, and in specific embodiments, it can be 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, or 5000 r / min; the time of the centrifugal separation is preferably 4-10 minutes, and in specific embodiments, it can be 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0064] In the present invention, when the animal-derived food sample to be tested is a high-fat food sample, the mixed extraction further comprises: subjecting the extract system obtained by the mixed extraction to solid-liquid separation and then freezing treatment, with the supernatant being the extract. In the present invention, the solid-liquid separation preferably includes centrifugation, and the speed of the centrifugation is preferably 3000-5000 r / min, and in specific embodiments, it can be 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, or 5000 r / min; the time of the centrifugation is preferably 4-10 minutes, and in specific embodiments, it can be 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. In the present invention, the temperature of the freezing treatment is preferably -16--20°C, and in specific embodiments, it can be -16°C, -17°C, -18°C, -19°C, or -20°C; the time of the freezing treatment is preferably 2-3 hours, and in specific embodiments, it can be 2 hours, 2.5 hours, or 3 hours. The present invention freezes the extract of high-fat food samples, which can cause free saturated fat impurities to solidify and precipitate under freezing conditions, thereby reducing the fat content in the extract and achieving a purification effect; however, the free saturated fat content in non-high-fat food samples is low, and the freezing treatment effect is not obvious.

[0065] After obtaining the extract, the present invention uses a PPR phospholipid-free solid phase extraction column to purify the extract to obtain a sample solution to be tested.

[0066] In the present invention, the PPR phospholipid removal solid phase extraction column preferably includes a CNW phospholipid removal PPR solid phase extraction column. The solid phase in the CNW phospholipid removal PPR solid phase extraction column has divinylbenzene, pyrrolidone and silanol groups, which are suitable for complex matrix processing containing a large amount of phospholipid interferents, and can achieve the effect of removing phospholipids. Phospholipids are components of protein and fat. Removing phospholipids can remove protein and fat accordingly, thereby improving the purification effect. Before using other columns, it is necessary to pre-leach and balance the column (discard the pre-leach solution), and after the column is mounted, it is necessary to rinse and collect the effluent. The present invention uses the CNW phospholipid removal PPR solid phase extraction column for purification. There is no need to pre-leach the column first. The effluent (i.e., the sample liquid to be tested) is directly collected after the column is mounted, and there is no need to wash the column again, so the purification operation is simpler.

[0067] After the purification is completed, the present invention preferably further comprises: mixing the purified liquid on a vortex mixer and then passing it through an organic phase microporous filter membrane to obtain a sample liquid to be tested. In the present invention, the pore size of the organic phase microporous filter membrane is preferably 0.22 μm.

[0068] After obtaining the sample liquid to be tested, the present invention performs high performance liquid chromatography-tandem mass spectrometry detection on the sample liquid to be tested to obtain the detection result of the imidazolinone compound.

[0069] In the present invention, the imidazolinone compounds include at least two of methoxazole, imazapyr, imazapyr, imidazoquinoline and imidazolin, specifically two, three, four or five of methoxazole, imazapyr, imazapyr, imidazoquinoline and imidazolin.

[0070] In the present invention, the HPLC-MS / MS detection includes HPLC separation and mass spectrometry detection. The HPLC separation conditions in the present invention include: a chromatographic column including a C18 column, which in a specific embodiment can be an Accucore aQ column; a column temperature preferably of 40°C; mobile phase A comprising an ammonium formate aqueous solution, wherein the concentration of ammonium formate in the aqueous solution is preferably 5 mmol / L, and the concentration of formic acid is preferably 0.1 v / v%; mobile phase B comprising methanol; a mobile phase flow rate preferably of 0.3 mL / min; an injection volume preferably of 2 μL; and a gradient elution method, the gradient elution program of which is shown in Table 1.

[0071] Table 1 Gradient elution program

[0072] Time / min Volume fraction of mobile phase A / % Volume fraction of mobile phase B / % 0 5.0 95.0 0.5 5.0 95.0 2.0 40.0 60.0 20.0 95.0 5.0 22.0 95.0 5.0 22.1 5.0 95.0 27.0 5.0 95.0

[0073] In the present invention, the conditions for mass spectrometry detection preferably include: the ion source mode is H-ESI; the scanning mode is positive ion scanning, multiple reaction monitoring mode; the sheath gas is nitrogen, the pressure is 50Arb; the auxiliary gas is nitrogen, the pressure is 20Arb; the sweep gas is nitrogen, the pressure is 1Arb; the spray voltage is 1kV; the vaporization chamber temperature is 415°C; the ion transfer tube temperature is 300°C; the retention time and mass spectrometry parameters of the imidazolinone compounds are shown in Table 2.

[0074] Table 2 Retention time and mass spectrometry parameters of imidazolinone compounds

[0075] Compound Retention time / min Precursor ion / (m / z) Product ion / (m / z) Collision energy / V Imidaclan 5.40 276 163* 27 Imidaclan 5.40 276 216 26 Imidacolin 4.64 262 217* 20 Imidacolin 4.64 262 220 18 imidazoquinolinic acid 7.13 312 252* 21 imidazoquinolinic acid 7.13 312 266 32 Imidacolin 6.30 290 177* 31 Imidacolin 6.30 290 248* 21 Imazamox 5.20 306 69* 28 Imazamox 5.20 306 86 28

[0076] In Table 2, * represents the quantitative ion.

[0077] In the present invention, the test results preferably include qualitative test results and / or quantitative test results. In the present invention, the method for obtaining the quantitative test results is preferably to obtain them by quantitatively using a standard curve, and the standard curve preferably includes a solvent standard curve or a matrix standard curve, and the standard curve uses the mass concentration of the imidazolinone compound as the independent variable and the corresponding peak area as the dependent variable. In the present invention, when the animal-derived food sample to be tested is an egg and / or a casing, it is preferably quantified by a solvent standard curve method, and when the animal-derived food sample to be tested is a sample of other animal-derived food, it is preferably quantified by a matrix standard curve method.

[0078] In the present invention, the method for drawing the solvent standard curve preferably includes the following steps: preparing a series of solvent mixed standard solutions, performing high performance liquid chromatography-tandem mass spectrometry detection on the series of solvent mixed standard solutions according to the conditions of the aforementioned high performance liquid chromatography-tandem mass spectrometry detection, and drawing the solvent standard curve with the mass concentration of the imidazolinone compound as the independent variable and the corresponding peak area as the dependent variable. In the present invention, the concentrations of the single imidazolinone compound standard in the series of solvent mixed standard solutions are 0 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L and 100 μg / L, respectively; the solvent in the series of solvent mixed standard solutions is preferably one or more of methanol, acetonitrile and acetone; the standards in the series of mixed standard solutions are imazamox standard, imazapic acid standard, imazapic acid standard, imidazoquinoline standard and imazethapyr standard.

[0079] In the present invention, the method for drawing the matrix standard curve preferably includes the following steps: performing sample pretreatment on a blank animal-derived food sample according to the preparation method of the sample solution to be tested to obtain a blank matrix solution; using the blank matrix solution to dilute the mixed standard solution to obtain a series of matrix standard solutions; performing high-performance liquid chromatography-tandem mass spectrometry detection on the series of matrix standard solutions according to the conditions of the aforementioned high-performance liquid chromatography-tandem mass spectrometry detection, and drawing a matrix standard curve with the mass concentration of the imidazolinone compound as the independent variable and the corresponding peak area as the dependent variable. In the present invention, the standards in the mixed standard solution are methoxazole standard, imazapyr standard, imazapyr standard, imidazoquinoline standard, and imidazopyr standard; the concentration of a single imidazolinone compound standard in the mixed standard solution is preferably 1 mg / L; the solvent in the mixed standard solution is preferably one or more of methanol, acetonitrile, and acetone. In the present invention, the concentrations of the single imidazolinone compound standard in the series of matrix standard solutions are preferably 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L and 100 μg / L, respectively.

[0080] In the present invention, when a chromatographic peak with a retention time consistent with that of an imidazolinone compound in a certain solvent mixed standard solution or a matrix standard solution is detected in the test sample solution (within a variation range of ±2.5%), and the deviation between the relative abundance ratio of the monitoring ion pair selected in the chromatogram of the test sample solution and the relative abundance ratio (k) of the ion of the standard solution of equivalent concentration does not exceed the range specified in Table 3, it can be determined that the corresponding compound is detected in the sample.

[0081] Table 3 Maximum allowable deviation of relative ion abundance in qualitative analysis

[0082] Relative ion abundance / % >50 20 to 50 and not 20 10 to 20 and not 10 ≤10 Allowable relative deviation / % ±20 ±25 ±30 ±50

[0083] In order to further illustrate the present invention, the detection method of imidazolinone compounds in animal-derived foods is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0084] In the following examples,

[0085] 1.1 Instruments and Reagents

[0086] Thermo TSQ Quantiva high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer; Lultra Turrax homogenizer; MS303TS / 02 electronic balance (d = 1 mg); SIGMA3-16P centrifuge; Heidolph Promax-2020 horizontal reciprocating shaker; IKAMS3 vortex mixer; and Milli-Q deionized water generator.

[0087] Methoxazolidinone standard solution (in acetonitrile), imazapicl standard solution (in acetonitrile), imazapicl standard solution (in acetonitrile), imazapicl standard solution (in acetone), imazapicl standard solution (in methanol) were all purchased from BePure at a concentration of 1000 mg / L. Acetonitrile, methanol, formic acid, acetic acid, and ammonium formate were all chromatographically grade. QuEChERS extraction salts (4.0 g anhydrous magnesium sulfate, 1.0 g sodium chloride, 1.0 g sodium citrate dihydrate, 0.5 g disodium citrate sesquihydrate) were used; a CNW dephospholipidated PPR solid-phase extraction cartridge (200 mg, 3 mL) was used; and a 0.22 μm organic filter membrane was used.

[0088] Animal-derived food samples: beef, chicken, tilapia, chicken liver, milk, eggs, and sausage casings were purchased from the market.

[0089] 1.2 Instrument working conditions

[0090] 1.2.1 HPLC conditions

[0091] The chromatographic column was an Accucore aQ column (150×2.1 mm, 2.6 μm); the column temperature was 40°C, and the injection volume was 2 μL; the mobile phase A was ammonium formate in aqueous solution (5 mmol / L ammonium formate, 0.1 v / v% formic acid), and the mobile phase B was methanol; the mobile phase flow rate was 0.30 mL / min; the elution mode was gradient elution, and the gradient elution program is shown in Table 1.

[0092] 1.2.2 Mass spectrometry conditions

[0093] Mass spectrometry analysis used H-ESI as the ion source; the scanning mode was positive ion scanning in multiple reaction monitoring mode; the sheath gas was nitrogen at a pressure of 50 Arb; the auxiliary gas was nitrogen at a pressure of 20 Arb; the sweep gas was nitrogen at a pressure of 1 Arb; and the spray voltage was 1.0 kV. The vaporizer temperature was set to 415°C, and the ion transfer tube temperature was 300°C. The retention times and mass spectrometric parameters for the imidazolinone compounds are shown in Table 2.

[0094] Example 1

[0095] 1. Sample pretreatment method and optimization

[0096] 1.1 Extraction and purification

[0097] 1.1.1 Sample preparation

[0098] Extraction solvent: 1 v / v% acetic acid-acetonitrile.

[0099] Meat and aquatic product sample preparation: Take 500g of the edible portion of the sample, crush it with a wall-breaking machine, mix it thoroughly, place it in a polyethylene bottle or bag, and store it at -18°C or above. Remove it and return it to room temperature before testing. Casing sample preparation: Randomly sample 500g, wash it with clean water, cut it to a length of less than 5mm, place it on a 200-mesh sieve to dry until no obvious water droplets appear, mix it thoroughly, place it in a polyethylene container, store it at -18°C or above, and return it to room temperature before testing.

[0100] Egg sample preparation: Randomly sample 500g, peel the whole egg, put it into a wall-breaking machine and mix it evenly, put it into a polyethylene bottle or bag, and store it at -18℃ or above. Take it out and return it to room temperature before testing.

[0101] Preparation of visceral samples: Randomly sample 500 g, crush with a wall-breaking machine, mix thoroughly, put into a polyethylene container, store at -18 ° C and store under the condition of 100 ° C. Take out and restore to room temperature before testing.

[0102] Raw milk: Use directly.

[0103] Extraction and cleanup of high-fat food samples (meat and aquatic products): 5.00 g of sample (accurate to 0.01 g) was weighed and placed in a 50 mL centrifuge tube. 10 mL of extraction solvent was added and homogenized in a homogenizer at 25°C and 12,000 rpm for 25 s. 10 mL of extraction solvent was then taken to clean the residue on the homogenizer blade. The combined extraction solutions were placed on a vortex mixer and vortex-extracted at 25°C and 2,500 rpm for 20 s. QuEChERS extraction salt was added and the samples were quickly shaken manually. The samples were then placed on a horizontal reciprocating shaker and extracted at 25°C and 350 rpm for 10 min. The samples were centrifuged at 4,000 rpm for 5 min and frozen at -18°C for 2 h. 2 mL of the supernatant was then transferred to a PPR phospholipid removal column for cleanup and elution. The eluate was collected, mixed on a vortex mixer, and passed through a 0.22 μm organic phase microporous filter membrane before being placed in an injection vial to obtain the sample solution to be tested.

[0104] Extraction and purification of non-high-fat food samples (casings, eggs, offal, raw milk): Weigh 5.00 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube. Add 10 mL of extractant and place it on a vortex mixer. Vortex extract at 25°C and 2500 r / min for 20 seconds. Add QuEChERS extraction salt and shake manually quickly to mix. Place it on a horizontal reciprocating shaker and extract at 25°C and 350 r / min for 10 minutes. Centrifuge at 4000 r / min for 5 minutes. Pipette 2 mL of supernatant and transfer it to a PPR phospholipid removal column for purification and elution. Collect the eluate, mix it on a vortex mixer, pass it through a 0.22 μm organic phase microporous filter membrane, and place it into a sample injection bottle for determination.

[0105] 1.1.2 Selection of extraction method

[0106] The QuEChERS extraction of imidazopyrin from beef was used as an example to optimize the extraction method. A 5.00 g beef sample (accurate to 0.01 g) was weighed into a 50 mL centrifuge tube. 10 mL of extractant was added and homogenized in a homogenizer at 25°C and 12,000 rpm for 25 seconds. Another 10 mL of extractant was then used to clean the residue from the homogenizer blade. The combined sample was vortexed and extracted for 20 seconds at 25°C and 2,500 rpm on a vortex mixer. QuEChERS extraction salts were added, manually shaken, and then placed on a horizontal reciprocating shaker. Extraction was performed at 25°C and 350 rpm for 10 minutes. The sample was centrifuged at 4,000 rpm for 5 minutes and then frozen at -18°C for 2 hours. The supernatant was used as the extract. The extractants and QuEChERS extraction salts used are shown in Table 4.

[0107] Table 4 Extractants and QuEChERS extraction salts

[0108]

[0109] By comparing the recovery results of the determination of imidazopyrine residues in beef, the experimental results are as follows Figure 1 As shown, the extraction method with the best recovery rate was finally determined: 1 v / v% acetic acid-acetonitrile was used as the extraction solvent, and 4.0 g anhydrous magnesium sulfate + 1.0 g sodium chloride + 1.0 g sodium citrate dihydrate + 0.5 g disodium citric acid sesquihydrate was used as the QuEChERS extraction salt.

[0110] 1.2 Selection of purification method

[0111] 1.2.1 Freezing

[0112] Meat matrix has a high fat content. Although most of the fat has been removed by extraction with 1v / v% acetic acid-acetonitrile, some of it will still enter the extract and affect the chromatographic performance. To further remove the fat, the extract was frozen at -18℃ for 6 hours. A fixed volume of extract was taken out every 0.5 hours, dried with nitrogen, and weighed. The weight of the soluble matter in the extract was recorded. After the freezing time was longer than 2 hours, there was no significant change in the percentage reduction of the mass content of the soluble matter in the extract compared to the mass content of the soluble matter at 0 hours of freezing (the mass content of the soluble matter was recorded as 100%). Figure 2 .

[0113] Experimental Group: Blank beef was pre-treated according to the extraction and cleanup procedures for medium- and high-fat food samples in 1.1 to obtain a frozen blank matrix solution. Control Group: The only difference from the experimental group was that the post-extraction freezing step was omitted, resulting in an unfrozen blank matrix solution. The imidazoquinolinic acid standard solution was diluted with both the frozen and unfrozen blank matrix solutions to obtain a 100 μg / L imidazoquinolinic acid blank matrix solution.

[0114] After comparison, it was found that the signal to noise ratio (S / N) of the imidazoquinolinic acid blank matrix solution prepared with the frozen blank matrix solution was higher than that of the imidazoquinolinic acid blank matrix solution prepared with the unfrozen blank matrix solution (such as Figure 3 This indicates that freezing at -18°C for 2 h can more effectively remove fat interference.

[0115] 1.2.2 Dispersive solid phase extraction cleanup and solid phase extraction cartridge cleanup

[0116] The extract obtained by the optimal extraction method is purified, and the eluate is collected, mixed on a vortex mixer, and then passed through a 0.22 μm organic phase microporous filter membrane to obtain a sample solution to be tested.

[0117] The two commonly used purification methods were compared: QuEChERS dispersed solid phase extraction (DSPE) and solid phase extraction (SPE).

[0118] The QuEChERS dispersive solid phase extraction method is simple to operate. The adsorption effects of five cleansing agents, graphitized carbon black (GCB), silica-bonded carbon 18 (C18), N-propylethylenediamine (primary secondary amine, PSA), neutral alumina and florisil on imidazopyr in beef were investigated. The results are shown in Figure 4 .Depend on Figure 4 The results showed that the five purifiers all had varying degrees of adsorption on imidazopyr, which may lead to low recovery rates in actual sample testing and fail to meet testing requirements.

[0119] The solid phase extraction cartridge has the advantages of good stability and good purification effect. The effects of five solid phase extraction cartridges commonly used for meat purification on the recovery rate of imidazopyrine in beef were investigated. The results are as follows: Figure 5 The types and specifications of the solid phase extraction cartridges are shown in Table 5. Figure 5The results showed that the purification effects of phospholipid-free PPR column, Florisil column and C18 column had a recovery rate of 60-120%.

[0120] Table 5 Solid Phase Extraction Cartridges

[0121] Serial number Solid phase extraction cartridge types and specifications 1 CNW dephospholipid PPR (200 mg, 3 mL) 2 PSA (200 mg, 3 mL) 3 Florisil (200 mg, 3 mL) 4 Neutral alumina (200 mg, 3 mL) 5 C18 (200 mg, 3 mL)

[0122] Further comparison of the purification effects of CNW phospholipid-free PPR solid phase extraction cartridge, Florisil cartridge and C18 cartridge was conducted. By comparing the activation balance and elution methods of different cartridges, it was found that CNW phospholipid-free PPR solid phase extraction cartridge not only effectively removed phospholipid impurities, but also only required the liquid to be purified to be directly loaded onto the cartridge for purification and collection. The operation was simple and convenient, saving reagent and time costs. At the same time, the recovery rate also met the requirements. Therefore, CNW phospholipid-free PPR solid phase extraction cartridge was selected for sample purification.

[0123] Example 2

[0124] Matrix effects

[0125] Matrix effect (ME) refers to the interference of the matrix on the analysis process and its influence on the analysis results.

[0126] 1.1 Drawing of solvent standard curve

[0127] Prepare a mixed standard solution of five imidazolinone standard solutions, which are then diluted with acetonitrile to create a series of solvent mixed standard solutions with individual imidazolinone concentrations of 0, 1 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L, respectively. Draw a solvent standard curve using the mass concentration of the imidazolinone compound as the abscissa and the corresponding peak area as the ordinate.

[0128] 1.2 Drawing of matrix standard curve

[0129] Blank samples were extracted and cleaned using the optimal pretreatment method. A blank matrix solution was prepared. Five imidazolinone mixed standard solutions were diluted with the blank matrix solution to prepare a series of matrix standard solutions with individual imidazolinone compounds at concentrations of 0, 1 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L, respectively. A matrix standard curve was plotted with the mass concentration of the imidazolinone compound as the abscissa and the corresponding peak area as the ordinate. The blank samples included egg, chicken liver, sausage casing, tilapia, milk, beef, and chicken.

[0130] 1.3 Calculation of matrix effects

[0131] The matrix effect is calculated as ME (%) = (A1-A2) / A2 × 100%, where A1 is the slope of the matrix standard curve and A2 is the slope of the solvent standard curve. When ME < 0, it is a matrix inhibition effect; when ME > 0, it is a matrix enhancement effect. When |ME| < 20% (i.e., the absolute value of ME < 20%), the matrix effect has a weak interference on the signal and can be ignored. The ME results of the 7 samples are shown in Table Figure 6 It can be seen that the MEs of the five imidazolinone compounds in eggs and casings are all within ±20%, so the solvent standard curve can be used for external standard method quantification. The other matrices (chicken liver, casings, tilapia, milk, beef and chicken) all have obvious matrix effects and are quantified using the matrix standard curve.

[0132] Example 3

[0133] 2.1 Selection and optimization of instrument conditions

[0134] Accurately weigh 0.1 mL each of 1000 mg / L imazamox standard solution, imazapic acid standard solution, imazapic acid standard solution, imidazoquinoline standard solution, and imazethapyr standard solution into the same volumetric flask, and dilute to 1 mL with acetonitrile to obtain a mixed standard solution with a concentration of 100 μg / mL.

[0135] Using the gradient elution program shown in Table 1, the separation effects of five imidazolinone compounds in a mixed standard solution were compared under two mobile phase systems (mobile phase A-mobile phase B, specifically 1 v / v% formic acid aqueous solution-methanol and ammonium formate formic acid aqueous solution-methanol, wherein the ammonium formate concentration in the ammonium formate formic acid aqueous solution was 5 mmol / L and the formic acid concentration was 0.1 v / v%). Figure 7 The peak height ratio diagram of the mixed standard solution in the ammonium formate / formic acid aqueous solution-methanol mobile phase system is shown in Figure 2. The results show that the peak shapes of the five imidazolinone compounds are symmetrical, with appropriate retention times and good resolution in the ammonium formate / formic acid aqueous solution-methanol mobile phase system.

[0136] Example 4

[0137] Methodological validation

[0138] 1.1 Standard curve, detection limit, and quantification limit

[0139] The solvent is acetonitrile, and the imidazolinone compounds are prepared into a standard curve with concentrations of 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L.

[0140] Accurately weigh 0.1 mL each of 1000 mg / L imazamox standard solution, imazapic acid standard solution, imazapic acid standard solution, imidazoquinoline standard solution, and imazethapyr standard solution into a single volumetric flask. Dilute to 1 mL with acetonitrile to obtain a mixed standard solution with a concentration of 100 μg / mL. Pipette 0.1 mL of the mixed standard solution into a 10 mL volumetric flask and dilute to 1 mL with acetonitrile to obtain a mixed standard solution with a concentration of 1.00 μg / mL. Pipette 0.01 mL, 0.05 mL, 0.1 mL, 0.5 mL, and 1 mL of the mixed standard solution into a 10 mL volumetric flask and dilute to acetonitrile to obtain a series of standard solutions with concentrations of 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively. Each series of standard solutions were tested by liquid chromatography-mass spectrometry, and the mass concentration of imidazolinone compounds was used as the horizontal axis and the corresponding peak area was used as the vertical axis to draw a standard curve. The linear regression equation was obtained. The results are shown in Table 6 and Figures 8-12 ,in, Figure 8 is the standard curve of imidazopyrine, Figure 9 is the standard curve of imazamox, Figure 10 is the standard curve of imazapiclone, Figure 11 is the standard curve of imazethapyr, Figure 12 This is the standard curve of imidazoquinolinic acid.

[0141] Table 6 Curves and correlation coefficients of imidazolinone pesticides

[0142]

[0143] Seven animal-derived foods, including beef, chicken, tilapia, chicken liver, milk, eggs, and sausage casings, were used as validation matrices. The detection limits of the five imidazolinone compounds in the above matrices are shown in Table 7.

[0144] Table 7 Detection limits of 5 imidazolinone compounds in different matrices

[0145]

[0146]

[0147] As shown in Tables 6 to 7, the linear relationship of the five imidazolinone compounds is good. The quantitative limit of the detection method provided by the present invention is 5 μg / kg at the minimum addition level. This quantitative limit can meet the detection needs, so no lower concentration is continued to be tested, and the actual detection limit will be smaller. The quantitative limit of the detection method provided by the present invention is 5 μg / kg (signal-to-noise ratio>10), and the detection limit of the five imidazolinone compounds in different matrices is 0.045~0.332 μg / kg (signal-to-noise ratio is 3), which is lower than the existing imidazolinone herbicide residue limit requirements in China and can meet daily detection requirements.

[0148] 1.2 Precision

[0149] Seven animal-derived foods, including beef, chicken, tilapia, chicken liver, milk, eggs, and sausage casings, were used as validation matrices. Recovery tests were conducted on five imidazolinone compounds at different concentrations. The determination was repeated six times at each concentration level to evaluate the accuracy and precision of the method (see Table 8).

[0150] Table 8 Laboratory verification data of recovery rate of blank samples spiked with imidazolinone pesticides

[0151]

[0152]

[0153] As shown in Table 8, at three addition levels (0.005 mg / kg, 0.010 mg / kg, and 0.100 mg / kg), the recoveries of the five imidazolinone compounds ranged from 67.8% to 120.1%, and the relative standard deviations ranged from 1.0% to 9.9%, meeting the actual test requirements.

[0154] Example 5

[0155] Analysis of animal-derived food samples

[0156] In order to meet the monitoring requirements for imidazopyr residues in imported animal meat at ports, the method established by the present invention was used to detect imidazopyr residues in hundreds of batches of imported beef, pork and other animal meats. The results showed that no imidazopyr residues were detected, thus reducing the safety risk of imported meat.

[0157] By optimizing different detection conditions, this paper has established a method for the determination of five imidazolinone compounds in animal-derived foods using a combination of QuEChERS extraction, solid-phase extraction cleanup, and LC-MS / MS. Using 1 v / v% acetic acid-acetonitrile as the extraction solvent, solid-phase extraction cleanup using a PPR dephospholipidated cartridge, LC-MS / MS analysis, and quantification using a matrix calibration curve external standard method, the method provided by this invention is rapid, simple, highly precise, and accurate, and meets domestic and international limit requirements.

[0158] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting imidazolinone compounds in animal-derived foods, characterized in that: The following steps are involved: The animal-derived food sample to be tested, an extractant, and a QuEChERS extraction salt are mixed and extracted to obtain an extract; the extractant is an acetic acid-acetonitrile solution; the QuEChERS extraction salt includes sodium citrate dihydrate, disodium citrate sesquihydrate, anhydrous magnesium sulfate, and sodium chloride; The animal-derived food samples to be tested include high-fat food samples and non-high-fat food samples, and the high-fat food samples include one or more of meat and aquatic products; When the animal-derived food sample to be tested is a high-fat food sample, the mixed extraction further comprises: performing solid-liquid separation on the extraction system obtained by the mixed extraction and then freezing the system, wherein the supernatant is the extraction solution; Purifying the extract using a PPR phospholipid-free solid phase extraction cartridge to obtain a sample solution to be tested; The sample solution to be tested is subjected to high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of the imidazolinone compound; The imidazolinone compound includes at least two of methoxam, imazapic acid, imazapic acid, imidazoquinoline acid and imazethapyr; The high performance liquid chromatography-tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection, the chromatographic column for the high performance liquid chromatography separation includes a C18 chromatographic column; the mobile phase A is ammonium formate and formic acid aqueous solution; the mobile phase B is methanol; the elution method is gradient elution, and the gradient elution procedure is as follows: From 0 to 0.5 min, the volume fraction of mobile phase A is 0.5%; from 0.5 to 2 min, the volume fraction of mobile phase A increases at a uniform rate from 0.5% to 40%; from 2 to 20 min, the volume fraction of mobile phase A increases at a uniform rate from 40% to 95%; from 20 to 22 min, the volume fraction of mobile phase A is 95%; from 22 to 22.1 min, the volume fraction of mobile phase A decreases at a uniform rate from 95% to 5%; from 22.1 to 27 min, the volume fraction of mobile phase A is 0.5%.

2. The detection method according to claim 1, wherein The non-high-fat food samples include offal, eggs, milk and sausage casings.

3. The detection method according to claim 1, wherein The volume fraction of acetic acid in the acetic acid-acetonitrile solution is 1%.

4. The detection method according to claim 1, wherein The solid-liquid ratio of the animal-derived food sample to be tested and the extractant is 1 g: 2 to 4 mL.

5. The detection method according to claim 1, wherein The mass ratio of the sodium citrate dihydrate to the disodium citrate sesquihydrate is 1:0.45-0.55; The mass ratio of the animal-derived food sample to be tested to sodium citrate dihydrate is 1:0.15-0.

25.

6. The detection method according to claim 1, characterized in that The mass ratio of the animal-derived food sample to be tested to anhydrous magnesium sulfate is 1:0.75-0.85; The mass ratio of the animal-derived food sample to be tested to sodium chloride is 1:0.15-0.

25.

7. The preparation method according to any one of claims 1 to 6, characterized in that The temperature of the mixed extraction is 18-25° C. The mixed extraction comprises: performing a first mixed extraction on the animal-derived food sample to be tested and the extractant, and then adding QuEChERS extraction salt to perform a second mixed extraction; The first mixed extraction includes vortex extraction, the rotation speed of the vortex extraction is 2000-3000 r / min, and the time is 15-25 s; When the food sample to be tested is a high-fat food sample, the vortex extraction further includes a homogenization extraction, and the homogenization extraction temperature is 18-25° C., the rotation speed is 10000-13500 r / min, and the time is 20-30 s; The second mixed extraction includes shaking extraction, the rotation speed of the shaking extraction is 300-400 r / min, and the time is 8-12 min.

8. The detection method according to claim 1, wherein The freezing treatment is performed at a temperature of -16 to -20°C and for a time of 2 to 3 hours.

9. The detection method according to claim 1, wherein The concentration of ammonium formate in the ammonium formate aqueous solution is 5 mmol / L, and the concentration of formic acid is 0.1 v / v%; The column temperature of the HPLC separation was 40° C., the mobile phase flow rate was 0.3 mL / min, and the injection volume was 2 μL.

10. The detection method according to claim 1, characterized in that The mass spectrometry detection conditions include: the ion source mode is H-ESI; the scanning mode is positive ion scanning, multiple reaction monitoring mode; the sheath gas is nitrogen, the value is 50Arb; the auxiliary gas is nitrogen, the value is 20Arb; the sweep gas is nitrogen, the value is 1Arb; the spray voltage is 1kV; the vaporization chamber temperature is 415°C; the ion transfer tube temperature is 300°C; the parent ion of the imazapic acid is 276, the daughter ions are 163 and 216, and the collision energies are 27V and 26V respectively; the imazapic acid The parent ion of the acid is 262, the daughter ions are 217 and 220, and the collision energies are 20V and 18V, respectively; the parent ion of the imidazoquinolinic acid is 312, the daughter ions are 252 and 266, and the collision energies are 21V and 32V, respectively; the parent ion of the imidazolic acid is 290, the daughter ions are 177 and 248, and the collision energies are 31V and 21V, respectively; the parent ion of the methoxazole is 306, the daughter ions are 69 and 86, and the collision energies are 28V and 28V, respectively.

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