1. A method for detecting residual quantity of tebuconazole amide and metabolite thereof is characterized by comprising the following steps:

s1, adding water into a sample to be tested to enable the water content of the sample to be tested to be larger than 75%, adding an extracting agent for extraction, salting out, carrying out solid-liquid separation after salting out, collecting filtrate, extracting, concentrating to obtain an extracting solution, and purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent to obtain a test solution;

s2, taking a blank sample of the same type of matrix without the tebuconazole amide and the metabolite thereof, processing according to the step S1 to obtain a blank sample matrix solution, adding a standard substance of the tebuconazole amide and the metabolite thereof into the blank sample matrix solution to prepare a standard solution, and finally preparing a matrix standard working solution containing at least 5 gradient concentrations of the tebuconazole amide and the metabolite thereof;

s3, performing qualitative detection, namely injecting at least 5 matrix standard working solutions with gradient concentrations in the step S2 and the test solution in the step S1, detecting by using HPLC-MS/MS, and performing qualitative verification on information according to chromatographic peak retention time and specific ions;

s4, quantitative detection, namely, injecting at least 5 matrix standard working solutions with gradient concentration in the step S2, detecting by using HPLC-MS/MS, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, and calculating the content of the tetrazolium amide and the metabolites thereof in the test solution by an external standard method.

2. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 1,

the extractant comprises chromatographically pure acetonitrile or chromatographically pure acetonitrile containing 0.1% of acetic acid, wherein the chromatographically pure acetonitrile containing 0.1% of acetic acid contains 1mL of chromatographically pure acetic acid with the purity of 99.9% in 1000mL of chromatographically pure acetonitrile;

the salting-out adopts NaCl and MgSO₄Salting out treatment is carried out;

the dispersed solid phase extractant adopts N-propyl ethylenediamine solid phase adsorbent (PSA).

3. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 2,

the specific steps of step S1 include: adding water into a sample to be tested, so that the water content of the sample to be tested is larger than 75%, uniformly mixing in a vortex mode for at least 30s, adding an extracting agent for extraction, uniformly mixing in a vortex mode for at least 2min, salting out, uniformly mixing in a vortex mode for at least 1min, performing solid-liquid separation in a centrifugal mode, wherein the rotating speed of the centrifugal mode is 4500 r/min-5500 r/min, the time is 3 min-7 min, collecting supernate, extracting an extraction layer, performing rotary evaporation concentration in a water bath at 40 ℃ -50 ℃ to obtain an extracting solution, purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent, performing centrifugal treatment again, wherein the rotating speed of the centrifugal treatment is 11000 r/min-13000 r/min, and the time is 3 min-5 min, and obtaining a test solution.

4. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 3,

the method also comprises passing the supernatant after centrifugation through a 0.22 μm organic filter before obtaining the test solution.

5. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 1,

the specific step of performing qualitative verification in step S3 includes: and comparing whether the retention time of the test solution is consistent with that of the matrix standard working solution, observing whether quantitative ions and qualitative ions have obvious chromatographic peaks or not in a mass spectrogram of the test solution after the background is subtracted, and whether the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration or not, judging that residues of the tetrazolium amide and metabolites thereof exist in the test solution if the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration, and judging that the residues of the tetrazolium amide and the metabolites thereof do not exist in the test solution if the two conditions cannot be met simultaneously.

6. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 5,

the detection conditions of HPLC-MS/MS high performance liquid chromatography in step S3 are as follows:

a chromatographic column: ZORBAX RRHD Eclipse Plus C18 column, 100mm × 3.0mm, 1.8 μm;

temperature of the column oven: 28-42 ℃;

mobile phase: the phase A is acetonitrile, the phase B is 0.1% formic acid water solution, and the mobile phase adopts linear gradient elution;

flow rate: 0.3mL/min to 0.5 mL/min;

sample introduction amount: 5 μ L.

7. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 6,

the linear gradient elution is carried out for 0-4 min,

the gradient change elution conditions of the phase A and the phase B are as follows: the volume ratio of the phase A to the phase B is 60: 40 at 0min, 60: 40 at 0.5min, 90: 10 at 0.6min, 90: 10 at 3min, 60: 40 at 3.1min, and 60: 40 at 4 min.

8. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 5,

the detection conditions of the HPLC-MS/MS mass spectrum in the step S3 are as follows:

type of ion source: ESI +;

ion source temperature: 500 ℃;

air curtain pressure: 40 psi;

spray gas pressure: 45 psi;

auxiliary heating gas pressure: 45 psi;

ionization voltage: 5500v

The detection mode is as follows: and (5) monitoring multiple reactions.

9. The method for detecting residual amount of tetrazolium amide and its metabolites according to claim 8,

the multiple reaction monitoring parameters include:

tetrazolium amide: retention time 1.96min, declustering voltage 30V, qualitative ion pair (m/z) 545/356 and 545/376, quantitative ion pair (m/z) 545/356, and collision voltage 19eV and 37 eV;

tetrazolium amide metabolites: the retention time was 2.34min, the declustering voltage was 30V, the qualitative ion pair (m/z) was 527/389 and 527/374, the quantitative ion pair (m/z) was 527/389, and the collision voltages were 29eV and 35 eV.

10. Use of the method for detecting residual amounts of tetrazolium amide and its metabolites according to any one of claims 1 to 8 in cereals.

Background

In recent years, grain pests are rapidly developed along with the scale of grain planting, great loss is brought to agricultural production, on the other hand, the continuous development of pest drug resistance also becomes a great problem for preventing the control of the pests, so that the conventional pesticide is difficult to control.

Tetrazolium amide (tetraniliprole, development code: BCS-CL73507)), the chemical name is: 1- (3-chloro-2-pyridinyl) -4 ' -cyano-2 ' -methyl-6 ' -methylcarbamoyl-3- { [5- (trifluoromethyl) -2H-tetrazol-2-yl]Methyl } -1H-pyrazole-5-carboxanilide, CAS accession No.: 1229654-66-3; the molecular formula is as follows: c₂₂H₁₆C_lF₃N₁₀O₂(ii) a Relative molecular mass: 544.88, having the formula:

tetrazoxamide is a novel broad-spectrum insecticide of anthranilamide benzamide. The pesticide is a new member in a family of diamide compounds such as chlorantraniliprole, cyantraniliprole and the like, and is a ryanodine receptor agonist; the international commission on Insecticide Resistance (IRAC) classified it as group 28. The tebuconazole amide can effectively prevent and control various Lepidoptera (Lepidoptera), Coleoptera (Coleoptera) and Diptera (Diptera) pests on grains, fruit trees, vegetables and other crops under low dosage, and particularly has obvious effect on preventing and controlling pests such as chilo suppressalis, beet armyworm, thrips and the like. The medicament has simple use mode, can be used for leaf surface treatment, soil treatment and seed treatment, and is a 200 g/L suspending agent for preventing and controlling beet armyworms on cabbages registered at present.

The technical product of the tebuconazole is low in toxicity to mammals (LD50 value is more than 2000mg/kg, brown rats) and birds (LD50 value is more than 2000mg/kg, 3 birds are tested in the test); the toxicity to aquatic vertebrates such as fish is relatively low (LC50 value is more than 10mg/L, 4 kinds of fish are tested in the test); high toxicity to aquatic invertebrates (minimum concentration EC50 value of 0.071mg/L, daphnia magna); high toxicity to benthonic animals (minimum concentration EC50 value of 0.23mg/L or LC50 value of 0.034mg/kg dry sediment, Chironomus); moderate toxicity to algal plants (minimum concentration EC50 value 1.4mg/L, Skeletonema costatum); relatively low toxicity to aquatic plants (ErC50 value >6.6mg/L, duckweed turgida); low toxicity to soil organism such as Lumbricus (LC50corr value >448mg/kg, dry soil, Eisenia Evosa); the product has high toxicity to bees, and after the product is exposed to the technical grade of the tebuconazole, the European bees are more sensitive than the European bumblebees (the LD50 values are 0.41-0.44 and 22-94 mu g/bee respectively). In conclusion, the tetrazolium amide as the pesticide has certain influence on human beings and the environment, and the residue of the tetrazolium amide in agricultural products can bring certain dietary intake risks, so that the establishment of a detection method for the tetrazolium amide and metabolites thereof in the agricultural products has very important significance. At present, no detection method for the tetrazolium amide and the metabolite thereof in agricultural products exists. In order to understand the digestion dynamics of the tebuconazole after being applied to the agricultural products and evaluate the safety of the tebuconazole in the agricultural products, a method for determining the residual quantity of the tebuconazole and the residual quantity of the metabolites of the tebuconazole in the agricultural products is urgently needed, and the method has important significance for improving the safety quality of food.

Disclosure of Invention

The invention provides a method for detecting residual amounts of tebuconazole and residues of tebuconazole and metabolites thereof and application thereof, and aims to solve the technical problems that the residual amounts of tebuconazole and residues of tebuconazole in agricultural products cannot be detected and the digestion dynamics of tebuconazole after the tebuconazole is applied to the agricultural products cannot be monitored in the prior art.

The technical scheme adopted by the invention is as follows:

a method for detecting residual quantity of tebuconazole amide and metabolite thereof comprises the following steps:

s1, adding water into a sample to be tested to enable the water content of the sample to be tested to be larger than 75%, adding an extracting agent for extraction, salting out, carrying out solid-liquid separation after salting out, collecting filtrate, extracting, concentrating to obtain an extracting solution, and purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent to obtain a test solution;

s2, taking a blank sample of the same type of matrix without the tebuconazole amide and the metabolite thereof, processing according to the step S1 to obtain a blank sample matrix solution, adding a standard substance of the tebuconazole amide and the metabolite thereof into the blank sample matrix solution to prepare a standard solution, and finally preparing a matrix standard working solution containing at least 5 gradient concentrations of the tebuconazole amide and the metabolite thereof;

s3, performing qualitative detection, namely injecting at least 5 matrix standard working solutions with gradient concentrations in the step S2 and the test solution in the step S1, detecting by using HPLC-MS/MS, and performing qualitative verification on information according to chromatographic peak retention time and specific ions;

s4, quantitative detection, namely, injecting at least 5 matrix standard working solutions with gradient concentration in the step S2, detecting by using HPLC-MS/MS, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, and calculating the content of the tetrazolium amide and the metabolites thereof in the test solution by an external standard method.

Further, the extractant comprises chromatographically pure acetonitrile or chromatographically pure acetonitrile containing 0.1% acetic acid and 0.1% acetic acidThe chromatographic purity acetonitrile of (1 mL) contains chromatographic purity acetic acid with the purity of 99.9 percent in 1000mL of chromatographic purity acetonitrile; salting out with NaCl and MgSO₄Salting out treatment is carried out; the dispersed solid phase extractant adopts N-propyl ethylenediamine solid phase adsorbent.

Further, the specific step of step S1 includes: adding water into a sample to be tested, so that the water content of the sample to be tested is larger than 75%, uniformly mixing in a vortex mode for at least 30s, adding an extracting agent for extraction, uniformly mixing in a vortex mode for at least 2min, salting out, uniformly mixing in a vortex mode for at least 1min, performing solid-liquid separation in a centrifugal mode, wherein the rotating speed of the centrifugal mode is 4500 r/min-5500 r/min, the time is 3 min-7 min, collecting supernate, extracting an extraction layer, performing rotary evaporation concentration in a water bath at 40 ℃ -50 ℃ to obtain an extracting solution, purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent, performing centrifugal treatment again, wherein the rotating speed of the centrifugal treatment is 11000 r/min-13000 r/min, and the time is 3 min-5 min, and obtaining a test solution.

Further, the method also comprises passing the supernatant after centrifugation through a 0.22 μm organic filter before obtaining the test solution.

Further, the detection conditions of HPLC-MS/MS in step S3 are as follows: a chromatographic column: ZORBAX RRHD Eclipse Plus C18 column, 100mm × 3.0mm, 1.8 μm; temperature of the column oven: 28-42 ℃; mobile phase: the phase A is acetonitrile, the phase B is 0.1% formic acid water solution, and the mobile phase adopts linear gradient elution; flow rate: 0.3mL/min to 0.5 mL/min; sample introduction amount: 5 μ L.

Further, linear gradient elution is carried out for 0-4 min, and gradient change elution conditions of the phase A and the phase B are as follows: the volume ratio of the phase A to the phase B is 60: 40 at 0min, 60: 40 at 0.5min, 90: 10 at 0.6min, 90: 10 at 3min, 60: 40 at 3.1min, and 60: 40 at 4 min.

Further, the specific step of performing qualitative verification in the line step S3 includes: and comparing whether the retention time of the test solution is consistent with that of the matrix standard working solution, observing whether quantitative ions and qualitative ions have obvious chromatographic peaks or not in a mass spectrogram of the test solution after the background is subtracted, and whether the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration or not, judging that residues of the tetrazolium amide and metabolites thereof exist in the test solution if the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration, and judging that the residues of the tetrazolium amide and the metabolites thereof do not exist in the test solution if the two conditions cannot be met simultaneously.

Further, the detection conditions of the mass spectrum of HPLC-MS/MS in step S3 are as follows: type of ion source: ESI +; ion source temperature: 500 ℃; air curtain pressure: 40 psi; spray gas pressure: 45 psi; auxiliary heating gas pressure: 45 psi; ionization voltage: 5500v assay format: and (5) monitoring multiple reactions.

Further, the multiple reaction monitoring parameters include: tetrazolium amide: retention time 1.96min, declustering voltage 30V, qualitative ion pair (m/z) 545/356 and 545/376, quantitative ion pair (m/z) 545/356, and collision voltage 19eV and 37 eV; tetrazolium amide metabolites: the retention time was 2.34min, the declustering voltage was 30V, the qualitative ion pair (m/z) was 527/389 and 527/374, the quantitative ion pair (m/z) was 527/389, and the collision voltages were 29eV and 35 eV.

According to another aspect of the invention, the application of the method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof in grains is also provided.

Further, the grains include corn or rice.

The invention has the following beneficial effects:

the invention relates to a method for detecting residual amounts of tebuconazole and residues of tebuconazole metabolites, which utilizes a dispersive solid-phase extraction technology to establish a sample pretreatment method which is simple, convenient, rapid and effective in avoiding matrix interference in a sample, combines the pretreatment method with HPLC-MS/MS to be used for qualitative confirmation and quantitative detection of the tebuconazole and the residues of the tebuconazole in grains, can rapidly detect the residual amounts of the tebuconazole and the residues of the tebuconazole at 20mg/kg, and can meet the residual limit amount of the tebuconazole and the residues of the tebuconazole in reported food safety detection of parts of America and Canada. The method for detecting the residual quantity of the tebuconazole and the residual quantity of the tebuconazole metabolites is high in recovery rate and good in repeatability, the average recovery rate reaches 89.8% -96.7%, the average relative standard deviation RSD is 1.8% -3.5%, the detection limit is lower than 0.002mg/kg, and the method has the advantages of being simple and convenient to operate, fast, accurate, high in sensitivity and good in repeatability. The digestion dynamics of the tebuconazole after the tebuconazole is applied to the agricultural products are monitored to evaluate the application safety of the tebuconazole in the agricultural products, so that the food safety is guaranteed, and a powerful technical support is provided for the healthy development of export trade.

In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is an HPLC-MS/MS selective ion chromatogram of a fresh corn test solution of tebuconazole amide and metabolite thereof (0.025 μ g/mL) of preferred embodiment 1 of the invention;

FIG. 2 is an HPLC-MS/MS selective ion chromatogram of a blank sample matrix solution of fresh corn without tebuconazole amide and metabolite thereof;

FIG. 3 is a HPLCMS/MS selective ion chromatogram of a fresh corn blank sample added with tebuconazole and metabolite standard thereof (0.05 mg/kg);

FIG. 4 is a matrix standard curve diagram of tebuconazole amide and its metabolite in accordance with preferred embodiment 1 of the present invention;

FIG. 5 is an HPLC-MS/MS selective ion chromatogram of a rice test solution of teflupyrad and its metabolites (0.1. mu.g/mL) according to a preferred embodiment 2 of the present invention;

FIG. 6 is an HPLC-MS/MS selective ion chromatogram of a rice blank sample matrix solution without tetrazolium amide and its metabolites;

FIG. 7 is a HPLCMS/MS selective ion chromatogram of a rice blank sample added with tebuconazole and metabolite standard thereof (0.5 mg/kg);

FIG. 8 is a matrix standard curve diagram of tebuconazole amide and its metabolite in accordance with the preferred embodiment 2 of the present invention;

FIG. 9 is an HPLC-MS/MS total ion chromatogram of a solvent standard solution of tebuconazole amide and its metabolite; and

FIG. 10 is a solvent calibration graph of the tetrazolium amide and its metabolites of comparative example 1.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

FIG. 1 is an HPLC-MS/MS selective ion chromatogram of a fresh corn test solution of tebuconazole amide and metabolite thereof (0.025 μ g/mL) of preferred embodiment 1 of the invention; FIG. 2 is an HPLC-MS/MS selective ion chromatogram of a blank sample matrix solution of fresh corn without tebuconazole amide and metabolite thereof; FIG. 3 is a HPLCMS/MS selective ion chromatogram of a fresh corn blank sample added with tebuconazole and metabolite standard thereof (0.05 mg/kg); FIG. 4 is a matrix standard curve diagram of tebuconazole amide and its metabolite in accordance with preferred embodiment 1 of the present invention; FIG. 5 is an HPLC-MS/MS selective ion chromatogram of a rice test solution of teflupyrad and its metabolites (0.1. mu.g/mL) according to a preferred embodiment 2 of the present invention; FIG. 6 is an HPLC-MS/MS selective ion chromatogram of a rice blank sample matrix solution without tetrazolium amide and its metabolites; FIG. 7 is a HPLCMS/MS selective ion chromatogram of a rice blank sample added with tebuconazole and metabolite standard thereof (0.5 mg/kg); FIG. 8 is a matrix standard curve diagram of tebuconazole amide and its metabolite in accordance with the preferred embodiment 2 of the present invention; FIG. 9 is an HPLC-MS/MS total ion chromatogram of a solvent standard solution of tebuconazole amide and its metabolite; FIG. 10 is a solvent calibration graph of the tetrazolium amide and its metabolites of comparative example 1.

The method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof comprises the following steps:

s1, adding water into a sample to be tested to enable the water content of the sample to be tested to be larger than 75%, adding an extracting agent for extraction, salting out, carrying out solid-liquid separation after salting out, collecting filtrate, extracting, concentrating to obtain an extracting solution, and purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent to obtain a test solution;

s2, taking a blank sample of the same type of matrix without the tebuconazole amide and the metabolite thereof, processing according to the step S1 to obtain a blank sample matrix solution, adding a standard substance of the tebuconazole amide and the metabolite thereof into the blank sample matrix solution to prepare a standard solution, and finally preparing a matrix standard working solution containing at least 5 gradient concentrations of the tebuconazole amide and the metabolite thereof;

s3, performing qualitative detection, namely injecting at least 5 matrix standard working solutions with gradient concentrations in the step S2 and the test solution in the step S1, detecting by using HPLC-MS/MS, and performing qualitative verification on information according to chromatographic peak retention time and specific ions;

s4, quantitative detection, namely, injecting at least 5 matrix standard working solutions with gradient concentration in the step S2, detecting by using HPLC-MS/MS, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, and calculating the content of the tetrazolium amide and the metabolites thereof in the test solution by an external standard method.

The invention relates to a method for detecting residual amounts of tebuconazole and residues of tebuconazole metabolites, which utilizes a dispersive solid-phase extraction technology to establish a sample pretreatment method which is simple, convenient, rapid and effective in avoiding matrix interference in a sample, combines the pretreatment method with HPLC-MS/MS to be used for qualitative confirmation and quantitative detection of the tebuconazole and the residues of the tebuconazole in grains, can rapidly detect the residual amounts of the tebuconazole and the residues of the tebuconazole at 20mg/kg, and can meet the residual limit amount of the tebuconazole and the residues of the tebuconazole in reported food safety detection of parts of America and Canada. The method for detecting the residual quantity of the tebuconazole and the residual quantity of the tebuconazole metabolites is high in recovery rate and good in repeatability, the average recovery rate reaches 89.8% -96.7%, the average relative standard deviation RSD is 1.8% -3.5%, the detection limit is lower than 0.002mg/kg, and the method has the advantages of being simple and convenient to operate, fast, accurate, high in sensitivity and good in repeatability. The digestion dynamics of the tebuconazole after the tebuconazole is applied to the agricultural products are monitored to evaluate the application safety of the tebuconazole in the agricultural products, so that the safety of the people food and the healthy development of the export trade are guaranteed and a powerful technical support is provided.

The sample to be tested is obtained by crushing agricultural products to free the tetrazolium amide and metabolites thereof. For example, crushing the agricultural products by using a multifunctional food processor XBLL-25A to break the cell wall, so as to realize the dissociation of the tetrazolium amide and the metabolites thereof and obtain a sample to be detected. According to the method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof, the safety of pesticide residues to human bodies is evaluated by extracting the tebuconazole amide and the metabolite thereof in agricultural products. After the tetrazolium amide and the metabolites thereof in the agricultural products are applied to crops, the tetrazolium amide is absorbed by the crops and enters cells of the crops, and part of the tetrazolium amide is combined with proteins or other substances in the cells, so that when the residues of the tetrazolium amide and the metabolites thereof are determined, the to-be-detected substances need to be crushed, the tetrazolium amide and the metabolites thereof are dissociated, and a to-be-detected sample is obtained. Then extracting the tetrazolium amide and the metabolites thereof from the solid by using an extracting agent, purifying by using a dispersed solid phase extraction technology, and determining the content of the tetrazolium amide and the metabolites thereof by using an HPLC-MS/MS method, wherein the HPLC-MS/MS method of the tetrazolium amide and the metabolites thereof in the agricultural products is interfered by the matrix of the chemical substances of the agricultural products, so that the quantification is required by using a matrix standard curve, and the quantitative determination by using the matrix standard curve is used for eliminating the matrix effect, reducing the interference of the matrix on the analysis process of the analytes and ensuring the accuracy of the analysis result. The safety of pesticide residues to human bodies is evaluated by quantitatively detecting the tetrazolium amide and the metabolites thereof in agricultural products.

And (4) quantitative detection, namely injecting at least 5 matrix standard working solutions with gradient concentration in the step S2, detecting by using HPLC-MS/MS, performing regression analysis on corresponding concentrations by using chromatographic peak areas, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, measuring the chromatographic peak areas of the tetrazolium amide and the metabolites thereof in the test solution under the same conditions, substituting the chromatographic peak areas into the matrix standard curve, and calculating to obtain the content of the tetrazolium amide and the metabolites thereof in the test solution.

In this example, the extractant comprises chromatographically pure acetonitrile or chromatographically pure acetonitrile containing 0.1% acetic acid, wherein the chromatographically pure acetonitrile containing 0.1% acetic acid contains 1mL of chromatographically pure acetic acid with a purity of 99.9% in 1000mL of chromatographically pure acetonitrile; salting out with NaCl and MgSO₄Salting out treatment is carried out; the dispersed solid phase extractant adopts N-propyl ethylenediamine solid phase adsorbent. The extraction agent is acetonitrile or acetonitrile containing 0.1% acetic acid, which is based on the high solubility of the tetrazolium amide and its metabolites in acetonitrile, and the acetonitrile is used as an extraction solvent in a complex matrix, so that the interference of protein and fat in a sample can be avoided. The graphitized carbon black is used as a dispersed solid phase extraction agent, so that the influence of pigments and sterols in a matrix can be removed, but early tests show that the graphitized carbon black has a large adsorption effect on the tetrazolium amide and metabolites thereof, and the single addition recovery rate is low (47.5-58.7%), so that the graphitized carbon black is not suitable for use. Therefore, the purification treatment is carried out by adopting an N-propyl ethylenediamine (PSA) solid phase adsorbent as a dispersed solid phase extracting agent, the purification treatment has strong adsorption effect on polysaccharides, peptides (including amino acids) and pigments, and the comparison of the purification effect of C18 and PSA on fresh corn shows that the influence of a chromatographic peak matrix obtained by PSA purification is smaller, and the purification effect is better than that of C18. Salting-out agents NaCl and MgSO₄10g of sample to be tested, 10mL of water, 20mL of extractant, 5g of NaCl and 1g of MgSO 4₄Salting out may be carried out by adding 1 to 5g of NaCl and 0.1 to 1.0g of MgSO₄The salting-out aims to promote the separation of the water phase and the organic phase, so that a saturated NaCl solution is formed after sufficient NaCl is added, and the saturated NaCl solution and acetonitrile are not mutually soluble, so that an acetonitrile layer containing a target compound and a sample layer containing no target compound are separated, and the next separation and purification are facilitated.

In this embodiment, the specific step of step S1 includes: adding water into a sample to be tested, so that the water content of the sample to be tested is larger than 75%, uniformly mixing in a vortex mode for at least 30s, adding an extracting agent for extraction, uniformly mixing in a vortex mode for at least 2min, salting out, uniformly mixing in a vortex mode for at least 1min, performing solid-liquid separation in a centrifugal mode, wherein the rotating speed of the centrifugal mode is 4500 r/min-5500 r/min, the time is 3 min-7 min, collecting supernate, extracting an extraction layer, performing rotary evaporation concentration in a water bath at 40 ℃ -50 ℃ to obtain an extracting solution, purifying a matrix in the extracting solution by using a dispersed solid-phase extracting agent, performing centrifugal treatment again, wherein the rotating speed of the centrifugal treatment is 11000 r/min-13000 r/min, and the time is 3 min-5 min, and obtaining a test solution.

The salting-out process aims to enable the water in the sample to be detected to reach a saturated NaCl solution state, the acetonitrile and the water are mutually soluble, but the acetonitrile and the saturated NaCl solution are not mutually soluble, when the NaCl in the water of the sample to be detected reaches the saturated solubility, the acetonitrile layer and the NaCl solution containing the sample (excluding the tetrazolium amide and the metabolites thereof) are layered, and because the solubility of the tetrazolium amide and the metabolites thereof in the acetonitrile is higher than that of the water, the tetrazolium amide and the metabolites thereof extracted through the acetonitrile enter the acetonitrile layer, so that the aim of extracting the tetrazolium amide and the metabolites thereof from the sample to be detected is fulfilled. Preferably, the solid-liquid separation is carried out by centrifugation, the rotation speed of the centrifugation is 4500 r/min-5500 r/min, the time is 3 min-7 min, the supernatant is collected, an extraction layer is extracted, acetonitrile is added into the centrifuged precipitate for repeated extraction for 1 time, and the extraction layer is combined after the centrifugation.

In this example, the method further comprises passing the centrifuged supernatant through a 0.22 μm organic filter before obtaining the test solution. The organic filter membrane with the diameter of 0.22 mu m can remove larger impurities in the supernatant and prevent the impurities from polluting the instrument.

In this embodiment, the specific step of performing qualitative verification in the line step S3 includes: and comparing whether the retention time of the test solution is consistent with that of the matrix standard working solution, observing whether quantitative ions and qualitative ions have obvious chromatographic peaks or not in a mass spectrogram of the test solution after the background is subtracted, and whether the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration or not, judging that residues of the tetrazolium amide and metabolites thereof exist in the test solution if the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with that of the matrix standard working solution with similar concentration, and judging that the residues of the tetrazolium amide and the metabolites thereof do not exist in the test solution if the two conditions cannot be met simultaneously. In the qualitative detection step, the ion abundance ratio of the quantitative ions and the qualitative ions and the ion abundance ratio of the matrix standard working solution with similar concentration are allowed to deviate +/-20% when the relative abundance is more than 50%; when the relative abundance is more than 20% to 50%, a deviation of +/-25% is allowed; when the relative abundance is more than 10% to 20%, deviation of +/-30% is allowed; the relative abundance is less than or equal to 10 percent, and the deviation of +/-50 percent is allowed.

In this example, the detection conditions of HPLC-MS/MS in step S3 are as follows: a chromatographic column: ZORBAX RRHD Eclipse Plus C18 column, 100mm × 3.0mm, 1.8 μm; temperature of the column oven: 28-42 ℃; mobile phase: the phase A is acetonitrile, the phase B is 0.1% formic acid water solution, and the mobile phase adopts linear gradient elution; flow rate: 0.3mL/min to 0.5 mL/min; sample introduction amount: 5 μ L.

In the embodiment, the linear gradient elution is carried out for 0-4 min, and the gradient change elution conditions of the phase A and the phase B are as follows: the volume ratio of the phase A to the phase B is 60: 40 at 0min, 60: 40 at 0.5min, 90: 10 at 0.6min, 90: 10 at 3min, 60: 40 at 3.1min, and 60: 40 at 4 min.

In this embodiment, the conditions for detecting the mass spectrum of HPLC-MS/MS in step S3 are as follows: type of ion source: ESI +; ion source temperature: 500 ℃; air curtain pressure: 40 psi; spray gas pressure: 45 psi; auxiliary heating gas pressure: 45 psi; ionization voltage: 5500v, assay format: and (5) monitoring multiple reactions. Through the previous experiments, the response value of the tetrazolium amide and the metabolite in the positive ion mode is determined to be higher, so that the positive ion mode is adopted.

In this embodiment, the multiple reaction monitoring parameters include: the retention time was 1.96min, the declustering voltage was 30V, the qualitative ion pair (m/z) was 545/356 and 545/376, the quantitative ion pair (m/z) was 545/356, and the collision voltages were 19eV and 37 eV.

According to another aspect of the invention, the application of the method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof in grains is also provided. The method for detecting the residual quantity of the tebuconazole amide and the tebuconazole metabolite thereof is used for detecting the grains, the liquid chromatography and the mass spectrum are combined for detection, the residual quantity of the tebuconazole amide and the tebuconazole metabolite thereof in a grain sample can be effectively detected, the method is simple and convenient to operate, is rapid, has a good separation effect, can meet the requirements of quantitative analysis on accuracy and precision, and has the advantages of high sensitivity, good repeatability, simplicity and convenience in operation, rapidness, accuracy and the like. Preferably, the grain comprises corn or rice.

Examples

The high performance liquid chromatography-mass spectrometer LC1290-MS-SCIEX 4500Q is purchased from Agilent and AB, USA;

the multifunctional food processor XBLL-25A is purchased from Shanghai Shuaijia electronic technology Co., Ltd;

sodium chloride, magnesium sulfate, analytically pure, purchased from national pharmaceutical group chemical reagents ltd;

acetonitrile, chromatographically pure, available from merck chemical technology (shanghai) ltd;

standard tetrazolium amide and its metabolites, 99.5% pure, purchased from dr.

Example 1

The method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof comprises the following steps:

s1, crushing a fresh corn sample by using a multifunctional food processor, accurately weighing 10g +/-0.01 mg of a sample to be detected into a 100mL plastic centrifugal tube with a plug, adding 10mL of water, adding 20mL of chromatographic pure acetonitrile into the sample to be detected, extracting, uniformly mixing for at least 2min by vortex, adding 5g of NaCl and 1g of MgSO 2₄Performing salting-out treatment, performing vortex mixing for at least 1min, centrifuging in a 4000r/min centrifuge for 5min, taking 1mL of supernatant fluid, purifying in a 2mL centrifuge tube filled with 40mg of PSA, performing vortex mixing for at least 30s, centrifuging in a 12000r/min centrifuge for 4min, and filtering the supernatant fluid with a 0.2 mu m organic filter membrane to obtain a test solution;

s2, accurately weighing 10 +/-0.01 mg of tebuconazole and a standard substance of a metabolite thereof in a 10mL volumetric flask, dissolving with acetonitrile and fixing the volume to 10mL, preparing to obtain 1000.0 mu g/mL standard stock solution, transferring 1.0mL of the standard stock solution into the 10mL volumetric flask, fixing the volume with acetonitrile to obtain 10.0 mu g/mL standard intermediate solution, diluting the 10.0 mu g/mL standard solution with acetonitrile step by step to prepare a series of mixed standard solutions with the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, taking a blank sample of the same type of matrix without tebuconazole and the metabolite thereof, processing according to the step S1 to obtain a blank sample matrix solution, adding a series of mixed standard solutions with a certain volume into the solution, uniformly mixing by vortex, and respectively preparing a matrix standard working solution with the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL;

s3, performing qualitative detection, namely injecting the matrix standard working solution prepared in the step S2 and the test solution prepared in the step S1 into the sample, wherein the matrix standard working solution is 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, detecting by using HPLC-MS/MS, comparing whether the retention time is consistent, observing whether quantitative ions and qualitative ions have obvious chromatographic peaks in a mass spectrogram of the test solution after background subtraction, judging whether the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with the ion abundance ratio of the matrix standard working solution with similar concentration, if so, judging that residues of tetrazolium amide and metabolites thereof exist in the test solution, and if the two conditions cannot be met simultaneously, judging that residues of tetrazolium amide and metabolites thereof do not exist in the test solution;

s4, carrying out quantitative detection, namely, injecting matrix standard working solution prepared in the step S2 and having the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, detecting by using HPLC-MS/MS, carrying out regression analysis on the corresponding concentrations by using chromatographic peak areas, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, measuring the chromatographic peak areas of the tetrazolium amide and the metabolites thereof in the test solution under the same conditions, substituting the chromatographic peak areas into the matrix standard curve, calculating to obtain the contents of the tetrazolium amide and the metabolites thereof in the test solution, and then calculating to obtain the residual quantity of the fresh corn samples and the metabolites thereof according to the mass of the samples represented by the test solution;

wherein, HPLC-MS/MS chromatographic conditions are as follows:

a chromatographic column: ZORBAX RRHD Eclipse Plus C18 column, 3.0X 100mm, 1.8 μm;

temperature of the sample chamber: 35 ℃;

mobile phase: 0.1% aqueous formic acid and acetonitrile;

mobile phase gradient elution conditions are shown in table 1,

TABLE 1 mobile phase gradient elution conditions

Flow rate: 0.4 mL/min;

sample introduction amount: 5 μ L.

The mass spectrum detection conditions comprise:

type of ion source: ESI +;

ion source temperature: 500 ℃;

air curtain pressure: 40 psi;

spray gas pressure: 45 psi;

auxiliary heating gas pressure: 45 psi;

ionization voltage: 5500v

The detection mode is as follows: multiple Reaction Monitoring (MRM), parameters are as in table 2 below.

TABLE 2 major MRM parameters of the components to be tested

(I) standard recovery and repeatability test

Adding standard solutions of the tebuconazole and the metabolite thereof with concentration levels of 5, 50 and 500 mu g/kg into fresh corn without the tebuconazole and the metabolite thereof, carrying out residual quantity measurement according to the step S4 after absorbing for 30min, comparing the measured concentration with the theoretical addition concentration of the pesticide to obtain the addition recovery rate of the pesticide, and carrying out parallel measurement for 5 times on each addition level to obtain the relative standard deviation.

(II) detection Limit test

Injecting standard solutions of the tebuconazole amide and the metabolites thereof into HPLC-MS/MS, and calculating the concentration of a target object corresponding to a 3-fold signal-to-noise ratio by using the signal-to-noise ratio of the chromatographic peak of the standard solution of the fresh corn matrix with the lowest concentration as the detection limit of the method.

Example 2

The method for detecting the residual quantity of the tebuconazole amide and the metabolite thereof comprises the following steps:

s1, crushing the rice sample by using a multifunctional food processor, accurately weighing 10g +/-0.01 mg of sample to be detected into a 100mL plastic centrifugal tube with a plug, adding 10mL of water, adding 20mL of chromatographic pure acetonitrile containing 0.1% of acetic acid into the sample to be detected, extracting, uniformly mixing for at least 2min in a vortex manner, adding 5g of NaCl and 1g of MgSO₄Performing salting-out treatment, performing vortex mixing for at least 1min, centrifuging in a 5000r/min centrifuge for 5min, taking 1mL of supernatant fluid, purifying in a 2mL centrifuge tube filled with 40mg of PSA, performing vortex mixing for at least 30s, centrifuging in a 12000r/min centrifuge for 5min, and filtering the supernatant fluid with a 0.2 mu m organic filter membrane to obtain a test solution;

s2, accurately weighing 10 +/-0.01 mg of tebuconazole and a standard substance of a metabolite thereof in a 10mL volumetric flask, dissolving with acetonitrile and fixing the volume to 10mL, preparing to obtain 1000.0 mu g/mL standard stock solution, transferring 1.0mL of the standard stock solution into the 10mL volumetric flask, fixing the volume with acetonitrile to obtain 10.0 mu g/mL standard intermediate solution, diluting the 10.0 mu g/mL standard solution with acetonitrile step by step to prepare a series of mixed standard solutions with the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, taking a blank sample of the same type of matrix without tebuconazole and the metabolite thereof, processing according to the step S1 to obtain a blank sample matrix solution, adding a series of mixed standard solutions with a certain volume into the solution, uniformly mixing by vortex, and respectively preparing a matrix standard working solution with the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL;

s3, performing qualitative detection, namely injecting the matrix standard working solution prepared in the step S2 and the test solution prepared in the step S1 into the sample, wherein the matrix standard working solution is 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, detecting by using HPLC-MS/MS, comparing whether the retention time is consistent, observing whether quantitative ions and qualitative ions have obvious chromatographic peaks in a mass spectrogram of the test solution after background subtraction, judging whether the ion abundance ratio of the quantitative ions and the qualitative ions is consistent with the ion abundance ratio of the matrix standard working solution with similar concentration, if so, judging that residues of tetrazolium amide and metabolites thereof exist in the test solution, and if the two conditions cannot be met simultaneously, judging that residues of tetrazolium amide and metabolites thereof do not exist in the test solution;

s4, carrying out quantitative detection, namely, injecting matrix standard working solution prepared in the step S2 and having the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, detecting by using HPLC-MS/MS, carrying out regression analysis on the corresponding concentrations by using chromatographic peak areas, drawing a matrix standard curve of the tetrazolium amide and the metabolites thereof, injecting the test solution in the step S1, detecting by using HPLC-MS/MS, measuring the chromatographic peak areas of the tetrazolium amide and the metabolites thereof in the test solution under the same conditions, substituting the chromatographic peak areas into the matrix standard curve, calculating to obtain the contents of the tetrazolium amide and the metabolites thereof in the test solution, and then calculating to obtain the residual amounts of the tetrazolium amide and the metabolites thereof in the rice sample according to the mass of the sample represented by the test solution;

wherein, HPLC-MS/MS chromatographic conditions are as follows:

a chromatographic column: ZORBAX RRHD Eclipse Plus C18 column, 3.0X 100mm, 1.8 μm;

temperature of the sample chamber: 38 ℃;

mobile phase: 0.1% aqueous formic acid and acetonitrile;

mobile phase gradient elution conditions are shown in table 3.

TABLE 3 mobile phase gradient elution conditions

Flow rate: 0.4 mL/min;

sample introduction amount: 5 μ L.

The mass spectrum detection conditions comprise:

type of ion source: ESI +;

ion source temperature: 500 ℃;

air curtain pressure: 40 psi;

spray gas pressure: 45 psi;

auxiliary heating gas pressure: 45 psi;

ionization voltage: 5500v

The detection mode is as follows: multiple Reaction Monitoring (MRM), parameters were as follows:

TABLE 4 major MRM parameters of the components to be tested

(I) standard recovery and repeatability test

Adding standard solutions of the tebuconazole and the metabolite thereof with concentration levels of 5, 50 and 500 mu g/kg to rice without the tebuconazole and the metabolite thereof, carrying out residue measurement according to the step S4 after absorbing for 30min, comparing the measured concentration with the theoretical addition concentration of the pesticide to obtain the addition recovery rate of the pesticide, and carrying out parallel measurement for 5 times on each addition level to obtain the relative standard deviation.

(II) detection Limit test

Injecting standard solutions of the tebuconazole amide and the metabolites thereof into HPLC-MS/MS, and calculating by using the signal-to-noise ratio of the chromatographic peak of the standard solution of the fresh corn matrix with the lowest concentration to obtain the target concentration corresponding to 3 times of the signal-to-noise ratio as the detection limit of the method.

Comparative example 1

Accurately weighing 10 +/-0.01 mg of tebuconazole amide and a standard substance of a metabolite thereof in a 10mL volumetric flask, dissolving the standard substance with acetonitrile and fixing the volume to 10mL, preparing 1000.0 mu g/mL standard stock solution, transferring 1.0mL of the standard stock solution into the 10mL volumetric flask, fixing the volume with acetonitrile to obtain 10.0 mu g/mL standard intermediate solution, gradually diluting the 10.0 mu g/mL standard solution with acetonitrile to prepare a series of mixed standard solutions with the concentrations of 1.0, 0.5, 0.1, 0.05 and 0.001 mu g/mL, injecting the standard solutions, detecting by HPLC-MS/MS, and drawing a solvent standard curve of the tebuconazole amide and the metabolite thereof, wherein the rest is the same as in example 1.

The measurement results in example 1 show that fig. 1 is an HPLC-MS/MS selective ion chromatogram of a fresh corn test solution containing tebuconazole amide and a metabolite thereof (0.025 mu g/mL), fig. 2 is an HPLC-MS/MS selective ion chromatogram of a fresh corn blank sample matrix solution without tebuconazole amide and a metabolite thereof, fig. 3 is an HPLCMS/MS selective ion chromatogram of a fresh corn blank sample added with tebuconazole amide and a metabolite standard substance (0.05mg/kg), which shows that tebuconazole amide and a metabolite thereof in the corn test solution are consistent with the retention time of chromatographic peaks of tebuconazole amide and a metabolite standard substance, and the ion abundance ratio of quantitative ions and qualitative ions is consistent with the ion abundance ratio of the standard substance with similar concentration.

FIG. 4 is a matrix calibration curve for the tetrazolium amide and its metabolites of example 1, with the formula: 8573051.47x +32806.28, R²＝0.9996。

The results of the spiking recovery and repeatability tests for example 1 are shown in table 5.

TABLE 5 recovery and reproducibility of tebuconazole amide and its metabolites in fresh corn

As can be seen from Table 5, the average recovery rate of the tetrazolium amide is 89.8-95.1%, the average Relative Standard Deviation (RSD) is 1.8-3.4%, the average recovery rate of the tetrazolium amide metabolite (BCS-CQ63359) is 84.0-91.8%, and the average Relative Standard Deviation (RSD) is 0.8-5.9% at 3 standard adding levels, which indicates that the method for detecting the residual quantity of the tetrazolium amide and the metabolite thereof has high recovery rate and good repeatability.

The detection limit test result of the example 1 shows that the detection limit of the tebuconazole and the metabolite thereof of the fresh corn is 0.13 mug/kg.

Results of the measurement in example 2: FIG. 5 shows an HPLC-MS/MS selective ion chromatogram of a rice test solution of tetrazolium amide and its metabolites (0.1. mu.g/mL); FIG. 6 is an HPLC-MS/MS selective ion chromatogram of a rice blank sample matrix solution without tetrazolium amide and its metabolites; FIG. 7 is an HPLCMS/MS selective ion chromatogram of a rice blank sample added with tebuconazole and a standard substance (0.5mg/kg) of a metabolite thereof, which shows that the tebuconazole and the metabolite thereof in a rice test solution are consistent with the retention time of chromatographic peaks of the tebuconazole and the standard substance of the metabolite thereof, and the ion abundance ratio of quantitative ions and qualitative ions is consistent with the ion abundance ratio of the standard substance with similar concentration.

FIG. 8 is a matrix calibration curve for the tetrazolium amide and its metabolites of example 2, with the formula: 9010410.71x +61886.34, R²＝0.9983。

The results of the spiking recovery and repeatability tests for example 2 are shown in table 6.

TABLE 6 recovery and reproducibility of tebuconazole amide and its metabolites in rice

As can be seen from Table 6, at 3 levels of standard addition, the average recovery rate of the tetrazolium amide is 90.1-96.7%, the average Relative Standard Deviation (RSD) is 2.6-3.5%, the average recovery rate of the tetrazolium amide metabolite (BCS-CQ63359) is 86.4-93.3%, and the average Relative Standard Deviation (RSD) is 1.2-1.7%, which indicates that the recovery rate of the method is high and the repeatability is good.

Detection limit test results of example 2, the detection limit of tebuconazole and its metabolite in rice is 0.1 mug/kg.

Measurement results of comparative example 1: the solvent standard curve of tebuconazole amide and metabolite thereof as shown in figure 10 is represented by the formula: 8133832.49x +16948.44, R²Reference is made to figure 9 for an HPLC-MS/MS total ion chromatogram of a solvent standard solution of tebuconazole amide and its metabolites 0.9996.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.