Method for identification and chiral resolution of enantiomers of three metabolites in indoxacarb rat

Abstract

The invention provides a method for identification and chiral resolution of enantiomers of three new metabolites in an indoxacarb rat, and belongs to the technical field of pesticide metabolite analysis. Three metabolite enantiomers in an indoxacarb rat are separated and identified by adopting a high performance liquid chromatography-mass spectrometry technology, a chromatographic column is a polysaccharide derivative chiral chromatographic column, a mobile phase A is an organic solvent, a mobile phase B is a 5mM ammonium acetate aqueous solution, the volume ratio of the mobile phase A to the mobile phase B is 80: 20-85: 15, the column temperature is 35 DEG C, and the flow velocity is 0.8 mL / min. The invention provides a method for effectively identifying and separating enantiomers of three metabolites in a chiral indoxacarb rat, and the method has the advantages of high sensitivity, simplicity in operation, high separation speed and the like.

Description

Technical Field

[0001] This invention belongs to the field of pesticide metabolite analysis technology, specifically involving the identification and chiral resolution method of three enantiomers of indoxacarb metabolites in rats, which is applicable to pesticide toxicology research, environmental monitoring and food safety assessment. Background Technology

[0002] Statistics show that chiral pesticides currently account for approximately 40% of the total pesticide use in my country, the vast majority of which are racemic mixtures of enantiomers, with insecticides having the highest proportion. It is noteworthy that although enantiomers of chiral pesticides possess similar physicochemical properties, they often exhibit significant stereoselectivity differences in biological activity, ecotoxicity, and environmental behavior. Therefore, in-depth research into the characteristics of chiral pesticides at the enantiomer level is of significant theoretical and practical importance for developing environmentally friendly, highly efficient, low-toxicity, optically pure monomeric pesticides.

[0003] After pesticide application, its active ingredients enter the environment or remain in agricultural products through various pathways, undergoing complex metabolic, degradation, and transformation processes to produce a series of structurally diverse metabolites. These metabolites not only have complex formation mechanisms, but their potential ecological risks are also difficult to fully assess. This makes the safety research of degradation products and metabolites a crucial aspect of modern pesticide risk management. In fact, both my country's residue definition for fipronil and the EU's residue definition for dimethyl polyphenols have incorporated the total amount of degradation products, metabolites, and their derivatives into their residue assessment systems, fully highlighting the importance of metabolite research.

[0004] Indoxacarb, a novel oxadiazine insecticide developed by DuPont, works by specifically blocking the Na+ receptors in insect nerve cells. + The pesticide exerts its insecticidal effect through a channel. It can be widely applied to various crops such as tea, cabbage, tomato, and cotton. Of particular note is that the indoxacarb molecule contains a chiral center, resulting in two enantiomers, S and R. Studies have found that only S-(+)-indoxacarb exhibits significant insecticidal activity, while R-(-)-indoxacarb shows almost no insecticidal activity and is considered ineffective. This characteristic makes establishing a chiral resolution method for indoxacarb and its metabolites particularly important, laying a methodological foundation for in-depth research on its stereoselective environmental behavior.

[0005]

[0006] Currently, research on indoxacarb degradation products and metabolites remains relatively scarce. The European Food Safety Authority (EFSA), in its 2019 risk assessment report on indoxacarb, specifically highlighted a significant data gap regarding systematic research on indoxacarb metabolites, particularly its metabolic pathways, transformation mechanisms, and bioaccumulation in mammals. This knowledge gap severely restricts the scientific community's comprehensive understanding and accurate assessment of indoxacarb's environmental fate (including its migration and transformation patterns in different environmental media) and potential ecotoxicological effects. Summary of the Invention

[0007] This invention provides a method for the identification and chiral resolution of three enantiomers of indoxacarb in rats, using high performance liquid chromatography-mass spectrometry to identify and separate the R and S configurations of the three metabolites.

[0008] The technical solution adopted in this invention is:

[0009] A method for identifying and chirally resolving the enantiomers of three indoxacarb metabolites in rats, including the following steps:

[0010] 1) Preparation of test solution: Weigh indoxacarb, dissolve in an organic solvent to prepare indoxacarb test solution with a concentration of 10-500 ng / mL; add acetone and rat blood to the test solution, incubate in a shaker at 37℃, and then add acetonitrile pre-cooled to -20℃ to terminate the reaction; collect rat tissues, add acetonitrile solution for sample processing, collect the samples, and perform HPLC-QTOF / MS analysis;

[0011] 2) Metabolic mass spectrometry was performed using an Agilent 6530 QTOF / MS mass spectrometer in both positive and negative ion electrospray ionization (ESI) modes, with automated MS / MS scanning. The full scan range was 50-1000 m / z, capillary voltage was ±3.5 Kv, dry gas flow rate was 9 L / min, temperature was 350℃, nebulizer gas pressure was 45 psi, sheath gas flow rate was 11 L / min, temperature was 400℃, nitrogen was used as the auxiliary gas and nebulizer gas, fragment voltage was 150 V, and collision energy (CE) was set to three gradients: 20 eV, 40 eV, and 60 eV.

[0012] 3) Chromatographic separation was performed using a 120 EC-C18 column with dimensions of 4.6 mm × 100 mm and a diameter of 2.7 μm. Mobile phase A was an acetonitrile-formic acid solution with a volume ratio of 100:0.1, and mobile phase B was a water-formic acid solution with a volume ratio of 100:0.1. The flow rate was 0.5 mL / min, the column temperature was 30℃, the injection chamber temperature was 4℃, the injection volume was 20 μL, and the total acquisition time was 60 min.

[0013] 4) High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to separate the enantiomers of three metabolites of indoxacarb in rats. The stationary phase was a chiral column of polysaccharide derivatives, the mobile phase A was an organic solvent, the mobile phase B was an aqueous solution of 5 mM ammonium acetate, the column temperature was 30-35℃, and the flow rate was 0.6-0.9 mL / min.

[0014] 5) Use an ultraviolet detector or a mass spectrometer for determination.

[0015] Furthermore, in the above-mentioned three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, in step 1), the organic solvent is one of methanol and acetonitrile.

[0016] Furthermore, in the above three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, in step 1), the concentration of the indoxacarb test solution is 500 ng / mL.

[0017] Furthermore, in step 2) of the above three methods for identifying enantiomers and chiral resolution of indoxacarb metabolites in rats, the metabolite mass spectrometry analysis mode is positive ion electrospray ionization (ESI) mode.

[0018] Furthermore, in step 3) of the above three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, the mobile phase is calculated by volume ratio, A:B is 5:95-100:0, and gradient elution is performed.

[0019] Furthermore, in the above-mentioned three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, in step 4), the organic solvent is acetonitrile.

[0020] Furthermore, in step 4) of the above-mentioned three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, the mass spectrometry conditions are as follows: sheath gas temperature 400℃, nozzle voltage 500 V, nebulizing gas pressure 45 psi, capillary voltage 3500 V in positive ion mode, drying gas temperature 350℃, and drying gas flow rate 7 L / min.

[0021] Furthermore, in step 4) of the above-mentioned three methods for the identification and chiral resolution of enantiomers of indoxacarb metabolites in rats, the mobile phase was calculated by volume ratio, A:B was 80:20-85:15, gradient elution was performed, the column temperature was 35℃, the flow rate was 0.8 mL / min, and the collection time was 10 min.

[0022] Furthermore, in step 5) of the above-mentioned three methods for identifying enantiomers and chiral resolution of indoxacarb metabolites in rats, mass spectrometry was used for determination.

[0023] The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in any one of the above methods, wherein the three enantiomers of indoxacarb metabolites in rats are S-IN-RM294 and R-IN-RM294, S-IN-RM493 and R-IN-RM493, and S-IN-RM541 and R-IN-RM541, respectively, and the structural formulas of S-IN-RM294 and R-IN-RM294, S-IN-RM493 and R-IN-RM493, and S-IN-RM541 and R-IN-RM541 are shown in (I) and (II), (III) and (IV), and (V) and (VI), respectively:

[0024]

[0025]

[0026] .

[0027] The technical solution of this application has the following beneficial effects: This invention uses high performance liquid chromatography-mass spectrometry to directly chirally separate the enantiomers of three indoxacarb metabolites in rats. The mobile phase consists of an aqueous solution of acetonitrile and 5 mM ammonium acetate. This method has the advantages of simple operation, high sensitivity, mild conditions, and short separation time. Attached Figure Description

[0028] Figure 1 It is the lysis pathway of indoxacarb.

[0029] Figure 2 It is the metabolite 1 cleavage pathway.

[0030] Figure 3 This is the chromatographic separation chromatogram of metabolite 1.

[0031] Figure 4 It is the metabolite 2 cleavage pathway.

[0032] Figure 5 This is the chromatographic separation chromatogram of metabolite 2.

[0033] Figure 6 It is the metabolite 3 cleavage pathway.

[0034] Figure 7 This is the chromatographic separation chromatogram of metabolite 3.

[0035] Figure 8 This is a structural diagram of the various metabolites of indoxacarb. Detailed Implementation

[0036] The advantages of the present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments.

[0037] In this embodiment, based on the rapid metabolism of indoxacarb in organisms, a systematic experiment was conducted on its metabolic decomposition process in blood and tissues to further elucidate the metabolic pathways of indoxacarb and its metabolites.

[0038] Preparation of test solution: Weigh indoxacarb, dissolve in an organic solvent to prepare an indoxacarb test solution with a concentration of 500 ng / mL.

[0039] The experiment used an Agilent 6530 QTOF / MS mass spectrometer, performing metabolite mass spectrometry analysis in both positive and negative ion electrospray ionization (ESI) modes, coupled with automated MS / MS scanning. The instrument parameters were set as follows: full scan range 50-1000 m / z, capillary voltage ± 3.5 KV, dry gas flow rate 9 L / min (350℃), nebulizer gas pressure 45 psi, sheath gas flow rate 11 L / min (400℃), nitrogen as auxiliary gas and nebulizer gas, fragment voltage 150 V, and collision energies (CE) set at three gradients: 20 eV, 40 eV, and 60 eV.

[0040] Chromatographic separation was performed using a 120 EC-C18 column (4.6 mm × 100 mm, 2.7 μm). The mobile phase consisted of acetonitrile-formic acid (A, 100:0.1, v / v) and water-formic acid (B, 100:0.1, v / v). The elution program was: 0–55 min, 5–100% A linear gradient; 55–60 min, 100% A isocratic elution. The chromatographic conditions were: flow rate 0.5 mL / min, column temperature 30 °C, injection chamber temperature 4 °C, injection volume 20 μL, and total acquisition time 60 min.

[0041] 1 mg of S-(+)-indoxacarb was added to a 2 mL EP tube, followed by 10 μL of acetone and 500 μL of rat blood. The mixture was incubated at 37°C for 30 min with a shaker, and then the reaction was terminated by adding 800 μL of acetonitrile pre-cooled to -20°C. The sample was processed, and the peak position of the S configuration of the metabolite was determined using UHPLC-QQQ / MS. The peak position of the R configuration of the metabolite was determined using the same method.

[0042] Male rats were administered S-(+)-indoxacarb (10 mg / kg) by gavage, and were euthanized by cervical dislocation 1 h later. Fourteen tissue samples were collected. The samples were processed, and the peak positions of the S-configuration metabolites were identified using UHPLC-QQQ / MS. First, Agilent Optimizer software was used to optimize mass spectrometry parameters such as the precursor ion, daughter ion, fragmentation voltage, and collision energy (CE) of the metabolites, using the metabolite's response value as an indicator. The peak positions of the R-configuration metabolites were determined using the same method.

[0043] The positive ion mode, which provides richer structural information, was chosen for mass spectrometry data acquisition of indoxacarb metabolites. Experimental results showed that three new pairs of indoxacarb enantiomers (metabolites 1-3) were identified in rat blood, stomach, and small intestine tissues, respectively. The retention times (R0) of these metabolites were [not specified in the original text]. t Detailed information on quasi-molecular ions, theoretical mass, predicted molecular formula, and characteristic fragment ions is summarized in Table 1.

[0044] Table 1 Qualitative analysis of metabolites

[0045] Serial Number name Retention time (min) <![CDATA[Precursor ion [M+H] + or [M+Na] + (m / z)]]> Theoretical molecular weight (m / z) Predicted molecular formula Error (ppm) Major fragment ions (m / z) 1 Indoxacarb 36.774 528.0784 528.0780 <![CDATA[C 22 H 17 N3O7ClF3]]> -0.78 293.0320,249.0420,218.0414,203.0183,150.0098 2 Metabolite 1 27.377 295.0849 295.0844 <![CDATA[C 14 H 16 N2O3Cl]]> -1.71 235.0625,150.0105,58.0652 3 Metabolite 2 33.922 494.1174 494.1170 <![CDATA[C 22 H 18 N3O7F3]]> 0.93 462.0915,259.0713,215.0813,116.0495 4 Metabolite 3 38.342 542.0952 542.0936 <![CDATA[C 23 H 19 N3O7ClF3]]> -2.89 510.0679,339.0741,307.0484,263.0592,218.0425,150.0105

[0046] Based on the mass spectrometry data in Table 1, a systematic structural analysis was performed on indoxacarb and its three newly identified enantiomers.

[0047] The indoxacarb molecule contains three fluorine atoms and one chlorine atom; therefore, the structure of its metabolites is primarily analyzed from the perspective of these groups. The quasi-molecular ion of indoxacarb is [M+H]. + Its m / z value is 528.0784 (molecular formula is C). 22 H 17 N3O7ClF3, theoretical molecular weight 528.0780). m / z is 293.0320 (C). 13 H 10 Fragment ions of N₂O₄Cl are generated through the loss of 4-(trifluoromethoxy)benzamide. Furthermore, the m / z is 249.0420 (C). 12 H 10 Fragment ions of N2O2Cl are generated through the loss of carbonyl groups. The fragment ion with m / z 150.0098 (C8H5NCl) is formed due to the cleavage of the six-membered nitrogen-oxygen ring, followed by hybridization and rearrangement. Another cleavage pathway produces a fragment ion with m / z 218.0414 (C9H7NO2F3), which is formed by the formation of 4-(trifluoromethoxy)benzamide, which further decomposes into a fragment ion with m / z 203.0183 (C8H4NO2F3). Detailed mass spectrometric cleavage pathways of indoxacarb are as follows... Figure 1 As shown.

[0048] These characteristic fragment ions provide important evidence for the structural identification of indoxacarb and its metabolites, and also lay a theoretical foundation for elucidating its metabolic transformation mechanism in organisms.

[0049] Mass spectrometry analysis of metabolite 1 (IN-RM294) showed that its quasi-molecular ion [M+H]+ had an m / z value of 295.0849 (molecular formula: C). 14 H16 N₂O₃Cl (theoretical molecular weight: 295.0844). The loss of the formyl group in this metabolite results in the formation of a fragment ion (C₂O₃Cl) with an m / z of 235.0625. 12 H 12 The fragment ion (N2OCl) undergoes a breakage of its six-membered nitrogen-oxygen ring, followed by hybridization and rearrangement, producing a fragment ion (C8H5NCl) with an m / z of 150.0105. This fragment ion (150.0105, C8H5NCl) further decomposes into a fragment ion (C3H8N) with an m / z of 58.0652. For details on the mass spectrometric fragmentation pathway of metabolite 1, please refer to [link to details]. Figure 2 .

[0050] Structural analysis revealed the presence of a chiral center in metabolite 1, thus necessitating the existence of two enantiomers. To accurately identify the enantiomeric configurations of the metabolite, S-(+)-indoxacarb and R-(-)-indoxacarb standards were incubated with rat blood in vitro. Experimental results showed that the metabolite produced by incubation of S-(+)-indoxacarb with blood had a retention time of 7.43 min, which was significantly shorter than that of the previous metabolite. Figure 3 The mid-E1 peak was completely consistent and identified as S-IN-RM294; while the retention time of the metabolite produced by R-(-)-indoxacarb incubation with blood was 9.24 min, which was consistent with... Figure 3 The E2 peak corresponds to and is identified as R-IN-RM294. This result provides reliable experimental evidence for the accurate identification of the enantiomers of metabolite 1.

[0051] Metabolite 2 (IN-RM493) has the chemical formula based on its quasi-molecular ion [M+H] at m / z 494.1174 in its MS / MS spectrum. + The inferred (molecular formula C) 22 H 18 N3O7F3 (theoretical molecular weight 494.1170). m / z 462.0915 (C 21 H 15 Fragment ions of N3O6F3 are produced due to the loss of methoxy groups. m / z 259.0834 (C 13 H 11 The fragment ions of N2O4 are generated by the loss of the 4-(trifluoromethoxy)aniline formyl group. Furthermore, m / z 215.0813 (C 12 H 11 Fragment ions of N2O2 are generated by the loss of a carbonyl group from the fragment ion at m / z 259.0834. Fragment ions at m / z 116.0495 (C8H6N) are generated due to the breaking of a six-membered nitrogen-oxygen ring, followed by hybridization and rearrangement. Structural analysis indicates that metabolite 2 is a product of the substitution of chlorine atoms with hydrogen atoms in the indoxacarb molecule; its cleavage pathway is described in [link to relevant documentation]. Figure 4 .

[0052] Given the presence of a chiral center in metabolite 2, this invention identified its enantiomers through in vivo experiments. Two groups of SD rats were administered S-(+)-indoxacarb and R-(-)-indoxacarb by gavage, respectively, and gastric tissue samples were collected for processing. The results showed that the retention time of the metabolite produced in the S-(+)-indoxacarb group was 6.74 min, which was similar to... Figure 5 The middle E1 peak completely corresponded to S-IN-RM493; while the metabolite produced in the R-(-)-indoxacarb group had a retention time of 7.61 min, which was consistent with... Figure 5 The peaks in the middle E2 region were consistent, and the result was identified as R-IN-RM493. This result provides reliable experimental evidence for the accurate identification of the enantiomers of metabolite 2.

[0053] Mass spectrometry analysis of metabolite 3 (IN-RM541) showed that its quasi-molecular ion [M+H] was present. + The m / z value is 542.0952 (molecular formula: C). 23 H 19 N3O7ClF3 (theoretical molecular weight: 542.0936). This metabolite's six-membered nitrogen-oxygen heterocycle cleaves and loses the CH4O group, yielding an m / z of 307.0484 (C). 14 H 12 Fragment ions were generated at N2O4Cl. Furthermore, a fragment ion with m / z 263.0592 (C) was formed by the loss of a carbonyl group. 13 H 12 Fragment ions at m / z 150.0105 (C8H5NCl) are produced by the cleavage of the six-membered nitrogen-oxygen ring, followed by hybridization and rearrangement. Another cleavage pathway involves the loss of the metabolite 3-methoxy group, resulting in fragment ions at m / z 510.0679 (C8H5NCl). 22 H 16 Fragment ions at N3O6ClF3. The fragment ion at m / z 218.0425 (C9H7NO2F3) is generated from 4-(trifluoromethoxy)anilineformyl. Structural analysis indicates that metabolite 3 is a product of the methoxy group being replaced by an ethoxy group in the indoxacarb molecule, and its cleavage pathway is shown in [reference needed]. Figure 6 .

[0054] Given that metabolite 3 contains a chiral center, this invention identified its enantiomers through in vivo experiments. Two groups of SD rats were administered S-(+)-indoxacarb and R-(-)-indoxacarb by gavage, respectively, and small intestinal tissue samples were collected for processing. The experimental results showed that the retention time of the metabolite produced in the S-(+)-indoxacarb group was 7.87 min, which was similar to... Figure 7The middle E1 peak completely corresponded to S-IN-RM541; while the metabolite produced in the R-(-)-indoxacarb group had a retention time of 9.13 min, which was consistent with... Figure 7 The peaks in the middle E2 region were consistent, identifying it as R-IN-RM541. This result provides reliable experimental evidence for the accurate identification of the enantiomer of metabolite 3.

[0055] The chromatographic conditions for the three metabolites were as follows: a Chiralpak IG column (250 mm × 4.6 mm, 5 μm, Daicel, Japan); a mobile phase consisting of acetonitrile (A) and an aqueous solution containing 5 mM ammonium acetate (B); the acetonitrile concentration linearly increased from 80% to 85% within 4 min and maintained at that concentration for 6 min; a flow rate of 0.8 mL / min; a column temperature of 35 °C; an injection chamber temperature of 4 °C; an injection volume of 20 μL; and a data acquisition time of 10 min. The mass spectrometry conditions were: sheath gas temperature of 400 °C; nozzle voltage of 500 V; nebulizer gas pressure of 45 psi; capillary voltage of 3500 V (positive ion mode); drying gas temperature of 350 °C; and drying gas flow rate of 7 L / min. Multiple reaction monitoring (MRM) mode was used to detect the enantiomeric forms of indoxacarb metabolites.

[0056] Table 2. Retention time, MRM, and detection parameters of indoxacarb metabolites

[0057] compound Retention time (min) Molecular weight (MW) Multiple reaction monitoring (m / z) Decomposition voltage (V) Collision energy (eV) S-IN-RM294 7.43 294 295→150 95 20 R-IN-RM294 9.24 294 295→150 95 20 S-IN-RM493 6.74 493 494→116 95 24 R-IN-RM493 7.61 493 494→116 95 24 S-IN-RM541 7.87 541 542→150 95 20 R-IN-RM541 9.13 541 542→150 95 20

[0058] This invention is the first to identify and isolate three pairs of novel indoxacarb metabolite enantiomers (IN-RM294, IN-RM493, and IN-RM541) in rats, and clarifies the metabolic pathway of indoxacarb. This method has advantages such as high sensitivity, simple operation, and rapid separation speed.

Claims

1. A method for identifying and chirally resolving the enantiomers of three indoxacarb metabolites in rats, characterized in that... Includes the following steps: 1) Preparation of test solution: Weigh indoxacarb, dissolve in an organic solvent to prepare indoxacarb test solution with a concentration of 10-500 ng / mL; add acetone and rat blood to the test solution, incubate in a shaker at 37℃, and then add acetonitrile pre-cooled to -20℃ to terminate the reaction; collect rat tissues, add acetonitrile solution for sample processing, collect the samples, and perform HPLC-QTOF / MS analysis; 2) Metabolic mass spectrometry was performed using an Agilent 6530 QTOF / MS mass spectrometer in both positive and negative ion electrospray ionization (ESI) modes, with automated MS / MS scanning. The full scan range was 50-1000 m / z, capillary voltage was ±3.5 Kv, dry gas flow rate was 9 L / min, temperature was 350℃, nebulizer gas pressure was 45 psi, sheath gas flow rate was 11 L / min, temperature was 400℃, nitrogen was used as the auxiliary gas and nebulizer gas, fragment voltage was 150 V, and collision energy (CE) was set to three gradients of 20 eV, 40 eV, and 60 eV. 3) Chromatographic separation was performed using a 120 EC-C18 column with dimensions of 4.6 mm × 100 mm and a diameter of 2.7 μm. Mobile phase A was an acetonitrile-formic acid solution with a volume ratio of 100:0.1, and mobile phase B was a water-formic acid solution with a volume ratio of 100:0.

1. The flow rate was 0.5 mL / min, the column temperature was 30℃, the injection chamber temperature was 4℃, the injection volume was 20 μL, and the total acquisition time was 60 min. 4) High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to separate the enantiomers of three metabolites of indoxacarb in rats. The stationary phase was a chiral column of polysaccharide derivatives, the mobile phase A was an organic solvent, the mobile phase B was an aqueous solution of 5 mM ammonium acetate, the column temperature was 30-35℃, and the flow rate was 0.6-0.9 mL / min. 5) Use an ultraviolet detector or a mass spectrometer for determination.

2. The method for identifying and chirally separating the three indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 1), the organic solvent is one of methanol and acetonitrile.

3. The method for identifying and chirally separating the three indoxacarb metabolites in rats as described in claim 1, characterized in that... In step 1), the concentration of the indoxacarb test solution is 500 ng / mL.

4. The method for identifying and chirally resolving the three indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 2), the metabolic mass spectrometry analysis mode is positive ion electrospray ionization (ESI) mode.

5. The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 3), the mobile phase is calculated by volume ratio, A:B is 5:95-100:0, and gradient elution is performed.

6. The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 4), the organic solvent is acetonitrile.

7. The method for identifying and chirally resolving the three indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 4), the mass spectrometry conditions are: sheath gas temperature 400℃, nozzle voltage 500 V, nebulizing gas pressure 45 psi, capillary voltage 3500 V in positive ion mode, drying gas temperature 350℃, and drying gas flow rate 7 L / min.

8. The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 4), the mobile phase is calculated by volume ratio, A:B is 80:20-85:15, gradient elution is used, the column temperature is 35℃, the flow rate is 0.8 mL / min, and the collection time is 10 min.

9. The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in claim 1, characterized in that, In step 5), a mass spectrometer detector is used for determination.

10. The method for identifying and chirally resolving the three enantiomers of indoxacarb metabolites in rats as described in any one of claims 1-9, characterized in that, The enantiomers of the three indoxacarb metabolites in rats are S-IN-RM294 and R-IN-RM294, S-IN-RM493 and R-IN-RM493, and S-IN-RM541 and R-IN-RM541, respectively. The structural formulas of S-IN-RM294 and R-IN-RM294, S-IN-RM493 and R-IN-RM493, and S-IN-RM541 and R-IN-RM541 are shown in (I) and (II), (III) and (IV), and (V) and (VI), respectively. 。

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