Method for quantitatively determining residual quantity of afidopyropen and metabolite M440I007 of afidopyropen in broken tea soup by PVPP (Polyvinyl Pyrrolidone) combined Cleanert TPT (Triphenyltin) solid-phase extraction method

Abstract

The invention discloses a method for quantitatively determining residual quantity of afidopyropen and metabolite M440I007 of afidopyropen in broken tea soup by PVPP (Polyvinyl Pyrrolidone) combined with Cleanert TPT (Triphenyltin) solid-phase extraction. The method comprises the steps of sample extraction, sample purification, UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) determination, blank test, result calculation and expression, judgment and the like. According to the method, firstly, a detection sample is purified through PVPP solid-phase dispersion and a Cleanert TPT solid-phase extraction column, and co-leaching substances which interfere with target object detection in the broken tea soup can be effectively purified; secondly, on the basis, specific UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) detection conditions aiming at a target object are adopted; and finally, the extraction efficiency is high, the purification effect is excellent, and the detection requirements in the aspects of recovery rate, detection limit, precision and the like can be met.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and specifically relates to a method for determining the residual amount of diprophyllin and its metabolite M440I007 in broken tea soup by combining PVPP with Cleanert TPT solid phase extraction technology. Background Art

[0002] Dimethoate is a new type of biogenic insecticide, which was registered on tea trees in September 2023 for the control of tea green leafhoppers. It will metabolize on tea leaves to produce M440I007, which is similar to its toxicity. At present, there are few studies on the detection methods of dimethoate and M440I007 in tea. Existing literature reports only focus on the detection in fresh tea leaves, dry tea, tea tree roots, stems and whole tea soup. This method cannot meet the recovery requirements in broken tea soup. Due to the lack of analytical methods for dimethoate and M440I007 in broken tea soup, it is impossible to monitor the residues, leaching rates and dietary risk assessments in broken tea soup. Therefore, it is of great significance to establish an analytical method for dimethoate and M440I007 in broken tea soup.

[0003] The molecular structures of abicyclon (A) and its metabolite M440I007 (B) are shown below: Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for the quantitative determination of the residues of cyclohexypropyl and its metabolite M440I007 in broken tea soup by using PVPP (cross-linked polyvinyl pyrrolidone) combined with Cleanert TPT solid phase extraction method. The method can effectively purify the co-extracted substances in the broken tea soup that interfere with the detection of the target object through solid phase dispersion of PVPP and purification by Cleanert TPT solid phase extraction column. The method is simple to operate, has excellent purification effect, can meet the requirements of residue detection in terms of recovery rate, detection limit and precision, and can provide technical support for the determination of cyclohexypropyl and M440I007 in broken tea soup.

[0005] In order to achieve the above object, the present invention specifically adopts the following technical solutions: A method for quantitatively determining the residues of diprofenopterin and its metabolite M440I007 in broken tea soup by using PVPP combined with Cleanert TPT solid phase extraction method, comprising the following steps: 1) Sample extraction Preparation of green tea, black tea and oolong tea soup: crush green tea, black tea and oolong tea, brew with water, filter to obtain tea soup; extract with acetonitrile, add PVPP (cross-linked polyvinyl pyrrolidone) for purification, take the supernatant after centrifugation, repeat the extraction once, combine the supernatant and evaporate to dryness by rotary evaporation; then re-dissolve with a mixed solvent of acetonitrile and benzene, filter through a membrane, and wait for purification; 2) Sample purification Take a Cleanert TPT column, pre-elute it with a mixed solvent of acetonitrile and benzene, load the complex solution obtained in step 1) onto the column, elute it with a mixed solvent of acetonitrile and benzene, collect all the column liquid and the eluate, rotary evaporate to dryness, and make up the volume with acetonitrile for UPLC-MS / MS testing; 3) UPLC-MS / MS determination The matrix-matched standard solution and sample solution were injected under the following LC-MS / MS conditions: A) HPLC-MS / MS instrument parameters: High performance liquid chromatography: Acquity UPLC HSS-T3 column was used for chromatographic separation; Mass spectrometry conditions: Multiple reaction monitoring mode MRM and ESI + The pattern was used to analyze abicyclon and M440I007; B) Mass spectrometric parameters of abicyclon and its metabolite M440I007 Table represents the quantitative ion; 4) Blank test Except that no sample is added, the above steps 1), 2), and 3) are followed; 5) Calculation and presentation of results Calculate the mass concentrations of abicyclon and metabolite M440I007 in the samples; 6) Judgment Draw a standard working curve. If the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix-matched standard solution, it can be determined that the target substance is present.

[0006] Furthermore, the step 1) sample extraction specifically includes: Green tea, black tea and oolong tea were crushed, brewed at a tea-water ratio of 1:50, and filtered to obtain tea soup; 20 mL of tea soup was taken into a 50 mL polytetrafluoroethylene centrifuge tube, 20 mL of acetonitrile was added, mixed, 200 mg of PVPP was added, vortexed for 5 min, 7 g of NaCl was added, vortexed for 2 min, centrifuged at 5000 r / min for 5 min, and all the supernatant was taken; the extraction was repeated once, the supernatants were combined and rotary evaporated to dryness; re-dissolved with 5 mL of a mixed solvent with a volume ratio of acetonitrile:benzene of 3:1, filtered through a 0.22 μm filter membrane, and purified.

[0007] Furthermore, the step 2) sample purification specifically includes: The Cleanert TPT column was pre-eluted with 5 mL of a mixed solvent of acetonitrile:benzene in a volume ratio of 3:1. The complex solution was loaded onto the column and eluted with 30 mL of a mixed solvent of acetonitrile:benzene in a volume ratio of 3:1. All the column liquid and the eluent were collected and rotary evaporated to dryness at 40°C. The volume was fixed with 1 mL of acetonitrile for UPLC-MS / MS testing.

[0008] Furthermore, the specifications of the chromatographic column in step 3) are 100 mm×2.1 µm, 1.8 µm; Waters, Milford, MA, USA.

[0009] Furthermore, in the step 3), the high performance liquid chromatography is as follows: the mobile phase is 0.1% formic acid acetonitrile A and water B, the flow rate is 0.25 mL / min, the gradient elution program is 60%-99% A 0.0-3.0 min, 99% A 3.0-4.5 min, 60% A 5.0-6.5 min; the column temperature is 40°C, and the injection volume is 5 μL.

[0010] Furthermore, the mass spectrometry conditions in step 3) are: electrospray voltage: 3.0 kV; ion source temperature: 150°C; desolvation temperature: 350°C; desolvation gas and cone backflush gas are nitrogen, with flow rates of 700 L / hr and 60 L / hr, respectively; collision gas (Ar) flow rate: 0.35 mL / min.

[0011] Furthermore, the formula for calculating the mass concentration of abicyclonil and metabolite M440I007 in the sample in step 5) is as follows, and the blank value must be deducted from the processing result: Where: X i ——The residual amount of the target substance in the sample, in milligrams per kilogram; C i ——The concentration of the target substance in solution obtained from the standard curve, in milligrams per liter; V – the final volume of the sample solution, in milliliters; m – the mass of the sample represented by the final sample solution, in grams.

[0012] Furthermore, the step 6) specifically includes: With mass concentration X as the horizontal axis and peak area ratio Y as the vertical axis, a 5-point standard working curve is drawn, and the sample is quantitatively determined using the standard working curve. The response value of the analyte in the sample solution meets the linear range of instrument detection; quantitative and qualitative ion pairs exist at the same time, and the signal-to-noise ratio is ≥3; the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix matching standard solution, and the allowable deviation is less than ±0.5%, then it can be determined that the target substance is contained.

[0013] The measuring method of the present invention has the following beneficial effects: The present invention firstly purifies the test sample through solid phase dispersion of PVPP and Cleanert TPT solid phase extraction column, which can effectively purify the co-extracted substances in the broken tea soup that interfere with the detection of the target object; secondly, on this basis, the present invention adopts UPLC-MS / MS detection conditions specific to the target object; finally, not only the extraction efficiency is high and the purification effect is excellent, but also the recovery rate, detection limit and precision can meet the detection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the mass spectrum of the standard sample of abicyclon and M440I007, with a concentration of 0.0008 mg / L; Figure 2 This is the effect of PVPP dosage on the recovery of abicyclon and M440I007. DETAILED DESCRIPTION

[0015] The present invention is further described below with reference to the examples.

[0016] Example 1: Establishment of mass spectrometry conditions ESI +In the full scan mode, the parent ions of cyclohexanone and M440I007 were obtained at m / z 594.2 and 1187.8, respectively. The cone voltage (20-80V) was further optimized, and the responses of cyclohexanone and M440I007 were the highest at 62 and 35 V, respectively. The parent ions were subjected to secondary mass spectrometry fragmentation product ion scanning, and the collision fragmentation energy (10-70 eV) was optimized to obtain the most abundant characteristic product ions of cyclohexanone ions m / z 148.0 and 68.9. The optimal collision energy was 52 V and 50 V, respectively, and there was no matrix interference at m / z 594.2>148.0 and 594.2>68.9. Therefore, the m / z 594.2>148.0 with the highest response was selected as the quantitative ion pair, and 594.2>68.9 was selected as the qualitative ion pair. The qualitative and quantitative ion pairs of the metabolite M440I007 were optimized in the same way. See attached for mass spectrum Figure 1 .

[0017] Example 2: Detection of diprofenoxam and M44I007 in broken tea soup by Cleanert TPT solid phase extraction The Cleanert TPT solid phase extraction method was directly used to detect the recovery rates of diprofen and M44I007 in broken tea soup, and the results are shown in Table 1. When only the Cleanert TPT column was used to purify the broken tea soup, the recovery rate of diprofen in the broken tea soup of the three types of tea was less than 77.18%, among which the lowest recovery rate in the oolong tea matrix was only 36.92%. The recovery rate of M440I007 in the broken tea soup of the three types of tea was less than 78.12%, among which the lowest recovery rate in the black tea matrix was only 62.49%, which could not meet the recovery rate requirements in pesticide residue analysis, and its pretreatment method needed to be optimized.

[0018] Table 1 shows the recoveries of diprofenac and M44I007 in broken tea soup by Cleanert TPT solid phase extraction (addition level was 0.2 or 2 mg / kg, n=3)

[0019] Example 3: Detection of diprofenoprene and M44I007 in broken tea soup by PVPP dispersed solid phase extraction Taking oolong tea soup as an example, the recovery rates of dimethoate and M44I007 in broken tea soup were compared using only PVPP dispersed solid phase extraction and PVPP combined with CleanertTPT solid phase extraction, and the results are shown in Table 2. When only PVPP dispersed solid phase extraction was used for purification, the recoveries of dimethoate and M440I007 were 67.04% and 65.53%, respectively; when PVPP was combined with Cleanert TPT solid phase extraction, the recoveries of the two target substances in the broken tea matrix were increased to more than 80%, so PVPP followed by Cleanert TPT solid phase extraction was used for pretreatment of the broken tea matrix.

[0020] Table 2 compares the recoveries of diprofenac and M44I007 in broken tea soup using PVPP dispersive solid phase extraction and PVPP combined with Cleanert TPT solid phase extraction (addition level 0.2 mg / kg, n=3)

[0021] Example 4: Optimization of PVPP dosage Taking oolong tea soup as an example, the PVPP dosage was set to 0, 50, 100, 200, 300 and 400 mg for preliminary purification in the tea soup. Combined with the Cleanert TPT solid phase extraction method, it was found that as the PVPP dosage increased from 0-50 mg to 200 mg, the recovery rate increased significantly (P<0.05). When the dosage of 200 mg PVPP was used, the recovery rate of diprofenoxam reached the highest of 88.15% (RSD=5.89%); there was no significant difference in the recovery rate of M440I007 when the dosage of 200-400 mg PVPP was used (P>0.05). Therefore, 200 mg PVPP was selected as the preliminary purification filler in the tea soup matrix.

[0022] 1. Reagents and Materials 1.1 Acetonitrile: chromatographic grade.

[0023] 1.2 Benzene: chromatographically pure.

[0024] 1.3 Anhydrous sodium chloride: analytical grade.

[0025] 1.4 PVPP (CAS: 25294-54-1) USP grade.

[0026] 1.5 Cleanert TPT solid phase extraction column: (1 g / 6 mL).

[0027] 1.6 Afidopyropen (CAS: 915972–17-7, C 33 H 39NO 9 ):98.7%.

[0028] 1.7 M440I007 Standard Material (C 65 H 76 N 2 O 18 ): purity 90.2%.

[0029] 1.8 Standard stock solution: Accurately weigh 0.010 g (accurate to 0.0001 g) of each standard substance (1.6 and 1.7) into a 50 mL volumetric flask, dilute to volume with acetonitrile to prepare a 100 mg / L stock standard solution, and store at -20 °C.

[0030] 1.9 Mixed standard solution: Accurately measure 5 mL (accurate to 0.01 mL) of the standard stock solution (1.8) into a 25 mL volumetric flask, dilute to volume with acetonitrile to prepare a 20 mg / L stock standard solution, and store at -20 °C.

[0031] 1.10 Preparation of standard intermediate solution: Accurately pipette an appropriate amount of standard stock solution (1.9), prepare a 4 mg / L mixed standard solution with acetonitrile and store in a -20 ℃ refrigerator away from light.

[0032] 1.11 Preparation of standard working solution: Dilute the standard intermediate solution (1.10) with acetonitrile to a standard working solution of appropriate concentration as required.

[0033] 1.12 Microporous filter membrane: 0.22μm, organic phase.

[0034] 2. Instruments and equipment 2.1 High performance liquid chromatography-mass spectrometry / mass spectrometer: equipped with (ESI) ion source.

[0035] 2.2 Analytical balance: sensitivity 0.00001g and 0.01g.

[0036] 2.3 Vortex instrument.

[0037] 2.4 Solid sample crusher.

[0038] 2.5 Centrifuge.

[0039] 2.6 Ultrasonic device.

[0040] 3. Determination steps 3.1 Sample extraction Preparation of green tea, black tea and oolong tea soup: Grind green tea, black tea and oolong tea, brew them at a tea-water ratio of 1:50, and filter to obtain tea soup. Take 20 mL of tea soup in a 50 mL polytetrafluoroethylene centrifuge tube, add 20 mL of acetonitrile, mix well, add 200 mg PVPP, vortex for 5 min, add 7 g NaCl, vortex for 2 min, centrifuge at 5000 r / min for 5 min, and take all the supernatant. Repeat the extraction once, combine the supernatants and evaporate to dryness. Redissolve with 5 mL of acetonitrile: benzene 3:1 (v:v), filter through a 0.22 μm filter membrane, and wait for purification.

[0041] 3.2 Sample purification The Cleanert TPT column was pre-eluted with 5 mL of acetonitrile:benzene 3:1 (v:v), the complex solution was loaded onto the column, and eluted with 30 mL of acetonitrile:benzene 3:1 (v:v), all the column liquid and eluent were collected, rotary evaporated to dryness at 40°C, and the volume was made up to 1 mL of acetonitrile for UPLC-MS / MS testing.

[0042] 3.3 Determination Since the test conditions depend on the instrument used, it is impossible to give universal parameters for chromatographic analysis. The following operating conditions have been proven to be suitable for the test. See Appendix 3 and Table 4 for specific parameters.

[0043] Table 3 Liquid chromatography parameters

[0044] Table 4 Mass spectrometry parameters

[0045] 4. Blank test The above steps were followed except that no sample was added.

[0046] 5. Calculation and presentation of results The contents of diprofenac and M440I007 in the broken tea soup were calculated according to the following formula, and the blank value must be deducted from the processing result: Where: X i ——The residual amount of the target substance in the sample, in milligrams per kilogram; C i ——The concentration of the target substance in solution obtained from the standard curve, in milligrams per liter; V – the final volume of the sample solution, in milliliters; m – the mass of the sample represented by the final sample solution, in grams.

[0047] The contents of acyclovir and M440I007 in the broken tea matrix were determined by UPLC-MS / MS. The peak areas of the standard samples of 0.0008 mg / L, 0.002 mg / L, 0.01 mg / L, 0.04 mg / L, 0.4 mg / L, 1 mg / L and 2 mg / L were determined respectively. The standard curves were drawn based on the response values ​​of acyclovir and M440I007 contents, which were y = 671719x -3764.9 (R 2 = 0.9999) and y = 1,251,345 x - 14,599 (R 2 = 1), the peak area of ​​the sample to be tested is 1000, and the standard curve is substituted to calculate, C 双丙环虫酯 and C M440I007 0.007 mg / L and 0.012 mg / L respectively, V is 1 mL, m is 0.4 g, substitute into the formula, X 双丙环虫酯 =0.018 mg / kg, X M440I007 =0.031 mg / kg.

[0048] 6. Determination limit The lower limit of determination of this method is 0.002 mg / kg.

[0049] 7. Recovery rate Three addition concentrations were set for the recovery experiment of this method. Five experiments were conducted for each addition concentration according to the experimental conditions determined by this method. The recovery and precision results are shown in Table 5.

[0050] Table 5 The range of added concentration and recovery rate of diprofenoxam and M440I007 in broken tea soup

[0051] Example 5: Application Example 5 days after the collection of abiprofen (5% abiprofen dispersible liquid, application rate 22.5 g ai / hm 2 ) fresh tea leaves were used to prepare oolong tea. The residual amount of cyclohexanone and its metabolites in the broken oolong tea soup was determined according to the method of the present invention, and the leaching rate was calculated. The leaching rate of cyclohexanone was 16.0%-20.7%; the leaching rate of the metabolite M440I007 was 4.3%-6.0%.

[0052] The above description is a preferred embodiment of the present invention, which is only used to explain the present invention and is not used to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction, characterized in that: The following steps are involved: 1) Sample extraction Preparation of green tea, black tea and oolong tea soup: crush green tea, black tea and oolong tea, brew with water, filter to obtain tea soup; extract with acetonitrile, add PVPP for purification, take the supernatant after centrifugation, repeat the extraction once, combine the supernatant and evaporate to dryness; then re-dissolve with a mixed solvent of acetonitrile and benzene, filter through a membrane, and wait for purification; 2) Sample purification Take a Cleanert TPT column, pre-elute it with a mixed solvent of acetonitrile and benzene, load the complex solution obtained in step 1) onto the column, elute it with a mixed solvent of acetonitrile and benzene, collect all the column liquid and the eluate, rotary evaporate to dryness, and make up the volume with acetonitrile for UPLC-MS / MS testing; 3) UPLC-MS / MS determination The matrix-matched standard solution and sample solution were injected under the following LC-MS / MS conditions: A) HPLC-MS / MS instrument parameters: High performance liquid chromatography: Acquity UPLC HSS-T3 column was used for chromatographic separation; Mass spectrometry conditions: Multiple reaction monitoring mode MRM and ESI + The pattern was used to analyze abicyclon and M440I007; B) Mass spectrometric parameters of abicyclon and its metabolite M440I007 Table represents the quantitative ion; 4) Blank test Except that no sample is added, the above steps 1), 2), and 3) are followed; 5) Calculation and presentation of results Calculate the mass concentrations of abicyclon and metabolite M440I007 in the samples; 6) Judgment Draw a standard working curve. If the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix-matched standard solution, it can be determined that the target substance is present.

2. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The step 1) sample extraction specifically includes: Green tea, black tea and oolong tea were crushed, brewed at a tea-water ratio of 1:50, and filtered to obtain tea soup; 20 mL of tea soup was taken into a 50 mL polytetrafluoroethylene centrifuge tube, 20 mL of acetonitrile was added, mixed, 200 mg of PVPP was added, vortexed for 5 min, 7 g of NaCl was added, vortexed for 2 min, centrifuged at 5000 r / min for 5 min, and all the supernatant was taken; the extraction was repeated once, the supernatants were combined and rotary evaporated to dryness; re-dissolved with 5 mL of a mixed solvent with a volume ratio of acetonitrile:benzene of 3:1, filtered through a 0.22 μm filter membrane, and purified.

3. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The step 2) sample purification specifically includes: The Cleanert TPT column was pre-eluted with 5 mL of a mixed solvent of acetonitrile:benzene in a volume ratio of 3:

1. The complex solution was loaded onto the column and eluted with 30 mL of a mixed solvent of acetonitrile:benzene in a volume ratio of 3:

1. All the column liquid and the eluent were collected and rotary evaporated to dryness at 40°C. The volume was fixed with 1 mL of acetonitrile for UPLC-MS / MS testing.

4. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The specifications of the chromatographic column in step 3) are 100 mm×2.1 µm, 1.8 µm; Waters, Milford, MA, USA.

5. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: In the step 3), the high performance liquid chromatography is as follows: the mobile phase is 0.1% formic acid acetonitrile A and water B, the flow rate is 0.25 mL / min, the gradient elution program is 60%-99% A 0.0-3.0 min, 99% A 3.0-4.5 min, 60% A 5.0-6.5 min; the column temperature is 40°C, and the injection volume is 5 μL.

6. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The mass spectrometry conditions in step 3) are as follows: electrospray voltage: 3.0 kV; ion source temperature: 150°C; desolvation temperature: 350°C; desolvation gas and cone backflush gas are nitrogen, with flow rates of 700 L / hr and 60 L / hr, respectively; Collision gas Ar flow rate: 0.35 mL / min.

7. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The formula for calculating the mass concentration of abicyclonil and metabolite M440I007 in the sample in step 5) is as follows, and the blank value must be deducted from the processing result: Where: X i ——The residual amount of the target substance in the sample, in milligrams per kilogram; C i ——The concentration of the target substance in solution obtained from the standard curve, in milligrams per liter; V – the final volume of the sample solution, in milliliters; m – the mass of the sample represented by the final sample solution, in grams.

8. The method for quantitatively determining the residues of diprofenoprene and its metabolite M440I007 in broken tea soup by PVPP combined with Cleanert TPT solid phase extraction as claimed in claim 1, characterized in that: The step 6) specifically includes: With mass concentration X as the horizontal axis and peak area ratio Y as the vertical axis, a 5-point standard working curve is drawn, and the sample is quantitatively determined using the standard working curve. The response value of the analyte in the sample solution meets the linear range of instrument detection; quantitative and qualitative ion pairs exist at the same time, and the signal-to-noise ratio is ≥3; the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix matching standard solution, and the allowable deviation is less than ±0.5%, then it can be determined that the target substance is contained.

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