Separation of butylfluorifen enantiomers and method for analyzing residues in agricultural products and tea beverages

Abstract

The application discloses a method for resolving an enantiomer of butylfluorfon and analyzing residues of the enantiomer in agricultural products and tea beverages. The resolving steps are as follows: preparing a solution of butylfluorfon racemate and monomers thereof; resolving the butylfluorfon racemate and confirming the monomers. The resolution can effectively separate chiral enantiomeric monomers of butylfluorfon, and can be used for analyzing activity, toxicity and residue metabolism of the monomers. The method for analyzing residues of chiral butylfluorfon enantiomers comprises the following steps: extracting and purifying a sample; separating and detecting butylfluorfon enantiomers by using a chromatographic column gradient elution and ultra-high performance liquid chromatography tandem mass spectrometry; calculating a standard curve, a linear correlation coefficient, a matrix effect and an enantiomeric fraction value of butylfluorfon enantiomers in different matrix solutions; and calculating an added recovery, a relative standard deviation, a detection limit and a quantitative limit of the method. The residue analysis method meets the requirement of residue analysis, and can provide an analysis method for detecting and analyzing residues of butylfluorfon enantiomers in agricultural products such as tea leaves and tea beverages.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chiral compound enantiomer resolution and pesticide residue analysis in agricultural products such as tea and tea beverages, and particularly relates to a method for resolution of bifenthrins enantiomers and residue analysis in agricultural products and tea beverages. BACKGROUND

[0002] Bifenthrins (C 24 H 24 F3NO4, CAS No.: 400882-07-7) is a new benzoyl acetonitrile acaricide developed by Nippon Soda Co., Ltd. of Japan, with the trade name Danisraba, which was launched in 2007. It has a high selective activity spectrum against spider mites and red spiders, and also has activity against other phytophagous mites, while being safe to predatory mites and other non-target organisms. In addition, the pesticide has good effectiveness against all developmental stages of the pest mites, and the activity against nymphs is higher than that against adult mites. Moreover, there is no cross-resistance with existing acaricides, and the control effect on pests that have developed resistance is more significant, which has good agricultural application prospects.

[0003] Bifenthrins is a chiral compound with an asymmetrically substituted C atom, consisting of a pair of enantiomers, namely (+)-bifenthrins and (-)-bifenthrins. Generally, one enantiomer has high target activity, while the other may be low, ineffective, or even have the opposite effect, or have different action modes and action sites, showing significant differences in biological activity, toxicity, absorption, transfer, enrichment, degradation, and clearance.

[0004] Currently, research on bifenthrins mainly focuses on racemates, and research on individual enantiomers is less, especially the resolution and residue analysis of enantiomers are still limited to normal phase high performance liquid chromatography (NP-HPLC) and ultra performance combined phase chromatography (UPC 2 ) separation, and there is no research on separation and resolution of bifenthrins enantiomers by reversed-phase liquid chromatography coupled with mass spectrometry for determination of bifenthrins enantiomer residues. The present application establishes a method for separation and determination of bifenthrins enantiomers based on reversed-phase liquid chromatography coupled with mass spectrometry, which not only provides more reliable data for evaluating the fate and potential toxicity of bifenthrins enantiomers, but also provides accurate information for further research and application of single enantiomer pure pesticides. SUMMARY

[0005] In view of the problems in the prior art, the present application aims to provide a technical solution of a method for resolving enantiomers of cyflumetofen and analyzing residues of the enantiomers in agricultural products and tea beverages. The method can resolve (+)- and (-)-enantiomers from a racemic cyflumetofen. Different extraction and purification methods are used to obtain racemic residue samples for different sample matrices, and the residues in the samples are determined by using an ultra-high performance liquid chromatograph coupled with a mass spectrometer.

[0006] The present application is implemented by the following technical solutions:

[0007] The first aspect of the present application provides a method for resolving enantiomers of cyflumetofen, which comprises the following steps:

[0008] 1) Preparation of cyflumetofen racemate and monomer solution: cyflumetofen racemate and monomer samples are weighed and prepared into stock solutions with acetonitrile for standby;

[0009] 2) Resolution of cyflumetofen racemate: the cyflumetofen racemate solution prepared in step 1) is used as a standard solution for injection, and the cyflumetofen racemate is eluted and resolved under certain chromatographic conditions by using an ultra-high performance liquid chromatograph to obtain two peak values, which are two enantiomers in the cyflumetofen racemate;

[0010] The ultra-high performance liquid chromatograph conditions are as follows: a 3μm Cellulose-3 chromatographic column with a size of 150×2mm, a column temperature of 45℃, an injection amount of 5μL, a flow rate of 0.2mL / min, a mobile phase A of 10mmol / L ammonium acetate aqueous solution, a mobile phase B of 0.1% formic acid in methanol, and a gradient elution program of 0-1.5min, 10-75% B; 1.5-5.45min, 75% B; 5.45-5.46min, 75%-50% B; 5.46-7.5min, 50% B; 7.5-9.0min, 50%-100% B, 100% B for 3min; 12.0-13.0, 100%-10% B;

[0011] 3) Confirmation of two monomers of cyflumetofen racemate: the cyflumetofen monomer solution and the racemate solution prepared in step 1) are used as standard solutions for injection, and the retention time of the peaks on the chromatogram is used to determine that (+)-cyflumetofen is eluted first and (-)-cyflumetofen is eluted later.

[0012] Further, the concentration of the cyflumetofen racemate solution in step 1) is 0.0025-200mg / L.

[0013] The second aspect of the present application provides a method for analyzing residues of cyflumetofen enantiomers in agricultural products and tea beverages, which comprises the following steps:

[0014] 1) sample extraction and purification, tea fresh leaves, black tea, green tea, tea soup or other agricultural products were extracted with 1% formic acid acetonitrile, multi-walled carbon nanotubes and nano zirconium oxide dispersion solid phase extraction purification, concentrated with acetonitrile, and detected by reverse phase ultra performance liquid chromatography tandem mass spectrometry with matrix external standard method;

[0015] 2) the prepared diflovidazin racemate solution was diluted with acetonitrile to a standard solution of 20 mg / L, and then the sample blank matrix treated in step 1) was prepared into matrix standard solutions of 2, 0.5, 0.1, 0.05, 0.01 and 0.005 mg / L, and the enantiomeric monomer concentration was half. Ultra performance liquid chromatography tandem mass spectrometry was used for sample analysis, each concentration was determined for 3 times, the concentration was taken as the abscissa x, and the peak area average value was taken as the ordinate y, to obtain the diflovidazin enantiomer standard curve, linear correlation coefficient, matrix effect and enantiomeric fraction value in different matrixes;

[0016] The ultra performance liquid chromatography conditions are as follows: 3 μm Cellulose-3 chromatographic column, size 150×2 mm; column temperature 45℃; injection volume 5 μL; flow rate 0.2 mL / min; mobile phase A is 10 mmol / L ammonium acetate aqueous solution, B is 0.1% formic acid methanol, gradient elution program is: 0-1.5 min, 10-75% B; 1.5-5.45 min, keep 75% B; 5.45-5.46 min, 75%-50% B; 5.46-7.5 min, keep 50% B; 7.5-9.0 min, 50%-100% B, 100% B keep 3 min; 12.0-13.0, 100%-10% B;

[0017] 3) calculate the addition recovery rate, relative standard deviation, method detection limit and quantification limit, meet the requirements of residual analysis; the diflovidazin enantiomers in the actual sample treated according to step 1) were calculated by matrix external standard method to calculate the residual amount.

[0018] Further, the tea fresh leaf extraction and purification in step 1) is as follows:

[0019] After grinding, 5 g of fresh leaves was weighed into a centrifuge tube, 8 mL of 2% formic acid water was added, and it was fully vortexed and mixed, then stood for 10 min, 15 mL of 1% formic acid acetonitrile was added, and it was vortexed and mixed, then oscillated for 5 min, ultrasonic for 10 min, 3 g of NaCl was added, vortexed and mixed, oscillated for 5 min, then centrifuged at 10000 rpm for 5 min, 4.8 mL of the upper organic phase solution was taken and added to a 10 mL centrifuge tube containing 64 mg of multi-walled carbon nanotubes and 24 mg of nano zirconium oxide, vortexed and purified for 1 min, then centrifuged, 3 mL of the supernatant was taken into a 50 mL chicken heart bottle, concentrated and dried, 1 mL of acetonitrile was added, ultrasonic assisted dissolution, 0.22 μm filter membrane was passed into the sample bottle, and UPLC-MS / MS was detected.

[0020] Further, the red green tea extraction purification in step 1) is specifically:

[0021] After grinding, 2 g of black tea was weighed into a centrifuge tube, 10 mL of 2% formic acid water was added, and it was fully vortexed and mixed, then stood for 10 min, 10 mL of 1% formic acid acetonitrile was added, and it was vortexed and mixed, then oscillated for 5 min, ultrasonic for 10 min, 3.5 g of NaCl was added, vortexed and mixed, oscillated for 5 min, then centrifuged at 10000 rpm for 5 min, 6 mL of the upper organic phase solution was taken and added to a 10 mL centrifuge tube containing 80 mg of multi-walled carbon nanotubes and 40 mg of nano zirconium oxide, vortexed and purified for 1 min, then centrifuged, 4 mL of the supernatant was taken into a 50 mL chicken heart bottle, concentrated and dried, 1 mL of acetonitrile was added, ultrasonic assisted dissolution, then frozen in a-24℃ refrigerator for 1.0 hour, taken out immediately after 10000 rpm centrifugation for 2 min, 0.22 μm filter membrane was passed into the sample bottle, and UPLC-MS / MS was detected.

[0022] Further, the tea soup extraction purification in step 1) is specifically:

[0023] The red green tea powder was added to boiling water according to the standard of tea water ratio 1:50, double layer filter paper was filtered twice after 10 min, and tea soup was obtained, 20 mL of tea soup was taken into a 50 mL centrifuge tube, 20 mL of 1% formic acid acetonitrile was added, vortexed and mixed, then oscillated for 5 min, 7 g of NaCl was added, vortexed and mixed, oscillated for 5 min, then centrifuged at 10000 rpm for 5 min, all the upper organic phase solution was taken, 20 mL of 1% formic acid acetonitrile was added for repeated extraction once, vortexed and mixed, oscillated for 5 min, ultrasonic for 10 min, then centrifuged, the supernatant of the two times was combined, concentrated and dried, 2 mL of acetonitrile was added, ultrasonic assisted dissolution, 1.2 mL was taken and added to a 2 mL centrifuge tube containing 16 mg of multi-walled carbon nanotubes and 8 mg of nano zirconium oxide, vortexed and purified for 1 min, then centrifuged at 12000 rpm for 5 min, 0.22 μm filter membrane was passed into the sample bottle, and UPLC-MS / MS was detected.

[0024] Further, the mass spectrometry conditions of the mass spectrometer in step 2) are as follows: electrospray positive ionization multiple reaction monitoring mode ESI + -MRM; electrospray capillary voltage 3.5 kV; ion source temperature 150℃; desolvation gas N2 temperature 350℃, flow rate 700 L / h; cone hole backflush gas N2 flow rate 50 L / h; collision gas Ar flow rate 0.30 mL / min; electron multiplier multiplication voltage 700 V; secondary parent ion residence time 0.1 s; butylfluoride mite enantiomer parent ion m / z 465, cone hole voltage 15 V, quantitative daughter ion m / z 173 and qualitative daughter ion m / z 249, collision energy 20 and 10 eV, respectively.

[0025] The application provides a method for effectively resolving butylfluoride mite enantiomers and applying the same to residue analysis of tea fresh leaves, red and green tea and red and green tea soup. The method is the first time to resolve butylfluoride mite chiral enantiomers by using reverse phase liquid chromatography tandem mass spectrometry at home and abroad. The method is suitable for residue analysis of butylfluoride mite enantiomers in tea and other agricultural products and tea beverages, has the characteristics of rapidness, sensitivity and accuracy, the linear correlation coefficients of standard curves of butylfluoride mite enantiomers in different matrices are all above 0.99, the average addition recovery is 76.3%-116.9%, the relative standard deviation is less than 6.4%, and the method limit of quantification is less than or equal to 0.0025 mg / kg. The method meets the requirements of residue analysis and can provide an analysis method for research and detection of butylfluoride mite enantiomers in tea fresh leaves, red and green tea and red and green tea soup and other agricultural products and tea beverages. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Chromatograms of butylfluoride mite racemate (2.0 mg / L) and its monomers (+)-butylfluoride mite and (-)-butylfluoride mite under ultra-high performance liquid chromatography tandem mass spectrometry;

[0027] Figure 2 Effects of different amounts of GCB, MWCNT and biochar on recovery rate (A) and matrix effect (B) of the target compound;

[0028] Figure 3 Effects of different amounts of nano zirconium oxide + MWCNT (A) and different amounts of PSA + MWCNT and nano zirconium oxide (B) on recovery rate of the target compound;

[0029] Figure 4 Response of the compound under two conditions of removing the precipitate and redissolving the precipitate after low-temperature freezing (the peak height of the former is higher, and the peak height of the latter is lower);

[0030] Figure 5Response of caffeine (A) and theanine (B) in the case of removing precipitate and re-dissolving precipitate after low temperature freezing (the peak height of re-dissolving precipitate is high, and the peak height of removing precipitate is low);

[0031] Figure 6 Effect of different mobile phase ratios on the resolution of the enantiomers of cyflumetofen;

[0032] Figure 7 Effect of different mobile phase ratios on the resolution of the enantiomers of cyflumetofen; DETAILED DESCRIPTION

[0033] The application will be further described in connection with the following specific examples. The examples described in the application are only used to illustrate and explain the application and do not constitute a limitation on the scope of the application.

[0034] Example:

[0035] 1. Experimental section

[0036] 1.1 Instruments and equipment

[0037] UPLC / Quattra Premier XE ultra-performance liquid chromatography-triple quadrupole mass spectrometer, ESI source, MassLynx 4.1 mass spectrometry workstation software (Waters Corporation, USA).

[0038] 1.2 Materials and reagents

[0039] Methanol and acetonitrile are chromatographically pure (Merck, Germany); chromatographically pure formic acid and analytically pure ammonia water (Macklin Biochemical Co., Ltd., Shanghai); chromatographically pure ammonium acetate (Anpel Scientific Technology Co., Ltd., Shanghai); analytically pure sodium chloride (Shanghai Shisihewei Chemical Co., Ltd.); multi-walled carbon nanotubes (>95%, I: 5-10 nm, O: 10-20 nm, Length: 10-30 um, Shanghai Macklin Biochemical Science and Technology Co., Ltd.); nano zirconium oxide (particle size 20-40 nm, Jiangsu Xianfeng Nanometer Material Technology Co., Ltd.); cyflumetofen: purity greater than 95%.

[0040] 1.3 Sample extraction and purification

[0041] 1.3.1 Fresh tea leaf: 5 g of fresh leaf was weighed into a centrifuge tube after grinding, 8 mL of 2% formic acid water was added, vortexed thoroughly, and then stood for 10 min. 15 mL of 1% formic acid acetonitrile was added, vortexed thoroughly, and then oscillated for 5 min, ultrasonic for 10 min, 3 g of NaCl was added, vortexed thoroughly, oscillated for 5 min, and then centrifuged at 10000 rpm for 5 min. 4.8 mL of the upper organic phase solution was taken and added to a 10 mL centrifuge tube containing 64 mg of multi-walled carbon nanotubes and 24 mg of nano zirconium oxide, vortexed for 1 min, and then centrifuged. 3 mL of the supernatant was taken into a 50 mL chicken heart bottle, concentrated to dryness, 1 mL of acetonitrile was added, ultrasonic assisted dissolution was performed, and then 0.22 μm filter membrane was used to filter into a sample bottle. UPLC-MS / MS was used for determination.

[0042] 1.3.2 Black-green tea: 2 g of black tea was weighed into a centrifuge tube after grinding, 10 mL of 2% formic acid water was added, vortexed thoroughly, and then stood for 10 min. 10 mL of 1% formic acid acetonitrile was added, vortexed thoroughly, and then oscillated for 5 min, ultrasonic for 10 min, 3.5 g of NaCl was added, vortexed thoroughly, oscillated for 5 min, and then centrifuged at 10000 rpm for 5 min. 6 mL of the upper organic phase solution was taken and added to a 10 mL centrifuge tube containing 80 mg of multi-walled carbon nanotubes and 40 mg of nano zirconium oxide, vortexed for 1 min, and then centrifuged. 4 mL of the supernatant was taken into a 50 mL chicken heart bottle, concentrated to dryness, 1 mL of acetonitrile was added, ultrasonic assisted dissolution was performed, and then frozen in a-24℃ refrigerator for 1.0 hour. After taking out, it was immediately centrifuged at 10000 rpm for 2 min, and then 0.22 μm filter membrane was used to filter into a sample bottle. UPLC-MS / MS was used for determination.

[0043] 1.3.3 Tea soup: black-green tea powder was added to boiling water according to the standard of tea to water ratio 1:50, double layer filter paper was used to filter twice after 10 min, and tea soup was obtained. 20 mL of tea soup was taken into a 50 mL centrifuge tube, 20 mL of 1% formic acid acetonitrile was added, vortexed thoroughly, oscillated for 5 min, 7 g of NaCl was added, vortexed thoroughly, oscillated for 5 min, and then centrifuged at 10000 rpm for 5 min. The whole upper organic phase solution was taken, 20 mL of 1% formic acid acetonitrile was added again, vortexed thoroughly, oscillated for 5 min, ultrasonic for 10 min, and then centrifuged. The two supernatants were combined, concentrated to dryness, 2 mL of acetonitrile was added, ultrasonic assisted dissolution was performed, 1.2 mL was taken and added to a 2 mL centrifuge tube containing 16 mg of multi-walled carbon nanotubes and 8 mg of nano zirconium oxide, vortexed for 1 min, and then centrifuged at 12000 rpm for 5 min. 0.22 μm filter membrane was used to filter into a sample bottle. UPLC-MS / MS was used for determination.

[0044] 1.4 Chromatography-mass spectrometry conditions

[0045] 1.4.1 Chromatography conditions: 3 μm Cellulose-3 (150 x 2 mm) separation, column temperature 45 °C, injection volume 5 μL, flow rate 0.2 mL / min, mobile phase A 10 mmol / L ammonium acetate aqueous solution, B 0.1% formic acid in methanol, gradient elution program: 0-1.5 min, 10-75% B; 1.5-5.45 min, 75% B; 5.45-5.46 min, 75%-50% B; 5.46-7.5 min, 50% B; 7.5-9.0 min, 50%-100% B, 100% B for 3 min; 12.0-13.0, 100%-10% B.

[0046] 1.4.2 Mass spectrometry conditions: electrospray positive ionization multiple reaction monitoring mode ESI + MRM; electrospray capillary voltage 3.5 kV; ion source temperature 150 °C; desolvation gas N2 temperature 350 °C, flow rate 700 L / h; cone pore backflush gas N2 flow rate 50 L / h; collision gas Ar flow rate 0.30 mL / min; electron multiplier multiplication voltage 700 V; secondary parent ion residence time 0.1 s; fluacyprim enantiomer parent ion m / z 465, cone pore voltage 15 V, quantitation daughter ion m / z 173 and qualitative daughter ion m / z 249, collision energy 20 and 10 eV respectively. The rest of the secondary mass spectrometry parameters of fluacyprim enantiomer were determined by tandem mass spectrometry MRM mode, see Table 1. The chromatograms of fluacyprim (CYF) racemate and its monomers (+)-fluacyprim and (-)-fluacyprim are shown in Figure 1 .

[0047] Table 1 Secondary mass spectrometry parameter conditions for detecting fluacyprim enantiomer by ultra-high performance liquid chromatography tandem mass spectrometry

[0048]

[0049] 1.5 Preparation of standard solutions and standard curve

[0050] Take 10 mg of fluacyprim racemate and its monomer standard respectively in a 50 mL volumetric flask, dissolve with acetonitrile and dilute to volume to prepare a 200 mg / L standard stock solution, store at -18 °C. Prepare a series of standard solutions (2.0, 0.5, 0.1, 0.05, 0.01 and 0.005 mg / L, enantiomeric monomer concentration is half) from the standard stock solution with acetonitrile and the tea fresh leaf, red-green tea and red-green tea soup matrix blank solutions obtained after sample extraction and purification, analyze by UPLC-MS / MS injection, determine 3 times for each concentration, take the concentration as the abscissa (x) and the peak area average value as the ordinate (y) to obtain the linear equation, correlation coefficient, matrix effect and enantiomeric fraction value of fluacyprim enantiomer.

[0051] The matrix effect (ME) was calculated by the following equation:

[0052] ME = (A / B - 1) x 100%

[0053] Where A is the slope of the matrix standard curve, and B is the slope of the solvent standard curve. If ME is greater than 0, it indicates that there is a matrix enhancement effect, otherwise, if ME is less than 0, it indicates that there is a matrix attenuation effect.

[0054] The enantiomer fraction (EF) was calculated by the following equation:

[0055] EF = E1 / (E1 + E2) x 100%

[0056] Where E1 and E2 are the first and second peaks of the chiral compound enantiomers in the chromatogram, respectively. The EF value ranges from 0 to 1, where EF = 0.5 indicates that the degradation rates of the two enantiomers are the same, and there is no enantioselectivity; when EF > 0.5, it indicates that (-)-cyflumetofen ester is preferentially degraded; when EF < 0.5, it indicates that the monomer is preferentially degraded in (+)-cyflumetofen ester.

[0057] 1.6 Recovery, precision and method limit of quantification

[0058] Weigh (take) tea fresh leaves, red green tea or red green tea tea soup blank samples determined to be free of cyflumetofen ester, add different levels of standard solution, vortex mix and place for 2 h to be closer to the actual sample pesticide residue situation, then add 2% formic acid water and 1% formic acid acetonitrile for extraction according to the sample extraction and purification method, repeat 5 times for each concentration; at the same time, add the corresponding concentration of standard solution to the blank tea fresh leaves, red green tea or red green tea tea soup sample solution after treatment, and dilute to volume to prepare the corresponding matrix standard solution for determination, and calculate the addition recovery, relative standard deviation, method detection limit and quantification limit.

[0059] 2 Results and discussion

[0060] 2.1 Optimization of pretreatment method

[0061] When purifying the extract, we first investigated the adsorption of cyflumetofen ester on new purification filler biomass activated carbon (biochar) and multi-walled carbon nanotubes (MWCNT) and traditional purification filler GCB, and its recovery, matrix effect as follows: Figure 2Biomass activated carbon can better adsorb impurities such as pigments in the matrix, and even 50mg purified sample is nearly colorless and transparent. However, it also has a greater adsorption of target compounds, resulting in a recovery rate that does not meet the analysis requirements. Multi-walled carbon nanotubes and GCB have less adsorption of target compounds, and the recovery rate can meet the requirements. However, from the color of the purified sample, the purification effect of GCB is not as good as that of multi-walled carbon nanotubes. Therefore, 20mg multi-walled carbon nanotubes are selected to adsorb impurities such as pigments in the sample.

[0062] On this basis, nano zirconium oxide (ZrO2) is added to adsorb lipids and heavy metals, and the results are shown in Figure 3 A. 10mg nano zirconium oxide has good effect. PSA is added to remove polar compounds such as fatty acids, and the results are shown in Figure 3 B. However, PSA has a strong adsorption of metabolite B-1, and 10mg will significantly reduce its recovery rate. Therefore, 20mg MWCNT+10mg ZrO2 combination is selected to purify 1.5mL sample.

[0063] The matrix composition of black tea and green tea is more complex than that of fresh tea leaves. The sample purified by the above method is easy to form a precipitate in the refrigerator at-24℃, which affects the accuracy of the results. Therefore, we compared the recovery rate and compound response under the conditions of removing the precipitate after low-temperature freezing and redissolving the precipitate, and further explored the composition of the precipitate and the freezing time. The results show that the removal of the precipitate not only does not cause the loss of cyflumetofen, but also improves the response during determination, as shown in Figure 4 . The precipitate under different freezing times (1, 2, 3, 4, 5, 6h) was compared, and it was found that there was no obvious increase in the precipitate after 1h. Therefore, the centrifugation effect is the best after freezing for 1h. After full scan, caffeine (m / z 195) and theanine (m / z 175) were extracted, and it was found that their contents were significantly reduced in the sample with the precipitate removed, as shown in Figure 5 .

[0064] 2.2 Optimization of chromatography-mass spectrometry conditions

[0065] By comparing 3μm Cellulose-3 in 10mmol / L ammonium acetate aqueous solution (A) and 0.1% formic acid methanol (B) under different mobile phase proportions and the same mobile phase proportion with different holding times on the separation effect of cyflumetofen enantiomers, respectively Figure 6 and Figure 7 . As Figure 5The third peak from the left shows good separation, but not complete separation. On this basis, we adjusted the retention time to achieve complete separation. The final choice of column and conditions in 1.4.1 is the analysis condition. The selection of mobile phase and mass spectrometry conditions is also based on the previous research results of the research group.

[0066] 3 Standard curve, sensitivity, matrix effect and detection limit

[0067] The UPLC-MS / MS was used to determine the solvent standard solution, tea fresh leaves, red and green tea, and red and green tea soup matrix standard solution in the concentration range of 0.0025-1.0 mg / L. The correlation linear equation, correlation coefficient, matrix effect, enantiomer fraction value, and detection limit are shown in Table 2. The results show that the linear relationship of the butufoside enantiomers is good in the above matrices, the correlation coefficients (R 2 ) are all above 0.985, which can meet the requirements, the detection limits of tea fresh leaves and red and green tea are both 1.0 μg / kg, and the detection limit of red and green tea soup is 0.1 μg / L. After purification of tea samples, the results show that there is still a certain matrix weakening effect. Therefore, matrix standard external standard method needs to be used for quantitative analysis.

[0068] 4 Recovery, precision and limit of quantification

[0069] According to the above, the butufoside enantiomers in tea fresh leaves, red and green tea, and red and green tea soup were tested for recovery at 5 parallel addition levels. The average addition recovery (A.R.), relative standard deviation (RSD), and limit of quantification are shown in Table 2. The results show that at different addition concentration levels, the addition recovery of (+)-butufoside is 76.3%-116.9%, and the RSD is 1.4%-6.4%; the addition recovery of (-)-butufoside is 82.9%-116.9%, and the RSD is 0.4%-5.9%. The limit of quantification of tea fresh leaves and red and green tea is 2.5 μg / kg, and the limit of quantification of red and green tea soup is 0.25 μg / L.

[0070] Table 2 Linear equation, correlation coefficient (R 2 ), matrix effect (ME), enantiomer fraction (EF), average recovery (A.R.), standard deviation (SD), relative standard deviation (RSD), detection limit (LOD), and limit of quantification (LOQ) of butufoside enantiomers in different tea matrices

[0071]

Claims

1. A method for enantiomeric separation of fenflurfen, characterized in that... Includes the following steps: 1) Preparation of racemic mixture and monomer solutions of dicofol: Weigh the racemic mixture and monomer samples of dicofol, and prepare stock solutions with acetonitrile for later use; 2) Resolution of racemic diflubenzuron: The racemic diflubenzuron solution prepared in step 1) was injected as a standard solution. The racemic diflubenzuron was eluted and resolved by ultra-high performance liquid chromatography under certain chromatographic conditions, resulting in two peaks, which are the two enantiomeric monomers in the racemic diflubenzuron. The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: Lux® 3μm Cellulose-3 column, 150×2 mm; column temperature 45℃; injection volume 5 μL; flow rate 0.2 mL / min; mobile phase A was 10 mmol / L ammonium acetate aqueous solution, and mobile phase B was 0.1% formic acid in methanol. The gradient elution program was as follows: 0–1.5 min, 10–75% B; 1.5–5.45 min, hold 75% B; 5.45–5.46 min, 75%–50% B; 5.46–7.5 min, hold 50% B; 7.5–9.0 min, 50%–100% B, hold 100% B for 3 min; 12.0–13.0 min, 100%–10% B. 3) Confirmation of the two monomers of the racemic mixture of dicofol: The dicofol monomer solution and the racemic mixture solution prepared in step 1) were used as standard solutions for injection. The retention time of the peaks on the chromatogram was used to determine that the one that eluted first was (+)-dicofol and the one that eluted later was (-)-dicofol.

2. The enantiomer separation method of fenfluridine as described in claim 1, characterized in that... In step 1), the concentration of the racemic solution of tebufenozide is 0.0025-200 mg / L.

3. A method for the residue analysis of enantiomeric benzoate in agricultural products and tea beverages, characterized in that... Includes the following steps: 1) Extraction and purification of samples: Fresh tea leaves, black tea, green tea, tea soup or other agricultural products were extracted with 1% formic acid and acetonitrile, and purified by solid-phase extraction with multi-walled carbon nanotubes and nano-zirconia. After concentration, the volume was adjusted with acetonitrile and the samples were detected by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry matrix external standard method. 2) Take the prepared racemic solution of fenfluridine and dilute it with acetonitrile to prepare a standard solution of 20 mg / L. Then, prepare matrix standard solutions of 2, 0.5, 0.1, 0.05, 0.01 and 0.005 mg / L respectively using the standard solution. The enantiomeric concentration is half. Inject the samples into the matrix using ultra-high performance liquid chromatography-tandem mass spectrometry. Each concentration is measured 3 times. Plot the concentration as the x-axis and the average peak area as the y-axis to obtain the enantiomeric standard curve, linear correlation coefficient, matrix effect and enantiomeric fraction value of fenfluridine in different matrices. The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: Lux® 3μm Cellulose-3 column, 150×2 mm; column temperature 45℃; injection volume 5 μL; flow rate 0.2 mL / min; mobile phase A was 10 mmol / L ammonium acetate aqueous solution, and mobile phase B was 0.1% formic acid in methanol. The gradient elution program was as follows: 0–1.5 min, 10–75% B; 1.5–5.45 min, hold 75% B; 5.45–5.46 min, 75%–50% B; 5.46–7.5 min, hold 50% B; 7.5–9.0 min, 50%–100% B, hold 100% B for 3 min; 12.0–13.0 min, 100%–10% B. 3) Calculate the recovery rate, relative standard deviation, limit of detection and limit of quantitation of the method to meet the requirements of residue analysis; calculate the residue of fenfluridine enantiomers in the actual samples treated in step 1) using the matrix external standard method.

4. The method for residue analysis of enantiomeric benzoate in agricultural products and tea beverages as described in claim 3, characterized in that... The extraction and purification of fresh tea leaves in step 1) specifically involves: After grinding, weigh 5 g of fresh leaves into a centrifuge tube, add 8 mL of 2% formic acid solution, vortex thoroughly, let stand for 10 min, then add 15 mL of 1% formic acid acetonitrile, vortex thoroughly, shake for 5 min, sonicate for 10 min, add 3 g of NaCl, vortex thoroughly, shake for 5 min, centrifuge at 10000 rpm for 5 min, take 4.8 mL of the upper organic phase solution and add it to a 10 mL centrifuge tube containing 64 mg of multi-walled carbon nanotubes and 24 mg of nano-zirconia, vortex for 1 min, centrifuge, take 3 mL of the supernatant into a 50 mL heart-shaped flask, concentrate to dryness, add 1 mL of acetonitrile to make up to volume, sonicate to dissolve, filter through a 0.22 μm filter membrane into a sample vial, and analyze by UPLC-MS / MS.

5. The method for residue analysis of fenpropathrin enantiomers in agricultural products and tea beverages as described in claim 3, characterized in that... The extraction and purification of red and green tea in step 1) specifically involves: After grinding, weigh 2 g of black tea into a centrifuge tube, add 10 mL of 2% formic acid solution, vortex thoroughly, let stand for 10 min, then add 10 mL of 1% formic acid acetonitrile, vortex and shake for 5 min, sonicate for 10 min, add 3.5 g of NaCl, vortex and shake for 5 min, centrifuge at 10000 rpm for 5 min, take 6 mL of the upper organic phase solution and add it to a 10 mL centrifuge tube containing 80 mg of multi-walled carbon nanotubes and 40 mg of nano-zirconia, vortex for 1 min, centrifuge, take 4 mL of the supernatant into a 50 mL heart-shaped flask, concentrate to dryness, add 1 mL of acetonitrile to make up to volume, dissolve with sonication, then freeze in a -24℃ freezer for 1.0 hour, immediately centrifuge at 10000 rpm for 2 min, filter through a 0.22 μm filter membrane into a sample vial, and analyze by UPLC-MS / MS.

6. The method for residue analysis of fenpropathrin enantiomers in agricultural products and tea beverages as described in claim 3, characterized in that... The tea infusion extraction and purification in step 1) specifically involves: Add red and green tea powder to boiling water at a tea-to-water ratio of 1:50 and brew for 10 minutes. Filter twice with double-layered filter paper to obtain tea liquor. Transfer 20 mL of tea liquor to a 50 mL centrifuge tube, add 20 mL of 1% formic acid acetonitrile, vortex and shake for 5 minutes. Add 7 g NaCl, vortex and shake for 5 minutes, centrifuge at 10000 rpm for 5 minutes, collect all the upper organic phase solution, add another 20 mL of 1% formic acid acetonitrile for extraction, vortex and shake for 5 minutes, sonicate for 10 minutes, centrifuge, combine the two supernatants, concentrate to dryness, add 2 mL of acetonitrile to make up to volume, sonicate to assist dissolution, take 1.2 mL and add to a 2 mL centrifuge tube containing 16 mg of multi-walled carbon nanotubes and 8 mg of nano-zirconia, vortex for 1 minute, centrifuge at 12000 rpm for 5 minutes, filter through a 0.22 μm filter membrane into a sample vial, and analyze by UPLC-MS / MS.

7. The method for residue analysis of fenpropathrin enantiomers in agricultural products and tea beverages as described in claim 3, characterized in that... The mass spectrometry conditions for the mass spectrometer in step 2) are: electrospray ionization positive multiple reaction detection mode (ESI). + -MRM; electrospray capillary voltage 3.5 kV; ion source temperature 150 ℃; desolvation gas N2 temperature 350 ℃, flow rate 700 L / h; cone backflush gas N2 flow rate 50 L / h; collision gas Ar flow rate 0.30 mL / min; electron multiplier voltage 700 V; secondary precursor ion residence time 0.1 s; fenfluridine enantiomer precursor ion m / z 465, cone voltage 15 V, quantitative daughter ion m / z 173 and qualitative daughter ion m / z 249, collision energies 20 and 10 eV, respectively.

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