A method for rapid determination of residues of the enantiomers of bifenthrin and flumioxazin in cabbage

Abstract

The present application relates to the technical field of vegetable biochemical detection, and particularly relates to a method for rapidly determining residues of bifenthrin and esfenvalerate enantiomers in cabbage. The sample is extracted by acetonitrile, purified by a solid-phase extraction column, separated by a CHIRALPAK OJ-H chiral chromatographic column, isocratically eluted at a flow rate of 1.0 mL / min using supercritical carbon dioxide-methanol as a mobile phase, detected at a wavelength of 203 nm, and quantified by an external standard method. The method quantification limits of the two bifenthrin enantiomers are both 0.1 mg / kg, the method quantification limits of the two esfenvalerate enantiomers are both 0.2 mg / kg, the linear range is 0.5-20 mg / L, the linear correlation coefficient is greater than 0.9992, the recovery rate range at three spiked levels (0.1, 0.2, 0.4, 1.0 and 2.0 mg / kg) is 80.4%-102%, and the relative standard deviation is 2.3%-6.8%. The method is convenient to operate, has good separation effect, is green and environmentally friendly, and can meet the needs of residue analysis of bifenthrin and esfenvalerate enantiomers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical detection of vegetables, and particularly relates to a method for rapidly determining residues of biphenthrin and esfenvalerate enantiomers in cabbage. BACKGROUND

[0002] Pyrethroids are a class of important synthetic insecticides with high efficiency, broad spectrum, low toxicity and biodegradability. Pyrethroids are synthetic derivatives of natural pyrethrin extracted from chrysanthemum, composed of chrysanthemic acid esters and halogenated derivatives of its acid and alcohol, with characteristics of high efficiency, low toxicity and stability. Pyrethroids have developed into the third largest class of insecticides globally after organophosphorus and carbamate insecticides. Most pyrethroid pesticides on the market are sold as racemates, with inactive enantiomers, which not only increases the use of pesticides, leading to increased residues in crops and food, but also has a negative impact on non-target organisms.

[0003] Biphenthrin and esfenvalerate are widely used pyrethroid insecticides, both of which have poor enantiomeric selectivity. Both of them contain a pair of enantiomers [7-12], the structures of which are (-)-(1S,3S)-cis-biphenthrin, (+)-(1R,3R)-cis-biphenthrin, (-)-(R,R)-tau-esfenvalerate and (+)-(R,S)-tau-esfenvalerate. The insecticidal activity of the four enantiomers is quite different. Compared with (-)-(1S,3S)-cis-biphenthrin, (+)-(1R,3R)-cis-biphenthrin has higher insecticidal toxicity and lower toxicity to the human body. The biological activity of (+)-(R,S)-tau-esfenvalerate is significantly higher than that of (-)-(R,R)-tau-esfenvalerate. In order to control the standard use of biphenthrin and esfenvalerate, various countries have established maximum residue limits of the two pesticides in cabbage. Table 1 lists the residue limits of biphenthrin and esfenvalerate in cabbage. However, these regulations only specify the safe limit of the racemate of biphenthrin and esfenvalerate, and do not accurately specify the residue limit of the enantiomer.

[0004] Table 1 Residue limits of biphenthrin and esfenvalerate in cabbage

[0005] Compound EU (mg / kg) China (mg / kg) Japan (mg / kg) USA (mg / kg) Bifenthrin 0.4 0.2 0.4 4 Spinetoram 0.3 0.5 0.5 None

[0006] At present, the determination methods for pyrethroid pesticides mainly include immunoassay, gas chromatography, gas chromatography-tandem mass spectrometry, high performance liquid chromatography, and liquid chromatography-tandem mass spectrometry. The above analysis methods have certain limitations for the efficient separation of a large number of isomers with similar structures and properties. Ultra performance liquid chromatography (UPC2 ) is a new separation and analysis technology developed on the basis of supercritical fluid chromatography (SFC), which uses supercritical fluid CO2 as the main mobile phase. Its diffusion coefficient and viscosity are closer to those of a gas, allowing for rapid and efficient separation. Its density is close to that of a liquid, so it has high solubility and can separate and analyze thermally unstable and relatively large molecular weight substances at room temperature. In recent years, UPC 2 technology has been widely used in the separation and determination of triazole pesticides, vitamins, and bisphenol compounds.

[0007] A method for resolving and determining carnitine enantiomers in health food based on UPC technology is disclosed in a Chinese invention patent (publication number CN113433257A, publication date: September 24, 2021) applied for by the applicant of the present application. The method uses UPC to resolve carnitine enantiomers and determines the residual amount of L-carnitine and D-carnitine enantiomers in health food. The sample is extracted with anhydrous ethanol under ultrasonic, and after derivatization, it is separated by an Acquity Trefoil CEL1 chiral chromatographic column, eluted with supercritical carbon dioxide-1% (v / v) ammonia methanol as the mobile phase, and quantified by the external standard method.

[0008] A method for rapidly determining the residual amount of fenpropathrin enantiomers in pears and their products is disclosed in a Chinese invention patent (publication number CN114858942A, publication date: August 5, 2022). The sample is extracted with ethyl acetate, purified by a C18 column, separated by an Acquity Trefoil AMY1 chiral chromatographic column, and eluted with different volume ratios of methanol containing 0.5% ammonia solution-super critical carbon dioxide as the mobile phase, and quantified by the external standard method. The detection limit is 0.2 mg / kg, the recovery rate is 81.4%-106%, and the RSD is 4.1%-7.2%. Actual samples were detected, and the detection amount was 0.22-0.25 mg / kg.

[0009] A method for rapidly determining cypermethrin and tau-fluvalinate enantiomer residues in cabbage is disclosed in Chinese invention patent (publication number CN114935611A, publication date: August 23, 2022). The method extracts the sample with acetonitrile, purifies it with a solid-phase extraction column, separates it with a CHIRALPAK AD-3 chiral chromatographic column, uses supercritical carbon dioxide-methanol as the mobile phase, performs gradient elution at a flow rate of 1.5 mL / min, detects at a wavelength of 230 nm, and quantifies by external standard method. The method quantitative limits of the two cypermethrin enantiomers are both 0.2 mg / kg, the linear range is 1.0-20 mg / L, the linear correlation coefficient is greater than 0.9992, the recovery rate at three spiked levels (0.2, 0.4 and 2.0 mg / kg) ranges from 80.6% to 105%, and the relative standard deviation is 2.6%-7.7%.

[0010] Based on this, it is particularly important to develop a method for rapidly determining cypermethrin and tau-fluvalinate enantiomer residues in cabbage, so as to effectively control the residue amount in food under the premise of ensuring the safety of pesticide use. SUMMARY

[0011] In order to solve the above technical problems, the purpose of the present application is to provide a method for rapidly determining cypermethrin and tau-fluvalinate enantiomer residues in cabbage, which is convenient to operate, has good separation effect, is green and environmentally friendly, and can meet the needs of cypermethrin and tau-fluvalinate enantiomer residue analysis.

[0012] In order to achieve the above purpose, the following technical solutions are adopted in the present application:

[0013] A method for rapidly determining cypermethrin and tau-fluvalinate enantiomer residues in cabbage, the structures of cypermethrin and tau-fluvalinate enantiomers are (-)-(1S,3S)-cis-cypermethrin, (+)-(1R,3R)-cis-cypermethrin, (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate, the method comprises the following steps:

[0014] 1) Sample extraction

[0015] Weigh the sample into a centrifuge tube, add acetonitrile for homogenization extraction, then add sodium chloride and mix well by vortex, centrifuge, and then take the supernatant into another centrifuge tube; add acetonitrile to the lower residue, repeat the extraction once, combine the two supernatants, concentrate to near dryness, dissolve with n-hexane, and then purify;

[0016] 2) Purification

[0017] The reconstituted solution was transferred to a Florisil column activated with n-hexane, eluted with n-hexane, and then eluted with a n-hexane: diethyl ether (7:3, v / v) solution, the eluate was blown to near dryness with nitrogen, dissolved with 1 mL of methanol, and the constant volume solution was filtered through a 0.22 μm filter membrane.

[0018] 3) High performance liquid chromatography conditions

[0019] The OJ-H chiral chromatographic column was selected to separate the four chrysanthemum ester enantiomers, methanol was selected as the cosolvent in the mobile phase, the system back pressure was 12.1 MPa, the column temperature was 31°C, and the detection wavelength was 203 nm.

[0020] 4) Quantitative determination

[0021] The standard working solution and the sample solution were injected under the set high performance liquid chromatography conditions, the mass concentration X was used as the abscissa, the peak area Y was used as the ordinate, and the compounds were quantitatively determined by the external standard method.

[0022] As preferred, 5 g of the sample was weighed to 0.01 g in a 50 mL centrifuge tube, 20 mL of acetonitrile was added for homogenate extraction, 3.0 g of sodium chloride was further added, vortexed and mixed, centrifuged at 4 000 r / min for 5 min, and the supernatant was taken to another 50 mL centrifuge tube; 20 mL of acetonitrile was added to the lower residue, and the extraction was repeated once, the supernatants of the two times were combined, concentrated to near dryness with a rotary evaporator, and dissolved with 10 mL of n-hexane.

[0023] As preferred, the reconstituted solution was transferred to a Florisil column activated with n-hexane, eluted with 5 mL of n-hexane, and then eluted with 10 mL of a n-hexane: diethyl ether (7:3, v / v) solution, the eluate was blown to near dryness with nitrogen, dissolved with 1 mL of methanol, and the constant volume solution was filtered through a 0.22 μm filter membrane.

[0024] As preferred, in step 3), the chromatographic column was CHIRALPAK OJ-H, 100 mm x 4.6 mm, 5 μm; the mobile phase was A: CO2 and B: methanol; the isocratic elution program was 0-5 min (14% B); the system back pressure was 12.1 MPa; the flow rate was 1.5 mL / min; the injection amount was 5 μL; the column temperature was 31°C; and the detection wavelength was 203 nm.

[0025] As preferred, the enantiomer standard stock solution was prepared as follows: 0.01 g of each of the bifenthrin and fluazaindolizine racemate standard was accurately weighed to 0.1 mg, dissolved with anhydrous ethanol and diluted to 10 mL to prepare a 1.0 g / L racemate standard stock solution; and the bifenthrin and fluazaindolizine racemate standard intermediate solution was prepared as follows: a certain amount of the racemate standard stock solution was accurately pipetted, diluted with anhydrous ethanol to a 10.0 mg / L standard intermediate solution.

[0026] As preferred, the enantiomer standard stock solution is prepared as follows: 0.01 g of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate standard is accurately weighed respectively to 0.1 mg, dissolved in methanol and diluted to 10 mL to prepare 1.0 g / L of enantiomer standard stock solution; the mixed standard working solution of the four ester enantiomers is prepared as follows: a certain amount of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate enantiomer standard stock solution is accurately pipetted respectively, and methanol is used for step-by-step dilution to 0.5, 1.0, 2.0, 4.0, 10.0, 20.0 mg / L of mixed standard working solution.

[0027] As preferred, the limit of quantification LOQ is calculated at a signal-to-noise ratio S / N = 10, and the LOQ of (-)-(1S,3S)-cis-bifenthrin and (+)-(1R,3R)-cis-bifenthrin is 0.1 mg / kg; the LOQ of (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate is 0.2 mg / kg.

[0028] As preferred, the recovery of the four ester enantiomers is 80.4% to 102%, and the relative standard deviation RSD is 2.3% to 6.8%.

[0029] The present application adopts the above technical scheme, for the first time, simultaneously resolves four kinds of bifenthrin and fluvalinate enantiomers by using ultra-high performance liquid chromatography, determines the residual amount of bifenthrin and fluvalinate enantiomers in cabbage, and studies the residual degradation dynamics of bifenthrin and fluvalinate enantiomers on cabbage. The method optimizes the instrument resolution conditions such as chromatographic column, column temperature, cosolvent, system back pressure and constant reagent, simultaneously investigates the stability of bifenthrin and fluvalinate enantiomer standard solution, and applies the established method to field sample detection. The results show that the method has high sensitivity and good stability, and can meet the needs of chiral enantiomer residue detection of bifenthrin and fluvalinate in cabbage. The method provides a reference for in-depth analysis of enantiomers with efficacy and low toxicity and enantiomers of pesticides with no efficacy and high toxicity in agricultural products, and has important significance for quality control and efficacy evaluation of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Spectrum of ester enantiomer standard solution.

[0031] Figure 2 Stability of four pyrethroid enantiomers standard solutions within 60 days.

[0032] Figure 3 Effect of different chromatographic columns on separation of four pyrethroid enantiomers. Figure 3 Example 1 (a) AD-3; Figure 3 Example 1 (b) IC; Figure 3 Example 1 (c) OJ-H.

[0033] Figure 4 Effect of different co-solvents on separation of four pyrethroid enantiomers. Figure 4 Example 2 (a) 0.5% (v / v) ammonia methanol solution; Figure 4 Example 2 (b) 0.5% (v / v) formic acid methanol solution; Figure 4 Example 2 (c) methanol.

[0034] Figure 5 Effect of different system back pressure on separation of four pyrethroid enantiomers. Figure 5 Example 3 (a) 10.3 MPa, Figure 5 Example 3 (b) 12.1 MPa, Figure 5 Example 3 (c) 13.8 MPa.

[0035] Figure 6 Effect of different chromatographic column temperature on separation of four pyrethroid enantiomers. Figure 6 Example 4 (a) 31 °C; Figure 6 Example 4 (b) 35 °C; Figure 6 Example 4 (c) 40 °C.

[0036] Figure 7 Effect of different constant volume reagents on separation of four pyrethroid enantiomers. Figure 7 Example 5 (a) methanol; Figure 7 Example 5 (b) acetonitrile; Figure 7 Example 5 (c) n-heptane.

[0037] Figure 8 Chromatograms of standard solution (a), blank cabbage sample (b) and added recovery (c). Chromatographic peaks: 1.

[0038] (-)-(1S,3S)-cis-bifenthrin, 2. (+)-(1R,3R)-cis-bifenthrin, 3. (-)-(R,R)-tau-fluvalinate, 4.

[0039] (+)-(R,S)-tau-fluvalinate.

[0040] Figure 9 Resolution of bifenthrin and fluvalinate racemates. Chromatographic peaks: 1. (-)-(1S,3S)-cis-bifenthrin, 2.

[0041] (+)-(1R,3R)-cis-bifenthrin, 3. (-)-(R,R)-tau-fluvalinate, 4. (+)-(R,S)-tau-fluvalinate.

[0042] Figure 10 Degradation curves of bifenthrin and fluvalinate enantiomers in cabbage leaves. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0044] 1. Instruments, materials and reagents

[0045] Acquity ultra-performance liquid chromatograph (Waters, USA) with a photodiode array detector (PDA); AE260 electronic balance (Mettler, Switzerland); R215 rotary evaporator (Buchi, Switzerland); ELGA CLXXX UV M2 ultrapure water purification system (Elga, UK); MS2 vortex mixer (Shanghai Medical Instrument Factory); N-EVAP TM 111 nitrogen blowing instrument (Tokyo Denshi, Japan).

[0046] Acetonitrile, methanol, formic acid, n-heptane, n-hexane (chromatographically pure, Scharlau, Spain); ammonia, diethyl ether (analytically pure); ultrapure water; high-purity carbon dioxide (99.999%); Florisil column (CNW, 5 g, 6 mL); cellulose derivative n-aphthyl column CHIRALPAK OJ-H (100 mm x 4.6 mm, 5 μm, spherical silica gel coated with chiral polymers (amylose or cellulose derivatives) on the surface); Chiralpak AD-3 (150 mm x 3.0 mm, 3 μm, filler is amylose-tris(3,5-dimethylphenylcarbamate)); Chiralpak IC (100 mm x 4.6 mm, 5 μm, silica gel covalently bonded with cellulose-tris(3,5-dichlorophenylcarbamate); other reagents used in the experiments are analytically pure unless otherwise specified.

[0047] 2 racemic standards (bifenthrin: CAS No. 82657-04-3, purity > 99.7%, Dr Ehrenstorfer Gmbh, Germany; flumethrin: CAS No. 102851-06-9, purity > 97.3%, Dr Ehrenstorfer Gmbh, Germany). Bifenthrin and flumethrin enantiomeric standards: (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-flumethrin, (+)-(R,S)-tau-flumethrin were obtained by separation and purification from bifenthrin and flumethrin racemic standards (Dr Ehrenstorfer Gmbh) respectively by Shanghai Qinnan Biotechnology Co., Ltd. The purity of each was greater than 97.0%.

[0048] 2. Preparation of standard stock solutions and working solutions

[0049] 2.1 Racemic standard stock solution

[0050] Accurately weigh 0.01 g (accurate to 0.1 mg) of bifenthrin and flumethrin racemic standard, respectively, dissolve in anhydrous ethanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution.

[0051] Bifenthrin and flumethrin racemic standard intermediate solution: accurately pipette a certain amount of racemic standard stock solution, dilute with anhydrous ethanol to 10.0 mg / L of standard intermediate solution.

[0052] 2.2 Enantiomeric standard stock solution

[0053] Accurately weigh 0.01 g (accurate to 0.1 mg) of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-flumethrin, (+)-(R,S)-tau-flumethrin standard, respectively, dissolve in methanol and dilute to 10 mL to prepare a 1.0 g / L enantiomeric standard stock solution.

[0054] Mixed standard working solution of 4 pyrethroid enantiomers: accurately pipette a certain amount of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-flumethrin, (+)-(R,S)-tau-flumethrin enantiomeric standard stock solution, respectively, dilute with methanol step by step to 0.5, 1.0, 2.0, 4.0, 10.0, 20.0 mg / L of mixed standard working solution.

[0055] 3. Sample pretreatment

[0056] 3.1 Sample extraction

[0057] Weigh 5g (accurate to 0.01g) of the sample into a 50mL centrifuge tube, add 20mL of acetonitrile, homogenously extract, then add 3.0g of sodium chloride, vortex to mix, centrifuge at 4 000r / min for 5min, then take the supernatant into another 50mL centrifuge tube; add 20mL of acetonitrile to the lower residue and repeat the extraction once, then combine the two supernatants, concentrate to near dryness using a rotary evaporator, dissolve with 10mL of n-hexane, and then purify.

[0058] 3.2 Purification

[0059] Transfer the redissolved solution to a Florisil column activated with n-hexane, wash with 5mL of n-hexane, then elute with 10mL of n-hexane:diethyl ether (7:3, v / v), concentrate the eluate to near dryness under nitrogen, dissolve with 1mL of methanol, and then filter the constant volume solution through a 0.22μm filter membrane and detect on the instrument.

[0060] 4. Chromatographic conditions

[0061] Chromatographic column: CHIRALPAK OJ-H (100mm x 4.6mm, 5μm); mobile phase: A is CO2 and B is methanol; isocratic elution program: 0-5min (14% B); system back pressure: 12.1MPa; flow rate: 1.5mL / min; injection volume: 5μL; column temperature: 31℃; detection wavelength: 203nm.

[0062] 5. Results and discussion

[0063] 5.1 Selection of detection wavelength

[0064] After scanning by the PDA detector, the ultraviolet spectra of the four chrysanthemum ester enantiomer standard solutions were extracted from the chromatogram. As shown in Fig. 1, the four chrysanthemum ester enantiomers all have the strongest absorption at 203nm and the highest sensitivity, so 203nm was selected as the detection wavelength in this experiment. Figure 1

[0065] 5.2 Investigation of the stability of chrysanthemum ester enantiomer standard solutions

[0066] Accurately pipette 1.0mL of the 5.0mg / L chrysanthemum ester enantiomer mixed standard working solution into 7 scratched 1.5mL UPC 2 ​The sample was injected into a special vial and then determined by the instrument. After the determination, the sample was transferred into 7 vials with aluminum caps, sealed with sealing film and stored at -18℃. The newly prepared standard solution of bifenthrin and fluazaindolyl at a concentration of 10.0 mg / L was compared with the determination results of 5.0 mg / L bifenthrin and fluazaindolyl enantiomers stored for 1, 3, 5, 7, 14, 30 and 60 days. The newly prepared standard solution was taken as 100%, and the change of the standard solution of bifenthrin and fluazaindolyl enantiomers less than 10% was taken as the reference. The results showed that the determination results of the four pyrethroid enantiomers were gradually reduced (see Figure 2 ). The content of the two bifenthrin enantiomers was reduced by more than 12% after being stored at -18℃ for 60 days, the content change was less than 10% after being stored for 30 days, and the content change was less than 5% after being stored for 14 days, indicating that the two bifenthrin enantiomers were relatively stable within 30 days. The content of the two fluazaindolyl enantiomers was reduced by more than 18% after being stored at -18℃ for 60 days, the content was reduced by more than 12% after being stored for 30 days, and the content change was less than 10% after being stored for 14 days, indicating that the two fluazaindolyl enantiomers were relatively stable within 14 days.

[0067] 5.3 Optimization of chromatographic column

[0068] In this experiment, three chiral separation chromatographic columns of CHIRALPAK AD-3, IC and OJ-H of DASIL Pharmaceutical Chiral Technology (Shanghai) Co., Ltd. were selected to investigate the separation effect of the four pyrethroid enantiomers. The results showed that when AD-3 and IC chiral chromatographic columns were used, only two or three chromatographic peaks appeared on the chromatogram, and the four pyrethroid enantiomers could not be completely separated. When OJ-H chiral chromatographic column was used, the separation degree was good, and the chromatographic peak shape was sharp (see Figure 3 ). Therefore, OJ-H chiral chromatographic column was selected to separate the four pyrethroid enantiomers.

[0069] 5.4 Selection of cosolvent in mobile phase

[0070] The organic solvent consumption of ultra-high performance liquid chromatography is small. Supercritical CO2 is usually used as the main mobile phase, and a small amount of organic solvent is used as a cosolvent to enhance the elution ability and selectivity of the target product. In this experiment, the effects of 0.5% (v / v) ammonia methanol solution, 0.5% (v / v) formic acid methanol solution and methanol on the separation of the four pyrethroid enantiomers were investigated. The results showed that when 0.5% (v / v) formic acid methanol solution was used as a cosolvent, the four pyrethroid enantiomers could not be separated. When 0.5% (v / v) ammonia methanol solution and methanol were used as cosolvents, the four pyrethroid enantiomers were completely separated within 5.0 min, and the peak shape was good (see Figure 4), but compared with 0.5% (v / v) ammonia-methanol solution as cosolvent, methanol as cosolvent is more convenient and less damage to the instrument and column. Therefore, methanol is selected as cosolvent in our lab.

[0071] 5.5 Selection of system back pressure

[0072] UPC 2 Using supercritical CO2 as mobile phase, the density of CO2 can be changed by adjusting the system back pressure and temperature, which can change the solubility, elution power and selectivity of CO2. CO2 will be in supercritical state when the temperature is above 31℃ and the pressure is above 7.38 MPa. Therefore, the effect of system back pressure on the separation of four kinds of chrysanthemum ester enantiomers was investigated in the range of 10.3-13.8 MPa. The results showed that with the increase of system back pressure, the retention time of the analyte was advanced (see Figure 5 ). When the system back pressure was 13.8 MPa, the separation degree of the chromatographic peak of (-)-(R,R)-tau-cyhalothrin and the interfering peak was too low. Compared with the system back pressure of 10.3 MPa, the chromatographic peak shape was sharper at the system back pressure of 12.1 MPa. Therefore, the back pressure of 12.1 MPa was selected in this study.

[0073] 5.6 Selection of column temperature

[0074] Considering that the maximum recommended operating temperature of CHIRALPAK OJ-H chiral chromatographic column is 40℃, and CO2 will be in supercritical state when the temperature is above 31℃ and the pressure is above 7.38 MPa, the effect of column temperature on the separation of four kinds of chrysanthemum ester enantiomers was investigated in the range of 31-40℃. Under the three column temperature conditions, the separation degree of the chromatographic peaks of four kinds of chrysanthemum ester enantiomers was good, and good baseline separation was achieved within 5.0 min, with fast analysis speed Figure 6 ). Considering the service life of the column, the optimal column temperature was selected as 31℃.

[0075] 5.7 Selection of constant volume reagent

[0076] Three constant volume reagents were used: methanol, acetonitrile, and n-heptane to separate the enantiomers of bifenthrin and cyhalothrin at 5.0 mg / L, and the results are shown in Figure 7 When acetonitrile was used as constant volume reagent, the peak shape of the target was poor; when methanol and n-heptane were used as constant volume reagent, the chromatographic peaks of four kinds of chrysanthemum ester enantiomers were completely separated within 5.0 min, and the peak shape was good. Compared with n-heptane, methanol has stronger solubility, smaller volatility, and is more convenient for subsequent analysis and detection. Therefore, methanol was finally selected as the constant volume reagent in this experiment.

[0077] 5.8 Selection of Florisil column eluent

[0078] The sample was usually eluted and purified with n-hexane-ethyl ether solution [33,34], and the elution effect of different volume ratios of n-hexane-ethyl ether (6:4, 7:3, 8:2, 9:1, v / v) was compared. Referring to the extraction method in Section 3.1, the extract was passed through a Florisil column, and then eluted with the three eluents and detected by the instrument. The results showed that with the increase of the proportion of ethyl ether, the recovery rate gradually increased, but the impurity interference peak liquid also increased. Considering comprehensively, when eluted with n-hexane: ethyl ether (7:3, v / v), there were relatively fewer interference peaks in the chromatogram, and the recovery rate reached more than 95.0%.

[0079] 5.9 Methodology investigation

[0080] 5.9.1 Linear range and limit of quantification

[0081] The series of mixed standard solutions of bifenthrin and esfenvalerate enantiomers were determined according to the above chromatographic conditions. The standard curve was drawn with the peak area (Y) of the standard as the vertical coordinate and the corresponding mass concentration (X) as the horizontal coordinate, and the regression equation and correlation coefficient were obtained. The results showed that the four enantiomers had good linear relationship in the mass concentration range of 0.5-20.0 mg / L, and the correlation coefficient was greater than 0.9992. By adding standard to the cabbage blank sample without bifenthrin and esfenvalerate, and determining according to the method, the limit of quantification (LOQ) was calculated according to the signal-to-noise ratio (S / N)=10, and the LOQ of (-)-(1S,3S)-cis-bifenthrin and (+)-(1R,3R)-cis-bifenthrin was 0.1 mg / kg; the LOQ of (-)-(R,R)-tau-esfenvalerate and (+)-(R,S)-tau-esfenvalerate was 0.2 mg / kg.

[0082] 5.9.2 Recovery and precision

[0083] Different concentration levels of four ester enantiomer standard solutions were added to the cabbage sample without bifenthrin and esfenvalerate for recovery and precision test, and the related chromatograms were shown in Figure 8 , and the results were shown in Table 2. The results showed that the recovery of four ester enantiomers was 80.4%-102%, and the relative standard deviation (RSD) was 2.3%-6.8%, which met the requirements of SANTE / 11312 / 2021

[35] for recovery, and could meet the determination of bifenthrin and esfenvalerate enantiomers in cabbage samples.

[0084] Table 2 Recovery and relative standard deviation of bifenthrin and esfenvalerate enantiomers in cabbage samples (n=6)

[0085]

[0086] 5.10 Application of the method

[0087] 5.10.1 Resolution of racemic standard

[0088] The purchased racemic standard of bifenthrin and tau-fluvalinate were resolved and determined by the method established in this paper. As shown in Fig. Figure 9 a, the four pyrethroid enantiomers were well separated and effectively resolved within 4.0 min, with resolution of R 1,2 = 1.9, R 2,3 = 4.8 and R 3,4 = 3.4, meeting the requirement of complete separation of R≥1.5

[36] . In order of retention time of chromatographic peaks, they were: (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate,

[0089] (+)-(R,S)-tau-fluvalinate. Figure 9 b, 9c, 9d, 9e). According to the standard curve drawn above, the contents of the four pyrethroid enantiomers in 10.0 mg / L of the racemic intermediate solution of bifenthrin and tau-fluvalinate in Section 2.1 were calculated by external standard quantification method, in which the contents of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate,

[0090] (+)-(R,S)-tau-fluvalinate were 5.36, 4.85, 5.34, 5.32 mg / L, respectively. The calculation results were basically consistent with the reported ratio of (-)-(1S,3S)-cis-bifenthrin to (+)-(1R,3R)-cis-bifenthrin in industrial racemic bifenthrin of 1.11:1.00 and the ratio of (-)-(R,R)-tau-fluvalinate to (+)-(R,S)-tau-fluvalinate in industrial racemic tau-fluvalinate of 1.00:1.00[37,38].

[0091] 5.10.2 Degradation dynamics analysis of two groups of pyrethroid enantiomers in cabbage

[0092] The established UPC 2The analysis method was used to study the dissipation of bifenthrin and tau-fluvalinate on cabbage leaves. Bifenthrin and tau-fluvalinate were diluted 2000 times and sprayed on cabbage leaves. The samples were collected at 4h, 1d, 3d, 5d, 7d and 10d after spraying, respectively. The residues of the two chiral enantiomers of bifenthrin and tau-fluvalinate were determined to obtain the degradation rules. The results showed that the residues of bifenthrin and tau-fluvalinate on cabbage were less than 0.5mg / kg( Figure 10 ) after 3 days of spraying at the usual concentration. The residues of bifenthrin and tau-fluvalinate on cabbage were mostly distributed on the outer layer, while the inner layer was not detected. This was related to the weak penetration of bifenthrin and tau-fluvalinate, which only contaminated the surface of the crop tissue. The half-lives of the four enantiomers were about 2.5 days. Through the half-life, it was found that there was no significant selectivity difference in the degradation of the four enantiomers of pyrethroid pesticides during the growth of cabbage leaves.

[0093] 5.10.3 Test of actual samples

[0094] To evaluate the effectiveness and practicality of the method, the contents of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate in 20 commercially available cabbage samples were determined. The results showed that no bifenthrin and tau-fluvalinate enantiomers were detected in the 20 cabbage samples.

[0095] 5.11 Comparison with other methods

[0096] The LOD, LOQ, linear range, separation time, recovery rate and RSD of this method were compared with other reported methods. The analysis time of this method is short (5min), the purification effect is good, the specificity is strong, and it can meet the daily detection requirements of bifenthrin and tau-fluvalinate. The limit of quantification (LOQ) of this method can meet the requirements of the maximum residue limit of national regulations. In addition, the linear range, recovery rate and RSD of this method are consistent with other methods.

[0097] Table 3 Comparison with other reported methods

[0098]

[0099] ​The foregoing is a description of the embodiments of the present application. The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the rapid determination of enantiomeric residues of bifenthrin and cypermethrin in cabbage, wherein the enantiomeric structures of bifenthrin and cypermethrin are (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-cypermethrin, and (+)-(R,S)-tau-cypermethrin, characterized in that... The method includes the following steps: 1) Sample extraction Weigh the sample into a centrifuge tube, add acetonitrile for homogenization and extraction, then add sodium chloride and vortex to mix. After centrifugation, take the supernatant into another centrifuge tube; add acetonitrile to the lower residue and repeat the extraction once. Combine the two supernatants, concentrate to near dryness, dissolve in n-hexane, and wait for purification. 2) Purification The reconstituted solution was transferred to a Florisil column activated with n-hexane, eluted with n-hexane, and then eluted with a solution of n-hexane:diethyl ether = 7:3 (v / v). The eluent was blown with nitrogen until nearly dry, dissolved in methanol, filtered through a filter membrane, and then analyzed by the instrument. 3) High-performance chromatographic conditions Four pyrethroid enantiomers were separated using an OJ-H chiral column. Methanol was selected as the co-solvent in the mobile phase. The system back pressure was 12.1 MPa, the column temperature was 31℃, and the detection wavelength was 203 nm. Column: CHIRALPAK OJ-H, 100 mm × 4.6 mm, 5 µm; Mobile phase: A is CO2, B is methanol; isocratic elution program: 0-5 min mobile phase contains 14% methanol by volume; flow rate: 1.5 mL / min; injection volume: 5 µL; 4) Quantitative determination Standard working solutions and sample solutions were injected under the set ultra-high performance chromatographic conditions. The mass concentration X was plotted on the x-axis and the peak area Y on the y-axis, with the external standard used to quantify the compound.

2. The method according to claim 1, characterized in that, In step 1), weigh 5 g of the sample, accurate to 0.01 g, and add 20 mL of acetonitrile to a 50 mL centrifuge tube. Extract by homogenization, then add 3.0 g of sodium chloride, vortex to mix, centrifuge at 4000 r / min for 5 min, and take the supernatant into another 50 mL centrifuge tube. Add 20 mL of acetonitrile to the lower residue, repeat the extraction once, combine the two supernatants, concentrate to near dryness using a rotary evaporator, and dissolve in 10 mL of n-hexane.

3. The method according to claim 1, characterized in that, In step 2), the reconstituted solution is transferred to a Florida silica column activated with n-hexane, eluted with 5 mL of n-hexane, and then eluted with 10 mL of a solution with a volume ratio of n-hexane:diethyl ether = 7:

3. The eluent is purged with nitrogen until nearly dry, and then dissolved in 1 mL of methanol. The diluted solution is then filtered through a 0.22 μm filter membrane.

4. The method according to claim 1, characterized in that, The enantiomeric standard stock solution was prepared as follows: 0.01 g (accurate to 0.1 mg) of each racemic standard of bifenthrin and lambda-cyhalothrin was accurately weighed, dissolved in anhydrous ethanol, and diluted to 10 mL to prepare a 1.0 g / L racemic standard stock solution. The intermediate standard solution of the racemic bifenthrin and lambda-cyhalothrin was prepared as follows: A certain amount of the racemic standard stock solution was accurately pipetted and diluted with anhydrous ethanol to a 10.0 mg / L intermediate standard solution.

5. The method according to claim 4, characterized in that, The enantiomeric standard stock solutions were prepared as follows: Accurately weigh 0.01 g (to a precision of 0.1 mg) of each of the following standards: (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-cyhalothrin, and (+)-(R,S)-tau-cyhalothrin. Dissolve each standard in methanol and dilute to a final volume of 10 mL to prepare a 1.0 mg / mL solution. The enantiomeric standard stock solutions of the four pyrethroids were prepared as follows: A certain amount of the enantiomeric standard stock solutions of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-flumethrin, and (+)-(R,S)-tau-flumethrin were accurately pipetted and diluted stepwise with methanol to prepare mixed standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L.

6. The method according to claim 1, characterized in that, With signal-to-noise ratio S / N =10 Calculate the limit of quantitation (LOQ), and find that the LOQ for (-)-(1S,3S)-cis-bifenthrin and (+)-(1R,3R)-cis-bifenthrin is 0.1 mg / kg; the LOQ for (-)-(R,R)-tau-flufenoxamyl and (+)-(R,S)-tau-flufenoxamyl is 0.2 mg / kg.

7. The method according to claim 1, characterized in that, The recoveries of the four pyrethroid enantiomers ranged from 80.4% to 102%, with relative standard deviations (RSDs) of 2.3% to 6.8%.

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