Method for rapidly determining bifenthrin and fluvalinate enantiomer residues in cabbage

Abstract

The invention relates to the technical field of biochemical detection of vegetables, in particular to a method for rapidly determining bifenthrin and fluvalinate enantiomer residues in cabbages. After a sample is extracted by acetonitrile and purified by a solid-phase extraction column, a CHIRALPAK OJ-H chiral chromatographic column is adopted for separation, supercritical carbon dioxide-methanol is taken as a mobile phase, isocratic elution is carried out at the flow speed of 1.0 mL / min, the detection wavelength is 203 nm, and quantification is carried out by an external standard method. The method quantitation limits of the two bifenthrin enantiomers are both 0.1 mg / kg, the method quantitation limits of the two fluvalinate enantiomers are both 0.2 mg / kg, the linear range is 0.5-20 mg / L, the linear correlation coefficient is larger than 0.9992, the recovery rate range is 80.4%-102% under three standard addition levels (0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 1.0 mg / kg and 2.0 mg / kg), and the relative standard deviation is 2.3%-6.8%. The method is convenient to operate, has a good separation effect, is green and environment-friendly, and can meet the requirements of residual analysis of bifenthrin and fluvalinate enantiomers.

Description

Technical Field

[0001] The invention relates to the technical field of vegetable biochemical detection, in particular to a method for rapidly determining the enantiomer residues of bifenthrin and fluvalinate in cabbage. Background Art

[0002] Pyrethroids are an important class of synthetic insecticides with high efficiency, broad spectrum, low toxicity and biodegradability. Pyrethroids are synthetic derivatives of natural pyrethrin extracted from chrysanthemums, composed of chrysanthemum esters and their acid and alcohol halogenated derivatives. They are highly effective, low toxic and stable, and have developed into the third largest class of insecticides in the world after organophosphorus and carbamate insecticides. At present, most pyrethroid pesticides on the market are sold as racemic forms, in which inactive enantiomers exist, which not only increases the amount of pesticides used, leading to increased residues in crops and food, but also has a negative impact on non-target organisms.

[0003] Bifenthrin and fluvalinate are currently widely used pyrethroid insecticides, both of which have enantiomeric toxicity selectivity differences. Both contain a pair of enantiomers [7-12], whose structures are (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate. The insecticidal activities of these four enantiomers vary greatly. Compared with (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin has higher insecticidal toxicity and lower toxicity to humans. The biological activity of (+)-(R,S)-tau-fluvalinate is significantly higher than that of (-)-(R,R)-tau-fluvalinate. In order to control the standardized use of bifenthrin and fluvalinate, various countries have established maximum residue limits for these two pesticides in cabbage. Table 1 lists the residue limits of bifenthrin and fluvalinate in cabbage. However, these regulations only stipulate the safe use limits of the racemic forms of bifenthrin and fluvalinate, and do not accurately stipulate the residue limits of their enantiomers.

[0004] Table 1 Residue limits of bifenthrin and fluvalinate in cabbage

[0005] Compound EU (mg / kg) China (mg / kg) Japan(mg / kg) United States (mg / kg) Bifenthrin 0.4 0.2 0.4 4 Fluvalinate 0.3 0.5 0.5 none

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

[0007] For example, the Chinese invention patent (publication number CN113433257A, publication date: 2021-09-24) applied by the applicant of this application discloses a method for separating and determining carnitine enantiomers in health foods based on ultra-high performance convergence chromatography technology. The method uses ultra-high performance convergence chromatography to separate carnitine enantiomers and determine the residual amount of L-carnitine and D-carnitine enantiomers in health foods. The sample is ultrasonically extracted with anhydrous ethanol, derivatized, separated by Acquity Trefoil CEL1 chiral chromatographic column, and gradient eluted with supercritical carbon dioxide-1% (v / v) ammonia methanol and other co-solvents as mobile phases, and quantified by external standard method.

[0008] The Chinese invention patent (publication number CN114858942A, publication date: 2022-08-05) discloses a method for rapid determination of cypermethrin enantiomer residues in pears and their products. The sample is extracted with ethyl acetate, purified by a C18 column, separated by an Acquity Trefoil AMY1 chiral chromatographic column, and eluted with a mobile phase of 0.5% ammonia in methanol solution-supercritical carbon dioxide in different volume ratios. The external standard method is quantitative, and the lower limit of determination is 0.2 mg / kg, the recovery rate of spiked addition is in the range of 81.4%-106%, and the RSD is 4.1%-7.2%. The actual sample was tested, and the detection amount was 0.22-0.25 mg / kg.

[0009] A Chinese invention patent (publication number CN114935611A, publication date: 2022-08-23) discloses a method for rapid determination of cypermethrin enantiomer residues in fruit and vegetable purees (apple puree, strawberry puree, tomato puree) by ultra-high performance convergence chromatography. The sample of this method is extracted with acetonitrile, purified by a solid phase extraction column, and separated by a CHIRALPAK AD-3 chiral chromatographic column, with supercritical carbon dioxide-methanol as the mobile phase, gradient elution at a flow rate of 1.5mL / min, detection wavelength of 230nm, and external standard method quantification. The method quantitative limits of the two cypermethrin enantiomers are both 0.2mg / kg, the linear range is 1.0-20mg / L, the linear correlation coefficient is greater than 0.9992, and the recovery range at three spike levels (0.2, 0.4 and 2.0mg / kg) is 80.6% to 105%, and the relative standard deviation is 2.6% to 7.7%.

[0010] Based on this, it is particularly important to develop a method for rapidly determining the enantiomer residues of bifenthrin and fluvalinate in cabbage in the present invention, so as to effectively control the residues in food under the premise of ensuring the safety of pesticide use. Summary of the invention

[0011] In order to solve the above technical problems, the purpose of the present invention is to provide a method for quickly determining the enantiomer residues of bifenthrin and fluvalinate in cabbage. The method is easy to operate, has good separation effect, is green and environmentally friendly, and can meet the needs of enantiomer residue analysis of bifenthrin and fluvalinate.

[0012] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

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

[0014] 1) Sample extraction

[0015] Weigh the sample into a centrifuge tube, add acetonitrile to homogenize and extract, then add sodium chloride and vortex to mix, centrifuge and take the supernatant into another centrifuge tube; add acetonitrile to the lower layer residue, repeat the extraction once, combine the supernatants of the two times, concentrate to near dryness, add n-hexane to dissolve, and wait for purification;

[0016] 2) Purification

[0017] The reconstituted solution was transferred to a Florisil column activated with n-hexane, washed with n-hexane, and then eluted with a solution of n-hexane: ether (7:3, v / v). The eluted solution was blown to near dryness with liquid nitrogen, dissolved in methanol, filtered through a membrane filter, and tested on an instrument.

[0018] 3) High performance convergence chromatography conditions

[0019] The OJ-H chiral chromatographic column was selected to separate the four pyrethroid enantiomers. Methanol was selected as the co-solvent 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 ultra-high performance convergence chromatography conditions, with mass concentration X as the abscissa and peak area Y as the ordinate, and the compounds were quantified by the external standard method.

[0022] Preferably, in the step 1), 5 g of the sample is weighed to an accuracy of 0.01 g, and 20 mL of acetonitrile is added to a 50 mL centrifuge tube for homogenous extraction. Then, 3.0 g of sodium chloride is added, vortexed and mixed, and after centrifugation at 4,000 r / min for 5 min, the supernatant is taken into another 50 mL centrifuge tube; 20 mL of acetonitrile is added to the lower layer residue, and the extraction is repeated once, and the supernatants are combined and concentrated to near dryness using a rotary evaporator, and 10 mL of n-hexane is added for dissolution.

[0023] Preferably, in step 2), the complex solution is transferred to a Florisil column activated with n-hexane, eluted with 5 mL of n-hexane, and then eluted with 10 mL of n-hexane: ether (7:3, v / v) solution, the eluted liquid nitrogen is blown to near dryness, 1 mL of methanol is made up to volume and dissolved, and the made up solution is filtered through a 0.22 μm filter membrane.

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

[0025] Preferably, the enantiomer standard stock solution is prepared as follows: accurately weigh 0.01 g, accurate to 0.1 mg, of the racemic standard of bifenthrin and fluvalinate, dissolve them in anhydrous ethanol and make up to 10 mL to prepare a 1.0 g / L racemic standard stock solution; the standard intermediate solution of bifenthrin and fluvalinate racemate is prepared as follows: accurately take a certain amount of the racemic standard stock solution and dilute it with anhydrous ethanol to a 10.0 mg / L standard intermediate solution.

[0026] Preferably, the enantiomeric standard stock solutions are prepared as follows: 0.01 g, accurate to 0.1 mg, of (-)-(1S, 3S)-cis-bifenthrin, (+)-(1R, 3R)-cis-bifenthrin, (-)-(R, R)-tau-fluvalinate, and (+)-(R, S)-tau-fluvalinate standards are accurately weighed, dissolved in methanol and diluted to 10 mL to prepare 1.0 g / L enantiomeric standard stock solutions; the enantiomeric stock solutions of the four pyrethroids are prepared. The mixed standard working solution of enantiomers was 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 solutions were accurately taken and diluted stepwise with methanol to a mixed standard working solution of 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L.

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

[0028] Preferably, the spiked recoveries of the four pyrethroid enantiomers are 80.4% to 102%, and the relative standard deviations (RSDs) are 2.3% to 6.8%.

[0029] The present invention adopts the above-mentioned technical scheme, and for the first time, ultra-high performance convergence chromatography is used to simultaneously separate four enantiomers of bifenthrin and fluvalinate, and the residual amounts of bifenthrin and fluvalinate enantiomers in cabbage are determined, and the residual digestion dynamics of bifenthrin and fluvalinate enantiomers on cabbage are studied. The method optimizes the instrument separation conditions such as the chromatographic column and column temperature, cosolvent, system back pressure, constant volume reagent, etc., and the stability of the standard solution of bifenthrin and fluvalinate enantiomers is investigated, and the established method is applied to the actual field sample detection. The results show that the method has high sensitivity and good stability, and can meet the needs of detecting the chiral enantiomer residues of bifenthrin and fluvalinate in cabbage. The method provides a reference for in-depth analysis of the enantiomers with efficacy and low toxicity and the enantiomers of pesticides with no efficacy and high toxicity in agricultural products, and is of great significance to the quality control and efficacy evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the spectrum of the pyrethroid enantiomer standard solution.

[0031] Figure 2 This study investigated the stability of four pyrethroid enantiomer standard solutions within 60 days.

[0032] Figure 3 This is the effect of different chromatographic columns on the separation of four pyrethroid enantiomers. Figure 3 in (a) AD-3; Figure 3 (b) IC; Figure 3 Middle (c) OJ-H.

[0033] Figure 4 This is the effect of different co-solvents on the separation of four pyrethroid enantiomers. Figure 4 (a) 0.5% (v / v) ammonia methanol solution; Figure 4 (b) 0.5% (v / v) formic acid methanol solution; Figure 4 (c) Methanol.

[0034] Figure 5 This is the effect of different system back pressures on the separation of four pyrethroid enantiomers. Figure 5 Medium (a) 10.3MPa, Figure 5 Middle (b) 12.1MPa, Figure 5 Middle (c) 13.8MPa.

[0035] Figure 6 The effect of different chromatographic column temperatures on the separation of four pyrethroid enantiomers. Figure 6 Medium (a) 31°C; Figure 6 Medium (b) 35°C; Figure 6 Medium (c) 40℃.

[0036] Figure 7 This is the effect of different volume-fixing reagents on the separation of four pyrethroid enantiomers. Figure 7 (a) methanol; Figure 7 (b) acetonitrile; Figure 7 (c) n-heptane.

[0037] Figure 8 The chromatograms are of standard solution (a), blank cabbage sample (b) and spiked 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] Fig. 9 For the separation of the racemic isoforms of bifenthrin and fluvalinate. Chromatographic peaks: 1. (-)-(1S,3S)-cis-bifenthrin, 2.

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

[0042] Fig.10 These are the degradation curves of bifenthrin and fluvalinate enantiomers in cabbage leaves. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] 1. Instruments, Materials, and Reagents

[0045] Acquity ultra-high performance convergence chromatograph (Waters, USA, with diode array detector (PDA)); AE260 electronic balance (Mettler, Switzerland); R215 rotary evaporator (Buchi, Switzerland); ELGA CLXXXUVM2 ultrapure water purification system (Elga, UK); MS2 vortex mixer (Shanghai Medical University Instrument Factory); N-EVAP TM 111 nitrogen blowdown apparatus (Rika Co., Ltd., Tokyo, Japan).

[0046] Acetonitrile, methanol, formic acid, n-heptane, n-hexane (chromatographic grade, Scharlau, Spain); ammonia water, ether (superior grade); ultrapure water; high-purity carbon dioxide (99.999%); Florisil column (CNW, 5 g, 6 mL); cellulose derivative normal phase chiral column CHIRALPAK OJ-H (100 mm × 4.6 mm, 5 μm, spherical silica gel coated with chiral polymer (amylose or fiber derivative) on the surface); DAICEL CHIRALPAK AD-3 (150 mm × 3.0 mm, 3 μm, filled with amylose-tris (3,5-dimethylphenylcarbamate)); DAICEL CHIRALPAK IC (100 mm × 4.6 mm, 5 μm, cellulose-tris (3,5-dichlorophenylcarbamate) covalently bonded to the silica gel surface); other reagents used in the experiments were of analytical grade unless otherwise specified.

[0047] Two racemic standards (bifenthrin: CAS No.: 82657-04-3, purity ≥ 99.7%, DrEhrenstorfer, Germany; fluvalinate: CAS No.: 102851-06-9, purity ≥ 97.3%, DrEhrenstorfer, Germany). Bifenthrin and fluvalinate enantiomer standards: (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate, (+)-(R,S)-tau-fluvalinate were obtained by Shanghai Qinlu Biotechnology Co., Ltd. from bifenthrin and fluvalinate racemic standards (DrEhrenstorfer), respectively, with a purity greater than 97.0%.

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

[0049] 2.1 Racemic standard stock solution

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

[0051] Standard intermediate solution of bifenthrin and fluvalinate racemates: Accurately pipette a certain amount of racemate standard stock solution and dilute it with anhydrous ethanol to a standard intermediate solution of 10.0 mg / L.

[0052] 2.2 Enantiomer 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-fluvalinate, and (+)-(R,S)-tau-fluvalinate standards, dissolve them in methanol and make up to 10 mL to prepare 1.0 g / L enantiomer standard stock solutions.

[0054] Mixed standard working solutions of four pyrethroid enantiomers: accurately pipette a certain amount of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate, and (+)-(R,S)-tau-fluvalinate enantiomer standard stock solutions, and dilute them stepwise with methanol to mixed standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L.

[0055] 3. Sample Pretreatment

[0056] 3.1 Sample extraction

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

[0058] 3.2 Purification

[0059] The complex solution was transferred to a Florisil column activated with n-hexane, rinsed with 5 mL of n-hexane, and then eluted with 10 mL of n-hexane: ether (7:3, v / v). The eluted liquid nitrogen was blown to near dryness, and 1 mL of methanol was made up to volume and dissolved. The made-up solution was filtered through a 0.22 μm filter membrane and tested on an instrument.

[0060] 4. Chromatographic conditions

[0061] Chromatographic column: CHIRALPAK OJ-H (100mm×4.6mm, 5μm); mobile phase: A is CO2, 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 with a PDA detector, the UV spectra of the four pyrethroid enantiomer standard solutions were extracted from the chromatogram. Figure 1 As shown, the four pyrethroid enantiomers all have the strongest absorption at 203nm and the highest sensitivity, so 203nm was selected as the detection wavelength in this experiment.

[0065] 5.2 Investigation on the stability of pyrethroid enantiomer standard solution

[0066] Accurately pipette 1.0 mL of 5.0 mg / L mixed standard working solution of pyrethroid enantiomers onto seven scratched 1.5 mL UPC 2The samples were put into special injection vials and measured on the machine. After the test, they were transferred to 7 injection vials sealed with aluminum caps, sealed with sealing film and stored at -18°C. The newly prepared standard solutions of bifenthrin and fluvalinate enantiomers with a mass concentration of 10.0 mg / L were compared with the measurement results of 5.0 mg / L bifenthrin and fluvalinate enantiomers after storage for 1, 3, 5, 7, 14, 30 and 60 days, respectively. The newly prepared standard solution was taken as 100%, and the change of bifenthrin and fluvalinate enantiomer standard solutions less than 10% was taken as the benchmark. The results showed that the measurement results of the four pyrethroid enantiomers all showed a gradually decreasing trend (see Figure 2 ), wherein the contents of the two enantiomers of bifenthrin decreased by more than 12% when placed at -18°C for 60 days, the contents changed by less than 10% when placed at 30 days, and the contents changed by less than 5% when placed at 14 days, indicating that the two enantiomers of bifenthrin were relatively stable within 30 days; the contents of the two enantiomers of fluvalinate decreased by more than 18% when placed at -18°C for 60 days, the contents decreased by more than 12% when placed at 30 days, and the contents changed by less than 10% when placed at 14 days, indicating that the two enantiomers of fluvalinate were relatively stable within 14 days.

[0067] 5.3 Optimization of chromatographic columns

[0068] In this experiment, three chiral separation columns, CHIRALPAK AD-3, IC, and OJ-H, from Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd., were selected to investigate the separation effect of four pyrethroid enantiomers. The results showed that when using AD-3 and IC chiral columns for separation, only 2 or 3 chromatographic peaks appeared on the chromatogram, and the four pyrethroid enantiomers were not completely separated; when using OJ-H chiral column for separation, the separation was good and the chromatographic peak shape was sharp (see Figure 3 ). Therefore, in this experiment, the OJ-H chiral column was selected to separate the four pyrethroid enantiomers.

[0069] 5.4 Selection of cosolvent in mobile phase

[0070] Ultra-high performance convergence chromatography consumes less organic solvents and uses supercritical CO2 as the main mobile phase. A small amount of organic solvent is usually used as a co-solvent to enhance the elution ability and selectivity of the target product. This experiment investigated the effects of different co-solvents such as 0.5% (v / v) ammonia methanol solution, 0.5% (v / v) formic acid methanol solution, and methanol on the separation of four pyrethroid enantiomers. The results showed that when 0.5% (v / v) formic acid methanol solution was used as a co-solvent, the four pyrethroid enantiomers failed to elute; when 0.5% (v / v) ammonia methanol solution and methanol were used as co-solvents, the chromatographic peaks of the four pyrethroid enantiomers were completely separated within 5.0 min, and the peak shape was good ( Figure 4), but compared with 0.5% (v / v) ammonia methanol solution as a co-solvent, methanol is more convenient to operate and less damaging to the instrument and chromatographic column. Therefore, this laboratory chose methanol as a co-solvent.

[0071] 5.5 System Backpressure Selection

[0072] UPC 2 Using supercritical CO2 as the mobile phase, the density of CO2 can be effectively changed by adjusting the system back pressure and temperature, thereby changing its solubility, elution ability and selectivity for substances. Since the temperature of CO2 exceeds 31°C and the pressure exceeds 7.38MPa, CO2 will enter the supercritical CO2 state. Therefore, this experiment investigates the effect of system back pressure in the range of 10.3 to 13.8MPa on the separation of four pyrethroid enantiomers. The results show that as the system back pressure increases, the retention time of the analyte is advanced (see Figure 5 ). When the system back pressure was 13.8 MPa, the separation between the chromatographic peak of (-)-(R,R)-tau-fluvalinate and the interference peak was too low. Compared with the system back pressure of 10.3 MPa, the chromatographic peak shape was sharper when the system back pressure was 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 highest recommended operating temperature of CHIRALPAK OJ-H chiral chromatographic column is 40℃, CO2 will enter the supercritical CO2 state only when the temperature of CO2 exceeds 31℃ and the pressure exceeds 7.38MPa. Therefore, this experiment investigated the effect of chromatographic column temperature in the range of 31-40℃ on the separation of four pyrethroid enantiomers. Under the three column temperature conditions, the chromatographic peak separation of the four pyrethroid enantiomers was good, and good baseline separation was achieved within 5.0min, with a fast analysis speed ( Figure 6 ). Taking the life of the chromatographic column into consideration, the optimal column temperature is 31°C.

[0075] 5.7 Selection of volume-fixing reagents

[0076] The enantiomers of 5.0 mg / L bifenthrin and fluvalinate were separated using three constant volume reagents: methanol, acetonitrile, and n-heptane. The results are as follows: Figure 7 As shown in the figure, when acetonitrile was used as the constant volume reagent, the peak shape of the target was poor; when methanol and n-heptane were used as constant volume reagents, the chromatographic peaks of the four pyrethroid enantiomers were completely separated within 5.0 min, and the peak shape was good. Compared with n-heptane, methanol has stronger solubility and lower volatility, which is convenient for subsequent analysis and detection. Therefore, methanol was finally determined as the constant volume reagent for this experiment.

[0077] 5.8 Selection of eluent for Florisil soil column

[0078] Normally, n-hexane ether solution is used to elute and purify pyrethroid pesticides in samples [33,34]. The experiment compared the elution effect of mixed solutions of n-hexane-ether with different volume ratios (6:4, 7:3, 8:2, 9:1, v / v) on the target. Referring to the extraction method in Section 3.1, the extract was eluted on a Florisil column, and then eluted with these three eluents and tested on the machine. The results showed that as the proportion of ether increased, the recovery rate gradually increased, but the impurity interference peak liquid increased accordingly. Taking all factors into consideration, when n-hexane: ether (7:3, v / v) was used for elution, the interference peaks in the chromatogram were relatively small, and the recovery rate reached more than 95.0%.

[0079] 5.9 Methodological Review

[0080] 5.9.1 Linear range and limit of quantitation

[0081] The series of mixed standard solutions of bifenthrin and fluvalinate enantiomers were determined under the above chromatographic conditions. The standard curve was drawn with the peak area (Y) of the standard as the ordinate and the corresponding mass concentration (X) as the abscissa, and the regression equation and correlation coefficient were obtained. The results showed that the four enantiomers showed a good linear relationship in the mass concentration range of 0.5 to 20.0 mg / L, and the correlation coefficient was greater than 0.9992. By adding standard samples to blank cabbage samples not containing bifenthrin and fluvalinate, the method was used for determination, and the limit of quantification (LOQ) was calculated with a signal-to-noise ratio (S / N) of 10. The LOQs of (-)-(1S,3S)-cis-bifenthrin and (+)-(1R,3R)-cis-bifenthrin were both 0.1 mg / kg; the LOQs of (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate were both 0.2 mg / kg.

[0082] 5.9.2 Recovery and precision

[0083] Four pyrethroid enantiomer standard solutions at different concentrations were added to cabbage samples without bifenthrin and fluvalinate to conduct spike recovery and precision tests. The relevant chromatograms are shown in Figure 8 The results are shown in Table 2. The results showed that the recoveries of the four pyrethroid enantiomers were 80.4% to 102%, and the relative standard deviations (RSDs) were 2.3% to 6.8%, which met the recovery requirements of SANTE / 11312 / 2021

[35] and were suitable for the determination of the enantiomers of bifenthrin and fluvalinate in cabbage samples.

[0084] Table 2 Spiked recoveries and relative standard deviations of bifenthrin and fluvalinate enantiomers in cabbage samples (n=6)

[0085]

[0086] 5.10 Application of the method

[0087] 5.10.1 Separation of racemic standard

[0088] The method established in this paper was used to separate and determine the purchased bifenthrin and fluvalinate racemic standards. Fig. 9 As shown in a, the separation effect of the four pyrethroid enantiomers was good, and effective separation was achieved within 4.0 min, with the resolution of R 1,2 =1.9, R 2,3 =4.8 and R 3,4 =3.4, which meets the requirement of complete separation of R≥1.5

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

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

[0090] The contents of (+)-(R,S)-tau-fluvalinate were 5.36, 4.85, 5.34, and 5.32 mg / L, respectively. The calculated results are consistent with the literature [37,38] that the ratio of (-)-(1S,3S)-cis-bifenthrin to (+)-(1R,3R)-cis-bifenthrin in the industrial bifenthrin racemate is 1.11:1.00, and the ratio of (-)-(R,R)-tau-fluvalinate to (+)-(R,S)-tau-fluvalinate in the fluvalinate racemate is 1.00:1.00.

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

[0092] Using the established UPC system for residues of chiral pesticides bifenthrin and fluvalinate in cabbage 2The analytical method was used to study the degradation law of bifenthrin and fluvalinate pesticides after spraying on cabbage leaves. 10% bifenthrin and 10% fluvalinate pesticides were diluted 2000 times respectively, and then sprayed on cabbage leaves. Samples were taken after 4h and 1, 3, 5, 7, and 10d after spraying, and the residues of two groups of chiral enantiomers of bifenthrin and fluvalinate in cabbage leaves were determined to obtain their degradation law. The results showed that under the usual concentration, the residues of bifenthrin and fluvalinate in cabbage were less than 0.5mg / kg ( Fig.10 ). The residues of bifenthrin and fluvalinate on cabbage were mostly distributed in the outer layer, while no residues were detected in the inner layer. This is related to the fact that bifenthrin and fluvalinate have weak permeability and only contaminate the surface of crop tissues. The half-life of the four enantiomers is about 2.5 days. The half-life shows that there is no obvious selectivity difference in the degradation of the four pyrethroid pesticide enantiomers during the growth of cabbage leaves.

[0093] 5.10.3 Testing of actual samples

[0094] In order to investigate the effectiveness and practicality of this method, the established method was used to determine 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. The results showed that bifenthrin and fluvalinate enantiomers were not detected in any of 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 those of other reported methods. The analysis time of this method was short (5min), the purification effect was good, the specificity was strong, and it could meet the daily detection requirements of bifenthrin and fluvalinate. The limit of quantification (LOQ) of this method could meet the requirements of the maximum residue limit of various countries' regulations. In addition, the linear range, recovery rate and RSD of this method were consistent with those of other methods.

[0097] Table 3. Comparison with other reported methods

[0098]

[0099] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A method for rapidly determining the residual enantiomers of bifenthrin and fluvalinate in cabbage, wherein the structures of the enantiomers of bifenthrin and fluvalinate are (-)-(1S, 3S)-cis-bifenthrin, (+)-(1R, 3R)-cis-bifenthrin, (-)-(R, R)- tau-fluvalinate and (+)-(R, S)- tau-fluvalinate, respectively, characterized in that: The method comprises the following steps: 1) Sample extraction Weigh the sample into a centrifuge tube, add acetonitrile to homogenize and extract, then add sodium chloride and vortex to mix, centrifuge and take the supernatant into another centrifuge tube; add acetonitrile to the lower layer residue, repeat the extraction once, combine the supernatants of the two times, concentrate to near dryness, add n-hexane to dissolve, 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 n-hexane: ether (7:3, v / v) solution. The eluted solution was blown to near dryness with liquid nitrogen, dissolved in methanol, filtered through a filter membrane, and tested on an instrument. 3) High performance convergence chromatography conditions The OJ-H chiral chromatographic column was selected to separate the four pyrethroid enantiomers. Methanol was selected as the co-solvent 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. 4) Quantitative determination The standard working solution and the sample solution were injected under the set ultra-high performance convergence chromatography conditions, with mass concentration X as the abscissa and peak area Y as the ordinate, and the compounds were quantified by the external standard method.

2. The method according to claim 1, characterized in that In step 1), weigh 5 g of the sample accurately to 0.01 g, put it into a 50 mL centrifuge tube, add 20 mL of acetonitrile, homogenize and extract, then add 3.0 g of sodium chloride, vortex mix, centrifuge at 4 000 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 add 10 mL of n-hexane to dissolve.

3. The method according to claim 1, characterized in that In step 2), the complex solution is transferred to a Florisil column activated with n-hexane, eluted with 5 mL of n-hexane, and then eluted with 10 mL of n-hexane: ether (7:3, v / v) solution. The eluted solution is blown to near dryness with liquid nitrogen, and dissolved with 1 mL of methanol. The fixed volume solution is filtered through a 0.22 μm filter membrane.

4. The method according to claim 1, characterized in that: Step 3) 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 (14% B); System back pressure: 12.1 MPa; Flow rate: 1.5 mL / min; Injection volume: 5 µL; Column temperature: 31°C; detection wavelength: 203 nm.

5. The method according to claim 1, characterized in that The enantiomer standard stock solutions were prepared as follows: 0.01 g, accurate to 0.1 mg, of the racemic standard of bifenthrin and fluvalinate were accurately weighed, dissolved in anhydrous ethanol and made up to 10 mL to prepare 1.0 g / L racemic standard stock solutions; the standard intermediate solutions of bifenthrin and fluvalinate racemates were prepared as follows: a certain amount of the racemic standard stock solutions were accurately aspirated and diluted with anhydrous ethanol to a standard intermediate solution of 10.0 mg / L.

6. The method according to claim 5, characterized in that The enantiomeric standard stock solutions were prepared as follows: 0.01 g, accurate to 0.1 mg, of (-)-(1S,3S)-cis-bifenthrin, (+)-(1R,3R)-cis-bifenthrin, (-)-(R,R)-tau-fluvalinate, and (+)-(R,S)-tau-fluvalinate standards were accurately weighed and dissolved in methanol to a volume of 10 mL to prepare 1.0 g / L enantiomer standard stock solution; the mixed standard working solutions of the four pyrethroid enantiomers were 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 solutions were accurately taken and diluted stepwise with methanol to 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L mixed standard working solutions.

7. The method according to claim 1, characterized in that Signal-to-Noise Ratio S / N =10 to calculate the limit of quantification LOQ, and the LOQs of (-)-(1S,3S)-cis-bifenthrin and (+)-(1R,3R)-cis-bifenthrin were both 0.1 mg / kg; the LOQs of (-)-(R,R)-tau-fluvalinate and (+)-(R,S)-tau-fluvalinate were both 0.2 mg / kg.

8. The method according to claim 1, characterized in that The recoveries of the four pyrethroid enantiomers were 80.4%-102%, and the relative standard deviations (RSD) were 2.3%-6.8%.

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