Rapid separation and quantitative determination method of dinotefuran enantiomer in cucumber sample, analysis system and application

Abstract

The invention relates to a method for determining pesticide residues, in particular to a method for rapidly separating and quantitatively determining dinotefuran enantiomers in a cucumber sample, an analysis system and application of the dinotefuran enantiomers. The method comprises the following steps: extracting a cucumber sample by using a 1% acetic acid-acetonitrile solution, purifying the cucumber sample by using a Pestii-Carb / PSA solid-phase extraction small column, fixing the volume by using isopropanol / n-heptane (2: 8, v / v), introducing the sample into a UPC2 system equipped with a CHIRALPAK AD-3 chiral chromatographic column, and realizing baseline separation of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran within 4 minutes under the conditions of supercritical CO2 / methanol gradient elution, proper column temperature and back pressure. The method is good in linearity within the range of 0.5-20.0 mg / L, the quantitation limits of the two enantiomers in the cucumber matrix are both 0.1 mg / kg, the adding standard recovery rate is 80.4%-106%, and the relative standard deviation is not larger than 7.5%. The method and the system have the advantages of high analysis speed, low solvent consumption and high sensitivity and precision, and are suitable for conventional monitoring and risk assessment of dinotefuran enantiomer residues in vegetables such as cucumbers and the like.

Description

Technical Field

[0001] This invention relates to methods for determining pesticide residues, and more particularly to a rapid method, analytical system, and application for the separation and quantitative determination of enantiostats in cucumber samples. Background Technology

[0002] Fipronil is a third-generation neonicotinoid insecticide, also known as "furanotinic acid." Its molecular structure does not contain chlorine atoms or aromatic rings; instead, it uses a tetrahydrofuran group to replace the chlorinated groups found in traditional neonicotinoid insecticides, resulting in superior systemic penetration and insecticidal activity. Fipronil has a broad insecticidal spectrum and is widely used on vegetables, rice, fruit trees, and other crops to control piercing-sucking and chewing pests such as planthoppers, aphids, and whiteflies. It is also highly effective against sanitary pests such as cockroaches and fleas. Research indicates that chirality is generated at the third carbon atom in the carbon structure of fipronil, giving it chirality.

[0003] The insecticidal activity and degradation rate of different enantiomers of dinotefuran vary considerably. (+)-(S)-dinotefuran exhibits higher insecticidal activity than (-)-(R)-dinotefuran, but its degradation rate is slower. In agricultural production, using a single, highly active (+)-(S)-dinotefuran would help reduce the amount of dinotefuran used and decrease pesticide pollution. Currently, dinotefuran is still produced and used in its racemic form. To regulate the standardized use of dinotefuran, GB 2763-2021 stipulates a maximum residue limit of 2 mg / kg for dinotefuran in cucumbers. Further research on the residue levels of the two enantiomers of dinotefuran in cucumbers would provide a more accurate basis for the analysis of dinotefuran enantiomer residues.

[0004] Currently, the main methods for detecting enantiomers of fipronil both domestically and internationally are enzyme-linked immunosorbent assay (ELISA), liquid chromatography (LC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, for chiral pesticides, traditional residue analysis methods do not distinguish between enantiomers, treating all enantiomers of chiral pesticides as the same substance and ignoring factors such as significant differences in activity and toxicity between enantiomers. Therefore, data obtained using traditional analytical methods cannot accurately assess the environmental risks of chiral pesticides. In this context, establishing residue analysis methods for chiral pesticides at the enantiomer level is particularly important. The emerging ultra-high performance phase chromatography (UPC) technique... 2 This novel chromatographic technique primarily uses supercritical CO2 as the main mobile phase, with small amounts of acetonitrile, methanol, isopropanol, and other co-solvents added. By adjusting the system back pressure, column temperature, and co-solvent ratio, the density and elution capacity of CO2 are altered, thereby precisely controlling the separation of target compounds. Therefore, this novel chromatographic separation technique exhibits excellent separation performance for structurally similar chiral compounds. Currently, UPC... 2 No reports have been found on the application of this technology to the separation and residue determination of enantiomers of fipronil. Summary of the Invention

[0005] The technical objective of this invention is to address the problems of existing methods for analyzing dinotefuran residues, such as "measuring only the total amount, not separating enantiomers, long analysis time, high solvent consumption, and significant matrix interference in vegetable matrices such as cucumbers." This invention provides a rapid separation and quantitative determination method and corresponding analytical system for dinotefuran enantiomers based on solid-phase extraction-ultra-high performance phase chromatography (UPC). This method involves sample extraction, purification, solvent adjustment, and UPC... 2 Optimized matching of chiral chromatographic separation conditions enables efficient baseline separation and low limit of quantitation determination of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran in cucumber samples within a short time, thus providing a rapid, green, sensitive and reliable technical means for monitoring dinotefuran enantiomer residues and assessing food safety risks.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A rapid method for the separation and quantitative determination of enantiostatin enantiomers in cucumber samples includes the following steps: S1) Sample extraction: Weigh 2-10g of cucumber sample, add 5-8 times the mass-volume ratio of 0.5-2% (volume fraction) acetic acid-acetonitrile solution, shake to extract and centrifuge, collect the supernatant, repeat the extraction of the residue once or twice, combine the extracts and concentrate under reduced pressure at 35-45℃ to near dryness, add 5-15mL of acetonitrile to redissolve, and obtain the extract to be purified; S2) Solid-phase extraction purification: The extract to be purified is loaded into a multifunctional solid-phase extraction column pre-activated with acetonitrile. The solid-phase extraction column contains a stacked packing material of graphitized carbon black and primary secondary amine adsorbent. The eluent is collected under gravity or negative pressure and eluted with 3-8 mL of acetonitrile. All eluents are combined and rotary evaporated to near dryness at 35-45°C. S3) Volume adjustment and injection solvent treatment: Add a mixed solvent of isopropanol and n-heptane to the evaporated residue to dissolve and adjust the volume, wherein the volume ratio of isopropanol to n-heptane is 5:5 to 0:10, preferably 2:8, to obtain the chromatographic injection solution. S4) Ultra-high performance phase chromatography (UHPLC) separation: The chromatographic injection solution is injected into an UHPLC system equipped with a chiral column. The chiral column is a linear starch-tris(3,5-dimethylphenylcarbamate) bonded stationary phase column with a packing particle size of 2–5 µm, a column length of 100–200 mm, and an inner diameter of 3.0 mm. Supercritical carbon dioxide is used as the main mobile phase A, and methanol as the co-solvent B. The system is operated at a back pressure of 10–15 MPa and a rotation speed of 30–40 °C. Under column temperature conditions of ℃, gradient elution was performed with a B phase volume fraction ranging from 10% to 30%. The B phase was maintained at 10% to 20% for 0 to 1 min, then linearly increased to 20% to 25% for 1 to 3 min, maintained at 20% to 25% for 3 to 4 min, and then decreased back to 10% to 20% for 4 to 6 min. This allowed (+)-S-fipronil and (-)-R-fipronil to achieve baseline separation within 4.0 min, with a resolution R ≥ 4.0. S5) Detection and Quantification: Chromatograms were acquired at an ultraviolet detection wavelength of 260–280 nm. An external standard working curve was established using a mixed standard solution of enantiomers of dinotefuran in the concentration range of 0.5–20.0 mg / L. The contents of (+)-S-dinotefuran and (-)-R-dinotefuran in cucumber were calculated by external standard method using the peak area of ​​the sample chromatogram. The method limit of quantitation was not higher than 0.1 mg / kg, and the spiked recoveries of the two enantiomers were between 80.4% and 106% with a relative standard deviation of not more than 8% at a spiked level of 0.1–1.0 mg / kg.

[0007] Preferably, the amount of cucumber sample weighed in S1) is 4-6g, the volume fraction of acetic acid in the acetic acid-acetonitrile solution is 1%, the volume of a single extraction is 15-25mL, and the ratio of the total liquid volume of two extractions to the sample mass is 6:1-10:1.

[0008] Preferably, in S2), the solid-phase extraction column is a Pesti-Carb / PSA column, the mass of graphitized carbon black filler is 300-700 mg, the mass of primary secondary amine filler is 300-700 mg, the total volume is 5-10 mL, the sample loading volume is 8-12 mL, and the elution volume is 4-6 mL.

[0009] Preferably, in step S3), the solvent for volume adjustment is isopropanol:n-heptane = 2:8 (volume ratio), the volume adjustment volume is 0.5-2.0 mL, and the injection volume is 3.0-7.0 μL.

[0010] Preferably, in S4): The chiral chromatographic column was a CHIRALPAKAD-3 column with a length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 3 μm. The system back pressure is 12.0–13.8 MPa, preferably about 12.4 MPa; The column temperature is 33–37°C, preferably 35°C; The total flow rate is 0.8–1.2 mL / min, preferably 1.0 mL / min; The B-phase gradient program satisfies the following: 15%B from 0 to 0.7 min, linearly increasing from 15% to 20%B from 0.7 to 1.2 min, maintaining 20%B from 1.2 to 1.6 min, increasing from 20% to 25%B from 1.6 to 2.2 min, maintaining 25%B from 2.2 to 3.0 min, linearly decreasing from 25% to 15%B from 3.0 to 4.0 min, and maintaining 15%B from 4.0 to 6.0 min.

[0011] Furthermore, the present invention also provides a dinotefuran enantiomeric residue analysis system for implementing the method, comprising: The sample pretreatment unit includes: The acidified acetonitrile extraction module is used to add 0.5-2% (volume fraction) acetic acid-acetonitrile solution to cucumber samples and complete the shaking, centrifugation and extraction liquid merging. The solid-phase extraction purification module is equipped with a multifunctional solid-phase extraction column filled with graphitized carbon black and primary secondary amine adsorbent, and is used to purify and elute the extract. The concentration and volume adjustment module is used to concentrate the purified liquid to near dryness under reduced pressure and then add isopropanol / n-heptane mixed solvent to adjust the volume. The ultra-high performance phase chromatography unit includes: Ultra-high efficiency phase chromatography pump and mixer for delivering supercritical CO2 and methanol mobile phases and achieving a programmable gradient of 10% to 30% B; Back pressure regulator is used to control the system back pressure at 10-15 MPa; Chiral chromatographic column with a linear starch-tris(3,5-dimethylphenylcarbamate) bonded stationary phase, column length 100-200 mm, inner diameter 3.0 mm, particle size 2-5 µm; A UV-Vis diode array detector is used to detect effluent components in the wavelength range of 260–280 nm. The data processing and quantification unit is used to establish an external standard working curve based on a mixed standard solution of 0.5–20.0 mg / L dinotefuran enantiomers, integrate the chromatographic peaks of (+)-S-dinotefuran and (-)-R-dinotefuran in the sample, and calculate their residual content in cucumber samples.

[0012] Preferably, the data processing and quantification unit is further configured as follows: Based on the residual concentrations of (+)-S-dimethalin and (-)-R-dimethalin measured in the sample, their enantiomeric ratio was calculated and compared with the theoretical ratio of the racemic industrial product. An over-limit warning message will be output when the content of any enantiomer in the sample exceeds 0.1 mg / kg or the total residue is close to or exceeds the maximum residue limit of 2 mg / kg.

[0013] Preferably, the sample pretreatment unit and the ultra-high performance chromatographic unit are electrically connected to the same controller, which pre-stores the temperature, back pressure and gradient elution program and is configured to automatically complete the linkage control of sample extraction, solid phase extraction purification, concentration and volume adjustment and ultra-high performance chromatographic separation according to the preset program.

[0014] Furthermore, the present invention also provides a method for assessing the enantiomeric residue risk of fipronil based on the aforementioned method, comprising: The residual contents of (+)-S-dimethomorph and (-)-R-dimethomorph in cucumber samples were determined according to the method described above; The total residue of the two enantiomers was calculated and compared with the maximum residue limit of 2 mg / kg for dinotefuran in cucumber to determine whether the sample met the pesticide residue limit requirements. Based on the degree to which the residual ratio of the two enantiomers deviates from the theoretical ratio of the racemic mixture, the chiral selective degradation or accumulation behavior of dinotefuran during cultivation and distribution is assessed, and the assessment results are used as the basis for the environmental risk and product quality control of chiral pesticides.

[0015] Furthermore, the present invention also provides an application method for monitoring enantiomeric residues of fipronil in vegetable or fruit products. The method is characterized by employing the aforementioned separation and quantification method, combined with the aforementioned analytical system, to routinely monitor vegetable and / or fruit samples, including cucumbers, to obtain the changing trends of the residue levels and enantiomeric ratios of each enantiomeric dichlorvos across different product batches. This method is used for: To guide the optimization of fipronil application dosage and interval; and / or, This provides a technical basis for the use of a single, highly active (+)-S-fipronil formulation to replace racemic mixtures in terms of residue behavior and food safety.

[0016] This invention utilizes a synergistic technology system comprising acidified acetonitrile extraction, Pesti-Carb / PSA solid-phase extraction purification, isopropanol / n-heptane (2:8) volume adjustment, and UPC²-CHIRALPAKAD-3 chiral column gradient elution. This system achieves efficient baseline separation of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran within 4 min in the complex matrix of cucumber, with a chromatographic resolution R≥4.0, symmetrical peak shape, and stable baseline. The method exhibits good linearity in the range of 0.5–20.0 mg / L (correlation coefficient R0). 2The limits of quantification (LOQ) for both enantiomers in cucumber matrix were 0.1 mg / kg (>0.999). Recovery rates at spiking levels of 0.1–1.0 mg / kg ranged from 80.4% to 106%, with intra-day / inter-day RSDs less than 7.5%, meeting the accuracy and precision requirements of SANTE / 11312 / 2021 for pesticide residue analysis methods. Compared to existing HPLC chiral methods and SFC-MS / MS methods, this invention significantly shortens analysis time, reduces organic solvent consumption and matrix interference intensity while maintaining sensitivity and accuracy. This makes routine residue monitoring of dinotefuran enantiomers in cucumber more efficient, environmentally friendly, and scalable, and can further support environmental behavior studies at the enantiomer level and risk assessments of chiral formulations. Attached Figure Description

[0017] Figure 1 Stability study of two enantiomer standard solutions of dinotefuran over 60 days.

[0018] Figure 2 The effects of different gradient separation conditions on the separation of two fipronil products. (A) Isocratic separation condition 1, (B) Isocratic separation condition 2, (C) Gradient separation condition 3, (D) Gradient separation condition 4.

[0019] Figure 3 Effects of different system back pressures on the enantiomeric separation of two fipronil species. (A) 12.4 MPa, (B) 13.8 MPa, (C) 15.2 MPa, (D) 17.2 MPa.

[0020] Figure 4 The effect of different dilution reagents on the enantiomeric separation of two fipronil species. (A) Methanol, (B) Anhydrous ethanol, (C) Acetonitrile, (D) Isopropanol, (E) n-Heptane.

[0021] Figure 5 Effects of different isopropanol:n-heptane ratios on the enantiomeric separation of two fipronil species. (A) Isopropanol:n-heptane (5:5, v:v), (B) Isopropanol:n-heptane (4:6, v:v), (C) Isopropanol:n-heptane (3:7, v:v), (D) Isopropanol:n-heptane (2:8, v:v), (E) Isopropanol:n-heptane (1:9, v:v), (F) Isopropanol:n-heptane (0:10, v:v).

[0022] Figure 6 Chromatograms of standard solution (A), blank cucumber sample (B), and added recovery (C). Peak 1: (+)-S-dimethalin; Peak 2: (-)-R-dimethalin.

[0023] Figure 7Resolution of the racemic mixture of dinotefuran. Chromatographic peak 1: (+)-S-dinotefuran; Chromatographic peak 2: (-)-R-dinotefuran.

[0024] Figure 8 Chromatogram of a positive cucumber sample. Peak 1: (+)-S-dimethalin; Peak 2: (-)-(R)-dimethalin. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0026] 1. Terminology and Reagent Description To facilitate understanding of this invention, some terms and reagents are explained first. This description is for illustrative purposes only and does not constitute a limitation of the claims of this invention.

[0027] 1.1 Fipronil and its enantiomers Fipronil is a third-generation neonicotinoid insecticide. Its molecular structure contains a chiral carbon atom, allowing it to form a pair of enantiomers, denoted as (+)-(S)-fipronil and (-)-(R)-fipronil. The enantiomers of fipronil described in this invention specifically refer to these two enantiomers and their residual forms in cucumber samples.

[0028] 1.2 Ultra-high performance phase chromatography (UPC²) UPC² is a chromatographic technique that uses supercritical or near-supercritical carbon dioxide as the main mobile phase and a small amount of organic modifier. It combines the advantages of supercritical fluid chromatography and liquid chromatography, and features fast analysis speed, low solvent consumption, and high separation efficiency.

[0029] 1.3 Chiral Columns The chiral chromatographic column used in this invention is a polysaccharide chiral column with a linear starch-tris(3,5-dimethylphenylcarbamate) bonded stationary phase, such as CHIRALPAKAD-3 (150 mm × 3.0 mm, 3 µm). This type of column has good chiral recognition ability for enantiomers of nitrogen-containing heterocyclic and neonicotinoid pesticides.

[0030] 1.4 Pesti-Carb / PSA solid phase extraction column These small columns typically have a layered structure, with fillers including graphitized carbon black (GCB) and primary secondary amine (PSA) or equivalent combinations thereof. GCB is mainly used to remove hydrophobic co-extractants such as pigments, while PSA is mainly used to remove polar interfering substances such as organic acids and sugars.

[0031] 1.5% Isopropanol / n-heptane mixed solvent This invention uses a 2:8 volume ratio of isopropanol to n-heptane as the solvent for volume adjustment and injection, in order to balance the solubility of the target analyte and the chromatographic peak shape, and reduce the effect of strong solvents on UPC. 2 The effects of separation.

[0032] Unless otherwise specified, all solvents used in this invention are of chromatographic grade or equivalent; all water used is ultrapure water.

[0033] 2. Materials and Methods 2.1 Instruments, Materials and Reagents Acquity ultra-high performance phase chromatography system (Waters, USA, with diode array detector (PDA)); ELGACLXXXUVM2 ultrapure water purification system (Elga, UK); AE260 electronic balance (Mettler, Switzerland); R215 rotary evaporator (Buchi, Switzerland); N-EVAP TM 111 Nitrogen Evaporator (Tokyo Rika Co., Ltd., Japan); MS2 Vortex Mixer (Shanghai Medical University Instrument Factory).

[0034] Methanol, acetonitrile, isopropanol (chromatographic grade, Scharlau, Spain); ultrapure water; high-purity carbon dioxide (99.999%); Cleanert Pesti-Carb / PSA solid-phase extraction column (Agela Technology Co., Ltd., Tianjin, 500mg / 500mg / 6mL); Daicel CHIRALPAKAD-3 (150mm×3.0mm, 3µm, packing material: amylose-tris(3,5-dimethylphenylcarbamate)); other reagents used in the experiments were of analytical grade unless otherwise specified.

[0035] Racemic standard (fipronil: CAS No.: 165252-70-0, purity ≥99.7%, Anpuyun Laboratory Supplies (Shanghai) Co., Ltd.). Enantiomer standards for fipronil: (+)-(S)-fipronil and (-)-(R)-fipronil were obtained by Shanghai Qinlu Biotechnology Co., Ltd. from the racemic standard of fipronil (Anpuyun Laboratory Supplies (Shanghai) Co., Ltd.), with purities greater than 98.0%.

[0036] 2.2 Preparation of Standard Stock Solution and Working Solution 2.2.1 Racemic Standard Stock Solution Accurately weigh 0.01 g (accurate to 0.1 mg) of dinotefuran racemic standard, dissolve it in isopropanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution.

[0037] Intermediate standard solution of racemic dinotefuran: Accurately pipette a certain amount of racemic dinotefuran standard stock solution and dilute it with isopropanol to a standard intermediate solution of 20.0 mg / L.

[0038] 2.2.2 Enantiomer Standard Stock Solution Accurately weigh 0.01 g (accurate to 0.1 mg) of (+)-(S)-fipronil and (-)-(R)-fipronil standards, dissolve them in isopropanol and dilute to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L.

[0039] Mixed standard working solutions of two enantiomers of dinotefuran: Accurately pipette a certain amount of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran enantiomer standard stock solutions, and dilute them stepwise with isopropanol:n-heptane (2:8, v:v) to 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L respectively to form mixed standard working solutions.

[0040] 2.3 Sample Pretreatment 2.3.1 Sample Extraction Weigh 5g of the sample (accurate to 0.01g) into a 50mL centrifuge tube, add 20mL of 1% acetic acid acetonitrile solution, shake to extract for 10min, vortex to mix, centrifuge at 4000r / min for 5min, and take the supernatant into another 50mL centrifuge tube; add 20mL of 1% acetic acid acetonitrile solution to the lower residue, repeat the extraction once, combine the two supernatants, concentrate to near dryness using a rotary evaporator, add 10mL of acetonitrile to dissolve, and wait for purification.

[0041] 2.3.2 Purification The reconstituted solution was transferred to a CleanertPesti-Carb / PSA solid-phase extraction column activated with acetonitrile. Once the acetonitrile had completely eluted from the column, 10 mL of the extracted solution was transferred back to the column, and the eluent was collected. Another 5 mL of acetonitrile was added for elution, and the eluent was collected. All eluents were rotary evaporated to near dryness at 40°C, dissolved and diluted to volume with 1 mL of isopropanol:n-heptane (2:8, v / v), and then filtered through a membrane for extraction.

[0042] 2.4 Chromatographic conditions Column: CHIRALPAKAD-3 (150mm×3.0mm, 3µm); Detection wavelength: 270nm; System back pressure: 12.4Mpa; Column temperature: 35℃; Mobile phase: A is CO2, B is methanol; Gradient elution program: 0~0.7min (15%B), 0.7~1.2min (15%~20%B), 1.2~1.6min (20%B), 1.6~2.2min (20%~25%B), 2.2~3min (25%B), 3.0~4.0min (25%B~15%B), 4.0~6.0min (15%B); Flow rate: 1.0mL / min; Injection volume: 5.0μL.

[0043] 2.5 Mobile phase elution conditions A is CO2, B is methanol: Isocratic separation condition 1: 0~10min (10%B); Isocratic separation condition 2: 0~5.0 min (15% B); Gradient separation condition 3: 0~2.0 min (10%B), 2.0~2.5 min (10%~15%B), 2.5~4.0 min (15%B), 4.0~4.5 min (15%~20%B), 4.5~6.0 min (20%B), 6.0~7.0 min (20%B~10%B), 7.0~8.0 min (10%B); Gradient separation condition 4: 0~1.5min (15%B), 1.5~2.0min (15%~20%B), 2.0~3.5min (20%B), 3.5~4.0min (20%~25%B), 4.0~5.5min (25%B), 5.5~6.5min (25%B~15%B), 6.5~7.0min (15%B); Gradient separation condition 5: 0~0.7min (15%B), 0.7~1.2min (15%~20%B), 1.2~1.6min (20%B), 1.6~2.2min (20%~25%B), 2.2~3min (25%B), 3.0~4.0min (25%B~15%B), 4.0~5.0min (15%B).

[0044] 3. Results and Discussion 3.1 Stability Study of Fipronil Enantiomer Standard Solutions Accurately transfer 1.0 mL of a mixed standard working solution of 10.0 mg / L dinotefuran enantiomers into seven scratched 1.5 mL LUPC2 vials for analysis. After analysis, transfer the solution to seven vials with aluminum caps, seal with sealing film, and store at -18°C. Compare the results of the freshly prepared 10.0 mg / L dinotefuran enantiomer standard solution with those obtained after 1, 3, 5, 7, 14, 30, and 60 days of storage. The freshly prepared standard solution is considered 100%, and a change in the dinotefuran enantiomer standard solution of less than 10% is used as the baseline. The results show that the results of both dinotefuran enantiomer determinations exhibit a gradual decreasing trend (see...). Figure 1 The contents of the two enantiomers of dinotefuran decreased by more than 15% after being stored at -18°C for 60 days, less than 10% after 30 days, and less than 5% after 14 days, indicating that the two enantiomers of dinotefuran were relatively stable within 30 days.

[0045] 3.2 Selection of chromatographic separation program To obtain the optimal chromatographic separation program, this invention investigated the effects of different isocratic and gradient chromatographic separation programs on the separation efficiency of (+)-S-dinotefuran and (-)-(R)-dinotefuran. The results showed that, compared to other chromatographic separation programs, when using gradient separation condition 5, the two chromatographic peaks had flat baselines, a separation time within 3.5 min, and sharp peak shapes. The peak resolution of the two dinotefuran isomers was R=4.4 (see [link to relevant documentation]). Figure 2 D). Therefore, this experiment selects gradient separation condition 5.

[0046] 3.3 Optimization of System Back Pressure UPC 2 Using supercritical CO2 as the mobile phase, the density of CO2 can be effectively altered by adjusting the temperature and system back pressure, thereby changing its selectivity, elution capacity, and solubility. CO2 only enters the supercritical state when its temperature exceeds 31℃ and its pressure exceeds 7.38 MPa. This experiment investigated the effect of system back pressure in the range of 12.4–17.2 MPa on the separation of two enantiomers of dinotefuran. The results showed that with increasing system back pressure, the retention time of the analytes decreased, while the separation degree and peak shape remained relatively similar (see...). Figure 3 To reduce the probability of instrument system blockage, this experiment selected a relatively low system back pressure of 12.4 MPa.

[0047] 3.4 Optimization of Volume Adjustment Reagent Five dilution reagents—methanol, anhydrous ethanol, acetonitrile, isopropanol, and n-heptane—were used to resolve the enantiomers of 10 mg / L dinotefuran. The results are as follows: Figure 4As shown, when methanol, anhydrous ethanol, acetonitrile, and isopropanol were used as volume-fixing reagents, the peak shape of the target analyte was poor; when n-heptane was used as volume-fixing reagent, the chromatographic peaks of the two enantiomers of dinotefuran were completely separated within 3.5 min, and the peak shape was good.

[0048] Considering the poor solubility of n-heptane, this experiment further investigated different ratios of isopropanol to n-heptane solutions (5:5, 4:6, 3:7, 2:8, 1:9, 0:10, v / v) to facilitate subsequent analysis and detection. Figure 5 The experimental results showed that as the isopropanol ratio decreased, the peak heights of the two enantiomers of dinotefuran gradually increased, reaching their highest point when the ratio was reduced to 2:8. However, as the isopropanol ratio continued to decrease, the peak heights gradually decreased. Therefore, isopropanol:n-heptane (2:8, v:v) was ultimately determined as the dilution reagent for this experiment.

[0049] 3.5 Optimization of Extraction Conditions Cucumber was chosen as the experimental subject. Fipronil is readily soluble in organic solvents such as acetonitrile and acetone. Therefore, the extraction effects of acetonitrile and acetone on the two enantiomers of fipronil were compared. The results showed that the recovery rates of the two extraction solvents were not significantly different. However, when using acetonitrile as the extraction solvent, its high polarity made it difficult to extract lipophilic substances from cucumber, thus reducing interfering components. Therefore, acetonitrile was chosen as the extraction solvent. Since fipronil is relatively stable under acidic conditions, a small amount of acid was added in the experiment to improve the recovery rate. Finally, a 1% acetic acid-acetonitrile solution was used for extraction.

[0050] 3.6 Methodological Examination 3.6.1 Linear range and limit of quantitation The mixed standard solutions of fipronil were analyzed as standard working solutions under the chromatographic conditions described in "2.4". A standard curve was plotted with the mass concentration of the standard solution as the abscissa (X) and the corresponding peak area as the ordinate (Y). The regression equation and correlation coefficient were then obtained. The two enantiomers showed a good linear relationship in the concentration range of 0.5–20.0 mg / L, with a correlation coefficient (R0). 2 The limit of quantitation (LOQ) was greater than 0.999. The LOQ for both (+)-S-dimethomorph and (-)-(R)-dimethomorph was 0.1 mg / kg, determined by adding a standard to a blank cucumber sample that did not contain dimethomorph.

[0051] 3.6.2 Recovery rate and precision The recovery rate and precision of the method were determined by adding a standard solution to a blank cucumber sample that did not contain dinotefuran. The addition levels of (+)-S-dinotefuran and (-)-R-dinotefuran were 0.1, 0.2, and 1.0 mg / kg, respectively. Six parallel determinations were performed, and the recovery rate and relative standard deviation (RSD) were calculated. The relevant chromatograms are shown in [Figure number missing]. Figure 6 The results are shown in Table 1. The recoveries of the two target compounds ranged from 80.4% to 106%, with relative standard deviations (RSDs) of [missing data]. n =6) The recovery rate ranges from 2.2% to 7.5%. This recovery rate and precision meet the requirements of SANTE / 11312 / 2021, satisfying the analytical requirements for cucumber samples and suitable for routine analytical testing.

[0052] Table 1. Spiking recoveries and relative standard deviations of enantiomeric dichlorvos in cucumber samples ( ) n =6)

[0053] 3.7 Application of the Method 3.7.1 Resolution of the racemic body The method established in this invention was used to separate and determine the purchased racemic standard of fipronil. For example... Figure 7 As shown in figure a, the two enantiomers of dinotefuran showed good separation, achieving effective separation within 3.5 min, with resolutions of [missing values]. R =3.8, which meets the requirements. R The requirement is ≥1.5 for complete separation. According to the retention time order of the chromatographic peaks, they are: (+)-S-dimethalin, (-)-R-dimethalin (…). Figure 7 (b, 7c). Based on the standard curves plotted above, the contents of the two enantiomers of dinotefuran in the 20.0 mg / L intermediate standard solution of dinotefuran in section 2.2.1 were calculated using the external standard quantification method. The contents of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran were 11.573 mg / L and 11.756 mg / L, respectively.

[0054] 3.7.2 Testing of actual samples The enantiomeric prochloraz of 30 commercially available cucumber samples was determined using optimized chromatographic conditions and pretreatment methods. Results showed that no enantiomeric prochloraz was detected in 29 cucumber samples, while two enantiomeric prochloraz were detected in one sample: (+)-(S)-dinotefuran at 0.198 mg / kg and (-)-(R)-dinotefuran at 0.202 mg / kg (see [link to sample details]). Figure 8 ).

[0055] 4. Conclusion This invention employs ultra-high performance phase-coordinated chromatography (UHPLC) to separate two enantiomers of dinotefuran and to determine the residue levels of these enantiomers in cucumber. Acetonitrile was used to extract the sample, and the residues were determined by C... 18 After purification of the sample, separation was performed using a CHIRALPAKAD-3 chiral column with supercritical CO2 and methanol as the mobile phase gradient elution and external standard method for quantification. Spiking recovery experiments were conducted in the range of 0.1–1.0 mg / kg. The recoveries of the two dinotefuran enantiomers were 80.4%–106%, with RSDs of 2.2%–7.5%. This method was used to determine the residues of the two dinotefuran enantiomers in 30 commercially available cucumber samples. One cucumber sample tested positive for (+)-(S)-dinotefuran and (-)-(R)-dinotefuran, with detected levels of 0.198 mg / kg and 0.202 mg / kg, respectively.

[0056] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined in the present invention may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A rapid method for the separation and quantitative determination of enantiostatin enantiomers in cucumber samples, characterized in that, Includes the following steps: S1) Sample extraction: Weigh 2-10g of cucumber sample, add 5-8 times the mass-volume ratio of 0.5-2% (volume fraction) acetic acid-acetonitrile solution, shake to extract and centrifuge, collect the supernatant, repeat the extraction of the residue once or twice, combine the extracts and concentrate under reduced pressure at 35-45℃ to near dryness, add 5-15mL of acetonitrile to redissolve, and obtain the extract to be purified; S2) Solid-phase extraction purification: The extract to be purified is loaded into a multifunctional solid-phase extraction column pre-activated with acetonitrile. The solid-phase extraction column contains a stacked packing material of graphitized carbon black and primary secondary amine adsorbent. The eluent is collected under gravity or negative pressure and eluted with 3-8 mL of acetonitrile. All eluents are combined and rotary evaporated to near dryness at 35-45°C. S3) Volume adjustment and injection solvent treatment: Add a mixed solvent of isopropanol and n-heptane to the evaporated residue to dissolve and adjust the volume, wherein the volume ratio of isopropanol to n-heptane is 1:9 to 3:7, preferably 2:8, to obtain the chromatographic injection solution. S4) Ultra-high performance phase chromatography (UHPLC) separation: The chromatographic injection solution is injected into an UHPLC system equipped with a chiral column. The chiral column is a linear starch-tris(3,5-dimethylphenylcarbamate) bonded stationary phase column with a packing particle size of 2–5 µm, a column length of 100–200 mm, and an inner diameter of 3.0 mm. Supercritical carbon dioxide is used as the main mobile phase A, and methanol as the co-solvent B. The system is operated at a back pressure of 10–15 MPa and a rotation speed of 30–40 °C. Under column temperature conditions of ℃, gradient elution was performed with a B phase volume fraction ranging from 10% to 30%. The B phase was maintained at 10% to 20% for 0 to 1 min, then linearly increased to 20% to 25% for 1 to 3 min, maintained at 20% to 25% for 3 to 4 min, and then decreased back to 10% to 20% for 4 to 6 min. This allowed (+)-S-fipronil and (-)-R-fipronil to achieve baseline separation within 4.0 min, with a resolution R ≥ 4.

0. S5) Detection and Quantification: Chromatograms were acquired at an ultraviolet detection wavelength of 260–280 nm. An external standard working curve was established using a mixed standard solution of enantiomers of dinotefuran in the concentration range of 0.5–20.0 mg / L. The contents of (+)-S-dinotefuran and (-)-R-dinotefuran in cucumber were calculated by external standard method using the peak area of ​​the sample chromatogram. The method limit of quantitation was not higher than 0.1 mg / kg, and the spiked recoveries of the two enantiomers were between 80.4% and 106% with a relative standard deviation of not more than 8% at a spiked level of 0.1–1.0 mg / kg.

2. The method according to claim 1, characterized in that, The amount of cucumber sample weighed in S1) is 4-6g, the volume fraction of acetic acid in the acetic acid-acetonitrile solution is 1%, the single extraction volume is 15-25mL, and the ratio of the total liquid volume of two extractions to the sample mass is 6:1-10:

1.

3. The method according to claim 1, characterized in that, The solid-phase extraction column in S2) is a Pesti-Carb / PSA column, with a graphitized carbon black filler mass of 300-700 mg, a primary secondary amine filler mass of 300-700 mg, a total volume of 5-10 mL, a loading volume of 8-12 mL, and an elution volume of 4-6 mL.

4. The method according to claim 1, characterized in that, The solvent used for volume adjustment in S3) is isopropanol:n-heptane = 2:8 (volume ratio), the volume adjustment volume is 0.5-2.0 mL, and the injection volume is 3.0-7.0 μL.

5. The method according to claim 1, characterized in that, In S4): The chiral chromatographic column was a CHIRALPAKAD-3 column with a length of 150 mm, an inner diameter of 3.0 mm, and a particle size of 3 μm. The system back pressure is 12.0–13.8 MPa, preferably about 12.4 MPa; The column temperature is 33–37°C, preferably 35°C; The total flow rate is 0.8–1.2 mL / min, preferably 1.0 mL / min; The B-phase gradient program satisfies the following: 15%B from 0 to 0.7 min, linearly increasing from 15% to 20%B from 0.7 to 1.2 min, maintaining 20%B from 1.2 to 1.6 min, increasing from 20% to 25%B from 1.6 to 2.2 min, maintaining 25%B from 2.2 to 3.0 min, linearly decreasing from 25% to 15%B from 3.0 to 4.0 min, and maintaining 15%B from 4.0 to 6.0 min.

6. A system for analyzing the enantiomeric residues of fipronil for implementing the method according to any one of claims 1 to 5, characterized in that, include: The sample pretreatment unit includes: The acidified acetonitrile extraction module is used to add 0.5-2% (volume fraction) acetic acid-acetonitrile solution to cucumber samples and complete the shaking, centrifugation and extraction liquid merging. The solid-phase extraction purification module is equipped with a multifunctional solid-phase extraction column filled with graphitized carbon black and primary secondary amine adsorbent, and is used to purify and elute the extract. The concentration and volume adjustment module is used to concentrate the purified liquid to near dryness under reduced pressure and then add isopropanol / n-heptane mixed solvent to adjust the volume. The ultra-high performance phase chromatography unit includes: Ultra-high efficiency phase chromatography pump and mixer for delivering supercritical CO2 and methanol mobile phases and achieving a programmable gradient of 10% to 30% B; Back pressure regulator is used to control the system back pressure at 10-15 MPa; Chiral chromatographic column with a linear starch-tris(3,5-dimethylphenylcarbamate) bonded stationary phase, column length 100-200 mm, inner diameter 3.0 mm, particle size 2-5 µm; A UV-Vis diode array detector is used to detect effluent components in the wavelength range of 260–280 nm. The data processing and quantification unit is used to establish an external standard working curve based on a mixed standard solution of 0.5–20.0 mg / L dinotefuran enantiomers, integrate the chromatographic peaks of (+)-S-dinotefuran and (-)-R-dinotefuran in the sample, and calculate their residual content in cucumber samples.

7. The analysis system according to claim 6, characterized in that, The data processing and quantification unit is further configured as follows: Based on the residual concentrations of (+)-S-dimethalin and (-)-R-dimethalin measured in the sample, their enantiomeric ratio was calculated and compared with the theoretical ratio of the racemic industrial product. An over-limit warning message will be output when the content of any enantiomer in the sample exceeds 0.1 mg / kg or the total residue is close to or exceeds the maximum residue limit of 2 mg / kg.

8. The analysis system according to claim 6 or 7, characterized in that, The sample pretreatment unit and the ultra-high performance chromatographic unit are electrically connected to the same controller. The controller is pre-stored with the temperature, back pressure and gradient elution program as described in any one of claims 1 to 5, and is configured to automatically complete the linkage control of sample extraction, solid phase extraction purification, concentration and volume adjustment and ultra-high performance chromatographic separation according to the preset program.

9. A method for assessing the risk of enantiomeric residues of dinotefuran based on the method described in any one of claims 1 to 5, characterized in that, include: The residual content of (+)-S-dimethomorph and (-)-R-dimethomorph in cucumber samples was determined by the method according to any one of claims 1 to 5; The total residue of the two enantiomers was calculated and compared with the maximum residue limit of 2 mg / kg for dinotefuran in cucumber to determine whether the sample met the pesticide residue limit requirements. Based on the degree to which the residual ratio of the two enantiomers deviates from the theoretical ratio of the racemic mixture, the chiral selective degradation or accumulation behavior of dinotefuran during cultivation and distribution is assessed, and the assessment results are used as the basis for the environmental risk and product quality control of chiral pesticides.

10. A method for monitoring enantiomeric residues of fipronil in vegetable or fruit products, characterized in that, Using the separation and quantification method according to any one of claims 1 to 5, combined with the analytical system according to any one of claims 6 to 9, routine monitoring is performed on vegetable and / or fruit samples, including cucumbers, to obtain the changing trends of the residue levels and enantiomeric ratios of each enantiomer of fipronil in different product batches, for the purpose of: To guide the optimization of fipronil application dosage and interval; and / or This provides a technical basis for the use of a single, highly active (+)-S-fipronil formulation to replace racemic mixtures in terms of residue behavior and food safety.

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