Method and system for rapidly determining residual quantity of dinotefuran enantiomers in rice through solid phase extraction-ultra performance convergence chromatography

Abstract

The invention relates to the technical field of pesticide residue detection, in particular to a method and system for rapidly determining dinotefuran enantiomer residue in rice through solid phase extraction-ultra performance convergence chromatography. The method comprises the following steps: extracting a rice sample twice by using acetonitrile, purifying by using a C18 solid-phase extraction column, redissolving by using isopropanol and n-heptane (2: 8, v / v), loading on a CHIRALPAK AD-3 chiral chromatographic column, separating under the conditions of CO2 / methanol gradient, column temperature of 31 DEG C and system back pressure of 13.8 MPa, detecting at 270 nm by using a diode array detector, and respectively quantifying (+)-(S)-dinotefuran and (-)-(R)-dinotefuran by using an external standard method. The two enantiomers have good linearity in the range of 0.5-20.0 mg / L (the correlation coefficient is greater than or equal to 0.9995), the method quantitation limit in rice is 0.1 mg / kg, the adding standard recovery rate is 80.2%-103%, and the relative standard deviation is 2.6%-7.1%. The method has the advantages of simplicity and convenience in pretreatment, high analysis speed, low organic solvent consumption, high enantiomer separation degree and the like, and is suitable for daily monitoring and supervision of dinotefuran enantiomer residues in rice.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection technology, and in particular to a method and system for rapidly determining the enantiomeric residue of fipronil in rice using solid-phase extraction-ultra-high performance phase chromatography. Background Technology

[0002] Dinotefuran is a novel neonicotinoid insecticide developed by Mitsui Chemicals, Inc. of Japan and launched in 2002. It is the only neonicotinoid that does not contain chlorine atoms or aromatic rings; its characteristic substituent is (tetrahydro-3-furan)-methyl, and it is considered a third-generation neonicotinoid insecticide. Its insecticidal mechanism involves acting on the acetylcholine receptor (AChR), competing with agonists (AC1) for binding sites on the AChR, causing insects to experience continuous nerve impulses, thereby blocking normal nerve transmission and leading to insect death. Dinotefuran is a highly effective, broad-spectrum insecticide suitable for various crops such as rice, vegetables, and fruits, and is effective in controlling pests such as planthoppers, aphids, whiteflies, and thrips. Currently, four products of this drug are registered in my country, mainly in soluble granules, water-dispersible granules, and wettable powder forms. The registered crops are rice and cucumber. GB 2763-2021 specifies the maximum residue limits (MRLs) of fipronil in rice, brown rice, and cucumber as 10 mg / kg, 5 mg / kg, and 2 mg / kg, respectively. Japan's MRL for this drug in brown rice is 2 mg / kg.

[0003] Zeng Lihong et al. [LH Zeng, YD Ouyang, XM Ou, GL Hou, J. Yan, J.Ou, GP Liang, QY Song, CL Nie, Determination of flonicamid, dinotefuran and 2 Kinds of metabolites in Peach by UPLC-MS / MS, Fine Chemical Intermediates, 51(2021)77-80.] analyzed dinotefuran in peaches using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Rhman et al. [MM Rahman, JH Park, AM Abd El-Aty, JH Choi, A.Yang, KH Park, MNUA Mahmud, GJ Im, JH Shim. Feasibility and application of an HPLC / UVD to determine dinotefuran and its shorter wavelength metabolite residues in melon with tandem mass confirmation, FoodChem. 136(2013)1038-1046.] The LC-MS / MS analysis method for dinotefuran in melon was reported. Rahman et al. studied the matrix effect on the recovery of dinotefuran and its metabolites in green tea during tandem mass spectrometry analysis in the literature [MM Rahman, AM Abd El-Aty, JH Choi, SW Kim, SC Shin, JH Shim. Consequences of the matrix effect on recovery of dinotefuran and its metabolites in green tea during tandem mass spectrometry analysis, Food Chem. 168(2015) 445-453. http: / / doi.org / 10.1016 / i.foodchem.2024.138792.]. Watanabe et al. studied the matrix effect on the analysis of dinotefuran residues in tea in the literature [E. Watanabe, K. Baba, S. Miyake].Analytical evaluation of enzyme-linked immunosorbent assay for neonicotinoid dinotefuran for potential application to quick and simple screening method in rice samples, Talanta. 84(2011)1107-1111. http: / / doi.org / 10.1016 / i.talanta.2011.03.019.] An enzyme-linked immunosorbent assay for dinotefuran was developed in 2011.

[0004] Currently, the main methods for determining fipronil are enzyme-linked immunosorbent assay (ELISA), liquid chromatography (LC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC offers relatively inexpensive instruments and high versatility, but it requires long detection times and consumes large amounts of organic solvents. LC-MS / MS has low detection limits and high sensitivity, but the instruments are expensive and exhibit significant matrix effects. In recent years, ultra-high performance chromatography (UPC) has become increasingly important. 2 The UPC method has attracted widespread attention due to its significant advantages in separation efficiency. This technique employs a sub-2 μm ultra-high efficiency column for separation, demonstrating superior performance in resolving structurally similar compounds compared to traditional liquid chromatography. Using supercritical CO2 as the main mobile phase, it offers unique advantages such as rapid diffusion, low viscosity, and low organic solvent consumption. Currently, no UPC has been observed... 2 Reports on the application of this technology to the determination of fipronil. Summary of the Invention

[0005] The technical objective of this invention is to provide a solid-phase extraction-ultra-high performance coupled phase chromatography (UPCLC) method suitable for rice matrix. Under the condition that conventional laboratories are only equipped with diode array detectors (PDA), this method can achieve effective separation and accurate quantification of two enantiomers of fipronil in a short time, while reducing the amount of organic solvent used, simplifying the pretreatment process, and improving the sensitivity, precision and environmental friendliness of the method, so as to meet the actual needs of daily monitoring and regulatory enforcement of fipronil enantiomer residues in rice.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for rapid determination of enantiomeric residues of fipronil in rice using solid-phase extraction-ultra-high performance phase chromatography includes the following steps: S1. Sample extraction: Take a rice sample, weigh 5.0±0.01g and place it in a 50mL centrifuge tube. Add 20mL of acetonitrile and shake to extract for 10min. Vortex to mix and centrifuge at 4000r / min for 5min. Transfer the supernatant. Add 20mL of acetonitrile to the residue and repeat the extraction once. Combine the two supernatants and concentrate them to near dryness by rotary evaporation. Add acetonitrile to dissolve to 10mL to obtain the extract to be purified. S2, Solid-phase extraction purification: The extract to be purified was transferred to a C14 container that had been sequentially activated with 3 mL of acetonitrile, 3 mL of water, and 2 mL of acetonitrile. 18 In a solid-phase extraction column, the eluent was collected and eluted with 5 mL of acetonitrile. All eluents were combined and evaporated to near dryness at 40 °C. The solution was then diluted to 1 mL with a mixed solvent of isopropanol:n-heptane = 2:8 (v / v) to obtain the solution for instrumental analysis. S3, Ultra-high performance phase chromatography separation: The solution used for analysis was injected into a CHIRALPAKAD-3 chiral column (150 mm × 3.0 mm, 3 μm) with amylose-tris(3,5-dimethylphenylcarbamate) as the stationary phase. Separation was performed at a column temperature of 31 °C and a system back pressure of 13.8 MPa, using supercritical CO2 as the main mobile phase (A) and methanol as the modifier (B), at a flow rate of 1.0 mL / min and an injection volume of 5.0 μL, following a gradient elution program: 0–1.5 m. The infusion rates were 1.5–2.0 min, 15% B; 2.0–3.5 min, 20% B; 3.5–4.0 min, 20%–25% B; 4.0–5.5 min, 25% B; 5.5–6.5 min, 25%–15% B; and 6.5–7.0 min, 15% B, which enabled baseline separation and sharp peaks in the enantiomers (+)-(S)-dinotefuran and (-)-(R)-dinotefuran within 3.5 min. S4. Detection and Quantification: A diode array detector was used to detect the chromatographic signal at a wavelength of 270 nm. The external standard method was used to quantify (+)-(S)-dinotefuran and (-)-(R)-dinotefuran, respectively. The linear correlation coefficient between the two enantiomers was not less than 0.9995 in the mass concentration range of 0.5–20.0 mg / L. The limit of quantitation in rice samples was 0.1 mg / kg, the spiked recovery rate was 80.2%–103%, and the intra-day and inter-day relative standard deviations were 2.6%–7.1%, thus achieving rapid and accurate determination of dinotefuran enantiomer residues in rice.

[0007] Preferably, in step S1, the sample extraction is performed using a combination of mechanical shaking and vortex mixing, with a shaking time of 10–20 min and a vortex mixing time of 1–3 min, in order to improve the extraction efficiency of fipronil enantiomers from the rice matrix.

[0008] Preferably, the C 18 The solid-phase extraction column has a packing mass of 500 mg and a column volume of 3 mL. The activation sequence is acetonitrile first, then water, then acetonitrile again. During the sample loading process, the flow rate is controlled at 1.0–2.0 mL per minute to balance purification efficiency and recovery rate.

[0009] Preferably, the isopropanol:n-heptane = 2:8 (v / v) mixed solvent is used both as the solvent for preparing the enantiomeric standard working solution and as the solvent for adjusting the volume of the purified sample, in order to improve the retention and peak shape stability on the CHIRALPAKAD-3 chiral column.

[0010] Preferably, when the method is used for batch testing of 40 rice samples, the chromatographic run time for a single sample does not exceed 7.0 min, and the intra-day and inter-day spiked recoveries of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran are 81.0%–96.5% and 80.2%–103% respectively under three spiking conditions: high (1.0 mg / kg), medium (0.2 mg / kg), and low (0.1 mg / kg), which meet the validation requirements of SANTE / 11312 / 2021 for the recovery and precision of pesticide residue analysis methods.

[0011] Furthermore, the present invention also provides a rapid determination system for solid-phase extraction-ultra-high performance coherence chromatography (UHPLC) for implementing the method, comprising: The sample pretreatment unit is used to extract and purify rice samples. The sample pretreatment unit includes an extraction module, which is used to add acetonitrile to a centrifuge tube containing rice samples for shaking-vortex extraction and centrifugation separation. The concentration module is used to concentrate the combined acetonitrile extract to near dryness by rotary evaporation and then reconstitute it with acetonitrile. C 18 The solid-phase extraction purification module is equipped with a C10 column containing 500 mg of packing material and a column volume of 3 mL. 18 Solid-phase extraction column, with an automatic activation program of acetonitrile-water-acetonitrile and an acetonitrile elution program, is used to remove lipids, pigments and co-extracted impurities from rice matrix; Ultra-high performance phase chromatography (UHPLC) separation unit, the UHPLC separation unit comprising: The supercritical CO2 supply module is used to provide high-purity CO2 as the main mobile phase. Modifier delivery module, used to deliver methanol and form a programmable gradient mobile phase with supercritical CO2; Gradient pump and back pressure regulator are used to maintain a system back pressure of 13.8 MPa and change the methanol volume fraction according to a preset gradient during chromatography operation; The chiral chromatography column module is equipped with a CHIRALPAKAD-3 chiral chromatography column (150mm×3.0mm, 3μm) and a constant temperature column oven to maintain a column temperature of 31℃. Detection and data processing unit, the detection and data processing unit comprising: A diode array detector is used to acquire chromatographic signals at a wavelength of 270 nm; The data processing module is used to store the external standard working curve and automatically calculate the residues of (+)-(S)-fipronil and (-)-(R)-fipronil in rice samples; The system is configured to operate according to the steps and chromatographic conditions of claims S1 to S4 in claim 1 to achieve baseline separation of the two enantiomers of dinotefuran within 3.5 min and achieve a method limit of quantitation of 0.1 mg / kg.

[0012] Preferably, an automatic sample injection module is provided between the sample pretreatment unit and the ultra-high performance phase chromatography (UHPLC) separation unit. This automatic sample injection module is suitable for directly aspirating samples from samples processed by CHPLC. 18 The sample solution was purified by solid-phase extraction and diluted to a final volume of isopropanol:n-heptane = 2:8 (v / v). The injection volume can be adjusted within the range of 1 to 10 μL.

[0013] Preferably, the data processing module pre-stores multi-point external standard calibration curves of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran in the range of 0.5 to 20.0 mg / L, and has the function of automatically calculating the linear correlation coefficient, detection limit and quantitation limit, for methodological quality control of each batch of tests.

[0014] Preferably, the system further includes a methodology validation module, which is configured to automatically generate spiked recovery rate and relative standard deviation based on the spiked level and number of repetitions input by the user, and compare them with preset acceptance criteria, thereby determining whether the current chromatographic operating conditions meet the requirements for the determination of enantiomeric residues of fipronil in rice.

[0015] Preferably, the system is integrated on the same rack and the sample pretreatment unit, ultra-high efficiency phase chromatography separation unit, and detection and data processing unit are uniformly controlled by the central control unit, which can realize continuous automatic analysis of multiple rice samples, with no less than 40 samples tested per day.

[0016] This invention achieves baseline separation of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran in the complex matrix of rice within approximately 3.5 min by synergistically optimizing the separation conditions of acetonitrile extraction-C18 solid-phase extraction purification and ultra-high performance coherent chromatography using a CHIRALPAK AD-3 chiral column. The single chromatographic run time is controlled within 7 min, significantly improving the detection throughput. The linear correlation coefficients between the two enantiomers are greater than 0.9995 in the range of 0.5–20.0 mg / L, the method limit of quantitation reaches 0.1 mg / kg, the spiked recovery rate is 80.2%–103%, and the intra-day and inter-day RSDs are 2.6%–7.1%, meeting and exceeding the accuracy and precision requirements of current pesticide residue analysis guidelines. Because it uses supercritical CO2 as the main mobile phase and only a small amount of methanol as a modifier, it significantly reduces the amount of organic solvent used compared with traditional normal-phase or reversed-phase HPLC, which has the advantages of being green and environmentally friendly. At the same time, the specific C18 purification process and the isopropanol:n-heptane (2:8, v / v) reconstitution system effectively suppress the interference of rice matrix co-extractants on chromatographic separation and ultraviolet detection, improve the selectivity and stability of the method, and enable the present invention not only to obtain information on the total residue of fipronil, but also to distinguish and quantify the two enantiomers separately, providing reliable technical support for conducting research on the behavior of fipronil enantiomers in grains and differential risk assessment. Attached Figure Description

[0017] Figure 1 Spectrum of enantiomeric standard solution of fipronil.

[0018] Figure 2 The effect of different chromatographic columns on the separation of two enantiomers of dinotefuran. (a) AD-3; (b) AMY1; (c) CEL1; (d) CEL2.

[0019] Figure 3 Effects of different cosolvents on the separation of two dinotefuran enantiomers. (a) 0.5% (v / v) ammonia-methanol solution; (b) methanol; (c) 0.5% (v / v) formic acid-methanol solution.

[0020] Figure 4 Effects of different column temperatures on the separation of two enantiomers of dinotefuran. (a) 31℃; (b) 35℃; (c) 40℃.

[0021] Figure 5 Effect of solid phase extraction columns on the purification efficiency of (+)-S-fipronil and (-)-(R)-fipronil. (a) C18 column; (b) Florisil column; (c) Supelclean ENVI-carb column.

[0022] Figure 6Chromatograms of the standard solution (a), blank rice sample (b), and recovered sample (c). Peak 1: (+)-S-dimethalin; Peak 2: (-)-(R)-dimethalin.

[0023] Figure 7 Chromatogram of a positive rice sample. Peak 1: (+)-S-dinotefuran; Peak 2: (-)-(R)-dinotefuran. Detailed Implementation

[0024] 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.

[0025] 1. Terminology Explanation In this invention, unless otherwise stated, the following terms have the following meanings: Dinotefuran: refers to a neonicotinoid insecticide molecule with the chemical name N-methyl-N-nitrosimino-N′-[(tetrahydro-3-furanyl)methyl]guanidine. It has a chiral center and can form two enantiomers, namely (+)-(S)-dinotefuran and (-)-(R)-dinotefuran.

[0026] Enantiomers: Two stereoisomers that have the same atomic structure and bond sequence, but are mirror images of each other and cannot be superimposed. The enantiomers of fipronil in this invention are such chiral compounds.

[0027] Ultra-high performance phase chromatography (UPC) 2 : refers to a chromatographic technique that uses supercritical or near-supercritical carbon dioxide as the main mobile phase, supplemented by a small amount of organic solvent as a modifier, and combined with an ultra-high efficiency chromatographic column for separation.

[0028] Solid phase extraction (SPE): refers to a pretreatment technique in which a sample solution is passed through a chromatographic column filled with a solid adsorbent to selectively retain or elute target compounds and matrix impurities, thereby achieving purification.

[0029] Limit of Quantitation (LOQ): The lowest analyte concentration that can be quantitatively determined under specified experimental conditions, with a signal-to-noise ratio (S / N) of 10. Where S is the signal intensity and N is the noise intensity.

[0030] Unless otherwise stated, the terms "mass concentration", "recovery rate", "relative standard deviation" and other terms used in this invention adopt the definitions commonly used in the field of pesticide residue analysis.

[0031] 2. Materials and Methods 2.1 Instruments, Materials and Reagents Acquity ultra-high performance phase chromatograph (Waters, USA, with diode array detector (PDA)); R215 rotary evaporator (Buchi, Switzerland); AE260 electronic balance (Mettler, Switzerland); MS2 vortex mixer (Shanghai Medical University Instrument Factory); ELGA CLXXXUVM2 ultrapure water purification system (Elga, UK); N-EVAP TM 111 Nitrogen Evaporator (Tokyo Rika Co., Ltd., Japan).

[0032] Isopropanol, acetonitrile, methanol (chromatographic grade, Scharlau, Spain); ultrapure water; high-purity carbon dioxide (99.999%); C 18 Solid-phase extraction (SPEE) columns (CNW, 500 mg, 3 mL); Florisil SPEE column (CNW, 5 g, 6 mL); Supelclean Envi-carb SPEE column (Supelco, 500 mg, 3 mL); Daicel CHIRALPAK AD-3 (150 mm × 3.0 mm, 3 µm, packing material: amylose-tris(3,5-dimethylphenylcarbamate)); Waters Acquity Trefoil AMY1 (150 mm × 3.0 mm, 2.5 µm, packing material: amylose-tris(3,5-dimethylphenylcarbamate)); Waters Acquity Trefoil CEL1 (150 mm × 3.0 mm, 2.5 µm, cellulose-tris(3,5-dimethylphenylcarbamate)); Waters Acquity Trefoil CEL2 (150 mm × 3.0 mm, 2.5 µm, packing material: cellulose-tris(3,5-dimethylphenylcarbamate)). µm, the packing material is cellulose-tris(3-chloro-4-methylphenylcarbamate) (Waters, Inc., USA); all other reagents used in the experiments were of analytical grade unless otherwise specified.

[0033] 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%.

[0034] 2.2 Preparation of Standard Stock Solution and Working 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 1.0 g / L enantiomeric standard stock solutions.

[0035] 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.

[0036] 2.3 Sample Pretreatment 2.3.1 Sample Extraction Weigh 5 g (accurate to 0.01 g) of the sample into a 50 mL centrifuge tube, add 20 mL of acetonitrile, shake to extract for 10 min, vortex to mix, centrifuge at 4000 r / min for 5 min, and transfer the supernatant to 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, dissolve in 10 mL of acetonitrile, and wait for purification.

[0037] 2.3.2 Purification Transfer the reconstituted solution to a C14 container activated with (3 mL acetonitrile, 3 mL water, 2 mL acetonitrile). 18 In a solid-phase extraction column, once the acetonitrile has completely eluted, transfer 10 mL of the extracted solution to the column and begin collecting the eluent. Add another 5 mL of acetonitrile for elution and collect the eluent. Evaporate all the eluent to near dryness at 40°C, dissolve and dilute to volume with 1 mL of isopropanol:n-heptane (2:8, v / v), and filter through a membrane for use in the extraction instrument.

[0038] 2.4 Chromatographic conditions Column: CHIRALPAK AD-3 (150 mm × 3.0 mm, 3 µm); Detection wavelength: 270 nm; System back pressure: 13.8 MPa; Column temperature: 31 °C; Mobile phase: A is CO2, B is methanol; Gradient elution program: 0–1.5 min (15% B), 1.5–2.0 min (15%–20% B), 2.0–3.5 min (20% B), 3.5–4.0 min (20%–25% B), 4.0–5.5 min (25% B), 5.5–6.5 min (25% B–15% B), 6.5–7.0 min (15% B); Flow rate: 1.0 mL / min; Injection volume: 5.0 μL.

[0039] Results and Discussion 3.1 Selection of detection wavelength According to the 3D spectrum obtained from the DAD full scan, fipronil exhibits the strongest UV absorption intensity and highest sensitivity at 270 nm (see...). Figure 1 Furthermore, at this wavelength, none of the mobile phases used in this experiment exhibited UV absorption. Therefore, the maximum UV absorption wavelength of fipronil is the optimal UV absorption wavelength, and 270 nm was selected as the detection wavelength in this experiment.

[0040] 3.2 Column Optimization This experiment selected four chiral separation columns—CHIRALPAK AD-3 from Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd., and Waters Acquity Trefoil AMY1, CEL1, and CEL2—to investigate their separation effects on two enantiomers of dinotefuran. The results showed that when using the CEL2 chiral column, no peak appeared on the chromatogram within 10 minutes, indicating that the two dinotefuran enantiomers could not be rapidly separated. When using the CEL1 chiral column, only one peak appeared on the chromatogram within 10 minutes, indicating that the two dinotefuran enantiomers completely overlapped, failing to achieve effective separation. When using the AMY1 and AD-3 chiral columns, the two dinotefuran enantiomers were completely separated within 10 minutes. However, compared to AMY1, the AD-3 chiral column showed better separation and sharper peak shapes (see...). Figure 2 The reason for this may be that the AD-3 chiral column stationary phase has many branches

[24] , and the two enantiomers of dinotefuran have a long retention time in the AD-3 column, thus achieving a good separation effect. Therefore, the AD-3 chiral chromatographic column was selected in this experiment to separate the two enantiomers of dinotefuran.

[0041] 3.3 Optimization of Cosolvents Ultra-high performance phase chromatography (UHPLC) consumes less organic solvent and uses supercritical CO2 as the main mobile phase. A small amount of organic solvent is typically used as a co-solvent to enhance the selectivity and elution ability of the target product. This experiment investigated the effects of different co-solvents, including 0.5% (v / v) ammonia-methanol solution, methanol, and 0.5% (v / v) formic acid-methanol solution, on the separation of two enantiomers of dinotefuran. The results showed that the separation effect and peak shape of the two dinotefuran enantiomers were almost identical with the three co-solvents (see...). Figure 3 However, compared to 0.5% (v / v) ammonia in methanol solution and 0.5% (v / v) formic acid in methanol solution as co-solvents, methanol is easier to handle and causes less damage to instruments and chromatographic columns. Therefore, our laboratory chose methanol as the co-solvent.

[0042] 3.4 Column Temperature Optimization Considering that the maximum recommended operating temperature of the CHIRALPAK AD-3 chiral column is 40℃, and CO2 only enters the supercritical CO2 state when the temperature exceeds 31℃ and the pressure exceeds 7.38 MPa, this experiment investigated the effect of column temperature in the range of 31~40℃ on the separation of two dinotefuran enantiomers. Under the three column temperature conditions, the chromatographic peak resolution of the two dinotefuran enantiomers was good, and good baseline separation was achieved within 3.5 min, indicating fast analysis speed (see...). Figure 4 Compared to 35℃ and 40℃, the separation of the two enantiomers of dinotefuran was higher at a column temperature of 31℃. Considering the column life, the optimal column temperature was selected as 31℃.

[0043] 3.5 Investigation of different purification columns Comparison with C 18 The purification effects of three solid-phase extraction columns, namely Florisil and Supelclean ENVI-carb, on two enantiomeric difenoconazoles in rice samples (see...) Figure 5 ).

[0044] The experiment compared C 18 The purification effects of different types of solid-phase extraction columns, including Florisil and Supelclean ENVI-carb, on rice sample extracts were investigated. Two enantiomer standard solutions of fipronil were added to rice samples that did not contain fipronil, and the samples were extracted twice with acetonitrile. The extracts were concentrated to near dryness and then reconstituted. The extracts were then purified using three different solid-phase extraction columns. The results showed that when using C1000C5 ... 18 During column purification, the recoveries of the two dinotefuran enantiomers were over 102% and 101%, respectively; when purified using a Florisil column, the recoveries were 97.7% and 100%, respectively; and when purified using a Supelclean ENVI-carb column, the recoveries were 43.2% and 45.6%, respectively. (Using C...) 18 The column-based purification method was superior to the Florisil column in removing waxes, lipids, and pigments; therefore, the experiment ultimately selected column C. 18 Solid-phase extraction columns are used as purification columns.

[0045] 3.6 Methodological Examination 3.6.1 Linear range and limit of quantitation A series of mixed standard solutions of dinotefuran enantiomers were determined under the chromatographic conditions described above. The peak area (Y) of the standards was plotted on the ordinate, corresponding to the mass concentration (...). XA standard curve was plotted with the x-axis as the abscissa, and the regression equation and correlation coefficient were obtained. The results showed that the two enantiomers exhibited a good linear relationship in the concentration range of 0.5–20.0 mg / L, with a correlation coefficient greater than 0.9995. The standard was added to a blank rice sample without fipronil, and the determination was performed according to this method, with the signal-to-noise ratio (SNR) as the criterion. S / N The limit of quantitation (LOQ) was calculated using 10, and the LOQ for both (+)-S-fipronil and (-)-(R)-fipronil was 0.1 mg / kg.

[0046] 3.6.2 Recovery rate and precision Two enantiomer standard solutions of dinotefuran at different concentrations were added to rice samples that did not contain dinotefuran. Spiking recovery and precision tests were performed. Relevant chromatograms are shown below. Figure 6 The results are shown in Table 1. The results showed that the spiked recoveries of the two enantiomers of fipronil were 80.2% to 103%, and the relative standard deviations (RSDs) were 2.6% to 7.1%, which met the recovery requirements of SANTE / 11312 / 2021

[25] and could meet the requirements for the determination of fipronil enantiomer content in rice samples.

[0047] Table 1. Spiking recoveries and relative standard deviations of enantiomeric difenoconazole in rice samples ( ) n =6)

[0048] 3.7 Testing of actual samples To evaluate the effectiveness and practicality of this method, the established method was used to determine the contents of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran in 40 commercially available rice samples. The results showed that dinotefuran enantiomers were not detected in 39 rice samples, while (+)-(S)-dinotefuran and (-)-(R)-dinotefuran were detected in 1 rice sample, with detected levels of 0.392 mg / kg and 0.408 mg / kg, respectively (see [link to study]. Figure 7 This situation is basically consistent with the ratio of (+)-(S)-finoteran and (-)-(R)-finoteran reported in the literature [24,27,28].

[0049] 3.8 Comparison with other methods The LOD, LOQ, linear range, separation time, recovery, and RSD of this application were compared with other reported methods (see Table 2). This application offers short analysis time, good purification effect, and high specificity, meeting the routine detection requirements for fipronil. The LOQ of this method meets the maximum residue limits required by regulations in various countries. Furthermore, the linear range, recovery, and RSD of this method are consistent with other methods.

[0050] Table 2 Comparison with other reporting methods

[0051] 4. Conclusion This application is the first to employ ultra-high performance phase chromatography (UHPLC) to separate two enantiomers of dinotefuran and to determine the residue levels of dinotefuran enantiomers in rice. The method optimized instrumental separation conditions, including detection wavelength, chromatographic column, co-solvent, and column temperature. The purification column of the pretreatment purification method was investigated. The established method was applied to the detection of actual samples, and the results showed that the method has high sensitivity and good stability, meeting the requirements for the detection of dinotefuran chiral enantiomer residues in rice. This method provides a reference for in-depth analysis of effective and low-toxicity enantiomers and ineffective and highly toxic pesticide enantiomer residues in agricultural products, and is of great significance for drug quality control and efficacy evaluation.

[0052] 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 this application 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 in this application, but is to be accorded the widest scope consistent with the principles and novelty disclosed in this application.

Claims

1. A method for rapid determination of enantiomeric residues of fipronil in rice using solid-phase extraction-ultra-high performance phase chromatography, characterized in that, Includes the following steps: S1. Sample extraction: Take a rice sample, weigh 5.0±0.01g and place it in a 50mL centrifuge tube. Add 20mL of acetonitrile and shake to extract. After vortexing and mixing, centrifuge at 4000r / min for 5min and transfer the supernatant. Add another 20mL of acetonitrile to the residue and repeat the extraction once. Combine the two supernatants and concentrate them to near dryness by rotary evaporation. Then add acetonitrile to dissolve to 10mL to obtain the extract to be purified. S2, Solid-phase extraction purification: The extract to be purified was transferred to a C14 container that had been sequentially activated with 3 mL of acetonitrile, 3 mL of water, and 2 mL of acetonitrile. 18 In a solid-phase extraction column, the eluent was collected and eluted with 5 mL of acetonitrile. All eluents were combined and evaporated to near dryness at 40 °C. The solution was then diluted to 1 mL with a mixed solvent of isopropanol:n-heptane = 2:8 (v / v) to obtain the solution for instrumental analysis. S3, Ultra-high performance phase chromatography separation: The solution used for analysis was injected into a CHIRALPAKAD-3 chiral column (150 mm × 3.0 mm, 3 μm) with amylose-tris(3,5-dimethylphenylcarbamate) as the stationary phase. Separation was performed at a column temperature of 31 °C and a system back pressure of 13.8 MPa, using supercritical CO2 as the main mobile phase (A) and methanol as the modifier (B), at a flow rate of 1.0 mL / min and an injection volume of 5.0 μL, following a gradient elution program: 0–1.5 m. The infusion rates were 1.5–2.0 min, 15% B; 2.0–3.5 min, 20% B; 3.5–4.0 min, 20%–25% B; 4.0–5.5 min, 25% B; 5.5–6.5 min, 25%–15% B; and 6.5–7.0 min, 15% B, which enabled baseline separation and sharp peaks in the enantiomers (+)-(S)-dinotefuran and (-)-(R)-dinotefuran within 3.5 min. S4. Detection and Quantification: A diode array detector was used to detect the chromatographic signal at a wavelength of 270 nm. The external standard method was used to quantify (+)-(S)-dinotefuran and (-)-(R)-dinotefuran, respectively. The linear correlation coefficient between the two enantiomers was not less than 0.9995 in the mass concentration range of 0.5–20.0 mg / L. The limit of quantitation in rice samples was 0.1 mg / kg, the spiked recovery rate was 80.2%–103%, and the intra-day and inter-day relative standard deviations were 2.6%–7.1%, thus achieving rapid and accurate determination of dinotefuran enantiomer residues in rice.

2. The method according to claim 1, characterized in that, In step S1, sample extraction is performed using a combination of mechanical shaking and vortex mixing. The shaking time is 10–20 min, and the vortex mixing time is 1–3 min, in order to improve the extraction efficiency of fipronil enantiomers from the rice matrix.

3. The method according to claim 1, characterized in that, The C 18 The solid-phase extraction column has a packing mass of 500 mg and a column volume of 3 mL. The activation sequence is acetonitrile first, then water, then acetonitrile again. During the sample loading process, the flow rate is controlled at 1.0–2.0 mL per minute to balance purification efficiency and recovery rate.

4. The method according to claim 1, characterized in that, The isopropanol:n-heptane = 2:8 (v / v) mixed solvent is used both as the solvent for preparing the enantiomeric standard working solution and as the solvent for adjusting the volume of the purified sample, in order to improve the retention and peak shape stability on the CHIRALPAKAD-3 chiral column.

5. The method according to claim 1, characterized in that, When the method is used for batch testing of 40 rice samples, the chromatographic run time for a single sample does not exceed 7.0 min. Under three spiking conditions (high (1.0 mg / kg), medium (0.2 mg / kg), and low (0.1 mg / kg), the intra-day and inter-day recoveries of (+)-(S)-dinotefuran and (-)-(R)-dinotefuran are 81.4%–101% and 81.5%–103%, respectively, which meets the validation requirements of SANTE / 11312 / 2021 for the recovery and precision of pesticide residue analysis methods.

6. A rapid determination system for solid-phase extraction-ultra-high performance coherence chromatography for implementing the method according to any one of claims 1-5, characterized in that, include: The sample pretreatment unit is used to extract and purify rice samples. The sample pretreatment unit includes an extraction module, which is used to add acetonitrile to a centrifuge tube containing rice samples for shaking-vortex extraction and centrifugation separation. The concentration module is used to concentrate the combined acetonitrile extract to near dryness by rotary evaporation and then reconstitute it with acetonitrile. C 18 The solid-phase extraction purification module is equipped with a C10 column containing 500 mg of packing material and a column volume of 3 mL. 18 Solid-phase extraction column, with an automatic activation program of acetonitrile-water-acetonitrile and an acetonitrile elution program, is used to remove lipids, pigments and co-extracted impurities from rice matrix; Ultra-high performance phase chromatography (UHPLC) separation unit, the UHPLC separation unit comprising: The supercritical CO2 supply module is used to provide high-purity CO2 as the main mobile phase. Modifier delivery module for delivering methanol and forming a programmable gradient mobile phase with supercritical CO2; Gradient pump and back pressure regulator are used to maintain a system back pressure of 13.8 MPa and change the methanol volume fraction according to a preset gradient during chromatography operation; The chiral chromatography column module is equipped with a CHIRALPAKAD-3 chiral chromatography column (150mm×3.0mm, 3μm) and a constant temperature column oven to maintain a column temperature of 31℃. Detection and data processing unit, the detection and data processing unit comprising: A diode array detector is used to acquire chromatographic signals at a wavelength of 270 nm; The data processing module is used to store the external standard working curve and automatically calculate the residues of (+)-(S)-fipronil and (-)-(R)-fipronil in rice samples; The system is configured to operate according to the steps and chromatographic conditions of claims S1 to S4 in claim 1 to achieve baseline separation of the two enantiomers of dinotefuran within 3.5 min and achieve a method limit of quantitation of 0.1 mg / kg.

7. The system according to claim 6, characterized in that, An automatic sample injection module is provided between the sample pretreatment unit and the ultra-high performance phase chromatography separation unit. The automatic sample injection module is suitable for directly aspirating samples from samples processed by C... 18 The sample solution was purified by solid-phase extraction and diluted to a final volume of isopropanol:n-heptane = 2:8 (v / v). The injection volume can be adjusted within the range of 1 to 10 μL.

8. The system according to claim 6 or 7, characterized in that, The data processing module pre-stores multi-point external standard calibration curves of (+)-(S)-fipronil and (-)-(R)-fipronil in the range of 0.5 to 20.0 mg / L, and has the function of automatically calculating the linear correlation coefficient, detection limit and quantitation limit, which is used for methodological quality control of each batch of tests.

9. The system according to claim 6, characterized in that, The system further includes a methodology validation module, which is configured to automatically generate spike recovery rate and relative standard deviation based on the spike level and number of repetitions input by the user, and compare them with preset acceptance criteria to determine whether the current chromatographic operating conditions meet the requirements for the determination of enantiomeric residues of fipronil in rice.

10. The system according to claim 6, characterized in that, The system is integrated on the same rack and the sample pretreatment unit, ultra-high efficiency phase chromatography separation unit, and detection and data processing unit are uniformly controlled by the central control unit. It can realize continuous automatic analysis of multiple rice samples, and the number of samples tested per day is no less than 40.

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