Method and system for separating and determining residual quantity of meconazole enantiomers in rice by adopting ultra-performance convergence chromatography

Abstract

The invention relates to the technical field of pesticide residue detection, in particular to a method and a system for separating and determining the residual quantity of meconazole enantiomers in rice by adopting ultra-performance convergence chromatography. By optimizing sample extraction and solid-phase extraction purification processes and combining ultra-performance convergence chromatography chiral separation conditions with supercritical carbon dioxide as a main mobile phase, rapid and effective separation of the two enantiomers and accurate and sensitive quantification under PDA detection are realized, so that matrix interference is reduced, analysis efficiency and result reliability are improved, and the method is suitable for industrial production. The application requirements of monitoring and supervising the residues of the meconazole enantiomers in the rice are met.

Description

Technical Field

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

[0002] Mefentrifluconazole (MFZ) is a triazole isopropanol fungicide with broad-spectrum, high-efficiency, and systemic properties, making it valuable for disease control in crops such as cereals. With the increasing use of this fungicide in agricultural production, the risk of its residues in agricultural products (especially rice, a staple food) and the resulting demand for food safety supervision are becoming increasingly prominent. Therefore, establishing reliable detection methods for MFZ residues in complex matrices such as rice is of great significance for agricultural product quality and safety monitoring, risk assessment, and regulatory enforcement.

[0003] Currently, conventional analytical methods for pesticide residue detection of triazole fungicides mainly include gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS / MS), and high performance liquid chromatography (HPLC) / ultra-high performance liquid chromatography (UHPLC) combined with ultraviolet or diode array detectors (UV / DAD / PDA). These methods are relatively mature in the quantitative analysis of total racemates and can meet the requirements of detection limits, accuracy, and throughput in some regulatory scenarios. However, MFZ is a typical chiral pesticide, containing a chiral carbon center in its molecule and existing in enantiomers. For chiral pesticides, the two enantiomers may differ in biological activity, metabolic / degradation behavior, ecotoxicity, and behavior in the environment and food chain. Characterizing only the total racemate may not reflect the true exposure level and risk characteristics at the enantiomer level, and is also not conducive to conducting more refined risk assessments and process control of chiral pesticides. Therefore, the enantiomeric separation and quantitative analysis of MFZ in food and agricultural product matrices has significant scientific and applied value.

[0004] In enantiomeric analysis, traditional chiral analysis often relies on chiral columns (e.g., stationary phases of polysaccharide derivatives) for normal-phase or reversed-phase liquid chromatography separation, followed by DAD / PDA or MS for detection. While this approach is relatively universal, conventional LC separation can face challenges such as long analysis times, high solvent consumption, and significant matrix-related effects on resolution and peak shape stability for compounds with similar structures, strong hydrophobicity, or multiple co-eluting interferences. Rice samples, in particular, are complex matrices with high starch content, a relatively "dry" basis, and multiple co-extractants. After extraction with organic solvents like acetonitrile, they often still contain lipids, pigments, surfactants, and other matrix components, easily leading to baseline fluctuations, increased interference peaks, accelerated column contamination, and decreased quantitative accuracy. For DAD / PDA systems relying on UV absorption signals for detection, these matrix interference problems are even more pronounced: on the one hand, UV detection lacks the selectivity information found in mass spectrometry; on the other hand, co-eluting impurities are more likely to affect integration and quantification. Therefore, methods for detecting enantiomeric residues in rice matrix require not only strong chromatographic separation capabilities, but also sample purification strategies and solvent systems that can effectively reduce interference and ensure peak shape and response stability.

[0005] In recent years, ultra-high performance phase chromatography (also often referred to as ultra-high performance supercritical fluid chromatography or UPC) has become increasingly popular. 2 Supercritical carbon dioxide (SC) has gradually become an important tool for chiral separation and rapid analysis of complex systems due to its advantages such as using supercritical carbon dioxide as the main mobile phase, low system viscosity, high diffusion coefficient, high column efficiency, fast separation speed, and relatively low organic solvent consumption. For structural analogs or enantiomer / diastereomer mixtures, combined phase chromatography with a sub-2-3 μm chiral stationary phase of polysaccharide derivatives often achieves higher separation efficiency and shorter analysis time. Against this backdrop, research and patent applications focusing on the combined phase chromatography separation and residue determination of chiral triazole fungicides are also gradually increasing.

[0006] For example, Chinese patent CN115494190A discloses a method for simultaneously separating and determining the enantiomeric residues of triazole and its metabolite triazoleol in fruit and vegetable purees using ultra-high performance combined phase chromatography (UHPLC). The method involves sample pretreatment with acetonitrile extraction followed by purification using an NH2 solid-phase extraction column. Chromatographic separation employs an Acquity Trefoil CEL2 chiral column with a gradient elution system of supercritical CO2 and "0.5% ammonia-methanol" as the mobile phase, and quantification using the external standard method. The methodological indicators, such as the limit of quantitation and recovery range, are provided. This patent embodies a typical combined route of "acetonitrile extraction + solid-phase purification + Trefoil polysaccharide chiral column + CO2 / alcohol modifier gradient + UV detection / external standard quantification," providing a reference for enantiomeric analysis of triazole chiral compounds in food matrices. However, its target is fruit and vegetable puree matrices, and the target compound is not MFZ. Furthermore, the type of purification column and the modifier system used are not entirely consistent with the matrix characteristics that dry grain samples such as rice may encounter.

[0007] For example, the literature [Xiaokang An, Xinglu Pan, Runan Li, et al. Enantioselective monitoring chiral fungicide mefentrifluconazole in tomato, cucumber, pepper and its pickled products by supercritical fluid chromatography tandem mass spectrometry. Food Chemistry, 2022, 376:131883. DOI:10.1016 / j.foodchem.2021.131883.] reports a method for rapid separation and analysis of MFZ enantiomers using supercritical fluid chromatography-tandem mass spectrometry (SFC-MS / MS). The subjects studied included vegetables and processed products such as tomatoes, cucumbers, peppers, and their pickled products. This literature indicates that baseline separation of MFZ enantiomers can be achieved within approximately 2 minutes, and provides validation results such as recovery and precision under different matrices. Furthermore, it discusses the enantiomer selectivity behavior of MFZ enantiomers in the field and during processing. This scheme demonstrates that the SFC route can achieve rapid separation of MFZ enantiomers and complete quantitative analysis in complex food matrices; however, its detection method is MS / MS, which has relatively high instrument costs and operation and maintenance requirements, and it mainly focuses on vegetable and processed product matrices, without directly addressing the interference control and quantitative feasibility issues of grain matrices such as rice under UV / array detection conditions.

[0008] Furthermore, the literature [Xu Jing, Haoyue Xue, Xiaoyan Sang, et al. Magnetic deepeutectic solvent-based dispersive liquid-liquid microextraction for enantioselectively determining chiral mefentrifluconazole in cereal samples via ultra-high-performance liquid chromatography. Food Chemistry, 2022, 391:133220. DOI:10.1016 / j.foodchem.2022.133220.] discloses a UHPLC-DAD method for enantioselective analysis of MFZ in cereal samples. This method achieves sample pretreatment by combining dispersive liquid-liquid microextraction with magnetic deep eutectic solvent (MDES), and applies this method to the enantioselective detection of cereal samples such as corn, rice, wheat, millet / millet, and sorghum. This literature indicates that quantitative analysis of MFZ enantiomers can be achieved for complex dry-based cereal samples through a combination of specific microextraction strategies and UHPLC-DAD detection. However, this method relies on a specific deep eutectic solvent system and microextraction process control, requiring a relatively unique pretreatment system, and the chromatographic platform is UHPLC rather than combined-phase chromatography. For applications that aim to leverage the advantages of UPC² in separation speed, solvent greening, and polysaccharide chiral column compatibility, the aforementioned UHPLC-DAD route remains difficult to directly replace.

[0009] Based on the above existing technologies, it can be seen that: on the one hand, the chiral separation capability based on the UPC² / combined phase chromatography platform has been verified in chiral pesticide systems such as triazoles, and existing literature has used a combination of Trefoil-type chiral columns, CO2-alcohol modifier gradients and external standard quantification in food matrices to determine enantiomeric residues; on the other hand, for the MFZ enantiomerics themselves, existing studies have used SFC-MS / MS to achieve rapid separation and complete quantification in vegetables and processed products, and some studies have used UHPLC-DAD to achieve enantiomeric detection in cereal (including rice) samples. However, based on the actual detection needs of rice matrix, there are still the following technical pain points that need to be addressed: (1) The compatibility between the platform and the detection method: In actual operation, regulatory and testing agencies need both high-throughput and green separation platforms (such as UPC² / combined phase chromatography platforms) and detection methods. 2While UHPLC-DAD (Ultra-High Performance Chromatography) is used in some applications, it also has applications primarily based on UV detection such as DAD / PDA (due to cost, maintenance, and availability factors). However, current MFZ enantiomer studies either focus on high-selectivity platforms such as SFC-MS / MS or employ UHPLC-DAD, which lacks the advantages of UHPLC in terms of separation efficiency and solvent systems. Therefore, how to achieve UHPLC-DAD is a crucial question. 2 On the combined phase chromatography platform, reliable quantification of MFZ enantiomers in rice samples by combining PDA detection still requires targeted method design and validation. (2) Rice matrix interference and method stability issues: As a high-starch grain matrix, rice has a significantly different composition of co-extracts compared to vegetable / fruit puree samples, and is more sensitive to UV detection. Even when using common routes such as acetonitrile extraction and solid phase extraction purification, systematic optimization is still needed in terms of purification column type, elution / elution conditions, solvent system, and combined phase chromatography gradient and back pressure / temperature parameters to balance the separation, peak shape, and response stability of the target enantiomers, and to minimize baseline drift and interference from stray peaks.

[0010] Therefore, based on the current state of technological development, it is still necessary to propose a method and system that is more suitable for the characteristics of rice matrix, can achieve rapid separation of MFZ enantiomers on an ultra-high efficiency phase chromatography platform, and obtain accurate and reliable quantitative results under PDA detection conditions, in order to meet the actual needs of grain quality and safety monitoring and regulatory enforcement. Summary of the Invention

[0011] The technical objective of this invention is to establish a method and system for detecting enantiomer residues of chlorfluazuron in rice matrix. By optimizing the sample extraction and solid-phase extraction purification process, and combining it with ultra-high efficiency phase chromatography chiral separation conditions using supercritical carbon dioxide as the main mobile phase, the invention achieves rapid and effective separation of the two enantiomers and accurate and sensitive quantification under PDA detection. This reduces matrix interference, improves analytical efficiency and result reliability, and meets the application requirements for monitoring and regulating enantiomer residues of chlorfluazuron in rice.

[0012] Firstly, in order to achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for separating and determining the enantiomeric residues of chlorfluazuron in rice using ultra-high performance phase chromatography (UHPLC), comprising the following steps: S1, Extraction: Weigh the rice sample, use acetonitrile as the extractant and add sodium chloride for salting out, centrifuge and take the supernatant; repeat the extraction and combine the extracts and concentrate to near dryness, redissolve with acetonitrile to obtain the solution to be purified; S2, Purification: The solution to be purified is loaded onto a hydrophilic-lipophilic balanced solid-phase extraction column activated with methanol and water. When the liquid surface is almost dry, it is washed with methanol:water = 3:7 (v / v) and the eluent is discarded. Then, it is eluted with 1% ammonia-methanol solution and the eluent is collected. The eluent is concentrated to near dryness at 40°C, dissolved and diluted to volume with n-heptane:isopropanol = 7:3 (v / v) and filtered to obtain the solution for use in the extraction process. S3, Ultra-high performance phase chromatography separation and detection: The above-mentioned solution was injected into an ultra-high performance phase chromatography system, using a chiral column containing a polysaccharide derivative of cellulose-tris(3-chloro-4-methylphenylcarbamate) as the packing material; gradient elution was performed at 35°C and 17.2 MPa back pressure using supercritical carbon dioxide as mobile phase A and methanol as the modifier mobile phase B. In the gradient elution, the volume fraction of modifier B varies between 15% and 25%, with a flow rate of 1.0 mL / min and an injection volume of 5.0 μL. The gradient elution program is as follows: 0–0.7 min 15% B; 0.7–1.2 min 15%–20% B; 1.2–1.6 min 20% B; 1.6–2.2 min 20%–25% B; 2.2–4.0 min 25% B; 4.0–5.0 min 25% B–15% B; 5.0–7.0 min 15% B. The two enantiomers of chlorfluazuron were separated; a diode array detector was used for detection at 230 nm, and the two enantiomers were quantified separately using the external standard method.

[0013] Preferably, the sample weight in the extraction step is 5 g; the amount of acetonitrile added each time is 20 mL, and the extracts are combined after two extractions. Preferably, the amount of sodium chloride added in the salting-out extraction is 3 g; the extraction shaking time is 20 min; and the centrifugation conditions are 4000 r / min and 5 min.

[0014] Preferably, the solution to be purified is obtained by rotary evaporating and concentrating the combined extracts to near dryness, followed by redissolving in 5 mL of acetonitrile.

[0015] Preferably, the solid-phase extraction column is an HLB solid-phase extraction column with a packing amount of 500 mg and a column volume of 6 mL; the activation method is to add 5 mL of methanol and 5 mL of water sequentially.

[0016] Preferably, the volume of the rinsing solution is 5 mL; the volume of the elution solution is 5 mL.

[0017] Preferably, the volumetric volume is 1 mL; the filtration is performed by passing the filter through an organic phase microporous membrane before filtration.

[0018] Preferably, the chiral chromatographic column is a chiral chromatographic column with a specification of 150 mm × 3.0 mm and 2.5 μm.

[0019] Preferably, the external standard method for quantification uses a mixed standard working solution of two enantiomers to establish a standard curve.

[0020] Secondly, the present invention also provides an ultra-high performance phase chromatography separation and determination system for implementing the method, comprising: The sample pretreatment module includes at least an extraction container, a mixing / shaking device, a centrifugation device, and a concentration device; The solid-phase extraction purification module includes at least a solid-phase extraction device that can be fitted with an HLB solid-phase extraction column; The ultra-high efficiency phase-matching chromatography module includes at least a supercritical carbon dioxide supply unit, a modifier supply unit, a back pressure adjustment unit, a column temperature control unit, and a chiral chromatographic column; The detection and data processing module includes at least a diode array detector and a data processing unit; The data processing unit is configured to control the system to perform gradient elution at 35°C and 17.2 MPa, using supercritical carbon dioxide as mobile phase A and methanol as modifier mobile phase B, and to acquire signals at 230 nm and complete the quantification of two enantiomers using the external standard method.

[0021] This invention combines acetonitrile salting-out extraction with HLB solid-phase extraction for purification. Addressing the matrix characteristics of rice—high starch content, numerous co-extractants, and susceptibility to UV interference—a purification strategy of "methanol:water (3:7) rinsing to remove matrix impurities + 1% ammonia-methanol selective elution of the target analyte" is employed. Furthermore, the system optimization using n-heptane:isopropanol (7:3) as the solvent for volume adjustment reduces impurity peaks and baseline drift, improves enantiomeric peak shape and height, and significantly enhances the stability and reproducibility of the method. Simultaneously, ultra-high performance co-phase chromatography (UHPLC) with supercritical CO2 as the main mobile phase and methanol as a modifier, under conditions of 35℃, 17.2 MPa, and gradient elution, combined with a Trefoil CEL2 chiral column, achieves rapid and efficient separation of two chlorfluazuron enantiomeric compounds. This method features short analysis time, high column efficiency, and low organic solvent consumption. Under PDA 230 nm detection and external standard quantification conditions, this method achieves a resolution within the range of 0.5–20.0 μL. It exhibits good linearity within the mg / L range (correlation coefficient ≥ 0.9996), with a limit of quantitation up to 0.1 mg / kg. Recovery rates of 80.4%–107% and relative standard deviations of 3.2%–8.1% are achieved at spiking levels of 0.1, 0.2, and 1.0 mg / kg. This enables accurate, sensitive, and repeatable determination of chlorfluazuron residues in rice at the enantiomeric level, meeting the comprehensive requirements of throughput, accuracy, and reliability for routine monitoring and regulatory enforcement. Furthermore, it provides more discriminative data support for enantiomeric differential exposure and risk assessment of chiral pesticides. Attached Figure Description

[0022] Figure 1 The molecular structural formula of chlorfluazuron is given.

[0023] Figure 2 The spectrum is shown for the enantiomeric standard solution of chlorfluazuron.

[0024] Figure 3 The effect of different co-solvents on the enantiomeric separation of two chlorfluazuron solutions was investigated. (A) 0.5% (v / v) ammonia-methanol solution; (B) methanol; (C) 0.5% (v / v) formic acid-methanol solution.

[0025] Figure 4 The effect of different system back pressures on the enantiomeric separation of two chlorfluazuron was investigated.

[0026] Figure 5 The effect of different dilution reagents on the enantiomeric separation of two chlorfluazuron products is investigated. (A) Methanol, (B) Anhydrous ethanol, (C) Acetonitrile, (D) Isopropanol, (E) n-Heptane.

[0027] Figure 6The effect of different n-heptane:isopropanol ratios on the enantiomeric separation of two chlorfluazuron. (A) n-heptane:isopropanol (5:5, v:v), (B) n-heptane:isopropanol (6:4, v:v), (C) n-heptane:isopropanol (7:3, v:v), (D) n-heptane:isopropanol (8:2, v:v), (E) n-heptane:isopropanol (9:1, v:v), (F) n-heptane:isopropanol (10:0, v:v).

[0028] Figure 7 Chromatograms of the standard solution (A), blank rice sample (B), and recovered solution (C). Peak 1: (+)-Clonofop-ethyl; Peak 2: (-)-Clonofop-ethyl.

[0029] Figure 8 The chromatogram shows a positive rice sample. Peak 1: (+)-chlorfluazuron; Peak 2: (-)-chlorfluazuron. Detailed Implementation

[0030] 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.

[0031] 1. Materials and Methods 1.1 Instruments, Materials and Reagents Acquity ultra-high performance phase chromatograph (Waters, USA, with diode array detector (PDA)); ME104E electronic balance (Mettler, Switzerland); R215 rotary evaporator (Buchi, Switzerland); ELGA CLXXXUVM2 ultrapure water purification system (Elga, UK); N-EVAP TM 111 Nitrogen Evaporator (Tokyo Rika Co., Ltd., Japan); MS2 Vortex Mixer (Shanghai Medical University Instrument Factory); MMS-3020 Horizontal Oscillator (Tokyo Rika Co., Ltd., Japan).

[0032] Anhydrous ethanol, acetonitrile, methanol, isopropanol, n-heptane (chromatographic grade, Scharlau, Spain); ultrapure water; high-purity carbon dioxide (99.999%); HLB column (Waters Oasis, 500 mg, 6 mL); Waters Acquity TrefoilCEL2 (150 mm × 3.0 mm, 2.5 µm, packing material: cellulose-tris(3-chloro-4-methylphenylcarbamate)) (Waters, USA); other reagents used in the experiments were of analytical grade unless otherwise specified.

[0033] Racemic standard (chlorfluazuron: CAS No.: 1417782-03-6, purity ≥98.0%, Shanghai Maclean's Biochemical Technology Co., Ltd.). Enantiomers of chlorfluazuron: (+)-chlorfluazuron and (-)-chlorfluazuron were isolated and purified by Shanghai Qinlu Biotechnology Co., Ltd. from the racemic standard of chlorfluazuron (Shanghai Maclean's Biochemical Technology Co., Ltd.), with purities greater than 98.0%.

[0034] 1.2 Preparation of Standard Stock Solution and Working Solution Accurately weigh 0.01 g (accurate to 0.1 mg) of (+)-chlorofludioxonil and (-)-chlorofludioxonil standards, dissolve them in methanol and dilute to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L.

[0035] Mixed standard working solutions of two enantiomers of chlorfluazuron: Accurately pipette a certain amount of (+)-chlorfluazuron and (-)-chlorfluazuron enantiomer standard stock solutions, and dilute them stepwise with methanol to 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L to form mixed standard working solutions.

[0036] 1.3 Sample Pretreatment 1.3.1 Sample Extraction Weigh 5 g (accurate to 0.01 g) of the sample into a 50 mL centrifuge tube, add 20 mL of acetonitrile and 3 g of sodium chloride, vortex to mix, shake to extract for 20 min, 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 5 mL of acetonitrile, and wait for purification.

[0037] 1.3.2 Purification The reconstituted solution was transferred to an HLB solid-phase extraction column activated with 5 mL of methanol and 5 mL of water. When the liquid surface was almost dry, 5 mL of methanol:water (3:7, v / v) was added for rinsing. The eluent was discarded and the column was dried under vacuum. 5 mL of 1% ammonia-methanol solution was added for elution. The eluent was collected and evaporated to near dryness in a 40°C water bath. The solution was then dissolved and diluted to volume with 1 mL of n-heptane:isopropanol solution (7:3, v:v). The solution was then passed through a membrane and used in the extraction instrument.

[0038] 1.4 Chromatographic conditions Column: Acquity Trefoil CEL2 (150 mm × 3.0 mm, 2.5 µm); Detection wavelength: 230 nm; System back pressure: 17.2 MPa; Column temperature: 35℃; Mobile phase: A is CO2, B is methanol; Gradient elution program: 0–0.7 min (15% B), 0.7–1.2 min (15%–20% B), 1.2–1.6 min (20% B), 1.6–2.2 min (20%–25% B), 2.2–4.0 min (25% B), 4.0–5.0 min (25% B–15% B), 5.0–7.0 min (15% B); Flow rate: 1.0 mL / min; Injection volume: 5.0 μL.

[0039] 2. Results and Discussion 2.1 Selection of detection wavelength After scanning with a PDA detector, the ultraviolet spectra of two enantiomer standard solutions of chlorfluazuron were extracted from the chromatogram. For example... Figure 2 As shown, there are obvious absorption peaks at both 230 nm and 280 nm. Compared with 280 nm, the chromatographic peak has the strongest absorption and the highest sensitivity at 230 nm. Therefore, 230 nm was selected as the detection wavelength in this experiment.

[0040] 2.2 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 elution capacity and selectivity 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 chlorfluazuron enantiomers. The results showed that when 0.5% (v / v) formic acid-methanol solution was used as a co-solvent, the chromatographic baseline was uneven and the peaks were very small. When 0.5% (v / v) ammonia-methanol solution or methanol was used as a co-solvent, the separation effect and peak shape of the two chlorfluazuron enantiomers were almost the same (see [link to relevant documentation]). Figure 3However, compared to a 0.5% (v / v) ammonia-methanol solution, using methanol as a co-solvent results in a cleaner chromatographic baseline, fewer interfering peaks, easier operation, and less damage to the instrument and column. Therefore, our laboratory chose methanol as the co-solvent.

[0041] 2.3 Optimization of System Back Pressure UPC 2 Supercritical CO2 was used as the mobile phase. Adjusting the system back pressure and temperature effectively altered the density of CO2, thereby changing its solubility, elution capacity, and selectivity. 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 10.3–17.2 MPa on the separation of two enantiomers of chlorfluazuron. The results showed that as the system back pressure increased, the retention time of the analytes decreased, while the separation degree and peak shape remained relatively similar (see...). Figure 4 In comparison, when 17.2 MPa is used as the system back pressure, the chromatographic peak height is higher and the peak shape is sharper. Therefore, 17.2 MPa was selected as the system back pressure in this experiment.

[0042] 2.4 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 chlorfluazuron. The results are as follows: Figure 5 As shown, compared with methanol, anhydrous ethanol, acetonitrile and isopropanol, when n-heptane was used as the volume-adjusting reagent, the chromatographic peaks of the two chlorfluazuron enantiomers were sharper and higher.

[0043] Considering the poor solubility of n-heptane, this experiment further investigated different ratios of n-heptane to isopropanol solutions (5:5, 6:4, 7:3, 8:2, 9:1, 10:0 n-heptane:isopropanol, v / v) to facilitate subsequent analysis and detection. The results showed that as the proportion of n-heptane increased, the peak heights of the two enantiomers of chlorfluazuron gradually increased (see...). Figure 6 When using a heptane:isopropanol solution (7:3, v:v), the chromatographic peak height reached its highest value. However, as the proportion of heptane continued to increase, the peak height showed a decreasing trend. Therefore, the heptane:isopropanol solution (7:3, v:v) was ultimately selected as the reagent for volume adjustment in the experiment.

[0044] 2.5 Methodological Examination 2.5.1 Linear range and limit of quantitation A series of mixed standard solutions of chlorfluazuron enantiomers were determined under the chromatographic conditions described above. A standard curve was plotted 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 two enantiomers exhibited a good linear relationship in the mass concentration range of 0.5–20.0 mg / L, with a correlation coefficient greater than 0.9996. By adding the standard to a blank rice sample without chlorfluazuron, the determination was performed according to this method. The limit of quantitation (LOQ) was calculated with a signal-to-noise ratio (S / N) = 10, and the LOQ for both (+)-chlorfluazuron and (-)-chlorfluazuron was 0.1 mg / kg (see Table 1).

[0045] Table 1. Linear range, linear equation, correlation coefficient, and limit of quantitation for each compound.

[0046] 2.5.2 Recovery rate and precision Two enantiomer standard solutions of chlorfluazuron at different concentrations were added to rice samples that did not contain chlorfluazuron. Spiking recovery and precision tests were performed. Relevant chromatograms are shown below. Figure 7 The results are shown in Table 2. The results indicate that the recoveries of the two enantiomers of chlorfluazuron were 80.4%–107%, and the relative standard deviations (RSDs) were 3.2%–8.1%, which meet the recovery requirements of SANTE / 11312 / 2021 and can satisfy the determination of chlorfluazuron enantiomer content in rice samples.

[0047] Table 2. Spiking recoveries and relative standard deviations of enantiomers of chlorfluazuron in rice samples (n=6)

[0048] 2.6 Testing of actual samples To examine the effectiveness and practicality of this method, the established method was used to determine the contents of (+)- and (-)-cloflufenicol in 20 commercially available rice samples. The results showed that cloflufenicol enantiomers were not detected in 19 rice samples, while (+)- and (-)-cloflufenicol were detected in one rice sample, with detected levels of 0.193 mg / kg and 0.207 mg / kg, respectively (see [link to study]). Figure 8 ).

[0049] 3. Conclusion This paper presents the first method using ultra-high performance phase chromatography (UHPLC) to simultaneously separate two enantiomers of chlorfluazuron and to determine the residue levels of chlorfluazuron enantiomers in rice. The method optimized instrumental separation conditions, including detection wavelength, co-solvent, system back pressure, and volumetric reagents. The established method was applied to the detection of actual samples, demonstrating high sensitivity and good stability, meeting the requirements for detecting chiral enantiomer residues of chlorfluazuron 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 significant importance for drug quality control and efficacy evaluation.

[0050] 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 herein 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 method for separating and determining the enantiomeric residues of chlorfluazuron in rice using ultra-high performance phase chromatography, characterized in that, The method includes the following steps: S1, Extraction: Weigh the rice sample, use acetonitrile as the extractant and add sodium chloride for salting out, centrifuge and take the supernatant; repeat the extraction and combine the extracts and concentrate to near dryness, redissolve with acetonitrile to obtain the solution to be purified; S2, Purification: The solution to be purified is loaded onto a hydrophilic-lipophilic balanced solid-phase extraction column activated with methanol and water. When the liquid surface is almost dry, it is washed with methanol:water = 3:7 (v / v) and the eluent is discarded. Then, it is eluted with 1% ammonia-methanol solution and the eluent is collected. The eluent is concentrated to near dryness at 40°C, dissolved and diluted to volume with n-heptane:isopropanol = 7:3 (v / v) and filtered to obtain the solution for use in the extraction process. S3, Ultra-high performance phase chromatography separation and detection: The above-mentioned solution was injected into an ultra-high performance phase chromatography system, using a chiral column containing a polysaccharide derivative of cellulose-tris(3-chloro-4-methylphenylcarbamate) as the packing material; gradient elution was performed at 35°C and 17.2 MPa back pressure using supercritical carbon dioxide as mobile phase A and methanol as the modifier mobile phase B. In the gradient elution, the volume fraction of modifier B varies between 15% and 25%, the flow rate is 1.0 mL / min, and the injection volume is 5.0 μL. The gradient elution program is as follows: 0–0.7 min: 15% B; 0.7–1.2 min: 15%–20% B; 1.2–1.6 min: 20% B; 1.6–2.2 min: 20%–25% B; 2.2–4.0 min: 25% B; 4.0–5.0 min: 25% B–15% B. 5.0–7.0 min is 15% B; The two enantiomers of chlorfluazuron were separated; a diode array detector was used for detection at 230 nm, and the two enantiomers were quantified separately using the external standard method.

2. The method according to claim 1, characterized in that, The sample weight in the extraction step is 5 g; the amount of acetonitrile added each time is 20 mL, and the extracts are combined after two extractions.

3. The method according to claim 1, characterized in that, The amount of sodium chloride added during the salting-out extraction was 3 g; the extraction shaking time was 20 min; and the centrifugation conditions were 4000 r / min for 5 min.

4. The method according to claim 1, characterized in that, The solution to be purified was obtained by rotary evaporating and concentrating the combined extracts to near dryness, followed by redissolving in 5 mL of acetonitrile.

5. The method according to claim 1, characterized in that, The solid-phase extraction column is an HLB solid-phase extraction column with a packing amount of 500 mg and a column volume of 6 mL; the activation method is to add 5 mL of methanol and 5 mL of water sequentially.

6. The method according to claim 1, characterized in that, The volume of the rinsing solution is 5 mL; the volume of the elution solution is 5 mL.

7. The method according to claim 1, characterized in that, The fixed volume is 1 mL; the filtration is performed by filtering through an organic phase microporous membrane before the material is processed.

8. The method according to claim 1, characterized in that, The chiral chromatographic column is a 150 mm × 3.0 mm, 2.5 μm chiral chromatographic column.

9. The method according to claim 1, characterized in that, The external standard method for quantification uses a mixed standard working solution of two enantiomers to establish a standard curve.

10. An ultra-high performance phase chromatography separation and determination system for implementing the method according to any one of claims 1 to 9, characterized in that, include: The sample pretreatment module includes at least an extraction container, a mixing / shaking device, a centrifugation device, and a concentration device; The solid-phase extraction purification module includes at least a solid-phase extraction device that can be fitted with an HLB solid-phase extraction column; The ultra-high efficiency phase-matching chromatography module includes at least a supercritical carbon dioxide supply unit, a modifier supply unit, a back pressure adjustment unit, a column temperature control unit, and a chiral chromatographic column; The detection and data processing module includes at least a diode array detector and a data processing unit; The data processing unit is configured to control the system to perform gradient elution at 35°C and 17.2 MPa, using supercritical carbon dioxide as mobile phase A and methanol as modifier mobile phase B, and to acquire signals at 230 nm and complete the quantification of two enantiomers using the external standard method.

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