Method and system for rapidly determining diniconazole enantiomer residual quantity in fruit and vegetable puree through solid phase extraction-ultra performance convergence chromatography

Abstract

The invention relates to a method for detecting pesticide residues in food, in particular to a method and a system for rapidly determining diniconazole enantiomer residues in fruit and vegetable puree through solid-phase extraction-ultra performance convergence chromatography. The method comprises the following steps of: shaking and extracting a sample by acetonitrile, centrifuging, combining supernatant, concentrating, redissolving by using acetone / normal hexane, activating by using a florisil solid-phase extraction column, leaching, eluting and purifying, concentrating, and performing constant-volume sample injection by acetonitrile. According to the method, an AMY1 chiral chromatographic column is used, supercritical CO2 / methanol is used as a mobile phase for gradient elution, PDA is used for detection at 250 nm at the column temperature of 40 DEG C, the back pressure of 17.2 MPa and the flow rate of 1.0 mL / min, and base line separation and external standard quantification of R and S enantiomers within 4 min are realized. The method is good in linearity, the quantitation limit is 0.1 mg / kg, the recovery rate is 80.1%-108%, the RSD is 4.2%-8.1, and the method is suitable for rapidly monitoring the diniconazole enantiomers in the fruit and vegetable puree.

Description

Technical Field

[0001] This invention relates to a method for detecting pesticide residues in food, and more particularly to a method and system for rapidly determining the enantiomeric residues of tebuconazole in fruit and vegetable purees using solid-phase extraction-ultra-high performance phase chromatography. Background Technology

[0002] Diconazole is a typical triazole chiral fungicide containing a chiral center in its molecule, and commercial formulations are usually used in racemic form. Numerous studies have shown that the two enantiomers of chiral pesticides often differ significantly in fungicidal activity, animal and human toxicity, and environmental metabolic behavior. One enantiomer may contribute the main efficacy, while the other may increase toxic side effects on non-target organisms or humans. Therefore, enantiomer-level residue analysis and risk assessment of chiral pesticides have become a research hotspot in the field of pesticide residues and food safety both domestically and internationally.

[0003] Traditional pesticide residue monitoring primarily uses "total residue" as an indicator, employing techniques such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS) for non-chiral detection of racemic compounds. However, this approach cannot distinguish between the content differences of two enantiomers. With increasingly stringent food safety standards and stricter regulations on infant formula, there is a growing demand for rapid, sensitive, and selective detection of chiral pesticide enantiomers in high-risk matrices such as fruit and vegetable purees. Fruit and vegetable purees, commonly used as infant formula, contain high proportions of sugars, organic acids, pectin, and natural pigments, exhibiting strong matrix effects and complex interfering components, posing a significant challenge to the accurate determination of trace pesticide enantiomers.

[0004] In sample pretreatment, solid-phase extraction (SPE) technology is widely used for pesticide residue detection in the environment and food due to its advantages such as good selectivity, stable purification effect, and convenient coupling with chromatographic systems. Chinese invention patent CN108802242B discloses a method for simultaneously determining the enantiomeric residues of six chiral pesticides in an aquatic environment. This method uses solid-phase extraction combined with dispersive liquid-liquid microextraction (DLI) for water sample pretreatment, and then utilizes chiral chromatographic columns and liquid chromatography-tandem mass spectrometry (LC-MS / MS) to simultaneously determine the enantiomeric residues of chiral pesticides such as hexaconazole, methamidophos, dichlorvos, metalaxyl, flutriafol, and cyproconazole. Although this patent achieves sensitive detection of multiple chiral pesticide enantiomeric residues in aquatic samples through SPE enrichment and chiral separation, its target is an aquatic matrix with relatively simple matrix composition. The pretreatment and separation conditions are difficult to directly transfer to fruit and vegetable puree samples with significantly higher sugar and pigment content, and it does not address the residue determination of tebuconazole enantiomeric residues in complex food matrices.

[0005] For triazole fungicides, an important category, Chinese invention patent CN111208234A proposes a rapid detection method for triazole fungicides in soil. This method utilizes an improved QuEChERS pretreatment technique to extract and purify soil samples, and combines this with liquid chromatography-mass spectrometry (LC-MS) to establish a standard curve, thereby achieving qualitative and quantitative analysis of various triazole fungicides in soil. This patent is representative in optimizing adsorbent ratios and reducing soil matrix interference, demonstrating that adsorbents such as PSA and Florisil are effective in removing pigments, organic acids, and macromolecular impurities. However, this method targets the detection of non-chiral total residues, does not achieve enantiomeric separation and quantification, does not involve novel separation techniques such as ultra-high performance phase chromatography (UHPLC), and does not consider the special characteristics of infant food matrices such as fruit and vegetable purees.

[0006] In recent years, supercritical fluid chromatography and ultra-high performance coherent chromatography (UPC) have become increasingly important. 2 Triadimefon technology, due to its advantages such as using supercritical CO2 as the main mobile phase, requiring less organic modifier, high separation efficiency, fast analysis speed, and easy integration with chiral stationary phase chromatographic columns, is widely used for the separation and residue determination of enantiomers of chiral pesticides. Against this backdrop, Chinese invention patent CN115494190A discloses a method for the simultaneous determination of enantiomers of triadimefon and its metabolite triadimefon in fruit and vegetable purees. This patent uses acetonitrile to extract samples of infant complementary food fruits and vegetables such as banana puree, pineapple puree, and grape puree. The extract is purified using an NH2 solid-phase extraction column, and then, using a supercritical CO2–0.5% ammonia-methanol mobile phase on an AcquityTrefoil CEL2 chiral chromatographic column, ultra-high performance phase chromatography (UPC) is employed to achieve rapid separation and quantification of triadimefon and triadimefon enantiomers. The method has a limit of quantitation of 0.05 mg / kg, a spiked recovery rate of 80.1%–106%, and an RSD of 3.3%–7.6%. This patent demonstrates UPC 2 Combining this technology with chiral chromatographic columns and solid-phase extraction purification can effectively address the need for enantiomeric residue analysis of triazole pesticides in complex food matrices such as fruit and vegetable purees. However, CN115494190A primarily targets the enantiomeric forms of triadimefon and its metabolite triazoleol, and does not cover tebuconazole, a chiral triazole fungicide with significantly different structures and physicochemical properties. Tebuconazole differs from triadimefon and triazoleol in terms of hydrophobicity, pKa, and partition behavior in fruit and vegetable purees. Directly applying its pretreatment conditions, solid-phase extraction packing materials, and mobile phase system can easily lead to insufficient target peak resolution, severe baseline interference, or low recovery rates. Furthermore, CN115494190A uses an NH2 solid-phase extraction column, which mainly focuses on the removal of polar interfering substances. For specific fruit and vegetable puree formulations containing large amounts of natural pigments and macromolecular organic matter, insufficient purification or strong matrix effects may still occur.

[0007] In summary, among the existing technologies, CN108802242B solves the problem of SPE enrichment and chiral separation of multiple chiral pesticide enantiomers in aquatic environments, but does not address complex food matrices such as fruit and vegetable purees; CN111208234A optimizes the improved QuEChERS process for rapid detection of total residues of triazole fungicides in soil, but does not perform enantiomer separation and analysis; although CN115494190A utilizes UPC... 2 The technology has achieved the separation and determination of triazolone and triazole enantiomers in fruit and vegetable purees, but it has not established a specific solid-phase extraction purification strategy and ultra-high performance phase chromatography separation conditions for the residue behavior of tebuconazole enantiomers in the fruit and vegetable puree matrix. Overall, existing patent literature lacks a specific technical solution for the rapid determination of tebuconazole enantiomer residues in fruit and vegetable purees using solid-phase extraction coupled with ultra-high performance phase chromatography, which takes into account efficient purification, rapid separation, and methodological reliability.

[0008] In the practice of producing fruit and vegetable purees and supervising the quality and safety of infant formula, if total residue analysis or enantiomeric analysis methods targeting only a few triazole pesticides are still used, the differences in exposure risk of tebuconazole enantiomers in these matrices are easily overlooked, and it is difficult to provide timely and targeted technical support for pesticide use guidance and product risk assessment. Therefore, it is necessary to improve the methods used in solid-phase extraction pretreatment, chiral column selection, and UPC. 2 The gradient procedure and detection wavelength were systematically optimized to establish a rapid determination method and supporting system for enantiomeric residues of tebuconazole suitable for fruit and vegetable pulp substrates, so as to make up for the shortcomings of existing technologies. Summary of the Invention

[0009] The purpose of this invention is to provide a rapid method for determining the residual amount of tebuconazole enantiomers in fruit and vegetable purees using solid-phase extraction-ultra-high performance phase chromatography (UPC). This method is used for the separation and determination of tebuconazole enantiomers. The instrumental chromatographic separation conditions and purification conditions for tebuconazole enantiomers in fruit and vegetable purees are optimized. Under optimal conditions, the established UPC... 2 The method was applied to and analyzed in actual fruit and vegetable pulp samples and racemic tebuconazole standards. This method features rapid analysis, good separation, and low organic solvent consumption, aiming to provide technical support for guiding the production process, quality control, and efficacy evaluation of tebuconazole pesticides, as well as for the quality and safety supervision of fruit and vegetable products.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A method for rapid determination of enantiomeric residues of tebuconazole in fruit and vegetable purees using solid-phase extraction-ultra-high performance phase chromatography includes the following steps: S1. Sample extraction: Weigh 5g of homogenized fruit and vegetable puree sample into a centrifuge tube, add 15-25mL of acetonitrile, vortex to mix, and then shake to extract for 15-25 minutes. Centrifuge at 3500-4500 rpm for 3-7 minutes to separate the supernatant. Repeat the above acetonitrile extraction steps for the residue at least once. Combine the supernatants and concentrate them to near dryness by rotary evaporation at 35-45℃. Add 5mL of a mixed solvent of acetone and n-hexane in a volume ratio of 1:20 to redissolve the supernatant to obtain the solution to be purified. S2, Solid-phase extraction purification: The solution to be purified was transferred to a Florisil solid-phase extraction column. Before use, the solid-phase extraction column was activated sequentially with 5 mL of acetone, 5 mL of n-hexane, and 5 mL of acetone / n-hexane = 1:20 (volume ratio). When the liquid level of the solution to be purified was close to the surface of the packing material, the interfering matrix was removed by rinsing with 8-12 mL of acetone / n-hexane = 1:20 (volume ratio). Then, the target analyte was eluted with 4-6 mL of acetone / n-hexane = 1:1 (volume ratio). All eluent was collected and evaporated to near dryness at 35-45°C by rotary evaporation. The solution was then diluted to volume with 0.8-1.2 mL of acetonitrile to obtain the test solution. S3. Chromatographic separation and detection: The test solution was injected into an ultra-high performance coherent chromatography system (UHPLC) and separated on a chiral column packed with amylose tris(3,5-dimethylphenylcarbamate). The chiral column was 150 mm long, 3.0 mm inner diameter, and 2.5 μm particle size. Supercritical carbon dioxide was used as mobile phase A, methanol as mobile phase B, the flow rate was 1.0 mL / min, the column temperature was 40 °C, and the system back pressure was 17.2 MPa. A gradient elution program with methanol volume fractions ranging from 15% to 30% was used for separation, and detection was performed at a wavelength of 250 nm using a diode array detector. Baseline separation of (-)-R-tebuconazole and (+)-S-tebuconazole was achieved within 4.0 minutes. The methanol gradient elution program was as follows: 0–1.0 minutes, 15% B; 1.0–1.1 minutes, 15%–25% B; 1.1–2.0 minutes, 25% B; 2.0–2.1 minutes, 25%–30% B; 2.1–2.5 minutes, 30% B; 2.5–2.8 minutes, 30%–15% B; 2.8–3.0 minutes, 15% B.

[0011] S4. Quantitative Calculation: A mixed standard working solution of two enantiomers of tebuconazole was used to establish an external standard calibration curve of mass concentration versus peak area in the concentration range of 0.5–20.0 mg / L. The residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in fruit and vegetable puree samples were calculated from the calibration curve using the peak areas of the corresponding chromatographic peaks in the samples. The method limit of quantitation was not higher than 0.1 mg / kg, and the spiked recoveries of the two enantiomers were 80.1%–108% and the relative standard deviations were 4.2%–8.1% in the spiked concentration range of 0.1–1.0 mg / kg.

[0012] Preferably, in step S1, the sample mass is 5.00±0.05g, the volume of acetonitrile added each time is 20±2mL, the shaking extraction time is 20±5 minutes, and the centrifugation conditions are 4000±500 rpm for 5±2 minutes.

[0013] According to the method described in claim 1, the Florisil solid-phase extraction column in step S2 has a packing amount of 5g, a column volume of 6mL, an acetone / n-hexane (1:20, volume ratio) elution volume of 10mL, an acetone / n-hexane (1:1, volume ratio) elution volume of 5mL, and all effluent collected during the entire purification process is combined and then concentrated and diluted to a fixed volume.

[0014] As a preferred method, a spiked recovery experiment was conducted by adding two enantiomer standard solutions of tebuconazole to blank samples of fruit and vegetable puree at three concentration levels of 0.1 mg / kg, 0.2 mg / kg, and 1.0 mg / kg. The intra-day and inter-day spiked recoveries were in the range of 80.1% to 108%, and the relative standard deviations were in the range of 4.2% to 8.1%, respectively.

[0015] Preferably, this method is also used to separate and determine the enantiomeric content of the racemic tebuconazole standard. The intermediate solution of the racemic tebuconazole is injected under the chromatographic conditions of step S3, and two symmetrical chromatographic peaks with a resolution of not less than 5.0 are obtained within 3.5 minutes, corresponding to (-)-R-tebuconazole and (+)-S-tebuconazole, respectively. The content ratio of the two enantiomeric components in the racemic mixture is then calculated.

[0016] Preferably, 0.01 g, accurate to 0.1 mg, of (-)-R-tebuconazole and (+)-S-tebuconazole standards are accurately weighed, dissolved in methanol, and diluted to 10 mL to prepare 1.0 g / L enantiomeric standard stock solutions; mixed standard working solutions of the two tebuconazole enantiomers are prepared by accurately pipetting a certain amount of (-)-R-tebuconazole and (+)-S-tebuconazole enantiomer standard stock solutions and diluting them stepwise with methanol to prepare mixed standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L.

[0017] Furthermore, the present invention also provides a rapid determination system for enantiomeric residues of tebuconazole for implementing the method, comprising: The sample pretreatment unit is used to weigh the fruit and vegetable puree sample, add acetonitrile, and then perform shaking extraction, centrifugation, vacuum concentration, and redissolution in a mixed solvent of acetone / n-hexane to obtain the solution to be purified. The solid phase extraction purification unit is equipped with a Florisil solid phase extraction column and supply channels for acetone, n-hexane and two volume ratios of acetone / n-hexane mixed solvents, respectively, for pre-column activation, rinsing and elution of the solution to be purified, so as to obtain the purified test solution. The ultra-high efficiency phase chromatography separation unit is equipped with a chiral chromatographic column with amylose tris(3,5-dimethylphenylcarbamate) as the stationary phase, a high-pressure delivery mechanism with supercritical carbon dioxide and methanol as the mobile phase, a back pressure regulator, and a column temperature control component, which are used to separate enantiomers in the purified test solution. The detection unit, connected to the outlet of the ultra-high efficiency phase chromatography separation unit, is a diode array detector with a detection wavelength of 250 nm, used to acquire chromatographic signals; The data processing and control unit is used to control the working parameters of the sample pretreatment unit, solid phase extraction purification unit and ultra-high efficiency phase chromatography separation unit, collect and process the chromatographic data output by the detection unit, and calculate the residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in the fruit and vegetable puree sample according to the external standard calibration curve.

[0018] Preferably, the ultra-high performance phase chromatography separation unit is a UPC. 2 The system uses a chiral chromatographic column, which is an AcquityTrefoilAMY1 column with a specification of 150mm×3.0mm and a particle size of 2.5 micrometers. The system back pressure is set to 17.2MPa by the back pressure regulator, the total flow rate of the mobile phase is 1.0mL / min, the column temperature is 40℃, and the data processing and control unit has a methanol gradient elution program pre-stored in it.

[0019] Preferably, the data processing and control unit includes: The storage module is used to store methodological parameters such as external standard calibration curve parameters, method limit of quantitation, and spiked recovery rate of the two tebuconazole enantiomers in the concentration range of 0.5–20.0 mg / L. The processor module is used to automatically calculate the residual amounts of the two enantiomers of tebuconazole in the sample by calling the calibration curve parameters based on the real-time collected sample chromatographic peak area, and generate a test report containing the enantiomer content and total amount. The human-computer interaction module is used to display chromatograms, method parameters and calculation results, and allows users to select the type of fruit and vegetable puree sample, the spike concentration level and the batch information.

[0020] Furthermore, the present invention also provides a computer-readable storage medium having a computer program stored thereon, wherein when executed by a processor, the computer program causes the processor to perform the following steps: The system calls upon the data processing and control unit to automatically set and control the mobile phase ratio, gradient elution program, flow rate, back pressure, and column temperature of the ultra-high efficiency phase chromatography separation unit. The system receives the chromatographic signal output from the diode array detector and identifies, integrates, and calculates the peak areas of the two enantiomers of tebuconazole. Based on the preset external standard calibration curve parameters, the peak area is converted into the residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in the fruit and vegetable puree sample to be tested, thereby completing the method for determining the residual amounts of tebuconazole enantiomers in fruit and vegetable puree by solid phase extraction-ultra-high performance phase chromatography.

[0021] This invention organically combines Florisil solid-phase extraction purification optimized for fruit and vegetable puree matrices with ultra-high performance co-phase chromatography separation conditions using a Trefoil AMY1 chiral column. The technical effects are as follows: Firstly, acetonitrile extraction combined with an acetone / n-hexane system and gradient elution using a Florisil column effectively removes a large amount of co-extracted impurities such as sugars, organic acids, pectin, and natural pigments from fruit and vegetable purees, significantly reducing matrix effects, resulting in a stable chromatographic baseline and a substantial reduction in interfering peaks. Secondly, under conditions of a column temperature of 40℃, a system back pressure of 17.2MPa, and a methanol gradient of 15%–30%, the two enantiomers of tebuconazoles R and S can be separated within 3.5–4.0 minutes. The method exhibits symmetrical peak shape and baseline separation with a resolution greater than 1.5, while maintaining a high peak response. This ensures good linearity within the range of 0.5–20.0 mg / L. The method's limit of quantitation in fruit and vegetable pulp samples can be stably reduced to 0.1 mg / kg, with spiked recoveries controlled at approximately 80%–108% and intra-day and inter-day RSDs controlled at approximately 4%–8%. This method not only meets the requirements of current pesticide residue analysis standards but also offers advantages such as fast analysis speed, low organic solvent and sample volume consumption, and suitability for batch detection. It provides reliable technical support for the efficient monitoring of enantiomeric residues of tebuconazole in fruit and vegetable pulp. Attached Figure Description

[0022] Figure 1 Spectrum of enantiomer standard solution of tebuconazole.

[0023] Figure 2 The effects of different separation conditions on the separation of two enantiomers of tebuconazole. (A) Isocratic separation condition 1, (B) Gradient separation condition 2, (C) Gradient separation condition 3.

[0024] Figure 3 The effect of different system back pressures on the enantiomeric separation of two tebuconazoles.

[0025] Figure 4 The effect of different column temperatures on the separation of two enantiomers of tebuconazole.

[0026] Figure 5 The effect of different purification methods on the purification efficiency of (-)-R-tebuconazole and (+)-S-tebuconazole. (A) Florisil column; (B) NH2 column.

[0027] Figure 6 Chromatograms of the standard solution (A), blank fruit and vegetable puree sample (B), and added recovery (C). Chromatogram peak 1: (-)-R-tebuconazole; Chromatogram peak 2: (+)-S-tebuconazole.

[0028] Figure 7 Resolution of the racemic mixture of tebuconazole. Chromatographic peak 1: (-)-R-tebuconazole; Chromatographic peak 2: (+)-S-tebuconazole.

[0029] Figure 8 Chromatogram of a positive fruit and vegetable puree sample. Chromatogram peak 1: (-)-R-tebuconazole; Chromatogram peak 2: (+)-S-tebuconazole. 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. Explanation of Terminology and Key Concepts 1.1 Fruit and vegetable puree: refers to a puree-like sample obtained by pulping and homogenizing fruits and / or vegetables as the main raw materials, including but not limited to apple puree, banana puree, pear puree, carrot puree, pumpkin puree and their mixtures; it can be commercially available infant complementary food or production process samples.

[0032] 1.2 Enantiomers of Tebuconazole: Tebuconazole molecules contain a chiral center and exist in two mirror-image enantiomers: (-)-R-tebuconazole and (+)-S-tebuconazole. These enantiomers possess different biological activities and potential risks, thus requiring separate quantification.

[0033] 1.3 Ultra-high performance phase chromatography (UPC) 2 ): Using supercritical carbon dioxide as the main mobile phase, combined with alcohol modifiers (such as methanol), it achieves efficient separation under high back pressure conditions; it is especially suitable for the rapid separation of chiral compounds.

[0034] 1.4 Solid-phase extraction (SPE) purification: The extract is loaded into a packed column (such as Florisil or amino NH2), and the purification effect is improved and the matrix effect is reduced by utilizing the adsorption of matrix interference and selective elution of target substances by the packed column.

[0035] 1.5 Limit of Quantification (LOQ): The lowest concentration level that can be reliably quantified in a spiked blank matrix with a signal-to-noise ratio of approximately 10 as the criterion. In this invention, it is 0.1 mg / kg.

[0036] 2. Materials and Methods 2.1 Instruments, Materials and Reagents Acquity ultra-high performance phase chromatograph (Waters, USA, with diode array detector (PDA)); AE260 electronic balance (Mettler, Switzerland); R215 rotary evaporator (Buchi, Switzerland); ELGACLXXXUVM2 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).

[0037] Anhydrous ethanol, acetonitrile, methanol, isopropanol, n-heptane (chromatographic grade, Scharlau, Spain); ultrapure water; high-purity carbon dioxide (99.999%); Florisil column (CNW, 5g, 6mL); amino (NH2) column (Ager, 500mg, 3mL); Waters AcquityTrefoilAMY1 (150mm × 3.0mm, 2.5µm, packed with amylose-tris(3,5-dimethylphenylcarbamate)); other reagents used in the experiments were of analytical grade unless otherwise specified.

[0038] Racemic standard (tebuconazole: CAS No.: 76714-88-0, purity ≥99.2%, Beijing Zhenxiang Technology Co., Ltd.). Enantiomer standards of tebuconazole: (-)-R-tebuconazole and (+)-S-tebuconazole were isolated and purified from the racemic standard of tebuconazole (Beijing Zhenxiang Technology Co., Ltd.) by Shanghai Qinlu Biotechnology Co., Ltd., with purities greater than 98.0%.

[0039] 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 tebuconazole racemic standard, dissolve it in methanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution.

[0040] Standard intermediate solution of racemic tebuconazole: Accurately pipette a certain amount of racemic standard stock solution and dilute it with methanol to a standard intermediate solution of 20.0 mg / L.

[0041] 2.2.2 Enantiomer Standard Stock Solution Accurately weigh 0.01 g (accurate to 0.1 mg) of (-)-R-tebuconazole and (+)-S-tebuconazole standards, dissolve them in methanol and dilute to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L.

[0042] Mixed standard working solutions of two tebuconazole enantiomers: Accurately pipette a certain amount of the (-)-R-tebuconazole and (+)-S-tebuconazole 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 respectively to form mixed standard working solutions.

[0043] 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 acetonitrile, vortex to mix, shake to extract for 20min, centrifuge at 4000r / min for 5min, and transfer the supernatant to another 50mL centrifuge tube; add 20mL of acetonitrile to the lower residue, repeat the extraction once, combine the two supernatants, concentrate to near dryness using a rotary evaporator, dissolve in 5mL of acetone:n-hexane (1:20, v / v) solution, and wait for purification.

[0044] 2.3.2 Purification The reconstituted solution was transferred to a Florisil solid-phase extraction column activated with (5 mL acetone, 5 mL n-hexane, 5 mL acetone:n-hexane (1:20, v / v)). When the liquid surface was almost dry, the solution to be purified was transferred to the column and washed with 10 mL acetone:n-hexane (1:20, v / v) solution. Then, 5 mL acetone:n-hexane (1:1, v / v) solution was added for further 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 acetonitrile, and then filtered through a membrane for extraction.

[0045] 2.4 Chromatographic conditions Column: AcquityTrefoil AMY1 (150mm×3.0mm, 2.5µm); Detection wavelength: 250nm; System back pressure: 17.2MPa; Column temperature: 40℃; Mobile phase: A is CO2, B is methanol; Gradient elution program: 0~1.0min (15%B), 1.0~1.1min (15%~25%B), 1.1~2.0min (25%B), 2.0~2.1min (25%~30%B), 2.1~2.5min (30%B), 2.5~2.8min (30%B~15%B), 2.8~3.0min (15%B); Flow rate: 1.0mL / min; Injection volume: 5.0μL.

[0046] 2.5 Mobile phase elution conditions A is CO2, B is methanol: Isocratic separation conditions 1: 0~1.4 min (15% B), 1.4~1.6 min (15%~20% B), 1.6~3.5 min (20% B), 3.5~3.6 min (20%~15% B), 3.6~4.0 min (15% B); Gradient separation condition 2: 0~1.2min (15%B), 1.2~1.4min (15%~25%B), 1.4~2.6min (25%B), 2.6~2.8min (25%~30%B), 2.8~3.0min (30%B), 3.0~3.1min (30%~15%B), 3.1~4.0min (15%B); Gradient separation condition 3: 0~1.0 min (15%B), 1.0~1.1 min (15%~25%B), 1.1~2.0 min (25%B), 2.0~2.1 min (25%~30%B), 2.1~2.5 min (30%B), 2.5~2.8 min (30%~15%B), 2.8~3.0 min (15%B).

[0047] 3. Results and Discussion 3.1 Selection of detection wavelength After scanning with a PDA detector, the UV spectra of the two enantiomer standard solutions of tebuconazole were extracted from the chromatogram. For example... Figure 1 As shown, there are obvious absorption peaks at both 210 nm and 250 nm, with the strongest absorption at 210 nm, indicating relatively high sensitivity. However, at this wavelength, there are many interference peaks at the enantiomer peak of tebuconazole. At 250 nm, the absorption is strong, and there are fewer interference peaks at the enantiomer peak of tebuconazole. Considering all factors, for the detection of tebuconazole, using a wavelength of 250 nm with higher absorbance and fewer impurities is more advantageous. Therefore, 250 nm was chosen as the detection wavelength in this experiment.

[0048] 3.2 Selection of chromatographic separation program To obtain the optimal chromatographic separation program, this study investigated the effects of different gradient chromatographic separation programs on the separation of (-)-R-tebuconazole and (+)-S-tebuconazole. The results showed that all two tebuconazole enantiomers could be completely separated within 4.0 min using all three separation programs. However, compared to other separation conditions, gradient separation condition 3 resulted in sharper peak shapes and better resolution for both chromatographic peaks (see [link to study program]). Figure 2 C). Therefore, this experiment selects gradient separation condition 3.

[0049] 3.3 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 10.3–20.7 MPa on the separation of two tebuconazole enantiomers. 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 3 In comparison, when 17.2 MPa is used as the system back pressure, the chromatographic peak shape is more symmetrical. Therefore, 17.2 MPa was selected as the system back pressure in this experiment.

[0050] 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 tebuconazole enantiomers. Under the three column temperature conditions, the chromatographic peak resolution of the two tebuconazole enantiomers was good, and good baseline separation was achieved within 3.5 min, indicating fast analysis speed (see...). Figure 4 In comparison, the peaks of the two tebuconazole enantiomers were sharper when the column temperature was 40℃, therefore 40℃ was chosen as the optimal column temperature.

[0051] 3.5 Investigation of Different Purification Methods This experiment compared the purification effects of two purification methods—Florisil column and NH2 column—on fruit and vegetable puree sample extracts. Two tebuconazole enantiomer standard solutions were added to fruit and vegetable puree samples that did not contain tebuconazole. The samples were extracted twice with acetonitrile by shaking. After the extracts were concentrated and reconstituted, they were treated using the two different purification methods. The results showed that when using the Florisil column for purification, the recoveries of the two tebuconazole enantiomers were 101% and 91.8%, respectively; when using the NH2 column for purification, the recoveries were 52.8% and 48.6%, respectively (see...). Figure 5 Therefore, this experiment ultimately selected the Florisil solid-phase extraction column, which has a higher recovery rate, as the purification column.

[0052] 3.6 Methodological Examination 3.6.1 Linear range and limit of quantitation A series of mixed standard solutions of (-)-R-tebuconazole and (+)-S-tebuconazole were determined. A standard curve was plotted with mass concentration on the x-axis (X) and the peak area of ​​the corresponding standard on the y-axis (Y), and the regression equation and correlation coefficient (R²) were obtained. 2 As shown in Table 1, both enantiomers of tebuconazole exhibited good linearity in the concentration range of 0.5–20.0 mg / L, with R... 2 All values ​​were greater than 0.9992. The limits of quantitation (LOQ, signal-to-noise ratio 10) were obtained by adding the standard to a blank sample of fruit and vegetable puree that did not contain tebuconazole. As shown in Table 1, the LOQ for both (-)-R-tebuconazole and (+)-S-tebuconazole was 0.1 mg / kg.

[0053] Table 1. Linear range, linear equation, correlation coefficient, and limit of quantitation for each compound.

[0054] 3.6.2 Recovery rate and precision Two enantiomer standard solutions of tebuconazole at different concentrations were added to fruit and vegetable puree samples that did not contain tebuconazole. Recovery and precision tests were performed. Relevant chromatograms are shown below. Figure 6 The results are shown in Table 2. The results indicate that the recoveries of the two enantiomers were 80.1%–108%, and the relative standard deviations (RSDs) were 4.2%–8.1%, which meet the recovery requirements of SANTE / 11312 / 2021 and can satisfy the determination of enantiomer content of tebuconazole in fruit and vegetable puree samples.

[0055] Table 2 Spiking recoveries and relative standard deviations of enantiomers of tebuconazole in fruit and vegetable puree samples ( n =6)

[0056] 3.7 Application of the Method 3.7.1 Resolution of the racemic body The method established in this paper was used to separate and determine the purchased racemic tebuconazole standard. Figure 7 As shown in figure a, the two enantiomers of tebuconazole showed good separation, achieving effective separation within 3.5 min, with resolutions of [missing values]. R =5.1, 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: (-)-R-tebuconazole, (+)-S-tebuconazole ( Figure 7 (b, 7c). Based on the standard curves plotted above, the contents of the two enantiomers of 20.0 mg / L of the racemic intermediate solution of tebuconazole in 2.2.1 were calculated using the external standard quantification method. The contents of (-)-R-tebuconazole and (+)-S-tebuconazole were 9.53 mg / L and 9.79 mg / L, respectively.

[0057] 3.7.2 Testing of actual samples To examine the practicality and effectiveness of the method, under optimal conditions, the established method was applied to detect enantiomers of tebuconazole in 20 randomly selected commercially available fruit and vegetable puree samples. The results showed that tebuconazole enantiomers were not detected in 19 of the fruit and vegetable puree samples; however, 1.06 mg / kg (-)-R-tebuconazole and 1.10 mg / kg (+)-S-tebuconazole were detected in one sample (see...). Figure 8 ).

[0058] 4. Conclusion Establish a UPC-based 2 A novel detection method was developed to separate two enantiomers of tebuconazole in fruit and vegetable puree samples. The optimal conditions for this method were determined: sample extraction with methanol, purification using a Florisil column, and separation using an AcquityTrefoil AMY1 chiral column with gradient elution of supercritical CO2 (mobile phase A) and methanol (mobile phase B), a system back pressure of 17.2 MPa, and a column temperature of 40 °C. These optimal conditions were then applied to subsequent experiments. Spiking recovery experiments were conducted in the range of 0.1–1.0 mg / kg, and the recoveries of the two tebuconazole enantiomers ranged from 80.1% to 108%, with RSDs of 4.2%–8.1%. The established method was used to analyze and determine actual fruit and vegetable puree samples and commercially available standards. The results show that this method is characterized by rapid analysis, high accuracy, and good separation effect, and can meet the needs of purity analysis and rapid quantification of tebuconazole in fruit and vegetable puree.

[0059] 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 rapid determination of enantiomeric residues of tebuconazole in fruit and vegetable purees using solid-phase extraction-ultra-high performance phase chromatography, characterized in that, Includes the following steps: S1. Sample extraction: Weigh 5g of homogenized fruit and vegetable puree sample into a centrifuge tube, add 15-25mL of acetonitrile, vortex to mix, and then shake to extract for 15-25 minutes. Centrifuge at 3500-4500 rpm for 3-7 minutes to separate the supernatant. Repeat the above acetonitrile extraction steps for the residue at least once. Combine the supernatants and concentrate them to near dryness by rotary evaporation at 35-45℃. Add 5mL of a mixed solvent of acetone and n-hexane in a volume ratio of 1:20 to redissolve the supernatant to obtain the solution to be purified. S2, Solid-phase extraction purification: The solution to be purified was transferred to a Florisil solid-phase extraction column. Before use, the solid-phase extraction column was activated sequentially with 5 mL of acetone, 5 mL of n-hexane, and 5 mL of acetone / n-hexane = 1:20 (volume ratio). When the liquid level of the solution to be purified was close to the surface of the packing material, the interfering matrix was removed by rinsing with 8-12 mL of acetone / n-hexane = 1:20 (volume ratio). Then, the target analyte was eluted with 4-6 mL of acetone / n-hexane = 1:1 (volume ratio). All eluent was collected and evaporated to near dryness at 35-45°C by rotary evaporation. The solution was then diluted to volume with 0.8-1.2 mL of acetonitrile to obtain the test solution. S3. Chromatographic separation and detection: The test solution was injected into an ultra-high performance coherent chromatography system (UHPLC) and separated on a chiral column packed with amylose tris(3,5-dimethylphenylcarbamate). The chiral column was 150 mm long, 3.0 mm inner diameter, and 2.5 μm particle size. Supercritical carbon dioxide was used as mobile phase A, methanol as mobile phase B, the flow rate was 1.0 mL / min, the column temperature was 40 °C, and the system back pressure was 17.2 MPa. A gradient elution program with methanol volume fractions ranging from 15% to 30% was used for separation, and detection was performed at a wavelength of 250 nm using a diode array detector. Baseline separation of (-)-R-tebuconazole and (+)-S-tebuconazole was achieved within 4.0 minutes. The methanol gradient elution program was as follows: 0–1.0 minutes, 15% B; 1.0–1.1 minutes, 15%–25% B; 1.1–2.0 minutes, 25% B; 2.0–2.1 minutes, 25%–30% B; 2.1–2.5 minutes, 30% B; 2.5–2.8 minutes, 30%–15% B; 2.8–3.0 minutes, 15% B; S4. Quantitative Calculation: A mixed standard working solution of two enantiomers of tebuconazole was used to establish an external standard calibration curve of mass concentration versus peak area in the concentration range of 0.5–20.0 mg / L. The residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in fruit and vegetable puree samples were calculated from the calibration curve using the peak areas of the corresponding chromatographic peaks in the samples. The method limit of quantitation was not higher than 0.1 mg / kg, and the spiked recoveries of the two enantiomers were 80.1%–108% and the relative standard deviations were 4.2%–8.1% in the spiked concentration range of 0.1–1.0 mg / kg.

2. The method according to claim 1, characterized in that, In step S1, the sample mass was 5.00±0.05g, the volume of acetonitrile added each time was 20±2mL, the shaking extraction time was 20±5 minutes, and the centrifugation conditions were 4000±500 rpm for 5±2 minutes.

3. The method according to claim 1, characterized in that, In step S2, the Florisil solid-phase extraction column has a packing volume of 5g, a column volume of 6mL, an acetone / n-hexane (1:20, volume ratio) elution volume of 10mL, an acetone / n-hexane (1:1, volume ratio) elution volume of 5mL, and all effluent collected during the entire purification process is combined and then concentrated and diluted to a fixed volume.

4. The method according to claim 1, characterized in that, Spiking recovery experiments were conducted by adding two enantiomer standard solutions of tebuconazole to blank samples of fruit and vegetable puree at three concentration levels of 0.1 mg / kg, 0.2 mg / kg, and 1.0 mg / kg. The intra-day and inter-day spiked recoveries were in the range of 80.1% to 108%, and the relative standard deviations were in the range of 4.2% to 8.1%, respectively.

5. The method according to claim 1, characterized in that, This method is also used to separate and determine the enantiomeric content of racemic tebuconazole standards. The intermediate solution of racemic tebuconazole is injected under the chromatographic conditions of step S3. Within 3.5 minutes, two symmetrical peaks with a resolution of not less than 5.0 are obtained, corresponding to (-)-R-tebuconazole and (+)-S-tebuconazole, respectively. The content ratio of the two enantiomeric compounds in the racemic mixture is then calculated.

6. The method according to claim 1, characterized in that, Accurately weigh 0.01 g, to the nearest 0.1 mg, of (-)-R-tebuconazole and (+)-S-tebuconazole standards, dissolve them in methanol and dilute to 10 mL to prepare 1.0 g / L enantiomeric standard stock solutions; for mixed standard working solutions of the two tebuconazole enantiomers: accurately pipette a certain amount of (-)-R-tebuconazole and (+)-S-tebuconazole enantiomer standard stock solutions, and dilute them stepwise with methanol to prepare mixed standard working solutions of 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L.

7. A rapid determination system for enantiomeric residues of tebuconazole for implementing the method of any one of claims 1 to 6, characterized in that, include: The sample pretreatment unit is used to weigh the fruit and vegetable puree sample, add acetonitrile, and then perform shaking extraction, centrifugation, vacuum concentration, and redissolution in a mixed solvent of acetone / n-hexane to obtain the solution to be purified. The solid phase extraction purification unit is equipped with a Florisil solid phase extraction column and supply channels for acetone, n-hexane and two volume ratios of acetone / n-hexane mixed solvents, respectively, for pre-column activation, rinsing and elution of the solution to be purified, so as to obtain the purified test solution. The ultra-high efficiency phase chromatography separation unit is equipped with a chiral chromatographic column with amylose tris(3,5-dimethylphenylcarbamate) as the stationary phase, a high-pressure delivery mechanism with supercritical carbon dioxide and methanol as the mobile phase, a back pressure regulator, and a column temperature control component, which are used to separate enantiomers in the purified test solution. The detection unit, connected to the outlet of the ultra-high efficiency phase chromatography separation unit, is a diode array detector with a detection wavelength of 250 nm, used to acquire chromatographic signals; The data processing and control unit is used to control the working parameters of the sample pretreatment unit, solid phase extraction purification unit and ultra-high efficiency phase chromatography separation unit, collect and process the chromatographic data output by the detection unit, and calculate the residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in the fruit and vegetable puree sample according to the external standard calibration curve.

8. The system according to claim 7, characterized in that, The ultra-high performance phase chromatography separation unit is a UPC. 2 The system includes a chiral chromatographic column of type AcquityTrefoil AMY1 with dimensions of 150 mm × 3.0 mm and a particle size of 2.5 micrometers. The system back pressure is set to 17.2 MPa by the back pressure regulator, the total flow rate of the mobile phase is 1.0 mL / min, the column temperature is 40 °C, and the data processing and control unit has a pre-stored methanol gradient elution program.

9. The system according to claim 7 or 8, characterized in that, The data processing and control unit includes: The storage module is used to store methodological parameters such as external standard calibration curve parameters, method limit of quantitation, and spiked recovery rate of the two tebuconazole enantiomers in the concentration range of 0.5–20.0 mg / L. The processor module is used to automatically calculate the residual amounts of the two enantiomers of tebuconazole in the sample by calling the calibration curve parameters based on the real-time collected sample chromatographic peak area, and generate a test report containing the enantiomer content and total amount. The human-computer interaction module is used to display chromatograms, method parameters and calculation results, and allows users to select the type of fruit and vegetable puree sample, the spike concentration level and the batch information.

10. A computer-readable storage medium having a computer program stored thereon, the computer program, when executed by a processor, causing the processor to perform the following steps: The system of any one of claims 7 to 9 calls the data processing and control unit to automatically set and control the mobile phase ratio, gradient elution program, flow rate, back pressure and column temperature of the ultra-high efficiency phase chromatography separation unit; The system receives the chromatographic signal output from the diode array detector and identifies, integrates, and calculates the peak areas of the two enantiomers of tebuconazole. Based on the preset external standard calibration curve parameters, the peak area is converted into the residual amounts of (-)-R-tebuconazole and (+)-S-tebuconazole in the fruit and vegetable puree sample to be tested, thereby completing the method for determining the residual amounts of tebuconazole enantiomers in fruit and vegetable puree by solid phase extraction-ultra-high performance phase chromatography as described in any one of claims 1 to 6.

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