Method and system for rapidly determining ethiprole enantiomer residual quantity in tomatoes through solid phase extraction-ultra performance convergence chromatography

Abstract

The invention relates to the technical field of vegetable and fruit pesticide residue detection, in particular to a method and a system for rapidly determining ethiprole enantiomer residue in tomatoes through solid phase extraction-ultra performance convergence chromatography. The method comprises the following steps: extracting a tomato sample with acetonitrile, concentrating, redissolving with dichloromethane / methanol, purifying by adopting an amino solid-phase extraction column, concentrating an eluent, fixing the volume with isopropanol / n-heptane, filtering, and loading on a machine; under the bonded phase chromatographic condition that supercritical carbon dioxide serves as a main mobile phase and methanol serves as a cosolvent, an Acquity Trefoil CEL2 chiral chromatographic column is used for achieving rapid baseline separation of two enantiomers, a PDA detector is used for detection at 250 nm, and an external standard method is used for quantification. The method is short in analysis time, low in organic solvent consumption, high in separation degree, good in linearity and low in quantification limit, the adding standard recovery rate and precision meet the pesticide residue detection requirements, and the method is suitable for rapid monitoring and quality safety supervision of ethiprole enantiomer residues in tomatoes.

Description

Technical Field

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

[0002] acetonitrile ( Ethiprole Acetaminophen belongs to the phenylpyrazole class of insecticides / acaricides. Its molecular structure is similar to that of fipronil, exhibiting high activity and a long residual effect. It has been used in the control of pests and diseases in various crops, including vegetables. Due to the different substituents attached to the sulfoxide atom in the acetonitrile molecule, it forms a pair of mirror-image, non-overlapping enantiomers. Enantiomers are often similar in physicochemical properties, but after entering organisms or environmental media, they may exhibit differences in biological activity, absorption and migration, metabolic transformation, accumulation behavior, and toxicological and ecological effects. Therefore, simply regulating and assessing the racemic mixture or total amount may mask the "selective residue / selective degradation" phenomenon of a particular enantiomer. In pesticide residue limit evaluation, processing factor studies, dietary exposure and risk assessment, the separation and determination of enantiomers is practically necessary.

[0003] In existing detection technology systems, the determination of pesticide residues in complex matrices such as fruits and vegetables generally adopts a process of "extraction-purification-chromatographic detection-quantitative evaluation". For enantiomeric analysis of chiral pesticides, traditional methods often rely on chiral liquid chromatography (HPLC / UPLC) or chiral liquid chromatography-mass spectrometry (LC-MS / MS), using a chiral stationary phase to achieve enantiomeric separation, followed by quantification using external or internal standards. However, these methods still have some common problems in practical engineering applications: First, chiral separation often requires a long analysis time or a high proportion of organic solvents, resulting in limited throughput and high costs; second, co-extractants such as pigments, lipids, and organic acids in fruit and vegetable matrices are prone to column contamination or matrix effects, leading to peak shape deterioration, decreased resolution, and even false positives / false negatives; third, for specific compounds and specific matrices, it is often necessary to re-screen the chiral column type, mobile phase system, and purification pathway, resulting in high method development and migration costs and making it difficult to quickly form standardized detection capabilities.

[0004] In recent years, ultra-high performance phase chromatography (UPC) has become increasingly important. 2 UPC (Ultra-Compound Chromatography), also known as a chromatographic system using compressed / supercritical carbon dioxide as the main mobile phase, has gained attention for separating enantiomers and isomers due to its advantages such as high diffusion coefficient, low viscosity, high mass transfer efficiency, fast separation speed, and low organic solvent consumption. It is particularly suitable for compounds with similar structures, strong hydrophobicity, or requiring normal-phase selectivity. However, in the context of pesticide residue detection, UPC... 2To obtain stable and reproducible enantiomeric quantitative results, the technology typically relies on "suitable pretreatment and purification strategies, appropriate solvent matching for instrumentation, and suitable chiral stationary phase and modifier system." If the purification method is not chosen properly, there may be a loss of selectivity for a certain enantiomeric component, or interference from co-extractants may lead to baseline instability, thereby affecting the accurate determination of enantiomeric proportions and residual amounts.

[0005] Chinese patent document CN115494190A discloses a method for simultaneously determining the enantiomeric residues of triazoles and their metabolites in fruit and vegetable purees: the sample is extracted with acetonitrile, purified using an NH2 solid-phase extraction column, separated using an Acquity Trefoil CEL2 chiral chromatographic column, and eluted using a supercritical CO2-methanol modifier system with gradient elution and external standard method for quantification, achieving a low limit of quantitation and good recovery rate. This document indicates that the overall technical route of "acetonitrile extraction + NH2 purification + CEL2 chiral column + gradient elution by co-phase chromatography" can be used for the determination of chiral pesticide enantiomeric residues in fruit and vegetable matrices. However, the target substances in CN115494190A are triazoles and their metabolites, and the retention and selectivity sources, UV response characteristics, and interaction patterns with matrix co-extractants are different from those of acetonitrile; moreover, its matrix is ​​fruit and vegetable puree (infant complementary food), and the composition and interference spectrum differ from those of tomatoes. Directly transferring this route to the "tomato-acetonitrile enantiomer" scenario still faces the challenge of re-optimizing issues such as chiral column selection, modifier selection, and matching of purification path with onboard solvent. In particular, it is necessary to avoid deviations caused by insufficient selective adsorption or elution of a particular enantiomer by the purification column.

[0006] Chinese patent document CN106841454B discloses a combined phase chromatography-tandem mass spectrometry (CMS-MS) method for the enantiomeric chiral component (nicotine) in a food matrix (boletus). The method involves sample pretreatment, purification, and chiral separation and detection on a combined phase chromatography platform, emphasizing the use of a combined phase chromatography system to achieve high sensitivity, strong specificity, and improved batch analysis capabilities. While this document demonstrates the feasibility of "complex edible matrices, chiral analysis, and combined phase chromatography" from a methodological framework perspective, its target analyte, extraction solvent system, purification mechanism, and detector configuration (MS / MS) differ from the PDA-based quantification of acetonitrile enantiomeric compounds in this invention. Furthermore, boletus and tomatoes differ significantly in water content, pigment composition, and organic acid profiles, making it difficult to simply equate matrix interference and purification strategies. Therefore, this method is insufficient to solve the specific engineering problems of rapid, stable, and low-consumption separation and quantification of acetonitrile enantiomeric compounds in tomato samples.

[0007] Furthermore, Chinese patent document CN108802242B discloses a method for simultaneously determining the enantiomeric residues of multiple chiral pesticides in an aquatic environment. This method employs enrichment and purification steps such as solid-phase extraction, and achieves enantiomeric separation that meets quantitative detection requirements under chiral separation conditions. This document reflects the need for "quantitative determination of chiral pesticide enantiomeric levels" in the regulatory and environmental monitoring fields, and also demonstrates that "enantiomeric quantification through pretreatment enrichment and purification, and chiral separation" is a well-known technical path. However, this method is designed for water samples, and compared to plant samples such as tomatoes, which are high in moisture, pigment, and have strong matrix effects, the pretreatment difficulties, sources of interference, and methodological control points are significantly different. Additionally, its separation platform and column system are not limited to UPC. 2 The specific combination of CEL2 chiral columns also lacks targeted technical inspiration for the goal of "rapid separation + low organic solvent consumption + high throughput" of acetaminophen enantiomers in tomato samples.

[0008] In summary, while existing technologies have made valuable explorations in (1) quantification of enantiomers in fruit and vegetable matrices using co-phase chromatography, (2) chiral analysis of complex edible matrices using co-phase chromatography, and (3) enrichment, purification, and chiral separation detection of enantiomer residues of chiral pesticides, a rapid quantitative method is still lacking for tomatoes, a typical vegetable matrix with high moisture content and strong pigment interference, and for acetaminophen, a target compound with a sulfoxide chiral center and potentially significantly different enantiomer behaviors. This method can achieve stable baseline separation of the two enantiomers within a short analysis time while also considering recovery rate, precision, and low organic solvent consumption. In particular, how to avoid the loss of enantiomer selectivity caused by different purification packing materials and achieve compatibility with the co-phase chromatography injection solvent system to ensure accurate and reliable enantiomer quantification results remains a key issue that needs further resolution in existing technologies. Based on this, it is necessary to propose a method and system for the rapid determination of acetaminophen enantiomer residues in tomatoes using solid-phase extraction-ultra-high performance co-phase chromatography to meet the actual needs of quality and safety supervision and testing institutions for high throughput, green practices, and standardization. Summary of the Invention

[0009] The technical objective of this invention is to provide a method and system for the rapid determination of acetaminophen enantiomer residues in tomatoes using solid-phase extraction-ultra-high performance co-phase chromatography. By constructing an acetonitrile extraction and NH2 solid-phase extraction purification process adapted to the tomato matrix, and combining it with co-phase chromatography chiral separation conditions using supercritical carbon dioxide as the main mobile phase, rapid baseline separation and accurate quantification of the two enantiomers of acetaminophen are achieved, thus meeting the high-throughput, low-solvent-consumption, and quality and safety regulatory detection requirements for acetaminophen enantiomer residues in tomato samples.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A method for rapid determination of enantiocyanate residues in tomatoes using solid-phase extraction-ultra-high performance combined phase chromatography, comprising the following steps: S1. Sample extraction: Take 3-10 g of homogenized tomato sample and place it in a centrifuge container. Add 15-30 mL of acetonitrile, vortex to mix, and then shake to extract for 10-30 min. Centrifuge and collect the supernatant. Repeat the extraction of the residue with acetonitrile at least once and combine the extracts. S2. Concentration and redissolution: The combined extracts are concentrated to near dryness by rotary evaporation, and redissolved by adding a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 90:10 to 99:1. S3. Solid-phase extraction purification: The complex solution obtained in S2 is purified using an amino (NH2) solid-phase extraction column. The amino solid-phase extraction column is activated with a dichloromethane / methanol mixed solution in the same ratio as S2 before loading the sample. After loading the sample, the solution is eluted with a dichloromethane / methanol mixed solution and the eluent is collected. S4. Volume adjustment and solvent matching: Concentrate the eluent to near dryness at a temperature not exceeding 45°C, adjust the volume with a mixed solution of isopropanol and n-heptane and filter, wherein the volume ratio of isopropanol to n-heptane is 1:9 to 3:7. S5. Enantiomer Separation and Detection: The solution obtained in S4 was injected into an ultra-high performance co-phase chromatography system. Under co-phase chromatography conditions with supercritical carbon dioxide as the main mobile phase, baseline separation of the two enantiomers of acetonitrile was performed on a cellulose chiral stationary phase column, and detection was performed in the wavelength range of 245–255 nm using a diode array detector. The chiral stationary phase column is an Acquity Trefoil CEL2 column; the back pressure of the combined phase chromatography system is 10-13 MPa, the column temperature is 31-40℃, the mobile phase includes phase A as carbon dioxide and phase B as methanol, the flow rate is 0.8-1.2 mL / min, and gradient elution is used to complete a separation and determination within 6 min; The gradient elution program was 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; 2.2–3 min, 25% B; 3.0–4.0 min, 25% B–15% B; 4.0–6.0 min, 15% B. S6. Quantification: The two enantiomers of acetonitrile were quantified using the external standard method to obtain the residual amounts of the two enantiomers of acetonitrile in the tomato samples.

[0011] Preferably, the centrifugation conditions in step S1 are 3000–5000 r / min for 3–10 min, and the total volume of acetonitrile extracted twice is 30–60 mL.

[0012] Preferably, the volume ratio of the dichloromethane / methanol mixed solution in step S2 is 95:5.

[0013] Preferably, the amino solid-phase extraction column in step S3 has a packing amount of 200–1000 mg, a column volume of 1–6 mL, and an elution volume of 3–10 mL.

[0014] Preferably, the volume ratio of the isopropanol / n-heptane mixed solution in step S4 is 2:8, the final volume is 0.5–2.0 mL, and the pore size of the filter membrane is 0.20–0.45 μm.

[0015] Preferably, the detection wavelength in step S5 is 250 nm.

[0016] Preferably, the external standard method uses a mixed standard working solution of two acetonitrile enantiomers to establish a standard curve. The mixed standard working solution has at least four concentration points selected from 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L, and the limit of quantitation is determined with a signal-to-noise ratio of 10.

[0017] As a further improvement, this application also provides a system for the rapid determination of enantiocyanate residues in tomatoes using solid-phase extraction-ultra-high performance phase chromatography (UPCLC) for implementing the method, comprising: A. Extraction module, used to extract tomato samples with acetonitrile and obtain a combined extract; B. Concentration and redissolution module, used to concentrate the combined extract to near dryness and redissolve it with a dichloromethane / methanol mixed solution; C. Solid phase extraction and purification module, including amino solid phase extraction column and its activation / elution flow path, used to purify the complex solution and output the eluent; D. Solvent matching and filtration module, used to concentrate the eluent, make up to volume with isopropanol / n-heptane mixed solution and filter to obtain the test solution; E. Ultra-high efficiency phase chromatography detection module, including supercritical carbon dioxide supply unit, back pressure control unit, column temperature control unit, injection unit, Acquity Trefoil CEL2 chiral column and diode array detector, used for the separation and detection of two enantiomers of acetonitrile; F. Data processing module, used to call the enantiomer standard curve and perform external standard quantitative calculation and result output of the residual amounts of the two enantiomers in tomato samples.

[0018] Preferably, the ultra-high performance phase chromatography detection module is configured with a back pressure of 10–13 MPa, a column temperature of 31–40 °C, mobile phase A being carbon dioxide, mobile phase B being methanol, a flow rate of 0.8–1.2 mL / min, and completes a separation and determination within 6 min.

[0019] Preferably, the data processing module is further configured to perform quality control judgment on the purification method: when only a single enantiomer peak appears in the chromatogram or the resolution of two peaks is less than 1.5, an alarm message of "purification or abnormal chromatographic conditions" is output, and the corresponding solid phase extraction column batch number, elution volume and gradient program parameters are recorded for traceability.

[0020] This invention, employing the aforementioned technical solution, achieves stable baseline separation and rapid quantification of two enantiomers of acetamiprid in the complex matrix of tomato, characterized by high water content and strong pigment interference. On one hand, NH2 solid-phase extraction effectively removes organic acids, pigments, and polar co-extractants from the tomato extract, significantly reducing matrix interference and background noise, avoiding enantiomer peak tailing, baseline drift, and quantitative deviation, thus improving the accuracy and reproducibility of the method. On the other hand, the use of an ultra-high efficiency combined phase chromatography system with supercritical carbon dioxide as the main mobile phase, coupled with a CEL2 chiral stationary phase, allows for the rapid separation and quantification of two enantiomers of acetamiprid within a short analysis time. This invention achieves clear enantiomeric separation and good peak shape, thereby significantly improving detection throughput, reducing organic solvent consumption and operating costs, and minimizing the risk of contamination to instruments and chromatographic columns. In terms of methodological performance, this invention maintains excellent linearity (high correlation coefficient) over a wide concentration range, the limit of quantitation meets the requirements for pesticide residue supervision, and the spiked recovery rate and precision are within the acceptable range. It can reliably detect and quantify enantiomeric residues of acetaminophen in actual commercially available tomato samples, providing a widely applicable technical means for quality control during the use of acetaminophen, evaluation of enantiomeric selective residues, and monitoring of the quality and safety of vegetables and fruits. Attached Figure Description

[0021] Figure 1 The effect of different chromatographic columns on the separation of two acetonitrile enantiomers.

[0022] Figure 2 The effect of different co-solvents on the separation of two acetonitrile enantiomers. (A) Methanol; (B) 0.5% (v / v) ammonia in methanol solution; (C) 0.5% (v / v) formic acid in methanol solution.

[0023] Figure 3 The effect of different column temperatures on the separation of two acetonitrile enantiomers.

[0024] Figure 4Effects of different purification methods on the purification efficiency of acetonitrile enantiomeric protons. (A) Florisil column; (B) NH2 column. Chromatographic peak 1: (+)-acetonitrile; Chromatographic peak 2: (-)-acetonitrile.

[0025] Figure 5 Resolution of the racemic mixture of acetonitrile. Chromatographic peak 1: (+)-acetonitrile; Chromatographic peak 2: (-)-acetonitrile.

[0026] Figure 6 Chromatogram of a positive tomato sample. Peak 1: (+)-acetaminophen; Peak 2: (-)-acetaminophen. Detailed Implementation

[0027] 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.

[0028] 1. Explanation of Terms and Abbreviations Enantiomers of acetonitrile: refers to the two enantiomers, (+)-acetonitrile and (-)-acetonitrile.

[0029] UPC 2 Ultra-high performance phase chromatography (a chromatographic system with compressed / supercritical carbon dioxide as the main mobile phase).

[0030] SPE: Solid phase extraction (preferably using an amino NH2 column for purification).

[0031] PDA: Diode Array Detector.

[0032] LOQ: Limit of Quantitation, evaluated with a signal-to-noise ratio (S / N) of 10.

[0033] RSD: Relative Standard Deviation.

[0034] Resolution Rs: Chromatographic resolution calculated based on the retention time and peak width of two adjacent peaks (the commonly used criterion is Rs≥1.5 for complete separation).

[0035] 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); MS2 vortex mixer (Shanghai Medical University Instrument Factory); R215 rotary evaporator (Buchi, Switzerland); ELGA CLXXXUVM2 ultrapure water purification system (Elga, UK); N-EVAP TM 111 Nitrogen Evaporator (Tokyo Rika Co., Ltd., Japan).

[0036] Acetonitrile, methanol, isopropanol, n-heptane (chromatographic grade, Scharlau, Spain); ultrapure water; high-purity carbon dioxide (99.999%); Florisil column (CNW, 5 g, 6 mL); amino (NH2) column (Agilt, 500 mg, 3 mL); Daicel CHIRALPAK AD-3 (150 mm × 3.0 mm, 3 µm, packed with amylose-tris(3,5-dimethylphenylcarbamate)); Waters Acquity Trefoil AMY1 (150 mm × 3.0 mm, 2.5 µm, packed with amylose-tris(3,5-dimethylphenylcarbamate)); Waters Acquity Trefoil CEL1 (150 mm × 3.0 mm, 2.5 µm, packed with cellulose-tris(3,5-dimethylphenylcarbamate)) (Waters, USA); Waters Acquity Trefoil CEL2 (150 mm × 3.0 mm, 2.5 µm, packed with cellulose-tris(3,5-dimethylphenylcarbamate)) (Waters, USA); ×3.0 mm, 2.5 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.

[0037] Racemic standard (acetaminophen: CAS No.: 181587-01-9, purity ≥98.0%, Shanghai Yuanye Biotechnology Co., Ltd.). Enantiomer standards of acetaminophen: (+)-acetaminophen and (-)-acetaminophen were isolated and purified from the racemic standard of acetaminophen (Shanghai Yuanye Biotechnology Co., Ltd.) by Shanghai Qinlu Biotechnology Co., Ltd., with purities greater than 98.0%.

[0038] 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 acetonitrile racemic standard, dissolve it in methanol and dilute to 10 mL to prepare a 1.0 g / L racemic standard stock solution.

[0039] Standard intermediate solution of racemic acetonitrile: 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.

[0040] 2.2.2 Enantiomer Standard Stock Solution Accurately weigh 0.01 g (accurate to 0.1 mg) of (+)-acetaminophen and (-)-acetaminophen standards, dissolve them in methanol and dilute to 10 mL to prepare an enantiomeric standard stock solution of 1.0 g / L.

[0041] Mixed standard working solutions of two acetaminophen enantiomers: Accurately pipette a certain amount of (+)-acetaminophen and (-)-acetaminophen 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 mixed standard working solutions.

[0042] 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, 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 dichloromethane:methanol (95:5, v / v) solution, and wait for purification.

[0043] 2.3.2 Purification Transfer the reconstituted solution to an amino solid-phase extraction column activated with 5 mL of dichloromethane:methanol (95:5, v / v). When the liquid surface is almost dry, transfer it to a 15 mL centrifuge tube for collection. Add the purified solution mentioned above and elute with 5 mL of dichloromethane:methanol (95:5, v / v) solution. 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 solution (2:8, v / v), and filter through a membrane for extraction.

[0044] 2.4 Chromatographic conditions Column: Acquity Trefoil CEL2 (150 mm × 3.0 mm, 2.5 µm); Detection wavelength: 250 nm; System back pressure: 12.1 MPa; Column temperature: 40℃; 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), 2.2–3 min (25% B), 3.0–4.0 min (25% B–15% B), 4.0–6.0 min (15% B); Flow rate: 1.0 mL / min; Injection volume: 5.0 μL.

[0045] 3. Results and Discussion 3.1 Optimization of chromatographic columns This application 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 the separation efficiency of two acetaminophen enantiomers. The results showed that when using the AMY1 chiral column, only one peak appeared on the chromatogram, and the two acetaminophen enantiomers completely overlapped. When using the AD-3 and CEL1 chiral columns, the two acetaminophen enantiomers failed to achieve baseline separation. When using the CEL2 chiral column, the two acetaminophen enantiomers were completely separated within 3.5 min (see [link to CEL2 column]). Figure 1 Therefore, this application selected a CEL2 chiral column to separate the two acetonitrile enantiomers.

[0046] 3.2 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 application investigated the effects of different co-solvents, including methanol, 0.5% (v / v) ammonia-methanol solution, and 0.5% (v / v) formic acid-methanol solution, on the separation of two acetonitrile enantiomers. The results showed that when 0.5% (v / v) formic acid-methanol solution was used as the co-solvent, the chromatographic baseline was uneven and the peaks were very small. When methanol or 0.5% (v / v) ammonia-methanol solution was used as the co-solvent, the separation effect and peak shape of the two acetonitrile enantiomers were almost the same (see [link to relevant documentation]). Figure 2 However, compared to a 0.5% (v / v) ammonia-methanol solution, using methanol as a co-solvent results in a slightly earlier peak elution time for the target analyte (0.5 min), is more convenient to operate, and causes less damage to the instrument and column. Therefore, this application laboratory has chosen methanol as the co-solvent.

[0047] 3.3 Column Temperature Optimization Considering that the maximum recommended operating temperature of the Acquity Trefoil CEL2 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 application investigated the effect of column temperature in the range of 31~40℃ on the separation of two acetonitrile enantiomers. Under the three column temperature conditions, the chromatographic peak resolution of the two acetonitrile enantiomers was good, and good baseline separation was achieved within 3.5 min, indicating fast analysis speed. Figure 3 In comparison, the peaks of the two acetonitrile enantiomers were sharper when the column temperature was 40℃, therefore 40℃ was chosen as the optimal column temperature.

[0048] 3.4 Investigation of different purification methods This experiment compared the purification effects of two purification methods—Florisil column and NH2 column—on tomato sample extracts. Two acetaminophen enantiomer standard solutions were added to tomato samples free of acetaminophen, and the samples were extracted twice with acetonitrile. After the extracts were concentrated and reconstituted, they were treated with the two different purification methods. The results showed that when using the Florisil column, only one peak (+)-acetaminophen was observed in the chromatogram, and the recovery rate was only 42.3%. When using the NH2 column, both acetaminophen enantiomers showed peaks, with recoveries of 98.2% and 85.6%, respectively (see [link to NH2 column purification method]). Figure 4 Therefore, the experiment ultimately selected the NH2 solid-phase extraction column as the purification column.

[0049] 3.5 Methodological Examination 3.5.1 Linear range and limit of quantitation A series of mixed standard solutions of acetamiprid enantiomers were determined under the chromatographic conditions described above. The peak area of ​​the standard was used as the chromatographic value. Y The vertical axis represents the mass concentration ( ). X A standard curve was plotted with α as the abscissa, and the regression equation and correlation coefficient were obtained. The results showed that the two enantiomers exhibited a good linear relationship within the concentration range of 0.5–20.0 mg / L, with a correlation coefficient greater than 0.9993. The standard was added to a blank tomato sample without acetonitrile, and the determination was performed according to this method, with the signal-to-noise ratio (SNR) as the metric. S / N The limit of quantitation (LOQ) was calculated using 10, and the LOQ for both (+)-acetaminophen and (-)-acetaminophen was 0.1 mg / kg (see Table 1).

[0050] Table 1. Linear range, linear equation, correlation coefficient, and limit of quantitation for each compound.

[0051] 3.5.2 Recovery rate and precision Two acetaminophen enantiomer standard solutions at different concentrations were added to tomato samples that did not contain acetaminophen, and spiked recovery and precision tests were conducted. The results are shown in Table 2. The results showed that the spiked recoveries of the two acetaminophen enantiomers were 81.2%–107%, and the relative standard deviations (RSDs) were 3.2%–7.8%, which met the recovery requirements of SANTE / 11312 / 2021 and can satisfy the determination of acetaminophen enantiomer content in tomato samples.

[0052] Table 2. Spike recoveries and relative standard deviations of acetaminophen enantiomeric enzymes in tomato samples (n=6)

[0053] 3.6 Application of the Method 3.6.1 Resolution of the racemic body The method established in this application was used to separate and determine the purchased racemic acetamiprid standard. Figure 5 As shown in figure a, the two enantiomeric acetonitrile species showed good separation, achieving effective separation within 2.5 min, with resolutions of [missing values]. R =1.6, 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: (+)-acetaminophen, (-)-acetaminophen (…). Figure 5 (b, 5c). Based on the standard curves plotted above, the contents of the two enantiomers of acetaminophen in the intermediate standard solution of 20.0 mg / L of the racemic acetaminophen in Section 2.2.1 were calculated using the external standard quantification method. The contents of (+)-acetaminophen and (-)-acetaminophen were 9.61 mg / L and 9.87 mg / L, respectively.

[0054] 3.6.2 Testing of actual samples To evaluate the effectiveness and practicality of this method, the established method was used to determine the contents of (+)-acetaminophen and (-)-acetaminophen in 30 commercially available tomato samples. The results showed that acetaminophen enantiomers were not detected in 29 tomato samples; however, 0.208 mg / kg (+)-acetaminophen and 0.204 mg / kg (-)-acetaminophen were detected in one tomato sample (see [link to sample description]). Figure 6 ).

[0055] 4. Conclusion This application establishes a UPC-based... 2A new detection method was developed to separate two enantiomers of acetaminophen from tomato samples. The optimal conditions for this method were determined: acetonitrile extraction, purification using an amino column, and separation using an Acquity Trefoil CEL2 chiral column with gradient elution of supercritical CO2 (mobile phase A) and methanol (mobile phase B), a system back pressure of 12.1 MPa, and a column temperature of 40℃. These optimal conditions were then used in subsequent experiments. Spiking recovery experiments were conducted in the range of 0.1–1.0 mg / kg, and the recoveries of the two acetaminophen enantiomers ranged from 81.2% to 107%, with RSDs of 3.2%–7.8%. The established method was used to analyze and determine the acetaminophen enantiomers in actual tomato samples and commercially available standards. The results show that this method is characterized by rapid analysis, high accuracy, and good separation, and can meet the needs of purity analysis and rapid quantification of acetaminophen in tomatoes.

[0056] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined in 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 acetaminophen enantiomeric residues in tomatoes using solid-phase extraction-ultra-high performance phase chromatography, characterized in that, The method includes the following steps: S1. Sample extraction: Take 3-10 g of homogenized tomato sample and place it in a centrifuge container. Add 15-30 mL of acetonitrile, vortex to mix, and then shake to extract for 10-30 min. Centrifuge and collect the supernatant. Repeat the extraction of the residue with acetonitrile at least once and combine the extracts. S2. Concentration and redissolution: The combined extracts are concentrated to near dryness by rotary evaporation, and redissolved by adding a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 90:10 to 99:

1. S3. Solid-phase extraction purification: The complex solution obtained in S2 is purified using an amino (NH2) solid-phase extraction column. The amino solid-phase extraction column is activated with a dichloromethane / methanol mixed solution in the same ratio as S2 before loading the sample. After loading the sample, the solution is eluted with a dichloromethane / methanol mixed solution and the eluent is collected. S4. Volume adjustment and solvent matching: Concentrate the eluent to near dryness at a temperature not exceeding 45°C, adjust the volume with a mixed solution of isopropanol and n-heptane and filter, wherein the volume ratio of isopropanol to n-heptane is 1:9 to 3:

7. S5. Enantiomer Separation and Detection: The solution obtained in S4 was injected into an ultra-high performance co-phase chromatography system. Under co-phase chromatography conditions with supercritical carbon dioxide as the main mobile phase, baseline separation of the two enantiomers of acetonitrile was performed on a cellulose chiral stationary phase column, and detection was performed in the wavelength range of 245–255 nm using a diode array detector. The chiral stationary phase column is an Acquity Trefoil CEL2 column; the back pressure of the combined phase chromatography system is 10-13 MPa, the column temperature is 31-40℃, the mobile phase includes phase A as carbon dioxide and phase B as methanol, the flow rate is 0.8-1.2 mL / min, and gradient elution is used to complete a separation and determination within 6 min; The gradient elution program was 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; 2.2–3 min, 25% B; 3.0–4.0 min, 25% B–15% B; 4.0–6.0 min, 15% B. S6. Quantification: The two enantiomers of acetonitrile were quantified using the external standard method to obtain the residual amounts of the two enantiomers of acetonitrile in the tomato samples.

2. The method according to claim 1, characterized in that, In step S1, the centrifugation conditions are 3000–5000 r / min for 3–10 min, and the total volume of acetonitrile extracted twice is 30–60 mL.

3. The method according to claim 1, characterized in that, The volume ratio of the dichloromethane / methanol mixed solution in step S2 is 95:

5.

4. The method according to claim 1, characterized in that, The amount of packing material in the amino solid-phase extraction column in step S3 is 200-1000 mg, the column volume is 1-6 mL, and the elution volume is 3-10 mL.

5. The method according to claim 1, characterized in that, The volume ratio of the isopropanol / n-heptane mixed solution in step S4 is 2:8, the final volume is 0.5–2.0 mL, and the pore size of the filter membrane is 0.20–0.45 μm.

6. The method according to claim 1, characterized in that, The detection wavelength in step S5 is 250 nm.

7. The method according to claim 1, characterized in that, The external standard method uses a mixed standard working solution of two acetonitrile enantiomers to establish a standard curve. The mixed standard working solution has at least four concentration points selected from 0.5, 1.0, 2.0, 4.0, 10.0, and 20.0 mg / L, and the limit of quantitation is determined with a signal-to-noise ratio of 10.

8. A system for rapidly determining the enantiomeric residues of acetamiprid in tomatoes using solid-phase extraction-ultra-high performance phase chromatography (UHPLC) for implementing the method described in any one of claims 1 to 7, characterized in that, include: A. Extraction module, used to extract tomato samples with acetonitrile and obtain a combined extract; B. Concentration and redissolution module, used to concentrate the combined extract to near dryness and redissolve it with a dichloromethane / methanol mixed solution; C. Solid phase extraction and purification module, including amino solid phase extraction column and its activation / elution flow path, used to purify the complex solution and output the eluent; D. Solvent matching and filtration module, used to concentrate the eluent, make up to volume with isopropanol / n-heptane mixed solution and filter to obtain the test solution; E. Ultra-high efficiency phase chromatography detection module, including supercritical carbon dioxide supply unit, back pressure control unit, column temperature control unit, injection unit, Acquity Trefoil CEL2 chiral column and diode array detector, used for the separation and detection of two enantiomers of acetonitrile; F. Data processing module, used to call the enantiomer standard curve and perform external standard quantitative calculation and result output of the residual amounts of the two enantiomers in tomato samples.

9. The system according to claim 8, characterized in that, The ultra-high performance phase chromatography detection module is configured with a back pressure of 10–13 MPa, a column temperature of 31–40 °C, mobile phase A being carbon dioxide, mobile phase B being methanol, a flow rate of 0.8–1.2 mL / min, and completes a separation and determination within 6 min.

10. The system according to claim 8, characterized in that, The data processing module is further configured to perform quality control judgment on the purification method: when only a single enantiomer peak appears in the chromatogram or the resolution of two peaks is less than 1.5, an alarm message of "purification or chromatographic conditions are abnormal" is output, and the corresponding solid phase extraction column batch number, elution volume and gradient program parameters are recorded for traceability.

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