Method for the determination of the chiral prothioconazole and its metabolites in food

Abstract

The application discloses a method for determining chiral prothioconazole and metabolites thereof in food. The method comprises the following steps: firstly, mixing an extractant and pure water to prepare an oil-in-water emulsion; the extractant is hexanoic acid, heptanoic acid, octanoic acid and nonanoic acid; then, adding the oil-in-water emulsion into a sample to be detected to quickly form a low-concentration oil-in-water emulsion and complete extraction; finally, adding an electrolyte solution to demulsify, collecting the upper extractant and performing ultra-high performance liquid chromatography analysis. The emulsion liquid microextraction technology established by the application has the advantages of simplicity, rapidness and environmental protection, and the analysis of chiral prothioconazole and metabolites thereof is realized by combining chiral ultra-high performance liquid chromatography.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing and relates to a method for detecting chiral prothioconazole and its metabolites in food. Specifically, it relates to a method for determining chiral prothioconazole and its metabolites in food using emulsion microextraction-ultra-high performance liquid chromatography. Background Technology

[0002] Triazole fungicides are widely used in agricultural production for disease control. Approximately 80% of triazole fungicides are chiral, possessing one or more chiral centers. Enantiomers can exhibit significant differences in biological activity and physiological toxicity. Prothioconazole, developed by Bayer in 2004, has become an important fungicide due to its excellent antibacterial activity. Prothioconazole is primarily biodegraded in soil, plants, and animals through desulfurization, transforming into desulfurized prothioconazole, which is more toxic to mammals than prothioconazole. Due to the presence of asymmetric carbon atoms, they have enantiomers: R- and S-prothioconazole and R- and S-desulfurized prothioconazole. Studies have shown that the R-enantiomer is more effective than the S-enantiomer in inhibiting the biosynthesis of pathogenic fungi and fungal toxins. Toxicological data indicate that the R-enantiomer is more toxic to Daphnia magna than the S-enantiomer. Therefore, establishing detection techniques for chiral prothioconazole and its chiral metabolites in environmental and food samples is of great significance.

[0003] Sample pretreatment is a crucial step in pesticide residue analysis, directly impacting the accuracy of detection results. Rezaee et al. developed dispersive liquid-liquid microextraction (DLME) in 2006. As the most commonly used DLME technique, it can complete the extraction process in a short time. However, a major drawback of dispersive liquid-liquid microextraction is the use of toxic dispersants. This method prepares a homogeneous mixture of extractant and dispersant, achieving dispersion of the extractant in the sample in the presence of the dispersant. Commonly used dispersants are acetonitrile, methanol, and acetone, which increases the amount of toxic solvent used during extraction. Furthermore, the dispersant ultimately dissolves in the sample, reducing the partition coefficient of the target analyte and thus affecting its transfer to the extractant. Liquid-liquid microextraction includes single-drop microextraction, hollow fiber liquid-phase microextraction, dispersive liquid-liquid microextraction, and homogeneous liquid-liquid microextraction. In this patent, we establish a novel liquid-liquid microextraction method that differs from traditional methods, avoiding the use of toxic dispersants. This method first prepares a high-concentration oil-in-water emulsion with the extractant and a small amount of pure water, then adds it to the sample to be tested, resulting in a low-concentration oil-in-water emulsion. At this point, the extractant and sample form an emulsion system, allowing for sufficient contact between the extractant and the sample, thus achieving rapid transfer of the target analyte. This patent names this previously unreported liquid-liquid microextraction technology "emulsion liquid-liquid microextraction." The principle of emulsion liquid-liquid microextraction is to achieve rapid extraction through the dilution process of the emulsion. The difference between emulsion liquid-liquid microextraction and dispersion liquid-liquid microextraction is that, instead of preparing a homogeneous solution of extractant and dispersant, an emulsion of extractant and pure water is prepared first. Then, by diluting the emulsion, the contact area is increased, enabling efficient extraction in a short time. Summary of the Invention

[0004] Based on this, this patent proposes a simple, rapid, and environmentally friendly liquid-liquid microextraction method, named emulsion liquid-liquid microextraction. This method differs from dispersion liquid-liquid microextraction, overcoming the drawbacks of requiring toxic dispersants and auxiliary dispersion equipment. A high-concentration emulsion prepared from an extractant and pure aqueous solution is added to the sample to obtain a low-concentration emulsion, enabling rapid extraction. Before ultra-high performance liquid chromatography analysis, an electrolyte solution is added to replace centrifugation for rapid demulsification.

[0005] (I) Problems to be solved

[0006] To address the aforementioned problems in the prior art, this invention provides an emulsion-liquid microextraction-ultra-high performance liquid chromatography method for the determination of chiral prothioconazole and its metabolites in food.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] This invention provides an emulsion-liquid microextraction-ultra-high performance liquid chromatography method for the determination of chiral prothioconazole and its metabolites in food, comprising the following steps:

[0010] The first step is to mix the extractant and pure water to form an oil-in-water emulsion; the extractant is hexanoic acid, and the volume ratio of the extractant to pure water is 1:3.

[0011] The second step involves adding the oil-in-water emulsion obtained in the first step to the sample to form a low-concentration oil-in-water emulsion and complete the extraction.

[0012] The third step involves adding an electrolyte solution to separate the organic and aqueous phases, and collecting the extractant phase for analysis by ultra-high performance liquid chromatography.

[0013] The fourth step involves the following specific conditions for ultra-high performance liquid chromatography (UHPLC): acetonitrile and 0.1% formic acid aqueous solution (85:15 v / v) are used as the mobile phase, the flow rate is 0.8 mL / min, the injection volume is 5 μL, and the detection wavelength is 220 nm. The prepared standard solution and the sample solution to be tested are injected and analyzed under the above HPLC conditions. A standard curve is plotted based on the concentration of prothioconazole and its metabolites in the sample and the peak area of ​​the chromatogram to achieve quantitative detection of prothioconazole and its metabolites in the sample.

[0014] Preferably, in the second step, the volume ratio of the oil-in-water emulsion prepared in the first step to the sample is 0.8:5.

[0015] In the method described above: in the first step, an extractant and pure water are added to a centrifuge tube and mixed by hand shaking eight times to form a high-concentration oil-in-water emulsion.

[0016] Furthermore, in the third step, an electrolyte solution is added to the low-concentration oil-in-water emulsion for demulsification. After standing until the solution becomes clear, the extractant is collected for analysis by ultra-high performance liquid chromatography. The electrolyte is sodium chloride, with a mass percentage concentration of 10%, and the volume ratio of the electrolyte solution to the sample is 1:10.

[0017] (III) Specific Measurement Methods

[0018] In the first step, an extraction solvent (hexanoic acid, heptanoic acid, octanoic acid, or nonanoic acid) and pure water are added to a 1.5 mL centrifuge tube and mixed by hand shaking eight times to form a high-concentration oil-in-water emulsion. The volume of the extraction solvent is 200 μL, and the volume of the pure water is 600 μL.

[0019] The second step involves adding a high-concentration oil-in-water emulsion to 5 mL of a water sample containing 1 mg / L of prothioconazole and dethioconazole to form a low-concentration oil-in-water emulsion and complete the extraction.

[0020] The third step involves adding an electrolyte solution to a low-concentration oil-in-water emulsion for demulsification. After standing until the solution becomes clear, the extractant is collected for ultra-high performance liquid chromatography (UHPLC) analysis. The electrolyte is sodium chloride, with a mass percentage concentration of 10% and a solution volume of 500 μL.

[0021] (iv) Beneficial effects

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention provides a rapid detection method for prothioconazole and its metabolites. The innovation of this invention is that it avoids the use of toxic dispersants such as acetonitrile, methanol and acetone, and does not use auxiliary extraction devices such as vortex and ultrasound. It can complete the extraction instantaneously (1s) when a low concentration emulsion is formed.

[0024] 2. This invention uses fatty acids as an extractant, replacing traditional toxic solvents such as dichloromethane, carbon tetrachloride, and chlorobenzene. Fatty acids are green, renewable, sustainable, biodegradable, and compatible with various analytical techniques, making them a good alternative to toxic extractants.

[0025] 3. This invention does not use time-consuming separation devices such as centrifuges. By adding an electrolyte solution, the separation of the extractant can be completed quickly (5 min) during deemulsification.

[0026] 4. Chiral ultra-high performance liquid chromatography was used to separate and detect four enantiomers of prothioconazole and its metabolites.

[0027] 5. This technology has the advantages of being simple, fast, and environmentally friendly. Attached Figure Description

[0028] Figure 1 Optimization of the types of extractants.

[0029] Figure 2 Optimization of extractant volume.

[0030] Figure 3 Optimization of pure water volume.

[0031] Figure 4 Optimization of electrolyte mass fraction.

[0032] Figure 5 Optimization of electrolyte solution volume.

[0033] Figure 6 Optimization of sample volume.

[0034] Figure 7 Optimization of the amount of salt.

[0035] Figure 8 Optimization of solution pH. Detailed Implementation

[0036] To better understand the present invention, it will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described herein are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0037] 1. In this invention, 200 μL of hexanoic acid and 600 μL of pure water are added to a 1.5 mL centrifuge tube and mixed by hand vortexing to form a high-concentration oil-in-water emulsion. Then, the high-concentration emulsion is added to 5 mL of a water sample containing 1 mg / L of prothioconazole and dethioconazole, forming a low-concentration oil-in-water emulsion within 1 second. Finally, 500 μL of 10% sodium chloride solution is added, and the organic and aqueous phases are separated within 5 minutes. The extractant phase is collected for ultra-high performance liquid chromatography (UHPLC) analysis.

[0038] 2. The specific conditions for ultra-high performance liquid chromatography (UHPLC) are as follows: acetonitrile and 0.1% formic acid aqueous solution (85:15 v / v) are used as the mobile phase; the flow rate is 0.8 mL / min; the injection volume is 5 μL; and the detection wavelength is 220 nm. The prepared standard solution and the sample solution are injected and analyzed under the above HPLC conditions. A standard curve is plotted based on the concentration and peak area of ​​prothioconazole and its metabolites in the sample, thus achieving quantitative detection of prothioconazole and its metabolites in the sample.

[0039] Example 1: Determination of chiral prothioconazole and its metabolites in food by emulsion-liquid microextraction-ultra-high performance liquid chromatography and optimization of conditions

[0040] The present invention is influenced by many factors and requires a series of optimization experiments. Therefore, single-factor optimization was carried out on the type of extractant, amount of extractant, volume of pure water, mass percentage concentration of electrolyte, volume of electrolyte solution, volume of sample, amount of salt, and pH value of solution.

[0041] 1. Optimization of the types of extractants

[0042] To find the optimal extractant, four fatty acids were investigated as extractants: hexanoic acid, heptanoic acid, octanoic acid, or nonanoic acid (Table 1). Other extraction conditions were as follows: 5 mL of aqueous samples containing 1 mg / L of prothioconazole and dethioconazole, 200 μL of extractant, 600 μL of pure water, and 500 μL of 10% sodium chloride solution as a demulsifier.

[0043] The specific steps are as follows: Add 200 μL of extraction solvent and 600 μL of pure water to a 1.5 ml centrifuge tube and mix by hand to form a high-concentration oil-in-water emulsion. Then, add the high-concentration emulsion to 5 mL of an aqueous sample containing 1 mg / L of prothioconazole and dethioconazole, forming a low-concentration oil-in-water emulsion within 1 second. Finally, add 500 μL of 10% sodium chloride solution, and separate the organic and aqueous phases within 5 minutes. Collect the extraction solvent phase for ultra-high performance liquid chromatography (UHPLC) analysis.

[0044] The specific conditions for ultra-high performance liquid chromatography (UHPLC) determination are as follows: acetonitrile and 0.1% formic acid aqueous solution (85:15 v / v) are used as the mobile phase; the flow rate is 0.8 mL / min; the injection volume is 5 μL; and the detection wavelength is 220 nm. The prepared standard solution and the sample solution are injected and analyzed under the above HPLC conditions. A standard curve is plotted based on the concentration and peak area of ​​prothioconazole and its metabolites in the sample, thus achieving quantitative detection of prothioconazole and its metabolite (dethioconazole) in the sample.

[0045]

[0046] C Found - Represents the concentration of the target analyte detected after adding a certain amount of standard to the sample solution; C Real - Represents the concentration of the target analyte detected in the sample solution without standard;

[0047] C Added - represents the concentration of the target substance added to the sample solution.

[0048] The results are as follows Figure 1 As shown, the highest recovery rate was achieved when using hexanoic acid. This is because the carbon chain of fatty acids affects amphiphilicity and emulsifying properties. Subsequent experiments used hexanoic acid as the extractant.

[0049] Table 1 Optimization of Extractant Types

[0050]

[0051] 2. Optimization of the amount of extractant

[0052] An appropriate extractant volume facilitates the separation of the extractant phase from the aqueous phase and ensures good sensitivity. The effect of extractant volume on recovery ranges from 150 to 250 μL (Table 2). Other extraction conditions are as follows: 5 mL of water sample containing 1 mg / L of prothioconazole and dethioconazole, hexanoic acid as the extractant, 600 μL of pure water, and 500 μL of 10% sodium chloride solution as the demulsifier. The specific method and other conditions are the same as in Example 1.

[0053] from Figure 2It can be seen that the recovery rate gradually increases within the range of 150-200 μL, reaching a maximum at 200 μL, and then decreases with increasing hexanoic acid content. Excessive extractant volume affects the interaction between the extractant phase and the aqueous phase. Subsequent experiments used 200 μL of hexanoic acid.

[0054] Table 2 Optimization of extractant volume

[0055]

[0056] 3. Optimization of pure water volume

[0057] The extractant and pure water together form a high-concentration emulsion. The volume ratio of the two affects the ease of emulsion formation and extraction efficiency. The volume of pure water varies between 200-1000 μL (Table 3). Other extraction conditions are as follows: 5 mL of water sample containing 1 mg / L of prothioconazole and dethioconazole, 200 μL of hexanoic acid as the extractant, and 500 μL of 10% sodium chloride solution as the demulsifier. The specific method and other conditions are the same as in Example 1.

[0058] like Figure 3 As shown, the recovery rate gradually increased in the range of 200-600 μL, reaching a maximum at 600 μL and then gradually decreased. This is because an appropriate volume ratio helps to generate a suitable emulsion and increases the sample surface charge, van der Waals forces, and contact area between the extractant and the analyte. Therefore, 600 μL was used in subsequent experiments.

[0059] Table 3 Optimization of pure water volume

[0060]

[0061] 4. Optimization of electrolyte mass percentage concentration

[0062] Electrolyte solutions promote flocculation and droplet collisions. Sodium chloride mass-volume fractions ranged from 0% to 20% (Table 4). Other extraction conditions were as follows: 5 mL of aqueous samples containing 1 mg / L of prothioconazole and dethioconazole, 200 μL of hexanoic acid as the extractant, 600 μL of pure water, and 500 μL of sodium chloride solution as the demulsifier. Specific methods and other conditions were the same as in Example 1.

[0063] like Figure 4 As shown, the recovery rate initially increased and then decreased. This is because the addition of electrolytes reduces the zeta potential and emulsion stability. Subsequent experiments used a 10% sodium chloride solution as a demulsifier.

[0064] Table 4 Optimization of electrolyte mass percentage concentration

[0065]

[0066] 5. Optimization of electrolyte solution volume

[0067] The volume of the demulsifier affects the rate and effectiveness of phase separation, and the volume of the demulsifier varies within the range of 100-500 μL (Table 5). Other extraction conditions are as follows: 5 mL of aqueous samples containing 1 mg / L each of prothioconazole and dethioconazole, 200 μL of hexanoic acid as the extractant, 600 μL of pure water, and 10% sodium chloride solution as the demulsifier. The specific method and other conditions are the same as in Example 1.

[0068] like Figure 5 As shown, the recovery rate gradually increases with the addition of demulsifier. Due to the decreasing stability of the emulsion, it changes from milky white to transparent, allowing for the collection of more extractant. The recovery rate gradually decreases when the demulsifier volume is 500-900 μL. Excessive demulsifier increases the viscosity of the sample solution, thus reducing analyte mass transfer. Subsequent experiments used 500 μL of demulsifier.

[0069] Table 5 Optimization of Electrolyte Solution Volume

[0070]

[0071] 6. Optimization of sample volume

[0072] exist Figure 6 In this study, the sample volume ranged from 5 to 13 mL (Table 6), and the concentrations of prothioconazole and dethioconazole were 1 mg / L. Other extraction conditions were as follows: 200 μL of hexanoic acid as the extractant, 600 μL of pure water, and 500 μL of 10% sodium chloride solution as the demulsifier. The specific method and other conditions were the same as in Example 1. The results showed that the recovery rate gradually decreased with increasing sample volume. This may be due to excessive dilution of the emulsion, which makes it difficult to transfer the analyte, leading to a decrease in extraction efficiency. Therefore, 5 mL was selected as a suitable sample volume.

[0073] Table 6 Optimization of Sample Volume

[0074]

[0075] 7. Optimization of salt quantity

[0076] like Figure 7As shown in Table 7, the effect of salt dosage (0-1000 mg) on ​​extraction efficiency was investigated. Other extraction conditions were as follows: 5 mL of water sample containing 1 mg / L of prothioconazole and dethioconazole, 200 μL of hexanoic acid as the extractant, 600 μL of pure water, and 500 μL of 10% sodium chloride solution as the demulsifier. The specific method and other conditions were the same as in Example 1. The results showed that the recovery rate gradually decreased with increasing salt dosage. Adding salt to the sample before extraction can increase the transfer of the analyte from the aqueous phase to the organic phase by reducing the solubility of the analyte. The recovery rate gradually decreased with increasing salt dosage. No salt was added in subsequent experiments.

[0077] Table 7 Optimization of Salt Amount

[0078]

[0079]

[0080] 8. Optimization of solution pH

[0081] The pH value of a sample affects the state of the analyte; analytes in a molecular state are more easily extracted into the extractant. Figure 8 As can be seen, the study was conducted within the pH range of 3-11 (Table 8). Other extraction conditions were as follows: 5 mL of water samples containing 1 mg / L of prothioconazole and dethioconazole, 200 μL of hexanoic acid as the extractant, 600 μL of pure water, and 500 μL of 10% sodium chloride solution as the demulsifier. The specific method and other conditions were the same as in Example 1. The results indicate that pH has a limited effect on extraction efficiency. Therefore, it was not necessary to adjust the pH of the sample solution in this experiment.

[0082] Table 8 Optimization of solution pH

[0083]

[0084]

[0085] Example 2: Determination of different concentrations of prothioconazole and its metabolites using the method of the present invention and verification of its effectiveness.

[0086] The specific methods for determining different concentrations of prothioconazole and its metabolites are as follows:

[0087] Add 200 μL of hexanoic acid and 600 μL of pure water to a 1.5 mL centrifuge tube and mix by hand to form a high-concentration oil-in-water emulsion. Then add the high-concentration emulsion to 5 mL of a water sample containing 1 mg / L of prothioconazole and dethioconazole. A low-concentration oil-in-water emulsion is formed within 1 second. Finally, add 500 μL of 10% sodium chloride solution and separate the organic phase and aqueous phase within 5 min. Collect the extractant phase for analysis by ultra-high performance liquid chromatography.

[0088] The specific conditions for ultra-high performance liquid chromatography (UHPLC) determination are as follows: acetonitrile and 0.1% formic acid aqueous solution (85:15 v / v) are used as the mobile phase; the flow rate is 0.8 mL / min; the injection volume is 5 μL; and the detection wavelength is 220 nm. The retention times of the target compounds S-prothioconazole, R-prothioconazole, S-dethioprothioconazole, and R-dethioprothioconazole are 7.8, 8.5, 8.9, and 9.4 min, respectively. The prepared standard solution and the sample solution to be tested are injected and analyzed under the above HPLC conditions. A standard curve is plotted based on the concentration of prothioconazole and its metabolites in the sample and the peak area of ​​the chromatogram, thus achieving quantitative detection of prothioconazole and its metabolites in the sample.

[0089] 1. Testing of actual samples

[0090] This embodiment uses the optimal conditions obtained in Example 1 to evaluate the application method of emulsion-liquid microextraction-ultra-high performance liquid chromatography for the determination of chiral prothioconazole and its metabolites in food. First, the reproducibility of the method was evaluated. Four food samples (water, fruit juice, tea, and vinegar) were tested with three different concentrations of prothioconazole and its metabolites mixed standards. After subtracting the blank, a standard curve was constructed (Table 9). Prothioconazole and its metabolites showed good linearity in the linear range of 0.01-1 mg / L. The limits of detection and quantitation were calculated using S / N = 3 and 10, respectively. The limits of detection and quantitation for prothioconazole and its metabolites were relatively low, at 0.003 and 0.01 mg / L, respectively. To examine the reproducibility of the method, spiked experiments were conducted on the three samples. Five parallel experiments were performed, and the relative standard deviation (RSD) was calculated to be 0.7-5.2% (Table 10), indicating that the method has good reproducibility.

[0091] Table 9. Methodological evaluation of prothioconazole and its metabolites in actual samples.

[0092]

[0093] Table 10 Analysis of prothioconazole and its metabolites in actual samples

[0094]

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for determining chiral prothioconazole and its metabolites, comprising emulsion liquid microextraction-ultra-high performance liquid chromatography (UPLC) for determining chiral prothioconazole and its metabolites in food, characterized in that: Includes the following steps: The first step involves adding the extractant and pure water to a centrifuge tube and mixing by hand shaking eight times to form a high-concentration oil-in-water emulsion. The extractant is hexanoic acid, and the volume ratio of the extractant to pure water is 1:

3. The second step involves adding the oil-in-water emulsion obtained in the first step to the sample at a volume ratio of 0.8:5 to form a low-concentration oil-in-water emulsion and completing the extraction. The third step involves adding an electrolyte solution to a low-concentration oil-in-water emulsion for demulsification, separating the organic and aqueous phases, allowing the solution to stand until it becomes clear, and then collecting the organic phase extractant for analysis by ultra-high performance liquid chromatography. The electrolyte is sodium chloride, with a mass percentage concentration of 10%, and the volume ratio of the electrolyte solution to the sample is 1:

10. The food is water; the metabolite is chiral dethioprothioconazole.

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