A method for detecting triazole pesticides in cereals based on green solvent-based dispersive liquid-liquid microextraction combined with high performance liquid chromatography

Abstract

The application provides a method for detecting triazole pesticides in grains based on green solvent-based dispersive liquid-liquid microextraction combined with high performance liquid chromatography, and belongs to the technical field of food safety detection. The nonanoic acid and iron hydroxide are used to prepare a magnetic eutectic solvent, the solvent has greenness and fast magnetic responsiveness, and can realize green and fast magnetic separation without centrifugation. The bio-based solvent isosorbide dimethyl ether is used as a green dispersant for the first time, which promotes the dispersion of the magnetic eutectic solvent and replaces the use of traditional toxic dispersants, and improves the environmental sustainability of the extraction process. The method is simple, convenient, green and environmentally friendly, the experimental results are accurate and reliable, and the green, rapid and efficient detection of triazole pesticides in different grain samples can be realized.

Description

Technical Field

[0001] This invention belongs to the technical field of food safety testing, and specifically discloses a method for detecting triazole pesticides in grains based on dispersion-liquid microextraction combined with high performance liquid chromatography using green solvents. Background Technology

[0002] Triazole pesticides, with their broad-spectrum activity and high efficiency, are among the most widely used pesticides for controlling fungal diseases in cereal crops. However, the widespread and long-term use of triazole pesticides inevitably leads to their residues and accumulation in grains. Residual triazole pesticides have endocrine-disrupting effects, reproductive toxicity, and potential carcinogenicity, posing potential risks to human health. Therefore, developing sensitive, reliable, and efficient analytical methods for detecting triazole pesticides in grains is crucial for ensuring food safety.

[0003] Grain matrices are complex and pesticide residue levels are low, making sample preparation crucial for improving analytical sensitivity, selectivity, and accuracy. Liquid phase microextraction (LPME) is a promising method due to its low solvent consumption, ease of operation, and strong enrichment capabilities. Among LME techniques, dispersive liquid-liquid microextraction (DLME) is the most widely used method due to its rapid kinetics and excellent extraction efficiency.

[0004] In dispersion-liquid microextraction, the extractant is dispersed into numerous droplets to maximize interfacial contact and enhance mass transfer. While the strategy of sufficiently dispersed droplets significantly improves extraction efficiency, these droplets are not easily collected completely. Prolonged centrifugation is typically required to promote phase separation, increasing sample pretreatment time and necessitating additional centrifugation equipment. To overcome this limitation, ferromagnetic metal elements are introduced into eutectic solvents to prepare stable magnetic eutectic solvents, potentially combining strong extraction capabilities with rapid magnetic responsiveness, enabling phase separation under an external magnetic field without the need for centrifugation. However, reported magnetic eutectic solvents are typically prepared using ferromagnetic metal chloride systems, primarily ferric chloride, but these solvents exhibit weak magnetic responsiveness and low magnetic collection efficiency.

[0005] In traditional dispersion-liquid microextraction, dispersion solvents such as acetonitrile, acetone, methanol, and ethanol are commonly used to promote the dispersion of the extractant in the water sample. However, these solvents have problems such as high toxicity and poor biodegradability, which impair environmental sustainability. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a method for detecting triazole pesticides in grains using dispersion-liquid microextraction combined with high-performance liquid chromatography based on green solvents. This method employs ferromagnetic metal hydroxides instead of ferromagnetic metal chlorides to prepare a magnetic eutectic solvent, enhancing its magnetic response performance. Furthermore, it utilizes isosorbide dimethyl ether, a green bio-based solvent, as a dispersant, thus establishing a completely green dispersion-liquid microextraction platform.

[0007] The above-mentioned method for detecting triazole pesticides in cereals using dispersion-liquid microextraction combined with high-performance liquid chromatography based on green solvents includes the following steps: S1, Add a bio-based solvent as a dispersant to the grain sample and perform vortex extraction to obtain a sample solution; S2, Add extractant and ultrapure water to the sample solution and perform vortex extraction. The extractant is a magnetic eutectic solvent prepared from fatty acids and ferromagnetic metal hydroxides. S3, using an external magnetic field to separate the magnetic eutectic solvent from the sample solution phase; S4, a magnetic eutectic solvent, was filtered through a filter membrane, brought to a final volume, and analyzed using high-performance liquid chromatography with a diode array detector.

[0008] In step S1, the bio-based solvent is isosorbide dimethyl ether, with 1000-2000 μL of isosorbide dimethyl ether per gram of grain sample.

[0009] In step S2, the fatty acid is one of valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid, and the ferromagnetic metal hydroxide is one of iron hydroxide, nickel hydroxide, and cobalt hydroxide.

[0010] In step S2, the fatty acid is nonanoic acid, and the ferromagnetic metal hydroxide is iron hydroxide.

[0011] In step S2, the molar ratio of nonanoic acid to ferric hydroxide is 1:1-10:1, and each gram of grain sample corresponds to 150-400 μL of magnetic eutectic solvent and 3-9 mL of ultrapure water.

[0012] Preferably, each gram of grain sample corresponds to 1500 μL of isosorbide dimethyl ether, 300 μL of magnetic eutectic solvent, and 7 mL of ultrapure water, with the molar ratio of nonanoic acid to ferric hydroxide in the magnetic eutectic solvent being 5:1.

[0013] In both steps S1 and S2, the vortex extraction time is 1 min.

[0014] In step S4, the diameter of the filter membrane is 0.22 μm, and the volume is adjusted to 300 μL; The analytical conditions were as follows: An Agilent 1260 high-performance liquid chromatograph equipped with a diode array detector and an HC-C18 column was used. The column length was 250 mm, the inner diameter was 4.6 mm, the inner diameter of the packing material was 3 μm, the column temperature was 30 ℃, the mobile phase ratio was acetonitrile:water mixed solution with a volume ratio of 51:49, the flow rate was 1.0 mL / min, the injection volume was 5 μL, and the detection wavelength was 220 nm.

[0015] The grain sample consisted of one of the following: wheat, rice, corn, buckwheat, and oats.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] 1. This invention is the first to use the green bio-based solvent isosorbide dimethyl ether as a dispersant, overcoming the dependence of traditional methods on toxic dispersants (acetonitrile, acetone, methanol and ethanol) and reducing the amount of toxic solvents used.

[0018] 2. This invention uses a novel magnetic eutectic solvent to replace the traditional chlorinated extractant, overcoming the dependence of traditional methods on toxic extractants.

[0019] 3. This invention achieves the separation of the extractant phase and the aqueous phase by applying an external magnetic field (magnet), overcoming the dependence on separation equipment such as centrifugation in traditional methods. The method of this invention is simple to operate, requires minimal equipment, and the magnet allows for the simultaneous separation of multiple samples.

[0020] 4. This invention is the first to use ferromagnetic metal hydroxides to replace ferromagnetic metal chlorides in the preparation of magnetic eutectic solvents, which enhances the magnetic response performance of magnetic eutectic solvents and shortens the separation time of magnetic eutectic solvents from 5-20 minutes to 5 seconds.

[0021] 5. This invention combines magnetic eutectic solvents with bio-based solvents to establish a dispersion-liquid microextraction platform that uses only green solvents. It has good applicability, accurate and reliable experimental results, and can realize green, rapid and efficient detection of triazole pesticides in different samples. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a flowchart of the method of the present invention.

[0024] Figure 2The effect of fatty acid type on recovery rate.

[0025] Figure 3 The effect of ferromagnetic metal hydroxide type on recovery rate.

[0026] Figure 4 The effect of the molar ratio of magnetic eutectic solvent on the recovery rate.

[0027] Figure 5 The effect of extractant volume on recovery rate.

[0028] Figure 6 The effect of dispersant type on recovery rate.

[0029] Figure 7 The effect of dispersant volume on recovery rate.

[0030] Figure 8 The effect of ultrapure water volume on recovery rate. Detailed Implementation

[0031] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: Optimization of Dispersion Liquid-Liquid Microextraction Conditions A. Fatty acid type (1) Experimental method: 1 g of wheat was weighed into a 10 mL centrifuge tube, and then 1500 μL of bio-based solvent isosorbide dimethyl ether was added as a dispersant. The mixture was vortexed for 1 min to obtain the sample solution. In a 15 mL centrifuge tube, the sample solution, 350 μL of a magnetic eutectic solvent prepared from fatty acids and ferric hydroxide (molar ratio 5:1) was added as the extractant, and 7 mL of ultrapure water was added. The mixture was vortexed for 1 min. The centrifuge tube was then placed next to a magnet. Within 5 s, the magnetic eutectic solvent, which had been dispersed into small droplets, aggregated and completely separated from the sample solution. Finally, the magnetic eutectic solvent was filtered through a 0.22 μm filter membrane and brought to a final volume of 300 μL. The solution was analyzed using high performance liquid chromatography with a diode array detector, and the recovery rate was calculated.

[0033] (2) Experimental results are as follows Figure 2 As shown.

[0034] (3) Results Analysis: The extractant plays a decisive role in the extraction process. Under the same extraction conditions, the effects of valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid fatty acids on the extraction efficiency were investigated. The results showed that the magnetic eutectic solvent prepared from valeric acid had high water solubility, but its volume was very small, making effective collection impossible. From hexanoic acid to nonanoic acid, the recovery rate gradually increased with increasing carbon chain length, while the recovery rate of decanoic acid decreased. Hexanoic acid, heptanoic acid, and octanoic acid have high polarity and weak interaction with triazole pesticides. Decanoic acid has high viscosity, which increases mass transfer resistance and hinders the distribution of triazole pesticides to the magnetic eutectic solvent. Therefore, nonanoic acid was selected as the most suitable fatty acid.

[0035] B. Ferromagnetic metal hydroxide types This embodiment innovatively utilizes ferromagnetic metal hydroxides as hydrogen bond acceptors and magnetic components to construct a novel magnetic eutectic solvent, which has not been reported before. Compared with traditional ferromagnetic metal chloride magnetic response systems, ferromagnetic metal hydroxides provide additional hydrogen bond binding sites, enhancing magnetic responsiveness and increasing magnetic separation rates, thus contributing to improved extraction and separation performance of eutectic solvents.

[0036] (1) Experimental method: The difference from (1) in A is that different types of ferromagnetic metal hydroxides (iron hydroxide, nickel hydroxide and cobalt hydroxide) are added respectively.

[0037] (2) Experimental results are as follows Figure 3 As shown.

[0038] (3) Results Analysis: The magnetic eutectic solvent can be rapidly separated from the aqueous phase within 5 seconds under the action of an external magnetic field, without the need for time-consuming centrifugation. To screen the optimal magnetic eutectic solvent that combines rapid and efficient extraction performance with green and safe properties, the effects of ferric hydroxide, nickel hydroxide, and cobalt hydroxide on the extraction effect were investigated. The results showed that the magnetic eutectic solvent prepared with ferric hydroxide had the highest recovery rate; the magnetic eutectic solvents prepared with nickel hydroxide and cobalt hydroxide were prone to extractant loss, ultimately resulting in a lower recovery rate. In addition, nickel and cobalt are relatively highly toxic and may pose a potential pollution risk to experimental personnel and the environment during the experimental operation, while iron is an essential trace element for the human body with extremely low physiological toxicity. Therefore, ferric hydroxide was selected as the ferromagnetic metal hydroxide for preparing the magnetic eutectic solvent.

[0039] C. Magnetic eutectic solvent molar ratio (1) Experimental method: The difference from (1) in A is that different molar ratios of fatty acids and ferromagnetic metal hydroxides (1:1, 3:1, 5:1, 7:1 and 10:1) are added respectively.

[0040] (2) Experimental results are as follows Figure 4 As shown.

[0041] (3) Results Analysis: The molar ratio of each component in the magnetic eutectic solvent is a key factor in balancing extraction performance and separation efficiency. The effects of nonanoic acid to ferric hydroxide molar ratios of 1:1, 3:1, 5:1, 7:1, and 10:1 on the extraction effect were investigated. The results showed that when the molar ratio changed from 1:1 to 5:1, the recovery rate of triazole pesticides increased significantly, reaching its maximum at a molar ratio of 5:1. Further increases in the ratio led to a decrease in recovery rate. As the molar ratio of nonanoic acid increased, the viscosity of the magnetic eutectic solvent system gradually decreased, which facilitated sufficient contact and interaction between the triazole pesticides and the extraction solvent. However, excessively high nonanoic acid content weakened the magnetic response performance of the magnetic eutectic solvent, increased the difficulty of two-phase separation, and resulted in incomplete collection of the magnetic eutectic solvent, ultimately leading to a decrease in recovery rate. Therefore, 5:1 was selected as the optimal molar ratio for this magnetic eutectic solvent.

[0042] D. Extractant volume (1) Experimental method: The difference from (1) in A is that different volumes of extractant (150, 200, 250, 300, 350 and 400 μL) were added respectively.

[0043] (2) Experimental results are as follows Figure 5 As shown.

[0044] (3) Results Analysis: The volume of the extractant is an important factor affecting the efficiency of extraction and the sensitivity of the method. The effects of 150, 200, 250, 300, 350, and 400 μL of extractant on the extraction effect were investigated. The results showed that when the extractant volume increased in the range of 150–300 μL, the recovery rate of triazole pesticides gradually increased, reaching the highest value at 300 μL. After further increasing the amount of extractant, the recovery rate remained basically stable. This may be because when the extractant volume is too small, it cannot be dispersed into a sufficient number of small droplets, resulting in incomplete extraction; when the extractant volume is too large, the size of the dispersed droplets is too large, reducing the effective interface area for mass transfer, leading to a decrease in extraction efficiency. Therefore, 300 μL was selected as the optimal extractant volume.

[0045] E. Types of dispersants (1) Experimental method: The difference from (1) in A is that different dispersants (isosorbide dimethyl ether, acetonitrile, acetone, methanol and ethanol) are added respectively.

[0046] (2) Experimental results are as follows Figure 6 As shown.

[0047] (3) Results Analysis: The type of dispersant is a key factor affecting the dispersion state of magnetic eutectic solvent droplets and the interfacial mass transfer efficiency. To screen a green dispersant that can achieve both efficient dispersion of the extractant and a high recovery rate, the extraction effect of the bio-based green solvent isosorbide dimethyl ether was investigated and compared with traditional dispersants such as acetonitrile, acetone, methanol, and ethanol. The results showed that isosorbide dimethyl ether had the highest recovery rate, which was better than the four traditional dispersants. Therefore, isosorbide dimethyl ether was selected as the optimal dispersant.

[0048] F. Dispersant volume (1) Experimental method: The difference from (1) in A is that different volumes of dispersant (1000, 1250, 1500, 1750 and 2000 μL) were added respectively.

[0049] (2) Experimental results are as follows Figure 7 As shown.

[0050] (3) Results Analysis: The volume of the dispersant affects both the dispersion effect of the extractant and the recovery efficiency of the subsequent extract phase. The effects of 1000, 1250, 1500, 1750, and 2000 μL of isosorbide dimethyl ether on the extraction effect were investigated. The results showed that as the volume of the dispersant increased, the recovery of triazole pesticides first increased and then decreased, reaching a peak at 1500 μL. This may be because when the volume of the dispersant is too small, it is not possible to uniformly disperse the extractant into tiny droplets, resulting in insufficient interfacial contact between the extractant and the sample solution; while when the volume is too large, a large amount of extractant will dissolve in the dispersant phase, resulting in the loss of extractant and triazole pesticides, ultimately leading to a decrease in recovery rate. Therefore, 1500 μL of isosorbide dimethyl ether was selected as the optimal dispersant volume.

[0051] G. Ultrapure water volume (1) Experimental method: The difference from (1) in A is that different volumes of ultrapure water (3, 4, 5, 6, 7, 8 and 9 mL) were added respectively.

[0052] (2) Experimental results are as follows Figure 8 As shown.

[0053] (3) Results Analysis: The volume of ultrapure water affects both the dispersion effect and phase separation efficiency of the extractant. The effects of 3, 4, 5, 6, 7, 8, and 9 mL on the extraction effect were investigated. The results showed that the recovery rate of triazole pesticides gradually increased with the increase of ultrapure water volume, reaching the maximum recovery rate at 7 mL. Further increases in ultrapure water volume resulted in a decreasing recovery rate. This may be because when the ultrapure water volume is small, the extractant dissolves, leading to volume loss and a decrease in recovery rate; while when the ultrapure water volume is too large, the dispersion effect of the extractant deteriorates, resulting in a decrease in recovery rate. Therefore, a volume of 7 mL of ultrapure water was selected for the experiment.

[0054] Example 2: Preparation of standard curves for triazole pesticides Experimental Methods: 1 g of sample (wheat, rice, corn, buckwheat, and oats) was weighed into a 10 mL centrifuge tube, followed by the addition of 1500 μL of the bio-based solvent isosorbide dimethyl ether as a dispersant. The mixture was vortexed for 1 min to obtain the sample solution. In a 15 mL centrifuge tube, the sample solution, 350 μL of a magnetic eutectic solvent prepared from nonanoic acid and ferric hydroxide (molar ratio 5:1) as the extractant, and 7 mL of ultrapure water were added, and the mixture was vortexed for 1 min. The centrifuge tube was then placed near a magnet. Within 5 s, the magnetic eutectic solvent, dispersed into small droplets, aggregated and completely separated from the sample solution. Finally, the magnetic eutectic solvent was filtered through a 0.22 μm filter membrane and brought to a final volume of 300 μL. Analytical analysis was performed using high-performance liquid chromatography with a diode array detector. A graph was plotted with triazole pesticide concentration on the x-axis and peak area on the y-axis to derive the linear equations and ranges for different matrices.

[0055] (2) The experimental results are shown in Table 1.

[0056] (3) Results analysis: This method is effective in the range of 0.001–0.1 μg g. -1 It showed a good linear relationship (R0) across the entire concentration range. 2 >0.99). The precision of the method was evaluated by intraday and interday relative standard deviations. The results showed that the intraday relative standard deviation (RSD) was 1.6%–4.5%, and the interday RSD was 1.7%–4.8%, indicating that the method has good linearity, repeatability and reproducibility for the detection of triadimefon, triazole and flusilazole pesticides in wheat, rice, corn, buckwheat and oat samples.

[0057] Example 3: Detection of actual samples (1) Experimental Methods: 1 g of sample (wheat, rice, corn, buckwheat, and oats) was weighed into a 10 mL centrifuge tube, followed by the addition of 1500 μL of the bio-based solvent isosorbide dimethyl ether as a dispersant, and vortexed extraction for 1 min. In a 15 mL centrifuge tube, the sample solution, 350 μL of a magnetic eutectic solvent prepared from nonanoic acid and ferric hydroxide (molar ratio 5:1) as the extractant, and 7 mL of ultrapure water were added, and vortexed extraction was performed for 1 min. The centrifuge tube was then placed next to a magnet. Within 5 s, the magnetic eutectic solvent, dispersed into small droplets, aggregated and completely separated from the sample solution. Finally, the magnetic eutectic solvent was filtered through a 0.22 μm filter membrane and brought to a final volume of 300 μL. HPLC-diode array detector was used for detection and analysis. The recovery rate was calculated using the following formula: .

[0058] C found : Represents the total pesticide concentration measured after adding a known concentration of standard to an actual sample; C real : Initial pesticide concentration measured in the actual sample; C added : The known concentration of pesticide added.

[0059] (2) The experimental results are shown in Table 2.

[0060] (3) Results Analysis: The method established in this invention was applied to the detection of actual samples of wheat, rice, corn, buckwheat, and oats to evaluate its practical feasibility in determining triazole pesticides in actual grain samples. Measurement values ​​of 0.001, 0.01, and 0.1 μg were set. -1 Three spiking concentration levels were used to recover the samples, with each sample group measured in triplicate. The results are shown in Table 2. The recoveries of triazole pesticides ranged from 75.0% to 101.9%, with RSDs ranging from 0.3% to 5.7%. This indicates that the method is suitable for the quantitative detection of triazole pesticides in wheat, rice, corn, buckwheat, and oat mashes.

[0061] 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 detecting triazole pesticides in cereals using dispersion-liquid microextraction combined with high-performance liquid chromatography based on green solvents, comprising the following steps: S1, Add a bio-based solvent as a dispersant to the grain sample and perform vortex extraction to obtain a sample solution; S2, Add extractant and ultrapure water to the sample solution and perform vortex extraction. The extractant is a magnetic eutectic solvent prepared from fatty acids and ferromagnetic metal hydroxides. S3, using an external magnetic field to separate the magnetic eutectic solvent from the sample solution phase; S4, a magnetic eutectic solvent, was filtered through a filter membrane, brought to a final volume, and analyzed using high-performance liquid chromatography with a diode array detector.

2. The method for detecting triazole pesticides in cereals based on dispersion-liquid microextraction combined with high-performance liquid chromatography using green solvents according to claim 1, characterized in that, In step S1, the bio-based solvent is isosorbide dimethyl ether, with 1000-2000 μL of isosorbide dimethyl ether per gram of grain sample.

3. The method for detecting triazole pesticides in cereals based on dispersion-liquid microextraction combined with high-performance liquid chromatography using green solvents according to claim 1, characterized in that, In step S2, the fatty acid is one of valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid, and the ferromagnetic metal hydroxide is one of iron hydroxide, nickel hydroxide, and cobalt hydroxide.

4. The method for detecting triazole pesticides in cereals based on dispersion-liquid microextraction combined with high-performance liquid chromatography using green solvents according to claim 3, characterized in that, In step S2, the fatty acid is nonanoic acid, and the ferromagnetic metal hydroxide is iron hydroxide.

5. The method for detecting triazole pesticides in cereals based on green solvent dispersion liquid-liquid microextraction combined with high performance liquid chromatography according to claim 4, characterized in that, In step S2, the molar ratio of nonanoic acid to ferric hydroxide is 1:1-10:1, and each gram of grain sample corresponds to 150-400 μL of magnetic eutectic solvent and 3-9 mL of ultrapure water.

6. The method for detecting triazole pesticides in cereals based on green solvent dispersion liquid-liquid microextraction combined with high performance liquid chromatography according to claim 1, characterized in that, Each gram of grain sample corresponds to 1500 μL of isosorbide dimethyl ether, 300 μL of magnetic eutectic solvent, and 7 mL of ultrapure water. The molar ratio of nonanoic acid to ferric hydroxide in the magnetic eutectic solvent is 5:

1.

7. The method for detecting triazole pesticides in cereals based on green solvent dispersion liquid-liquid microextraction combined with high performance liquid chromatography according to claim 1, characterized in that, In both steps S1 and S2, the vortex extraction time is 1 min.

8. The method for detecting triazole pesticides in cereals based on green solvent dispersion liquid-liquid microextraction combined with high performance liquid chromatography according to claim 1, characterized in that, In step S4, the diameter of the filter membrane is 0.22 μm, and the volume is adjusted to 300 μL; The analytical conditions were as follows: An Agilent 1260 high-performance liquid chromatograph equipped with a diode array detector and an HC-C18 column was used. The column length was 250 mm, the inner diameter was 4.6 mm, the inner diameter of the packing material was 3 μm, the column temperature was 30 ℃, the mobile phase ratio was acetonitrile:water mixed solution with a volume ratio of 51:49, the flow rate was 1.0 mL / min, the injection volume was 5 μL, and the detection wavelength was 220 nm.

9. The method for detecting triazole pesticides in cereals based on green solvent dispersion liquid-liquid microextraction combined with high performance liquid chromatography according to claim 1, characterized in that, The grain sample consisted of one of the following: wheat, rice, corn, buckwheat, and oats.

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