A method for detecting triazole pesticides in cereals by bio-based solvent liquid phase microextraction combined with high performance liquid chromatography

Abstract

The application provides a method for detecting triazole pesticides in grains by a bio-based solvent liquid phase microextraction combined with high performance liquid chromatography. The bio-based solvent tetrahydro-2-methyl furan is used in the application, direct and rapid extraction of a grain solid matrix is realized, and a conventional organic solvent extraction step of the solid matrix is eliminated. Ammonium sulfate is used to induce phase separation of the bio-based solvent, and the use of a dispersing agent such as acetonitrile and a centrifuge is avoided. The method is simple and convenient, green and environmentally friendly, the experimental results are accurate and reliable, and rapid detection of pesticide residues in solid 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 using a combination of bio-based solvent liquid-phase microextraction and high-performance liquid chromatography. Background Technology

[0002] As a fundamental energy source in the human diet, the scale of grain cultivation continues to expand with the increasing global demand for nutrition. Various pesticide formulations are required in the stages of field cultivation, harvesting, transportation, storage, and processing to control pests and diseases and ensure crop yield and quality. Triazole fungicides, as a typical representative of azole fungicides, play a crucial role in controlling fungal diseases in grains, effectively controlling pathogens such as powdery mildew, brown rust, and leaf spot. However, the widespread use of triazole pesticides has made them a significant concern affecting food security and human health.

[0003] Given the complexity of cereal solid matrices, efficient sample pretreatment techniques are essential for analytical detection. Traditional sample pretreatment methods generally suffer from limitations such as cumbersome operation, lengthy processing time, and high consumption of organic solvents. Traditional liquid-phase microextraction (LPME) methods typically require the use of large amounts of highly toxic organic solvents to first extract the target analyte from the solid sample, followed by the use of a second organic solvent as the extractant to extract the target analyte from the first organic solvent. This often requires at least two steps. Therefore, achieving direct extraction of triazole pesticides from cereal solid samples is of great significance for the development of sample pretreatment techniques. Summary of the Invention

[0004] This invention proposes a method for detecting triazole pesticides in grains using a combination of bio-based solvent liquid-phase microextraction and high-performance liquid chromatography, achieving direct extraction of triazole pesticides from solid grain samples.

[0005] The above-mentioned method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography includes the following steps: S1, ultrapure water and a bio-based solvent were added to the grain sample for extraction. The bio-based solvent was tetrahydro-2-methylfuran. S2, add salt to the liquid system of step S1, let it stand until the salt is fully dissolved to complete the salting-out separation, and the liquid system is divided into an upper organic phase and a lower aqueous phase; S3, the upper organic phase is taken, filtered through a filter membrane, and then detected and analyzed using high performance liquid chromatography-diode array detector.

[0006] In the above method, the grain sample is one of wheat, corn, or oats.

[0007] In step S2, the salt is one of ammonium sulfate, sodium chloride, sodium lactate, and sodium acetate.

[0008] In the above method, each gram of grain sample corresponds to 4–14 mL of ultrapure water, 500 μL–1000 μL of tetrahydro-2-methylfuran, and 2.2–3.4 g of ammonium sulfate.

[0009] Preferably, each gram of grain sample corresponds to 10 mL of ultrapure water, 1000 μL of tetrahydro-2-methylfuran, and 3 g of ammonium sulfate.

[0010] In step S2, after adding ammonium sulfate, the standing time is 0–5 min.

[0011] Preferably, the standing time after adding ammonium sulfate is 3 minutes.

[0012] In step S3, the diameter of the filter membrane is 0.22 μm; The analytical conditions were as follows: An Agilent 1260 high-performance liquid chromatograph equipped with a diode array detector and a C18-HD column was used. The column length was 250 mm, the inner diameter was 4.6 mm, the packing particle size was 5 μm, the column temperature was 30 °C, the mobile phase ratio was acetonitrile:water mixed solution with a volume ratio of 60:40, the flow rate was 1.0 mL / min, the injection volume was 5 μL, and the detection wavelength was 220 nm.

[0013] Compared with the prior art, the present invention has the following beneficial effects.

[0014] 1. This invention simplifies the steps of traditional liquid-phase microextraction (LPME) of solid samples. Traditional LME first uses a large amount of organic solvent, such as acetonitrile, to extract the solid sample, and then uses a small amount of organic solvent to extract the target analyte from the first step. This invention uses an aqueous solution of a bio-based solvent to complete the extraction of solid samples in one step, resulting in fewer extraction steps, simpler operation, shorter processing time, and less organic solvent usage.

[0015] 2. This invention uses only an aqueous solution of a single green bio-based solvent as the extractant, while traditional methods require the use of multiple highly toxic organic solvents to extract solid samples. This invention reduces the amount of solvent used, lowers the toxicity of the extractant, and avoids the negative impact of using dispersants on extraction efficiency.

[0016] 3. This invention uses only a small amount of ammonium sulfate to separate the upper extractant from the lower water and solid sample, which is simpler to operate and takes less time than traditional centrifugation methods.

[0017] 4. This invention does not require the use of dispersants such as acetonitrile or methanol, extraction equipment such as vortex or ultrasonic extraction, or separation equipment such as centrifugation. It avoids the consumption of toxic solvents and dependence on complex equipment, and the experimental results are accurate and reliable, enabling rapid detection. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a flowchart of the detection method of the present invention.

[0020] Figure 2 The effect of extractant type on recovery rate.

[0021] Figure 3 The effect of extractant volume on recovery rate.

[0022] Figure 4 The effect of salt type on recovery rate.

[0023] Figure 5 The effect of salt dosage on recovery rate.

[0024] Figure 6 The effect of settling time on recovery rate.

[0025] Figure 7 The effect of water volume on recovery rate. Detailed Implementation

[0026] Extractants are crucial in extraction systems. In recent years, renewable bio-based solvents prepared from biomass raw materials have received widespread attention as a new generation of green extractants. Tetrahydro-2-methylfuran is a low-toxicity, highly biodegradable bio-based solvent obtained by catalytic conversion of oat hulls and corn cobs rich in pentoses after dehydration. According to research, no reports have been found on using the bio-based solvent tetrahydro-2-methylfuran to replace traditional organic solvents in cereal matrices or in liquid-phase microextraction technology for triazole pesticides. This invention uses tetrahydro-2-methylfuran as a novel functional extractant, achieving phase separation from the aqueous phase and the cereal by adding specific salts, providing a technical basis for establishing a novel green extraction system for cereal solid samples.

[0027] 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.

[0028] Example 1: Optimization of conditions for bio-based solvent liquid-phase microextraction A. Type of extractant (1) Experimental method: 0.5 g of wheat was weighed into a 10 mL centrifuge tube, and 5 mL of ultrapure water and 500 μL of tetrahydro-2-methylfuran were added for extraction. Then, 1.5 g of ammonium sulfate was added, and after standing for 3 min, the system was separated into two phases: the extract phase (upper organic phase) and the aqueous phase. Finally, the upper organic phase was filtered through a 0.22 μm filter membrane and analyzed by high performance liquid chromatography-diode array detector to calculate the recovery rate.

[0029] (2) Experimental results are as follows Figure 2 As shown.

[0030] (3) Results analysis: The extractant plays a decisive role in the extraction process.

[0031] Under the same extraction conditions, the effects of eutectic solvents (butyric acid / urea), acetonitrile, and the bio-based solvent tetrahydro-2-methylfuran on the extraction efficiency were investigated. The results showed that the acetonitrile system could not be separated into two phases, while the bio-based solvent tetrahydro-2-methylfuran showed the best extraction efficiency. Therefore, the green bio-based solvent tetrahydro-2-methylfuran was selected as the extractant.

[0032] B. Extractant volume (1) Experimental method: The difference from (1) in A is that different volumes of tetrahydro-2-methylfuran (250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL) were added respectively.

[0033] (2) Experimental results are as follows Figure 3 As shown.

[0034] (3) Results Analysis: The volume of the extraction solvent is a key parameter determining the extraction efficiency and the final enrichment coefficient. Insufficient volume leads to incomplete extraction, while excessive volume dilutes the extract phase, resulting in low recovery. The results show that the recovery rate of triazole pesticides increases with increasing extractant volume. This enhancement is driven by the increased interfacial area, which promotes efficient mass transfer of hydrophobic analytes. Therefore, the extractant volume used was 500 µL.

[0035] C. Salt type (1) Experimental method: The difference from (1) in A is that different types of salts (ammonium sulfate, sodium chloride, sodium lactate and acetic acid) are added respectively.

[0036] (2) Experimental results are as follows Figure 4 As shown.

[0037] (3) Results Analysis: Salting out regulates ionic strength to reduce the solubility of triazole pesticides in the aqueous phase of the sample, thereby achieving phase separation. To optimize the salting out effect, the effects of four salts (ammonium sulfate, sodium chloride, sodium lactate, and sodium acetate) on the extraction recovery rate were evaluated. The results showed that ammonium sulfate had the highest extraction recovery rate for triazole pesticides, followed by sodium chloride, while sodium lactate and sodium acetate had significantly lower recovery rates. The superior performance of ammonium sulfate stems from its ability to reduce the number of free water molecules (used to dissolve the solute), thereby promoting the transfer of the target analyte to the extraction phase. Therefore, ammonium sulfate was selected as the salting out agent.

[0038] D. Dosage of salt (1) Experimental method: The difference from (1) in A is that different amounts of salt (1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g and 1.7 g) were added respectively.

[0039] (2) Experimental results are as follows Figure 5 As shown.

[0040] (3) Results Analysis: The amount of salt used is a key parameter for controlling ionic strength, achieving phase separation, and ultimately determining the volume of the extract phase. The effect of ammonium sulfate dosage in the range of 1.1 to 1.7 g on the extraction recovery rate of triazole pesticides was investigated. When the salt dosage increased from 1.1 g to 1.5 g, the pesticide recovery rate showed an upward trend. This may be because the increased salt concentration significantly enhanced the ionic strength of the system and reduced the number of free water molecules, thereby promoting obvious phase separation. However, when the salt dosage was further increased to 1.6 g, the recovery rate tended to stabilize, while it decreased when it increased to 1.7 g. This downward trend may be attributed to the increased viscosity of the sample solution caused by excessive salt, which hindered the mass transfer efficiency and diffusion rate of the target analyte. Therefore, 1.5 g of ammonium sulfate was selected as the optimal salt dosage.

[0041] E. Settling time (1) Experimental method: The difference from (1) in A is that they are left to stand for different times (0 min, 1 min, 2 min, 3 min and 5 min).

[0042] (2) Experimental results are as follows Figure 6 As shown.

[0043] (3) Results Analysis: Sufficient settling time ensures extraction equilibrium between the bio-based solvent aqueous solution and the solid sample, and guarantees complete separation of the bio-based solvent phase from the aqueous phase and the wheat sample. This study investigated the effect of different settling times (0–5 min) on the extraction recovery rate. With increasing settling time, the extraction recovery rate showed a stable upward trend, reaching its maximum value at 3 min, indicating that the extraction equilibrium and phase separation process were completely completed at this point. Extending the settling time beyond 3 min did not further improve the extraction recovery rate, which is inconsistent with the goal of rapid detection. Therefore, 3 min was selected as the optimal settling time.

[0044] F. Water volume (1) Experimental method: The difference from (1) in A is that different volumes of water (2 mL, 3 mL, 4 mL, 5 mL, 6 mL and 7 mL) are added respectively.

[0045] (2) Experimental results are as follows Figure 7 As shown.

[0046] (3) Results Analysis: The recovery rate showed a steady upward trend when the water volume increased from 2 mL to 5 mL. At 5 mL, the salt concentration was sufficient to achieve extraction equilibrium. However, when the water volume was further increased to 6 mL and 7 mL, the recovery rate decreased. This may be because the excessive water volume diluted the ionic strength of the system, weakening the salting-out ability of ammonium sulfate; in addition, the increase in free water molecules hindered the transfer of triazole pesticides to tetrahydro-2-methylfuran, thereby reducing the partition coefficient. When the water volume increased to 8 mL, the system could not achieve phase separation, possibly because the ionic strength was too low to drive the phase separation of tetrahydro-2-methylfuran. Therefore, a water volume of 5 mL was selected for the experiment.

[0047] Example 2: Preparation of standard curves for triazole pesticides (1) Experimental method: Weigh 0.5 g of spiked sample (wheat, corn, oats), add 5 mL of ultrapure water and 500 μL of tetrahydro-2-methylfuran for extraction. Add 1.5 g of ammonium sulfate, let stand for 3 min, and the system is separated into two phases: the extract phase (upper organic phase) and the aqueous phase. The upper organic phase is filtered through a 0.22 μm filter membrane and detected and analyzed by high performance liquid chromatography-diode array detector. Plot a graph with triazole pesticide concentration as the x-axis and peak area as the y-axis to obtain the linear detection range for different matrices. Calculate the limit of quantitation (LOQ) and limit of detection (LOD) under this optimized condition according to the formula: LOQ = 10σ / K and LOD = 3σ / K (σ is the standard deviation of the blank sample; K is the slope of the working curve).

[0048] (2) The experimental results are shown in Table 1.

[0049] (3) Results analysis: This method showed good linear correlation (R0) in wheat, corn and oat samples. 2 >0.99), with a linear concentration range of 0.001–0.5 μg / mL. -1 Based on a signal-to-noise ratio (S / N) of 3 and 10, the limit of detection (LOD) and limit of quantitation (LOQ) were 0.0003 μg / mL. -1 and 0.001 μg mL -1 The relative standard deviation (RSD) for intraday precision (three repeated injections within a single day) was 1.4%–3.5%, and the RSD for interday precision (testing over three consecutive days) was 1.5%–3.4%. These experimental results indicate that this method has high sensitivity and repeatability for the detection of triazole, tebuconazole, and propiconazole pesticides in wheat, corn, and oat samples.

[0050] Example 3: Detection of actual samples (1) Experimental method: Weigh 0.5 g of spiked sample (wheat, corn, oats), add 5 mL of ultrapure water and 500 μL of tetrahydro-2-methylfuran for extraction. Add 1.5 g of ammonium sulfate, let stand for 3 min, and the system will separate into two phases: the extract phase (upper organic phase) and the aqueous phase. Filter the upper organic phase through a 0.22 μm filter membrane and analyze by high performance liquid chromatography-diode array detector. Calculate the recovery rate according to the following formula: .

[0051] C found : Indicates the total concentration of the analyte obtained by adding a standard of known concentration to an actual sample; C real : Initial concentration of the analyte in the actual sample; C added : Add the known concentration of the analyte.

[0052] (2) The experimental results are shown in Table 2.

[0053] (3) Results analysis: Spiking recovery experiments of triazole pesticides were conducted on wheat, corn and oat samples, with spiked concentrations of 0.001, 0.01 and 0.5 μg / mL, respectively. -1 As shown in Table 2, the spiked recoveries ranged from 84.7% to 97.9%, and the relative standard deviations (RSDs) were 0.4% to 2.5%, indicating that the method is reliable and applicable for the extraction of triazole pesticides from wheat, corn, and oat samples. This method can also be widely used for other solid samples.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting triazole pesticides in cereals using a bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography, characterized in that, Includes the following steps: S1, ultrapure water and a bio-based solvent were added to the grain sample for extraction. The bio-based solvent was tetrahydro-2-methylfuran. S2, add salt to the liquid system of step S1, let it stand until the salt is fully dissolved to complete the salting-out separation, and the liquid system is divided into an upper organic phase and a lower aqueous phase; S3, the upper organic phase is taken, filtered through a filter membrane, and then detected and analyzed using high performance liquid chromatography-diode array detector.

2. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 1, characterized in that, The grain sample consisted of one of the following: wheat, corn, or oats.

3. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 1, characterized in that, In step S2, the salt is one of ammonium sulfate, sodium chloride, sodium lactate, and sodium acetate.

4. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 3, characterized in that, Each gram of grain sample corresponds to 4–14 mL of ultrapure water, 500 μL–1000 μL of tetrahydro-2-methylfuran, and 2.2–3.4 g of ammonium sulfate.

5. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 4, characterized in that, Each gram of grain sample corresponds to 10 mL of ultrapure water, 1000 μL of tetrahydro-2-methylfuran, and 3 g of ammonium sulfate.

6. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 5, characterized in that, In step S2, after adding ammonium sulfate, the standing time is 0–5 min.

7. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 6, characterized in that, In step S2, after adding ammonium sulfate, the standing time is 3 minutes.

8. The method for detecting triazole pesticides in cereals using bio-based solvent liquid-phase microextraction combined with high-performance liquid chromatography according to claim 1, characterized in that, In step S3, the diameter of the filter membrane is 0.22 μm; The analytical conditions were as follows: An Agilent 1260 high-performance liquid chromatograph equipped with a diode array detector and a C18-HD column was used. The column length was 250 mm, the inner diameter was 4.6 mm, the packing particle size was 5 μm, the column temperature was 30 °C, the mobile phase ratio was acetonitrile:water mixed solution with a volume ratio of 60:40, the flow rate was 1.0 mL / min, the injection volume was 5 μL, and the detection wavelength was 220 nm.

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