Determination method of phenoxy carboxylic acid pesticide residues

Abstract

The invention provides a method for determining phenoxy carboxylic acid pesticide residues. The problem of low extraction efficiency of polar phenoxy carboxylic acid molecules in a traditional micro-extraction technology is effectively solved by utilizing the complexing action of TBP. Meanwhile, the kapok fiber is used as a hydrophobic carrier to firmly retain the TBP extraction agent, so that the bottlenecks of difficult emulsification formation and phase separation and complicated operation in the conventional liquid-liquid microextraction are thoroughly overcome, and the stability and reliability of the extraction process are ensured. The technology is integrated in an injector platform, the whole process of sample loading, extraction, washing and elution can be completed only through simple piston push-pull operation, the operation is extremely simple, convenient and efficient, only microliter TBP organic solvent is consumed, the dosage of toxic reagents and generation of waste liquid are remarkably reduced, the method can efficiently enrich and purify trace target objects in complex matrixes, and the method is suitable for large-scale industrial production. By combining HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) analysis, the international strict maximum residual limit detection requirement is met, and the method has high sensitivity, high selectivity and good reliability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide detection, and particularly relates to a determination method of phenoxy acid pesticide residues. BACKGROUND

[0002] Phenoxy acid herbicides (also known as phenoxyacetic acid herbicides) as a class of small polar molecule organic compounds, due to their key role in increasing crop yield, have been widely used in agricultural production. However, the overuse of phenoxy acid herbicides may lead to their residual deposition on agricultural products, posing a serious threat to human health. In order to ensure the quality of agricultural products and protect human safety, it is of great practical significance to detect the residues of phenoxy acid herbicides in agricultural products.

[0003] For the pretreatment of phenoxy acid herbicide residues, a variety of sample preparation techniques with different characteristics have been reported. In the early analysis research, liquid-liquid extraction (LLE) is often used as a conventional pretreatment method. However, this classic method is not only inefficient, but also often requires the consumption of a large amount of toxic organic reagents. In order to meet the principles of green analytical chemistry, the derivative form of LLE, liquid-liquid microextraction (LLME), has emerged. LLME overcomes the shortcomings of LLE, only needs microliter level of extraction solvent, and has attracted widespread attention due to its high enrichment capacity and extremely low consumption of organic solvent. So far, a variety of LLME methods have been developed, including dispersive liquid-liquid microextraction (DLLME), ultrasonic-assisted emulsification microextraction (USAEME), supramolecular solvent-based liquid-phase microextraction (SUPRAS-LPME) and homogeneous liquid-liquid microextraction (HLLME), for the analysis of phenoxy acid herbicide residues in various types of samples. However, the existing methods still have certain limitations, especially the problems of emulsion formation and phase separation process, both of which can affect the reliability of the sample pretreatment process. SUMMARY

[0004] The technical problem solved by the present application is to provide a determination method of phenoxy acid pesticide residues.

[0005] The technical solution adopted by the present application is:

[0006] A determination method of phenoxy acid pesticide residues (KF-SLME-TBP) uses tri-n-butyl phosphate (TBP) as an extractant, carries out liquid-phase microextraction (KF-SLME) in a syringe using kapok fiber as a supporting medium, and combines HPLC-UV to determine phenoxy acid pesticide residues.

[0007] Tributyl phosphate (TBP) is a moderate Lewis base with P=O functionality that can provide lone pair electrons to form hydrogen-bonded complexes with Lewis acids. Intermolecular forces are generated between kapok fibers and tributyl phosphate (TBP), and TBP and target compounds to be detected are combined through complexation reaction, and based on complexation, TBP exhibits excellent affinity for small polar molecule organic compounds. At the same time, the long carbon chain structure of TBP endows it with strong hydrophobicity, making it a very promising extractant in KF-SLME technology.

[0008] Preferably, in the method for determining residues of phenoxy carboxylic acid pesticides, the syringe is a commercially available syringe with a part of the needle cut off / cut away, the kapok fiber is placed in the needle of the syringe, and the kapok fiber is soaked with the extractant to form a syringe integrated platform.

[0009] Preferably, in the method for determining residues of phenoxy carboxylic acid pesticides, a streamlined workflow is performed by simply pushing and pulling the syringe on the syringe integrated platform, and the specific steps are as follows:

[0010] (1) Using kapok fiber as a support medium and tributyl phosphate (TBP) as an extractant, 0.5-4 ml of sample solution is extracted with every 25 μL of tributyl phosphate (TBP);

[0011] (2) Extracting phenoxy carboxylic acid herbicides from a hydrophilic liquid and performing quantitative analysis by high performance liquid chromatography (HPLC-UV) equipped with an ultraviolet detector.

[0012] Preferably, in the method for determining residues of phenoxy carboxylic acid pesticides, the hydrophilic liquid is water or fruit and vegetable homogenate.

[0013] Preferably, in the method for determining residues of phenoxy carboxylic acid pesticides, 2.5 ml of sample solution is extracted with every 25 μL of tributyl phosphate (TBP), the pH of the sample solution is adjusted to 1, extraction is performed by pushing and pulling the syringe, and desorption is performed with 150 μL of methanol.

[0014] Preferably, in the method for determining residues of phenoxy carboxylic acid pesticides, extraction is performed by pushing and pulling 4 times, and desorption is performed for 6 cycles.

[0015] In the method for determining residues of phenoxy carboxylic acid pesticides, kapok fiber has inherent lipophilicity and is used as a support medium for fixed extractant. The fixed extractant selectively extracts target analytes when in contact with aqueous samples, and then after removing the sample matrix, the target analytes can be easily eluted using a desorption solvent. Since the extractant is firmly retained in the kapok fiber, efficient phase separation can be easily achieved.

[0016] The beneficial effects of the present application are:

[0017] The determination method of the phenoxy carboxylic acid pesticide residue is based on the complex extraction principle, and effectively solves the problem of low extraction efficiency of traditional micro-extraction technology on polar phenoxy carboxylic acid molecules by using the complexation of TBP (forming hydrogen bonds through P=O groups). At the same time, kapok fiber is used as a hydrophobic carrier to firmly retain TBP extractant, which completely overcomes the bottleneck of emulsion formation and phase separation difficulty, complicated operation in conventional liquid-liquid micro-extraction (such as DLLME), and ensures the stability and reliability of the extraction process. The technology is integrated into a syringe platform, and the whole process of sample loading, extraction, washing and elution can be completed by simply pushing and pulling the piston, which is extremely simple and efficient, only consumes microliter (μL) TBP organic solvent, significantly reduces the amount of toxic reagent and waste liquid, and meets the green analysis chemistry principle. The method can efficiently enrich and purify trace (ng / g level) target substances in complex matrix (such as agricultural products, fruit juice, etc.), combined with HPLC-UV analysis, meets the international strict maximum residue limit detection requirements, has high sensitivity, high selectivity and good reliability, provides a simple, efficient, environmentally friendly and accurate analysis solution for agricultural product safety monitoring, and can be used as a simplified and efficient sample preparation technology for water sample analysis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is an extraction flow chart.

[0019] Figure 2 It is an extraction effect comparison chromatogram, wherein (A) is a chromatogram after treatment and extraction, (B) is a chromatogram before extraction, and (C) is a chromatogram of a treated blank aqueous solution without phenoxy carboxylic acid pesticide.

[0020] Figure 3 It is a single factor extraction process condition analysis chart.

[0021] Figure 4 It is a Box-Behnken design-response surface analysis chart.

[0022] Figure 5 It is a green degree evaluation chart of the extraction pretreatment detection method. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions of the present application, the technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments.

[0024] Example 1

[0025] A determination method of phenoxy carboxylic acid pesticide residue is as follows:

[0026] 1. Experimental method

[0027] 1.1. Solution preparation

[0028] Weigh standards of 2-Methyl-4-chlorophenoxyacetic acid (MCPA), 2-(4-chlorophenoxy)-2-methylpropionic acid (MCPP), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), and 2,4-dichlorophenoxybutyric acid (2,4-DB) and prepare a 1 mg / mL stock standard solution in methanol. Dilute an appropriate amount of the stock standard solution with water and mix thoroughly to obtain a 5 μg / mL mixed standard solution.

[0029] 1.2 Sample processing

[0030] Seven commercially available liquids (three teas, three juices, and one oral solution) were selected as samples, namely: green juice, apple and hawthorn juice, peach juice, jasmine tea, lipid-lowering tea, laxative tea, and compound platycodon ammonium chloride oral solution.

[0031] Commercially available solid samples were ground into a uniform powder using a mortar and pestle. After sieving, 1 g of the powder was weighed and added to 10 mL of deionized water. After vortex mixing, ultrasonic extraction was performed for 10 minutes, and the supernatant was collected after centrifugation. All liquid samples were stored in a dark place at 4°C for subsequent analysis. For tea, juice and oral liquid, they were first centrifuged at 14,000 g for 10 minutes, the supernatant was collected, and diluted with deionized water at a ratio of 1:10 (v / v) to obtain the sample solution. To prepare the spiked sample, phenoxycarboxylic acid pesticides were added to the homogenized mixture by adding the calculated amount of standard solution to ensure that the final concentration based on the weight of the fresh sample was the desired concentration. The spiked mixture was thoroughly mixed using a vortex mixer and allowed to equilibrate at room temperature for 30 minutes to ensure that the pesticide was properly incorporated into the matrix. The prepared spiked sample was ready for subsequent use.

[0032] 1.3. Device construction and extraction process Figure 1 shown

[0033] Device construction: Cut off about 2 mm of the plastic needle of the syringe, place 2.5 mg of kapok fiber into the syringe needle, add 10 μL of extractant to the kapok fiber, and then firmly insert the needle into the 2.5 mL syringe.

[0034] Extraction process: Take 2.5 mL of the sample solution with adjusted pH in a 5 mL centrifuge tube, extract by pulling and pushing the syringe for three times, and each time pulling the syringe to the top, ensuring a certain amount of gas inside to reduce the residual liquid when pushing out. Each cycle is kept for about 100 seconds, making the liquid flow out in droplets.

[0035] Desorption: Add 100 μL of methanol to a 1.5 mL centrifuge tube, and desorb by sucking and pushing the methanol for 5 times. Finally, put the desorbed sample solution into the sample vial for subsequent HPLC-UV detection.

[0036] 1.4, Analysis method and chromatographic conditions

[0037] Waters E2695 HPLC high performance liquid chromatograph and ultraviolet detector were used for detection, COSMOSIL 5C 18 -MS-Ⅱ(4.6 ID x 250 mm) chromatographic column, mobile phase was phosphate buffer (7 mmol / L, PH = 4.8)-methanol (40:60, v / v), column temperature was 30℃, flow rate was 1 mL / min, ultraviolet detection wavelength was 230 nm, and sample amount was 10 μL.

[0038] 2, Experimental results

[0039] 2.1, Optimization of extraction conditions

[0040] In order to improve the extraction efficiency, the following six main parameters that may affect the extraction recovery of phenoxy acid herbicides were optimized in this experiment. The chromatograms before and after extraction are shown in Figure 2 These parameters include the type of extractant, the volume of extractant, the pH value of sample solution, the number of times of pushing and pulling the syringe during extraction and desorption, and the volume of desorption solvent methanol Figure 3 ). Single factor analysis method was used to optimize these parameters, and each test was repeated four times. The average extraction recovery and standard deviation of each group were compared to determine the optimal level of each factor, as shown in Figure 3 and 4 Finally, 25 μL of TBP was used to extract 2.5 mL of sample solution, the pH of sample solution was adjusted to 1, and the extraction was performed by pushing and pulling 4 times. 150 μL of methanol was used for desorption, and the cycle was repeated 6 times.

[0041] 2.1.1, Type of extractant

[0042] Common extractants n-octanol, n-hexane, tri-n-octylamine and extractant TBP based on molecular complexation method were used to treat sample solution in the same way, and the extraction effect was compared. TBP was finally selected as the extractant in this experiment.

[0043] 2.1.2, TBP dosage

[0044] The dosage of TBP was increased from 5 to 30 μL, and the extraction efficiency of the seven phenoxy carboxylic acid pesticides was improved with the increase of TBP dosage. However, considering the influence of TBP dosage on economic cost and environment, and the fact that excessive TBP cannot be well absorbed by kapok fibers, resulting in loss of TBP and affecting the repeatability of the experiment, the dosage of TBP was adjusted to 25 μL in the subsequent experiment.

[0045] 2.1.3, pH of sample solution

[0046] Since phenoxy carboxylic acid substances are usually weak acids, they will dissociate in water to form corresponding anions and hydrogen ions. Their pKa values are low, and they usually exist in ionic form in neutral and alkaline environments, and the degree of dissociation is closely related to the pH of the solution, so the pH of the solution has a great influence on the extraction efficiency. In this experiment, the pH of the sample solution was adjusted by adding sulfuric acid and phosphate buffer, and the extraction efficiency at different pH values of 1, 2, 4, 6, 8 and 10 was compared. It was found that the extraction efficiency gradually decreased with the increase of pH, and the extraction efficiency was better when the substance existed in molecular form in acidic environment. Therefore, the pH of the extraction stock solution should be adjusted to 1 in the experiment.

[0047] 2.1.4, Push-pull times during extraction

[0048] The extraction times were investigated, and each group of samples was subjected to 2, 3, 4, 5, 6 and 8 push-pull cycles, and the extraction efficiency was compared. The extraction efficiency increased significantly when the push-pull times were 2-3, but the extraction efficiency tended to be stable or even decreased when the push-pull times were further increased. Considering that excessive push-pull times would result in loss of extractant, 3 cycles were finally selected for extraction.

[0049] 2.1.5, Push-pull times during desorption

[0050] The influence of different push-pull cycle times during desorption was investigated, and 2-7 push-pull cycles were performed for groups 1-6 samples, respectively. It was found that the extraction efficiency gradually increased and tended to be stable, and reached a maximum value at five cycles. Therefore, 5 push-pull cycles were selected in the subsequent experiment.

[0051] 2.1.6, Desorption solvent volume

[0052] Different volumes of methanol 100, 150, 200, 250, 300 and 400 μL were used for elution to investigate the influence of desorption solvent volume on the desorption efficiency. The experimental cost was considered and the recovery rates were compared and evaluated, so 150 μL of methanol was selected for desorption in the subsequent experiment.

[0053] 2.1.7 Response surface methodology based on Box-Behnken design

[0054] According to the results of the single factor experiment, three variables with greater influence, such as the volume of extractant, the number of push-pull cycles of extraction and desorption, were selected for Box-Behnken design (BBD) study, and the total peak area of the five phenoxy acid pesticides was used as the response of the design experiment. The three significant variables were studied, each variable was divided into three levels: (+1) high level, (0) medium level, and (-1) low level. BBD design was used to conduct 17 experiments to evaluate the influence of the volume of extractant, the number of push-pull cycles of extraction and desorption on the average extraction recovery of phenoxy acid pesticide residues. All variables and their corresponding results are summarized in Table 1. The results in Table 1 were analyzed by multiple regression analysis and analysis of variance (ANOVA), and the results of the analysis of variance are shown in Table 2.

[0055] The results of the analysis of variance are shown in Table 2. The non-fitting degree with F value of 3.44 and P value of 0.1320 indicates that there is no significant non-fitting at 95% confidence level. This evaluation generates a simulated equation representing the relationship between the average extraction recovery (Y) and factors such as the volume of TBP (A), the number of extraction times (B), and the number of desorption times (C): Y = 52.94 + 9.74A + 4.4B + 5.42C - 2.21AB - 1.41AC + 0.4435BC - 2.82A 2 -1.19B2-4.27C 2 .

[0056] As Figure 4 shown in the three-dimensional response surface graph in the relationship between independent variables and response, through single factor experiment and multivariate BBD optimization evaluation, the optimal conditions of the extraction process are as follows: the volume of TBP is 25 μL, the number of push-pull cycles of extraction is 4, and the number of push-pull cycles of desorption is 6.

[0057] Table 1 Response surface analysis based on Box-Behnken design

[0058]

[0059] Table 2 Analysis of variance based on the quadratic model of response surface.

[0060]

[0061]

[0062] 2.2, Matrix effect

[0063] In the actual sample detection, complex matrix can interfere with the detection of target analyte. In this study, 1 μg / mL of standard was added to the blank sample to explore the matrix effect. The matrix effect was determined by calculating the ratio of the peak area of the spiked sample to the peak area of the same concentration standard aqueous solution. The results are shown in Table 3, and the matrix has almost no interference with the detection. Therefore, any one sample is subsequently selected as a blank matrix.

[0064] Table 3 Average matrix effect of the method (1 μg / mL, n = 3)

[0065]

[0066] 2.3, Methodology validation

[0067] In this study, the established method was verified for linearity, limit of detection (LOD), limit of quantification (LOQ), precision and stability.

[0068] As shown in Table 4, the analysis method under the optimized experimental conditions (i.e. 25 μL of TBP was selected for extraction, the pH of the extract was adjusted to 1, 4 times of extraction was performed by push-pull, 150 μL of methanol was used for desorption, and 6 times of circulation) was summarized, the standard stock solution prepared with methanol was detected at 0.01-10 μg / mL, and the peak areas corresponding to different concentrations were fitted to draw a standard curve, and the R 2 value indicates a good linear relationship. The detection limit (LOD, S / N = 3) and the limit of quantification (LOQ, S / N = 10) of each compound were calculated by the signal-to-noise ratio.

[0069] As shown in Table 5, the intra-day precision and inter-day precision of the analysis method were evaluated by the size of the relative recovery RSD.

[0070] As shown in Table 6, the samples placed at different times 0 h, 12 h, 24 h, 7 days, and at different temperature environments for 24 h were analyzed and detected to verify the stability of the samples, and the peak area and RSD value were calculated within the required range.

[0071] The above results show that the supported liquid extraction (SLE) method is suitable for the detection of phenoxy acid herbicide residues in liquid.

[0072] Table 4 Linear relationship, correlation coefficient, detection limit, and limit of quantification of five phenoxy acid herbicides

[0073]

[0074] Table 5 Precision study of five phenoxycarboxylic acid herbicides based on HPLC detection

[0075]

[0076]

[0077] Table 6 Stability analysis of five phenoxycarboxylic acid herbicides based on HPLC detection

[0078]

[0079] 2.4 Authenticity and Accuracy

[0080] Due to limited sample size and strict market regulations, no phenoxyacetic acid pesticide residues were detected in any samples. To further evaluate the quantitative analysis capability of this method, a single-blind experiment was conducted using a 0.25 μg / mL standard. The results, shown in Table 7, show that the detection level closely matches the spiked level, demonstrating the effectiveness of this method in accurately analyzing and determining phenoxyacetic acid pesticide residues in real samples.

[0081] Table 7 Detection levels of five phenoxycarboxylic acid herbicides added to different matrix samples

[0082]

[0083]

[0084] 2.5 Greenness Assessment

[0085] The greenness is assessed using six indicators: AGREE (Analytical Green Metric), AGREEprep, BAGI (Blue Applicability Grade Index), GAPI (Green Analytical Procedure Index), ComplexGAPI, and SPMS (Sample Preparation Sustainability Metric).

[0086] like Figure 5 As shown, the AGREE evaluation score is 0.9. It is a clock-like circle that displays the total score and color. A score of more than 0.6 indicates compliance with the green standard. Each part is coded into three color levels based on the degree of greenness in the analysis stage: green, yellow, or red, representing low, medium, and high negative impacts on the environment, respectively. The AGREEprep score is 0.8, and its evaluation rules are the same as AGREE. BAGI is an asteroid icon with a number in the center. The color indicates whether it meets the green analysis standard: white indicates non-compliance, light blue indicates low compliance, and blue indicates relatively compliance. Its blue applicability level index is 80. The color meanings of GAPI and ComplexGAPI are the same as AGREE. The SPMS result output is also consistent with AGREE.

[0087] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various changes and modifications made by those skilled in the art to the present application without departing from the spirit of the present application should fall within the scope of the present application.

Claims

1. A method for determining phenoxycarboxylic acid pesticide residues, characterized in that: Phenoxycarboxylic acid pesticide residues were determined by liquid phase microextraction using tributyl phosphate as the extractant and kapok fiber as the support medium in a syringe, combined with HPLC-UV.

2. The method for determining phenoxycarboxylic acid pesticide residues according to claim 1, wherein: The syringe is a commercially available syringe with a portion of the needle cut off. Kapok fibers are placed in the syringe needle, and the kapok fibers are soaked in an extractant to form a syringe integrated platform.

3. The method for determining phenoxycarboxylic acid pesticide residues according to claim 2, wherein: The streamlined workflow is implemented through simple push-pull operations on the syringe integration platform, with the following steps: (1) Using kapok fiber as the support medium and tributyl phosphate as the extractant, 0.5-4 ml of sample solution was extracted with every 25 μL of tributyl phosphate; (2) Phenoxycarboxylic acid herbicides were extracted from hydrophilic liquids and quantitatively analyzed using high performance liquid chromatography equipped with a UV detector.

4. The method for determining phenoxycarboxylic acid pesticide residues according to claim 3, wherein: The hydrophilic liquid is water or fruit and vegetable homogenate.

5. The method for determining phenoxycarboxylic acid pesticide residues according to claim 3, wherein: 2.5 ml of sample solution was extracted with 25 μL of tributyl phosphate, the pH of the sample solution was adjusted to 1, extraction was performed by pushing and pulling the syringe, and desorption was performed with 150 μL of methanol.

6. The method for determining phenoxycarboxylic acid pesticide residues according to claim 5, wherein: Extraction was performed by pushing and pulling 4 times and desorption cycle was performed 6 times.

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