Method for rapidly detecting parathion-methyl residue based on emulsion microextraction

Abstract

The invention relates to a method for rapidly detecting parathion-methyl residues based on emulsion microextraction, and belongs to the technical field of food safety detection.Natural and healthy oleic acid is used for replacing common dangerous chemicals to serve as an extracting agent, the requirement of'green chemistry 'is met, harm to the environment and the human body is reduced, and the extraction efficiency is improved. Meanwhile, the low-concentration sodium hydroxide aqueous solution is applied to emulsion micro-extraction for the first time, due to the saponification capacity of the sodium hydroxide aqueous solution, oil-in-water emulsion is formed by rapidly whipping oleic acid and the sodium hydroxide aqueous solution, parathion-methyl generates p-nitrophenol with a stronger spectral signal under the alkaline condition, and the detection sensitivity is high. And the p-nitrophenol is efficiently transferred into a trace amount of extractant phase from a food matrix, so that rapid enrichment of p-nitrophenol in a sample is realized. The spectrophotometric method is adopted, complex pretreatment steps and expensive detection equipment are not needed, and the method is suitable for on-site rapid detection.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically discloses a method for quickly detecting methyl parathion residues based on emulsion liquid-liquid microextraction. Background Art

[0002] Methyl parathion, as a highly effective and broad-spectrum organophosphorus insecticide, has played an important role in global agricultural production since its introduction. The compound inhibits the activity of cholinesterase in insects and effectively blocks nerve conduction, thereby quickly killing or repelling a variety of pests that harm crops, greatly improving the yield and quality of crops and ensuring the stability of global food supply. However, with the widespread use of methyl parathion in agricultural production, the problems caused by its irrational use have become increasingly prominent, especially in the field of food safety. Due to the lack of scientific guidance on the use of drugs by some farmers, there are phenomena such as excessive use, improper application time, and insufficient safety interval, resulting in the residue of methyl parathion on the surface of agricultural products exceeding the safety standard. These residues may not only directly pose a potential threat to human health, such as causing damage to the nervous system and affecting the endocrine system, but may also accumulate in the food chain, further amplifying its harm. Methyl parathion is a typical organophosphorus pesticide that is widely used in agricultural production, but its residues pose a potential threat to the environment and human health. If someone ingests methyl parathion accidentally, eats it by mistake, or intentionally, it may cause poisoning or even death. Therefore, it is necessary to detect the residues of methyl parathion in food.

[0003] Traditional pretreatment methods include liquid-liquid extraction (LLE) and solid phase extraction (SPE). However, these methods have limitations such as being time-consuming and labor-intensive. Unlike traditional extraction methods, emulsified liquid–liquid microextraction (Emulsive Liquid–Liquid Microextraction) is based on diluting the O / W emulsion to complete the extraction. The advantages of emulsified liquid–liquid microextraction are fast speed, simple operation, good versatility and flexibility; but in emulsified liquid–liquid microextraction, the only suitable extractant is hexanoic acid, which limits the application of this method. Different types of target analytes require different types of extractants, but this extraction method has limited emulsification effect on many extractants. Therefore, it is necessary to improve the emulsified liquid–liquid microextraction to be suitable for new extractants and expand the scope of application of the emulsified liquid–liquid microextraction method.

[0004] Although traditional detection methods such as gas chromatography (GC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are sensitive and accurate, they have limitations such as expensive equipment, complex operation, long time consumption, high detection cost and unsuitability for rapid on-site detection. Therefore, it is particularly important to develop a simple, fast, economical and suitable detection method for on-site monitoring. Summary of the invention

[0005] Based on the defects of the prior art, the present invention proposes an innovative methyl parathion detection technology, which uses an alkaline solution with saponification ability as an emulsifier in emulsion liquid-liquid microextraction, expands the types of extractants suitable for emulsion liquid-liquid microextraction, and realizes oleic acid and linoleic acid as green extractants. The emulsion generated by the extractant-alkaline solution is injected into a sample containing methyl parathion to generate an O / W emulsion, and the derivative of methyl parathion, p-nitrophenol, is analyzed by an enzyme marker to quantitatively detect the methyl parathion residues in water, juice, tea, and coffee, in order to further reduce the detection cost and improve the detection efficiency while ensuring the detection accuracy, and provide strong support for the effective monitoring of methyl parathion residues in food, thereby ensuring the safety and stability of the food supply chain.

[0006] The above-mentioned method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction comprises the following steps:

[0007] S1, mixing an extractant and an alkaline solution to generate an extractant-alkaline solution emulsion;

[0008] S2, mixing the extractant-alkaline solution emulsion with a sample containing methyl parathion to obtain an emulsion containing p-nitrophenol;

[0009] S3, centrifuging the emulsion containing p-nitrophenol, collecting the extractant containing p-nitrophenol and transferring it into an ELISA plate for detection by an ELISA instrument;

[0010] S4, preparing a standard solution of p-nitrophenol, plotting a standard working curve with the mass concentration of the standard solution as the abscissa and the corresponding absorbance as the ordinate, and obtaining a linear equation of p-nitrophenol;

[0011] S5, substituting the absorbance of p-nitrophenol in the sample obtained by the pre-treatment in step S3 into the linear equation in step S4 to obtain the mass concentration of methyl parathion corresponding to p-nitrophenol in the sample.

[0012] In step S1, the extractant is one of oleic acid and linoleic acid; the ratio between the volume of the extractant and the volume of the sample is 17-21:400-1000.

[0013] In step S1, the extractant is oleic acid.

[0014] In step S1, the ratio between the volume of the extractant and the volume of the sample is 19:500.

[0015] In step S1, the alkaline solution is one of a sodium hydroxide aqueous solution, a sodium carbonate aqueous solution, and a sodium bicarbonate aqueous solution; the concentration of the alkaline solution is 0-0.005 mol / L; and the ratio between the volume of the alkaline solution and the volume of the sample is 0-8:40-100.

[0016] In step S1, the alkaline solution is an aqueous sodium hydroxide solution.

[0017] In step S1, the concentration of the alkaline solution is 0.002 mol / L; the ratio between the volume of the alkaline solution and the volume of the sample is 7:50.

[0018] In step S1, the extractant and the alkaline solution are mixed 2 to 7 times; preferably, the alkaline solution and the extractant are mixed using a pipette, and the mixing number is 5 times;

[0019] In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 3 min;

[0020] In step S3, ultraviolet detection is performed using an enzyme marker; the absorbance is recorded at 317 nm.

[0021] The sample was one of water, juice, tea or coffee containing methyl parathion.

[0022] Compared with the prior art, the present invention has the following beneficial effects.

[0023] 1. The present invention overcomes the limitations of the selection of extractants based on the saponification ability of alkaline solutions, broadens the types of extractants suitable for emulsion liquid-liquid microextraction, realizes the rapid emulsification of a variety of extractants, and establishes a green, simple and rapid emulsion liquid-liquid microextraction.

[0024] 2. The present invention is based on the fact that methyl parathion can be hydrolyzed into p-nitrophenol under alkaline conditions provided by an alkaline solution, and the derivatization of methyl parathion is simultaneously completed during the extraction process, which greatly enhances the spectral signal and establishes a low-cost spectral method for detecting methyl parathion in food.

[0025] 3. The present invention optimizes the types of extractants and alkaline solutions, adopts oleic acid as the extractant, adopts sodium hydroxide aqueous solution as the alkaline solution, forms an oil-in-water emulsion based on oleic acid and sodium hydroxide aqueous solution, and allows methyl parathion to generate p-nitrophenol under alkaline conditions, thereby efficiently transferring from the food matrix to a trace amount of extraction phase, achieving rapid extraction and enrichment of samples, and analyzing and detecting methyl parathion in food in combination with spectrophotometry.

[0026] 4. The present invention does not use toxic reagents in the sample pretreatment stage, but only uses low-cost natural healthy oleic acid and low-concentration sodium hydroxide aqueous solution, which is in line with the principles of green chemistry.

[0027] 5. The present invention improves the traditional liquid-liquid microextraction, uses oleic acid and low-concentration sodium hydroxide solution to produce emulsification to replace traditional vortex ultrasound and other operations to accelerate the reaction, does not require traditional large-scale instruments to participate in the detection, and realizes the rapid detection of methyl parathion, and the test results can be obtained within 5 seconds.

[0028] 6. The present invention uses less reagents and is a green, rapid and simple method that can be applied to the analysis of methyl parathion in a variety of food samples. The recovery rate is between 86.8% and 102.9%, and the relative standard deviation is less than or equal to 4.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 is the effect of the type of extractant on the recovery of methyl parathion;

[0031] Figure 2 is the effect of the volume of the extractant on the recovery of methyl parathion;

[0032] Figure 3 is the effect of the type of alkaline solution on the recovery rate of methyl parathion;

[0033] Figure 4 is the effect of the concentration of the alkaline solution on the recovery of methyl parathion;

[0034] Figure 5 is the effect of the volume of alkaline solution on the recovery of methyl parathion;

[0035] Figure 6 is the effect of the number of mixing times on the recovery of methyl parathion;

[0036] Figure 7 is the effect of sample volume on the recovery of methyl parathion. DETAILED DESCRIPTION

[0037] The strong alkalinity of sodium hydroxide aqueous solution helps to destroy the tension of the oil-water interface, allowing the originally immiscible fatty acids and water to form an emulsion. Sodium hydroxide has a strong saponification ability and can react with oils and fats to generate corresponding fatty acid salts and glycerol. This saponification reaction is an important step in the emulsification process, which helps to disperse oils and fats into the water phase to form a stable emulsion. At the same time, due to the saponification effect of sodium hydroxide, it can replace some emulsifiers, thereby reducing costs and improving production efficiency, and only a very low concentration is required to achieve a good emulsification effect. Oleic acid is a very healthy fatty acid that performs outstandingly in protecting cardiovascular health, anti-inflammatory, improving insulin sensitivity, and supporting skin and brain health. However, oleic acid is weakly acidic, and a weakly acidic environment is conducive to demulsification but not to emulsification. The emulsification effect of oleic acid is very poor and it is difficult to form an emulsion with water. Therefore, it is only necessary to add an appropriate alkaline solution to replace the existing toxic extractant with non-toxic oleic acid and apply it to the field of emulsion liquid-liquid microextraction. Oleic acid is used as an extractant and sodium hydroxide solution is used as an alkaline solution to be whipped and mixed, so that oleic acid and sodium hydroxide are in more complete contact, and the generated surfactant sodium oleate has a dispersing effect, which reduces the interfacial tension between oil and water, so that oleic acid droplets can be stably dispersed in water to form an emulsion. At the same time, methyl parathion can be quickly hydrolyzed into p-nitrophenol with a stronger spectral signal under alkaline conditions. The principle of using sodium hydroxide as both an emulsifier and a derivatization agent to improve the method of emulsion liquid-liquid microextraction has not been reported.

[0038] Based on the above theory, this embodiment proposes a method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction, which includes two parts: one is to extract samples using the emulsion liquid-liquid microextraction method, and optimize the conditions to obtain the best extraction conditions; the other is to use the best extraction and detection conditions to measure 4 samples.

[0039] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Among them, methyl parathion standard, purity ≥ 99%; p-nitrophenol standard, purity ≥ 99%; sodium hydroxide aqueous solution: dissolved in 10 mL of water to prepare a 0.002 mol / L aqueous solution, stored at -20°C away from light.

[0041] Example 1

[0042] A. Draw a standard curve

[0043] A standard p-nitrophenol was used for detection, and a standard working curve was drawn with the concentration of the standard in water (0.01, 0.05, 0.1, 0.5 and 1 mg / L) as the abscissa and the corresponding absorbance as the ordinate, yielding a linear equation y=0.7313x+1.9744.

[0044] The specific operation is as follows: 700 μL of 0.002 mol / L sodium hydroxide aqueous solution and 190 μL of oleic acid are added to a 1.5 mL centrifuge tube in sequence, and mixed with a pipette 5 times to generate a high-concentration emulsion. Subsequently, it is transferred to a 10 mL centrifuge tube containing 5 mL of water to generate a low-concentration emulsion of the extractant and water, and then centrifuged at 4000 rpm for 3 minutes, the extractant is collected, and directly transferred to a 96-well ELISA plate, and a standard solution of p-nitrophenol is added, and the ELISA instrument is used for detection, and its absorbance is determined at 317 nm.

[0045] B. Optimization of reaction conditions

[0046] Methyl parathion was added to water to obtain a preset concentration of methyl parathion-water as a sample.

[0047] 1. Optimization of extraction agent types

[0048] The affinity of the extractant to the aqueous sodium hydroxide solution will affect the emulsification efficiency and further affect the extraction recovery of the pesticide. The present embodiment selects two green solvents as model extractants, including oleic acid and linoleic acid. Oleic acid and linoleic acid belong to unsaturated fatty acids, which play an important role in the industrial production of plasticizers, paints, medicines, foods, soaps and biodiesel. They are derived from triglycerides and can be obtained by hydrolyzing vegetable oils and animal fats. They have high biodegradability, low flammability, reproducibility and non-toxicity.

[0049] The specific operation is to add 700 μL of 0.002 mol / L sodium hydroxide aqueous solution and 190 μL of oleic acid or linoleic acid to a 1.5 mL centrifuge tube in turn, mix with a pipette 5 times, and generate a high-concentration emulsion. Then, transfer it to a 10 mL centrifuge tube containing 5 mL of sample to generate a low-concentration emulsion of the extractant and the sample, and quickly extract the p-nitrophenol generated in the sample through full contact. Then centrifuge at 4000 rpm for 3 minutes, collect the extractant and transfer it to a 96-well ELISA plate for detection by an ELISA instrument. Figure 1 The experimental results show that when oleic acid is used as the extractant, the recovery rate of p-nitrophenol generated in the sample is the highest. In the presence of sodium hydroxide aqueous solution, linoleic acid also has a certain emulsification ability, but its emulsification stability is slightly inferior to oleic acid. Therefore, oleic acid is determined to be the best type of extractant for subsequent experiments.

[0050] 2. Optimization of extractant volume

[0051] Too small a volume of extractant will result in incomplete emulsification, while an excess of extractant will affect the stability of the emulsion liquid-liquid microextraction.

[0052] The above method 1 was followed, except that oleic acid was used as the extractant, and the volumes of the extractant were 170, 180, 190, 200, and 210 μL, respectively. The results are shown in Figure 2 The larger the amount of extractant, the higher the recovery rate, reaching the maximum value when the amount of oleic acid was 190 μL, and then gradually decreased. Therefore, 190 μL of extractant was determined as the optimal volume for subsequent experiments.

[0053] 3. Optimization of alkaline solution types

[0054] Since the alkaline solution helps to destroy the tension of the oil-water interface, the originally immiscible oil and water can form an emulsion, and the alkaline solution can form an emulsion with oleic acid, thereby achieving rapid extraction. Since the sodium hydroxide aqueous solution, the sodium carbonate aqueous solution, and the sodium bicarbonate aqueous solution all have good saponification ability, these three types of alkaline solutions are selected in this embodiment to determine the optimal alkaline solution type.

[0055] According to the above operation method 1, the difference is that the extraction agent is oleic acid, the volume of the extraction agent is 190μL, and the types of alkaline solutions are selected from sodium hydroxide aqueous solution, sodium carbonate aqueous solution, and sodium bicarbonate aqueous solution for treatment. The results show that Figure 3 In the experiment, when sodium hydroxide aqueous solution was selected as the alkaline solution, the recovery rate of p-nitrophenol generated in the sample was the highest. This may be because the alkalinity of sodium hydroxide is much stronger than that of sodium carbonate and sodium bicarbonate. In the saponification reaction, the strong base can more effectively generate water-soluble fatty acids and glycerol. In contrast, although sodium carbonate and sodium bicarbonate also have a certain alkalinity, they are strong bases and weak acid salts, and their alkalinity is relatively weak. Their saponification reaction rate is relatively slow and may not be complete. Therefore, sodium hydroxide aqueous solution was determined to be the best alkaline solution type for subsequent experiments.

[0056] 4. Optimization of alkaline solution concentration

[0057] During the emulsification process, the active substances generated by the reaction of sodium hydroxide and oleic acid form a protective film on the oil-water interface to prevent the aggregation and sedimentation of oil droplets. The concentration of the sodium hydroxide aqueous solution determines the stability and integrity of this protective film, thereby improving the emulsification effect. When the concentration of the sodium hydroxide aqueous solution is low, the number of active substances generated by the reaction with oleic acid is small. The amount of these active substances on the oil-water interface is insufficient, and it is difficult to form a stable emulsion, resulting in poor emulsification effect. The appropriate concentration of sodium hydroxide aqueous solution can ensure that the generated active substances reach a hydrophilic-lipophilic balance at the oil-water interface, which is conducive to the formation of a stable water-in-oil emulsion. If the concentration of the sodium hydroxide aqueous solution is too high, the excess sodium hydroxide will ionize a large number of sodium ions and hydroxide ions in the water. These ions may produce a salt effect and affect the stability of the emulsion.

[0058] The above operation method 1 is used, except that oleic acid is used as the extractant, the volume of the extractant is 190 μL, the alkaline solution is sodium hydroxide aqueous solution, and the concentration of sodium hydroxide aqueous solution is treated according to 0, 0.001, 0.002, 0.003, 0.004, and 0.005 mol / L, respectively. The results are shown in Figure 4 The recovery rate increased with the increase of the concentration of sodium hydroxide aqueous solution, reaching the highest recovery rate at a concentration of 0.002 mol / L, and then slightly decreased. Therefore, 0.002 mol / L was determined as the optimal concentration of sodium hydroxide aqueous solution in subsequent experiments.

[0059] 5. Optimization of alkaline solution volume

[0060] The larger the volume of the alkaline solution, the more complete its reaction with oleic acid is, and the more oleic acid is consumed, resulting in a decrease in the remaining volume of the extractant, which in turn leads to a worse extraction effect in subsequent experiments.

[0061] The above operation method 1 is used, except that oleic acid is used as the extractant, the volume of the extractant is 190 μL, the alkaline solution is sodium hydroxide aqueous solution, the concentration of the sodium hydroxide aqueous solution is 0.002 mol / L, and the volume of the sodium hydroxide aqueous solution is treated according to 0, 500, 600, 700, and 800 μL, respectively. The results are shown in Figure 5 The recovery rate increased with the increase of the volume of sodium hydroxide aqueous solution, reaching the highest recovery rate at a volume of 700 μL, and then slightly decreased. This may be because the ratio of the extractant and the sodium hydroxide aqueous solution is optimal at this time, and the formed water-in-oil emulsion is the most stable. Therefore, 700 μL was determined as the optimal volume of the sodium hydroxide aqueous solution in subsequent experiments.

[0062] 6. Optimization of mixing times

[0063] By whipping and mixing, emulsification can be promoted, the contact between the extractant and the analyte can be enhanced, and the extraction time can be saved. Therefore, the number of mixing times will affect the recovery rate. According to the above operation method 1, the difference is that the extractant is oleic acid, the volume of the extractant is 190μL, the alkaline solution is sodium hydroxide aqueous solution, the concentration of sodium hydroxide aqueous solution is 0.002mol / L, the volume of sodium hydroxide aqueous solution is 700μL, and the mixing times are 2, 3, 4, 5, 6, and 7 times respectively. The results are shown in Figure 6 The recovery rate increased with the increase of mixing times, reached the maximum value when the mixing times was 5 times, and then decreased slightly. Therefore, 5 times was determined as the optimal mixing times for subsequent experiments.

[0064] 7. Optimization of sample volume

[0065] The recovery and sensitivity of emulsion liquid-liquid microextraction are both affected by the sample volume.

[0066] The above operation method 1 is used, except that oleic acid is used as the extractant, the volume of the extractant is 190 μL, the alkaline solution is sodium hydroxide aqueous solution, the concentration of sodium hydroxide aqueous solution is 0.002 mol / L, the volume of sodium hydroxide aqueous solution is 700 μL, the mixing times is 5 times, and the sample volumes are processed according to 4, 5, 6, 8, 9, and 10 mL, respectively. The results are shown in Figure 7 With the increase of sample volume, the recovery rate first maintained a high level and then gradually decreased. This may be attributed to the excessive dilution of the emulsion, which made the transfer of analytes worse and the extraction efficiency decreased. Therefore, 5mL was determined as the optimal sample volume for subsequent experiments.

[0067] Example 2 Detection of actual samples

[0068] (1) Sample pretreatment

[0069] The purchased water, juice, tea and coffee were filtered through a 0.45 μm filter for later use, and methyl parathion was added to the water, juice, tea and coffee respectively to obtain methyl parathion-water, methyl parathion-juice, methyl parathion-tea and methyl parathion-coffee of preset concentrations as samples.

[0070] (2) Sample extraction

[0071] 700 μL of 0.002 mol / L sodium hydroxide aqueous solution and 190 μL of oleic acid were added to a 1.5 mL centrifuge tube in sequence and mixed 5 times with a pipette to generate a high-concentration emulsion. Subsequently, it was transferred to a 10 mL centrifuge tube containing 5 mL of sample to generate a low-concentration emulsion of the extractant and the sample, and the p-nitrophenol generated in the sample was quickly extracted through full contact. Subsequently, the extractant was collected by centrifugation at 4000 rpm for 3 minutes, and the extractant was directly transferred to a 96-well ELISA plate for detection by an ELISA instrument, and its absorbance was determined at 317 nm.

[0072] (3) Draw the linear equation for p-nitrophenol

[0073] Use the standard substance of p-nitrophenol to carry out the test, draw the standard working curve with the concentration of the standard substance as the horizontal axis and the corresponding absorbance as the vertical axis, and obtain the linear equation.

[0074] The specific operation is as follows: 700 μL of 0.002 mol / L sodium hydroxide aqueous solution and 190 μL of oleic acid are added to a 1.5 mL centrifuge tube in sequence, and mixed 5 times with a pipette to generate a high-concentration emulsion. Subsequently, it is transferred to a 10 mL centrifuge tube containing 5 mL of water to generate a low-concentration emulsion of the extractant and water, followed by centrifugation at 4000 rpm for 3 min, the extractant is collected, and directly transferred to a 96-well ELISA plate, and a standard solution of p-nitrophenol is added, and the ELISA instrument is used for detection, and its absorbance is determined at 317 nm to obtain the linear equation of p-nitrophenol-water. Repeat the above operation to obtain the linear equations of p-nitrophenol-juice, p-nitrophenol-tea, and p-nitrophenol-coffee.

[0075] (4) Recovery rate of methyl parathion in samples

[0076] Substitute the absorbance of the sample to be tested in step (2) into the linear equation obtained in step (3) to obtain the concentration of methyl parathion in the sample to be tested. According to Y = (AB) / C * 100%, A is the measured mass concentration of methyl parathion in the spiked sample, B is the measured mass concentration of methyl parathion in the actual sample, and C is the known mass concentration of methyl parathion added to the sample, the recovery rate Y can be obtained.

[0077] According to the above steps, the spiked recoveries of methyl parathion at three concentrations (1, 0.1 and 0.01 mg / L) in water, juice, tea and coffee were tested and analyzed. The results are shown in Table 1. The spiked recoveries were between 86.8% and 102.9%, and the relative standard deviation (RSD) was less than or equal to 4.5%. This shows that the established method is accurate and reliable and is suitable for the residual analysis of methyl parathion in food.

[0078] Table 1 Spiked recovery and relative standard deviation in actual samples

[0079]

[0080] Example 3 Method Verification

[0081] The method used in this embodiment is: 700 μL of 0.002 mol / L sodium hydroxide aqueous solution and 190 μL of oleic acid are added to a 1.5 mL centrifuge tube in sequence, and mixed 5 times with a pipette to generate a high-concentration emulsion. Subsequently, it is transferred to a 10 mL centrifuge tube containing 5 mL of sample to generate a low-concentration emulsion of the extractant and the sample, and the p-nitrophenol generated in the sample is quickly extracted by full contact. Subsequently, the extractant is collected by centrifugation at 4000 rpm for 3 min, and directly moved into a 96-well ELISA plate, and the ELISA instrument is used for detection, and its absorbance is determined at 317 nm.

[0082] This example studies the feasibility, linear range, determination coefficient, precision, detection limit, quantitative limit and other characteristics of the method. Different concentrations of 0.01, 0.05, 0.1, 0.5, 1 mg / L of p-nitrophenol were added to four food samples (water, juice, tea, coffee). The calibration standard curve was drawn with the mass concentration of methyl parathion as the abscissa and the absorbance of the water-in-oil emulsion as the ordinate. The results are shown in Table 2.

[0083] Table 2 Method evaluation

[0084]

[0085] The results showed that in the concentration range of 0.01-1 mg / L, the absorbance showed a good positive linear correlation with the mass concentration of methyl parathion, and the determination coefficient was 0.991-0.997. The intra-day and inter-day relative standard deviations were 1.7-3.8% and 1.0-5.1%, respectively. The detection limit and quantification limit were 0.003 and 0.01 mg / L, respectively. The results showed that the established extraction and detection method of methyl parathion met the analytical requirements of food samples.

[0086] Example 4 Comparison of this method with reported detection methods for methyl parathion

[0087] Comparing this method with the extraction methods published in the past three years (Table 3), the emulsion liquid-liquid microextraction can be completed with a pipette in less than 1 minute, and does not require traditional pretreatment equipment. It is a simple and fast method. On the other hand, only 190 μL of oleic acid and 700 μL of sodium hydroxide aqueous solution are used in the entire microextraction process. The reagent dosage is small and green, avoiding the large-scale use of methanol and acetonitrile and does not require large-scale analytical instruments such as chromatography and mass spectrometry. In summary, compared with other microextraction methods, the emulsion liquid-liquid microextraction method has significant advantages such as high efficiency, simple operation, rapidity, high sensitivity, and low cost.

[0088] Table 3 Comparison of the method of the present invention with other methods for analyzing methyl parathion in food samples

[0089]

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction, characterized in that: The steps include: S1, mixing an extractant and an alkaline solution to generate an extractant-alkaline solution emulsion; S2, mixing the extractant-alkaline solution emulsion with a sample containing methyl parathion to obtain an emulsion containing p-nitrophenol; S3, centrifuging the emulsion containing p-nitrophenol, collecting the extractant containing p-nitrophenol and transferring it into an ELISA plate for detection by an ELISA instrument; S4, preparing a standard solution of p-nitrophenol, plotting a standard working curve with the mass concentration of the standard solution as the abscissa and the corresponding absorbance as the ordinate, and obtaining a linear equation of p-nitrophenol; S5, substituting the absorbance of p-nitrophenol in the sample obtained by the pre-treatment in step S3 into the linear equation in step S4 to obtain the mass concentration of methyl parathion corresponding to p-nitrophenol in the sample.

2. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 1, characterized in that: In step S1, the extractant is one of oleic acid and linoleic acid; The ratio between the volume of the extractant and the volume of the sample is 17-21:400-1000.

3. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 2, characterized in that: In step S1, the extractant is oleic acid.

4. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 2 or 3, characterized in that: In step S1, the ratio between the volume of the extractant and the volume of the sample is 19:

500.

5. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 1, characterized in that: In step S1, the alkaline solution is one of a sodium hydroxide aqueous solution, a sodium carbonate aqueous solution, and a sodium bicarbonate aqueous solution; The concentration of the alkaline solution is 0 to 0.005 mol / L; The ratio between the volume of the alkaline solution and the volume of the sample is 0-8:40-100.

6. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 5, characterized in that: In step S1, the alkaline solution is an aqueous sodium hydroxide solution.

7. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 5 or 6, characterized in that: In step S1, the concentration of the alkaline solution is 0.002 mol / L; The ratio between the volume of the alkaline solution and the volume of the sample is 7:

50.

8. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 2 or 5, characterized in that: In step S1, the extractant and the alkaline solution are mixed 2 to 7 times; In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 3 min; In step S3, ultraviolet detection is performed using an enzyme marker; the absorbance is recorded at 317 nm.

9. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 8, characterized in that: In step S1, the alkaline solution and the extractant are mixed by a pipette, and the mixing times are 5 times.

10. The method for rapid detection of methyl parathion residues based on emulsion liquid-liquid microextraction according to claim 1, characterized in that: The sample was one of water, juice, tea or coffee containing methyl parathion.

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