Method for detecting pesticide residues in olive oil by using dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry

Abstract

The invention provides a method for detecting pesticide residues in olive oil through dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry. The method comprises the following steps: (1) sample pretreatment; (2) preparing a standard solution; (3) gas chromatography time-of-flight mass spectrometry analysis; (4) drawing a standard working curve; and (5) quantitative analysis. The method is simple and rapid to operate, high in sensitivity and good in precision and accuracy, and can be used for simultaneously detecting 180 kinds of pesticide residues with different polarities and different chemical properties in the olive oil matrix mixed standard solution; moreover, in the sample pretreatment process, the reagent consumption is low, the consumable cost is low, and the method is green and environment-friendly, can meet the requirement for detecting various pesticide residues in the olive oil matrix mixed standard solution, and can also provide reference for detection of other oil pesticide residues and pretreatment development of complex matrixes.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for detecting pesticide residues in olive oil by combining dispersed liquid-liquid microextraction and gas chromatography time-of-flight mass spectrometry. Background Art

[0002] Olive oil is cold-pressed from fresh olive fruits. It is rich in oleic acid, linoleic acid and other natural nutrients that are beneficial to the human body. In recent years, olive oil has gradually entered the Chinese consumer market and has shown a rapid development trend. Since pesticides are used to prevent pests during the planting and growth of olives, and some pesticides are not easy to degrade, there may be pesticide residues in olive oil. The potential risks cannot be ignored. Targeted monitoring of pesticide residue risks is an important measure to ensure the safety of olive oil. Therefore, it is necessary to establish a fast, efficient and high-throughput screening method for multi-pesticide residues in olive oil.

[0003] At present, the main pretreatment methods for pesticide residues in olive oil or vegetable oil samples with high oil content are solid phase extraction (SPE), gel permeation chromatography (GPC), QuEChERS, etc. Dispersive liquid-liquid microextraction (DLLME) has the advantages of high enrichment multiples, simple and rapid operation, low consumables cost, and environmental protection; however, DLLME technology is mainly used for the detection and analysis of relatively simple liquid samples and one or a class of pesticide residues. There are few reports on the use of DLLME technology to detect hundreds of pesticide residues of different polarities and chemical properties in the complex matrix of olive oil. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting pesticide residues in olive oil by using dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry to solve the above-mentioned technical problems.

[0005] In order to solve the above technical problems, the specific technical solutions adopted by the present invention are as follows:

[0006] The present invention provides a method for detecting pesticide residues in olive oil by using dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry, the method comprising the following steps:

[0007] (1) Sample pretreatment: Weigh an appropriate amount of olive oil sample and place it in a centrifuge tube, add acetonitrile solution acidified with formic acid, vortex and centrifuge; take the supernatant after centrifugation and place it in another centrifuge tube, add the extractant and aqueous phase, vortex and centrifuge, and take the droplets at the bottom of the centrifuge tube for later use;

[0008] (2) Preparation of standard solution: taking a certain amount of pesticide standard and preparing it with ethyl acetate to prepare a pesticide mixed standard intermediate solution; diluting the mixed standard intermediate solution step by step with the blank olive oil matrix extract treated in step (1) to prepare a series of matrix mixed standard solutions of mass concentration;

[0009] (3) Gas chromatography time-of-flight mass spectrometry analysis: performing gas chromatography time-of-flight mass spectrometry analysis on the sample prepared in step (1);

[0010] (4) Drawing of standard working curve: The matrix mixed standard solution with a series of mass concentrations obtained in step (2) is subjected to gas chromatography time-of-flight mass spectrometry analysis; the peak areas of various pesticides obtained are linearly analyzed with the concentrations of the corresponding pesticides to calculate the standard working curve and regression equation of each pesticide;

[0011] (5) Quantitative analysis: Substitute the peak area of ​​each pesticide obtained in step (3) into the corresponding regression equation obtained in step (4) to obtain the concentration of each pesticide in the sample and calculate the residual amount of each pesticide in the sample.

[0012] Furthermore, in step (1), the amount of formic acid used is 0.5% to 10% (volume percentage) of the mixed solution.

[0013] Furthermore, the amount of formic acid used accounts for 2% (volume percentage) of the mixed solution.

[0014] Furthermore, in step (1), the extractant is one of chloroform and carbon tetrachloride.

[0015] Furthermore, in step (1), the amount of the aqueous phase is 6 to 10 mL.

[0016] Furthermore, the amount of the aqueous phase is 9 mL.

[0017] Furthermore, in step (3), the chromatographic conditions are as follows: using an HP-5MS UI chromatographic column with a specification of 30m×0.25mm×0.25μm, the carrier gas is helium with a purity of ≥99.999%, and the gas flow rate is 1mL / min; the injection port temperature is 280°C; the heating program is as follows: the initial temperature is 50°C, maintained for 2min, heated to 120°C at a rate of 40°C / min, then heated to 255°C at a rate of 3°C / min, and then heated to 300°C at a rate of 10°C / min, and maintained for 6min; non-split injection, the injection volume is 1μL.

[0018] Furthermore, in step (3), the mass spectrometry conditions are as follows: ion source type: electron bombardment source, electron energy is 70 eV, ion source temperature is 230°C; full scan mode, scan range is 35-500 m / z, scan rate is 5 spectra / s; quadrupole temperature is 150°C; collision gas is nitrogen with a purity of 99.999%.

[0019] The method for detecting pesticide residues in olive oil by dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry of the present invention has the following advantages:

[0020] (1) This method is simple to operate, rapid, highly sensitive, and has good precision and accuracy. It can simultaneously detect up to 180 pesticide residues of different polarities and chemical properties in a mixed standard solution of olive oil matrix;

[0021] (2) The sample pretreatment process consumes less reagents, has low consumables cost and is environmentally friendly, which can meet the needs of detecting multiple pesticide residues in olive oil matrix mixed standard solutions. It can also provide a reference for the detection of pesticide residues in other oils and the development of pretreatment of complex matrices. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a bar graph showing the effect of different acetonitrile acidification concentrations on the recovery rates of 180 pesticides in the sample pretreatment stage of the embodiment of the present invention;

[0023] Figure 2 This is a bar graph showing the effect of different aqueous phase volumes on the recovery rates of 180 pesticides during the sample pretreatment stage in an embodiment of the present invention;

[0024] Figure 3 This is a bar graph showing the effects of different extractants on the recovery rates of 180 pesticides in the sample pretreatment stage of an embodiment of the present invention;

[0025] Figure 4 This is an extracted ion chromatogram of a matrix mixed standard solution of 180 pesticides according to an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to better understand the purpose, structure and function of the present invention, the following is a further detailed description of a method for detecting pesticide residues in olive oil by dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry of the present invention in conjunction with the accompanying drawings.

[0027] An embodiment of the present invention provides a method for detecting pesticide residues in olive oil by using dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry, the method comprising the following steps: (1) sample pretreatment; (2) preparation of a standard solution; (3) gas chromatography time-of-flight mass spectrometry analysis; (4) drawing of a standard working curve; and (5) quantitative analysis.

[0028] The detection method of the present invention is simple to operate, rapid, highly sensitive, and has good precision and accuracy, and can simultaneously detect up to 180 pesticide residues of different polarities and chemical properties in olive oil.

[0029] Specifically, the sample pretreatment process includes: weighing 1.0 g of olive oil sample (all commercially available extra virgin olive oil) and placing it in a 15 mL centrifuge tube, adding 2 mL of acetonitrile solution acidified with formic acid, vortexing for 3 minutes, and refrigerated centrifuging at 5000 r / min for 3 minutes at -5°C; taking 1 mL of the supernatant after centrifugation and placing it in another 15 mL centrifuge tube, adding 200 μL of the extractant and the aqueous phase, vortexing for 3 minutes, and refrigerated centrifuging at 5000 r / min for 3 minutes at -5°C, and taking the droplets at the bottom of the centrifuge tube for use.

[0030] The preparation process of the standard solution includes: measuring a certain amount of 180 pesticide standards with a mass concentration of 100 μg / mL and preparing a pesticide mixed standard intermediate solution with a mass concentration of 5 μg / mL with ethyl acetate; diluting the mixed standard intermediate solution with the blank olive oil matrix extract treated in step (1) step by step to prepare a series of matrix mixed standard solutions with mass concentrations (specifically, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1 and 0.2, 0.5, 0.8, 1.2 μg / mL).

[0031] 1. Optimization of sample pretreatment process

[0032] The embodiment of the present invention compares and analyzes the influence of different acetonitrile acidification concentrations on the recovery rate of 180 pesticides in the olive oil matrix mixed standard solution during the sample pretreatment stage: wherein, in the comparative example, the formic acid contents added in Example 1, Example 2, Example 3, Example 4 and Example 5 are 0%, 0.5%, 1%, 2%, 5% and 10% (volume percentage), respectively, and the remaining steps are the same.

[0033] The main component of olive oil is oil, which will cause strong interference to subsequent analysis. However, the amount of oil that can be dissolved in acetonitrile is very small, and the solubility of most pesticides is relatively large; and some pesticides may be sensitive to pH. Therefore, the extraction effect of acetonitrile solutions containing 0, 0.5, 1, 2, 5, and 10% (volume percentage) formic acid on 180 pesticides was analyzed. Figure 1As shown, the number of pesticides with a recovery rate of 70% to 120% of the 180 pesticides in the olive oil matrix mixed standard solution first increases and then decreases with the increase in the volume of formic acid input, indicating that the degree of acetonitrile acidification has a great influence on the pesticide recovery rate. In particular, when the acetonitrile solution containing 2% (volume percentage) formic acid is used for extraction, the number of pesticides with a recovery rate of 70% to 120% is the largest, accounting for 76.67% of all the tested pesticides, so the subsequent examples choose to use the acetonitrile solution containing 2% formic acid for extraction.

[0034] After determining that the acetonitrile solution containing 2% formic acid is used for treatment, the examples of the present invention also compare and analyze the effects of different aqueous phase volumes on the recoveries of 180 pesticides in the olive oil matrix mixed standard solution during the sample pretreatment stage: wherein the volumes of aqueous phase added in Examples 6, 7, 8, 9 and 10 are 6, 7, 8, 9 and 10 mL, respectively, and the remaining steps are consistent.

[0035] Different volumes of dispersant, extractant and aqueous phase in the DLLME system will affect the extraction efficiency of pesticide components. Therefore, in order to extract the target analytes from the sample as much as possible, the extraction effect of different aqueous phase volumes on 180 pesticides in the olive oil matrix mixed standard solution was also analyzed. Figure 2 As shown, with the increase of the volume of the aqueous phase, the number of pesticides with a recovery rate of 70% to 120% among the 180 pesticides increased or decreased significantly, indicating that the extraction efficiency of the pesticide residue items is inseparable from the volume of the aqueous phase in the process of rapid enrichment. In addition, the extraction efficiency is low when the amount of water added is 6, 7, and 8 mL, which may be related to insufficient extraction; when 9 mL of water is added, the recovery rate of the pesticide is the highest, accounting for 77.78% of all the tested pesticides, so the volume of the aqueous phase in the subsequent examples is 9 mL.

[0036] After determining that the acetonitrile solution containing 2% formic acid is used for treatment and the volume of the added aqueous phase is 9 mL, the embodiment of the present invention also compares and analyzes the effects of different extractants on the recoveries of 180 pesticides in the olive oil matrix mixed standard solution during the sample pretreatment stage: wherein the extractants in Example 11 and Example 12 are chloroform and carbon tetrachloride, respectively, and the remaining steps are the same.

[0037] like Figure 3 As shown in the figure, when carbon tetrachloride is selected as the extractant, 73.33% of the 180 pesticides have a recovery rate within the range of 70% to 120%, while when chloroform is selected as the extractant, the percentage increases to 76.67%. Therefore, chloroform is used as the extractant in the subsequent examples.

[0038] 2. Matrix Effect

[0039] The matrix effect refers to the enhancement or weakening of the detection signal caused by the remaining analytes except the sample during the analysis process. The embodiment of the present invention conducted a comparative analysis of the matrix effects (ME=matrix standard solution response value / solvent standard solution response value) of 180 pesticides. When ME is lower than 0.8, it is a matrix inhibition effect, when ME is between 0.8 and 1.2, it is a weak matrix effect, and when ME is greater than 1.2, it is a matrix enhancement effect. The results showed that 2% of the 180 pesticides showed a matrix inhibition effect, 58% of the pesticides showed a weak matrix effect, and 40% of the pesticides showed a matrix enhancement effect. Therefore, the embodiments of the present invention all use a blank matrix to prepare a mixed standard solution for quantification. In the present invention, the extracted ion chromatograms of the matrix mixed standard solution of 180 pesticides are shown in Figure 4 .

[0040] The gas chromatography time-of-flight mass spectrometry analysis process includes: performing gas chromatography time-of-flight mass spectrometry analysis on the sample prepared in step (1); the drawing process of the standard working curve includes: performing gas chromatography time-of-flight mass spectrometry analysis on the matrix mixed standard solution of the series of mass concentrations obtained in step (2); performing linear analysis on the peak area of ​​various pesticides obtained and the concentration of the corresponding pesticides, and calculating the standard working curve and regression equation of each pesticide. The chromatographic conditions are: using an HP-5MS UI chromatographic column with a specification of 30m×0.25mm×0.25μm, the carrier gas is helium with a purity of ≥99.999%, and the gas flow rate is 1mL / min; the injection port temperature is 280℃; the heating program is: the initial temperature is 50℃, maintained for 2min, and the temperature is increased to 120℃ at a rate of 40℃ / min, then increased to 255℃ at a rate of 3℃ / min, and then increased to 300℃ at a rate of 10℃ / min, and maintained for 6min; non-split injection, the injection volume is 1μL. The mass spectrometry conditions were as follows: ion source type: electron bombardment source, electron energy of 70 eV, ion source temperature of 230°C; full scan mode, scan range of 35-500 m / z, scan rate of 5 spectra / s; quadrupole temperature of 150°C; collision gas was nitrogen with a purity of 99.999%.

[0041] The quantitative analysis process includes: substituting the peak area of ​​each pesticide obtained in step (3) into the corresponding regression equation obtained in step (4), obtaining the concentration of each pesticide in the sample, and calculating the residual amount of each pesticide in the sample.

[0042] When calculating the residues of each pesticide in the sample, two methods are used for qualitative and quantitative determination: non-targeted screening and targeted screening.

[0043] Non-targeted screening:

[0044] First, according to the set chromatographic conditions and mass spectrometry conditions, a single standard blank matrix sample of each pesticide compound was injected to obtain the retention time, mass spectrum, CAS and molecular formula of each pesticide compound.

[0045] The target information (compound name, retention time, mass spectrum, CAS, molecular formula, molecular weight) obtained was input into the "Library Editor" of the "Agilent Masshunter" software to establish a pesticide residue library for the olive oil blank matrix; the standard information of the library was obtained from the standard prepared from the olive oil blank matrix, which made the target matching in the olive oil sample more accurate and highly avoided the occurrence of false positives.

[0046] The olive oil samples after pretreatment were processed in the same way as above, and preliminary data analysis was performed to obtain the total ion current map, which was then compared and searched with the established olive oil blank matrix pesticide residue library, and those with a matching degree greater than 70 were identified as suspected targets.

[0047] Ion extraction was performed on the suspected target, and the target was further determined based on the abundance ratio of the characteristic ions of the pesticide residues in the olive oil sample and the established olive oil blank matrix pesticide residue library. If the error was ≤15%, it was determined to be the target to be determined, and then the target to be determined was quantitatively analyzed.

[0048] The screened target substances to be determined were configured into a series of blank matrix mixed standard solutions for gas chromatography time-of-flight mass spectrometry analysis, and the "Agilent Masshunter" software was used to establish a processing method for the blank matrix standard curve of the target substance to be determined based on the retention time, quantitative ion, and qualitative ion. This method was used to analyze the data of suspected positive olive oil samples, and ultimately qualitative and quantitative results were obtained.

[0049] Targeted screening:

[0050] First, according to the set chromatographic conditions and mass spectrometry conditions, a single blank matrix standard of each pesticide compound is injected, and then the retention time, quantitative ion, and qualitative ion information of each pesticide compound are obtained.

[0051] Hundreds of mixed standard intermediate solutions with a series of mass concentrations (specifically 0.002, 0.005, 0.01, 0.02, 0.05, 0.1 and 0.2, 0.5, 0.8, 1, 2 μg / mL) of blank matrix mixed standard solutions were prepared and injected and analyzed under the above conditions.

[0052] Agilent Masshunter software was used to establish hundreds of blank matrix mixed standard curves based on retention time, quantitative ion, and qualitative ion for each compound; and this processing method was used to simultaneously perform qualitative and quantitative analysis on unknown substances in olive oil samples.

[0053] 3. Methodological validation analysis

[0054] The present invention is used to determine the retention time (t R ), quantitative ion, qualitative ion, linear range, detection limit (LOD), lower limit of quantification (LOQ), spike recovery rate (Recov.) and relative standard deviation (RSD) were calculated, and the results are shown in Table 1. Table 1 Retention time, quantitative ion, qualitative ion, linear range, detection limit, lower limit of quantification, spike recovery rate and relative standard deviation of 180 pesticides in olive oil

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] The linear range of the method of the present invention is investigated by the standard curve of the target analyte, as shown in Table 1. The linear range of 87.8% pesticides is 0.02-2.00 μg / mL, the linear range of 12.2% pesticides is 0.1-2.0 μg / mL, and the correlation coefficient (R 2 ) were all greater than 0.99, and the 180 pesticides had a good relationship within their respective linear ranges, meeting the detection requirements. The detection limit (LOD) was 3 times the signal-to-noise ratio (S / N=3), and the quantification limit (LOQ) was 10 times the signal-to-noise ratio (S / N=10). The results showed that the detection limit of the 180 pesticides was 0.002-0.020 mg / kg, and the quantification limit was 0.007-0.067 mg / kg. Among them, the quantification limit of 135 pesticides reached 0.007 mg / kg, and the quantification limit of 23 pesticides reached 0.017 mg / kg, accounting for 87.78% of the total number. The quantification limit of the remaining 5% of the pesticides was 0.033 mg / kg, and the quantification limit of 7% of the pesticides was 0.067 mg / kg.

[0061] In the blank olive oil matrix, three levels of 0.2, 0.5, and 0.8 mg / kg were selected for spike recovery experiments, and each level was repeated 6 times to analyze the accuracy and precision of 180 pesticides. The results showed that under the above three spike levels, pesticides with a recovery rate of 70% to 120% accounted for 76.3% of all the tested pesticides, and pesticides with a relative standard deviation (RSD) of less than 10% accounted for more than 94.4% of the total (see Table 1). This shows that the method has good accuracy and precision and can meet the requirements of multi-pesticide residue analysis.

[0062] 4. Actual olive oil sample testing

[0063] In order to verify the repeatability of this method again, commercially available extra virgin olive oil samples were subjected to repeatability testing and analysis. The results are shown in Table 2.

[0064] Table 2 Detection of pesticide residues in commercial olive oil samples

[0065]

[0066] The present invention tested 10 commercially available olive oil samples and detected a total of 5 pesticides, with a detection amount of 0.0057-0.0427 mg / kg. Among them, dichlorvos was detected in 4 olive oil samples, with a content of 0.0111-0.0124 mg / kg; 3 pesticides, dichlorvos, carbofuran and cypermethrin, were detected in 1 olive oil sample (see Table 2). Compared with the current GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food", the residues of dichlorvos, carbofuran, triadimenol and cypermethrin do not exceed their maximum residue limit values, and cypermethrin has not yet formulated a corresponding maximum residue limit value.

[0067] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for detecting pesticide residues in olive oil by dispersive liquid-liquid microextraction-gas chromatography time-of-flight mass spectrometry, characterized in that: The following steps are involved: (1) Sample pretreatment: Weigh an appropriate amount of olive oil sample and place it in a centrifuge tube, add acetonitrile solution acidified with formic acid, vortex and centrifuge; take the supernatant after centrifugation and place it in another centrifuge tube, add the extractant and aqueous phase, vortex and centrifuge, and take the droplets at the bottom of the centrifuge tube for later use; (2) Preparation of standard solution: taking a certain amount of pesticide standard and preparing it with ethyl acetate to prepare a pesticide mixed standard intermediate solution; diluting the mixed standard intermediate solution step by step with the blank olive oil matrix extract treated in step (1) to prepare a series of matrix mixed standard solutions of mass concentration; (3) Gas chromatography time-of-flight mass spectrometry analysis: performing gas chromatography time-of-flight mass spectrometry analysis on the sample prepared in step (1); (4) Drawing of standard working curve: The matrix mixed standard solution with a series of mass concentrations obtained in step (2) is subjected to gas chromatography time-of-flight mass spectrometry analysis; the peak areas of various pesticides obtained are linearly analyzed with the concentrations of the corresponding pesticides to calculate the standard working curve and regression equation of each pesticide; (5) Quantitative analysis: Substitute the peak area of ​​each pesticide obtained in step (3) into the corresponding regression equation obtained in step (4) to obtain the concentration of each pesticide in the sample and calculate the residual amount of each pesticide in the sample.

2. The method according to claim 1, characterized in that In step (1), the amount of formic acid used accounts for 0.5% to 10% (volume percentage) of the mixed solution.

3. The method according to claim 2, characterized in that The amount of formic acid used accounts for 2% (volume percentage) of the mixed solution.

4. The method according to claim 1, characterized in that: In step (1), the extractant is one of chloroform and carbon tetrachloride.

5. The method according to claim 1, characterized in that In step (1), the amount of the aqueous phase is 6 to 10 mL.

6. The method according to claim 5, characterized in that The amount of the aqueous phase used is 9 mL.

7. The method according to claim 1, characterized in that In step (3), the chromatographic conditions are as follows: using an HP-5MS UI chromatographic column with a specification of 30m×0.25mm×0.25μm, the carrier gas is helium with a purity of ≥99.999%, and the gas flow rate is 1mL / min; the injection port temperature is 280°C; the heating program is as follows: the initial temperature is 50°C, maintained for 2min, heated to 120°C at a rate of 40°C / min, then heated to 255°C at a rate of 3°C / min, and then heated to 300°C at a rate of 10°C / min, and maintained for 6min; non-split injection, the injection volume is 1μL.

8. The method according to claim 1, characterized in that In step (3), the mass spectrometry conditions are as follows: ion source type: electron bombardment source, electron energy is 70 eV, ion source temperature is 230°C; full scan mode, scan range is 35-500 m / z, scan rate is 5 spectra / s; quadrupole temperature is 150°C; collision gas is nitrogen with a purity of 99.999%.

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