A method for detecting 11 acidic herbicides in rice

Abstract

The present invention provides a method for detecting 11 kinds of acidic herbicides in rice, belonging to the technical field of food quality detection. It adopts dispersed solid phase extraction-gas chromatography tandem mass spectrometry detection, including sample pretreatment and GC-MS / MS detection. The detection method of the present invention uses trimethyldiazomethane derivatization to perform gas chromatography-tandem mass spectrometry (GC-MS / MS) determination. Compared with other methods, tandem mass spectrometry has high sensitivity and strong anti-interference ability. The use of solid phase dispersant purification can greatly shorten the detection cycle and reduce the consumption of organic solvents. The establishment of this method can meet the needs of daily detection and rapid screening of acidic herbicides in rice, and has extremely strong practical significance.

Description

Technical Field

[0001] The invention relates to a method for detecting 11 kinds of acidic herbicides in rice, belonging to the technical field of food quality detection. Background Art

[0002] Rice, also known as rice, is a food made from rice seeds after cleaning, husking, milling, and finishing. When planting rice, rice and weeds are in a competitive relationship. They will compete for living space, nutrients, water, etc. Weeds will directly affect the growth of rice. The more weeds there are, the weaker the growth of rice. Conversely, the fewer weeds, the better the growth of rice. In order to increase the yield of rice and the efficiency of weed control, pesticides are often used for weed control, and the residues of pesticide parent, degradation products, metabolites, derivatives, etc. in rice. Irrational use of pesticides will affect the safety of consumers' edible agricultural products. In severe cases, it will cause consumers to become ill, develop deformities, and even directly lead to poisoning and death. Acidic herbicides have good weed control effects and a wide range of applications, so they are used by most farmers. However, these herbicides are often highly toxic and easily soluble in water, which is more harmful to humans and organisms.

[0003] At present, the main methods reported for detecting such pesticides are gas chromatography [Jin Kuiqi, Liu Yupeng, Jin Wenjiu, Sun Yang, Pan Long, Shi Xinnuo. Study on the analysis of dicamba content in degradation liquid by derivatization gas chromatography [J]. Anhui Agricultural Sciences, 2013, 41(09): 3864-3865] [Zhang Li, Zhang Yongtao, Gui Jianye, Zuo Haiying, Li Xiaoya. Discussion on the main elements of derivatization gas chromatography for chlorinated acid herbicides in water [J]. China Environmental Monitoring, 2011, 27(04): 45-49], gas chromatography-mass spectrometry [SN / T 2228-2008, Detection method for residues of 31 acidic herbicides in imported and exported foods. Gas chromatography-mass spectrometry [S].] Liu Bojing. Detection of five herbicide residues in cereals by gas chromatography-mass spectrometry [J]. Journal of the Chinese Cereals and Oils Association, 2008(01):185-188.], HPLC [Zhang Jiping, Jiang Xindi, Huang Wei, Qin Qian, Zhou Qiao. Detection of two phenoxycarboxylic acid herbicides in environmental water samples by dispersive liquid-liquid microextraction based on molecular complexation coupled with HPLC [J]. Chromatography, 2018, 36(05):458-463] and HPLC-tandem mass spectrometry [GB / T 20769-2008, Determination of 450 pesticides and related chemicals residues in fruits and vegetables by liquid chromatography-tandem mass spectrometry [S]] [Cheng Jing, Ding Lei, Jiang Junshu, Lu Yeju. Determination of phenoxycarboxylic acid herbicide residues in cereal agricultural products by liquid chromatography-tandem mass spectrometry [J]. Food Science, 2010, 31(20):389-393.]. Zhang Li, et al., Determination of acidic herbicides in water by pre-derivatization-gas chromatography-mass spectrometry, PTCA (PART B: CHEM. ANAL.) Vol. 47, 2011, discloses a method for detecting acidic herbicides in water.

[0004] Since acidic herbicides are poorly volatile and highly polar, they are difficult to measure directly and accurately by chromatographic analysis. Furthermore, due to the differences in the detection objects and targets, the retrieval results are unpredictable. Summary of the invention

[0005] The invention provides a method for detecting 11 kinds of acidic herbicides in rice.

[0006] The present invention provides a method for detecting 11 kinds of acidic herbicides in rice, which adopts dispersed solid phase extraction-gas chromatography tandem mass spectrometry for detection, and comprises the following steps:

[0007] a. Sample pretreatment:

[0008] Take the sample in a centrifuge tube, add ultrapure water to mix, let it stand, adjust the pH of the sample solution to less than or equal to 2 with sulfuric acid solution, add ethyl acetate to shake and extract, centrifuge at 4000-8000r / min for 5-11min; take the supernatant and add it to a centrifuge containing anhydrous magnesium sulfate, C18 Place in a centrifuge tube, vortex mix, 4000-8000r / min 5 -11min;

[0009] Accurately draw 2 mL of supernatant into a derivatization bottle and blow it dry with nitrogen, add methanol, isooctane, trimethylsilylated diazomethane n-hexane solution, cover the bottle tightly and derivatize at 60-80℃ for 5-10 min, take it out and cool it to room temperature, blow it dry with nitrogen, dissolve the residue with 1 mL n-hexane, pass the membrane and detect it on the analyzer;

[0010] b. GC-MS / MS detection, the detection conditions are:

[0011] Chromatographic conditions:

[0012] Column: HP-5 MS UI Agilent, 30 m × 250 μm × 0.25 μm;

[0013] Carrier gas: high-purity helium with a purity of ≥99.999%; injection port temperature: 280 °C; injection volume: 1 μL; injection mode: splitless injection; flow rate: 1.0 mL / min; program temperature rise: 60 °C for 1 min, then increase the temperature to 170 °C at 40 °C / min, and then increase the temperature to 310 °C at 10 °C / min and maintain for 2 min;

[0014] Mass spectrometry conditions: ion source: Xtr-EI; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; electron energy: 70 eV; transfer line temperature: 280 ℃; collision gas: high-purity nitrogen with a purity of ≥99.999%; quenching gas: high-purity helium with a purity of ≥99.999%; solvent delay time: 5.0 min; monitoring mode: dynamic multiple reaction ion monitoring mode (dMRM).

[0015] Further preferably, the mass volume ratio of the sample to ultrapure water in step a is 1:2.

[0016] Further preferably, the pH in step a=2.

[0017] Further preferably, the centrifugation condition in step a is: 4200 r / min.

[0018] Further preferably, the anhydrous magnesium sulfate, C 18 The mass ratio is: 3-5:1.

[0019] Further preferably, the volume ratio of methanol, isooctane and trimethylsilylated diazomethane to n-hexane in step a is 1:1:1.

[0020] Further preferably, the derivatization temperature in step a is 70° C. and the derivatization time is 5 min.

[0021] Further preferably, the 11 acidic herbicides are: dicamba, dicamba, 2-Methyl-4-chloro-4-propionic acid, 2,4-D, naphthylacetic acid, 2,4,5-TP, 2,4,5-T, 2,4-D butyric acid, chlorothiazide, and quizalofop-p-ethyl.

[0022] The beneficial effects of the present invention are:

[0023] The detection method of the present invention is determined by gas chromatography-tandem mass spectrometry (GC-MS / MS) through trimethyldiazomethane derivatization. Compared with other methods, tandem mass spectrometry has high sensitivity and strong anti-interference ability. The use of solid phase dispersant purification can greatly shorten the detection cycle and reduce the consumption of organic solvents. The establishment of this method can meet the needs of daily detection and rapid screening of acidic herbicides in rice, and has extremely strong practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Total ion maps of 11 acidic herbicides;

[0025] Figure 2 Effect of extraction solution pH on extraction efficiency;

[0026] Figure 3 Graph showing the effect of derivatization temperature on methyl esterification of compounds;

[0027] Figure 4 Effect of derivatization time on methyl esterification of compounds;

[0028] Figure 5 Matrix effect diagram of 11 acidic herbicides. DETAILED DESCRIPTION

[0029] Example 1 Method for detecting 11 acidic herbicides in rice according to the present invention

[0030] 1. Experimental part

[0031] 1.1 Instruments and reagents

[0032] GC-MS / MS (Agilent 8890-7000D); high-speed centrifuge (eppendorf 5804R, Germany); 11 kinds of herbicides (Shanghai Anpu Laboratory Technology); nitrogen blowdown instrument (Yirao Technology); pH meter (METTLER TOLEDO); ultrapure water machine (Merck Milli-Q); C 18Dispersant (Shenzhen Comma Biotechnology); anhydrous magnesium sulfate (Guangzhou Guanghua Technology); sulfuric acid (Chengdu Jinshan Chemical Reagent); ethyl acetate (Opsun); 2.0 mol / L trimethylsilylated diazomethane n-hexane solution (Shanghai Aladdin Biochemical Technology); 11 kinds of acidic herbicides 1000 mg / L (Shanghai Anpu) see Table 1

[0033] 1.2 Preparation of standard solution

[0034] Take appropriate amount of standard solution respectively, dilute it with methanol to make 10 mg / L mixed standard stock solution, and store it at -4℃.

[0035] 1.3 Sample pretreatment

[0036] Weigh 5 g of sample (accurate to 0.01 g) accurately into a 50 mL centrifuge tube, add 10 mL of ultrapure water, mix well, and let stand for 30 minutes. Adjust the pH of the sample solution to 2 with sulfuric acid solution, add 10 mL of ethyl acetate, shake and extract for 10 minutes, and centrifuge at 4200 r / min for 5 minutes. Take the supernatant and add it to a centrifuge containing 1200 mg of anhydrous magnesium sulfate, 400 mg of C 18 Place in a 15 mL centrifuge tube, vortex mix for 1 min, and centrifuge at 4200 r / min for 5 min. Accurately pipette 2 mL of supernatant into a derivatization bottle and blow it to near dryness with nitrogen, add 0.2 mL of methanol, 0.2 mL of isooctane, and 0.2 mL of trimethylsilylated diazomethane hexane solution, cover the bottle tightly, and derivatize at 70 °C for 5 min. Take it out, cool it to room temperature, blow it to dryness with nitrogen, dissolve the residue with 1 mL of hexane, and pass it through a membrane for detection.

[0037] 1.4 GC-MS / MS instrument conditions

[0038] Chromatographic conditions: chromatographic column HP-5 MS UI (Agilent, 30 m×250 μm× 0.25 μm); carrier gas: high-purity helium (purity ≥99.999%); inlet temperature: 280 ℃; injection volume: 1 μL; injection mode: splitless injection; flow rate: 1.0 mL / min; programmed temperature: 60 ℃ for 1 min, increase the temperature to 170 ℃ at 40 ℃ / min, and then increase the temperature to 310 ℃ at 10 ℃ / min and maintain for 2 min.

[0039] Mass spectrometry conditions: ion source: Xtr-EI; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; electron energy: 70 eV; transfer line temperature: 280 ℃; collision gas: high-purity nitrogen (purity ≥99.999%); quenching gas: high-purity helium (purity ≥99.999%); solvent delay time: 5.0 min; monitoring mode: dynamic multiple reaction ion monitoring mode (dMRM).

[0040] Results and Analysis

[0041] 2.1 Optimization of mass spectrometry conditions

[0042] The mass spectrometry conditions for triple quadrupole generally include parameters for parent ions, daughter ions, and their collision energies. This method uses the database provided by Agilent and the Agilent GC-TQ optimizer software to determine the characteristic ion fragments and collision energies. The quantitative ions, qualitative ions, and collision energy results of 11 herbicides are shown in Table 1, and the total ion current under dynamic multiple reaction monitoring mode scanning is shown in Figure 1 .

[0043] Table 1 Retention time, monitoring ion pairs, and collision energy of 11 pesticides

[0044] Compound Retention time (min) Quantitative ion pair (m / z) Collision energy (eV) Qualifying ion pair (m / z) Collision energy (eV) Qualifying ion pair (m / z) Collision energy (eV) Dicamba 6.376 205.0 -> 190.0 15 205.0 -> 177.0 10 234.0 -> 203.0 5 grass bud fear 6.540 207.0 -> 107.9 40 207.0 -> 178.9 15 209.0 -> 181.0 20 2-Methyl-4-chloro 6.644 141.1 -> 77.1 15 141.1 -> 113.1 5 155.1 -> 125.1 10 2,4-Dichloropropionic acid 7.004 162.0 -> 63.1 30 162.0 -> 98.0 15 164.0 -> 63.1 30 2,4-D 7.164 234.0 -> 199.0 10 111.0 -> 75.0 15 175.0 -> 111.0 10 Naphthaleneacetic acid 7.746 141.1 -> 115.0 20 141.1 -> 89.0 40 200.0 -> 115.0 20 2,4,5-TP 8.136 196.1 -> 97.0 30 198.1 -> 97.0 30 282.1 -> 196.0 10 2,4,5-T 8.373 233.0 -> 190.0 15 109.0 -> 74.0 25 208.9 -> 145.0 15 2,4-D butyric acid 8.920 162.0 -> 63.0 35 101.0 -> 59.0 25 162.0 -> 98.0 20 Chlorophthalic acid 10.233 298.9 -> 221.0 25 300.9 -> 223.0 25 331.8 -> 300.9 10 Quizalofop-p-ethyl 16.973 163.0 -> 100.0 20 163.0 -> 136.0 10 242.9 -> 208.1 10

[0045] 2.2 Optimization of pre-treatment conditions

[0046] 2.2.1 Selection of extraction solvent

[0047] This method selected common laboratory organic solvents such as methanol, acetonitrile, n-hexane and ethyl acetate for comparison, and found that n-hexane had low extraction efficiency. Although ethyl acetate, methanol and acetonitrile could effectively extract the compounds, the latter two were miscible with water, and if the water was not effectively removed in the subsequent derivatization process, the derivatization efficiency would be affected. Therefore, this method selected ethyl acetate as the extraction solvent.

[0048] 2.2.2 Effect of pH on recovery during extraction

[0049] Since the acidic herbicide in this method has a high solubility in water, its -COOH is prone to ionization reaction in water. Simple salting-out effect cannot fundamentally change its solubility. Adding a certain amount of acid to increase H + The concentration of the herbicide in water is increased to promote the ionization equilibrium of the herbicide in the reverse direction to ensure that the herbicide exists in a molecular state, thereby effectively extracting it. The results show that (such as Figure 2 ), so this method chooses to dissolve the sample in water, adjust the pH to 2, and then use organic solvent to extract.

[0050] 2.2.3 Effect of derivatization temperature on the results

[0051] Under the same experimental conditions, 200 μL trimethylsilyldiazomethane was reacted with 0.2 mL of a 1 mg / L mixed standard solution at different temperatures for 10 min, and GC-MS / MS was used to determine the effect of the derivatization temperature on the derivatization effect. Figure 3 ): Except for 2,4,5-T, 2,4-D butyric acid and quizalofop-p-butyl, the peak areas of the derivatization products of other compounds increased with the increase of derivatization temperature. However, when the derivatization temperature was greater than 70°C, the peak areas changed little, and the derivatization temperature was finally selected to be 70°C.

[0052] 2.2.4 Effect of derivatization time on results

[0053] Under the same experimental conditions, 200 μL trimethylsilyldiazomethane was reacted with 0.2 mL of a 1 mg / L mixed standard solution at 70°C for different time periods. The results were analyzed by GC-MS / MS, with the derivatization time as the abscissa and the peak area of ​​the derivatized product as the ordinate, to investigate the effect of the derivatization time on the derivatization effect. Figure 4 ): With the increase of derivatization time, the peak area of ​​the derivatization product of each compound increased, but when the derivatization time was greater than 5 min, the peak area changed little, and the final derivatization time was 5 min.

[0054] 2.3 Method evaluation

[0055] 2.3.1 Matrix Effect

[0056] The matrix effect is generally caused by the competition between the co-eluent in the matrix and the target substance, resulting in enhanced or inhibited signals. The matrix effect calculation formula is as follows:

[0057] like Figure 5 The results showed that the 11 pesticides showed a large matrix effect. Therefore, the test used a matrix matching standard curve for quantitative analysis to improve the accuracy and precision of the analysis results.

[0058] 2.3.2 Linear range, detection limit and quantification limit of the method

[0059] Under the optimized experimental conditions, 2 mL of blank rice extract solution was pipetted, and an appropriate amount of mixed standard stock solution was added. The sample was derivatized according to the derivatization method for sample pretreatment, and a series of matrix-matched standard solutions of 5 to 200 μg / L were prepared for detection and a standard curve was drawn [the horizontal axis was the mass concentration of the 11 herbicides (x, μg / L), and the vertical axis was the quantitative ion peak area (y)]. The detection limit (LOD) of the 11 herbicides in the matrix was calculated with a signal-to-noise ratio of 3 times, and the minimum added concentration of the 11 herbicides in the matrix was the limit of quantification (LOQ). The results showed (see Table 2) that in the range of 5 to 200 μg / L, the mass concentration of the 11 herbicides and their peak areas showed a good linear relationship, and the correlation coefficients all showed: R>0.99, the detection limit of the 11 herbicides was 0.11 to 1.15 μg / kg, and the limit of quantification was 10 μg / kg.

[0060] Table 2 Detection limit, quantification limit, recovery rate and relative standard deviation of 11 pesticides

[0061]

[0062] in conclusion

[0063] By optimizing the extraction solvent, extraction pH, derivatization time and temperature, it was determined that the sample was extracted with ethyl acetate in a sample solution with pH = 2, and then derivatized with 2.0 mol / L trimethylsilylated diazomethane n-hexane solution at 70°C for 5 min for methyl esterification. A gas chromatography-tandem mass spectrometry (GC-MS / MS) screening method for 11 acidic herbicides was established using the database provided by Agilent and Agilent GC-TQ optimizer software. After a series of methodological verifications such as matrix effect, addition recovery and repeatability, it was proved that this method can quickly, accurately and simply detect 11 acidic herbicide pesticide residues in rice.

Claims

1. A method for detecting 11 kinds of acidic herbicides in rice, characterized in that: It is detected by dispersed solid phase extraction-gas chromatography tandem mass spectrometry, including the following steps: a. Sample pretreatment: Take the sample in a centrifuge tube, add ultrapure water to mix, let it stand, adjust the pH of the sample solution to less than or equal to 2 with sulfuric acid solution, add ethyl acetate to shake and extract, centrifuge at 4000-8000r / min for 5-11min; take the supernatant and add it to a centrifuge containing anhydrous magnesium sulfate, C 18 Place in a centrifuge tube, vortex mix, 4000-8000r / min 5 -11min; Accurately draw 2 mL of supernatant into a derivatization bottle and blow it dry with nitrogen, add methanol, isooctane, trimethylsilylated diazomethane n-hexane solution, cover the bottle tightly and derivatize at 60-80℃ for 5-10 min, take it out and cool it to room temperature, blow it dry with nitrogen, dissolve the residue with 1 mL n-hexane, pass the membrane and detect it on the analyzer; b. GC-MS / MS detection, the detection conditions are: Chromatographic conditions: Column: HP-5 MS UI Agilent, 30 m × 250 μm × 0.25 μm; Carrier gas: high-purity helium with a purity of ≥99.999%; Injection port temperature: 280 °C; Injection volume: 1 μL; Injection method: splitless injection; Flow rate: 1.0 mL / min; temperature program: 60 °C for 1 min, then increase the temperature to 170 °C at 40 °C / min, then increase the temperature to 310 °C at 10 °C / min and maintain for 2 min; Mass spectrometry conditions: ion source: Xtr-EI; ion source temperature: 230 °C; quadrupole temperature: 150 °C; electron energy: 70 eV; transfer line temperature: 280 °C; Collision gas: high-purity nitrogen with a purity of ≥99.999%; Quenching gas: high-purity helium with a purity of ≥99.999%; solvent delay time: 5.0 min; monitoring mode: dynamic multiple reaction ion monitoring mode dMRM; The 11 kinds of acidic herbicides are: dicamba, dicamba, 2-Methyl-4-chloropropionic acid, 2,4-D, naphthylacetic acid, 2,4,5-TP, 2,4,5-T, 2,4-D butyric acid, chlorophthalic acid, and quizalofop-p-ethyl.

2. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: The mass volume ratio of the sample to ultrapure water in step a is 1:

2.

3. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: The pH described in step a is 2.

4. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: The centrifugal condition in step a is: 4200 r / min.

5. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: anhydrous magnesium sulfate described in step a, C 18 The mass ratio is: 3-5:

1.

6. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: The volume ratio of methanol, isooctane and trimethylsilylated diazomethane-n-hexane described in step a is 1:1:

1.

7. The method for detecting 11 kinds of acidic herbicides in rice according to claim 1, characterized in that: The derivatization temperature in step a is 70° C. and the derivatization time is 5 min.

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