A high-sensitivity method for detecting quinclorac in grains and honey

Abstract

The application provides a high-sensitivity detection method for quinclorac in grains and honey, comprising S1 extraction, S2 derivatization, S3 redissolution and GCMSMS and LCMSMS on-machine analysis. Through the technical scheme of the application, the application adopts iodine ethane to react with quinclorac under alkaline conditions to generate a new compound 3,7-dichloro-8-quinoline carboxylic acid ethyl ester; the derivatization reaction speed is fast, and the reaction can be completed in 60 minutes; the used derivatization reagent is safe and low in cost. The application quantitatively detects the derivatization product 3,7-dichloro-8-quinoline carboxylic acid ethyl ester of quinclorac by GCMSMS, is high in sensitivity, far lower in quantitative limit than the quantitative limit in the prior art method, and can meet the stringent requirements of the market on the detection of quinclorac in food.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of herbicide detection, in particular to a high-sensitivity detection method for quinclorac in grain and honey. BACKGROUND

[0002] Quinclorac (3,7-dichloro-8-quinolinecarboxylic acid), also known as fast kill, is a hormone-type low-toxicity herbicide developed by BASF in 1984, mainly used for the control of barnyard grass in rice fields. With the large-scale application of quinclorac in rice production, its harm to animals and plants has attracted more and more attention. The research of the Joint Meeting of the Pesticide Residue Experts of the Food and Agriculture Organization of the United Nations / World Health Organization (FAO / WHO) (JMPR) shows that quinclorac can be metabolized into quinclorac methyl ester in plants.

[0003] The national standard GB2763-2021 defines the residue of quinclorac as quinclorac and stipulates that the maximum residue limit of quinclorac in rice is 0.1 mg / kg. The national standard method GB23200.43-2016 can meet the detection requirements of substrates with specified limits such as grain. However, with the improvement of consumers' awareness of food safety and their own health, organic food is more and more favored by consumers. The requirement for quinclorac residue in organic grain certification is that it cannot be detected. In addition, Japan is the most important market for the export of Chinese bee products. Since Japan has not set the maximum residue limit of quinclorac for honey substrates, according to the positive list system, honey purchasers generally require that the residue of quinclorac in honey be less than 0.01 mg / kg. For the above reasons, laboratories are required to develop a more sensitive detection method for quinclorac.

[0004] The detection methods of quinclorac residues in food mainly include gas chromatography, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Compared with liquid chromatography-tandem mass spectrometry, the instrument cost of gas chromatography and gas chromatography-mass spectrometry is low, and the instrument popularization rate is higher. Because quinclorac has strong polarity and high boiling point, it needs to be derivatized before detection by gas chromatography and gas chromatography-mass spectrometry. The national standard method GB 23200.43-2016 adopts diazomethane derivatization to generate quinclorac methyl ester, which is detected by gas chromatography; the industry standard method SN / T 2228-2008 adopts trimethylsilane diazomethane derivatization to generate quinclorac methyl ester, which is detected by gas chromatography-mass spectrometry; and the patent CN 109212108 A "Gas chromatography-mass spectrometry method for simultaneously determining three trace quinoline herbicides in rice and soybeans" also adopts trimethylsilane diazomethane derivatization and gas chromatography-mass spectrometry detection. The above three detection methods can realize the accurate quantitative analysis of quinclorac, but their shortcomings are also obvious. Because quinclorac methyl ester is not only the derivative product of the above methods, but also the metabolite of quinclorac, after detecting a positive result, it is necessary to further determine whether the positive result comes from quinclorac or quinclorac methyl ester, which increases the additional workload. In addition, the national standard GB 23200.43-2016 and the industry standard SN / T 2228-2008 use a large amount of acetone, which is a precursor of toxic drugs, bringing inconvenience to the management of the laboratory.

[0005] In addition to the above methyl esterization derivatization of quinclorac, the patent CN 116660424 A "Method for detecting quinclorac in honey" uses five-fluorobenzyl bromide for reaction, and the derivative product is detected by gas chromatography-mass spectrometry. The derivatization reaction of this method needs 12 hours, which is too long and is not conducive to the rapid analysis of samples. SUMMARY

[0006] In order to make up for the shortcomings of the prior art, the present application provides a high-sensitivity detection method for quinclorac in grains and honey.

[0007] The present application is realized by the following technical scheme: a high-sensitivity detection method for quinclorac in grains and honey, specifically comprising the following steps:

[0008] S1 extraction: weigh the sample into a centrifugal tube, add water and 1 mol / L sulfuric acid solution, vortex to immerse the sample, stand, add sodium chloride and ethyl acetate, oscillate, centrifuge; remove all supernatant, add ethyl acetate again, oscillate, centrifuge, combine the supernatant, and dilute to 30 mL with ethyl acetate; remove 10 mL of the extract to a 40℃ water bath and nitrogen blow to dryness;

[0009] S2 derivatization: add 0.5 mL sodium carbonate saturated acetonitrile solution to dissolve the residue, then add 80~300 μL iodoethane, seal and react in a 25~60℃ water bath for 45~120 minutes, then dry under nitrogen in a 35℃ water bath;

[0010] S3 redissolution: for qualitative and quantitative analysis of quinclorac by GCMSMS, add 1 mL ethyl acetate-n-hexane mixed solution (30 / 70, v / v) and 1 mL 1% formic acid-water solution, ultrasonic, vortex, centrifuge at 3000 r / m for 5 minutes, pass the supernatant through a 0.22 μm nylon filter membrane, and analyze by LCMSMS; for confirmation analysis of positive results of quinclorac by LCMSMS, redissolve and dilute to 1 mL with 1% formic acid-methanol, ultrasonic, vortex, pass through a 0.22 μm nylon filter membrane, and analyze by LCMSMS.

[0011] As a preferred solution, step S1 specifically comprises the following steps: weigh 3 g of sample into a 50 mL centrifuge tube, add 15 mL of water and 0.5 mL of 1 mol / L sulfuric acid solution, vortex to soak the sample, stand for 30 minutes, add 3 g of sodium chloride and 15 mL of ethyl acetate, oscillate at 1000 r / m for 1 minute, then centrifuge at 4000 r / m for 5 minutes; remove all the supernatant into a 50 mL graduated cylinder, add 15 mL of ethyl acetate, oscillate at 1000 r / m for 1 minute, then centrifuge at 4000 r / m for 5 minutes, combine the supernatants, and dilute to 30 mL with ethyl acetate; remove 10 mL of the extract to dry under nitrogen in a 40℃ water bath.

[0012] As a preferred solution, the water bath reaction temperature in step S2 derivatization is 40℃.

[0013] As a preferred solution, the water bath reaction time in step S2 derivatization is 60 minutes.

[0014] As a preferred solution, the volume of iodoethane added in step S2 derivatization is 100 μL.

[0015] As a preferred solution, the sample in step S1 is rice or honey.

[0016] As a preferred solution, the instrument method of GCMSMS in step S3 is: chromatographic conditions: chromatographic column: HP-5ms 30 m x 0.25 mm x 0.25 µm; carrier gas: helium; inlet temperature: 280 ℃; splitless injection; injection volume: 1 µL; programmed temperature conditions: 60 ℃ for 1 minute, increased to 170 ℃ at a rate of 40 ℃ / min, and then increased to 310 ℃ at a rate of 10 ℃ / min, and maintained for 3 minutes; mass spectrometry conditions: ionization voltage: 70 V; acquisition mode: multiple reaction monitoring mode (MRM); transfer line temperature: 250 ℃; ion source temperature: 280 ℃; compound mass spectrometry parameters are shown in Table 1;

[0017] Table 1 Mass spectrometry parameters of quinclorac derivatives on GCMSMS

[0018]

[0019] Note: * quantitative ion.

[0020] As a preferred solution, the instrument method of LCMSMS in step S3 is: chromatographic conditions: mobile phase A - 0.1% formic acid aqueous solution; mobile phase B - methanol. Gradient elution conditions are as follows: 0-0.6 min, 90% A; 0.6-2.0 min, 90-50% A; 2.0-6.0 min, 50%-30% A; 6.0-8.0 min, 30%-0% A; 8.0-11.0 min, 0% A; 11.0-11.2 min, 0%-90% A; 11.2-14.0 min, 90% A. The flow rate is 0.3 mL / min, the column temperature is 40 ℃, and the injection volume is 1 µL; mass spectrometry conditions: data are collected in AJS positive ion (ESI+) mode and multiple reaction monitoring (MRM) mode. Ion source parameters are: capillary voltage, 3,000 V; drying gas temperature, 250 ℃; drying gas flow rate, 15 L / min; atomization gas pressure, 35 psi; sheath gas temperature, 350 ℃; sheath gas flow rate, 11 L / min. Compound mass spectrometry parameters are shown in Table 2;

[0021] Table 2 Mass spectrometry parameters of quinclorac derivatives on LCMSMS

[0022]

[0023] Note: * quantitative ion.

[0024] The present application has the following beneficial effects compared with the prior art due to the adoption of the above technical solutions:

[0025] 1. The present application adopts iodoethane to verify quinclorac, generating a new compound 3,7-dichloro-8-quinoline carboxylic acid ethyl ester. The derivatization reaction is fast, and can be completed in 60 minutes. The derivatization reagent used is safe and low in cost. According to the official quotation of Aladdin reagent, the quotation of the derivatization reagent trimethylsilyl diazomethane used in SN / T 2228-2008 and CN 109212108 A "A gas chromatography-mass spectrometry method for simultaneously determining three trace quinoline herbicides in rice and soybeans" is: 100 mL specification 1472.90 yuan (RMB); the quotation of the derivatization reagent pentafluorobenzyl bromide used in CN 116660424 A "A detection method for quinclorac in honey" is: 25 g specification 3004.9 yuan (RMB); the quotation of the derivatization reagent iodoethane used in the present application is: 100 mL specification 259.90 yuan (RMB), which is only 1 / 5 of the price of trimethylsilyl diazomethane and 1 / 40 of the price of pentafluorobenzyl bromide for the same amount (mass / volume).

[0026] 2. The derivatization product 3,7-dichloro-8-quinoline carboxylic acid ethyl ester of quinclorac is quantitatively detected by GCMSMS, which is high in sensitivity and far lower in quantification limit than the quantification limit in the existing literature method, and can meet the stringent requirements of the market for the detection of quinclorac in food. Table 3 shows the quantification limits of the existing literature reported methods and the quantification limit of the present application, which proves the high sensitivity advantage of the present application.

[0027] Table 3 Quantification limits of existing literature reported methods

[0028]

[0029] Additional aspects and advantages of the present application will become apparent from the following description, or will be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0030] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0031] Figure 1 Reaction scheme for the derivatization reaction of quinclorac with iodoethane;

[0032] Figure 2 Primary mass spectrum of the quinclorac derivative on GCMSMS;

[0033] Figure 3 Secondary mass spectrum of the quinclorac derivative on GCMSMS for characteristic ion m / z 197.0

[0034] Figure 4Secondary mass spectrum of dichloroquinox acid derivative at m / z 199.0 characteristic ion on GCMSMS

[0035] Figure 5 Secondary mass spectrum of dichloroquinox acid derivative at m / z 224.0 characteristic ion on GCMSMS

[0036] Figure 6 Secondary mass spectrum of dichloroquinox acid derivative at m / z 226.0 characteristic ion on GCMSMS

[0037] Figure 7 Primary mass spectrum of dichloroquinox acid derivative on LCMSMS,

[0038] Figure 8 Secondary mass spectrum of dichloroquinox acid derivative at m / z 270.0 parent ion on LCMSMS

[0039] Figure 9 Response of derivative product under different derivative temperatures;

[0040] Figure 10 Response value of derivative product under different derivative reaction time;

[0041] Figure 11 Response value of derivative product under different derivative solution volume;

[0042] Figure 12 MRM chromatogram of dichloroquinox acid derivative on GCMSMS (0.2 μg / L);

[0043] Figure 13 MRM chromatogram of dichloroquinox acid derivative on LCMSMS (0.1 μg / L). DETAILED DESCRIPTION

[0044] In order to enable the above objects, features and advantages of the present application to be more clearly understood, the following will further describe the present application with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0045] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in different ways from those described herein, and the scope of the present application is not limited to the specific embodiments disclosed below.

[0046] The following will be described with reference to the accompanying drawings and specific embodiments. Figures 1 to 13 The high-sensitivity detection method of dichloroquinox acid in grains and honey according to the embodiments of the present application will be specifically described.

[0047] The application provides a high-sensitivity detection method for quinclorac in grains and honey, and specifically comprises the following steps:

[0048] S1 extraction: 3 g of sample is taken into a 50 mL centrifuge tube, 15 mL of water and 0.5 mL of 1 mol / L sulfuric acid solution are added, the sample is immersed by vortexing, and then is left to stand for 30 minutes, 3 g of sodium chloride and 15 mL of ethyl acetate are added, oscillation is carried out at 1000 r / m for 1 minute, and then centrifugation is carried out at 4000 r / m for 5 minutes; all the supernatant is removed into a 50 mL measuring cylinder, 15 mL of ethyl acetate is further added, oscillation is carried out at 1000 r / m for 1 minute, and then centrifugation is carried out at 4000 r / m for 5 minutes, the supernatant is combined, and is diluted to 30 mL with ethyl acetate; 10 mL of the extraction solution is removed into a 40℃ water bath and is blown to dryness by nitrogen;

[0049] S2 derivation: 0.5 mL of a saturated sodium carbonate acetonitrile solution is added to re-dissolve the residue, 100 μL of iodoethane is further added, and then sealing is carried out in a 40℃ water bath for 60 minutes, and then the solution is blown to near dryness by nitrogen in a 35℃ water bath;

[0050] S3 re-dissolution: for a GCMSMS method (used for qualitative and quantitative analysis of quinclorac), 1 mL of an ethyl acetate-n-hexane mixed solution (30 / 70, v / v) and 1 mL of a 1% formic acid-water solution are added, ultrasonic treatment is carried out, vortex mixing is carried out, centrifugation is carried out at 3000 r / m for 5 minutes, the supernatant is filtered through a 0.22 μm nylon filter membrane, and then is analyzed by using a machine; for an LCMSMS method (used for verification analysis of positive results of quinclorac), a 1% formic acid-methanol solution is used for re-dissolution and dilution to 1 mL, ultrasonic treatment is carried out, vortex mixing is carried out, the solution is filtered through a 0.22 μm nylon filter membrane, and then is analyzed by using a machine.

[0051] Establishment of a GCMSMS detection method for quinclorac derivatives: the instrument method of GCMSMS in step S3 is as follows: chromatographic conditions: chromatographic column: HP-5ms 30 m x 0.25 mm x 0.25 µm; carrier gas: helium; inlet temperature: 280 ℃; non-split injection; injection volume: 1 µL; programmed temperature conditions: 60 ℃ for 1 minute, increased to 170 ℃ at a rate of 40 ℃ / min, and then increased to 310 ℃ at a rate of 10 ℃ / min, and kept for 3 minutes; mass spectrometry conditions: ionization voltage: 70 V; acquisition mode: multiple reaction monitoring mode (MRM); transfer line temperature: 250 ℃; ion source temperature: 280 ℃; compound mass spectrometry parameters are shown in Table 1;

[0052] Table 1 Mass spectrometry parameters of quinclorac derivatives on GCMSMS

[0053]

[0054] Note: * quantitative ion.

[0055] The standard solution of quinclorac was prepared by derivatization. 10 μL of quinclorac working solution with a concentration of 1000 mg / L was taken into a 15 mL plastic centrifuge tube and dried by nitrogen blowing. 500 μL of sodium carbonate saturated acetonitrile solution was added and mixed by vortex; 500 μL of derivatization solution was added; the reaction was carried out in a water bath at 80°C for 1 hour, and dried by nitrogen blowing to near dryness. 1 mL of ethyl acetate-n-hexane mixed solution (30 / 70, v / v) and 1 mL of 1% formic acid-water solution were added, ultrasonic, vortex mixed, centrifuged at 3000 r / m for 5 minutes, and the supernatant was transferred to an injection vial.

[0056] The derivatization reaction mechanism of quinclorac is shown in Figure 1 . Quinclorac reacts with iodoethane under alkaline conditions to form 3,7-dichloro-8-quinoline carboxylic acid ethyl ester.

[0057] The molecular formula of quinclorac is C 10 H5Cl2NO2, the relative molecular mass is 242.06, and the CAS number is 84087-01-4;

[0058] The molecular formula of iodoethane is C2H5I, the relative molecular mass is 155.97, and the CAS number is 75-03-6;

[0059] The molecular formula of the derivative product 3,7-dichloro-8-quinoline carboxylic acid ethyl ester is C 12 H9Cl2NO2, the relative molecular mass is 270.11.

[0060] The parent ion scan of quinclorac derivative was carried out in full scan mode to obtain the mass spectrum of quinclorac derivative (see Figure 2 ). As can be seen from the figure, the characteristic fragment ions in the mass spectrum of quinclorac derivative are m / z 197, m / z / 199, m / z 224, m / z 226 and m / z 161.

[0061] The four characteristic ions above were selected as parent ions, respectively, in product ion mode, different collision energies were set for scanning, m / z 197 could obtain fragment ions m / z 127 (optimal CE = 40 eV) and m / z 162 (optimal CE = 20 eV); m / z 199 could obtain fragment ions m / z 127 (optimal CE = 40 eV) and m / z 162 (optimal CE = 20 eV) and m / z 164 (optimal CE = 20 eV); m / z 224 could obtain fragment ions m / z 161 (optimal CE = 28 eV) and m / z 196 (optimal CE = 18 eV); m / z 226 could obtain fragment ions m / z 161 (optimal CE = 26 eV), m / z 163 (optimal CE = 26 eV) and m / z 198 (optimal CE = 22 eV), see Figures 3~6 At the optimal collision energy, the sensitivity (signal-to-noise ratio) of m / z 224→161 was the highest, so m / z 224→161 was selected as the quantitative ion pair, and other ion pairs, including m / z 197→m / z 162, m / z 197→m / z 127, m / z 199→m / z 127, m / z 199→m / z 164, m / z 199→m / z 162, m / z 224→m / z 196, m / z 226→161, m / z 226→163 and m / z 226→198 could be used as qualitative ion pairs. Since setting too many ions would occupy the instrument's acquisition time, and usually 1 quantitative ion and 2 qualitative ions could meet the needs of the qualitative analysis of the target, the highest sensitivity m / z 197→m / z 162 and m / z 197→127 ion pairs were selected from the above qualitative ions for method validation and qualitative analysis in daily detection.

[0062] LCMSMS confirmation method of quinclorac derivatives: The instrument method of LCMSMS in step S3 is as follows: Chromatographic conditions: mobile phase A - 0.1% formic acid aqueous solution; mobile phase B - methanol. Gradient elution conditions are as follows: 0-0.6 min, 90% A; 0.6-2.0 min, 90-50% A; 2.0-6.0 min, 50%-30% A; 6.0-8.0 min, 30%-0% A; 8.0-11.0 min, 0% A; 11.0-11.2 min, 0%-90% A; 11.2-14.0 min, 90% A. The flow rate is 0.3 mL / min, the column temperature is 40°C, the injection volume is 1 μL; mass spectrometric conditions: data are collected in AJS positive ion (ESI+) mode and multiple reaction monitoring (MRM) mode. Ion source parameters are as follows: capillary voltage, 3,000 V; dry gas temperature, 250°C; dry gas flow rate, 15 L / min; atomizing gas pressure, 35 psi; sheath gas temperature, 350°C; sheath gas flow rate, 11 L / min. Compound mass spectrometric parameters are shown in Table 2;

[0063] Table 2 Mass spectrometric parameters of quinclorac derivatives on LCMSMS

[0064]

[0065] Note: * quantitative ion.

[0066] A quinclorac standard solution is prepared by derivatization. 1 mL of a 1 mg / L quinclorac working solution is taken into a 15 mL plastic centrifuge tube and dried by nitrogen blowing. 500 μL of a saturated sodium carbonate solution in acetonitrile is added, vortexed; 500 μL of a derivatization solution is added; the mixture is reacted in a water bath at 80°C for 1 hour, dried by nitrogen blowing, 1 mL of a 1% formic acid-methanol solution is added, ultrasonicated, vortexed, filtered through a 0.22 μm nylon filter, and analyzed on the instrument.

[0067] In full scan mode, the parent ion scan of quinclorac derivatives is performed, and [M+H]+ m / z 270.0 and the isotopic peak m / z 272.0, and [M+Na]+ m / z 292.0 and the isotopic peak m / z 294.0 are found in the mass spectrum, as shown in Figure 7 ; in product ion mode, the most abundant m / z 270.0 is set as the parent ion, and different collision energies are scanned to obtain fragment ions m / z 161 (optimal CE = 55 eV), m / z 195.9 (optimal CE = 38 eV), and m / z 224 (optimal CE = 38 eV), as shown in Figure 8The response of three fragment ions at the optimal collision energy is arranged in descending order of sensitivity as follows: m / z 161 > m / z 195.9 > m / z 224.

[0068] Example: selection of derivatization solvent

[0069] The reaction solvent medium is the primary factor affecting the derivatization reaction. Five solvents, including methanol, acetone, acetonitrile, N, N-dimethylformamide, and dimethyl sulfoxide, were tested. It was found that the derivatization reaction could not be carried out in methanol and acetone, and could be carried out in acetonitrile, N, N-dimethylformamide, and dimethyl sulfoxide. However, N, N-dimethylformamide and dimethyl sulfoxide solvents are not easy to evaporate, and the sample cannot be dried by nitrogen blowing for solvent replacement after derivatization. In addition, a deep red by-product is produced after the reaction in dimethyl sulfoxide, which cannot be removed. Therefore, acetonitrile is finally selected as the solvent medium.

[0070] However, the response of the reaction product of quinclorac and iodoethane in pure acetonitrile is relatively low, which cannot meet the requirement of detection sensitivity. It is suspected that the yield of the reaction of quinclorac and iodoethane in pure acetonitrile is low. The effects of adding two commonly used basic substances, triethylamine and sodium carbonate, on the reaction in acetonitrile were compared. It was found that when 0.5% triethylamine was added to the acetonitrile solution, the derivatization reaction of quinclorac and iodoethane could be completed at room temperature for 30 minutes, with fast reaction speed and high efficiency. However, triethylamine is not friendly to mass spectrometry. Therefore, sodium carbonate is preferred in this patent, and a saturated solution of sodium carbonate in acetonitrile is used as the reaction solvent medium. Furthermore, three main conditions, including the verification temperature, the derivatization time, and the volume of the derivatization solution, are optimized.

[0071] Example: optimization of derivatization conditions

[0072] Firstly, the derivatization temperature is optimized. The responses of the standard solution after reaction at room temperature (about 25°C) and at 40°C, 60°C, 80°C, and 95°C are compared. The experimental results are shown in Table 1. Figure 9 As shown in Table 1, when the temperature reaches 40°C from room temperature (about 25°C), the response of the quinclorac derivative is the highest, and the response gradually decreases with the further increase of the temperature. When the temperature reaches 80°C and 95°C, the response is the lowest, which may be related to the evaporation of the solution at high temperature. According to the query, the boiling point of iodoethane is 72°C, and the boiling point of acetonitrile is 81.6°C. At high temperature, especially at 95°C, the volume of the solution in the derivatization test tube is significantly reduced. Therefore, the derivatization reaction is suitable to be carried out at a temperature of 25 to 60°C. In order to achieve the optimal derivatization effect, the derivatization experiment is carried out at a relatively mild condition of 40°C.

[0073] Secondly, the derivatization time is optimized. The responses of the standard solution after reaction at 40°C water bath for 15, 30, 45, 60, 90, and 120 minutes are compared. The experimental results are shown in Table 2. Figure 10: The response changed little after 45 minutes, indicating that the optimal derivatization time was 45-120 minutes. The derivatization reaction time was selected as 60 minutes in the experiment.

[0074] Finally, the volume of the derivatization solution was optimized. The responses of the standard solution after derivatization with 20, 40, 60, 80, 100, 150, 200, 250, and 300 μL of iodoethane were compared. The experimental results are shown in Table 3. Figure 11 : The response value gradually increased slowly as the volume of the derivatization solution increased from 20 μL to 80 μL, and the response value changed little after 80 μL, indicating that the optimal addition volume of the derivatization solution was 80-300 μL. In order to obtain more stable results, the volume of the derivatization reagent was selected as 100 μL in the experiment.

[0075] Results

[0076] 1. Linearity

[0077] Rice and honey were used as matrix samples for sample pretreatment, and matrix-matched standard samples of 0.5, 1, 2, 5, 10, 20, and 50 μg / L were prepared. GC-MS / MS analysis was performed, and the samples were quantified by the external standard method. The peak area was used as the vertical coordinate (y), and the mass concentration was used as the horizontal coordinate (x) to draw the matrix-matched standard curve. The results showed that the linear equation of the rice matrix was y = 6122.6929x + 2859.9902, the determination coefficient R 2 was 0.9977, and the linear equation of the honey matrix was y = 9461.3428x + 3606.7965, the determination coefficient R 2 was 0.9991; indicating that the linearity of the quinclorac derivative in the two matrices was good.

[0078] 2. Matrix effect

[0079] The matrix effect (ME) represents the influence of co-eluting components on the ionization efficiency of the analyte (matrix enhancement or matrix suppression), which ultimately affects the accuracy of quantitative analysis. The ME value was calculated by the following equation: ME = (peak area of matrix-matched standard sample / peak area of solvent standard sample - 1) × 100%. The results showed that the ME value of the rice matrix was 18.0%, indicating a weak matrix enhancement effect. The ME value of the honey matrix was 0.1%, and the matrix effect could be ignored.

[0080] 3. Limit of quantification

[0081] The pre-treatment concentration multiple of the method is 1, and the matrix effect is small, so the signal-to-noise ratio of the solvent standard is used to determine the quantitative limit of the method. Prepare solvent standards of different concentrations (0.1, 0.2, 0.5, 1 μg / L), and determine the quantitative limit of the method according to the signal-to-noise ratio (S / N) of the analyte ≥10. After calculation, the quantitative limit of the GCMSMS quantitative method is 0.2 μg / kg, and the quantitative limit of the LCMSMS confirmation method can be as low as 0.1 μg / kg. The chromatogram of the quinclorac derivative at the quantitative limit concentration level is shown in Figure 12 and Figure 13 .

[0082] 4. Accuracy and precision

[0083] In rice and honey matrices, standard solutions were added at levels of 1, 10 and 50 μg / kg, respectively, and each concentration level was determined in parallel for 5 times. The results: the average recovery rate was 87.6%~103.8%; the relative standard deviation (RSD) was 1.9%~9.9%, and the accuracy and precision met the standard requirements of the pesticide residue detection method verification.

[0084] Table 4. Spiked recovery rate and relative standard deviation (n=5)

[0085]

[0086] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "a specific embodiment" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0087] The above is only the preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the high sensitive detection of quinclorac in cereals and honey, characterized by , specifically comprising the following steps: S1 extraction: weigh the sample into a centrifuge tube, add water and 1 mol / L sulfuric acid solution, vortex the sample to soak, stand, add sodium chloride and ethyl acetate, shake, centrifuge; remove all supernatant, add ethyl acetate again, shake, centrifuge, combine the supernatant, and dilute to 30 mL with ethyl acetate; remove 10 mL of the extract to a 40℃ water bath and nitrogen blow to dryness; S2 derivatization: add 0.5 mL of a saturated sodium carbonate acetonitrile solution to redissolve the residue, then add 80-300 μL of iodoethane, seal and react in a 25-60℃ water bath for 45-120 minutes, then nitrogen blow to near dryness in a 35℃ water bath; S3 redissolution: for GC-MS / MS qualitative and quantitative analysis of quinclorac, add 1 mL of ethyl acetate-hexane mixed solution and 1 mL of 1% formic acid-water solution, ultrasonic, vortex, centrifuge at 3000 r / m for 5 minutes, pass the supernatant through a 0.22 μm nylon filter membrane, and analyze on the machine; the instrument method of GC-MS / MS is: chromatographic conditions: chromatographic column: HP-5ms 30 m x 0.25 mm x 0.25 μm; carrier gas: helium; inlet temperature: 280℃; no split injection; injection volume: 1 μL; programmed temperature conditions: 60℃ for 1 minute, increased to 170℃ at 40℃ / min, then increased to 310℃ at 10℃ / min, and kept for 3 minutes; mass spectrometry conditions: ionization voltage: 70V; acquisition mode: multiple reaction monitoring mode; transfer line temperature: 250℃; ion source temperature 280℃; compound mass spectrometry parameters are shown in Table 1; Table 1 Mass spectrometry parameters of quinclorac derivative on GC-MS / MS Note: * quantitative ion; For LC-MS / MS used for confirmation analysis of positive results of quinclorac, redissolve and dilute to 1 mL with 1% formic acid-methanol, ultrasonic, vortex, pass through a 0.22 μm nylon filter membrane, and analyze on the machine; the instrument method of LC-MS / MS is: chromatographic conditions: mobile phase A-0.1% formic acid aqueous solution; mobile phase B-methanol; gradient elution conditions as follows: 0-0.6 min, 90% A; 0.6-2.0 min, 90-50% A; 2.0-6.0 min, 50%-30% A; 6.0-8.0 min, 30%-0% A; 8.0-11.0 min, 0% A; 11.0-11.2 min, 0%-90% A; 11.2-14.0 min, 90% A; flow rate is 0.3 mL / min, column temperature is 40℃, injection volume is 1 μL; mass spectrometry conditions: data are collected in AJS positive ion mode and multiple reaction monitoring mode; ion source parameters are: capillary voltage, 3,000 V; drying gas temperature, 250℃; drying gas flow rate, 15 L / min; atomization gas pressure, 35psi; sheath gas temperature, 350℃; sheath gas flow rate, 11 L / min; compound mass spectrometry parameters are shown in Table 2; Table 2 Mass spectrometry parameters of quinclorac derivatives on LC-MS / MS Note: * Quantitative ion.

2. A method for the high sensitive determination of quinclorac in cereals and honey according to claim 1, characterized in that The step S1 specifically comprises the following steps: weighing 3 g of sample into a 50 mL centrifuge tube, adding 15 mL of water and 0.5 mL of 1 mol / L sulfuric acid solution, vortexing to soak the sample, standing for 30 minutes, adding 3 g of sodium chloride and 15 mL of ethyl acetate, oscillating at 1000 r / m for 1 minute, and then centrifuging at 4000 r / m for 5 minutes; removing all supernatant into a 50 mL measuring cylinder, adding 15 mL of ethyl acetate, oscillating at 1000 r / m for 1 minute, and then centrifuging at 4000 r / m for 5 minutes, combining the supernatant, and diluting to 30 mL with ethyl acetate; and removing 10 mL of the extract to a 40℃ water bath for nitrogen blowing to dryness.

3. A method for the high sensitive determination of quinclorac in cereals and honey according to claim 1, characterized in that The water bath reaction temperature in the step S2 derivation is 40℃.

4. A method for the high sensitive determination of quinclorac in cereals and honey according to claim 1, characterized in that The water bath reaction time in the step S2 derivation is 60 minutes.

5. A method for the high sensitive determination of quinclorac in cereals and honey according to claim 1, characterized in that The volume of iodomethane added in the step S2 derivation is 100 μL.

6. A method for the high sensitive determination of quinclorac in cereals and honey according to claim 1, characterized in that The sample in the step S1 is rice.

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