Liquid chromatography-tandem mass spectrometry determination method for herbicide imazapyr and haloxyfop-R-methyl in aquatic products

Abstract

The invention relates to a liquid chromatogram-tandem mass spectrum determination method for herbicide imazapyr and haloxyfop-R-methyl in aquatic products, through a simple sample pretreatment method and an instrument analysis method, the detection sample range is increased to various aquatic product matrixes such as fishes, shrimps, crabs and shellfishes, and the method is simple, convenient, rapid and high in sensitivity. The method has the advantages of low reagent dosage, high sensitivity, low cost, high accuracy and good stability. According to the method, a technical means is provided for evaluating the toxic effect of the Spartina alterniflora herbicide, i.e., the melatryn and the cyhalofop-ethyl on aquatic organisms and monitoring and evaluating the risk of the Spartina alterniflora.

Description

Technical Field

[0001] The invention belongs to the field of aquatic product detection, and particularly relates to a liquid chromatography-tandem mass spectrometry method for determining herbicides imazapyr and haloxyfop-ethyl in aquatic products. Background Art

[0002] In recent years, sustained population growth has brought major challenges to increasing crop production. In order to meet the growing demand for food, pesticides are overused in the process of crop cultivation. Excessive pesticides enter water, soil and air, causing serious damage to biological and human health. Imazapyr (25% imazapyr aqueous solution) and haloxyfop-P-methyl (10.8% high-efficiency fluazifop-methyl emulsifiable concentrate) are two herbicides that have the characteristics of fast weed control, strong systemic effect and high safety to crops. Therefore, they are widely used in weed control of crops such as corn and sunflowers and in the control of Spartina alterniflora. The effective herbicidal ingredient of Imazapyr is imazapyr, which is a broad-spectrum herbicide and a commonly used herbicide. Its half-life in the soil is longer than that of haloxyfop-P-methyl. The effective weed-killing ingredient of haloxyfop-P-methyl is high-efficiency fluazifop-methyl, which is a non-broad-spectrum herbicide. It has a good treatment effect on grass weeds. Its efficacy is stable and is less affected by adverse environmental conditions such as low temperature and rain. Imidazolin and halofop-butyl are generally not susceptible to photochemical degradation or thermal decomposition and are relatively stable in the environment. The long-term and irrational use of such herbicides has caused them to pollute the water environment at an alarming rate, and they are often detected as pollutants in the environment. In recent years, Spartina alterniflora has received great attention due to its large-scale invasion around the world. Spartina alterniflora mainly invades coastal tidal wetlands and estuary areas. During the process of spraying Kangmijing and Gaoxuegaineng to control Spartina alterniflora, some unabsorbed drugs enter the adjacent sea areas, which may have adverse effects on nearshore marine aquaculture and marine ecological environment. Studies have found that imidazolin and halofop-butyl have certain bioaccumulation, and excessive use of pesticides poses a threat to aquatic animals and human health. Therefore, many countries have set limits on pesticide residues in food. In my country's "GB 2763-2021 National Food Safety Standard Maximum Residue Limits of Pesticides in Foods", it is clearly stipulated that the limit of imazapyr in different foods is 0.01-5 mg / kg, and the maximum limit of high-efficiency fluazifop-p-ethyl is 0.02-3 mg / kg.

[0003] At present, the methods for detecting the herbicides imazapyr and haloxyfop-ethyl include GC, FIA / SEC, SERS, LC, LC-MS, etc., but the detection range is limited to cereals, vegetables, water bodies and soil, etc., and the detection method for the residues of imazapyr and haloxyfop-ethyl in aquatic products (fish, shrimps, crabs, shellfish, etc.) has not been reported. All of the above have brought great inconvenience to the evaluation of the toxic effects of the herbicides Kangmijing and Gaicaoneng in aquatic organisms and their risk monitoring and assessment. Therefore, it is urgent to develop a detection method with simple pre-treatment, strong selectivity and high sensitivity. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a liquid chromatography-tandem mass spectrometry method for determining the herbicides imazapyr and haloxyfop-ethyl in aquatic products.

[0005] The present invention provides a liquid chromatography-tandem mass spectrometry method for determining the herbicides imazapyr and haloxyfop-ethyl in aquatic products, comprising:

[0006] (1) adding acidified acetonitrile to the pretreated water product, vortexing, ultrasonicating, centrifuging, and taking the supernatant to obtain a liquid to be purified;

[0007] (2) The liquid to be purified, anhydrous sodium sulfate and dispersed solid phase material C 18 The mixture was mixed, vortexed, and rotary evaporated to dryness after purification, and an acetonitrile aqueous solution was added for oscillation, and filtered to obtain a treated water product, which was then determined by liquid chromatography-tandem mass spectrometry.

[0008] Preferably, the aquatic product in step (1) comprises one or more of fish, shrimp, crab and shellfish; the pretreated aquatic product is obtained by mincing and mixing the edible part of the aquatic product, such as removing the head, bones and internal organs of the aquatic product, mincing and mixing the edible parts respectively, to obtain the pretreated aquatic product, and storing it below -18°C.

[0009] Preferably, the volume percentage concentration of the acid in the acidified acetonitrile (extraction reagent) in step (1) is 0.1%-1%, more preferably 1% acidified acetonitrile, which can effectively improve the recovery rate of imidazopyric acid; the acidified acetonitrile is formic acid acetonitrile; the acidified acetonitrile is added in 1 to 3 times.

[0010] Furthermore, adding 1% formic acid and acetonitrile for two extractions can effectively improve the recovery rate of the analyte.

[0011] The preparation process of the acidified acetonitrile is: taking acetonitrile and adding formic acid; wherein the volume ratio of acetonitrile to formic acid is 990:10.

[0012] In the step (1), the vortexing speed is 2000-2500 r / min, and the vortexing time is 5-10 min; the ultrasonic time is 10-20 min; the centrifugation speed is 4000-8000 r / min, and the centrifugation time is 2-5 min.

[0013] Furthermore, in step (1), the vortex speed is 2000-2500r / min, the vortex time is 5-10min, preferably 2500r / min vortexing for 10min, which can disperse the fish meat to the greatest extent and fully contact it with the extraction reagent, thereby improving the recovery rate of the analyte; the ultrasonic time is 10-20min, preferably 10min, which is mainly because the recovery rates of the analytes after 10min and 20min of ultrasound are similar, and selecting 10min of ultrasound can save pretreatment time and improve detection efficiency; the centrifugal speed is 4000-8000r / min, preferably 4000r / min, and the centrifugal time is 2-5min, preferably 5min, which is mainly because 8000r / min has higher requirements for instruments and equipment, while selecting 4000r / min centrifugal 5min can achieve the ideal effect, so the preferred centrifugal speed is 4000r / min and the centrifugal time is 5min.

[0014] Preferably, the vortex time in the step (2) is 0.5-3 min; and the filtration is performed using a 0.22 μm polytetrafluoroethylene filter membrane.

[0015] Preferably, the ratio of the pretreated water product to the acidified acetonitrile in step (1) is 1-2 g: 5-10 mL;

[0016] The aquatic product, anhydrous sodium sulfate and dispersed solid phase material C pretreated in step (2) 18 The ratio is 1-2g:200-500mg:100-300mg.

[0017] Furthermore, in step (2), the QuEChERS purification method is used, and anhydrous sodium sulfate and octadecylsilane dispersed solid phase extraction material (C 18 ), this step effectively reduces the purification steps, greatly saves pretreatment time, reduces the use of organic reagents, is more environmentally friendly, and after testing, the QuEChERS method is suitable for the pretreatment of pesticide residues in animal-derived foods, which can reduce the impact of sample matrix on instrumental analysis.

[0018] Preferably, the determination by liquid chromatography-tandem mass spectrometry in step (2) comprises:

[0019] S1: preparing a mixed standard series of solutions of the herbicides imazapyr and haloxyfop-ethyl at different concentrations, and performing liquid chromatography-tandem mass spectrometry determination to obtain a standard curve;

[0020] S2: Perform liquid chromatography-tandem mass spectrometry testing on the treated water products.

[0021] Preferably, the concentration ranges of the herbicides imazapyr and haloxyfop-ethyl in S1 are both 0.5 μg / L to 50 μg / L.

[0022] The mixed standard series solutions of different concentrations of the herbicides imazapyr and haloxyfop-butyl are prepared in S1 and include:

[0023] a: Preparation of standard stock solutions of herbicides imazapyr and haloxyfop-butyl: 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) nicotinic acid and methyl 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy] propionate were dissolved in methanol and added to a 10 mL volumetric flask, and the volume was adjusted to the mark with methanol, and the mixture was mixed to obtain standard stock solutions of herbicides imazapyr and haloxyfop-butyl, respectively, and the solutions were stored at -18°C in the dark; b: Preparation of mixed standard intermediate solutions of herbicides imazapyr and haloxyfop-butyl: The standard stock solutions of herbicides imazapyr and haloxyfop-butyl were placed in a 50 mL volumetric flask, diluted with methanol and adjusted to the mark, and the mixture was mixed to obtain standard stock solutions of herbicides imazapyr and haloxyfop-butyl, respectively, and the solutions were stored at -18°C in the dark. c: Preparation of mixed standard working solution of herbicide imazapyr and high-efficiency haloxyfop-butyl: place the mixed standard intermediate solution of herbicide imazapyr and high-efficiency haloxyfop-butyl in a 100mL volumetric flask, dilute and make up to the scale with methanol, mix well, and store at -18℃ away from light; d: 0.1mL, 0.25mL, 0.5mL, 1.0mL, 2.5mL, and 5.0mL of the mixed standard working solution of herbicide imazapyr and high-efficiency haloxyfop-butyl were placed in 10mL volumetric flasks, respectively, diluted and made up to the volume with acetonitrile aqueous solution to obtain a mixed standard series solution with a concentration gradient of 0.5μg / L, 1μg / L, 2μg / L, 5μg / L, 10μg / L, 20μg / L, and 50μg / L.

[0024] Further, a: Preparation of standard stock solutions of herbicides imazapyr and halpyrazole: 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) nicotinic acid and methyl 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy) phenoxy] propionate were dissolved in methanol, respectively, and after dissolution, they were transferred to a 10 mL brown volumetric flask, and the volume was adjusted to the mark with methanol, and the solution was mixed to prepare a concentration of 1.0 mg / mL. The standard stock solutions of herbicides imazapyr and haloxyfop-butyl were stored at -18℃ away from light, and the validity period was 1 year; b: Preparation of mixed standard intermediate solution of herbicides imazapyr and haloxyfop-butyl: Accurately pipette 1.00mL of each of the standard stock solutions of herbicides imazapyr and haloxyfop-butyl, place them in a 50mL volumetric flask, dilute with methanol and make up to the mark, mix well, and the concentration is 20.0μg / mL, store at -18℃ away from light, and The validity period is 3 months; c: Preparation of mixed standard working solution of herbicide imazapyr and high-efficiency haloxyfop-butyl: Accurately pipette 1.00mL of mixed standard intermediate solution of herbicide imazapyr and high-efficiency haloxyfop-butyl, place it in a 100mL volumetric flask, dilute it with methanol and make up to the mark, mix well, the concentration is 200ng / mL, store at -18℃ away from light, the validity period is 1 month; d: Accurately pipette 0.025mL, 0.05mL, 0. 1 mL, 0.25 mL, 0.5 mL, 1.0 mL, and 2.5 mL of the mixed standard working solution of herbicides imazapyr and haloxyfop-butyl are placed in 10 mL volumetric flasks, respectively, and diluted and fixed to volume with 80% acetonitrile aqueous solution, and used immediately after preparation; a series of mixed standard solutions with a concentration gradient of 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, and 50 μg / L are obtained;

[0025] The preparation method of 80% acetonitrile aqueous solution is as follows: take 400 mL of acetonitrile, dilute it to 500 mL with water, and mix it to obtain an acetonitrile aqueous solution with a concentration of 80%;

[0026] Preferably, the liquid chromatography-tandem mass spectrometry determination process in S1 and S2 includes: liquid chromatography conditions: Capcell PAK C 18 MG II chromatographic column (100 mm × 2.0 mm, 5 μm), column temperature: 40 °C, flow rate: 0.4 mL / min, injection volume: 10 μL, mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile;

[0027] Preferably, the mass spectrometry conditions are: positive ion scanning, spray voltage: 5500 V, ion transmission capillary temperature: 550° C., curtain gas pressure CUR of 35 psi, nebulizer gas pressure GS1 of 55 psi, auxiliary heating gas pressure GS2 of 55 psi, and collision gas CAD of 5 psi.

[0028] Preferably, different collision energies are used as mass spectrometry conditions for different ion fragments, and the mass spectrometry conditions in S1 and S2 are as follows:

[0029]

[0030] The standard curve in S1 is drawn with the peak area of ​​the characteristic ion mass of the herbicide imazapyr as the ordinate and the concentration of the herbicide imazapyr as the abscissa. Figure 2 shown.

[0031] The standard curve in S1 is drawn with the high-efficiency haloxyfop-butyl characteristic ion mass peak area in the standard solution as the ordinate and the high-efficiency haloxyfop-butyl concentration as the abscissa, such as Figure 3 shown.

[0032] Preferably, the liquid chromatography-tandem mass spectrometer in S2 qualitatively and quantitatively detects the herbicides imazapyr and fluazifop-ethyl in aquatic products; wherein the detection limit (LOD) of the qualitative determination method is 0.25 μg / kg, and the limit of quantification (LOQ) of the quantitative determination is 0.5 μg / kg; wherein the quantification is performed by a standard curve method.

[0033] After the aquatic product is pretreated, the edible part is taken, homogenized, extracted twice with acidified acetonitrile, vortexed, ultrasonicated, centrifuged, the supernatant is taken, anhydrous sodium sulfate and octadecylsilane (C 18 ) After the adsorbent is purified, it is rotary evaporated to dryness, and then dissolved in acetonitrile aqueous solution, filtered with a microporous filter membrane, and then detected by liquid chromatography-tandem mass spectrometry. The present invention only requires a small amount of acidified acetonitrile extraction (such as 10 mL of acidified acetonitrile extraction for every 2 g of aquatic product), and the purification step only requires a small amount of C 18 Dispersed solid phase materials save purification time compared with existing methods, and greatly reduce the use of organic reagents, which is an efficient and environmentally friendly method. Therefore, the present invention not only increases the detection sample range to various aquatic product matrices such as fish, shrimps, crabs, and shellfish through simple sample pretreatment methods and instrumental analysis methods, but also has a simple and fast method, with low reagent dosage, high sensitivity, low cost, high accuracy, and good stability. This method provides technical means for evaluating the toxic effects of the herbicides Kangmijing and Gaicaoneng on aquatic organisms and their risk monitoring and assessment.

[0034] Beneficial Effects

[0035] (1) The present invention only needs to use a small amount of acidified acetonitrile for extraction, and the purification step adopts the QuEChERS purification method, and the liquid chromatography-tandem mass spectrometry method is used for determination, which saves the extraction and purification time compared with the traditional method, and greatly reduces the use of organic reagents, and is an efficient and environmentally friendly method. Therefore, the present invention not only increases the detection sample range to a variety of aquatic product matrices such as fish, shrimp, crab and shellfish through a simple sample pretreatment method and instrumental analysis method, but also the method is simple and fast, the reagent dosage is small, and the cost of the experimental process is reduced.

[0036] (2) Under different samples and different spike levels, the recoveries of the herbicides imazapyr and haloxyfop-butyl were in the range of 70-110%, and the RSD was no more than 10%, indicating that the method has high accuracy and good stability.

[0037] (3) The detection limit of the method of the present invention is only 0.25 μg / kg, which greatly improves the detection sensitivity and can be used for routine monitoring and risk assessment of the herbicide imazapyr and haloxyfop-butyl residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the quantitative ion chromatogram of the herbicide imazapyr and high-efficiency haloxyfop-butyl standard solution of the present invention.

[0039] Figure 2 It is a standard curve diagram of the herbicide imazapyr of the present invention (including linear equation and correlation coefficient).

[0040] Figure 3 It is a standard curve diagram of the herbicide high-efficiency haloxyfop-methyl of the present invention (including linear equation and correlation coefficient). DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0042] The reagents and preparation methods used in the method provided by the present invention are as follows:

[0043] Unless otherwise specified, all reagents used are analytical grade, and the experimental water is first-grade water specified in GB / T 6682; Formic acid (CH3COOH): chromatographic grade; Acetonitrile (CH3CN): chromatographic grade; 80% acetonitrile aqueous solution: dilute 400mL acetonitrile to 500mL with water and mix well;

[0044] Mobile phase A (0.1% formic acid in water): Take 1 mL of formic acid, dilute to 1000 mL with water, and mix well.

[0045] The standard products of the herbicides imazapyr and haloxyfop-butyl used in the method provided by the present invention are as follows:

[0046] Imazapyr (C 13 H 15 N3O3, CAS No.: 81334-34-1) standard (purity ≥ 98%);

[0047] Haloxyfop-P-methyl, C 16 H 13 ClF3NO4, CAS No.: 72619-32-0) standard (purity ≥ 95%).

[0048] Example 1

[0049] The liquid chromatography-tandem mass spectrometry method for the determination of the herbicides imazapyr and haloxyfop-ethyl in aquatic products comprises the following steps:

[0050] 1) Preparation of standard stock solutions of herbicides imazapyr and haloxyfop-butyl: 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) nicotinic acid and methyl 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy] propionate were dissolved in methanol, respectively, and the solution was transferred to a 10 mL brown volumetric flask, and the volume was made up to the mark with methanol, and the mixture was mixed to prepare standard stock solutions of herbicides imazapyr and haloxyfop-butyl with a concentration of 1.0 mg / mL, respectively, and the solutions were stored at -18°C away from light, and the validity period was 1 year;

[0051] 2) Prepare a mixed standard intermediate solution using the standard stock solution obtained in step 1): accurately pipette 1.00 mL of each of the herbicide imazapyr and the high-efficiency haloxyfop-butyl standard stock solution, place in a 50 mL volumetric flask, dilute with methanol and make up to the mark, mix well, the concentration is 20.0 μg / mL, store at -18°C away from light, and the validity period is 3 months;

[0052] 3) Prepare a mixed standard working solution using the mixed standard intermediate solution obtained in step 2): accurately pipette 1.00 mL of the mixed standard intermediate solution of herbicides imazapyr and haloxyfop-butyl, place it in a 100 mL volumetric flask, dilute it with methanol and make up to the mark, mix well, the concentration is 200 ng / mL, store it at -18°C away from light, and the validity period is 1 month;

[0053] 4) using the mixed standard working solution of the herbicide imazapyr and the high-efficiency haloxyfop-butyl obtained in step 3) to prepare a mixed standard series solution, specifically comprising the following steps: accurately pipetting 0.025mL, 0.05mL, 0.1mL, 0.25mL, 0.5mL, 1.0mL, and 2.5mL of the mixed standard working solution of the herbicide imazapyr and the high-efficiency haloxyfop-butyl, respectively placing them in 10mL volumetric flasks, diluting and fixing the volume with 80% acetonitrile aqueous solution, and preparing and using them immediately; obtaining a mixed standard series solution with a concentration gradient of 0.5μg / L, 1μg / L, 2μg / L, 5μg / L, 10μg / L, 20μg / L, and 50μg / L;

[0054] The preparation method of 80% acetonitrile aqueous solution is as follows: take 400 mL of acetonitrile, dilute it to 500 mL with water, and mix well to obtain 80% acetonitrile aqueous solution.

[0055] 5) Liquid chromatography conditions: using Capcell PAK C 18 MG II chromatographic column (100 mm × 2.0 mm, 5 μm), column temperature: 40 °C, flow rate: 0.4 mL / min, injection volume: 10 μL, mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile, and the specific gradient elution program is shown in Table 1;

[0056] 6) Positive ion scanning, spray voltage: 5500 V, ion transmission capillary temperature: 550°C, 35 psi air curtain gas pressure CUR, 55 psi nebulizer gas pressure GS1, 55 psi auxiliary heating gas pressure GS2, 5 psi collision gas CAD, different collision energies were used as mass spectrometry conditions for different ion fragments, and the multi-reflection monitoring MRM parent ion, daughter ion and collision energy are shown in Table 2;

[0057] 7) The mixed standard series solution obtained in step 4) is used to prepare a standard curve using the liquid chromatography determination conditions set in step 5) and the mass spectrometry determination conditions set in step 6), and qualitative and quantitative determinations of the herbicides imazapyr and haloxyfop-butyl in aquatic products are performed.

[0058] Table 1 Gradient elution program

[0059]

[0060]

[0061] The provided liquid chromatography-tandem mass spectrometry method for the determination of the herbicides imazapyr and haloxyfop-ethyl is suitable for the determination of the herbicides imazapyr and haloxyfop-ethyl in various aquatic products such as fish, shrimp, crab, and shellfish.

[0062] Table 2 Qualifying ions, quantitative ions and collision energies of the herbicides imazapyr and haloxyfop-butyl

[0063]

[0064] The detection limit (LOD) and quantification limit (LOQ) of this method are: when the solid sample weight is 2 g and the fixed volume of the extract is 1.00 mL, the detection limit and quantification limit of the herbicides imazapyr and haloxyfop-butyl are 0.25 μg / kg and 0.5 μg / kg, respectively.

[0065] The principle of the liquid chromatography-tandem mass spectrometry determination method for determining the herbicides imazapyr and haloxyfop-butyl provided by the present invention is that the sample is ultrasonically extracted with acidified acetonitrile, centrifuged, the supernatant is taken, purified by the QuEChERS method, rotary evaporated to dryness at 48°C, redissolved in an acetonitrile aqueous solution, and determined by a liquid chromatography-tandem mass spectrometer in a multiple reaction monitoring mode (positive ion mode).

[0066] The method for drawing the standard curve of the method provided by the present invention is as follows:

[0067] The mixed standard series solution of the test substance was injected into the liquid chromatography-tandem mass spectrometer, and the peak area of ​​the characteristic ions of the herbicides imazapyr and haloxyfop-butyl was used as the ordinate, and the concentration of the herbicides imazapyr and haloxyfop-butyl was used as the abscissa to prepare a standard curve, such as Figure 2 and Figure 3 shown.

[0068] The qualitative determination method provided by the present invention is as follows:

[0069] Under the same conditions, the retention time and relative abundance of characteristic ions of the sample solution and the standard solution with similar concentration are compared for qualitative analysis. If the relative deviation of the retention time of the analyte in the sample and the standard solution is no more than ±2.5%, and the relative abundance tolerance of the detected characteristic ions meets the requirements of Table 3, it can be determined that the corresponding analyte exists in the sample.

[0070] Table 3 Permissible deviation of relative ion abundance in qualitative confirmation

[0071]

[0072]

[0073] The quantitative determination method provided by the present invention is as follows:

[0074] Take the sample solution and the mixed standard series solution for determination, draw the standard working curve with the peak area in the standard solution as the ordinate and the concentration of the mixed standard series solution as the abscissa, and use the standard working curve to quantify the sample. The response values ​​of the analyte in the sample solution and the standard series solution should be within the linear range of the instrument. When the concentration of the sample exceeds the linear range, it needs to be diluted according to the measured concentration and re-measured.

[0075] like Figure 1 The following are the characteristic ion chromatograms of the standard solutions of the herbicides imazapyr and haloxyfop-butyl, both of which have a concentration of 5 ng / mL. Figure 1 It can be seen that the peak shapes of the characteristic ion peaks of the herbicides imazapic acid and fluazifop-butyl are symmetrical, without problems such as tailing and impurity interference, indicating that the target compounds can be qualitatively and quantitatively identified under the mass spectrometry and chromatographic conditions.

[0076] The blank experiment of the present invention is to use the same determination steps to operate except that no sample is added.

[0077] The instruments and consumables used in the present invention are as follows:

[0078] Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI);

[0079] Analytical balance: sensitivity 0.00001g and 0.01g;

[0080] Homogenizer;

[0081] Vortex mixer;

[0082] Ultrasonic extractor;

[0083] Centrifuge: speed ≥4000r / min;

[0084] Polytetrafluoroethylene hydrophobic filter membrane (Jinlong): 0.22μm.

[0085] The content of the herbicides imazapyr and haloxyfop-ethyl in the sample of the present invention is expressed as a mass fraction X in micrograms per kilogram (μg / kg), calculated according to formula (1), and the blank value needs to be deducted from the calculation result.

[0086]

[0087] Where:

[0088] C—mass concentration of the substance to be tested in the sample solution obtained from the standard curve, in micrograms per liter (μg / L);

[0089] V—volume of the solution, in milliliters (mL);

[0090] n—the dilution multiple of the sample solution after exceeding the linear range;

[0091] m—sample mass, in grams (g).

[0092] Note: It is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions. The blank value must be deducted from the calculated result and the result must be rounded to three significant figures.

[0093] Before analyzing the actual sample, the laboratory must achieve the required precision and accuracy level. Verify the reliability of the analytical method by analyzing the spiked sample. Take no less than 3 blank samples and add the standard series solution respectively. Analyze the prepared spiked sample according to the above method. The recovery rate of each target compound should be in the range of 70-120%, and the precision (RSD) should not exceed 15%. If a standard reference material with a similar matrix to the sample is available, the standard reference material can be used instead of the spiked sample for precision and accuracy testing.

[0094] After adding acidified acetonitrile during extraction, the extraction needs to be fully shaken to ensure that the tissue is completely and evenly dispersed in the extraction reagent.

[0095] From the structures of the herbicides imazapyr and halpyrazole, we can see that both pesticides are highly polar compounds. Therefore, the experiment compared the extraction effects of acetonitrile, methanol, ethyl acetate, and acetone as extraction solvents. At the same time, n-hexane, PSA adsorbent, C 18 Adsorbents and ODS-C 18 The results showed that the use of 1% formic acid acetonitrile as an extractant and the addition of C 18 The adsorbent was used for purification, which had simple steps, small amount of reagents, high sensitivity, low cost, high accuracy and good stability. The effects of different extractants and purifiers on the recovery of the two herbicides in Chinese sea bass are shown in Table 4.

[0096] Table 4 Effects of different extractants and purifiers on the recovery of two herbicides in Chinese sea bass (1 μg / kg, n=3).

[0097]

[0098] During the extraction process, if the sample is not dispersed well, you can use a homogenizer or a glass rod to crush and disperse it so that it can fully contact with the extraction reagent.

[0099] During the rotary evaporation concentration process, the temperature should not be too high (50°C). The temperature must be low when starting to heat, and the temperature should be raised slowly to avoid violent boiling of the solution, otherwise the recovery rate will be affected.

[0100] When dissolving the residue with 80% acetonitrile aqueous solution, it is necessary to vortex thoroughly before passing through the membrane machine.

[0101] A method blank test is required for each batch of samples.

[0102] It is recommended that a quality control sample be included with each batch of samples. The quality control sample can be a standard reference or a spiked sample with a known concentration. The measured value of the target compound should be within 70-120% of the standard value based on the spiked concentration.

[0103] Use calibration solution to promptly calibrate the mass spectrometer to ensure that the mass number of the monitored ions does not change.

[0104] In order to ensure the accuracy of the analysis results, it is required that 10% of the parallel samples be measured when analyzing each batch of samples, and the relative deviation of the parallel sample results should not exceed 15%.

[0105] During the measurement process, it is recommended to intersperse the measurement of the same standard solution with the sample measurement to check the stability of the instrument.

[0106] Example 2

[0107] In this embodiment, the liquid chromatography-tandem mass spectrometry method for determining the herbicides imazapyr and haloxyfop-ethyl in various aquatic products such as fish, shrimp, crab, and shellfish comprises the following steps:

[0108] B1: Pre-processing of aquatic products such as fish, shrimp, crab, shellfish, bullfrog, turtle, etc., specifically:

[0109] Fish: remove scales, shells, viscera, and take out edible parts such as muscle; Shrimp: remove head, shells, and intestinal glands, and take out muscle parts; Shellfish: remove shells and take out edible parts; Crab: remove shells and take out edible parts;

[0110] Then mince, mix homogenously and set aside;

[0111] B2: All samples were stored below -18°C;

[0112] B3: Weigh 2 g of the sample from step B2 (accurate to 0.01 g) as a test sample, place it in a 50 mL centrifuge tube, add 10 mL of 1% formic acid acetonitrile to extract twice, vortex mix at 2200 r / min for 10 min, ultrasonicate in a water bath at room temperature for 10 min, centrifuge at 4000 r / min for 5 min, take the supernatant, add 400 mg of anhydrous sodium sulfate and 200 mg of C 18Adsorbent, vortex at 2200r / min for 1min, transfer to a chicken heart bottle after purification, rotary evaporate to dryness at 48℃, add 1mL, 80% acetonitrile aqueous solution, vortex and oscillate for 1min, pass the reconstituted solution through a 0.22μm PTFE filter into a sample injection bottle, and provide liquid chromatography-tandem mass spectrometry for determination. The method detection limit for fish, shrimp, crab, and shellfish is 0.25μg / kg, and the quantitative limit is 0.5μg / kg. The method recovery rate is 79-105%, and the precision is 0.5-10.1%.

Claims

1. A method for determining the herbicides imazapyr and haloxyfop-ethyl in aquatic products by liquid chromatography-tandem mass spectrometry, comprising: (1) adding acidified acetonitrile to the pretreated water product, vortexing, ultrasonicating, centrifuging, and taking the supernatant to obtain a liquid to be purified; (2) The liquid to be purified, anhydrous sodium sulfate and dispersed solid phase material C 18 After mixing, vortexing, purification, rotary evaporation, adding acetonitrile aqueous solution and shaking, filtering, the treated water product is obtained, and then measured by liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that: The aquatic product in step (1) includes one or more of fish, shrimp, crab, and shellfish; the pretreated aquatic product is obtained by mincing and mixing the edible part of the aquatic product.

3. The method according to claim 1, characterized in that: The volume percentage concentration of the acidified acetonitrile in step (1) is 0.1%-1%; the acidified acetonitrile is formic acid acetonitrile; the acidified acetonitrile is added in 1 to 3 times.

4. The method according to claim 1, characterized in that: In the step (1), the vortexing speed is 2000-2500 r / min, and the vortexing time is 5-10 min; the ultrasonic time is 10-20 min; the centrifugation speed is 4000-8000 r / min, and the centrifugation time is 2-5 min; The vortex time in the step (2) is 0.5-3 min; the filtration is performed by using a polytetrafluoroethylene filter membrane.

5. The method according to claim 1, characterized in that: The ratio of the pretreated aquatic product and the acidified acetonitrile in step (1) is 1-2 g: 5-10 mL; the pretreated aquatic product, anhydrous sodium sulfate and dispersed solid phase material C in step (2) 18 The ratio is 1-2g:200-500mg:100-300mg.

6. The method according to claim 1, characterized in that: The determination by liquid chromatography-tandem mass spectrometry in step (2) comprises: S1: preparing a mixed standard series of solutions of the herbicides imazapyr and haloxyfop-ethyl at different concentrations, and performing liquid chromatography-tandem mass spectrometry determination to obtain a standard curve; S2: Perform liquid chromatography-tandem mass spectrometry testing on the treated water products.

7. The method according to claim 1, characterized in that: The concentration ranges of the herbicides imazapyr and haloxyfop-ethyl in S1 are both 0.5 μg / L to 50 μg / L.

8. The method according to claim 1, characterized in that: The liquid chromatography-tandem mass spectrometry determination process in S1 and S2 includes: liquid chromatography conditions: Capcell PAK C 18 MG II column, column temperature: 40°C, flow rate: 0.4 mL / min, injection volume: 10 μL, mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile; Mass spectrometry conditions: positive ion scanning, spray voltage: 5500 V, ion transmission capillary temperature: 550°C, curtain gas pressure CUR of 35 psi, nebulizer gas pressure GS1 of 55 psi, auxiliary heating gas pressure GS2 of 55 psi, collision gas CAD of 5 psi.

9. The method according to claim 1, characterized in that: The mass spectrometry conditions in S1 and S2 are as follows:

10. The method according to claim 1, characterized in that: In S2, liquid chromatography-tandem mass spectrometry is used to qualitatively and quantitatively determine the herbicides imazapyr and fluazifop-ethyl in aquatic products; the detection limit of the qualitative determination method is 0.25 μg / kg, and the quantification limit of the quantitative determination is 0.5 μg / kg; and the quantification is performed by the standard curve method.

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