Rapid detection method for clofenoxine, ovastatin, novaluron, pendimethalin and metalaxyl in aquatic products

Abstract

The invention relates to a rapid detection method for clofenoxine, ordentine, novaluron, pendimethalin and metalaxyl in aquatic products, which can realize simultaneous detection of five compounds of clofenoxine, ordentine, novaluron, pendimethalin and metalaxyl in aquatic products, and has the advantages of simple, rapid and sensitive detection and good purification effect.

Description

Technical Field

[0001] The invention belongs to the field of pesticide residue detection methods, and particularly relates to a rapid detection method for chlorfenapyr, fenthion, fenfluramide, pendimethalin and metalaxyl in aquatic products. Background Art

[0002] Aquatic product testing is an important part of ensuring the safety and quality of aquatic products. The development of aquatic product quality and safety testing technology can be traced back to the early 20th century. Initially, it mainly relied on sensory judgment. Later, with the advancement of science and technology, instruments and equipment began to be used in aquatic product testing, such as vacuum arc sterilization lamps and ultraviolet disinfection lamps. In recent years, PCR technology, enzyme-linked immunosorbent assay and other technologies have been widely used in the detection of trace harmful substances in aquatic products.

[0003] Most of the detection standards in the existing technology are only applicable to fish meat, and can only detect one or two harmful substances. The detection targets are limited, and there are defects such as long detection time and poor sensitivity. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a rapid detection method for chlorfenapyr, emathine, fenfluramide, pendimethalin and metalaxyl in aquatic products.

[0005] The present invention provides a method for detecting chlorfenapyr, fenthion, fenfluramide, pendimethalin and metalaxyl, comprising:

[0006] (1) Add acetonitrile to the sample for homogenization, then add anhydrous sodium sulfate, vortex mix, and centrifuge to obtain an extract;

[0007] (2) Anhydrous magnesium sulfate, PSA, C 18 Vortex-mix the extract from step (1), centrifuge, and filter;

[0008] (3) Liquid chromatography-tandem mass spectrometry was performed.

[0009] The sample in step (1) is aquatic product; wherein the aquatic product includes one or more of fish, shrimp, crab, turtle, shellfish and sea cucumber.

[0010] Preferably, in step (1), the ratio of sample, acetonitrile and anhydrous sodium sulfate is 3-5 g: 8-10 mL: 8-10 g;

[0011] Preferably, in the step (1), the homogenization time is 0.5-1 min; the vortex mixing is 2-3 min; and the centrifugation is 8000 r / min for 5-10 min.

[0012] Preferably, in step (2), anhydrous magnesium sulfate, PSA, C 18The ratio of extract is 1200mg:400mg:400mg:8mL;

[0013] Preferably, the filtration in step (2) is performed using a microporous nylon filter membrane.

[0014] Preferably, the particle diameter of the PSA is 40 μm to 60 μm; 18 The particle diameter is 40 μm to 60 μm; the pore size of the microporous nylon filter membrane is 0.22 μm.

[0015] Preferably, in step (3), the liquid chromatography-tandem mass spectrometer is used for determination, wherein the liquid chromatography determination conditions are: chromatographic column: C18 chromatographic column; column temperature: 40°C; injection volume: 2 μL; flow rate: 0.40 mL / min; mobile phase: A is 0.1% formic acid solution, B is acetonitrile; mobile phase gradient elution conditions:

[0016] Time / min A / ％ B / ％ 0.0 95 5.0 0.2 95 5.0 2.5 5.0 95 3.5 5.0 95 3.6 95 5.0 5.0 95 5.0 .

[0017] Preferably, in step (3), the phase chromatography-tandem mass spectrometer is used for determination, wherein the mass spectrometry determination conditions are as follows: ionization mode: electrospray ion source ESI; scanning mode: positive ion scanning; monitoring mode: multiple reaction monitoring MRM; capillary voltage: 3.5 kV; ion source temperature: 150° C.; desolvation temperature: 500° C.; and the specific parameters of parent ion, daughter ion, cone voltage, and collision energy are as follows:

[0018]

[0019] The detection method also includes the preparation of a matrix mixed standard working curve solution; wherein the preparation of the matrix mixed standard working curve solution includes: preparing a blank matrix according to steps (1) and (2) to obtain a blank matrix extract, and using the blank matrix extract to dilute the mixed standard solution to obtain a series of matrix mixed standard working curve solutions with concentration gradients.

[0020] Preferably, the concentrations of the matrix mixed standard working curve solutions of the series of concentration gradients are 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, and 50 μg / L;

[0021] The matrix mixed standard working curve solution is measured by liquid chromatography-tandem mass spectrometry to establish a standard curve.

[0022] After the detection, qualitative and quantitative analysis is performed; wherein the qualitative analysis is to compare the retention time of the analyte with the retention time and relative ion abundance of the corresponding target in the matrix matching standard solution to perform qualitative analysis on the five components of chlorfenapyr, fenthiocarb, pendimethalin, and metalaxyl;

[0023] The quantitative analysis is to quantify the samples using the standard curve method.

[0024] Beneficial Effects

[0025] In the present invention, the mass spectrometry detection uses positive ions as the ionization mode, and adding a certain amount of formic acid in the liquid chromatography system can improve the detection sensitivity of the positive ion mode. After experiments, 0.1% formic acid aqueous solution-acetonitrile was selected as the mobile phase system for gradient elution, so that all five pesticides obtained good peak shape and sensitivity;

[0026] The method of the invention can realize the simultaneous detection of five compounds, namely, chlorfenapyr, fenthion, pendimethalin and metalaxyl, in aquatic products. The detection method of the invention is simple, rapid, highly sensitive, has a good purification effect, a stable recovery rate and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the characteristic ion chromatogram of standard solution of clofenac, fenfluramide, pendimethalin and metalaxyl. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1

[0030] 1. Reagents and Materials

[0031] Unless otherwise specified, all reagents were of analytical grade and water was first-grade water in accordance with GB / T 6682.

[0032] Reagents:

[0033] 1.1.1 Acetonitrile (CH3CN): chromatographic grade.

[0034] 1.1.2 Formic acid (HCOOH): chromatographically pure.

[0035] 1.1.3 Anhydrous sodium sulfate (Na2SO4).

[0036] 1.1.4 Anhydrous magnesium sulfate (MgSO4).

[0037] 1.2 Solution preparation

[0038] 1.2.1 Formic acid aqueous solution (0.1%): Take 1 mL of formic acid, dilute to 1000 mL with water, and mix well.

[0039] 1.3 Standards

[0040] The contents of clofenac, fenfluramide, pendimethalin and metalaxyl were all ≥ 95.0%. See Table 1 for details.

[0041] Table 1 English names, molecular formulas and CAS numbers of clofenac, fenfluramide, pendimethalin and metalaxyl

[0042] Compound English name Molecular formula CAS Number Clofenac Lufenuron <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> H8Cl2F8N2O3<h2 style=";text-align:left;direction:ltr"> 103055-07-8 Entametacin Emamectinbenzoate <![CDATA[C 49 H 77 NO 13 ]]> 155569-91-8 Tebuconazole Teflubenzuron <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> H6Cl2F4N2O2<h2 style=";text-align:left;direction:ltr"> 83121-18-0 Pendimethalin Pendimethalin <![CDATA[C 13 H 19 N3O4]]> 40487-42-1 Metalaxyl Metalaxyl <![CDATA[C 15 H 21 NO4]]> 57837-19-1

[0043] 1.4 Preparation of standard solution

[0044] 1.4.1 Standard stock solution (1000 mg / L): Accurately weigh about 10 mg (accurate to 0.01 mg) of each of chlorfenapyr, fenthion, fenfluramide, pendimethalin and metalaxyl, respectively, add appropriate amount of acetonitrile to dissolve them and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 1000 mg / L. Store at below -18°C away from light, with a validity period of 1 year.

[0045] 1.4.2 Mixed standard intermediate solution (10.0 mg / L): Accurately measure 1 mL of each of the above standard stock solutions in a 100 mL volumetric flask, dilute to volume with acetonitrile, and prepare a mixed standard intermediate solution with a concentration of 10.0 mg / L. Store at below -18°C away from light, with a validity period of 3 months.

[0046] 1.4.3 Mixed standard working solution (1.0 mg / L): Accurately measure 1 mL of the mixed standard intermediate solution into a 10 mL volumetric flask, dilute to volume with acetonitrile to prepare a mixed standard working solution with a concentration of 1.0 mg / L. Store at below -18°C away from light, with a validity period of 1 month.

[0047] 1.5 Materials

[0048] 1.5.1 Ethylenediamine-N-propylsilane silica gel (PSA): particle diameter is 40μm~60μm.

[0049] 1.5.2 Octadecylsilane bonded silica gel (C 18 ): The particle diameter is 40μm~60μm.

[0050] 1.5.3 Microporous nylon filter membrane: 0.22μm×13mm, organic, or equivalent.

[0051] 2 Instruments and equipment

[0052] 2.1 Liquid chromatography-tandem mass spectrometry: equipped with an electrospray ionization source (ESI).

[0053] 2.2 Analytical balance: sensitivity 0.00001g and 0.01g.

[0054] 2.3 Homogenizer.

[0055] 2.4 Vortex mixer.

[0056] 2.5 Centrifuge: speed not less than 8000r / min.

[0057] 3. Determination steps

[0058] 3.1 Extraction

[0059] Weigh 5 g of the sample (accurate to ±0.05 g), place it in a 50 mL polypropylene centrifuge tube, add 10 mL of acetonitrile, homogenize for 1 min, add 10 g of anhydrous sodium sulfate, vortex mix for 3 min, centrifuge at 8000 r / min for 10 min, and take 8 mL of supernatant for later use.

[0060] 3.2 Purification

[0061] Weigh 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA and 400 mg of C 18 The above extract was added to a 15 mL centrifuge tube, thoroughly vortexed and centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a microporous nylon filter membrane for determination by liquid chromatography-tandem mass spectrometry.

[0062] 3.3 Preparation of matrix-matched standard curve

[0063] Take a blank sample and process it according to steps 4.1 to 4.2 to obtain a blank matrix solution. Accurately measure an appropriate amount of the mixed standard working solution and dilute it with the blank matrix solution to a matrix-matched standard solution with a mass concentration of 1μg / L, 2μg / L, 5μg / L, 10μg / L, 20μg / L, and 50μg / L for determination by liquid chromatography-tandem mass spectrometry. Draw a matrix-matched standard curve with the measured characteristic ion mass chromatographic peak area as the ordinate and the mass concentration of the matrix-matched standard solution as the abscissa. Calculate the regression equation and correlation coefficient.

[0064] Aquatic products themselves include a variety of aquatic organisms such as fish, shrimp, and crabs. The matrix effects of each organism are different, so matrix-matched standard solutions are used for quantification.

[0065] Table 2: Matrix effects

[0066]

[0067] Table 3 Linearity (1μg / L~50μg / L)

[0068] Compound Linear equations <![CDATA[Correlation coefficient r 2 > Flufenac Y=637.89x-166.69 0.995380 Entametacin Y=30655.6x-17618.4 0.998076 Tebuconazole Y=1134.77x-637.468 0.999873 Pendimethalin Y=763.035x-121.861 0.999058 Metalaxyl Y=43078.1x-15661.2 0.998943

[0069] 3.4 Determination

[0070] 3.4.1 Liquid chromatography reference conditions

[0071] a) Chromatographic column: C 18 Chromatographic column (2.1mm×100mm, 1.8μm), or equivalent;

[0072] b) Column temperature: 40°C;

[0073] c) Injection volume: 2 μL;

[0074] d) Flow rate: 0.40 mL / min;

[0075] e) Mobile phase: A is 0.1% formic acid solution, B is acetonitrile, and the gradient elution conditions are shown in Table 4.

[0076] Table 4 Mobile phase gradient elution conditions

[0077] Time / min A / ％ B / ％ 0.0 95 5.0 0.2 95 5.0 2.5 5.0 95 3.5 5.0 95 3.6 95 5.0 5.0 95 5.0

[0078] 3.4.2 Mass spectrometry reference conditions

[0079] a) Ionization mode: electrospray ionization (ESI);

[0080] b) Scanning mode: positive ion scanning;

[0081] c) Monitoring method: multiple reaction monitoring (MRM);

[0082] d) Capillary voltage: 3.5 kV;

[0083] e) Ion source temperature: 150°C;

[0084] f) Desolventizing temperature: 500°C;

[0085] g) Other parameters such as parent ion, daughter ion, cone voltage, collision energy, etc. are shown in Table 5.

[0086] Table 5 Monitoring ions of the analytes

[0087]

[0088] 3.4.3 Determination method

[0089] 3.4.3.1 Qualitative determination

[0090] Under the same test conditions, the retention time of the analyte in the sample solution and the retention time of the corresponding target in the matrix-matched standard solution should be within ±2.5%, and the detected relative ion abundance should be consistent with the relative ion abundance of the matrix-matched standard solution with equivalent concentration, and the allowable deviation should meet the requirements of Table 6.

[0091] Table 6 Maximum allowable deviation of relative ion abundance in qualitative confirmation (in percent)

[0092] Relative ion abundance ＞50 20～50 10～20 ≤10 Maximum allowed deviation ±20 ±25 ±30 ±50

[0093] 3.4.3.2 Quantitative determination

[0094] Take the sample solution and the corresponding matrix-matched standard solution and measure them under the same measurement conditions, perform single-point or multi-point calibration, and quantify by chromatographic peak area according to the external standard method. The response values ​​of the target analyte in the matrix-matched standard curve solution and the sample solution should be within the linear range of the instrument detection. Under the above-mentioned chromatography-mass spectrometry conditions, the characteristic ion mass chromatogram of the analyte is shown in Figure 1 .

[0095] 3.5 Blank test

[0096] Except for not adding sample, the above measurement steps are followed.

[0097] 4 Calculation and presentation of results

[0098] The residual amount of the substance to be tested in the sample is calculated according to the standard curve or formula (1).

[0099]

[0100] Where:

[0101] X——the value of the residual amount of the substance to be tested in the sample, in micrograms per kilogram (μg / kg);

[0102] C S ——The numerical value of the concentration of the analyte in the matrix matching standard solution, in micrograms per liter (μg / L); A——The chromatographic peak area of ​​the analyte in the sample solution;

[0103] A S ——Chromatographic peak area of ​​the analyte in the matrix matching standard solution;

[0104] V——the value of the constant volume of the sample solution, in milliliters (mL);

[0105] m – the numerical value of the test sample mass, in grams (g).

[0106] 5. Sensitivity, accuracy and precision of detection methods

[0107] 5.1 Sensitivity

[0108] The detection limit of this method is 1μg / kg, and the quantification limit is 2μg / kg.

[0109] 5.2 Accuracy

[0110] The recovery rate of this method at the concentration addition level of 2μg / kg to 20μg / kg is 60% to 120%.

[0111] Table 7:

[0112]

[0113] 5.3 Precision

[0114] The intra-batch relative standard deviation of this method is ≤15%, and the inter-batch relative standard deviation is ≤15%.

[0115] Comparative Example 1

[0116] The anhydrous sodium sulfate in the extraction part 4.1 of Example 1 was replaced with magnesium sulfate. It was found that a small amount of magnesium sulfate could remove moisture, but it was easy to form agglomerates and generate severe heat when added, and the recovery rates of fenvalerate and flubendiamide were also affected.

[0117] Comparative Example 2

[0118] Regarding the purification part 4.2 in Example 1, the purification method is compared.

[0119] 1. Prime HLB, which is commonly used in solid phase extraction, was used for testing. The results showed that it adsorbed all five pesticides, with a recovery rate between 1% and 70%, which did not meet the minimum recovery rate requirement of 60% for testing.

[0120] Table 8: Prime HLB recovery

[0121] Compound Flufenac Entametacin Tebuconazole Pendimethalin Metalaxyl Recovery rate (%) 4.105 50.70 9.727 1.679 64.19

[0122] 2. Use PSA and C18 to remove impurities such as sugars, organic acids, fats, pigments, etc. in the sample. According to the test, the recovery rate is between 50% and 80% without adding magnesium sulfate, and the recovery rate can reach 90% to 98% after adding magnesium sulfate. The first step of sodium sulfate dehydration is not sufficient, resulting in a decrease in the recovery rate. The present invention selects 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA and 400 mg of C18 corresponding to 8 mL of extract as a purification method.

[0123] Table 9: Recovery rate without adding magnesium sulfate

[0124] Compound Flufenac Entametacin Tebuconazole Pendimethalin Metalaxyl Recovery rate (%) 4.105 50.70 9.727 1.679 64.19

[0125] Table 10 magnesium sulfate recovery

[0126] Compound Flufenac Entametacin Tebuconazole Pendimethalin Metalaxyl Recovery rate (%) 107.7 98.95 98.60 99.20 100.4

Claims

1. A method for detecting clofenac, fenfluramide, pendimethalin and metalaxyl, comprising: (1) Add acetonitrile to the sample for homogenization, then add anhydrous sodium sulfate, vortex mix, and centrifuge to obtain an extract; (2) Anhydrous magnesium sulfate, PSA, C 18 Vortex-mix the extract from step (1), centrifuge, and filter; (3) Liquid chromatography-tandem mass spectrometry was performed.

2. The detection method according to claim 1, characterized in that: The sample in step (1) is aquatic product; wherein the aquatic product includes one or more of fish, shrimp, crab, turtle, shellfish and sea cucumber.

3. The detection method according to claim 1, characterized in that: In the step (1), the ratio of sample, acetonitrile and anhydrous sodium sulfate is 3-5 g: 8-10 mL: 8-10 g; The homogenization time in step (1) is 0.5-1 min; the vortex mixing is 2-3 min; and the centrifugation is 8000 r / min for 5-10 min.

4. The detection method according to claim 1, characterized in that: In the step (2), anhydrous magnesium sulfate, PSA, C 18 The ratio of extract is 1200mg:400mg:400mg:8mL; The filtration in step (2) is performed by using a microporous nylon filter membrane.

5. The detection method according to claim 4, characterized in that: The particle diameter of the PSA is 40 μm to 60 μm; 18 The particle diameter is 40 μm to 60 μm; the pore size of the microporous nylon filter membrane is 0.22 μm.

6. The detection method according to claim 1, characterized in that: The liquid chromatography-tandem mass spectrometer in step (3) is used for determination, wherein the liquid chromatography determination conditions are: chromatographic column: C18 chromatographic column; Column temperature: 40°C; injection volume: 2 μL; flow rate: 0.40 mL / min; Mobile phase: A is 0.1% formic acid solution, B is acetonitrile; mobile phase gradient elution conditions: 。 7. The detection method according to claim 1, characterized in that: In the step (3), the phase chromatography-tandem mass spectrometer is used for determination, wherein the mass spectrometer determination conditions are as follows: ionization mode: electrospray ion source ESI; scanning mode: positive ion scanning; monitoring mode: multiple reaction monitoring MRM; capillary voltage: 3.5 kV; ion source temperature: 150° C.; desolvation temperature: 500° C.; the specific parameters of parent ion, daughter ion, cone voltage, and collision energy are as follows:

8. The detection method according to claim 1, characterized in that: The detection method also includes the preparation of a matrix mixed standard working curve solution; The preparation of the matrix mixed standard working curve solution includes: preparing a blank matrix according to steps (1) and (2) to obtain a blank matrix extract, and using the blank matrix extract to dilute the mixed standard solution to obtain a series of matrix mixed standard working curve solutions with concentration gradients.

9. The detection method according to claim 8, characterized in that: The concentrations of the matrix mixed standard working curve solutions of the series of concentration gradients are 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, and 50 μg / L; The matrix mixed standard working curve solution is measured by liquid chromatography-tandem mass spectrometry to establish a standard curve.

10. The detection method according to claim 1, characterized in that: After the test, qualitative and quantitative analysis was performed; the qualitative analysis was performed on the five components of chlorfenapyr, fenthion, fenfluramide, pendimethalin and metalaxyl by comparing the retention time of the analyte with the retention time and relative ion abundance of the corresponding target in the matrix matching standard solution; The quantitative analysis is to quantify the samples using the standard curve method.

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