A rapid detection method for chlorfenapyr, emathine, fenfluramide, pendimethalin and metalaxyl in aquatic products

Abstract

The present invention relates to a method for rapid detection of chlorfenac, emathine, fenfluramide, pendimethalin and metalaxyl in aquatic products. The method can realize simultaneous detection of five compounds, namely chlorfenac, emathine, fenfluramide, pendimethalin and metalaxyl in aquatic products. The method has 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, emathine, fenfluramide, pendimethalin and metalaxyl in aquatic products. Background Art

[0002] Aquatic product testing is a crucial step in ensuring the safety and quality of aquatic products. The development of aquatic product quality and safety testing technology dates back to the early 20th century. Initially, it relied primarily on sensory evaluation. Later, with technological advancements, instrumentation and equipment began to be applied to aquatic product testing, such as vacuum arc sterilization lamps and ultraviolet disinfection lamps. In recent years, techniques such as PCR and enzyme-linked immunosorbent assays have been widely used to detect trace amounts of harmful substances in aquatic products.

[0003] Most of the existing detection standards 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, emathine, fenfluramide, pendimethalin and metalaxyl, comprising:

[0006] (1) Add acetonitrile to the sample and homogenize it, then add anhydrous sodium sulfate and vortex mix it, and centrifuge it to obtain the 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, 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.

[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 spectrometry 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 spectrometry is performed, wherein the mass spectrometry conditions are as follows: ionization mode: electrospray ionization 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 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 diluting the mixed standard solution with the blank matrix extract 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 in 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 is completed, qualitative and quantitative analysis is performed; wherein the qualitative analysis is to perform qualitative analysis on the five components of chlorfenapyr, fenvalerate, fenfluramide, pendimethalin and metalaxyl based on the retention time of the analyte and the retention time and relative ion abundance of the corresponding target in the matrix matching standard solution;

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

[0024] Beneficial effects

[0025] In this invention, mass spectrometry detection uses positive ionization mode. Adding a certain amount of formic acid to the liquid chromatography system can improve the detection sensitivity in positive ion mode. Experiments have shown that gradient elution using a 0.1% formic acid aqueous solution in acetonitrile as the mobile phase system resulted in good peak shape and sensitivity for all five pesticides.

[0026] The method of the present invention can realize the simultaneous detection of five compounds, namely, chlorfenapyr, fenfluramide, pendimethalin and metalaxyl, in aquatic products. The detection method of the present 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 The characteristic ion chromatograms of standard solutions of clofenac, emathine, fenfluramide, pendimethalin and metalaxyl are shown. DETAILED DESCRIPTION

[0028] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, 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 equally within the scope limited by the appended claims of the application.

[0029] Example 1

[0030] 1. Reagents and Materials

[0031] Unless otherwise specified, all reagents were of analytical grade and water was grade 1 water in accordance with GB / T 6682.

[0032] Reagents:

[0033] 1.1.1 Acetonitrile (CH3CN): chromatographically pure.

[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 Indole Emamectinbenzoate <![CDATA[C 49 H 77 NO 13 ]]> 155569-91-8 Teflubenzuron 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 approximately 10 mg (accurate to 0.01 mg) each of chlorfenapyr, fenfluramide, pendimethalin, and metalaxyl. Dissolve each in acetonitrile and dilute to volume in a 10 mL volumetric flask to prepare a 1000 mg / L standard stock solution. Store below -18°C in the dark. Valid for 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 into a 100 mL volumetric flask. Dilute to volume with acetonitrile to prepare a mixed standard intermediate solution with a concentration of 10.0 mg / L. Store below -18°C in the dark. Valid for 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 and dilute to volume with acetonitrile to prepare a 1.0 mg / L mixed standard working solution. Store below -18°C in the dark. Valid for one month.

[0047] 1.5 Materials

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

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

[0050] 1.5.3 Microporous nylon filter membrane: 0.22 μm × 13 mm, 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 Measurement 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 rpm for 10 min, and take 8 mL of the 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. The supernatant was filtered through a microporous nylon filter membrane and subjected to liquid chromatography-tandem mass spectrometry analysis.

[0062] 3.3 Preparation of matrix-matched standard curve

[0063] Prepare a blank sample by processing according to steps 4.1-4.2 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 produce matrix-matched standard solutions with concentrations of 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, and 50 μg / L. Determine the matrix-matched standard solution using liquid chromatography-tandem mass spectrometry. Plot a matrix-matched standard curve using the measured characteristic ion mass chromatographic peak area as the ordinate and the matrix-matched standard solution concentration 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[Coefficient of correlation r 2 > Flufenac Y=637.89x-166.69 0.995380 Indole Y=30655.6x-17618.4 0.998076 Teflubenzuron 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 (2.1 mm × 100 mm, 1.8 μm), 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: 3.5kV;

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

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

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

[0086] Table 5 Monitoring ions of 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 compound 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. The allowable deviation should meet the requirements of Table 6.

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

[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 analyte in the sample, in micrograms per kilogram (μg / kg);

[0102] C S ——The numerical value of the concentration of the analyte in the matrix-matched 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-matched 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 1 μg / kg, and the quantification limit is 2 μg / kg.

[0109] 5.2 Accuracy

[0110] The recovery rate of this method at the spiked concentration level of 2 μg / kg to 20 μg / 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 section 4.1 of Example 1 was replaced with magnesium sulfate. Experiments have shown that a small amount of magnesium sulfate can remove moisture. However, magnesium sulfate easily forms lumps and generates severe heat when added, and also affects the recovery rate of fenvalerate and flubendiamide.

[0117] Comparative Example 2

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

[0119] 1. Prime HLB, a common solid phase extraction reagent, was used for testing. The results showed that it adsorbed all five pesticides, with recovery rates ranging from 1% to 70%, which did not meet the minimum recovery rate requirement of 60% for testing.

[0120] Table 8: Prime HLB recovery

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

[0122] 2. PSA and C18 were used to remove impurities such as sugars, organic acids, fats, and pigments from the sample. Testing showed that without magnesium sulfate, the recovery rate ranged from 50% to 80%, while with the addition of magnesium sulfate, the recovery rate reached 90% to 98%. Inadequate sodium sulfate removal in the first step resulted in a decrease in recovery. Therefore, the present invention uses 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA, and 400 mg of C18 per 8 mL of extract as a purification method.

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

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

[0125] Table 10 adds magnesium sulfate recovery rate

[0126] Compound Flufenac Indole Teflubenzuron 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 and homogenize it, then add anhydrous sodium sulfate and vortex mix it, and centrifuge it to obtain the extract; (2) Anhydrous magnesium sulfate, PSA, C 18 Vortex-mix the extract from step (1), centrifuge, and filter; (3) performing liquid chromatography-tandem mass spectrometry determination; The liquid chromatography-tandem mass spectrometry determination in step (3) is performed, 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: In step (3), the determination was performed by phase chromatography-tandem mass spectrometry, wherein the mass spectrometry determination conditions were as follows: ionization mode: electrospray ionization 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:

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 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 the 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; The C 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 detection method further comprises 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 diluting the mixed standard solution with the blank matrix extract to obtain a series of matrix mixed standard working curve solutions with concentration gradients.

7. The detection method according to claim 6, 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.

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

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