CN107860841B - Method for detecting fipronil and its metabolite in poultry egg - Google Patents
Method for detecting fipronil and its metabolite in poultry egg Download PDFInfo
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Abstract
The invention provides a method for detecting fipronil and its metabolites in poultry eggs, which comprises the steps of optimally selecting an extraction buffer solution, extracting fipronil and its metabolites from egg samples by the extraction buffer solution, and purifying the samples by ethanol. And concentrating the sample liquid to remove ethanol, purifying and purifying by a QuEChERS method, and determining by a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method. The method can effectively detect the poultry egg sample and identify whether the poultry egg sample is polluted by the insecticide fipronil. The detection method can meet the requirement of measuring fipronil and its metabolites in poultry eggs. The standard substance in the research is added into blank eggs, and the processing process of the standard substance is consistent with that of the sample to be detected, so that errors caused by matrix effect are eliminated.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for detecting fipronil and a metabolite thereof in poultry eggs.
Background
Eggs exported from Holland in 7 months in 2017 are firstly found to contain fipronil components, the 'poisonous egg' event is outbreak, fermentation and spread to Europe in many countries, a large number of eggs polluted by insecticide fipronil are laid, problem hens are slaughtered, and economic loss is heavy. Fipronil was also detected in hong kong, china in both egg samples imported from the netherlands. Since the limit of fipronil in eggs is not clearly regulated in China and a detection method of fipronil in eggs is not regulated, relevant government departments cannot effectively evaluate the quality safety of domestic native eggs, and the real-time and deep understanding, control and supervision of the risk of whether eggs are polluted by fipronil in the domestic market are directly influenced.
As China has no legal method for determining fipronil in poultry eggs, the laboratory can only refer to the determination method of fipronil in fruits and vegetables or the determination method of fipronil in food, such as SN/T1982-2007. However, the detection of the sample is not good according to the existing method, and the method only aims at fipronil and does not contain fipronil metabolites (such as fipronil, fipronil thioether and fipronil sulfone), so a more complete detection method is needed.
Disclosure of Invention
The invention provides a method for detecting fipronil and its metabolite in poultry eggs, thereby effectively detecting poultry egg samples and identifying whether the poultry egg samples are polluted by the insecticide fipronil.
The method comprises the following steps:
1) preparation of samples to be tested
Breaking eggshells of poultry eggs to be detected, and crushing contents to be used as samples to be detected;
2) preparation of the extract
Adding a sample to be detected into an extraction buffer solution, and performing oscillation extraction; adding absolute ethyl alcohol into the extracting solution to precipitate impurities, and then centrifuging to obtain a supernatant; transferring the supernatant into a heart-shaped flask, and removing ethanol by water bath reduced pressure rotary evaporation; after the rotary evaporation is finished, transferring the aqueous solution into a centrifuge tube, rinsing a heart bottle with acetonitrile, merging the heart bottle into the centrifuge tube, adding magnesium sulfate and sodium acetate for oscillation treatment, adding excessive sodium chloride, oscillating to saturate water components, centrifuging, absorbing supernatant, adding the supernatant into a purification tube containing magnesium sulfate and PSA, centrifuging again after vortex oscillation, transferring supernatant into the centrifuge tube added with anhydrous sodium sulfate for oscillation dehydration, and filtering with a 0.22 mu m organic phase needle type filter membrane to obtain detection liquid for gas chromatography-mass spectrometry;
the centrifugation is carried out for 5min at 4000 r/min;
the mass ratio of the magnesium sulfate to the sodium acetate is 4: 1;
the volume ratio of the absolute ethyl alcohol, the washing buffer solution and the ammonia water in the extraction buffer solution is 10:10:1,
wherein the washing buffer solution comprises 0.004mo L/L sodium dihydrogen phosphate dihydrate (NaH)2PO4.2H2O), 0.016mo L/L disodium hydrogen phosphate dodecahydrate (Na)2HPO4.12H2O), 0.15mo sodium chloride (NaCl) L/L, 0.1% Tween 20;
3) gas chromatography-mass spectrometry
Using an HP-5 MS capillary chromatographic column, keeping the temperature of a sample inlet at 280 ℃, in a non-flow-dividing mode, carrying gas He, in a constant-pressure mode, keeping the sample volume at 1.0 mu L, keeping the temperature of a column box at 80 ℃ for 1min, raising the temperature by 20 ℃/min to 200 ℃ in a programmed mode, keeping the temperature for 4min, continuing raising the temperature by 20 ℃/min to 280 ℃ in a programmed mode, raising the temperature by 10 ℃/min to 310 ℃ in a programmed mode again, keeping the temperature for 2min, keeping the temperature of an ion source at 300 ℃, keeping the ionization voltage at 70ev, keeping the temperature of a quadrupole rod at 180 ℃, keeping the temperature of an auxiliary channel at 280 ℃, keeping the emission voltage at 1500ev, adopting a scanning mode of a multi-reaction ion monitoring mode (MRM), keeping the solvent delay at 5min, detecting ions;
comparing the retention time of the sample chromatogram with the retention time of the standard solution, and comparing the characteristic ions of chromatographic peaks with the characteristic ions of chromatographic peaks of the standard substance with corresponding concentrations for qualitative judgment;
comparing the relative abundance of ions in the sample solution with that of ions in the matrix matching standard solution, performing an external standard method according to the quantitative ions, and performing quantitative judgment through a formula obtained from a standard curve;
4) standard Curve preparation
Diluting fipronil and its metabolite standard substance with acetone to prepare standard mixed solution, adding the standard mixed solution into blank egg sample to prepare standard substance to-be-detected samples with different standard concentration points; detecting according to the sequence from step 1) to step 3);
the standard concentrations are 5ng/m L, 10ng/m L, 20ng/m L, 40ng/m L, 60ng/m L, 80ng/m L and 100ng/m L;
5) calculation formula and data processing
Performing curve regression on response values of fipronil and metabolite standard solutions thereof at various concentration points to serve as quantitative standards, automatically calculating the pesticide concentration of a sample solution according to the response values of samples, converting the calculation result into mass fraction, wherein the fipronil residual quantity is the sum of fipronil and metabolite (fipronil, fipronil thioether and fipronil sulfone) residues, expressed as fipronil, and the unit is expressed in milligrams per kilogram (mg/kg) in terms of W, and the calculation formula is as follows:
W＝ρ×V/(m×1000)
wherein rho is the concentration (ng/m L) of the sample solution to be measured obtained by regression according to a standard curve;
m-mass (g) of sample
V-constant volume (m L).
The detection method can meet the requirement of measuring fipronil and its metabolites in poultry eggs. The standard substance in the research is added into blank eggs, and the processing process of the standard substance is consistent with that of the sample to be detected, so that errors caused by matrix effect are eliminated.
Drawings
FIG. 1: acetonitrile extraction centrifuge chart according to SN/T1982-2007 method;
FIG. 2: the invention extracts the centrifugal chart of the method;
FIG. 3: GC-MS/MS total ion current chromatogram of fipronil and metabolite standard thereof;
FIG. 4: a standard curve diagram of fluoronitrile;
FIG. 5: fipronil standard curve chart;
FIG. 6 is a graph of fipronil sulfone standard curve;
FIG. 7: fipronil thioether standard curve diagram;
FIG. 8: mass spectrum of the compound;
Detailed Description
When fipronil and its metabolites are measured in poultry eggs according to the method in SN/T1982-2007, the applicant finds that the interference of lipids and phospholipids in poultry egg samples on the target substances is large. The existing acetonitrile extraction can dissolve a large amount of lipid substances, so that the subsequent purification work can bring great pressure, the purification degree is low, the chromatographic column can be polluted, and the service life is influenced. Meanwhile, the sample after acetonitrile extraction is still flocculent and contains a plurality of target objects, so that the recovery rate is low. In order to solve the problems, the applicant optimally selects the extraction buffer solution, and purifies the sample by using ethanol after the extraction buffer solution extracts fipronil and metabolites thereof in the egg sample. And concentrating the sample liquid to remove ethanol, purifying and purifying by a QuEChERS method, and determining by a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method.
The reagents and instruments used in the method of the invention are as follows:
1. fipronil, fipronil sulfide and fipronil sulfone standard substances are purchased from Beijing Yanxiang Limited company, and the model specifications are 100 mu g/m L and 1.2m L of fipronil sulfone in methanol, 101 mu g/m L and 1.1m L of fipronil sulfide in acetone, 100 mu g/m L and 1m L of fipronil in acetone, 100 mu g/m L and 1.5m L of fipronil in n-hexane and acetonitrile (chromatographically pure, Vokay and national drug group chemical reagent Limited company);
2. absolute ethyl alcohol (analytical purity, chemical reagents of the national drug group, ltd.), and concentrated ammonia water (analytical purity, chemical reagents of the national drug group, ltd.).
3. Preparation of a washing buffer: 0.622g of sodium dihydrogen phosphate dihydrate (NaH) was weighed out separately2PO4.2H2O), 5.735g of disodium hydrogen phosphate dodecahydrate (Na)2HPO4.12H2O), 9g of sodium chloride (NaCl) were distilledDissolving in water, transferring the total amount to a volumetric flask of 1000m L, adding 1m L Tween 20, metering to the scale, and mixing.
4. The extraction buffer was prepared by mixing 100m L absolute ethanol, 100m L wash buffer and 10m L concentrated aqueous ammonia.
5. QuEChERS extraction package, brand CNW, cat no: 2, ca8115.b000, content: 6g of magnesium sulfate and 1.5g of sodium acetate.
6. QuEChERS purification tube, brand CNW, cat #: CA8321.0001, content: magnesium sulfate 1.2g, PSA400 mg.
7. Ceramic proton (agilent company, usa) cat #: 5982-9313.
8. GC (Agilent 7890B-7000C Agilent, Agilent);
9. rotary evaporator (type Hei-VAP Precision, heidolph, germany);
10. centrifuges (centrifuge, eppendorf, germany);
11. vortex shaker (MS3basic, IKA, Germany).
Example 1: establishment of extraction method
1) Sample preparation
Breaking egg shell, pulverizing the content in multifunctional wall-breaking food processor, and making into sample to be tested. Placing into subpackage container, and storing at-18 deg.C for use.
2) Sample pretreatment
Weighing 10.00g of sample into a plastic centrifuge tube with a plug of 100m L, adding 10m L of water, accurately adding 40m L of acetonitrile, covering the plug, violently oscillating for 10 minutes, adding 5g of sodium chloride, violently oscillating for 10 minutes, centrifuging for 10 minutes at 3000r/min, taking 20m L of upper-layer extract, transferring into a centrifuge tube with a plug of 50m L, adding 10m L of n-hexane, shaking for 3 minutes, statically layering, discarding the upper-layer n-hexane phase, collecting the lower-layer acetonitrile phase into a 100m L chicken heart bottle, concentrating to near dryness in a water bath at 40 ℃, adding 1.0m L of acetone plus n-hexane (3+7) to dissolve residues, pouring the sample liquid into a PSA column pre-leached by 5m 5 of acetone plus (3+7), eluting by 10m L of acetone plus n-hexane (3+7), collecting all eluates in a 100m 6 of acetone plus n-hexane plus (3+7), diluting to a volume, pouring into a water bath at 40m 387 of 5m, dissolving the acetone plus n-hexane in a water bath at 3871, and measuring the lower-hexane phase by a mass spectrometer, and concentrating by a water bath at 40 ℃ of 583 + 5 m.
The results show that the egg sample floats in the solution in a large area of cotton-like shape after acetonitrile extraction and centrifugation by the SN/T1982-2007 method (figure 1), which is directly related to the recovery rate of the spiked sample. The sample is detected to have the standard recovery rate of only about 50% -70%, which is related to the cotton floc of the sample, and indicates that the cotton floc sample may contain the target.
In order to solve the problem, the extraction buffer solution is optimally selected, and the specific scheme is as follows:
taking 2.00g of egg sample into a 50m L plastic centrifuge tube with a plug, adding 10m L extraction buffer solution, carrying out vortex oscillation for 2 minutes after the plug is covered, adding 30m L anhydrous ethanol, carrying out vigorous shaking for 5 minutes after the plug is covered, carrying out centrifugation for 5 minutes at 4000r/min, as shown in fig. 2, comparing with an SN/T1982 plus 2007 method for extracting a centrifuged picture, using an extract solution for dissolving and extracting, ethanol for precipitating protein and the like, enabling the egg sample to be located at the bottom layer of the solution in a small-volume solid state, obviously improving the recovery rate of the added standard, pouring a supernatant into a 100m L chicken heart bottle, carrying out decompression and rotation on the sample solution in a 51 ℃ water bath, removing ethanol, finishing the rotation, transferring the aqueous solution into another 50m L centrifuge tube, adding 5m L acetonitrile for rinsing the original chicken heart bottle, combining the rinsing solution into the 50m L min, repeating the operation, placing a ceramic homogeneous mass spectrum into the centrifuge tube, adding the content in an extraction packet, carrying out the shake for 2 minutes after the plug is covered, adding 3 g sodium chloride, shaking for 2min, carrying out the sample, carrying out vortex oscillation for 2min, adding the sample into a sample, carrying out chromatography, adding the sample, carrying out filtration, carrying out chromatography for 2min, adding 10.6 min, carrying out purification, carrying out chromatography, carrying out a vortex analysis, adding a sample layer, adding 10m 2min, carrying out a vortex analysis, adding a sample, carrying out a.
3) Gas chromatography-mass spectrometry conditions
The capillary chromatographic column comprises HP-5 MS (Crolenked 5% Phenyl Methyl Siloxane 30m × 0.32.32 mm × 0.25.25 mu m), a sample inlet temperature of 280 ℃, a non-flow-dividing mode, carrier gas of He, a constant-pressure mode, a sample feeding amount of 1.0 mu L, a column box temperature of initial 80 ℃ for 1min, a temperature programmed from 20 ℃/min to 200 ℃ for 4min, a temperature programmed from 20 ℃/min to 280 ℃ and a temperature programmed from 10 ℃/min to 310 ℃ again for 2min, an ion source temperature of 300 ℃, an ionization voltage of 70ev, a quadrupole rod temperature of 180 ℃, an auxiliary channel temperature of 280 ℃, an emission voltage of 1500ev, a scanning mode of a multi-reaction ion monitoring mode (MRM), a solvent delay of 5min, a monitoring ion determination and retention time of fipronil and metabolites thereof, and shown in Table 1.
TABLE 1 Retention time, detection limits, monitoring ion and Collision energies of the Components
Quantitative ions
TABLE 2 allowable deviation Range of relative abundance of ions in sample solutions
| Relative abundance% | Tolerance deviation% |
| ＞50 | ±20 |
| ＞20～50 | ±25 |
| ＞10～20 | ±30 |
| ≤10 | ±50 |
4) Preparation of standard solution and preparation of standard curve
In this experiment, 5 μ L, 10 μ L, 20 μ L, 40 μ L, 60 μ L, 80 μ L and 100 μ L were added to obtain 5 μ 8655, 10 μ L, 20 μ L, 40 μ L, 60 μ L, 80 μ L and 100 μ L mixed standard, respectively, to obtain 5ng/m L, 10ng/m L, 20ng/m L, 40ng/m L, 60ng/m L, 80ng/m L and 100ng/m L GC-MS/MS chromatogram of 80ng/m L standard, mass spectrum 8, FIG. 4 is a fipronil standard curve diagram, FIG. 5 is a fipronil standard curve diagram, 6 is a fipronil standard thioether curve diagram, and FIG. 7 is a fipronil standard curve diagram.
5) Calculation formula and data processing
Performing curve regression on response values of various concentration points of a fipronil and metabolite standard solution thereof to serve as a quantitative standard, automatically calculating the pesticide concentration of a sample solution according to the response values of samples, converting the calculation result into a mass fraction, wherein the fipronil residual quantity is the sum of fipronil and metabolite (fipronil, fipronil thioether and fipronil sulfone) residues, expressed as fipronil, and the unit is expressed in milligrams per kilogram (mg/kg) in terms of W, and the calculation formula is as follows:
W＝ρ×V/(m×1000)
wherein rho is the concentration (ng/m L) of the sample solution to be measured obtained by regression according to a standard curve;
m-mass (g) of sample
V-constant volume (m L)
6) Linear range and detection limit
The fipronil and its metabolite are quantified by external standard method, and each series of standard working solutions show good linear relationship (r/m L, 10ng/m L, 20ng/m L, 40ng/m L, 60ng/m L, 80ng/m L and 100ng/m L) at concentration points2>0.995). Pre-treating and processing a sample on a computer to obtain 3 times and 10 times of signal-to-noise ratio, wherein the detection limit and the quantification limit of the method are respectively as follows: 0.005mg/kg and 0.015mg/kg。
Example 2: censored sample detection
The fipronil content of 15 egg and duck egg samples is determined by using the method established in example 1, and one of the egg samples is detected by detecting fipronil sulfone component, specifically, the sample is thoroughly homogenized and crushed into liquid by using a multifunctional broken-wall food processor, 2.00g of the sample is weighed into a 50m L stoppered plastic centrifuge tube, 10m L extraction buffer is added, the sample is covered and shaken for 2 minutes, 30m L absolute ethyl alcohol is added, the sample is covered and shaken vigorously for 5 minutes, the sample is centrifuged for 5 minutes at 4000r/min, the supernatant is poured into a 100m L heart bottle, the sample liquid in the heart bottle is swirled in a 51 ℃ water bath under reduced pressure to remove the ethyl alcohol, the sample liquid is transferred into another 50m L centrifuge tube, 5m L acetonitrile is added to moisten the original heart bottle, the washing liquid is combined into the 50m L centrifuge tube, the operation is repeated once, a stirred proton is put into the centrifuge tube, the ceramic proton is added, the extracted, the sodium sulfate is added into another 50m L centrifuge tube, the sample, the sodium sulfate is added, the sodium sulfate concentration of the sample is measured, the sodium sulfate is calculated as the average concentration of the sample after the sample is measured by using a 0212 mg of the sample, the sodium sulfate, the sample is calculated, the sodium sulfate concentration of the sodium sulfate is calculated after the sodium sulfate is calculated, the sodium sulfate is calculated, the sodium sulfate concentration of the sodium sulfate is calculated, the sodium sulfate is calculated, the sodium sulfate is found by using a theoretical concentration of the sodium sulfate is calculated, the sodium sulfate is calculated, the sodium sulfate is calculated, the sodium sulfate is measured, the sodium sulfate is calculated, the sodium sulfate is calculated.
Claims (5)
1. A method for detecting fipronil and its metabolites, namely, fipronil thioether and fipronil sulfone in poultry eggs is characterized by comprising the following steps:
1) preparation of samples to be tested
Breaking eggshells of poultry eggs to be detected, and crushing contents to be used as samples to be detected;
2) preparation of the extract
Adding a sample to be detected into an extraction buffer solution, and performing oscillation extraction; adding absolute ethyl alcohol into the extracting solution to precipitate impurities, and then centrifuging to obtain a supernatant; pouring the supernatant into a heart-shaped bottle, and removing ethanol by a water bath reduced pressure rotary evaporation mode; after the rotary evaporation is finished, transferring the aqueous solution into a centrifuge tube, rinsing a heart bottle with acetonitrile, merging the heart bottle into the centrifuge tube, adding magnesium sulfate and sodium acetate for oscillation treatment, adding excessive sodium chloride, oscillating to saturate water components, centrifuging, absorbing supernatant, adding the supernatant into a purification tube containing magnesium sulfate and PSA, centrifuging again after vortex oscillation, transferring the supernatant into the centrifuge tube added with anhydrous sodium sulfate for oscillation dehydration, and filtering a sample solution through a 0.22-micrometer organic phase needle type filter membrane to obtain a detection solution for gas chromatography-mass spectrometry;
3) gas chromatography-mass spectrometry
The method comprises the following steps of (1) using an HP-5 MS capillary chromatographic column, keeping the temperature of a sample inlet at 280 ℃, in a non-flow-dividing mode, carrying gas He, in a constant-pressure mode, keeping the sample volume at 1.0 mu L, keeping the temperature of a column box at 80 ℃ for 1min, carrying out programmed temperature rise at 20 ℃/min to 200 ℃ for 4min, continuing to carry out programmed temperature rise at 20 ℃/min to 280 ℃ and carrying out programmed temperature rise at 10 ℃/min to 310 ℃ for 2min again, keeping the temperature of an ion source at 300 ℃, carrying out ionization voltage at 70ev, keeping the temperature of a quadrupole rod at 180 ℃, keeping the temperature of an auxiliary channel at 280 ℃, carrying out emission voltage at 1500ev, carrying out a scanning mode in a multi-reaction ion monitoring mode MRM, delaying a solvent for 5min, and monitoring ions to determine;
comparing the retention time of the sample chromatogram with the retention time of the standard solution, and comparing the characteristic ions of chromatographic peaks with the characteristic ions of chromatographic peaks of the standard substance with corresponding concentrations for qualitative judgment;
comparing the relative abundance of ions in the sample solution with that of ions in the matrix matching standard solution, performing an external standard method according to the quantitative ions, and performing quantitative judgment through a formula determined from a standard curve;
the extraction buffer solution in the step 2) is a mixed solution of absolute ethyl alcohol, a washing buffer solution and ammonia water, wherein the volume ratio of the absolute ethyl alcohol to the washing buffer solution to the ammonia water is 10:10: 1;
the washing buffer solution comprises 0.004mo sodium dihydrogen phosphate dihydrate L/L, 0.016mo L/L disodium hydrogen phosphate dodecahydrate, 0.15mo sodium chloride L/L and 0.1% Tween 20.
2. The method of claim 1, wherein the formula is established by:
1) standard Curve preparation
Diluting fipronil and its metabolite standard substance with acetone to prepare standard mixed solution, adding the standard mixed solution into blank egg sample to prepare working curve with different standard concentration points; the standard sample and the sample to be detected are detected according to the sequence of the steps 1-3 in the claim 1;
2) calculation formula and data processing
Performing curve regression by using response values of various concentration points of a fipronil and metabolite standard solution thereof as a quantitative standard, automatically calculating the pesticide concentration of a sample solution according to the response value of a sample, converting the calculation result into a mass fraction, wherein the fipronil residual quantity is the sum of fipronil and metabolite residues thereof, expressed as fipronil, and the unit is expressed in milligrams per kilogram in terms of W, and the calculation formula is as follows:
W＝ρ×V/(m×1000)
wherein rho-is regressed according to a standard curve to obtain the concentration ng/m L of the sample solution to be measured;
m-mass g of sample,
V-constant volume m L.
3. The method of claim 1, wherein the centrifugation in step 2) is performed at 4000r/min for 5 min.
4. The method according to claim 1, wherein the mass ratio of magnesium sulfate to sodium acetate in step 2) is 4: 1.
5. The method of claim 2, wherein the concentration of the standard solution of fipronil and its metabolites is 5ng/m L, 10ng/m L, 20ng/m L, 40ng/m L, 60ng/m L, 80ng/m L, 100ng/m L.
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