CN111879870A - Method for measuring residual quantity of de-isopropylaminophos in poultry eggs - Google Patents
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Abstract
The invention establishes a method for measuring the residual quantity of the de-isopropylaminophos in the poultry eggs for the first time. The specific method is that acetonitrile is used as an extraction solvent, and after degreasing and purification by a filter column, detection is carried out by a liquid chromatogram-tandem mass spectrometer, and quantification is carried out by an external standard method. The method has the advantages that the correlation coefficient of the regression equation reaches more than 0.99, the determination lower limit is 5.0 mu g/kg, the method is simple and convenient to operate, high in sensitivity, strong in anti-interference capability, accurate in qualitative and quantitative determination, and capable of providing technical support for relevant supervision departments in China.
Description
Technical Field
The invention belongs to the technical field of veterinary drug detection, and particularly relates to a method for determining the residual quantity of de-isopropylaminophos in poultry eggs.
Background
In GB 31650 plus 2019 maximum residue limit of veterinary drugs in national food Standard for food safety issued in 9 months in 2019, the residue marker is increased by the dopaminergic phosphorus: at present, no standard method for detecting the residual quantity of the deisopropyldopaminergic phosphorus in animal-derived food exists, and no literature report of any relevant detection method is found. The residue marker is revised according to the maximum residue limit standard of European Union veterinary drugs, and the dopaminergic phosphorus is metabolized into the de-isopropylamine phosphorus in the animal body, so that the detection of the de-isopropylamine phosphorus is increased to more accurately reflect the residue of the dopaminergic phosphorus in the animal body.
Acetonitrile is used as an extraction solvent, and after degreasing and purification by an enhanced degreasing filter column, the acetonitrile is detected by a liquid chromatogram-tandem mass spectrometer, and the acetonitrile is quantified by an external standard method. The correlation coefficient of the regression equation reaches above 0.99, the determination limit is 5.0 mu g/kg, the method is simple and convenient to operate, high in sensitivity, strong in anti-interference capability, accurate in qualitative and quantitative determination, and capable of providing technical support for relevant supervision departments in China.
Disclosure of Invention
In view of the technical problem of detection of the existing veterinary drugs, the invention provides a method for determining the residual quantity of the de-isopropylaminophos in poultry eggs.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for measuring the residual quantity of the de-isopropylaminophos in poultry eggs comprises the following specific steps: taking poultry eggs, removing shells, and homogenizing to be used as test materials; taking a homogeneous blank sample as a blank sample; taking a homogeneous blank sample, and adding a standard working solution with a proper concentration to serve as a blank addition test material; weighing 2g of test material, adding 12mL of acetonitrile into a 50mL centrifuge tube, uniformly mixing by vortex, performing ultrasonic treatment for 10min, horizontally oscillating for 20min, centrifuging for 5min at 4000r/min, taking supernatant, adding 12mL of acetonitrile into residues, repeatedly extracting once, combining the supernatants, fixing the volume to 25mL, uniformly mixing by vortex, taking 12.5mL into a graduated test tube A, blowing the volume to 2.5mL by 42 ℃ water bath nitrogen, vortex for 1min, adding 1mL of water, and vortex for 1min for later use; passing the standby liquid through an EMR-Lipid column, eluting under the action of gravity, collecting eluent in another graduated test tube B, adding 1mL of 70% acetonitrile aqueous solution into the graduated test tube A, swirling for 30s, transferring into the EMR-Lipid column, collecting eluent in the graduated test tube B, and pumping under negative pressure; blowing the eluent to 2.5mL in water bath nitrogen at 42 ℃, adding water to fix the volume to 4mL, uniformly mixing by vortex, and performing liquid chromatography-tandem mass spectrometry; the preparation method of the matrix matching standard curve comprises the following steps: taking a blank sample, processing the blank sample according to the extraction and purification method to obtain a blank matrix solution, accurately measuring a proper amount of the iso-propyl-dopaminergic phosphorus removal standard working solution, diluting the blank matrix solution to prepare a series of iso-propyl-dopaminergic phosphorus removal matrix matching mixed standard solutions with the concentrations of 1.0, 2.0, 5.0, 10 and 20ng/mL, and performing liquid chromatography-tandem mass spectrometry; and drawing a standard curve by taking the measured peak area of the characteristic ion as a vertical coordinate and the corresponding concentration of the standard solution as a horizontal coordinate, and solving a regression equation and a correlation coefficient.
The liquid chromatography conditions are as follows: a chromatographic column: c18; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.3 mL/min; mobile phase: a: aqueous solution, B: acetonitrile; the gradient elution procedure was: 0min, 90% of water solution and 10% of acetonitrile; 4min, 10% of aqueous solution and 90% of acetonitrile; 6min, 10% of aqueous solution and 90% of acetonitrile; 6.1min, 90% of water solution and 10% of acetonitrile; 10min, 90% of water solution and 10% of acetonitrile; the mass spectrum conditions are as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: monitoring multiple reactions; spraying voltage: 5500V; ion source temperature: 400 ℃; air curtain pressure: 35; collision gas pressure: medium; atomizing gas pressure: 50; auxiliary heater pressure: 60, adding a solvent to the mixture; all the gases are nitrogen; declustering voltage DP: -70V; port voltage EP: 10V; collision cell outlet voltage CXP: 10V; qualitative ion pair and collision energy: 237.9>193.9, collision energy-18V; 237.9>153.9, collision energy-17V; and (3) quantitative ion pair: 237.9> 153.9.
The qualitative determination method comprises the following steps: judging that the corresponding object to be detected exists in the sample according to the fact that the retention time of the sample chromatogram is consistent with that of the standard, and the relative abundance of the characteristic ions of each spectral peak is consistent with that of the characteristic ions of each spectral peak of the standard with corresponding concentration; the maximum allowable deviation range of relative ion abundance in qualitative confirmation is: relative ion abundance > 50%, allowable relative deviation ± 20%; relative ion abundance > 20% to 50%, allowable relative deviation ± 25%; relative ion abundance > 10% to 20%, allowed relative deviation ± 30%; the relative ion abundance is less than or equal to 10 percent, and the allowable relative deviation is +/-50 percent.
The quantitative determination method comprises the following steps: and taking the sample solution and the corresponding matrix matching standard solution, performing single-point or multi-point calibration, and quantifying according to an external standard method, wherein the response value of the deisopropyldopaminergic phosphorus in the standard solution and the sample solution is in a linear range detected by an instrument.
The measurement result is calculated and expressed by adopting an external standard method for quantification and calculated according to the following formula,
in the formula:
x is the residual quantity of the de-isopropylaminophos in the unit of mu g/kg in a sample;
a-the peak area of the de-isopropylaminophos in the sample solution;
the concentration of the de-isopropylaminophos in the Cs-matrix matching standard solution is ng/mL;
as-substrate matches the peak area of the de-isopropylaminophos in the standard solution;
v1-total volume of extraction solution, unit is mL;
v2-accurately measure the volume of the extraction solution for detection, the unit is mL;
v3-volume of constant volume of sample solution, unit is mL;
m is the mass of the sample in g.
The method for confirming the sensitivity of the measurement comprises the following steps: determining a quantitative limit based on the value of the signal-to-noise ratio; adding 5.0 mu g/kg of de-isopropylaminophos standard solution into a blank poultry egg, measuring the ratio of a signal to noise, and taking the concentration as a quantitative limit when S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirements of a residual detection method; the determination method of the accuracy of the determination comprises the following steps: accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, wherein each concentration is 6 in parallel, and measuring after the sample is processed according to the pretreatment process, and calculating the standard recovery rate; the determination method of the precision of the measurement comprises the following steps: accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, making 6 parallels for each concentration, processing according to the sample pretreatment process, determining, and calculating the indoor relative standard deviation.
The invention has the following beneficial effects:
the method establishes a determination method for the residual quantity of the de-isopropylaminophos in the poultry eggs by adopting the liquid chromatography-tandem mass spectrometry for the first time, and fills the technical blank in the field. The pretreatment and separation technology of the sample is original, and the problems solved by the method comprise: the metabolism of the dopaminergic phosphorus is de-isopropyl dopaminergic phosphorus, the chemical structure is changed from ester into acid, the polarity is obviously increased, so the pretreatment method and the instrument detection method are different from the original medicine. As the compound has no detection method or reference, the standard substance has no ready-made commodity, and only can be designed and synthesized by a standard substance company, and the extraction solvent, the purification means and the instrument conditions need to be originally designed, particularly, the poultry eggs contain high protein and high fat, and impurities which interfere with detection are well removed by adopting an enhanced fat removal filter column. The method is scientific and accurate in quantification and strong in anti-interference capability, and can meet the latest technical requirements of relevant supervision departments in China. The correlation coefficient of the regression equation of the invention reaches more than 0.999, the determination lower limit is 5.0 mug/kg, the recovery rate on different addition concentration levels is 90.9-97.8%, and the relative standard deviation in a laboratory is less than or equal to 10%.
Drawings
FIG. 1-a is a standard solution of deisopropyldopaminergic phosphorus with a characteristic ion mass chromatogram (5.0. mu.g/L) (237.9>153.9)
FIG. 1-b is a characteristic ion mass chromatogram of a deisopropyldopaminergic phosphorus standard solution (5.0. mu.g/L) (237.9>193.9)
FIG. 2-a egg blank characteristic ion mass chromatogram (237.9>153.9)
FIG. 2-b egg blank characteristic ion mass chromatogram (237.9>193.9)
FIG. 3-a egg blank addition deisopropylammonium phosphate sample characteristic ion mass chromatogram (5.0. mu.g/kg)
(237.9>153.9)
FIG. 3-b is a characteristic ion mass chromatogram of a blank egg sample with addition of deisopropylammonium phosphate (5.0. mu.g/kg)
(237.9>193.9)
FIG. 4-a Positive ion mode deisopropylbaraphosphate standard solution characteristic ion mass chromatogram (10.0. mu.g/L) (240.1>156)
FIG. 4-b anion mode deisopropylbaraphosphate standard solution characteristic ion mass chromatogram (10.0. mu.g/L) (237.9>153.9)
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the present invention will be described in detail with reference to specific embodiments and drawings, wherein the drawings only apply to the following embodiments, and other drawings can be obtained by a person skilled in the art according to the method of the present invention. The scope of the invention is not limited to the following examples.
Example 1: establishing method
1. Reagents and materials
1.1 reagents
The reagents used below, unless otherwise noted, are analytical reagents; the water is first-grade water meeting the GB/T6682 specification.
Acetonitrile (CH3 CN): and (4) carrying out chromatographic purification.
1.2 preparation of solution
70% acetonitrile in water: acetonitrile 70mL is taken, water is added for dissolution, the mixture is diluted to 100mL, and the mixture is mixed evenly.
1.3 Standard substance
The content of the de-isopropyl dopamine phosphorus (Desisopropyl-propetathos, C7H14NO4PS, CAS number: 99606-11-8) is more than or equal to 98.0 percent.
1.4 Standard solution preparation
Removing an isopropyl dopamine phosphorus standard stock solution: accurately weighing 10mg of each of the iso-propyl dopaminergic phosphorus standard products, dissolving the iso-propyl dopaminergic phosphorus standard products in a 100mL measuring flask by using a proper amount of acetonitrile, diluting the acetonitrile to a scale, and preparing the iso-propyl dopaminergic phosphorus standard stock solution with the concentration of 100 mg/L. Storing at below-20 deg.C for 12 months.
Removing the standard working solution of the isoprotylbaraphosphate: accurately measuring 1.00mL of each iso-propyl dopaminergic phosphorus standard stock solution, diluting the stock solutions to a scale with acetonitrile in a 100mL measuring flask, and preparing the mixed standard working solution with the concentration of the iso-propyl dopaminergic phosphorus of 1.00 mu g/mL. Storing at below-20 deg.C for 6 months.
1.5 materials
EMR-Lipid enhanced Lipid removal filter column: 300mg/3mL, or equivalent.
2. Apparatus and device
Liquid chromatography-tandem mass spectrometer: a power distribution spray ion source; analytical balance: the sensory quantity is 0.00001 g; balance: the sensory quantity is 0.01 g; a homogenizer; a vortex mixer; a horizontal oscillator; a high speed centrifuge; a nitrogen blowing instrument; a solid phase extraction device.
3. Preparation and preservation of samples
3.1 preparation of sample
Taking a proper amount of fresh eggs, removing shells, and homogenizing.
-taking a homogeneous test sample as a test sample.
-taking the homogeneous blank sample as a blank sample.
And taking the homogeneous blank sample, and adding a standard working solution with a proper concentration to be used as a blank addition sample.
3.2 preservation of samples
Storing at below-20 deg.C.
4. Measurement procedure
4.1 extraction
Weighing 2g (accurate to +/-0.02 g) of a sample, adding 12mL of acetonitrile into a 50mL centrifuge tube, performing vortex mixing, performing ultrasonic treatment for 10min, horizontally oscillating for 20min, centrifuging for 5min at 4000r/min, taking supernatant, adding 12mL of acetonitrile into residues, repeatedly extracting once, combining supernatants, performing constant volume to 25mL, performing vortex mixing, taking 12.5mL into a graduated test tube A, blowing nitrogen in a water bath at 42 ℃ to 2.5mL, performing vortex for 1min, adding 1mL of water, and performing vortex for 1min for later use.
4.2 purification
And (3) passing the standby liquid through an EMR-Lipid column, eluting under the action of gravity, collecting eluent in another graduated test tube B, adding 1mL of 70% acetonitrile aqueous solution into the graduated test tube A, swirling for 30s, transferring the mixture into the EMR-Lipid column, collecting eluent in the graduated test tube B, and draining at negative pressure. Blowing the eluent into 2.5mL in water bath at 42 ℃, adding water to fix the volume to 4mL, uniformly mixing by vortex, and performing liquid chromatography-tandem mass spectrometry.
4.3 preparation of matrix matching Standard Curve
Taking a blank sample, processing the blank sample according to the extraction and purification method to prepare a blank matrix solution, accurately measuring a proper amount of the iso-propyl dopaminergic phosphorus removal standard working solution, diluting the blank matrix solution to prepare a series of iso-propyl dopaminergic phosphorus removal matrix matching mixed standard solutions with the concentrations of 1.0, 2.0, 5.0, 10 and 20ng/mL, and performing liquid chromatography-tandem mass spectrometry. And drawing a standard curve by taking the measured peak area of the characteristic ion as a vertical coordinate and the corresponding concentration of the standard solution as a horizontal coordinate. And solving a regression equation and a correlation coefficient.
4.4 determination
4.4.1 reference conditions for liquid chromatography
a) A chromatographic column: c18(100 mm. times.3.0 mm, 2.7 μm), or equivalent; b) column temperature: 40 ℃; c) sample introduction amount: 10 mu L of the solution; d) flow rate: 0.3 mL/min; e) mobile phase: a: aqueous solution, B: acetonitrile, gradient elution procedure see table 1.
TABLE 1 gradient elution procedure
| Time, min | A,％ | B,％ |
| 0 | 90 | 10 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 6.1 | 90 | 10 |
| 10 | 90 | 10 |
4.4.2 reference conditions for Mass Spectrometry
a) An ion source: electrospray ion source (ESI); b) the scanning mode is as follows: scanning negative ions; c) the detection mode is as follows: multiple Reaction Monitoring (MRM); d) spray voltage (IS): 5500V; e) ion source Temperature (TEM): 400 ℃; f) air curtain pressure (nitrogen): 35; g) collision gas pressure (nitrogen): medium; h) atomization gas pressure (nitrogen): 50;
i) auxiliary heater pressure (nitrogen): 60, adding a solvent to the mixture; j) declustering voltage DP: -70V; k) entrance voltage EP: 10V;
l) collision cell exit voltage CXP: 10V; m) qualitative ion-pair and collision energy: 237.9>193.9, collision energy-18V; 237.9>153.9, collision energy-17V; n) quantitative ion pairs: 237.9> 153.9. Note: for different mass spectrometry instruments, instrument parameters may differ, and the mass spectrometry parameters should be optimized before measurement.
4.4.3 qualitative determination
And comparing and determining the retention time of the sample chromatogram with the retention time of the standard, and the characteristic ions of each spectral peak with the characteristic ions of each spectral peak of the standard at corresponding concentrations. The relative deviation of the retention time of the sample from the standard is no more than 5%; the relative abundance of the characteristic ions of the sample is consistent with that of the mixed standard solution with the same concentration, and the deviation of the relative abundance does not exceed the specification of the table 2, so that the corresponding detected object exists in the sample.
TABLE 2 maximum permissible deviation of relative ion abundance in qualitative confirmation
| Relative ion abundance | ＞50％ | More than 20% to 50% | More than 10% to 20% | ≤10％ |
| Allowable relative deviation | ±20％ | ±25％ | ±30％ | ±50％ |
4.4.4 quantitative determination
Taking the sample solution and the corresponding matrix matching standard solution, performing single-point or multi-point calibration, and quantifying according to an external standard method, wherein the response value of the deisopropyldopaminergic phosphorus in the standard solution and the sample solution is in a linear range detected by an instrument. Under the conditions of the chromatography-mass spectrometry, the mass spectrograms of the liquid chromatography of the deisopropyldopaminergic phosphorus standard solution and the sample solution are shown in the attached drawings of 1-a, 1-b, 2-a, 2-b, 3-a and 3-b of the specification.
4.4.5 blank test
The parallel operation was carried out by exactly the same procedure except that no sample was added.
4.4.6 results calculation and presentation
Quantifying by adopting an external standard method, and calculating according to the formula (1):
in the formula:
x is the residual quantity of the deisopropyldopaminergic phosphorus in the sample, and the unit is microgram per kilogram (mu g/kg);
a-the peak area of the de-isopropylaminophos in the sample solution;
the Cs-matrix matches the concentration of deisopropyldopaminergic in standard solution in nanograms per milliliter (ng/mL);
as-substrate matches the peak area of the de-isopropylaminophos in the standard solution;
v1 — total volume of extraction solution in milliliters (mL);
v2 — volume of exact amount of extraction solution for detection in milliliters (mL);
v3 — volume of sample solution to volume in milliliters (mL);
m is the mass of the sample in grams (g).
Note: the blank value is subtracted from the calculation result, the measurement result is represented by the arithmetic mean value of the parallel measurement, and three significant figures are reserved.
Sensitivity, accuracy and precision of detection method
4.4.6 sensitivity
The quantitative limit is determined based on the value of the signal-to-noise ratio (S/N). Adding 5.0 mu g/kg of the de-isopropylaminophos standard solution into the blank eggs, measuring the ratio of signals to noise, and taking the concentration as the quantitative limit when the S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirements of the residual detection method.
The experimental results are as follows: the limit of quantitation of this method is 5.0. mu.g/kg.
4.4.7 accuracy
Accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, making 6 samples in parallel for each concentration, processing and determining according to the sample pretreatment process, and calculating the standard recovery rate.
The experimental results are as follows: the recovery rate of the method on the addition concentration level of 5.0 mu g/kg-50 mu g/kg is 90.9% -97.8%.
4.4.8 precision
Accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, making 6 parallels for each concentration, processing according to the sample pretreatment process, determining, and calculating the indoor relative standard deviation.
The experimental results are as follows: the relative standard deviation in the laboratory of the method is less than or equal to 10 percent.
5. Examples of the invention
The method is used for daily detection, 50 detected egg samples are obtained, and no isopropyl-dopaminergic phosphorus is detected.
Example 2: selection of sample extraction solvent
The de-isopropylaminophos is insoluble in water and easily soluble in organic solvents. For organophosphorus drugs, common plant samples are mainly extracted by organic solvents such as ethyl acetate, acetone and the like. Because the eggs contain a large amount of lipid substances, the polarity of ethyl acetate and acetone is smaller than that of acetonitrile, the solubility to fat is higher, fat-soluble impurities in the extracting solution are more, the purification is not facilitated, and solvent conversion is needed before purification. Acetonitrile was therefore chosen as the extraction solvent for this experiment. When the liquid chromatography is used for detection, the specificity of the selective ion pair is strong, even if impurities such as fat and protein exist, the impurities cannot be seen on a spectrogram, but the nitrogen blowing is obvious, because hundreds of microliters of lipids cannot be blown dry, an instrument is easy to pollute, and a chromatographic column is easy to block.
Example 3: optimization of purification mode
Although acetonitrile is used as an extraction solvent, the extraction solution contains relatively few lipid substances, the lipid substances are still main interference substances for measuring residues in poultry egg matrixes, the sensitivity of analytical substances is reduced, a chromatographic system is blocked, the service life of the chromatographic system is shortened, and therefore, the degreasing is the key point of sample purification. The commonly used organophosphorus medicament solid phase extraction columns comprise a C18 reversed phase solid phase extraction column, a Carb/NH2 (carbon black/amino) composite column, a Carb/PSA composite column and the like, and the captivaEMR-Lipid extraction column is adopted in the experiment to further realize the most thorough Lipid removal and analyte recovery, so that the Lipid can be removed on the premise of not losing the analyte. Compared with the traditional solid phase extraction, the whole purification process omits the processes of activation, balance, leaching and the like, the operation steps are very simple, and the pretreatment efficiency is obviously improved. The method has good degreasing effect and simple operation. The ratio of acetonitrile to water is adjusted to be directly passed through. Nevertheless, the analysis process faces some difficulties: the proportion of acetonitrile water in the sample liquid has a large influence on the purification effect, the proportion of acetonitrile is more than 80%, the degreasing efficiency is reduced, the proportion of acetonitrile is too small, the recovery rate of an analyte is influenced, and finally the proportion of acetonitrile is 70% in the experiment, so that the degreasing effect and the recovery rate of the analyte are optimal.
Example 4: selection of Mass Spectrometry conditions
Electrospray ionization (ESI) is the most common ion source for liquid chromatography-tandem mass spectrometry, and adopts standard solution with higher concentration to perform full scanning of parent ions in positive and negative ion modes respectively to determine excimer ions, then carrying out full scanning on the daughter ions to obtain the daughter ions with stronger secondary mass spectrum response, wherein common organophosphorus medicaments have higher response in a positive ion mode, and the de-isopropylaminophos has higher response in a positive ion mode and a negative ion mode, however, in the positive ion mode, two chromatographic peaks appear in the chromatogram of the standard solution, as shown in FIGS. 4-a and 4-b, while more interfering peaks exist in the chromatogram of the labeled sample, and the baseline noise is higher, therefore, the test selects the negative ion mode, not only can obtain higher response, but also has no interference and higher signal to noise ratio, and is more beneficial to detecting the residual quantity of the dl-isopolylamine phosphate in a complex matrix.
Claims (6)
1. A method for measuring the residual quantity of the de-isopropylaminophos in poultry eggs is characterized by comprising the following specific steps: taking poultry eggs, removing shells, and homogenizing to be used as test materials; taking a homogeneous blank sample as a blank sample; taking a homogeneous blank sample, and adding a standard working solution with a proper concentration to serve as a blank addition test material; weighing 2g of test material, adding 12mL of acetonitrile into a 50mL centrifuge tube, uniformly mixing by vortex, performing ultrasonic treatment for 10min, horizontally oscillating for 20min, centrifuging for 5min at 4000r/min, taking supernatant, adding 12mL of acetonitrile into residues, repeatedly extracting once, combining the supernatants, fixing the volume to 25mL, uniformly mixing by vortex, taking 12.5mL into a graduated test tube A, blowing the volume to 2.5mL by 42 ℃ water bath nitrogen, vortex for 1min, adding 1mL of water, and vortex for 1min for later use; passing the standby liquid through an EMR-Lipid column, eluting under the action of gravity, collecting eluent in another graduated test tube B, adding 1mL of 70% acetonitrile aqueous solution into the graduated test tube A, swirling for 30s, transferring into the EMR-Lipid column, collecting eluent in the graduated test tube B, and pumping under negative pressure; blowing the eluent to 2.5mL in water bath nitrogen at 42 ℃, adding water to fix the volume to 4mL, uniformly mixing by vortex, and performing liquid chromatography-tandem mass spectrometry; the preparation method of the matrix matching standard curve comprises the following steps: taking a blank sample, processing the blank sample according to the extraction and purification method to obtain a blank matrix solution, accurately measuring a proper amount of the iso-propyl-dopaminergic phosphorus removal standard working solution, diluting the blank matrix solution to prepare a series of iso-propyl-dopaminergic phosphorus removal matrix matching mixed standard solutions with the concentrations of 1.0, 2.0, 5.0, 10 and 20ng/mL, and performing liquid chromatography-tandem mass spectrometry; and drawing a standard curve by taking the measured peak area of the characteristic ion as a vertical coordinate and the corresponding concentration of the standard solution as a horizontal coordinate, and solving a regression equation and a correlation coefficient.
2. The assay of claim 1, wherein the liquid chromatography conditions are: a chromatographic column: c18; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.3 mL/min; mobile phase: a: aqueous solution, B: acetonitrile; the gradient elution procedure was: 0min, 90% of water solution and 10% of acetonitrile; 4min, 10% of aqueous solution and 90% of acetonitrile; 6min, 10% of aqueous solution and 90% of acetonitrile; 6.1min, 90% of water solution and 10% of acetonitrile; 10min, 90% of water solution and 10% of acetonitrile; the mass spectrum conditions are as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: monitoring multiple reactions; spraying voltage: 5500V; ion source temperature: 400 ℃; air curtain pressure: 35; collision gas pressure: medium; atomizing gas pressure: 50; auxiliary heater pressure: 60, adding a solvent to the mixture; all the gases are nitrogen; declustering voltage DP: -70V; port voltage EP: 10V; collision cell outlet voltage CXP: 10V; qualitative ion pair and collision energy: 237.9>193.9, collision energy-18V; 237.9>153.9, collision energy-17V; and (3) quantitative ion pair: 237.9> 153.9.
3. The assay of claim 1, wherein the qualitative assay is: judging that the corresponding object to be detected exists in the sample according to the fact that the retention time of the sample chromatogram is consistent with that of the standard, and the relative abundance of the characteristic ions of each spectral peak is consistent with that of the characteristic ions of each spectral peak of the standard with corresponding concentration; the maximum allowable deviation range of relative ion abundance in qualitative confirmation is: relative ion abundance > 50%, allowable relative deviation ± 20%; relative ion abundance > 20% to 50%, allowable relative deviation ± 25%; relative ion abundance > 10% to 20%, allowed relative deviation ± 30%; the relative ion abundance is less than or equal to 10 percent, and the allowable relative deviation is +/-50 percent.
4. The assay of claim 1, wherein the quantitative assay method comprises: and taking the sample solution and the corresponding matrix matching standard solution, performing single-point or multi-point calibration, and quantifying according to an external standard method, wherein the response value of the deisopropyldopaminergic phosphorus in the standard solution and the sample solution is in a linear range detected by an instrument.
5. The assay of claim 1, wherein the results of said assay are calculated and expressed, quantified using an external standard method, according to the following formula,
in the formula:
x is the residual quantity of the de-isopropylaminophos in the unit of mu g/kg in a sample;
a-the peak area of the de-isopropylaminophos in the sample solution;
the concentration of the de-isopropylaminophos in the Cs-matrix matching standard solution is ng/mL;
as-substrate matches the peak area of the de-isopropylaminophos in the standard solution;
v1-total volume of extraction solution, unit is mL;
v2-accurately measure the volume of the extraction solution for detection, the unit is mL;
v3-volume of constant volume of sample solution, unit is mL;
m is the mass of the sample in g.
6. The method of measuring according to claim 1, wherein the method of confirming the sensitivity of the measurement is: determining a quantitative limit based on the value of the signal-to-noise ratio; adding 5.0 mu g/kg of de-isopropylaminophos standard solution into a blank poultry egg, measuring the ratio of a signal to noise, and taking the concentration as a quantitative limit when S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirements of a residual detection method; the determination method of the accuracy of the determination comprises the following steps: accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, wherein each concentration is 6 in parallel, and measuring after the sample is processed according to the pretreatment process, and calculating the standard recovery rate; the determination method of the precision of the measurement comprises the following steps: accurately weighing 2.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the deisopropyldopamine-phosphorus being 5.0, 10 and 50 mu g/kg respectively, making 6 parallels for each concentration, processing according to the sample pretreatment process, determining, and calculating the indoor relative standard deviation.
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| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
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| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
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