CN108982701B - Method for determining residual mefenacet in aquatic product material - Google Patents

Method for determining residual mefenacet in aquatic product material Download PDF

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CN108982701B
CN108982701B CN201810953401.7A CN201810953401A CN108982701B CN 108982701 B CN108982701 B CN 108982701B CN 201810953401 A CN201810953401 A CN 201810953401A CN 108982701 B CN108982701 B CN 108982701B
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CN108982701A (en
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欧阳少伦
林峰
蓝草
邵琳智
吴映璇
姚仰勋
邹游
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Inspection and Quarantine Technology Center of Guangdong Entry Exit Inspection and Quarantine Bureau
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Abstract

The invention discloses a method for determining a mefenacet residue in an aquatic product material and application thereof. The specific method comprises the steps of directly mincing and homogenizing a sample, adopting ethyl acetate as an extraction solvent, concentrating a part of extracting solution to a constant volume, purifying by a solid phase extraction column, detecting by a liquid chromatography-tandem mass spectrometer, and quantifying by an external standard method. The invention provides a standard method for detecting the residual mieheitin in aquatic product materials for the first time, and fills the technical blank in the field. The correlation coefficient of the regression equation of the invention reaches more than 0.99, the determination lower limit is 0.5 mug/kg, the recovery rate at the addition concentration level of 0.5 mug/kg-5.0 mug/kg is 80-120%, and the relative standard deviation in the laboratory is less than or equal to 20%. The method has the advantages of simple operation, high sensitivity, strong anti-interference capability and scientific and accurate qualitative and quantitative determination, and can provide technical support for relevant supervision departments in China to establish the maximum residual limit of the mefenatine in the aquatic products.

Description

Method for determining residual mefenacet in aquatic product material
Technical Field
The invention relates to the technical field of detection of veterinary drug residues, and particularly relates to an aquatic product material, in particular to a method for determining the residue of mefenatine in tilapia, shrimps or crabs.
Background
The residue of veterinary drugs in aquatic products directly influences the health of human bodies.
Emamectin (Emamectin), alias Emamectin, with the molecular formula C49H75NO13Molecular weight 886.51, and its structural formula is shown in formula (I):
Figure BDA0001770452550000011
because of its high efficiency, broad-spectrum anthelmintic action. The food code commission (CAC)2015 increased the maximum residual limit due to methacetin, allowed use in seafood and set the maximum residual limit: 100 mu g/kg of muscle of salmon and trout. The Japanese affirmation List also stipulates that the maximum residual limit of the extract in aquatic animals such as fish fillet, crab, frozen shrimp meat, etc. is 0.5. mu.g/kg.
Because the highest residual limit of the mefenadine is newly increased by the food code committee (CAC) in 2015, no standard for the detection of the residual amount of the mefenadine in aquatic products exists so far, no document report of any related detection method is found, the detection method of the mefenadine in aquatic products is lacked, and the technical support for the establishment of the highest residual limit of the mefenadine for related regulatory gates in China is difficult to provide, the invention discloses a liquid-mass combination method for detecting the residual amount of the mefenadine in aquatic products, wherein ethyl acetate is used as an extracting solution, a mixed cation exchange solid-phase extraction column is used for purification, a Waters Atlantis selected and used for T3(4.6mM multiplied by 100mM, the particle size is 3 mu m) chromatographic column separation, an acetonitrile-5 mM formic acid aqueous solution is used for a mobile phase, gradient elution is adopted, and a qualitative ion pair is selected: 886.5>158.3, collision energy 121V; 886.5>81.9, impact energy 42V; selecting 886.5>158.3 with little interference as a quantitative ion pair, and adopting an external standard method for quantification. The method is simple and convenient, can effectively eliminate the matrix effect, has good accuracy and precision, and provides technical support for relevant supervision departments in China to the detection of the mefenatine.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the residual quantity of the mefenacet in an aquatic product aiming at the technical defect of the detection of the residual quantity of the mefenacet.
The invention aims to solve the technical problem of providing the application of the detection method, in particular to the application in the aspect of measuring the residual quantity of the mefenatine in tilapia, shrimps or crabs.
The purpose of the invention is realized by the following technical scheme:
a method for determining the concentration of emetine in an aquatic product material, the method comprising: sample pretreatment; extracting with ethyl acetate twice, concentrating and dissolving part of the extractive solution, and purifying with solid phase extraction column to obtain test solution; preparing a series of standard solutions of the mefenacet standard working solution, and determining by using a liquid chromatography-tandem mass spectrometer; drawing a standard curve by taking the characteristic ion mass chromatographic peak area of the mefenatine as the ordinate and the standard solution concentration as the abscissa, and solving a regression equation and a correlation coefficient; detecting the liquid to be tested by using a liquid chromatogram-tandem mass spectrometer, and quantifying by using an external standard method; the sample pretreatment method comprises the following steps: taking an edible tissue of a water product, mincing and homogenizing, and taking a sample to be tested after homogenizing as a test material; taking the homogenized blank sample as a blank sample; taking the homogenized blank sample, and adding a standard working solution with a proper concentration to serve as a blank addition test material; detecting the obtained test sample, blank sample and blank additive sample or storing at below-18 deg.C; the method for extracting by using secondary ethyl acetate comprises the following steps: weighing a homogenized sample, adding 10mL of ethyl acetate, horizontally oscillating for 15min, centrifuging, taking supernatant into a 25mL colorimetric tube, adding 10mL of ethyl acetate into residue, horizontally oscillating for 30min, centrifuging, combining two extracting solutions, fixing the volume to 25mL, shaking up, accurately sucking 5mL of the extracting solution into a glass test tube, concentrating at 45 ℃ with nitrogen until the extracting solution is dry, adding 5mL of a fixing solution, oscillating for 2min to obtain a solution to be purified; the solution is obtained by uniformly mixing acetonitrile and 2% formic acid aqueous solution according to the volume ratio of 25: 75; the conditions of the centrifugation treatment are: treating at 4500r/min for 5 min; the homogenization treatment condition is that the homogenization treatment is carried out for 30s at 12000 r/min.
The preparation method of the series of standard solutions of the imipramine standard working solution comprises the following steps:
100 μ g/mL of nemadectin standard stock solution: accurately weighing 10mg of an imietidine standard substance, dissolving the imietidine standard substance in a 100mL measuring flask by using acetonitrile, diluting the solution to a scale, and preparing into an imietidine standard storage solution with the concentration of 100 mu g/mL, and storing the solution in a dark place below-18 ℃;
5 μ g/mL of nemadectin standard intermediate: precisely measuring 5.00mL of a 100 mu g/mL emethentine standard stock solution, diluting the stock solution to a scale with acetonitrile in a 100mL measuring flask to prepare a 5 mu g/mL emethentine standard intermediate solution, and storing the intermediate solution in a dark place at the temperature below-18 ℃;
100ng/mL of a nemadectin standard working solution: precisely measuring 1.00mL of a 5 mu g/mL emetine standard intermediate solution, diluting the intermediate solution to the scale with acetonitrile in a 50mL measuring flask to prepare an acetonitrile standard working solution with the concentration of 100ng/mL, and storing the working solution away from light at the temperature below-18 ℃.
The purification of the solid phase extraction column is to pass 5mL of the solution to be purified through the solid phase extraction column which is activated by 3mL of methanol and 3mL of 2% formic acid aqueous solution, and the flow rate is maintained at 0.5 mL/min; then eluting with 4mL of 2% formic acid aqueous solution and 4mL of eluent in sequence, eluting with 4mL of eluent again, maintaining the flow rate at 0.5mL/min, and collecting the eluent. The eluate was dried at 45 ℃ with nitrogen and the residue was dried over 1.0mL of a 1: dissolving acetonitrile of 1 and 5mM formic acid solution, filtering with a 0.22 μm filter membrane to obtain a solution to be tested, and performing liquid chromatography-tandem mass spectrometry; the leacheate comprises 10% ammonia water solution with the volume ratio of 80% and 20% methanol with the volume ratio; the eluent comprises 90% of water by volume and 10% of ammonia water by volume.
The liquid chromatography conditions are as follows: and (3) chromatographic column: waters Atlantis T3, 4.6mm × 100mm, particle size 3 μm; mobile phase: acetonitrile-5 mM formic acid aqueous solution, gradient elution conditions are shown in Table 1; flow rate: 0.3 mL/min; column temperature: 40 ℃; sample introduction amount: 5 μ L.
The mass spectrum conditions are as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning positive ions; the detection mode is as follows: monitoring multiple reactions; spray voltage IS: 5500V; and (4) CUR: 35; CAD: medium; auxiliary heating gas temperature: 600 ℃; GS 1: 60, adding a solvent to the mixture; GS 2: 60, adding a solvent to the mixture; declustering voltage DP: 173V; entrance voltage EP: 12V; collision cell outlet voltage CXP: 10V; residence time: 0.1 s; qualitative ion pair and collision energy: 886.5>158.3, impact energy 121V; 886.5>81.9, impact energy 42V; and (3) quantitative ion pair: 886.5> 158.3.
The qualitative detection method of the determination method comprises the following steps: the retention time of a sample chromatogram is compared with the retention time of a corresponding standard substance, and characteristic ions of a chromatographic peak are compared with characteristic ions of a chromatographic peak of a standard solution with a corresponding concentration for qualitative comparison. The relative deviation of the retention time of the sample and the standard substance is not more than 5 percent; the relative abundance of the characteristic ions of the sample is consistent with that of the standard solution with the equivalent concentration, and the deviation of the relative abundance does not exceed the specification of the table 2, so that the corresponding object to be detected exists in the sample.
The quantitative detection method of the determination method comprises the following steps: taking sample solution and standard solution, and calculating by peak area according to external standard method.
The result calculation method of the measuring method comprises the following steps:
standard curve As＝a×cs+ b calibration: by finding a and b, then
Figure BDA0001770452550000031
Figure BDA0001770452550000032
The amount of residual statin in the sample was calculated according to formula (III):
Figure BDA0001770452550000033
Figure BDA0001770452550000034
in the formula:
as-the peak area of mefenatine in the standard solution;
cs— concentration of emetine in standard solution in nanograms per milliliter (ng/mL);
c-concentration of emetine in the sample solution in nanograms per milliliter (ng/mL);
a-peak area of mefenatine in the sample;
x-residual amount of mefenatine in the test sample in units of micrograms per kilogram (. mu.g/kg);
v — volume of dissolution residue in milliliters (mL);
m-sample mass under test in grams (g);
d- — dilution factor;
the dilution factor in the above formula is 1; wherein blank value is subtracted from the calculated result, and the measured result is measured in parallel
The arithmetic mean of (d) indicates that three significant digits are retained.
The aquatic product comprises tilapia, shrimp or crab.
The invention has the following beneficial effects:
the invention provides a standard method for detecting the residual mefenatine in aquatic products for the first time, and fills the technical blank in the field. The invention directly rubs the sample and homogenizes the slurry, optimizes the extraction and purification treatment steps and conditions of the solid-phase extraction column, has simple sample treatment operation and does not need to add complex chemical reagents. Based on the solution to be detected obtained by the sample pretreatment, the invention provides a detection method and detection conditions pertinently, and successfully establishes a standard method for detecting the residual emetine in the aquatic products.
The correlation coefficient of the regression equation reaches more than 0.99, the determination lower limit is 0.5 mu g/kg, the recovery rate on the addition concentration level of 0.5 mu g/kg-5.0 mu g/kg is 80-120 percent, and the relative standard deviation in a laboratory is less than or equal to 20 percent.
The method has the advantages of simple operation, high sensitivity, strong anti-interference capability and scientific and accurate qualitative and quantitative determination, and can provide technical support for the relevant supervision department in China to determine the maximum residual quantity of the mefenacet.
Drawings
FIG. 1. characteristic ion mass chromatogram of the efetin standard solution (0.5. mu.g/L), (886.5> 81.9);
FIG. 2. characteristic ion mass chromatogram of the efetin standard solution (0.5. mu.g/L), (886.5> 158.3);
FIG. 3. tilapia mossambica blank sample characteristic ion mass chromatogram, (886.5> 81.9);
FIG. 4. tilapia mossambica blank sample characteristic ion mass chromatogram, (886.5> 158.3);
FIG. 5. mass chromatogram of characteristic ions of tilapia mossambica blank addicting nemadectin sample (0.5 μ g/kg), (886.5> 81.9);
FIG. 6. mass chromatogram of characteristic ions of tilapia mossambica blank addicting nemetin sample (0.5 μ g/kg), (886.5> 158.3);
FIG. 7. shrimp blank sample characteristic ion mass chromatogram, (886.5> 81.9);
FIG. 8, ion mass chromatogram characteristic of shrimp blanks, (886.5> 158.3);
FIG. 9. shrimp blank addition nemadectin sample characteristic ion mass chromatograms (0.5. mu.g/kg), (886.5> 81.9);
FIG. 10. shrimp blank addition nemadectin sample characteristic ion mass chromatograms (0.5. mu.g/kg), (886.5> 158.3);
FIG. 11 mass chromatogram of characteristic ions of positive Tilapia oreochromitin sample (3.1. mu.g/kg), (886.5> 81.9);
FIG. 12 positive Tilapia oreochromitin sample characteristic ion mass chromatogram (3.1. mu.g/kg), (886.5> 158.3).
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 those skilled in the art according to the method of the present invention. The scope of protection of the invention is not limited to the examples described below.
Example 1: nemacidin detection in tilapia and shrimp
1. Reagent
The reagents used were analytical reagents unless otherwise noted. The water is first-grade water meeting the GB/T6682 regulation; due to the fact that the standard substance of mefenacet: the content is more than or equal to 98 percent; acetonitrile: carrying out chromatographic purification; ethyl acetate: carrying out chromatographic purification; formic acid: and (4) carrying out chromatographic purification.
5mM aqueous formic acid: mu.L of formic acid was taken, dissolved in water and diluted to 1000 mL.
Solution determination: 25mL of acetonitrile and 75mL of 2% formic acid aqueous solution are taken and mixed uniformly for standby.
Mobile phase solution: 50mL of acetonitrile and 50mL of 5mM formic acid aqueous solution were mixed well for use.
100 μ g/mL of nemadectin standard stock solution: weighing 10mg of the imipenem standard substance precisely, dissolving the imipenem standard substance in a 100mL measuring flask by using acetonitrile, diluting the solution to a scale, and preparing the solution into a 100 mu g/mL imipenem standard stock solution, and storing the solution at the temperature of below-18 ℃ in a dark place.
5 μ g/mL of nemadectin standard intermediate: precisely measuring 5.00mL of the standard because-extine stock solution with the concentration of 100 mu g/mL, diluting the standard because-extine stock solution with the concentration of 5 mu g/mL into a 100mL measuring flask by using acetonitrile to scale, and preparing the standard because-extine intermediate solution with the concentration of 5 mu g/mL for storage at the temperature of minus 18 ℃ in a dark place.
100ng/mL of the standard working solution of the emetine: precisely measuring 1.00mL of a 5-mu g/mL concentration noreptin standard intermediate solution, diluting the intermediate solution to a scale with acetonitrile in a 50-mL measuring flask to prepare an acetonitrile standard working solution with the concentration of 100ng/mL, and storing the working solution at the temperature of below-18 ℃ in a dark place.
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 meat tissue triturator, a vortex oscillator, a horizontal oscillator, a tissue homogenizer, a centrifuge, a nitrogen blower; centrifuging the tube: 50 mL; centrifuging the tube: 1.5 mL; and (3) filtering the membrane: 0.22 μm.
3. Preparation and preservation of samples
3.1. Preparation of sample
Taking blank or tested edible tissue of tilapia or shrimp, mincing, and homogenizing. Storing at below-18 deg.C.
And taking the homogenized test sample as a test material.
The blank sample after homogenization was taken as a blank sample.
And (3) taking the blank sample after homogenization, adding a standard working solution with appropriate concentration to be used as a blank addition sample.
3.2 extraction
Weighing 5g +/-0.05 g of a sample, adding 10mL of ethyl acetate into a 50mL centrifuge tube, horizontally oscillating for 15min, centrifuging for 5min at 4500r/min, taking supernatant into a 25mL colorimetric tube, adding 10mL of ethyl acetate into residues, horizontally oscillating for 30min, centrifuging for 5min at 4500r/min, combining two extracting solutions, fixing the volume to 25mL, shaking up, accurately sucking 5mL of the extracting solution into a glass test tube, blowing nitrogen at 45 ℃ for concentration to be dry, adding 5mL of a fixing solution, and oscillating for 2min to obtain the liquid to be purified.
3.3 purification
The solution to be purified was passed through a solid phase extraction column which had been activated with 3mL of methanol and 3mL of 2% aqueous formic acid, the flow rate being maintained at 0.5 mL/min. Then, the mixture was sequentially eluted with 4mL of a 2% formic acid aqueous solution and 4mL of an eluent (10% aqueous ammonia solution + methanol, 80+20, v/v), and then eluted with 4mL of an eluent (water + aqueous ammonia, 90+10, v/v) at a flow rate of 0.5mL/min, and the eluent was collected. The eluate was dried at 45 ℃ with nitrogen, and the residue was dissolved in 1.0mL of a mobile phase solution (acetonitrile +5mM formic acid, volume ratio 1: 1), filtered through a 0.22 μm filter to obtain a test solution, and subjected to liquid chromatography-tandem mass spectrometry.
4 determination of
4.1 preparation of Standard Curve
Precisely measuring a proper amount of a 100ng/mL mefenacet standard working solution, diluting the solution with a constant volume solution to prepare a series of standard solutions with the concentration of mefenacet of 0, 0.2, 0.5, 1.0, 5.0 and 10.0 mu g/L, and determining the concentration by using a liquid chromatography-tandem mass spectrometer. Drawing a standard curve by taking the peak area of the characteristic ion mass chromatogram of the mefenacet as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate, and solving a regression equation and a correlation coefficient;
4.2 liquid chromatography-tandem mass spectrometer determination conditions:
4.2.1 the liquid chromatography conditions were:
and (3) chromatographic column: waters Atlantis T3(4.6 mm. times.100 mm, particle size 3 μm);
mobile phase: acetonitrile-5 mM formic acid in water, gradient elution conditions are shown in Table 1
Flow rate: 0.3 mL/min;
column temperature: at 40 ℃;
sample introduction amount: 5 mu L of the solution;
TABLE 1 gradient elution conditions
Time (min) Acetonitrile/%) 5mmol/L aqueous formic acid solution/%
0.0 50 50
3.0 100 0
5.0 100 0
5.1 50 50
11.0 50 50
4.2.2 Mass Spectrometry conditions were:
an ion source: an electrospray ion source; the scanning mode is as follows: scanning positive ions; the detection mode is as follows: monitoring multiple reactions; spray voltage IS: 5500V; and (4) CUR: 35; CAD: medium; auxiliary heating gas temperature: 600 ℃; GS 1: 60, adding a solvent to the mixture; GS 2: 60; declustering voltage DP: 173V; entrance voltage EP: 12V; collision cell outlet voltage CXP: 10V; residence time: 0.1 s; qualitative ion pair and collision energy: 886.5>158.3, impact energy 121V; 886.5>81.9, impact energy 42V; and (3) quantitative ion pair: 886.5> 158.3.
4.3. Assay method
4.3.1 qualitative determination: and comparing and determining the retention time of a sample chromatogram with the retention time of a corresponding standard substance, and comparing and determining the characteristic ions of chromatographic peaks with the characteristic ions of chromatographic peaks of a standard solution with corresponding concentration. The relative deviation of the retention time of the sample and the standard sample is not more than 5 percent; the relative abundance of the characteristic ions of the sample is consistent with that of the standard solution with the equivalent concentration, and the deviation of the relative abundance does not exceed the specification of the table 2, so that the corresponding object to be detected exists in the sample.
TABLE 2 allowable deviation Range of relative ion abundance
Relative ion abundance% ＞50 ＞20～50 ＞10～20 ≤10
Allowable relative deviation% ±20％ ±25 ±30 ±50
4.3.2 quantitative determination:
taking a sample solution and a standard solution, and calculating according to the peak area by an external standard method. The response value of the deintene in the standard solution and the sample solution is within the linear range detected by the instrument. Under the conditions of the chromatography-mass spectrometry, characteristic ion mass chromatograms of the standard solution, the blank sample solution and the blank additive sample solution are shown in figures 1-10.
4.3.3 blank test
The parallel operation was carried out by exactly the same procedure except that no sample was added.
4.3.4 calculation of results
Calibration of a standard curve: from As＝a×cs+ b to find a and b, then
Figure BDA0001770452550000071
The amount of residual statin in the sample was calculated according to formula (III):
Figure BDA0001770452550000072
In the formula:
as-peak area of emetine in standard solution;
cs-concentration of emetine in standard solution in nanograms per milliliter (ng/mL);
c-concentration of emetine in the sample solution in nanograms per milliliter (ng/mL);
A-Peak area of emetine in the sample;
x-residual amount of mefenatine in the test sample in units of micrograms per kilogram (. mu.g/kg);
v- — volume of dissolved residue in milliliters (mL);
m-sample mass under test in grams (g);
d- — dilution factor.
The dilution factor in the above formula is 1. Wherein blank values need to be deducted from the calculation results, the measurement results are expressed by the arithmetic mean value of parallel measurement, and three significant figures are reserved.
In this embodiment, a characteristic ion mass chromatogram of a mietin standard solution is shown in fig. 1, a characteristic ion mass chromatogram of a mietin standard solution is shown in fig. 2, a characteristic ion mass chromatogram of a tilapia blank sample is shown in fig. 3, a characteristic ion mass chromatogram of a tilapia blank sample is shown in fig. 4, a characteristic ion mass chromatogram of a tilapia blank addition mietin sample is shown in fig. 5, a characteristic ion mass chromatogram of a tilapia blank addition mietin sample is shown in fig. 6, a characteristic ion mass chromatogram of a shrimp blank sample is shown in fig. 7, a characteristic ion mass chromatogram of a shrimp blank sample is shown in fig. 8, a characteristic ion mass chromatogram of a shrimp blank addition mietin sample is shown in fig. 9, a characteristic ion mass chromatogram of a shrimp blank addition mietin sample is shown in fig. 10, and a characteristic ion mass chromatogram of an actually detected tilapia positive sample is shown in fig. 11, the mass chromatogram of the characteristic ions of the actual positive sample of tilapia is shown in figure 12.
Example 2: sensitivity, accuracy and precision testing
1 sensitivity test
1.1 determination of quantitation Limit
The limit of quantitation is determined from the value of the signal-to-noise ratio (S/N). Respectively adding 0.5 mu g/kg of emetine standard solution into blank tilapia and shrimps, measuring the ratio of signals to noise, and determining the concentration as the 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 the residual detection method. The experimental results are as follows: the limit of quantitation of the method is 0.5. mu.g/kg.
1.2 accuracy test
Accurately weighing 5.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with concentration of the drug containing the phentermine being 0.5, 1.0 and 5.0 mu g/kg respectively, wherein each concentration is 6 in parallel, processing according to the sample pretreatment process, determining, and calculating the standard addition recovery rate. The experimental results are as follows: the recovery rate of the method on the addition concentration level of 0.5 mu g/kg-5.0 mu g/kg is 80% -120%.
1.3 precision test
Accurately weighing 5.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with concentration of the drug of the phentermine being 0.5, 1.0 and 5.0 mu g/kg respectively, making 6 samples in parallel at 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 20 percent.
The above examples are only preferred embodiments of the present invention, it should be noted that: it will be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the spirit of the invention, and it is intended that all such modifications and equivalents fall within the scope of the invention as defined in the claims.

Claims (8)

1. A method for determining the residual concentration of mefenadine in aquatic product materials, which is characterized by comprising the following steps: sample pretreatment; extracting with ethyl acetate twice, concentrating and dissolving part of the extractive solution, and purifying with solid phase extraction column to obtain test solution; preparing a series of standard solutions of a standard working solution of the mefenatine, and determining by using a liquid chromatography-tandem mass spectrometer; drawing a standard curve by taking the characteristic ion mass chromatographic peak area of the mefenatine as the ordinate and the standard solution concentration as the abscissa, and solving a regression equation and a correlation coefficient; detecting the liquid to be tested by using a liquid chromatogram-tandem mass spectrometer, and quantifying by using an external standard method; the sample pretreatment method comprises the following steps: taking an edible tissue of a water product, mincing and homogenizing, and taking a sample to be tested after homogenizing as a test material; taking the homogenized blank sample as a blank sample; taking the homogenized blank sample, and adding a standard working solution with a proper concentration to serve as a blank addition test material; detecting the obtained test sample, blank sample and blank additive sample or storing at below-18 deg.C; the method for extracting by using secondary ethyl acetate comprises the following steps: weighing a homogenized sample, adding 10mL of ethyl acetate, horizontally oscillating for 15min, centrifuging, taking supernatant into a 25mL colorimetric tube, adding 10mL of ethyl acetate into residue, horizontally oscillating for 30min, centrifuging, combining two extracting solutions, fixing the volume to 25mL, shaking up, accurately sucking 5mL of the extracting solution into a glass test tube, concentrating at 45 ℃ with nitrogen until the extracting solution is dry, adding 5mL of a fixing solution, oscillating for 2min to obtain a solution to be purified; the solution is obtained by uniformly mixing acetonitrile and 2% formic acid aqueous solution according to the volume ratio of 25: 75; the conditions of the centrifugation treatment are: treating at 4500r/min for 5 min; the homogenization condition is that the homogenization treatment is carried out for 30s at 12000 r/min;
The solid phase extraction column is a mixed cation exchange solid phase extraction column;
the purification of the solid phase extraction column is to pass 5mL of the solution to be purified through the solid phase extraction column which is activated by 3mL of methanol and 3mL of 2% formic acid aqueous solution, and the flow rate is maintained at 0.5 mL/min; then, sequentially eluting with 4mL of 2% formic acid aqueous solution and 4mL of eluent, eluting with 4mL of eluent, maintaining the flow rate at 0.5mL/min, and collecting the eluent; the eluate was dried at 45 ℃ with nitrogen and the residue was dried over 1.0mL of a 1: 1, dissolving acetonitrile and a 5mM formic acid solution, filtering by using a 0.22 mu m filter membrane to obtain a solution to be tested, and carrying out liquid chromatography-tandem mass spectrometry; the leacheate comprises 10% ammonia water solution with the volume ratio of 80% and methanol with the volume ratio of 20%; the eluent comprises 90% of water by volume and 10% of ammonia water by volume.
2. The method of claim 1, wherein the preparation of the series of standard solutions of the efecitin standard working solution comprises:
100 μ g/mL of the standard stock solutions of emetine: accurately weighing 10mg of an imietidine standard substance, dissolving the imietidine standard substance in a 100mL measuring flask by using acetonitrile, diluting the solution to a scale, and preparing into an imietidine standard storage solution with the concentration of 100 mu g/mL, and storing the solution in a dark place below-18 ℃;
5 μ g/mL of the nemadectin standard intermediate: precisely measuring 5.00mL of a standard because-extinct stock solution with the concentration of 100 mu g/mL, diluting the stock solution with acetonitrile to a scale in a 100mL measuring flask, and preparing a standard because-extinct intermediate solution with the concentration of 5 mu g/mL, and storing the intermediate solution at the temperature of minus 18 ℃ in a dark place;
100ng/mL of the standard working solution of the emetine: precisely measuring 1.00mL of a 5-mu g/mL concentration noreptin standard intermediate solution, diluting the intermediate solution to a scale with acetonitrile in a 50-mL measuring flask to prepare an acetonitrile standard working solution with the concentration of 100ng/mL, and storing the working solution at the temperature of below-18 ℃ in a dark place.
3. The assay of claim 1 wherein the liquid chromatography conditions are: a chromatographic column: waters Atlantis T3, 4.6mm × 100mm, particle size 3 μm; mobile phase: acetonitrile-5 mM formic acid aqueous solution, gradient elution conditions are shown in Table 1; flow rate: 0.3 mL/min; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution;
TABLE 1 gradient elution conditions
Figure 411854DEST_PATH_IMAGE001
。
4. The assay of claim 1, wherein the mass spectrometry conditions are: an ion source: an electrospray ion source; the scanning mode is as follows: scanning positive ions; the detection mode is as follows: monitoring multiple reactions; spray voltage IS: 5500V; and (4) CUR: 35; CAD: medium; auxiliary heating gas temperature: 600 ℃; GS 1: 60, adding a solvent to the mixture; GS 2: 60, adding a solvent to the mixture; declustering voltage DP: 173V; entrance voltage EP: 12V; collision cell outlet voltage CXP: 10V; residence time: 0.1 s; qualitative ion pair and collision energy: 886.5>158.3, impact energy 121V; 886.5>81.9, impact energy 42V; and (3) quantitative ion pair: 886.5> 158.3.
5. The assay method according to claim 1, wherein the qualitative detection method of the assay method is: comparing the retention time of a sample chromatogram with the retention time of a corresponding standard sample, and comparing the characteristic ions of a chromatographic peak with the characteristic ions of a chromatographic peak of a standard solution with corresponding concentration for qualitative purpose, wherein the relative deviation of the retention time of the sample and the standard sample is not more than 5%; the relative abundance of the sample characteristic ions is consistent with that of the standard solution with the equivalent concentration, and the deviation of the relative abundance does not exceed the specification of the table 2, so that the corresponding measured object in the sample can be judged;
TABLE 2 allowable deviation Range of relative ion abundance
Figure 537415DEST_PATH_IMAGE002
。
6. The method according to claim 1, wherein the quantitative determination method comprises: taking sample solution and standard solution, and calculating by peak area according to external standard method.
7. The assay of claim 1, wherein the results of the assay are calculated
The method comprises the following steps:
calibration of a standard curve: determining a and b from As ═ a × cs + b, then
Figure 670587DEST_PATH_IMAGE003
（II）
The amount of residual statin in the sample was calculated according to formula (III):
Figure 5492DEST_PATH_IMAGE004
（III）
in the formula:
as-the peak area of mefenatine in the standard solution;
cs — concentration of emetine in standard solution in nanograms per milliliter (ng/mL);
c-concentration of emetine in the sample solution in nanograms per milliliter (ng/mL);
A-Peak area of emetine in the sample;
x- — -the residual amount of mefenatine in the test sample in micrograms per kilogram (μ g/kg);
v- — volume of dissolved residue in milliliters (mL);
m-mass of test sample in grams (g);
d- (dilution factor);
the dilution factor in the above formula is 1; wherein, blank values need to be deducted from the calculation results, the measurement results are represented by arithmetic mean values of parallel measurement, and three significant figures are reserved.
8. The assay of claim 1, wherein the aquatic product comprises tilapia, shrimp, or crab.
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