CN115266989A - Quantitative detection method for decoquinate residue in chicken tissues or eggs - Google Patents
Quantitative detection method for decoquinate residue in chicken tissues or eggs Download PDFInfo
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- CN115266989A CN115266989A CN202210915665.XA CN202210915665A CN115266989A CN 115266989 A CN115266989 A CN 115266989A CN 202210915665 A CN202210915665 A CN 202210915665A CN 115266989 A CN115266989 A CN 115266989A
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- 241000287828 Gallus gallus Species 0.000 title claims abstract description 133
- JHAYEQICABJSTP-UHFFFAOYSA-N decoquinate Chemical group N1C=C(C(=O)OCC)C(=O)C2=C1C=C(OCC)C(OCCCCCCCCCC)=C2 JHAYEQICABJSTP-UHFFFAOYSA-N 0.000 title claims abstract description 107
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Abstract
The invention relates to the field of veterinary drug residue detection, in particular to a quantitative detection method for decoquinate residue in chicken tissues or eggs, which comprises the following steps: weighing chicken tissues or egg tissues, adding acetonitrile, carrying out vortex mixing, carrying out ultrasonic extraction, extracting supernatant after centrifugation, adding acetonitrile into the residual residues, carrying out repeated centrifugation, combining two extracting solutions, adding acetonitrile saturated normal hexane into the extracting solution for degreasing, carrying out vortex mixing, removing the upper normal hexane layer after centrifugation, placing the lower layer liquid under nitrogen flow for drying, re-dissolving the dried sample by using an acetonitrile aqueous solution, purifying the re-dissolved solution by using an HLB (hydrophile-lipophile balance) solid phase extraction column, and drying and concentrating the eluent under nitrogen flow; adding trichloromethane into the concentrated and dried sample for redissolution, sequentially adding pyridine and acetic anhydride after redissolution, filtering after derivatization reaction, and detecting the filtrate by adopting GC-MS/MS. The invention has the advantages of more accurate qualitative and quantitative determination, good accuracy and sensitivity, high recovery rate and good repeatability.
Description
Technical Field
The invention relates to the field of veterinary drug residue detection, and particularly relates to a quantitative detection method for decoquinate residue in chicken tissues or eggs.
Background
At present, the domestic and foreign pretreatment methods for decoquinate residues in related animal food mainly comprise a liquid-liquid extraction method and a solid-phase extraction method, and extraction reagents comprise organic solvents such as acetonitrile, trichloromethane, ethyl acetate and the like. Due to the high boiling point of decoquinate, direct detection by gas chromatography or gas chromatography-mass spectrometry is not feasible. Therefore, the acetic anhydride is used as a derivatization reagent, and the acetic anhydride is used for performing acetylation reaction to perform derivatization on the decoquinate, so that the chemical structure is changed, and the boiling point of the decoquinate is reduced. So far, no report has been found at home and abroad about the research on extracting decoquinate residue from chicken tissues and eggs and carrying out derivatization reaction on the decoquinate residue and acetic anhydride to generate a decoquinate derivative product, namely acetyl decoquinate (C26H 37O 6N).
The detection method for decoquinate residues at home and abroad mainly comprises a high performance liquid chromatography-ultraviolet detection method, a high performance liquid chromatography-diode array detection method, a high performance liquid chromatography-fluorescence detection method and a liquid chromatography-mass spectrometry combined method, but the methods for detecting decoquinate residues in animal food by applying GC, GC-MS and GC-MS/MS are not reported at home and abroad.
Disclosure of Invention
Aims to solve the problem of extracting and purifying decoquinate residue and derivatization from chicken muscle, chicken liver, chicken kidney and egg. In the experiment, liquid-liquid extraction and solid-phase extraction methods are adopted to extract the residues of decoquinate in chicken muscles, chicken livers, chicken kidneys and eggs; acetylation of decoquinate with acetic anhydride, the derivation of decoquinate is shown by the equation:
in order to detect the residual decoquinate in chicken tissues and eggs by using a gas chromatography-tandem mass spectrometry method, the invention provides a pre-column derivatization-gas chromatography-tandem mass spectrometry (GC-MS/MS) detection method. Proved by methodology parameters, the method can realize accurate qualitative and quantitative determination, has high recovery rate and accuracy and good sensitivity, and meets the requirement of veterinary drug residue detection.
A method for quantitatively detecting decoquinate residues in chicken tissues or eggs comprises the following steps:
weighing chicken tissues or egg tissues, adding acetonitrile, performing vortex mixing, performing ultrasonic extraction, performing centrifugation, extracting supernatant, adding acetonitrile into the residual residues, performing repeated extraction and centrifugation, combining the two extracting solutions, adding acetonitrile saturated normal hexane into the extracting solution, degreasing, performing vortex mixing, performing centrifugation, removing the upper normal hexane layer, placing the lower liquid layer under nitrogen flow for drying, re-dissolving the dried sample by using acetonitrile aqueous solution, purifying the re-dissolved solution by using an HLB solid-phase extraction column, and drying and concentrating the eluent under nitrogen flow; adding trichloromethane into the concentrated and dried sample for redissolution, sequentially adding pyridine and acetic anhydride after redissolution, carrying out derivatization reaction, filtering, and detecting the filtrate by adopting GC-MS/MS.
Further, the chicken tissue is chicken muscle, chicken liver or chicken kidney; the egg is whole egg, egg white or egg yolk.
Further, the GC-MS/MS detection gas chromatography conditions are as follows: TG-5MS is used as a capillary chromatographic column; high-purity helium gas is used as carrier gas, and the flow rate of the carrier gas column is 1.0mL/min.
Further, the temperature programming step of GC-MS/MS detection is as follows: maintaining the initial temperature at 100 deg.C for 1min; raising the temperature to 220 ℃ at a speed of 30 ℃/min, and keeping the temperature for 1min; raising the temperature to 290 ℃ at 30 ℃/min and keeping the temperature for 13min.
Further, the mass spectrum conditions of GC-MS/MS detection are as follows: electron bombardment ion source; ionization energy of 70eV; high-purity argon is used as collision gas; the ion source temperature is 280 ℃; the temperature of the transmission line is 280 ℃; the solvent delay time is 3.0min; qualitative in a full scanning mode and quantitative in a reaction monitoring mode.
Further, the purification step of the HLB solid-phase extraction column comprises the following steps: activating the HLB solid phase extraction column with 3mL of methanol, balancing with 3mL of water, enabling the compound solution to pass through the column at a constant speed, then sequentially leaching with 3mL of 30% acetonitrile and 3mL of water, draining for 5min, and eluting the target with 3mL of acetonitrile.
Further, performing pre-column derivatization before GC-MS/MS detection, wherein the pre-column derivatization comprises the following steps: adding 1mL of chloroform into the eluent after being dried and concentrated by nitrogen for redissolution, sequentially adding pyridine and acetic anhydride, sealing, reacting for 3.5 hours at room temperature in a dark place, filtering the derivative product solution after the reaction is finished, and detecting and analyzing the filtrate by GC-MS/MS.
Further, the residual amount of the decoquinate in the chicken tissues or the eggs is calculated by adopting the peak area detected by GC-MS/MS.
Further, the acetonitrile concentration is 80%.
Further, the detection formulas of chicken muscle, chicken liver, chicken kidney, whole chicken egg, egg white or egg yolk are respectively as follows:
samples of chicken tissues (chicken muscle, chicken liver, chicken kidney) and eggs (whole egg, egg white, egg yolk) were extracted and purified by liquid-liquid extraction and solid-phase extraction, acetic anhydride and pyridine were derivatized, and the derivatized product (acetyldecoquinate) was detected by GC-MS/MS. Gas chromatography conditions: TG-5MS (30.0 m.times.0.25 μm.times.0.25mm i.d.) was used as a capillary column; high purity helium (> 99.999%,60 psi) is used as carrier gas, and the flow rate of the carrier gas column is 1.0mL/min. The temperature programming step is that the initial temperature is 100 ℃, and the temperature is kept for 1min; raising the temperature to 220 ℃ at a speed of 30 ℃/min, and keeping the temperature for 1min; raising the temperature to 290 ℃ at 30 ℃/min and keeping the temperature for 13min. The temperature of a sample inlet is 280 ℃; a non-shunting sample injection mode; the flow rate of the flow distribution is 50.0mL/min; the non-shunting time is 1.0min; a constant current mode; the flow rate of the carrier gas is 1.0mL/min;2min later, the valve is opened, the carrier gas saving time is 2min, and the carrier gas saving flow is 20.0mL/min; sample introduction volume: 1.0. Mu.L. Mass spectrum conditions: electron impact ion source (EI); ionization energy of 70eV; high-purity argon (99.999 percent, 40 psi) is used as collision gas; the ion source temperature is 280 ℃; the temperature of the transmission line is 280 ℃; the solvent delay time is 3.0min; qualitative in full SCAN (SCAN) mode, quantitative in Auto SRM (Auto SRM) mode.
A method for extracting and purifying decoquinate residue from chicken tissues and eggs and performing derivatization comprises liquid-liquid extraction, solid-phase extraction and purification, and pre-column derivatization by using acetic anhydride as a derivatization reagent. The extraction and purification process comprises three steps: extracting decoquinate residue in chicken tissues by acetonitrile, extracting decoquinate residue in eggs by using an acetonitrile water solution with the concentration of 80%, degreasing by using acetonitrile saturated n-hexane, blow-drying an extracting solution at the temperature of 50 ℃ under a nitrogen flow, re-dissolving a blow-dried sample by using an acetonitrile water solution with the concentration of 30%, purifying a fourth re-solution by using an HLB solid-phase extraction column (60 mg/3 mL), and blow-drying and concentrating eluent by using nitrogen. And (3) derivatization reaction process: redissolving the concentrated sample by using chloroform after drying nitrogen, sequentially adding acetic anhydride and pyridine after redissolving, sealing under the condition of room temperature, carrying out light-resistant reaction for 3.5h to carry out decoquinate derivatization, and carrying out confirmation analysis on a derivative product by using gas chromatography-tandem mass spectrometry to confirm that the decoquinate derivative product is acetyl decoquinate (C) 26 H 37 O 6 N)。
The steps of extraction and purification are: accurately weighing (2.0 +/-0.01) g of well-homogenized sample, putting the well-homogenized sample into a 50mL polypropylene centrifuge tube, adding 5mL of acetonitrile (adding 5mL of 80% acetonitrile aqueous solution into an egg sample), uniformly mixing the mixture by vortex for 10min, then carrying out ultrasonic oscillation extraction for 10min, centrifuging the mixture at the speed of 5500r/min at 4 ℃ for 10min, and transferring the supernatant into a new centrifuge tube. Adding 5mL of extracting solution into the residual residue, repeatedly extracting for 1 time, centrifuging at 8000r/min for 10min, and mixing the two extracting solutions; adding 10mL acetonitrile saturated n-hexane into the extractive solution, vortex mixing for 5min, centrifuging at 8000r/min for 5min, and discarding n-hexane fat layer. The extract after the fat removal is dried under nitrogen flow at 50 ℃ and redissolved with 5mL of 30% acetonitrile solution for later use.
Solid phase extraction and purification: activating the HLB solid phase extraction column with 3mL of methanol, balancing with 3mL of water, allowing the redissolution to pass through the column at uniform speed, eluting with 3mL of 30% acetonitrile and 3mL of water, draining for 5min, and eluting the target with 3mL of acetonitrile
In conclusion, the optimal pretreatment conditions were selected: when the mass of the sample is 2.0g, the total amount of acetonitrile or 80% acetonitrile aqueous solution extracted twice by the extraction reagent is 10mL.
Using acetic anhydride as a derivatization reagent to perform pre-column derivatization: drying the eluent at 50 ℃ in a nitrogen blowing instrument, redissolving the eluent by trichloromethane, sequentially adding 300 mu L of pyridine and 150 mu L of acetic anhydride, sealing the mixture, reacting the mixture for 3.5 hours in a dark place at room temperature, filtering the product solution derived after the reaction is finished by a 0.22 mu m organic phase needle filter, and detecting and analyzing the filtrate by GC-MS/MS.
Advantageous effects
The invention compares the influence of the combination of liquid-liquid extraction and solid-phase extraction on the result. The result shows that the derivative product obtained by adopting the extraction method combining liquid-liquid extraction and solid-phase extraction has good chromatographic peak shape, few interfering impurity peaks and high response value of the sample derivative product. Meanwhile, the influence of acetonitrile, ethyl acetate (1: 1) and 4% acetonitrile acetic acid solution on the extraction effect of the target substances in chicken tissues (chicken muscles, chicken livers and chicken kidneys) and eggs (whole eggs, egg white and egg yolks) is compared, and the result shows that the recovery rate of the extraction result is highest when the acetonitrile is used as an extraction reagent for the chicken tissues and the eggs. Finally, the influence of acetonitrile with different concentrations on the extraction effect is further compared, and the result shows that the extraction effect of 100% acetonitrile on the target object in the chicken tissue is the best, but the extraction effect of 80% acetonitrile aqueous solution on the target object in the eggs is higher than that of 100% acetonitrile. Therefore, in the experiment, the extraction reagent taking acetonitrile as the chicken tissue and the extraction reagent taking 80% acetonitrile in water as the egg are finally selected, and the residues of the decoquinate in the chicken tissue and the egg are extracted and purified by a method combining liquid-liquid extraction and solid-phase extraction.
In the invention, TG-5MS (30.0 m multiplied by 0.25 mu m multiplied by 0.25mm i.d.) capillary chromatographic columns are adopted, qualitative analysis is carried out by adopting a full Scanning (SCAN) mode, quantitative analysis is carried out by selecting a reaction monitoring (Auto SRM) mode, and finally the chromatographic peak shape of the obtained decoquinate derivative product is good (namely high sensitivity), the retention time of an analyte is moderate, and no interference of other impurity peaks exists.
The invention provides a method for detecting decoquinate residue by using pre-column derivatization-gas chromatography-tandem mass spectrometry (GC-MS/MS). The method has high recovery rate, precision and sensitivity and good repeatability, and is suitable for application and popularization in batch sample analysis. Proved by methodology parameters, the method can realize accurate qualitative and quantitative determination, has high recovery rate and accuracy and good sensitivity, and meets the requirement of veterinary drug residue detection.
Compared with the reported analysis method, the pretreatment method of the test is simple to operate, and the test cost is saved. The method has the advantages of more accurate qualitative and quantitative determination, good accuracy and sensitivity, high recovery rate and good repeatability, provides a new technical support for detecting the decoquinate residue in the poultry product, and provides a scientific basis for establishing GC-MS/MS detection standards of the decoquinate residue in animal-derived food.
Drawings
FIG. 1 shows a total ion current chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of a blank chicken muscle sample (A) and a blank chicken muscle sample added with 100.0 μ g/kg decoquinate standard (B);
FIG. 2 shows a total ion current chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of a blank chicken liver sample (A) and a blank chicken liver sample added with 200.0 μ g/kg decoquinate standard (B);
FIG. 3 shows the total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatogram (MC) of a blank chicken kidney sample (A) and a blank chicken kidney sample (B) added with 200.0. Mu.g/kg decoquinate standard (B);
FIG. 4 shows a total ion current chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of a blank whole egg sample (A) and a blank whole egg sample with 100.0 μ g/kg decoquinate standard (B);
FIG. 5 shows a total ion current chromatogram (TIC) and a Mass Chromatogram (MC) of qualitative and qualitative ions of a blank egg white sample (A) and a blank egg white sample with 100.0. Mu.g/kg decoquinate standard (B) added;
FIG. 6 shows a total ion current chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of a blank yolk sample (A) and a blank yolk sample (B) with 100.0. Mu.g/kg decoquinate standard (B);
FIG. 7 standard curve of addition of decoquinate to a chicken muscle matrix blank;
FIG. 8 is a standard curve of adding decoquinate to a blank chicken liver matrix;
FIG. 9 standard curve of decoquinate addition to chicken kidney substrate blank;
FIG. 10 is a standard curve of addition of decoquinate to a blank whole egg matrix;
FIG. 11 is a standard curve of addition of decoquinate to a blank egg white base;
FIG. 12 standard curve for decoquinate addition to a blank egg yolk substrate;
FIG. 13 effect of amount of acetic anhydride on the response of derivatized products;
FIG. 14 effect of pyridine dosage on derivative product response;
FIG. 15 effect of time of derivatization on the response value of the derivatized product;
FIG. 16 effect of different concentrations of extraction reagent on decoquinate recovery in chicken tissues;
FIG. 17 effect of different concentrations of extraction reagents on decoquinate recovery from chicken eggs.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The source, trade name and composition of the reagents used are indicated at the first appearance and the same reagents used thereafter are the same as indicated for the first time unless otherwise specified.
(I) raising and sample collection of experimental livestock and poultry
The test was conducted with approval from the ethical committee of the university of Yangzhou and the Jinghai poultry group, jiangsu, inc.
12 70 days old Haiyang yellow chickens are randomly selected from Jiangsu Jinghai poultry group limited company, wherein the male and female half chickens are raised in a single cage and freely drink water during the whole raising period, and the feeds are all full-value feeds without any medicine (the feeds are provided by feed factories of Jiangsu Jinghai poultry group limited company). Slaughtering after 1 month of feeding, and collecting breast muscle, leg muscle, liver and kidney of the chicken as blank experiment samples. The collected sample is cut into small pieces which are easy to handle by using clean surgical scissors, and then the homogenate is further smashed by a homogenizer. And packaging the homogenized sample, sealing and storing in a freezer at-34 ℃ for later use.
20 Jinghai yellow chickens (Jiangsu Jinghai poultry group, inc.) with the age of 30 weeks were randomly selected, and were raised in a single cage during the test period with free drinking. The prefeed began within the first half month of the trial, eggs were collected on day 16, and all experimental animals received complete feed without any added drugs from the beginning of the prefeed until the end of the trial (provided by feed factory, kyoto Hai poultry group, jiangsu). Collecting eggs from four to five afternoon every day, continuously collecting for 15 days, processing the collected eggs into egg white, yolk and whole egg, homogenizing, respectively placing in centrifuge tubes, and storing in freezer at-34 deg.C as blank egg sample.
(II) the extraction, purification, concentration and derivatization steps of the invention
(1) Accurately weighing (2.0 +/-0.01) g of the homogenized sample, putting the sample into a 50mL polypropylene centrifuge tube, adding 5mL of acetonitrile (5 mL of 80% acetonitrile aqueous solution is added into an egg sample), carrying out vortex mixing for 10min, then carrying out ultrasonic extraction for 10min, carrying out high-speed centrifugation at the speed of 5500r/min at 4 ℃ for 10min, and transferring the supernatant into a new centrifuge tube. Adding 5mL acetonitrile into the residue (adding 5mL 80% acetonitrile water solution into egg sample), extracting repeatedly for 1 time, centrifuging at 8000r/min for 10min, mixing the two extractive solutions,
and adding 10mL of acetonitrile saturated n-hexane into the extracting solution for degreasing, placing the extracting solution on a vortex mixer for vortex mixing for 5min, then carrying out high-speed centrifugation for 5min at the speed of 8000r/min at 4 ℃, removing the n-hexane layer on the upper layer, drying the liquid on the lower layer under 50 ℃ nitrogen flow, and re-dissolving the dried sample with 5mL of 30% acetonitrile aqueous solution for later use.
(2) Activating the HLB solid phase extraction column with 3mL of methanol, balancing with 3mL of water, enabling the compound solution to pass through the column at a constant speed, then sequentially leaching with 3mL of 30% acetonitrile and 3mL of water, draining for 5min, and eluting the target with 3mL of acetonitrile. The eluate was collected in a 10mL glass centrifuge tube and blown dry at 50 ℃ under a nitrogen stream.
(3) Adding 1mL of trichloromethane into the concentrated sample for redissolution, carrying out vortex mixing for 1min, sequentially adding 300 mu L of pyridine and 150 mu L of acetic anhydride, carrying out light-shielding reaction for 3.5h at room temperature, directly filtering the solution by using a 0.22 mu m organic phase nylon needle filter, and directly detecting and analyzing the filtrate by using a GC-MS/MS instrument.
(III) GC-MS/MS analysis conditions
1. Gas chromatography conditions
A chromatographic column: TG-5MS (30.0 m.times.0.25 μm.times.0.25mm i.d.5% -dimethyl-95% -dimethyl siloxane); carrier gas: high purity helium (> 99.999%,60 psi), carrier gas column flow rate: 1.0mL/min.
A temperature programming step: maintaining the initial temperature at 100 deg.C for 1min; raising the temperature to 220 ℃ at a speed of 30 ℃/min, and keeping the temperature for 1min; raising the temperature to 290 ℃ at a speed of 30 ℃/min, and keeping the temperature for 13min; see table 1 for details. Sample inlet temperature: 280 ℃; shunting mode: no shunt sampling; flow splitting: 50.0mL/min; non-shunting time: 1.0min; carrier gas mode: a constant current mode; flow rate of carrier gas: 1.0mL/min;2min later, the valve is opened, the time is saved by 2min for carrier gas, and the flow is saved by 20.0mL/min for carrier gas; sample introduction volume: 1.0. Mu.L.
TABLE 1 temperature programmed step
2. Conditions of Mass Spectrometry
Ionization mode: electron impact ion source (EI); electron beam energy (ionization energy): 70eV; collision gas: high purity argon (> 99.999%,40 psi); ion source temperature: 280 ℃; transmission line temperature: 280 ℃; solvent retardation: 3.0min; and (3) data acquisition mode: qualitative in a full SCAN (SCAN) mode, and quantitative in an Auto SRM (Auto SRM) mode. The molecular weight and mass spectral parameters of decoquinate derivatives are shown in table 2.
Table 2 retention time and related mass spectral parameters of decoquinate-derived products
Note: * Quantitative ion pair
(IV) quantitative method
1. Determination of detection and quantitation limits
Accurately weighing (2.0 + -0.01) g of homogenized blank sample, and preparing blank matrix extract according to the method in the second step. Diluting the low-concentration decoquinate standard working solution by blank matrix extracting solution step by step, and detecting according to the established GC-MS/MS method. The analysis was repeated 6 times per concentration and the mean signal to noise ratio (S/N) was calculated. When the S/N is more than or equal to 3, the corresponding decoquinate concentration is the limit of detection (LOD); when the S/N is more than or equal to 10, the corresponding decoquinate concentration is the limit of quantitation (LOQ).
According to the above test of adding and recovering 6 parallel blank samples, the LOD of decoquinate in chicken muscle, chicken liver, chicken kidney, whole chicken egg, egg white and egg yolk is 2.2, 4.3, 3.7, 1.8, 1.4 and 2.4 mug/kg respectively, and the LOQ is 4.9, 8.2, 6.3, 3.4, 2.1 and 4.9 mug/kg respectively under the existing conditions.
2. Drawing of standard curve
Respectively and accurately weighing 15 parts (2.0 +/-0.01) g of homogenized blank sample, preparing blank extracting solutions of 6 different matrixes according to the method (II), and placing the extracting solutions in a refrigerator at the temperature of-34 ℃ for later use. Diluting the decoquinate standard working solution into different concentrations by using the prepared blank extracting solution, wherein the concentration of the finally diluted matrix matching standard solution is equivalent to the addition concentration of blank chicken muscle, and the addition concentration is quantitative Limit (LOQ), 200.0, 400.0, 800.0, 1600.0 and 3200.0 mu g/kg; correspondingly adding LOQ, 300.0, 600.0, 1200.0, 2400.0 and 4800.0 mu g/kg into blank chicken liver and chicken kidney; the concentrations added to the whole egg, egg white and egg yolk samples were quantitative Limit (LOQ), 50, 100, 150, 200 and 250. Mu.g/kg, respectively.
And (3) sequentially detecting and analyzing the processed concentration samples under an optimized GC-MS/MS method. Each concentration was repeated 6 times and averaged. And (3) taking the concentration of the decoquinate standard working solution added in a blank matrix as an abscissa (x), and taking the peak area of the quantitative ion pair m/z 231.1 & gt 230.1 of the acetyl decoquinate as an ordinate (y), and establishing a matrix standard curve to be used as a quantitative curve of the sample to be detected.
As can be seen from Table 3 and accompanying figures 7-12, the addition concentration of decoquinate in the matrix extract of the blank chicken muscle sample is in the range of LOQ-3200.0 mug/kg, the addition concentration of decoquinate in the matrix extract of the blank chicken liver and chicken kidney is in the range of LOQ-4800.0 mug/kg, the addition concentration of decoquinate in the matrix extract of the blank chicken whole egg, egg white and egg yolk is in the range of LOQ-250 mug/kg, the quantitative ion of the derivative product of decoquinate has a good linear relationship with the concentration (x axis) and the peak area (y axis) of m/z 231.1 > 230.1, and the linear regression equation, the coefficient of determination and the linear range thereof are shown in Table 3. If the concentration analyzed exceeds the linear range of the sample, the concentration analyzed needs to be diluted to be within the range, and the detected result is multiplied by the dilution factor to obtain the concentration of the original sample.
<xnotran> 3 , , , (n = 6) </xnotran>
3. Measurement of recovery and precision
Accurately weighing (2.0 +/-0.01) g of well-homogenized blank sample, processing the blank sample according to the method of the step (II), adding a proper amount of decoquinate standard working solution to enable the final addition concentration of the blank sample to be LOQ, 0.5MRL, 1.0MRL and 2.0MRL, setting 6 parallel addition concentrations, extracting, purifying and deriving the blank sample by the method of the step (II), detecting the obtained filtrate by GC-MS/MS, bringing the detection result into a blank matrix standard curve to obtain the concentration, and comparing the concentration with the concentration of an actually added analyte to obtain the sample standard recovery rate.
The total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatogram (MC) of the blank chicken muscle sample (A) and the blank chicken muscle sample added with 100.0 μ g/kg decoquinate standard (B) are shown in figure 1; the total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the blank chicken liver sample (A) and the blank chicken liver sample added with 200.0 μ g/kg decoquinate standard (B) are shown in figure 2; the total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the blank chicken kidney sample (A) and the blank chicken kidney sample added with 200.0 μ g/kg decoquinate standard (B) are shown in figure 3; the total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatogram (MC) of the blank whole egg (A) and the blank whole egg sample added with 100.0 μ g/kg decoquinate standard (B) are shown in figure 4; the total ion current chromatogram (TIC) and the Mass Chromatogram (MC) of qualitative and quantitative ions of the blank egg white sample (A) and the blank egg white sample added with 100.0 μ g/kg decoquinate standard (B) are shown in figure 5; the total ion current chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the blank egg yolk sample (A) and the blank egg yolk sample with 100.0 μ g/kg decoquinate standard (B) added are shown in FIG. 6.
Precision within day (batch): on the same day, 4 samples of the added concentrations (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) were assayed at different time points using the same instrument and the same standard curve, and the RSD obtained was determined 6 times for each added concentration.
Daytime (batch) precision: on different days of the week, 4 samples of the additive concentration (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) were assayed with the same instrument and with different standard curves (standard curves were plotted every day), and the RSD obtained was repeated 6 times for each additive concentration.
Under the condition, the adding recovery rate and the precision of the decoquinate extracted from the chicken tissues and the eggs by the method are shown in tables 4 and 5.
Table 4 recovery and precision of decoquinate addition in chicken blank muscle, chicken liver and chicken kidney (n = 6)
Note: maximum residual limit of alpha
TABLE 5 recovery and precision of decoquinate addition to empty hen whole egg, egg white, egg yolk (n = 6)
Note: maximum residual limit of alpha
4. Determination of the limits of determination (CC. Alpha.) and the detection Capacity (CC. Beta.)
The determination of the limit and the detection tolerance are important indexes for evaluating the pesticide residue detection method in European Union 2002/657/EC. The certainty limit (CC α) is the highest level at which an accurate analysis of the target can be performed in the detection method, and the probability that the detection result of the analyte in the sample does not meet the prescribed conclusion (false positive) can be derived. For veterinary drugs with explicitly specified MRL, CC β is the concentration at which the MRL concentration of the target can be accurately detected in the sample, and the probability that the MRL sample can be detected can be calculated; for veterinary drugs for which MRL is not specified, CC β refers to the lowest concentration of the target that can be accurately detected in the sample, and the probability that a negative sample will not meet the specification (false negative) can be calculated. There are clear maximum residual limit standards (MRL) for decoquinate remaining in chicken muscle, chicken liver and chicken kidney, 20 blank samples were randomly taken for each substrate, 100. Mu.L of 20.0. Mu.g/mL standard working solution was added to the blank chicken muscle samples, 100. Mu.L of 40.0. Mu.g/mL standard working solution was added to the chicken liver and chicken kidney samples, the samples were processed according to the method (II), and the Standard Deviation (SD) was calculated by analyzing under the condition (III). The calculation formulas of CC α and CC β are CC α = MRLs +1.64 × SD (α = 5%) and CC β = CC α +1.64 × SD (β = 5%), respectively.
Under these conditions, the results of the present method for determining the limit of decoquinate in chicken, chicken liver and chicken kidney and the detection of the volume are shown in Table 6. As can be seen from Table 6, when the concentration of the detected sample is higher than 1032.0. Mu.g/kg when detecting the chicken muscle sample, the probability that the positive sample does not meet the specification is 95%; when the concentration of the test sample is 1034.1 μ g/kg or greater, a 95% probability is that 1000 μ g/kg of sample can be detected. When the chicken liver sample is detected, when the concentration of the detected sample is higher than 2042.0 mug/kg, the probability that the positive sample does not meet the regulation is 95 percent; when the concentration of the test sample is greater than or equal to 2049.0 μ g/kg, a 95% probability is that 2000 μ g/kg of sample can be detected. When the chicken liver sample is detected, when the concentration of the detected sample is higher than 2047.0 mug/kg, the probability that the positive sample does not meet the regulation is 95 percent; when the concentration of the test sample is greater than or equal to 2054.1 μ g/kg, a 95% probability is that 2000 μ g/kg of sample can be detected.
Table 6 definition of decoquinate in chicken, chicken liver and chicken kidney and detection capacity (μ g/kg) (n = 20)
According to the requirements in the European Union 2002/657/EC resolution, the CC alpha of the forbidden drug can be calculated by adopting a calibration curve method. Randomly extracting a blank sample, adding a target object according to equidistant gradient to enable the target object to be equal to or higher than the minimum amount required by the reaction, drawing a curve graph according to the added concentration after analyzing the sample, and adding 2.23 times of standard deviation of the laboratory internal reproducibility to the corresponding concentration of a Y-axis intercept (Y-axis extrapolation method) to obtain CC alpha (alpha = 1%); a limit-determining level of decoquinate standards was added to at least 20 blank samples, and the samples were analyzed for the standard deviation of laboratory internal reproducibility of the mean assay content with CC α plus 1.64 times the CC α concentration, i.e., CC β (β = 5%).
Decoquinate is a prohibited anticoccidial drug during the egg producing period and is not equivalent to a prohibited drug, so the test calculates the CC α and CC β of decoquinate in whole chicken eggs, egg white and egg yolk at the LOQ level.
Under these conditions, the results of the method of the present invention for the CC α and CC β of decoquinate in whole chicken eggs, egg white and egg yolk are shown in Table 7. As can be seen from Table 7, when decoquinate is detected in whole chicken eggs, the probability that the positive sample does not meet the regulation is 99% when the concentration of the detected sample is higher than 4.7 mug/kg; when the concentration of the tested sample is more than or equal to 5.4 mug/kg, the probability that the negative sample does not meet the regulation is 5 percent; when the decoquinate in the egg white is detected, the concentration of the detected sample is higher than 2.5 mug/kg, and the probability that the positive sample does not meet the regulation is 99 percent; the concentration of the tested sample is more than or equal to 3.3 mug/kg, and the probability that the negative sample does not meet the regulation is 5 percent; when the concentration of the detected sample is higher than 5.5 mug/kg when detecting the decoquinate residue in the egg yolk, the probability that the positive sample does not meet the specification is 99 percent; when the concentration of the test sample is 6.2. Mu.g/kg or more, the probability that the negative sample does not meet the specification is 5%.
TABLE 7 determination of decoquinate in Whole egg, egg white and egg yolk limit and detection capacity (. Mu.g/kg) (n = 20)
5. Determination of matrix Effect
After the GC-MS/MS method was established, the matrix effect was evaluated using the slope of the standard curve. When each matrix was prepared to match the standard curve, the corresponding solvent standard curve was prepared at the same time. Diluting decoquinate standard stock solution into standard working solution of 9.8, 400.0, 800.0, 1600.0, 3200.0 and 6400.0ng/mL from high to low step by using trichloromethane, performing derivatization by using optimized test conditions, analyzing by using GC-MS/MS, recording peak area, preparing a solvent standard curve, and calculating the matrix effect of decoquinate in chicken muscle; and diluting the decoquinate standard stock solution into standard working solutions of 16.4, 600.0, 1200.0, 2400.0, 4800.0 and 9600.0ng/mL by trichloromethane step by step, preparing a solvent standard curve, and calculating the matrix effect in the chicken liver. And diluting the decoquinate standard stock solution into standard working solution of 12.6, 600.0, 1200.0, 2400.0, 4800.0 and 9600.0ng/mL by trichloromethane step by step, preparing a solvent standard curve, and calculating the matrix effect in the chicken kidney. (the formula for the calculation of the matrix effect is ME = S matrix match standard curve slope/S solvent standard curve slope-1). Times.100%). When ME is between 20 percent and 20 percent, the weak matrix effect exists; when the ratio of ME is between 50% and ME is between 20% and ME is less than 50%, the matrix effect is medium; if ME is less than or equal to-50% or ME is more than 50%, it is strong substrate effect.
Under these conditions, the matrix effect of decoquinate in chicken muscle, liver and kidney by the method of the present invention is shown in Table 8. As can be seen from Table 8, using GC-MS/MS, the matrix effect of decoquinate in chicken muscle, chicken liver and chicken kidney was between-20% and 20%. Weaker matrix inhibition effects are prevalent in the three matrices, with the liver having the strongest matrix inhibition effect.
TABLE 8 matrix Effect of decoquinate in Chicken muscle, liver and Kidney
The matrix effect of decoquinate in eggs (whole egg, egg white and egg yolk) was evaluated according to the chicken tissue method described above. Diluting decoquinate standard stock solution into 6.8, 100, 200, 300, 400 and 500ng/mL series standard solution from high concentration to low concentration step by using trichloromethane, making a standard curve, and calculating the matrix effect of the whole egg of the chicken; diluting the standard stock solution from high concentration to low concentration to 4.2, 100, 200, 300, 400 and 500ng/mL series of standard solutions, making a standard curve, and calculating the matrix effect of the egg white; diluting the standard stock solution from high concentration to low concentration to 9.8, 100, 200, 300, 400 and 500ng/mL series of standard solutions, making a standard curve, and calculating the matrix effect of the egg yolk. The results are shown in Table 9, and it can be seen from Table 9 that in this method, decoquinate generally exhibited a matrix-inhibiting effect in whole chicken eggs, egg white and egg yolk.
TABLE 9 ground Effect of decoquinate in Whole egg, egg white and egg yolk of chickens
Table 3-6Matrix effects of decoquinate in whole chicken eggs,egg whites and egg yolks
(V) optimization of the conditions of derivation and selection of the reagents for extraction
1. Optimization of derivatization conditions
The optimal acetic anhydride dosage is as follows: in order to ensure the accuracy of the quantitative result, the optimal conditions for the reaction of decoquinate with acetic anhydride are optimized and finally determined. First, the amount of acetic anhydride used is optimized. The amount of acetic anhydride used is plotted on the abscissa and the peak area of the derivative product is plotted on the ordinate (see FIG. 13). As can be seen from FIG. 13, the peak area of the derived product showed a rising trend when the amount of acetic anhydride added was 50 to 150. Mu.L, and the peak area of the derived product reached the maximum when the amount added was 150. Mu.L; the addition amount is in the range of 150-250 mu L, the peak area is unstable and has a descending trend; when the amount of the compound added is 250 to 350. Mu.L, the peak area gradually increases again, but the peak area is always smaller than that when the amount is 150. Mu.L. When the amount of acetic anhydride added was increased to 400. Mu.L, the peak area of the derivative product gradually decreased. Therefore, the optimal dose of acetic anhydride to react with decoquinate is 150. Mu.L.
The optimal pyridine dosage is as follows: the amount of pyridine used was plotted on the abscissa and the peak area of the derivative product was plotted on the ordinate (see FIG. 14). As can be seen from FIG. 14, the peak area of the derived product reached a maximum at 300. Mu.L of pyridine added, and the peak area of the derived product did not change much with the increase in the amount of pyridine. Therefore, the optimal amount of pyridine used in this test is 300. Mu.L.
Optimal derivatization time: after the amounts of acetic anhydride and pyridine are determined, the optimal time for the reaction of acetic anhydride and decoquinate is optimized. And drawing a curve by taking the derivation time as an abscissa and taking the peak area of the quantitative ion pair of the derived product as an ordinate. As shown in FIG. 15, when the time for the derivatization ranged from 0.5h to 3.5h, the peak area of the derivatized product increased slowly with the lapse of time and reached a maximum at 3.5h, and then the peak area of the derivatized product became gradually stable without any major change.
In summary, as can be seen from fig. 13 to 15, the optimal derivation conditions of decoquinate and acetic anhydride are: the reaction was carried out with 150. Mu.L of acetic anhydride under 300. Mu.L of pyridine with exclusion of light for 3.5h.
2. Effect of different extraction reagents on decoquinate recovery in Chicken tissues and eggs
Comparing the effect of acetonitrile, acetonitrile/ethyl acetate (1: 1) and 4% acetonitrile acetic acid solution on the extraction of target substances from chicken tissues (chicken muscle, chicken liver, chicken kidney) and eggs (whole egg, egg white, egg yolk), see Table 10 for details.
TABLE 10 Effect of different concentrations of extraction reagents on decoquinate recovery (%) in chicken tissues and eggs (n = 5)
As is clear from Table 10, the recovery rate of the extraction results was the highest when the chicken tissues and eggs were extracted with acetonitrile as the extraction reagent. Further comparing the effect of different concentrations of acetonitrile on the extraction effect, the results show that 100% acetonitrile has the best extraction effect on the target in chicken tissues (see fig. 16), but 80% acetonitrile water solution has higher extraction effect on the target in eggs than 100% acetonitrile (see fig. 17). Therefore, in the experiment, the extraction reagent taking acetonitrile as the chicken tissue and the extraction reagent taking 80% acetonitrile in water as the egg are finally selected, and the residues of the decoquinate in the chicken tissue and the egg are extracted and purified by a method combining liquid-liquid extraction and solid-phase extraction.
Claims (10)
1. A method for quantitatively detecting decoquinate residues in chicken tissues or eggs is characterized by comprising the following steps: weighing chicken tissues or egg tissues, adding acetonitrile, carrying out vortex mixing, carrying out ultrasonic extraction, extracting supernatant after centrifugation, adding acetonitrile into the residual residues, carrying out extraction centrifugation, combining the two extracting solutions, adding acetonitrile saturated normal hexane into the extracting solution for degreasing, carrying out vortex mixing, removing the upper normal hexane layer after centrifugation, placing the lower layer liquid under nitrogen flow for drying, re-dissolving the dried sample by using an acetonitrile aqueous solution, purifying the re-dissolved solution by using an HLB (hydrophile-lipophile balance) solid phase extraction column, and drying and concentrating the eluent under nitrogen flow; adding trichloromethane into the concentrated and dried sample for redissolution, sequentially adding pyridine and acetic anhydride after redissolution, carrying out derivatization reaction, filtering, and detecting the filtrate by adopting GC-MS/MS.
2. The method for quantitatively detecting decoquinate residues in chicken tissues or eggs according to claim 1, wherein the chicken tissues are chicken muscles, chicken livers or chicken kidneys; the egg is whole egg, egg white or egg yolk.
3. The method for quantitatively detecting the residual decoquinate in the chicken tissues or the eggs as claimed in claim 1, wherein the GC-MS/MS detection has the following gas chromatography conditions: TG-5MS is used as a capillary chromatographic column; high-purity helium gas is used as carrier gas, and the flow rate of the carrier gas column is 1.0mL/min.
4. The method for quantitatively detecting the residual decoquinate in the chicken tissues or the eggs as claimed in claim 1, wherein the temperature programming steps of GC-MS/MS detection are as follows: maintaining the initial temperature at 100 deg.C for 1min; raising the temperature to 220 ℃ at a speed of 30 ℃/min, and keeping the temperature for 1min; raising the temperature to 290 ℃ at 30 ℃/min and keeping the temperature for 13min.
5. The method for quantitatively detecting the residual decoquinate in the chicken tissues or the eggs as claimed in claim 1, wherein the mass spectrum conditions of GC-MS/MS detection are as follows: electron bombardment ion source; ionization energy of 70eV; high-purity argon is used as collision gas; the ion source temperature is 280 ℃; the transmission line temperature is 280 ℃; the solvent delay time is 3.0min; qualitative in a full scanning mode and quantitative in a reaction monitoring mode.
6. The method for quantitatively detecting the residual decoquinate in chicken tissues or eggs as claimed in claim 1, wherein the purification step of the HLB solid-phase extraction column comprises: activating the HLB solid phase extraction column with 3mL of methanol, balancing with 3mL of water, allowing the re-solution to pass through the column at a constant speed, leaching with 3mL of 30% acetonitrile and 3mL of water in sequence, draining for 5min, and eluting the target with 3mL of acetonitrile.
7. The method for quantitatively detecting decoquinate residues in chicken tissues or eggs according to claim 1, wherein the GC-MS/MS detection is preceded by pre-column derivatization, which comprises the following steps: adding chloroform into the eluent after being dried and concentrated by nitrogen for redissolving, sequentially adding pyridine and acetic anhydride, sealing, reacting for 3.5 hours at room temperature in a dark place, filtering the derivative product solution after the reaction is finished, and detecting and analyzing the filtrate by GC-MS/MS.
8. The method for quantitatively detecting the residual decoquinate in chicken tissues or eggs as claimed in claim 1, wherein the residual decoquinate in chicken tissues or eggs is calculated by using the peak area detected by GC-MS/MS.
9. The method for quantitatively determining decoquinate residues in chicken tissues or eggs according to claim 1, wherein the acetonitrile concentration is 80%.
10. The method for quantitatively detecting the residual decoquinate in the chicken tissues or the eggs, according to claim 1, is characterized in that the detection formulas of chicken muscles, chicken livers, chicken kidneys, whole chicken eggs, egg white or egg yolks are respectively as follows:
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