Method for detecting residual quantity of ethoxyaniline in poultry tissues

Abstract

The invention provides a method for detecting the residual quantity of ethoxyaniline in poultry tissues, and belongs to the technical field of analysis and detection. According to the method, acetonitrile is used as an extracting agent, so that the extraction rate of the intermediate ethoxyaniline in the poultry tissues is increased; according to the method, solid-phase extraction and purification are carried out on an extracting solution, so that the influence of impurities on a test result can be reduced, and the accuracy and reproducibility of a detection result are improved; according to the method disclosed by the invention, the m-ethoxyaniline standard working solution and the sample solution are respectively determined by adopting liquid chromatography-mass spectrometry / mass spectrometry, and when the deviation between the retention time of the sample solution and the retention time of the m-ethoxyaniline standard working solution is within + / -2.5%, the m-ethoxyaniline standard working solution is determined; when the relative ion abundance of the sample solution is consistent with the relative ion abundance of the m-ethoxyaniline standard working solution, qualitatively determining m-ethoxyaniline in the sample solution; according to the method, an external standard method is adopted for quantification, and the content of the intermediate ethoxyaniline in the poultry tissue sample can be accurately calculated.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a method for detecting ethoxyaniline residues in poultry tissues. Background Art

[0002] Ethopabate, also known as methyl 4-acetamido-2-ethoxybenzoate, ethhopabate, or coccidiostat, is an anticoccidial drug widely used in poultry farming. Currently, different countries have established maximum residue limits (MRLs) for ethhopabate in edible poultry tissues. For example, the United States and my country both set MRLs for chicken muscle at 500 μg / kg; chicken liver at 1500 μg / kg; and chicken kidney at 1500 μg / kg. Japan sets MRLs for ethhopabate in poultry tissues at 40 μg / kg for chicken muscle, liver, kidney, fat, and edible offal; 5 mg / kg for muscle and fat of other poultry, and 20 mg / kg for liver, kidney, and edible offal of other poultry. Furthermore, ethhopabate is not approved for use in the EU. Since my country has a record of using ethopabenz, the EU requires that ethopabenz be included in the residue monitoring plan. Ethoxyamide has been added to the 2023 export animal food safety risk monitoring project, and the lower limit of determination of ethopabenz in the edible tissue of poultry is required to be 20μg / kg.

[0003] Currently, my country has only published a standard method for the determination of ethopabate residues in edible poultry tissues. However, this standard has a high limit of quantification (LOQ) of 50 μg / kg for poultry muscle and 100 μg / kg for poultry liver and kidney, which does not meet the technical requirements for food safety risk monitoring of exported animal-derived foods. Separately, related prior art discloses the use of liquid chromatography-tandem mass spectrometry to determine the content of 20 anticoccidial drugs in chicken using positive / negative ion segmented scanning, with a determination limit of 0.005 to 0.05 mg / kg.

[0004] While there have been reports of ethopabate detection both domestically and internationally, these methods target the technical compound and have high limits of quantification. Furthermore, long-term exposure to the metabolite m-ethoxyaniline, an ethopabate metabolite, may also increase cancer risk. Therefore, a method for detecting m-ethoxyaniline residues in edible poultry tissues is needed to safely monitor food safety risks in exported animal-derived foods. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting ethoxyaniline residues in poultry tissues. The method provided by the present invention has a low limit of quantification and can meet the detection needs of safety risk monitoring of exported animal-derived food.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting ethoxyaniline residues in poultry tissues, comprising the following steps:

[0008] homogenizing the poultry tissue to obtain a poultry tissue sample;

[0009] mixing the poultry tissue sample with acetonitrile and performing extraction to obtain an extract;

[0010] The extract is subjected to solid phase extraction purification to obtain a sample solution; the adsorbent for the solid phase extraction purification is a neutral alumina column;

[0011] Qualitative determination: Using liquid chromatography-mass spectrometry / mass spectrometry, under identical test conditions, a standard working solution of m-ethoxyaniline and the sample solution are measured to determine the retention time of the standard working solution of m-ethoxyaniline, the relative ion abundance of the standard working solution of m-ethoxyaniline, the retention time of the sample solution, and the relative ion abundance of the sample solution; if the deviation between the retention time of the sample solution and the retention time of the standard working solution of m-ethoxyaniline is within ±2.5%, and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the standard working solution of m-ethoxyaniline, the presence of m-ethoxyaniline in the sample solution is determined;

[0012] The conditions for the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution are: when the relative ion abundance is greater than 50%, the allowable relative deviation is ±20%; when the relative ion abundance is between 20% and 50%, the allowable relative deviation is ±25%; when the relative ion abundance is between 10% and 20%, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10%, the allowable relative deviation is ±50%;

[0013] Quantitative determination: Liquid chromatography-mass spectrometry / mass spectrometry was used to measure the m-ethoxyaniline standard working solution and the sample solution under the same test conditions. A standard working curve was then drawn with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate, and quantification was performed using the external standard method.

[0014] Preferably, the poultry tissue comprises one or more of poultry muscle, liver and kidney.

[0015] Preferably, the purity of the acetonitrile is chromatographically pure.

[0016] Preferably, the ratio of the mass of the poultry tissue sample to the volume of acetonitrile is (2.00-5.00) g: (10-45) mL.

[0017] Preferably, the concentration of the m-ethoxyaniline standard working solution is 0.25-10.00 μg / L.

[0018] Preferably, the liquid chromatography conditions of the liquid chromatography-mass spectrometry / mass spectrometry determination include: the chromatographic column is a C18 column with an inner diameter of 4.6 mm, a column length of 100 mm, and a particle size of 3 μm; the mobile phase is acetonitrile-formic acid aqueous solution; mobile phase A is acetonitrile, and mobile phase B is formic acid aqueous solution, and the concentration of the formic acid aqueous solution is 5 mmol / L; the flow rate is 0.35 mL / min; the column temperature is 40°C; and the injection volume is 10 μL.

[0019] Preferably, the elution gradient conditions of the mobile phase include: 0-2 min, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%; 2-7 min, the volume content of mobile phase A linearly increases to 85%, and the volume content of mobile phase B linearly increases to 15%; 7-9 min, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%; 9-9.1 min, the volume content of mobile phase A linearly decreases to 20%, and the volume content of mobile phase B linearly decreases to 80%; 9.1-14 min, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%.

[0020] Preferably, a single-point calibration is used when drawing the standard working curve, and the content X of ethoxyaniline in the poultry tissue sample is calculated as shown in formula (1):

[0021]

[0022] In the formula (1):

[0023] A is the chromatographic peak area response value corresponding to the sample solution;

[0024] A s is the chromatographic peak area response value corresponding to the standard working solution of m-ethoxyaniline;

[0025] C s is the concentration of ethoxyaniline in the standard working solution of m-ethoxyaniline, in ng / mL;

[0026] V is the constant volume of the sample solution, in mL;

[0027] m is the mass of the poultry tissue sample corresponding to the sample solution, in g;

[0028] The unit of X is μg / kg.

[0029] Preferably, multi-point calibration is used when drawing the standard working curve, and the content X of the intermediate ethoxyaniline in the poultry tissue sample is calculated using chromatography data processing software or formula (2):

[0030]

[0031] In the formula (2):

[0032] C i is the concentration of ethoxyaniline in the sample solution obtained from the standard working curve of m-ethoxyaniline, in ng / mL;

[0033] V is the constant volume of the sample solution, in mL;

[0034] m is the mass of the poultry tissue sample corresponding to the sample solution, in g;

[0035] The unit of X is μg / kg.

[0036] Preferably, the mass spectrometry conditions of liquid chromatography-mass spectrometry / mass spectrometry in the qualitative determination and the quantitative determination are the same, and the mass spectrometry conditions include: curtain gas pressure of 33-37 psi; ion source spray voltage of 2800-3200 V; ion source temperature of 500-600°C; nebulizing gas pressure of 50-60 psi; and auxiliary heating gas pressure of 55-65 psi.

[0037] The invention provides a method for detecting the residual amount of meta-ethoxyaniline in poultry tissues, comprising the following steps: the invention performs homogenization on the poultry tissues and then mixes the mixture with acetonitrile for extraction, wherein the homogenization can break the poultry tissues and fully dissolve the meta-ethoxyaniline; the invention uses acetonitrile as an extractant, which has a good dissolving effect on the meta-ethoxyaniline and can improve the extraction rate of the meta-ethoxyaniline in the poultry tissues; the invention performs solid-phase extraction purification on the extract and limits the adsorbent of the solid-phase extraction purification to a neutral alumina column, thereby reducing the influence of impurities on the test results and improving the accuracy of the test results; the invention adopts liquid chromatography-mass spectrometry / mass spectrometry to respectively determine the meta-ethoxyaniline standard working solution and the sample solution, and when the retention time of the sample solution is When the deviation of the retention time of the m-ethoxyaniline standard working solution is within ±2.5% and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the m-ethoxyaniline standard working solution, and a method for determining the consistency of the relative ion abundance is defined, the presence of m-ethoxyaniline in the sample solution is determined, and the m-ethoxyaniline in the sample solution is qualitatively analyzed. The method adopts liquid chromatography-mass spectrometry / mass spectrometry to respectively measure the m-ethoxyaniline standard working solution and the sample solution, then draws a standard working curve with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate, performs single-point calibration or multi-point calibration, and adopts external standard method for quantitative analysis, thereby being able to quantitatively analyze the content of m-ethoxyaniline in poultry tissue samples. The results of the examples show that the quantitative limit of m-ethoxyaniline in chicken, chicken liver and chicken fat according to the method provided by the present invention is 10 μg / kg, which has a low quantitative limit; in addition, the recovery rate of chicken samples is 80.1% to 89.2%, with a relative standard deviation of 3.1% to 5.9%, the recovery rate of chicken liver samples is 73.9% to 94.7%, with a relative standard deviation of 1.2% to 5.6%, and the recovery rate of chicken fat samples is 90.1% to 103%, with a relative standard deviation of 4.0% to 11.7%, with excellent accuracy and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a full scan of the secondary mass spectrum of m-ethoxyaniline in Example 1 of the present invention;

[0039] Figure 2 This is a liquid chromatography-mass spectrometry / mass spectrometry multiple reaction monitoring (MRM) chromatogram of the m-ethoxyaniline standard solution in Example 1 of the present invention;

[0040] Figure 3 This is a chromatogram of m-ethoxyaniline in a blank chicken liver sample from Example 1 of the present invention;

[0041] Figure 4 This is the standard working curve of the m-ethoxyaniline standard working solution in Example 1 of the present invention;

[0042] Figure 5 This is the chromatogram of m-ethoxyaniline in the blank chicken sample of Example 2 of the present invention;

[0043] Figure 6 This is a chromatogram of m-ethoxyaniline in a blank chicken fat sample from Example 3 of the present invention;

[0044] Figure 7 The chromatogram of the blank chicken standard spiked sample m-ethoxyaniline (low concentration) of the present invention;

[0045] Figure 8 The chromatogram of the blank chicken liver standard spiked with m-ethoxyaniline (low concentration) of the present invention;

[0046] Figure 9 A chromatogram of a blank chicken fat standard spiked sample of m-ethoxyaniline (low concentration) of the present invention;

[0047] Figure 10 The chromatograms are compared between the sample solutions prepared by the methods of Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0048] The present invention provides a method for detecting ethoxyaniline residues in poultry tissues, comprising the following steps:

[0049] homogenizing the poultry tissue to obtain a poultry tissue sample;

[0050] mixing the poultry tissue sample with acetonitrile and performing extraction to obtain an extract;

[0051] The extract is subjected to solid phase extraction purification to obtain a sample solution; the adsorbent for the solid phase extraction purification is a neutral alumina column;

[0052] Qualitative determination: Using liquid chromatography-mass spectrometry / mass spectrometry, under identical test conditions, a standard working solution of m-ethoxyaniline and the sample solution are measured to determine the retention time of the standard working solution of m-ethoxyaniline, the relative ion abundance of the standard working solution of m-ethoxyaniline, the retention time of the sample solution, and the relative ion abundance of the sample solution; if the deviation between the retention time of the sample solution and the retention time of the standard working solution of m-ethoxyaniline is within ±2.5%, and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the standard working solution of m-ethoxyaniline, the presence of m-ethoxyaniline in the sample solution is determined;

[0053] The conditions for the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution are: when the relative ion abundance is greater than 50%, the allowable relative deviation is ±20%; when the relative ion abundance is between 20% and 50%, the allowable relative deviation is ±25%; when the relative ion abundance is between 10% and 20%, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10%, the allowable relative deviation is ±50%;

[0054] Quantitative determination: Liquid chromatography-mass spectrometry / mass spectrometry was used to measure the m-ethoxyaniline standard working solution and the sample solution under the same test conditions. A standard working curve was then drawn with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate, and quantification was performed using the external standard method.

[0055] In the present invention, the poultry tissue preferably includes one or more of poultry muscle, liver, kidney, and fat, and more preferably one or more of chicken muscle (referred to as chicken in the embodiments of the present invention), liver, and kidney. The present invention does not particularly limit the source of the poultry tissue; commercially available products known to those skilled in the art may be used.

[0056] The present invention has no particular limitation on the method of homogenization. Conventional homogenization methods can be used to sufficiently disrupt the poultry tissue. In an embodiment of the present invention, the homogenization method can be: homogenizing the poultry tissue using a homogenizer.

[0057] After obtaining the poultry tissue sample, the present invention mixes the poultry tissue sample with acetonitrile and performs extraction to obtain an extract.

[0058] In the present invention, the purity of the acetonitrile is preferably chromatographically pure. The use of acetonitrile of the above purity is more conducive to fully extracting m-ethoxyaniline from poultry tissue samples.

[0059] In the present invention, the ratio of the mass of the poultry tissue sample to the volume of acetonitrile is preferably (2.00-5.00) g: (10-45) mL, more preferably 2.00 g: 10 mL. In the present invention, the mass of the poultry tissue sample is preferably accurate to 0.01 g.

[0060] In the present invention, the extraction method preferably includes vortexing, shaking, and centrifuging performed sequentially. In the present invention, the vortexing time is preferably 1 to 3 minutes, more preferably 1 to 2 minutes. In the present invention, the shaking time is preferably 10 to 20 minutes, more preferably 15 minutes. In the present invention, the centrifugal speed is preferably 3000 to 8000 r / min, more preferably 4000 to 5000 r / min; the centrifugal time is preferably 3 to 10 minutes, more preferably 5 to 8 minutes. By adopting the above-mentioned extraction method, the present invention is more conducive to improving the extraction rate of m-ethoxyaniline.

[0061] In the present invention, the extraction is preferably performed 1 to 3 times, more preferably 2 times.

[0062] The present invention preferably settles the solution obtained by the extraction to obtain an extract. The present invention can control the volumes of the extracts obtained from different batches to be the same by settling the volume, which is beneficial for having similar concentrations during subsequent solid phase extraction purification, thereby improving the accuracy and reproducibility of the test results. The present invention does not specifically limit the volume of the set volume, and can be adjusted according to the model of the adsorbent for solid phase extraction purification used. In an embodiment of the present invention, the adsorbent for solid phase extraction purification is a neutral alumina column, and the volume after settling can be 20mL.

[0063] After obtaining the extract, the present invention performs solid phase extraction purification on the extract to obtain a sample solution.

[0064] In the present invention, the adsorbent for solid phase extraction purification is a neutral alumina column. The present invention uses a neutral alumina column as the adsorbent for solid phase extraction purification, which has a good recovery rate and the effect of removing impurity peaks, and forms less flocculent precipitation after the sample solution is refrigerated.

[0065] In the present invention, the solid phase extraction purification method preferably includes: activating a neutral alumina column with an acetonitrile solution, then transferring the extract into the acetonitrile-activated neutral alumina column to obtain a filtrate; washing the column with an acetonitrile solution to obtain a column wash filtrate; combining the filtrate and the column wash filtrate, adding water to the fixed volume, and filtering to obtain a sample solution.

[0066] The present invention has no particular limitation on the activation method, and any conventional method for activating a neutral alumina column can be used. In an embodiment of the present invention, the acetonitrile solution can be pure acetonitrile, and the volume of the acetonitrile solution can be 3 mL.

[0067] In the present invention, the volume of the extract is preferably 1 to 3 mL, more preferably 2 mL.

[0068] In an embodiment of the present invention, the acetonitrile solution used for column washing may be pure acetonitrile, and the volume of the acetonitrile solution may be 1 mL.

[0069] In an embodiment of the present invention, the volume of the fixed volume may be 4 mL.

[0070] In the present invention, the pore size of the filtration membrane is preferably 0.22 μm.

[0071] After obtaining the sample solution, the present invention adopts liquid chromatography-mass spectrometry / mass spectrometry to respectively measure the m-ethoxyaniline standard working solution and the sample solution under the same test conditions to obtain the retention time of the m-ethoxyaniline standard working solution, the relative ion abundance of the m-ethoxyaniline standard working solution, the retention time of the sample solution, and the relative ion abundance of the sample solution.

[0072] In the present invention, the concentration of the m-ethoxyaniline standard working solution is preferably 0.25 to 10.00 μg / L. The present invention does not particularly limit the method for preparing the m-ethoxyaniline standard working solution; conventional methods for preparing standard solutions can be used to adjust the concentration of the m-ethoxyaniline standard working solution to within the above range. In embodiments of the present invention, the concentration of the m-ethoxyaniline standard working solution can be 0.25 μg / L, 0.50 μg / L, 1.00 μg / L, 2.00 μg / L, 5.00 μg / L, and 10.00 μg / L.

[0073] In the present invention, the liquid chromatography conditions for the liquid chromatography-mass spectrometry / mass spectrometry determination preferably include: a chromatographic column is preferably a C18 column; an inner diameter is preferably 4.6 mm; a column length is preferably 100 mm; a particle size is preferably 3 μm; the mobile phase is preferably acetonitrile-formic acid aqueous solution; mobile phase A is preferably acetonitrile, and mobile phase B is preferably formic acid aqueous solution, and the concentration of the formic acid aqueous solution is preferably 5 mmol / L; the flow rate is preferably 0.35 mL / min; the column temperature is preferably 40°C; and the injection volume is preferably 10 μL. The present invention uses a C18 column chromatographic column, and m-ethoxyaniline has a certain degree of retention on the C18 column, and the peak shape is symmetrical in the acetonitrile-formic acid aqueous system. The present invention uses acetonitrile-formic acid aqueous solution as the mobile phase. Compared with methanol and different concentrations of formic acid, ammonium formate, ammonium acetate and aqueous solution, when methanol is the organic phase, the response of m-ethoxyaniline is significantly lower than acetonitrile; after adding buffer salts such as ammonium formate and ammonium acetate to the aqueous phase, the response of m-ethoxyaniline decreases significantly, and the sensitivity is not as good as pure water. When a small amount of formic acid is added to the aqueous phase, the response of m-ethoxyaniline does not change significantly, but the chromatographic peak type improves, and the baseline noise decreases. As the formic acid concentration increases, the response decreases significantly. The present invention compares retention time, response value, chromatographic peak type and baseline noise, and selects acetonitrile-formic acid aqueous solution as the mobile phase, which is more conducive to improving detection efficiency and the accuracy of the test results. The present invention controls the chromatographic conditions within the above range, which is more conducive to improving detection efficiency and the accuracy of the test results.

[0074] In the present invention, the elution gradient conditions of the mobile phase preferably include: 0-2 min, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%; 2-7 min, the volume content of mobile phase A linearly increases to 85%, and the volume content of mobile phase B linearly increases to 15%; 7-9 min, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%; 9-9.1 min, the volume content of mobile phase A linearly decreases to 20%, and the volume content of mobile phase B linearly decreases to 80%; 9.1-14 min, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%. The elution gradient conditions of the mobile phase used in the present invention are more conducive to improving the separation degree, so that the various components in the test sample are separated under appropriate conditions, thereby improving peak shape, improving column efficiency, reducing analysis time, and improving detection efficiency and the accuracy of detection results.

[0075] In the present invention, the mass spectrometry conditions for the liquid chromatography-mass spectrometry / mass spectrometry coupled analysis in the qualitative and quantitative determinations are the same, and preferably include: a curtain gas pressure of preferably 33 to 37 psi, more preferably 35 psi; an ion source spray voltage of preferably 2800 to 3200 V, more preferably 3000 V; an ion source temperature of preferably 500 to 600° C., more preferably 550° C.; a nebulizing gas pressure of preferably 50 to 60 psi, more preferably 55 psi; and an auxiliary heating gas pressure of preferably 55 to 65 psi, more preferably 60 psi. Controlling the mass spectrometry conditions within the above ranges is beneficial to the sensitivity, selectivity, and accuracy of mass spectrometry analysis, thereby obtaining more reliable analysis results.

[0076] In an embodiment of the present invention, the method for mass spectrometry condition optimization is preferably: the m-ethoxyaniline standard solution of 0.05mg / L is continuously injected into the ESI ion source with a peristaltic pump at a flow rate of 10 μL / min, and under positive ion detection mode, a mass spectrometry analysis (Q1 scan) is performed to obtain quasi-molecular ion peak [M+1], and the declustering voltage is optimized. Secondary mass spectrometry analysis (daughter ion scan) is performed to the quasi-molecular ion peak, and fragment ion information is obtained, and collision energy is optimized to obtain secondary mass spectrum. The ion pair with high response value and low baseline noise is selected as the monitoring ion pair (qualitative ion pair), and the ion pair with the strongest signal is selected as the quantitative ion pair. In addition, the parameters such as ion source temperature (TEM), spray voltage (IS), curtain gas (CUR), atomizing gas (GS1), auxiliary heating gas (GS2), collision gas (CAD) are optimized. MRM mode data collection is adopted to optimize the declustering voltage (DP), collision gas energy (CE), entrance voltage (EP), collision cell outlet voltage (CXP) of each ion pair. Multiple reaction monitoring conditions are obtained. The present invention adopts this optimization method to make the mass spectrometry conditions more conducive to improving the sensitivity, selectivity and accuracy of the analysis results, thereby obtaining more reliable analysis results.

[0077] In the present invention, the liquid chromatography conditions for measuring the sample solution by liquid chromatography-mass spectrometry / mass spectrometry are the same as the liquid chromatography conditions for measuring the standard working solution of m-ethoxyaniline by liquid chromatography-mass spectrometry / mass spectrometry described in the above technical solution, and will not be repeated here.

[0078] In the present invention, the mass spectrometry conditions for measuring the sample solution by liquid chromatography-mass spectrometry / mass spectrometry are the same as the mass spectrometry conditions for measuring the m-ethoxyaniline standard working solution by liquid chromatography-mass spectrometry / mass spectrometry described in the above technical solution, and will not be repeated here.

[0079] In the present invention, the deviation between the retention time of the sample solution and the retention time of the m-ethoxyaniline standard working solution is within ±2.5%, and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the m-ethoxyaniline standard working solution, thereby determining the presence of m-ethoxyaniline in the sample solution. By controlling the retention time of the sample solution and the retention time of the m-ethoxyaniline standard working solution to be within the above deviation range and controlling the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution, the present invention can be used to qualitatively determine the presence of m-ethoxyaniline in the sample solution, thereby achieving qualitative determination of ethoxyaniline in poultry tissues.

[0080] In the present invention, the conditions for the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution are: when the relative ion abundance is greater than 50%, the allowable relative deviation is ±20%; when the relative ion abundance is between 20% and 50%, the allowable relative deviation is ±25%; when the relative ion abundance is between 10% and 20%, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10%, the allowable relative deviation is ±50%.

[0081] After determining the presence of m-ethoxyaniline in the sample solution, the present invention uses liquid chromatography-mass spectrometry / mass spectrometry to respectively measure the m-ethoxyaniline standard working solution and the sample solution, then draws a standard working curve with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate, and adopts the external standard method for quantification.

[0082] The present invention has no special limitation on the operation method of the external standard method for quantification, and a conventional external standard method for quantification can be used.

[0083] In the present invention, the liquid chromatography conditions and chromatographic conditions for respectively determining the m-ethoxyaniline standard working solution and the sample solution by liquid chromatography-mass spectrometry / mass spectrometry are the same as the liquid chromatography conditions and mass spectrometry conditions for determining the m-ethoxyaniline standard working solution by liquid chromatography-mass spectrometry / mass spectrometry described in the above technical solution, and will not be repeated here.

[0084] When drawing the standard working curve, single-point calibration is used. The calculation method of the content X of ethoxyaniline in the poultry tissue sample is preferably as shown in formula (1):

[0085]

[0086] In the formula (1):

[0087] A is the chromatographic peak area response value corresponding to the sample solution;

[0088] A sis the chromatographic peak area response value corresponding to the standard working solution of m-ethoxyaniline;

[0089] C s is the concentration of ethoxyaniline in the standard working solution of m-ethoxyaniline, in ng / mL;

[0090] V is the constant volume of the sample solution, in mL;

[0091] m is the mass of the poultry tissue sample corresponding to the sample solution, in g;

[0092] The unit of X is μg / kg.

[0093] In the present invention, during single-point calibration, the concentration of the standard working solution is preferably 0.5 μg / L.

[0094] When drawing the standard working curve, multi-point calibration is used, and the content X of the intermediate ethoxyaniline in the poultry tissue sample is calculated according to the chromatography data processing software or preferably according to formula (2):

[0095]

[0096] In the formula (2):

[0097] C i is the concentration of ethoxyaniline in the sample solution obtained from the standard working curve of m-ethoxyaniline, in ng / mL;

[0098] V is the constant volume of the sample solution, in mL;

[0099] m is the mass of the poultry tissue sample corresponding to the sample solution, in g;

[0100] The unit of X is μg / kg.

[0101] In the present invention, the concentrations of the standard working solutions are preferably 0.25 μg / L, 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 5.0 μg / L and 10 μg / L.

[0102] The present invention adopts the external standard method for quantification, and can quantitatively analyze and detect the residual amount of ethoxyaniline in poultry tissues.

[0103] The method provided by the present invention is simple to operate and easy to control, can detect the residual amount of ethoxyaniline in poultry tissues, has a low limit of quantification, and the detection results have excellent accuracy and reproducibility.

[0104] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0105] In an embodiment of the present invention, the preparation method of the m-ethoxyaniline standard working solution is as follows: an appropriate amount of 1.00 mg / L m-ethoxyaniline standard solution is taken and prepared with an acetonitrile-water (1+1, v / v) solution to prepare m-ethoxyaniline standard working solutions with concentrations of 0.25 μg / L, 0.50 μg / L, 1.00 μg / L, 2.00 μg / L, 5.00 μg / L and 10.00 μg / L.

[0106] Example 1

[0107] A method for detecting ethoxyaniline residues in poultry tissues, comprising the following steps:

[0108] The poultry tissue (chicken liver) is homogenized to obtain a poultry tissue sample;

[0109] 2.00 g (accurate to 0.01 g) of the poultry tissue sample was placed in a 50 mL centrifuge tube, 10 mL of chromatographically pure acetonitrile was added, the mixture was vortexed for 1 min, shaken for 15 min, and then centrifuged at 4000 r / min for 5 min. The supernatant was collected and placed in another 50 mL centrifuge tube. 8 mL of chromatographically pure acetonitrile was added to the residue, and the extraction was repeated once. The mixture was vortexed for 1 min, shaken for 15 min, and centrifuged at 4000 r / min for 5 min. The supernatants from the two centrifugations were combined and the volume was made up to 20 mL with chromatographically pure acetonitrile to obtain an extract.

[0110] Transfer 2 mL of the extract into a neutral alumina column activated with 3 mL of acetonitrile to obtain a filtrate; wash the column with 1 mL of acetonitrile to obtain a column wash filtrate; combine the filtrate and the column wash filtrate, add water to make the volume 4 mL, and filter through a 0.22 μm filter membrane to obtain a sample solution;

[0111] Qualitative determination: The conditions for liquid chromatography-mass spectrometry / mass spectrometry determination include liquid chromatography conditions and mass spectrometry conditions:

[0112] The liquid chromatography-mass spectrometry / mass spectrometry liquid chromatography conditions are:

[0113] The chromatographic column was a C18 column with an inner diameter of 4.6 mm, a column length of 100 mm, and a particle size of 3 μm;

[0114] The mobile phase was acetonitrile-formic acid aqueous solution; A was acetonitrile, B was 5 mmol / L formic acid aqueous solution, and the gradient elution conditions were as shown in Table 1:

[0115] Table 1 Mobile phase elution gradient conditions

[0116] Time / min Mobile phase A / % Mobile phase B / % 0 20 80 2 20 80 7 85 15 9 85 15 9.1 20 80 14 20 80

[0117] Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 10 L;

[0118] The optimization process of the mass spectrometry conditions for the liquid chromatography-mass spectrometry / mass spectrometry determination is as follows: a 0.05 mg / L m-ethoxyaniline standard solution is continuously injected into the ESI ion source at a flow rate of 10 μL / min using a peristaltic pump, and the m-ethoxyaniline standard solution is subjected to primary mass spectrometry analysis (Q1 scan) in positive ion detection mode to obtain a quasi-molecular ion peak [M+1], and the declustering voltage is optimized. The quasi-molecular ion peak is subjected to secondary mass spectrometry analysis (daughter ion scan) to obtain fragment ion information, and the collision energy is optimized. The full scan of the secondary mass spectrum of m-ethoxyaniline is shown in FIG. Figure 1 The ion pair with high response value and low baseline noise was selected as the monitoring ion pair (qualitative ion pair), and the ion pair with the strongest signal was selected as the quantitative ion pair. The multiple reaction monitoring conditions are shown in Table 2. The chromatogram of the liquid chromatography-mass spectrometry / mass spectrometry multiple reaction monitoring (MRM) of the m-ethoxyaniline standard solution is shown in Figure 2 shown; in Figure 2 In the figure, the left image represents the quantitative ion pair, and the right image represents the qualitative ion pair. Furthermore, the ion source temperature (TEM), spray voltage (IS), curtain gas (CUR), nebulizer gas (GS1), auxiliary heater gas (GS2), and collision gas (CAD) were optimized. The optimized results were: curtain gas pressure of 35 psi; ion source spray voltage of 3000 V; ion source temperature of 550°C; nebulizer gas pressure of 55 psi; and auxiliary heater gas pressure of 60 psi. Data were acquired in MRM mode, and the declustering voltage (DP), collision gas energy (CE), entrance voltage (EP), and collision cell exit voltage (CXP) for each ion pair were optimized.

[0119] Table 2 Multiple reaction monitoring conditions

[0120]

[0121] In Table 2, ions marked with “*” are quantification ions.

[0122] According to the above-mentioned liquid chromatography-mass spectrometry / mass spectrometry test conditions of liquid chromatography and mass spectrometry, the standard working solution of m-ethoxyaniline was measured by liquid chromatography-mass spectrometry / mass spectrometry to obtain a chromatogram of the standard working solution of m-ethoxyaniline, and the retention time and relative ion abundance of the standard working solution of m-ethoxyaniline were obtained; the sample solution was measured by liquid chromatography-mass spectrometry / mass spectrometry to obtain a chromatogram of the sample solution (i.e., the blank chicken liver sample m-ethoxyaniline) as shown in FIG. Figure 3 As shown, in Figure 3 In the figure, the left figure represents the quantitative ion pair, and the right figure represents the qualitative ion pair; the retention time and relative ion abundance of the sample solution are obtained; the deviation of the retention time of the sample solution and the retention time of the m-ethoxyaniline standard working solution is within ±2.5%, and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the m-ethoxyaniline standard working solution, thereby determining that m-ethoxyaniline exists in the sample solution.

[0123] The conditions for the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution are: when the relative ion abundance is greater than 50%, the allowable relative deviation is ±20%; when the relative ion abundance is between 20% and 50%, the allowable relative deviation is ±25%; when the relative ion abundance is between 10% and 20%, the allowable relative deviation is ±30%; and when the relative ion abundance is ≤10%, the allowable relative deviation is ±50%. The test results indicate that m-ethoxyaniline is present in the sample solution.

[0124] Quantitative determination: Liquid chromatography-mass spectrometry / mass spectrometry was used to determine the concentrations of the m-ethoxyaniline standard working solution (concentrations were 0.25 μg / L, 0.50 μg / L, 1.00 μg / L, 2.00 μg / L, 5.00 μg / L and 10.00 μg / L) and the sample solution under the same test conditions as above. The standard working curve was then drawn with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate. Figure 4 As shown, the regression equation is: y = 8.56832e5x + 24896.85821r = 0.99993; Figure 4 From the regression equation, it can be seen that when the concentration of m-ethoxyaniline is in the range of 0.25μg / L to 10μg / L, the linear relationship is good. When the concentration of the analyte in the sample exceeds this linear range, the dilution factor of the sample can be appropriately increased.

[0125] Perform multi-point calibration and adopt external standard method for quantification;

[0126] Calculate the content X of ethoxyaniline in the sample according to formula (2):

[0127]

[0128] In the formula (2):

[0129] C i is the concentration of ethoxyaniline in the sample solution obtained from the standard working curve of m-ethoxyaniline, in ng / mL;

[0130] V is the constant volume of the sample solution, in mL;

[0131] m is the mass of the poultry tissue sample corresponding to the sample solution, in g;

[0132] The unit of X is μg / kg;

[0133] When calculating the content X of ethoxyaniline in the sample according to the method of formula (2), a blank value is deducted. The method for deducting the blank value is: first, a blank value is obtained by a blank experiment. The method of the blank experiment is according to the above-mentioned method for preparing the sample solution, which differs from the above-mentioned method for preparing the sample solution in that no poultry tissue sample is added. Then, the test results are obtained according to the test conditions of liquid chromatography-mass spectrometry / mass spectrometry. The blank value is calculated according to formula (2), and the blank value is deducted by the content of ethoxyaniline in the sample solution.

[0134] Example 2

[0135] The difference from Example 1 is that the poultry tissue used is chicken;

[0136] The sample solution was measured by liquid chromatography-mass spectrometry / mass spectrometry to obtain a chromatogram of the sample solution (i.e., blank chicken sample m-ethoxyaniline). Figure 5 As shown, in Figure 5 In the middle, the left panel represents the quantitative ion transition, and the right panel represents the qualitative ion transition;

[0137] The remaining steps are the same as those in Example 1.

[0138] Example 3

[0139] The difference from Example 1 is that the poultry tissue used is chicken fat;

[0140] The sample solution was measured by liquid chromatography-mass spectrometry / mass spectrometry to obtain a chromatogram of the sample solution (i.e., blank chicken fat sample meta-ethoxyaniline). Figure 6 As shown, in Figure 6 In the middle, the left panel represents the quantitative ion transition, and the right panel represents the qualitative ion transition;

[0141] The remaining steps are the same as those in Example 1.

[0142] Test Case

[0143] (1) The quantitative limit and recovery rate of ethoxyaniline in poultry tissues were determined by adding m-ethoxyaniline to different matrices (chicken, chicken liver and chicken fat); wherein the concentrations of m-ethoxyaniline added to chicken were 10 μg / kg, 40 μg / kg, 500 μg / kg and 1000 μg / kg, respectively; the concentrations of m-ethoxyaniline added to chicken liver were 10 μg / kg, 40 μg / kg, 1500 μg / kg and 3000 μg / kg, respectively; and the concentrations of m-ethoxyaniline added to chicken fat were 10 μg / kg, 40 μg / kg and 80 μg / kg, respectively. Then, the above-mentioned different matrices were detected using the method of Example 1; the chromatogram of the sample solution (i.e., the chromatogram of the blank chicken standard sample with m-ethoxyaniline added (low concentration 10 μg / kg)) was obtained as shown below. Figure 7 The chromatogram of blank chicken liver standard spiked sample m-ethoxyaniline (low concentration 10 μg / kg) is shown in Figure 8 The chromatogram of blank chicken fat standard spiked sample with m-ethoxyaniline (low concentration 10 μg / kg) is shown in Figure 9 As shown. Figures 7-9 In the figure, the left panel represents the quantification ion transition, and the right panel represents the qualification ion transition.

[0144] The indoor verification data were obtained after 6 parallel measurements and are shown in Table 3.

[0145] Table 3 Indoor verification data

[0146]

[0147] As shown in Table 3, the recovery rate of chicken samples was 80.1% to 89.2%, with a relative standard deviation of 3.1% to 5.9%. The recovery rate of chicken liver samples was 73.9% to 94.7%, with a relative standard deviation of 1.2% to 5.6%. The recovery rate of chicken fat samples was 90.1% to 103%, with a relative standard deviation of 4.0% to 11.7%, which met the relevant requirements of SN / T0001-2016 and GB 5009.295-2023. In addition, the method provided by the present invention can obtain a stable signal-to-noise ratio (S / N ≥ 10) that meets the quantitative requirements, with a limit of quantification of 10 μg / kg.

[0148] Comparative Example 1

[0149] The difference from Example 1 is that a degreasing column is used instead of a neutral alumina column, and the remaining steps are the same as those in Example 1.

[0150] Comparative Example 2

[0151] The difference from Example 1 is that a PRIME HLB column is used instead of a neutral alumina column, and the remaining steps are the same as those in Example 1.

[0152] Comparative Example 3

[0153] The difference from Example 1 is that an LC-Si column is used instead of a neutral alumina column, and the remaining steps are the same as those in Example 1.

[0154] Test Example 2

[0155] The matrix of animal-derived food is relatively complex, and the acetonitrile extraction solution needs to be further purified before entering the instrument detection. The neutral alumina column, EMR degreasing column, PRIMEHLB column, LC-Si column and liquid-liquid extraction purification were compared through Example 1 and Comparative Examples 1 to 3. These solid phase extraction columns are all pass-through type, adsorb impurities, and do not retain the analyte. The results show that the PRIMEHLB column adsorbs the analyte with a recovery rate of 0. The LC-Si column has a good effect in removing impurity peaks, but the recovery rate is low, only 60%. The use of n-hexane liquid-liquid extraction for degreasing has a recovery rate of about 80%. The neutral alumina column and EMR degreasing column have the best recovery rate, about 90%, but the EMR degreasing column has a poor effect in removing impurity peaks, and also increases the impurity peaks with a retention time between 7 and 9 minutes, and there is a small amount of flocculent precipitation after the sample liquid is refrigerated. Finally, the neutral alumina column was selected, which not only effectively removed the impurity peaks, clarified the sample liquid, and had a good recovery rate and an ideal purification effect. The chromatographic comparison diagram of the sample solution prepared by the method of Example 1 and Comparative Example 1 is shown as follows. Figure 10 As shown. Figure 10 In the figure, the left figure is a chromatogram of the sample solution obtained by the method of Example 1, and the right figure is a chromatogram of the sample solution obtained by the method of Comparative Example 1.

[0156] Test Example 3

[0157] The matrix effect was investigated as follows: ME = (slope of the matrix-matched standard curve / slope of the solvent standard curve - 1) × 100%. When ME > 0, it was a matrix-enhanced effect; when ME < 0, it was a matrix-inhibitory effect; when |ME| < 20%, it was a weak matrix effect and the matrix effect was not significant; when |ME| < 50%, it was a moderate matrix effect; and when |ME| > 50%, it was a strong matrix effect and the matrix effect was considered significant.

[0158] Different types of substances have different matrix effects. The matrix effect results of chicken, chicken liver and chicken fat are shown in Table 4.

[0159] Table 4 Matrix effect

[0160]

[0161] As can be seen from Table 4, the sample solution obtained by extraction provided by the present invention has a very weak matrix effect on the target compound, and there is no need to use a matrix-matched standard curve to correct the influence of the matrix effect on the method recovery rate.

[0162] At present, the United States, Japan and my country have all stipulated the maximum residue limit of ethopabenzyl in animal-derived foods, with a minimum value of 40 μg / kg. However, considering that the EU has not approved the use of this drug, it is necessary to provide a detection method that can be lower than the 40 μg / kg limit of quantification. As can be seen from the above results, the limit of quantification of ethoxyaniline in chicken, chicken liver and chicken fat in the method provided by the present invention is 10 μg / kg, which has a lower limit of quantification and can meet the detection requirements of export animal-derived food safety risk monitoring. In addition, the detection results of the method provided by the present invention have excellent accuracy and reproducibility.

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting ethoxyaniline residues in poultry tissues, comprising the following steps: homogenizing the poultry tissue to obtain a poultry tissue sample; mixing the poultry tissue sample with acetonitrile and performing extraction to obtain an extract; The extract is subjected to solid phase extraction purification to obtain a sample solution; the adsorbent for the solid phase extraction purification is a neutral alumina column; Qualitative determination: Using liquid chromatography-mass spectrometry / mass spectrometry, under identical test conditions, a standard working solution of m-ethoxyaniline and the sample solution are measured to determine the retention time of the standard working solution of m-ethoxyaniline, the relative ion abundance of the standard working solution of m-ethoxyaniline, the retention time of the sample solution, and the relative ion abundance of the sample solution; if the deviation between the retention time of the sample solution and the retention time of the standard working solution of m-ethoxyaniline is within ±2.5%, and the relative ion abundance of the sample solution is consistent with the relative ion abundance of the standard working solution of m-ethoxyaniline, the presence of m-ethoxyaniline in the sample solution is determined; The conditions for the relative ion abundance of the sample solution to be consistent with the relative ion abundance of the m-ethoxyaniline standard working solution are: when the relative ion abundance is greater than 50%, the allowable relative deviation is ±20%; when the relative ion abundance is between 20% and 50%, the allowable relative deviation is ±25%; when the relative ion abundance is between 10% and 20%, the allowable relative deviation is ±30%; when the relative ion abundance is ≤10%, the allowable relative deviation is ±50%; Quantitative determination: Liquid chromatography-mass spectrometry / mass spectrometry was used to measure the m-ethoxyaniline standard working solution and the sample solution under the same test conditions. A standard working curve was then drawn with the concentration of the m-ethoxyaniline standard working solution as the abscissa and the quantitative ion peak area as the ordinate, and quantification was performed using the external standard method.

2. The method according to claim 1, characterized in that The poultry tissue includes one or more of poultry muscle, liver, kidney and fat.

3. The method according to claim 1, characterized in that The purity of the acetonitrile is chromatographically pure.

4. The method according to claim 1 or 3, characterized in that The ratio of the mass of the poultry tissue sample to the volume of acetonitrile is (2.00-5.00) g: (10-45) mL.

5. The method according to claim 1, wherein The concentration of the m-ethoxyaniline standard working solution is 0.25-10 μg / L.

6. The method according to claim 1, characterized in that The liquid chromatography conditions for the liquid chromatography-mass spectrometry / mass spectrometry determination include: The chromatographic column was a C18 column with an inner diameter of 4.6 mm, a column length of 100 mm, and a particle size of 3 μm; the mobile phase was acetonitrile-formic acid aqueous solution; mobile phase A was acetonitrile, and mobile phase B was formic acid aqueous solution, and the concentration of the formic acid aqueous solution was 5 mmol / L; the flow rate was 0.35 mL / min; the column temperature was 40°C; and the injection volume was 10 μL.

7. The method according to claim 6, characterized in that The elution gradient conditions of the mobile phase include: from 0 to 2 minutes, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%; from 2 to 7 minutes, the volume content of mobile phase A linearly increases to 85%, and the volume content of mobile phase B linearly increases to 15%; from 7 to 9 minutes, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%; from 9 to 9.1 minutes, the volume content of mobile phase A linearly decreases to 20%, and the volume content of mobile phase B linearly decreases to 80%; from 9.1 to 14 minutes, the volume content of mobile phase A is 20%, and the volume content of mobile phase B is 80%.

8. The method according to claim 1, characterized in that Single-point calibration was used when drawing the standard working curve. The calculation method of the content X of ethoxyaniline in the poultry tissue sample was as shown in formula (1): In the formula (1): A is the chromatographic peak area response value corresponding to the sample solution; A s is the chromatographic peak area response value corresponding to the standard working solution of m-ethoxyaniline; C s is the concentration of ethoxyaniline in the standard working solution of m-ethoxyaniline, in ng / mL; V is the constant volume of the sample solution, in mL; m is the mass of the poultry tissue sample corresponding to the sample solution, in g; The unit of X is μg / kg.

9. The method according to claim 1, characterized in that When drawing the standard working curve, multi-point calibration is used, and the content X of ethoxyaniline in the poultry tissue sample is calculated using the chromatography data processing software or according to formula (2): In the formula (2): C i is the concentration of ethoxyaniline in the sample solution obtained from the standard working curve of m-ethoxyaniline, in ng / mL; V is the constant volume of the sample solution, in mL; m is the mass of the poultry tissue sample corresponding to the sample solution, in g; The unit of X is μg / kg.

10. The method according to claim 1, characterized in that The mass spectrometry conditions of the liquid chromatography-mass spectrometry / mass spectrometry combined determination in the qualitative determination and the quantitative determination are the same, and the mass spectrometry conditions include: a curtain gas pressure of 33 to 37 psi; an ion source spray voltage of 2800 to 3200 V; an ion source temperature of 500 to 600°C; a nebulizing gas pressure of 50 to 60 psi; and an auxiliary heating gas pressure of 55 to 65 psi.

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