Determination method and application of samidazuril and deacetylated samidazuril

Abstract

The invention provides a determination method and application of samidazuril and deacetylated samidazuril. The determination method comprises the following steps: S1, preparing a standard solution; s2, pretreatment of a sample: weighing the homogenized tissue or blank sample, firstly adding a 10% Na2CO3 solution, performing vortex mixing uniformly, adding acetonitrile of which the amount is 9 times that of the 10% Na2CO3 solution, performing vortex mixing uniformly, performing ultrasonic treatment, performing vortex treatment, and performing centrifugation to obtain supernate; taking supernate to pass through a rapid filtration purification column, collecting filtrate, diluting, uniformly mixing, centrifuging, and filtering by a filtering membrane; s3, respectively drawing standard substance curves and matrix matching standard curves with different concentrations; s4, detecting by adopting a liquid chromatography-mass spectrometry method, and analyzing by applying a multi-ion reaction monitoring method; and S5, determining the homogenized sample to be tested by a liquid chromatography-tandem mass spectrometer. The method is suitable for quantitative detection of samipril and deacetylated samipril in animal-derived food, the quantification limit is as low as 5 mu g / kg, the recovery rate is high, and the relative standard deviation is low; the sample pretreatment is simple, and the sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the field of food detection, and in particular relates to a determination method and application of samizuril and deacetyl samizuril. Background Art

[0002] Samizurin is a new type of triazine anticoccidial drug independently developed by the veterinary anti-infective drug innovation team of the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. It is mainly used to prevent and treat coccidiosis in poultry. In poultry and livestock farming, coccidiosis is a common and harmful parasitic disease that can cause slow growth, reduced feed conversion rate, decreased egg production and even death in poultry. Samizurin is mainly metabolized into 6 metabolites in the body. Among them, the metabolite deacetyl samizurin has the longest residual time and the largest residual amount in kidney tissue, while the original drug has a higher residual amount in other tissues.

[0003] Samizurin and deacetyl Samizurin can prevent and treat coccidiosis by interfering with the metabolic process of coccidia and inhibiting their growth and reproduction. They are highly effective, low toxic, and not prone to drug resistance, and play an important role in protecting poultry health and improving breeding efficiency.

[0004] On February 1, 2021, "Announcement No. 350 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China" approved samizuril as a new veterinary drug for the first time, and established the acceptable daily intake (ADI) and maximum residue limits (MRLs) in chicken tissues of samizuril. Therefore, when using samizuril, it is necessary to strictly abide by the relevant medication regulations and measurement requirements to ensure its safe and effective use and reduce possible problems such as drug resistance and drug residues.

[0005] The detection methods for samizuril reported in the literature are all high performance liquid chromatography (HPLC), which is mostly used to determine the content of samizuril in feed or chicken tissue. But in reality, in addition to poultry, many animals may be infected with coccidiosis, such as rabbits, cattle, sheep, pigs, etc. Therefore, samizuril can act on a variety of animals as an anticoccidial drug. The matrix components of animal tissues are extremely complex, and the matrix effect is obvious, which greatly interferes with HPLC detection. The measurement method of substances in a single type of animal tissue is difficult to be used with other animal tissues.

[0006] Therefore, the existing methods are difficult to simultaneously determine deacetyl samizuril and the samizuril content in other livestock tissues, and have narrow applicability.

[0007] Therefore, it is of great significance to establish a universal detection method for samizulil and deacetyl samizuloid in animal-derived foods, improve the accuracy and sensitivity of samizulil and deacetyl samizuloid, and reduce the lower limit of quantitative detection. Summary of the invention

[0008] In view of the problem that the prior art lacks a highly universal method for detecting samizuril and deacetyl samizuril in animal-derived foods, the present invention provides a method for determining samizuril and deacetyl samizuril and uses thereof.

[0009] The technical solution of the present invention is as follows: A method for determining samizuril and deacetyl samizuril comprises the following steps:

[0010] S1. Preparation of standard solution

[0011] The samizuril and desacetyl samizuril standards were prepared into 1.0 mg / mL samizuril and desacetyl samizuril standard stock solutions, and diluted into 10 μg / mL and 1.0 μg / mL standard working solutions for later use;

[0012] S2. Sample pretreatment

[0013] Take an appropriate amount of fresh or thawed blank sample or test muscle, sebum, liver, or kidney tissue, mince and homogenize it before pre-treating the sample;

[0014] S21. Weigh the homogenized tissue or blank sample and place it in a centrifuge tube with a lid. First, add 10% Na2CO3 solution and vortex to mix. Then, add 9 times the amount of acetonitrile as the 10% Na2CO3 solution and vortex to mix. Ultrasonicate, vortex, and centrifuge to obtain a supernatant.

[0015] S22, take the supernatant and filter it through a rapid filtration purification column, collect the filtrate, accurately measure the extract, dilute it with 3 times water, mix it, centrifuge it, take the upper layer of liquid and filter it through a filter membrane, and take the filtrate to obtain the corresponding sample;

[0016] S3. Standard curve drawing

[0017] The standard curves and matrix-matched standard curves of different concentrations of samizuril and deacetyl samizuril were drawn respectively; the standard curve was prepared by diluting the samizuril and deacetyl samizuril standard working solutions into a series of standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL; the matrix-matched standard curve was prepared by diluting the samizuril and deacetyl samizuril standard working solutions into a series of matrix-matched standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL using the blank sample pre-treated in step S2;

[0018] S4. Liquid chromatography-mass spectrometry conditions

[0019] Liquid chromatography-mass spectrometry was used for detection and multiple ion reaction monitoring was used for analysis;

[0020] The liquid chromatography column is a C18 column, the mobile phase is acetonitrile + 0.1% formic acid aqueous solution, the flow rate is 0.3mL / min; the column temperature is 30°C, the injection volume is 10μL; the elution program is: 0-1min, 23%A+77%B; 2-3min, 40%A+60%B; 4-5min, 23%A+77%B; A is acetonitrile, and B is 0.1% formic acid aqueous solution;

[0021] Mass spectrometer voltage: 5500V, curtain gas: 30 psi, IS voltage: 5500V, ion source temperature: 550℃, nebulizer gas: 55psi, auxiliary gas: 55psi; multiple ion reaction monitoring parameters: samizurin parent ion 353.1m / z; daughter ion 147.1*m / z, collision energy 24V; daughter ion 108.1m / z, collision energy 24V; deacetyl samizurin parent ion 311.2 m / z, daughter ion 79.9*m / z, collision energy 34V; daughter ion 108.1 m / z, collision energy 18V;

[0022] S5. Determination of samples

[0023] The homogenized test sample is measured by liquid chromatography-tandem mass spectrometry, and the retention time of the test sample chromatogram is compared with the retention time of the standard solution, and the characteristic ions of each chromatographic peak are compared with the characteristic ions of each chromatographic peak of the standard solution of the corresponding concentration for qualitative analysis;

[0024] Then take the test sample and the corresponding matrix matching standard solution, take single point or multi-point calibration, and perform quantitative measurement according to the external standard method.

[0025] Furthermore, the analyte residue in the test sample is calculated as follows:

[0026] Single point calibration or standard curve calibration is performed by:

[0027] ! As=aC s +b

[0028] The residual amount of samizuril or deacetyl samizuril was calculated according to the following formula:

[0029]

[0030] Where: X——the residual amount of samizuril or deacetyl samizuril in the sample, in micrograms per kilogram (μg / kg);

[0031] Cs——the concentration of samizuril or desacetyl samizuril in the matrix matching solution, in nanograms per milliliter (ng / mL);

[0032] C——the concentration of samizuril or deacetyl samizuril in the sample solution, in nanograms per milliliter (ng / mL);

[0033] As——peak area of ​​samizuril or desacetyl samizuril in matrix matching solution;

[0034] A——peak area of ​​samizuril or deacetyl samizuril in the sample solution;

[0035] V——volume of the sample after dilution with water, in milliliters (mL);

[0036] m – the mass of the sample, in grams (g).

[0037] Furthermore, in step S1, the solvent used to prepare the standard stock solution and standard working solution of samizuril and deacetylsamizuril is methanol.

[0038] Furthermore, the rapid filtration purification column in step S2 is a CAFS Clean-up Column (200 mg, 3 mL).

[0039] Furthermore, the pre-treatment conditions of the sample in step S2 are:

[0040] S21. Weigh 5.0 g (accurate to ±0.02 g) of the homogenized tissue or blank sample into a 50 mL centrifuge tube with a lid, add 1 mL of 10% Na2CO3 solution, vortex to mix, add 10 mL of acetonitrile, vortex to mix, sonicate for 30 min, vortex, and centrifuge at 5000 r / min for 10 min to obtain the supernatant;

[0041] S22. Take 1 mL of the supernatant, filter it through a rapid filtration purification column, collect the filtrate, accurately measure 0.25 mL of the extract into a 2 mL centrifuge tube with a cap, dilute it with 0.75 mL of water, mix well, centrifuge at 12000 r / min for 10 min, take the upper layer of liquid and filter it through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain the corresponding sample.

[0042] Furthermore, step S5 further includes step S6:

[0043] After the test sample is tested, a homogenized blank sample is added to the liquid chromatography-tandem mass spectrometer to confirm that it does not contain substances that interfere with the components to be tested.

[0044] Furthermore, in step S4, the relative deviation of the retention time of the test sample and the standard solution is no more than 2.5%; the relative abundance of characteristic ions of the test sample is consistent with the relative abundance of the standard solution with equivalent concentration.

[0045] Furthermore, when the relative ion abundance is greater than 50%, the maximum allowable deviation is ±20%; when the relative ion abundance is greater than 20% to 50%, the maximum allowable deviation is ±25%; when the relative ion abundance is greater than 10% to 20%, the maximum allowable deviation is ±30%; when the relative ion abundance is ≤10%, the maximum allowable deviation is ±50%.

[0046] The above-mentioned determination method of samizuril and deacetyl samizuril is used for the quantitative detection of samizuril and deacetyl samizuril in animal-derived foods.

[0047] Furthermore, animal-derived foods include: muscle, sebum, liver and kidney of chicken, pig, sheep and rabbit.

[0048] The advantages of the present invention are: it is suitable for the quantitative detection of samizuril and deacetyl samizuril in animal-derived foods; it adopts rapid filtration column purification, and after high-performance liquid chromatography separation, it is highly sensitively detected by multiple ion reaction monitoring (MRM) of a tandem quadrupole, and quantified by an external standard method. The determination method of the present invention has been verified to show that the method has extremely high sensitivity and stability, with a quantitative limit as low as 5 μg / kg, and the recovery rate and relative standard deviation method of the method can meet the quantitative detection of samizuril in various complex matrices. Compared with other methods, it has the advantages of simple sample pretreatment, high safety factor, high sensitivity, fast detection speed, good selectivity, and accurate quantitative results. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the standard chromatogram of samizuril;

[0050] Figure 2 This is the standard chromatogram of acetyl-samizurin;

[0051] Figure 3 This is a comparison chart of the recoveries of spiked samples with different pretreatment solvents. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part 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 creative work are within the scope of protection of the present invention.

[0053] The present invention uses samizuril and deacetyl samizuril as residue markers to study the residue detection method in animal tissues (muscle, sebum, liver and kidney of chicken, pig, sheep and rabbit).

[0054] The reagents used in the determination method of samizuril and deacetyl samizuril of the present invention are all analytically pure, and the water is first-grade water in accordance with GB / T 6682; wherein acetonitrile and formic acid are chromatographically pure. The reference substance is samizuril: C 18 H 16 N4O4 content ≥99.5%, deacetyl samizurin: C 16 H 14 N4O3 content ≥97.5%.

[0055] Specific measurement methods include:

[0056] S1. Preparation of standard solution

[0057] Standard stock solution of samizuril and desacetyl samizuril (1.0 mg / mL): Accurately weigh 10.0 mg each of samizuril and desacetyl samizuril reference substances, place them in 10 mL volumetric flasks, dissolve them in an appropriate amount of methanol and dilute to the scale to prepare standard stock solutions of samizuril and desacetyl samizuril with a concentration of 1.0 mg / mL; store in a dark place below 4°C, with a shelf life of 1 month.

[0058] Continue to dilute the samizuril standard stock solution and deacetyl samizuril standard stock solution into 10 μg / mL and 1.0 μg / mL standard working solutions for standby use.

[0059] S2. Sample pretreatment

[0060] Take an appropriate amount of fresh or thawed blank sample or test muscle, sebum, liver, or kidney tissue, mince and homogenize.

[0061] The sample pretreatment process is as follows:

[0062] S21. Weigh 5.0 g (accurate to ±0.02 g) of the homogenized tissue or blank sample into a 50 mL centrifuge tube with a lid, add 1 mL of 10% Na2CO3 solution, vortex to mix, add 10 mL of acetonitrile, vortex to mix, sonicate for 30 min, vortex, and centrifuge at 5000 r / min for 10 min to obtain the supernatant;

[0063] S22. Take 1 mL of the supernatant and pass it through a rapid filtration purification column (the purification column does not need to be activated), collect the filtrate, accurately measure 0.25 mL of the extract into a 2 mL centrifuge tube with a cap, add 0.75 mL of water to dilute, mix well, centrifuge at 12000 r / min for 10 min, take the upper layer of liquid and filter it through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain the corresponding sample.

[0064] The samples obtained are divided into three categories: one is the homogeneous test sample, the second is the homogeneous blank sample, and the third is the homogeneous blank sample + the standard working solution with appropriate concentration added.

[0065] S3. Standard curve drawing

[0066] S31. Solvent standard curve: Accurately measure appropriate amounts of samizuril and desacetyl samizuril standard working solutions, dilute them with 25% acetonitrile aqueous solution, and prepare a series of standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL for determination by liquid chromatography-tandem mass spectrometry. Both samizuril and desacetyl samizuril have good linear relationships, with correlation coefficients greater than 0.99.

[0067] S32. Matrix matching standard curve: Take blank samples of each tissue to be tested and process them into homogeneous blank sample test pieces according to the method of step S2. Accurately measure appropriate amounts of samizuril and deacetylsamizuril standard working solutions, dilute them with blank sample test pieces respectively, and prepare a series of matrix matching standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL. Prepare them before use and provide them for liquid chromatography-tandem mass spectrometry determination.

[0068] S4. Liquid chromatography-mass spectrometry conditions

[0069] Liquid chromatography-mass spectrometry was used for detection and multiple ion reaction monitoring was used for analysis.

[0070] Liquid chromatography column: Chromatographic column: C18 (50 mm × 2.1 mm, particle size 1.7 μm), or equivalent. Mobile phase flow rate: 0.3 mL / min. Chromatographic column temperature: 30 ° C, injection volume: 10 μL, gradient elution program: 0-1 min, 23% A + 77% B; 2-3 min, 40% A + 60% B; 4-5 min, 23% A + 77% B; A is acetonitrile, B is 0.1% formic acid aqueous solution.

[0071] Mass spectrometer voltage: 5500V, curtain gas: 30psi, IS voltage: 5500V, ion source temperature: 550℃, nebulizer gas: 55psi, auxiliary gas: 55psi. MRM mass spectrometer parameters: Samizurin parent ion 353.1m / z; daughter ion 147.1*m / z, collision energy 24V; daughter ion 108.1m / z, collision energy 24V. Deacetyl Samizurin parent ion 311.2m / z, daughter ion 79.9*m / z, collision energy 34V; daughter ion 108.1m / z, collision energy 18V.

[0072] The above parameters are affected by instruments of different manufacturers and models and can be adjusted or optimized as needed.

[0073] The reason for selecting acetonitrile + 0.1% formic acid aqueous solution as the mobile phase is that in addition to considering the separation effect of the chromatographic system, attention must also be paid to the efficiency of ionization when the separated components enter the mass spectrometer in order to obtain the best resolution and the highest sensitivity. In the preliminary experiment, the present invention selects methanol or acetonitrile as the strong elution mobile phase, uses water as the weak elution mobile phase, and adopts the ESI+ method for determination.

[0074] When applying the multi-ion reaction monitoring method for sample analysis, it was found that methanol as a strong elution mobile phase can retain the target substance well due to its high viscosity, but the column pressure is easy to be too high. When acetonitrile is used as a strong elution mobile phase, the target substance can be effectively separated in a shorter time, with good peak shape and moderate column pressure, which will not reduce the service life of the chromatographic column. Therefore, acetonitrile is used as a strong elution mobile phase in the subsequent method development process.

[0075] When using mass spectrometry for analysis and detection, a certain concentration of formic acid, acetic acid, ammonium formate or ammonium acetate and other reagents are usually added to the mobile phase to promote the ionization of the substance to a certain extent. Therefore, during the experiment, it was considered to add different ionization-promoting agents to the mobile phase. The experiment found that when 0.1% formic acid was added to the mobile phase, the peak areas of samizuril and deacetyl samizuril were the largest and the sensitivity was the highest. When 10mmol / L ammonium formate or ammonium acetate was added to the mobile phase, the ionization efficiency was not significantly improved. Therefore, taking the above factors into consideration, the present invention finally adopted the ESI+ method for data acquisition, with acetonitrile and 0.1% formic acid aqueous solution as the mobile phase.

[0076] S5. Determination of samples

[0077] S51, Qualitative determination

[0078] The homogenized test sample was measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the qualitative analysis was performed by comparing the retention time of the test sample chromatogram with the retention time of the standard solution, and the characteristic ions of each chromatographic peak with the characteristic ions of each chromatographic peak of the standard solution of the corresponding concentration. The relative deviation of the retention time of the test sample and the standard solution is no more than 2.5%; the relative abundance of the characteristic ions of the test sample is consistent with the relative abundance of the standard solution of equivalent concentration. The standard chromatograms of samizuril and acetyl samizuril are shown in the figure. Figure 1 , 2 shown.

[0079] Table 1 Maximum allowable error of relative ion abundance in qualitative determination

[0080]

[0081] S52, quantitative determination

[0082] The ratio of the quantitative ion area of ​​the test sample and the standard solution is used as the single-point calibration quantification.

[0083] Take the test sample and the corresponding matrix-matched standard solution, take single-point or multi-point calibration, and quantify according to the external standard method. The response values ​​of samizuril and deacetylsamizuril in the matrix-matched standard solution and the test sample should be within the linear range of instrument detection.

[0084] S6. Blank test

[0085] After the test sample is tested, a homogenized blank sample is added to the liquid chromatography-tandem mass spectrometer to confirm that it does not contain substances that interfere with the components to be tested.

[0086] Calculation of the residual amount of samizuril or desacetyl samizuril:

[0087] The residue of the substance to be tested in the test sample is calculated according to the following formula:

[0088] Single point calibration or standard curve calibration is performed by:

[0089] As=aC s +b

[0090] The residual amount of samizuril or deacetyl samizuril was calculated according to the following formula:

[0091] Where:

[0092] X——the residual amount of samizuril or deacetyl samizuril in the sample, in micrograms per kilogram (μg / kg);

[0093] Cs——the concentration of samizuril or desacetyl samizuril in the matrix matching solution, in nanograms per milliliter (ng / mL);

[0094] C——the concentration of samizuril or deacetyl samizuril in the sample solution, in nanograms per milliliter (ng / mL);

[0095] As——peak area of ​​samizuril or desacetyl samizuril in matrix matching solution;

[0096] A——peak area of ​​samizuril or deacetyl samizuril in the sample solution;

[0097] V——volume of the sample after dilution with water, in milliliters (mL);

[0098] m – the mass of the sample, in grams (g).

[0099] Note: The blank value must be deducted from the calculation result. The measurement result is expressed as the arithmetic mean of parallel measurements, retaining three significant figures.

[0100] The invention performs gradient dilution on the standard solutions of samizuril and deacetyl samizuril, and respectively performs sampling and detection under optimized LC-MS / MS instrument conditions, and takes the sampling solution concentration when the signal-to-noise ratio S / N=3 as the minimum detection concentration, and takes the sampling solution concentration when the signal-to-noise ratio S / N=10 as the minimum quantitative concentration, and then calculates the quantitative limits of samizuril and deacetyl samizuril in muscle, sebum, liver and kidney according to the dilution multiple in the sample pretreatment process, and obtains that the quantitative limits of samizuril and deacetyl samizuril in muscle, sebum, liver and kidney are all 5 μg / kg. The detection requirements of samizuril in different animal-derived foods can be met.

[0101] Different concentrations of samizurin and deacetyl samizurin were added to chicken, pork, mutton and rabbit meat samples to evaluate the recovery rate and repeatability of the method. The addition levels used in the experiment were 5, 10 and 50 μg / kg, respectively. After mixing, the samples were treated according to the sample pretreatment method and then tested by LC-MS / MS. The average recovery rates of the three addition levels were calculated to be more than 78.6%, and the relative standard deviation RSD was less than 10%. These results show that the present invention has good recovery rate and repeatability at different addition levels.

[0102] In order to further illustrate the advantages of the determination method of samizuril and desacetyl samizuril of the present invention, the following comparative examples are provided for illustration.

[0103] As mentioned in the background technology, the matrix components of animal tissues are extremely complex and the matrix effect is obvious. The present invention is applied to the determination of samizuril and deacetyl samizuril in a variety of different animals. One of the key points is to find a better pretreatment method for different animal tissues, which not only affects the content and recovery process of the test components samizuril and deacetyl samizuril, but also overcomes the adverse effects caused by the matrix effect. Therefore, the selection of pretreatment methods and treatment reagents is more critical.

[0104] Comparative Example 1

[0105] The pre-treatment solvent of the sample is pure acetonitrile solvent:

[0106] Weigh 5g of the homogenized sample with an electronic balance, put it into a 50mL centrifuge tube, add 10mL of acetonitrile extract for extraction and separation, vortex with a vortexer for 30s, and then put the centrifuge tube into ultrasound for 20min. After the ultrasound is over, take it out and put it into a high-speed centrifuge, centrifuge it at 5000r / min for 5min, get the supernatant after centrifugation, and let it stand for purification. Take 1mL of the supernatant and pass it through the column into a 15mL centrifuge tube, use a pipette to accurately take 0.25mL of the supernatant after the column into a 2mL centrifuge tube with a cap, add 0.75mL of water to dilute, mix well, centrifuge at 12000r / min for 10min, take the upper layer of liquid and filter it through a 0.22μm filter membrane, and put it into a sample injection vial.

[0107] The samples treated as above were measured by LC-MS / MS for the recoveries of samizuril and acetyl samizuril in the samples. The recoveries of three different concentrations (5, 10 and 50 μg / kg) were tested.

[0108] Comparative Example 2

[0109] The pre-treatment solvent of the sample is anhydrous sodium sulfate + acetonitrile solvent:

[0110] Weigh 5g of the homogenized sample with an electronic balance, put it into a 50mL centrifuge tube, add 2-5g of anhydrous sodium sulfate, and then add 10mL of acetonitrile for extraction and separation. Vortex with a vortexer for 30s, then put the centrifuge tube into ultrasound for 20min. After the ultrasound is over, take it out and put it into a high-speed centrifuge, centrifuge it at 5000r / min for 5min, and get the supernatant after centrifugation. Let it stand for purification. Take 1mL of the supernatant and pass it through the column into a 15mL centrifuge tube. Use a pipette to accurately take 0.25mL of the supernatant after the column into a 2mL centrifuge tube with a cap, add 0.75mL of water to dilute, mix well, centrifuge it at 12000r / min for 10min, take the upper layer of liquid and filter it through a 0.22μm filter membrane, and put it into a sample injection vial.

[0111] The samples treated as above were measured by LC-MS / MS for the recoveries of samizuril and acetyl samizuril in the samples. The recoveries of three different concentrations (5, 10 and 50 μg / kg) were tested.

[0112] Figure 3 The figure is a comparison chart of the recovery rates of different pre-treatment solvents, comparing the effects of the pre-treatment of Comparative Examples 1 and 2 and the 10% Na2CO3 solution + acetonitrile solvent of the present invention on the recovery rates of different concentrations of samizuril and acetylsamizuril. It can be found that the treatment form of 10% Na2CO3 + acetonitrile of the present invention can obtain the highest recovery rate of samizuril and acetylsamizuril, indicating that the extraction degree of the residual samizuril and acetylsamizuril in the sample is the highest, which can effectively overcome the matrix effect.

[0113] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for determining samizuril and deacetyl samizuril, characterized in that: The steps include: S1. Preparation of standard solution The samizuril and desacetyl samizuril standards were prepared into 1.0 mg / mL samizuril and desacetyl samizuril standard stock solutions, and diluted into 10 μg / mL and 1.0 μg / mL standard working solutions for later use; S2. Sample pretreatment Take an appropriate amount of fresh or thawed blank sample or test muscle, sebum, liver, kidney tissue, mince and homogenize it before pre-treatment of the sample; S21. Weigh the homogenized tissue or blank sample and place it in a centrifuge tube with a lid. First, add 10% Na2CO3 solution and vortex to mix. Then, add 9 times the amount of acetonitrile as the 10% Na2CO3 solution and vortex to mix. Ultrasonicate, vortex, and centrifuge to obtain a supernatant. S22, take the supernatant and filter it through a rapid filtration purification column, collect the filtrate, accurately measure the extract, dilute it with 3 times of water, mix it, centrifuge it, take the upper liquid filter membrane to filter it, and take the filtrate to obtain the corresponding sample; S3. Standard curve drawing The standard curves of samizuril and deacetyl samizuril at different concentrations and the matrix matching standard curve are drawn respectively; the standard curve is prepared by diluting the samizuril and deacetyl samizuril standard working solutions into a series of standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL respectively; the matrix matching standard curve is prepared by diluting the samizuril and deacetyl samizuril standard working solutions into a series of matrix matching standard solutions of 1, 2, 5, 10, 20, 50 and 100 ng / mL respectively using the blank sample pre-treated in step S2; S4. Liquid chromatography-mass spectrometry conditions Liquid chromatography-mass spectrometry was used for detection and multiple ion reaction monitoring was used for analysis; The liquid chromatography column is a C18 column, the mobile phase is acetonitrile + 0.1% formic acid aqueous solution, the flow rate is 0.3mL / min; the column temperature is 30°C, the injection volume is 10μL; the elution program is: 0-1min, 23%A+77%B; 2-3min, 40%A+60%B; 4-5min, 23%A+77%B; A is acetonitrile, and B is 0.1% formic acid aqueous solution; Mass spectrometer voltage: 5500V, curtain gas: 30psi, IS voltage: 5500V, ion source temperature: 550℃, nebulizer gas: 55psi, auxiliary gas: 55psi; multiple ion reaction monitoring parameters: samizurin parent ion 353.1m / z; daughter ion 147.1*m / z, collision energy 24V; daughter ion 108.1m / z, collision energy 24V; deacetyl samizurin parent ion 311.2m / z, daughter ion 79.9*m / z, collision energy 34V; daughter ion 108.1m / z, collision energy 18V; S5. Determination of samples The homogenized test sample is measured by liquid chromatography-tandem mass spectrometry, and the retention time of the test sample chromatogram is compared with the retention time of the standard solution, and the characteristic ions of each chromatographic peak are compared with the characteristic ions of each chromatographic peak of the standard solution of the corresponding concentration for qualitative analysis; Then take the test sample and the corresponding matrix matching standard solution, take single point or multi-point calibration, and perform quantitative measurement according to the external standard method.

2. The method for determining samizuril and deacetyl samizuril according to claim 1, characterized in that: The residue of the test sample is calculated as follows: Single point calibration or standard curve calibration is performed by: As=aC s + b The residual amount of samizuril or deacetyl samizuril was calculated according to the following formula: Where: X——the residual amount of samizuril or deacetyl samizuril in the sample, in micrograms per kilogram (μg / kg); Cs——the concentration of samizuril or desacetyl samizuril in the matrix matching solution, in nanograms per milliliter (ng / mL); C——the concentration of samizuril or deacetyl samizuril in the sample solution, in nanograms per milliliter (ng / mL); As——peak area of ​​samizuril or desacetyl samizuril in matrix matching solution; A——peak area of ​​samizuril or deacetyl samizuril in the sample solution; V——volume of the sample after dilution with water, in milliliters (mL); m – the mass of the sample, in grams (g).

3. The method for determining samizuril and deacetyl samizuril according to claim 1, characterized in that: In step S1, the solvent for preparing the standard stock solution and standard working solution of samizuril and deacetylsamizuril is methanol.

4. The method for determining samizuril and deacetyl samizuril according to claim 1, characterized in that: The rapid filtration purification column in step S2 is CAFS Clean-up Column (200 mg, 3 mL).

5. The method for determining samizuril and deacetyl samizuril according to claim 1 or 4, characterized in that: The pretreatment conditions of the sample in step S2 are: S21. Weigh 5.0 g (accurate to ±0.02 g) of the homogenized tissue or blank sample into a 50 mL centrifuge tube with a lid, add 1 mL of 10% Na2CO3 solution, vortex to mix, add 10 mL of acetonitrile, vortex to mix, sonicate for 30 min, vortex, and centrifuge at 5000 r / min for 10 min to obtain the supernatant; S22. Take 1 mL of the supernatant, filter it through a rapid filtration purification column, collect the filtrate, accurately measure 0.25 mL of the extract into a 2 mL centrifuge tube with a cap, dilute it with 0.75 mL of water, mix well, centrifuge at 12000 r / min for 10 min, take the upper layer of liquid and filter it through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain the corresponding sample.

6. The method for determining samizuril and deacetyl samizuril according to claim 1, characterized in that: Step S5 also includes step S6: after the test sample is tested, a homogenized blank sample is added to the liquid chromatography-tandem mass spectrometer to confirm that it does not contain substances that interfere with the components to be tested.

7. The method for determining samizuril and deacetyl samizuril according to claim 1, characterized in that: In step S4, the relative deviation of the retention time of the test sample and the standard solution is no more than 2.5%; the relative abundance of characteristic ions of the test sample is consistent with the relative abundance of the standard solution with equivalent concentration.

8. The method for determining samizuril and deacetyl samizuril according to claim 1 or 7, characterized in that: When the relative ion abundance is greater than 50%, the maximum allowable deviation is ±20%; when the relative ion abundance is greater than 20% to 50%, the maximum allowable deviation is ±25%; when the relative ion abundance is greater than 10% to 20%, the maximum allowable deviation is ±30%; when the relative ion abundance is ≤10%, the maximum allowable deviation is ±50%.

9. Use of the method for determining samizuril and deacetylsamizuril as claimed in any one of claims 1 to 8 for quantitative detection of samizuril and deacetylsamizuril in animal-derived food.

10. The use according to claim 8, characterized in that Animal-derived foods include: muscle, sebum, liver and kidney of chicken, pig, sheep and rabbit.

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