Synchronous determination method for anthelmintics in feed

Abstract

The invention relates to a method for synchronously determining an anthelmintic in a feed. The anthelmintic comprises three kinds of nitro-iodonitrile, rafoxanide and closantel. Comprising the following steps: S1, extracting a to-be-detected feed sample with acetonitrile to obtain a to-be-purified solution; s2, purifying the to-be-purified solution by adopting a Prime HLB solid-phase extraction column, collecting all effluent, fixing the volume by using water, uniformly mixing, and passing through a microfiltration membrane to obtain a to-be-purified sample solution. According to the method, acetonitrile is adopted to extract the anthelmintic in the feed, so that the recovery rate is high, and the repeatability is good; a Prime HLB solid-phase extraction small column is adopted to purify a sample, most lipids and other impurities in the feed can be removed, the method has good selectivity on target compounds, operation is easy and convenient, the steps of activation, balancing, impurity leaching and the like are not needed, the detection time is greatly shortened, and the detection efficiency is improved. The method fills the blank of the method for detecting the nitro-iododinitrile, the rafoxanide and the closantel in the feed at present, and can provide reliable technical support for feed quality safety monitoring.

Description

Technical Field

[0001] The invention relates to the field of residual drug detection in feed, and in particular to a synchronous determination method of anti-helminth drugs in feed. Background Art

[0002] Fasciola hepatica is a zoonotic foodborne parasite that mainly parasitizes the bile duct of mammals. The toxins it secretes have hemolytic effects. After infection, it can cause liver damage and bleeding, posing a huge threat to animal husbandry and human health. Nitrofenoxanil, iodoxanil, and closantel are phenolic and salicylic acid anilide antihelmintics. This type of drug is mainly used to kill liver worms, roundworms, tapeworms, blood hairworms, etc. Because of its small dosage, convenience, and high efficiency, it is widely used in animal husbandry, especially herbivorous animal husbandry. However, the results of animal experimental studies show that iodoxanil has obvious embryotoxicity, closantel has certain toxic and side effects on the liver and kidneys, and high doses of nitrofenoxanil can cause damage to the heart, nerves, muscles, and respiration. In order to ensure consumer health and the quality and safety of livestock products, many countries have established maximum residue limits for nitrofenoxanil, iodoxanil, and closantel in animal food and milk. my country also has strict control over veterinary drug residues. GB31650-2019 "National Food Safety Standard Maximum Residues of Veterinary Drugs in Food" stipulates that the residue limits of nitrothion, iodofenoxamide, and closantel in milk are 20, 10, and 45 μg / kg, respectively.

[0003] Feed quality and safety are the premise and fundamental guarantee for animal food safety. The above three anthelmintics are very easy to be mixed into the feed when used in herbivorous animal husbandry. This is likely to be enriched in animal tissues through food chain transmission, environmental accumulation, and contaminated feed, causing food quality and safety risks and posing a threat to consumer health.

[0004] However, after reviewing the literature, it was found that there are few studies on the detection methods of anthelmintics in the existing technology. The existing studies are mainly aimed at the detection of animal tissues and dairy products, not including feed matrices, and the types of drugs detected are also relatively small. In other words, to date, there is no detection standard for nitrothion, iodine sulfoxide, and closantel in feed in my country, and there is no relevant literature report on the simultaneous detection of nitrothion, iodine sulfoxide, and closantel in feed.

[0005] Therefore, in view of the lack of existing methods for determining antihelminthic drugs in feed and the need for feed quality and safety monitoring, it is necessary to establish rapid, sensitive and accurate sample pretreatment technology and instrument detection methods for the above three antihelminthic drugs in feed. Summary of the invention

[0006] In view of this, the present invention provides a synchronous determination method of anthelmintics in feed, which comprises the following steps: adding acetonitrile to the feed to be tested, extracting nitrofosinate, iodoxime and closantel, purifying the sample extract by using a Prime HLB solid phase extraction column after nitrogen blowing and concentration, and detecting the sample by liquid chromatography-tandem mass spectrometry (LC-MS / MS), thereby establishing an analysis method for nitrofosinate, iodoxime and closantel in feed, providing technical support for feed quality and safety monitoring, and having important research significance and practical application value.

[0007] The invention discloses a synchronous determination method of anthelmintics in feed, wherein the anthelmintics are nitrothione, closantel and closantel. The method comprises the following steps:

[0008] S1, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified;

[0009] S2, purifying the solution to be purified by using a Prime HLB solid phase extraction column, collecting all the effluent, making up to volume with water, mixing and then passing through a microporous filter membrane to obtain a sample solution to be tested.

[0010] In the present invention, the drug to be tested in the feed sample is firstly diluted out by acetonitrile extraction, and at the same time, 95% of common matrix interferences such as phospholipids and proteins in complex samples can be removed by Prime HLB solid phase extraction cartridges using water-wettable strong hydrophilic polymers as adsorbents. Moreover, the operation process is simpler, and a higher recovery rate can be obtained without activation and equilibration steps, so that the present invention constructs an analytical method for three anti-helminth drugs in feed, provides technical support for feed quality and safety monitoring, and has important research significance and practical application value.

[0011] In the method disclosed in the present invention, before step S1, it also includes step S0, preparing a matrix-matching series of standard working solutions based on the corresponding standard products of each drug to be tested; after step S2, it also includes step S3, subjecting the matrix-matching series of standard working solutions and the test sample solution to liquid chromatography-tandem mass spectrometry, drawing a working curve with the concentration of the matrix-matching series of standard working solutions as the horizontal coordinate and the measured chromatographic peak area of ​​each drug to be tested as the vertical coordinate, and quantifying each drug to be tested using the external standard method.

[0012] In the method disclosed in the present invention, in step S3, in the step of liquid chromatography-tandem mass spectrometry, the chromatographic column of the liquid chromatography is C 18 The column has a mobile phase system of mobile phase A and mobile phase B, wherein mobile phase A is formic acid aqueous solution, mobile phase B is methanol, and gradient elution is adopted.

[0013] In the method disclosed in the present invention, in the liquid chromatography, the flow rate of the mobile phase system is 0.1-0.5 mL / min, and the gradient elution program of the mobile phase system is:

[0014] 0.0~1.0min, the volume percentage of the mobile phase A is 65%;

[0015] From 1.0 to 4.0 min, the volume percentage of the mobile phase A is uniformly reduced from 65% to 5%;

[0016] 4.0~7.0min, the volume percentage of the mobile phase A is 5%;

[0017] 7.0-7.1 min, the volume percentage of the mobile phase A is increased uniformly from 5% to 65%;

[0018] 7.1-10.0 min, the volume percentage of the mobile phase A is 65%.

[0019] The initial methanol ratio is set to 35%, which appropriately increases the organic phase ratio in the mobile phase to shorten the sample detection time and solve the problem of late peak time due to the small polarity of the anthelmintic; 1-4min, the methanol ratio gradually increases, which can make each test object flow out from the chromatographic column in sequence; when 4-7min, the methanol ratio is 95%, and the non-polar impurities in the sample can flow out as much as possible with the help of a high proportion of organic phase, avoiding accumulation in the chromatographic column and contaminating the chromatographic column; and when 7-10min, it returns to the initial ratio, and after 3min of equilibrium, the next injection of the sample can be made. Based on the entire gradient elution program set by the present invention, it is possible to ensure that the three anthelmintic drugs of nitroaniline, iodine ether sulfonamide, and closantel iodine sulfonamide peak at the right time and obtain a good peak shape, while avoiding the accumulation of other impurities in the chromatographic column, ensuring that good repeatability can still be obtained when repeatedly injecting samples.

[0020] In the method disclosed in the present invention, mobile phase A is 0.1% formic acid aqueous solution.

[0021] In the method disclosed in the present invention, in the liquid chromatography, the column oven temperature is 40° C. and the injection volume is 5 μL.

[0022] In the method disclosed in the present invention, the measurement conditions of mass spectrometry in liquid chromatography-tandem mass spectrometry are: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is negative ion scanning, the electrospray voltage is 4.5Kv, and the desolvation temperature is 450°C.

[0023] In the method disclosed in the present invention, in the step S0, an appropriate amount of a mixed standard solution of nitrothione, iodine ether sulfamethoxazole and closantel iodine sulfamethoxazole is taken, and a blank matrix solution is used to prepare a matrix-matched series of standard working solutions with concentrations of 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 500 ng / mL, respectively.

[0024] In the method disclosed in the present invention, in step S1, it includes:

[0025] Step S11, taking 1-5 g of the feed sample to be tested, placing it in a 50 mL centrifuge tube, adding 5-15 mL of acetonitrile, ultrasonically extracting for 15 min, centrifuging at 4000 r / min for 3-5 min, and taking the supernatant into another centrifuge tube;

[0026] Step S12, the lower layer residue is extracted again with 5-15 mL of acetonitrile, and the supernatant is taken;

[0027] Step S13, combining the supernatants obtained in step S11 and step S12, vortexing, adding anhydrous magnesium sulfate and anhydrous sodium acetate, vortexing and shaking, to obtain a final supernatant;

[0028] Step S14, the supernatant finally obtained is blown to about 2-5 mL with nitrogen in a 40° C. water bath, water is added to make the volume to 5 mL, and mixed to obtain a solution to be purified.

[0029] Among them, preferably, in step S11, 5 g of the feed sample to be tested is taken, placed in a 50 mL centrifuge tube, 10 mL of acetonitrile is added, ultrasonic extraction is performed for 15 min, centrifuged at 4000 r / min for 5 min, and the supernatant is taken into another centrifuge tube; in step S12, the lower layer residue is repeatedly extracted with 10 mL of acetonitrile again, and the supernatant is taken; and then in step S14, the final supernatant is blown to about 4 mL in a 40°C water bath with nitrogen, water is added to make the volume to 5 mL, and mixed to obtain a solution to be purified.

[0030] Since the sample to be tested is pretreated with acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate, the interfering substances such as fat, protein, pigment in the supernatant obtained in the end are greatly removed. At the same time, the Prime HLB solid phase extraction column is used for purification, which can further remove 95% of common matrix interfering substances in complex samples, such as phospholipids, proteins, etc., so that the residues of nitroaniline, iodine ether sulfonamide and closantel iodine sulfonamide can be released to the greatest extent. That is, by pretreating the sample to be tested with acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate in the present invention and by means of the Prime HLB solid phase extraction column, the loss of the analyte can be reduced to the greatest extent while removing impurities to the greatest extent.

[0031] In the method disclosed in the present invention, in step S3, the liquid to be purified is completely passed through the Prime HLB column, all the effluent is collected, the volume is made up to 10 mL with water, mixed, and passed through a 0.22 μm microporous filter membrane to obtain a sample solution to be tested.

[0032] Beneficial effects: In the method of the present invention, acetonitrile extraction, Prime HLB solid phase extraction column and liquid chromatography-tandem mass spectrometry form a close whole, and have a synergistic effect with each other, which is specifically embodied in that acetonitrile is used to extract three anthelmintics (nitroaniline, iodine ether sulfonamide, closantel) in feed, with high recovery rate and good repeatability; after nitrogen blowing and concentration, the Prime HLB solid phase extraction column is used to purify the sample, which can remove most of the lipids and other impurities in the feed, has good selectivity for the target compound, and is simple to operate, does not require activation, equilibration and eluting impurities and other steps, and greatly shortens the detection time; the three anthelmintics are simultaneously quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the detection sensitivity, accuracy and stability are all high, which fills the gap in the current detection method for nitroaniline, iodine ether sulfonamide and closantel in feed, and can provide reliable technical support for feed quality and safety monitoring.

[0033] The method provided by the present invention has good extraction and purification effects, high sensitivity, good accuracy and stability, and is easy to operate, greatly shortens the detection time, and is suitable for high-throughput detection of three anthelmintic drug residues of nitrothionine, iodoxime and closantel in feed samples.

[0034] The method for synchronous determination of anthelmintics in feed of the present invention is disclosed in detail below in conjunction with the embodiments shown in the accompanying drawings and the reference numerals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The present invention is a flowchart of the steps of the synchronous determination method of anthelmintics in feed disclosed in the present invention.

[0036] Figure 2 It is the chemical structural formula of nitroiodine, iodofenoxamide and closantel.

[0037] Figure 3 The chromatograms are shown when water and acetonitrile are used as mobile phases.

[0038] Figure 4 Chromatogram of 0.1% formic acid in water and acetonitrile as the mobile phase.

[0039] Figure 5 Chromatogram of 0.1% formic acid in water and methanol as the mobile phase.

[0040] Figure 6 Extracted ion chromatograms with 0.1% formic acid in water and methanol as the mobile phase.

[0041] Figure 7 Chromatogram of blank feed matrix after Prime HLB cleanup.

[0042] Figure 8 Added sample chromatogram for Prime HLB purified feed. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0044] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0045] The present invention provides a synchronous determination method that can be accurate, fast and simple for the determination of three anthelmintic drug residues of nitroaniline, iodine sulfonamide and closantel, that is, firstly, acetonitrile is added to the feed to be tested, nitroaniline, iodine sulfonamide and closantel are extracted, and the sample is purified by Prime HLB solid phase extraction column after nitrogen blowing and concentration, and detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and quantitatively measured by matrix matching standard solution external standard method. With the help of the method of the present invention, not only the extraction and purification effects are good, the sensitivity is high, the accuracy and stability are good, and the operation is simple, the detection time is greatly shortened, and it is suitable for high-throughput detection of three anthelmintic drug residues of nitroaniline, iodine sulfonamide and closantel in feed samples.

[0046] In the specific embodiment of the present invention, the following instruments and reagents are used: liquid chromatography-mass spectrometry (SCIEX 5500+, SCIEX, USA), electronic analytical balance (ME204E, Mettler Toledo, Switzerland), centrifuge (Hettich, Germany), nitrogen blowdown instrument (Beijing Tongtai Lian Technology Development Co., Ltd.). Nitroiodine phenol nitrile (purity>95%), iodoether sulfonamide (purity>95%), closantel iodine sulfonamide (purity>95%), chromatographically pure methanol, acetonitrile and formic acid. Prime HLB solid phase extraction cartridge (200 mg / 6 mL). Specific embodiment 1

[0048] In this embodiment, the method for synchronously determining three anthelmintics in feed comprises the following steps:

[0049] S0, prepare a matrix-matched series of standard working solutions based on the corresponding standards of each drug to be tested;

[0050] S1, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified;

[0051] S2, purifying the solution to be purified by using a Prime HLB solid phase extraction column, collecting all the effluent, making up to volume with water, mixing and passing through a microporous filter membrane to obtain a sample solution to be tested;

[0052] S3, subjecting the matrix-matching series standard working solutions and the test sample solutions to liquid chromatography-tandem mass spectrometry, plotting a working curve with the concentration of the matrix-matching series standard working solutions as the abscissa and the measured chromatographic peak area of ​​each test drug as the ordinate, and quantifying each test drug using the external standard method.

[0053] Specifically, in step S0, firstly, a 1 mg / mL standard stock solution is prepared, specifically, 10.0 mg of three anthelmintic drug standards of nitroaniline, iodoxanamide and closantel are accurately weighed in a 10 mL volumetric flask, dissolved to the scale with acetonitrile, mixed, and stored below -18°C. The specific specifications of the three anthelmintic drug standards are shown in Table 1, and the structural formula is shown in Figure 2 .

[0054] Table 1 Specific specifications of three anti-helminthic drugs

[0055] English name Chinese name Molecular formula CSA Number Nitroxinil Nitroiodine <![CDATA[C 7 H 3 IN 2 O 3 ]]> 1689-89-0 Rafoxanide Iodothiocarbamide <![CDATA[C 19 H 11 Cl 2 I 2 NO 3 ]]> 22662-39-1 Closantel Closantel <![CDATA[C 22 H 14 Cl 2 I 2 N 2 O 2 ]]> 57808-65-8

[0056] In step S0, a mixed standard intermediate solution (100 μg / mL) is then prepared: 1 mL of each of the above standard stock solutions is accurately transferred into a 10 mL volumetric flask, diluted to the mark with acetonitrile, and stored at 2° C. to 8° C.

[0057] Next, prepare the blank matrix solution: weigh 5.0g of blank feed (without three anti-helminthic drugs, accurate to 0.01g), place in a 50mL centrifuge tube, add 10mL of acetonitrile, vortex mix for 1min, ultrasonically extract for 15min, centrifuge at 4000r / min for 5min, and take the supernatant into another centrifuge tube. Repeat the extraction of the lower layer residue with 10mL of acetonitrile once, combine the supernatants, blow nitrogen gas to about 4mL in a 40℃ water bath, add water to make up to 5mL, mix, purify through Prime HLB column, collect all the effluent, make up to 10mL with water, mix, and filter through 0.22μm microporous filter membrane to obtain the blank matrix solution.

[0058] The mixed standard intermediate solution was then diluted stepwise with blank matrix solution to obtain a matrix-matched standard series working solutions with concentrations of 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL, respectively.

[0059] In step S1, 5 g of the feed sample to be tested is accurately weighed, placed in a 50 mL centrifuge tube, 10 mL of acetonitrile is added, vortexed for 1 min, ultrasonically extracted for 15 min, centrifuged at 4000 r / min for 5 min, and the supernatant is taken into another centrifuge tube. The lower layer residue is repeatedly extracted once with 10 mL of acetonitrile, the supernatants are combined, anhydrous magnesium sulfate and anhydrous sodium acetate are added after vortexing, and vortexed and shaken to obtain the final supernatant, and the final supernatant is blown to about 4 mL in a 40°C water bath with nitrogen, and water is added to make up to 5 mL, mixed, and used as the liquid to be purified.

[0060] The actual technical problem to be solved in this application is to provide a method for determining the residual matrix effect of three anthelmintics, nitroanilide, iodine sulfonamide and closantel in feed by a liquid phase-tandem mass spectrometry scheme. In the field of drug detection, the elimination method of the matrix effect of different detection matrices is different. The detection object of the present invention is animal feed, specifically the determination of the residual three anthelmintics, nitroanilide, iodine sulfonamide and closantel in animal feed, and the animal feed is rich in plant protein, plant oil and starch, etc., which have an adverse effect on the determination. Therefore, in step S1, a series of pre-treatment means are used to eliminate the influence of the matrix effect of the feed matrix during drug detection. Specifically, acetonitrile is used as an extractant in the present invention, and anhydrous magnesium sulfate and anhydrous sodium acetate are added for pretreatment, so that the obtained supernatant is not free of dry meat substances such as lipids, proteins, pigments, etc., so that the three anthelmintics, nitroanilide, iodine sulfonamide and closantel, are released to the greatest extent.

[0061] Moreover, after extraction, in the next purification step, Prime HLB solid phase extraction cartridges are used for purification, which can further remove most of the lipids and other impurities in the feed while further eliminating the matrix effect of drug detection in the feed matrix. At the same time, it has good selectivity for the target compound, and is easy to operate, without the steps of activation, equilibration and elution of impurities, which greatly shortens the detection time. Specifically, in step S2, all the liquid to be purified is passed through the PrimeHLB solid phase extraction cartridge, and all the effluent is collected, diluted to 10mL with water, mixed, and passed through a 0.22μm microporous filter membrane as the sample solution to be tested. Different purification columns have different selectivities for different analytes. In the determination of three anthelmintics, namely, nitrothionine, iodoxime and closantel, the Prime HLB is set in the present invention. On the premise of achieving the above-mentioned effects, it is not only possible to reduce the loss of the analytes to the greatest extent and obtain a higher recovery rate, but also the three anthelmintics, namely, nitrothionine, iodoxime and closantel, can be completely separated, that is, it further has a good separation degree, so that it can have a peak with good separation degree when performing mass spectrometry determination in the next step.

[0062] On the basis of the above steps S0 to S2, step S3 is performed, that is, liquid chromatography-tandem mass spectrometry analysis is performed.

[0063] Specifically, the liquid chromatography conditions include: the chromatographic column is a C18 column, the column oven temperature is 40°C, the injection volume is 5 μL, the mobile phase system is mobile phase A and mobile phase B, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is methanol, the flow rate of the mobile phase system is 0.3 mL / min, and the gradient elution program is shown in Table 2.

[0064] Table 2 Gradient elution program of mobile phase system

[0065]

[0066]

[0067] That is, the elution procedure of the mobile phase system in the present invention is:

[0068] 0.0~1.0min, the volume percentage of the mobile phase A is 65%;

[0069] From 1.0 to 4.0 min, the volume percentage of the mobile phase A is uniformly reduced from 65% to 5%;

[0070] 4.0~7.0min, the volume percentage of the mobile phase A is 5%;

[0071] 7.0-7.1 min, the volume percentage of the mobile phase A is increased uniformly from 5% to 65%;

[0072] 7.1-10.0 min, the volume percentage of the mobile phase A is 65%.

[0073] In addition, the mass spectrometry detection conditions include: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is a negative ion mode, the electrospray voltage is 4.5Kv, the desolvation temperature is 450°C, the curtain gas is 35psi, the spray gas is 50psi, and the collision gas is 9psi. The multiple reaction monitoring ion pairs, cone voltage and collision energy in the present invention are shown in Table 3.

[0074] Table 3 Multiple reaction monitoring ion pairs, cone voltage and collision energy of three anthelmintics

[0075]

[0076] Then, the matrix-matched standard series working solutions and the test sample solutions were subjected to liquid chromatography tandem mass spectrometry detection to obtain the peak area of ​​each anthelmintic drug, and the concentration of the series of matrix standard working solutions was used as the abscissa, and the peak area of ​​the chromatographic peak of the quantitative ion of the anthelmintic drug was used as the ordinate to draw a standard curve to obtain the standard curve regression equation shown in Table 4, and the sample content was quantified.

[0077] Table 4 Standard curve regression equations for anthelmintic drugs

[0078]

[0079]

[0080] The results showed that the detection method provided by the present invention had a good linear relationship between the three anthelmintics in the range of 5 ng / mL to 500 ng / mL, and the correlation coefficients (r) were all greater than 0.995.

[0081] Optimization of the mobile phase system in specific embodiment 2

[0082] In order to verify the advantages of using water and methanol as the mobile phase system for the determination of the three anthelmintics in the feed of the present invention, a specific embodiment 2 is specially designed. In this embodiment, the three anthelmintics standard solutions are detected on the liquid chromatography tandem mass spectrometry according to the conditions of Example 1, and the mobile phase systems composed of mobile phase A and mobile phase B are water and acetonitrile, 0.1% formic acid water and acetonitrile, and 0.1% formic acid water and methanol, respectively. Figure 3 This is the chromatogram when water and acetonitrile are used as mobile phases. Figure 4 The chromatogram when 0.1% formic acid water and acetonitrile are used as the mobile phase. Figure 5 The chromatogram when 0.1% formic acid water and methanol are used as the mobile phase. Figure 6 Extracted ion chromatograms with 0.1% formic acid in water and methanol as the mobile phase.

[0083] It can be seen that, with 0.1% formic acid water and methanol as the mobile phase, the three anthelmintics have good separation, symmetrical peak shape, high response value, and meet the detection requirements. That is, because 0.1% formic acid water and methanol are set as the mobile phase in the present invention, the three anthelmintics can achieve good separation, and the peak shape is symmetrical, which satisfies the detection requirements very well.

[0084] Specific Example 3 Selection of sample solvent

[0085] In order to verify the advantages of using acetonitrile as a solvent for the determination of three anthelmintics in feed in the present invention, a specific embodiment 3 is specially designed. In this embodiment:

[0086] (1) First, prepare the spiked sample: weigh 5.0 g of blank compound feed (accurate to 0.01 g), place it in a 50 mL centrifuge tube, and add 20 μL of the mixed standard intermediate solution of Example 1 to obtain a spiked sample.

[0087] (2) Then, the test solution was measured according to the extraction and purification steps and liquid chromatography tandem mass spectrometry detection conditions of Example 1, except that the extraction solvents were methanol, acetonitrile, and acetonitrile + acetone (6:4) solution. The recovery rates of the three anthelmintics are shown in Table 5.

[0088] Table 5 Recovery of anthelmintics under different extraction solvents (%)

[0089] Extraction solvent type Nitroiodine Iodothiocarbamide Closantel Methanol 68.3 71.2 79.4 Acetonitrile 89.5 92.2 86.9 Acetonitrile + acetone (6:4) 81.2 85.4 77.6

[0090] It can be seen that when acetonitrile was used as the extraction solvent, the extraction recoveries of the three anthelmintics were the highest, meeting the requirements for residue detection.

[0091] Specific Example 4 Selection of Purification Conditions

[0092] The spiked samples were prepared in the manner of Example 3, and the test solution was measured on the machine according to the extraction and purification steps and liquid chromatography tandem mass spectrometry detection conditions of Example 1, except that HLB, Prime HLB and C18 solid phase extraction columns were used in the purification process. The recovery rates of the three anthelmintics under different purification conditions are shown in Table 6.

[0093] Table 6 Recovery of anthelmintics under different purification conditions (%)

[0094] Purification column type Nitroiodine Iodothiocarbamide Closantel HLB 69.3 65.4 61.9 C18 71.2 73.6 70.8 Prime HLB 90.6 92.4 87.3

[0095] It can be seen that the recovery rate and purification effect of Prime HLB solid phase extraction cartridges are satisfactory. Compared with ordinary solid phase extraction cartridges, Prime HLB cartridges omit the steps of activation and elution during operation, which is easy and quick to operate and greatly shortens the detection time. The blank feed matrix chromatogram purified by Prime HLB is shown in Figure 7 , the chromatogram of the feed supplement sample purified by Prime HLB is shown in Figure 8 .

[0096] Specific Example 5 Method Sensitivity Determination

[0097] The detection limit and quantification limit of the three anthelmintics were determined by adding spikes to a blank feed matrix. The specific steps were as follows: the mixed standard intermediate solution prepared in Example 1 was added to a blank feed matrix (containing no anthelmintics), and extraction, purification and machine determination were performed according to Example 1. The detection limit was determined by a signal-to-noise ratio S / N ≥ 3, and the quantification limit was determined by a signal-to-noise ratio S / N ≥ 10. The detection limits of the three anthelmintics were all 2.0 μg / kg, and the quantification limits were all 5.0 μg / kg.

[0098] Specific Example 6 Accuracy and Precision Determination

[0099] Spiked samples with concentrations of 5.0 μg / kg, 25.0 μg / kg, and 100.0 μg / kg were prepared respectively, and 6 parallel samples were set for each concentration point. The extraction, purification, and measurement were performed according to Example 1. The recovery and precision results are shown in Table 7.

[0100] Table 7 Accuracy and precision test results

[0101]

[0102] It can be seen that the average recovery rate of spiked feed samples is between 86.0% and 92.5% (n=6), and the relative standard deviation of precision is ≤6.43%. The method provided by the present invention has high sensitivity, good accuracy and reproducibility, and is suitable for simultaneous detection of three anthelmintics in feed samples.

[0103] Based on the above experimental results, it can be seen that the detection limit of the detection method provided by the present invention is 2.0 μg / kg, the quantitative limit is 5.0 μg / kg, the average recovery rate is between 86.0% and 92.5% (n=6), and the precision relative standard deviation is ≤6.43%. That is, compared with the prior art, the detection method of the present invention can realize the quantitative detection of the three anthelmintics of nitroaniline, iodine ether sulfonamide and closantel, and has a lower detection limit and quantitative limit, which is more conducive to the detection of the three anthelmintics of nitroaniline, iodine ether sulfonamide and closantel in animal feed and the accurate quantification of low content.

[0104] In summary, the method provided by the present invention has good extraction and purification effects, high sensitivity, good accuracy and stability, and is easy to operate, greatly shortening the detection time, and is suitable for high-throughput detection of three anthelmintic drug residues of nitrothionil, iodoxamide and closantel in feed samples.

[0105] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein, for example. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for synchronous determination of anthelmintics in feed, characterized in that: The anthelmintics are nitrothionine, closantel and closantel; the method comprises the following steps: S1, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified; S2, purifying the solution to be purified by using a Prime HLB solid phase extraction column, collecting all the effluent, making up to volume with water, mixing and then passing through a microporous filter membrane to obtain a sample solution to be tested.

2. The method for synchronous determination of anthelmintics in feed according to claim 1, characterized in that: Before step S1, the method further includes step S0, preparing a matrix-matched series of standard working solutions based on the corresponding standard products of each drug to be tested; After step S2, the method further includes step S3, wherein the matrix-matching series standard working solutions and the test sample solutions are subjected to liquid chromatography-tandem mass spectrometry, a working curve is drawn with the concentration of the matrix-matching series standard working solutions as the abscissa and the chromatographic peak area of ​​each drug to be tested as the ordinate, and each drug to be tested is quantified using the external standard method.

3. The method for synchronous determination of anthelmintics in feed according to claim 2, characterized in that: In step S3, in the step of liquid chromatography-tandem mass spectrometry, the chromatographic column of the liquid chromatography is C 18 The column has a mobile phase system of mobile phase A and mobile phase B, wherein mobile phase A is formic acid aqueous solution, mobile phase B is methanol, and gradient elution is adopted.

4. The method for synchronous determination of anthelmintics in feed according to claim 3, characterized in that: In the liquid chromatography, the flow rate of the mobile phase system is 0.1-0.5 mL / min, and the gradient elution program of the mobile phase system is: 0.0~1.0min, the volume percentage of the mobile phase A is 65%; From 1.0 to 4.0 min, the volume percentage of the mobile phase A is uniformly reduced from 65% to 5%; 4.0~7.0min, the volume percentage of the mobile phase A is 5%; 7.0-7.1 min, the volume percentage of the mobile phase A is increased uniformly from 5% to 65%; 7.1-10.0 min, the volume percentage of the mobile phase A is 65%.

5. The method for synchronous determination of anthelmintics in feed according to claim 4, characterized in that: The mobile phase A is 0.1% formic acid aqueous solution.

6. The method for synchronous determination of anthelmintics in feed according to claim 4, characterized in that: In the liquid chromatography, the column oven temperature was 40 °C and the injection volume was 5 μL.

7. The method for synchronous determination of anthelmintics in feed according to claim 4, characterized in that: The mass spectrometry measurement conditions in the liquid chromatography-tandem mass spectrometry are as follows: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is negative ion scanning, the electrospray voltage is 4.5 Kv, and the desolvation temperature is 450°C.

8. The method for synchronous determination of anthelmintics in feed according to any one of claims 2 to 7, characterized in that: In the step S0, an appropriate amount of a mixed standard solution of nitrothione, iodine ether sulfamethoxazole and closantel iodine sulfamethoxazole is taken, and a blank matrix solution is used to prepare a matrix-matched series of standard working solutions with concentrations of 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 500 ng / mL, respectively.

9. The method for synchronous determination of anthelmintics in feed according to any one of claims 1 to 7, characterized in that: In the step S1, it includes: Step S11, taking 1-5 g of the feed sample to be tested, placing it in a 50 mL centrifuge tube, adding 5-15 mL of acetonitrile, ultrasonically extracting for 15 min, centrifuging at 4000 r / min for 3-5 min, and taking the supernatant into another centrifuge tube; Step S12, the lower layer residue is extracted again with 5-15 mL of acetonitrile, and the supernatant is taken; Step S13, combining the supernatants obtained in step S11 and step S12, vortexing, adding anhydrous magnesium sulfate and anhydrous sodium acetate, vortexing and shaking, to obtain a final supernatant; Step S14, the supernatant finally obtained is blown to about 2-5 mL with nitrogen in a 40° C. water bath, water is added to make the volume to 5 mL, and mixed to obtain a solution to be purified.

10. The method for synchronous determination of anthelmintics in feed according to any one of claims 2 to 7, characterized in that: In step S3, the liquid to be purified is passed through the Prime HLB column, all the effluent is collected, the volume is made up to 10 mL with water, mixed, and filtered through a 0.22 μm microporous filter membrane to obtain a sample solution to be tested.

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