CN107843678B

CN107843678B - A method for detecting the content of thiamphenicol in animal feed by thin layer chromatography combined with high performance liquid chromatography

Info

Publication number: CN107843678B
Application number: CN201711278289.3A
Authority: CN
Inventors: 杨波; 杨金晶; 孙桂芝
Original assignee: Wuhan Bioengineering Institute
Current assignee: Wuhan Bioengineering Institute
Priority date: 2017-12-06
Filing date: 2017-12-06
Publication date: 2020-04-14
Anticipated expiration: 2037-12-06
Also published as: CN107843678A

Abstract

The invention discloses a method for detecting the content of thiamphenicol in animal feed by thin layer chromatography combined with high performance liquid chromatography, and belongs to the field of food safety risk monitoring. The method of the invention comprises the following steps: the animal feed is extracted with ethyl acetate, acetonitrile-saturated n-hexane is degreased, and the volume ratio of dichloromethane:acetone:ammonia (25%-28%) is equal to (3-5):(5-7 ): (0.05-0.25) as the developing solution, the samples were separated by thin-layer chromatography, and then detected by high-performance liquid chromatography. The invention significantly improves the anti-interference ability of other substances in the animal feed, reduces the detection cost, reduces the consumption of organic solvents, and has reliable quantitative analysis results, and is suitable for the basic detection units to conduct daily monitoring of thiamphenicol in the feed. .

Description

Method for detecting content of thiamphenicol in animal feed by combining thin-layer chromatography with high performance liquid chromatography

Technical Field

The invention relates to the field of food safety risk monitoring, in particular to a method for detecting thiamphenicol content in animal feed by combining thin-layer chromatography with high performance liquid chromatography.

Background

Thiamphenicol is an amide alcohol antibacterial drug. Compared with chloramphenicol, thiamphenicol has the advantages of wide antibacterial spectrum, good antibacterial effect, high safety and the like, so that thiamphenicol is taken as a substitute of chloramphenicol and is widely used for treating respiratory diseases of livestock and poultry. Mixed feed administration is the common practice of thiamphenicol in veterinary clinics. However, in actual production, an excessive addition of thiamphenicol to the feed sometimes occurs. Toxicology studies indicate that thiamphenicol has significant toxicity to the hematopoietic system. Excessive addition of thiamphenicol can cause the thiamphenicol to remain in animal-derived food, and endanger the health of consumers and the sustainable development of the breeding industry. To prevent the abuse of thiamphenicol, it is necessary to establish a quantitative analysis method that is reliable and suitable for the detection mechanism of the substrate.

The quantitative analysis method for detecting thiamphenicol in animal feed by adopting enzyme-linked immunoassay technology, high performance liquid chromatography and liquid chromatography-mass spectrometry combined technology has been reported in documents. The high performance liquid chromatography has the advantages of reliable detection result and low detection cost, and is particularly suitable for basic detection units. However, the conventional high performance liquid chromatography lacks an effective sample purification step, and the sample pretreatment process is too complicated, so that improvement is required. For example, in the HPLC determination method for determining the content of chloramphenicol, thiamphenicol and florfenicol in feed disclosed in the spectral laboratory 2006 and the HPLC determination method for determining the content of thiamphenicol in feed disclosed in the feed research 2010, ethyl acetate, water and n-hexane are adopted to repeatedly extract thiamphenicol in feed in the sample pretreatment link, the sample pretreatment is complicated, and the liquid-liquid extraction method cannot effectively remove impurities such as amino acid and pigment coexisting in feed, and possibly interferes with subsequent chromatographic analysis.

Thin layer chromatography is a classical sample separation technique. Compared with the solid phase extraction technology commonly used in food safety analysis, the thin layer chromatography has the advantages of good separation effect, low cost and environmental protection, and is very suitable for separating and purifying trace compounds in complex matrixes such as biological samples, animal feeds and the like. Li Chun Xiang adopts thin layer chromatography to purify complex biological sample matrix in the text of 'research on novel method for measuring Sudan red in food by combining novel separation technology and high performance liquid chromatography'; wufuda et al also combine thin-layer chromatography with high performance liquid chromatography for the detection of zearalenone in feeds. However, thin-layer chromatography is used for analyzing thiamphenicol in animal feed, and no document is reported at home and abroad.

The thin layer development conditions are key techniques for thin layer chromatography. The conditions for spreading the thin layer depend on the physicochemical properties of the target compound itself and the characteristics of the sample matrix. Thin layer analysis of thiamphenicol in pharmaceutical formulations has been reported in the literature. For example, in the text of "thin-layer chromatography for identifying florfenicol and thiamphenicol" by Yangyujia, chloroform-methanol-ethyl acetate-water (15:6:15:1) is used as a developing agent. Because the composition of the animal feed is far more complex than that of the bulk drugs and the pharmaceutical preparations, endogenous interfering substances and thiamphenicol in the feed cannot be separated by the expansion condition.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a reliable, simple, convenient and rapid method for detecting the content of thiamphenicol in animal feed by combining thin-layer chromatography with high performance liquid chromatography.

The purpose of the invention is realized by the following technical scheme:

a method for detecting the content of thiamphenicol in animal feed by combining thin-layer chromatography with high performance liquid chromatography comprises the following steps:

(1) ethyl acetate was added to the ground animal feed, vortexed and centrifuged.

(2) Taking supernatant, drying by air flow, adding acetonitrile, vortexing, adding acetonitrile-saturated n-hexane, vortexing, centrifuging, and discarding the upper n-hexane layer. Wherein the volume ratio of the supernatant to the acetonitrile-saturated n-hexane is (1-5) to (0.05-0.1) to (1-5).

(3) And respectively spotting the acetonitrile layer (feed extract) and the thiamphenicol standard substance on different positions of the same GF-254 silica gel thin-layer plate at the lower layer, and spreading the mixture to the top in a mixed solution of dichloromethane, acetone and ammonia water in a volume ratio of (3-5) - (5-7) - (0.05-0.25). The concentration of the ammonia water is preferably 25% -28%.

(4) Taking out the GF-254 silica gel thin layer plate, observing at 254-360nm after the solvent is volatilized, marking the approximate position of the thiamphenicol in the feed extracting solution according to the development distance of the thiamphenicol standard, and scraping the silica gel at the corresponding position.

(5) Adding the acetonitrile-water mixed solution into the scraped silica gel, whirling, centrifuging, taking the supernatant, and filtering by a filter membrane. Wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 20:80-80: 20.

(6) And detecting the content of thiamphenicol in the filtered sample by high performance liquid chromatography. Wherein the mobile phase of the high performance liquid chromatography is a mixed solution of acetonitrile and water according to the volume ratio of 20:80-80: 20.

Preferably, in step (1): the mass volume ratio of the animal feed to the ethyl acetate is 2-5g:20-100 mL; the particle size of the ground animal feed is 1-10 mm; the vortex time is 1-5min, and the centrifugation condition is 3000-.

Preferably, in step (2): the temperature for drying by air flow is 45-60 ℃; the time of the two vortexes is 1-2 min; the centrifugation condition is 3000-8000g for 2-5 min.

Preferably, the volume ratio of the dichloromethane, the acetone and the ammonia water in the step (3) is equal to 3:7: 0.25.

Preferably, in step (5): the vortex time is 1-3 min; the centrifugation condition is 3000-8000g for 5-10min, and the filter membrane is a 0.22 μm organic filter membrane.

Preferably, the conditions of the high performance liquid chromatography in the step (6) are as follows: the chromatographic separation is completed in a Waters Symmetry C18 chromatographic column (250mm × 4.6mm I.D, 5 μm), the column temperature is 32-40 deg.C, the flow rate is 0.5-1mL/min, and the detection wavelength is 220-230 nm.

More preferably, the method for detecting the content of thiamphenicol in the animal feed by combining thin-layer chromatography with high performance liquid chromatography comprises the following steps:

(1) grinding the animal feed, and sieving with a sieve of 1-10 mm; weighing 2-5g of ground and sieved animal feed sample, adding 20-100mL of ethyl acetate, carrying out vortex for 1-5min, and centrifuging for 2-5min at 8000g of 3000-.

(2) Removing 1-5mL of supernatant, and drying with 45-60 ℃ air flow; adding 50-100 μ L acetonitrile, and vortexing for 1-2 min; adding 1-5mL of acetonitrile saturated n-hexane, vortexing for 1-2min, centrifuging for 2-5min at 3000-8000g, and discarding the upper n-hexane layer.

(3) Spotting the lower layer of acetonitrile layer (feed extract) and thiamphenicol standard sample on different positions of a GF-254 silica gel thin layer plate (10cm multiplied by 10cm), and spreading the mixture to the top in a mixed solution with the volume ratio of dichloromethane, acetone, ammonia water (25-28%) equal to (3-5), (5-7) and (0.05-0.25).

(5) Adding 1-2mL of mixed solution of acetonitrile and water in a volume ratio of 20:80-80:20 into silica gel, vortexing for 1-3min, centrifuging for 5-10min at 3000-8000g, collecting supernatant, and filtering with 0.22 μm organic filter membrane.

(6) And detecting the content of thiamphenicol in the filtered sample by high performance liquid chromatography.

A method for separating thiamphenicol from animal feed by thin layer chromatography comprises the steps (1) to (4) of the method.

In the method, the thiamphenicol in the animal feed is quantified by adopting a standard curve, and the method specifically comprises the following steps: weighing a standard substance containing a proper amount of thiamphenicol, adding the standard substance into a 10mL volumetric flask, dissolving the standard substance with acetonitrile, fixing the volume, shaking up the solution, and filtering the solution through a 0.20 mu m filter membrane for preparing a test standard substance solution; wherein the proper amount of thiamphenicol is 5-20 mg. Taking a test sample, diluting the test sample into a series of concentrations by a mobile phase step by step, feeding 10-50 mu L of sample into each concentration sample, repeating the steps for three times, taking the concentration of thiamphenicol as a horizontal coordinate and taking a peak area as a vertical coordinate to obtain a regression equation y which is 162296x-1493.2, wherein x is the concentration of thiamphenicol and y is the peak area; substituting the area of the thiamphenicol peak detected by the high performance liquid chromatography of the detected sample into the linear equation, and calculating to obtain the content of the thiamphenicol in the feed.

The invention has the following advantages and beneficial effects: the method adopts the mixed solution of dichloromethane, acetone and ammonia water (25-28 percent) in volume ratio of (3-5) to (5-7) to (0.05-0.25) as the developing solution to purify the sample by adopting the thin-layer chromatography, obviously improves the anti-interference capability of the detection method on other substances in the animal feed, simultaneously reduces the detection cost, reduces the using amount of organic solvents, has reliable quantitative analysis result, and is suitable for basic detection units to carry out daily monitoring on the thiamphenicol in the feed.

Drawings

FIG. 1 is a chromatogram of a blank feed and a thiamphenicol standard substance when a developing solution is a mixed solution of chloroform, methanol, ethyl acetate and water in a volume ratio of 15:6:15: 1.

FIG. 2 is a chromatogram of space-time white feed and thiamphenicol standard substance when developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25% -28%) in a volume ratio of 2:8: 0.25.

FIG. 3 is a chromatogram of a blank feed and a thiamphenicol standard substance when a developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25% -28%) in a volume ratio of 3:7: 0.25.

FIG. 4 is a chromatogram of a blank feed and a thiamphenicol standard substance when the developing solution is a mixed solution with a volume ratio of dichloromethane to acetone equal to 3: 7.

FIG. 5 is a chromatogram of a blank feed and a thiamphenicol standard substance when a developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25% -28%) in a volume ratio of 4:6: 0.25.

FIG. 6 is a chromatogram of a blank feed and a thiamphenicol standard substance when a developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25% -28%) in a volume ratio of 5:5: 0.25.

FIG. 7 is chromatograms of different samples of examples 2-4, wherein (a) is thiamphenicol standard (0.1. mu.g/mL), (b) is a blank chicken feed, (c) is a chicken feed supplemented with 1mg/kg of thiamphenicol, (d) is a blank pig feed, (e) is a pig feed supplemented with 1mg/kg of thiamphenicol, (f) is a blank fish feed, and (g) is a fish feed supplemented with 1mg/kg of thiamphenicol.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

EXAMPLE 1 investigation of the Effect of different thin layer development conditions on the specificity of the method of the invention

Method and device

(1) Grinding the animal feed, and sieving by a 10mm sieve; weighing 2g of ground and sieved animal feed sample, adding 20mL of ethyl acetate, vortexing for 2min, and centrifuging for 5min at 3000 g. Wherein the animal feed comprises pig, chicken and fish feed. The pig feed comprises 63.4 percent of corn (mass percentage, the same below), 25 percent of soybean meal, 4.9 percent of wheat bran, 1.9 percent of fish meal, 2 percent of soybean oil, 0.3 percent of salt and 2.5 percent of mineral; the chicken feed comprises 56.8% of corn, 25% of soybean meal, 11.5% of fish meal, 3.5% of soybean oil, 0.2% of salt, 0.5% of stone powder and 2.5% of mineral; the fish feed comprises 38% of rapeseed meal, 15% of soybean meal, 10% of peanut meal, 13.5% of wheat, 10% of cottonseed meal, 12% of rice bran and 1.5% of soybean oil.

(2) 1mL of supernatant is removed and dried by air flow at 45 ℃; adding 50 μ L acetonitrile, and vortexing for 1 min; adding 5mL of acetonitrile-saturated n-hexane, vortexing for 1min, centrifuging 3000g for 5min, and discarding the upper n-hexane layer.

(3) Spotting the lower layer acetonitrile layer (feed extract) and thiamphenicol standard substance at different positions of GF-254 silica gel thin layer plate (10cm × 10cm), and spreading in 6 different spreading solutions to the top.

(4) Taking out the GF-254 silica gel thin layer plate, observing at 254nm after the solvent is evaporated, marking the approximate position of thiamphenicol in the feed extracting solution according to the development distance of the standard substance, and scraping the silica gel at the corresponding position.

(5) Adding 1mL of mixed solution of acetonitrile and water in a volume ratio of 21.7:78.3 into silica gel, vortexing for 2min, centrifuging for 5min at 8000g, collecting supernatant, and filtering with 0.22 μm organic filter membrane.

(6) And detecting the content of thiamphenicol in the filtered sample by high performance liquid chromatography. Detection was done in a Waters 1525HPLC system, which included a 1525 binary pump, 2489UV detector and 2707 autosampler. The chromatographic separation was carried out on a Waters Symmetry C18 column (250 mm. times.4.6 mm I.D., 5 μm) at a column temperature of 32 ℃; the mobile phase is a mixed solution consisting of acetonitrile and water according to the volume ratio of 21.7:78.3, the flow rate is 0.6mL/min, the acetonitrile in the mobile phase is chromatographically pure, and the water is purified water; the detection wavelength was 225 nm.

The thiamphenicol is quantified by adopting a standard curve, a thiamphenicol standard product with a series of concentrations is detected by high performance liquid chromatography, the thiamphenicol concentration is taken as a horizontal coordinate, the peak area is taken as a vertical coordinate, and a regression equation y is 162296x-1493.2, wherein x is the thiamphenicol concentration, and y is the peak area; substituting the area of the thiamphenicol peak detected by the high performance liquid chromatography of the detected sample into the linear equation, and calculating to obtain the content of the thiamphenicol in the feed.

Second, result in

(1) When the developing solution is a mixed solution of chloroform, methanol, ethyl acetate and water in a volume ratio of 15:6:15:1, the chromatogram of the blank feed and the thiamphenicol standard substance is shown in fig. 1, and it can be known from fig. 1 that interference exists near the retention time of the thiamphenicol under the developing condition, and the chromatographic peak of the thiamphenicol standard substance is close to the impurity peak (at the retention time of 12 min) in the feed and is not taken as a candidate developing condition.

(2) When the developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25-28%) in a volume ratio of 2:8:0.25, a chromatogram of a blank feed and a thiamphenicol standard substance is shown in fig. 2, and it can be known from fig. 2 that under the developing condition, no obvious interference exists near the retention time of thiamphenicol, but the chromatographic peak of the thiamphenicol standard substance is close to the impurity peak (retention time at 11.8 min) in the feed, and the chromatographic peak is not used as a candidate developing condition.

(3) When the developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25-28%) in a volume ratio of 3:7:0.25, the chromatogram of the blank feed and the thiamphenicol standard substance is shown in fig. 3, and it can be seen from fig. 3 that under the developing condition, the thiamphenicol is free of interference around the retention time and is the optimal developing condition.

(4) When the developing solution is a mixed solution with a volume ratio of dichloromethane to acetone being equal to 3:7, the chromatogram of the blank feed and the thiamphenicol standard substance is shown in fig. 4, and it can be known from fig. 4 that strong interference exists near the thiamphenicol retention time under the developing condition, which indicates that the retention of weak acidic impurities in the feed can be enhanced by adding ammonia water, and the specificity of the method is remarkably improved.

(5) When the developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25-28%) in a volume ratio of 4:6:0.25, a chromatogram of a blank feed and a thiamphenicol standard substance is shown in fig. 5, and it can be known from fig. 5 that under the developing condition, significant interference exists near the retention time of thiamphenicol, and a chromatographic peak of the thiamphenicol standard substance is close to a peak of impurities in the feed (retention time at 11.8 min), so that the developing solution is not used as a candidate developing condition.

(6) When the developing solution is a mixed solution of dichloromethane, acetone and ammonia water (25-28%) in a volume ratio of 5:5:0.25, a chromatogram of a blank feed and a thiamphenicol standard substance is shown in fig. 6, and it can be known from fig. 6 that under the developing condition, no obvious interference exists near the retention time of thiamphenicol, but the chromatographic peak of the thiamphenicol standard substance is close to the impurity peak (retention time at 11.8 min) in the feed, and the chromatographic peak is not used as a candidate developing condition.

Example 2 testing of the accuracy and precision of Thiamphenicol in Chicken feed

A thiamphenicol standard sample is added into 15 parts of blank chicken feed (the composition of the chicken feed comprises 56.8 percent (mass percentage content, the same below) of corn, 25 percent of soybean meal, 11.5 percent of fish meal, 3.5 percent of soybean oil, 0.2 percent of salt, 0.5 percent of stone powder and 2.5 percent of mineral substances) to obtain added samples with the concentrations of 0.1, 1.0 and 10.0mg/kg, the operation is repeated for 3 batches for each concentration, and 5 samples are repeated for each batch. When the test was performed under the optimum development conditions in example 1 (the chromatogram is shown in FIG. 7), the average recovery of thiamphenicol was found to be 88.4-111.3%, the intra-day relative standard deviation was less than 7.5%, and the inter-day relative standard deviation was less than 6.3%. The accuracy and precision test results are shown in table 1.

TABLE 1 accuracy and precision results of the method in Chicken feed

The results show that the detection method has higher accuracy and precision and can be used for determining the content of thiamphenicol in chicken feed.

Example 3 accuracy and precision testing of thiamphenicol in pig feed.

Standard thiamphenicol is added into 15 parts of blank pig feed (the composition of the pig feed comprises 63.4 percent (mass percentage content, the same below) of corn, 25 percent of soybean meal, 4.9 percent of wheat bran, 1.9 percent of fish meal, 2 percent of soybean oil, 0.3 percent of salt and 2.5 percent of mineral substances) to obtain added samples with the concentrations of 0.1, 1.0 and 10.0mg/kg, the operation is repeated for 3 batches for each concentration, and 5 samples are repeated for each batch. The recovery rate of thiamphenicol was determined to be 81.6-115.3%, the relative standard deviation in days was less than 8.7%, and the relative standard deviation in days was less than 10.1% when the assay was performed under the optimal development conditions in example 1 (the chromatogram is shown in fig. 7). The accuracy and precision test results are shown in table 2.

TABLE 2 accuracy and precision results of the method in pig feed

The results show that the detection method has higher accuracy and precision and can be used for measuring the content of thiamphenicol in the pig feed.

Example 4 testing of the accuracy and precision of Thiamphenicol in Fish feed

Adding thiamphenicol standard samples into 15 parts of blank fish feed (the fish feed comprises 38 percent (mass percentage content, the same below) of rapeseed meal, 15 percent of soybean meal, 10 percent of peanut meal, 13.5 percent of wheat, 10 percent of cottonseed meal, 12 percent of rice bran and 1.5 percent of soybean oil) to obtain added samples with the concentrations of 0.1, 1.0 and 10.0mg/kg, repeating the operation for 3 batches for each concentration, and repeating 5 samples for each batch. When the test was performed under the optimum development conditions in example 1 (the chromatogram is shown in FIG. 7), the average recovery of thiamphenicol was found to be 80.8-102.3%, the intra-day relative standard deviation was less than 8.0%, and the inter-day relative standard deviation was less than 7.3%. The accuracy and precision test results are shown in table 3.

TABLE 3 accuracy and precision results of the method in Fish feed

The results show that the detection method has higher accuracy and precision and can be used for measuring the content of thiamphenicol in the fish feed.

The above-mentioned embodiments are intended to illustrate the technical solutions and advantages of the present invention, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modifications, additions, equivalents, etc. made within the scope of the principles of the present invention should be included in the scope of the present invention.

Claims

1. a method for detecting thiamphenicol content in animal feed in conjunction with thin-layer chromatography combined with high performance liquid chromatography, is characterized in that: may further comprise the steps:

(1) adding ethyl acetate to the animal feed after grinding, and centrifuging after vortexing;

(2) take the supernatant, blow dry with air flow, add acetonitrile first, vortex, then add acetonitrile-saturated n-hexane, vortex, centrifuge, discard the upper n-hexane; wherein, supernatant, acetonitrile, acetonitrile-saturated n-hexane The volume ratio of n-hexane is 1-5:0.05-0.1:1-5;

(3) Spot the lower acetonitrile layer and thiamphenicol standard on different positions of the same GF-254 silica gel thin-layer plate respectively, in a mixture of dichloromethane:acetone:ammonia equal to 3:7:0.25 by volume Expand to the top in the liquid; the concentration of the ammonia water is 25%-28%;

(4) Take out the GF-254 silica gel thin-layer plate, and inspect it at 254-360 nm after the solvent is evaporated. Mark the position of thiamphenicol in the feed extract according to the expansion distance of the thiamphenicol standard, and scrape the corresponding position of silicone;

(5) add acetonitrile-water mixed solution to the scraped silica gel, vortex, centrifuge, take supernatant, filter through filter membrane; wherein, in the acetonitrile-water mixed solution, the volume ratio of acetonitrile and water is 20:80-80 :20;

(6) the thiamphenicol content of the filtered sample is detected by high performance liquid chromatography; wherein, the high performance liquid chromatography mobile phase is that the mobile phase is a mixed solution composed of acetonitrile and water in a volume ratio of 20:80-80:20 The conditions of high performance liquid chromatography are: chromatographic separation is completed in a Waters Symmetry C18 chromatographic column, the column temperature is 32-40°C, the flow rate is 0.5-1mL/min, and the detection wavelength is 220-230nm.

2. method according to claim 1, is characterized in that: in step (1): the mass volume ratio of animal feed and ethyl acetate is 2-5g:20-100mL.

3. method according to claim 1, is characterized in that: the particle diameter of the animal feed after grinding in step (1) is 1-10mm; The time of vortex is 1-5min, and the centrifugal condition is 3000-8000g centrifugal 2-5min.

4. method according to claim 1, is characterized in that: in step (2): the temperature of air flow drying is 45-60 ℃; The time of twice vortex is 1-2min; The centrifugal condition is 3000 -8000g centrifugation for 2-5min.

5. method according to claim 1, is characterized in that: in step (5): the time of vortex is 1-3min; The condition of centrifugal is 3000-8000g centrifugal 5-10min, and filter membrane is 0.22 μm organic filter membrane .

6. method according to claim 1 is characterized in that: comprise the following steps:

(1) The animal feed is ground and passed through a 1-10 mm sieve; 2-5 g of the ground and sieved animal feed sample is weighed, 20-100 mL of ethyl acetate is added, vortexed for 1-5 min, and centrifuged at 3000-8000 g for 2-5 min;

(2) Pipette 1-5 mL of supernatant, blow dry at 45-60°C with air flow; add 50-100 μL of acetonitrile, vortex for 1-2 min; add 1-5 mL of acetonitrile-saturated n-hexane, vortex for 1-2 min, 3000 -8000g centrifugation for 2-5min, discard the upper n-hexane;

(3) Spot the lower acetonitrile layer and the thiamphenicol standard on different positions of the GF-254 silica gel thin-layer plate, in a volume ratio of dichloromethane:acetone:25%-28% ammonia equal to 3:7:0.25 Expand to the top in the mixture;

(4) Take out the GF-254 silica gel thin-layer plate, and inspect it at 254-360 nm after the solvent is evaporated. Mark the approximate position of thiamphenicol in the feed extract according to the expansion distance of the thiamphenicol standard, and scrape Silica gel at the corresponding position;

(5) Add 1-2mL of a mixture of acetonitrile:water in a volume ratio of 20:80-80:20 to the silica gel, vortex for 1-3min, centrifuge at 3000-8000g for 5-10min, take the supernatant, filter it with a 0.22 μm organic filter membrane filtration;

(6) The thiamphenicol content of the filtered sample was detected by high performance liquid chromatography.

7. A method for separating thiamphenicol in animal feed by thin-layer chromatography, characterized in that it comprises steps (1)-(4) of the method according to any one of claims 1-6.