CN109709258B - Method for detecting florfenicol total residues in edible tissues of pigs and application - Google Patents
Method for detecting florfenicol total residues in edible tissues of pigs and application Download PDFInfo
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Abstract
The invention discloses a method for detecting florfenicol total residues in edible tissues of pigs and application thereof. The method comprises the following steps: hydrolyzing the pig edible tissue homogenate by a high-concentration hydrochloric acid solution, washing by ethyl acetate, extracting by the ethyl acetate under an alkaline condition, concentrating an extract, degreasing by normal hexane, purifying by thin-layer chromatography by taking a mixed solution of the ethyl acetate, acetone and ammonia water as a developing solution, and detecting by adopting high performance liquid chromatography. The florfenicol combined residue in the edible tissues of the pigs is fully released and converted into florfenicol amine, so that a false negative result possibly caused by the conventional method is avoided. In addition, the tissue extracting solution is purified by the thin-layer chromatography, so that endogenous substances contained in the tissue are effectively removed, the interference of the tissue extracting solution on chromatographic analysis is avoided, the detection cost is reduced, the using amount of an organic solvent is reduced, the result is reliable, and the method is suitable for daily monitoring of a basic detection mechanism.
Description
Technical Field
The invention belongs to the field of veterinary drug residue detection, and particularly relates to a method for detecting florfenicol total residue in edible tissues of pigs and application of the florfenicol total residue.
Background
Florfenicol has the advantages of broad spectrum, high efficiency and low toxicity, and is widely used for preventing and treating the respiratory tract infection of pigs in China. Toxicology studies, however, suggest that florfenicol and metabolites may induce hepatotoxicity, reproductive toxicity, embryotoxicity, hematologic toxicity, and immunotoxicity. Improper use can result in their remaining in the edible tissue of pigs, compromising consumer health, and affecting the sustainable development of the swine industry. Therefore, a reliable quantitative analysis method for monitoring florfenicol residues in edible tissues of pigs is urgently needed to be established.
Florfenicol is metabolized extensively in pigs. Part of the metabolites can form a firm binding residue with biological macromolecules (such as proteins) and accumulate in edible tissues of pigs for a long time. These bound residues cannot be directly extracted by the solvent and need to be hydrolyzed by strong acids to be released from the tissue and converted to florfenicol amine itself. Considering the food safety risk that florfenicol-bound residues may carry, the european drug administration stipulates that the residual marker for florfenicol is the total residue of florfenicol, including florfenicol prototypes and various metabolites, calculated as florfenicol amine. The maximum residual limits of florfenicol in the pig muscle, liver and kidney were 300, 2000, 500. mu.g/kg, respectively.
A large number of documents report methods for detecting florfenicol and florfenicol amine in foods of animal origin based on physicochemical analysis (e.g. chromatography, chromatography-mass spectrometry) and immunoassay (e.g. enzyme-linked immunoassay). Most of these methods do not involve a hydrolysis step in the sample pretreatment procedure that is effective in releasing the bound residue, and therefore only a portion of the residue can be detected. The method for monitoring florfenicol residues in livestock and poultry products can cause false negative results and cause serious misjudgment. Taking the study of Imran et al as an example, chickens were orally administered for 5 days at a dose of 30mg/kg, and the concentration of florfenicol residue detected in the kidney after 7 days of drug holiday as extractable content was 125.04. mu.g/kg, which was below the maximum residue limit (750. mu.g/kg), and was judged to be acceptable. In fact, 806.50. mu.g/kg of florfenicol-bound residue was not found in this batch. Once the livestock and poultry products flow into the market, the food safety is threatened, and if the livestock and poultry products are sold to the European Union, the livestock and poultry breeding enterprises in China can face huge compensation. Therefore, it is necessary to establish a quantitative analysis method for detecting the florfenicol total residues in the edible tissues of livestock and poultry.
To date, only a few documents report methods for the detection of florfenicol total residue (calculated as florfenicol amine). The methods mainly adopt liquid chromatography-tandem mass spectrometry and ultra-high performance liquid chromatography-tandem mass spectrometry. The mass spectrum detector used in the method has the advantages of high sensitivity and high specificity, but is easily interfered by matrix effect, and is expensive, high in operation and maintenance cost and not beneficial to popularization to a basic detection mechanism. The high performance liquid chromatography is reliable in quantification, the sensitivity can meet the requirement of florfenicol residue monitoring, the popularization rate of a basic layer detection mechanism is high, and the high performance liquid chromatography-tandem mass spectrometry and the ultra-high performance liquid chromatography-tandem mass spectrometry can be used as a substitute and a supplement of the liquid chromatography-tandem mass spectrometry and the ultra-high performance liquid chromatography-tandem mass spectrometry. However, high performance liquid chromatographs are mostly equipped with uv detectors, which are far less specific than mass spectrometric detectors. Porcine edible tissue contains a large amount of endogenous substances that can be co-extracted with florfenicol amine into the sample to be tested. Since these endogenous interferents tend to have ultraviolet absorption, their presence can cause severe interference with chromatographic detection. In order to ensure the reliability of the detection result, the systematic and deep research on the sample purification step is very important.
For the residue detection of florfenicol and florfenicol amine, common sample purification technologies include solid phase extraction technologies (such as high performance liquid chromatography for detecting amide alcohols and penicillins in fish meat established by Evagelopouou et al, gas chromatography-mass spectrometry combined method for detecting amide alcohols drugs in pig muscle and liver established by Shen et al, liquid chromatography-tandem mass spectrometry for detecting florfenicol total residues in cattle, horses and pig edible tissues established by Fedeniuk et al, liquid chromatography-tandem mass spectrometry for detecting florfenicol total residues in chicken edible tissues established by Imran et al) and immunoaffinity chromatography (such as liquid chromatography-tandem mass spectrometry for detecting amide alcohols drugs in pig muscle established by Luo et al). The filler for solid phase extraction is expensive, and most of the filler has poor selectivity, so that the ideal purification effect is difficult to obtain. The immunoaffinity chromatography uses a specific antibody as an adsorbent of florfenicol and florfenicol amine, has good purification effect, but has long antibody preparation period, high cost and poor antibody stability, and can not realize commercialization at present. Thin layer chromatography is a classical chromatographic separation technique. Compared with solid phase extraction and immunoaffinity chromatography, the thin-layer chromatography has the advantages of good purification effect, low analysis cost, small organic solvent consumption and the like. The thin-layer chromatography is used for purifying and analyzing florfenicol amine in edible tissues of pigs, and is hopeful to fill up short plates with poor specificity of an ultraviolet detector, so that the application of the high performance liquid chromatography in veterinary drug residue analysis is widened.
The thin layer developing condition is the key technology of thin layer chromatography purification. The conditions for spreading the thin layer depend on the physicochemical properties of the target compound itself and the characteristics of the sample matrix. The thin-layer chromatography purification of florfenicol in animal feed is reported (such as high performance liquid chromatography established by Yang et al for detecting florfenicol in animal feed), and the thin-layer separation of florfenicol amine in edible tissue hydrolysate of pigs is not reported. The conditions for thin-layer separation of florfenicol in animal feed are not necessarily suitable for the separation and purification of florfenicol amine in edible tissues of pigs. Florfenicol amine is a hydrolysis product of florfenicol. Due to the side chain containing primary amine groups, strong adsorption of florfenicol amine on the thin layer chromatography plate may occur, which will change the chromatographic behavior. Meanwhile, the edible tissue composition of the pig is complex, and particularly after the tissue is hydrolyzed, the contained endogenous interferents are sufficiently released. Their presence increases the difficulty of screening thin layer development conditions.
Disclosure of Invention
The invention aims to make up for the defects and shortcomings of the prior detection technology and provides a more reliable detection method for florfenicol total residues in edible tissues of pigs, which is suitable for a basic detection mechanism.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, there is provided a method of detecting florfenicol total residue in porcine edible tissue, comprising the steps of:
(1) taking edible pig tissues, and homogenizing in a tissue homogenizer;
(2) adding a high-concentration hydrochloric acid solution into the tissue homogenate, and placing the homogenate in a water bath after vortexing until the tissue is completely hydrolyzed; wherein the molar concentration of the hydrochloric acid solution is 4-8N, the mass-volume ratio of the tissue homogenate to the hydrochloric acid solution is (1-2) g, (2-8) mL, the water bath temperature is 80-100 ℃, and the water bath time is 2-24 h; florfenicol and metabolites are completely hydrolyzed into florfenicol amine under the condition;
(3) after cooling, adding ethyl acetate into the hydrolyzed sample, carrying out vortex, centrifuging and discarding the ethyl acetate; wherein the volume ratio of the hydrolyzed sample to the ethyl acetate is (3-6) to (4-40);
(4) adding a high-concentration sodium hydroxide solution into the residual sample to adjust the pH value to 10-12; wherein, the mass percentage concentration of the sodium hydroxide is 20 percent to 50 percent, and the adding volume is 0.5mL to 5 mL;
(5) adding ethyl acetate into the alkalized sample, whirling, centrifuging, and transferring the ethyl acetate into another clean centrifugal tube; wherein the volume ratio of the alkalized sample to the ethyl acetate is (3-8) to (4-10);
(6) repeating the above operation once with equal amount of ethyl acetate, and combining ethyl acetate;
(7) transferring ethyl acetate into a centrifugal tube containing a proper amount of glacial acetic acid solution in batches, and volatilizing the ethyl acetate by air flow; wherein, the volume percentage concentration of the glacial acetic acid solution is 1 to 10 percent, and the volume is 0.2 to 1 mL;
(8) adding a proper amount of normal hexane into the residual glacial acetic acid solution, carrying out vortex and centrifugation, and removing the normal hexane; wherein the volume ratio of the residual glacial acetic acid solution to the normal hexane is (0.2-1) to (4-10);
(9) blowing the residual glacial acetic acid solution to the volume of about 0.1mL by using an air flow;
(10) respectively spotting the glacial acetic acid solution and the florfenicol amine standard substance at different positions of the same GF-254 silica gel thin layer plate, and spreading to the top end in a mixed solution of ethyl acetate, acetone and ammonia water in a volume ratio of (1-5) to (5-9) to (0.05-0.5); the concentration of the ammonia water in percentage by volume is preferably 25% -28%;
(11) taking out the GF-254 silica gel thin layer plate, inspecting at 254-360nm after the solvent is volatilized, marking the approximate position of the florfenicol amine in the tissue extracting solution according to the expansion distance of the florfenicol amine standard substance, and scraping the silica gel at the corresponding position;
(12) adding 0.5-5mL of acetonitrile-glacial acetic acid mixed solution with the volume ratio of 20:80-80:20 into the scraped silica gel, swirling, centrifuging, taking supernatant, and filtering through a filter membrane; wherein, the volume percentage concentration of the glacial acetic acid in the acetonitrile-glacial acetic acid mixed solution is 1-10%;
(13) detecting the content of florfenicol amine in the filtered sample by high performance liquid chromatography; wherein the high performance liquid chromatography mobile phase is a mixed solution of acetonitrile and phosphate buffer solution according to the volume ratio of 30:70-70: 30; the phosphate buffer solution contains 3 to 10 mass percent of sodium dodecyl sulfate, 1 to 4 volume percent of disodium hydrogen phosphate dihydrate, 0.05 to 2 volume percent of triethylamine and 83 to 98 volume percent of phosphoric acid.
Preferably, in step (1): the revolution of homogenate is 3000-10000r/min, and the time is 2-5 min.
Preferably, in step (2): the vortex time was 1-3 min.
Preferably, in step (3): the vortex time is 1-3 min; the centrifugation condition is 3000-8000g for 5-10 min.
Preferably, in step (5): the vortex time is 1-3 min; the centrifugation condition is 3000-8000g for 5-10 min.
Preferably, in step (7): the temperature of the air flow is 35-60 ℃.
Preferably, in step (8): the vortex time is 1-3 min; the centrifugation condition is 3000-8000g for 5-10 min.
Preferably, in step (9): the temperature of the air flow is 35-60 ℃.
Preferably, in step (12): the filter was a 0.22 μm organic filter.
Preferably, the conditions of the high performance liquid chromatography in the step (13) are as follows: the chromatographic separation is carried out in Waters Symmetry C18The chromatographic column (250mm multiplied by 4.6mm I.D, 5 μm) is completed, the column temperature is 32-40 ℃, the flow rate is 0.5-1mL/min, and the detection wavelength is 220-230 nm.
More preferably, the method for detecting the florfenicol total residue in the edible tissues of the pigs comprises the following steps:
(1) making pig edible tissue into homogenate;
(2) weighing 2-5g of tissue homogenate, adding 2-16mL of 4-8N hydrochloric acid solution, vortexing for 1-3min, and placing in a water bath at 90-100 ℃ for 6-12 h; florfenicol and its metabolites are completely hydrolyzed into florfenicol amine under the conditions;
(3) cooling the sample to room temperature, adding 8-40mL of ethyl acetate, performing vortex for 1-3min, centrifuging for 5-10min at 3000-8000g, and discarding the ethyl acetate;
(4) adding 0.5-5mL of sodium hydroxide solution with the mass percent concentration of 20% -50% into the rest sample to adjust the pH value to 10-12;
(5) adding 5-20mL of ethyl acetate into the alkalized sample, performing vortex for 1-3min, centrifuging for 5-10min at 3000-8000g, and transferring the ethyl acetate into another clean centrifugal tube;
(6) repeating the above operation once with equal amount of ethyl acetate, and combining ethyl acetate;
(7) transferring ethyl acetate into a centrifugal tube containing 0.2-1mL of glacial acetic acid solution with the volume percentage concentration of 1% -10% in batches, and volatilizing the ethyl acetate in air flow at 35-60 ℃;
(8) adding 2-10mL of normal hexane into the rest glacial acetic acid solution, carrying out vortex for 1-3min, centrifuging for 5-10min at 3000-8000g, and discarding the normal hexane;
(9) blowing the residual glacial acetic acid solution to the volume of about 0.1mL at 35-60 ℃ by using air flow;
(10) respectively spotting the glacial acetic acid solution and the florfenicol amine standard substance at different positions of the same GF-254 silica gel thin layer plate, and spreading the solution to the top in a mixed solution of ethyl acetate, acetone and ammonia water with the volume ratio of (1-5) to (5-9) to (0.05-0.5);
(11) taking out the GF-254 silica gel thin layer plate, inspecting at 254-360nm after the solvent is volatilized, marking the approximate position of the florfenicol amine in the tissue extracting solution according to the expansion distance of the florfenicol amine standard substance, and scraping the silica gel at the corresponding position;
(12) adding 0.5-5mL of mixed solution of acetonitrile and glacial acetic acid solution (1-10%) in a volume ratio of 20:80-80:20 into silica gel, swirling for 1-3min, centrifuging for 5-10min at 8000g of 3000-;
(13) detecting the amount of florfenicol amine in the filtered sample by high performance liquid chromatography, wherein the amount of florfenicol amine represents the amount of florfenicol total residues;
in a second aspect, there is provided a method for the thin layer chromatography separation of florfenicol total residue in porcine edible tissue, comprising steps (1) - (12) of any of the methods described above.
In a third aspect, the method for detecting florfenicol total residue in porcine edible tissue is provided for detecting florfenicol residue in porcine edible tissue.
Preferably, the edible tissue of swine is swine muscle, liver and kidney.
In the method, florfenicol and metabolites thereof in the edible tissues of the pigs are converted into florfenicol amine through hydrolysis. The concentration of florfenicol total residue in the edible tissues of swine is therefore expressed as the concentration of florfenicol amine. The concentration is quantified by a standard curve, specifically: weighing a standard substance containing a proper amount of florfenicol amine, adding the standard substance into a 10mL volumetric flask, dissolving with acetonitrile, fixing the volume, and shaking up for later use; wherein the proper amount of florfenicol amine is 5-20 mg. Taking a test sample, diluting the test sample into a series of concentrations step by using a mobile phase, feeding a sample by 10-50 mu L of each concentration sample, repeating the sampling for three times, taking the concentration of the florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate to obtain a regression equation y which is 282116x +9413.7, wherein x is the concentration of the florfenicol amine and y is the peak area; substituting the florfenicol amine peak area detected by the high performance liquid chromatography of the sample into the linear equation, and calculating to obtain the florfenicol amine residual concentration in the edible tissue of the pig.
The invention has the following advantages and beneficial effects: the florfenicol combined residues in the edible tissues of the pigs are released through strong acid hydrolysis, so that the method can be used for determining the florfenicol total residues in the tissues, and the false negative result is avoided; in the pretreatment process of the sample, a hydrolysis product is washed by ethyl acetate and n-hexane is used for degreasing, so that a large amount of fat and other endogenous interferents are removed; when tissue extract is further purified by thin-layer chromatography, a mixed solution with the volume ratio of ethyl acetate, acetone and ammonia water (25-28%) being equal to (1-5) to (5-9) to (0.05-0.5) is used as a developing solution to purify a sample, so that the anti-interference capability of the detection method on other substances in the edible tissue of the pig is remarkably improved, the detection cost is reduced, the using amount of an organic solvent is reduced, the quantitative analysis result is reliable, and the method is suitable for basic detection units to perform daily monitoring on the florfenicol total residues in the edible tissue of the pig.
Drawings
FIG. 1 is a comparison of the purification effect of the muscle of the blank pig treated by the various sample purification techniques in example 1;
wherein, (a) is florfenicol amine standard substance (2 mu g/mL), (b) is a chromatogram for direct analysis of blank pig muscle extract, (c) is a chromatogram for solid-phase extraction and purification of blank pig muscle extract, and (d) is a chromatogram for thin-layer chromatography and purification of blank pig muscle extract. As can be seen from the figure, the blank pig muscle extract has no impurity peak near the retention time of florfenicol amine after being purified by thin-layer chromatography, and the method has good specificity.
FIG. 2 is a comparison of the decontamination effect of the blank pig liver treated by the various sample decontamination techniques of example 1;
wherein, (a) is florfenicol amine standard substance (2 mug/mL), (b) is chromatogram for direct analysis of blank pig liver extract, (c) is chromatogram of blank pig liver extract after solid phase extraction and purification, and (d) is chromatogram of blank pig liver extract after thin layer chromatography and purification. As can be seen from the figure, after the blank pig liver extract is purified by thin-layer chromatography, no impurity peak exists near the retention time of florfenicol amine, and the method specificity is good.
FIG. 3 is a comparison of the decontamination effect of the kidney of the blank pigs treated by the various sample decontamination techniques of example 1. Wherein (a) is florfenicol amine standard substance (2 mu g/mL), (b) is a chromatogram for direct analysis of blank pig kidney extract, (c) is a chromatogram obtained after solid phase extraction and purification of blank pig kidney extract, and (d) is a chromatogram obtained after thin layer chromatography purification of blank pig kidney extract. As can be seen from the figure, the blank pig kidney extract has no impurity peak near the retention time of florfenicol amine after being purified by thin-layer chromatography, and the method has good specificity.
Figure 4 is a chromatogram of different samples from example 2, wherein (a) is florfenicol amine standard (2 μ g/mL), (b) is blank pig liver, (c) is pig liver supplemented with 0.5-fold maximum residual limit of florfenicol, (d) is blank pig kidney, (e) is pig kidney supplemented with 0.5-fold maximum residual limit of florfenicol, (f) is blank pig muscle, (g) is pig muscle supplemented with 0.5-fold maximum residual limit of florfenicol.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
EXAMPLE 1 comparison of the decontamination efficacy of various sample decontamination techniques
Method and device
(1) Homogenizing muscle, liver and kidney of the blank pig.
(2) Accurately weigh each 2g of the homogenate and repeat 15 samples per tissue. 4mL of 6N hydrochloric acid solution is added, vortexed for 2min, and placed in a water bath at 90-100 ℃ for 6 h.
(3) The sample was cooled to room temperature, then 20mL of ethyl acetate was added, vortexed for 2min, and centrifuged at 5000g for 5min, discarding the ethyl acetate.
(4) And adding 1.7mL of 50% sodium hydroxide solution by mass into the rest sample to adjust the pH value to 10-12.
(5) Add 8mL of ethyl acetate to the alkalinized sample, vortex for 2min, centrifuge for 5min at 5000g, transfer ethyl acetate to another clean centrifuge tube.
(6) The above procedure was repeated once with an equal amount of ethyl acetate, and the ethyl acetate was combined.
(7) The ethyl acetate was transferred in portions into centrifuge tubes containing 0.5mL of a 5% strength by volume glacial acetic acid solution and evaporated in an air stream at 50 ℃.
(8) Adding 5mL of n-hexane into the rest glacial acetic acid solution, vortexing for 2min, centrifuging for 5min at 5000g, and discarding the n-hexane.
(9) The remaining glacial acetic acid solution was treated in the following three ways (5 samples repeated in each way): firstly, blowing the glacial acetic acid solution to be dry in 45 ℃ air flow without purification, adding 1mL of redissolved residue of a mobile phase, and filtering the redissolved residue with a 0.22 mu m organic filter membrane to be detected; secondly, solid phase extraction and purification are carried out, 1.5mL of 5% glacial acetic acid solution is added into the rest glacial acetic acid solution, vortex is carried out for 2min, well balanced Waters Oasis MCX solid phase extraction small columns (60mg,3mL) are added, then 2mL of 5% glacial acetic acid solution and 5% glacial acetic acid solution-methanol mixed solution with the volume ratio of 50:50 are sequentially used for washing, finally 2mL of ammonia water-methanol mixed solution with the volume ratio of 10:90 are used for elution, the eluent is dried by air flow at 45 ℃, 1mL of re-dissolved residue of mobile phase is added, and the re-dissolved residue passes through an organic filter membrane with the diameter of 0.22 mu m and is to be tested; ③ purifying by thin-layer chromatography, and blowing the residual glacial acetic acid solution to the volume of about 0.1mL by air flow at 45 ℃. Then the glacial acetic acid solution and the florfenicol amine standard substance are respectively spotted on different positions of the same GF-254 silica gel thin layer plate, and are developed to the top end in a mixed solution with the volume ratio of ethyl acetate, acetone and ammonia water being equal to 2:8: 0.5. Taking out the GF-254 silica gel thin layer plate, observing at 254nm after the solvent is evaporated, marking the approximate position of the florfenicol amine in the tissue extracting solution according to the expansion distance of the florfenicol amine standard product, and scraping the silica gel at the corresponding position. Adding 1mL of mixed solution of acetonitrile and glacial acetic acid solution (5%) with the volume ratio of 30:70 into silica gel, vortexing for 2min, centrifuging for 5min at 8000g, collecting supernatant, filtering with 0.22 μm organic filter membrane, and testing.
(10) The samples were tested in a Waters 1525 high performance liquid chromatography system comprising a 1525 binary pump, a 2489UV detector and a 2707 autosampler. The chromatographic separation is carried out in Waters Symmetry C18The reaction is finished in a chromatographic column (250mm multiplied by 4.6mm I.D, 5 mu m), and the temperature of the column is 32 ℃; the mobile phase is a mixed solution consisting of acetonitrile and phosphate buffer solution (every 500mL of the phosphate buffer solution contains 0.34g of sodium dodecyl sulfate, 0.78g of disodium hydrogen phosphate dihydrate, 0.5mL of phosphoric acid and 0.5mL of triethylamine) according to the volume ratio of 33.3:66.7, the flow rate is 0.6mL/min, acetonitrile in the mobile phase is chromatographically pure, and water is purified water; the detection wavelength was 225 nm.
Second, result in
The null pig muscle, liver and kidney homogenates were processed according to the sample pretreatment procedure described above, and the resulting typical chromatograms are shown in fig. 1-3. The results show that: under the chromatographic conditions, the retention time of the florfenicol amine standard substance is 9.407min, and the peak shape is sharp and symmetrical; the extracting solution of the muscle, the liver and the kidney of the blank pig is directly analyzed without solid phase extraction or thin-layer chromatography purification, a large number of impurity peaks exist near the retention time of florfenicol amine, the method specificity is poor, and the florfenicol total residue in the tissues cannot be accurately quantified; the extraction liquid of the muscle, the liver and the kidney of the blank pig is analyzed after solid phase extraction and purification, the florfenicol amine still has obvious interference near the retention time, the method specificity is poor, and the florfenicol total residue in the tissues can not be accurately quantified; the extracting solution of the muscle, the liver and the kidney of the blank pig is purified by thin-layer chromatography and then analyzed, no impurity interference exists near the retention time of florfenicol amine, the method specificity is good, and the florfenicol total residues in the tissues can be accurately quantified.
Example 2 testing of the accuracy and precision of florfenicol Total residue in porcine muscle
Method and device
(1) Accurately weighing 2g of the pig muscle homogenate, adding a proper amount of florfenicol into the homogenate, and converting to ensure that the muscle homogenate contains 0.5 time, 1 time and 1.5 times of florfenicol amine with maximum residual limit amount, 5 samples are repeated at each concentration, and three batches are continuously repeated.
(3) The above samples were treated according to the hydrolysis, washing, extraction, concentration and thin-layer chromatography purification procedures shown in example 1 and analyzed according to the chromatographic conditions shown in example 1. Quantifying florfenicol amine by adopting a standard curve, detecting a series of florfenicol amine standard products with concentration by high performance liquid chromatography, and obtaining a regression equation y which is 282116x +9413.7 by taking the concentration of the florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate, wherein x is the concentration of the florfenicol amine and y is the peak area; substituting the florfenicol amine peak area detected by the high performance liquid chromatography of the detected sample into the linear equation, and calculating to obtain the concentration of the florfenicol amine in the muscle of the pig.
Second, result in
Method a typical chromatogram for detecting florfenicol total residue (expressed as florfenicol amine) in the muscle of pigs is shown in fig. 4, and the mean recovery of florfenicol amine is measured to be 80.86-108.81%, the relative standard deviation in the day is less than 12.24%, and the relative standard deviation in the day is less than 9.82%. The accuracy and precision test results are shown in table 1.
Table 1 accuracy and precision results of the method in pig muscle
The result shows that the detection method has higher accuracy and precision and can be used for determining the florfenicol total residues in the pig muscle.
Example 3 accuracy and precision testing of florfenicol Total residue in pig liver
Method and device
(1) Accurately weighing 2g of pig liver homogenate, adding a proper amount of florfenicol into the homogenate, and converting to ensure that the liver homogenate contains 0.5 time, 1 time and 1.5 times of florfenicol amine with maximum residual limit amount, 5 samples are repeated at each concentration, and three batches are continuously repeated.
(3) The above samples were treated according to the hydrolysis, washing, extraction, concentration and thin-layer chromatography purification procedures shown in example 1 and analyzed according to the chromatographic conditions shown in example 1. Quantifying florfenicol amine by adopting a standard curve, detecting a series of florfenicol amine standard products with concentration by high performance liquid chromatography, and obtaining a regression equation y which is 282116x +9413.7 by taking the concentration of the florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate, wherein x is the concentration of the florfenicol amine and y is the peak area; substituting the florfenicol amine peak area of the detected sample detected by high performance liquid chromatography into the linear equation, and calculating to obtain the florfenicol amine concentration in the pig liver.
Second, result in
Method a typical chromatogram for detecting florfenicol total residue (expressed as florfenicol amine) in pig liver is shown in figure 4, and the average recovery rate of florfenicol amine is measured to be 81.37-108.14%, the relative standard deviation in days is less than 13.08%, and the relative standard deviation in days is less than 11.16%. The accuracy and precision test results are shown in table 2.
TABLE 2 accuracy and precision results of the method in pig liver
The result shows that the detection method has higher accuracy and precision and can be used for determining the florfenicol total residues in the pig liver.
Example 4 accuracy and precision testing of florfenicol Total residue in pig Kidney
Method and device
(1) Accurately weighing 2g of pig kidney homogenate, adding a proper amount of florfenicol into the homogenate, and converting to ensure that the kidney homogenate contains 0.5 time, 1 time and 1.5 times of florfenicol amine with maximum residual limit amount, 5 samples are repeated at each concentration, and three batches are continuously repeated.
(3) The above samples were treated according to the hydrolysis, washing, extraction, concentration and thin-layer chromatography purification procedures shown in example 1 and analyzed according to the chromatographic conditions shown in example 1. Quantifying florfenicol amine by adopting a standard curve, detecting a series of florfenicol amine standard products with concentration by high performance liquid chromatography, and obtaining a regression equation y which is 282116x +9413.7 by taking the concentration of the florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate, wherein x is the concentration of the florfenicol amine and y is the peak area; substituting the florfenicol amine peak area of the detected sample detected by high performance liquid chromatography into the linear equation, and calculating to obtain the florfenicol amine concentration in the pig kidney.
Second, result in
Method a typical chromatogram for detecting florfenicol total residue (expressed as florfenicol amine) in pig kidney is shown in fig. 4, and the average recovery rate of florfenicol amine is measured to be 80.21-102.50%, the relative standard deviation in day is less than 7.53%, and the relative standard deviation in day is less than 6.52%. The accuracy and precision test results are shown in table 3.
TABLE 3 accuracy and precision results of the method in pig Kidney
The result shows that the detection method has higher accuracy and precision and can be used for determining the florfenicol total residues in the pig kidney.
EXAMPLE 5 analysis of actual samples from different sources
Method and device
(1) Pork tenderloin, liver and kidney were purchased from 3 different supermarkets in wuhan city. The pretreatment procedure described in example 1 was followed and the chromatographic conditions described in example 1 were followed. Quantifying florfenicol amine by adopting a standard curve, detecting a series of florfenicol amine standard products with concentration by high performance liquid chromatography, and obtaining a regression equation y which is 282116x +9413.7 by taking the concentration of the florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate, wherein x is the concentration of the florfenicol amine and y is the peak area; substituting the florfenicol amine peak area detected by the high performance liquid chromatography of the detected sample into the linear equation, and calculating to obtain the concentration of the florfenicol amine in the sample.
Second, result in
The results of the florfenicol total residue assay in muscle, liver and kidney of pigs from different sources are shown in table 4. Through determination, florfenicol amine is detected in 2 parts of livers, wherein the residual concentration of the florfenicol amine contained in the livers is lower than the maximum residual limit specified in European Union and China.
TABLE 4 florfenicol Total residue (in florfenicol amine) in porcine edible tissues from different sources
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 (4)
1. A method for detecting florfenicol total residue in edible pig tissue is characterized by comprising the following steps:
(1) taking edible pig tissue, and homogenizing in a tissue homogenizer at a revolution of 3000-10000r/min for 2-5 min;
(2) adding high-concentration hydrochloric acid solution into the tissue homogenate, and placing in a water bath after vortexing until the tissue is completely hydrolyzed, wherein the time of vortexing is 1-3 min; wherein the final molar concentration of the hydrochloric acid solution is 4-8N, the mass-volume ratio of the tissue homogenate to the hydrochloric acid solution is (1-2) g, (2-8) mL, the water bath temperature is 80-100 ℃, and the water bath time is 2-24 h; florfenicol and metabolites are completely hydrolyzed into florfenicol amine under the condition;
(3) after cooling, adding ethyl acetate into the hydrolyzed sample, carrying out vortex for 1-3min, centrifuging for 5-10min at 3000-; wherein the volume ratio of the hydrolyzed sample to the ethyl acetate is (3-6) to (4-40);
(4) adding a high-concentration sodium hydroxide solution into the residual sample to adjust the pH value to 10-12; wherein, the mass percentage concentration of the sodium hydroxide solution is 20-50%, and the adding volume is 0.5-5 mL;
(5) adding ethyl acetate into the alkalized sample, performing vortex for 1-3min, centrifuging for 5-10min at 3000-8000g, and transferring the ethyl acetate into another clean centrifugal tube; wherein the volume ratio of the alkalized sample to the ethyl acetate is (3-8) to (4-10);
(6) repeating the above operation once with equal amount of ethyl acetate, and combining ethyl acetate;
(7) transferring ethyl acetate into a centrifugal tube containing a proper amount of glacial acetic acid solution in batches, volatilizing the ethyl acetate by air flow, wherein the volatilizing temperature of the air flow is 35-60 ℃; wherein, the volume percentage concentration of the glacial acetic acid solution is 1 to 10 percent, and the volume is 0.2 to 1 mL;
(8) adding a proper amount of normal hexane into the residual glacial acetic acid solution, carrying out vortex for 1-3min, centrifuging for 5-10min at 3000-8000g, and discarding the normal hexane; wherein the volume ratio of the residual glacial acetic acid solution to the normal hexane is (0.2-1) to (4-10);
(9) blowing the residual glacial acetic acid solution to the volume of about 0.1mL by using air flow, wherein the temperature of the air flow for volatilizing is 35-60 ℃;
(10) respectively spotting the glacial acetic acid solution and the florfenicol amine standard substance at different positions of the same GF-254 silica gel thin layer plate, and spreading to the top end in a mixed solution with the volume ratio of ethyl acetate, acetone and ammonia water being 2:8: 0.5;
(11) taking out the GF-254 silica gel thin layer plate, inspecting at 254-360nm after the solvent is volatilized, marking the approximate position of the florfenicol amine in the tissue extracting solution according to the expansion distance of the florfenicol amine standard substance, and scraping the silica gel at the corresponding position;
(12) adding 0.5-5mL of acetonitrile-glacial acetic acid mixed solution with the volume ratio of 20:80-80:20 into the scraped silica gel, swirling, centrifuging, taking supernatant, and filtering through a 0.22 mu m organic filter membrane, wherein the volume percentage concentration of the glacial acetic acid in the acetonitrile-glacial acetic acid mixed solution is 1-10%;
(13) detecting the content of florfenicol amine in the filtered sample by high performance liquid chromatography; wherein the high performance liquid chromatography mobile phase is a mixed solution of acetonitrile and phosphate buffer solution according to the volume ratio of 33.3: 66.7; every 500mL of phosphate buffer solution contains 0.34g of sodium dodecyl sulfate, 0.78g of disodium hydrogen phosphate dihydrate, 0.5mL of triethylamine and 0.5mL of phosphoric acid;
the conditions of the high performance liquid chromatography are as follows: the chromatographic separation is carried out in Waters Symmetry C18The chromatographic column is completed, the temperature of the chromatographic column is 32-40 ℃, the flow rate is 0.5-1mL/min, and the detection wavelength is 220-230 nm.
2. A quantitative detection method for florfenicol total residues in edible tissues of pigs is characterized by comprising the following steps:
1) detecting florfenicol total residues in edible tissues of pigs: steps (1) - (13) of the method for detecting florfenicol total residue in porcine edible tissue according to claim 1 are employed;
2) weighing 5-20mg of florfenicol amine standard substance, adding into a 10mL volumetric flask, dissolving with acetonitrile, fixing the volume, and shaking up for later use; taking a sample to be tested, diluting the mobile phase in the step (13) in the step 1) step by step into a series of concentrations, feeding a sample by 10-50 mu L of sample per concentration, repeating the sampling for three times, taking the concentration of florfenicol amine as a horizontal coordinate and taking a peak area as a vertical coordinate to obtain a regression equation y = 282116x +9413.7, wherein x is the concentration of florfenicol amine and y is the peak area; substituting the florfenicol amine peak area of the sample detected in the step (13) in the step 1) by the high performance liquid chromatography into the linear equation, and calculating to obtain the florfenicol amine residual concentration in the edible tissues of the pigs.
3. A method for separating florfenicol total residue from porcine edible tissue by thin layer chromatography, comprising steps (1) - (12) of the method of claim 1.
4. Use of the method of detecting florfenicol total residue in porcine edible tissue according to claim 1 or the quantitative detection method according to claim 2 for detecting florfenicol residue in porcine edible tissue.
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| CN111693639B (en) * | 2020-06-12 | 2022-11-18 | 扬州大学 | Confirmation analysis method for detecting penicillin G residue in poultry tissue, poultry egg or pork |
| CN115248276B (en) * | 2022-06-23 | 2023-11-24 | 江苏省农业科学院 | Method for measuring phenobarbital drug residual quantity in pig edible tissues |
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