CN109946393B - Method for detecting ciprofloxacin residue in livestock and poultry body - Google Patents
Method for detecting ciprofloxacin residue in livestock and poultry body Download PDFInfo
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Abstract
The invention relates to the technical field of animal residue detection, and discloses a method for detecting ciprofloxacin residues in livestock and poultry bodies, which comprises the steps of sample pretreatment and HPLC analysis, wherein the livestock and poultry hair is used as a sample, the sampling is convenient, no destructiveness is caused, no harm or pain is caused to the animals, and the storage is easy; trifluoroacetic acid acidified methanol is selected as an extracting solution, so that on one hand, the extraction efficiency is improved, on the other hand, calcium in the hair of the livestock and poultry can be precipitated, the matrix interference is reduced, and the accuracy of the detection result is effectively improved.
Description
Technical Field
The invention relates to the technical field of veterinary residue detection, in particular to a method for detecting ciprofloxacin residue in livestock and poultry.
Background
Quinolone drugs (quinolones) are relatively new broad-spectrum antibacterial drugs, and have become main drugs for treating bacterial infectious diseases due to strong antibacterial power, good oral absorption and no cross drug resistance with other antibacterial drugs. The quinolone medicaments are divided into first, second, third and fourth generations according to the difference of the invention and antibacterial property, ciprofloxacin is the third generation quinolone medicaments, has the characteristics of wide antibacterial range, large antibacterial activity, strong tissue penetrating power and the like, and is widely applied to the production of livestock and poultry products. However, the drugs have serious and persistent adverse reactions, including tendon injury, central nervous system disorder and the like, can also cause cancer, mutation and anaphylactic reaction, and the livestock and poultry products with residual ciprofloxacin after long-term consumption can certainly cause serious harm to human health. Therefore, the rapid and sensitive detection of ciprofloxacin in the livestock and poultry products has positive significance in the aspect of guaranteeing food safety.
At present, the tissues of slaughtered animals such as muscles, livers and the like or animal wastes are usually taken as samples for detecting veterinary drug residues, but the contents of proteins and fats in the muscles or livers are high, and the wastes contain a large amount of metal ions and other impurities, so that the extraction of a target object is influenced, the reliability and the stability of a detection result are low, in addition, in order to reduce the interference of a matrix effect on chromatographic analysis, the extract is generally required to be purified, the operation is complex, and the workload of pretreatment is increased.
Disclosure of Invention
Therefore, the invention aims to provide the method for detecting the ciprofloxacin residue in the livestock and poultry body, which can be used for sampling the living body, and has the advantages of simple operation, rapidness, sensitivity and high reliability and stability of the detection result.
Therefore, the invention provides a method for detecting ciprofloxacin residues in livestock and poultry, which comprises the steps of sample pretreatment and HPLC analysis, wherein the sample pretreatment comprises the following steps:
adding the extracting solution into clean and dry livestock and poultry hair, extracting at least for 24 hours at the temperature of not less than 65 ℃, taking supernate, and removing organic matters in the supernate to obtain a sample to be detected;
the extracting solution is trifluoroacetic acid acidified methanol, and the concentration of the trifluoroacetic acid in the methanol is 0.2 mol/L.
Further, the preparation of the sample to be tested is carried out in an anaerobic or oxygen-limited environment.
Furthermore, the removal of the organic matters in the supernatant is carried out by nitrogen blowing treatment at 35-45 ℃.
Further, before adding the extracting solution into the livestock and poultry hair, the method also comprises the following steps:
the method comprises the following steps of taking hair in the neck area of livestock and poultry, washing the hair with pure water for at least 3 times, cutting the hair into 4-6 mm small sections, drying the small sections at the temperature of 40-60 ℃, and sealing the small sections for later use.
Further, the chromatographic conditions for the HPLC analysis were:
and (3) chromatographic column: c18250 mm × 4.6nm, and the filler is octadecylsilane chemically bonded silica with particle size of 5 μm;
mobile phase: acetonitrile-0.05 mol/L phosphoric acid solution (v: v ═ 15: 85);
flow rate: 0.8 mL/min;
sample introduction amount: 10 mu L of the solution;
column temperature: 30 ℃;
a detector: and the fluorescence detector has an excitation wavelength of 280nm and an emission wavelength of 450 nm.
Further, the method also comprises the preparation of ciprofloxacin standard solution and sample solution to be detected before the HPLC analysis, and specifically comprises the following steps:
taking a ciprofloxacin standard, adding the mobile phase to dilute into ciprofloxacin standard solutions with at least 3 different concentrations;
and taking the sample to be detected, adding the mobile phase, and fully dissolving the sample to be detected to obtain the sample solution to be detected.
Further, the HPLC analysis comprises the steps of:
sucking 10 mu L of ciprofloxacin standard solutions with different concentrations, injecting the ciprofloxacin standard solutions into an HPLC analyzer, and drawing a standard curve graph of ciprofloxacin peak area-solution concentration according to a measurement result;
and sucking 10 mu L of the sample solution to be detected, injecting the sample solution into an HPLC analyzer, and calculating to obtain the content of ciprofloxacin in the sample solution to be detected according to the peak area and the standard curve graph.
The technical scheme of the invention has the following advantages:
1. the method for detecting ciprofloxacin residues in the livestock and poultry takes the livestock and poultry hair as a sample, is convenient to sample, has no destructiveness, can not cause any damage and pain of animals, is easy to store, and has the advantages that compared with the method taking the tissues of the slaughtered animals or the excrement of the animals as the sample, the matrix interference influence of the livestock and poultry hair as the sample is small, further purification is not needed, the operation is simple, the detection period is shortened, and the workload and the investment cost of early-stage treatment are reduced; the acidified methanol is selected as the extracting solution, so that on one hand, the extraction efficiency is improved, on the other hand, the calcium in the livestock hair can be precipitated, the matrix interference is reduced, and the accuracy of the detection result is effectively improved.
2. According to the method for detecting ciprofloxacin residues in livestock and poultry, the sample to be detected is prepared in an anaerobic or oxygen-limited environment, so that the stability of the sample is improved, and the possibility of deterioration or pollution of the sample is effectively avoided.
3. According to the method for detecting ciprofloxacin residues in livestock and poultry, the hair in the neck area of the livestock and poultry is selected as a sampling object, the ciprofloxacin accumulation is high and stable, a longer detection time window is provided, and the reliability and stability of the detection result are improved. The hair of the livestock and poultry is cleaned, lipid and other exogenous interference are effectively removed, and the accuracy of the detection result is further improved.
4. According to the method for detecting the ciprofloxacin residue in the livestock and poultry, provided by the invention, the residual amount of the ciprofloxacin in the hair of the livestock and poultry can be accurately detected by optimally combining the conditions of the mobile phase, the distribution ratio of each component, the flow speed, the temperature and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an HPLC chromatogram of a sample solution to be tested in example 1 of the present invention;
FIG. 2 is an HPLC chromatogram of a spiked sample solution (spiked concentration 5. mu.g/mL) in example 1 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
Example 1
The embodiment provides a method for detecting ciprofloxacin residue in livestock and poultry, which comprises the following steps:
(1) taking hair in the neck area of the chicken, washing the hair for 3 times by using pure water, cutting the hair into small sections of 4-6 mm, drying the small sections at 50 ℃, putting the small sections into a self-sealing bag and placing the self-sealing bag into a drying dish for later use.
(2) Weighing 0.15g of the treated chicken feather, placing the chicken feather into a spiral glass anaerobic tube, adding 10mL of trifluoroacetic acid acidified methanol (the concentration of trifluoroacetic acid is 0.2mol/L), heating to 70 ℃, extracting for 24 hours, taking 5mL of supernatant, and drying under the condition of nitrogen at 40 ℃ to obtain a sample to be detected.
(3) Preparing ciprofloxacin standard solution, sample solution to be detected and labeled sample solution
Weighing 5.0mg of ciprofloxacin standard, placing the ciprofloxacin standard in a 5mL volumetric flask, dissolving the ciprofloxacin standard by using a mobile phase (acetonitrile-0.05 mol/L phosphoric acid, v: 15:85), fixing the volume to a scale mark, and shaking up to obtain a 1mg/mL cyclobarbituric Saxinus stock solution. Accurately sucking 1mL of the ciprofloxacin stock solution into a 10mL volumetric flask, dissolving and diluting the ciprofloxacin stock solution to a scale mark by using a mobile phase (acetonitrile-0.05 mol/L phosphoric acid, v: 15:85), diluting the ciprofloxacin stock solution into a working solution with the concentration of 100 mu g/mL, diluting the working solution according to the method to obtain standard solutions with the concentrations of 0.05 mu g/mL, 0.1 mu g/mL, 0.5 mu g/mL, 1.0 mu g/mL, 5.0 mu g/L, 10 mu g/mL and 50 mu g/mL, and storing the standard solutions at 0 ℃ in a dark place for later use;
adding 1mL of mobile phase (acetonitrile-0.05 mol/L phosphoric acid, v: v is 15:85) into the sample to be detected, and fully dissolving to obtain a sample solution to be detected;
weighing 0.15g of the chicken feather treated in the step (1), adding 0.1mL of 5 mu g/mL standard solution, standing for 2h, adding 10mL of trifluoroacetic acid acidified methanol (the concentration of trifluoroacetic acid is 0.2mol/L), heating to 65 ℃, extracting for 24h, taking 5mL of supernate, drying at 40 ℃ under the condition of nitrogen to obtain a sample to be tested, and adding 1mL of mobile phase (acetonitrile-0.05 mol/L phosphoric acid, v: 15:85) to obtain the sample solution to be tested.
(4) HPLC analysis
Chromatographic analysis conditions: a chromatographic column: c18250 mm × 4.6nm, octadecylsilane chemically bonded silica as filler, and particle size of 5 μm; mobile phase: acetonitrile-0.05 mol/L phosphoric acid solution (v: v ═ 15: 85); flow rate: 0.8 mL/min; sample injection amount: 10 mu L of the solution; column temperature: 30 ℃; a detector: and the fluorescence detector has an excitation wavelength of 280nm and an emission wavelength of 450 nm.
And respectively sucking 10 mu L of ciprofloxacin standard solutions of 0.05 mu g/mL, 0.1 mu g/mL, 0.5 mu g/mL, 1.0 mu g/mL, 5.0 mu g/L, 10 mu g/mL, 50 mu g/mL and 100 mu g/mL, injecting the ciprofloxacin standard solutions into an HPLC analyzer, drawing a standard curve graph of ciprofloxacin peak area-solution concentration according to the measurement results, fitting, and solving a regression equation and a correlation coefficient. The result shows that the linear relation is good and the correlation coefficient is more than 0.999 within the concentration range of 0.05-100 mug/mL.
And (3) sucking 10 mu L of sample solution to be detected, injecting the sample solution into an HPLC analyzer, analyzing a map shown in a figure 1, obtaining the concentration of ciprofloxacin in the sample solution to be detected according to the ciprofloxacin peak area and the standard curve graph, and calculating to obtain the residual amount of ciprofloxacin in the chicken feather shown in the following table 1.
And (3) sucking 10 mu L of the standard sample solution, injecting the standard sample solution into an HPLC analyzer, obtaining an analysis map shown in figure 2, obtaining the concentration of ciprofloxacin in the standard sample solution according to the ciprofloxacin peak area and the standard curve graph, and calculating to obtain the standard recovery rate shown in the following table 1.
TABLE 1 ciprofloxacin residual and spiking recovery
(remarks: 1 is sample solution to be measured; 2 is labeled sample solution).
As can be seen from the data in the above table, the detection method of the present invention has good accuracy.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (3)
1. The method for detecting ciprofloxacin residues in livestock and poultry comprises the steps of sample pretreatment and HPLC analysis, and is characterized in that the sample pretreatment comprises the following steps:
adding the extracting solution into clean and dry livestock and poultry hair, extracting at least for 24 hours at the temperature of not less than 65 ℃, taking supernate, and removing organic matters in the supernate to obtain a sample to be detected;
the extracting solution is trifluoroacetic acid acidified methanol, and the concentration of the trifluoroacetic acid in the methanol is 0.2 mol/L;
the preparation of the sample to be detected is carried out in an oxygen-free or oxygen-limited environment;
the step of removing the organic matters in the supernatant is carried out by nitrogen blowing treatment at 35-45 ℃; before the extracting solution is added into the livestock and poultry hair, the method also comprises the following steps:
taking hair in the neck area of livestock and poultry, washing with pure water for at least 3 times, cutting into 4-6 mm small sections, drying at 40-60 ℃, and sealing for later use; the chromatographic conditions for the HPLC analysis were:
and (3) chromatographic column: c18250 mm × 4.6nm, and the filler is octadecylsilane chemically bonded silica with a particle size of 5 μm;
mobile phase: acetonitrile-0.05 mol/L phosphoric acid solution, wherein the volume ratio of the acetonitrile to the 0.05mol/L phosphoric acid solution is 15: 85;
flow rate: 0.8 mL/min;
sample injection amount: 10 mu L of the solution;
column temperature: 30 ℃;
a detector: and the fluorescence detector has an excitation wavelength of 280nm and an emission wavelength of 450 nm.
2. The method according to claim 1, further comprising the preparation of ciprofloxacin standard solution and sample solution to be tested before the HPLC analysis, and specifically comprising the following steps:
taking a ciprofloxacin standard, adding the mobile phase to dilute into ciprofloxacin standard solutions with at least 3 different concentrations;
and taking the sample to be detected, adding the mobile phase, and fully dissolving the sample to be detected to obtain the sample solution to be detected.
3. The method of claim 2, wherein the HPLC analysis comprises the steps of:
sucking 10 mu L of ciprofloxacin standard solutions with different concentrations, injecting the ciprofloxacin standard solutions into an HPLC analyzer, and drawing a standard curve graph of ciprofloxacin peak area-solution concentration according to a determination result;
and (3) sucking 10 mu L of the sample solution to be detected, injecting the sample solution to be detected into an HPLC analyzer, and calculating to obtain the content of the ciprofloxacin in the sample solution to be detected according to the peak area and the standard curve graph.
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