CN109946393B - A kind of method for detecting ciprofloxacin residues in livestock and poultry - Google Patents

Abstract

The invention relates to the technical field of animal residue detection, and discloses a method for detecting ciprofloxacin residues in livestock and poultry, including sample pretreatment and HPLC analysis. , it will not cause any harm and pain to animals, and is easy to store. Compared with the tissue of slaughtered animals or the feces of animals, the matrix of the present invention taking the hair of livestock and poultry as the sample has little interference effect, and no further purification is required. It is simple, shortens the detection period, and reduces the workload and input cost of pre-processing; the use of trifluoroacetic acid acidified methanol as the extraction solution improves the extraction efficiency on the one hand, and on the other hand, it can also precipitate calcium in the hair of livestock and poultry. Reduce matrix interference and effectively improve the accuracy of detection results.

Description

A kind of method for detecting ciprofloxacin residues in livestock and poultry

technical field

The invention relates to the technical field of animal residue detection, in particular to a method for detecting ciprofloxacin residues in livestock and poultry.

Background technique

Quinolone is a new class of broad-spectrum antibacterial drugs. Because of its strong antibacterial activity, good oral absorption, and no cross-resistance with other antibacterial drugs, it has become the main drug for the treatment of bacterial infectious diseases. Quinolones are classified into first, second, third, and fourth generations according to their invention order and different antibacterial properties. Ciprofloxacin is the third generation quinolone, which has a wide antibacterial range, high antibacterial activity and strong tissue penetration. It is widely used in the production of livestock and poultry products. However, such drugs have serious and persistent adverse reactions, including tendon damage, central nervous system disorders, etc., and can also cause cancer, mutagenesis, and allergic reactions. Long-term consumption of livestock and poultry products with ciprofloxacin residues will definitely cause harm to human health. serious injury. Therefore, realizing the rapid and sensitive detection of ciprofloxacin in livestock and poultry products has positive significance in ensuring food safety.

At present, the detection of veterinary drug residues usually takes the tissues of slaughtered animals such as muscle, liver, etc. or animal feces as samples, but the protein and fat content in muscle or liver are high, and the feces contain a large amount of metal ions and other impurities. It affects the extraction of the target compound, which will lead to low reliability and stability of the detection results. In addition, in order to reduce the interference of the matrix effect on the chromatographic analysis, it is generally necessary to purify the extract. workload.

SUMMARY OF THE INVENTION

Therefore, the present invention aims to provide a method for detecting ciprofloxacin residues in livestock and poultry that can be sampled in vivo, which is simple to operate, rapid and sensitive, and has high reliability and stability of detection results.

To this end, the present invention provides a method for detecting ciprofloxacin residues in livestock and poultry, including the pretreatment and HPLC analysis of the sample, and the pretreatment of the sample includes the following steps:

Take clean and dry livestock and poultry hair, add extracting solution, extract at least 24 hours at a temperature of not lower than 65 °C, take the supernatant, remove the organic matter in the supernatant, and obtain the sample to be tested;

The extraction solution is acidified methanol with trifluoroacetic acid, and the concentration of the trifluoroacetic acid in 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.

Further, the removal of organic matter in the supernatant is performed by nitrogen blowing at 35-45°C.

Further, the following steps are also included before adding the extract to the hair of the livestock and poultry:

Take the hair of the neck area of livestock and poultry, wash it with pure water at least 3 times, cut it into small pieces of 4-6mm, dry it at 40-60°C, and seal it for later use.

Further, the chromatographic conditions of the HPLC analysis are:

Chromatographic column: C18 250mm×4.6nm, the filler is octadecylsilane bonded silica gel, and the particle size is 5μm;

Mobile phase: acetonitrile-0.05mol/L phosphoric acid solution (v:v=15:85);

Flow rate: 0.8mL/min;

Injection volume: 10 μL;

Column temperature: 30℃;

Detector: Fluorescence detector with excitation wavelength of 280 nm and emission wavelength of 450 nm.

Further, the preparation of ciprofloxacin standard solution and sample solution to be tested is also included before the HPLC analysis, which specifically includes the following steps:

Get ciprofloxacin standard substance, add described mobile phase and dilute into at least 3 kinds of ciprofloxacin standard solutions of different concentrations;

Take the sample to be tested, add the mobile phase, and fully dissolve the sample to be tested to obtain the sample solution to be tested.

Further, the HPLC analysis comprises the steps:

Draw 10 μL of the ciprofloxacin standard solution of different concentrations and inject it into the HPLC analyzer, and draw the standard curve diagram of ciprofloxacin peak area-solution concentration according to the measurement result;

Pipette 10 μL of the sample solution to be tested and inject it into the HPLC analyzer, and calculate the content of ciprofloxacin in the sample solution to be tested according to the peak area and the standard curve.

The technical scheme of the present invention has the following advantages:

1. The method for detecting ciprofloxacin residues in livestock and poultry provided by the present invention takes the hair of livestock and poultry as a sample, which is convenient for sampling, has no destructiveness, does not cause any injury and pain of animals, is easy to store, and is used after slaughtering. Compared with animal tissue or animal feces as the sample, the present invention takes the animal hair as the sample, and the matrix interference effect is small, and no further purification is required, the operation is simple, the detection period is shortened, and the workload and input cost of the preliminary treatment are reduced; The use of acidified methanol as the extraction solution improves the extraction efficiency on the one hand, and on the other hand, it can also precipitate calcium in the hair of livestock and poultry, reduce matrix interference, and effectively improve the accuracy of detection results.

2. In the method for detecting ciprofloxacin residues in livestock and poultry provided by the present invention, the sample to be tested is prepared in an anaerobic or oxygen-limited environment, which improves the stability of the sample and effectively avoids the possibility of sample deterioration or contamination .

3. The method for detecting ciprofloxacin residues in livestock and poultry provided by the present invention selects the hair in the neck region of livestock and poultry as the sampling object, and the cumulative amount of ciprofloxacin is high, highly stable, and has a longer detection time window. , which improves the reliability and stability of the detection results. The cleaning of livestock and poultry hair can effectively remove lipids and other exogenous interference, and further improve the accuracy of the detection results.

4. The method for detecting ciprofloxacin residues in livestock and poultry provided by the present invention can accurately detect the residual amount of ciprofloxacin in livestock and poultry hair by optimizing the combination of conditions such as the mobile phase and the distribution ratio of each component, flow rate, temperature, etc. The method has the characteristics of simple operation and high accuracy, and is suitable for quantitative analysis of ciprofloxacin residues in livestock and poultry.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the HPLC chromatogram of the sample solution to be tested in the embodiment of the present invention 1;

FIG. 2 is the HPLC chromatogram of the spiked sample solution (spike concentration is 5 μg/mL) in Example 1 of the present invention.

Detailed ways

The following examples are provided for a better understanding of the present invention, and are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by combining with the features of other prior art shall fall within the protection scope of the present invention.

If the specific experimental steps or conditions are not indicated in the examples, it can be carried out according to the operations or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.

Example 1

The present embodiment provides a method for detecting ciprofloxacin residues in livestock and poultry, comprising the following steps:

(1) Take the hair of the chicken neck area, wash it three times with pure water, cut it into small pieces of 4-6 mm, dry it at 50°C, put it in a ziplock bag and place it in a drying dish for use.

(2) Weigh 0.15 g of the above-treated chicken feathers, place them in a spiral glass anaerobic tube, add 10 mL of trifluoroacetic acid to acidify methanol (the concentration of trifluoroacetic acid is 0.2 mol/L), heat to 70°C for extraction for 24 hours, and take 5 mL of the supernatant was dried under nitrogen at 40°C to obtain the sample to be tested.

(3) Preparation of ciprofloxacin standard solution, sample solution to be tested and sample solution for adding standard

Weigh 5.0 mg of standard ciprofloxacin, put it in a 5 mL volumetric flask, dissolve it with mobile phase (acetonitrile-0.05 mol/L phosphoric acid, v:v=15:85) and dilute to the mark, shake well to get 1 mg /mL cyclopyrifloxacin stock solution. Accurately pipette 1mL of the above ciprofloxacin stock solution into a 10mL volumetric flask, dissolve and dilute to the scale line with mobile phase (acetonitrile-0.05mol/L phosphoric acid, v:v=15:85), and dilute to a concentration of 100μg/mL The working solution was diluted according to the above method to obtain standard solutions with concentrations of 0.05 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 1.0 μg/mL, 5.0 μg/L, 10 μg/mL and 50 μg/mL, Store at 0°C in the dark for future use;

Add 1 mL of mobile phase (acetonitrile-0.05mol/L phosphoric acid, v:v=15:85) to the above-mentioned sample to be tested, and fully dissolve to obtain the sample solution to be tested;

Weigh 0.15g chicken feathers treated in step (1), add 0.1mL 5μg/mL standard solution, let stand for 2h, add 10mL trifluoroacetic acid acidified methanol (the concentration of trifluoroacetic acid is 0.2mol/L), heat to 65 Extraction at ℃ for 24h, take 5 mL of supernatant, dry it under nitrogen at 40 ℃, to obtain the sample to be tested, add 1 mL of mobile phase (acetonitrile-0.05mol/L phosphoric acid, v:v=15:85), that is, the obtained the spiked sample solution.

(4) HPLC analysis

Chromatographic analysis conditions: chromatographic column: C18 250mm×4.6nm, filler is octadecylsilane bonded silica gel, particle size is 5μm; mobile phase: acetonitrile-0.05mol/L phosphoric acid solution (v:v=15:85 ); flow rate: 0.8 mL/min; injection volume: 10 μL; column temperature: 30 °C; detector: fluorescence detector, excitation wavelength is 280 nm, emission wavelength is 450 nm.

Pipette 10 μL of ciprofloxacin standard solution of 0.05 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 1.0 μg/mL, 5.0 μg/L, 10 μg/mL, 50 μg/mL and 100 μg/mL respectively, and inject them into HPLC analysis According to the measurement results, draw the standard curve of ciprofloxacin peak area-solution concentration, fit, and find the regression equation and correlation coefficient. The results showed that in the concentration range of 0.05～100μg/mL, the linear relationship was good, and the correlation coefficients were all above 0.999.

Draw 10 μL of the sample solution to be tested, inject it into the HPLC analyzer, and see Figure 1 for the analysis spectrum. According to the peak area of ciprofloxacin and the above standard curve, the concentration of ciprofloxacin in the sample solution to be tested is obtained, and the ciprofloxacin in chicken feathers is calculated by calculating. The residual amount is shown in Table 1 below.

Draw 10 μL of the spiked sample solution and inject it into the HPLC analyzer. The analytical spectrum is shown in Figure 2. According to the ciprofloxacin peak area and the above standard curve graph, the concentration of ciprofloxacin in the spiked sample solution is obtained, and the spiked recovery rate is calculated. See Table 1 below.

Table 1 Ciprofloxacin Residual Amount and Standard Recovery Rate

(Note: 1 is the sample solution to be tested; 2 is the sample solution with added standard).

As can be seen from the data in the above table, the detection method of the present invention has good accuracy.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (3)

1. a method for detecting ciprofloxacin residues in livestock and poultry, comprising the pretreatment of sample and HPLC analysis, it is characterized in that, the pretreatment of described sample comprises the steps: Take clean and dry livestock and poultry hair, add extracting solution, extract at least 24 hours at a temperature of not lower than 65 °C, take the supernatant, remove the organic matter in the supernatant, and obtain the sample to be tested; The extraction solution is acidified methanol with trifluoroacetic acid, and the concentration of the trifluoroacetic acid in methanol is 0.2 mol/L; The preparation of the sample to be tested is carried out in an anaerobic or oxygen-limited environment; The removal of the organic matter in the supernatant is performed by nitrogen blowing at 35-45°C; the following steps are also included before adding the extract to the animal hair: Take the hair of the neck area of livestock and poultry, wash it at least 3 times with pure water, cut it into 4~6mm small pieces, dry it at 40~60°C, and seal it for use; the chromatographic conditions of the HPLC analysis are: Chromatographic column: C18 250mm×4.6nm, the filler is octadecylsilane bonded silica gel, and the particle size is 5μm; Mobile phase: acetonitrile-0.05mol/L phosphoric acid solution, the volume ratio of acetonitrile and 0.05mol/L phosphoric acid solution is 15:85; Flow rate: 0.8mL/min; Injection volume: 10 μL; Column temperature: 30℃; Detector: Fluorescence detector with excitation wavelength of 280 nm and emission wavelength of 450 nm. 2. method according to claim 1, is characterized in that, also comprises the preparation of ciprofloxacin standard solution and test sample solution before carrying out described HPLC analysis, specifically comprises the steps: Get ciprofloxacin standard substance, add described mobile phase and dilute into at least 3 kinds of ciprofloxacin standard solutions of different concentrations; Take the sample to be tested, add the mobile phase, and fully dissolve the sample to be tested to obtain the sample solution to be tested. 3. method according to claim 2, is characterized in that, described HPLC analysis comprises the steps: Draw 10 μL of the ciprofloxacin standard solution of different concentrations and inject it into the HPLC analyzer, and draw the standard curve diagram of ciprofloxacin peak area-solution concentration according to the measurement result; Pipette 10 μL of the sample solution to be tested and inject it into the HPLC analyzer, and calculate the content of ciprofloxacin in the sample solution to be tested according to the peak area and the standard curve.

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