Method for detecting antibiotic residues in chicken

Abstract

The application provides a method for detecting antibiotic residues in chicken, and belongs to the technical field of food detection. The method comprises the following steps: (1) freezing pretreatment of the chicken sample; (2) liquid phase extraction of the pretreated chicken sample, wherein the liquid phase extraction adopts a solution system, and the solution system comprises acidified acetonitrile, EDTA-2Na solution and phosphate; (3) enrichment and purification of the chicken extract, wherein the enrichment and purification adopts a series connection of an HLB solid phase extraction column, a C18 solid phase extraction column and an MCX solid phase extraction column; (4) drawing of standard curves of various antibiotics; and (5) detection of the sample to be measured by using a portable Raman spectrometer, and calculation of the content of each antibiotic in the sample according to the standard curves. The method uses freezing pretreatment, solution system extraction and the mode of enrichment and purification of three solid phase extraction columns, so that the extraction efficiency of the antibiotics in the chicken sample is significantly improved, and the detection accuracy of the antibiotics is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a method for detecting antibiotic residues in chicken. Background Art

[0002] With the rapid development of my country's livestock and poultry farming industry, the scale of livestock farming is growing. In the animal husbandry process, antibiotics play an indispensable role in the prevention and treatment of diseases, as well as in reducing the growth cycle of livestock and poultry. However, improper or abused use of antibiotics leads to their residues in animal-derived foods. Studies have shown that antibiotics can be transferred and accumulated in chicken. Residual antibiotics may directly cause disease through low-dose exposure or indirectly cause harm through antibiotic resistance, thereby adversely affecting humans. Therefore, it is of great significance to detect antibiotic residues in animal-derived foods.

[0003] Chicken is rich in protein and lipids, which have a great influence on the results of antibiotic residue analysis. Therefore, establishing an effective antibiotic separation and enrichment method is particularly important for accurately determining the content of antibiotics in chicken. At present, the commonly used extraction methods in chicken sample pretreatment mainly include liquid-liquid extraction, microwave extraction, accelerated solvent extraction, supercritical fluid extraction and solid phase extraction. Among them, although the liquid-liquid extraction method is simple to operate and low in cost, its selectivity is poor and may cause more impurities to be extracted together, increasing the difficulty of subsequent purification steps. The substances to be separated in the two-phase aqueous system are affected by forces such as ionic bonds, hydrogen bonds, hydrophobic interactions and van der Waals forces, as well as the system environment, thus showing better selectivity. This method has been used to separate various proteins, enzymes and other substances in organisms.

[0004] Although the two-phase aqueous system has the advantage of high selectivity for the extraction and separation of specific substances in animal-derived foods, for the small molecule organic solvent-inorganic salt two-phase aqueous system, how to choose the appropriate combination of organic solvent and inorganic salt to obtain the best extraction effect is still a problem that needs to be studied. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting antibiotic residues in chicken, so as to solve the problem that when extracting antibiotics from chicken, the extraction selectivity is low, resulting in inaccurate detection results.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting antibiotic residues in chicken comprises the following steps:

[0008] (1) Pre-freezing of chicken samples

[0009] (2) Liquid phase extraction of pretreated chicken samples

[0010] The pretreated chicken sample was cut into pieces and crushed into minced meat, which was then added to the solution system and stirred. The mass volume ratio of the minced meat to the solution system was (1-2) g:10 mL. Ultrasonic treatment was also applied during stirring. After completion, the supernatant was collected, the resulting residue was redissolved in the solution system, and the supernatant was collected again. The supernatants were combined to obtain a chicken extract.

[0011] The solution system includes acidified acetonitrile, EDTA-2Na solution and phosphate;

[0012] (3) Enrichment and purification of chicken extract

[0013] An HLB solid phase extraction column, a C18 solid phase extraction column, and an MCX solid phase extraction column are connected in series, activated with methanol, and balanced with water. The chicken extract is passed through the solid phase extraction column, the filtrate is discarded, and the extract is washed with an eluent. The washed liquid is discarded, and the extract is eluted with an eluent. The eluent is blown dry and redissolved in a methanol-water solution to obtain a chicken concentrate, which is the sample to be tested;

[0014] (4) Draw standard curves for each antibiotic;

[0015] (5) The samples were tested using a Raman spectrometer, and the content of each antibiotic in the sample was calculated based on the standard curve.

[0016] To improve the extraction and purification of antibiotics from chicken and thereby enhance detection accuracy, the present invention utilizes a freeze pretreatment, extraction with a specifically formulated solution system, and enrichment and purification using three solid-phase extraction columns in series. The results demonstrate improved detection accuracy. The reason for this improvement is that ice crystals formed during the freeze pretreatment rupture cells in the chicken, releasing more antibiotics. The acidified acetonitrile in the solution effectively extracts both fat- and water-soluble compounds, particularly antibiotics. The presence of trifluoroacetic acid enhances extraction efficiency and increases antibiotic solubility. EDTA-2Na is a chelating agent that complexes metal ions, preventing interference with the extraction process and improving extraction efficiency. Phosphate stabilizes the solution pH, preventing denaturation or degradation of antibiotics during extraction. Furthermore, phosphate enhances the solubility of antibiotics in the extract. Solid-phase extraction columns effectively enrich and purify antibiotics from the extract, removing impurities. HLB columns are suitable for the extraction of polar and non-polar compounds, C18 columns for non-polar compounds, and MCX columns for acidic and basic compounds. Different solid phase extraction columns have different selective adsorption capabilities, which can be used to specifically adsorb specific types of antibiotics and improve purity. The multiple columns in series in the present invention can further improve extraction efficiency and purity.

[0017] Preferably, in step (1), the freezing pretreatment process is as follows: freezing the chicken sample at-35 to-20 DEG C for 12 to 24 hours, then thawing, and continuing to store at 4 to 8 DEG C for 3 to 5 days.

[0018] Preferably, in step (2), the ultrasonic treatment power is 500 to 800 W, and the time is 30 to 40 min.

[0019] Preferably, in step (2), the acidified acetonitrile is an acetonitrile solution containing 0.5 to 0.8% trifluoroacetic acid, the concentration of the EDTA-2Na solution is 0.1 M, the volume ratio of the acidified acetonitrile and the EDTA-2Na solution is (1 to 2):(8 to 9), the phosphates include trisodium phosphate and monosodium phosphate according to a mass ratio of (3 to 5):(0.5 to 1.5), and the mass-volume ratio of the phosphates and the acidified acetonitrile is (1.5 to 2.5) g:(8 to 9) mL.

[0020] Preferably, in step (3), the volume percentage of methanol in the methanol aqueous solution is 2%.

[0021] Preferably, in step (3), the weight ratio and the volume ratio of the HLB solid-phase extraction column, the C18 solid-phase extraction column and the MCX solid-phase extraction column are all (1 to 3):1:(1 to 3).

[0022] Preferably, in step (3), the column passing speed of the chicken extract is 3 to 5 mL / min, the elution liquid is a 5% methanol aqueous solution, and the eluent is methanol and 10% ammonia methanol solution.

[0023] Preferably, in step (4), each antibiotic is added to the solution system to configure each antibiotic solution with different concentrations, the liquid phase extraction of step (2) and the enrichment and purification of step (3) are performed on each antibiotic solution with different concentrations respectively, then the portable Raman spectrometer is used for detection, and a standard curve is drawn.

[0024] Compared with the prior art, the application has the following advantages and beneficial effects:

[0025] 1. The freezing pretreatment, liquid phase extraction and ultrasonic assistance can efficiently extract the antibiotics in the chicken, and improve the extraction efficiency.

[0026] 2. The enrichment and purification step of the multi-column series can effectively remove impurities, improve the purity of the target antibiotic, and reduce background interference.

[0027] 3. The whole detection process of the application is simple to operate and suitable for on-site rapid detection. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] This embodiment provides a method for detecting antibiotic residues in chicken, characterized by comprising the following steps:

[0031] (1) Pre-freezing of chicken samples

[0032] 1.5 g of chicken sample was frozen at -30 °C for 18 h, thawed, and stored at 6 °C for another 4 days;

[0033] (2) Liquid phase extraction of pretreated chicken samples

[0034] The pretreated chicken sample was cut into pieces and crushed into minced meat, which was then added to the solution system and stirred. The mass volume ratio of the minced meat to the solution system was 1.5 g:10 mL. Ultrasonic treatment was applied while stirring. The ultrasonic treatment power was 600 W and the time was 35 min. After completion, the supernatant was collected, and the resulting residue was redissolved in 10 mL of the solution system. The supernatant was collected again and the supernatants were combined to obtain a chicken extract.

[0035] The solution system includes acidified acetonitrile, EDTA-2Na solution and phosphate;

[0036] The acidified acetonitrile is an acetonitrile solution containing 0.6% trifluoroacetic acid, the concentration of the EDTA-2Na solution is 0.1 M, the volume ratio of the acidified acetonitrile to the EDTA-2Na solution is 1.5:8.5, the phosphate includes trisodium phosphate and sodium monohydrogen phosphate in a mass ratio of 4:1, and the mass volume ratio of the phosphate to the acidified acetonitrile is 2 g:8.5 mL;

[0037] (3) Enrichment and purification of chicken extract

[0038] The HLB solid phase extraction column (60 mg, 3 mL), the C18 solid phase extraction column (60 mg, 3 mL) and the MCX solid phase extraction column (60 mg, 3 mL) are connected in series by using an adapter, activated by using 3 mL of methanol, and balanced by using 3 mL of water. The chicken extract is passed through the solid phase extraction column at a speed of 4 mL / min, and the filtrate is discarded. The elution is performed by using an eluent, which is 1 mL of 5% (v / v) methanol solution, and the eluted liquid is discarded. Then, the elution is performed by using an eluent, which is 6 mL of methanol and 6 mL of 10% (v / v) ammonia methanol solution. The eluent is blown dry under a nitrogen atmosphere in a 4°C water bath, redissolved in 1 mL of 0.2% (v / v) methanol solution, vortexed at 1000 r / min for 1 min, and then the chicken concentrate is obtained, which is the sample to be detected and used for machine determination.

[0039] (4) Drawing of standard curves of each antibiotic

[0040] Each antibiotic is added to the solution system to prepare each antibiotic solution with different concentrations. The liquid phase extraction of step (2) and the enrichment and purification of step (3) are performed on each antibiotic solution with different concentrations, and then the portable Raman spectrometer is used for detection to draw the standard curve.

[0041] (5) Detection of the sample to be detected by using the portable Raman spectrometer, and calculation of the content of each antibiotic in the sample according to the standard curve.

[0042] The detection of the following representative antibiotics is performed according to the method of Example 1:

[0043] Example 2

[0044] Drawing of the standard curve of penicillin: Penicillin solutions with different concentrations (0.1 μg / L, 1.0 μg / L, 10.0 μg / L, 100.0 μg / L, 1000 μg / L, 10000 μg / L) are prepared, and then 20 mL of the penicillin solutions with different concentrations are subjected to the liquid phase extraction of step (2) and the enrichment and purification of step (3) to obtain penicillin concentrates with different concentrations. 10 μL of the penicillin concentrates with different concentrations are subjected to detection by using the portable Raman spectrometer, and the standard curve is drawn.

[0045] 10 μL of the chicken concentrate to be detected are subjected to detection by using the portable Raman spectrometer, and the content of penicillin in the sample to be detected is calculated according to the standard curve.

[0046] Example 3

[0047] The difference from Example 2 is that the erythromycin content of the chicken sample to be detected is detected.

[0048] Example 4

[0049] The difference from Example 2 is that the ciprofloxacin content in the chicken sample to be tested is detected.

[0050] Example 5

[0051] The difference from Example 2 is that the tetracycline content of the chicken sample to be tested is detected.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that the chicken pretreatment process is different. In this comparative example, no freezing treatment is performed, and the chicken sample is directly cut into pieces and crushed for liquid phase extraction.

[0054] Comparative Example 2

[0055] The difference between this comparative example and Example 1 is that the composition of the solution system is different. In this comparative example, the solution system is acetonitrile.

[0056] Comparative Example 3

[0057] The difference between this comparative example and Example 1 is that the ultrasonic treatment in step (2) is different, and no ultrasonic treatment is performed in this comparative example.

[0058] The liquid phase extraction process of the pretreated chicken sample in this comparative example is as follows:

[0059] The pretreated chicken sample was cut into pieces and crushed into minced meat, which was then added to the solution system and stirred. The mass volume ratio of the minced meat and the solution system was 1.5 g:10 mL. The mixture was stirred for 35 minutes. After completion, the supernatant was taken, and the residue was redissolved in 10 mL of the solution system. The supernatant was taken again and the supernatants were combined to obtain the chicken extract.

[0060] Comparative Example 4

[0061] The difference between this comparative example and Example 1 is that the ultrasonic treatment parameters in step (2) are different, and the ultrasonic power is 1500W.

[0062] Comparative Example 5

[0063] The difference between this comparative example and Example 1 is that the type of solid phase extraction column in step (3) is different, and this comparative example does not contain a C18 solid phase extraction column.

[0064] The process of enriching and purifying the chicken extract in this comparative example is as follows:

[0065] An HLB solid phase extraction column (60 mg, 3 mL) and an MCX solid phase extraction column (60 mg, 3 mL) were connected in series using an adapter. When used, they were activated with 3 mL of methanol and balanced with 3 mL of water. The chicken extract was passed through the solid phase extraction column at a flow rate of 4 mL / min. The filtrate was discarded and the extract was washed with 1 mL of a 5% (v / v) methanol aqueous solution. The washed liquid was discarded and eluted with 6 mL of methanol and 6 mL of a 10% (v / v) ammonia methanol solution. The eluent was dried with nitrogen in a 4°C water bath and redissolved in 1 mL of a 0.2% (v / v) methanol aqueous solution. The chicken concentrate was mixed at 1000 r / min for 1 min to obtain the test sample for determination on the instrument.

[0066] Comparative Example 6

[0067] The difference between this comparative example and Example 1 is that the connection method of the solid phase extraction column in step (3) is different.

[0068] The process of enriching and purifying the chicken extract in this comparative example is as follows:

[0069] An MCX solid phase extraction column (60 mg, 3 mL), a C18 solid phase extraction column (60 mg, 3 mL), and an HLB solid phase extraction column (60 mg, 3 mL) were connected in series using an adapter. When used, they were activated with 3 mL of methanol and balanced with 3 mL of water. The chicken extract was passed through the solid phase extraction column at a flow rate of 4 mL / min. The filtrate was discarded and eluted with 1 mL of 5% (v / v) methanol aqueous solution. The eluted liquid was discarded and eluted with 6 mL of methanol and 6 mL of 10% (v / v) ammonia methanol solution. The eluent was blown dry with nitrogen in a 4°C water bath, redissolved in 1 mL of 0.2% (v / v) methanol aqueous solution, and vortexed at 1000 r / min for 1 min to obtain a chicken concentrate, which was the sample to be tested and used for analysis on the instrument.

[0070] The above embodiment was tested as follows:

[0071] 1. Linear relationship investigation

[0072] Plotting curves between the logarithm of the response value (log I) and the logarithm of the concentration (log C) of the detection methods of Examples 2-5 above, the resulting regression equations (Table 1) all have good linear relationships, indicating that the detection method of this embodiment can effectively detect various antibiotics in chicken samples.

[0073] Table 1 Results of linear relationship investigation of Examples 2-5

[0074] Regression equation <![CDATA[相关系数R 2 ]]> Example 2 lgI = 0.8144 lgC + 1.5451 0.9982 Example 3 lgI = 0.9325 lgC + 1.219 0.9941 Example 4 lgI = 0.8946 lgC + 1.687 0.9953 Example 5 lgI = 0.8558 lgC + 2.014 0.9926

[0075] 2. Repeatability inspection

[0076] A 100.0 ng / mL standard solution of each antibiotic was prepared and tested using the methods of Examples 2-5, respectively. The test was repeated 5 times, and the concentration and RSD value of each antibiotic were calculated based on the linear relationship, as shown in Table 2.

[0077] Table 2 Repeatability test results of Examples 2-5

[0078] 1 2 3 4 5 SD RSD Example 2 97.25 102.37 101.58 97.93 98.95 1.183 0.0119 Example 3 101.55 98.78 100.38 99.84 101.75 0.545 0.00542 Example 4 101.95 98.54 101.23 101.23 99.85 0.612 0.0061 Example 5 97.68 101.27 98.74 98.74 98.54 1.018 0.0102

[0079] It can be seen from the data results in Table 2 that the RSD values ​​of the method of this embodiment are around 1%, which are all less than 5%. This shows that the detection method of this embodiment has good repeatability.

[0080] In addition, according to the data in Table 1 and Table 2, combined with the triple signal-to-noise ratio method (LSD=3SD / S), the detection limits (LSDs) of penicillin, erythromycin, ciprofloxacin, and tetracycline can be calculated to be 4.36 μg / L, 1.75 μg / L, 2.05 μg / L, and 3.57 μg / L, respectively.

[0081] Application Examples

[0082] Freshly slaughtered broilers were purchased from a local market as the test chicken. 100 g of chicken tissue was taken as the test chicken sample. Chicken concentrate was extracted according to the methods of Example 1 and Comparative Examples 1 to 6. The process was repeated twice to obtain two groups of test chicken concentrate samples. A 20 μg / L penicillin standard solution was added to the first group of test chicken concentrate samples. The spiked sample value was calculated using the above method. The sample value was calculated using the second group of test chicken concentrate samples (a blank group without any antibiotics added). The spiked recovery was then calculated. The measurement was repeated three times for each group and the average value was taken. The calculation formula is: spiked recovery = (spiked sample value - sample value) ÷ spiked amount × 100%. The recoveries of erythromycin, ciprofloxacin, and tetracycline standards were calculated using the same method. The results are shown in Table 3.

[0083] Table 3 Spike recovery results (n=3)

[0084]

[0085]

[0086] The above performance test results show that the detection method of Example 1 has a high extraction efficiency for antibiotics in chicken. The spiked recoveries of four antibiotics, penicillin, erythromycin, ciprofloxacin and tetracycline, are between 95% and 105%, indicating that the detection method has a good recovery rate. This is mainly because the present invention uses a specific solution system, a specific chicken pretreatment method, and a specific solid phase extraction column enrichment and purification process in the chicken antibiotic extraction process, resulting in higher antibiotic extraction efficiency and purity, and thus higher detection accuracy.

[0087] The comparative examples, however, lacked the necessary technical solutions, resulting in significantly inferior performance tests compared to the examples. In Comparative Example 1, the chicken was not pre-frozen. The results show that the spiked recoveries determined by the detection method were approximately 90% or 107%, significantly lower than those in Example 1, demonstrating that freezing chicken significantly impacts the accuracy of antibiotic detection. In Comparative Example 2, the solution system was replaced with pure acetonitrile. The results show that this impacted the spiked recoveries and further decreased the accuracy of chicken antibiotic detection, demonstrating that the present invention's use of a specific solution system for chicken sample treatment can improve antibiotic extraction efficiency. In Comparative Example 3, ultrasonic treatment was not used. The results show that the accuracy of the detection method decreased, demonstrating that combining solution extraction with ultrasonic treatment can maximize the extraction efficiency of antibiotics in chicken. In Comparative Example 4, the ultrasonic power was increased. The results show that the accuracy of antibiotic detection in chicken also decreased, indicating that excessive ultrasonic power can damage the structure of the antibiotics, leading to poor detection accuracy. Therefore, ultrasonic treatment at a specific power is crucial for improving the extraction efficiency and detection accuracy of chicken antibiotics. In Comparative Example 5, only two solid-phase extraction columns were used. The results show that the accuracy of antibiotic detection in chicken decreased, indicating that the enrichment and purification using three fixed extraction columns in the present invention can improve the purity of antibiotics, thereby improving detection accuracy. In Comparative Example 6, the order of the three fixed extraction columns was adjusted. The results show that the accuracy of antibiotic detection in chicken also decreased, indicating that the specific serial order of the three solid-phase extraction columns has a significant impact on improving the accuracy of antibiotic detection in chicken. These experimental results further demonstrate the importance of the technical solution defined in the present invention for its technical effectiveness.

[0088] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting antibiotic residues in chicken, wherein the antibiotics are penicillin, erythromycin, ciprofloxacin and tetracycline, characterized in that: The following steps are involved: (1) Pre-freeze the chicken samples; (2) Liquid phase extraction of pretreated chicken samples; The pretreated chicken sample was cut into pieces and crushed into minced meat, which was then added to the solution system and stirred. The mass volume ratio of the minced meat to the solution system was (1-2) g:10 mL. Ultrasonic treatment was also applied during stirring. After completion, the supernatant was collected, the resulting residue was redissolved in the solution system, and the supernatant was collected again. The supernatants were combined to obtain a chicken extract. The solution system includes acidified acetonitrile, EDTA-2Na solution and phosphate; The acidified acetonitrile is an acetonitrile solution containing 0.5-0.8% trifluoroacetic acid, the concentration of the EDTA-2Na solution is 0.1 M, the volume ratio of the acidified acetonitrile to the EDTA-2Na solution is (1-2):(8-9), the phosphate includes trisodium phosphate and sodium monohydrogen phosphate in a mass ratio of (3-5):(0.5-1.5), and the mass volume ratio of the phosphate to the acidified acetonitrile is (1.5-2.5) g:(8-9) mL; (3) Enriching and purifying the chicken extract; An HLB solid-phase extraction column, a C18 solid-phase extraction column, and an MCX solid-phase extraction column were connected in series using an adapter, activated with methanol, and balanced with water. The chicken extract was passed through the solid-phase extraction column, the filtrate was discarded, and the extract was rinsed with eluent. The eluted liquid was discarded and eluted with eluent. The eluent was dried and redissolved in methanol-water solution to obtain a chicken concentrate, which was the sample to be tested. (4) Draw standard curves for each antibiotic; (5) Using Raman spectrometer to test the sample, calculate the content of each antibiotic in the sample according to the standard curve; The freezing pretreatment process is as follows: the chicken sample is frozen at -35 to -20°C for 12 to 24 hours, then thawed and stored at 4 to 8°C for 3 to 5 days.

2. The method for detecting antibiotic residues in chicken according to claim 1, wherein: In step (2), the power of ultrasonic treatment is 500-800W, and the time is 30-40 minutes.

3. The method for detecting antibiotic residues in chicken according to claim 1, wherein: In step (3), the volume percentage of methanol in the methanol aqueous solution is 2%.

4. The method for detecting antibiotic residues in chicken according to claim 1, wherein: In step (3), the weight ratio and volume ratio of the HLB solid phase extraction column, the C18 solid phase extraction column and the MCX solid phase extraction column are all (1-3):1:(1-3).

5. The method for detecting antibiotic residues in chicken according to claim 1, wherein: In step (3), the chicken extract passes through the column at a speed of 3 to 5 mL / min, the eluent is a 5% methanol aqueous solution, and the elution liquid is a methanol and 10% ammonia methanol solution.

6. The method for detecting antibiotic residues in chicken according to claim 1, wherein: In step (4), each antibiotic is added to the solution system to prepare each antibiotic solution of different concentrations, and each antibiotic solution of different concentrations is subjected to liquid phase extraction in step (2) and enrichment and purification in step (3), and then detected using a portable Raman spectrometer to draw a standard curve.

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