Method for detecting residual quantity of antibiotics in chicken

Abstract

The invention provides a method for detecting antibiotic residues in chicken, and belongs to the technical field of food detection. The detection method comprises the following steps: (1) carrying out freezing pretreatment on a chicken sample; (2) the pretreated chicken sample is subjected to liquid phase extraction, a solution system is adopted for liquid phase extraction, and the solution system comprises acidified acetonitrile, an EDTA-2Na solution and phosphate; (3) enriching and purifying the chicken extracting solution, wherein an HLB solid-phase extraction column, a C18 solid-phase extraction column and an MCX solid-phase extraction column which are connected in series are adopted for enriching and purifying; (4) drawing a standard curve of each antibiotic; and (5) detecting the sample to be detected by using the portable Raman spectrometer, and calculating the content of each antibiotic in the sample according to the standard curve. According to the method, freezing pretreatment, solution system extraction and enrichment and purification of three solid-phase extraction columns are adopted for the sample, so that the extraction efficiency of the antibiotics in the chicken sample is remarkably improved, and the detection accuracy of the antibiotics is further 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 livestock and poultry farming in my country, the scale of farming is getting larger and larger. In the process of animal breeding, antibiotics play an indispensable role in the prevention and treatment of diseases and in order to reduce the growth cycle of livestock and poultry. However, improper or abuse of antibiotics causes their residues in animal-derived foods. Studies have shown that antibiotics can be transferred and accumulated in chicken. Residual antibiotics may directly cause diseases through low-dose exposure, or indirectly cause harm through antibiotic resistance, thereby having adverse effects on 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, it is particularly important to establish an effective antibiotic separation and enrichment method 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, the selectivity of this method is poor, which may cause more impurities to be extracted together, increasing the difficulty of subsequent purification steps. The separated substances in the two-phase aqueous system are affected by ionic bonds, hydrogen bonds, hydrophobic interactions, van der Waals forces and other forces as well as the system environment, thus showing better selectivity. This method has been used to separate various proteins and enzymes in organisms.

[0004] Although the aqueous two-phase 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 aqueous two-phase 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 is cut into pieces and crushed into minced meat, and then added into the solution system for stirring, the mass volume ratio of the minced meat to the solution system is (1-2) g: 10 mL, and ultrasonic treatment is applied while stirring. After completion, the supernatant is taken, the obtained residue is redissolved using the solution system, the supernatant is taken again, and the supernatants are 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, the eluent is used for elution, the eluted liquid is discarded, and the eluent is used for elution. The eluent is blown dry and redissolved in a methanol aqueous 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 samples was calculated based on the standard curve.

[0016] In order to improve the extraction and purification process of antibiotics in chicken and thus improve the detection accuracy, the present invention successively uses freezing pretreatment, a solution system of a specific composition for extraction, and three solid phase extraction columns in series for enrichment and purification in the chicken processing process. It can be seen from the results that the detection accuracy is improved. Analysis of the reasons: The ice crystals formed by freezing pretreatment can rupture the cells in the chicken, thereby releasing more antibiotics. The acidified acetonitrile in the solution system can effectively extract fat-soluble and water-soluble compounds, especially antibiotics. The presence of trifluoroacetic acid can enhance the extraction effect and improve the solubility of antibiotics. EDTA-2Na is a chelating agent that can complex metal ions to prevent metal ions from interfering with the extraction process and improve the extraction efficiency. Phosphates can maintain the pH value of the solution stable and prevent antibiotics from denaturing or degrading during the extraction process. At the same time, phosphates can also help antibiotics in the extract to dissolve better. The solid phase extraction column can effectively enrich and purify the antibiotics in the extract and remove impurities. The HLB column is suitable for the extraction of polar and non-polar compounds, the C18 column is suitable for non-polar compounds, and the MCX column is suitable for acidic and alkaline compounds. Different solid phase extraction columns have different selective adsorption capabilities, and can specifically adsorb specific types of antibiotics to improve purity. The multiple columns in series in the present invention can further improve the extraction efficiency and purity.

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

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

[0019] Preferably, in step (2), the acidified acetonitrile is an acetonitrile solution containing 0.5-0.8% trifluoroacetic acid, the concentration of the EDTA-2Na solution is 0.1M, 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.

[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 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).

[0022] Preferably, 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 eluting liquid is methanol and 10% ammonia methanol solution.

[0023] Preferably, in step (4), each antibiotic is added to a solution system to prepare antibiotic solutions of different concentrations, and each antibiotic solution of different concentration is subjected to liquid phase extraction of step (2) and enrichment and purification of step (3), and then detected using a portable Raman spectrometer to draw a standard curve.

[0024] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0025] 1. The present invention can efficiently extract antibiotics from chicken through freezing pretreatment, liquid phase extraction and ultrasound assistance, thereby improving extraction efficiency.

[0026] 2. The present invention adopts a multi-column series enrichment and purification step to effectively remove impurities, improve the purity of the target antibiotics, and reduce background interference.

[0027] 3. The entire detection process of the present invention is easy to operate and suitable for on-site rapid detection. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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, which is 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, and then added into the solution system for stirring. The mass volume ratio of minced meat to the solution system was 1.5 g: 10 mL. Ultrasonic treatment was applied while stirring. The power of ultrasonic treatment was 600 W and the time was 35 min. After completion, the supernatant was taken, and the residue was redissolved with 10 mL of the solution system. The supernatant was taken 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] An HLB solid phase extraction column (60 mg, 3 mL), a C18 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, 3 mL of methanol was used for activation and 3 mL of water was used for balance. The chicken extract was passed through the solid phase extraction column at a rate of 4 mL / min. The filtrate was discarded, and the eluent was used for elution. The eluent was 1 mL of 5% (v / v) methanol aqueous solution. The liquid after elution was discarded, and the eluent was used for elution. The eluent was 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 determination on the machine;

[0039] (4) Draw the standard curve of each antibiotic

[0040] Adding each antibiotic into the solution system to prepare each antibiotic solution of different concentrations, performing the liquid phase extraction of step (2) and the enrichment and purification of step (3) on each antibiotic solution of different concentrations, and then performing detection using a portable Raman spectrometer to draw a standard curve;

[0041] (5) The samples were tested using a portable Raman spectrometer and the content of each antibiotic in the samples was calculated based on the standard curve.

[0042] The following representative antibiotics were tested according to the method of Example 1:

[0043] Example 2

[0044] Drawing of penicillin standard curve: prepare penicillin solutions of different concentrations (0.1 μg / L, 1.0 μg / L, 10.0 μg / L, 100.0 μg / L, 1000 μg / L, 10000 μg / L), then take 20 mL of penicillin solutions of different concentrations for liquid phase extraction in step (2) and enrichment and purification in step (3) to obtain penicillin concentrates of different concentrations, respectively detect 10 μL of penicillin concentrates of different concentrations using a portable Raman spectrometer, and draw a standard curve.

[0045] 10 μL of the chicken concentrate to be tested was detected using a portable Raman spectrometer, and the content of penicillin in the sample to be tested was calculated based on the standard curve.

[0046] Example 3

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

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that the chicken pretreatment process is different, no freezing treatment is performed in this comparative example, 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, then added into the solution system for stirring, the mass volume ratio of minced meat to the solution system was 1.5g:10mL, stirred for 35min, and after completion, the supernatant was taken, the residue was redissolved with 10mL 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] The HLB solid phase extraction column (60 mg, 3 mL) and the MCX solid phase extraction column (60 mg, 3 mL) were connected in series with 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 speed of 4 mL / min. The filtrate was discarded and eluted with an eluent, which was 1 mL of a 5% (v / v) methanol aqueous solution. The liquid after elution was discarded and eluted with an eluent, which was 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, redissolved in 1 mL of a 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 machine determination.

[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, 3 mL of methanol was used for activation and 3 mL of water was used for balance. The chicken extract was passed through the solid phase extraction column at a speed of 4 mL / min. The filtrate was discarded and eluted with an eluent, which was 1 mL of a 5% (v / v) methanol aqueous solution. The liquid after elution was discarded and eluted with an eluent, which was 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, 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 machine determination.

[0070] The above embodiment is tested as follows:

[0071] 1. Linear relationship investigation

[0072] The logarithm of the response value (lg I) and the logarithm of the concentration (lgC) of the detection method of the above Examples 2-5 were plotted, and the obtained regression equations (Table 1) all had a good linear relationship, indicating that the detection method of this embodiment can effectively detect various antibiotics in chicken samples.

[0073] Table 1 Linear relationship investigation results of Examples 2-5

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

[0075] 2. Repeatability test

[0076] A 100.0 ng / mL standard solution of each antibiotic was prepared, and the methods of Examples 2-5 were used for detection, respectively. The detection was repeated 5 times, and the concentration and RSD value of each antibiotic were calculated by 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 value of the method of this embodiment is about 1%, which is 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 LSD 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 the local market as the chicken to be tested, 100 g of chicken tissue was taken as the chicken sample to be tested, and chicken concentrate was extracted according to the method of Example 1 and Comparative Examples 1 to 6; the process was repeated twice to obtain 2 groups of chicken concentrate samples to be tested; 20 μg / L penicillin standard solution was added to the first group of chicken concentrate samples to be tested, and the spiked sample measurement value was calculated by the above method, and the sample measurement value was calculated by the second group of chicken concentrate samples to be tested (blank group without any antibiotics added), thereby calculating the spiked recovery rate, and each group was repeated 3 times to take the average value, wherein the calculation formula is: spiked recovery rate = (specified sample measurement value - sample measurement value) ÷ spiked amount × 100%, and the same method was used to calculate the recovery rates of erythromycin, ciprofloxacin, and tetracycline standards, and the results are shown in Table 3.

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

[0084]

[0085]

[0086] It can be seen from the above performance test results that the detection method of Example 1 has a high extraction efficiency for antibiotics in chicken, and the spiked recoveries of four antibiotics, penicillin, erythromycin, ciprofloxacin and tetracycline, are between 95% and 105%, indicating that the recovery rate of the detection method is good. 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, and the antibiotic extraction efficiency and purity are higher, thereby the detection accuracy is higher.

[0087] However, the comparative example is obviously inferior to the embodiment in the corresponding performance test because the necessary technical scheme is not adopted. In comparative example 1, the chicken is not frozen pre-treated. It can be seen from the results that the recovery rate of the spiked test determined by its detection method is about 90% or about 107%, which is obviously worse than that of embodiment 1, proving that the freezing treatment of chicken has an important influence on the accuracy of antibiotic detection. In comparative example 2, the solution system is replaced with pure acetonitrile. It can be seen from the results that the recovery rate of the spiked test is affected, and the accuracy of chicken antibiotic detection is further reduced, indicating that the present invention uses a specific solution system to treat chicken samples, which can improve the extraction efficiency of antibiotics. Ultrasonic treatment is not used in comparative example 3. It can be seen from the results that the accuracy of its detection method is reduced, indicating that the extraction of solution system combined with ultrasonic treatment can maximize the extraction efficiency of antibiotics in chicken. In comparative example 4, the power of ultrasound is increased. It can be seen from the results that the detection accuracy of antibiotics in chicken is also reduced, indicating that excessive power may destroy the structure of antibiotics, thereby causing the accuracy of the test results to deteriorate. Therefore, the ultrasonic treatment of the present invention at a specific power is very important for improving the extraction efficiency of chicken antibiotics and improving the detection accuracy. In Comparative Example 5, only two solid phase extraction columns were used. From the results, it can be seen that the detection accuracy of antibiotics in chicken is deteriorated, indicating that the enrichment and purification of the three fixed extraction columns in the present invention can improve the purity of antibiotics, thereby improving the detection accuracy. In Comparative Example 6, the order of the three fixed extraction columns was adjusted. From the results, it can be seen that the detection accuracy of antibiotics in chicken is deteriorated, indicating that the specific series order of the three solid phase extraction columns has an important influence on improving the detection accuracy of the antibiotic content in chicken. The above experimental results further prove the importance of the technical solution defined in the present invention for its technical effect.

[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 the scope of protection of the present invention.

Claims

1. A method for detecting antibiotic residues in chicken, characterized in that: The following steps are involved: (1) Pre-freezing of chicken samples (2) Liquid phase extraction of pretreated chicken samples The pretreated chicken sample is cut into pieces and crushed into minced meat, and then added into the solution system for stirring, the mass volume ratio of the minced meat to the solution system is (1-2) g: 10 mL, and ultrasonic treatment is applied while stirring. After completion, the supernatant is taken, the obtained residue is redissolved using the solution system, the supernatant is taken again, and the supernatants are combined to obtain a chicken extract; The solution system includes acidified acetonitrile, EDTA-2Na solution and phosphate; (3) Enrichment and purification of chicken extract 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, the eluent is used for elution, the eluted liquid is discarded, and the eluent is used for elution. The eluent is blown dry and redissolved in a methanol aqueous solution to obtain a chicken concentrate, which is the sample to be tested; (4) Draw standard curves for each antibiotic; (5) The samples were tested using a Raman spectrometer and the content of each antibiotic in the samples was calculated based on the standard curve.

2. The method for detecting antibiotic residues in chicken according to claim 1, characterized in that: In step (1), the freezing pretreatment process is: freezing the chicken sample at -35 to -20°C for 12 to 24 hours, thawing it, and continuing to store it at 4 to 8°C for 3 to 5 days.

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

4. The method for detecting antibiotic residues in chicken according to claim 1, characterized in that: In step (2), the acidified acetonitrile is an acetonitrile solution containing 0.5-0.8% trifluoroacetic acid, the concentration of the EDTA-2Na solution is 0.1M, 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.

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

6. The method for detecting antibiotic residues in chicken according to claim 1, characterized in that: 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).

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

8. The method for detecting antibiotic residues in chicken according to claim 1, characterized in that: 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 of step (2) and enrichment and purification of step (3), and then detected by a portable Raman spectrometer to draw a standard curve.

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