CN107632090B - Method for rapidly detecting various antibiotic residues in livestock and poultry manure - Google Patents
Method for rapidly detecting various antibiotic residues in livestock and poultry manure Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting various antibiotic residues in livestock and poultry manure, which takes an improved QuEchERs technology as a sample pretreatment means, takes a high-sensitivity Waters acquisition UPLC-AB5500 liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer as a detection means, establishes a rapid screening and confirming technology for simultaneously detecting various commonly used antibiotic drug residues in the livestock and poultry manure, and has the advantages of short detection period, high detection efficiency, good reliability and the like.
Description
Technical Field
The invention relates to the technical field of antibiotic detection, in particular to a method for rapidly detecting various antibiotic residues in livestock and poultry manure.
Background
The development of intensive animal husbandry leads to the use of a large amount of veterinary drugs, and antibiotic drugs such as tetracyclines, macrolides, sulfonamides, quinolones and the like are widely applied to the livestock and poultry raising industry because of the effects of preventing and treating diseases, promoting the growth of animals, improving the feed conversion rate and the like. The drugs are usually difficult to absorb by the intestines and stomach of animals, and about 30-90% of the drugs are directly released into the environment through livestock and poultry manure in the original mode. Because livestock and poultry breeding is mostly dispersed around rural areas and towns in China, livestock and poultry manure cannot be effectively treated, so that a large amount of residual veterinary drugs enter the environment, environmental and ecological risks such as biotoxicity or drug resistance genes generated by pathogenic bacteria can be brought, and harm can be generated to the health of people.
The residual veterinary drugs can enter soil and underground water environment through the use of breeding wastewater and excrement, and become a novel concerned environmental organic pollutant. Barnes et al (2008) performed sampling and detection of 47 groundwater in 18 states in the United states, and detected organic pollutants in 81% of water samples, wherein the pollutants generated by livestock and poultry medication account for 23% of the total detected amount. As a large country for producing and using veterinary drugs in China, the relevant investigation of veterinary drug residues in livestock and poultry excrement is still incomplete, the reports of relevant documents are few, and the establishment of an analysis and determination method for various veterinary drug residues in livestock and poultry excrement is the basis for developing the migration and accumulation of veterinary drug residues in the environment and evaluating the environmental risk.
the method for detecting the drug residues reported at home and abroad mainly comprises a liquid chromatography (HPLC), a capillary electrophoresis-mass spectrometry combined method (CE-MS), a liquid chromatography-tandem mass spectrometry combined method (LC-MS/MS), an enzyme linked immunosorbent assay and the like, the veterinary drug residue detection technology in China is rapidly developed, and more detection technical researches are carried out, such as a ROSA method for rapidly detecting β -lactam and tetracycline drug residues in milk from an SN/T3256-2012 outlet, a macrolide antibiotic residue detection method in SN/T1777.2-2007 animal-derived food, a 2 nd part-high performance liquid chromatography tandem mass spectrometry method, GB29692-2013 food safety national standard-determination of more residues of quinolones in milk-high performance liquid chromatography, and determination of sulfonamide residues in GB/T21316-2007 animal-derived food.
Disclosure of Invention
In view of the above, the invention discloses a method for rapidly detecting multiple antibiotic residues in livestock and poultry manure, so as to at least solve the problems of single detection type, long detection period, low detection efficiency and the like in the existing detection technology.
The technical scheme provided by the invention is specifically a method for rapidly detecting various antibiotic residues in livestock and poultry manure, and is characterized by comprising the following steps:
1) preparing an internal standard substance and various antibiotic samples into standard stock solution;
2) preparing a mixed standard working solution by using a standard stock solution;
3) setting parameters of a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer;
4) generating an internal standard substance and standard curve models of each antibiotic in a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer;
5) pretreating a sample of the livestock and poultry manure to be detected for later use;
6) analyzing the livestock and poultry manure to be detected subjected to sample pretreatment in the step 5) in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer to obtain a detection result according to the parameters of the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer set in the step 3) through the internal standard substance and each antibiotic standard curve model generated in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in the step 4).
Preferably, the antibiotics are tetracycline antibiotics, quinolone antibiotics, sulfonamide antibiotics and macrolide antibiotics;
the internal standard substance is D5-enrofloxacin.
More preferably, the tetracycline antibiotics are one or more of oxytetracycline, tetracycline and chlortetracycline;
the quinolone antibiotics are one or more of enrofloxacin, ofloxacin, ciprofloxacin, norfloxacin, oxolinic acid and flumequine;
the sulfa antibiotic is one or more of sulfadoxine, sulfadiazine, sulfamethoxazole, sulfamethazine, sulfaphenazine, sulfamethoxazole, sulfadimethoxine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamoyl and sulfaquinoxaline;
the macrolide antibiotics are one or more of lincomycin, spiramycin, kitasamycin, tylosin, clindamycin and josamycin.
Further preferably, the pretreatment of the sample of the livestock and poultry manure to be detected in the step 5) is specifically as follows:
5.1 weighing the livestock and poultry manure to be detected, adding Na into the livestock and poultry manure to be detected, and conveniently centrifuging the livestock and poultry manure2EDTA and 1% acetonitrile solution of formic acid, and oscillating;
5.2, adding anhydrous sodium sulfate and sodium chloride into the vibrated centrifugal tube, uniformly mixing by vortex, centrifuging after ultrasonic treatment, and taking supernatant;
5.3, adding anhydrous sodium sulfate and a C18 adsorbent into the supernatant, horizontally oscillating, centrifuging, standing to precipitate protein, and taking the supernatant into a test tube;
and 5.4, concentrating the supernatant in a test tube under nitrogen until the supernatant is dry, adding 0.1% acetonitrile formate to a constant volume solution, uniformly mixing by vortex, and filtering through a 0.22-micron filter membrane for later use.
Further preferably, the liquid chromatography-triple quadrupole-linear ion trap tandem mass spectrometer in the step 3) is specifically set according to the following parameters,
the liquid chromatography conditions were:
a chromatographic column: ACQUITY UPLCTMBEH C18(100mm×2.1mm i.d.，1.7μm)；
Column temperature: 40 ℃;
temperature of the sample chamber: room temperature;
sample introduction volume: 10 mu L of the solution;
flow rate: 0.2 mL/min;
mobile phase: methanol (a) and 0.1% aqueous formic acid (B), gradient elution procedure: 0min, 5% A, 2min, 5% A, 8min, 25% A, 14min, 42% A, 30min, 95% A, 32min, 95% A, 35min, 5% A;
the mass spectrum conditions are as follows:
an ion source: ESI +;
ion source temperature: 550 ℃;
ion spray voltage: 5500V;
inlet voltage: 10V;
atomizing gas 50 Psi;
auxiliary heating gas: 50 Psi;
air curtain air: 30 Psi;
and (3) monitoring mode: the MRM mode is employed.
Further preferably, in the step 6), the internal standard substance generated in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in the step 4) and each antibiotic standard curve model are used, the livestock manure to be detected after the sample pretreatment in the step 5) is analyzed in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer to obtain a detection result,
6.1, qualitative analysis:
selecting 1 parent ion and 2 daughter ions for qualitative analysis of each antibiotic, wherein during the qualitative analysis, the retention time ratio of an object to be detected and an internal standard substance in a livestock and poultry manure sample to be detected is respectively compared with the retention time ratio of the antibiotic and the internal standard substance in each mixed standard working solution, the deviation is within +/-2.5%, the relative abundance of each component qualitative ion in the livestock and poultry manure sample to be detected is respectively compared with the relative abundance of the qualitative ion of the antibiotic in each mixed standard working solution, and if the deviation does not exceed the specified range, the corresponding object to be detected exists in the livestock and poultry manure sample to be detected;
6.2, quantitative determination:
for each antibiotic, 1 parent ion was selected and 1 daughter ion was quantitatively determined by the internal standard method.
Further preferably, the specific calculation formula of the quantitative determination in step 6.2 is as follows:
in the formula:
x is the residual quantity of the detected object in the livestock and poultry excrement sample to be detected, and the unit is microgram per kilogram (microgram/kg);
cs is the concentration of antibiotic in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
a is the chromatographic peak area of the measured object in the livestock and poultry manure sample solution to be detected;
as is the chromatographic peak area of the antibiotic in the corresponding mixed standard working solution;
ci is the concentration of an internal standard substance in the livestock and poultry manure sample solution to be detected, and the unit is nanogram per milliliter (ng/mL);
csi is the concentration of internal standard in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
asi is the chromatographic peak area of the internal standard substance in the corresponding mixed standard working solution;
ai is the chromatographic peak area of an internal standard substance in the livestock and poultry manure sample solution to be detected;
v is the final constant volume of the livestock and poultry manure sample solution to be detected, and the unit is milliliter (mL);
and m is the mass of the livestock and poultry manure sample to be detected and has the unit of gram (g).
The method for rapidly detecting the residues of various antibiotics in the livestock and poultry manure provided by the invention takes an improved QuEchERs technology as a sample pretreatment means, and takes a high-sensitivity Waters acquisition UPLC-AB5500 liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer as a detection means to establish a rapid screening and confirmation technology for simultaneously detecting the residues of various commonly used antibiotics in the livestock and poultry manure.
The method for rapidly detecting the residues of various antibiotics in the livestock and poultry manure provided by the invention has the following characteristics:
1. the detectable antibiotic variety basically covers the antibiotics with the requirement of limited residual quantity at home and abroad and the common antibiotic residual varieties in domestic animal and poultry feces, and the method has low detection limit and completely meets the requirement of limited residual quantity of antibiotics in the environment at home and abroad.
2. By taking a tandem mass spectrometry system Waters acquisition UPLC-AB5500 liquid chromatogram-triple quadrupole-linear acceleration ion trap tandem mass spectrometer as a detection means, the method can realize the simultaneous and rapid detection of multiple antibiotics, and save time (the current standard detection method is mainly used for detecting single-class antibiotics, and has low efficiency and long time); in addition, the instrument has high sensitivity and accurate qualitative and quantitative determination, greatly reduces the detection limit of the method, and can completely reach the international specified limit for antibiotic residues in farmland environment. Therefore, the method completely overcomes the defects of the existing detection method, and is an analysis technology with high efficiency and high reliability.
3. The improved QuEChERS rapid pretreatment technology is adopted in the pretreatment of the sample, the extraction is sufficient, the purification effect is good, the pretreatment process of the analysis of the antibiotic residues in the livestock and poultry manure is greatly simplified on the basis of improving the automation degree and the reproducibility of the pretreatment method, and the simultaneous detection of various antibiotic residues in the livestock and poultry manure is realized.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a total ion current graph model of 29 antibiotic mixed standard solutions generated in a liquid chromatography-triple quadrupole-linear ion trap tandem mass spectrometer in an embodiment of the present disclosure, wherein 1-sulfadiazine, 2-sulfathiazole, 3-sulfamethazine, 4-oxytetracycline, 5-tetracycline, 6-ofloxacin, 7-norfloxacin, 8-sulfamethazine, 9-sulfamethoxypyridazine, 10-sulfachlorpyridazine, 11-lincomycin, 12-aureomycin, 13-neonomycin, 14-sulfamonomethoxine, 15-ciprofloxacin, 16-norfloxacin, 17-sulfadoxine, 18-sulfisoxazole, 19-sulfamoyl, 20-sulfaphenazole, 21-sulfadimethoxine, 22-oxolinic acid, 23-sulfaquinoxaline, 24-spiramycin, 25-flumequine, 26-clindamycin, 27-tylosin, 28-kitasamycin and 29-josamycin;
fig. 2-28 are multiple reaction monitoring chromatogram models of 29 veterinary and internal standard substances generated in a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in accordance with an embodiment of the present disclosure.
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of methods consistent with certain aspects of the invention, as detailed in the appended claims.
According to the limit requirement of various antibiotic residues in livestock and poultry excrement at home and abroad and the current use situation of the existing antibiotic veterinary drugs, the technical scheme provided by the embodiment is a method for simultaneously qualitatively and quantitatively detecting various antibiotic residues in the livestock and poultry excrement under the laboratory detection condition, and the detection process specifically comprises the following steps:
1. the instrument comprises the following steps:
liquid chromatography tandem mass spectrometry (Waters Acquity/AB5500), us Waters corporation/us AB corporation; low temperature centrifuges (beckmann coulter, usa); Vortex-Genie 2 Vortex shaker (scientific industries, USA); electronic analytical balance MP200, balance factory of shanghai precision scientific instruments company; electronic analytical balance AE163, Mettler, switzerland; nitrogen-blown concentrators (EVAP 112); Milli-Q ultra pure water machine (Millipore).
2. Reagents and materials:
the methanol and the acetonitrile are both chromatographically pure;
formic acid, disodium ethylene diamine tetraacetate (Na)2EDTA), anhydrous sodium sulfate, anhydrous magnesium sulfate, sodium chloride and sodium acetate are analytically pure;
c18, PAX, PSA, NH2 Tianjin Bonna-Agela.
3. And (3) standard substance:
tetracyclines (3): oxytetracycline, tetracycline, chlortetracycline; quinolones (6 species): enrofloxacin, ofloxacin, ciprofloxacin, norfloxacin, oxolinic acid, flumequine; sulfonamides (14 types): sulfadoxine, sulfadiazine, sulfamethoxazole, sulfamethazine, sulfamethoxazole, sulfadimethoxine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, sulfamoyl, sulfaquinoxaline; macrolides (6): lincomycin, spiramycin, kitasamycin, tylosin, clindamycin and josamycin with the purity of more than or equal to 95.0 percent, Germany Dr.
Internal standard substance: d5-enrofloxacin, purity not less than 95.0%, Germany Dr.
4. Standard stock solutions:
weighing a proper amount of each antibiotic standard substance, selecting methanol for dilution according to the solubility of each antibiotic standard substance to prepare a standard stock solution, and refrigerating and storing at 0-4 ℃.
5. Mixing standard working solution:
according to the response condition of each antibiotic on the instrument, different amounts of stock solutions are absorbed, diluted by methanol to prepare a mixed standard working solution, and refrigerated and stored at 0-4 ℃.
6. Sample pretreatment:
weighing 2.0g of livestock and poultry manure to be detected, facilitating the weighing in a 50mL plastic centrifugal tube, and adding 0.1g of Na2EDTA and 10mL of 1.0% (v/v) acetonitrile formate aqueous solution, oscillating for 1min, wherein the extracting solution is optimized in the step, the extracting solution commonly used in the traditional QuEChERS method is acetonitrile, but considering that the livestock and poultry manure contains a large amount of organic matters and fat, and simultaneously looking up literature analysis, designing Na2The extraction liquid of acidic acetonitrile (containing 1 percent of formic acid) of EDTA is subjected to experiments and is compared with the extraction liquid of pure acetonitrile, and the result shows that the recovery rate of tetracycline antibiotics is lower when only acetonitrile is used as an extraction solvent, which is probably related to that the substances are easily chelated with nitrogen, phosphorus and potassium ions in livestock and poultry feces to form insoluble salts, and Na is added2When the acidic acetonitrile (containing 1% formic acid) of EDTA is used as extraction liquid, the chelation of the compounds with cations is inhibited, and the recovery rate is higher, so Na is selected2Acid acetonitrile of EDTA (containing 1% formic acid) as an extracting solution;
adding 4.0g of anhydrous sodium sulfate and 1.0g of sodium chloride into the shaken centrifugal tube, uniformly mixing for 15s in a vortex manner, carrying out ultrasonic treatment for 15min, then centrifuging for 5min at 9000r/min, and taking 6mL of supernatant, wherein the removal of water in the livestock and poultry manure is very important for the treatment of samples because the livestock and poultry manure contains a large amount of water. The water-absorbing agents used are anhydrous magnesium sulfate and anhydrous sodium sulfate, which are compared experimentally. The result shows that the addition of the anhydrous magnesium sulfate can reduce the recovery rate of quinolone veterinary drugs, sulfonamide veterinary drugs, tetracycline veterinary drugs and macrolide veterinary drugs to different degrees, wherein the reduction of the tetracycline veterinary drugs is the most obvious. This is due to MgSO4Mg in (1)2+The compound is combined with tetracycline veterinary drugs to form chelate, thereby influencing the extraction of the tetracycline veterinary drugs. Therefore, the water absorbing agent was finally determined to be anhydrous sodium sulfate;
adding 900mg of anhydrous sodium sulfate and 150mg of C18 adsorbent into the supernatant, after horizontally oscillating for 5min, fully contacting the adsorbent with the extracting solution, centrifuging for 5min at 9000r/min, standing for 10min to precipitate protein, and taking 3mL of supernatant into a 10mL test tube, wherein the inventor selects 4 adsorbents of C18, NH2, PSA and PAX experimentally to compare the purifying effect and the influence on the compound recovery rate. As a result, the influence of 4 adsorbents on the recovery rate of most antibiotics is not obviously different, the PSA adsorbent has better purification effect on most target compounds, but has lower recovery rate on tetracycline compounds with higher polarity, and the adsorption is related to the adsorption of the PSA on the tetracycline antibiotics, which is probably the result of hydrogen bonding and is influenced by factors such as the water content of the substrate, the pH value and the like. Compared with PSA, the C18 adsorbent has better recovery rate for all target compounds including tetracycline compounds, and the matrix inhibition is obviously improved, so the C18 adsorbent is selected for purification;
concentrating the supernatant test tube at 40 ℃ in nitrogen until the supernatant test tube is dry, adding 1ml of 0.1% formic acid acetonitrile (9: 1) constant volume solution, uniformly mixing by vortex, and filtering through a 0.22 mu m filter membrane for later use, wherein in simultaneous qualitative and quantitative analysis of various veterinary drug antibiotic residues, the constant volume solution is closely related to the response and peak shape of a target veterinary drug, so that the constant volume solution universally applicable to all drugs needs to be preferably selected. Different volume ratios of 0.1% formic acid to acetonitrile (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) were selected as constant volume solutions for comparative experiments. And calculating the average response value of 29 antibiotics in a certain proportion of constant volume liquid. By analyzing the average value of the drug response of the constant volume solutions in each proportion, the average response is the highest when the ratio of 0.1% formic acid to acetonitrile is 10:0 and 9: 1. By observing the peak shape of the compound, except the volume fixing liquid with the ratio of 9:1, the volume fixing liquid with other ratios all affect the peak shape of part of veterinary drugs. Combining two aspects, finally determining that the constant volume solution is 0.1% formic acid: acetonitrile (9:1, V/V).
7. Detecting by an instrument:
1) liquid chromatography conditions:
a chromatographic column: ACQUITY UPLCTM BEH C18(100 mm. times.2.1 mm i.d., 1.7 μm); column temperature: 40 ℃; temperature of the sample chamber: room temperature; sample introduction volume: 10 mu L of the solution; flow rate: 0.2 mL/min; mobile phase: methanol (A) and 0.1% aqueous formic acid (B) were eluted with a gradient under the conditions shown in Table 1.
TABLE 1 gradient elution conditions
2) Mass spectrum conditions:
an ion source: ESI +; ion source temperature: 550 ℃; ion spray voltage: 5500V; inlet voltage: 10V; atomizing gas 50 Psi; auxiliary heating gas: 50 Psi; air curtain air: 30 Psi; and (3) monitoring mode: the MRM mode is employed. The parameters of the 29 veterinary antibiotics, such as detected ions, declustering voltage, collision energy and the like, are detailed in table 2.
Mass spectrum condition optimization results of 229 veterinary antibiotics and internal standard substances in table
Note: is a quantitative ion
Wherein, fig. 1 is a total ion current graph model for generating 29 antibiotic mixed standard solutions in a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer; fig. 2-28 are multiple reaction monitoring chromatogram models of 29 veterinary drugs and internal standard generated in a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer.
8. Qualitative and quantitative analysis
Referring to eu directive 2002/657/EC, 1 parent ion, 2 daughter ions per antibiotic were selected for characterization, with 1 parent ion, and 1 daughter ion being quantitative. During qualitative determination, the deviation of the retention time ratio of the substance to be determined and the internal standard substance in the animal manure sample to be determined and the retention time ratio of the antibiotics in the internal standard substance in each mixed standard working solution is within +/-2.5%, the relative abundance of the qualitative ions of each component in the animal manure sample to be determined and the relative abundance of the qualitative ions of the antibiotics in each mixed standard working solution are respectively compared, and if the deviation does not exceed the specified range, the corresponding substance to be determined exists in the animal manure sample to be determined. The maximum allowable deviation from ion abundance in the qualitative confirmation is shown in table 3.
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative confirmation
And (4) quantitatively measuring by adopting an internal standard method. The internal standard substance is D5-enrofloxacin. Selecting mixed standard working solutions with similar concentrations according to the estimated content of the antibiotics to be detected in the livestock and poultry manure sample solution to be detected, requiring that the response values of the antibiotics in the mixed standard working solutions and the livestock and poultry manure sample solution to be detected are within the linear range detected by an instrument, performing equal-volume sample injection measurement on the mixed standard working solutions and the livestock and poultry manure sample solution to be detected, and quantifying by an internal standard method. The calculation formula is as follows:
in the formula:
x is the residual quantity of the detected object in the livestock and poultry excrement sample to be detected, and the unit is microgram per kilogram (microgram/kg);
cs is the concentration of antibiotic in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
a is the chromatographic peak area of the measured object in the livestock and poultry manure sample solution to be detected;
as is the chromatographic peak area of the antibiotic in the corresponding mixed standard working solution;
ci is the concentration of an internal standard substance in the livestock and poultry manure sample solution to be detected, and the unit is nanogram per milliliter (ng/mL);
csi is the concentration of internal standard in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
asi is the chromatographic peak area of the internal standard substance in the corresponding mixed standard working solution;
ai is the chromatographic peak area of an internal standard substance in the livestock and poultry manure sample solution to be detected;
v is the final constant volume of the livestock and poultry manure sample solution to be detected, and the unit is milliliter (mL);
and m is the mass of the livestock and poultry manure sample to be detected and has the unit of gram (g).
9. Actual sample detection
The inventor randomly extracts cow dung, pig dung, horse dung and chicken dung from Shenyang and surrounding areas for detection by using the detection method, detects more than ten batches of the detection times, detects ciprofloxacin, ofloxacin, norfloxacin, chlortetracycline and lincomycin in classified livestock and poultry dung, and detects the conditions shown in the table.
TABLE 29 detection of antibiotics in the feces of livestock and poultry (mg/kg)
The actual sample detection results in the above table may be that part of the antibiotics used in the animal raising process may remain in the feces, which may affect the quality of the product. The establishment and the application of the method can quickly screen various antibiotics in the livestock and poultry manure, thereby effectively monitoring the antibiotic residue and the farmland environmental pollutant residue in the livestock and poultry breeding process.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
It will be understood that the invention is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the invention is limited only by the appended claims.
Claims (4)
1. A method for rapidly detecting various antibiotic residues in livestock and poultry manure is characterized by comprising the following steps:
1) preparing an internal standard substance and various antibiotic samples into standard stock solution;
2) preparing a mixed standard working solution by using a standard stock solution;
3) setting parameters of a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer;
4) generating an internal standard substance and standard curve models of each antibiotic in a liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer;
5) pretreating a sample of the livestock and poultry manure to be detected for later use;
6) analyzing the livestock and poultry manure to be detected subjected to sample pretreatment in the step 5) in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer to obtain a detection result according to the parameters of the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer set in the step 3) through the internal standard substance and each antibiotic standard curve model generated in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in the step 4);
the pretreatment of the sample of the livestock and poultry manure to be detected in the step 5) is specifically as follows:
5.1 weighing the livestock and poultry manure to be detected, adding Na into the livestock and poultry manure to be detected, and conveniently centrifuging the livestock and poultry manure2EDTA and 1% acetonitrile solution of formic acid, and oscillating;
5.2, adding anhydrous sodium sulfate and sodium chloride into the vibrated centrifugal tube, uniformly mixing by vortex, centrifuging after ultrasonic treatment, and taking supernatant;
5.3, adding anhydrous sodium sulfate and a C18 adsorbent into the supernatant, horizontally oscillating, centrifuging, standing to precipitate protein, and taking the supernatant into a test tube;
5.4, concentrating the supernatant in a test tube under nitrogen until the supernatant is dry, adding 0.1% acetonitrile formate to a constant volume solution, uniformly mixing by vortex, and filtering with a 0.22-micron filter membrane for later use;
the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in the step 3) is specifically set according to the following parameters,
the liquid chromatography conditions were:
a chromatographic column: ACQUITY UPLCTMBEH C18(100mm×2.1mmi.d.，1.7μm)；
Column temperature: 40 ℃;
temperature of the sample chamber: room temperature;
sample introduction volume: 10 mu L of the solution;
flow rate: 0.2 mL/min;
mobile phase: methanol (a) and 0.1% aqueous formic acid (B), gradient elution procedure: 0min, 5% A, 2min, 5% A, 8min, 25% A, 14min, 42% A, 30min, 95% A, 32min, 95% A, 35min, 5% A;
the mass spectrum conditions are as follows:
an ion source: ESI +;
ion source temperature: 550 ℃;
ion spray voltage: 5500V;
inlet voltage: 10V;
atomizing gas 50 Psi;
auxiliary heating gas: 50 Psi;
air curtain air: 30 Psi;
and (3) monitoring mode: adopting an MRM mode;
in the step 6), the livestock and poultry manure to be detected after sample pretreatment in the step 5) is analyzed in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer through the internal standard substance generated in the liquid chromatogram-triple quadrupole-linear ion trap tandem mass spectrometer in the step 4) and the standard curve models of all antibiotics to obtain a detection result,
6.1, qualitative analysis:
selecting 1 parent ion and 2 daughter ions for qualitative analysis of each antibiotic, wherein during the qualitative analysis, the retention time ratio of an object to be detected and an internal standard substance in a livestock and poultry manure sample to be detected is respectively compared with the retention time ratio of the antibiotic and the internal standard substance in each mixed standard working solution, the deviation is within +/-2.5%, the relative abundance of each component qualitative ion in the livestock and poultry manure sample to be detected is respectively compared with the relative abundance of the qualitative ion of the antibiotic in each mixed standard working solution, and if the deviation does not exceed the specified range, the corresponding object to be detected exists in the livestock and poultry manure sample to be detected;
6.2, quantitative determination:
for each antibiotic, 1 parent ion was selected and 1 daughter ion was quantitatively determined by the internal standard method.
2. The method for rapidly detecting the residues of various antibiotics in the livestock and poultry manure according to claim 1, wherein the antibiotics are tetracycline antibiotics, quinolone antibiotics, sulfonamide antibiotics and macrolide antibiotics;
the internal standard substance is D5-enrofloxacin.
3. The method for rapidly detecting the residues of a plurality of antibiotics in the livestock and poultry manure according to claim 2, wherein the tetracycline antibiotics are one or more of oxytetracycline, tetracycline and aureomycin;
the quinolone antibiotics are one or more of enrofloxacin, ofloxacin, ciprofloxacin, norfloxacin, oxolinic acid and flumequine;
the sulfa antibiotic is one or more of sulfadoxine, sulfadiazine, sulfamethoxazole, sulfamethazine, sulfaphenazine, sulfamethoxazole, sulfadimethoxine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamoyl and sulfaquinoxaline;
the macrolide antibiotics are one or more of lincomycin, spiramycin, kitasamycin, tylosin, clindamycin and josamycin.
4. The method for rapidly detecting the residues of various antibiotics in the livestock and poultry manure as claimed in claim 1, wherein the specific calculation formula of the quantitative determination in the step 6.2 is as follows:
in the formula:
x is the residual quantity of the detected object in the livestock and poultry excrement sample to be detected, and the unit is microgram per kilogram (microgram/kg);
cs is the concentration of antibiotic in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
a is the chromatographic peak area of the measured object in the livestock and poultry manure sample solution to be detected;
as is the chromatographic peak area of the antibiotic in the corresponding mixed standard working solution;
ci is the concentration of an internal standard substance in the livestock and poultry manure sample solution to be detected, and the unit is nanogram per milliliter (ng/mL);
csi is the concentration of internal standard in the corresponding mixed standard working solution in nanograms per milliliter (ng/mL);
asi is the chromatographic peak area of the internal standard substance in the corresponding mixed standard working solution;
ai is the chromatographic peak area of an internal standard substance in the livestock and poultry manure sample solution to be detected;
v is the final constant volume of the livestock and poultry manure sample solution to be detected, and the unit is milliliter (mL);
and m is the mass of the livestock and poultry manure sample to be detected and has the unit of gram (g).
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