CN112505195A - Method for accurately and sensitively detecting antibiotics in environmental sample - Google Patents
Method for accurately and sensitively detecting antibiotics in environmental sample Download PDFInfo
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Abstract
The invention relates to the technical field of mass spectrometry detection, and relates to a method for accurately and sensitively detecting antibiotics in an environmental sample. The method adopts a proper pretreatment method for liquid samples and solid samples in the environmental samples, purifies the pretreated environmental sample extract, dries nitrogen, performs constant volume filtration, and detects the antibiotic types in the environmental samples to be detected by utilizing a high performance liquid chromatography-mass spectrometry combined technology. The method disclosed by the invention is simple to operate and high in accuracy, can realize quick and sensitive detection of 5 major classifications and 26 antibiotics remained in the environmental sample by one-time operation, and can be used for screening the antibiotic residues of water quality, soil and sediments in the environment.
Description
Technical Field
The invention relates to the technical field of high performance liquid chromatography mass spectrometry detection of antibiotics in environmental samples, in particular to a method for accurately and sensitively detecting antibiotics in environmental samples.
Background
The antibiotics are mainly used in medical health, livestock and poultry breeding, aquaculture industry and the like, and are widely used, so that the phenomenon of overuse of the antibiotics is very common while the life quality of people is improved. Large amounts of antibiotics remain in the environment and accumulate over time, causing damage to microbial communities in the water and affecting aquatic life. Meanwhile, the ecological system can be damaged, food is conveyed into the human body, and high risks exist for both ecology and human health. In addition, the long-term residue of antibiotics in the environment can improve the drug resistance of germs, and poses great threat to the global health. Quantitative analysis is carried out on the antibiotic residue in the environment through a scientific detection method, and the method has great significance for objectively evaluating the risk of the antibiotic and further researching the migration and transformation of the antibiotic.
In the existing report, ultrasonic extraction is used for extracting antibiotics in soil and sediments, an EDTA-Mclvaine solution is used as a buffer solution, and supernatant is extracted through centrifugation and combination after multiple ultrasonic extractions.
Disclosure of Invention
The invention aims to provide a method for accurately and sensitively detecting antibiotics in an environmental sample, so as to solve the problems of high detection limit, high antibiotic omission ratio and narrow antibiotic detection spectrum when the environmental sample is detected in the prior art.
The invention discovers that the proper enrichment method, extraction mode, organic solvent suitable for extraction and extraction condition are selected in the pretreatment process of the environmental sample, so that the sample to be detected can obtain more comprehensive antibiotic enrichment, and the accurate, quick and sensitive detection of antibiotic residues in the environmental sample, especially in environmental water quality, soil and sediments, can be realized by adopting the high performance liquid chromatography tandem mass spectrometry.
The sample in the environment is a liquid sample, such as various water quality samples in the environment; solid samples such as soil, sediment, etc.
If the environmental sample is a liquid sample, the liquid sample is added into a brown glass bottle when being collected, and 0.5g of Na is added into every 1L of the liquid sample2-EDTA, 0.1M sulfuric acid to adjust pH to 4.0; 10mL of methanol was added.
Placing the collected sample in a sampling box, transporting the sample back to a laboratory on the same day, transferring the sediments and soil samples from the glass bottle on the same day, storing the sediments and soil samples in a freezer at the temperature of-20 ℃, and waiting for freeze drying and subsequent pretreatment operation; the water quality samples are pretreated on the same day.
In the embodiment of the invention, after the solid sample is transported back to the laboratory, the solid sample is firstly placed in a freeze dryer for freeze drying for 24 hours, and the moisture of the sediment is fully removed. The dried sample is suitably ground and sieved through a 10 mesh sieve to remove non-soil components on the sieve. In order to increase the specific surface area of subsequent ASE extraction, a sample sieved by a 10-mesh sieve is further ground, then sieved by a 60-mesh sieve, and then placed in a valve bag. In preparation for pre-treatment of the sample.
Specifically, the pretreatment is performed by (1) a method of pretreating a liquid sample when the environmental sample is a liquid sample: adding substitute Kabaoxin (20 μ l, concentration of 1ug/ml) before solid-phase extraction of the liquid sample, and sequentially activating the solid-phase extraction column with ethyl acetate, methanol and ultrapure water; eluting the sample by using methanol after extraction; or
(2) If the environmental sample is a solid sample, the method for pretreating the solid sample comprises the following steps: grinding a dried solid sample, and then carrying out pretreatment by using an ASE extraction technology, wherein an extraction solvent is EDTA-mellvaine/methanol.
(3) And (3) detecting the pretreated liquid sample or solid sample by adopting high performance liquid chromatography-tandem mass spectrometry LC/MS/MS.
The preparation method of the EDTA-Mclvaine solution comprises the following steps: 37.2g disodium edetate, 27.5g disodium hydrogen phosphate and 12.9g citric acid were dissolved in 1L water. Mixing EDTA-Mclvaine solution and methanol according to the volume ratio of 8-12% to prepare the EDTA-mellvaine methanol solution.
In the step (1), the ethyl acetate, the methanol and the ultrapure water are sequentially activated at the use volume ratio of 1:1: 2. The flow rate for solid phase extraction was controlled at 3 mL/min. And (3) leaching the wall of the HLB column by using ultrapure water after extraction, vacuumizing the solid-phase extraction column to remove water after leaching, eluting the sample in a container by using 3X 3mL (3 times of elution and 3mL of methanol each time), staying the methanol in the tube for 3-6min each time to sufficiently elute the antibiotics, and then controlling the flow rate to be 1 mL/min.
The activation of the solid phase extraction column is to open a carbon group chain bonded on silica gel to fully act, methanol is used in the process to enable the carbon chain to be mutually soluble, the activation by adding ethyl acetate in the steps is an improvement measure of the invention, the activation in the prior art is usually by using methanol and water, but the invention discovers that the solid phase extraction column is activated by using ethyl acetate firstly, and then activated by using formic acid and water to better improve the enrichment efficiency of the small column, and the water is used for being compatible with the sample solution.
In the step (2), the extraction solvent is 8-12% of EDTA-mellvaine methanol solution, and the% is volume percentage.
Further, in the step (1), the sample obtained after elution is a purified solution, the purified solution is dried, 200 μ l (mixed standard of sulfanilamide D5, enrofloxacin D5, demeclocycline, roxithromycin D7 and chloramphenicol D5) of an internal standard mixture with the concentration of 500 μ g/ml is added, and whether the volume ratio of the five internal standard substances is 1:1:1:1:1) is determined by using an initial mobile phase 0.5% formic acid-water solution/acetonitrile (95/5, V/V) to obtain a sample to be detected of the liquid sample after pretreatment;
the embodiment of the invention is that after the internal standard is added, the initial mobile phase 1% formic acid-water solution/acetonitrile (95/5, V/V) is used for fixing the volume to 1ml, the mixture is evenly mixed by vortex and then transferred to a sample injection bottle, and the sample injection bottle is sealed and refrigerated for analysis.
Or, the ASE extraction condition in the step (2) is 50 +/-10 ℃, 1500psi, preheating for 5min, static extraction for 10-15min, washing volume of 60%, and circulating for 2 times.
Preferably, in the step (2), the extraction solvent is 8-12% of EDTA-mellvaine: extracting with methanol solution at 50 deg.C under 1500psi for 5min, statically extracting for 10min, washing with 60% volume, and circulating for 2 times.
In the embodiment of the invention, the solid sample is extracted by the following method: taking a cleaned and dried extraction tank, adding an ASE filter membrane at the bottom of the extraction tank after the extraction tank is assembled, sequentially filling 5g of diatomite and 5g of sediment sample into the extraction tank, and adding substitute kappa oxygen (20 mu l of which the concentration is 1ug/ml) and 5g of diatomite, wherein the diatomite needs to be burned at 400 ℃ for 4 hours in advance, and the volume of the filled diatomite occupies more than 2/3 of the extraction tank without compaction. The loaded extraction cells are sequentially placed on ASE, extraction bottles which are numbered and correspond to the extraction cells are placed at the bottoms of the extraction cells, and the extraction bottles are cleaned, dried and sealed by aluminum foil paper before use. The solvent bottle was checked for sufficient amount of extraction solvent and sufficient nitrogen. The extraction solvent is selected from 8-12% EDTA-Mellvaine-methanol solution, and the extraction conditions are set at 50 + -10 deg.C and 1500psi (preheating for 5min, static extraction for 10-15min, washing volume for 60%, and circulation for 2 times).
Furthermore, in the step (2), the extraction liquid is purified and concentrated to a constant volume; the purification comprises concentrating the extractive solution to about 5ml, adding 5ml methanol-water solution (20/80, V/V), enriching with solid phase extraction HLB column, eluting with methanol, and blowing nitrogen to clean;
and the concentration constant volume is to perform nitrogen blowing concentration on the purified liquid in a full-automatic nitrogen blowing instrument until the purified liquid is less than 0.5ml, take out the purified liquid and continue nitrogen blowing until the purified liquid is completely dried, add an internal standard, perform constant volume to 1ml by using an initial mobile phase 1% formic acid-water solution/acetonitrile (95/5, V/V), and filter the purified liquid into a sample bottle by using a microporous filter membrane to obtain a pre-treated sample to be detected.
After a liquid sample or a solid sample is pretreated, a sample to be detected is detected by adopting a high performance liquid chromatography-tandem mass spectrometry LC/MS/MS (liquid chromatography-tandem mass spectrometry), and an Agilent 1260 high performance liquid chromatography system is adopted by adopting the combination of the liquid chromatography and the tandem mass spectrometry, and comprises a quaternary infusion pump and an automatic sample injector (Agilent company in America); 6460 Triple Quad type liquid chromatography/tandem mass spectrometer equipped with electrospray ionization source (ESI) and MassHunter data processing software (Agilent).
A chromatographic column: ZORBAX Eclipse Plus C18 column, 1.8 μm particle size, 2.1mm by 50mm I.D, Agilent Inc., USA;
flow rate of carrier gas: 0.4 mL/min; column temperature: 35 ℃; MS ion source: an electrospray ionization source (AJS ESI), wherein the ion injection voltage is-3500V during negative ion mode detection, and the ion injection voltage is 4000V during positive ion mode detection; ion source temperature: 320 ℃; flow rate: 8.0 l/min; atomizer pressure: 40 psi; temperature of sheath gas: 350 ℃; the flow rate of the sheath gas: 10 l/min; the scanning mode was multiplex reaction monitoring (Dynamic MRM).
Gradient elution: water (A) and acetonitrile (B) are used as mobile phases, the proportion of acetonitrile is respectively increased from 5% to 30% within 0-5min, from 35% to 35% within 5-6min, from 40% within 6-7min, from 60% within 7-9min, from 95% within 9-10min and from 95% within 10-12min, and the gradient conditions of the mobile phases with the percentages being volume percentages are detailed in table 1.
TABLE 1 gradient conditions of mobile phase
The selection of the internal standard substance and the substitute substance is necessary to be substances which have similar properties and do not exist in the natural world, the invention finally selects the most suitable internal standard substance and substitute substance through a large amount of complicated screening and verification work, and finally determines that the internal standard substance is a mixture of sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5 through repeated experiments, and the substitute substance is carbalox, and the mass spectrum condition of the substitute substance is searched to improve the detection accuracy.
TABLE 2 internal standard and surrogate instrument parameters
The invention uses the ASE rapid solvent extraction device, improves the antibiotic extraction efficiency by controlling stable temperature and pressure, reduces intermediate links and is more convenient. By consulting the physicochemical properties of 26 antibiotic substances and considering hydrophilic and hydrophobic antibiotic substances, the comprehensive consideration is to use methanol which can be uniformly mixed with an aqueous solution as an organic solvent to be mixed with an EDTA-Mclvaine solution as an extracting solution, the method has the advantages of good solubility, environmental protection and economic performance, and the optimal extraction conditions are screened by groping the optimal mixing volume ratio to finally obtain the comprehensive method with optimal extraction efficiency, least solvent, most environment-friendly process and lowest toxicity.
The method has the advantages that the method adopts a proper pretreatment method for liquid samples and solid samples in environmental samples, specifically, 8% -12% of EDTA-mellvaine methanol solution is determined as ASE extraction organic solvent, the optimal extraction condition is screened, after pretreatment of the sample to be detected in the mode, the internal standard is determined to be a mixture of sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5, the substitute is carbadox, the mass spectrum condition is searched, and the detection accuracy is improved. The method can realize the rapid and sensitive detection of 29 common antibiotics in 5 categories (sulfonamides, quinolones, tetracyclines, macrolides and chloramphenicol) remained in an environmental sample by adopting a high performance liquid chromatography tandem mass spectrometry method, has the detection limit of sulfadiazine in environmental water as low as 0.017 mu g/L and the detection limit of sulfadiazine in soil as low as 0.009 mu g/kg, has good detection precision and accuracy for the 29 common antibiotics, and can be used for the antibiotic residue screening of water quality, soil and sediments in the environment.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The preparation method of the EDTA-mellvaine methanol solution comprises the following steps: EDTA-Mclvaine solution: 37.2g disodium edetate, 27.5g disodium hydrogen phosphate and 12.9g citric acid were dissolved in 1L water. Mixing EDTA-Mclvaine solution and methanol according to the volume ratio of 8-12% to prepare the EDTA-mellvaine methanol solution.
Example 1 method for detecting antibiotics in environmental samples
1. The sample is a liquid sample
(1) When collecting the liquid sample in the environment, the liquid sample is added into a brown glass bottle, and 0.5g of Na is added into every 1L of the liquid sample2-EDTA, 0.1M sulfuric acid to adjust pH to 4.0; 10mL of methanol was added. Placing the collected sample in a sampling box, transporting the sample back to a laboratory on the same day, transferring the sediments and soil samples from the glass bottle on the same day, storing the sediments and soil samples in a freezer at the temperature of-20 ℃, and waiting for freeze drying and subsequent pretreatment operation; the water quality samples are pretreated on the same day.
(2) The method for pretreating the liquid sample comprises the following steps: adding substitute Kabaoxin (20 μ l, concentration of 1ug/ml) before solid-phase extraction of the liquid sample, and sequentially activating the solid-phase extraction column with ethyl acetate, methanol and ultrapure water; after extraction, the sample was eluted with methanol. The volume ratio of the ethyl acetate to the methanol to the ultrapure water is 1:1:2, and the activation is sequentially carried out. The flow rate for solid phase extraction was controlled at 3 mL/min. And (3) leaching the wall of the HLB column by using ultrapure water after extraction, vacuumizing the solid-phase extraction column to remove water after leaching, eluting the sample in a container by using 3X 3mL (3 times of elution and 3mL of methanol each time), staying the methanol in the tube for 3-6min each time to sufficiently elute the antibiotics, and then controlling the flow rate to be 1 mL/min.
Drying the purified liquid obtained after elution, adding 200 mul (mixed standard of sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5) of an internal standard mixture with the concentration of 500 mug/ml, fixing the volume to 1ml by using 0.5% formic acid-water solution/acetonitrile (95/5, V/V) of an initial mobile phase, transferring the mixture into a sample inlet bottle after vortex mixing, and sealing and refrigerating the mixture for analysis to obtain a sample to be detected of the liquid sample after pretreatment.
2. The sample is a solid sample
(1) After the solid sample is transported back to the laboratory, the solid sample is firstly placed in a freeze dryer for freeze drying for 24 hours, and the moisture of the sediment is fully removed. The dried sample is suitably ground and sieved through a 10 mesh sieve to remove non-soil components on the sieve. In order to increase the specific surface area of subsequent ASE extraction, a sample sieved by a 10-mesh sieve is further ground, then sieved by a 60-mesh sieve, and then placed in a valve bag. In preparation for pre-treatment of the sample.
(2) Pretreating a solid sample
When the solid sample is pretreated, the method for activating the small column is the same as the pretreatment method of the liquid sample.
Taking a cleaned and dried extraction tank, adding an ASE filter membrane at the bottom of the extraction tank after the extraction tank is assembled, sequentially filling 5g of diatomite and 5g of sediment sample into the extraction tank, and adding substitute kappa oxygen (20 mu l of which the concentration is 1ug/ml) and 5g of diatomite, wherein the diatomite needs to be burned at 400 ℃ for 4 hours in advance, and the volume of the filled diatomite occupies more than 2/3 of the extraction tank without compaction. The loaded extraction cells are sequentially placed on ASE, extraction bottles which are numbered and correspond to the extraction cells are placed at the bottoms of the extraction cells, and the extraction bottles are cleaned, dried and sealed by aluminum foil paper before use. The solvent bottle was checked for sufficient amount of extraction solvent and sufficient nitrogen. The extraction solvent is selected from 8-12% EDTA-Mellvaine-methanol solution, and the extraction conditions are set at 50 + -10 deg.C and 1500psi (preheating for 5min, static extraction for 10-15min, washing volume for 60%, and circulation for 2 times).
Purifying the extract, concentrating and fixing the volume; the purification comprises concentrating the extractive solution to about 5ml, adding 5ml methanol-water solution (20/80, V/V), enriching with solid phase extraction HLB column, eluting with methanol, and blowing nitrogen to clean;
and the concentration constant volume is to perform nitrogen blowing concentration on the purified liquid in a full-automatic nitrogen blowing instrument until the purified liquid is less than 0.5ml, take out the purified liquid and continue nitrogen blowing until the purified liquid is completely dried, add an internal standard, perform constant volume to 1ml by using an initial mobile phase 1% formic acid-water solution/acetonitrile (95/5, V/V), and filter the purified liquid into a sample bottle by using a microporous filter membrane to obtain a pre-treated sample to be detected.
3. After liquid samples and solid samples are pretreated, samples to be detected are detected by adopting high performance liquid chromatography-tandem mass spectrometry LC/MS/MS,
(1) conditions of liquid chromatography
The liquid chromatogram-tandem mass spectrum combination comprises the following steps: an Agilent 1260 high performance liquid chromatography system comprising a quaternary infusion pump, an autosampler (Agilent Corp.); 6460 Triple Quad type liquid chromatography/tandem mass spectrometer equipped with electrospray ionization source (ESI) and MassHunter data processing software (Agilent).
A chromatographic column: ZORBAX Eclipse Plus C18 column, 1.8 μm particle size, 2.1mm by 50mm I.D., Agilent Inc., USA;
flow rate of carrier gas: 0.4 mL/min; column temperature: 35 ℃; MS ion source: an electrospray ionization source (AJS ESI), wherein the ion injection voltage is-3500V during negative ion mode detection, and the ion injection voltage is 4000V during positive ion mode detection; ion source temperature: 320 ℃; flow rate: 8.0 l/min; atomizer pressure: 40 psi; temperature of sheath gas: 350 ℃; the flow rate of the sheath gas: 10 l/min; the scanning mode was multiplex reaction monitoring (Dynamic MRM).
Gradient elution: water (A) and acetonitrile (B) are used as mobile phases, the proportion of acetonitrile is respectively increased from 5% to 30% within 0-5min, from 35% to 35% within 5-6min, from 40% within 6-7min, from 60% within 7-9min, from 95% within 9-10min and from 95% within 10-12min, and the percentages are volume percentages.
(2) Mass spectrum conditions: the mass spectrum of the compound was specified in Table 3 below.
TABLE 3 antibiotic instrumental parameters
(3) Standard curve
Weighing single standard substance and substitute 0.1g of substance to be measured, diluting to 10ml with methanol, preparing into stock solution of 10mg/ml, placing into brown glass bottle, and sealing and storing at 4 deg.C; taking 1ml stock solution, adding to 10ml volume to prepare 1000 mug/ml stock solution, and placing into a brown glass bottle to be stored at a low temperature of 4 ℃ for later use. Internal standard substances (sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5 are purchased and mixed in equal volume to obtain the internal standard substance of the invention), are all liquid and are diluted to 10 mu g/ml as stock solution.
Respectively taking 10 mu l to 10ml volumetric flasks of stock solutions of the to-be-detected substances containing 26 known antibiotics, and fixing the volume with methanol to prepare a using solution of 1 mu g/ml. The internal standard substances are all taken from 500 mu L to 10ml volumetric flasks, and the methanol is added to 10ml to prepare 500 mu g/L use solution. The substitute was diluted to 10. mu.g/ml and 1. mu.g/ml 2 use solutions. The series of curves is formulated as shown in table 4 below, with the solvent as the initial mobile phase, i.e., 5/95 acetonitrile/water solution.
TABLE 4 Standard Curve preparation
(4) Detection limit
The minimum detection limit of the liquid chromatography-tandem mass spectrometry on the antibiotic substances is shown in table 5, and the detection limit is calculated according to annex A of HJ 168-2010. Namely, adding the mixed standard solution into a blank machine, processing according to all steps of sample analysis, and ensuring that the concentration of an analyzed sample is within the range of the detection limit of the method calculated by 3-5 times.
TABLE 5 detection limits of antibiotics in water and soil by the method of the present invention
(5) Precision and accuracy
Taking the mixed standard concentrations (80ng/L,400ng/L and 720ng/L) of 0.1,0.5 and 0.9 times of the highest point of the standard curve, taking 6 parts of the same parallel in each concentration, adding the same into a blank soil matrix, and pretreating the same as the samples. Calculating RSD by 6 samples with the same concentration, wherein the RSD is the precision, and the result is the mean value of the precision of the high, medium and low concentrations; accuracy is the mean of recovery for three concentrations plus standard. The parallel sample refers to the same sample, which ensures the uniform processing of the whole procedure, so as to verify the precision of the experimental process; and the standard adding refers to adding a target object with a known concentration into a sample, and calculating the recovery efficiency of the added target object by detecting the concentration of the sample and the concentration of the standard added sample so as to determine the accuracy of the experimental process.
TABLE 6 precision and accuracy of antibiotics in water and soil
Example 2 in the process of sampling environmental water quality, the liquid sample is added with methanol for fixation, and the stable preservation effect is better
The other links of the pretreatment of the water quality sample are the same, and 0.5g of Na is added into every 1L of liquid sample in the early stage sampling2-EDTA, 0.1M sulfuric acid to pH 4.0, methanol 10 mL; and methanol was not added (0.5 g Na per 1L liquid sample)2-EDTA; pH adjusted to 4.0 with 0.1M sulfuric acid) as a control, see example 1 for other methods, the results are as follows:
TABLE 7 Water quality sample Collection fixative addition contrast test
Example 3 optimization of sample pretreatment method after environmental water sampling
Before the acquired liquid sample is enriched by adopting an HLB solid-phase extraction column, ethyl acetate is added to be used as an activating reagent, so that the enrichment efficiency of the small column can be improved.
This example sets ethyl acetate and no ethyl acetate as comparative conditions during liquid sample pre-treatment during activation of the HLB solid phase extraction cartridge only, see example 1 for additional methods, with the following results:
TABLE 8 Water quality sample enrichment link contrast test
EXAMPLE 4 screening of extraction solvent in sample pretreatment method
(1) Grinding and sieving
After the sediment sample is transported back to the laboratory, the sediment sample should be first freeze-dried in a freeze-dryer for 24 hours to sufficiently remove the water from the sediment. The dried sample is suitably ground and sieved through a 10 mesh sieve to remove non-soil components on the sieve. In order to increase the specific surface area of subsequent ASE extraction, a sample sieved by a 10-mesh sieve is further ground, then sieved by a 60-mesh sieve, and then placed in a valve bag.
(2) ASE extraction
Taking a cleaned and dried extraction tank, adding an ASE filter membrane at the bottom of the extraction tank after the extraction tank is assembled, sequentially filling 5g of diatomite and 5g of sediment sample into the extraction tank, and adding substitute kappa oxygen (20 mu l of which the concentration is 1ug/ml) and 5g of diatomite, wherein the diatomite needs to be burned at 400 ℃ for 4 hours in advance, and the volume of the filled diatomite occupies more than 2/3 of the extraction tank without compaction. The loaded extraction cells are sequentially placed on ASE, extraction bottles which are numbered and correspond to the extraction cells are placed at the bottoms of the extraction cells, and the extraction bottles are cleaned, dried and sealed by aluminum foil paper before use. The solvent bottle was checked for sufficient amount of extraction solvent and sufficient nitrogen. The extraction solvent is 10% of EDTA-Mellvine-methanol (a comparison group of 3 extraction solvents is set at the same time, namely a comparison group 1 is set, the solvent is only EDTA-MMellvaine, a comparison group 2 is set, the solvent is EDTA-MMellvaine-n-hexane, and a comparison group 3 is set, namely EDTA-MMellvaine-acetone (the concentration is 10%)), the extraction conditions are set to be 50 +/-10 ℃, 1500psi (preheating is carried out for 5min, static extraction is carried out for 10-15min, the washing volume is 60%, and the circulation is carried out for 2 times).
(3) Purifying, concentrating and fixing volume
And (3) blowing and concentrating ASE extraction liquid nitrogen by using a full-automatic nitrogen blowing instrument, adding 5ml of methanol-water solution (20/80, V/V) to start purification when blowing to the vicinity of 5ml, enriching by using an HLB (hydrophile-lipophile balance) small column, eluting by using methanol, blowing nitrogen to be clean and dry, fixing the volume to 1ml by using initial mobile phase water/acetonitrile (95/5, V/V), filtering by using a microporous filter membrane into a sample bottle, and detecting.
Different extraction solvents are selected for pretreatment of soil samples, other methods are shown in example 1, and the recovery rate results are shown in table 9 below.
TABLE 9 Effect of soil sample extraction solvent selection on sample recovery
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for accurately and sensitively detecting antibiotics in environmental samples is characterized in that,
(1) if the environmental sample is a liquid sample, after the liquid sample is collected, a fixing agent is added, and then the liquid sample is pretreated: adding a substitute carbachol oxygen before solid-phase extraction of a liquid sample, and sequentially activating a solid-phase extraction column by using ethyl acetate, methanol and ultrapure water; eluting the sample by using methanol after enrichment; adding an internal standard substance after elution, and fixing the volume;
(2) if the environmental sample is a solid sample, the method for pretreating the solid sample comprises the following steps: grinding a dried solid sample, adding a substitute carbalkoxy, and performing pretreatment by using an ASE extraction technology, wherein an extraction solvent is a solution prepared by mixing EDTA-mellvaine and methanol;
(3) and (3) detecting the pretreated liquid sample or solid sample by adopting high performance liquid chromatography-tandem mass spectrometry LC/MS/MS.
2. The method of claim 1, wherein if the environmental sample is a liquid sample, the fixative is added after the liquid sample is collected by adding 0.5g Na to 1L of the liquid sample2-EDTA; adjusting the pH to 4.0 with 0.1M sulfuric acid; 10mL of methanol was added.
3. The method according to claim 1, wherein the volume ratio of the ethyl acetate, the methanol and the ultrapure water in the step (1) is (0.7-1.3): (0.7-1.3): (1.7-2.3), controlling the flow rate of solid phase extraction to be 2.5-3.5 mL/min; leaching the wall of the HLB column by ultrapure water after extraction, drying the solid-phase extraction column in vacuum after leaching to remove water, eluting the sample in a container by adopting methanol, staying the methanol in the tube each time to fully elute the antibiotics, and then controlling the flow rate to be 0.8-1.2 mL/min;
or, in the step (2), the extraction solvent is a methanol solution containing 8-12% of EDTA-mellvaine, and the% is volume percentage.
4. The method according to claim 1, wherein the internal standard substance is a mixture of sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5, preferably an internal standard mixture with a concentration of 500 μ g/L; the volume ratio of five of the internal standard mixtures is 1:1:1:1: 1.
5. The method according to claim 1, wherein in the step (1), the eluent obtained after elution is blown to be clean and dry, and an internal standard substance is added to be fixed in volume by using an initial mobile phase of 0.5% formic acid-water solution/acetonitrile (95/5, V/V) and is to be detected;
or, the ASE extraction condition in the step (2) is 50 +/-10 ℃, 1500psi, preheating for 5min, static extraction for 10-15min, washing volume of 60%, and circulating for 2 times.
6. The method according to any one of claims 1 to 5, wherein in the step (2), the extraction solvent is 10% EDTA-mellvaine in methanol, the extraction conditions are 60 ℃, 1500psi, preheating is carried out for 5min, static extraction is carried out for 10-15min, the washing volume is 60%, and the circulation is carried out for 2 times, wherein the% is volume percentage.
7. The method according to claim 6, wherein in the step (2), the extraction liquid is purified and concentrated to a constant volume; after the extract liquid is blown to be about 5ml, methanol-water solution (20/80, V/V) is added, the HLB column is used for enrichment, methanol is used for elution, and then nitrogen is blown to be clean and dry;
the concentration constant volume is to blow and concentrate purified liquid nitrogen to less than 0.5ml, then take out and continue to blow the nitrogen until the purified liquid nitrogen is clean and dry, add an internal standard substance, use 0.5% formic acid-water solution/acetonitrile (95/5, V/V) of initial mobile phase to fix the volume to 1ml, filter the liquid into a sample bottle by a microporous filter membrane to obtain a pre-treated sample to be detected; the internal standard substance is an internal standard mixture of sulfanilamide D5, enrofloxacin D5, demethyl aureomycin, roxithromycin D7 and chloramphenicol D5, the volume ratio of the five internal standard substances is 1:1:1:1: 1.
8. The method according to claim 7, wherein the gradient elution stage of detection is performed by using 0.1% formic acid-water solution as mobile phase of solution A and acetonitrile as mobile phase of solution B, wherein the acetonitrile ratio is respectively increased from 5% to 30% in 0-5min, from 5% to 35% in 5-6min, from 40% in 6-7min, from 60% in 7-9min, from 95% in 9-10min and from 95% in 10-12min, and the percentages are volume percentages.
9. The method according to claim 7, wherein the gradient condition of the mobile phase is:
10. the method according to any one of claims 1 to 9, wherein the mass spectrum condition parameters of the internal standard substance and the substitute substance for antibiotic detection by high performance liquid chromatography-tandem mass spectrometry LC/MS/MS are as follows:
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113063864A (en) * | 2021-03-17 | 2021-07-02 | 湖北省地质实验测试中心(国土资源部武汉矿产资源监督检测中心) | Method for extracting and measuring sulfonamide antibiotics in solid-state environment sample by using aqueous solvent |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014190944A1 (en) * | 2013-05-31 | 2014-12-04 | 南京祥中生物科技有限公司 | Biochip and method for simultaneously and visually detecting multiple antibiotics, illegal additives, and biotoxins |
| CN104698107A (en) * | 2015-03-24 | 2015-06-10 | 中国地质大学（武汉） | Pretreatment method of using accelerated solvent extraction on various antibiotics remained in soil |
| CN105241990A (en) * | 2015-09-25 | 2016-01-13 | 沈阳药科大学 | Method for determining 10 kinds of antibiotics in water environment through combination of sample pre-treatment technology and HPLC-MS |
| CN107543877A (en) * | 2017-06-09 | 2018-01-05 | 上海市环境科学研究院 | A kind of method that SPE liquid chromatography tandem mass spectrometry determines six 24 kinds of antibiotic of class in water body simultaneously |
| CN108318613A (en) * | 2018-01-11 | 2018-07-24 | 四川国测检测技术有限公司 | The detection method of antibiotic in a kind of environmental sample |
| CN110658271A (en) * | 2019-09-05 | 2020-01-07 | 南方科技大学 | Method for simultaneously determining residual amounts of five antibiotics in water sample |
| CN111505149A (en) * | 2020-04-30 | 2020-08-07 | 华东理工大学 | Method for detecting sulfanilamide and β -lactam antibiotics in livestock and poultry breeding wastewater |
-
2020
- 2020-12-25 CN CN202011563945.6A patent/CN112505195A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014190944A1 (en) * | 2013-05-31 | 2014-12-04 | 南京祥中生物科技有限公司 | Biochip and method for simultaneously and visually detecting multiple antibiotics, illegal additives, and biotoxins |
| CN104698107A (en) * | 2015-03-24 | 2015-06-10 | 中国地质大学（武汉） | Pretreatment method of using accelerated solvent extraction on various antibiotics remained in soil |
| CN105241990A (en) * | 2015-09-25 | 2016-01-13 | 沈阳药科大学 | Method for determining 10 kinds of antibiotics in water environment through combination of sample pre-treatment technology and HPLC-MS |
| CN107543877A (en) * | 2017-06-09 | 2018-01-05 | 上海市环境科学研究院 | A kind of method that SPE liquid chromatography tandem mass spectrometry determines six 24 kinds of antibiotic of class in water body simultaneously |
| CN108318613A (en) * | 2018-01-11 | 2018-07-24 | 四川国测检测技术有限公司 | The detection method of antibiotic in a kind of environmental sample |
| CN110658271A (en) * | 2019-09-05 | 2020-01-07 | 南方科技大学 | Method for simultaneously determining residual amounts of five antibiotics in water sample |
| CN111505149A (en) * | 2020-04-30 | 2020-08-07 | 华东理工大学 | Method for detecting sulfanilamide and β -lactam antibiotics in livestock and poultry breeding wastewater |
Non-Patent Citations (5)
| Title |
|---|
| 夏天骄等: "基于固相萃取-高效液相色谱法的畜禽粪便中四环素类抗生素残留量检测", 《安全与环境学报》 * |
| 孙宝利等: "ASE-HPLC-MS/MS法测定土壤中四环素类抗生素残留", 《农业环境与发展》 * |
| 孙广大等: "固相萃取-超高压液相色谱-串联质谱同时分析环境水样中四环素类和喹诺酮类抗生素", 《色谱》 * |
| 屈芳等: "高效液相色谱-串联质谱法测定水果中的8种环境雌激素", 《中国食品卫生杂志》 * |
| 张营等: "《现代仪器分析技术及其在环境领域中的应用》", 31 December 2019, 辽宁大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113063864A (en) * | 2021-03-17 | 2021-07-02 | 湖北省地质实验测试中心(国土资源部武汉矿产资源监督检测中心) | Method for extracting and measuring sulfonamide antibiotics in solid-state environment sample by using aqueous solvent |
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