CN114720571B - Method for detecting 15 antibiotics in fish body - Google Patents

Method for detecting 15 antibiotics in fish body Download PDF

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CN114720571B
CN114720571B CN202011528420.9A CN202011528420A CN114720571B CN 114720571 B CN114720571 B CN 114720571B CN 202011528420 A CN202011528420 A CN 202011528420A CN 114720571 B CN114720571 B CN 114720571B
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phase
solution
antibiotics
sample
internal standard
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CN114720571A (en
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胡双庆
沈根祥
张洪昌
李贞金
郭文宏
朱英
李雨薇
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Shanghai Academy of Environmental Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G01N30/8634—Peak quality criteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Abstract

The invention provides a method for detecting 15 antibiotics in fish bodies, which comprises the following steps: sequentially adding an internal standard substance and an extraction solution into a fish sample, performing ultrasonic extraction, adding the obtained extraction solution into a solid phase extraction column for enrichment, then eluting and degreasing, determining the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, determining 15 antibiotics in the sample solution according to the retention time, quantifying by adopting an internal standard curve method, and determining the content of the 15 antibiotics in the sample solution. The method for detecting 15 antibiotics in fish bodies provided by the invention can be used for rapidly, efficiently, sensitively and accurately detecting 7 kinds of 15 antibiotics in fish bodies at the same time, greatly improves the detection efficiency, and has the advantages of high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection results and the like.

Description

Method for detecting 15 antibiotics in fish body
Technical Field
The invention belongs to the technical field of organic pollutant residue detection, relates to a method for detecting 15 antibiotics in fish bodies, and in particular relates to a method for detecting 15 antibiotics in fish bodies by combining ultrasonic extraction-solid phase extraction with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).
Background
In recent years, the aquaculture industry in China is rapidly developed, the scale and intensification of the aquaculture industry become the trend of cultivation, and the occurrence frequency of aquatic animal diseases is also improved. In order to prevent and treat diseases of aquatic products and accelerate growth and reproduction of the aquatic products, the aquaculture industry widely and largely uses antibiotics. The ionic form of the antibiotic has stronger affinity to protein and DNA, and the input antibiotic can accumulate in aquatic products through bioaccumulation. Although the residual level of the antibiotics in the aquatic products is only trace, long-time ingestion can cause lesions of nerves, hematopoietic systems, liver, kidney viscera, gastrointestinal tracts and the like, toxic and side effects of antibiotic allergic reactions and the like, so that the residual detection of the antibiotics in the aquatic products has important significance for human diet exposure risk assessment and ecological risk assessment.
The aquatic products contain abundant proteins and fats, which has a great challenge in accurately and efficiently determining the antibiotic content in the aquatic products. Therefore, establishing an effective separation and enrichment method is particularly important for accurately measuring the content of antibiotics in aquatic products. The extraction method commonly used in the sample pretreatment mainly comprises a Soxhlet extraction method, an accelerated solvent extraction method, an ultrasonic extraction method, a microwave extraction method and the like. The ultrasonic extraction method is to accelerate the release, diffusion and dissolution of effective substances in cells by utilizing cavitation, mechanical effect, thermal effect and the like of ultrasonic waves, has the advantages of simple operation, high extraction efficiency, low temperature, short time and the like, and is particularly suitable for animal source samples. Meanwhile, the antibiotics have multiple types and large structural characteristic difference, the selection of the extraction solvent has direct influence on the recovery rate, the recovery rate of various antibiotics is improved to the greatest extent possible, and the content of other impurities coexisting with the antibiotics is reduced. The detection methods commonly used at present mainly comprise High Performance Liquid Chromatography (HPLC) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The HPLC-MS/MS has strong separation capability of HPLC, high sensitivity of MS and extremely strong qualitative identification capability, is one of the most effective means for detecting trace antibiotic residues in complex matrixes in the environment at present, and has the advantages of wide analysis range, strong separation capability, reliable qualitative analysis result, low detection limit, quick analysis time, high degree of automation and the like. The components of the complex mixture can be accurately, qualitatively and quantitatively determined.
At present, a complete and reliable analysis method for separating, enriching and co-detecting various antibiotics in aquatic products does not exist, and the existing method generally has the problems of instability, more interference, limited types and quantity of antibiotics to be detected, limited recovery rate and the like. Therefore, a need exists for a complete and reliable assay including separation enrichment and co-detection that rapidly, efficiently, sensitively, and accurately detects as much antibiotic residue as possible in aqueous products to meet increasingly stringent residue limiting requirements.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for detecting 15 antibiotics in fish, which can simultaneously detect the residual condition of 15 antibiotics in fish at one time, has the advantages of stable detection result, small interference, rapidness, high efficiency, high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection result, and the like.
To achieve the above and other related objects, the present invention provides a method for detecting 15 antibiotics in fish bodies, comprising: sequentially adding an internal standard substance and an extraction solution into a fish sample, performing ultrasonic extraction, adding the obtained extraction solution into a solid phase extraction column for enrichment, then eluting and degreasing, determining the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, determining 15 antibiotics in the sample solution according to the retention time, and determining the content of the 15 antibiotics in the sample solution by adopting an internal standard curve method.
Preferably, the 15 antibiotics include sulfadiazine SD (CAS number 68-35-9), sulfamethyidine SM1 (CAS number 127-79-7), sulfadimidine SM2 (CAS number 57-68-1), sulfam-dimethoxy pyrimidine SDM (CAS number 122-11-2), sulfamethoxazole SMX (CAS number 723-46-6), trimethoprim TMP (CAS number 738-70-5) of sulfa potentiators, enrofloxacin ENR (CAS number 93106-60-6) of fluoroquinolones, ciprofloxacin hydrochloride CIP (CAS number 93107-08-5), norfloxacin (CAS number 70458-96-7), furazolidone AOZ (CAS number 67-45-8) of nitrofurans, aureomycin hydrochloride CTC (CAS number 64-72-2) of tetracyclines, oxytetracycline hydrochloride OTC (CAS number 2058-46-0) of tetracycline hydrochloride DOX (CAS number 24390-14-5), and quinolone hydrochloride of benzoquinone (CAS number 3524-34-28) of fluorostyril (35-28).
Preferably, the fish sample is homogenized. The homogenization is to clean the collected fish body sample, absorb the surface moisture with gauze, cut the back muscle of the fish body, separate the fish skin, and homogenate the fish body sample after adding water with the same quality. The homogenized fish sample was frozen at-20 ℃.
Preferably, the internal standard comprises sulfamethoxazole-D 4 SMX-D 4 (CAS number 1020719-86-1), trimethoprim-D 3 TMP-D 3 (CAS number 1189923-38-3), norfloxacin-D 5 NOR-D 5 (CAS number 1015856-57-1), furazolidone-D 4 AOZ-D 4 (CAS number 1217222-76-8), tetracycline-D 6 TC-D 6 (CAS number 60-54-8) olaquindox-D 4 OLA-D 4 (CAS number 1189487-82-8) and florfenicol-D 3 FF-D 3 (CAS number 73231-34-2).
More preferably, in the internal standard, sulfamethoxazole-D 4 SMX-D 4 As internal standard of sulfadiazine SD, sulfadiazine SM1, sulfadimidine SM2, sulfam-dimethoxy pyrimidine SDM, sulfa-methyl oxazole SMX, trimethoprim-D 3 TMP-D 3 norfloxacin-D as an internal standard for trimethoprim TMP 5 NOR-D 5 Furazolidone-D as an internal standard for enrofloxacin ENR, CIP, norfloxacin NOR 4 AOZ-D 4 As an internal standard for furazolidone AOZ, tetracycline-D 6 TC-D 6 As an internal standard for aureomycin CTC, oxytetracycline OTC, doxycycline hydrochloride DOX, olaquindox-D 4 OLA-D 4 florfenicol-D as an internal standard for olaquindox OLA 3 FF-D 3 As an internal standard for florfenicol FF.
Given the large differences in structure and properties of the different classes of antibiotics, the addition of internal standards avoids possible losses during pretreatment.
Preferably, the ratio of the mass of the fish sample to the mass of the 7 internal standard additions is 5.00+/-0.02: 1X 10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 。
Preferably, the fish sample is added with an internal standard and is kept stand for 5-15min, preferably 10min, in dark after being uniformly mixed.
Preferably, the extraction solution comprises acetonitrile and phosphate buffer sequentially added to the fish sample.
More preferably, the volume ratio of acetonitrile to phosphate buffer is added is 0.95-1.05:1, preferably 1:1.
More preferably, the pH of the phosphate buffer is 2.95-3.05, preferably 3.
More preferably, the acetonitrile is added to the fish sample and vortexed for 0.5-1.5min, preferably 1min. So as to denature the protein and release the antibiotic component bound thereto.
More preferably, the acetonitrile phosphate buffer is added to the fish sample and vortexed for 0.5-1.5min, preferably 1min. Providing a relatively stable pH environment for the antibiotic component.
Preferably, the ratio of the mass g of the fish sample addition to the volume mL of the extraction solution addition is 5.00±0.02:18-22, preferably 5.00±0.02:20.
preferably, the time of the ultrasonic extraction is 15-25min, preferably 20min.
Preferably, the extract is centrifuged, the supernatant is filtered, and the pH of the extract is adjusted after dilution with water.
More preferably, the centrifugation time is 4-6min, and the centrifugation speed is 9000-11000 r.min -1 . Further preferably, the centrifugation time is 5min, and the centrifugation speed is 10000 r.min -1 。
More preferably, the filtration is carried out using a glass fiber filter membrane having a pore size of 0.6 to 0.8. Mu.m, preferably 0.7. Mu.m.
More preferably, the dilution is to a volume ratio of acetonitrile of less than 10%.
More preferably, the pH is adjusted to 2.5-3. Can use 4 mol.L -1 And adjusting by hydrochloric acid.
Preferably, the solid phase extraction column is an Oasis HLB column. The Oasis HLB column is produced by Waters company, is a hydrophilic-lipophilic polymer packing column, and the adsorbent is a macroporous copolymer formed by polymerizing lipophilic divinylbenzene and hydrophilic N-vinyl pyrrolidone according to a certain proportion, and is a universal adsorbent suitable for acidic, neutral and alkaline compounds.
Preferably, the solid phase extraction column is activated by adopting methanol and water in sequence before enrichment, wherein the dosage of the methanol and the water in the activation is 1-2 times of the volume of the solid phase extraction column filler; and after enrichment, the solid phase extraction column is sequentially leached by water and methanol solution, and air pushing is carried out, wherein the dosage of the water and the methanol solution in leaching is 1-2 times of the volume of the solid phase extraction column filler.
More preferably, the dosage of the methanol, the water and the methanol solution is 6-12mL.
More preferably, the methanol solution is an aqueous methanol solution with a volume percent concentration of less than or equal to 10 percent. In the aqueous methanol solution, the ratio of methanol to water is not particularly limited. The purpose is to wash away the excess impurities in the solid phase extraction column, but the proportion of methanol in the aqueous solution used for leaching is not more than 10% in order to prevent the possibility of simultaneously washing out the target.
More preferably, the air pushing is to discharge the leaching solution remained in the solid phase extraction column in a nitrogen air pushing mode.
Preferably, the solid phase extraction column is eluted by methanol, and the dosage of the methanol is 2-3 times of the volume of the solid phase extraction column packing.
More preferably, the methanol is used in an amount of 10-15mL.
Preferably, the flow rates of activation, loading, leaching and elution in the solid phase extraction column are not more than 5 mL-min -1 。
Preferably, the degreasing is to blow-dry the eluent in a water bath with nitrogen, add the dissolution liquid to dissolve residues, add n-hexane to vortex and mix, then centrifuge, take the supernatant and filter, thus obtaining the sample solution to be measured. Acetonitrile is a nonpolar solvent, and can extract a large amount of fat impurities, so that n-hexane is required for further degreasing.
More preferably, the temperature of the water bath is 35-45 ℃, preferably 40 ℃.
More preferably, the dissolution liquid is selected from one of methanol, acetonitrile or aqueous methanol solution with the volume percentage concentration of 70-80%.
More preferably, the amount of the dissolution liquid is 0.5 to 1.5mL, preferably 1.0mL.
More preferably, the amount of n-hexane is 1.5-2.5mL, preferably 2.0mL.
More preferably, the vortex mixing is for a period of 20 to 40s, preferably 30s.
More preferably, the centrifugation time is 4-6min, and the centrifugation speed is 9000-11000 r.min -1 . Further preferably, the centrifugation time is 5min, and the centrifugation speed is 10000 r.min -1 。
More preferably, the filtration is performed using a needle filter having a membrane of 0.22 μm pore size of an organic phase (nylon) filter.
Preferably, the sample solution is measured by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), comprising the following steps:
1) Preparing a standard solution: 15 antibiotic standard substances are taken, and an internal standard substance and a methanol solution are added to prepare a standard solution;
2) Sample detection: the positive ion mode and the negative ion mode of an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method are adopted to respectively detect the sample solution and the standard solution in the step 1), the obtained liquid chromatogram of the sample solution is compared with the liquid chromatogram of the standard solution, the common characteristic peak is identified according to the relative retention time for qualitative determination, and then the concentration of 15 antibiotics in the sample solution is determined by quantifying according to the chromatographic peak area of the common characteristic peak through an internal standard curve method.
More preferably, in step 1), the concentration of 7 internal standard substances in the standard solution is 100.0. Mu.g.L -1 。
More preferably, in step 1), the methanol solution is an aqueous methanol solution with a volume percentage concentration of 65-75%, and the volume percentage concentration is preferably 70%.
More preferably, in step 1), the concentration of 15 antibiotics in the standard solution is in the range of 1.37-500. Mu.g.L -1 。
More preferably, in step 2), in the ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, the florfenicol is detected by using a negative ion mode, and 14 other antibiotics other than florfenicol are detected by using a positive ion mode.
More preferably, in step 2), in the ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), the positive ion mode is detected by using a Waters AcquityUPLCIclass ultra performance liquid chromatography tandem Waters TQ-XS mass spectrometer.
Further preferably, in the detection of the positive ion mode, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: a C18 column; column temperature: 35-45 ℃; sample injection amount: 1-2 mu L; flow rate: 0.3-0.4mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The mobile phase A is formic acid aqueous solution with the volume percentage concentration of 0.4-0.6%; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
Most preferably, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1×column length 50mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 1.5. Mu.L; flow rate: 0.35mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the The mobile phase A is formic acid aqueous solution with the volume percentage concentration of 0.5%; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
Most preferably, the specific procedure of the gradient elution is:
0-1min, phase A: the volume ratio of the phase B is 92:8-92:8, 8;
1-1.2min, phase A: the volume ratio of the phase B is 92:8-80:20, a step of;
1.2-2.5min, phase A: the volume ratio of the phase B is 80:20-80:20, a step of;
2.5-2.7min, phase A: the volume ratio of the phase B is 80:20-5:95;
2.7-4.5min, phase A: the volume ratio of the phase B is 5:95-5:95;
4.5-4.7min, phase A: the volume ratio of the phase B is 5:95-92:8, 8;
4.7-7.5min, phase A: the volume ratio of the phase B is 92:8-92:8.
further preferably, in the detection of the positive ion mode, the mass spectrum (MS/MS) is determined under the following conditions:
ionization mode: electrospray ion source (ESI), positive ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the scanning time is 0.1s; the collision gas is argon; the desolventizing gas temperature is 500 ℃; the flow rate of the desolventizing air flow is 1000 L.Hr -1 The method comprises the steps of carrying out a first treatment on the surface of the The cone voltage was 3500v.
Most preferably, the mass-to-charge ratios of parent ions, the mass-to-charge ratios of child ions, the cone voltages, and the collision energies of the 14 antibiotics and the corresponding internal standard other than florfenicol are shown in table 1.
Table 1 conditions for mass spectrometric detection of 14 antibiotics and internal standard
Note that: * Representing quantitative ions; * Represents the collision energy corresponding to the quantitative ion.
More preferably, in step 2), in the ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, the negative ion mode is detected by using an shimadzu 30A ultra performance liquid chromatography tandem AB 5500Q-trap mass spectrometer (AB company in the united states).
Further preferably, when the negative ion mode is detected, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: a C18 column; column temperature: 35-45 ℃; sample injection amount: 1-2 mu L; flow rate: 0.3-0.4mL/min; the mobile phase A is water; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
Most preferably, the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are:
chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1X column length 100mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 1.5. Mu.L; flow rate: 0.35mL/min; the mobile phase A is water; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
Most preferably, the specific procedure of the gradient elution is:
0-1min, phase A: the volume ratio of the phase B is 90:10-90:10;
1-1.2min, phase A: the volume ratio of the phase B is 90:10-60:40, a step of performing a;
1.2-3min, phase A: the volume ratio of the phase B is 60:40-60:40, a step of performing a;
3-3.5min, phase A: the volume ratio of the phase B is 60:40-10:90;
3.5-5min, phase A: the volume ratio of the phase B is 10:90-10:90;
5-5.2min, phase A: the volume ratio of the phase B is 10:90-90:10;
5.2-7.5min, phase A: the volume ratio of the phase B is 90:10-90:10.
further preferably, in the detection in the negative ion mode, the mass spectrum (MS/MS) is determined under the following conditions:
ionization mode: electrospray ion source (ESI), negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, and the auxiliary heating gas GS2 was 50psi.
Most preferably, the mass-to-charge ratios of parent ions, the mass-to-charge ratios of daughter ions, the cone voltage, and the collision energy of the florfenicol and the corresponding internal standard are shown in Table 2.
Table 2 mass spectrometric detection conditions for florfenicol and internal standard
Note that: * Representing quantitative ions; * Represents the collision energy corresponding to the quantitative ion.
Preferably, in step 2), the internal standard curve method includes the following steps:
a1 Respectively carrying out positive ion mode and negative ion mode analysis of a high performance liquid chromatography-mass spectrometry method on standard solutions containing 15 antibiotics with a series of different concentrations to obtain linear relations of the ratios of chromatographic peak areas of 15 antibiotic components under different concentrations to the ratios of different concentrations of corresponding components to the internal standard concentration, drawing corresponding standard curves, and carrying out regression operation by using a weighted least square method to obtain regression equations of the standard curves of 15 antibiotic components;
a2 Analyzing the sample solution by using a high performance liquid chromatography-mass spectrometry method, substituting the obtained ratio of the chromatographic peak areas of the 15 antibiotic components under different concentrations to the chromatographic peak areas of the internal standard into the regression equation of the standard working curve of the corresponding components in the step A1), and calculating the concentration of the 15 antibiotic components in the sample solution according to the known concentration of the internal standard.
More preferably, in step A1) or A2), the ratio of the chromatographic peak areas of 15 antibiotic components at different concentrations to the chromatographic peak areas of the internal standard is taken as the ordinate (Y-axis) and the ratio of the different concentrations of the corresponding components to the concentration of the internal standard is taken as the abscissa (X-axis) in the standard working curve.
The water used in the present invention is ultrapure water.
As described above, the method for detecting 15 antibiotics in fish provided by the invention adopts the ultrasonic extraction-solid phase extraction combined with high performance liquid chromatography tandem mass spectrometry technology to optimize the extraction method, the enrichment and purification conditions and the liquid quality detection conditions, and has the following beneficial effects:
(1) The method for detecting 15 antibiotics in fish provided by the invention adopts ultrasonic extraction to extract the antibiotics in fish, and has the advantages of high extraction efficiency, less extractant consumption and short extraction time.
(2) According to the method for detecting 15 antibiotics in the fish body, provided by the invention, the composite solution of the organic solvent and the buffer solution is used as the extracting solution, so that the target object is in a relatively stable pH environment in the extracting process, and compared with the method for extracting the target object by using only the organic solvent, the method has the advantages of more stable recovery rate of the target object, high extraction efficiency and small matrix effect.
(3) According to the method for detecting 15 antibiotics in the fish body, the Oasis HLB solid phase extraction column is selected to enrich and purify 7 kinds of 15 antibiotics, so that the recovery rate is high and stable, the selectivity is high, the adsorption capacity is high, the interferents can be effectively separated, and the purpose of separating and enriching the target substances is achieved.
(4) According to the method for detecting 15 antibiotics in the fish body, the internal standard curve method is adopted to quantify the antibiotics, the linear relation of the measurement result is good, the relative deviation is small, and the analysis precision is improved.
(5) The method for detecting 15 antibiotics in fish provided by the invention adopts the ultra-high performance liquid chromatography tandem mass spectrometer to carry out quantitative analysis detection, has high sensitivity, and the detection limit of 15 antibiotics is lower than 2 mug.L -1 Can meet the detection requirement of trace antibiotics in fish meat. In addition, the tandem triple quadrupole mass spectrometer performs qualitative analysis according to characteristic fragment ions generated by collision of corresponding parent ions, has strong selectivity, and eliminates interference of other impurity signals in the sample.
(6) According to the method for detecting 15 antibiotics in the fish body, the internal standard substance for indicating the recovery rate is added before the extracting solution is added, so that the loss of the antibiotics in the whole pretreatment process can be more accurately represented, meanwhile, the same kind of internal standard substance for indicating the recovery rate is selected for the same kind of antibiotics in consideration of the loss of different kinds of antibiotics in the pretreatment process. And the internal standard is properly selected, so that the final detection result is more real and reliable. In the existing trace antibiotic detection method, most of the trace antibiotics are calibrated by using only one internal standard substance, and the detected result has some deviation.
(7) The method for detecting 15 antibiotics in the fish body provided by the invention has the advantages of small using amount of organic reagent and environmental friendliness. The method can simultaneously detect the residual condition of 15 antibiotics in the fish meat at one time, the detection process is less in time consumption, and the cost of manpower and material resources is saved.
(8) The method for detecting 15 antibiotics in fish bodies provided by the invention can be used for rapidly, efficiently, sensitively and accurately detecting 7 kinds of 15 antibiotics in fish bodies at the same time, greatly improves the detection efficiency, has stable detection results and small interference, and has the advantages of high recovery rate, high sensitivity, high stability, low detection limit, more true and reliable detection results and the like.
Drawings
FIG. 1 is a graph showing the labeled recovery of 15 antibiotics from fish flesh measured using 3 different extraction solutions in accordance with the present invention.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The reagents or apparatus used in the examples below are conventional products available commercially without the manufacturer's knowledge.
Example 1
1. Sample pretreatment
Cleaning the collected fish body sample, sucking the surface water with gauze, cutting back muscle of the fish body, separating fish skin, adding water with the same mass into the fish body sample, homogenizing, rapidly freezing at-20deg.C, and further extracting.
5.00g of homogenized fish sample were weighed into 30mL glass centrifuge tubes, and 100ng of sulfamethoxazole-D was added, respectively 4 trimethoprim-D 3 norfloxacin-D 5 Furazolidone-D 4 Tetracycline-D 6 olaquindox-D 4 And florfenicol-D 3 The 7 internal standard substances are fully and uniformly mixed and then kept stand for 10min in a dark place. And adding 10mL of acetonitrile into the centrifuge tube, mixing for 1min by vortex, denaturing the protein by the acetonitrile, releasing the combined drug, adding 10mL of phosphate buffer solution with pH=3, mixing for 1min by vortex, providing a relatively stable pH environment for a target object by the buffer solution, and performing ultrasonic extraction for 20min. Then, 10000 r.min -1 Centrifuging at rotation speed for 5min, collecting supernatant, filtering with glass fiber filter membrane with pore diameter of 0.7 μm, collecting extractive solution, diluting with distilled water to 250mL, and concentrating with 4mol.L -1 The pH was adjusted to 2.5 with hydrochloric acid.
Using ReekoFotector Plus full-automatic solid phase extraction instrument, selecting the solid phase extraction column as Oasis HLB column. Methanol (5 mL. Min.) was used in sequence before enrichment -1 6 mL), water (5 mL. Min -1 6 mL) and then activating the mixture, and then mixing the mixture with 250mL of the extract at 3.5 mL/min -1 Loading onto column for enrichment, and sequentially adding ultrapure water (5 mL. Min -1 6 mL), 5% by volume aqueous methanol solution (5 mL. Min) -1 Eluting with 6mL, air-drying, and eluting with methanol (5 mL. Min) -1 10 mL), an eluent was obtained. Drying the eluent in water bath at 40deg.C under nitrogen, adding 1.0mL of 70% methanol water solution to dissolve residues, transferring into 4mL centrifuge tube, adding 2.0mL of n-hexane, mixing for 30s at 10000 r.min -1 Centrifuging for 5min, removing upper n-hexane layer, collecting supernatant, and passing through organic solvent with pore diameter of 0.22 μmThe phase (nylon) needle filter is filtered into a brown sample bottle to obtain a sample solution to be measured.
2. Standard solution
15 antibiotic standard substances are taken, 7 internal standard substances and 70 percent methanol aqueous solution with volume percentage concentration are added to prepare a series of standard solutions with different concentrations. Wherein the 15 antibiotics comprise sulfadiazine SD, sulfadiazine SM1, sulfadimidine SM2, sulfam-dimethoxypyrimidine SDM, sulfamethoxazole SMX, trimethoprim TMP, enrofloxacin ENR, ciprofloxacin CIP, norfloxacin NOR, furazolidone AOZ, aureomycin CTC, terramycin OTC, doxycycline hydrochloride DOX, olaquindox OLA, and florfenicol FF. The concentration of 7 internal standard substances in the standard solution is 100.0 mug.L -1 . The concentration of 15 antibiotics in a series of standard solutions with different concentrations is 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 500 mug.L respectively -1 。
3. Detection of
The positive ion mode and the negative ion mode of an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method are adopted to respectively detect a sample solution and a standard solution, the obtained liquid chromatogram of the sample solution is compared with the liquid chromatogram of the standard solution, the characterization of a common characteristic peak is identified according to the relative retention time, and then the quantification is carried out according to the chromatographic peak area of the common characteristic peak by an internal standard curve method, so that the concentration of 15 antibiotics in the sample solution is determined.
Specifically, standard solutions containing 15 antibiotics with a series of different concentrations are respectively subjected to positive ion mode and negative ion mode analysis by a high performance liquid chromatography-mass spectrometry method, linear relations of the ratios of chromatographic peak areas of 15 antibiotic components under different concentrations to the ratios of the different concentrations of the corresponding components to the internal standard are obtained, corresponding standard curves are drawn, regression operation is carried out by a weighted least square method, and regression equations of the standard curves of the 15 antibiotic components are obtained. And analyzing the sample solution by using a high performance liquid chromatography-mass spectrometry method, substituting the obtained ratio of the chromatographic peak areas of the 15 antibiotic components under different concentrations to the chromatographic peak areas of the internal standard into a regression equation of a standard working curve of the corresponding components, and calculating the concentration of the 15 antibiotic components in the sample solution according to the known concentration of the internal standard.
In an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, the florfenicol is detected by adopting a negative ion mode, and 14 antibiotics except the florfenicol are detected by adopting a positive ion mode.
The positive ion mode was detected using a Waters AcquityUPLCIclass ultra high performance liquid chromatography tandem Waters TQ-XS mass spectrometer.
The measurement conditions of ultra high performance liquid chromatography (UPLC) were: chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1×column length 50mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 1.5. Mu.L; flow rate: 0.35mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the The mobile phase A is formic acid aqueous solution with the volume percentage concentration of 0.5%; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
The specific procedure of gradient elution is:
0-1min, phase A: the volume ratio of the phase B is 92:8-92:8, 8;
1-1.2min, phase A: the volume ratio of the phase B is 92:8-80:20, a step of;
1.2-2.5min, phase A: the volume ratio of the phase B is 80:20-80:20, a step of;
2.5-2.7min, phase A: the volume ratio of the phase B is 80:20-5:95;
2.7-4.5min, phase A: the volume ratio of the phase B is 5:95-5:95;
4.5-4.7min, phase A: the volume ratio of the phase B is 5:95-92:8, 8;
4.7-7.5min, phase A: the volume ratio of the phase B is 92:8-92:8.
the measurement conditions of mass spectrometry (MS/MS) were: ionization mode: electrospray ion source (ESI), positive ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the scanning time is 0.1s; the collision gas is argon; the desolventizing gas temperature is 500 ℃; the flow rate of the desolventizing air flow is 1000 L.Hr -1 The method comprises the steps of carrying out a first treatment on the surface of the The cone voltage was 3500v. The parent ion mass to charge ratio, child ion mass to charge ratio, cone hole voltage, and collision energy of 14 antibiotics and corresponding internal standard substances except florfenicol are shown in table 1.
The negative ion mode is detected by using an Shimadzu 30A ultra-high performance liquid chromatography (UHF-HPLC) tandem AB 5500Q-trap mass spectrometer (AB company in America).
The measurement conditions of ultra high performance liquid chromatography (UPLC) were: chromatographic column: waters Acquity UPLC BEH C18 column (inner diameter 2.1X column length 100mm, particle size 0.7 μm); column temperature: 40 ℃; sample injection amount: 1.5. Mu.L; flow rate: 0.35mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the The mobile phase A is water; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution.
The specific procedure of gradient elution is:
0-1min, phase A: the volume ratio of the phase B is 90:10-90:10;
1-1.2min, phase A: the volume ratio of the phase B is 90:10-60:40, a step of performing a;
1.2-3min, phase A: the volume ratio of the phase B is 60:40-60:40, a step of performing a;
3-3.5min, phase A: the volume ratio of the phase B is 60:40-10:90;
3.5-5min, phase A: the volume ratio of the phase B is 10:90-10:90;
5-5.2min, phase A: the volume ratio of the phase B is 10:90-90:10;
5.2-7.5min, phase A: the volume ratio of the phase B is 90:10-90:10.
the measurement conditions of mass spectrometry (MS/MS) were: ionization mode: electrospray ion source (ESI), negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, and the auxiliary heating gas GS2 was 50psi. The mass-to-charge ratio of parent ion, the mass-to-charge ratio of child ion, the cone voltage and the collision energy of florfenicol and the corresponding internal standard are shown in Table 2.
Example 2
The fish sample has complex matrix and contains protein, fat, saccharide, inorganic salt, water and other compounds. The sample matrixes can influence the extraction efficiency of the to-be-detected object, so that the detection result is error, and meanwhile, the instrument is polluted, so that the detection efficiency and the accuracy are directly influenced. Therefore, in veterinary drug residue analysis, the pretreatment technology of the sample is very critical, and the main purpose is to separate the target compound from the sample matrix and remove the interfering impurities. Meanwhile, the target antibiotics are various in types and large in physical and chemical property difference, and in order to maximize the extraction efficiency of various antibiotics in fish meat, the invention researches the extraction effect of 3 different extracting solutions on target objects by adopting a single factor test.
Two sets of fish samples were prepared for comparison experiments, one set being a standard sample set and the other set being a blank sample set, each set being 3 replicates, as in step 1 of example 1. Accurately weighing 5.00g of fish sample in a 30mL glass centrifuge tube, respectively adding 50, 100 and 200ng of mixed standard solution of 15 antibiotics into the fish sample as standard sample group to make the final concentration of 15 antibiotics added into fish be 20 ng.g -1 、40ng·g -1 、80ng·g -1 . The blank sample group was not added with any antibiotic.
Ultrasonic extraction was performed on standard and blank sample sets, respectively, as in step 1 of example 1, using 3 different extraction solvents, respectively, (1) acetonitrile-ph=3 phosphate buffer solution (V: v=1:1); (2) acetonitrile-ph=3 citric acid buffer (V: v=1:1); (3) acetonitrile, i.e., a mixed solution of acetonitrile and acetic acid in a volume ratio of 99:1, was acidified. Solid Phase Extraction (SPE) was then performed as in step 1 of example 1. The sample solution obtained was tested according to step 3 of example 1 to determine the labeled recovery of 15 antibiotics in fish meat, the results are shown in FIG. 1. As can be seen from fig. 1, acetonitrile, ph=3 phosphate buffer solution (V: v=1:1) was used as the extraction solvent, and the recovery rate was the highest and the effect was the best. Therefore, acetonitrile: ph=3 phosphate buffer solution (V: v=1:1) was selected as the extraction solution for ultrasonic extraction in the actual detection of fish meat.
Example 3
The 15 antibiotics mixed standard solution was diluted with 70% methanol-water solution to a series of concentration gradients (1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 500. Mu.g.L according to step 2 of example 1 -1 ) The internal standard is 100.0 mug.L -1 Is added to the standard, and the response value (response value=peak area of target object/internal standardPeak area) is used as an ordinate to draw a 7-point standard curve to obtain a standard curve and a correlation coefficient (R) 2 ). Meanwhile, the detection limit and the quantitative limit of the instrument are determined by adopting a signal-to-noise ratio method (S/N), the detection Limit (LOD) is estimated by using a signal-to-noise ratio of 3 times, and the quantitative Limit (LOQ) is estimated by using a signal-to-noise ratio of 10 times. The standard curves, correlation coefficients, detection limits and quantification limits for the 15 antibiotics are shown in table 3.
As can be seen from Table 6, 15 antibiotic components have good linear relationship, correlation coefficient (R 2 ) Are all greater than 0.98.
TABLE 3 Table 3
Example 4
The fish samples were subjected to the standard recovery test as in steps 1 and 3 of example 1. Setting 3 groups of standard adding concentrations of 20, 40 and 80ng g respectively -1 3 replicates were run for each set of samples and the normalized Recovery (RE) and Relative Standard Deviation (RSD) were calculated.
The calculation formula of the addition standard recovery rate (RE%) is as follows:wherein C is 0 To mix the concentration of standard solution, mug.L -1 ；C 1 To detect the concentration of the sample without adding the mixed standard solution, mug.L -1 ；C 2 To add the detection concentration of the mixed standard solution sample, μg.L -1 ；V 0 The volume of the mixed standard solution is L; v (V) 1 The volume is fixed before the machine is started to carry out the operation of adding no mixed standard solution sample, and L; v (V) 2 And (3) fixing the volume and L before loading the mixed standard solution sample.
The method examines the standard recovery rate of 3 concentrations of 15 antibiotics in fish meat, and the result is shown in a table 4. The standard deviation of the labeled recovery rate of each antibiotic is lower than 10 percent and the labeled recovery rate of each antibiotic is lower than 61.1 to 124.9 percent, which indicates that different substrate interferences exist for different antibiotics; the influence of matrix interference can be reduced by adding an internal standard to calculate the recovery rate of the addition standard and correcting the test result by using the recovery rate of the addition standard.
TABLE 4 labeling recovery and relative standard deviation for different labeling concentrations in fish samples
Example 5
Collecting black carp and catfish in a certain aquaculture area in the Shanghai, extracting, enriching and purifying samples according to the step 1 of the embodiment 1, and detecting and analyzing the actual concentration of the samples according to the step 3 of the embodiment 1 to examine the applicability of the method to different kinds of fish samples. The 15 antibiotic contents in the muscle samples of the black carp and the catfish obtained by actual detection are shown in table 5. Experimental results show that the method can be applied to determination of the content of various antibiotics in fish meat, and has good applicability.
TABLE 5 content of 15 antibiotics in muscle samples of herring and catfish
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (3)

1. A method of detecting 15 antibiotics in fish bodies comprising: sequentially adding an internal standard substance and an extraction solution into a fish sample, performing ultrasonic extraction, adding the obtained extraction solution into a solid phase extraction column for enrichment, then eluting and degreasing, determining the obtained sample solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, determining 15 antibiotics in the sample solution according to the retention time, and determining the content of the 15 antibiotics in the sample solution by adopting an internal standard curve method;
the 15 antibiotics comprise sulfadiazine, sulfamethazine, sulfadimidine, sulfamonomethoxine, sulfamethoxine, trimethoprim, enrofloxacin, ciprofloxacin hydrochloride, norfloxacin, furazolidone, aureomycin hydrochloride, oxytetracycline hydrochloride, doxycycline hydrochloride, olaquindox and florfenicol;
the internal standard substance is composed of sulfamethoxazole-D 4 trimethoprim-D 3 norfloxacin-D 5 Furazolidone-D 4 Tetracycline-D 6 olaquindox-D 4 And florfenicol-D 3 Composition; in the internal standard, sulfamethoxazole-D 4 As an internal standard for sulfadiazine, sulfa-methyl pyrimidine, sulfa-dimethyl pyrimidine, sulfa-m-dimethoxy pyrimidine and sulfamethoxazole, trimethoprim-D 3 norfloxacin-D as an internal standard for trimethoprim 5 Furazolidone-D as an internal standard for enrofloxacin, ciprofloxacin, norfloxacin 4 As an internal standard for furazolidone, tetracycline-D 6 As an internal standard for aureomycin hydrochloride, oxytetracycline hydrochloride and doxycycline hydrochloride, olaquindox-D 4 florfenicol-D as an internal standard for olaquindox 3 As an internal standard for florfenicol;
adding an internal standard into the fish sample, uniformly mixing, and standing in a dark place for 5-15min;
the extracting solution comprises acetonitrile and phosphate buffer solution which are sequentially added into the fish sample; the ratio of the added volume of acetonitrile to the added volume of the phosphate buffer solution is 0.95-1.05:1; the pH value of the phosphate buffer solution is 2.95-3.05;
adding the acetonitrile into the fish sample, and then vortex mixing for 0.5-1.5min;
centrifuging the extract, taking supernatant, filtering, diluting the obtained extract with water, and regulating pH;
the centrifugal time is 4-6min, and the centrifugal rotating speed is 9000-11000 r.min -1 ；
The filtration is carried out by adopting a glass fiber filter membrane;
the volume ratio of the acetonitrile is reduced to below 10 percent;
the pH value is regulated to 2.5-3;
the solid phase extraction column is an Oasis HLB small column;
the solid phase extraction column is eluted by methanol, and the dosage of the methanol is 2-3 times of the volume of the solid phase extraction column filler;
the degreasing step is to blow-dry eluent in a water bath with nitrogen, add a dissolving solution to dissolve residues, add n-hexane to vortex and mix, then centrifuge, take a supernatant and filter to obtain a sample solution to be measured; the dissolving solution is selected from methanol, acetonitrile or 70-80% methanol aqueous solution by volume percentage concentration;
the temperature of the water bath is 35-45 ℃; the vortex mixing time is 20-40s; the centrifugal time is 4-6min, and the centrifugal rotating speed is 9000-11000 r.min -1 ；
The sample solution is measured by adopting an ultra-high performance liquid chromatography tandem mass spectrometry method, and the method comprises the following steps of:
1) Preparing a standard solution: 15 antibiotic standard substances are taken, and an internal standard substance and a methanol solution are added to prepare a standard solution;
2) Sample detection: respectively detecting the sample solution and the standard solution in the step 1) by adopting a positive ion mode and a negative ion mode of an ultra-high performance liquid chromatography tandem mass spectrometry, comparing the obtained liquid chromatogram of the sample solution with the liquid chromatogram of the standard solution, identifying the nature of a common characteristic peak according to the relative retention time, quantifying according to the chromatographic peak area of the common characteristic peak by an internal standard curve method, and determining the concentration of 15 antibiotics in the sample solution;
in the step 2), in the ultra performance liquid chromatography tandem mass spectrometry, 14 antibiotics except florfenicol are detected by adopting a positive ion mode, and the positive ion mode is detected by adopting a Waters AcquityUPLCIclass ultra performance liquid chromatography tandem Waters TQ-XS mass spectrometer;
the measurement conditions of the ultra-high performance liquid chromatography are as follows: chromatographic column: waters Acquity UPLC BEH C18 column 18; column temperature: 40 ℃; sample injection amount: 1.5 mu L; flow rate: 0.35mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the The mobile phase A is formic acid aqueous solution with the volume percentage concentration of 0.5%; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-1min, phase A: the volume ratio of the phase B is 92:8-92:8, 8;1-1.2min, phase A: the volume ratio of the phase B is 92:8-80:20, a step of; 1.2-2.5min, phase A: the volume ratio of the phase B is 80:20-80:20, a step of; 2.5-2.7min, phase A: the volume ratio of the phase B is 80:20-5:95;2.7-4.5min, phase A: the volume ratio of the phase B is 5:95-5:95;4.5-4.7min, phase A: the volume ratio of the phase B is 5:95-92:8, 8;4.7-7.5min, phase A: the volume ratio of the phase B is 92:8-92:8, 8;
the measurement conditions of the mass spectrum are as follows: ionization mode: electrospray ion source ESI, positive ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the scanning time is 0.1s; the collision gas is argon; the desolventizing gas temperature is 500 ℃; the flow rate of the desolventizing air flow is 1000L Hr -1 The method comprises the steps of carrying out a first treatment on the surface of the The taper hole voltage is 3500v;
in the step 2), in the ultra performance liquid chromatography tandem mass spectrometry, the florfenicol is detected by adopting an anion mode, and the anion mode is detected by adopting an Shimadzu 30A ultra performance liquid chromatography tandem AB 5500Q-trap mass spectrometer;
the measurement conditions of the ultra-high performance liquid chromatography are as follows: chromatographic column: waters Acquity UPLC BEH C18 column 18; column temperature: 40 ℃; sample injection amount: 1.5 mu L; flow rate: 0.35mL/min; the mobile phase A is water; the mobile phase B is acetonitrile; the analysis time was 7.5min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-1min, phase A: the volume ratio of the phase B is 90:10-90:10;1-1.2min, phase A: the volume ratio of the phase B is 90:10-60:40, a step of performing a; 1.2-3min, phase A: the volume ratio of the phase B is 60:40-60:40, a step of performing a; 3-3.5min, phase A: the volume ratio of the phase B is 60:40-10:90;3.5-5min, phase A: the volume ratio of the phase B is 10:90-10:90;5-5.2min, phase A: the volume ratio of the phase B is 10:90-90:10;5.2-7.5min, phase A: the volume ratio of the phase B is 90:10-90:10;
the measurement conditions of the mass spectrum are as follows: ionization mode: electrospray ion source ESI, negative ion detection mode, triple quaternary rod mass analyzer; scanning mode: multi-reaction monitoring mode MRM; the collision gas is nitrogen; the ion source temperature was 550 ℃, the ionization voltage was-4500V, the curtain gas CUR was 35psi, the spray gas GS1 was 50psi, and the auxiliary heating gas GS2 was 50psi.
2. A method of detecting 15 antibiotics in fish according to claim 1, wherein the obtaining of the sample solution comprises any one or more of the following conditions:
a1 The mass ratio of the fish sample to the addition of 7 internal standards was 5.00.+ -. 0.02: 1X 10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ：1×10 -7 ；
A2 The ratio of the mass of the fish sample added to the volume of the extraction solution added is 4.98-5.02:18-22 g/mL;
a3 The ultrasonic extraction time is 15-25min;
a4 Before enrichment, the solid phase extraction column is activated by adopting methanol and water in sequence, wherein the dosage of the methanol and the water in the activation is 1-2 times of the volume of the solid phase extraction column filler;
a5 And (3) sequentially eluting and air pushing the solid phase extraction column by using water and methanol solution after enrichment, wherein the use amount of the water and the methanol solution in the eluting is 1-2 times of the volume of the solid phase extraction column filler.
3. A method of detecting 15 antibiotics in fish according to claim 1, comprising any one or more of the following conditions in step 1):
b1 Concentrations of 7 internal standard substances in the standard solution are 100.0 mug.L -1 ；
B2 The methanol solution is 65-75% methanol aqueous solution by volume percent;
b3 The concentration range of 15 antibiotics in the standard solution is 1.37-500 mug.L -1 。
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