CN116990421A - Method for detecting residual quantity of nosiheptide in animal-derived food - Google Patents
Method for detecting residual quantity of nosiheptide in animal-derived food Download PDFInfo
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- CN116990421A CN116990421A CN202311255457.2A CN202311255457A CN116990421A CN 116990421 A CN116990421 A CN 116990421A CN 202311255457 A CN202311255457 A CN 202311255457A CN 116990421 A CN116990421 A CN 116990421A
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- nosiheptide
- acetonitrile
- organic solvent
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- 238000000034 method Methods 0.000 title claims abstract description 50
- MQWDKYHFGBWGQZ-JQTJYXGUSA-N nosiheptide Chemical compound N([C@H](C(=O)N\C(C=1SC=C(N=1)C(=O)N[C@@H]1CC(O)C(=O)OCC=2C=CC=C3NC(=C(C3=2)C)C(=O)SC[C@H](NC(=O)C=2N=C1SC=2)C=1SC=C(N=1)C1=N2)=C/C)[C@@H](C)O)C(=O)C(N=3)=CSC=3C1=CC(=O)\C2=C1/NC(C(=O)NC(=C)C(N)=O)=CS1 MQWDKYHFGBWGQZ-JQTJYXGUSA-N 0.000 title claims abstract description 46
- FPTCMHOCGKKRGQ-WYOWUDGCSA-N Multhiomycin Natural products CC=C1NC(=O)[C@@H](NC(=O)c2csc(n2)c3cc(O)c(nc3c4csc(n4)[C@H]5CSC(=O)c6[nH]c7cccc(COC(=O)[C@@H](O)C[C@H](NC(=O)c8csc1n8)c9nc(cs9)C(=O)N5)c7c6C)c%10nc(cs%10)C(=O)N[C@@H](C)C(=O)N)[C@H](C)O FPTCMHOCGKKRGQ-WYOWUDGCSA-N 0.000 title claims abstract description 39
- 101800003864 Nosiheptide Proteins 0.000 title claims abstract description 39
- 229950006423 nosiheptide Drugs 0.000 title claims abstract description 39
- 241001465754 Metazoa Species 0.000 title claims abstract description 30
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N methyl cyanide Natural products CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 130
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 239000012224 working solution Substances 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 4
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- 210000001835 viscera Anatomy 0.000 claims description 4
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 230000007246 mechanism Effects 0.000 claims description 3
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 3
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 25
- 238000004811 liquid chromatography Methods 0.000 abstract description 8
- 239000000523 sample Substances 0.000 description 43
- 239000012496 blank sample Substances 0.000 description 13
- 238000011084 recovery Methods 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 10
- 238000012544 monitoring process Methods 0.000 description 10
- 241000287828 Gallus gallus Species 0.000 description 9
- 235000013330 chicken meat Nutrition 0.000 description 9
- 235000019688 fish Nutrition 0.000 description 8
- 235000015277 pork Nutrition 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 6
- 238000000746 purification Methods 0.000 description 5
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 235000013594 poultry meat Nutrition 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
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- 238000002372 labelling Methods 0.000 description 2
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- 210000000813 small intestine Anatomy 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- ACTOXUHEUCPTEW-BWHGAVFKSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2s,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-BWHGAVFKSA-N 0.000 description 1
- SUBWVHAACKTENX-XLGVTROISA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4s,5s,6r)-4-(dimethylamino)-3,5-dihydroxy-6-methyloxan-2-yl]oxy-10-[(2r,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-oxo-1-oxacyclohexadeca-11,13-dien-7-yl]acetaldehyde Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O)[C@@H](C)O1)N(C)C)O)OC)[C@H]1CC[C@H](N(C)C)[C@@H](C)O1 SUBWVHAACKTENX-XLGVTROISA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 239000004187 Spiramycin Substances 0.000 description 1
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
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- 239000000427 antigen Substances 0.000 description 1
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- 229910052717 sulfur Inorganic materials 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28011—Other properties, e.g. density, crush strength
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
-
- 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
- G01N30/89—Inverse chromatography
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- 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
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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- General Physics & Mathematics (AREA)
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Abstract
The application discloses a method for detecting nosiheptide residual quantity in animal-derived food, which belongs to the technical field of food detection and comprises the following steps: s1, preparing and storing a sample: taking an original sample, uniformly mashing the original sample by a high-speed tissue masher, separating out a proper amount of samples by a quartering method, equally dividing the samples into two parts, respectively filling the two parts into a clean container, sealing the clean container, marking the sealed container, taking one part as the sample and taking the other part as the reserved sample, and storing the sample at the temperature of minus 20 ℃; s2, sample extraction: weighing a homogeneous sample in a centrifuge tube, adding an extracting solution, performing vortex oscillation and centrifugation, taking a supernatant, repeatedly extracting, combining the extracting solutions, adding water to a certain volume, performing vortex mixing to obtain a mixed extracting solution, adding acetonitrile saturated n-hexane into the mixed extracting solution, performing centrifugation, and collecting an acetonitrile layer for later use. Solves the problem that false positive is easy to occur in the detection by adopting the liquid chromatography method because the detection is mainly carried out by adopting the liquid chromatography method in the prior art. The method has the advantages of being simple in operation and greatly improving the detection efficiency and accuracy.
Description
Technical Field
The application relates to the technical field of food detection, in particular to a method for detecting nosiheptide residues in animal-derived foods.
Background
Nosiheptide is a sulfur-containing polypeptide antibiotic which is obtained by culturing actinomycetes separated from Argentina soil in 1961 and is discovered for the first time by the French lop company, can inhibit the growth of gram-positive bacteria and the secretion of small intestine virus antigens, promote the growth of small intestine cells, obviously promote the growth of animals such as chickens, pigs and fishes, has the characteristics of high efficiency, low toxicity and low residues, is not easy to generate drug resistance, and is approved to be used in livestock and poultry cultivation in Europe and Japan at present. In 1998, china approved the nosiheptide as a novel veterinary drug of three types in China, and the Japanese patent publication No. 168 clearly uses nosiheptide as a feed drug additive, and is widely applied to livestock and poultry raising industry. The drug withdrawal period of nosiheptide chicken was specified in bulletin 278 of the Ministry of agriculture to be 7 days, and the egg laying period was disabled. In order to ensure the safety of animal-derived foods, international disputes have formulated a residual quantity and a detection method of nosiheptide, for example, japanese positive list formulation has prescribed that the highest residual quantity of nosiheptide in chicken and pig muscle, liver and other tissues is 30 mug/kg, and as the national standards of the residual quantity and the detection method of nosiheptide in animal tissues have not been formulated yet, in order to perfect a nosiheptide safe drug administration system, the establishment of a detection method of nosiheptide residue in animal-derived foods is urgently needed.
There is only one local standard for residue determination gas chromatography mass spectrometry of nosiheptide in animal tissue of DB34/T1371-2011 in China, and the method is suitable for residue determination of nosiheptide in animal muscle, liver and kidney. Derivatization is required in the pretreatment of the method due to the adoption of gas chromatography mass spectrometry, and the egg of the drug is forbidden in the application range due to the lack of the egg laying period of the poultry. In addition, NY/T3480-2019 'high performance liquid chromatography for measuring nosiheptide in feed' is only applicable to feed detection, and false positive is easy to occur by adopting liquid chromatography detection.
Therefore, the establishment of a high-efficiency, accurate and sensitive detection method for the residual quantity of nosiheptide in animal-derived foods has important significance.
Disclosure of Invention
Therefore, the application provides a method for detecting the residual quantity of nosiheptide in animal-derived food, which aims to solve the problem that false positive is easy to occur in the detection by adopting a liquid chromatography method because the detection is mainly carried out by adopting the liquid chromatography method in the prior art.
In order to achieve the above object, the present application provides the following technical solutions:
the application provides a method for detecting the residual quantity of nosiheptide in animal-derived food, which comprises the following steps:
s1, preparing and storing a sample: taking an original sample, uniformly mashing the original sample by a high-speed tissue masher, separating out a proper amount of samples by a quartering method, equally dividing the samples into two parts, respectively filling the two parts into a clean container, sealing the clean container, marking the sealed container, taking one part as the sample and taking the other part as the reserved sample, and storing the sample at the temperature of minus 20 ℃;
s2, sample extraction: weighing a homogeneous sample in a centrifuge tube, adding an extracting solution, performing vortex oscillation and centrifugation, taking a supernatant, repeatedly extracting, combining the extracting solutions, adding water to a certain volume, performing vortex mixing to obtain a mixed extracting solution, adding acetonitrile saturated n-hexane into the mixed extracting solution, performing centrifugation, and collecting an acetonitrile layer for later use;
s3, purifying: activating a composite solid-phase extraction column, taking a collecting liquid in S2, passing through the composite solid-phase extraction column, sequentially eluting with a strong-polarity organic solvent and a weak-polarity organic solvent for the first time, vacuumizing after the eluent completely passes through the composite solid-phase extraction column, adding the eluent consisting of the strong-polarity organic solvent and the weak-polarity organic solvent into the composite solid-phase extraction column, vacuumizing after the eluent completely passes through the composite solid-phase extraction column to remove residual eluent, drying the eluent by nitrogen, fixing the volume to 1mL, fully oscillating and uniformly mixing, and filtering by using a filter membrane to obtain a liquid to be detected;
the composite solid phase extraction column used in the step S3 is an HLB solid phase extraction column series modified C18 solid phase extraction column;
s4, detecting the filtrate by a liquid chromatography-mass spectrometry method;
s5, preparing a matrix matching standard curve: preparing blank extracting solution according to the steps of extraction and purification, and preparing a mechanism standard working solution.
Further, the S2 sample pretreatment specific operation is:
accurately weighing 2g of homogeneous sample, adding 8mL of acetonitrile extract into 50mL of polypropylene centrifuge tube with a spiral cover, carrying out vortex oscillation for 10min, centrifuging for 5min at 9500r/min, taking supernatant into another 50mL of centrifuge tube, adding 8mL of acetonitrile, carrying out vortex oscillation for 10min, centrifuging for 5min, combining the two extracts, adding water to fix the volume to 20mL, carrying out vortex mixing for 1min, accurately sucking 10mL of mixed extract, adding 3mL of acetonitrile saturated n-hexane, carrying out up-down light shaking for 20 times, carrying out 9500r/min centrifugation for 5min, and collecting an acetonitrile layer for later use.
Further, the specific operation steps of the step S3 are as follows:
sequentially activating an HLB solid phase extraction column with 5mL of methanol, water and sodium dihydrogen phosphate buffer solution in advance, allowing an extracting solution to pass through the activated column at a flow rate of 1mL/min, sequentially eluting the column with 5mL of water and 5mL of methanol-water (2:8, v/v), completely passing the eluting solution through the HLB solid phase extraction column, vacuumizing for more than 5min, and sequentially eluting with 4mL of strong-polarity organic solvent and 4mL of weak-polarity organic solvent;
and (3) activating the modified C18 solid-phase extraction column by using 8mL of acetonitrile, passing the eluent through the modified C18 solid-phase extraction column, performing secondary elution by using an eluent formed by mixing 4mL of a strong-polarity organic solvent and a weak-polarity organic solvent, drying the eluent by blowing nitrogen at 45 ℃, determining the volume to 1mL by using a mobile phase, and filtering by using a 0.22 mu m filter membrane after full shaking and uniform mixing to obtain the liquid to be detected.
Further, the parameters of the liquid chromatography-tandem mass spectrometry in S4 include:
chromatographic column: acccooreRP-MS (100 mm. Times.2.1 mm,2.6 μm); column temperature: 30 ℃; sample injection volume: 20mL; phenomenex Luna C18 (150 mm. Times.2 mm. D,5 μm) reversed-phase chromatography column; mobile phase a:0.01mol/L ammonium acetate solution, mobile phase B: acetonitrile; flow rate: 200. Mu.L/min;
gradient elution procedure: 0-1min,30% mobile phase B,1-5min, mobile phase B linearly increasing to 90%, and maintaining for 1min; the mobile phase B was linearly reduced to 30% for 6-6.1min and maintained for 1.9min.
Further, the conditions for ionization of the electrospray ion source include:
the electrospray ionization adopts a negative ion mode; electrospray voltage is-4500V, atomizer flow: 3L/min; heating air flow: 10L/min; interface temperature: 300 ℃; desolventizing temperature: 526 deg.c; DL temperature: 250 ℃; heating block temperature: 400 ℃; dry air flow: 10L/min.
Further, the filler of the modified C18 solid phase extraction column is a C18 carbon chain silica filler of which the surface is bonded with octadecyltriethoxy chlorosilane, vinyl trimethoxysilane and carbodecaborane.
Further, the strong polar organic solvent forming the eluent is one or more of tetrahydrofuran, chloroform, methanol, ethanol or acetone, the weak polar organic solvent is one or more of cyclohexane, n-pentane, n-heptane, n-hexane or hexane, and the ratio of the strong polar organic solvent to the weak polar organic solvent is 1:3.
further, the acetonitrile content of the constant volume liquid adopted by the constant volume is 50-70%, and the concentration of formic acid is 1-3%.
Further, the filtration membrane in S3 is a polytetrafluoroethylene membrane (PTFE-Q).
Further, the raw samples in S1 include muscle, viscera, fish, shrimp, milk, and egg.
The application has the following advantages:
1. the purification step of the application is mainly aimed at the impurities with strong retention on the reversed phase solid phase extraction column such as pigment, fat and the like, and the interference substances are further removed by the composite solid phase extraction pretreatment technology, so that the detection accuracy is improved, the residue of nosiheptide in animal-derived food can be rapidly and accurately measured, the detection efficiency and accuracy are improved, the recovery rate is good, the operation is simple, and the method can be widely applied to the daily supervision and detection field of nosiheptide residue in animal-derived food.
2. The octadecyl triethoxy chlorosilane, vinyl trimethoxy silane and carbodecaborane are bonded on the surface of a silica gel filler of a C18 carbon chain to be used as the filler of a modified C18 solid-phase extraction column, so that compounds with different polarities can be selectively adsorbed, the compounds with different polarities can be better separated, and as the groups have certain polarities and hydrophilicities, more target compounds can be adsorbed, the adsorption capacity and stability of the target compounds on the column are improved, the loss of the target compounds is reduced, and the recovery rate and the purity of the target compounds are improved.
3. The detection method provided by the application is linear within the concentration range of 1.0-20ng/mLGood correlation coefficient (R 2 ) Are all greater than 0.99; under the standard adding levels of 1.0 mug/kg, 2.0 mug/kg and 10 mug/kg, the average standard adding recovery rate is 81.3-96.6 percent, the relative standard deviation is 2.22-9.97 percent, the technical requirements of residual detection can be met, and the method has the advantages of high efficiency, accuracy and sensitivity in detection.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the application, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present application, should fall within the ambit of the technical disclosure.
FIG. 1 shows the extraction effect of nosiheptide with different extraction solvents provided by the application;
FIG. 2 is a graph showing the effect of adding acid and base to the extraction solvent provided by the application;
FIG. 3 is a graph of response intensity for different flow versus series of concentrations provided by the present application;
FIG. 4 is a multi-reaction monitoring (MRM) chromatogram of chicken labeled and blank samples;
FIG. 5 is a multi-reaction monitoring (MRM) chromatogram of fish labeling samples and blank samples;
FIG. 6 is a multi-reaction monitoring (MRM) chromatogram of a milk-labeled sample and a blank sample;
FIG. 7 is a multi-reaction monitoring (MRM) chromatogram of shrimp meat labeled and blank samples;
FIG. 8 is a multi-reaction monitoring (MRM) chromatogram of an egg labeling sample and a blank sample;
FIG. 9 is a multi-reaction monitoring (MRM) chromatogram of pig liver labeled and blank samples;
FIG. 10 is a multi-reaction monitoring (MRM) chromatogram of pork labeled and blank samples;
FIG. 11 is a multi-reaction monitoring (MRM) chromatogram of chicken liver labeled and blank samples;
FIG. 12 shows the effect of different extraction solutions provided by the present application on the extraction efficiency of egg samples.
Detailed Description
Other advantages and advantages of the present application will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The application provides a method for detecting the residual quantity of nosiheptide in animal-derived food, which comprises the following steps:
s1, preparing and storing a sample: taking an original sample, uniformly mashing the original sample by a high-speed tissue masher, separating out a proper amount of samples by a quartering method, equally dividing the samples into two parts, respectively filling the two parts into a clean container, sealing the clean container, marking the sealed container, taking one part as the sample and taking the other part as the reserved sample, and storing the sample at the temperature of minus 20 ℃;
s2, sample extraction: weighing a homogeneous sample in a centrifuge tube, adding an extracting solution, performing vortex oscillation and centrifugation, taking a supernatant, repeatedly extracting, combining the extracting solutions, adding water to a certain volume, performing vortex mixing to obtain a mixed extracting solution, adding acetonitrile saturated n-hexane into the mixed extracting solution, performing centrifugation, and collecting an acetonitrile layer for later use;
s3, purifying: activating a composite solid-phase extraction column, taking a collecting liquid in S2, passing through the composite solid-phase extraction column, sequentially eluting with a strong-polarity organic solvent and a weak-polarity organic solvent for the first time, vacuumizing after the eluent completely passes through the composite solid-phase extraction column, adding the eluent consisting of the strong-polarity organic solvent and the weak-polarity organic solvent into the composite solid-phase extraction column, vacuumizing after the eluent completely passes through the composite solid-phase extraction column to remove residual eluent, drying the eluent by nitrogen, fixing the volume to 1mL, fully oscillating and uniformly mixing, and filtering by using a filter membrane to obtain a liquid to be detected;
s3, the composite solid phase extraction column used in the step S is an HLB solid phase extraction column series modified C18 solid phase extraction column;
s4, detecting the filtrate by a liquid chromatography-mass spectrometry method;
s5, preparing a matrix matching standard curve: preparing blank extracting solution according to the steps of extraction and purification, and preparing a mechanism standard working solution.
S2, sample pretreatment specific operation is as follows:
accurately weighing 2g of homogeneous sample, adding 8mL of acetonitrile extract into 50mL of polypropylene centrifuge tube with a spiral cover, carrying out vortex oscillation for 10min, centrifuging for 5min at 9500r/min, taking supernatant into another 50mL of centrifuge tube, adding 8mL of acetonitrile, carrying out vortex oscillation for 10min, centrifuging for 5min, combining the two extracts, adding water to fix the volume to 20mL, carrying out vortex mixing for 1min, accurately sucking 10mL of mixed extract, adding 3mL of acetonitrile saturated n-hexane, carrying out up-down light shaking for 20 times, carrying out 9500r/min centrifugation for 5min, and collecting an acetonitrile layer for later use.
S3, the specific operation steps are as follows:
sequentially activating an HLB solid phase extraction column with 5mL of methanol, water and sodium dihydrogen phosphate buffer solution in advance, allowing an extracting solution to pass through the activated column at a flow rate of 1mL/min, sequentially eluting the column with 5mL of water and 5mL of methanol-water (2:8, v/v), completely passing the eluting solution through the HLB solid phase extraction column, vacuumizing for more than 5min, and sequentially eluting with 4mL of strong-polarity organic solvent and 4mL of weak-polarity organic solvent;
and (3) activating the modified C18 solid-phase extraction column by using 8mL of acetonitrile, passing the eluent through the modified C18 solid-phase extraction column, performing secondary elution by using an eluent formed by mixing 4mL of a strong-polarity organic solvent and a weak-polarity organic solvent, drying the eluent by blowing nitrogen at 45 ℃, determining the volume to 1mL by using a mobile phase, and filtering by using a 0.22 mu m filter membrane after full shaking and uniform mixing to obtain the liquid to be detected.
The parameters of the liquid chromatography-tandem mass spectrometry in S4 include:
chromatographic column: acccooreRP-MS (100 mm. Times.2.1 mm,2.6 μm); column temperature: 30 ℃; sample injection volume: 20mL; phenomenex Luna C18 (150 mm. Times.2 mm. D,5 μm) reversed-phase chromatography column; mobile phase a:0.01mol/L ammonium acetate solution, mobile phase B: acetonitrile; flow rate: 200. Mu.L/min;
gradient elution procedure: 0-1min,30% mobile phase B,1-5min, mobile phase B linearly increasing to 90%, and maintaining for 1min; the mobile phase B was linearly reduced to 30% for 6-6.1min and maintained for 1.9min.
Conditions for ionization of electrospray ion sources include:
the electrospray ionization adopts a negative ion mode; electrospray voltage is-4500V, atomizer flow: 3L/min; heating air flow: 10L/min; interface temperature: 300 ℃; desolventizing temperature: 526 deg.c; DL temperature: 250 ℃; heating block temperature: 400 ℃; dry air flow: 10L/min.
The filler of the modified C18 solid phase extraction column is C18 carbon chain silica filler of which the surface is bonded with octadecyltriethoxy chlorosilane, vinyl trimethoxysilane and carbodecaborane.
The strong polar organic solvent forming the eluent is one or more of tetrahydrofuran, chloroform, methanol, ethanol or acetone, the weak polar organic solvent is one or more of cyclohexane, n-pentane, n-heptane, n-hexane or hexane, and the ratio of the strong polar organic solvent to the weak polar organic solvent is 1:3.
the acetonitrile content of constant volume liquid adopted for constant volume is 50-70%, and the concentration of formic acid is 1-3%.
The filter membrane in S3 is a polytetrafluoroethylene (PTFE-Q) membrane.
The raw samples in S1 include muscle, viscera, fish, shrimp, milk and egg.
Materials and methods
1. Instrument and reagent
SHIMADZU LCMS-8050 type liquid chromatography tandem quadrupole mass spectrometer, distribution spray ion source (Shimadzu corporation), LC-30A high performance liquid chromatograph (Agilent corporation, U.S.); an Acccore RP-MS (100 mm. Times.2.1 mm,2.6 μm) column; HLB solid phase extraction cartridge (Waters company); a reverse phase chromatographic column; centrifuge (type 3K18, sigma Germany); solid phase extraction vacuum apparatus (Agilent company, usa); nitrogen-blown concentrator (TurboVapLV model, biotage, sweden); vortex oscillators (model V-230, company Barnstead International, usa); gilson pipettes (specification: 10-100. Mu.L, 20-200. Mu.L, 10-1000. Mu.L, 1000-5000. Mu.L, gilson, france).
Methanol (Merck, germany), acetonitrile (Merck, germany), formic acid (Merck, germany), ammonium acetate (Aldrich chemical company, usa) as chromatographic purity; dimethyl sulfoxide, anhydrous sodium sulfate, and n-hexane (american world limited) are analytically pure; high purity water from Millipore pure water system (Millipore corporation, usa). Standard substance: nosiheptide (purity 77.6% of Bepure company).
2. Liquid chromatography and mass spectrometry conditions
Chromatographic column: acccooreRP-MS (100 mm. Times.2.1 mm,2.6 μm); column temperature: 30 ℃; sample injection volume: 20mL; reversed phase chromatography column Phenomenex Luna C (150 mm x 2 mm. D.,5 μm); mobile phase a:0.01mol/L ammonium acetate solution, mobile phase B: acetonitrile; flow rate: 200. Mu.L/min. Gradient elution procedure: 0-1min,30% mobile phase B,1-5min, mobile phase B linearly increasing to 90%, and maintaining for 1min; the mobile phase B was linearly reduced to 30% for 6-6.1min and maintained for 1.9min.
Electrospray ionization negative ion mode (Electro spray ionization, esi+); multiple reaction monitoring (Multiple teaction monitoring, MRM); atomizer flow rate: 3L/min; heating air flow: 10L/min; interface temperature: 300 ℃; desolventizing temperature: 526 deg.c; DL temperature: 250 ℃; heating block temperature: 400 ℃; dry air flow: 10L/min; other mass spectral parameters are shown in table 1.
TABLE 1 mass spectral parameters of spiramycin and neospiramycin
Example 1
The method for detecting the residual quantity of nosiheptide in animal-derived food provided by the embodiment comprises the following steps:
s1, preparing and storing a sample: taking an original sample, uniformly mashing the original sample by a high-speed tissue masher, separating out a proper amount of samples by a quartering method, equally dividing the samples into two parts, respectively filling the two parts into a clean container, sealing the clean container, marking the sealed container, taking one part as the sample and taking the other part as the reserved sample, and storing the sample at the temperature of minus 20 ℃;
s2, sample pretreatment: accurately weighing muscle, viscera, fish, shrimp, milk and egg, 2g (accurate to 0.01 g) of homogeneous sample, adding 8mL of acetonitrile extract into 50mL of polypropylene centrifuge tube with spiral cover, carrying out vortex oscillation for 10min, centrifuging for 5min at 9500r/min, taking supernatant into another 50mL centrifuge tube, adding 8mL of acetonitrile for re-extraction, carrying out vortex oscillation for 10min, centrifuging for 5min, combining the two extracts, adding water to constant volume to 20mL, carrying out vortex mixing for 1min, accurately sucking 10mL of mixed extract, adding 3mL of acetonitrile saturated n-hexane, carrying out up-down light shaking for 20 times, carrying out centrifugation for 5min at 9500r/min, and collecting an acetonitrile layer for standby.
S3, purifying: sequentially activating an HLB solid phase extraction column with 5mL of methanol, water and sodium dihydrogen phosphate buffer solution in advance, allowing the extract to pass through the activated column at a flow rate of 1mL/min, sequentially eluting the column with 5mL of water and 5mL of methanol-water (2:8, v/v), completely passing the eluate through the small column, vacuumizing for more than 5min, and eluting with 4mL of methanol;
activating the modified C18 solid phase extraction column by using 8mL of acetonitrile, passing the eluent through the modified C18 solid phase extraction column, eluting again, drying the eluent by blowing nitrogen at 45 ℃, using a mobile phase to reach 1mL, fully vibrating and uniformly mixing, and filtering by using a 0.22 mu m filter membrane to obtain filtrate, wherein the filtrate can be directly used for liquid chromatography-mass spectrometry/mass spectrometer measurement.
S4, after filtrate is obtained, liquid chromatography-mass spectrometry detection is carried out on the filtrate;
chromatographic column: acccooreRP-MS (100 mm. Times.2.1 mm,2.6 μm); column temperature: 30 ℃; sample injection volume: 20mL; mobile phase a:0.01mol/L ammonium acetate solution, mobile phase B: acetonitrile; flow rate: 200. Mu.L/min. Gradient elution procedure: 0-1min,30% mobile phase B,1-5min, mobile phase B linearly increasing to 90%, and maintaining for 1min; the mobile phase B was linearly reduced to 30% for 6-6.1min and maintained for 1.9min.
The mass spectrometry conditions were ion source: electrospray ionization negative ion mode (Electro spray ionization, esi+); multiple reaction monitoring (Multiple teaction monitoring, MRM); atomizer flow rate: 3L/min; heating air flow: 10L/min; interface temperature: 300 ℃; desolventizing temperature: 526 deg.c; DL temperature: 250 ℃; heating block temperature: 400 ℃; dry air flow: 10L/min;
s5, preparing a matrix matching standard curve: accurately weighing 2g of homogeneous blank muscle tissue, preparing blank sample extracting solution according to the extraction and purification steps, preparing matrix standard working solution with serial concentration by using the blank sample extracting solution, and preparing corresponding standard working solution with concentration of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0 ng/mL. And under the optimal working condition of the instrument, sampling the standard working solution. The standard working curve is quantified according to an external standard method, and the response values of the to-be-measured objects in the sample solution are all in the linear range measured by the instrument. Setting nosiheptide concentration as x abscissa and corresponding chromatographic peak area as y ordinate, and performing linear regression analysis to obtain regression equation and correlation coefficient (R 2 ）。
TABLE 2 liquid chromatography-Mass Spectrometry Linear equation and correlation coefficient
Recovery rate and precision:
accurately weighing 2g of homogeneous blank animal muscle, adding a proper amount of nosiheptide standard working solution with different concentrations, performing a standard adding experiment according to the measurement conditions, injecting sample six times under the optimized conditions to obtain signal to noise ratio of six times of sample injection respectively, and taking the signal to noise ratio of 10 times as the corresponding mass concentration of a target as the quantitative Limit (LOQ) of the method, wherein the quantitative limit of the method is 1.0 mug/kg.
In the embodiment, chicken liver, pork liver, egg, milk, fish, shrimp meat and the like are used as sample matrixes, and a standard adding method is adopted to test the recovery rate and the precision of the added samples according to an optimized experimental method. Under the standard adding levels of 1.0 mug/kg, 2.0 mug/kg and 10 mug/kg, the average standard adding recovery rate is 81.3-96.6% and the relative standard deviation is 2.22-9.97%, which shows that the method has higher recovery rate and precision and meets the requirement of veterinary drug residue detection in animal tissues.
Example 2
Nosiheptide is soluble in acetonitrile, methanol, ethanol and other organic reagents, and insoluble in water. Pork is taken as a sample matrix, 10 mug/kg of nosiheptide standard solution is added, 15mL of acetonitrile, methanol and ethanol are respectively added for extraction, extraction efficiencies of three different organic solvents of acetonitrile, methanol and ethanol are examined, and the result is shown in figure 1. As can be seen from the figure, acetonitrile is the best extraction solvent for nosiheptide compounds in the three extraction reagents. Nosiheptide contains phenolic hydroxyl groups and a plurality of amino groups, and the pH value of an extraction solvent may have an influence on the extraction efficiency.
For this purpose, pork was used as a sample substrate, and the effect of adding 1% formic acid, 1% ammonia water and pure acetonitrile to the extraction solvent was examined in this example, and the results are shown in fig. 2. As can be seen from the figure, the extraction efficiency of nosiheptide is over 80% by adding formic acid or ammonia water with a certain concentration into acetonitrile and pure acetonitrile is the best, so that the pure acetonitrile is finally selected as the extraction solvent.
Example 3
In order to examine the influence of different flows on nosiheptide compounds, acetonitrile-0.01% formic acid water, acetonitrile-0.01% ammonia water, acetonitrile-0.002 mol/L ammonium acetate, acetonitrile-0.005 mol/L ammonium acetate and acetonitrile-0.010 mol/L ammonium acetate are selected as mobile phases, 50% acetonitrile water (containing 1% formic acid) is used for preparing series standard solutions, the concentrations of which are 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL, and the response signal intensity of the series concentrations in each mobile phase is respectively measured, wherein the measurement result is shown in figure 3, and the nosiheptide response is highest in the acetonitrile-0.010 mol/L ammonium acetate mobile phase system, so that acetonitrile-0.010 mol/L ammonium acetate is finally adopted as the mobile phase system.
Example 4
The method provided by the application is used for analyzing blank samples, and chicken, fish, milk, shrimp meat, eggs, pork liver, pork and chicken liver are respectively prepared to prepare labeled samples and blank samples (the labeled concentration is 1.0 mug/kg).
After purification, the blank sample has no impurity peak interference measurement at the peak position of nosiheptide, and the specificity of the method can meet the detection requirement, as shown in fig. 4-11.
Example 5
The acetonitrile is adopted as an extraction solvent, and the sample matrixes such as pork, pork liver, chicken liver, fish, shrimp, milk and the like are extracted to obtain ideal recovery rate, but the recovery rate of the egg is below 40%; therefore, this example uses a 1% acetonitrile formate solution as the extraction solution, and can obtain a desired recovery rate, as shown in FIG. 12. For this purpose, a 1% acetonitrile formate solution was used for the egg sample extraction solution.
Conclusion(s)
The method combines the composite solid phase extraction pretreatment technology with the liquid chromatography-tandem mass spectrometry technology, further removes interfering substances, establishes a high-efficiency, accurate and sensitive method for detecting the nosiheptide residues in animal-derived foods, and improves the detection efficiency and accuracy. Through the optimization of the detection method and the extraction solution, the solid phase extraction condition, the liquid chromatography and the mass spectrum condition in the embodiment 1-embodiment 4, the method has good linear relation in the range of 0-20 mug/kg, the correlation coefficient R is more than 0.995, the average standard adding recovery rate is 81.3-96.6% under the standard adding levels of 1.0 mug/kg, 2.0 mug/kg and 10 mug/kg, the relative standard deviation is 2.22-9.97%, the quantitative limit is 1.0 mug/kg, the technical requirement of residual detection can be met, and through continuous fumbling and trial discovery, the 1% acetonitrile formate solution is adopted as the extraction solution, and the ideal recovery rate can be obtained through egg sample extraction. For this reason, a 1% acetonitrile formate solution was used for the egg sample extraction solution, and it was widely used in the field of daily supervision and detection of nosiheptide residues in animal foods.
While the application has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.
Claims (9)
1. A method for detecting the residual quantity of nosiheptide in animal-derived food, which is characterized by comprising the following steps:
s1, preparing and storing a sample: taking an original sample, uniformly mashing the original sample by a high-speed tissue masher, separating out a proper amount of samples by a quartering method, equally dividing the samples into two parts, respectively filling the two parts into a clean container, sealing the clean container, marking the sealed container, taking one part as the sample and taking the other part as the reserved sample, and storing the sample at the temperature of minus 20 ℃;
s2, sample extraction: weighing a homogeneous sample in a centrifuge tube, adding an extracting solution, performing vortex oscillation and centrifugation, taking a supernatant, repeatedly extracting, combining the extracting solutions, adding water to a certain volume, performing vortex mixing to obtain a mixed extracting solution, adding acetonitrile saturated n-hexane into the mixed extracting solution, performing centrifugation, and collecting an acetonitrile layer for later use;
s3, purifying: activating a composite solid-phase extraction column, taking a collecting liquid in S2, passing through the composite solid-phase extraction column, sequentially eluting with a strong-polarity organic solvent and a weak-polarity organic solvent for the first time, vacuumizing after the eluent completely passes through the composite solid-phase extraction column, adding the eluent consisting of the strong-polarity organic solvent and the weak-polarity organic solvent into the composite solid-phase extraction column, vacuumizing after the eluent completely passes through the composite solid-phase extraction column to remove residual eluent, drying the eluent by nitrogen, fixing the volume to 1mL, fully oscillating and uniformly mixing, and filtering by using a filter membrane to obtain a liquid to be detected;
the composite solid phase extraction column used in the step S3 is an HLB solid phase extraction column series modified C18 solid phase extraction column;
s4, detecting the filtrate by a liquid chromatography-mass spectrometry method;
s5, preparing a matrix matching standard curve: preparing blank extracting solution according to the extracting and purifying steps, and preparing a mechanism standard working solution;
the specific operation steps of the S3 are as follows:
sequentially activating an HLB solid-phase extraction column with 5mL of methanol, water and sodium dihydrogen phosphate buffer solution in advance, allowing the extract to pass through the activation column at a flow rate of 1mL/min, sequentially eluting the column with 5mL of water and 5mL of methanol-water, completely allowing the eluate to pass through the HLB solid-phase extraction column, vacuumizing for more than 5min, and sequentially eluting with 4mL of strong-polarity organic solvent and 4mL of weak-polarity organic solvent;
and (3) activating the modified C18 solid-phase extraction column by using 8mL of acetonitrile, passing the eluent through the modified C18 solid-phase extraction column, performing secondary elution by using an eluent formed by mixing 4mL of a strong-polarity organic solvent and a weak-polarity organic solvent, drying the eluent by blowing nitrogen at 45 ℃, determining the volume to 1mL by using a mobile phase, and filtering by using a 0.22 mu m filter membrane after full shaking and uniform mixing to obtain the liquid to be detected.
2. The method for detecting the residual amount of nosiheptide in an animal-derived food as claimed in claim 1, characterized in that the S2 sample pretreatment is specifically operated as:
accurately weighing 2g of homogeneous sample, adding 8mL of acetonitrile extract into 50mL of polypropylene centrifuge tube with a spiral cover, carrying out vortex oscillation for 10min, centrifuging for 5min at 9500r/min, taking supernatant into another 50mL of centrifuge tube, adding 8mL of acetonitrile, carrying out vortex oscillation for 10min, centrifuging for 5min, combining the two extracts, adding water to fix the volume to 20mL, carrying out vortex mixing for 1min, accurately sucking 10mL of mixed extract, adding 3mL of acetonitrile saturated n-hexane, carrying out up-down light shaking for 20 times, carrying out 9500r/min centrifugation for 5min, and collecting an acetonitrile layer for later use.
3. The method for detecting the residual amount of nosiheptide in animal-derived food according to claim 1, characterized in that the parameters of the liquid chromatography-tandem mass spectrometry in S4 include:
chromatographic column: acccooreRP-MS; column temperature: 30 ℃; sample injection volume: 20mL; phenomenex Luna C18 reverse phase chromatography column; mobile phase a:0.01mol/L ammonium acetate solution, mobile phase B: acetonitrile; flow rate: 200. Mu.L/min;
gradient elution procedure: 0-1min,30% mobile phase B,1-5min, mobile phase B linearly increasing to 90%, and maintaining for 1min; the mobile phase B was linearly reduced to 30% for 6-6.1min and maintained for 1.9min.
4. A method for detecting the residual amount of nosiheptide in an animal-derived food as claimed in claim 3 characterised in that the conditions for ionising the electrospray ion source include:
the electrospray ionization adopts a negative ion mode; electrospray voltage is-4500V, atomizer flow: 3L/min; heating air flow: 10L/min; interface temperature: 300 ℃; desolventizing temperature: 526 deg.c; DL temperature: 250 ℃; heating block temperature: 400 ℃; dry air flow: 10L/min.
5. The method for detecting the residual amount of nosiheptide in animal-derived food according to claim 2, wherein the filler of the modified C18 solid-phase extraction column is a C18 carbon chain silica filler in which octadecyltriethoxy chlorosilane, vinyl trimethoxysilane and carbodecaborane are bonded to the surface of the C18 carbon chain silica filler.
6. The method for detecting the residual amount of nosiheptide in animal-derived foods as claimed in claim 3, wherein the strong polar organic solvent constituting the eluent is one or more of tetrahydrofuran, chloroform, methanol, ethanol or acetone, the weak polar organic solvent is one or more of cyclohexane, n-pentane, n-heptane, n-hexane or hexane, and the ratio of the strong polar organic solvent to the weak polar organic solvent is 1:3.
7. the method for detecting the residual amount of nosiheptide in animal-derived food as claimed in claim 3, characterized in that the acetonitrile content of the constant volume liquid adopted for the constant volume is 50-70% and the formic acid concentration is 1-3%.
8. The method for detecting the residual amount of nosiheptide in an animal-derived food as claimed in claim 3, characterized in that the S3 filter membrane is a polytetrafluoroethylene membrane.
9. The method for detecting the residual amount of nosiheptide in an animal-derived food as claimed in claim 1, characterized in that the raw sample in S1 includes muscle, viscera, fish, shrimp, milk and egg.
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