CN108931593A - The valued methods of Sulfonamides residual substrate standard substance in pork - Google Patents
The valued methods of Sulfonamides residual substrate standard substance in pork Download PDFInfo
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- CN108931593A CN108931593A CN201810532814.8A CN201810532814A CN108931593A CN 108931593 A CN108931593 A CN 108931593A CN 201810532814 A CN201810532814 A CN 201810532814A CN 108931593 A CN108931593 A CN 108931593A
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The present invention provides the valued methods of Sulfonamides residual substrate standard substance in pork, and the method includes the pretreated steps of substrate standard substance；The pretreatment includes：(1) it extracts：The extraction that Extraction solvent carries out object is added into substrate standard substance, obtained supernatant crosses anhydrous sodium sulfate column, obtains extract；(2) it purifies：Step (1) extract is dry through water-bath rotary evaporation, it is dissolved using mobile phase constant volume, is purified using the liquid-liquid distribution method of purification, pretreated substrate standard substance is obtained after filtering；Wherein, the anhydrous sodium sulfate column uses acetonitrile to infiltrate in advance；The mobile phase uses n-hexane to be saturated in advance.Method of the invention carries out the extract of constant volume Sulfonamides residual substrate standard substance using the mobile phase of n-hexane saturation, sulfa drugs when reducing liquid-liquid distribution in extracting solution is assigned in organic phase, the rate of recovery of sulfa drugs is between 95.7%-104.8%, and relative standard deviation is less than 5% (n=6).
Description
Technical field
The invention belongs to technical field of analysis and detection, it is related to the definite value of Sulfonamides residual substrate standard substance in pork
Method.
Background technique
Sulfonamides are the general names of a kind of antibacterials with P-aminobenzene-sulfonamide structure, have antibacterial pedigree
Extensively, strong antibacterial, it is cheap the advantages that, be widely used in preventing and treating the infectious diseases of food-borne animal.So
And the animal-derived food containing Sulfonamides is eaten for a long time and easily causes allergic reaction, urinary system function is influenced, is tied
The symptoms such as brilliant urine, blood urine, destroy the ecological balance of body normal flora, cause the adverse consequences such as resistance.Sulfamido beast
Medicine is in addition to " clenbuterol hydrochloride ", and residual quantity is easiest to exceeded one of veterinary drug, currently, China, Codex Alimentary Commission
(CAC), sulfa drugs is classified as animal feeding and limits the drug used in the process by European Union and the U.S., and limits animal sources food
The maximum residue limit (MRL) of sulfa drugs is 0.1mg/kg in product.The Ministry of Agriculture of China also strictly monitors Sulfonamides
Sulfonamides have been classified as national basic veterinary drug residual always since implementation national basic veterinary drug in 1999 remains Supervisory Surveillance Program by residual quantity
One of monitor control index.
Sulfonamides residual substrate standard substance is generally obtained by way of cultivation administration, object (sulfanilamide (SN)) and base
The combining form of body (pork) and authentic sample are completely the same, not only it is possible to prevente effectively from matrix is to object in analytic process
It influences, and can guarantee the consistency and comparativity of sulfanilamide (SN) testing result in different time and space, be to realize that sulfanilamide (SN) is quasi-
The material base for determining amount and tracing to the source.It establishes a kind of sulfanilamide (SN) valued methods that accuracy is high and is developing Sulfonamides residual matrix
It is of great significance in standard substance.
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the most important confirmation method of current sulfanilamide (SN) detection, but by
Fat content and protein content in pork is higher, and pretreatment process is cumbersome, significantly affect measurement result reliability and
Accuracy.The recovery of standard addition of LC-MS/MS reported in the literature is generally 70%-110%, and as a result standard deviation is generally 15%-
30%, the definite value for being unable to satisfy substrate standard substance requires (" sulfanilamide (SN) in ultra performance liquid chromatography-tandem mass spectrometry measurement pork
Class drug residue ", red bayberry etc.,《Food safety quality testing journal》, volume 8, the 3633-3638 pages, 2017).
101949898 A of CN discloses a kind of detection method of residual quantity of multiple alkaline drugs in animal derived food, institute
It is solid by acetonitrile, isopropanol and the extraction of citric acid solution vortex mixed, hydrophily polystyrene-divinylbenzene to state method
Phase extraction column and cation exchange Solid Phase Extraction column purification and liquid chromatography-mass spectrography measurement come detect pork, pork liver, egg, shrimp,
A variety of alkaline drugs such as beta-receptor agonist class, sulfamido, Benzodiazepines, nitro glyoxaline, benzimidazole, three in milk
The residual quantity of phenylmethane class, however this method does not remove the moisture in Extraction solvent, significantly affects the relative recovery of drug
And relative standard deviation, the relative recovery of drug are only 63.1%-109.0%, relative standard deviation 1.6%-23.0%,
It is unable to satisfy the definite value requirement of substrate standard substance.
102175784 A of CN discloses Solid Phase Extraction-liquid chromatography-mass spectrography/mass spectrography while measuring 54 kinds of medicines in pork
The remaining method of object, the method are extracted by acetonitrile, n-hexane degreasing, then use C18Solid phase extraction column purification, liquid chromatogram-
Mass spectrum/mass spectrography (LC-MS/MS) measurement, Isotope Internal Standard Dilution Technique internal standard method and quantified by external standard method, rate of recovery range are
21.1%-121%, relative standard deviation can be used as screening technique applied to sulfamido, nitroimidazole in pork less than 19.8%
Class, quinolones, macrolides, LIN Kesheng and praziquantel measure while amounting to 54 kinds of medicament residues, however the party
The rate of recovery and relative standard deviation of method are still up for advanced optimizing.
Therefore it provides the detection method that a kind of accuracy is good, the rate of recovery is high, relative standard deviation is small, is used for sulfamido beast
Medicine remains the definite value of substrate standard substance, is of great significance in field of food inspection.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides the definite values of Sulfonamides residual substrate standard substance in pork
Method, the method improve the rate of recovery and precision, realize sulfamido by improving and optimizing to pretreatment process
The high precision definite value of residue of veterinary drug substrate standard substance.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the method includes matrix standards the present invention provides a kind of valued methods of substrate standard substance
The step of preprocessing substance；
The pretreatment includes：
(1) it extracts：The extraction that Extraction solvent carries out object is added into substrate standard substance, obtained supernatant crosses nothing
Aqueous sodium persulfate column, obtains extract；
(2) it purifies：Step (1) extract is dry through water-bath rotary evaporation, it is dissolved, is used using mobile phase constant volume
The liquid-liquid distribution method of purification is purified, and pretreated substrate standard substance is obtained after filtering；
Wherein, the anhydrous sodium sulfate column uses acetonitrile to infiltrate in advance；
The mobile phase uses n-hexane to be saturated in advance.
In the present invention, moisture removal is removed by anhydrous sodium sulfate column is crossed after substrate standard substance progress homogeneous extraction, avoids nothing
Caking phenomenon occurs for aqueous sodium persulfate, improves the homogenieity of substrate standard substance, the anhydrous sodium sulfate column of use uses second in advance
Nitrile infiltration, the acetonitrile and sulfa drugs further avoided in extracting solution are adsorbed on anhydrous sodium sulfate column, improve sulfanilamide (SN)
The rate of recovery of class drug.
The present invention dissolves extract after water-bath rotary evaporation is dry using the mobile phase constant volume of n-hexane saturation, not only
The content of hydrone in extract is reduced, and avoids object distribution in organic phase, improves the recycling of object
Rate.
Preferably, step (1) described Extraction solvent include in acetonitrile, ethyl acetate or acetonitrile solution any one or
At least two combination, preferably acetonitrile.
Preferably, the number of step (1) described extraction is 1-4 times, such as can be 1 time, 2 times, 3 times or 4 times, preferably
3-4 times.
Preferably, the dosage of step (1) described anhydrous sodium sulfate be 20g-100g, such as can be 20g, 50g, 70g or
100g, preferably 70g-100g.
Preferably, the temperature of step (2) described water-bath is 40 DEG C -60 DEG C, such as can be 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C
Or 60 DEG C, preferably 50 DEG C.
Preferably, step (2) mobile phase is the mixed liquor of acetonitrile and 0.1% formic acid.
Preferably, the volume ratio of the acetonitrile and 0.1% formic acid is 5:(90-100), such as can be 5:90,5:95 or
5:100, preferably 5:95.
Preferably, step (2) described mobile phase uses n-hexane to be saturated in advance.
The present invention, as mobile phase, is conducive to improve object using -0.1% formic acid mixed liquor of acetonitrile of n-hexane saturation
The rate of recovery.
Preferably, the solvent that step (2) the liquid-liquid distribution method of purification uses is n-hexane.
The present invention extracts the purification of object using simple liquid-liquid distribution method of purification substitution Solid phase extraction method, no
Only avoid absorption of the solid-phase extraction column center pillar material to sulfa drugs, it is thus also avoided that the solid-phase extraction column quality of commercialization is irregular
The big problem of standard deviation caused by uneven, the liquid-liquid distribution method of purification uses n-hexane as purifying solvent, in pork
Fat and protein there is apparent removal to act on, significantly improve the rate of recovery of sulfa drugs.
Preferably, step (2) filtering is carried out using filter membrane.
Preferably, the aperture of the filter membrane is 0.22 μm.
Preferably, the filter membrane includes nylon and/or polytetrafluoroethylene (PTFE), preferably polytetrafluoroethylene (PTFE).
Preferably, further include the steps that internal standard reagent is added before step (1) the addition Extraction solvent.
In the present invention, in order to reduce influence of the matrix effect to definite value accuracy, it is added into substrate standard substance same
The plain internal standard reagent in position, improves method accuracy, after Isotopic Internal Standard reagent is added, substrate standard substance can be placed in 4 DEG C of rings
0-24h is balanced in border, improves Isotopic Internal Standard reagent and matrix combination degree, closer to Natural Samples state.
Preferably, further include the steps that detecting using liquid chromatography-mass spectrography after step (2).
Preferably, the chromatographic column of the liquid chromatogram uses gradient elution mode.
Preferably, the mobile phase of the gradient elution uses 0.1% aqueous formic acid-acetonitrile.
Preferably, the change of gradient of the gradient elution is：
0-1min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
1min-5min, 0.1% aqueous formic acid：95.0%-20.0%, acetonitrile：5.0%-80.0%；
5min-5.1min, 0.1% aqueous formic acid：20.0%-95.0%, acetonitrile：80.0%-5.0%；
5.1min-7min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%.
Preferably, the mass spectrographic parameter includes：
Electron spray positive electron (ESI+) ionizes mode；
Spray voltage：3.00kv；
Ion source temperature：100℃；
Desolvation temperature：350℃；
Taper hole air blowing flow：150L/hr；
Desolventizing gas flow：650L/hr.
As optimal technical scheme, the present invention provides a kind of valued methods of substrate standard substance, the method includes
Following steps；
(1) it extracts：After Isotopic Internal Standard reagent is added into substrate standard substance, Extraction solvent is added and carries out object
It extracts 1-4 times, by obtained supernatant after the 20g-100g anhydrous sodium sulfate column infiltrated in advance with acetonitrile, obtains extract；
(2) it purifies：Step (1) extract is dry through 40 DEG C of -60 DEG C of water-bath rotary evaporations, use volume ratio for 5:
The dissolution of the mobile phase constant volume of the acetonitrile of (90-100) and 0.1% formic acid distributes the method for purification using liquid-liquid and is carried out only by n-hexane
Change processing, obtains pretreated substrate standard substance after filtering using 0.22 μm of filter membrane；
(3) it detects：Using liquid chromatography-mass spectrometry, step (2) the pretreated substrate standard substance is quantified
Detection；
Wherein, the chromatographic column of the liquid chromatogram uses gradient elution mode, and the mobile phase of gradient elution uses 0.1% first
The change of gradient of aqueous acid-acetonitrile, gradient elution is：
0-1min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
1min-5min, 0.1% aqueous formic acid：95.0%-20.0%, acetonitrile：5.0%-80.0%；
5min-5.1min, 0.1% aqueous formic acid：20.0%-95.0%, acetonitrile：80.0%-5.0%；
5.1min-7min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
The mass spectrographic parameter includes：
Electron spray positive electron (ESI+) ionizes mode；
Spray voltage：3.00kv；
Ion source temperature：100℃；
Desolvation temperature：350℃；
Taper hole air blowing flow：150L/hr；
Desolventizing gas flow：650L/hr.
Second aspect, the present invention provide a kind of sulfa drug residue matrix in pork of method as described in relation to the first aspect
Application in standard substance definite value.
Preferably, the sulfa drugs includes sulphadiazine and/or sulfadimidine.
According to the present invention, sulfa drugs is in charcuterie, and residual quantity is easiest to exceeded one of veterinary drug, of the invention
Method using in charcuterie sulphadiazine and/or sulfadimidine as target detection thing；Meanwhile those skilled in the art
Member is it is to be understood that method of the invention is equally applicable to the setting examination of other sulfa drugs or veterinary drug.
The third aspect is 95.7%-104.8%, relative standard deviation less than 5% (n the present invention provides a kind of rate of recovery
=6) valued methods of Sulfonamides residual substrate standard substance in pork.
Compared with prior art, the present invention has the advantages that：
(1) method of the invention removes the anhydrous sodium sulfate column that acetonitrile infiltration is crossed after substrate standard substance progress homogeneous extraction
Moisture removal, the acetonitrile and sulfa drugs avoided in extracting solution are adsorbed on anhydrous sodium sulfate column；
(2) method of the invention carries out constant volume Sulfonamides using the mobile phase of n-hexane saturation and remains matrix reference substance
The extract of matter, sulfa drugs when reducing liquid-liquid distribution in extracting solution are assigned in organic phase；
(3) method of the invention replaces Solid phase extraction method to extract object using the simple liquid-liquid distribution method of purification
Purification, not only avoid absorption of the solid-phase extraction column center pillar material to sulfa drugs, it is thus also avoided that the Solid Phase Extraction of commercialization
The big problem of standard deviation caused by column quality is irregular, the liquid-liquid distribution method of purification are molten as purifying using n-hexane
Agent, in pork fat and protein there is apparent removal to act on；
(4) method of the invention is to sulphadiazine and sulfadimidine in 10.0ng/mL-200.0ng/mL concentration range
Interior is in good linear relationship, and linearly dependent coefficient is all larger than 0.999, and the detection limit and quantitative limit of method are respectively 0.5 μ g/kg
With 2.0 μ g/kg, for the rate of recovery within the scope of basic, normal, high 3 pitch-based spheres between 95.7%-104.8%, relative standard is inclined
Difference is less than 5% (n=6)；
(4) method of the invention Sulfonamides residual substrate standard substance uniformity testing, stability prison in pork
It surveys and there is significant application value in definite value.
Detailed description of the invention
Figure 1A is sulfadimidine (SM2) and isotope labelling sulfadimidine (D4-SM2) in different Solid Phase Extraction
The rate of recovery of column and liquid-liquid distribution extracting process, Figure 1B are sulphadiazine (SDZ) and isotope labelling sulphadiazine (D4-SDZ)
In the rate of recovery of different solid-phase extraction columns and liquid-liquid distribution extracting process；
Fig. 2 is the rate of recovery that different solvents extract sulfonamide of the addition in blank pork sample；
Fig. 3 is influence of the different extraction times to testing result；
Fig. 4 A is sulfadimidine (SM2) and isotope labelling sulfadimidine (D4-SM2) on different filter membranes
The rate of recovery, Fig. 4 B are the rate of recovery of sulphadiazine (SDZ) and isotope labelling sulphadiazine (D4-SDZ) on different filter membranes.
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
Key instrument and reagent：
UPLC H-Class/Xevo TQ-S ultra performance liquid chromatography/triple level four bars mass spectrometer (U.S. Waters
Company)；Vortex3000 type vortex mixer (Wiggens company of the U.S.)；500 dispersion machine of Power Gen (U.S. Fisher
Scientific company)；5810R type supercentrifuge (German Eppendorf company)；N-1300D type Rotary Evaporators (Japan
EYELA company)；24 solid-phase extraction devices (Supelco company of the U.S.)；Multidigit nitrogen concentrating instrument (U.S. Organomation
Company)；MCX solid phase extraction column (60mg/3mL, Waters, US)；HLB solid phase extraction column (60mg/3mL, the U.S.
Waters company)；C18 solid phase extraction column (500mg/6mL, German CNW company)；Aluminium oxide solid phase extraction column (500mg/
6mL, German CNW company)；Milli-Q ultrapure water production system (Millipore company of the U.S.)；
Sulfadimidine veterinary drug purity rubric substance (GBW (E) 060900, purity 99.8%, expanded uncertainty U=
0.2%, Coverage factor k=2, China National Measuring Science Research Inst.)；Sulphadiazine veterinary drug purity rubric substance (GBW (E) 060901,
Purity 99.6%, expanded uncertainty U=0.4%, Coverage factor k=2, China National Measuring Science Research Inst.)；Isotope labelling sulphur
Amine diformazan pyrimidine standard items (D4-sulfadimidine (D4-SM2), purity 98%, Canadian Toronto Research
Chemicals company)；(D4-sulfadiazine (D4-SDZ), purity 98% add and take isotope labelling sulphadiazine standard items
Big Toronto Research Chemicals company)；Methanol (chromatographically pure, German MERCK company)；Acetonitrile (chromatographically pure, Germany
MERCK company)；Formic acid (chromatographically pure, Sigma Co., USA)；(analysis is pure, and Chinese medicines group chemical reagent is limited for anhydrous sodium sulfate
Company)；N-hexane (chromatographically pure, Sigma Co., USA)；Experimental water is prepared by Milli-Q ultrapure water production system；
The preparation of blank pork：The pig that drug is raised is not added from farm's order to obtain；
The preparation of Sulfonamides residual substrate standard substance candidate in pork：It is ordered from farm and does not add drug feeding
Feeding pig obtains positive sample according to the dosage intravenous injection administration being pre-designed.
The preparation of 1 standard working solution of embodiment
The preparation of Standard Stock solutions：It accurately weighs respectively pure for 100% sulfadimidine veterinary drug through purity conversion
Degree standard substance and each 10.0mg of sulphadiazine veterinary drug purity rubric substance (accurately to 0.1mg), are completely dissolved with methanol, are shifted
Into 10mL brown volumetric flask, with methanol constant volume, shake up, be configured to mass concentration be 1mg/mL Standard Stock solutions, -20 DEG C
Refrigerator is kept in dark place, and validity period 12 months；It accurately weighs respectively phonetic for 100% isotope labelling sulfanilamide (SN) diformazan through purity conversion
Pyridine and each 1.0mg of isotope labelling sulphadiazine (accurately to 0.1mg), are completely dissolved with methanol, are transferred to 10mL brown capacity
It in bottle, with methanol constant volume, shakes up, is configured to the isotope labelling Standard Stock solutions that mass concentration is 0.1mg/mL, -20 DEG C of ice
Case is kept in dark place, and validity period 12 months；
The preparation of standard mixed solution：Sulfadimidine standard reserving solution and the storage of sulphadiazine standard are accurately pipetted respectively
Standby each 1mL of liquid, with methanol constant volume, shakes up in 100mL brown volumetric flask, and it is mixed to be configured to the standard that mass concentration is 10 μ g/mL
Solution is closed, -20 DEG C of refrigerators are kept in dark place, and validity period 6 months；The storage of isotope labelling sulfadimidine standard is accurately pipetted respectively
Standby liquid and each 1mL of isotope labelling sulphadiazine standard reserving solution with methanol constant volume, shake up, match in 100mL brown volumetric flask
The isotope labelling standard mixed solution that mass concentration is 1 μ g/mL is made, -20 DEG C of refrigerators are kept in dark place, and validity period 6 months；
The preparation of standard working solution：A certain amount of standard mixed solution is drawn, with -0.1% aqueous formic acid (5 of acetonitrile:
95) it is diluted to the standard that concentration is respectively 10.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL and 200.0ng/mL
Working solution, every milliliter of standard working solution is interior to contain D4-SM2 and D4-SDZ Isotopic Internal Standard 100.0ng.
The pretreatment of 2 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
20mL acetonitrile, homogenate is added in mixing 1min on turbine mixer for the isotope labelling standard mixed solution of 1 μ g/mL
It is centrifuged 2min under 2min, 9000r/min (4 DEG C), obtained supernatant is crossed into 70g anhydrous sodium sulfate column and (is soaked in advance using acetonitrile
Profit) wherein moisture is removed, and be transferred in 100mL chicken heart bottle, repetitive operation 3 times, by obtained extracting solution in 50 DEG C of water-baths
Rotary evaporation is dry, accurate mobile phase (- 0.1% aqueous formic acid (5 of acetonitrile that 3mL n-hexane saturation is added:95)), whirlpool is mixed
It is even, it is transferred in 15mL centrifuge tube, it is to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear carries out LC-MS/MS measurement through 0.22 μm of polytetrafluoroethylene (PTFE) filtering with microporous membrane.
The pretreatment of 3 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
20mL acetonitrile, homogenate is added in mixing 1min on turbine mixer for the isotope labelling standard mixed solution of 1 μ g/mL
It is centrifuged 2min under 2min, 9000r/min (4 DEG C), obtained supernatant is crossed into 100g anhydrous sodium sulfate column and (is soaked in advance using acetonitrile
Profit) wherein moisture is removed, and be transferred in 100mL chicken heart bottle, repetitive operation 4 times, by obtained extracting solution in 50 DEG C of water-baths
Rotary evaporation is dry, accurate mobile phase (- 0.1% aqueous formic acid (5 of acetonitrile that 3mL n-hexane saturation is added:95)), whirlpool is mixed
It is even, it is transferred in 15mL centrifuge tube, it is to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear is filtered through 0.22 μm of nylon micro porous filter membrane, carries out LC-MS/MS measurement.
The pretreatment of 4 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
20mL acetonitrile, homogenate is added in mixing 1min on turbine mixer for the isotope labelling standard mixed solution of 1 μ g/mL
It is centrifuged 2min under 2min, 9000r/min (4 DEG C), obtained supernatant is crossed into 50g anhydrous sodium sulfate column and (is soaked in advance using acetonitrile
Profit) wherein moisture is removed, and be transferred in 100mL chicken heart bottle, repetitive operation 2 times, by obtained extracting solution in 50 DEG C of water-baths
Rotary evaporation is dry, accurate mobile phase (- 0.1% aqueous formic acid (5 of acetonitrile that 3mL n-hexane saturation is added:95)), whirlpool is mixed
It is even, it is transferred in 15mL centrifuge tube, it is to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear carries out LC-MS/MS measurement through 0.22 μm of polytetrafluoroethylene (PTFE) filtering with microporous membrane.
The pretreatment of 5 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
20mL acetonitrile, homogenate is added in mixing 1min on turbine mixer for the isotope labelling standard mixed solution of 1 μ g/mL
It is centrifuged 2min under 2min, 9000r/min (4 DEG C), obtained supernatant is crossed into 20g anhydrous sodium sulfate column and (is soaked in advance using acetonitrile
Profit) wherein moisture is removed, and be transferred in 100mL chicken heart bottle, it operates 1 time, obtained extracting solution is rotated in 50 DEG C of water-baths
Evaporation drying, accurate mobile phase (- 0.1% aqueous formic acid (5 of acetonitrile that 3mL n-hexane saturation is added:95)), whirlpool mixes,
It is transferred in 15mL centrifuge tube, it is to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear is filtered through 0.22 μm of nylon micro porous filter membrane, carries out LC-MS/MS measurement.
The pretreatment of 6 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
20mL ethyl acetate, homogenate is added in mixing 1min on turbine mixer for the isotope labelling standard mixed solution of 1 μ g/mL
It is centrifuged 2min under 2min, 9000r/min (4 DEG C), obtained supernatant is crossed into 70g anhydrous sodium sulfate column and (is soaked in advance using acetonitrile
Profit) wherein moisture is removed, and be transferred in 100mL chicken heart bottle, it operates 3 times, obtained extracting solution is rotated in 60 DEG C of water-baths
Evaporation drying, accurate mobile phase (- 0.1% aqueous formic acid (5 of acetonitrile that 3mL n-hexane saturation is added:90)), whirlpool mixes,
It is transferred in 15mL centrifuge tube, it is to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear carries out LC-MS/MS measurement through 0.22 μm of polytetrafluoroethylene (PTFE) filtering with microporous membrane.
The pretreatment of 7 substrate standard substance of embodiment
(1) sample preparation：Pig two sides trunk muscle is taken, size sliced meat appropriate are cut into, is twisted 3 times with the meat grinder that aperture is 5mm,
And it manually mixes thoroughly；
(2) it extracts：3.00g is accurately weighed (to be accurately placed in 50mL centrifuge tube to 0.01g) pork, 300 μ L concentration are added
For the isotope labelling standard mixed solution of 1 μ g/mL, in mixing 1min on turbine mixer, 20mL volume fraction, which is added, is
30% acetonitrile solution is centrifuged 2min under homogenate 2min, 9000r/min (4 DEG C), obtained supernatant is crossed the anhydrous sulphur of 70g
Sour sodium column (infiltrates) removal wherein moisture using acetonitrile in advance, and is transferred in 100mL chicken heart bottle, operates 3 times, mentions what is obtained
Take liquid rotary evaporation in 40 DEG C of water-baths dry, (- 0.1% formic acid of acetonitrile is water-soluble for the accurate mobile phase that 3mL n-hexane saturation is added
Liquid (5:100)), whirlpool mixes, and is transferred in 15mL centrifuge tube, to be clean；
(3) it purifies：5mL n-hexane, vortex 1min, in 9000r/min (4 are added in the extracting solution obtained to step (2)
DEG C) under be centrifuged 2min, upper layer n-hexane is discarded after stratification, adds 5mL n-hexane, is repeated the above steps until subnatant
Body clear carries out LC-MS/MS measurement through 0.22 μm of polytetrafluoroethylene (PTFE) filtering with microporous membrane.
Comparative example 1
Compared with Example 2, the anhydrous sodium sulfate column of comparative example 1 does not use acetonitrile to infiltrate, other conditions and 2 phase of embodiment
Together.
Comparative example 2
Compared with Example 2, directly add after the substrate standard substance addition isotope labelling standard mixed solution of comparative example 2
Enter 70g anhydrous sodium sulfate and remove moisture removal, other conditions are same as Example 2.
Comparative example 3
Compared with Example 2, the extract of comparative example 3 uses MCX Solid Phase Extraction column purification, other conditions and embodiment 2
It is identical.
Comparative example 4
Compared with Example 2, the extract of comparative example 3 uses Al2O3Solid Phase Extraction column purification, other conditions and embodiment 2
It is identical.
Comparative example 5
Compared with Example 2, the extract of comparative example 3 uses HLB Solid Phase Extraction column purification, other conditions and embodiment 2
It is identical.
Comparative example 6
Compared with Example 2, the extract of comparative example 3 uses C18Solid Phase Extraction column purification, other conditions and 2 phase of embodiment
Together.
Embodiment 8LC-MS/MS measurement
The pretreated substrate standard substance that embodiment 2-7 and comparative example 1-6 is obtained carries out LC-MS/MS measurement, instrument
Parameter is as follows with determination condition：
(1) chromatographic condition
Chromatographic column：ACQUITY UPLC BEH C18(2.1 × 50mm, 1.7 μm, Waters)；
Column temperature：35℃；
Mobile phase：A phase：0.1% aqueous formic acid, B phase：Acetonitrile, gradient elution program are shown in Table 1；
Flow velocity：0.3mL/min；
Sampling volume：2μL；
1 eluent gradient elution program of table
(2) Mass Spectrometry Conditions
According to the molecular structure of Sulfonamides, select electrospray ionisation source (electrospray ionization,
ESI), multiple-reaction monitoring (multiple reaction monitoring, MRM) mode detects, and mass spectrometry parameters are：Electron spray is just
Electronics (ESI+) ionizes mode；
Spray voltage：3.00kv；
Ion source temperature：100℃；
Desolvation temperature：350℃；
Taper hole air blowing flow：150L/hr；
Desolventizing gas flow：650L/hr；
Qualitative ion pair, quota ion pair, collision energy are shown in Table 2；
Qualitative ion pair, the quota ion pair, collision energy of 2 sulfa drugs of table
The influence factor of the sulfa drugs rate of recovery
(1) usage mode and dosage of anhydrous sodium sulfate
In pork in the examination criteria of sulfa drugs, the moisture in anhydrous sodium sulfate removal extracting solution is generallyd use,
Prevent extracting solution from emulsifying, the usage mode of different anhydrous sodium sulfates has been respectively adopted in embodiment 2, comparative example 1 and comparative example 2,
As a result, it has been found that：Compared with Example 2, the anhydrous sodium sulfate column of comparative example 1 does not use acetonitrile to shift to an earlier date the acetonitrile in rinse extracting solution
It can be attracted in anhydrous sodium sulfate column with sulfonamide, extracting solution is caused to be demulsified insufficient result；Comparative example 2 is in matrix mark
Anhydrous sodium sulfate is added before quasi- substance homogeneous, forms big ingot after anhydrous sodium sulfate water suction, has seriously affected homogenizing step,
It cannot be guaranteed that substrate standard substance is by abundant homogeneous；
When the dosage difference of anhydrous sodium sulfate, also there is certain influence on the extraction effect of extracting solution：Work as anhydrous slufuric acid
When the dress column amount of sodium is 20g-50g, the easy crystallization of anhydrous sodium sulfate is blocking, influences the demulsification and subsequent purification effect of extracting solution
Fruit；When the dress column amount of anhydrous sodium sulfate is increased to 70g-100g, the moisture in extracting solution can be sufficiently removed, is conducive to subsequent
Purification process.
(2) purification style
The purification of sample is to reduce one of the major measure of matrix effect, in pork sample Oil content and Protein content compared with
Height needs to remove the interference of fat and protein in extraction process.Embodiment 2 and comparative example 3-6 compare Solid phase extraction
(MCX column, Al2O3Column, HLB column, C18Column) and liquid-liquid distribution extracting and purifying (LLE) sulfadimidine and sulphadiazine are returned
The influence of yield, as shown in Figure 1A and 1B, HLB column and C18Column is lower to the rate of recovery of sulfadimidine and sulphadiazine,
Relative standard deviation is big, and stability is poor；MCX column and Al2O3The rate of recovery of column increases, but relative standard deviation is big, stablizes
Property is poor；The rate of recovery highest of LLE method, stability is good, this may be to have certain absorption to sulfanilamide (SN) due to solid-phase extraction column
Effect, causes the rate of recovery to reduce.
(3) Extraction solvent
The drug rate of recovery of embodiment 2, embodiment 6 and embodiment 7 is as shown in Fig. 2, acetonitrile, ethyl acetate and 30% acetonitrile
Aqueous solution all has certain extraction effect to sulfadimidine (SM2) and sulphadiazine (SDZ)：Wherein, acetonitrile is to two kinds
The extraction effect of drug is best, and for the rate of recovery close to 100%, the extraction efficiency of ethyl acetate is weaker than acetonitrile, 30% acetonitrile solution
Water content is higher, will affect the moisture removal effect of anhydrous sodium sulfate.
(4) extraction time
It, can be with after the extract concentrations of embodiment 1-4 are as shown in figure 3, extract substrate standard substance 1-4 times using acetonitrile
Higher drug concentration is obtained, for SM2, the SM2 concentration that 2-4 times is relatively extracted 1 acquisition is extracted and is significantly improved；For SDZ,
The SDZ that 3-4 times is relatively extracted 1-2 acquisition is extracted to be significantly improved.
(5) selection of filter membrane
In embodiment 2-7, (100.0ng/mL isotope labelling is included using 100.0ng/mL sulfonamide standard solution
Sulfanilamide (SN) diformazan density (D4-SM2) and 100.0ng/mL isotope labelling sulphadiazine (D4-SDZ)), compare 2 kinds of nylon membranes and 4
Influence of the kind polytetrafluoroethylene film to sulfonamide and its Isotopic Internal Standard rate of recovery.
As shown in Figure 4 A and 4 B shown in FIG., polytetrafluoroethylene film is higher to the rate of recovery of drug compared with nylon membrane, and the rate of recovery is most
Up to 98%, relative standard deviation is smaller；Nylon membrane has certain suction to sulfa drugs to the rate of recovery about 90% of drug
Attached effect.
Matrix effect evaluation
Matrix effect is to influence one of the principal element of LC-MS/MS result precision, and isotope dilution mass spectrometry is to eliminate
Or reduce the main method of matrix effect, but object and isotopic label by matrix effect influenced to deposit
In difference, result is caused to generate deviation, is measured especially for the high accuracy of substrate standard substance definite value, study matrix effect
It has very important significance.
The ratio that experiment is responded in calibration solution and matrix solution respectively by detection sulfanilamide (SN) and isotope labelling sulfanilamide (SN)
ME indicates matrix effect, and the matrix effect factor (θ) is the ratio of sulfanilamide (SN) matrix effect and isotope labelling sulfanilamide (SN) matrix effect.
It the results are shown in Table 3, it can be seen that in pork sample substrate, sulfanilamide (SN) and isotope labelling sulfanilamide (SN) occur more sternly
The matrix suppression of weight, and the degree that the matrix being subject to inhibits is inconsistent.As shown in Table 3, the matrix effect of sulphadiazine
It is 0.712, the matrix effect of isotope labelling sulphadiazine is 0.705, and the matrix effect factor is 1.011, is illustrated if only used
The calibration solution that solution is prepared as a result will be higher when being measured with isotope dilution mass spectrometry to sulphadiazine in pork
1.1%；Similarly, if the calibration solution only prepared with solution, with isotope dilution mass spectrometry to sulfadimidine in pork
When measuring, as a result by higher 1.7%.
3 sulphadiazine of table and isotope labelling sulphadiazine, sulfadimidine and isotope labelling sulfadimidine
Matrix effect and correction factor
Quantitative calibration technique study
In order to eliminate influence of the matrix effect to measurement result to the full extent, the present invention compares standard solution calibration side
Method, matrix matching calibration method and standard solution θ bearing calibration, wherein matrix matching standard method is divided into two kinds：Blank sample
Middle addition standard solution carries out sample pre-treatments and obtains matrix matching calibration solution A；Blank extracting solution adds standard solution, obtains
It obtains matrix matching and calibrates solution B.
Table 4 lists the measurement result of 4 kinds of quantitative calibration methods of sulphadiazine and sulfadimidine, phonetic for sulfanilamide (SN)
For pyridine and sulfadimidine, differed not using standard solution θ bearing calibration with the result of matrix matching solution calibration method B
Greatly, and matrix matching solution calibration method A with both result differ larger, and the relative standard deviation of testing result compares
Greatly, it may be possible to since standard solution and isotope labelling solution being added before sample pre-treatments, by a series of pretreatment process,
The regression analysis of standard curve is affected, bigger so as to cause measurement result uncertainty, relative standard deviation as a result is big.Cause
This, comprehensively considers the accuracy, precision and timeliness of method, and experimental selection standard solution θ bearing calibration carries out quantitative calibration.
Four kinds of quantitative calibration method measurement results of 4 sulphadiazine of table and sulfadimidine compare
The range of linearity
Standard working solution is prepared according to embodiment 1, respectively with the ratio of sulfanilamide (SN) and corresponding isotope labelling sulfanilamide (SN) peak area
Value is ordinate (y), and the concentration (ng/mL) of sulfanilamide (SN) is that abscissa (x) draws standard curve, and sulphadiazine is in 10.0ng/mL-
It is in good linear relationship in 200.0ng/mL concentration range：Y=1.222x-0.467, linearly dependent coefficient r are 0.9994；Sulphur
Amine diformazan pyrimidine is in good linear relationship in 10.0ng/mL-200.0ng/mL concentration range：Y=3.160x-0.562, line
Property correlation coefficient r be 0.9996.
Detection limit, quantitative limit and the rate of recovery
According to the ratio of sulfanilamide (SN) quota ion peak area and corresponding isotope labelling sulfanilamide (SN) peak area, calculation method detection limit
With quantitative limit：With S/N>3 be method detection limit, with S/N>10 be method quantitative limit.
It is verified using mark-on reclaims, the experimental results showed that, when adding concentration is 0.5 μ g/kg, S/N>3, sulfanilamide (SN) is phonetic
The detection of pyridine and sulfadimidine in pork sample is limited to 0.5 μ g/kg；When adding concentration is 2.0 μ g/kg, S/N>10,
The detection of sulphadiazine and sulfadimidine in pork sample is limited to 2.0 μ g/kg, show that sulphadiazine and sulfanilamide (SN) diformazan are phonetic
Quantitative limit of the pyridine in pork sample is respectively 2.0 μ g/kg.
Select blank pork sample, addition sulphadiazine and sulfadimidine hybrid standard substance, pitch-based sphere difference
For 50.0 μ g/kg, 100.0 μ g/kg and 150.0 μ g/kg, each concentration level takes 6 Duplicate Samples, according to optimal experimental method
The rate of recovery and relative standard deviation of sulphadiazine and sulfadimidine are measured, the results are shown in Table 5, and the rate of recovery is
Between 95.7%-104.8%, relative standard deviation illustrates that this method is phonetic to sulphadiazine in pork and sulfanilamide (SN) diformazan less than 5%
The measurement of pyridine has good accuracy and precision, meets Sulfonamides in pork and remains substrate standard substance valued methods
Requirement.
5 sulfadimidine of table, four kinds of quantitative calibration method measurement results compare
Method application
Sulfonamides remain matrix standard in the pork prepared using preprocess method and analysis condition to this laboratory
Substance candidate has carried out uniformity detection：15 bottles are randomly selected from every kind of candidate, to each sample replication 3 times,
Experimental result statistical analysis is carried out according to method of analysis of variance, the results showed that every kind of standard substance candidate is all uniform.
In conclusion the present invention carries out homogeneous by improving and optimizing to pretreatment process, by substrate standard substance
The anhydrous sodium sulfate column that acetonitrile infiltration is crossed after extraction removes moisture removal, and the acetonitrile and sulfa drugs avoided in extracting solution is adsorbed in
On anhydrous sodium sulfate column, replaces conventional Solid phase extraction method to extract the purification of object using the liquid-liquid distribution method of purification, keep away
Absorption of the solid-phase extraction column center pillar material to sulfa drugs is exempted from, using n-hexane as purifying solvent, to the fat in pork
There is apparent removal to act on protein；In the present invention, sulphadiazine and sulfadimidine are in 10.0ng/mL-200.0ng/
It is in good linear relationship in mL concentration range, linearly dependent coefficient is all larger than 0.999, detection limit and the quantitative limit difference of method
For 0.5 μ g/kg and 2.0 μ g/kg, the rate of recovery within the scope of basic, normal, high 3 pitch-based spheres between 95.7%-104.8%,
Relative standard deviation is less than 5% (n=6), and Sulfonamides remain substrate standard substance uniformity testing, stability in pork
There is significant application value in monitoring and definite value.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of valued methods of substrate standard substance, which is characterized in that it is pretreated that the method includes substrate standard substances
Step；
The pretreatment includes：
(1) it extracts：The extraction that Extraction solvent carries out object, the excessively anhydrous sulphur of obtained supernatant are added into substrate standard substance
Sour sodium column, obtains extract；
(2) it purifies：Step (1) extract is dry through water-bath rotary evaporation, it is dissolved using mobile phase constant volume, using liquid-
The liquid distribution method of purification is purified, and pretreated substrate standard substance is obtained after filtering；
Wherein, the anhydrous sodium sulfate column uses acetonitrile to infiltrate in advance；
The mobile phase uses n-hexane to be saturated in advance.
2. the method according to claim 1, wherein step (1) described Extraction solvent includes acetonitrile, ethyl acetate
In acetonitrile solution any one or at least two combination, preferably acetonitrile；
Preferably, the number of step (1) described extraction is 1-4 times, preferably 3-4 times；
Preferably, the dosage of step (1) described anhydrous sodium sulfate is 20g-100g, preferably 70g-100g.
3. method according to claim 1 or 2, which is characterized in that the temperature of step (2) described water-bath is 40 DEG C -60 DEG C；
Preferably, step (2) mobile phase is the mixed liquor of acetonitrile and 0.1% formic acid；
Preferably, the volume ratio of the acetonitrile and 0.1% formic acid is 5:(90-100).
4. method according to claim 1-3, which is characterized in that step (2) the liquid-liquid distribution method of purification is adopted
Solvent is n-hexane；
Preferably, step (2) filtering is carried out using filter membrane；
Preferably, the aperture of the filter membrane is 0.22 μm；
Preferably, the filter membrane includes nylon and/or polytetrafluoroethylene (PTFE), preferably polytetrafluoroethylene (PTFE).
5. method according to claim 1-4, which is characterized in that before step (1) the addition Extraction solvent
Further include the steps that internal standard reagent is added.
6. method according to claim 1-5, which is characterized in that further include using liquid phase after step (2)
The step of chromatography-mass spectroscopy detects.
7. method according to claim 1-6, which is characterized in that the chromatographic column of the liquid chromatogram uses gradient
Type of elution；
Preferably, the mobile phase of the gradient elution uses 0.1% aqueous formic acid-acetonitrile；
Preferably, the change of gradient of the gradient elution is：
0-1min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
1min-5min, 0.1% aqueous formic acid：95.0%-20.0%, acetonitrile：5.0%-80.0%；
5min-5.1min, 0.1% aqueous formic acid：20.0%-95.0%, acetonitrile：80.0%-5.0%；
5.1min-7min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%.
8. method according to claim 1-7, which is characterized in that the mass spectrographic parameter includes：
Electron spray positive electron ionizes mode；
Spray voltage：3.00kv；
Ion source temperature：100℃；
Desolvation temperature：350℃；
Taper hole air blowing flow：150L/hr；
Desolventizing gas flow：650L/hr.
9. method according to claim 1-8, which is characterized in that the described method comprises the following steps：
(1) it extracts：After Isotopic Internal Standard reagent is added into substrate standard substance, the extraction that Extraction solvent carries out object is added
1-4 times, by obtained supernatant after the 20g-100g anhydrous sodium sulfate column infiltrated in advance with acetonitrile, obtain extract；
(2) it purifies：Step (1) extract is dry through 40 DEG C of -60 DEG C of water-bath rotary evaporations, use volume ratio for 5:(90-
100) the mobile phase constant volume dissolution of acetonitrile and 0.1% formic acid is carried out at purification using the liquid-liquid distribution method of purification by n-hexane
Reason obtains pretreated substrate standard substance after filtering using 0.22 μm of filter membrane；
(3) it detects：Using liquid chromatography-mass spectrometry, quantitative inspection is carried out to step (2) the pretreated substrate standard substance
It surveys；
Wherein, the chromatographic column of the liquid chromatogram uses gradient elution mode, and the mobile phase of gradient elution uses 0.1% formic acid water
The change of gradient of solution-acetonitrile, gradient elution is：
0-1min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
1min-5min, 0.1% aqueous formic acid：95.0%-20.0%, acetonitrile：5.0%-80.0%；
5min-5.1min, 0.1% aqueous formic acid：20.0%-95.0%, acetonitrile：80.0%-5.0%；
5.1min-7min, 0.1% aqueous formic acid：95.0%, acetonitrile：5.0%；
The mass spectrographic parameter includes：
Electron spray positive electron ionizes mode；
Spray voltage：3.00kv；
Ion source temperature：100℃；
Desolvation temperature：350℃；
Taper hole air blowing flow：150L/hr；
Desolventizing gas flow：650L/hr.
10. a kind of, such as the described in any item methods of claim 1-9, sulfa drug residue substrate standard substance is fixed in pork
Application in value；
Preferably, the sulfa drugs includes sulphadiazine and/or sulfadimidine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655549A (en) * | 2019-01-24 | 2019-04-19 | 梧州市食品药品检验所 | A kind of method of quick detection fish Malachite Green |
| CN114088858A (en) * | 2021-11-19 | 2022-02-25 | 江苏省淡水水产研究所 | Detection method of lincosamide antibiotics |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090103323A (en) * | 2008-03-28 | 2009-10-01 | 한국과학기술연구원 | Determination of Antibiotics by High Performance Liquid Chromatography and Mass Spectrometry |
| CN102175784A (en) * | 2011-01-19 | 2011-09-07 | 浙江出入境检验检疫局检验检疫技术中心 | Method for synchronously detecting 54 medicament residues in pork by virtue of solid phase extraction-liquid chromatogram-mass spectrum/mass spectrometry |
| CN104764844A (en) * | 2015-03-18 | 2015-07-08 | 谱尼测试科技股份有限公司 | Detection method of measuring residue amount of 35 veterinary drugs in meat of poultry and livestock |
| CN105891389A (en) * | 2016-04-08 | 2016-08-24 | 广东省农业科学院农产品公共监测中心 | Pretreatment method for improving accuracy of measuring sulfonamide residues in meat |
| CN107817306A (en) * | 2017-10-27 | 2018-03-20 | 中山出入境检验检疫局检验检疫技术中心 | Method that is a kind of while detecting 64 kinds of residues of veterinary drug in aquatic products |
-
2018
- 2018-05-29 CN CN201810532814.8A patent/CN108931593A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090103323A (en) * | 2008-03-28 | 2009-10-01 | 한국과학기술연구원 | Determination of Antibiotics by High Performance Liquid Chromatography and Mass Spectrometry |
| CN102175784A (en) * | 2011-01-19 | 2011-09-07 | 浙江出入境检验检疫局检验检疫技术中心 | Method for synchronously detecting 54 medicament residues in pork by virtue of solid phase extraction-liquid chromatogram-mass spectrum/mass spectrometry |
| CN104764844A (en) * | 2015-03-18 | 2015-07-08 | 谱尼测试科技股份有限公司 | Detection method of measuring residue amount of 35 veterinary drugs in meat of poultry and livestock |
| CN105891389A (en) * | 2016-04-08 | 2016-08-24 | 广东省农业科学院农产品公共监测中心 | Pretreatment method for improving accuracy of measuring sulfonamide residues in meat |
| CN107817306A (en) * | 2017-10-27 | 2018-03-20 | 中山出入境检验检疫局检验检疫技术中心 | Method that is a kind of while detecting 64 kinds of residues of veterinary drug in aquatic products |
Non-Patent Citations (6)
| Title |
|---|
| 侯建波等: "液相色谱-串联质谱-同位素稀释法同时测定猪肉中54种药物残留", 《质谱学报》 * |
| 刘薇等: "环境样品中硝基苯类化合物测定的预处理方法概述", 《环境科学与管理》 * |
| 杨总: "同位素稀释质谱法测定奶粉基体中药物残留的基质效应研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 杨总等: "同位素稀释液相色谱－串联质谱法准确测定猪肉中的氯霉素", 《色谱》 * |
| 蒋施等: "超高效液相色谱-串联质谱法测定动物组织中19种兽药残留量", 《理化检验-化学分册》 * |
| 陈永平等: "UPLC法测定水产品中磺胺类药物改进", 《河北渔业》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655549A (en) * | 2019-01-24 | 2019-04-19 | 梧州市食品药品检验所 | A kind of method of quick detection fish Malachite Green |
| CN114088858A (en) * | 2021-11-19 | 2022-02-25 | 江苏省淡水水产研究所 | Detection method of lincosamide antibiotics |
| CN114088858B (en) * | 2021-11-19 | 2023-09-01 | 江苏省淡水水产研究所 | Detection method of lincosamide antibiotics |
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Application publication date: 20181204 |