CN107037140A - A kind of method of cynnematin residual quantity in efficient detection aquatic products - Google Patents
A kind of method of cynnematin residual quantity in efficient detection aquatic products Download PDFInfo
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Abstract
The present invention relates to a kind of method of cynnematin residual quantity in efficient detection aquatic products, methods described includes：After the extracted solvent extraction of sample, multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bar mass spectrographs are determined under multiple-reaction monitoring pattern, and making standard curve using matrix matching legal system carries out quantified by external standard method.Testing cost of the present invention is low, and method is accurate, sensitive and quick, can provide technical support for the residual monitoring of cephalosporins in aquatic products.
Description
Technical field
The invention belongs to test and analyze technical field, it is related to a kind of method for detecting cynnematin residual quantity, more particularly to
A kind of testing cost is low, and accurate, the sensitive and quick multi-walled carbon nanotube purification-ultra performance liquid chromatography-mass spectrography of method is surveyed
Determine the method for cynnematin residual quantity in aquatic products.
Background technology
Cynnematin is also known as cephaloridnum, is semi-synthetic containing cephem and a mould class beta-lactam of alkene two in molecule
Antibiotic, with broad-spectrum antibacterial action, be mainly used in clinical medicine treatment gram- bacteria for example staphylococcus, streptococcus pneumonia,
Various infection caused by Escherichia coli and salmonella etc., be also widely used in animal farming industry treatment livestock and poultry animal urethra,
Intestines and stomach and respiratory tract infection.The residual that cynnematin causes is excessively used in animal product to pass to human body by food chain
Health is impacted, and is caused allergic reaction, three cause the harm such as effect and bacterial drug resistance enhancing.Therefore countries in the world are raiseeed part
Poultry product-derived proposes cynnematin residue limits requirement.
In recent years, with the appearance of new disease epidemic situation in culture fishery and fisherman's blindness use antibiotic the problem of, some
The preferable people of clinical effectiveness with or veterinary medicine also begin to be used in aquaculture, in the market is also occurred in that for used for aquiculture
Input containing cynnematin.
Required there is presently no cynnematin residue limits in aquatic products, also lack corresponding detection method.Head in food
The method of spore rhzomorph residue detection mainly has microbial method, immunization, liquid chromatography, Liquid Chromatography/Mass Spectrometry and Capillary Electrophoresis
Method.Some related national standards are also primarily adapted for use in the bases such as livestock and poultry animal muscle, liver, kidney, egg, milk and honey
Matter, GB/T 22960-2008 only provide that 5 kinds of cynnematins are determined suitable for globe fish and eel.And found in real work
When GB/T 22960-2008 are used for aquatic products measure, on the one hand because aquatic products moisture content is high, the second after n-hexane degreasing purification
Boiling explosion phenomenon is also easy to produce during nitrile extract solution revolving, and sample is difficult to be evaporated under reduced pressure to net dry, is brought to follow-up constant volume
Error, on the other hand because aquatic products species is more, matrix components are complicated, and the mutual interference thing such as protein, fat is more, only by just oneself
Alkane liquid abstraction impurity removal effect is not good, and matrix interference is serious during mass spectroscopy, and particularly low concentration sample measure effect is poor, reclaims
Rate is relatively low.It is therefore desirable to set up the cephalosporins multi-residue determination of a kind of quick, accurate and suitable a variety of aquatic products matrix
Method.SPE SPE is a kind of sample pre-treatments purification techniques commonly used in wild animal resources.The SPE pillars of commercialization
Expensive, testing cost is higher.
Multi-walled carbon nanotube (MWCNTs) is a kind of functional material, can pass through noncovalent interaction power and target molecule knot
Close, high adsorption capacity, and have the advantages that specific surface area is high, chemical property is good, mechanical and heat endurance is high and the cost of material is low,
Preferable application has been obtained in heavy metal adsorption and organic matter detection and analysis work.Have not yet to see is used for cephalo by MWCNTs
Rhzomorph residue detection analyzes the research of pre-treatment.
The content of the invention
It is an object of the invention to deposited for the national standard method for solving cynnematin residue detection in existing aquatic products
Applicable variety is narrow, the defect that species to be measured are on the low side and the method rate of recovery is relatively low and a kind of low, the method that provides testing cost
Accurately, cynnematin is residual in sensitive and quick multi-walled carbon nanotube purification-ultra performance liquid chromatography-mass spectrometric determination aquatic products
The method of allowance.
To achieve these goals, the present invention uses following technical scheme：
A kind of method of cynnematin residual quantity in efficient detection aquatic products, methods described includes：The extracted solvent extraction of sample,
After multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bar mass spectrographs are how anti-
Answer and determined under monitoring pattern, making standard curve using matrix matching legal system carries out quantified by external standard method.
Preferably, the preparation method of multi-walled carbon nanotube solid-phase extraction column is：Weigh 2g multi-wall carbon nano-tubes pipe powder in
In 500mL single-necked flasks, upper liquid, solid are removed after adding the 50mL concentrated sulfuric acids and 100mL concentrated nitric acids, sonic oscillation 1h, standing
Thing is washed till neutrality with pure water repeatedly, and fine powder is ground to form after 80 DEG C of dryings；Fill before post, clean the poly- of 6mL with pure water, methanol respectively
Propylene SPE posts blank pipe and 20 μm of polyethylene post sieve plates, after pillar and sieve plate are dried, first seal SPE column bottoms, then claim with sieve plate
45-80mg multi-walled carbon nanotubes are taken in pillar, pad upper prop sieve plate compacting compresses downwards degree by adjusting upper sieve plate and controlled
Multi-walled carbon nanotube depth of packing is in 0.6cm；The multi-walled carbon nanotube SPE posts loaded are loaded in hermetic bag, are used before use
Preceding use 5mL methanol, 5mL water and the activation of 5mL phosphate buffers.
Preferably, the Extraction solvent is acetonitrile, acetonitrile-aqueous solution or methanol-metaphosphoric acid aqueous solution.
Preferably, the cynnematin be cefoperazone, Cefquinome, cefalonium, cephazoline, cefapirin,
Ceftiofur, Cefpirome and cefalexin.Cefoperazone (Cefoperazone, CEFOP)；Cefquinome (Cefquinome,
CEFQU)；Cefalonium (Cefalonium, CEFAL)；Cephazoline (Cefazolin, CEFAZ)；Cefapirin
(Cefapirin,CEFAP)；Ceftiofur (Ceftiofur, CEFTI)；Cefpirome (Cefpirome, CEFPI)；Cephalo ammonia
Benzyl (Cefalexin, CEFALE).
Preferably, the Extraction solvent is acetonitrile-aqueous solution.
Preferably, the processing method of sample is：5.0g samples are weighed in 50mL centrifuge tubes, adding 20mL volume ratios is
4:1 acetonitrile-aqueous solution, vortex oscillation extracts 2min, and 5000r/min centrifugation 6min, supernatant is transferred to 100mL hearts bottle
In, 15mL acetonitrile-aqueous solutions are added in residue and repeat to extract operation once, merges extract solution twice, adds 4mL saturation chlorinations
Sodium solution removes acetonitrile after 40 DEG C of rotary evaporations, and 10mL 0.10mol/L phosphate buffer solutions, whirlpool are added into heart bottle
Rotation, which mixes 1min, makes analyte fully dissolve, and whole loadings cross MWCNTs pillars, then uses 6mL phosphate buffer solutions, 6mL respectively
Water wash, is finally eluted with 6ml acetonitriles and receives eluent and dried up in 45 DEG C of nitrogen, is fully dissolved with 2mL pure water, crosses 0.22 μm
Determined after aqueous phase filter membrane for UPLC-MS/MS instrument.
Preferably, chromatogram and Mass Spectrometry Conditions
Chromatographic column：Acquity Xselect CSH C18Post 2.1 × 50mm, 1.7 μm；40 DEG C of column temperature；The μ L of sample size 10；Flow velocity：
0.3mL/min；Mobile phase A is 0.05-0.2% aqueous formic acids, and B is acetonitrile, gradient elution：0-3.0min, 90%-60%A；
3.0-4.0min, 60%-40%A；4.0-5.0min, 40%-90%A；5.0-6.5min, 90%A；
Mass Spectrometry Conditions：Electric spray ion source, positive ion mode；Capillary voltage 3.50kv；120 DEG C of ion source temperature；Desolventizing
380 DEG C of temperature degree；Desolventizing gas flow 600L/h；Taper hole throughput 50L/h；Multiple-reaction monitoring scan pattern.
Preferably, multiple-wall carbon nanotube is before single-necked flask is put into, it is 65-75wt% by multi-walled carbon nanotube and concentration
Lactic acid solution it is well mixed after heating stirring, wherein the mass ratio of the multi-walled carbon nanotube and lactic acid solution is 100:4-6,
Heating-up temperature is 80-95 DEG C, heat time 40-60min；It is described to be gradually added into quality to reaction solution during heating stirring
Multi-walled carbon nanotube 20-25% concentration is 30wt% hydrogen peroxide solution；PH is adjusted after heating stirring terminates to neutrality, most
After be dried in vacuo.
Cynnematin belongs to existing free carboxy in beta-lactam antibiotic, structural formula, and amino is carried again, is amphoteric compound
Matter, polarity is smaller, and most-often used in chromatographic isolation is anti-phase column packing.Investigation and comparison Waters Acquity UPLC
BEH C18(2.1 × 50mm, 1.7 μm) and Waters Acquity Xselect CSH C18Two kinds of (2.1 × 50mm, 1.7 μm)
Chromatographic column separating effect.As a result show that 2 kinds of chromatographic columns all have preferable reservation to 8 kinds of cynnematins, using the powered hydridization in surface
The Waters Acquity Xselect CSH C of granule technology18Chromatographic column is to two kinds of analytes of cefoperazone and cephazoline
Peak shape improves significantly, therefore present invention selection Waters Acquity Xselect CSH C18It is used as analytical column.
Extraction solvent for cynnematin has acetonitrile, acetonitrile-aqueous solution and methanol-metaphosphoric acid aqueous solution etc..The present invention
Acetonitrile, acetonitrile-water, methanol, the metaphosphoric acid of methanol -0.2% are compared as the extraction effect of extractant.Protein in aquatic products,
The macromolecular substances such as lipid are more, and when being extracted with methanol and methanol aqueous solution, extract solution is more muddy, causes sample purification difficult,
And the rate of recovery of cephazoline and cefapirin is relatively low.Acetonitrile can cause protein denaturation precipitation as organic solvent, and dissolution is miscellaneous
Matter is few, and extract solution is relatively clarified.It is water-soluble stronger because commercialization cynnematin is generally to contain salt pref.Carried using acetonitrile-aqueous solution
Drug solubility can be improved by taking.
The beneficial effects of the invention are as follows testing cost of the present invention is low, method is accurate, sensitive and quick, can be head in aquatic products
The residual monitoring of spore bacteriums provides technical support.
Embodiment
Below by specific embodiment, technical scheme is described in further detail.It should be appreciated that this hair
Bright implementation is not limited to the following examples, and any formal accommodation and/or change made to the present invention will all fall
Enter the scope of the present invention.
In the present invention, if not refering in particular to, all part, percentage are unit of weight, equipment and raw material for being used etc.
It is commercially available or commonly used in the art.Method in following embodiments, is the normal of this area unless otherwise instructed
Rule method.
Instrument and reagent
AcquityTMUltra performance liquid chromatography-Quattro Premier XE triple quadrupole bar matter mass spectrograph (match somebody with somebody ESI ion guns,
Waters, US)；IKA T18 basic models tissue mashing machine (German IKA companies)；(German IKA is public for MS2 eddy mixers
Department)；The supercentrifuges of Centrifuge 5810 (German Eppendorf companies)；R-201 types Rotary Evaporators (Shanghai section Xing Yi
Device Co., Ltd)；12 passage solid-phase extraction devices (Supelco companies of the U.S.).
Cefquinome sulfate (purity >=95.0%), Cefpirome Sulfate hydrate (purity >=98.5%), cefalexin
Monohydrate (purity >=99.0%) and Ceftiofur Hydrochloride (purity >=78.0%) are given birth to for Dr.Ehrenstorfer companies of Germany
Production, brizolina (purity >=89.1%), cefalonium hydrate (purity >=99.6%), cefapirin sodium (purity >=
98.0%) produced with cefoperazone dihydrate (purity >=99.9%) for Sigma Co., USA；Acetonitrile, methanol (chromatographically pure,
German Merk companies)；Ammonium acetate, formic acid (Sigma Co., USA)；(purity is more than 97%, diameter 40-60nm, length to MWCNTs
5-15 μm, Nanometer Port Co., Ltd., Shenzhen)；Water is ultra-pure water (Millipore companies of the U.S.) prepared by Milli-Q.
Cynnematin standard reserving solution：8 kinds of appropriate cynnematin standard substances are weighed, with methanol-water (v/v, 4：1) enter
Row dissolving and dilution, are made into the standard reserving solution that concentration is 100 μ g/mL, are stored at -18 DEG C.
Cynnematin hybrid standard uses liquid：8 kinds of appropriate cynnematin standard reserving solutions are pipetted respectively, are carried out with pure water
Dilution, is made into two kinds of hybrid standards that concentration is 10.0 μ g/mL and 0.10 μ g/mL and uses liquid, stored at -18 DEG C.
0.10mol/L phosphate buffer solutions：17.4g dipotassium hydrogen phosphate pure water is weighed to dissolve and be diluted to 1000mL,
Adjust pH 8.5.
Embodiment 1
A kind of method of cynnematin residual quantity in efficient detection aquatic products, methods described includes：The extracted solvent extraction of sample,
After multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bar mass spectrographs are how anti-
Answer and determined under monitoring pattern, making standard curve using matrix matching legal system carries out quantified by external standard method.
The preparation method of multi-walled carbon nanotube solid-phase extraction column is：2g multi-wall carbon nano-tubes pipe powder is weighed in 500mL single port
In flask, upper liquid is removed after adding the 50mL concentrated sulfuric acids and 100mL concentrated nitric acids, sonic oscillation 1h, standing, solids is repeatedly with pure
Neutrality is washed to, fine powder is ground to form after 80 DEG C of dryings；Fill before post, clean 6mL polypropylene SPE posts with pure water, methanol respectively
Blank pipe and 20 μm of polyethylene post sieve plates, after pillar and sieve plate are dried, first seal SPE column bottoms with sieve plate, then to weigh 45mg more
Wall carbon nano tube is in pillar, pad upper prop sieve plate compacting, and degree is compressed downwards by adjusting upper sieve plate controls multi-wall carbon nano-tube
Tube packing thickness is in 0.6cm；The multi-walled carbon nanotube SPE posts loaded are loaded in hermetic bag, and 5mL first is used before using preceding
Alcohol, 5mL water and the activation of 5mL phosphate buffers.
Multiple-wall carbon nanotube mixes multi-walled carbon nanotube with concentration before single-necked flask is put into for 65wt% lactic acid solution
Uniform rear heating stirring is closed, wherein the mass ratio of the multi-walled carbon nanotube and lactic acid solution is 100:4, heating-up temperature is 80
DEG C, heat time 40min；It is the multi-walled carbon nanotube 20% to be gradually added into quality to reaction solution during heating stirring
Concentration is 30wt% hydrogen peroxide solution；PH is adjusted after heating stirring terminates to neutrality, is finally dried in vacuo.
The processing method of sample is：5.0g samples are weighed in 50mL centrifuge tubes, it is 4 to add 20mL volume ratios:1 second
Nitrile-the aqueous solution, vortex oscillation extracts 2min, 5000r/min centrifugation 6min, and supernatant is transferred in 100mL hearts bottle, in residue
Add 15mL acetonitrile-aqueous solutions to repeat to extract operation once, merge extract solution twice, add after 4mL saturated nacl aqueous solutions
Acetonitrile is removed in 40 DEG C of rotary evaporations, 10mL 0.10mol/L phosphate buffer solutions are added into heart bottle, is vortexed and mixes
1min makes analyte fully dissolve, and whole loadings cross MWCNTs pillars, then respectively with 6mL phosphate buffer solutions, the pouring of 6mL water
Wash, finally eluted with 6ml acetonitriles and receive eluent and dried up in 45 DEG C of nitrogen, fully dissolved with 2mL pure water, cross 0.22 μm of aqueous phase
Determined after filter membrane for UPLC-MS/MS instrument.
Chromatogram and Mass Spectrometry Conditions
Chromatographic column：Acquity Xselect CSH C18Post 2.1 × 50mm, 1.7 μm；40 DEG C of column temperature；The μ L of sample size 10；Flow velocity：
0.3mL/min；Mobile phase A is 0.05% aqueous formic acid, and B is acetonitrile, gradient elution：0-3.0min, 90%-60%A；3.0-
4.0min, 60%-40%A；4.0-5.0min, 40%-90%A；5.0-6.5min, 90%A；
Mass Spectrometry Conditions：Electric spray ion source, positive ion mode；Capillary voltage 3.50kv；120 DEG C of ion source temperature；Desolventizing
380 DEG C of temperature degree；Desolventizing gas flow 600L/h；Taper hole throughput 50L/h；Multiple-reaction monitoring scan pattern.Parent ion, son from
The multiple-reaction monitoring conditions such as son, taper hole voltage and collision energy are shown in Table 1.
Embodiment 2
A kind of method of cynnematin residual quantity in efficient detection aquatic products, methods described includes：The extracted solvent extraction of sample,
After multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bar mass spectrographs are how anti-
Answer and determined under monitoring pattern, making standard curve using matrix matching legal system carries out quantified by external standard method.
The preparation method of multi-walled carbon nanotube solid-phase extraction column is：2g multi-wall carbon nano-tubes pipe powder is weighed in 500mL single port
In flask, upper liquid is removed after adding the 50mL concentrated sulfuric acids and 100mL concentrated nitric acids, sonic oscillation 1h, standing, solids is repeatedly with pure
Neutrality is washed to, fine powder is ground to form after 80 DEG C of dryings；Fill before post, clean 6mL polypropylene SPE posts with pure water, methanol respectively
Blank pipe and 20 μm of polyethylene post sieve plates, after pillar and sieve plate are dried, first seal SPE column bottoms with sieve plate, then to weigh 80mg more
Wall carbon nano tube is in pillar, pad upper prop sieve plate compacting, and degree is compressed downwards by adjusting upper sieve plate controls multi-wall carbon nano-tube
Tube packing thickness is in 0.6cm；The multi-walled carbon nanotube SPE posts loaded are loaded in hermetic bag, and 5mL first is used before using preceding
Alcohol, 5mL water and the activation of 5mL phosphate buffers.
Multiple-wall carbon nanotube mixes multi-walled carbon nanotube with concentration before single-necked flask is put into for 70wt% lactic acid solution
Uniform rear heating stirring is closed, wherein the mass ratio of the multi-walled carbon nanotube and lactic acid solution is 100:5, heating-up temperature is 85
DEG C, heat time 45min；It is the multi-walled carbon nanotube 22% to be gradually added into quality to reaction solution during heating stirring
Concentration is 30wt% hydrogen peroxide solution；PH is adjusted after heating stirring terminates to neutrality, is finally dried in vacuo.
The processing method of sample is：5.0g samples are weighed in 50mL centrifuge tubes, it is 4 to add 20mL volume ratios:1 second
Nitrile-the aqueous solution, vortex oscillation extracts 2min, 5000r/min centrifugation 6min, and supernatant is transferred in 100mL hearts bottle, in residue
Add 15mL acetonitrile-aqueous solutions to repeat to extract operation once, merge extract solution twice, add after 4mL saturated nacl aqueous solutions
Acetonitrile is removed in 40 DEG C of rotary evaporations, 10mL 0.10mol/L phosphate buffer solutions are added into heart bottle, is vortexed and mixes
1min makes analyte fully dissolve, and whole loadings cross MWCNTs pillars, then respectively with 6mL phosphate buffer solutions, the pouring of 6mL water
Wash, finally eluted with 6ml acetonitriles and receive eluent and dried up in 45 DEG C of nitrogen, fully dissolved with 2mL pure water, cross 0.22 μm of aqueous phase
Determined after filter membrane for UPLC-MS/MS instrument.
Chromatogram and Mass Spectrometry Conditions
Chromatographic column：Acquity Xselect CSH C18Post 2.1 × 50mm, 1.7 μm；40 DEG C of column temperature；The μ L of sample size 10；Flow velocity：
0.3mL/min；Mobile phase A is 0.2% aqueous formic acid, and B is acetonitrile, gradient elution：0-3.0min, 90%-60%A；3.0-
4.0min, 60%-40%A；4.0-5.0min, 40%-90%A；5.0-6.5min, 90%A；
Mass Spectrometry Conditions：Electric spray ion source, positive ion mode；Capillary voltage 3.50kv；120 DEG C of ion source temperature；Desolventizing
380 DEG C of temperature degree；Desolventizing gas flow 600L/h；Taper hole throughput 50L/h；Multiple-reaction monitoring scan pattern.Parent ion, son from
The multiple-reaction monitoring conditions such as son, taper hole voltage and collision energy are shown in Table 1.
Embodiment 3
A kind of method of cynnematin residual quantity in efficient detection aquatic products, methods described includes：The extracted solvent extraction of sample,
After multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bar mass spectrographs are how anti-
Answer and determined under monitoring pattern, making standard curve using matrix matching legal system carries out quantified by external standard method.
The preparation method of multi-walled carbon nanotube solid-phase extraction column is：2g multi-wall carbon nano-tubes pipe powder is weighed in 500mL single port
In flask, upper liquid is removed after adding the 50mL concentrated sulfuric acids and 100mL concentrated nitric acids, sonic oscillation 1h, standing, solids is repeatedly with pure
Neutrality is washed to, fine powder is ground to form after 80 DEG C of dryings；Fill before post, clean 6mL polypropylene SPE posts with pure water, methanol respectively
Blank pipe and 20 μm of polyethylene post sieve plates, after pillar and sieve plate are dried, first seal SPE column bottoms with sieve plate, then to weigh 50mg more
Wall carbon nano tube is in pillar, pad upper prop sieve plate compacting, and degree is compressed downwards by adjusting upper sieve plate controls multi-wall carbon nano-tube
Tube packing thickness is in 0.6cm；The multi-walled carbon nanotube SPE posts loaded are loaded in hermetic bag, and 5mL first is used before using preceding
Alcohol, 5mL water and the activation of 5mL phosphate buffers.
The processing method of sample is：5.0g samples are weighed in 50mL centrifuge tubes, it is 4 to add 20mL volume ratios:1 second
Nitrile-the aqueous solution, vortex oscillation extracts 2min, 5000r/min centrifugation 6min, and supernatant is transferred in 100mL hearts bottle, in residue
Add 15mL acetonitrile-aqueous solutions to repeat to extract operation once, merge extract solution twice, add after 4mL saturated nacl aqueous solutions
Acetonitrile is removed in 40 DEG C of rotary evaporations, 10mL 0.10mol/L phosphate buffer solutions are added into heart bottle, is vortexed and mixes
1min makes analyte fully dissolve, and whole loadings cross MWCNTs pillars, then respectively with 6mL phosphate buffer solutions, the pouring of 6mL water
Wash, finally eluted with 6ml acetonitriles and receive eluent and dried up in 45 DEG C of nitrogen, fully dissolved with 2mL pure water, cross 0.22 μm of aqueous phase
Determined after filter membrane for UPLC-MS/MS instrument.
Chromatogram and Mass Spectrometry Conditions
Chromatographic column：Acquity Xselect CSH C18Post 2.1 × 50mm, 1.7 μm；40 DEG C of column temperature；The μ L of sample size 10；Flow velocity：
0.3mL/min；Mobile phase A is 0.1% aqueous formic acid, and B is acetonitrile, gradient elution：0-3.0min, 90%-60%A；3.0-
4.0min, 60%-40%A；4.0-5.0min, 40%-90%A；5.0-6.5min, 90%A；
Mass Spectrometry Conditions：Electric spray ion source, positive ion mode；Capillary voltage 3.50kv；120 DEG C of ion source temperature；Desolventizing
380 DEG C of temperature degree；Desolventizing gas flow 600L/h；Taper hole throughput 50L/h；Multiple-reaction monitoring scan pattern.
The multiple-reaction monitoring condition such as parent ion, daughter ion, taper hole voltage and collision energy is shown in Table 1.
The mass spectrum multiple-reaction monitoring condition of the cynnematin of table 1
The range of linearity and quantitative limit
Standard curve is made using bare substrate matching method, the sample concentration for taking signal to noise ratio S/N=10 is method quantitative limit.8 kinds
The cynnematin range of linearity, curvilinear equation, coefficient correlation and quantitative limit are shown in Table 2.Each component concentration is responded with its mass spectrum
Good linear relationship, correlation coefficient r >=0.995, quantitative limit scope is between 2~10 μ g/kg.
The quantitation curves and quantitative limit of 28 kinds of cynnematins of table
The rate of recovery and precision of method
Recovery test is added using blank large yellow croaker and Penaeus Vannmei sample, average recovery rate and RSD, investigation side is calculated
The degree of accuracy of method and precision (table 3).8 kinds of cynnematin average recovery rates be 67.9%~93.5%, RSD be 3.29%~
12.5%, the preci-sion and accuracy of method can meet medicament residue detection requirement.
The blank sample TIANZHU XINGNAO Capsul of table 3 and precision measurement result (n=4)
Actual sample is determined
Cynnematin Risk Monitoring analysis is carried out to 36 portions of Zhejiang marine product using this method, wherein 1 part of seawater fish sample has head
Cefalexin is detected, and concentration is 5.73 μ g/kg.Learn before the plant 1 month that once used cephalo piece treats fish disease through investigation.
Claims (8)
1. a kind of method of cynnematin residual quantity in efficient detection aquatic products, it is characterised in that methods described includes：Sample is passed through
After Extraction solvent extraction, multi-walled carbon nanotube Solid phase extraction, using ultra performance liquid chromatography C18Post separation, triple level Four bars
Mass spectrograph is determined under multiple-reaction monitoring pattern, and making standard curve using matrix matching legal system carries out quantified by external standard method.
2. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1, it is characterised in that
The preparation method of multi-walled carbon nanotube solid-phase extraction column is：2g multi-wall carbon nano-tubes pipe powder is weighed in 500mL single-necked flasks, plus
Upper liquid is removed after entering the 50mL concentrated sulfuric acids and 100mL concentrated nitric acids, sonic oscillation 1h, standing, during solids is washed till with pure water repeatedly
Property, grind to form fine powder after 80 DEG C of dryings；Fill before post, clean 6mL polypropylene SPE posts blank pipe and 20 μ with pure water, methanol respectively
M polyethylene post sieve plates, after pillar and sieve plate are dried, SPE column bottoms are first sealed with sieve plate, then weigh many wall carbon of 45-80mg and are received
Mitron is in pillar, pad upper prop sieve plate compacting, and degree is compressed downwards by adjusting upper sieve plate controls multi-wall carbon nano-tube tube packing
Thickness is in 0.6cm；The multi-walled carbon nanotube SPE posts loaded are loaded in hermetic bag, and 5mL methanol, 5mL water are used before using preceding
With the activation of 5mL phosphate buffers.
3. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1, it is characterised in that
The Extraction solvent is acetonitrile, acetonitrile-aqueous solution or methanol-metaphosphoric acid aqueous solution.
4. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1, it is characterised in that
The cynnematin is cefoperazone, Cefquinome, cefalonium, cephazoline, cefapirin, Ceftiofur, Cefpirome
With cefalexin.
5. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1, it is characterised in that
The Extraction solvent is acetonitrile-aqueous solution.
6. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1 or 3 or 5, it is special
Levy and be, the processing method of sample is：5.0g samples are weighed in 50mL centrifuge tubes, it is 4 to add 20mL volume ratios:1 acetonitrile-
The aqueous solution, vortex oscillation extracts 2min, and 5000r/min centrifugation 6min, supernatant is transferred in 100mL hearts bottle, in residue again
Add 15mL acetonitrile-aqueous solutions to repeat to extract operation once, merge extract solution twice, add 4mL saturated nacl aqueous solutions after
40 DEG C of rotary evaporations remove acetonitrile, and 10mL 0.10mol/L phosphate buffer solutions are added into heart bottle, is vortexed and mixes 1min
Analyte is fully dissolved, whole loadings cross MWCNTs pillars, then use 6mL phosphate buffer solutions, 6mL water wash respectively, most
Eluted afterwards with 6ml acetonitriles and receive eluent and dried up in 45 DEG C of nitrogen, fully dissolved with 2mL pure water, cross 0.22 μm of aqueous phase filter membrane
Determined afterwards for UPLC-MS/MS instrument.
7. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 1 or 2 or 3 or 4, its
It is characterised by, chromatogram and Mass Spectrometry Conditions
Chromatographic column：Acquity Xselect CSH C18Post 2.1 × 50mm, 1.7 μm；40 DEG C of column temperature；The μ L of sample size 10；Flow velocity：
0.3mL/min；Mobile phase A is 0.05-0.2% aqueous formic acids, and B is acetonitrile, gradient elution：0-3.0min, 90%-
60%A；3.0-4.0min, 60%-40%A；4.0-5.0min, 40%-90%A；5.0-6.5min, 90%A；
Mass Spectrometry Conditions：Electric spray ion source, positive ion mode；Capillary voltage 3.50kv；120 DEG C of ion source temperature；Desolventizing
380 DEG C of temperature degree；Desolventizing gas flow 600L/h；Taper hole throughput 50L/h；Multiple-reaction monitoring scan pattern.
8. the method for cynnematin residual quantity in a kind of efficient detection aquatic products according to claim 2, it is characterised in that
Multiple-wall carbon nanotube mixes multi-walled carbon nanotube with concentration before single-necked flask is put into for 65-75wt% lactic acid solution
Even rear heating stirring, wherein the mass ratio of the multi-walled carbon nanotube and lactic acid solution is 100:4-6, heating-up temperature is 80-95
DEG C, heat time 40-60min；Quality is gradually added into for the multi-walled carbon nanotube 20- to reaction solution during heating stirring
25% concentration is 30wt% hydrogen peroxide solution；PH is adjusted after heating stirring terminates to neutrality, is finally dried in vacuo.
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| CN108387536A (en) * | 2018-01-31 | 2018-08-10 | 佛山市南海东方澳龙制药有限公司 | Cefquinome sulfate breast injection（Lactation period）The rapid assay methods of content |
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| CN114674950B (en) * | 2022-03-18 | 2024-01-09 | 衢州市疾病预防控制中心 | Method for quantitatively determining anesthetic |
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