CN109709258A - A kind of method and application detecting Florfenicol total residual object in pig edible tissue - Google Patents
A kind of method and application detecting Florfenicol total residual object in pig edible tissue Download PDFInfo
- Publication number
- CN109709258A CN109709258A CN201910091751.1A CN201910091751A CN109709258A CN 109709258 A CN109709258 A CN 109709258A CN 201910091751 A CN201910091751 A CN 201910091751A CN 109709258 A CN109709258 A CN 109709258A
- Authority
- CN
- China
- Prior art keywords
- florfenicol
- ethyl acetate
- tissue
- concentration
- amine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses the methods and application of Florfenicol total residual object in a kind of detection pig edible tissue.The method of the present invention includes: the homogenate of pig edible tissue by high salt concentration aqueous acid hydrolysis, ethyl acetate washing, it is extracted under alkaline condition by ethyl acetate, extract is by concentration, n-hexane degreasing, and using ethyl acetate: acetone: the mixed liquor of ammonium hydroxide carries out thin-layer chromatography purification as developping solution, then is detected using high performance liquid chromatography.The present invention sufficiently releases Florfenicol Bound residues in pig edible tissue, and is translated into florfenicol amine, avoids " false negative " result that existing method may cause.In addition, the present invention purifies tissue extract with thin-layer chromatography, endogenous material contained in tissue is effectively removed, its interference to chromatography is avoided, testing cost is reduced, reduces the dosage of organic solvent, as a result reliable, it is suitable for testing agency, base and carries out daily monitoring.
Description
Technical field
The invention belongs to field of veterinary drug residue detection, and in particular to Florfenicol is always residual in a kind of detection pig edible tissue
Stay the method and application of object.
Background technique
Florfenicol has the advantages that wide spectrum, efficient, low toxicity, is widely used in prevention and treatment respiratory tract infection of pigs in China.However
Toxicologic study shows Florfenicol and metabolin can induce hepatotoxicity wind agitation, genotoxicity, embryotoxicity, hematotoxicity and exempt from
Epidemic disease toxicity.Unreasonable use can lead to them and remain in pig edible tissue, endanger consumer health, and influence pig breeding industry can
Sustainable development.For this purpose, Florfenicol is residual in monitoring pig edible tissue there is an urgent need to establish reliable quantitative analysis method
It stays.
Florfenicol can be metabolized extensively in pig body.Part metabolin can form jail with large biological molecule (such as protein)
Though Bound residues be accumulated in pig edible tissue for a long time.These Bound residues can not need directly by solvent extraction
It could be released from tissue by strong acid hydrolysis, while itself is converted into florfenicol amine.In view of Florfenicol combines
The possible food safety risk of residue, European drug administration provide that the residual marker of Florfenicol is with fluorobenzene Buddhist nun
Examine the Florfenicol total residual object of amine calculating, including Florfenicol prototype and various metabolins.Fluorine in pig muscle, liver and kidney
The maximum residue limit that benzene Buddhist nun examines is respectively 300,2000,500 μ g/kg.
Lot of documents reports (such as enzyme-linked to exempt from based on physico-chemical analysis (such as chromatography, color-matter joint technology) and immunoassay
Epidemic disease detection technique) detection animal derived food in Florfenicol and florfenicol amine method.Wherein most methods
The hydrolysing step that can be released effectively Bound residues is not included in sample pre-treatments program, therefore can only detection part residue.
Using these methods monitoring animal products in Florfenicol residual will lead to " false negative " as a result, causing serious erroneous judgement.
By taking the research of Imran et al. as an example, according to dosage continuous oral 5 days of 30mg/kg, not medicine was detected from kidney chicken after 7 days
The fluoride protector object concentration that can be extracted is 125.04 μ g/kg, is lower than maximum residue limit (750 μ g/kg), it should determine
For qualification.And in fact, the Florfenicol Bound residues in this batch of sample there are also 806.50 μ g/kg are undiscovered.In this way
Animal products once come into the market and will threaten food safety, if selling to European Union, the breeding enterprise in China may be made to face
Huge reparation.It can be seen that establishing the quantitative analysis method ten of Florfenicol total residual object in detectable livestock and poultry edible tissue
Divide necessity.
Up to the present, only a small amount of document report detection of Florfenicol total residual object (being calculated with florfenicol amine)
Method.These methods are based on Liquid Chromatography-Tandem Mass Spectrometry and ultra performance liquid chromatography-tandem mass spectrometry.Method is used
Mass detector have the advantages that high sensitivity and high specific, but be also easy interference by matrix effect, and price
Valuableness, operation and maintenance cost are higher, are unfavorable for promoting to testing agency, base.High performance liquid chromatography is quantitative reliable, sensitive
Property can satisfy fluoride protector monitoring requirement, and the popularity rate of testing agency, base height, can be used as liquid chromatogram-
Tandem mass spectrometry and ultra performance liquid chromatography-tandem mass spectrometry substitution and supplement.However, high performance liquid chromatograph is equipped with mostly
UV detector, their specificity can not show a candle to mass detector.Pig edible tissue contains a large amount of endogenous material, they
It can be with florfenicol amine by coextraction, into sample to be detected.Due to these endogenous chaff interferents often have it is ultraviolet
It absorbs, their presence, which can detect chromatography, causes serious interference.To guarantee the reliable of testing result, sample purification is walked
Rapid progress system, in-depth study are most important.
For the residue detection of Florfenicol and florfenicol amine, common sample purification technology includes solid phase extraction techniques
(high performance liquid chromatography for detecting amphenicols and penicillins in the flesh of fish that such as Evaggelopoulou et al. is established, Shen
Et al. the gas chromatography-mass spectrometry of amphenicols drug in the detection pig muscle and liver established, Fedeniuk et al. builds
Vertical detection ox, horse, in pig edible tissue Florfenicol total residual object Liquid Chromatography-Tandem Mass Spectrometry, Imran et al. builds
The Liquid Chromatography-Tandem Mass Spectrometry of Florfenicol total residual object in vertical detection edible chicken tissues) and immune affinity chromatographic is (such as
The Liquid Chromatography-Tandem Mass Spectrometry of amphenicols drug in the detection pig muscle that Luo et al. is established).The filler valence of Solid Phase Extraction
Lattice are expensive, and wherein most Fillers selection is poor, it is difficult to obtain ideal clean-up effect.Immune affinity chromatographic is with specificity
Adsorbent of the antibody as Florfenicol and florfenicol amine, good purification, but the Antibody preparation period is long, and it is at high cost and anti-
Body stability is poor, could not also realize commercialization at present.Thin-layer chromatography is a kind of chromatographic separation technology of classics.With Solid Phase Extraction and
Immune affinity chromatographic is compared, and thin-layer chromatography has many advantages, such as that good purification, analysis cost are low, consumption of organic solvent is few.It will be thin
Layer chromatography is expected to the short slab of polishing UV detector poor specificity for the purification analysis of florfenicol amine in pig edible tissue,
Widen application of the high performance liquid chromatography in wild animal resources.
Thin-layer developing condition is the key technology of thin-layer chromatography purification.Thin-layer developing condition depends on target compound itself
Physicochemical property and the characteristics of sample substrate.The thin-layer chromatography of Florfenicol purifies existing document report (such as in animal feed
The high performance liquid chromatography of Florfenicol in the detection animal feed that Yang et al. is established), and pig edible tissue is hydrolyzed
In object florfenicol amine thin layer separation there is no literature reported on.The thin layer separation condition of Florfenicol might not in animal feed
Florfenicol amine isolates and purifies in suitable pig edible tissue.Florfenicol amine is the hydrolysate of Florfenicol.Due to side
Chain contains primary amine groups, and stronger absorption may occur on chromatographic sheet for florfenicol amine, this will change its chromatographic behavior.Together
When, pig edible tissue complicated component, especially after tissue is hydrolyzed, contained by endogenous chaff interferent sufficiently released
It puts.Their presence increases the difficulty of screening thin-layer developing condition.
Summary of the invention
The shortcomings that the present invention is directed to make up existing detection technique and deficiency provide a kind of more reliable and suitable base's detection
The detection method of Florfenicol total residual object in the pig edible tissue of mechanism.
The purpose of the invention is achieved by the following technical solution:
In a first aspect, providing a kind of method for detecting Florfenicol total residual object in pig edible tissue, including following step
It is rapid:
(1) pig edible tissue is taken, homogenate is made in tissue refiner；
(2) high salt concentration acid solution is added in tissue homogenate, vortex is placed in water-bath to tissue complete hydrolysis；Its
In, the molar concentration of hydrochloric acid solution is 4-8N, and tissue is homogenized and the mass volume ratio of hydrochloric acid solution is (1-2) g:(2-8) mL, water
Bath temperature is 80-100 DEG C, water bath time 2-24h；Florfenicol and metabolin with this condition complete hydrolysis at Florfenicol
Amine；
(3) after cooling, ethyl acetate is added in sample after hydrolyzing, is vortexed, centrifugation discards ethyl acetate；Wherein,
The volume ratio of sample and ethyl acetate after hydrolysis is (3-6): (4-40)；
(4) high-concentration sodium hydroxide solution is added in remaining sample and adjusts pH to 10-12；Wherein, sodium hydroxide quality
Percent concentration is 20%-50%, and addition volume is 0.5-5mL；
(5) ethyl acetate is added in the sample after alkalization, is vortexed, centrifugation, transfer ethyl acetate to another clean centrifugation
In pipe；Wherein, the volume ratio of the sample after alkalization and ethyl acetate is (3-8): (4-10)；
(6) primary with equivalent ethyl acetate repetition aforesaid operations, combined ethyl acetate；
(7) in batches in transfer ethyl acetate to the centrifuge tube containing appropriate glacial acetic acid solution, air stream volatilizes ethyl acetate；
Wherein, the concentration of volume percent of glacial acetic acid solution is 1%-10%, volume 0.2-1mL；
(8) appropriate n-hexane is added in remaining glacial acetic acid solution, is vortexed, centrifugation discards n-hexane；Wherein, remaining
Glacial acetic acid solution and the volume ratio of n-hexane are (0.2-1): (4-10)；
(9) remaining glacial acetic acid solution is blown to volume about 0.1mL with air stream；
(10) by above-mentioned glacial acetic acid solution and florfenicol amine standard items difference point sample in same GF-254 silica gel thin-layer
The different location of plate, in volume ratio ethyl acetate: acetone: ammonium hydroxide is equal to (1-5): (5-9): the mixed liquor expansion of (0.05-0.5)
To top；The concentration of volume percent of the ammonium hydroxide is preferably 25%-28%；
(11) GF-254 silica gel thin-layer plate is taken out, is inspected under 254-360nm after solvent volatilizes, according to Florfenicol
The approximate location of florfenicol amine, scrapes the silica gel of corresponding position in the development distance tagged tissue extracting solution of amine standard items；
(12) acetonitrile-glacial acetic acid mixed liquor 0.5-5mL of volume ratio 20:80-80:20, whirlpool are added in the silica gel of scraping
Rotation, centrifugation, takes supernatant, passes through membrane filtration；Wherein, in acetonitrile-glacial acetic acid mixed solution glacial acetic acid concentration of volume percent
For 1%-10%；
(13) filtered sample is passed through to the content of high performance liquid chromatography detection florfenicol amine；Wherein, efficient liquid phase
Chromatogram flow phase is acetonitrile and phosphate buffer is the mixed liquor of 30:70-70:30 composition by volume；Phosphate buffer
In containing mass percent concentration be 3%-10% dodecyl sodium sulfate, 1%-4% phosphate dihydrate disodium hydrogen, volume basis
Specific concentration is the triethylamine and 83%-98% phosphoric acid of 0.05%-2%.
Preferably, in step (1): the revolution for organizing homogenate is 3000-10000r/min, time 2-5min.
Preferably, in step (2): the time of vortex is 1-3min.
Preferably, in step (3): the time of vortex is 1-3min；The condition of centrifugation is that 3000-8000g is centrifuged 5-
10min。
Preferably, in step (5): the time of vortex is 1-3min；The condition of centrifugation is that 3000-8000g is centrifuged 5-
10min。
Preferably, in step (7): the temperature that air stream volatilizes is 35-60 DEG C.
Preferably, in step (8): the time of vortex is 1-3min；The condition of centrifugation is that 3000-8000g is centrifuged 5-
10min。
Preferably, in step (9): the temperature that air stream volatilizes is 35-60 DEG C.
Preferably, in step (12): filter membrane is 0.22 μm of organic filter membrane.
Preferably, in step (13) high performance liquid chromatography condition are as follows: chromatographic isolation is in Waters Symmetry C18Color
It composes in column (250mm × 4.6mm I.D, 5 μm) and completes, column temperature is 32-40 DEG C, flow velocity 0.5-1mL/min, and Detection wavelength is
220-230nm。
It is furthermore preferred that in a kind of detection pig edible tissue Florfenicol total residual object method, including it is following
Step:
(1) homogenate is made in pig edible tissue；
(2) tissue homogenate 2-5g is weighed, the hydrochloric acid solution 2-16mL of 4-8N is added, vortex 1-3min is placed in water-bath 90-
Water-bath 6-12h in 100 DEG C；Florfenicol and its metabolin with this condition complete hydrolysis at florfenicol amine；
(3) sample is cooled to room temperature, and ethyl acetate 8-40mL is then added, and vortex 1-3min, 3000-8000g are centrifuged 5-
10min discards ethyl acetate；
(4) the sodium hydroxide solution 0.5-5mL that mass percent concentration is 20%-50% is added in remaining sample to adjust
PH to 10-12；
(5) ethyl acetate 5-20mL is added in the sample after alkalization, vortex 1-3min, 3000-8000g are centrifuged 5-
10min is shifted in ethyl acetate to another clean centrifuge tube；
(6) primary with equivalent ethyl acetate repetition aforesaid operations, combined ethyl acetate；
(7) glacial acetic acid solution that transfer ethyl acetate is 1%-10% to concentration of volume percent containing 0.2-1mL in batches
Centrifuge tube in, volatilize ethyl acetate in 35-60 DEG C of air stream；
(8) 2-10mL n-hexane is added in remaining glacial acetic acid solution, vortex 1-3min, 3000-8000g are centrifuged 5-
10min discards n-hexane；
(9) remaining glacial acetic acid solution is blown to volume about 0.1mL in 35-60 DEG C of air stream；
(10) by above-mentioned glacial acetic acid solution and florfenicol amine standard items difference point sample in same GF-254 silica gel thin-layer
The different location of plate, in volume ratio ethyl acetate: acetone: ammonium hydroxide is equal to (1-5): (5-9): opening up in the mixed liquor of (0.05-0.5)
It drives to top；
(11) GF-254 silica gel thin-layer plate is taken out, is inspected under 254-360nm after solvent volatilizes, according to Florfenicol
The approximate location of florfenicol amine, scrapes the silica gel of corresponding position in the development distance tagged tissue extracting solution of amine standard items；
(12) volume ratio second eyeball is added in silica gel: glacial acetic acid solution (1%-10%) is the mixed liquor of 20:80-80:20
0.5-5mL, vortex 1-3min, 3000-8000g centrifugation 5-10min, takes supernatant, with 0.22 μm of organic membrane filtration；
(13) filtered sample passes through the amount of high performance liquid chromatography detection florfenicol amine, with the amount generation of florfenicol amine
The amount of table Florfenicol total residual object；
Second aspect provides a kind of method of Florfenicol total residual object in TLC separation pig edible tissue, packet
The step of including above-mentioned any one the method (1)-(12).
The third aspect, the method for providing Florfenicol total residual object in above-mentioned detection pig edible tissue can for detecting pig
The residual of Florfenicol in feeding habits tissue.
Preferably, the pig edible tissue is pig muscle, liver and kidney.
In the above method, Florfenicol and its metabolin in pig edible tissue are florfenicol amine through hydrolysis.
Therefore the concentration of Florfenicol total residual object is indicated in pig edible tissue with the concentration of florfenicol amine.The concentration is by standard song
Line is quantified, specifically: it weighs the standard items containing appropriate florfenicol amine and is added in 10mL volumetric flask, with the dissolution of second eyeball, determine
Hold, shake up, it is spare；Wherein " appropriate " florfenicol amine refers to 5-20mg.It takes for test-object product, series is diluted to step by step with mobile phase
Concentration, every concentration mark product are with 10-50 μ L sample introduction, are vertical sit with peak area using Florfenicol amine concentration as abscissa in triplicate
Mark, obtains regression equation y=282116x+9413.7, and wherein x is Florfenicol amine concentration, and y is peak area；Institute's sample is passed through
High performance liquid chromatography detection to florfenicol amine peak area bring above-mentioned linear equation into, be computed and pig edibility group can be obtained
Knit middle florfenicol amine residual concentration.
The invention has the advantages that and the utility model has the advantages that the present invention discharges fluorobenzene in pig edible tissue by strong acid hydrolysis
Buddhist nun examines Bound residues, enables the method to the Florfenicol total residual object in measurement tissue, avoids appearance " false negative "
Result；A large amount of fat is eliminated by ethyl acetate washing hydrolysis product and n-hexane degreasing in sample pretreatment process
With other endogenous chaff interferents；When thin-layer chromatography further purifies tissue extract, with volume ratio ethyl acetate: acetone: ammonia
Water (25%-28%) is equal to (1-5): (5-9): the mixed liquor of (0.05-0.5) purifies sample as developping solution, significantly improves
Detection method reduces testing cost to the anti-interference abilities of other substances in pig edible tissue, reduces organic molten
The dosage of agent, quantitative analysis results are reliable, suitable for base's detection unit to the Florfenicol total residual pig edible tissue
Object carries out daily monitoring.
Detailed description of the invention
Fig. 1 is that the clean-up effect of various sample purification technical treatment blank pig muscles in embodiment 1 compares；
Wherein, (a) is florfenicol amine standard substance (2 μ g/mL), (b) is directly analyzed for blank pig muscle extracting solution
Chromatogram (c) is chromatogram of the blank pig muscle extracting solution after Solid phase extraction, (d) extracts for blank pig muscle
The liquid chromatogram purified by thin-layer chromatography.As seen from the figure, blank pig muscle extracting solution after thin-layer chromatography purifies
Nearby free from admixture peak, method specificity are good for florfenicol amine retention time.
Fig. 2 is that the clean-up effect of various sample purification technical treatment blank pig livers in embodiment 1 compares；
Wherein, (a) is florfenicol amine standard substance (2 μ g/mL), (b) is directly analyzed for blank pig liver extracting solution
Chromatogram (c) is chromatogram of the blank pig liver extracting solution after Solid phase extraction, (d) extracts for blank pig liver
The liquid chromatogram purified by thin-layer chromatography.As seen from the figure, blank pig liver extracting solution after thin-layer chromatography purifies
Nearby free from admixture peak, method specificity are good for florfenicol amine retention time.
Fig. 3 is that the clean-up effect of various sample purification technical treatment blank Ren sus domesticas in embodiment 1 compares.Wherein (, a) it is
Florfenicol amine standard substance (2 μ g/mL), (b) chromatogram directly analyzed for blank Ren sus domestica extracting solution (c) are blank pig
Chromatogram of the kidney extracting solution after Solid phase extraction (d) purifies for blank Ren sus domestica extracting solution by thin-layer chromatography
Chromatogram afterwards.As seen from the figure, blank Ren sus domestica extracting solution is after thin-layer chromatography purifies in florfenicol amine retention time
Neighbouring free from admixture peak, method specificity are good.
Fig. 4 is the chromatogram of different samples in embodiment 2, wherein (a) is florfenicol amine standard substance (2 μ g/mL),
(b) it is blank pig liver, is (c) pig liver of the Florfenicol of 0.5 times of maximum residue limit of addition, (d) is blank Ren sus domestica,
(e) it is the Ren sus domestica for adding the Florfenicol of 0.5 times of maximum residue limit, (f) is blank pig muscle, is (g) 0.5 times of addition
The pig muscle of the Florfenicol of maximum residue limit.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
The comparison of the various sample purification technology clean-up effects of embodiment 1
One, method
(1) homogenate is made in blank pig muscle, liver, kidney.
(2) each 2g of above-mentioned homogenate is accurately weighed, every kind of tissue repeats 15 samples.The hydrochloric acid solution 4mL of 6N is added, is vortexed
2min is placed in water-bath 6h in 90-100 DEG C of water-bath.
(3) sample is cooled to room temperature, and ethyl acetate 20mL is then added, and vortex 2min, 5000g centrifugation 5min discards second
Acetoacetic ester.
(4) the sodium hydroxide solution 1.7mL that mass percent concentration is 50% is added in remaining sample and adjusts pH to 10-
12。
(5) ethyl acetate 8mL is added in the sample after alkalization, vortex 2min, 5000g are centrifuged 5min, shift acetic acid second
In ester to another clean centrifuge tube.
(6) primary with equivalent ethyl acetate repetition aforesaid operations, combined ethyl acetate.
(7) centrifuge tube for the glacial acetic acid solution that transfer ethyl acetate is 5% to concentration of volume percent containing 0.5mL in batches
It is interior, ethyl acetate is volatilized in 50 DEG C of air streams.
(8) 5mL n-hexane is added in remaining glacial acetic acid solution, vortex 2min, 5000g are centrifuged 5min, discard just oneself
Alkane.
(9) remaining glacial acetic acid solution handles (each way repeats 5 samples) according to following three kinds of modes respectively: 1. not
Purification, glacial acetic acid solution is dried up in 45 DEG C of air streams, and mobile phase 1mL is added and redissolves residue, crosses 0.22 μm of organic filter membrane, to
It surveys；2. 5% glacial acetic acid solution 1.5mL, vortex 2min are added into remaining glacial acetic acid solution with Solid phase extraction, it is added
Waters Oasis MCX solid phase extraction column (60mg, 3mL) through having balanced, then successively with 5% glacial acetic acid solution, volume
It is finally mixed with the ammonia water-methanol that volume ratio is 10:90 than the 5% glacial acetic acid solution-methanol mixed solution 2mL washing for 50:50
Solution 2mL elution is closed, eluent is dried up in 45 DEG C of air streams, and mobile phase 1mL is added and redissolves residue, crosses 0.22 μm of organic filter membrane,
It is to be measured；3. being purified with thin-layer chromatography, remaining glacial acetic acid solution is blown to volume about 0.1mL in 45 DEG C of air streams.Then by above-mentioned ice
Acetic acid solution and florfenicol amine standard items difference point sample are in the different location of same GF-254 silica gel thin-layer plate, in volume ratio
Ethyl acetate: acetone: ammonium hydroxide is expanded to top in the mixed liquor equal to 2:8:0.5.GF-254 silica gel thin-layer plate is taken out, to molten
Agent is inspected under 254nm after volatilizing, according to Florfenicol in the development distance tagged tissue extracting solution of florfenicol amine standard items
The approximate location of amine scrapes the silica gel of corresponding position.Volume ratio second eyeball is added in silica gel: glacial acetic acid solution (5%) is 30:70
Mixed liquor 1mL, vortex 2min, 8000g are centrifuged 5min, take supernatant, to be measured with 0.22 μm of organic membrane filtration.
(10) above-mentioned sample detects in 1525 highly effective liquid phase chromatographic system of Waters, the system include 1525 binary pumps,
2489UV detector and 2707 autosamplers.Chromatographic isolation is in Waters Symmetry C18Chromatographic column (250mm × 4.6mm
I.D, 5 μm) in complete, column temperature is 32 DEG C；Mobile phase is that (every 500mL phosphate buffer contains acetonitrile with phosphate buffer
0.34g dodecyl sodium sulfate, 0.78g phosphate dihydrate disodium hydrogen, 0.5mL phosphoric acid, 0.5mL triethylamine) be by volume 33.3:
The mixed liquors of 66.7 compositions, flow velocity 0.6mL/min, the second eyeball in mobile phase is chromatographically pure, water is purified water；Detection wavelength is
225nm。
Two, result
According to above-mentioned sample pre-treatments program processing blank pig muscle, liver and kidney homogenate, gained typical case chromatogram is such as
Shown in Fig. 1-3.The result shows that: florfenicol amine standard substance retention time is 9.407min, peak type under above-mentioned chromatographic condition
It is sharp symmetrical；Blank pig muscle, liver, kidney extracting solution without Solid Phase Extraction or thin-layer chromatography purification directly analysis,
Florfenicol amine retention time nearby has a large amount of impurity peaks, method poor specificity, can not fluorobenzene in the above-mentioned tissue of accurate quantitative analysis
Buddhist nun examines total residual object；Blank pig muscle, liver, kidney extracting solution pass through Solid phase extraction post analysis, in florfenicol amine
Retention time nearby still significantly interferes with, method poor specificity, can not Florfenicol in the above-mentioned tissue of accurate quantitative analysis it is always residual
Stay object；Blank pig muscle, liver, kidney extracting solution by thin-layer chromatography purify post analysis, in florfenicol amine retention time
Neighbouring free from admixture interference, method specificity is good, can be with the Florfenicol total residual object in the above-mentioned tissue of accurate quantitative analysis.
The accuracy and precision test of Florfenicol total residual object in 2 pig muscle of embodiment
One, method
(1) pig muscle homogenate 2g is accurately weighed, appropriate Florfenicol is added in homogenate, is converted, is made in muscle refining
Florfenicol amine containing 0.5 times, 1 times and 1.5 times maximum residue limit, each concentration repeat 5 samples, continuously repeat three
Batch.
(3) above-mentioned sample is handled according to hydrolysis, washing shown in embodiment 1, extraction, concentration and thin-layer chromatography decontamination procedure
Product, and analyzed according to chromatographic condition shown in embodiment 1.Florfenicol amine is quantified using standard curve, passes through efficient liquid
Phase chromatography detects series of concentrations florfenicol amine standard items, using Florfenicol amine concentration as abscissa, using peak area as ordinate,
Regression equation y=282116x+9413.7 is obtained, wherein x is Florfenicol amine concentration, and y is peak area；By institute's sample through height
The florfenicol amine peak area that effect liquid phase chromatogram detects brings above-mentioned linear equation into, is computed and fluorobenzene in pig muscle can be obtained
Buddhist nun examines amine concentration.
Two, result
Method detects typical chromatogram such as Fig. 4 institute of Florfenicol total residual object (indicating with florfenicol amine) in pig muscle
Show, the average recovery rate for measuring florfenicol amine is 80.86-108.81%, and in a few days relative standard deviation is less than 12.24%, day
Between relative standard deviation less than 9.82%.Accuracy and precision test result is as shown in table 1.
The accuracy and precision result of method in 1 pig muscle of table
The above result shows that detection method of the invention accuracy and precision with higher, can be used in pig muscle
The measurement of Florfenicol total residual object.
The accuracy and precision test of Florfenicol total residual object in 3 pig liver of embodiment
One, method
(1) pig liver homogenate 2g is accurately weighed, appropriate Florfenicol is added in homogenate, is converted, is made in liver homogenate
Florfenicol amine containing 0.5 times, 1 times and 1.5 times maximum residue limit, each concentration repeat 5 samples, continuously repeat three
Batch.
(3) above-mentioned sample is handled according to hydrolysis, washing shown in embodiment 1, extraction, concentration and thin-layer chromatography decontamination procedure
Product, and analyzed according to chromatographic condition shown in embodiment 1.Florfenicol amine is quantified using standard curve, passes through efficient liquid
Phase chromatography detects series of concentrations florfenicol amine standard items, using Florfenicol amine concentration as abscissa, using peak area as ordinate,
Regression equation y=282116x+9413.7 is obtained, wherein x is Florfenicol amine concentration, and y is peak area；By institute's sample through height
The florfenicol amine peak area that effect liquid phase chromatogram detects brings above-mentioned linear equation into, is computed and fluorobenzene in pig liver can be obtained
Buddhist nun examines amine concentration.
Two, result
Method detects typical chromatogram such as Fig. 4 institute of Florfenicol total residual object (indicating with florfenicol amine) in pig liver
Show, the average recovery rate for measuring florfenicol amine is 81.37-108.14%, and in a few days relative standard deviation is less than 13.08%, day
Between relative standard deviation less than 11.16%.Accuracy and precision test result is as shown in table 2.
The accuracy and precision result of method in 2 pig liver of table
The above result shows that detection method of the invention accuracy and precision with higher, can be used in pig liver
The measurement of Florfenicol total residual object.
The accuracy and precision test of Florfenicol total residual object in 4 Ren sus domestica of embodiment
One, method
(1) Ren sus domestica homogenate 2g is accurately weighed, appropriate Florfenicol is added in homogenate, is converted, is made in kidney homogenate
Florfenicol amine containing 0.5 times, 1 times and 1.5 times maximum residue limit, each concentration repeat 5 samples, continuously repeat three
Batch.
(3) above-mentioned sample is handled according to hydrolysis, washing shown in embodiment 1, extraction, concentration and thin-layer chromatography decontamination procedure
Product, and analyzed according to chromatographic condition shown in embodiment 1.Florfenicol amine is quantified using standard curve, passes through efficient liquid
Phase chromatography detects series of concentrations florfenicol amine standard items, using Florfenicol amine concentration as abscissa, using peak area as ordinate,
Regression equation y=282116x+9413.7 is obtained, wherein x is Florfenicol amine concentration, and y is peak area；By institute's sample through height
The florfenicol amine peak area that effect liquid phase chromatogram detects brings above-mentioned linear equation into, is computed and fluorobenzene in Ren sus domestica can be obtained
Buddhist nun examines amine concentration.
Two, result
Method detects typical chromatogram such as Fig. 4 institute of Florfenicol total residual object (indicating with florfenicol amine) in Ren sus domestica
Show, the average recovery rate for measuring florfenicol amine is 80.21-102.50%, and in a few days relative standard deviation is less than 7.53%, in the daytime
Relative standard deviation is less than 6.52%.Accuracy and precision test result is as shown in table 3.
The accuracy and precision result of method in 3 Ren sus domestica of table
The above result shows that detection method of the invention accuracy and precision with higher, can be used in Ren sus domestica
The measurement of Florfenicol total residual object.
The analysis of the actual sample of 5 separate sources of embodiment
One, method
(1) griskin, liver, kidney are bought from different supermarket, Wuhan City 3.According to preceding place shown in embodiment 1
Program processing is managed, and is analyzed according to chromatographic condition shown in embodiment 1.Florfenicol amine is quantified using standard curve, is led to
High performance liquid chromatography detection system column concentration florfenicol amine standard items are crossed, using Florfenicol amine concentration as abscissa, with peak area
For ordinate, regression equation y=282116x+9413.7 is obtained, wherein x is Florfenicol amine concentration, and y is peak area；It will be surveyed
Sample through high performance liquid chromatography detection to florfenicol amine peak area bring above-mentioned linear equation into, be computed can be obtained it is above-mentioned
Florfenicol amine concentration in sample.
Two, result
The results are shown in Table 4 for Florfenicol total residual analyte detection in the pig muscle of separate sources, liver and kidney.By surveying
It is fixed, detect that florfenicol amine, the residual concentration of florfenicol amine contained therein are below European Union and China's rule in 2 parts of livers
Fixed maximum residue limit.
Florfenicol total residual object in the pig edible tissue of 4 separate sources of table (in terms of florfenicol amine)
Technical solution of the present invention and beneficial effect is described in detail in embodiment described above, it should be understood that
Above is only a specific embodiment of the present invention, it is not intended to restrict the invention, it is all to be done in spirit of the invention
Any modification, supplementary, and equivalent replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method of Florfenicol total residual object in detection pig edible tissue, which comprises the following steps:
(1) pig edible tissue is taken, homogenate is made in tissue refiner；
(2) high salt concentration acid solution is added in tissue homogenate, vortex is placed in water-bath to tissue complete hydrolysis；Wherein, salt
The whole molar concentration of acid solution is 4-8N, and tissue is homogenized and the mass volume ratio of hydrochloric acid solution is (1-2) g:(2-8) mL, water-bath
Temperature is 80-100 DEG C, water bath time 2-24h；Florfenicol and metabolin with this condition complete hydrolysis at Florfenicol
Amine；
(3) after cooling, ethyl acetate is added in sample after hydrolyzing, is vortexed, centrifugation discards ethyl acetate；Wherein, it hydrolyzes
The volume ratio of sample and ethyl acetate afterwards is (3-6): (4-40)；
(4) high-concentration sodium hydroxide solution is added in remaining sample and adjusts pH to 10-12；Wherein, the matter of sodium hydroxide solution
Amount percent concentration is 20%-50%, and addition volume is 0.5-5mL；
(5) ethyl acetate is added in the sample after alkalization, is vortexed, centrifugation is shifted in ethyl acetate to another clean centrifuge tube；
Wherein, the volume ratio of the sample after alkalization and ethyl acetate is (3-8): (4-10)；
(6) primary with equivalent ethyl acetate repetition aforesaid operations, combined ethyl acetate；
(7) in batches in transfer ethyl acetate to the centrifuge tube containing appropriate glacial acetic acid solution, air stream volatilizes ethyl acetate；Its
In, the concentration of volume percent of glacial acetic acid solution is 1%-10%, volume 0.2-1mL；
(8) appropriate n-hexane is added in remaining glacial acetic acid solution, is vortexed, centrifugation discards n-hexane；Wherein, remaining ice second
The volume ratio of acid solution and n-hexane is (0.2-1): (4-10)；
(9) remaining glacial acetic acid solution is blown to volume about 0.1mL with air stream；
(10) by above-mentioned glacial acetic acid solution and florfenicol amine standard items difference point sample in same GF-254 silica gel thin-layer plate
Different location, in volume ratio ethyl acetate: acetone: ammonium hydroxide is equal to (1-5): (5-9): the mixed liquor of (0.05-0.5) is expanded to top
End；The concentration of volume percent of the ammonium hydroxide is preferably 25%-28%；
(11) GF-254 silica gel thin-layer plate is taken out, is inspected under 254-360nm after solvent volatilizes, according to florfenicol amine mark
The approximate location of florfenicol amine, scrapes the silica gel of corresponding position in the development distance tagged tissue extracting solution of quasi- product；
(12) acetonitrile-glacial acetic acid mixed liquor 0.5-5mL of volume ratio 20:80-80:20 is added in the silica gel of scraping, is vortexed, from
The heart takes supernatant, and by membrane filtration, filter membrane is preferably 0.22 μm of organic filter membrane；Wherein, ice in acetonitrile-glacial acetic acid mixed solution
The concentration of volume percent of acetic acid is 1%-10%；
(13) filtered sample is passed through to the content of high performance liquid chromatography detection florfenicol amine；Wherein, high performance liquid chromatography
Mobile phase is acetonitrile and phosphate buffer is the mixed liquor of 30:70-70:30 composition by volume；Contain in phosphate buffer
Having mass percent concentration is the dodecyl sodium sulfate of 3%-10%, and 1%-4% phosphate dihydrate disodium hydrogen, percent by volume is dense
Degree is the triethylamine and 83%-98% phosphoric acid of 0.05%-2%.
2. the method for Florfenicol total residual object in detection pig edible tissue according to claim 1, which is characterized in that
The revolution of tissue homogenate is 3000-10000r/min, time 2-5min in step (1).
3. the method for Florfenicol total residual object in detection pig edible tissue according to claim 1, which is characterized in that
In step (2): the time of vortex is 1-3min.
4. the method for Florfenicol total residual object in detection pig edible tissue according to claim 1, which is characterized in that
Step (3), (5), (8) mesoscale eddies time be 1-3min；The condition of centrifugation is that 3000-8000g is centrifuged 5-10min.
5. the method for Florfenicol total residual object in detection pig edible tissue according to claim 1, which is characterized in that
The temperature that air stream volatilizes in step (7), (9) is 35-60 DEG C.
6. the method for Florfenicol total residual object in detection pig edible tissue according to claim 1, which is characterized in that
The condition of high performance liquid chromatography in step (13) are as follows: chromatographic isolation is in Waters Symmetry C18Chromatographic column (250mm ×
4.6mm I.D, 5 μm) in complete, column temperature is 32-40 DEG C, flow velocity 0.5-1mL/min, Detection wavelength 220-230nm.
7. the method for detecting Florfenicol total residual object in pig edible tissue described in any one according to claim 1~6,
Characterized by comprising the following steps:
(1) homogenate is made in pig edible tissue；
(2) tissue homogenate 2-5g is weighed, the hydrochloric acid solution 2-16mL of 4-8N is added, vortex 1-3min is placed in 90-100 DEG C of water-bath
Middle water-bath 6-12h；Florfenicol and its metabolin with this condition complete hydrolysis at florfenicol amine；
(3) sample is cooled to room temperature, and ethyl acetate 8-40mL is then added, and vortex 1-3min, 3000-8000g are centrifuged 5-
10min discards ethyl acetate；
(4) in remaining sample be added mass percent concentration be 20%-50% sodium hydroxide solution 0.5-5mL adjust pH to
10-12；
(5) ethyl acetate 5-20mL is added in the sample after alkalization, vortex 1-3min, 3000-8000g are centrifuged 5-10min, turn
It moves in ethyl acetate to another clean centrifuge tube；
(6) primary with equivalent ethyl acetate repetition aforesaid operations, combined ethyl acetate；
(7) in batches transfer ethyl acetate to concentration of volume percent containing 0.2-1mL be 1%-10% glacial acetic acid solution from
In heart pipe, ethyl acetate is volatilized in 35-60 DEG C of air stream；
(8) 2-10mL n-hexane is added in remaining glacial acetic acid solution, vortex 1-3min, 3000-8000g are centrifuged 5-10min,
Discard n-hexane；
(9) remaining glacial acetic acid solution is blown to volume about 0.1mL in 35-60 DEG C of air stream；
(10) by above-mentioned glacial acetic acid solution and florfenicol amine standard items difference point sample in same GF-254 silica gel thin-layer plate
Different location, in volume ratio ethyl acetate: acetone: ammonium hydroxide is equal to (1-5): (5-9): being expanded in the mixed liquor of (0.05-0.5)
Top；
(11) GF-254 silica gel thin-layer plate is taken out, is inspected under 254-360nm after solvent volatilizes, according to florfenicol amine mark
The approximate location of florfenicol amine, scrapes the silica gel of corresponding position in the development distance tagged tissue extracting solution of quasi- product；
(12) volume ratio second eyeball is added in silica gel: glacial acetic acid solution (1%-10%) is the mixed liquor 0.5- of 20:80-80:20
5mL, vortex 1-3min, 3000-8000g centrifugation 5-10min, takes supernatant, with 0.22 μm of organic membrane filtration；
(13) filtered sample passes through the amount of high performance liquid chromatography detection florfenicol amine, represents fluorine with the amount of florfenicol amine
Benzene Buddhist nun examines the amount of total residual object.
8. the quantitative detecting method of Florfenicol total residual object in a boar edible tissue, which is characterized in that including following step
It is rapid:
1) Florfenicol total residual object in pig edible tissue is detected: using detection pig described in claim 1~6 any one
Step (1)~(13) in edible tissue in the method for Florfenicol total residual object；
2) standard items for weighing 5-20mg florfenicol amine are added in 10mL volumetric flask, with the dissolution of second eyeball, constant volume, are shaken up, spare；
Take for test-object product, series of concentrations be diluted to step by step with (13) step mobile phase in step 1), every concentration mark product with 10-50 μ L into
Sample using Florfenicol amine concentration as abscissa, using peak area as ordinate, obtains regression equation y=282116x+ in triplicate
9413.7, wherein x is Florfenicol amine concentration, and y is peak area；By (13) step institute sample in step 1) through high-efficient liquid phase color
It composes the florfenicol amine peak area detected and brings above-mentioned linear equation into, be computed and fluorobenzene Buddhist nun in pig edible tissue can be obtained
Examine amine residual concentration.
9. a kind of method of Florfenicol total residual object in TLC separation pig edible tissue, which is characterized in that including power
Benefit requires step (1)-(12) in 1~7 any one the method.
10. the method for Florfenicol total residual object is being examined in detection pig edible tissue described in claim 1~7 any one
Survey the remaining application of Florfenicol in pig edible tissue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910091751.1A CN109709258B (en) | 2019-01-30 | 2019-01-30 | Method for detecting florfenicol total residues in edible tissues of pigs and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910091751.1A CN109709258B (en) | 2019-01-30 | 2019-01-30 | Method for detecting florfenicol total residues in edible tissues of pigs and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109709258A true CN109709258A (en) | 2019-05-03 |
| CN109709258B CN109709258B (en) | 2021-07-20 |
Family
ID=66262030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910091751.1A Active CN109709258B (en) | 2019-01-30 | 2019-01-30 | Method for detecting florfenicol total residues in edible tissues of pigs and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109709258B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122754A (en) * | 2019-11-14 | 2020-05-08 | 浙江工业大学 | Method for detecting total amount of florfenicol and metabolites thereof in poultry eggs |
| CN111693639A (en) * | 2020-06-12 | 2020-09-22 | 扬州大学 | Confirmation analysis method for detecting penicillin G residue in poultry tissue, poultry egg or pork |
| CN115248276A (en) * | 2022-06-23 | 2022-10-28 | 江苏省农业科学院 | Method for determining phenobarbital drug residue in edible porcine tissues |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101726553A (en) * | 2009-12-22 | 2010-06-09 | 扬州大学 | Method for detecting thiamphenicol, florfenicol and residues of metabolite florfenicol amine in egg simultaneously |
| RO126606A0 (en) * | 2010-08-31 | 2011-08-30 | Maria Neagu | Analytical () method for identifying, dosing fluorfenicol and the chemically related impurities in the pharmaceutical products fluorfenidem 10%-oral solution, fluorfenidem 50 and fluorfenicol asactive pharmaceutical ingredient |
| CN105137068A (en) * | 2015-07-24 | 2015-12-09 | 烟台昊清生物科技有限公司 | Florfenicol on-site test paper, and preparation and use methods thereof |
-
2019
- 2019-01-30 CN CN201910091751.1A patent/CN109709258B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101726553A (en) * | 2009-12-22 | 2010-06-09 | 扬州大学 | Method for detecting thiamphenicol, florfenicol and residues of metabolite florfenicol amine in egg simultaneously |
| RO126606A0 (en) * | 2010-08-31 | 2011-08-30 | Maria Neagu | Analytical () method for identifying, dosing fluorfenicol and the chemically related impurities in the pharmaceutical products fluorfenidem 10%-oral solution, fluorfenidem 50 and fluorfenicol asactive pharmaceutical ingredient |
| CN105137068A (en) * | 2015-07-24 | 2015-12-09 | 烟台昊清生物科技有限公司 | Florfenicol on-site test paper, and preparation and use methods thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHRISTOPHER L.W RZESINSKI ET AL: "Determination of Florfenicol Amine in Channel Catfish Muscle by Liquid Chromatography", 《JOURNAL OF AOAC INTERNATIONAL》 * |
| KAIZHOU XIE ET AL: "Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| M. R. QIAN ET AL: "Diffusion- limited PBPK model for predicting pulmonary pharmacokinetics of florfenicol in pig", 《J. VET. PHARMACOL. THERAP. 》 * |
| SUXIA ZHANG ET AL: "Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography–tandem mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY B》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122754A (en) * | 2019-11-14 | 2020-05-08 | 浙江工业大学 | Method for detecting total amount of florfenicol and metabolites thereof in poultry eggs |
| CN111693639A (en) * | 2020-06-12 | 2020-09-22 | 扬州大学 | Confirmation analysis method for detecting penicillin G residue in poultry tissue, poultry egg or pork |
| CN115248276A (en) * | 2022-06-23 | 2022-10-28 | 江苏省农业科学院 | Method for determining phenobarbital drug residue in edible porcine tissues |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109709258B (en) | 2021-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109709258A (en) | A kind of method and application detecting Florfenicol total residual object in pig edible tissue | |
| Ufelmann et al. | Human and rat hepatocyte toxicity and protein phosphatase 1 and 2A inhibitory activity of naturally occurring desmethyl-microcystins and nodularins | |
| Bergmark et al. | Determination of hemoglobin adducts in humans occupationally exposed to acrylamide | |
| Chen et al. | Nodularins in poisoning | |
| Tang et al. | Rapid in vivo determination of fluoroquinolones in cultured puffer fish (Takifugu obscurus) muscle by solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry | |
| Shephard et al. | Fate of a single dose of 14C‐labelled fumonisin B1 in vervet monkeys | |
| Baptista et al. | Determination of the non protein amino acid β-N-methylamino-l-alanine in estuarine cyanobacteria by capillary electrophoresis | |
| CN102507564A (en) | Kit for quickly detecting nitrite in edible bird's nest and application of kit in edible bird's nest detection | |
| CN104597194B (en) | The high performance liquid chromatography-fluorescence method of 3-chlorine-1,2-propylene glycol | |
| Lu et al. | Levels of phthalates, bisphenol-A, nonylphenol, and microplastics in fish in the estuaries of northern Taiwan and the impact on human health | |
| CN105092753A (en) | Application of combination amine metabolism marker and kit and detection method thereof | |
| Chang et al. | Analytical detection of sulfonamides and organophosphorus insecticide residues in fish in Taiwan | |
| CN101936985B (en) | Method for detecting diethylstilbestrol and special chemiluminescent immunoassay kit thereof | |
| CN105116063A (en) | Multi-detection method of residual of cephalo-type drugs in milk product | |
| Aranganathan et al. | Spectral and microscopic analysis of fulvic acids isolated from marine fish waste and sugarcane bagasse co-compost | |
| CN101446574B (en) | LC-MS quantitative detection method for melamine and tricyanic acid in human urine | |
| CN108008057A (en) | The assay method of teracycline antibiotic residues amount in a kind of poultry | |
| CN110687241A (en) | Method for detecting roxarsone and 8 metabolites thereof in animal body | |
| CN109596753A (en) | A method of nitrite in cubilose is detected based on bag filter purification techniques | |
| CN110715986A (en) | Method for determining glucosamine hydrochloride in health food | |
| CN110954688A (en) | Method for determining bioavailability of nickel in soil and food for mice | |
| CN105510460A (en) | Method for quantitative detection of gluconic acids and glucono lactone | |
| CN114018886B (en) | Method for indirectly detecting sulfanilamide veterinary drug | |
| Even et al. | Determination of cadmium and lead in Northern pike from the Missouri River | |
| CN106442818A (en) | Magnetic separation based method for rapidly determining residual amount of sulfonamide antibiotics in animal muscle tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |