CN106645477B - A kind of remaining method of detection florfenicol amine and application - Google Patents
A kind of remaining method of detection florfenicol amine and application Download PDFInfo
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- CN106645477B CN106645477B CN201611193127.5A CN201611193127A CN106645477B CN 106645477 B CN106645477 B CN 106645477B CN 201611193127 A CN201611193127 A CN 201611193127A CN 106645477 B CN106645477 B CN 106645477B
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- florfenicol amine
- detection
- ethyl acetate
- methanol
- acetonitrile
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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
- G01N30/14—Preparation by elimination of some components
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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
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention belongs to field of veterinary drug residue detection, and in particular to a kind of remaining method of detection florfenicol amine.This method is to use high performance liquid chromatography mass spectrometer detection florfenicol amine to remain later using florfenicol amine residue in acetonitrile ethyl acetate extraction sample；The acetonitrile ethyl acetate is that acetonitrile and ethyl acetate are mixed by 1: 1 volume ratio.Sample is extracted, purifies, after purification, and high performance liquid chromatography mass spectrometer is utilized to detect florfenicol amine residual quantity.The method matrix covering of the present invention is extensive, easy to operate, high sensitivity, reproducible, detection limitation is low, greatly reduces impurity interference, and big to instrument and chromatographic column protectiveness, and the maintenance of instrument and reproducibility is made to be greatly improved.
Description
Technical field
The invention belongs to field of veterinary drug residue detection, and in particular to a kind of remaining method of detection florfenicol amine and answer
With.
Background technology
Currently, the method mostly used greatly for animal-derived food detection florfenicol amine is：Enzyme-linked immunization is efficient
Liquid chromatography, the detection of these methods are limited to 1mg/kg or higher, and some is analyzed using liquid matter method, pre-treatment C18
Column or the distribution purification of liquid liquid, impurity purification are not thorough and just for single-matrix：Egg, matrix cover not comprehensive, Bu Nengshi
Answer practical application request.
Invention content
For the problems of the prior art, the purpose of the present invention is to provide a kind of for a variety of matrix, easy to operate, clever
Sensitivity is high, reproducible, detection limits the low remaining method of detection florfenicol amine.
To achieve the above objectives, the technical solution adopted by the present invention is that：
A kind of remaining method of detection florfenicol amine is extracted florfenicol amine in sample using acetonitrile ethyl acetate and is remained
Object uses high performance liquid chromatography mass spectrometer detection florfenicol amine to remain later；The acetonitrile ethyl acetate be acetonitrile and
Ethyl acetate is mixed by 1: 1 volume ratio.
On the basis of said program, the remaining method and step of the detection florfenicol amine is as follows：
1) extraction purification
2.00g samples are weighed, 15mL acetonitrile ethyl acetate is added, ultrasonic 20min, 4000r/min centrifuge 5min, take supernatant
Liquid, repetitive operation merge supernatant in heart bottle twice, and 5% acetic acid solutions of 3mL, rotation are added in supernatant after merging
It steams to no reflux；Remaining liq is poured into centrifuge tube, then is poured into centrifuge tube after cleaning heart bottle with 4mL5% acetic acid solutions；Add
Enter 10mL n-hexanes, vortex oscillation 1min, 4000r/min centrifuge 5min, remove n-hexane layer, extracting solution is spare；
2) column purification is purified
After 2mL methanol and 2mL water balances activation PCX decontaminating columns, loading is eluted 2 times with 3mL5% formic acid methanol solutions,
It drains；Then it uses 6mL20% ammonium hydroxide methanol solutions to elute, receives eluent, rotate to dry；2mL methanol aqueous solutions are added to be vortexed
Constant volume；0.22 μm of filter membrane is crossed, it is to be measured；
3) high performance liquid chromatography mass spectrometer detection florfenicol amine is utilized to remain
Chromatographic parameter is：Chromatographic column：1.8 μm of XDB-C18,4.6mm*50mm；Column temperature：40 degree；Flow velocity：0.3mL/min；
Sample size：2μL；Mobile phase is：A：0.1% formic acid water, B：Methanol；
Mass spectrometry parameters are：Ion source：The sources ESI+；Gas Temp：300℃；Gas Flow：10L/min；Nebulizer：
45psi；Sheath Gas Tem：375℃：Sheath Gas flow：11L/min.
On the basis of said program, the detection florfenicol amine residual method is applied to detection animal derived food
The residual of middle florfenicol amine.
On the basis of said program, the animal derived food is muscle, liver, milk and eggs.
Beneficial effects of the present invention：
Present invention florfenicol amine to be detected belongs to polar compound, and acetonitrile polarity is also very big, is mixed according to similar
Principle, with pure acetonitrile extract sample polar impurity it is more, to interfere the purification of florfenicol amine, and ethyl acetate belongs to inclined
The substance of low pole is mixed as Extraction solvent using the two, can improve the extraction efficiency of target compound and reduce pole
The interference of property impurity；5% acetic acid aqueous solution is added when revolving can make florfenicol amine at ionic condition, dissolve in aqueous solution,
It prepares to cross PCX decontaminating columns in next step；N-hexane is nonpolar solvent, and n-hexane is added in extraction can be by the non-pole in matrix
Property impurity further extracts；There is PCX decontaminating columns benzene sulfonic acid type functional group, the florfenicol amine of adsorbable ionization to use
5% formic acid methanol solution elutes, and can further remove the polar impurity in matrix；Ammonium hydroxide methanol solution has higher ion strong
Degree, when elution, can replace florfenicol amine from PCX decontaminating columns.
This method carries out Instrumental Analysis using UPLC-MSMS, and matrix covers the animal sources such as muscle, liver, milk, eggs food
Product, low detection limit is 1 μ g/kg；It is extracted using acetonitrile ethyl acetate in the present invention, n-hexane removal of impurities, compared to its other party
Method, it is easy to operate；Using PCX solid-phase extraction columns, selectivity is strong, and the rate of recovery is high, effectively removes impurity；Use high performance liquid chromatography
Mass spectrometer, and multiple-reaction monitoring (MRM) acquisition method is used, compared to single high performance liquid chromatography, there is sensitivity
High, reproducible, detection limits low advantage, and greatly reduces impurity interference, this is big to instrument and chromatographic column protectiveness, makes instrument
The maintenance of device and reproducibility are greatly improved.
Description of the drawings
The efficient liquid phase mass spectrogram of Fig. 1 florfenicol amines, wherein ordinate are response, abscissa m/z；
The measurement of Fig. 2 florfenicol amine retention times, wherein ordinate are response, and abscissa is retention time；
The quantitative limits of Fig. 3 florfenicol amines measures, and wherein ordinate is response, and abscissa is retention time, 3 figures are upper,
In, under respectively represent the S/N of 3 daughter ions.
The standard curve of Fig. 4 florfenicol amines, wherein ordinate are peak area response, and abscissa is that florfenicol amine is dense
Degree.
Specific implementation mode
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.
With reference to specific embodiment, and with reference to the data further detailed description present invention.Following embodiment only be
It illustrates the present invention, rather than limits the scope of the invention in any way.
Embodiment
1. the preparation of standard solution
The preparation of 1.1 storing solutions
Precise 10.00mg florfenicol amines, are dissolved into methanol, constant volume to 10mL, the deposit as 1000 μ g/mL
Liquid (actual concentrations should be multiplied by the purity of florfenicol amine), be placed in 4 DEG C it is stored refrigerated；
The preparation of 1.2 standard intermediate fluids
It is accurate to measure 1mL florfenicol amines, it is dissolved into methanol, constant volume to 10mL, as the storing solution of 100 μ g/mL, sets
It is stored refrigerated in 4 DEG C；
The preparation of 1.3 standard working curves
Be configured to 1 with standard intermediate fluid, 2,5,10, the standard liquid of 20ng/mL (use methanol+water (v/v=1:1) constant volume), make
For the standard solution of working curve.
2. sample treatment
2.1 extraction purification
15mL acetonitrile ethyl acetate (v/v=1 is added in 50mL centrifuge tubes in accurate weighing 2.00g ± 0.01g samples:1),
Ultrasonic 20min, 4000r/min centrifuge 5min, take supernatant in 100mL heart bottles, repetitive operation merges supernatant twice, adds
Enter 5% acetic acid solutions of 3mL, revolving to no reflux.Remaining liq is poured into new 50mL centrifuge tubes, then with 5% acetic acid solutions of 4mL
The heart bottle is cleaned, and is poured into new 50mL centrifuge tubes.10mL n-hexanes, vortex oscillation 1min, 4000r/min centrifugation is added
5min, removes n-hexane layer, and extracting solution is spare.
2.2 purification column purifications
After 2mL methanol and 2mL water balances activation PCX decontaminating columns, said extracted liquid is added in PCX decontaminating columns and is purified,
It is used in combination 3mL5% formic acid methanol solutions to elute 2 times, drains.Then it uses 20% ammonium hydroxide methanol solutions of 6mL to elute, receives eluent,
It rotates to dry.2mL methanol aqueous solutions (v/v=1 is added:1) vortex constant volume.0.22 μm of filter membrane is crossed, it is to be measured.
3. high performance liquid chromatography mass spectrometer detects florfenicol amine residual quantity
The high performance liquid chromatography mass spectrometer used is Agilent LC-MSMS 1290/6460, and chromatographic parameter is：Color
Compose column：1.8 μm of XDB-C18,4.6mm*50mm；Column temperature：40 degree；Flow velocity：0.3mL/min；Sample size：2μL；Mobile phase is：A：
0.1% formic acid water, B：Methanol；The gradient of mobile phase is as shown in table 1：
1 eluent gradient of table
| Time(min) | B% |
| 0.00 | 20 |
| 1.00 | 20 |
| 3.00 | 90 |
| 4.00 | 90 |
| 4.01 | 20 |
| 5.50 | 20 |
Mass spectrometry parameters are：Ion source：The sources ESI+；Gas Temp：300℃；Gas Flow：10L/min；Nebulizer：
45psi；Sheath Gas Tem：375℃：Sheath Gas flow：11L/min.
4. result and analysis
4.1 specificity
As shown in Figure 1, machine on 1ppm florfenicol amines, respectively obtains 3 ion pairs, wherein ordinate is by mass spectrum optimization
Response, abscissa m/z.
Mass spectral results are as shown in table 2：
2 mass spectral results of table
50ppb florfenicol amines determine retention time by C18 chromatography post separations.As shown in Fig. 2, retention time is about
1.5min, and the peak shape of chromatography is preferable, noiseless peak occurs.
4.2 quantitative limit
Addition is equivalent to the standard items that sample contains 1.0 μ g/kg and calculates its letter through machine testing in pre-treatment in matrix
It makes an uproar than (S/N) > 10, as shown in Figure 3.
Measure the quantitative limit of this method：1.0μg/kg.
4.3 standard curve
Precision draws florfenicol amine standard items, and it is 2,5,10,20 to be diluted to series concentration with 50% methanol aqueous solution,
50ng/mL standard solution, difference sample introduction measures, and using peak area response as ordinate, a concentration of abscissa of florfenicol amine is drawn
Standard curve is as shown in Figure 4：The regression equation for obtaining florfenicol amine is Y=2970.653983*X-2691.191137, R2=
0.9996, by related coefficient as it can be seen that in 2ng/mL~50ng/mL standard curve ranges of linearity, UPLC-MSMS methods measure linear
Relationship is good, this standard curve can be used for accurate quantitative analysis.
The addition recycling of 4.4 blank samples
Blank sample addition recycling is as shown in table 3：
The addition recycling of 3 blank sample of table
The high pork of fat content, low beef, egg, the duck liver of supermarket's purchase, milk in the market are chosen in addition recycling
Quality Control experiment is done, the rate of recovery illustrates that this method has good stability between 70%-95%.
5. conclusion
By being measured to the florfenicol amine in animal sources matrix with UPLC-LCMSMS methods, determination acetonitrile：Second
Acetoacetic ester=1:1 volume ratio extracts, and PCX pillars are purified, and florfenicol amine chromatographic peak can be made to reach baseline separation,
Peak shape is good, and detection limits low, high sensitivity, and recycling is stablized, and the present invention is the florfenicol amine residual quantity in animal sources matrix
Measurement establishes a kind of reliably analysis method.
Claims (3)
1. a kind of remaining method of detection florfenicol amine, it is characterised in that：Steps are as follows：
1) extraction purification
2.00g samples are weighed, 15mL acetonitrile ethyl acetate is added, the acetonitrile ethyl acetate is that acetonitrile and ethyl acetate press 1: 1
Volume ratio mix, ultrasonic 20min, 4000r/min centrifuge 5min, take supernatant, repetitive operation to merge supernatant twice
In heart bottle, 5% acetic acid solutions of 3mL, revolving to no reflux are added in supernatant after merging；Remaining liq is poured into
Centrifuge tube, then poured into centrifuge tube after cleaning heart bottle with 5% acetic acid solutions of 4mL；10mL n-hexanes, vortex oscillation is added
1min, 4000r/min centrifuge 5min, remove n-hexane layer, and extracting solution is spare；
2) column purification is purified
After 2mL methanol and 2mL water balances activation PCX decontaminating columns, loading is eluted 2 times with 3mL5% formic acid methanol solutions, is taken out
It is dry；Then it uses 20% ammonium hydroxide methanol solutions of 6mL to elute, receives eluent, rotate to dry；2mL methanol aqueous solutions are added and are vortexed and determine
Hold；0.22 μm of filter membrane is crossed, it is to be measured；
3) high performance liquid chromatography mass spectrometer detection florfenicol amine is utilized to remain
Chromatographic parameter is：Chromatographic column：1.8 μm of XDB-C18,4.6mm*50mm；Column temperature：40 degree；Flow velocity：0.3mL/min；Sample introduction
Amount：2μL；Mobile phase is：A：0.1% formic acid water, B：Methanol；Wherein, the gradient of mobile phase is：
Mass spectrometry parameters are：Ion source：The sources ESI+；Gas Temp：300℃；Gas Flow：10L/min；Nebulizer：45psi；
Sheath Gas Tem：375℃：Sheath Gas flow：11L/min.
2. detecting the remaining method of florfenicol amine according to claim 1, it is characterised in that：For detecting animal derived food
The residual of florfenicol amine in product.
3. detecting the remaining method of florfenicol amine according to claim 2, it is characterised in that：The animal derived food is
Muscle, liver, milk and eggs.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760938A (en) * | 2018-08-01 | 2018-11-06 | 扬州大学 | Chloramphenicol, Thiamphenicol, Florfenicol and its how remaining analysis method of metabolite florfenicol amine in a kind of efficient detection birds, beasts and eggs |
| CN110108826A (en) * | 2019-05-24 | 2019-08-09 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of florfenicol amine in cream or dairy products |
| CN112345661B (en) * | 2020-10-21 | 2022-11-15 | 北京精益捷检测科技有限公司 | Method for determining content of florfenicol in veterinary drug gardenia flaviviridae oral liquid |
| CN112461823A (en) * | 2020-11-10 | 2021-03-09 | 苏州慧源安食检测科技有限公司 | Rapid detection method of florfenicol |
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| 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 |
| CN101747246A (en) * | 2009-12-22 | 2010-06-23 | 扬州大学 | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs |
| CN102565269A (en) * | 2012-03-05 | 2012-07-11 | 扬州大学 | Method for simultaneously detecting chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues in eggs |
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| 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 |
| CN101747246A (en) * | 2009-12-22 | 2010-06-23 | 扬州大学 | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs |
| CN102565269A (en) * | 2012-03-05 | 2012-07-11 | 扬州大学 | Method for simultaneously detecting chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues in eggs |
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Effective date of registration: 20200618 Address after: Hangzhou City, Zhejiang province 310000 city of Shen Jia Lu No. 319 room 416 Patentee after: HANGZHOU HAIRUNTAIHE DETECTING TECHNOLOGY Co.,Ltd. Address before: 266000, Qingdao Agricultural University, 700 the Great Wall Road, Chengyang District, Shandong, Qingdao Patentee before: QINGDAO HARRENS-QAU TESTING Ltd. |