CN105784857B - Analysis test method for Ractopamine - Google Patents
Analysis test method for Ractopamine Download PDFInfo
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- CN105784857B CN105784857B CN201510855637.3A CN201510855637A CN105784857B CN 105784857 B CN105784857 B CN 105784857B CN 201510855637 A CN201510855637 A CN 201510855637A CN 105784857 B CN105784857 B CN 105784857B
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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
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Abstract
The present invention provides the analysis test method for Ractopamine, the extraction of AAO-MIP nanotube films and detection method associated with high performance liquid chromatography are established, and is realized to β in complex sample2The detection of adrenoceptor agonists.
Description
[technical field]
The present invention relates to a kind of analysis test methods for Ractopamine.
[background technique]
Ractopamine (Ractopamine, RAC) belongs to β2Adrenoceptor agonists class drug, can promote animal
Muscle growth and protein packing, still, being excessively used will lead to it and accumulates in animal tissue, and possible human health generates negative
Face is rung.Therefore, carrying out detection to the residual quantity of Ractopamine in sample (RAC) is particularly important.
Detection method currently used for Ractopamine has gas chromatography, high performance liquid chromatography (HPLC), gas phase color
Spectrum-Mass Spectrometry, Liquid Chromatography-Mass Spectrometry, molecularly imprinted polymer extraction-liquid chromatogram combination and molecular engram are poly-
Close object electrochemical sensor etc..No matter with which kind of analysis method, to reduce detection limit and eliminating interference, the step of enrichment and elution
It is all required.
Molecular imprinting technology can design and construct the system that the three-dimensional hole of specific recognition is realized to target molecule.Have
Relevant studies have shown that has substantial portion of research that the MIP of Ractopamine is used as adsorbent.Although the operation letter of these methods
Just, and β can selectively be extracted2Adrenoceptor agonists, but adsorbance is not high.Therefore, it establishes a kind of for prison
Survey β2Easy, highly selective, efficient, the stable preprocess method of adrenoceptor agonists is particularly important.
[summary of the invention]
The purpose of the present invention is be to have overcome the deficiencies of the prior art and provide the analysis test side for Ractopamine
Method.
In order to solve above-mentioned technical problem, the present invention is adopted the following technical solutions:
The present invention is the analysis test method for Ractopamine, and steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
C, pork sample is prepared；
D, the Ractopamine of 2.0 μ g/g is added in pork sample, the Ractopamine analog of 20 μ g/g, institute is added
The Ractopamine analog stated is clenbuterol (Clenbuterol), Terbutaline (Terbutaline), adrenaline
(Epinephrine) or dopamine (Dopamine)；
E, 10~20mL acetonitrile is added in pork sample and is saturated hexane solution；
F, the solution of potassium carbonate of 1~3mL 4mol/L is added in pork sample, makes protein denaturation, take out fatty acid and
Water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
H, mixture is acutely shaken, the concussion time is 5~10 minutes, and acetonitrile layer is precipitated；
I, 20~25mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it by said mixture at 3~8 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates second
Nitrile phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 2~5mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 5~10mL methanol and AAO-MIP nanotube films is used to extract
It takes, AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, analyzes Ractopamine in sample and the like mark
The content of quasi- object.
In step N, the Ractopamine (RAC) is detected with fluorescence detector, the clenbuterol
(Clenbuterol), Terbutaline (Terbutaline), adrenaline (Epinephrine) or dopamine (Dopamine) are adopted
It is detected with UV detector.
Compared with prior art, the beneficial effects of the present invention are: the present invention provides the analysis surveys for Ractopamine
Method for testing, the method for establishing the extraction of AAO-MIP nanotube films with HPLC combination detection, and realize to β in complex sample2Kidney
The detection of upper parathyrine receptor stimulating agent.
The present invention is described in further detail with specific embodiment for explanation with reference to the accompanying drawing:
[Detailed description of the invention]
Fig. 1 is HPLC chromatogram of the mark-on sample at 223nm.
[specific embodiment]
The present invention is the analysis test method for Ractopamine, and steps are as follows:
O, pellumina is subjected to the processing of ATRP initiator surface modification；
P, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
Q, pork sample is prepared；
R, the Ractopamine of 2.0 μ g/g is added in pork sample, the Ractopamine analog of 20 μ g/g, institute is added
The Ractopamine analog stated is clenbuterol, Terbutaline, adrenaline or dopamine；
S, 10~20mL acetonitrile is added in pork sample and is saturated hexane solution；
T, the solution of potassium carbonate of 1~3mL 4mol/L is added in pork sample, makes protein denaturation, take out fatty acid and
Water；
U, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
V, mixture is acutely shaken, the concussion time is 5~10 minutes, and acetonitrile layer is precipitated；
W, 20~25mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
X, it by said mixture at 3~8 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates second
Nitrile phase；
Y, by the mixture residue Na after separating acetonitrile2SO4It is dried；
Z, the mixture residue after drying is washed with the acetonitrile of 2~5mL；
AA, the acetonitrile in mixture residue is evaporated, then is dissolved with 5~10mL methanol and AAO-MIP nanotube films is used to extract
It takes, AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
BB, sample residue extracted is detected with HPLC, analyzes the Ractopamine and the like in sample
The content of reference substance.
In step N, the Ractopamine (RAC) is detected with fluorescence detector, the clenbuterol
(Clenbuterol), Terbutaline (Terbutaline), adrenaline (Epinephrine) or dopamine (Dopamine) are adopted
It is detected with UV detector.
The present invention provides the analysis test method for Ractopamine, establish the extraction of AAO-MIP nanotube films with
The method of HPLC combination detection, and realize to β in complex sample2The detection of adrenoceptor agonists.
Embodiment one
For the analysis test method of Ractopamine, steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
C, pork sample is prepared；
D, the Ractopamine of 1.5 μ g/g is added in pork sample, the Clenbuterol of 15 μ g/g is added；
E, 10mL acetonitrile is added in pork sample and is saturated hexane solution；
F, the solution of potassium carbonate of 1mL 4mol/L is added in pork sample, makes protein denaturation, takes out fatty acid and water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
H, mixture is acutely shaken, the concussion time is 5 minutes, and acetonitrile layer is precipitated；
I, 20mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it by said mixture at 3 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates acetonitrile
Phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 2mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 5mL methanol and AAO-MIP nanotube films is used to extract,
AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, the Ractopamine and Clenbuterol in analysis sample
Content.
Embodiment two
For the analysis test method of Ractopamine, steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
C, pork sample is prepared；
D, the Ractopamine of 1.8 μ g/g is added in pork sample, the Terbutaline of 18 μ g/g is added；
E, 15mL acetonitrile is added in pork sample and is saturated hexane solution；
F, the solution of potassium carbonate of 2mL 4mol/L is added in pork sample, makes protein denaturation, takes out fatty acid and water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
H, mixture is acutely shaken, the concussion time is 8 minutes, and acetonitrile layer is precipitated；
I, 22mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it at 5 DEG C by said mixture, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates acetonitrile
Phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 3mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 8mL methanol and AAO-MIP nanotube films is used to extract,
AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, the Ractopamine and Terbutaline in analysis sample
Content.
Embodiment three
For the analysis test method of Ractopamine, steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
C, pork sample is prepared；
D, the Ractopamine of 2.0 μ g/g is added in pork sample, the adrenaline of 20 μ g/g is added；
E, 18mL acetonitrile is added in pork sample and is saturated hexane solution；
F, the solution of potassium carbonate of 3mL 4mol/L is added in pork sample, makes protein denaturation, takes out fatty acid and water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
H, mixture is acutely shaken, the concussion time is 10 minutes, and acetonitrile layer is precipitated；
I, 24mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it by said mixture at 7 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates acetonitrile
Phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 5mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 10mL methanol and AAO-MIP nanotube films is used to extract,
AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, analyzes the Ractopamine and adrenergic in sample
Content.
Example IV
For the analysis test method of Ractopamine, steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP
Nanotube films；
C, pork sample is prepared；
D, the Ractopamine of 2.0 μ g/g is added in pork sample, the dopamine of 20 μ g/g is added；E, in pork sample
Middle addition 20mL acetonitrile is saturated hexane solution；
F, the solution of potassium carbonate of 3mL 4mol/L is added in pork sample, makes protein denaturation, takes out fatty acid and water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixing is made
Object；
H, mixture is acutely shaken, the concussion time is 10 minutes, and acetonitrile layer is precipitated；
I, 25mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it by said mixture at 8 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates acetonitrile
Phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 5mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 10mL methanol and AAO-MIP nanotube films is used to extract,
AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, analyzes containing for the Ractopamine and dopamine in sample
Amount.
This experiment establishes the side of the extraction of AAO-MIP nanotube films and HPLC combination detection Ractopamine and the like
Method.The range of linearity, related coefficient, detection limit, the reproducibility of analysis method by optimization are shown in Table one.
The extraction of AAO-MIP nanotube films and HPLC combination detection Ractopamine (RAC), clenbuterol in following table
(CLEN), Terbutaline (TER), the range of linearity of adrenaline (EP) and dopamine (DA), detection limit (LOD), related coefficient
(R2) and relative standard deviation (RSD)
As shown above, the range of linearity of Ractopamine is that the range of linearity of 10-1000 μ g/L, CLEN, EP and DA are
The range of linearity of 100-1000 μ g/L, TER are 200-1000 μ g/L.The range of detection limit is in 0.074-0.25 μ g/L.Opposite mark
Quasi- deviation range is 2.79-4.34%.It is above-mentioned statistics indicate that this novel method is to β2Adrenoceptor agonists
It is showed in detection good.
Following table shows the mark-on of pork sample results, it can be seen that beta-2-adrenoreceptor agonists are dense at two
The recovery of standard addition spent under gradient is respectively 86.32%-96.95% and 87.84%-95.73%.Relative standard deviation range exists
2.67%-5.72%.The result shows that testing result of this method for beta-2-adrenoreceptor agonists in complex sample
It is accurate believable.
The β of pork sample2Adrenoceptor agonists recovery of standard addition average value (n=3)
This experiment establishes the extraction of AAO-MIP nanotube films and is combined with HPLC, examines for beta-2-adrenoreceptor agonists
The method of survey.(HPLC chromatogram (a) 200ug/ β 2- adrenal gland of the mark-on sample at 223nm is denoted in figure as shown in Figure 1
The mixed liquor of plain receptor stimulating agent titer and pork sample extracting solution, (b) after AAO-MIPs membrane extraction, then desorb plus
Mark sample liquid.Note: (1) Clenbuterol (2) Terbutaline (3) adrenaline (4) Ractopamine), Ractopamine it is linear
Range is 10-1000 μ g/L, and the range of linearity of Clenbuterol, adrenaline and dopamine is 100-1000 μ g/L, Terbutaline
The range of linearity be 200-1000 μ g/L.The range of detection limit is in 0.074-0.25 μ g/L.RSD range is 2.79-4.34%.This
Method is successfully applied to β in the pork sample of mark-on2The detection of adrenoceptor agonists, under two concentration gradients
Recovery of standard addition is respectively 86.32%-96.95% and 87.84%-95.73%.RSD range is in 2.67%-5.72%.As a result
The method for showing that this experiment is established can be to β in pork sample2Adrenoceptor agonists realize effectively detection.
It is aobvious for those skilled in the art by the disclosure although the present invention is described in detail referring to above example
And be clear to, and in the case where not departing from the principle of the present invention and scope that the claim limits, it can be right
The present invention makes a variety of changes or modifies.Therefore, the detailed description of the embodiment of the present disclosure is only used to explain, rather than is used to limit
The present invention, but it is defined by the subject-matter of the claims the range of protection.
Claims (2)
1. being used for the analysis test method of Ractopamine, which is characterized in that steps are as follows:
A, pellumina is subjected to the processing of ATRP initiator surface modification；
B, the method ractopamine synthesis of atom transfer radical polymerization is utilized on pellumina, prepares AAO-MIP nanometers
Periosteum；
C, pork sample is prepared；
D, the Ractopamine of 2.0 μ g/g is added in pork sample, the Ractopamine analog of 20 μ g/g is added, it is described
Ractopamine analog is clenbuterol, Terbutaline, adrenaline or dopamine；
E, 10~20mL acetonitrile is added in pork sample and is saturated hexane solution；
F, the solution of potassium carbonate of 1~3mL 4mol/L is added in pork sample, makes protein denaturation, takes out fatty acid and water；
G, acetonitrile solution, solution of potassium carbonate are mixed with pork sample, removes the fat in pork sample, mixture is made；
H, mixture is acutely shaken, the concussion time is 5~10 minutes, and acetonitrile layer is precipitated；
I, 20~25mL is added and is saturated NaCl solution, emulsified with preventing from being mixed object；
J, it by said mixture at 3~8 DEG C, is centrifuged 20 minutes in 5000 turns of centrifuge per minute, isolates acetonitrile phase；
K, by the mixture residue Na after separating acetonitrile2SO4It is dried；
L, the mixture residue after drying is washed with the acetonitrile of 2~5mL；
M, the acetonitrile in mixture residue is evaporated, then is dissolved with 5~10mL methanol and AAO-MIP nanotube films is used to extract,
AAO-MIP nanotube films use methanol: acetic acid (v/v)=9:1 is as eluted template；
N, sample residue extracted is detected with HPLC, analyzes the Ractopamine in sample and the like reference substance
Content.
2. the analysis test method according to claim 1 for Ractopamine, which is characterized in that in step N, institute
It states Ractopamine to be detected with fluorescence detector, the clenbuterol, Terbutaline, adrenaline or dopamine are using ultraviolet
Photodetector detection.
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| CN106383193A (en) * | 2016-11-22 | 2017-02-08 | 无锡艾科瑞思产品设计与研究有限公司 | Detection method for phenylethanolamine in pork |
| CN107179357A (en) * | 2017-03-23 | 2017-09-19 | 苏州农业职业技术学院 | The detection method of antibiotic residue in poultry meat |
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| CN104910339A (en) * | 2015-06-23 | 2015-09-16 | 嘉兴学院 | Magnetic molecular imprinting polyion liquid for detecting ractopamine as well as preparation method and application of magnetic molecular imprinting polyion liquid |
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