CN104807918B - The method of the Ractopamine content in high-acruracy survey pig urine - Google Patents
The method of the Ractopamine content in high-acruracy survey pig urine Download PDFInfo
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- CN104807918B CN104807918B CN201510243042.2A CN201510243042A CN104807918B CN 104807918 B CN104807918 B CN 104807918B CN 201510243042 A CN201510243042 A CN 201510243042A CN 104807918 B CN104807918 B CN 104807918B
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Abstract
The invention discloses the method for Ractopamine content in a kind of high-acruracy survey pig urine, comprise the following steps: step 1: sample pretreatment；Collect pig urine, step 2: after supernatant is joined SLE post, stand 2～4 minutes；Step 3: use binary eluant, eluent that SLE post is eluted at least twice, receive eluent, obtain the eluent containing Ractopamine；Step 4: the eluent using high performance liquid chromatography to obtain step 3 detects；Wherein, the described mixture that binary eluant, eluent is isopropanol and methyl acetate, wherein the weight ratio of isopropanol and methyl acetate is 0.5～1:1.It is an object of the invention to provide the method for Ractopamine content in the high-acruracy survey pig urine that a kind of separative efficiency is high, simple to operate.
Description
Technical field
The present invention relates to the detection field of animal feed, the Ractopamine content in a kind of high-acruracy survey pig urine
Method.
Background technology
Sample pre-treatments is the key link of analyte detection process, is to concentrate determined trace components, improves the sensitive of method
Degree, and remove the important means analyzing the noisy material of system.Extraction is to isolate target compound from sample collective
A technology, be one of most important method in sample pretreatment.At present, liquid-liquid extraction remains most widely used sample
One of product preparation method, but, the complex operation of traditional liquid-liquid extraction is time-consuming, organic solvent consumption big, and easily
The defect of emulsion occurs so that there is certain limitation in actual applications.
1998, scientist proposed on the basis of traditional liquid-liquid extraction, utilizes and a kind of has adsorptivity and bigger
The many empty fillers of inertia of specific surface area are as medium, the preparation method of a kind of new and effective sample of foundation-medium liquid-liquid extraction
(supported liquid extraction is called for short SLE), SLE abstraction technique selects a kind of porous inert material exactly
-calcined diatomite as medium, replaces separatory funnel, increases two active areas when contacting, it is achieved efficiently, quickly
Extraction process.
After on calcined diatomite in aqueous sample is loaded onto fixing polypropylene syringe pipe, (general through certain time
It is divided into a few minutes), aqueous phase solution is distributed in filling surface with the form of liquid film of thin layer, afterwards, by organic solvent from post
Pipe upper end adds, and occurs immediately when immiscible two-phase extracts while diatomite surface contacts, and last extract is at weight
Flow out under power effect, in the case of necessity, less pressure can be applied, and aqueous phase retains in media as well.The efflux of access
Directly solvent is dried up, after redissolution, i.e. can be used for next step and analyze detection.
The Ministry of Agriculture 1025 bulletin-11-2008 discloses beta-receptor activator multi-residue determination in pig urine, and it uses enzymolysis
Rear isopropanol-ethyl acetate extracts, and can be obtained the testing result being correlated with by internal standard method, but applies in reality
In, then detected by chromatogram after using conventional method extraction after using enzymolysis and sometimes cannot detect residual item.How to improve
The certainty of measurement of the Ractopamine content in pig urine is the problem that those skilled in the art need to consider.
Summary of the invention
It is an object of the invention to provide the Ractopamine in the high-acruracy survey pig urine that a kind of separative efficiency is high, simple to operate
The method of content.
The technical scheme is that the method for Ractopamine content in a kind of high-acruracy survey pig urine, including following step
Rapid:
Step 1: sample pretreatment；Collect pig urine, be firstly added β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out
Enzymolysis, enzymolysis adds the protein in trichloroacetic acid precipitation pig urine after terminating, the Ph scope adding 4 times of pig urine volumes is
Ammoniacal liquor-the ammonium chloride buffer of 8.0～10.0, centrifugal after eddy mixer mixing；Take the supernatant after being centrifuged；
Step 2: after supernatant joins SLE post, stands 2～4 minutes；
Step 3: use binary eluant, eluent that SLE post is eluted at least twice, receive eluent, obtain containing Ractopamine
Eluent；
Step 4: the eluent using high performance liquid chromatography to obtain step 3 detects, uses variable concentrations simultaneously
Ractopamine standard liquid draws standard regression curve, and the design parameter of described high performance liquid chromatography is:
Chromatographic column: C18 post, 150mm*3.9mmID
Column temperature: room temperature
Flowing phase: water and the mixed liquor of acetonitrile, wherein the volume ratio of water and acetonitrile is 100:12；
Detector: UV detector；
Detection wavelength: 226nm；
Sample size: 50 μ L；
Wherein, the described mixture that binary eluant, eluent is isopropanol and methyl acetate, wherein isopropanol and the weight of methyl acetate
Amount ratio is 0.5～1:1.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, described pig urine is 1ml, buffering
Solution is 4ml, and the supernatant taken in step 2 is 0.5ml, in described step 3, and wash-out every time used during wash-out
Agent is 0.5ml.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, the urine of every milliliter adds β-salt
Sour grapes alditol glycosides enzyme/aryl sulfatase 12 μ L.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, described trichloroacetic acid and the matter of pig urine
Amount ratio is 1:98～100.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, in described step 1, whirlpool mixes
The time of device mixed processing is 0.5～1.5 minute.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, in described step 1, described from
The concrete operations that the heart processes are: by the solution of blended process in centrifuge with the speed of 9000～11000 revs/min from
The heart 0.5～1.5 minutes.
In the method for the Ractopamine content in above-mentioned high-acruracy survey pig urine, described SLE post is secret Shandong section
The medium liquid-liquid extraction SLE post that skill Co., Ltd produces.
Beneficial effects of the present invention is as follows:
Use SLE method to separate the Ractopamine in pig urine, first carry out enzymolysis and make protein separate with Ractopamine, use
Cushioning liquid mixes, and then isolates Ractopamine by SLE post, and extractant is isopropanol and methyl acetate, this
The separative efficiency of invention is good, disengaging time is short, the eluent being obtained by extraction in combination with HPLC method measurement, it is possible to accurately
The content of the Ractopamine in measurement pig urine, the eluant, eluent of the present invention is compared with traditional eluant, eluent, and its certainty of measurement improves
11.28%.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme is described in further detail, but does not constitute this
Bright any restriction.
Embodiment 1
Step 1: take pig urine 1ml, be firstly added 12 μ L β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis,
Enzymolysis time is 3-4 hour, and enzymolysis adds the protein in trichloroacetic acid precipitation pig urine after terminating, the Ph adding 4ml is
Ammoniacal liquor-the ammonium chloride buffer of 9, centrifugal after eddy mixer mixing；Take the supernatant 0.5ml after being centrifuged；Trichloroacetic acid and
The mass ratio of pig urine is 1:99.
Step 2: after supernatant joins SLE post, stands 3 minutes；
Step 3: use binary eluant, eluent that SLE post is eluted 2 times, receive eluent, obtain washing containing Ractopamine
De-liquid；The eluant, eluent every time consumed is 0.5ml, and the described mixture that binary eluant, eluent is isopropanol and methyl acetate is different
The weight ratio of propyl alcohol and methyl acetate is 1:1.
Embodiment 2
Step 1: take pig urine 1ml, be firstly added 12 μ L β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis,
Enzymolysis time is 3-4 hour, and enzymolysis adds the protein in trichloroacetic acid precipitation pig urine after terminating, the Ph adding 4ml is
Ammoniacal liquor-the ammonium chloride buffer of 9, centrifugal after eddy mixer mixing；Take the supernatant 0.5ml after being centrifuged；Trichloroacetic acid and
The mass ratio of pig urine is 1:100.
Step 2: after supernatant joins SLE post, stands 3 minutes；
Step 3: use binary eluant, eluent that SLE post is eluted 2 times, receive eluent, obtain washing containing Ractopamine
De-liquid；The eluant, eluent every time consumed is 0.5ml, and the described mixture that binary eluant, eluent is isopropanol and methyl acetate is different
The weight ratio of propyl alcohol and methyl acetate is 0.8:1.
The preparation of standard sample
Weigh Ractopamine standard items (purity >=99%) 0.0100g, be placed in 1000ml volumetric flask, by weight ratio be
The solvent of the isopropanol of 1:1 and the mixture of methyl acetate dissolves constant volume, and its concentration is 10 μ g/ml；
Take Ractopamine solution that above-mentioned concentration is 10 μ g/ml in 1000ml volumetric flask, preparation become 2 μ g/L,
5 μ g/L, 8 μ g/L, 10 μ g/L, the standard sample of 20 μ g/L.
Comparative example 1
Being substantially the same with embodiment 1, different places is, in step 3, what eluant, eluent was isopropanol with ethyl acetate is mixed
Compound, its volume ratio is 40:60.
Analyze:
Detection method particularly as follows:
Instrument: LC-2010 type high performance liquid chromatograph (Japan's Shimadzu)
Chromatographic column: C18 post, 150mm*3.9mmID
Column temperature: room temperature
Flowing phase: water: acetonitrile=100:12 (volume ratio)；
Detector: UV detector；
Detection wavelength: 226nm；
Sample size: 50 μ L
Test with 2 μ g/L, 5 μ g/L, 8 μ g/L, 10 μ g/L, the standard sample of 20 μ g/L, regression equation
For Y=13531X+1.0, R2=0.9994, X are testing concentration, and Y is peak area.
Take with a pig urine 1kg, prepare 10 parts of extraction sample A according to the method for embodiment 1 and be numbered A1-A10, according to
The method of comparative example 1 is prepared 10 parts of extraction sample B and is numbered B1-B10.
According to above-mentioned detection method sample detection, obtain following testing result:
| Embodiment 1 | Concentration (μ g/L) | Comparative example 1 | Concentration (μ g/L) |
| A1 | 2.015 | B1 | 1.854 |
| A2 | 2.114 | B2 | 1.867 |
| A3 | 2.127 | B3 | 1.865 |
| A4 | 2.078 | B4 | 1.899 |
| A5 | 2.065 | B5 | 1.797 |
| A6 | 2.100 | B6 | 1.823 |
| A7 | 1.954 | B7 | 1.835 |
| A8 | 1.967 | B8 | 1.780 |
| A9 | 2.085 | B9 | 1.844 |
| A10 | 1.994 | B10 | 1.857 |
By upper table it can be seen that the extraction yield of the solution of the present invention is higher than the extraction yield of comparative example 1.
The present invention also uses liquid-liquid extraction method (LLE) to carry out extraction test, is 60:40 isopropanol and second when using volume ratio
During the mixing extractant of acetoacetic ester, it is difficult to detect if carrying out detection with high performance liquid chromatography.
The above-described presently preferred embodiments of the present invention that is only, all made in the range of the spirit and principles in the present invention any repair
Change, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (7)
1. the method for the Ractopamine content in a high-acruracy survey pig urine, it is characterised in that comprise the following steps:
Step 1: sample pretreatment；Collect pig urine, be firstly added β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out
Enzymolysis, enzymolysis adds the protein in trichloroacetic acid precipitation pig urine after terminating, adds 4 times of pigs urinate the pH scope of volumes be 8.0～
Ammoniacal liquor-the ammonium chloride buffer of 10.0, centrifugal after eddy mixer mixing；Take the supernatant after being centrifuged；
Step 2: after supernatant joins SLE post, stands 2～4 minutes；
Step 3: use binary eluant, eluent that SLE post is eluted at least twice, receive eluent, obtain containing Ractopamine
Eluent；
Step 4: the eluent using high performance liquid chromatography to obtain step 3 detects, and uses the Lay of variable concentrations simultaneously
Gram dopamine standard liquid draws standard regression curve, and the design parameter of described high performance liquid chromatography is:
Chromatographic column: C18 post, 150mm × 3.9mmID
Column temperature: room temperature
Flowing phase: water and the mixed liquor of acetonitrile, wherein the volume ratio of water and acetonitrile is 100:12；
Detector: UV detector；
Detection wavelength: 226nm；
Sample size: 50 μ L；
Wherein, the described mixture that binary eluant, eluent is isopropanol and methyl acetate, wherein isopropanol and the weight ratio of methyl acetate
It is 0.5～1:1.
The method of the Ractopamine content in high-acruracy survey pig the most according to claim 1 urine, it is characterised in that institute
The pig urine stated is 1ml, and cushioning liquid is 4ml, and the supernatant taken in step 2 is 0.5ml, in described step 3, every time
Eluant, eluent used during wash-out is 0.5ml.
The method of the Ractopamine content in high-acruracy survey pig the most according to claim 2 urine, it is characterised in that every
The urine of milliliter adds β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase 12 μ L.
The method of the Ractopamine content in high-acruracy survey pig the most according to claim 2 urine, it is characterised in that institute
The mass ratio of the trichloroacetic acid stated and pig urine is 1:98～100.
The method of the Ractopamine content in high-acruracy survey pig the most according to claim 1 urine, it is characterised in that institute
In the step 1 stated, the time of eddy mixer mixed processing is 0.5～1.5 minute.
The method of the Ractopamine content in high-acruracy survey pig the most according to claim 5 urine, it is characterised in that institute
In the step 1 stated, the concrete operations of described centrifugal treating are: by the solution of blended process in centrifuge with 9000～
The centrifugation 0.5 of 11000 revs/min～1.5 minutes.
7. according to the method for the Ractopamine content in the arbitrary described high-acruracy survey pig urine of claim 1 to 6, its feature
Being, described SLE post is the medium liquid-liquid extraction SLE post that secret Shandong Science and Technology Ltd. produces.
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Address after: 543000 198 West Ring Road, Wuzhou, the Guangxi Zhuang Autonomous Region Patentee after: WUZHOU FOOD AND DRUG INSPECTION INSTITUTE Address before: 543000 198 West Ring Road, Wuzhou, the Guangxi Zhuang Autonomous Region Patentee before: WUZHOU INSTITUTE FOR FOOD AND DRUG CONTROL, GUANGXI ZHUANG AUTONOMOUS REGION |
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Application publication date: 20150729 Assignee: Cenxi Longyuan Breeding Co.,Ltd. Assignor: WUZHOU FOOD AND DRUG INSPECTION INSTITUTE Contract record no.: X2022450000210 Denomination of invention: A method for measuring ractopamine in pig urine with high accuracy Granted publication date: 20160831 License type: Common License Record date: 20221202 |
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