CN104820047A - Method for simultaneously separating ractopamine, clenbuterol and salbutamol in feed by SLE (supported liquid extraction) process - Google Patents

Method for simultaneously separating ractopamine, clenbuterol and salbutamol in feed by SLE (supported liquid extraction) process Download PDF

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Publication number
CN104820047A
CN104820047A CN201510238888.7A CN201510238888A CN104820047A CN 104820047 A CN104820047 A CN 104820047A CN 201510238888 A CN201510238888 A CN 201510238888A CN 104820047 A CN104820047 A CN 104820047A
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sle
ractopamine
clenbuterol
salbutamol
pig urine
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CN104820047B (en
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陈学松
陈启钊
叶小强
郭振旺
吴岚
邓水德
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Wuzhou Institutes for Food and Drug Control
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Wuzhou Institutes for Food and Drug Control
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Abstract

The invention discloses a method for simultaneously separating ractopamine, clenbuterol and salbutamol in feed by an SLE (supported liquid extraction) process, which comprises the following steps: 1. sample pretreatment: collecting pig urine, adding beta-glucuronidase hydrochloride/aryl sulphatase to perform enzymolysis, adding trichloroacetic acid after the enzymolysis finishes to precipitate proteins in the porcine urine, adding 4 times of by volume of ammonium acetate buffer solution with the pH value of 5.5-6 into the porcine urine, mixing in a swirl mixer, and centrifugating; taking the centrifugated supernate; 2. adding the supernate into an SLE column, and standing for 2-4 minutes; and 3. eluting the SLE column at least twice by a binary eluting agent, and receiving the eluate to obtain the eluate containing ractopamine, clenbuterol and salbutamol. The invention aims to provide a method for simultaneously separating ractopamine, clenbuterol and salbutamol in pig urine by an SLE process.

Description

Adopt SLE method to be separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously
Technical field
The present invention relates to animal tissue and excremental detection field, particularly a kind of SLE of employing method be separated simultaneously pig urine in Ractopamine, clenbuterol, salbutamol method.
Background technology
Sample pre-treatments is the key link of analyte detection process, is concentrated determined trace components, the sensitivity of raising method, and the important means of removing to the noisy material of analytic system.Extraction is the technology isolating target compound from sample collective, is one of most important method in sample pretreatment.At present, liquid-liquid extraction remains one of most widely used sample preparation methods, but the complex operation of traditional liquid-liquid extraction is time-consuming, organic solvent consumption is large, and easily there is the defect of emulsion, make that there is certain limitation in actual applications.
1998, scientist proposes on the basis of traditional liquid-liquid extraction, utilize a kind of how empty filler of inertia with adsorbability and larger specific surface area as medium, preparation method-medium the liquid-liquid extraction (supported liquid extraction is called for short SLE) of a kind of new and effective sample set up, SLE abstraction technique selects a kind of poriness inert material-calcined diatomite as medium exactly, replace separating funnel, increase two active areas when contacting, realize efficiently, extraction process fast.
Be loaded onto when aqueous sample after on the calcined diatomite in fixing polypropylene syringe pipe, through certain hour (being generally divided into a few minutes), aqueous phase solution is distributed in filling surface with the form of liquid film of thin layer, afterwards, organic solvent is added from column jecket upper end, occur immediately when immiscible two-phase extracts while zeyssatite surface contact, last extract flows out under gravity, in necessary situation, less pressure can be applied, and aqueous phase retains in media as well.Solvent directly dries up by the efflux of access, after redissolution, namely can be used for next step and analyzes detection.
No. 1025, Ministry of Agriculture bulletin-11-2008 discloses beta-receptor activator multi-residue determination in pig urine, after it adopts enzymolysis, isopropyl alcohol-ethyl acetate extracts, and the testing result of being correlated with can be obtained by internal standard method, but then detected by chromatogram after the extraction of employing classic method after in actual applications, adopting enzymolysis and sometimes cannot detect residual item.
Summary of the invention
The employing SLE method that the object of the present invention is to provide a kind of separation efficiency high, simple to operate be separated simultaneously pig urine in Ractopamine, clenbuterol, salbutamol method.
Technical scheme of the present invention is: a kind of SLE of employing method be separated simultaneously pig urine in Ractopamine, clenbuterol, salbutamol method, comprise the following steps:
Step 1: sample pretreatment; Collect pig urine, first add β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis, add the protein in trichloroacetic acid precipitation pig urine after enzymolysis terminates, the Ph scope adding 4 times of pig urine volumes is the ammonium acetate buffer of 5.5 ~ 6, centrifugal after eddy mixer mixing; Get centrifugal after supernatant;
Step 2: after supernatant being joined SLE post, leaves standstill 2 ~ 4 minutes;
Step 3: adopt binary eluant, eluent to SLE post wash-out at least twice, receive eluent, obtain the eluent containing Ractopamine, clenbuterol, salbutamol;
Wherein, described binary eluant, eluent is the potpourri of normal butyl alcohol and tetrahydrofuran, and wherein the weight ratio of normal butyl alcohol and tetrahydrofuran is 1:1 ~ 2:1.
Be separated in the method for the Ractopamine in pig urine, clenbuterol, salbutamol in above-mentioned employing SLE method simultaneously, described pig urine is 1ml, and buffer solution is 4ml, and the supernatant got in step 2 is 0.5ml, in described step 3, eluant, eluent used during each wash-out is 0.5ml.
Be separated in above-mentioned employing SLE method in the method for the Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, in the urine of every milliliter, add β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase 12 μ L.
Be separated in above-mentioned employing SLE method in the method for the Ractopamine in pig urine, clenbuterol, salbutamol, the mass ratio of described trichloroacetic acid and pig urine is 1:98 ~ 100 simultaneously.
Be separated in above-mentioned employing SLE method in the method for the Ractopamine in pig urine, clenbuterol, salbutamol, in described step 1, the time of eddy mixer hybrid processing is 0.5 ~ 1.5 minute simultaneously.
Be separated in the method for the Ractopamine in pig urine, clenbuterol, salbutamol in above-mentioned employing SLE method simultaneously, in described step 1, the concrete operations of described centrifugal treating are: by the solution through hybrid processing in centrifuges with the centrifugation 0.5 ~ 1.5 minute of 9000 ~ 11000 revs/min.
Be separated in above-mentioned employing SLE method in the method for the Ractopamine in pig urine, clenbuterol, salbutamol, described SLE post is the medium liquid-liquid extraction SLE post that secret Shandong Science and Technology Ltd. produces simultaneously.
Beneficial effect of the present invention is as follows:
Adopt Ractopamine, clenbuterol, the salbutamol in SLE method separation pig urine, first carrying out enzymolysis makes protein be separated with Ractopamine, buffer solution is adopted to mix, then Ractopamine is isolated by SLE post, extractant is normal butyl alcohol and tetrahydrofuran, separation efficiency of the present invention is good, disengaging time is short, is conducive to the measurement in later stage.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in further detail, but does not form any limitation of the invention.
Embodiment 1
Step 1: get pig urine 1g, first add 12 μ L β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis, enzymolysis time is 3-4 hour, the protein in trichloroacetic acid precipitation pig urine is added after enzymolysis terminates, the Ph adding 4ml is the ammonium acetate buffer of 5.6, centrifugal after eddy mixer mixing; Get centrifugal after supernatant 0.5ml; The mass ratio of trichloroacetic acid and pig urine is 1:99.
Step 2: after supernatant being joined SLE post, leaves standstill 3 minutes;
Step 3: adopt binary eluant, eluent to SLE post wash-out 2 times, receive eluent, obtain the eluent containing Ractopamine, clenbuterol, salbutamol; The eluant, eluent of each consumption is 0.5ml, and described binary eluant, eluent is the potpourri of normal butyl alcohol and tetrahydrofuran, and the weight ratio of isopropyl alcohol and tetrahydrofuran is 1:1.
Embodiment 2
Step 1: get pig urine 1g, first add 12 μ L β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis, enzymolysis time is 3-4 hour, the protein in trichloroacetic acid precipitation pig urine is added after enzymolysis terminates, the Ph adding 4ml is the ammonium acetate buffer of 5.8, centrifugal after eddy mixer mixing; Get centrifugal after supernatant 0.5ml; The mass ratio of trichloroacetic acid and pig urine is 1:100.
Step 2: after supernatant being joined SLE post, leaves standstill 3 minutes;
Step 3: adopt binary eluant, eluent to SLE post wash-out 2 times, receive eluent, obtain the eluent containing Ractopamine, clenbuterol, salbutamol; The eluant, eluent of each consumption is 0.5ml, and described binary eluant, eluent is the potpourri of normal butyl alcohol and tetrahydrofuran, and the weight ratio of isopropyl alcohol and tetrahydrofuran is 2:1.
The preparation of standard sample
Take Ractopamine standard items (purity >=99%) 0.0100g, be placed in 1000ml volumetric flask, be the dissolution with solvents constant volume of the normal butyl alcohol of 1:1 and the potpourri of tetrahydrofuran by weight ratio, its concentration is 10 μ g/ml;
Get above-mentioned concentration be the Ractopamine solution of 10 μ g/ml in 1000ml volumetric flask, preparation becomes the standard sample of 2 μ g/L, 5 μ g/L, 8 μ g/L, 10 μ g/L, 20 μ g/L.
Take clenbuterol standard items (purity >=99%) 0.0100g, be placed in 1000ml volumetric flask, be the dissolution with solvents constant volume of the normal butyl alcohol of 1:1 and the potpourri of tetrahydrofuran by weight ratio, its concentration is 10 μ g/ml;
Get above-mentioned concentration be the clenbuterol solution of 10 μ g/ml in 1000ml volumetric flask, preparation becomes the standard sample of 2 μ g/L, 5 μ g/L, 8 μ g/L, 10 μ g/L, 20 μ g/L.
Take salbutamol standard items (purity >=99%) 0.0100g, be placed in 1000ml volumetric flask, be the dissolution with solvents constant volume of the normal butyl alcohol of 1:1 and the potpourri of tetrahydrofuran by weight ratio, its concentration is 10 μ g/ml;
Get above-mentioned concentration be the albuterol solution of 10 μ g/ml in 1000ml volumetric flask, preparation becomes the standard sample of 2 μ g/L, 5 μ g/L, 8 μ g/L, 10 μ g/L, 20 μ g/L.
Analyze:
Detection method is specially:
Instrument: LC-2010 type high performance liquid chromatograph (Japanese Shimadzu)
Chromatographic column: C18 post, 150mm*3.9mmID
Column temperature: room temperature
Mobile phase: water: acetonitrile=80:20 (volume ratio);
Detecting device: UV detecting device;
Determined wavelength: 217nm;
Sample size: 20 μ L
Above-mentioned detection method is adopted respectively standard sample to be drawn Ractopamine respectively, clenbuterol, the regression equation of salbutamol, simultaneously, the sample of embodiment 1 is adopted to detect by above-mentioned parameter, according to the Ractopamine under 217nm condition, clenbuterol, the regression equation of salbutamol measures Ractopamine, clenbuterol, the content of salbutamol, wherein, salbutamol appearance time is 7.1min, Ractopamine appearance time is 9.3min, the appearance time of clenbuterol is 9.9min, in urine, clenbuterol content is 8.7 μ g/L, Ractopamine content is 6.1 μ g/L, salbutamol content is 3.7 μ g/L.
Above-describedly be only preferred embodiment of the present invention, all do within the scope of the spirit and principles in the present invention any amendment, equivalently to replace and improvement etc., all should be included within protection scope of the present invention.

Claims (7)

1. adopt SLE method be separated simultaneously pig urine in Ractopamine, clenbuterol, salbutamol a method, it is characterized in that, comprise the following steps:
Step 1: sample pretreatment; Collect pig urine, first add β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase and carry out enzymolysis, add the protein in trichloroacetic acid precipitation pig urine after enzymolysis terminates, the Ph scope adding 4 times of pig urine volumes is the ammonium acetate buffer of 5.5 ~ 6, centrifugal after eddy mixer mixing; Get centrifugal after supernatant;
Step 2: after supernatant being joined SLE post, leaves standstill 2 ~ 4 minutes;
Step 3: adopt binary eluant, eluent to SLE post wash-out at least twice, receive eluent, obtain the eluent containing Ractopamine, clenbuterol, salbutamol;
Wherein, described binary eluant, eluent is the potpourri of normal butyl alcohol and tetrahydrofuran, and wherein the weight ratio of normal butyl alcohol and tetrahydrofuran is 1:1 ~ 2:1.
2. employing SLE method according to claim 1 is separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, it is characterized in that, described pig urine is 1ml, buffer solution is 4ml, the supernatant got in step 2 is 0.5ml, in described step 3, eluant, eluent used during each wash-out is 0.5ml.
3. employing SLE method according to claim 2 is separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, it is characterized in that, adds β-hydrochloric acid grape alditol glycosides enzyme/aryl sulfatase 12 μ L in the urine of every milliliter.
4. employing SLE method according to claim 2 is separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, and it is characterized in that, the mass ratio of described trichloroacetic acid and pig urine is 1:98 ~ 100.
5. employing SLE method according to claim 1 is separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, and it is characterized in that, in described step 1, the time of eddy mixer hybrid processing is 0.5 ~ 1.5 minute.
6. employing SLE method according to claim 5 is separated the method for Ractopamine in pig urine, clenbuterol, salbutamol simultaneously, it is characterized in that, in described step 1, the concrete operations of described centrifugal treating are: by the solution through hybrid processing in centrifuges with the centrifugation 0.5 ~ 1.5 minute of 9000 ~ 11000 revs/min.
7. be separated the method for the Ractopamine in pig urine, clenbuterol, salbutamol according to the arbitrary described employing SLE method of claim 1 to 6, it is characterized in that, described SLE post is the medium liquid-liquid extraction SLE post that secret Shandong Science and Technology Ltd. produces simultaneously.
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