CN103196885B - The detection method of clenbuterol hydrochloride in one boar hair - Google Patents
The detection method of clenbuterol hydrochloride in one boar hair Download PDFInfo
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- CN103196885B CN103196885B CN201310090410.5A CN201310090410A CN103196885B CN 103196885 B CN103196885 B CN 103196885B CN 201310090410 A CN201310090410 A CN 201310090410A CN 103196885 B CN103196885 B CN 103196885B
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Abstract
The present invention relates to the detection method of clenbuterol hydrochloride in a boar hair, the present invention is using Surface enhanced raman spectroscopy (SERS) as detection method, using nm of gold as SERS substrate, pig hair is carried out concentration and separation by pre-treatment to clenobuterol hydrochloride (clenbuterol hydrochloride) residual on hair, by Portable Raman spectrometer, obtain the SERS spectrogram of special sieve in hydrochloric acid Crow.In the pig hair that the present invention adopts, the detection method of clenobuterol hydrochloride, easy and simple to handle, can realize the quick detection to clenobuterol hydrochloride in pig hair.
Description
Technical field
The present invention relates to detection field, relate to a kind of detection method of clenbuterol hydrochloride particularly.
Background technology
Clenbuterol hydrochloride is the general designation of a class medicine with adrenocepter agonism, and also known as B-excitant, be a series ofly can play the medicines promoting lean meat growth, suppress fat meat growth, hydrochloric acid Ke Lunte is one of the most frequently used at present clenbuterol hydrochloride medication medication.Clenobuterol hydrochloride (clenbuterol, CLE) is also known as clenbuterol hydrochloride, gram heavy breathing element, and be the crystalline powder of white or off-white color, odorless, bitter, fusing point 161e, water-soluble, ethanol, is slightly soluble in acetone, is insoluble to ether.Within 1964, first in U.S.'s synthesis, Main Function is lax tracheal smooth muscle, is medically used for the treatment of asthma.American scientist in 1984 surprisingly finds clenobuterol hydrochloride to be added on pasture in feed, can obviously promote that carcass grows, and reduces carcass lipid precipitation, improves lean meat percentage.Subsequently, clenbuterol hydrochloride is widely used in aquaculture by the state such as American-European.China adds at the initial stage nineties in mixed fodder, widely applies, become the hidden danger that animal derived food/clenbuterol hydrochloride is residual after the pig industry through drug in some provinces is on probation.
Mainly containing chromatography to the detection method of clenobuterol hydrochloride residue in animal tissue in the world contains high performance liquid chromatography (HPLC), GC-MS(gas chromatography-mass spectrography) (GC-MS), liquid phase-mass spectrometry (LC-MS), thin-layered chromatography and the infrared coupling method of gas chromatography-Fourier etc. at present.But because the requirement of chromatography to instrument and equipment, operating personnel is higher, consuming time longer, often as confirming the method for inspection in testing process.Immunoassay comprises enzyme-linked immunosorbent assay, radio immunoassay and drips golden immunological technique.Because radioimmunology exists, instrument price is higher, the not tractable shortcoming of radioactive waste, applies less.When enzyme-linked immunosorbent assay detects, have false positive and occur, need to adopt high performance liquid chromatography or gas chromatography-mass spectrography to confirm.The method of current clenbuterol hydrochloride routine testing is: gather pig urine, adopt euzymelinked immunosorbent assay (ELISA) or the screening of gold test strip method, but there is false positive issue equally, and directly gather method that pig urine examination surveys and there is medicament residue cycle short problem, be difficult to realize the real-time follow-up to medicament residue situation and monitoring.
Summary of the invention
Namely object of the present invention is the method for quick providing clenbuterol hydrochloride in a boar hair, by collection in worksite pig hair, and by simple pre-treatment, can realize the quick detection to clenobuterol hydrochloride in pig hair.
Technical scheme of the present invention is as follows:
In one boar hair, the detection method of clenbuterol content, comprises the steps:
1) hair is cleared up: take 0.200g hair and add 3-5ml0.5-2 mole of alkali lye, clear up 5-15 minute for 70-90 DEG C, centrifugal, gets supernatant stand-by;
2) liquid-liquid extraction: add 8-12ml organic solvent in supernatant, carries out liquid-liquid extraction, gets subnatant after static layering, then subnatant is added 4-6ml0.05-0.2MHCl again extracts, and gets supernatant liquor, be sample extracting solution after static layering;
3) Solid-Phase Extraction: get PCX solid-phase extraction column, after water and methyl alcohol activate, sample extracting solution crosses post, with the mixed solution drip washing pillar of water and methyl alcohol volume ratio 1:1, drain extraction column, wash post and collect with 3-5% ammoniated methanol, water-bath is blown from nitrogen and (is generally blown to residue 50 μ about l to closely dry, become concentrated liquid), take out cooling;
4) test after the liquid after above-mentioned concentrating and nano metal particles mixing, obtain SERS(Surface enhanced raman spectroscopy) spectrogram.
Wherein, the alkali lye described in step 1) comprises NaOH or potassium hydroxide etc.
Wherein, centrifugal described in step 1), getting the stand-by concrete operations of supernatant is: 6000-9000 leaves heart 2-10 minute, inclines and supernatant, then adds 0.5-2 mole of alkali lye 0.8-1.2mL, and 6000-9000 leaves heart 2-10 minute, merges supernatant stand-by.
Wherein, step 2) described in organic solvent to comprise in methylene chloride, acetone, hexane a kind of.
Wherein, step 3) bath temperature is 30-60 DEG C.
Metal nanoparticle described in step 4) comprises golden nanometer particle, Nano silver grain or copper nano-particle.
Wherein, the test described in step 4) is: in the liquid after above-mentioned concentrating, add 5-30 μ l nano gold sol, mixing, and be placed on Raman sample platform, utilize Portable Raman spectrometer to detect.
Surface enhanced raman spectroscopy (SERS) technology is due to up to monomolecular detection sensitivity and be easy to obtain the advantages such as full wave vibrational spectrum and just developing into a kind of analysis detection means with wide application prospect.The present invention is using Surface enhanced raman spectroscopy (SERS) as detection method, using nano metal particles as SERS substrate, pig hair is carried out concentration and separation by pre-treatment to clenobuterol hydrochloride residual on hair, by Portable Raman spectrometer, obtain the SERS spectrogram of special sieve in hydrochloric acid Crow.Advantage of the present invention is as follows:
1, detection sample of the present invention is pig hair, because the growth cycle of hair is long, compares, have the advantage of length of traceable time with the method gathering pig urine examination survey;
2, Portable Raman spectrometer is utilized to detect, easy and simple to handle, quick;
3, possess good Impurity removal effect, Detection results is good, and detection accuracy is high, does not have false positive situation and occurs, erroneous judgement situation can be avoided in testing process to occur.
4, highly sensitive, lowest detectable limit can reach 1ppm.
Accompanying drawing explanation
Fig. 1 is the curve map of embodiment of the present invention 1CLE standard items;
Fig. 2 is the result figure of the embodiment of the present invention 2,3.Three curves are from the bottom to top respectively the spectrogram of CLE standard items, blank pig hair, pig hair to be measured.Hair sample after liquid-liquid extraction and SPE, can be detected the characteristic peak of CLE at 1280cm-1 place.
Fig. 3 is the result figure of the embodiment of the present invention 1,3, is respectively the detection figure of 500ppbCLE, pig hair blank, 1ppmCLE and 2ppmCLE from the bottom to top, illustrates that lowest detectable limit of the present invention can reach 1ppm.
Embodiment
The preparation of embodiment 1CLE standard items
Take water as solution, formulation content is the CLE standard items of 0.25ppm, 0.5ppm1ppm2ppm4ppm8ppm and 16ppm respectively, adopt B & WTEKiRaman spectrometer, 780nm exciting light, adopts instrumental quantitative analysis software to concentration production standard curve, as shown in Figure 1.It all has good linear relationship within the scope of 0-16ppm.
Embodiment 2 pig hair to be measured
1. hair is cleared up: take 0.200g hair and add 4ml1 moles of NaOH, clears up 10 minutes for 80 DEG C, and 8000 leave the heart 5 minutes.Incline and supernatant, then add 1MNaOH solution 1mL, 8000 leave the heart 5 minutes, merge supernatant stand-by.
2. liquid-liquid extraction: add 10ml methylene chloride in supernatant, carries out liquid-liquid extraction, gets subnatant after static layering, then subnatant is added 5ml0.1MHCl again extracts, and gets supernatant liquor and carry out SPE(Solid-Phase Extraction after static layering.
3. Solid-Phase Extraction: get PCX solid-phase extraction column (Tianjin Bonaaijieer Technology Co., Ltd), with 5mL water, the activation of 5mL methyl alcohol, sample extracting solution crosses post, with 5mL water, 5mL methyl alcohol: water is the mixed solution drip washing pillar of 1:1, drain extraction column 2 minutes, wash post with 4% ammoniated methanol and collect, 60 DEG C of water-baths are blown to 50ul from nitrogen, take out cooling.
4. test after the liquid after above-mentioned 50ul being concentrated and the mixing of 10ul nm of gold, obtain SERS spectrogram.
Test condition is: instrument: B & WTEKiRaman; Power: 40mw; Integral time: 10s(A), 20s
The blank pig hair of embodiment 3 (other standard method is determined as blank)
1. hair is cleared up: take 0.200g hair and add 4ml1 moles of NaOH, clears up 10 minutes for 80 DEG C, and 8000 leave the heart 5 minutes.Incline and supernatant, then add 1MNaOH solution 1mL, 8000 leave the heart 5 minutes, merge supernatant stand-by.
2. liquid-liquid extraction: add 10ml methylene chloride in supernatant, carries out liquid-liquid extraction, gets subnatant after static layering, then subnatant is added 5ml0.1MHCl again extracts, and gets supernatant liquor and carry out SPE(Solid-Phase Extraction after static layering.
3. Solid-Phase Extraction: get PCX solid-phase extraction column, with 5mL water, the activation of 5mL methyl alcohol, sample extracting solution crosses post, with 5mL water, 5mL methyl alcohol: water is the mixed solution drip washing pillar of 1:1, drains extraction column 2 minutes, washes post and collect with 4% ammoniated methanol, 60 DEG C of water-baths are blown to 50ul from nitrogen, take out cooling.
4. test after the liquid after above-mentioned 50ul being concentrated and the mixing of 10ul nm of gold, obtain SERS spectrogram.
Test condition instrument: B & WTEKiRaman; Power: 40mw; Integral time: 10s(A), 20s
The testing result of embodiment 2,3 is shown in Fig. 2.
Above are only specific embodiments of the invention, but design concept of the present invention is not limited thereto, all changes utilizing this design the present invention to be carried out to unsubstantiality, all should belong to the behavior of invading scope.
Claims (5)
1. the detection method of clenbuterol hydrochloride in a boar hair, comprises the steps:
1) hair is cleared up: take 0.200g hair and add 3-5ml0.5-2 mole of alkali lye, clear up 5-15 minute for 70-90 DEG C, 6000-9000 leaves heart 2-10 minute, incline and supernatant, add 0.5-2 mole of alkali lye 0.8-1.2mL again, 6000-9000 leaves heart 2-10 minute, merges supernatant stand-by;
2) liquid-liquid extraction: add 8-12ml organic solvent in supernatant, carries out liquid-liquid extraction, gets subnatant after static layering, then subnatant is added 4-6ml0.05-0.2MHCl again extracts, and gets supernatant liquor, be sample extracting solution after static layering; Described organic solvent comprises the one in methylene chloride, acetone, hexane;
3) Solid-Phase Extraction: get PCX solid-phase extraction column, after water and methyl alcohol activate, sample extracting solution crosses post, with the mixed solution drip washing pillar of water and methyl alcohol volume ratio 1:1, drain extraction column, wash post with 3-5% ammoniated methanol and collect, in water-bath, nitrogen blows becomes concentrated liquid to closely dry, takes out cooling;
4) test after the liquid after above-mentioned concentrating and nano metal particles mixing, obtain SERS spectrogram.
2. the detection method of clenbuterol hydrochloride in a boar hair as claimed in claim 1, is characterized in that: step 1) described in alkali lye comprise NaOH or potassium hydroxide.
3. the detection method of clenbuterol hydrochloride in a boar hair as claimed in claim 1, is characterized in that: step 3) bath temperature is 30-60 DEG C.
4. the detection method of clenbuterol hydrochloride in a boar hair as claimed in claim 1, is characterized in that: step 4) described in metal nanoparticle comprise golden nanometer particle, Nano silver grain or copper nano-particle.
5. the detection method of clenbuterol hydrochloride in a boar hair as claimed in claim 1, it is characterized in that: step 4) described in test be: toward above-mentioned concentrated after liquid in add 5-30 μ l nano gold sol, mixing, and be placed on Raman sample platform, utilize Portable Raman spectrometer to detect.
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| CN104422607A (en) * | 2013-09-05 | 2015-03-18 | 杭州优玛达生物科技有限公司 | Hair pretreatment method |
| CN103630524B (en) * | 2013-10-10 | 2016-06-08 | 杨建夫 | The detection device of the chemical substance quantitative and qualitative analysis of intelligent type automatic |
| CN103776997B (en) * | 2014-02-26 | 2016-03-02 | 南京农业大学 | A kind of method extracted from pig hair and detect cortisol |
| CN107490504B (en) * | 2017-07-19 | 2019-05-14 | 山东大学 | A kind of method of pyrroles's adduct content in measurement hair |
| CN107478496A (en) * | 2017-08-24 | 2017-12-15 | 山东省城市供排水水质监测中心 | The extraction detection method of organophosphor in a kind of water |
| CN112630207B (en) * | 2020-12-24 | 2021-12-28 | 江南大学 | Method for rapidly detecting zilpaterol residue in pork |
| CN113759041B (en) * | 2021-08-30 | 2023-04-11 | 浙江省检验检疫科学技术研究院 | Method for separating and detecting clenbuterol enantiomer residues in pig urine by ultra-high performance synthetic phase chromatography |
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| 应用表面增强拉曼光谱技术快速检测尿样中的β-***;翟福丽等;《分析化学研究报告》;20120531;第40卷(第5期);第718页第1,2.1节,第719页第2.2.1~2.2.2节 * |
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