CN106353437A - Method for detecting thiamphenicol - Google Patents
Method for detecting thiamphenicol Download PDFInfo
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- CN106353437A CN106353437A CN201610982203.4A CN201610982203A CN106353437A CN 106353437 A CN106353437 A CN 106353437A CN 201610982203 A CN201610982203 A CN 201610982203A CN 106353437 A CN106353437 A CN 106353437A
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- thiamphenicol
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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
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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
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Abstract
The invention discloses a method for detecting thiamphenicol, which comprises the following steps: weighing 1-5 g of a to-be-detected sample, adding 10-20 mL of ethyl acetate, mixing by vortex, ultrasonically extracting for 20 min, centrifuging for 8-20 min, collecting the supernatant, and drying the supernatant by blowing at 38-42 DEG C to obtain an extract A; adding 0.5-3 mul of methanol to dissolve the extract A, then adding 3-8 mL of deionized water to dilute and mixing by vortex, adding 3-8 mL of n-hexane, mixing by vortex, centrifuging, and discarding the supernatant to obtain an extract B; enabling the extract B to pass through a solid phase extraction column, rinsing with 3-10 mL of water, draining, adding 5-10 mL of methanol for elution, collecting eluate, drying at 38-42 DEG C by blowing with nitrogen gas, and dissolving the nitrogen gas dried residue with an acetonitrile solution, and filtering with a filter membrane to obtain a to-be-detected solution; and measuring by using liquid chromatography. The detection method disclosed by the invention has good reproducibility and high precision, is convenient in operation, and can well detect the quality of thiamphenicol.
Description
Technical field
The present invention relates to detection and analysis technical field, specifically a kind of thiamphenicol detection method.
Background technology
Thiamphenicol, for sensitive organism as the respiratory tract caused by hemophilus influenza, escherichia coli, Salmonella etc.,
Urinary tract, intestinal etc. infect.The main side effects of thiamphenicol are suppression hematopoietic functions, and irreversible aplastic occurs
Anemia.People and young animal are all very sensitive to thiamphenicol.Thiamphenicol is classified as food by European Union, China and many countries
Animal disables veterinary drug, and in animal food, regulation thiamphenicol must not detect.But, some illegal use thiamphenicols
Phenomenon very serious, have resulted in serious international influence.It is big that the detection method that thiamphenicol remains at present is broadly divided into 3
Class: microbiological assay, immunoassay and chemical method.These methods all cannot be avoided in ethyl acetate extraction process at present
The emulsion producing, can make the extraction of thiamphenicol not exclusively, and typically to avoid this problem using internal standard method at present
The detection bringing is inaccurate, but because internal standard substance is expensive, detection single sample cost can be higher.
Content of the invention
It is an object of the invention to provide the high thiamphenicol detection method of a kind of favorable reproducibility, precision, on solving
State the problem proposing in background technology.
For achieving the above object, the following technical scheme of present invention offer:
A kind of thiamphenicol detection method, comprises the following steps:
1) weigh 1-5g testing sample, add 10-20ml ethyl acetate, be vortexed and mix, supersound extraction 20min, 10000r/min
Lower centrifugation 8-20min, takes supernatant, dries up supernatant in 40 DEG C of ± 2 DEG C of nitrogen, prepared extract a；
2) add 0.5-3ml methanol dissolving extract a, be vortexed after being subsequently added into the dilution of 3-8ml deionized water and mix, add 3-
8ml normal hexane, is vortexed and mixes, and is centrifuged 3-10min, discards supernatant liquid, prepared extract b under 3000-4000r/min；
3) extract b is crossed solid-phase extraction column, then use 3-10ml water wash, drain, add 5-10ml methanol-eluted fractions, collection is washed
De- liquid, dries up in 40 DEG C of ± 2 DEG C of nitrogen, after the acetonitrile solution dissolved nitrogen air-blowing being 10-20% with the mass fraction of 1-5ml is done
Residue, membrane filtration, prepared prepare liquid；
4) utilize liquid chromatography for measuring.
As the further scheme of the present invention: filter membrane adopts 0.22 μm of microporous filter membrane.
As the further scheme of the present invention: liquid chromatography adopt methanol, the potassium dihydrogen phosphate of 0.05mol/l,
Triethylamine is mobile phase, and the potassium dihydrogen phosphate of methanol: 0.05mol/l: the mass ratio of triethylamine is 20:80:0.05,
Ph to 3.0 is adjusted with phosphoric acid after mixing.
As the further scheme of the present invention: also include preparing standard curve, using chromatograph of liquid analytical standard liquid
Chromatographic peak result and standard curve, quantitative thiamphenicol residual.
As the further scheme of the present invention: with octadecylsilane chemically bonded silica as filler；Detection wavelength is
226nm；Column temperature: 30 DEG C.
Compared with prior art, the invention has the beneficial effects as follows:
The detection method favorable reproducibility of the present invention, precision are high, easy to operate, can be good at detecting the quality of thiamphenicol,
Really ensure that clinical application safely, effectively, controlled；The present invention is applied to the quality testing of thiamphenicol.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of thiamphenicol detection method, comprise the following steps:
1) weigh 1g testing sample, add 10ml ethyl acetate, be vortexed and mix, supersound extraction 20min, it is centrifuged under 10000r/min
8min, takes supernatant, dries up supernatant in 40 DEG C of ± 2 DEG C of nitrogen, prepared extract a.
2) add 0.5ml methanol dissolving extract a, be vortexed after being subsequently added into the dilution of 3ml deionized water and mix, add
3ml normal hexane, is vortexed and mixes, and is centrifuged 3min, discards supernatant liquid, prepared extract b under 3000r/min.
3) extract b is crossed solid-phase extraction column, then use 3ml water wash, drain, add 5ml methanol-eluted fractions, collect eluting
Liquid, dries up in 40 DEG C of ± 2 DEG C of nitrogen, the residue after the acetonitrile solution dissolved nitrogen air-blowing being 10% with the mass fraction of 1ml is dry,
It is vortexed and mix, 0.22 μm of filtering with microporous membrane, prepared prepare liquid.
4) utilize liquid chromatography for measuring: liquid chromatography adopt methanol, the potassium dihydrogen phosphate of 0.05mol/l, three
Ethamine is mobile phase, and the potassium dihydrogen phosphate of methanol: 0.05mol/l: the mass ratio of triethylamine is 20:80:0.05, mixes
Ph to 3.0 is adjusted with phosphoric acid after even.With octadecylsilane chemically bonded silica as filler；Detection wavelength is 226nm；Column temperature: 30
℃.
Prepare standard curve, using chromatographic peak result and the standard curve of chromatograph of liquid analytical standard liquid, quantitative first
Hyrazin remains.
Embodiment 2
In the embodiment of the present invention, a kind of thiamphenicol detection method, comprise the following steps:
1) weigh 5g testing sample, add 20ml ethyl acetate, be vortexed and mix, supersound extraction 20min, it is centrifuged under 10000r/min
20min, takes supernatant, dries up supernatant in 40 DEG C of ± 2 DEG C of nitrogen, prepared extract a.
2) add 3ml methanol dissolving extract a, be vortexed after being subsequently added into the dilution of 8ml deionized water and mix, add 8ml
Normal hexane, is vortexed and mixes, and is centrifuged 10min, discards supernatant liquid, prepared extract b under 4000r/min.
3) extract b is crossed solid-phase extraction column, then use 10ml water wash, drain, add 10ml methanol-eluted fractions, collection is washed
De- liquid, dries up in 40 DEG C of ± 2 DEG C of nitrogen, the remnants after the acetonitrile solution dissolved nitrogen air-blowing being 20% with the mass fraction of 5ml is dry
Thing, is vortexed and mixes, 0.22 μm of filtering with microporous membrane, prepared prepare liquid.
4) utilize liquid chromatography for measuring: liquid chromatography adopt methanol, the potassium dihydrogen phosphate of 0.05mol/l, three
Ethamine is mobile phase, and the potassium dihydrogen phosphate of methanol: 0.05mol/l: the mass ratio of triethylamine is 20:80:0.05, mixes
Ph to 3.0 is adjusted with phosphoric acid after even.With octadecylsilane chemically bonded silica as filler；Detection wavelength is 226nm；Column temperature: 30
℃.
Prepare standard curve, using chromatographic peak result and the standard curve of chromatograph of liquid analytical standard liquid, quantitative first
Hyrazin remains.
Embodiment 3
In the embodiment of the present invention, a kind of thiamphenicol detection method, comprise the following steps:
1) weigh 3g testing sample, add 15ml ethyl acetate, be vortexed and mix, supersound extraction 20min, it is centrifuged under 10000r/min
10min, takes supernatant, dries up supernatant in 40 DEG C of ± 2 DEG C of nitrogen, prepared extract a.
2) add 2ml methanol dissolving extract a, be vortexed after being subsequently added into the dilution of 5ml deionized water and mix, add 5ml
Normal hexane, is vortexed and mixes, and is centrifuged 5min, discards supernatant liquid, prepared extract b under 4000r/min.
3) extract b is crossed solid-phase extraction column, then use 8ml water wash, drain, add 8ml methanol-eluted fractions, collect eluting
Liquid, dries up in 40 DEG C of ± 2 DEG C of nitrogen, the residue after the acetonitrile solution dissolved nitrogen air-blowing being 15% with the mass fraction of 3ml is dry,
It is vortexed and mix, 0.22 μm of filtering with microporous membrane, prepared prepare liquid.
4) utilize liquid chromatography for measuring: liquid chromatography adopt methanol, the potassium dihydrogen phosphate of 0.05mol/l, three
Ethamine is mobile phase, and the potassium dihydrogen phosphate of methanol: 0.05mol/l: the mass ratio of triethylamine is 20:80:0.05, mixes
Ph to 3.0 is adjusted with phosphoric acid after even.With octadecylsilane chemically bonded silica as filler；Detection wavelength is 226nm；Column temperature: 30
℃.
Prepare standard curve, using chromatographic peak result and the standard curve of chromatograph of liquid analytical standard liquid, quantitative first
Hyrazin remains.
1st, actual sample measures
Have chosen representative sterilized milk, milk powder, ice cream as practical measurement sample, by above corresponding sample treatment
Method is processed, and sample introduction measures.It is separately added into 0.5ng/ml, 1.5ng/ml, 2.5ng/ml thiamphenicol standard solution again, press
Sample treatment is processed, sample introduction.
The test liquid having prepared is utilized chromatographic peak result and the standard curve of chromatograph of liquid analytical standard liquid.
Show that in sample, quantitative detection is limited to (in terms of breast): thiamphenicol detection limit: 0.1 μ g/kg.
Add thiamphenicol standard solution to carry out determination of recovery rates from above-mentioned sample, response rate scope 62%～
Between 100%, meet the requirement of " gb/t27404-2008 Good Laboratory is feared to control specification food Physico-chemical tests ".
Prepare the positive of thiamphenicol as testing sample, retest 6 times, precision scope 0.01～
15%, meet " gb/t27404-2008 Good Laboratory is feared to control specification food Physico-chemical tests ".
Recording thiamphenicol measured value in milk powder using said method is 0.256, and in sterilized milk, thiamphenicol measured value is
0.136, in ice cream, thiamphenicol measured value is 0.108.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment only wraps
Containing an independent technical scheme, only for clarity, those skilled in the art should for this narrating mode of description
Using description as an entirety, the technical scheme in each embodiment can also form those skilled in the art through appropriately combined
Understandable other embodiment.
Claims (5)
1. a kind of thiamphenicol detection method is it is characterised in that comprise the following steps:
1) weigh 1-5g testing sample, add 10-20ml ethyl acetate, be vortexed and mix, supersound extraction 20min, 10000r/min
Lower centrifugation 8-20min, takes supernatant, dries up supernatant in 40 DEG C of ± 2 DEG C of nitrogen, prepared extract a；
2) add 0.5-3ml methanol dissolving extract a, be vortexed after being subsequently added into the dilution of 3-8ml deionized water and mix, add 3-
8ml normal hexane, is vortexed and mixes, and is centrifuged 3-10min, discards supernatant liquid, prepared extract b under 3000-4000r/min；
3) extract b is crossed solid-phase extraction column, then use 3-10ml water wash, drain, add 5-10ml methanol-eluted fractions, collection is washed
De- liquid, dries up in 40 DEG C of ± 2 DEG C of nitrogen, after the acetonitrile solution dissolved nitrogen air-blowing being 10-20% with the mass fraction of 1-5ml is done
Residue, membrane filtration, prepared prepare liquid；
4) utilize liquid chromatography for measuring.
2. thiamphenicol detection method according to claim 1 is it is characterised in that filter membrane adopts 0.22 μm of microporous filter membrane.
3. thiamphenicol detection method according to claim 1 it is characterised in that liquid chromatography adopt methanol,
The potassium dihydrogen phosphate of 0.05mol/l, triethylamine are mobile phase, and the potassium dihydrogen phosphate of methanol: 0.05mol/l: three
The mass ratio of ethamine is 20:80:0.05, adjusts ph to 3.0 with phosphoric acid after mixing.
4. thiamphenicol detection method according to claim 1, it is characterised in that also including preparing standard curve, utilizes
The chromatographic peak result of chromatograph of liquid analytical standard liquid and standard curve, quantitative thiamphenicol residual.
5. thiamphenicol detection method according to claim 1 is it is characterised in that with octadecylsilane chemically bonded silica be
Filler；Detection wavelength is 226nm；Column temperature: 30 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610982203.4A CN106353437A (en) | 2016-11-09 | 2016-11-09 | Method for detecting thiamphenicol |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610982203.4A CN106353437A (en) | 2016-11-09 | 2016-11-09 | Method for detecting thiamphenicol |
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| CN106353437A true CN106353437A (en) | 2017-01-25 |
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| CN201610982203.4A Pending CN106353437A (en) | 2016-11-09 | 2016-11-09 | Method for detecting thiamphenicol |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107843678A (en) * | 2017-12-06 | 2018-03-27 | 武汉生物工程学院 | A kind of method of Thiamphenicol content in thin-layer chromatography combined highly effective liquid chromatographic detection animal feed |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107843678A (en) * | 2017-12-06 | 2018-03-27 | 武汉生物工程学院 | A kind of method of Thiamphenicol content in thin-layer chromatography combined highly effective liquid chromatographic detection animal feed |
| CN107843678B (en) * | 2017-12-06 | 2020-04-14 | 武汉生物工程学院 | Method for detecting content of thiamphenicol in animal feed by combining thin-layer chromatography with high performance liquid chromatography |
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