CN104280483A - Pretreatment method for detecting chloromycetin in animal product - Google Patents
Pretreatment method for detecting chloromycetin in animal product Download PDFInfo
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Abstract
The invention belongs to the technical field of food safety, and particularly relates to a pretreatment method for detecting chloromycetin in an animal product. The pretreatment method disclosed by the invention comprises the following operations: weighing of a to-be-detected sample, extraction, cyclotron oscillation, centrifugation, purification, derivatization and the like, wherein the used apparatus comprises a centrifugal machine, an oscillator, a constant-temperature drying oven, a pressure blowing concentrator and the like. A sample solution prepared by using the pretreatment method can be directly detected by virtue of a gas chromatography-mass spectrometry method. Compared with an existing method, the pretreatment method has the advantages of being low in operation cost, high in repeatability, high in accuracy, high in sensitivity and the like, has strong operability, is expected to be popularized and applied on a large scale in the industries such as agriculture, quality inspection departments, and entry and exit inspection and quarantine, and has significant economic benefits. According to the pretreatment method, whether chloromycetin is contained in meat can be accurately detected, and the detected animal product is safe to eat and accords with the requirements of the national standard in China.
Description
Technical field
The invention belongs to technical field of food safety, be specifically related to the pre-treating method that in a kind of animal product, chloromycetin detects.
Background technology
Chloromycetin is a kind of microbiotic of high-efficiency broad spectrum, be widely used in the preventing and treating of various animal infectious disease, because chloromycetin has serious toxic and side effect, long-term trace takes in chloromycetin not only can make the multiple bacterial strain such as Escherichia coli, salmonella produce drug resistance, and can cause the imbalance of animal body normal flora, resistibility reduction, the various disease of easy infection.Chloromycetin remaining in edible animal, by food chain enrichment in human body, therefore constitutes potential hazard to the health of the mankind.Many countries and regions have been forbidden in succession or strictly restriction uses chloromycetin, on September 13rd, 1999, the Ministry of Agriculture of China has issued the notice of " animal food herbal medicine maximum residue limit(MRL) ", defines chloromycetin and must not detect in the edible tissues of all food animals.But due to chloromycetin good antimicrobial effect, cheap, the phenomenon of current illegal use happens occasionally.Therefore, set up high sensitivity, high specificity, simple and easy to do and economic residual chloromycetin analytical approach become a urgent task.
In existing detection animal product, the method for chloromycetin content comprises chloromycetin detection method and comprises microbial method, chromatography, immunoassay.Microbial method is easy to operate, and expense is low, but sensitivity is low, poor specificity; Immunoassays have highly sensitive, high specificity, to instrument and equipment and the advantage such as personnel qualifications is low and sample pre-treatments is simple, be suitable for on-site supervision and the screening of extensive sample, but its influence factor is more, easily there is false positive results, antibody batch difference, measurement result also there will be difference etc.; Chromatography is accurately reliable, highly sensitive, and detection limit can reach 0.1 μ gPkg, but pre-treatment step is many, and the recovery is on the low side, so it is low to be badly in need of a kind of running cost, possesses high reappearance, high accuracy, highly sensitive pre-treating method.
Summary of the invention
Pre-treating method of the present invention comprises the operations such as the weighing of testing sample, extraction, cyclotron oscillation, centrifugal, purification, derivatization; Instrument comprises hydro-extractor, oscillator, thermostatic drying chamber and Nitrogen evaporator etc.; The sample solution prepared with this pre-treating method can detect by direct gas chromatography-mass spectrography.This pre-treating method compared with the conventional method, there is the advantages such as low, the high reappearance of running cost, high accuracy, high sensitivity, there is very strong operability, be expected to, at industry large-scale promotion applications such as agricultural, quality testing department and inspection and quarantining for import/export, to there is significant economic benefit.The present invention accurately can detect that it is safe that the animal product after testing eats, and meets the requirement of China's national standard whether containing chloromycetin in meat.
For achieving the above object, the invention provides the pre-treating method that in a kind of animal product, chloromycetin detects, be made up of following steps:
(1) extract; Take the meat after 3 ~ 8g homogenate or eggs are placed in 100mL centrifuge tube, add 15 ~ 25mL ethyl acetate, homogeneous 0.5 ~ 1.5min, the centrifugal 3 ~ 8min of cyclotron oscillation 15 ~ 25min, 2500 ~ 3500r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 10 ~ 20mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up;
(2) purify; Add 0.5 ~ 1.5mL methyl alcohol ultrasonic dissolution residue, adding 15 ~ 25mL massfraction is successively 3% ~ 8% sodium chloride and 5 ~ 15mL normal hexane; Cyclotron oscillation 5 ~ 15min, discards normal hexane layer; Add 10 ~ 20mL ethyl acetate again, cyclotron oscillation 5 ~ 15min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 0.5 ~ 1.5mL;
(3) derivatization; Aforesaid liquid is moved in 5mL tool plug centrifuge tube, wash former centrifuge tube with 1 ~ 3mL ethyl acetate, combined ethyl acetate solution, with 50 DEG C ~ 60 DEG C vaporized nitrogens to dry; In residue, add 90 ~ 110 μ L normal hexanes successively, 90 ~ 110 μ L silylating reagents, mixing 20 ~ 40s, 110 DEG C ~ 130 DEG C reaction 20 ~ 40min in thermostatic drying chamber, nitrogen dries up; Add 0.3 ~ 0.6mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
Meat after described step (1) more preferably takes 5.0g homogenate or eggs are placed in 100mL centrifuge tube, add 20mL ethyl acetate, homogeneous 1min, the centrifugal 5min of cyclotron oscillation 20min, 3000r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 15mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up.
((2) more preferably add 1mL methyl alcohol ultrasonic dissolution residue to described step, add 20mL 5% sodium chloride and 10mL normal hexane successively; Cyclotron oscillation 10min.Discard normal hexane layer; Add 15mL ethyl acetate again, cyclotron oscillation 10min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 1mL.
Aforesaid liquid more preferably moves in 5mL tool plug centrifuge tube by described step (3), washes former centrifuge tube, combined ethyl acetate solution with 2mL ethyl acetate, with 55 DEG C of vaporized nitrogens to dry; In residue, add 100 μ L normal hexanes successively, 100 μ L silylating reagents, mixing 30s, 120 DEG C of reaction 30min in thermostatic drying chamber, nitrogen dries up; Add 0.50mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
The advantages such as that the present invention has is quantitatively accurate, highly sensitive, detection limit is low, simple to operate, analysis time is short, applied widely, can meet standard and the outlet relevant requirements of domestic food safety, compared with prior art, have following features:
1. running cost is low.Other patented technologies are generally adopt solid phase extraction method to purify, and improve testing cost.Liquid-liquid extraction method is taked in this patent innovation, under guarantee clean-up effect prerequisite, greatly reduces pre-treatment testing cost (solid-phase extraction column about 30 yuan), has broad application prospects.
2. simple to operate, analysis time is short.General personnel can grasp through simple training, and the pre-treatment time is shorter than other patented technology methods, can be more suitable for the needs meeting enterprise and all kinds of testing agency and detect fast.
3. applied widely.The advantages such as other patented technology methods are generally only applicable to chicken or pork is compared, and the technology of the present invention method the experiment proved that, can be applicable to widely comprise the animal products such as meat, eggs, pluck, applied widely.
Embodiment
For making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1:
The pre-treating method that in animal product, chloromycetin detects, is made up of following steps:
(1) extract; Take the meat after 5.0g homogenate or eggs are placed in 100mL centrifuge tube, add 20mL ethyl acetate, homogeneous 1min, the centrifugal 5min of cyclotron oscillation 20min, 3000r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 15mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up;
(2) purify; Add 1mL methyl alcohol ultrasonic dissolution residue, add 20mL 5% sodium chloride and 10mL normal hexane successively; Cyclotron oscillation 10min.Discard normal hexane layer; Add 15mL ethyl acetate again, cyclotron oscillation 10min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 1mL;
(3) derivatization; Aforesaid liquid is moved in 5mL tool plug centrifuge tube, wash former centrifuge tube with 2mL ethyl acetate, combined ethyl acetate solution, with 55 DEG C of vaporized nitrogens to dry; In residue, add 100 μ L normal hexanes successively, 100 μ L silylating reagents, mixing 30s, 120 DEG C of reaction 30min in thermostatic drying chamber, nitrogen dries up; Add 0.50mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
Embodiment 2:
The pre-treating method that in animal product, chloromycetin detects, is made up of following steps:
(1) extract; Take the meat after 3g homogenate or eggs are placed in 100mL centrifuge tube, add 25mL ethyl acetate, homogeneous 0.5min, the centrifugal 8min of cyclotron oscillation 25min, 2500r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 10mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up;
(2) purify; Add 1.5mL methyl alcohol ultrasonic dissolution residue, adding 15mL concentration is successively 8% sodium chloride and 5mL normal hexane; Cyclotron oscillation 15min, discards normal hexane layer; Add 10mL ethyl acetate again, cyclotron oscillation 15min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 0.5mL;
(3) derivatization; Aforesaid liquid is moved in 5mL tool plug centrifuge tube, wash former centrifuge tube with 3mL ethyl acetate, combined ethyl acetate solution, with 50 DEG C of vaporized nitrogens to dry; In residue, add 110 μ L normal hexanes successively, 90 μ L silylating reagents, mixing 40s, 110 DEG C of reaction 40min in thermostatic drying chamber, nitrogen dries up; Add 0.3mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
Embodiment 3:
The pre-treating method that in animal product, chloromycetin detects, is made up of following steps:
(1) extract; Take the meat after 8g homogenate or eggs are placed in 100mL centrifuge tube, add 15mL ethyl acetate, homogeneous 1.5min, the centrifugal 3min of cyclotron oscillation 15min, 3500r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 20mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up;
(2) purify; Add 0.5mL methyl alcohol ultrasonic dissolution residue, adding 25mL concentration is successively 3% sodium chloride and 15mL normal hexane; Cyclotron oscillation 5min, discards normal hexane layer; Add 20mL ethyl acetate again, cyclotron oscillation 5min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 1.5mL;
(3) derivatization; Aforesaid liquid is moved in 5mL tool plug centrifuge tube, wash former centrifuge tube with 1mL ethyl acetate, combined ethyl acetate solution, with 60 DEG C of vaporized nitrogens to dry; In residue, add 90 μ L normal hexanes successively, 110 μ L silylating reagents, mixing 20s, 130 DEG C of reaction 20min in thermostatic drying chamber, nitrogen dries up; Add 0.6mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
Below the present invention be described in detail, the above, be only the preferred embodiment of the present invention, when not limiting the scope of the present invention, namely allly does impartial change according to the application's scope and modify, all should still belong in covering scope of the present invention.
Claims (4)
1. the pre-treating method that in animal product, chloromycetin detects, is characterized in that being made up of following steps:
(1) extract; Take the meat after 3 ~ 8g homogenate or eggs are placed in 100mL centrifuge tube, add 15 ~ 25mL ethyl acetate, homogeneous 0.5 ~ 1.5min, the centrifugal 3 ~ 8min of cyclotron oscillation 15 ~ 25min, 2500 ~ 3500r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 10 ~ 20mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up;
(2) purify; Add 0.5 ~ 1.5mL methyl alcohol ultrasonic dissolution residue, adding 15 ~ 25mL massfraction is successively 3% ~ 8% sodium chloride and 5 ~ 15mL normal hexane; Cyclotron oscillation 5 ~ 15min, discards normal hexane layer; Add 10 ~ 20mL ethyl acetate again, cyclotron oscillation 5 ~ 15min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 0.5 ~ 1.5mL;
(3) derivatization; Aforesaid liquid is moved in 5mL tool plug centrifuge tube, wash former centrifuge tube with 1 ~ 3mL ethyl acetate, combined ethyl acetate solution, with 50 DEG C ~ 60 DEG C vaporized nitrogens to dry; In residue, add 90 ~ 110 μ L normal hexanes successively, 90 ~ 110 μ L silylating reagents, mixing 20 ~ 40s, 110 DEG C ~ 130 DEG C reaction 20 ~ 40min in thermostatic drying chamber, nitrogen dries up; Add 0.3 ~ 0.6mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
2. the pre-treating method that in a kind of animal product as claimed in claim 1, chloromycetin detects, it is characterized in that: described step (1) is placed in 100mL centrifuge tube for the meat after taking 5.0g homogenate or eggs, add 20mL ethyl acetate, homogeneous 1min, the centrifugal 5min of cyclotron oscillation 20min, 3000r/min; Get supernatant to proceed in another 100mL centrifuge tube, use 15mL ethyl acetate again, then extract once in sample, merge supernatant, nitrogen dries up.
3. the pre-treating method that in a kind of animal product as claimed in claim 1, chloromycetin detects, is characterized in that: described step (2), for adding 1mL methyl alcohol ultrasonic dissolution residue, adds 20mL 5% sodium chloride and 10mL normal hexane successively; Cyclotron oscillation 10min.Discard normal hexane layer; Add 15mL ethyl acetate again, cyclotron oscillation 10min, stratification, draw upper strata and proceed in 15mL tool plug centrifuge tube, nitrogen blows to 1mL.
4. the pre-treating method that in a kind of animal product as claimed in claim 1, chloromycetin detects, it is characterized in that: described step (3) is for move in 5mL tool plug centrifuge tube by aforesaid liquid, former centrifuge tube is washed with 2mL ethyl acetate, combined ethyl acetate solution, with 55 DEG C of vaporized nitrogens to dry; In residue, add 100 μ L normal hexanes successively, 100 μ L silylating reagents, mixing 30s, 120 DEG C of reaction 30min in thermostatic drying chamber, nitrogen dries up; Add 0.50mL toluene to dissolve, with 0.25 μm of filtering with microporous membrane, gained filtrate is liquid to be measured.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849119A (en) * | 2015-05-29 | 2015-08-19 | 福州大学 | Pretreatment method of residue detection of eight glucocorticoid drugs in chicken |
| CN105181875A (en) * | 2015-10-15 | 2015-12-23 | 北京市检验检疫科学技术研究院 | Method and sample pretreatment method for detecting content of chloramphenicol in silkworm faeces |
| CN105891359A (en) * | 2016-03-31 | 2016-08-24 | 广州京诚检测技术有限公司 | Detection method for residual quantity of chloramphenicol |
| CN106526012A (en) * | 2016-11-03 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection method for chloramphenicol in fodder |
| CN109406249A (en) * | 2018-11-30 | 2019-03-01 | 江苏美正生物科技有限公司 | A kind of pre-treating method of food safety medicament residue detection |
| CN111458438A (en) * | 2020-04-21 | 2020-07-28 | 深圳市深大检测有限公司 | Method for detecting content of chloramphenicol in poultry |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849119A (en) * | 2015-05-29 | 2015-08-19 | 福州大学 | Pretreatment method of residue detection of eight glucocorticoid drugs in chicken |
| CN105181875A (en) * | 2015-10-15 | 2015-12-23 | 北京市检验检疫科学技术研究院 | Method and sample pretreatment method for detecting content of chloramphenicol in silkworm faeces |
| CN105181875B (en) * | 2015-10-15 | 2017-02-01 | 北京市检验检疫科学技术研究院 | Method and sample pretreatment method for detecting content of chloramphenicol in silkworm faeces |
| CN105891359A (en) * | 2016-03-31 | 2016-08-24 | 广州京诚检测技术有限公司 | Detection method for residual quantity of chloramphenicol |
| CN106526012A (en) * | 2016-11-03 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection method for chloramphenicol in fodder |
| CN109406249A (en) * | 2018-11-30 | 2019-03-01 | 江苏美正生物科技有限公司 | A kind of pre-treating method of food safety medicament residue detection |
| CN109406249B (en) * | 2018-11-30 | 2022-10-14 | 江苏美正生物科技有限公司 | Pretreatment method for food safety drug residue detection |
| CN111458438A (en) * | 2020-04-21 | 2020-07-28 | 深圳市深大检测有限公司 | Method for detecting content of chloramphenicol in poultry |
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