CN101718755A - Method for measuring residual quantity of diethylstilbestrol in aquatic products by utilizing liquid chromatogram fluorescent method - Google Patents
Method for measuring residual quantity of diethylstilbestrol in aquatic products by utilizing liquid chromatogram fluorescent method Download PDFInfo
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- CN101718755A CN101718755A CN 200910223269 CN200910223269A CN101718755A CN 101718755 A CN101718755 A CN 101718755A CN 200910223269 CN200910223269 CN 200910223269 CN 200910223269 A CN200910223269 A CN 200910223269A CN 101718755 A CN101718755 A CN 101718755A
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Abstract
The invention discloses a method for measuring residual quantity of diethylstilbestrol in aquatic products by utilizing a liquid chromatogram fluorescent method, comprising the following steps: (1) weighing 100g of ground aquatic product samples in a 50mL plastic centrifuge tube, adding 15-25mL of acetic ether, homogenizing for 25-30s, centrifuging for 5-15min with the speed of 7000r/min, moving the acetic ether layer to a 100mL pear-shaped flask, utilizing 1-3mL of mixed solvent of normal hexane and acetic ether (85+15) to dissolve residue, and then stirring for 5-8min; (2) activating an SLH column by utilizing 4-6mL of normal hexane and 4-6mL of acetic ether, adding extracting solution on the SLH column, utilizing 8-12mL of mixed solution of normal hexane and acetic ether (80+20) to elute by controlling the flow rate not greater than 1.5-2.5mL/min, collecting eluent, carrying water bath at the temperature of 40-60 DEG C, and then carrying out nitrogen drying till the original state; and (3) adding 0.5-1.5mL of sulfuric acid to the centrifuge tube of nitrogen drying, then adding 7-9mL of water in the centrifuge tube, utilizing 1mL of mobile phase for metering volume so as to be measured by an efficient liquid phase chromatogram.
Description
Technical field
A kind of method of using diethylstilbestrol residual quantity in the liquid chromatography fluorescence spectrometry aquatic products.
Background technology
Diethylstilbestrol belongs to hormone medicine, is added in the feed, and the effect that promotes breed live body growths such as fish is arranged, and has in aquaculture at present widely and uses.
Diethylstilbestrol has stronger class estrogen action as a kind of chemical substance of synthetic.Through the scientific experimentation conclusive evidence, diethylstilbestrol is a kind of carcinogen, and is long-term edible, can the health of humans and animals be worked the mischief, in addition carcinogenic, so life-threatening.World's majority state has forbidden using diethylstilbestrol in feed.No. 193 bulletin regulation of China Ministry of Agriculture, by on May 15th, 2002, diethylstilbestrol and salt thereof, ester formulation stopped in all food animals and use.
To detect in all food animals whether contain the diethylstilbestrol composition in order satisfying, to need practicable detection method.
In the past, the residual detection method of diethylstilbestrol had ELLISA method, HPLC ultraviolet method, GC-MS method, LC-MS/MS method.The survey of HPLC ultraviolet method is limited to 0.25mg/kg.Though it is lower that the ELLISA method is surveyed limit, be specialty because of its method of operating is complicated with crossing, only be suitable as a kind of screening technique and use.GC-MS method and LC-MS/MS method cost costliness can't be popularized use aborning.
At present, still do not have DES (diethylstilbestrol) method for detecting residue with low cost, that the survey limit can satisfy user demand again, can not satisfy the demand that social progress and market become increasingly prosperous.
Summary of the invention
The present invention designs a kind of method of using diethylstilbestrol residual quantity in the liquid chromatography fluorescence spectrometry aquatic products, by diethylstilbestrol is carried out the sulfonation hydrolysis process, it is generated send out the reactant that fluorescence is arranged, with the high performance liquid chromatography fluorescence detector it is analyzed then, thereby obtain the sensitivity and the recovery that can compare favourably with the LC-MS/MS method, be fit to the mensuration of diethylstilbestrol residual quantity in the aquatic products.
The present invention is with low cost, method is succinct, survey limit can satisfy user demand, is adapted at promoting the use of in the production practices.
The instrument that the present invention uses is as follows:
Waters 2695 high performance liquid chromatographs are joined 2475 fluorescence detectors, supercentrifuge (LG-10A), ultrasonic cleaner, Nitrogen evaporator, Rotary Evaporators, vacuum plant and mechanical pump or the automatic Solid-Phase Extraction of ASPEC XL4 system, SLH post, SPE device, positive solid phase extraction column (SLH).
The reagent that the present invention uses is as follows:
Diethylstilbestrol standard items (99.5%, U.S. SIGMA-ALDRICH company), sulfuric acid (analyzing pure), cetab (analyzing pure), formic acid (chromatographically pure), ethyl acetate (chromatographically pure), methyl alcohol (chromatographically pure).
Chromatographic condition of the present invention is as follows:
Chromatographic column: Sunfire C184.6mm * 150mm, 5 μ m; Moving phase: A is a methyl alcohol, and B is the 2mmol/L cetab; A: B=40: 60; Flow velocity: 0.8mL/min; Column temperature: 35 ℃; Excitation wavelength: emission wavelength: sample size: 20 μ L.
Method of the present invention is as follows:
(1) takes by weighing the aquatic products sample of 10g rubbing in the 50mL plastic centrifuge tube, add ethyl acetate 15-25mL, homogeneous 25-30s, the centrifugal 5-15min of 7000r/min pipettes ethyl acetate layer in 100mL pyriform bottle, residue adds 10mL ethyl acetate again, repeat to extract once, centrifugal back combined ethyl acetate layer is lower than 1-3% in 40-50 ℃ of rotary evaporation to water cut, with 1-3mL normal hexane-ethyl acetate (85+15) mixed solvent dissolution residual substance, stirred 5-8 minute;
(2) with 4-6ml normal hexane, 4-6mL ethyl acetate activation SLH post, extract is added on the SLH post, and the control flow velocity is no more than 1.5-2.5mL/min; Use the 1-3mL normal hexane, 1-3ml normal hexane-ethyl acetate (85+15) mixed solution drip washing SLH post, with 8-12mL normal hexane-ethyl acetate (80+20) mixed solution wash-out, flow speed control is no more than 1.5-2.5mL/min, collect eluent, 40-60 ℃ of water-bath nitrogen dries up to original state;
(3) in the centrifuge tube that nitrogen dries up, add 0.5-1.5mL sulfuric acid, whirlpool mixing 0.3-0.7min, place 80-100min, add 7-9mL water in centrifuge tube, whirlpool mixing 0.3-0.7min places 25-35min, add the 4-6mL acetic acid ethyl acetate extract, extract 40-60 ℃ of water-bath nitrogen dries up the water cut to 2-4%, with 1mL moving phase constant volume, gets final product for high-performance liquid chromatogram determination.
Use when of the present invention, compound concentration is 1.0,2.0,5.0,10.0, the diethylstilbestrol standard solution of 50.0,100.0 μ g/L, by processing step of the present invention and instrument condition, can obtain the linear relationship between diethylstilbestrol concentration (Y) and the peak area (x), correlation coefficient r is greater than 0.99.Calculate greater than 3 by signal to noise ratio (S/N ratio), the detection limit of the diethylstilbestrol in the aquatic products can reach 0.1 μ g/Kg.
The present invention is with low cost, method is succinct, it is low to survey limit, be adapted at promoting the use of in the production practices, and be the technological means that needs in the market, positive social effect and social demand are arranged.
Embodiment
The present invention will be further described now to lift an example.
Embodiment:
The diethylstilbestrol residual quantity is an example in the sea cucumber to measure:
(1) takes by weighing the aquatic products sample of 10g rubbing in the 50mL plastic centrifuge tube, add ethyl acetate 20mL, homogeneous 30s, the centrifugal 10min of 7000r/min pipettes ethyl acetate layer in 100mL pyriform bottle, residue adds 10mL ethyl acetate again, repeat to extract once, centrifugal back combined ethyl acetate layer is extremely done in 45 ℃ of rotary evaporations, with 2mL normal hexane-ethyl acetate (85+15) mixed solvent dissolution residual substance, stirred 6 minutes;
(2) with 5ml normal hexane, 5mL ethyl acetate activation SLH post, extract is added on the SLH post, and the control flow velocity is no more than 2mL/min; Use the 2mL normal hexane, 2ml normal hexane-ethyl acetate (85+15) mixed solution drip washing SLH post, with 10mL normal hexane-ethyl acetate (80+20) mixed solution wash-out, flow speed control is no more than 2mL/min, collects eluent, and 50 ℃ of water-bath nitrogen dry up to original state;
(3) in the centrifuge tube that nitrogen dries up, add 1mL sulfuric acid, whirlpool mixing 0.5min, place 90min, add 8mL water in centrifuge tube, whirlpool mixing 0.5min places 30min, add the 5mL acetic acid ethyl acetate extract, 50 ℃ of water-bath nitrogen of extract dry up, and with 1mL moving phase constant volume, get final product for high-performance liquid chromatogram determination.
Example proves, adds standard solution in the sea cucumber sample, reclaims according to method of the present invention, repeats 5 times, and calculate recovery rate and precision, result show that average recovery rate was all greater than 70% when addition was 0.1～0.5 μ g/kg level; Relative standard deviation is all less than 10%.
Claims (1)
1. method of using diethylstilbestrol residual quantity in the liquid chromatography fluorescence spectrometry aquatic products is characterized in that:
(1) takes by weighing the aquatic products sample of 10g rubbing in the 50mL plastic centrifuge tube, add ethyl acetate 15-25mL, homogeneous 25-30s, the centrifugal 5-15min of 7000r/min pipettes ethyl acetate layer in 100mL pyriform bottle, residue adds 10mL ethyl acetate again, repeat to extract once, centrifugal back combined ethyl acetate layer is lower than 1-3% in 40-50 ℃ of rotary evaporation to water cut, with 1-3mL normal hexane-ethyl acetate (85+15) mixed solvent dissolution residual substance, stirred 5-8 minute;
(2) with 4-6ml normal hexane, 4-6mL ethyl acetate activation SLH post, extract is added on the SLH post, and the control flow velocity is no more than 1.5-2.5mL/min; Use the 1-3mL normal hexane, 1-3ml normal hexane-ethyl acetate (85+15) mixed solution drip washing SLH post, with 8-12mL normal hexane-ethyl acetate (80+20) mixed solution wash-out, flow speed control is no more than 1.5-2.5mL/min, collect eluent, 40-60 ℃ of water-bath nitrogen dries up to original state;
(3) in the centrifuge tube that nitrogen dries up, add 0.5-1.5mL sulfuric acid, whirlpool mixing 0.3-0.7min, place 80-100min, add 7-9mL water in centrifuge tube, whirlpool mixing 0.3-0.7min places 25-35min, add the 4-6mL acetic acid ethyl acetate extract, extract 40-60 ℃ of water-bath nitrogen dries up the water cut to 2-4%, with 1mL moving phase constant volume, gets final product for high-performance liquid chromatogram determination.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175792A (en) * | 2010-12-24 | 2011-09-07 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102183606A (en) * | 2011-03-02 | 2011-09-14 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octyl phenol and bisphenol A in water body sediment together |
| CN102359961A (en) * | 2011-08-18 | 2012-02-22 | 南京量子化工科技有限公司 | Kit for detecting estrol or beta-adrenocepter agonist compounds, its method and application |
| CN103995068A (en) * | 2014-03-26 | 2014-08-20 | 江汉大学 | Method for detecting ethinyl estradiol, diethylstilbestrol, nonylphenol and estradiol benzoate |
| CN109001311A (en) * | 2018-06-29 | 2018-12-14 | 浙江省海洋水产研究所 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products |
| CN109738542A (en) * | 2019-01-29 | 2019-05-10 | 江苏康达检测技术股份有限公司 | The detection method of diethylstilbestrol residual quantity in a kind of aquatic products |
-
2009
- 2009-11-23 CN CN 200910223269 patent/CN101718755B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175792A (en) * | 2010-12-24 | 2011-09-07 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102175792B (en) * | 2010-12-24 | 2012-04-25 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octylphenol and bisphenol A together in water environment |
| CN102183606A (en) * | 2011-03-02 | 2011-09-14 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octyl phenol and bisphenol A in water body sediment together |
| CN102183606B (en) * | 2011-03-02 | 2012-04-25 | 北京师范大学 | Method for detecting estrogen, nonyl phenol, octyl phenol and bisphenol A in water body sediment together |
| CN102359961A (en) * | 2011-08-18 | 2012-02-22 | 南京量子化工科技有限公司 | Kit for detecting estrol or beta-adrenocepter agonist compounds, its method and application |
| CN102359961B (en) * | 2011-08-18 | 2014-11-19 | 南京量子化工科技有限公司 | Kit for detecting estrol or beta-adrenocepter agonist compounds, its method and application |
| CN103995068A (en) * | 2014-03-26 | 2014-08-20 | 江汉大学 | Method for detecting ethinyl estradiol, diethylstilbestrol, nonylphenol and estradiol benzoate |
| CN109001311A (en) * | 2018-06-29 | 2018-12-14 | 浙江省海洋水产研究所 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products |
| CN109738542A (en) * | 2019-01-29 | 2019-05-10 | 江苏康达检测技术股份有限公司 | The detection method of diethylstilbestrol residual quantity in a kind of aquatic products |
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