CN105319291A - Method for detecting trichlorfon chiral enantiomers in aquatic products - Google Patents
Method for detecting trichlorfon chiral enantiomers in aquatic products Download PDFInfo
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- CN105319291A CN105319291A CN201410379997.6A CN201410379997A CN105319291A CN 105319291 A CN105319291 A CN 105319291A CN 201410379997 A CN201410379997 A CN 201410379997A CN 105319291 A CN105319291 A CN 105319291A
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Abstract
The invention relates to a method for detecting trichlorfon chiral enantiomers in aquatic products. The method is characterized by including the following steps that firstly, fish tissue is weighed and minced and placed in a centrifugal machine for centrifugal separation after tissue homogenate and continuous homogenization and stands still, and supernate is obtained for use after standing; secondly, the supernate in the first step is processed with anhydrous sodium sulfate, passes C18 and PSA serial connection columns, an acetonitrile solution is added after flowing of the supernate is completed for leaching, the supernate is collected to a volumetric flask, nitrogen is blown till the supernate is nearly dried, a mobile phase of normal hexane and isopropanol is used for setting the volume to be 2 mL, the supernate passes a 0.22 organic filtering membrane to obtain a solution A to be detected, and the size proportion of normal hexane and isopropanol of the mobile phase is 85-95 to 15-5; thirdly, an efficient liquid phase chromatographic instrument is used for detecting the solution A, a CHIRALCEL IC chiral column is selected in detection, the column temperature of the chiral column is 20-35 DEEG C, the flow speed during detection is 0.7 mL/min-1.0 mL/min, and the wavelength is 207 nm. By means of the method, resolution of the trichlorfon chiral enantiomers can be successfully achieved, and the residual quantity of the trichlorfon chiral enantiomers in the aquatic products is detected.
Description
Technical field
The present invention relates to a kind of method detecting metrifonate chiral enantiomer in aquatic products.
Background technology
Metrifonate [O, O-dimethyl-(2, 2, the chloro-1-hydroxyethyl of 2-tri-) phosphonate ester] belong to chirality organophosphorus insecticide, there is an asymmetric carbon atom, a pair chiral enantiomer, it is a kind of organophosphorus pesticide of low toxicity, be widely used in fishery cultivating, it is the choice drug preventing fish endoparasite, use also more in the sea-farming of Zhejiang Province and Zhoushan sea area, but, metrifonate in seawater can lodge in cultivation fish body, even if fish before consumption also will through poach, the techniques such as immersion, but the fish residual with metrifonate are still troubling, blood cholinesterase activity decrease can be caused after metrifonate is poisoning, threaten health.
At present, many in the residue detection research of aquatic products for metrifonate raceme, but still do not remain for metrifonate enantiomorph and carry out analyzing, and so in aquatic products, the data of the risk assessment institute foundation of metrifonate medicament residue are then not exclusively true.Therefore the research of enantiomers of chiral drugs disassemble technique is carried out, selectivity for metrifonate conventional in aquaculture remains behavior and carries out systematic study, greatly will advance chiral drug progress of research used for aquiculture and application, be metrifonate enantiomer-specific structure formula below:
The present invention adopts high performance liquid chromatography Chiral Stationary Phases to detect the residual quantity of metrifonate enantiomorph in aquatic products, in breeding water body, chiral drug residue detection provides theoretical foundation.
Summary of the invention
Technical matters to be solved by this invention is the present situation for prior art, the detection method of metrifonate chiral enantiomer in a kind of aquatic products is provided, the method successfully can realize the fractionation of metrifonate chiral enantiomer, thus the residual quantity of metrifonate enantiomorph in detection aquatic products, in aquatic products, chiral drug residue detection provides theoretical foundation.
The present invention solves the problems of the technologies described above adopted technical scheme: the detection method of metrifonate chiral enantiomer in a kind of aquatic products, is characterized in that comprising the following steps:
(1) take 3.0g structure of fish muscle to rub, put into 50mL centrifuge tube, add acetonitrile 10 ~ 15mL, deionized water 5 ~ 10mL, tissue homogenate 15000 ~ 20000r/min, add 3.0g sodium chloride after 30s and continue homogeneous 30s, put into hydro-extractor afterwards with the centrifugal 5min of 3500 ~ 5000r/min, after leaving standstill, get supernatant substitute;
(2) by the supernatant in step (1) through anhydrous sodium sulfate process, get 5mL and cross C18 and PSA series connection pillar, the drip washing of 2 × 4mL acetonitrile solution is added after having flowed, nature flow velocity, is collected into 10mL volumetric flask, and nitrogen blows near dry, 2mL is settled to the mobile phase of normal hexane+isopropyl alcohol, cross 0.22 organic filter membrane, obtain liquid A to be measured, the normal hexane of mobile phase and isopropyl alcohol volume ratio are 85 ~ 95:15 ~ 5;
(3) high performance liquid chromatograph is adopted to detect above-mentioned solution A; Select CHIRALCELIC chiral column during detection, the filler of this chiral column is 5 μm of Silica Surface coating celluloses-three [3,5-dichlorophenyl carbamate]; The column temperature of described chiral column is 20 DEG C ~ 35 DEG C; Flow velocity during detection is 0.7mL/min ~ 1.0mL/min, and wavelength is 207nm.
As preferably, described step 2 and 3) mobile phase also containing ethanol, it is separated with the impurity in water sample metrifonate enantiomorph as mobile phase than for 90:8 ~ 9.5:2 ~ 0.5 with ethanol contend with normal hexane, isopropyl alcohol.
Preferred again, described step 2 and 3) the volume ratio of normal hexane, isopropyl alcohol and ethanol be 90:8.5:1.5.
Preferably, flow velocity when adopting high performance liquid chromatograph to detect described solution A in described step (3) is 1mL/min.
Preferably, when adopting high performance liquid chromatograph to detect described solution A, the column temperature of described chiral column is 25 DEG C.
Finally, described step 2) in C18 and PSA connect pillar use the drip washing of 6 ~ 8mL acetonitrile solution in advance.
Compared with prior art, the present invention is for detecting metrifonate chiral enantiomer in aquatic products, successfully achieve the fractionation of metrifonate chiral enantiomer, thus the residual quantity of metrifonate enantiomorph in aquatic products is detected, wherein, enantiomorph and be good linear relationship with its response in 0.5 μ g/g ~ 60 μ g/g concentration range, regression equation and related coefficient are respectively Y=0.462X-0.005, R
2=0.9995; Y=0.573X-0.008, R
2=0.9997, the sample average recovery is respectively 87.01% and 82.95%, and day to day precision is respectively 6.01% and 6.98%, and quantitative limit is respectively 0.33 μ g/g and 0.27 μ g/g.
Accompanying drawing explanation
Fig. 1 is the chromatogram of metrifonate in the embodiment of the present invention;
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
A detection method for metrifonate chiral enantiomer in aquatic products, comprises the following steps:
(1) take 3.0g structure of fish muscle to rub, put into 50mL centrifuge tube, add acetonitrile 10 ~ 15mL, deionized water 5 ~ 10mL, add 3.0g sodium chloride after tissue homogenate (15000 ~ 20000r/min) 30s and continue homogeneous 30s, put into hydro-extractor afterwards with the centrifugal 5min of 3500 ~ 5000r/min, after leaving standstill, get supernatant substitute;
(2) by the supernatant in step (1) through anhydrous sodium sulfate process, get 5mL and cross C18 and PSA series connection pillar (using the drip washing of 6 ~ 8mL acetonitrile solution in advance), the drip washing of 2 × 4mL acetonitrile solution is added after having flowed, nature flow velocity, be collected into 10mL volumetric flask, nitrogen blows near dry, is that 90:8.5:1.5 (v:v:v) is settled to 2ml and crosses 0.22 μm of organic filter membrane, obtains liquid A to be measured by the ratio of normal hexane, isopropyl alcohol and ethanol;
(3) high performance liquid chromatograph is adopted to detect above-mentioned solution A; Select CHIRALCELIC chiral column during detection, the filler of this chiral column is 5 μm of Silica Surface coating celluloses-three [3,5-dichlorophenyl carbamate]; The column temperature of described chiral column is 20 DEG C ~ 35 DEG C; Flow velocity during detection is 0.7mL/min ~ 1.0mL/min, and wavelength is 207nm.
As preferably, in described step (3) mobile phase, the volume ratio of normal hexane, isopropyl alcohol and absolute ethyl alcohol is 90:8.5:1.5.
In the present embodiment, the mobile phase that normal hexane and isopropyl alcohol volume ratio are 95:5,90:10 and 85:15 is used to carry out chiral resolution to metrifonate respectively in step (2 and 3), split result is as shown in table 1, can find out, along with the increase of isopropanol ratios in mobile phase, mobile phase polarity strengthens, and therefore degree of separation reduces, use the best of mobile phase to consist of normal hexane and isopropyl alcohol volume fraction be respectively 90 and 10, under this proportion of mobile phase condition, Chiral Separation is respond well.
Table 1
During owing to detecting, flow velocity difference splits effect possibility difference, the present embodiment select volume fraction be respectively 90 and 10 normal hexane and isopropyl alcohol as under the prerequisite of mobile phase, have detected the fractionation situation between enantiomorph when flow velocity is 0.7mL/min, 0.8mL/min, 0.9mL/min, 1.0mL/min respectively, split result is as shown in table 2, is separated required time the shortest when flow velocity is 1mL/min.Therefore flow velocity when the present embodiment detects is preferably 1mL/min.
Table 2
Keep other chromatographic condition constant, the present embodiment has also investigated the impact of column temperature on metrifonate enantiomorph chiral separation.Table 3 is metrifonate and interior target split result under different column temperature, can find out, along with column temperature raises, degree of separation has downtrending, but metrifonate enantiomorph and interior mark all can reach baseline separation under following temperature, because the too high meeting of column temperature makes the lost of life of chromatographic column, therefore, the present embodiment preferably close to 25 DEG C of room temperature as optimum column temperature.
Table 3
When carrying out sample determination, because in right metrifonate chromatographic peak and aquatic products, the chromatographic peak of impurity can not realize baseline separation, therefore in mobile phase, add a certain proportion of ethanol, make it realize baseline separation, result is as shown in table 4.When normal hexane: isopropyl alcohol: when ethanol is 90:8.5:1.5, right metrifonate can be separated with the magazine in aquatic products, also can ensure that experiment completes in the short period of time simultaneously.
Table 4
Get blank fish blood plasma, add metrifonate control series product respectively, be configured to be equivalent to S-(+)-trichlorfon and R-(-)-trichlorfon plasma concentration is 0.5,1,10,20,30,40,50,60 μ g/g respectively, preparation standard curve.Using the testing concentration in blood plasma as horizontal ordinate, using peak area as ordinate, obtain typical curve.S-(+)-trichlorfon is good linear relationship with its response within the scope of 0.5 μ g/g ~ 60 μ g/g, and represent concentration with X, Y represents peak area.Regression equation and the related coefficient of levo-enantiomer are respectively Y=0.462X-0.005, R
2=0.9995; R-(-)-trichlorfon concentration is good linear relationship with its response within the scope of 0.5 μ g/g ~ 60 μ g/g, and represent concentration with Y, X represents right metrifonate peak area.Regression equation and related coefficient are Y=0.573X-0.008, R
2=0.9997.In blood plasma blank sample, add the metrifonate standard solution of 0.7 μ g/g, 2 μ g/g and 8 these 3 concentration of μ g/g, carry out recovery experiment by this law.Each interpolation concentration operation repetitive 3 times, calculate the recovery and precision, the sample average recovery of S-(+)-metrifonate and R-(-)-metrifonate is respectively 87.01% and 82.95%, day to day precision is respectively 6.01% and 6.98%, and quantitative limit is respectively 0.33 μ g/g and 0.27 μ g/g.
Claims (6)
1. the detection method of metrifonate chiral enantiomer in aquatic products, is characterized in that comprising the following steps:
(1) take 3.0g structure of fish muscle to rub, put into 50mL centrifuge tube, add acetonitrile 10 ~ 15mL, deionized water 5 ~ 10mL, tissue homogenate 15000 ~ 20000r/min, add 3.0g sodium chloride after 30s and continue homogeneous 30s, put into hydro-extractor afterwards with the centrifugal 5min of 3500 ~ 5000r/min, after leaving standstill, get supernatant substitute;
(2) by the supernatant in step (1) through anhydrous sodium sulfate process, get 5mL and cross C18 and PSA series connection pillar, the drip washing of 2 × 4mL acetonitrile solution is added after having flowed, nature flow velocity, is collected into 10mL volumetric flask, and nitrogen blows near dry, 2mL is settled to the mobile phase of normal hexane+isopropyl alcohol, cross 0.22 organic filter membrane, obtain liquid A to be measured, the normal hexane of mobile phase and isopropyl alcohol volume ratio are 85 ~ 95:15 ~ 5;
(3) high performance liquid chromatograph is adopted to detect above-mentioned solution A; Select CHIRALCELIC chiral column during detection, the filler of this chiral column is 5 μm of Silica Surface coating celluloses-three [3,5-dichlorophenyl carbamate]; The column temperature of described chiral column is 20 DEG C ~ 35 DEG C; Flow velocity during detection is 0.7mL/min ~ 1.0mL/min, and wavelength is 207nm.
2. the detection method of metrifonate chiral enantiomer in aquatic products according to claim 1, it is characterized in that: described step 2 and 3) mobile phase also containing ethanol, it is separated with the impurity in water sample metrifonate enantiomorph as mobile phase than for 90:8 ~ 9.5:2 ~ 0.5 with ethanol contend with normal hexane, isopropyl alcohol.
3. the detection method of metrifonate chiral enantiomer in aquatic products according to claim 2, is characterized in that: described step 2 and 3) the volume ratio of normal hexane, isopropyl alcohol and ethanol be 90:8.5:1.5.
4. the detection method of metrifonate chiral enantiomer in aquatic products according to claim 1, is characterized in that: flow velocity when adopting high performance liquid chromatograph to detect described solution A in described step (3) is 1mL/min.
5. the detection method of metrifonate chiral enantiomer in the aquatic products according to claim 1 or 2 or 3 or 4, is characterized in that: when adopting high performance liquid chromatograph to detect described solution A, the column temperature of described chiral column is 25 DEG C.
6. in the aquatic products according to claim 1 or 2 or 3 or 4, the detection method of metrifonate chiral enantiomer, is characterized in that: described step 2) in C18 and PSA connect pillar use the drip washing of 6 ~ 8mL acetonitrile solution in advance.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115060838A (en) * | 2022-06-27 | 2022-09-16 | 云南中烟工业有限责任公司 | Method for efficiently separating lactic acid enantiomer based on column series technology |
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| CN102628844A (en) * | 2012-04-09 | 2012-08-08 | 湖南省食品药品检验研究院 | Content determining method for trichlorfon in dried fish |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102628844A (en) * | 2012-04-09 | 2012-08-08 | 湖南省食品药品检验研究院 | Content determining method for trichlorfon in dried fish |
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| CN115060838A (en) * | 2022-06-27 | 2022-09-16 | 云南中烟工业有限责任公司 | Method for efficiently separating lactic acid enantiomer based on column series technology |
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