CN107655984A - Nitrofuran metabolites method for detecting residue in a kind of poultry - Google Patents
Nitrofuran metabolites method for detecting residue in a kind of poultry Download PDFInfo
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Abstract
The present invention is applied to poultry medicament residue detection technique field, there is provided Nitrofuran metabolites method for detecting residue in a kind of poultry, comprises the following steps：A, standard reserving solution is prepared；B, sample liquid is extracted；C, sample liquid derivatization, extraction processing；D, Solid phase extraction；E, the sample liquid obtained in step D is subjected to liquid-phase chromatographic analysis, analysis condition is as follows：Chromatographic column：CN posts, 250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile+isopropanol+the heptane sulfonic acid sodium salt of glacial acetic acid+0.05%, volume ratio 10：10：0.1：80；Flow velocity：1.0mL/min；Detection wavelength：280nm；Column temperature：30℃；F, test substance concentration is calculated with standard curve.The present invention, by Experimental Comparison, draws optimization measure scheme, detection limit reaches 5.0ng/mL and mark-on reclaims effect is preferable in terms of the Detection wavelength of chromatographiccondition, chromatographic column, flowing are equal five.
Description
Technical field
The present invention relates to poultry medicament residue detection technique field, more particularly to Nitrofuran metabolites are residual in a kind of poultry
Stay detection method.
Background technology
Nitrofuran metabolites are the artificial synthesized extensive pedigree antibiotics with 5- nitro basic structures, because it has extensively
Spectrum antibacterial action and growth promoting function were once widely used in prevention and treatment of diseases in the cultivation industries such as livestock and poultry and aquatic products, it
Mainly include 4 kinds of furazolidone, furaltadone, nitrofurazone, furantoin etc..Wherein furazolidone and furaltadone price be just
Preferably, it is curative for effect, it is widely used in the prevention and treatment of domestic animal, the dysentery of poultry, enteritis.It is white that nitrofurazone is mainly used in poultry
The prevention and treatment of dysentery and coccidiosis of rabbit, but because toxicity is larger, it is typically used as external sterilizing medicine.Furantoin is mainly used
The prevention and treatment of diseases in aquaculture.
Show that Nitrofuran metabolites and its metabolin have very big harm by studying for a long period of time, mainly with potential cause
Cancer and induction organism produce the material of mutation.The half-life period of nitrofurans is very short, degradable in a few hours in animal body,
But its metabolite can combine closely with protein, stable residue is formed, the residence time is for several weeks, or even is steaming
Boil, toast, also can not effectively be degraded in sweet broken and microwave heating process.Therefore the metabolite of Nitrofuran metabolites is detected,
Preferable supervisory function bit can just be played.European Union 2377/90/EEC has completely forbidden itrofurans antibacterials as growth stimulator
It is used in bactericide in animal feed, European Union requirements are using metabolin as target analytes, detection nitrofuran active compound residual.Nitro
Furans metabolite is 3- amino -2- uh oxazolidinyl ketone (AOZ), 5- methyl morpholine -3- amino -2- uh oxazolidinyl ketone respectively
(AMOZ), semicarbazides (SEM), 1-aminohydantoin (AHD).
Because the chronic toxicity of Nitrofuran metabolites and countries in the world are to its disabling, its retention analysis in vivo is ground
Studying carefully turns into focus, and single residue detection development of such medicine is rapider, the analysis side of the nitrofurans residual of document report
The shortcomings of the methods of HPLC, SPE-HPLC, LC-MS in method, to determine composition single because of it, or detection sensitivity is not high without
Can meet to detect a kind of matrix whether the needs simultaneously containing four kinds of medicines, therefore development can be detected same simultaneously with a kind of method
Four kinds of medicines in matrix are then very necessary.At present, Nitrofuran metatolites method for detecting residue mainly have liquid chromatogram-
Mass spectrography and liquid chromatogram-tandem mass spectrometry etc..
Pang Guofang with 0.2mol/L hydrochloric acid hydrolyze poultry tissues in protein bound metabolites of nitrofuran, with 2- nitros
37 DEG C of benzaldehyde derivative 16 hours.After supernatant adjusts pH=7.4, with EN SPE column purifications, ethyl acetate elution, mobile phase
Constant volume.Detection limit AMOZ, AOZ are that 5 μ g/kg, SEM, AHD are respectively 10 μ g/kg and 5 μ g/kg；Rosa Draisci reports use
Nitrofurazone, furaltadone in efficient liquid phase-Diode Array Detector egg, and with liquid phase-mass-spectrometric technique it is carried out
Checking；The former report of Jiang uses nitrofuran in high performance liquid chromatography-electrospray multi-stage mass while quick, Accurate Determining honey
Metabolin.Method is that metabolites of nitrofuran passes through o-nitrobenzaldehyde derivatization in acid condition, and mark-on sample average returns
Yield reaches 64%~79%, and detection line is limited to 10 μ g/kg.
In summary, prior art there will naturally be inconvenience and defect in actual use, and it is therefore necessary to improved.
The content of the invention
For it is above-mentioned the defects of, it is an object of the invention to provide Nitrofuran metabolites residue detection side in a kind of poultry
Method, it passes through experiment pair in terms of the Detection wavelength of liquid-phase chromatographic analysis condition, chromatographic column, mobile phase, flow velocity, column temperature five
Than drawing optimization measure scheme, detection limit reaches 5.0ng/mL and mark-on reclaims effect is preferable.
To achieve these goals, the present invention provides Nitrofuran metabolites method for detecting residue in a kind of poultry, including
Following steps：
A, standard reserving solution is prepared；
Weigh Furaxone metabolite, AMOZ, Furacilin metabolite and Cistofuran metabolite standard
Each 10mg of product, add methanol dissolving and constant volume is in 100mL brown volumetric flasks, preserved at -18 DEG C；
B, sample liquid is extracted；
Weigh sample to be placed in centrifuge tube, add solids extract agent and liquid extraction agent, centrifuging and taking supernatant after mixing, weight
Multiple extraction once, merges supernatant；
C, sample liquid derivatization, extraction processing；
D, Solid phase extraction；
By C18- CN mixing decontaminating column successively with after 5mL methanol, the activation of 5mL water, add 5% methanol water-soluble liquid with
1mL/min speed crosses post, with 5mL water wash pillar and is drained after sample liquid is all excessively complete, efflux is discarded, with 2mL methanol
Elution, receive nitrogen at whole 40 DEG C of eluents and dry up；Residue 1.0mL acetonitrile dissolves, and vibration mixes, with 0.22 μm
Membrane filtration；
E, the sample liquid obtained in step D is subjected to liquid-phase chromatographic analysis, analysis condition is as follows：
Chromatographic column：CN posts, 250mm × 4.6mm, 5 μm；
Mobile phase：Acetonitrile+isopropanol+the heptane sulfonic acid sodium salt of glacial acetic acid+0.05%, volume ratio 10：10：0.1：80；
Flow velocity：1.0mL/min；
Detection wavelength：280nm；
Sample size：20μL；
Column temperature：30℃；
F, test substance concentration is calculated with standard curve.
According to Nitrofuran metabolites method for detecting residue in the poultry of the present invention, the solids extract agent is acidic oxidation
The mixed-powder of aluminium and silica gel.
According to Nitrofuran metabolites method for detecting residue in the poultry of the present invention, the liquid extraction agent is three chloroethenes
Acid-methanol solution.
It is specially according to Nitrofuran metabolites method for detecting residue, the derivatization reagent in the poultry of the present invention：Inhale
100 μ L 2- chlorobenzaldehydes are taken to be dissolved in the mixed liquor of 5mL glacial acetic acids and 20mL methanol.
The present invention provides Nitrofuran metabolites method for detecting residue in a kind of poultry, and this method needs to prepare determinand first
The standard reserving solution of matter analysis foundation as a comparison, sample liquid extraction then is carried out to sample, and the sample liquid of extraction is derived
Change, extraction processing, after the completion of above-mentioned steps, sample liquid and standard reserving solution are subjected to liquid-phase chromatographic analysis, pass through standard reserving solution
The standard curve of gained chromatogram obtains regression equation, passes through the test substance concentration of regression equation calculation sample.The present invention is right
Than prior art, derivatization treatment has been carried out in preceding processing, analyte is responded in ultra-violet (UV) band sensitiveer, and establish liquid
Analysis of hplc optimum analysis condition：Acetonitrile：Isopropanol：Glacial acetic acid：The volume ratio of 0.05% heptane sulfonic acid sodium salt is 10:
10:0.1:80；Flow velocity is 1.0mL/min；Detection wavelength：280nm.As a result show, this method is simple to operate, extraction is quick, accurate
True property is high, repeatability is strong, the analysis measure of Nitrofuran metabolites residual quantity suitable for poultry.
Brief description of the drawings
Fig. 1 is metabolites of nitrofuran standard liquid chromatogram.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The invention provides Nitrofuran metabolites method for detecting residue in a kind of poultry.This method makes a concrete analysis of process bag
Include following steps：
Step 1: prepare standard reserving solution.
Accurately weigh Furaxone metabolite, AMOZ, Furacilin metabolite and Cistofuran metabolite
Each 10mg of standard items, add methanol dissolving and constant volume is in 100mL brown volumetric flasks, preserved at -18 DEG C, be standby.
Step 2: sample liquid is extracted.
Weigh sample to be placed in centrifuge tube, add solids extract agent and liquid extraction agent, centrifuging and taking supernatant after mixing, weight
Multiple extraction once, merges supernatant.
By taking 10g samples as an example, sample is placed in centrifuge tube, solids extract agent is added and liquid extraction agent, extractant adds
For dosage depending on example weight, the addition of solids extract agent herein and liquid extraction agent is respectively 7g and 10mL, by sample with
Extractant whirlpool is mixed after ultrasonic 30min in 40 DEG C of water-baths, is then centrifuged 10min under 6000r/min rotating speeds, is taken supernatant
Liquid, 10mL liquid extraction agent is added in residue, repeat extraction once, merge supernatant.
Step 3: sample liquid derivatization, extraction processing.
Derivatization reagent is added in supernatant, is ultrasonically treated in water bath with thermostatic control, taking-up is cooled to room temperature；Then pH is adjusted
Extracted, repeated once to 7.0, and with ethyl acetate, combined ethyl acetate layer depressurizes rotary evaporation extremely in water-bath
It is dry, then add methanol aqueous solution dissolved residue.
Specifically, by taking the supernatant of 10g samples as an example, derivatization reagent 0.5mL is added, it is ultrasonic in 40 DEG C of waters bath with thermostatic control
60min, taking-up are cooled to room temperature, and pH most 7.0 is adjusted with NaOH, add the extraction of 15mL ethyl acetate, 4000r/min centrifugations
10min, ethyl acetate layer is taken out in pear shape bottle, 15mL ethyl acetate is added, repeats once, combined ethyl acetate layer
And rotary evaporated to dryness is depressurized in 35 DEG C of water-baths, add 5mL 5% methanol aqueous solution dissolved residue.
Step 4: Solid phase extraction.
By C18- CN mixing decontaminating column successively with after 5mL methanol, the activation of 5mL water, add 5% methanol water-soluble liquid with
1mL/min speed crosses post, with 5mL water wash pillar and is drained after sample liquid is all excessively complete, efflux is discarded, with 2mL methanol
Elution, receive nitrogen at whole 40 DEG C of eluents and dry up；Residue 1.0mL acetonitrile dissolves, and vibration mixes, with 0.22 μm
Membrane filtration.
Step 5: the sample liquid obtained in step 4 is carried out into liquid-phase chromatographic analysis, analysis condition is as follows：
Chromatographic column：CN posts, 250mm × 4.6mm, 5 μm.
Mobile phase：Acetonitrile+isopropanol+the heptane sulfonic acid sodium salt of glacial acetic acid+0.05%, volume ratio 10：10：0.1：80.
Flow velocity：1.0mL/min.
Detection wavelength：280nm.
Sample size：20μL.
Column temperature：30℃.
Step 6: calculate test substance concentration with standard curve.
Sample liquid injection high performance liquid chromatograph obtained by step 4 is analyzed, reference standard Regression Equations and chromatogram
Figure, ask for the test substance concentration of sample.Standard curve regression equation, coefficient correlation and the range of linearity are shown in Table 1；Chromatogram is shown in figure
1。
Also need to illustrate, the solids extract agent in step 2 is the mixed-powder of acidic alumina and silica gel；Liquid extraction
Agent is trichloroacetic acid-methanol solution；Derivatization reagent in other step 3 for 100 μ L 2- chlorobenzaldehydes and 5mL glacial acetic acids and
The mixed dissolution liquid of 20mL methanol.
It is related to preferred, the determination explanation of parameter in analysis continuous mode：
(1) Detection wavelength
By being scanned to 400ng/mL mixed standard solutions, two are carried it is determined that absorbing, respectively in 265~300nm
And 320~370nm, at 300nm, though impurity peaks are relatively fewer, sensitivity is inadequate, and AHD, SEM do not reach wanting for quantitative limit
Ask, the results showed that：The rate of recovery of 4 components and sensitivity can meet testing requirements at wavelength 280nm, therefore select
Detection wavelengths of the 280nm as this method.
(2) selection of chromatographic column and mobile phase
By the use of existing method as reference, C is compared18The separating effect of post and CN posts to four kinds of components.C18Chromatographic column
It is larger due to disturbing, it is impossible to, can be compared with by the change to mobile phase when efficiently separating 4 kinds of target components, and selecting CN posts
4 components are separated well.Primarily determine that from 10:The composition of 90 acetonitrile and 0.05% sodium heptanesulfonate as mobile phase, point
It is not highly desirable from degree, then by constantly fine setting, experiment, addition is micro to be advantageous to keep the acetic acid of pH value of solution, promotes
The isopropanol that peak shape changes, when the volume ratio of acetonitrile, isopropanol, glacial acetic acid, 0.05% heptane sulfonic acid sodium salt is 10:10:
0.1:When 80, the separating degree of standard substance is 2.13 and peak shape is it is clear that the ratio for thereby determining that mobile phase is 10:10:
0.1:80。
(3) selection of flow velocity
The size of flow velocity directly affects the pressure of chromatographic column and detecting system, the separating degree of each component, elution time, is flowing
, it is necessary to select the flow velocity of optimal separating degree and most short analysis time after dynamic phase is selected.Flow velocity is too small, and analysis time is oversize,
Chromatographic peak can be very wide；Flow velocity is too big, and the pressure of system is too high, and can cause each chromatogram overlap of peaks and be easily damaged chromatogram
Post.Therefore, this experimental selection flow velocity be respectively 0.6,0.8,1.0,1.2,1.4mL/min tested, the results showed that work as flow velocity
The requirement of experiment can sufficiently be met in 1.0mL/min.
(4) selection of column temperature
After mobile phase and flow velocity determine, the detection time of chromatographic peak can be controlled by detecting the selection of column temperature.Now with
20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C are tested, the results showed that, when detection temperature is 30 DEG C, the retention time of each component shifts to an earlier date,
And the separation between each component is not influenceed, therefore selected 30 DEG C are used as optimum detection temperature.
(5) equation of linear regression
Precision measures that standard reserving solution is appropriate, a series of standard liquid with methanol dilution into various concentrations, respectively sample introduction 2
It is secondary, linear regression is carried out to sharp side product Y with concentration of standard solution (ng/mL) X, calculation formula is:
Regression equation, coefficient correlation and the range of linearity of gained standard curve, 1 is shown in Table, gained chromatogram is shown in Fig. 1.
Regression equation, coefficient correlation and the range of linearity of the standard curve of table 1
With 3 times of signal-to-noise ratio computations, the minimum detectability for determining this method is 5.0 μ g/kg, it can thus be seen that using this method
Can be with the content of metabolites of nitrofuran in effective detection poultry.
In order to better illustrate the superiority of the present invention, experiment is measured to its average recovery and precision respectively：
(1) average recovery
Respectively using negative sample chicken as test object, sample 10g is weighed, while does parallel test, respectively in sample
In be mixed into the standard mixed solution of two kinds of concentration of 5.0 μ g/kg and 50.0 μ g/kg (1. adding 50 μ L, 2. 500 μ L concentration be 100 μ
G/mL standard reserving solution).
), it is sufficiently mixed uniformly, is tested according to the operating procedure of the present invention, measurement result and the rate of recovery are shown in Table 2, by
2 visible measurement result of table meets the requirement of medicament residue detection and analysis.
2 four kinds of Nitrofuran metatolites TIANZHU XINGNAO Capsuls of table
(2) precision
Experiment is carried out using negative chicken meat sample, when the pitch-based sphere of 4 kinds of Nitrofuran metatolites is 20 μ g/kg, even
Continuous 6 measure, RSD are respectively：AOZ 3.9%, SEM 4.8%, AMOZ 4.7%, AHD4.2%, show that stability of instrument is good
It is good.
In order to prove the dependable with function of the present invention, now by liquid chromatogram-series connection general in existing detection work
Mass spectrography and ELISA carry out contrast test explanation with the present invention, take negative chicken meat sample to add 4 kinds of itrofurans generations
Thank to thing, pitch-based sphere is 10 μ g/kg, using the high performance liquid chromatography (LC-UV) of the present invention with leading to existing detect in work
Liquid chromatogram-tandem mass spectrometry (LC-MS/MS) and ELISA (ELISA) are compared, the measurement result such as institute of table 3
Show.
The result of the test of the different measuring methods of table 3
It was found from measurement result, due to linear and quantitative, measurement result has different, LC-UV methods and LC-
The measurement result error of MS/MS methods is less than 3%, and the error of ELISA method and LC-MS/MS method measurement results is less than 4%, determines knot
Fruit is preferable.
In summary, the present invention provides Nitrofuran metabolites method for detecting residue in a kind of poultry, and this method needs first
The standard reserving solution analysis foundation as a comparison of test substance is prepared, sample liquid extraction then is carried out to sample, and to the sample of extraction
Liquid performs the derivatization, extraction processing, after the completion of above-mentioned steps, sample liquid and standard reserving solution is carried out into liquid-phase chromatographic analysis, passed through
The standard curve of chromatogram obtains regression equation obtained by standard reserving solution, and the test substance by regression equation calculation sample is dense
Degree.Present invention contrast prior art, has carried out derivatization treatment in preceding processing, analyte is responded in ultra-violet (UV) band cleverer
It is quick, and establish liquid-phase chromatographic analysis optimum analysis condition：Acetonitrile：Isopropanol：Glacial acetic acid：0.05% heptane sulfonic acid sodium salt
Volume ratio is 10:10:0.1:80；Flow velocity is 1.0mL/min；Detection wavelength：280nm.As a result show, this method is simple to operate,
Extraction is quick, accuracy is high, repeatability is strong, the analysis measure of Nitrofuran metabolites residual quantity suitable for poultry.
Certainly, the present invention can also have other various embodiments, ripe in the case of without departing substantially from spirit of the invention and its essence
Know those skilled in the art when can be made according to the present invention it is various it is corresponding change and deformation, but these corresponding change and become
Shape should all belong to the protection domain of appended claims of the invention.
Claims (4)
1. Nitrofuran metabolites method for detecting residue in a kind of poultry, it is characterised in that comprise the following steps：
A, standard reserving solution is prepared；
It is each to weigh Furaxone metabolite, AMOZ, Furacilin metabolite and Cistofuran metabolite standard items
10mg, add methanol dissolving and constant volume is in 100mL brown volumetric flasks, preserved at -18 DEG C；
B, sample liquid is extracted；
Weigh sample to be placed in centrifuge tube, add solids extract agent and liquid extraction agent, centrifuging and taking supernatant after mixing, repetition carries
Take once, merge supernatant；
C, sample liquid derivatization, extraction processing；
D, Solid phase extraction；
By C18- CN mixing decontaminating column with after 5mL methanol, the activation of 5mL water, adds 5% methanol water-soluble liquid with 1mL/min successively
Speed cross post, with 5mL water wash pillar and drained after sample liquid is all excessively complete, discard efflux, eluted, connect with 2mL methanol
Nitrogen at 40 DEG C of portion's eluent is received to dry up；Residue 1.0mL acetonitrile dissolves, and vibration mixes, with 0.22 μm of filter membrane mistake
Filter；
E, the sample liquid obtained in step D is subjected to liquid-phase chromatographic analysis, analysis condition is as follows：
Chromatographic column：CN posts, 250mm × 4.6mm, 5 μm；
Mobile phase：Acetonitrile+isopropanol+the heptane sulfonic acid sodium salt of glacial acetic acid+0.05%, volume ratio 10：10：0.1：80；
Flow velocity：1.0mL/min；
Detection wavelength：280nm；
Sample size：20μL；
Column temperature：30℃；
F, test substance concentration is calculated with standard curve.
2. Nitrofuran metabolites method for detecting residue in poultry according to claim 1, it is characterised in that the solid
Extractant is the mixed-powder of acidic alumina and silica gel.
3. Nitrofuran metabolites method for detecting residue in poultry according to claim 1, it is characterised in that the liquid
Extractant is trichloroacetic acid-methanol solution.
4. Nitrofuran metabolites method for detecting residue in poultry according to claim 1, it is characterised in that the derivative
Changing reagent is specially：100 μ L 2- chlorobenzaldehydes are drawn to be dissolved in the mixed liquor of 5mL glacial acetic acids and 20mL methanol.
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| CN (1) | CN107655984A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680410A (en) * | 2018-05-17 | 2018-10-19 | 北京和合医学诊断技术股份有限公司 | Metabolism group tissue samples processing method |
| CN115372528A (en) * | 2022-09-20 | 2022-11-22 | 北京云鹏鹏程医药科技有限公司 | Detection method for simultaneously determining multiple impurities in nitrofurantoin |
-
2017
- 2017-08-03 CN CN201710654476.0A patent/CN107655984A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680410A (en) * | 2018-05-17 | 2018-10-19 | 北京和合医学诊断技术股份有限公司 | Metabolism group tissue samples processing method |
| CN115372528A (en) * | 2022-09-20 | 2022-11-22 | 北京云鹏鹏程医药科技有限公司 | Detection method for simultaneously determining multiple impurities in nitrofurantoin |
| CN115372528B (en) * | 2022-09-20 | 2023-06-23 | 北京云鹏鹏程医药科技有限公司 | Detection method for simultaneously measuring various impurities in nitrofurantoin |
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