CN107121515A - A kind of method for extracting dinitolmide and its metabolite residual simultaneously from chicken tissues - Google Patents
A kind of method for extracting dinitolmide and its metabolite residual simultaneously from chicken tissues Download PDFInfo
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- CN107121515A CN107121515A CN201710480242.9A CN201710480242A CN107121515A CN 107121515 A CN107121515 A CN 107121515A CN 201710480242 A CN201710480242 A CN 201710480242A CN 107121515 A CN107121515 A CN 107121515A
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- 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
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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 present invention relates to field of veterinary drug residue detection, and in particular to a kind of method for extracting dinitolmide and its ANOT of metabolite 3 residuals simultaneously from chicken tissues.This method is to be ground chicken tissues sample and diatomite to be packed into abstraction pool, 80 DEG C, under the conditions of 1500psi, and online degreasing extracts (acetonitrile is used as extractant) with dinitolmide and its ANOT of metabolite 3 and is carried out continuously；Neutral alumina solid phase extraction column is crossed after extract processing；Eluted with methanol acetone solution；Eluent is concentrated to dryness with offline concentrating instrument negative pressure of vacuum, and methylene chloride acetone solution vortex oscillation dissolved residue is used before upper machine, and normal temperature centrifugation, supernatant liquid crosses 0.22 μm of filter membrane, and filtrate uses for analysis.The extracting method, which saves extractant, sample substrate, influences small, extraction efficiency and the rate of recovery high, and whole process is quick, convenient and automates.
Description
Technical field
The present invention relates to field of veterinary drug residue detection, and in particular to one kind from chicken tissues simultaneously extract dinitolmide and its
The method of metabolite 3-ANOT residuals.
Background technology
When the residue of veterinary drug in animal derived food is detected, only medicine is extracted from sample,
Efficient detection method may be set up.Dinitolmide is faint yellow or khaki pulverulent solids, odorless, bitter, third
Dissolved in ketone, in ethanol slightly soluble, the soluble,very slightly in chloroform or ether is insoluble in water.Its in chicken tissues metabolism production
Thing is 3- amino -5- nitro toluoyl amine (3-ANOT), and 3-ANOT is soluble in acetone, acetonitrile, first alcohol and water.Both at home and abroad on
It is mainly liquid-liquid extraction method, extractant to extract dinitolmide and its metabolite 3-ANOT extracting method simultaneously from chicken tissues
Generally phosphate buffer and acetonitrile solution, this method generally existing：Emulsion, weight as a result easily occur in extraction process
Existing property is poor；The height of the rate of recovery depends greatly on operating personnel and grasps qualification of the technology etc..But at present
Untill, method while extract dinitolmide and its metabolite 3-ANOT residual is followed the example of from chicken tissues using accelerated solvent extraction
At home and abroad there is not been reported.Because accelerated solvent extraction is followed the example of with solvent load is few, rate of extraction is fast, efficiency high, the rate of recovery
The advantages of high, favorable reproducibility and high automaticity, surname extraction, ultrasonic extraction are may replace, (separatory funnel) is shaken by hand, is boiled
Boiling method and other conventional extraction process.So this research is made using the organic solvent acetonitrile that polarity is larger, protein precipitation effect is good
For extractant, under suitable temperature, pressure condition, followed the example of using accelerated solvent extraction from chicken tissues while extracting dinitro support
The residual of amine and its metabolite 3-ANOT.
The content of the invention
To solve from chicken tissues while the problem of extracting dinitolmide and its metabolite 3-ANOT, this research is provided
It is a kind of to extract dinitolmide and its metabolite 3-ANOT new extracting method --- accelerated solvent extraction simultaneously from chicken tissues
Follow the example of.The extracting method, which saves extractant, sample substrate, influences small, extraction efficiency and the rate of recovery high, and whole process it is quick,
Convenient and automation.
Efficiently to extract the residual of dinitolmide and its metabolite 3-ANOT in chicken tissues simultaneously, this research is molten to accelerating
The extraction temperature and static extracting number of times of agent abstraction instrument are optimized.
The technical scheme is that：
A kind of method for extracting dinitolmide and its metabolite residual simultaneously from chicken tissues, comprises the following steps：
(1) take the chicken tissues sample after homogeneous to be ground with diatomite and be packed into abstraction pool, 80 DEG C, 1500psi conditions
Under, online degreasing is carried out continuously with dinitolmide and its metabolite 3-ANOT extractions；The extraction is used as extractant using acetonitrile.
(2) extract is blown to dry with dry bath nitrogen evaporator nitrogen, is dissolved with dichloromethane-acetone solution, as sample solution, excessively in
Property aluminum oxide solid phase extraction column；
(3) solid phase extraction column is first balanced with acetone, the activation of dichloromethane-acetone solution successively, and sample solution is treated after loading
After at the uniform velocity draining off, then the polypropylene centrifuge tube rinsed with dichloromethane-acetone solution after loading, and insert solid phase as leacheate
In the sample cell for extracting pillar, it is set at the uniform velocity all to flow out, it is final to be eluted with methanol-acetone solution after after pillar drying.
(4) eluent is concentrated to dryness with offline concentrating instrument negative pressure of vacuum, is vortexed and shaken with dichloromethane-acetone solution before upper machine
Dissolved residue is swung, normal temperature centrifugation, supernatant liquid crosses 0.22 μm of filter membrane, and filtrate uses for analysis.
In the inventive method, the online degreasing n-hexane is used as grease removal agent.
Wherein, in step (2) dichloromethane-acetone the ︰ 2 of volume ratio 8；The volume ratio of dichloromethane-acetone in step (3)
7 ︰ 3；The ︰ 2 of volume ratio 8 of methanol-acetone；The ︰ 2 of volume ratio 8 of dichloromethane-acetone in step (4).
The filtrate obtained by the inventive method only uses for gas-chromatography-tandem mass spectrometry (GC-MS/MS) analysis.
The present invention compare different extractants (acetonitrile, acetoneand ethyl acetate), Different Extraction Method (liquid-liquid extraction method and
Accelerated solvent extraction is followed the example of) influence (referring to Tables 1 and 2) to extraction recovery, final choice acetonitrile solution is extractant, is accelerated
Solvent extraction extracts dinitolmide and its metabolite 3-ANOT residuals in chicken tissues.Dinitolmide and its metabolite 3-
ANOT extraction recovery reaches more than 81.96%.Refer to table 4 and table 5.
Brief description of the drawings
Fig. 1 is influence of the temperature to dinitolmide extraction efficiency in chicken muscle.
Fig. 2 is influence of the temperature to 3-ANOT extraction efficiencies in chicken muscle.
Embodiment
1. raising and the sample collection of experimental chicken
The 112d yellow chicken 10 (male and female half and half) in capital sea is chosen from industry Group Co., Ltd of Jiangsu Jinghai Poultry Group Co., Ltd., single cage is raised,
The complete feed without any medicine of big feed factory is raised in feeding from Yangzhou, and free water is butchered after raising 2 weeks.Butcher
Afterwards, chest muscle meat, liver, kidney, the skin+fat for examination chicken are taken respectively, are blended and are fully mixed as blank sample respectively
Product, are dispensed, and sealing is preserved in -35 DEG C of refrigerators.
The 196d yellow chicken laying hen 20 in capital sea is chosen from industry Group Co., Ltd of Jiangsu Jinghai Poultry Group Co., Ltd., single cage is raised, feeding
The complete feed without any medicine of big feed factory, free water are raised from Yangzhou.Raise after 2 weeks, it is continuous to collect 10d chickens
Egg, is placed in temporarily preserving in 4 DEG C of refrigerators.After sample collection is finished, after distinguishing shell breaking and being homogenized egg white, yolk and shell egg
Respectively as blank egg white, yolk and shell egg sample, dispense, sealing is preserved in -35 DEG C of refrigerators.
2. the optimization of Extracting temperature and extraction times
From Fig. 1 and Fig. 2, when temperature is 80 DEG C, extraction efficiency of the acetonitrile to dinitolmide in chicken muscle and 3-ANOT
Highest, impurity interference is less.Therefore the Extracting temperature of this experiment is set to 80 DEG C.In addition, this research also compares static extracting
Count the extraction efficiency of dinitolmide and its metabolite 3-ANOT in chicken tissues when being respectively 1 and 2.As a result show：Static extracting
When number of times is 2, dinitolmide and its metabolite the 3-ANOT rate of recovery are higher in chicken tissues.
3. the comparison of Different Extraction Method and Extraction solvent
As shown in Table 1, acetonitrile to dinitolmide extraction recovery (>86%) be significantly higher than ethyl acetate (>60.%) with third
Ketone (>40%).The extraction recovery that accelerated solvent extraction is followed the example of is higher than liquid-liquid extraction method.
The comparison (%) of the Different Extraction Method of table 1 and Extraction solvent to dinitolmide extraction recovery
As shown in Table 2, acetonitrile to 3-ANOT extraction recoveries (>83%) be significantly higher than ethyl acetate (>And acetone 70%)
(>60%).The extraction recovery that accelerated solvent extraction is followed the example of is higher than liquid-liquid extraction method.
The comparison (%) of the Different Extraction Method of table 2 and Extraction solvent to 3-ANOT extraction recoveries
4. the present invention is extracted and purifying step
1) the good blank tissue samples of (2.0 ± 0.02) g homogeneous are accurately weighed, are ground in mortar with 3.0g or so diatomite
It is milled to after powdery mixing, is packed into 22mL stainless steel abstraction pools；
2) under the conditions of 80 DEG C and 1500psi, each abstraction pool extraction once (5min), is gone first by n-hexane extraction
Fat is used, and collection liquid is discarded, and reuses pure acetonitrile extraction 2 times (10min), and extraction is used, and extract is collected in 60mL receiving flasks, is shifted
To 50mL polypropylene centrifuge tubes；
3) will be equipped with the 50mL of extract polypropylene centrifuge tube be placed in nitrogen on 40 DEG C of dry bath nitrogen evaporator be blown to it is dry, then
Dissolved with 2mL dichloromethane-acetone (8 ︰ 2, V/V) solution, be used as sample solution；
4) 10.0mL acetone and 10.0mL dichloromethane-acetones (7 ︰ 3, V/V) solution are used respectively successively by neutral alumina
Solid phase extraction column is activated；
5) sample solution is added into the solid phase extraction column after activation, after at the uniform velocity draining off, with 2mL dichloromethane-acetones (7 ︰
3, V/V) polypropylene centrifuge tube for the 50mL that solution is rinsed after loading, and insert as leacheate the sample cell of solid phase extraction column
In, it is at the uniform velocity all flowed out；
6) after after solid phase extraction column drying, with 5 elutions of 15mL methanol-acetones (8 ︰ 2, V/V) solution point, eluent is received
Combine in 50mL polypropylene centrifuge tubes；
7) centrifuge tube is placed in centrifuge concentrator, 40 DEG C of negative pressure of vacuum are concentrated to dryness；
8) with 2mL dichloromethane-acetone (8 ︰ 2, V/V) dissolved residue (liver specimens 16mL dichloromethane-acetone
(8 ︰ 2, V/V) dissolved residue, egg and egg yolk sample 18mL dichloromethane-acetone (8 ︰ 2, V/V) dissolved residue),
Vortex concussion mixes matrix, 12000 × g, and normal temperature centrifugation 10min, supernatant liquid crosses 0.22 μm of filter membrane, and filtrate supplies GC-MS/MS
Analysis.
5. the measure of the rate of recovery and precision
It is accurate weigh 2.0 ± 0.02g it is homogeneous after blank tissue samples in mortar, be separately added into dinitolmide and 3-
ANOT mixing reference substances working solution it is appropriate (correspondence added in chicken muscle, egg white and yolk concentration be LOQ, 1500.0,
3000.0、6000.0μg/Kg；It is the μ g/ of LOQ, 3000.0,6000.0,12000.0 that correspondence adds concentration in chicken liver and kidney
Kg；It is the μ g/Kg of LOQ, 1000.0,2000.0,4000.0 that correspondence adds concentration in cock skin skin+fat；), ground with diatomite
Mixing is packed into 22mL abstraction pools, and each pitch-based sphere setting 6 is parallel, after extracted and purified treatment (in addition to LOQ levels,
Machine liquid is respectively with bare substrate liquid dilution 50,100,40 times, liver bare substrate liquid on chicken muscle, kidney and skin+fat
10 times of dilution, egg white, yolk and shell egg respectively with bare substrate liquid dilution 50,10,10 times), in suitable gas-chromatography and matter
(using high-purity helium as carrier gas, flow velocity is 0.8mL/min, temperature programming pattern, Splitless injecting samples, sample introduction body under the conditions of analysis of spectrum
Product：1.0μL；Using electron impact ion source, full scan mode is qualitative, and Selective reaction monitoring mode is quantified), carry out GC-MS/MS
Analysis, calculates TIANZHU XINGNAO Capsul.
Withinday precision is determined：Surveyed with a few days different time with 6 repetitions of same standard curve and same instrument
The sample of fixed above-mentioned addition concentration, tries to achieve in a few days precision (in batch).
Day to day precision is determined：Not on the same day with 6 replications of different standard curves and same instrument in one week
The sample of above-mentioned addition concentration, tries to achieve precision (between batch) in the daytime.
Chromatographic column：VF-5ht(30m×0.25mmi.d.×0.1μm)；Carrier gas：High-purity helium (>99.999%, 60psi),
Carrier gas column flow rate：0.8mL/min；Temperature programming parameter：120 DEG C of holdings 1min, 20 DEG C/min rise to 240 DEG C, keep 1min, 10
DEG C/min rises to 320 DEG C, keeps 1min.Injector temperature：340℃；Shunt mode：Splitless injecting samples, bypass flow：
50.0mL/min；Valve opening after 2min (time 2min is saved in carrier gas, and flow 20mL/min is saved in carrier gas)；Do not shunt the time：
1.0min；Sampling volume：1.0μL.
Ionization pattern：Electron bombardment (EI)；Beam energy (ionization energy)：70eV；Collision gas：High-purity argon gas (>
99.999%, 40psi)；Ion source temperature：320℃；Transmission line temperature：320℃；Solvent delay：4.0min；Gathered data side
Formula：Full scan (Full Scan) mode is qualitative, and Selective reaction monitoring (Auto SRM) mode is quantitative.Dinitolmide and its metabolism
Product 3-ANOT molecular mass and mass spectrometry parameters is shown in Table 3.
The molecular weight and mass spectrometry parameters of the dinitolmide of table 3 and its metabolite 3-ANOT
Note：*Quota ion pair
By dinitolmide quota ion pair m/z118.0 ＞ 64.0,3-ANOT quota ion pairs m/ obtained by addition sample
Z178.0 ＞ 104.0 peak area substitutes into standard curve and tries to achieve concentration, is tried to achieve compared with the concentration of the analyte of actual interpolation
TIANZHU XINGNAO Capsul.
On this condition, the inventive method extracts the addition recovery of dinitolmide and its metabolite 3-ANOT in chicken tissues
Rate and precision are shown in Table 4 and table 5.
From table 4 and table 5, when dinitolmide and its metabolite 3-ANOT pitch-based spheres be respectively quantitative limit (LOQ),
When 0.5MRL (MRL), 1MRL, 2MRL, dinitolmide and its metabolite 3-ANOT's is averaged in chicken tissues sample
The rate of recovery is between 81.96%-94.31%, and relative standard deviation is between 1.72%-5.37%, in a few days relative standard
Deviation is between 1.87%-5.74%, and relative standard deviation is between 3.34%-7.36% in the daytime.
The TIANZHU XINGNAO Capsul of dinitolmide and precision (n=6) in the chicken tissues of table 4
Note：A. MRL
3-ANOT TIANZHU XINGNAO Capsul and precision (n=6) in the chicken tissues of table 5
Note：A. MRL.
Claims (3)
1. a kind of method for extracting dinitolmide and its metabolite residual simultaneously from chicken tissues, it is characterised in that including following
Step：
(1) take the chicken tissues sample after homogeneous and diatomite to be ground and be packed into abstraction pool, 80 DEG C, under the conditions of 1500psi,
Line degreasing is carried out continuously with dinitolmide and its metabolite 3-ANOT extractions；The extraction is used as extractant using acetonitrile；
(2) acetonitrile extraction liquid is blown to dry with dry bath nitrogen evaporator nitrogen, is dissolved with dichloromethane-acetone solution, as sample solution, excessively in
Property aluminum oxide solid phase extraction column；
(3) solid phase extraction column is first balanced with acetone, the activation of dichloromethane-acetone solution successively, treats sample solution at the uniform velocity after loading
After draining off, then the polypropylene centrifuge tube rinsed with dichloromethane-acetone solution after loading, and insert SPE as leacheate
In the sample cell of pillar, it is set at the uniform velocity all to flow out, it is final to be eluted with methanol-acetone solution after after pillar drying；
(4) eluent is concentrated to dryness with offline concentrating instrument negative pressure of vacuum, molten with dichloromethane-acetone solution vortex oscillation before upper machine
Residue is solved, normal temperature centrifugation, supernatant liquid crosses 0.22 μm of filter membrane, and filtrate uses for analysis.
2. according to the method described in claim 1, it is characterised in that the online degreasing n-hexane is used as grease removal agent.
3. according to the method described in claim 1, it is characterised in that the ︰ 2 of volume ratio 8 of dichloromethane-acetone in step (2)；Step
Suddenly in (3) dichloromethane-acetone the ︰ 3 of volume ratio 7；The ︰ 2 of volume ratio 8 of methanol-acetone；Dichloromethane-acetone in step (4)
The ︰ 2 of volume ratio 8.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311448A (en) * | 2014-09-21 | 2015-01-28 | 浙江汇能动物药品有限公司 | Preparation method of dinitolmide |
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- 2017-06-22 CN CN201710480242.9A patent/CN107121515A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311448A (en) * | 2014-09-21 | 2015-01-28 | 浙江汇能动物药品有限公司 | Preparation method of dinitolmide |
Non-Patent Citations (2)
| Title |
|---|
| 中华人民共和国国家质量监督检验检疫总局: "《中华人民共和国出入境检验检疫行业标准SN/T 2453-2010》", 10 January 2010 * |
| 木合他拜尔等: "QuEChERS－超高效液相色谱－高分辨串联质谱技术检测鸡肉中6种抗球虫药物", 《色谱》 * |
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Application publication date: 20170901 |