CN107941953A - The detection method of amantadine medicament residue in a kind of veterinary drug - Google Patents
The detection method of amantadine medicament residue in a kind of veterinary drug Download PDFInfo
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- CN107941953A CN107941953A CN201711235020.7A CN201711235020A CN107941953A CN 107941953 A CN107941953 A CN 107941953A CN 201711235020 A CN201711235020 A CN 201711235020A CN 107941953 A CN107941953 A CN 107941953A
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- G—PHYSICS
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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
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Abstract
The present invention provides a kind of detection method of amantadine medicament residue in veterinary drug, the amantadine medicament residue in veterinary drug is measured with liquid chromatography tandem mass spectrometry, whens this method sample pre-treatments eliminates purification process, has the processing routine simple, quickly, the features such as accuracy rate is high is measured；Analyzed from veterinary drug source, effective guarantee aquaculture drug safety, improve Animal Food Security.
Description
Technical field
The present invention relates to technical field of analytical chemistry, the detection side of amantadine medicament residue in especially a kind of veterinary drug
Method.
Background technology
Amantadine is to be used to treating the people of influenza earliest with antiviral class medicine, is once widely used in poultry farming
In.Preventive medicine is used as in approval in 1966 in the world, curative is confirmed as on the basis of preventive medicine within 1976.Domestic 20th century
The end of the nineties, veterinary clinic was once used for the prevention and treatment of animal virosis.Since it transplants the effective animal peace of rear shortage for animals
Complete test data, amantadine are applied to veterinary clinic as antiviral drugs, and shortage is scientific and normal, safely and effectively tests number
According to not only bringing undesirable consequence to animal epidemic control for animal viral epidemic disease, and influence National Animal epidemic disease and prevent
Control policy, people's its virus drug resistance after the edible animal product with amantadine medicament residue can increase.For this reason, the Ministry of Agriculture
No. 560 bulletin of issue in 2005, clearly forbids using amantadine during animal-breeding.In actual production, especially
It is to still suffer from illegally to use phenomenon during broiler chicken and breeding layer chicken.
At present, in medicine and blood plasma amantadine detection mainly using high performance liquid chromatography (HPLC), liquid chromatogram-string
Join mass spectrography (LC-MS/MS), electrochemical process, fluorescence spectrophotometry, gas chromatography etc., while in animal blood plasma and chicken
There is related detecting method report in the animal derived foods such as liver, chicken.Amantadine in chicken gizzard is realized with LC-MS/MS methods
Measure.Residue detection of the amantadine in egg not yet establishes effective method.But in existing detection method and standard
The overwhelming majority rarely has what is analyzed from veterinary drug source all just for animal derived food.This method is by optimizing extraction and cleaning
Condition, optimizes chromatography and Mass Spectrometry Conditions, improves separating effect, can detect animal beast as qualitative, quantitative confirmation method
Amantadine residual in medicine.
The content of the invention
Detection method of the technical problems to be solved by the invention in the amantadine medicament residue in a kind of veterinary drug is provided.
In order to solve the above technical problems, the technical scheme is that：
The detection method of amantadine medicament residue in a kind of veterinary drug, with liquid chromatography-tandem mass spectrometry veterinary drug
Amantadine medicament residue, comprise the following steps that：
(1) standard working solution and the sample solution sample introduction under the conditions of the liquid chromatography-tandem mass spectrometry of setting, with mass concentration
X is abscissa, and the ratio Y of peak area is ordinate, draws 5 standard working curves, sample is carried out with standard working curve
It is quantitative, in sample solution the response of medicine should in the range of linearity that instrument detects, wherein, set liquid chromatogram-string
It is as follows to join Mass Spectrometry Conditions：
1. liquid phase chromatogram condition
Chromatographic column：Hypersil Gold C18,100mm×2.1mm,1.9μm
Mobile phase：0.1% formic acid water/methanol；Flow velocity：0.3mL/min；Column temperature：35℃；
Sample size：10μL
Liquid phase gradient condition
2. Mass Spectrometry Conditions
(2) under above-mentioned chromatographic condition, with the presence or absence of corresponding measured object, it is necessary to meet following condition in judgement sample：
The mass chromatography peak retention time occurred in sample solution is consistent with mixed-matrix standard working solution, it is allowed to deviation be less than ±
2.5%, the medicine corresponding to the chromatographic peak is in the relative abundance of the setting mass spectrometry ion extraction standard work suitable with concentration
Make that the relative ion abundance of liquid is consistent, relative abundance deviation is no more than the regulation of setting, then can determine that containing the medicine.
Preferably, in above-mentioned veterinary drug amantadine medicament residue detection method, the preparation method of the sample solution is
Sample is directly extracted with two step of methanol ethyl acetate mixed solution, and nitrogen crosses 0.22 μm of miillpore filter after blowing redissolution.
The detection method of amantadine medicament residue in above-mentioned veterinary drug, the preparation method of the sample solution, specific steps
It is as follows：
(1) 1g samples accurately are weighed, is accurate to 0.01g, be placed in 50mL polystyrene centrifuge tubes, add 4mL methanol acetic acids
Ethyl ester mixed solution, whirling motion mix, and centrifugal force 4000g-6000g centrifuges 10min under room temperature, take out supernatant take to
In centrifuge tube clean 10mL；
(2) residue continues to repeat to extract with 4mL methanol ethyl acetates mixed solution, merges extracting solution twice and is taken out after mixing
1.6mL into clean centrifuge tube, 55 DEG C of nitrogen be blown to it is dry, with 1mL redissolve liquid redissolve, after redissolution sample liquid through dilute 10 times of solution mistakes
0.22 μm of miillpore filter, you can measured for liquid chromatography-tandem mass spectrometry instrument.
The beneficial effects of the invention are as follows：
The detection method of amantadine medicament residue in above-mentioned veterinary drug, with liquid chromatography-tandem mass spectrometry veterinary drug
Amantadine medicament residue, whens this method sample pre-treatments eliminates purification process, has that processing routine is simple, quickly, surveys
Determine the features such as accuracy rate is high；Analyzed from veterinary drug source, effective guarantee aquaculture drug safety, improve Animal Food Security.
Brief description of the drawings
Fig. 1 detects amantadine total ion current figure for veterinary drug negative sample and positive；
3 kinds of Extraction solvents of Fig. 2 extract amantadine result total ion current figure respectively.
Embodiment
In order to make those skilled in the art be better understood from technical scheme, with reference to embodiment
Technical solution of the present invention is described in further detail.
Embodiment 1
First, extract
The addition recovery experiment progress method validation that 4 kinds of different veterinary drug samples do 3 kinds of various concentrations is chosen, is treated in veterinary drug
The Extraction solvent of medicine is surveyed, 2% formic acid methanol, 2% formic acid acetonitrile and methanol ethyl acetate mixed solution, 3 kinds of examinations are respectively adopted
Agent carries out the selection of extraction conditions.Test result indicates that being remained for the amantadine in veterinary drug, methanol ethyl acetate mixing is molten
The extraction efficiency of liquid is better than other two kinds of reagents.
Concrete operations：
(1) 1g samples accurately are weighed, are accurate to 0.01g, be placed in 50mL polystyrene centrifuge tubes, add 4mL Extraction solvents,
Whirling motion mixes, centrifugal force 4000g-6000g, centrifuges 10min under room temperature, takes out supernatant and takes to centrifuge tube clean 10mL
In；
(2) residue continues to repeat to extract with 4mL Extraction solvents, merges and 1.6mL is taken out after extracting solution mixes twice to clean
In centrifuge tube, 55 DEG C of nitrogen are blown to dry, redissolve liquid with 1mL and redissolve, sample liquid crosses the filter of 0.22 μm of micropore through diluting 10 times of solution after redissolution
Film, you can measured for liquid chromatography-tandem mass spectrometry instrument.
2nd, measure
This experiment quantitative approach is quantified by external standard method.Amantadine 100ng/mL standard working solutions are taken out from refrigerator, it is extensive
It is multiple to after room temperature with 10% methanol, 0.1% formic acid water do solvent compound concentration be respectively 0.5ng/mL, 1ng/mL, 2ng/mL,
The amantadine standard solution of 5ng/mL, 10ng/mL, parallel 3 pin of sample introduction of each concentration, by its gained peak area ratio average
Y is ordinate, and corresponding standard concentration X draws standard curve for abscissa.According to the medicament contg of measured object in sample, choosing
Amantadine standard working solution similar in response is taken to be carried out at the same time analysis.Medicine in amantadine standard working solution and prepare liquid
Response should all be in instrument linear response range.The curve ranges if content is above standard, it is dilute using matrix blank solution
Release suitable concentration post analysis.
Specific detection parameters are as follows：
1. liquid phase chromatogram condition
Chromatographic column：Hypersil Gold C18,100mm×2.1mm,1.9μm
Mobile phase：0.1% formic acid water/methanol；Flow velocity：0.3mL/min；Column temperature：35℃；
Sample size：10μL
Liquid phase gradient condition
2. Mass Spectrometry Conditions
Under above-mentioned chromatographic condition, with the presence or absence of corresponding measured object, it is necessary to meet following condition in judgement sample：Sample
The mass chromatography peak retention time occurred in solution is consistent with mixed-matrix standard working solution, it is allowed to which deviation is less than ± 2.5%, should
The phase of medicine corresponding to chromatographic peak in the relative abundance of the setting mass spectrometry ion extraction standard working solution suitable with concentration
Consistent to abundance of ions, relative abundance deviation is no more than the regulation of setting, then can determine that containing the medicine.
3rd, case result is embodied
This experiment chooses 4 kinds of veterinary drugs progress amantadines and does addition recovery test, the specific rate of recovery and precision result altogether
Such as following table：
By above case as it can be seen that the present invention uses the amantadine medicine in liquid chromatography-tandem mass spectrometry veterinary drug
Residual, sample cross miillpore filter after blowing redissolution, then use liquid by water-soluble filtering, the extraction of two step of methanol ethyl acetate, nitrogen first
Phase chromatography-tandem mass spectrometry is measured, appearance standard measure, and the rate of recovery is 78%~94%；Relative standard deviation is less than 10%.
The detection method sample treatment program of the present invention is simple, quick, eliminates sample purification process, reduces testing cost, and
Remaining amantadine in veterinary drug can fast and accurately be determined.The method measure amantadine lowest limit is 0.5 μ g/kg, completely full
The requirement of sufficient medicament residue detection.
Above-mentioned reference embodiment carries out the detection method of amantadine medicament residue in a kind of veterinary drug detailed
Thin description, is illustrative rather than limited, several embodiments can be included according to limited scope, therefore do not taking off
From changing and modifications under present general inventive concept, should belong within protection scope of the present invention.
Claims (3)
- A kind of 1. detection method of amantadine medicament residue in veterinary drug, it is characterised in that：Surveyed with Liquid Chromatography-Tandem Mass Spectrometry Determine the amantadine medicament residue in veterinary drug, comprise the following steps that：(1) standard working solution and the sample solution sample introduction under the conditions of the liquid chromatography-tandem mass spectrometry of setting, using mass concentration X as Abscissa, the ratio Y of peak area is ordinate, draws 5 standard working curves, sample is determined with standard working curve Amount, in sample solution the response of medicine should in the range of linearity that instrument detects, wherein, set liquid chromatography-tandem Mass Spectrometry Conditions are as follows：1. liquid phase chromatogram conditionChromatographic column：Hypersil Gold C18,100mm×2.1mm,1.9μmMobile phase：0.1% formic acid water/methanol；Flow velocity：0.3mL/min；Column temperature：35℃；Sample size：10μLLiquid phase gradient condition2. Mass Spectrometry Conditions(2) under above-mentioned chromatographic condition, with the presence or absence of corresponding measured object, it is necessary to meet following condition in judgement sample：Sample The mass chromatography peak retention time occurred in solution is consistent with mixed-matrix standard working solution, it is allowed to which deviation is less than ± 2.5%, should The phase of medicine corresponding to chromatographic peak in the relative abundance of the setting mass spectrometry ion extraction standard working solution suitable with concentration Consistent to abundance of ions, relative abundance deviation is no more than the regulation of setting, then can determine that containing the medicine.
- 2. the detection method of amantadine medicament residue in veterinary drug according to claim 1, it is characterised in that：The sample The preparation method of solution is directly extracted for sample with two step of methanol ethyl acetate mixed solution, and nitrogen crosses 0.22 μm of micropore after blowing redissolution Filter membrane.
- 3. the detection method of amantadine medicament residue in veterinary drug according to claim 2, it is characterised in that：The sample The preparation method of solution, comprises the following steps that：(1) 1g samples accurately are weighed, is accurate to 0.01g, be placed in 50mL polystyrene centrifuge tubes, add 4mL methanol ethyl acetates Mixed solution, whirling motion mix, centrifugal force 4000g-6000g, centrifuge 10min under room temperature, and taking-up supernatant, which is taken to 10mL, to be done In net centrifuge tube；(2) residue continues to repeat to extract with 4mL methanol ethyl acetates mixed solution, merges extracting solution twice and is taken out after mixing 1.6mL into clean centrifuge tube, 55 DEG C of nitrogen be blown to it is dry, with 1mL redissolve liquid redissolve, after redissolution sample liquid through dilute 10 times of solution mistakes 0.22 μm of miillpore filter, you can measured for liquid chromatography-tandem mass spectrometry instrument.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150275302A1 (en) * | 2014-04-01 | 2015-10-01 | Teva Pharmaceutical Industries, Ltd. | Determination of single nucleotide polymorphisms useful to predict response for rasagiline |
| CN105203654A (en) * | 2015-09-11 | 2015-12-30 | 山东省分析测试中心 | Method for measuring content of 11 illegally added medicaments in veterinary drug powder |
| CN107202839A (en) * | 2017-05-16 | 2017-09-26 | 河南省兽药饲料监察所 | The screening method of 122 kinds of OTCs in a kind of veterinary drug preparation |
-
2017
- 2017-11-30 CN CN201711235020.7A patent/CN107941953A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150275302A1 (en) * | 2014-04-01 | 2015-10-01 | Teva Pharmaceutical Industries, Ltd. | Determination of single nucleotide polymorphisms useful to predict response for rasagiline |
| CN105203654A (en) * | 2015-09-11 | 2015-12-30 | 山东省分析测试中心 | Method for measuring content of 11 illegally added medicaments in veterinary drug powder |
| CN107202839A (en) * | 2017-05-16 | 2017-09-26 | 河南省兽药饲料监察所 | The screening method of 122 kinds of OTCs in a kind of veterinary drug preparation |
Non-Patent Citations (2)
| Title |
|---|
| 刘雪红等: "超高效液相色谱-串联质谱法检测清瘟败毒散中非法添加金刚烷胺的研究", 《中国兽药杂志》 * |
| 花锦等: "高效液相色谱-串联质谱法测定兽药制剂中的金刚烷胺", 《湖北农业科学》 * |
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