CN108469484A - A kind of method that unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue - Google Patents
A kind of method that unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue Download PDFInfo
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Abstract
The present invention provides a kind of methods that unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue, belong to detection of veterinary drugs in food technical field, including：Sample introduction sample progress Liquid Chromatography-Tandem Mass Spectrometry detection is obtained into the content of aminoglycoside medicaments in sample introduction sample according to the liquid chromatogram of the standard curve of aminoglycoside medicaments content and sample introduction sample；The chromatographic column of the liquid chromatogram is SIELC Obelisc R.Method provided by the invention is under conditions of without using ion-pairing agent, chromatography reservation can equally be enhanced, the content of aminoglycoside medicaments in animal muscle tissue can be detected, the detection limit and quantitative limit of drug are respectively within the scope of 1~10 μ g/kg and 2~20 μ g/kg；In batch and betweenrun precision is respectively between 2~14.4% and 3.7~19.8%.
Description
Technical field
The invention belongs to detection of veterinary drugs in food technical fields more particularly to a kind of unused ion-pairing agent to measure animal flesh
The method of aminoglycoside medicaments in meat tissue.
Background technology
Aminoglycoside antibiotics is a kind of broad-spectrum antibiotic, is all had to Gram-negative bacteria and gram-positive bacteria aobvious
The inhibitory or killing effect of work is widely used in treating various animality diseases, also be often added in feed, for promoting growth of animal to send out
It educates.However, there is such drug side effects, continuous medication and the unreasonable uses such as ototoxicity, renal toxicity can lead to amino sugar
Aminoglycoside excess residual in edible animal causes the mankind by food chain potentially hazardous.
It reports the detection method about aminoglycoside medicaments in animal derived food both at home and abroad at present, mainly there is liquid phase color
Spectrum-series connection spectrometry etc., but since aminoglycoside medicaments have high polarity, it is difficult to be protected on traditional reverse-phase chromatographic column
It stays, it is therefore desirable to ion-pairing agent is added in mobile phase, to enhance chromatography reservation.But the use of ion-pairing agent not only can be right
Chromatographic column causes irreversible damage, but also will produce serious ion inhibition and corrosive ions source.
Invention content
The purpose of the present invention is to provide a kind of unused ion-pairing agents to measure aminoglycoside in animal muscle tissue
The method of drug can equally enhance chromatography reservation, can detect animal muscle group under conditions of without using ion-pairing agent
Knit the content of middle aminoglycoside medicaments.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides the sides that a kind of unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue
Method, including：Sample introduction sample is subjected to liquid chromatography-tandem mass spectrometry detection, according to the standard curve of aminoglycoside medicaments content
With the liquid chromatogram of sample introduction sample, the content of aminoglycoside medicaments in sample introduction sample is obtained；
The chromatographic column of the liquid chromatogram is SIELC Obelisc R；
The mobile phase of the liquid chromatogram is made of mobile phase A and Mobile phase B, and the mobile phase A is acetonitrile, the flowing
Phase B is formic acid solution or acetic acid solution；The volumn concentration of the formic acid solution is 0.5~1.5%, the acetic acid solution
Volumn concentration is 1.5~2.5%；
The gradient elution program of the liquid chromatogram：The volume content of 0~0.5min, mobile phase A are mobile phase volume
85%；The volume content of 0.5~3.8min, mobile phase A are reduced to 65% by 85%；3.8~4.3min, the volume of mobile phase A
Content is reduced to 55% by 65%；The volume content of 4.3~4.5min, mobile phase A are 55%；4.5~6.5min, mobile phase A
Volume content be reduced to 0% by 55%；The volume content of 6.5~11min, mobile phase A are 0%；11~12min, mobile phase
The volume content of A increases to 85% by 0%；The volume content of 12~21min, mobile phase A are 85%.
Preferably, the condition of the liquid chromatogram includes：
The flow velocity of the mobile phase is 0.4~0.8ml/min；
The column temperature of the liquid chromatogram is 25~40 DEG C；
The sampling volume of the liquid chromatogram is 5~20 μ l.
Preferably, the condition of the tandem mass spectrum includes：
The ion source of the tandem mass spectrum is electron spray ionisation ion source；
The electron spray voltage of the tandem mass spectrum is 4000~5500V；
The ion source temperature of the tandem mass spectrum is 450~650 DEG C；
The gas curtain atmospheric pressure of the tandem mass spectrum is 20~35psi；
The atomization gas pressure of the tandem mass spectrum is 45~65psi；
The assist gas pressure power of the tandem mass spectrum is 45~65psi；
The residence time of the tandem mass spectrum is 20~100s.
Preferably, the aminoglycoside medicaments include actiline, apramycin, kanamycin B, tobramycin, prestige he
One or more of mycin, hygromycin B, gentamicin, amikacin and spectinomycin.
Preferably, the preparation method of the sample introduction sample includes：
1) animal muscle tissue is mixed with phosphate extracting solution, carries out first and be ultrasonically treated 8~12min, obtains first
Sonicates obtain the first precipitation and the first clear liquid after centrifuging first Sonicates；
2) the first precipitation that the step 1) obtains is mixed with phosphate extracting solution, the second supersound process of progress 8~
12min obtains the second Sonicates, after second Sonicates are centrifuged, obtains the second precipitation and second clearly
Liquid；
3) the first clear liquid that the step 1) obtains is mixed with the second clear liquid that the step 2) obtains, it is clear obtains mixing
Liquid；
4) the mixing clear liquid for obtaining the step 3) is added in MCX columns, uses acetic acid solution and water wash successively, then use ammonia
Methanol solution is eluted, and eluent is obtained；
5) eluent that the step 4) obtains is dried up through nitrogen, obtains dried object, the dried object and acetic acid is molten
Liquid mixes, and obtains third mixture, and after the third mixture is filtered, obtained liquid phase component is as sample introduction sample.
Preferably, the phosphate extracting solution includes per 1L：1.0~2.0g of potassium dihydrogen phosphate, disodium ethylene diamine tetraacetate
0.1~0.2g, 10~50g of trichloroacetic acid.
Preferably, the quality of the step 1) animal muscle tissue and the volume ratio of phosphate extracting solution are (1~3) g:
(4~6) ml.
Preferably, the conditional sampling that the step 1) and step 2) centrifuge includes：The rotating speed of the centrifugation
Time for 8000~10000rpm, the centrifugation is 8~12min.
Preferably, the volumn concentration of the step 5) acetic acid solution is 1~4%.
Preferably, the volumn concentration of ammonia is preferably 15~25% in the step 4) methanolic ammonia solution.
The present invention provides a kind of methods of aminoglycoside medicaments in measurement animal muscle tissue, without using ion pair
Under conditions of reagent, it can equally enhance chromatography reservation, the content of aminoglycoside medicaments in animal muscle tissue can be detected.
The result of the embodiment of the present invention is shown：Under conditions of without using ion-pairing agent, it can equally enhance chromatography guarantor
It stays, the content of aminoglycoside medicaments in animal muscle tissue can be detected, the detection limit and quantitative limit of drug are respectively 1~10
Within the scope of μ g/kg and 2~20 μ g/kg, show that this method has very high sensitivity, trace in Accurate Determining animal tissue can be quantified
Measure horizontal antibacterial medicine residue；In batch and betweenrun precision is respectively between 2~14.4% and 3.7~19.8%, shows have
There are preferable accuracy and precision.
Description of the drawings
Fig. 1 is the SRM chromatograms of blank chicken meat sample；
Fig. 2 is the mixed standard solution (50 μ g/kg) of 10 kinds of aminoglycoside medicaments of addition in blank chicken meat sample
SRM chromatographies；
Fig. 3 is the SRM chromatograms of blank beef sample；
Fig. 4 is the mixed standard solution (50 μ g/kg) of 10 kinds of aminoglycoside medicaments of addition in blank beef sample
SRM chromatographies；
Fig. 5 is the SRM chromatograms of blank pork sample；
Fig. 6 is the mixed standard solution (50 μ g/kg) of 10 kinds of aminoglycoside medicaments of addition in blank pork sample
SRM chromatographies.
Specific implementation mode
The present invention provides the sides that a kind of unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue
Method, including：Sample introduction sample is subjected to liquid chromatography-tandem mass spectrometry detection, according to the standard curve of aminoglycoside medicaments content
With the liquid chromatogram and mass spectrogram of sample introduction sample, the content of aminoglycoside medicaments in sample introduction sample is obtained；The liquid phase color
The chromatographic column of spectrum is SIELC Obelisc R；The mobile phase of the liquid chromatogram is made of mobile phase A and Mobile phase B, the stream
Dynamic phase A is acetonitrile, and the Mobile phase B is formic acid solution or acetic acid solution；The volumn concentration of the formic acid solution is 0.5
~1.5%, the volumn concentration of the acetic acid solution is 1.5~2.5%；The gradient elution program of the liquid chromatogram：0~
0.5min, the volume content of mobile phase A are the 85% of mobile phase volume；0.5~3.8min, the volume content of mobile phase A by
85% is reduced to 65%；The volume content of 3.8~4.3min, mobile phase A are reduced to 55% by 65%；4.3~4.5min, flowing
The volume content of phase A is 55%；The volume content of 4.5~6.5min, mobile phase A are reduced to 0% by 55%；6.5~11min,
The volume content of mobile phase A is 0%；The volume content of 11~12min, mobile phase A increase to 85% by 0%；12~21min,
The volume content of mobile phase A is 85%.
In the present invention, the chromatographic column of the liquid chromatogram is SIELC Obelisc R.In the present invention, the chromatography
The specification of column SIELC Obelisc R is preferably 5 μm, 150mm × 2.1mm i.d..In the present invention, the chromatographic column SIELC
Obelisc R have reverse phase, ion exchange and hydrophilic interaction simultaneously.
In the present invention, the aminoglycoside medicaments preferably include actiline, apramycin, kanamycin B, appropriate cloth
One or more of mycin, ribostamycin, hygromycin B, gentamicin, amikacin and spectinomycin.
In the present invention, the animal muscle tissue comes preferably from pork, beef, chicken, mutton or the flesh of fish.In this hair
In bright, provided again after the preferred rubbing of the animal muscle tissue, the degree that the present invention rubs the animal muscle tissue does not have
Particular determination, using the conventional rubbing degree of those skilled in the art.
In the present invention, the condition of the liquid chromatogram includes：
The mobile phase of the liquid chromatogram is made of mobile phase A and Mobile phase B, and the mobile phase A is acetonitrile, the flowing
Phase B is formic acid solution or acetic acid solution；
The flow velocity of the mobile phase is 0.4~0.8ml/min；
The column temperature of the liquid chromatogram is 25~40 DEG C；
The sampling volume of the liquid chromatogram is 5~20 μ l；
The gradient elution program of the liquid chromatogram：The volume content of 0~0.5min, mobile phase A are mobile phase volume
85%；The volume content of 0.5~3.8min, mobile phase A are reduced to 65% by 85%；3.8~4.3min, the volume of mobile phase A
Content is reduced to 55% by 65%；The volume content of 4.3~4.5min, mobile phase A are 55%；4.5~6.5min, mobile phase A
Volume content be reduced to 0% by 55%；The volume content of 6.5~11min, mobile phase A are 0%；11~12min, mobile phase
The volume content of A increases to 85% by 0%；The volume content of 12~21min, mobile phase A are 85%.
In the present invention, the mobile phase of the liquid chromatogram is made of mobile phase A and Mobile phase B, and the mobile phase A is preferred
For acetonitrile, the Mobile phase B is preferably formic acid solution or acetic acid solution.In the present invention, the volume basis of the formic acid solution
Content is preferably 0.5~1.5%, more preferably 0.8~1.2%, most preferably 1.0%；The volume basis of the acetic acid solution
Content is preferably 1.5~2.5%, more preferably 1.8~2.2%, most preferably 2.0%.
In the present invention, the flow velocity of the mobile phase is preferably 0.5~0.7ml/min, more preferably 0.6ml/min.
In the present invention, the column temperature of the liquid chromatogram is preferably 25~40 DEG C, more preferably 30~35 DEG C, most preferably
32℃。
In the present invention, the sampling volume of the liquid chromatogram is preferably 5~20 μ l, more preferably 10~15 μ l, more excellent
It is selected as 12 μ l.
In the present invention, the gradient elution program of the liquid chromatogram is preferably：0~0.5min, the volume of mobile phase A
Content is the 85% of mobile phase volume；The volume content of 0.5~3.8min, mobile phase A are reduced to 65% by 85%；3.8~
The volume content of 4.3min, mobile phase A are reduced to 55% by 65%；The volume content of 4.3~4.5min, mobile phase A is
55%；The volume content of 4.5~6.5min, mobile phase A are reduced to 0% by 55%；The volume of 6.5~11min, mobile phase A contain
Amount is 0%；The volume content of 11~12min, mobile phase A increase to 85% by 0%；The volume of 12~21min, mobile phase A contain
Amount is 85%.
In the present invention, the Mass Spectrometry Conditions of the tandem mass spectrum include：
The ion source of the tandem mass spectrum is electron spray ionisation ion source；
The electron spray voltage of the tandem mass spectrum is 4000~5500V；
The ion source temperature of the tandem mass spectrum is 450~650 DEG C；
The gas curtain atmospheric pressure of the tandem mass spectrum is 20~35psi；
The atomization gas pressure of the tandem mass spectrum is 45~65psi；
The assist gas pressure power of the tandem mass spectrum is 45~65psi；
The residence time of the tandem mass spectrum is 20~100s.
In the present invention, the ion source of the tandem mass spectrum is preferably electron spray ionisation ion source.
In the present invention, the electron spray voltage of the tandem mass spectrum is preferably 4000~5500V, more preferably 4500~
5200V, most preferably 4800~5000V.
In the present invention, the ion source temperature of the tandem mass spectrum is preferably 450~650 DEG C, more preferably 500~600
DEG C, most preferably 520~580 DEG C.
In the present invention, the gas curtain atmospheric pressure of the tandem mass spectrum is preferably 20~35psi, more preferably 25~
30psi, most preferably 25psi.
In the present invention, the atomization gas pressure of the tandem mass spectrum is preferably 45~65psi, more preferably 50~
60psi, more preferably 55~58psi.
In the present invention, the assist gas pressure power of the tandem mass spectrum is preferably 45~65psi, more preferably 50~
60psi, more preferably 55~58psi.
In the present invention, the residence time of the tandem mass spectrum is preferably 20~100s, most preferably 30~90s, optimal
It is selected as 40~80s.
In the present invention, the preparation method of the sample introduction sample includes：
1) animal muscle tissue is mixed with phosphate extracting solution, carries out first and be ultrasonically treated 8~12min, obtains first
Sonicates obtain the first precipitation and the first clear liquid after centrifuging first Sonicates；
2) the first precipitation that the step 1) obtains is mixed with phosphate extracting solution, the second supersound process of progress 8~
12min obtains the second Sonicates, after second Sonicates are centrifuged, obtains the second precipitation and second clearly
Liquid；
3) the first clear liquid that the step 1) obtains is mixed with the second clear liquid that the step 2) obtains, it is clear obtains mixing
Liquid；
4) the mixing clear liquid for obtaining the step 3) is added in MCX columns, uses acetic acid solution and water wash successively, then use ammonia
Methanol solution is eluted, and eluent is obtained；
5) eluent that the step 4) obtains is dried up through nitrogen, obtains dried object, the dried object and acetic acid is molten
Liquid mixes, and obtains third mixture, and after the third mixture is filtered, obtained liquid phase component is sample introduction sample.
The present invention mixes animal muscle tissue with phosphate extracting solution, carries out first and is ultrasonically treated 8~12nin, obtains
First Sonicates obtain the first precipitation and the first clear liquid after centrifuging first Sonicates.
In the present invention, the quality of the animal muscle tissue be preferably (1~3) with the volume ratio of phosphate extracting solution
g:(4~6) ml, more preferably (1.5~2.5) g:(4.5~5.5) ml, more preferably 3g:5ml.
In the present invention, the described first time being ultrasonically treated was preferably 8~12min, more preferably 9~11min, most
Preferably 10min.The present invention is not particularly limited other conditions such as ultrasonic power etc. of the supersound process, using this field
The condition that technical staff routinely selects.
In the present invention, the described first condition centrifuged preferably includes：The rotating speed of the centrifugation is preferably
8000~10000rpm, more preferably 8500~9500rpm, most preferably 9000rpm；The time of the centrifugation is preferably
8~12min, more preferably 9~11min, most preferably 10min.
In the present invention, the phosphate extracting solution is preferably included per 1L：1.0~2.0g of potassium dihydrogen phosphate, ethylenediamine tetraacetic
Acetic acid 0.1~0.2g of disodium, 10~50g of trichloroacetic acid more preferably include 1.2~1.5g of potassium dihydrogen phosphate, ethylenediamine tetra-acetic acid
0.12~0.18g of disodium, 15~30g of trichloroacetic acid most preferably include potassium dihydrogen phosphate 1.36g, disodium ethylene diamine tetraacetate
0.15g, trichloroacetic acid 20g.In an embodiment of the present invention, the preparation method of the phosphate extracting solution is preferably by di(2-ethylhexyl)phosphate
The 980mL water dissolutions of hydrogen potassium, sequentially add disodium ethylene diamine tetraacetate and trichloroacetic acid, fully dissolve mixing, use ultra-pure water
It is settled to 1L.
The present invention mixes the obtain first precipitation with phosphate extracting solution, carries out second and is ultrasonically treated 8~12min, obtains
To the second Sonicates the second precipitation and the second clear liquid are obtained after centrifuging second Sonicates.
In the present invention, in second mixture, the quality of the first precipitation is with the Mass Calculation of animal muscle tissue, phosphorus
The volume of hydrochlorate extracting solution and the quality of animal muscle tissue with than preferably (4~6) ml:(1~3) g, more preferably (4.5~
5.5)ml:(1.5~2.5) g, more preferably 5ml:3g.
In the present invention, the described second time being ultrasonically treated was preferably 8~12min, more preferably 9~11min, most
Preferably 10min.The present invention is not particularly limited other conditions such as ultrasonic power etc. of the supersound process, using this field
The condition that technical staff routinely selects.
In the present invention, the described second condition centrifuged preferably includes：The rotating speed of the centrifugation is preferably
8000~10000rpm, more preferably 8500~9500rpm, most preferably 9000rpm；The time of the centrifugation is preferably
8~12min, more preferably 9~11min, most preferably 10min.
The present invention mixes the first clear liquid that the step 1) obtains with the second clear liquid that the step 2) obtains, and is mixed
Close clear liquid.The present invention is not particularly limited the mixing by the way of, using the mixing side of those skilled in the art's routine
Formula.In the present invention, the mixing clear liquid preferably adds after ammonium hydroxide adjusts pH value in MCX columns, and the pH value is preferred
It is 5.0~6.0, more preferably 5.2~5.8, most preferably 5.5.
The mixing clear liquid that the present invention obtains the step 3) is added in MCX columns, uses acetic acid solution and water wash successively, then
It is eluted with methanolic ammonia solution, obtains eluent.
In the present invention, the MCX columns reuse after being activated preferably through alcohol and acid solution, the volume hundred of the acid solution
Point content is preferably 1~3%, more preferably 1.5~2.5%, most preferably 2%；The alcohol is preferably methanol or ethyl alcohol, described
Acid solution is preferably formic acid solution or acetic acid solution.In the present invention, the alcohol with mix clear liquid volume ratio be preferably (2~
4):(96~98) mL, more preferably (2.5~3.5):(96.5~97.5), most preferably 3:97；The acid solution with mix
The volume ratio of clear liquid is preferably (2~4):(96~98), more preferably (2.5~3.5):(96.5~97.5), most preferably 3:
97.In the present invention, the MCX columns have hydrophobic interaction and strong ion exchange.
The present invention will the mixing clear liquid be added MCX columns in, successively use acetic acid solution and water wash, the acetic acid solution with
The volume ratio for mixing clear liquid is preferably (2~4) (96~98), more preferably (2.5~3.5):(96.5~97.5), most preferably
It is 3:97；The water is preferably (2~4) with the volume ratio for mixing clear liquid:(96~98), more preferably (2.5~3.5):
(96.5~97.5), most preferably 3:97.In the present invention, the volumn concentration of the acetic acid solution is preferably 1~3%,
More preferably 1.5~2.5%, most preferably 2%.
In the present invention, the volumn concentration of ammonia is preferably 15~25% in the methanolic ammonia solution, more preferably
18~22%, most preferably 20%.In the present invention, the volume ratio of the methanolic ammonia solution and acetic acid solution be preferably (4~
6):(2~4), more preferably (4.5~5.5):(2.5~3.5), most preferably 5:3.
In the present invention, the flow velocity of the eluant, eluent of the MCX columns is preferably 0.5~1.5ml/min, more preferably 0.8~
1.2ml/min, most preferably 1ml/min.
The present invention dries up the eluent that the step 4) obtains through nitrogen, obtains dried object, by the dried object and second
Acid solution mixes, and obtains third mixture, and after the third mixture is filtered, obtained liquid phase component is as sample introduction sample.
In the present invention, the mass ratio of the volume of the acetic acid solution and dried object is preferably (1~4) ml:(40~
160) mg, more preferably (1.5~3.5) ml:(60~140) mg, most preferably 3ml:80mg.In the present invention, the acetic acid
The volumn concentration of solution is preferably 1~3%, more preferably 1.5~2.5%, most preferably 2%.
After the present invention filters third mixture, sample to be tested is obtained, the filtering preferably uses 0.22 μm of filter membrane to carry out
Filtering.
Sample introduction sample is carried out liquid chromatography-tandem mass spectrometry detection by the present invention, according to the mark of aminoglycoside medicaments content
The liquid chromatogram of directrix curve and sample introduction sample obtains the content of aminoglycoside medicaments in sample introduction sample.
In the present invention, the preparation of the standard solution of the aminoglycoside medicaments is preferably：By the aminoglycoside medicine
For object 10mg with ultra-pure water constant volume in 10ml brown volumetric flasks, mixing obtains the Standard Stock solutions of a concentration of 1mg/ml, and
The hybrid standard working solution of appropriate mass concentration is diluted to ultra-pure water as needed.
In the present invention, the preparation method of the standard curve of the aminoglycoside medicaments content preferably includes：By 2g skies
White sample obtains sample introduction sample according to the method described in above-mentioned technical proposal, Standard Stock solutions is configured to sample introduction sample dense
Degree is respectively the standard working solution of 5,10,20,50,100,200,400,500 μ g/L, upper machine testing.With object in blank base
A concentration of abscissa in quality sample, object peak area are ordinate, draw the standard curve of aminoglycoside medicaments content.
The present invention preferably adds target analytes in blank animal muscle tissue, according to the method described in above-mentioned technical proposal obtain into
All product are tested, and corresponding sample adds a concentration of detection and limits and quantify when being higher than 3 and 10 respectively with signal-to-noise ratio (S/N)
Limit.
In the present invention, when the aminoglycoside medicaments are preferably actiline, apramycin, kanamycin B, appropriate cloth
When mycin, ribostamycin, hygromycin B, gentamicin, amikacin or spectinomycin, the animal muscle tissue is preferably chicken
When meat, beef or pork, the standard curve of the aminoglycoside medicaments content, the range of linearity and related coefficient are as shown in table 1.
1 drug matrices of table match standard curve, the range of linearity and related coefficient (n=5)
Ammonia in animal muscle tissue is measured to a kind of unused ion-pairing agent provided by the invention with reference to embodiment
The method of base glycoside drug is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Involved solution percentage concentration, is concentration expressed in percentage by volume unless otherwise indicated in the present embodiment.
1. the preparation of standard solution
It is big mould that precision weighs actiline, apramycin, kanamycin B, tobramycin, ribostamycin, hygromycin B, celebrating
Each 10mg of element, amikacin, spectinomycin, with ultra-pure water constant volume in 10mL brown volumetric flasks, it is dense to obtain each drug for mixing
Degree is the Standard Stock solutions of 1mg/mL, and the hybrid standard for being diluted to appropriate mass concentration with ultra-pure water as needed works
Liquid, -20 DEG C are kept in dark place, the term of validity 1 month.
2. the preparation of phosphate extracting solution
Accurately weigh potassium dihydrogen phosphate 1.36g, with 980mL water dissolutions, sequentially add disodium ethylene diamine tetraacetate 0.15g and
Trichloroacetic acid 20g, fully dissolves mixing, and 1L is settled to ultra-pure water.
3. the preparation of sample
2g animal muscle tissues are weighed in 50mL polypropylene centrifuge tubes, 5mL phosphate extracting solutions, ultrasonic extraction is added
After 10min, 9000rpm centrifuges 10min, obtains the first precipitation and the first clear liquid, and the first precipitation is surpassed with 5mL phosphate extracting solutions
After sound extracts 10min, 9000rpm centrifuges 10min, obtains the second precipitation and the second clear liquid, and the first clear liquid and the second clear liquid are closed
And obtain mixing clear liquid.Will mixing clear liquid ammonium hydroxide to adjust pH value be the mixing clear liquid after adjusted pH value to 5.5.It will adjust
Mixing clear liquid after section pH value adds to the MCX pillars for having used 3mL methanol and 2% acetic acid water of 3mL to activate, and coutroi velocity is
1mL/min；3mL acetic acid aqueous solutions and 3mL water wash are used successively；Finally 5mL20% ammonia methanol (volumn concentration) is used to elute,
It collects eluent and is dried up with nitrogen, obtain dried object, dried object 2% acetic acid aqueous solutions of 4mL dissolve, and obtain third mixing
Object, third mixture obtain sample to be tested, sample to be tested is for liquid phase color through 0.22 μm of membrane filtration after removing insoluble impurities
Spectrum-tandem mass spectrometer detection.
4. chromatographic condition
Chromatographic column：SIELC Obelisc R, 5 μm, 150mm × 2.1mm i.d.；Mobile phase is by mobile phase A and Mobile phase B
Composition, mobile phase A are acetonitrile, and Mobile phase B is 1% formic acid solution；Flow velocity：0.6mL/min；Column temperature：40℃；Sampling volume：5μ
The gradient elution program of L, drug are shown in Table 2.
The gradient elution program of 2 drug of table
| Time (min) | Acetonitrile (%) | 1% formic acid (%) | Flow velocity (mL/min) |
| 0 | 85 | 15 | 0.6 |
| 0.5 | 85 | 15 | 0.6 |
| 3.8 | 65 | 35 | 0.6 |
| 4.3 | 55 | 45 | 0.6 |
| 4.5 | 55 | 45 | 0.6 |
| 6.5 | 0 | 100 | 0.6 |
| 11 | 0 | 100 | 0.6 |
| 12 | 85 | 15 | 0.6 |
| 21 | 85 | 15 | 0.6 |
5. Mass Spectrometry Conditions
Ion source：Electron spray ionisation ion source；Scan mode：Cation scans；Detection mode：Multiple-reaction monitoring pattern；
Electron spray voltage：5500V；Ion source temperature：550℃；Gas curtain atmospheric pressure：20psi；Atomization gas pressure：55psi；Assist gas pressure
Power：60psi.The retention time and optimization Mass Spectrometry Conditions of aminoglycoside medicaments are shown in Table 3.
The mass spectrometry parameters of 3 aminoglycoside medicaments of table
6. standard curve, detection limit and quantitative limit
2g blank samples accurately are weighed in 50mL polypropylene centrifuge tubes, according to above-mentioned sample extraction with after purification, are obtained
Matrix extracting solution.By Standard Reserving Solution with matrix extracting solution be configured to various concentration it is horizontal (5,10,20,50,100,200,
400,500 μ g/L) standard working solution, upper machine testing.With a concentration of abscissa of the object in bare substrate sample, target
Object peak area is ordinate, draws matrix matching standard curve, the results are shown in Table 4.
4 drug matrices of table match standard curve, the range of linearity and related coefficient (n=5)
Add target analytes in blank muscle samples, tested according to above-mentioned sample-pretreating method, respectively with
Signal-to-noise ratio (S/N) is higher than sample corresponding when 3 and 10 and adds a concentration of detection limit and quantitative limit.It is testing as can be known from Table 3
The matrix matching standard curve of each analyte is linearly good in concentration range, and related coefficient is all higher than 0.99.The inspection of all drugs
Survey limit and quantitative limit within the scope of 1~10 μ g/kg and 2~20 μ g/kg, show that this method has very high sensitivity, energy respectively
The antibacterial medicine residue of trace level in quantitative Accurate Determining animal tissue.
7. the rate of recovery and precision
The mixing of the aminoglycoside medicaments of three various concentration levels (25,50 and 100 μ g/kg) is added in blank sample
Standard solution, after extraction described above and purifying step, the rate of recovery and precision of each concentration pitch-based sphere are shown in Table 5.
5 rate of recovery of table and precision
As known from Table 4, under 25,50 and 100 μ g/kg, tri- concentration pitch-based spheres, the average recovery rate phase of neomycin is removed
To relatively low outer (being more than 60%), the average recovery rate of other drugs is above 65%, in batch and betweenrun precision respectively 2~
Between 14.4% and 3.7~19.8%.Show that method provided by the present application has preferable accuracy and precision, disclosure satisfy that
The requirement of animal tissue's sample veterinary drug residue detection and analysis.
As seen from the above embodiment, the present invention provides a kind of sides of aminoglycoside medicaments in measurement animal muscle tissue
Method can equally enhance chromatography reservation, can detect amino in animal muscle tissue under conditions of without using ion-pairing agent
The content of glycoside drug has preferable accuracy and precision, disclosure satisfy that animal tissue's sample veterinary drug residue detection
The requirement of analysis.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method that unused ion-pairing agent measures aminoglycoside medicaments in animal muscle tissue, including：By sample introduction
Sample carries out liquid chromatography-tandem mass spectrometry detection, according to the liquid of the standard curve of aminoglycoside medicaments content and sample introduction sample
Phase chromatogram obtains the content of aminoglycoside medicaments in sample introduction sample；
The chromatographic column of the liquid chromatogram is SIELC Obelisc R；
The mobile phase of the liquid chromatogram is made of mobile phase A and Mobile phase B, and the mobile phase A is acetonitrile, the Mobile phase B
For formic acid solution or acetic acid solution；The volumn concentration of the formic acid solution is 0.5~1.5%, the body of the acetic acid solution
Product percentage composition is 1.5~2.5%；
The gradient elution program of the liquid chromatogram：0~0.5min, the volume content of mobile phase A are the 85% of mobile phase volume；
The volume content of 0.5~3.8min, mobile phase A are reduced to 65% by 85%；3.8~4.3min, the volume content of mobile phase A by
65% is reduced to 55%；The volume content of 4.3~4.5min, mobile phase A are 55%；4.5~6.5min, the volume of mobile phase A
Content is reduced to 0% by 55%；The volume content of 6.5~11min, mobile phase A are 0%；11~12min, the volume of mobile phase A
Content increases to 85% by 0%；The volume content of 12~21min, mobile phase A are 85%.
2. according to the method described in claim 1, it is characterized in that, the condition of the liquid chromatogram includes：
The flow velocity of the mobile phase is 0.4~0.8ml/min；
The column temperature of the liquid chromatogram is 25~40 DEG C；
The sampling volume of the liquid chromatogram is 5~20 μ l.
3. according to the method described in claim 1, it is characterized in that, the condition of the tandem mass spectrum includes：
The ion source of the tandem mass spectrum is electron spray ionisation ion source；
The electron spray voltage of the tandem mass spectrum is 4000~5500V；
The ion source temperature of the tandem mass spectrum is 450~650 DEG C；
The gas curtain atmospheric pressure of the tandem mass spectrum is 20~35psi；
The atomization gas pressure of the tandem mass spectrum is 45~65psi；
The assist gas pressure power of the tandem mass spectrum is 45~65psi；
The residence time of the tandem mass spectrum is 20~100s.
4. according to the method described in claim 1, it is characterized in that, the aminoglycoside medicaments include actiline, peace it is general mould
One kind in element, kanamycin B, tobramycin, ribostamycin, hygromycin B, gentamicin, amikacin and spectinomycin or
It is several.
5. according to the method described in claim 1, it is characterized in that, the preparation method of the sample introduction sample includes：
1) animal muscle tissue is mixed with phosphate extracting solution, carries out first and is ultrasonically treated 8~12min, obtain the first ultrasound
Processed material obtains the first precipitation and the first clear liquid after centrifuging first Sonicates；
2) the first precipitation that the step 1) obtains is mixed with phosphate extracting solution, carries out second and is ultrasonically treated 8~12min,
The second Sonicates are obtained, after second Sonicates are centrifuged, obtain the second precipitation and the second clear liquid；
3) the first clear liquid that the step 1) obtains is mixed with the second clear liquid that the step 2) obtains, obtains mixing clear liquid；
4) the mixing clear liquid obtained the step 3) is added in MCX columns, uses acetic acid solution and water wash successively, then with ammonia methanol
Solution is eluted, and eluent is obtained；
5) eluent that the step 4) obtains is dried up through nitrogen, obtains dried object, the dried object is mixed with acetic acid solution
It closes, obtains third mixture, after the third mixture is filtered, obtained liquid phase component is as sample introduction sample.
6. according to the method described in claim 5, it is characterized in that, the phosphate extracting solution includes per 1L：Potassium dihydrogen phosphate
1.2~2.0g, 0.1~0.2g of disodium ethylene diamine tetraacetate, 10~50g of trichloroacetic acid.
7. according to the method described in claim 6, it is characterized in that, the quality and phosphate of the step 1) animal muscle tissue
The volume ratio of extracting solution is (1~3) g:(4~6) ml.
8. according to the method described in claim 5, it is characterized in that, the conditional sampling that the step 1) and step 2) centrifuge
Include：The rotating speed of the centrifugation is 8000~10000rpm, and the time of the centrifugation is 8~12min.
9. according to the method described in claim 5, it is characterized in that, the volumn concentration of the step 5) acetic acid be 1~
4%.
10. according to the method described in claim 5, it is characterized in that, in the step 4) methanolic ammonia solution ammonia volume hundred
Point content is preferably 15~25%.
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