CN106290666B - A kind of streptomysin detection method - Google Patents
A kind of streptomysin detection method Download PDFInfo
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- CN106290666B CN106290666B CN201610982166.7A CN201610982166A CN106290666B CN 106290666 B CN106290666 B CN 106290666B CN 201610982166 A CN201610982166 A CN 201610982166A CN 106290666 B CN106290666 B CN 106290666B
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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
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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
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention discloses a kind of streptomysin detection method, sample to be tested is carried out to be acidified to pH1.5-4.5, flocculant is added and is stirred, then carries out ceramic membrane filter；PH is adjusted to 6.0-8.0 by obtained filtrate with sodium hydroxide, carries out Dynamic Adsorption with macroporous absorbent resin to get saturated resin；Saturated resin is desorbed with the aqueous sulfuric acid of 3-15%；The mixed bed desalination of obtained stripping liquid spent ion exchange resin composition neutralizes to obtain refined liquid；Be filtered, be dried with nitrogen to refined liquid with the nanofiltration membrane of 200-500Da, count the solution dissolved residue of acetonitrile and water containing 0.2% formic acid by volume with 0.5ml, wherein count by volume, acetonitrile: water=70:30 is measured for LC-MS/MS.After the present invention carries out treatment and purification to sample to be tested, numerous interference impurity are eliminated, reagent dosage is few, and without using ion-pairing agent, detection is quick, high sensitivity, accurate, economy.
Description
Technical field
The present invention relates to technical field of analysis and detection, specifically a kind of streptomysin detection method.
Background technique
Streptomysin (streptomycin) is a kind of aminoglycoside antibiotics, molecular formula C21H39N7O12.Streptomysin is
A kind of antibiotic extracted from the culture solution of grey streptomycete.Belong to Aminoglycoside alkali compounds, it and tubercle bacillus thallus
Ribonucleoprotein body protein matter combines, and plays the role of interfering the synthesis of tubercle bacillus protein, to kill or inhibit
The effect of growth of bacillus tubercle.Since the pain reaction of streptomysin intramuscular injection is smaller, be suitable for clinical use, as long as using pair
Proper as selecting, dosage is again proper, and most patients can be with long term injections (general 2 months or so).So application is tens of
It is still the mainstay in antituberculosis therapy over year.The aldehyde radical of strepto- saccharide part is reduced into primary alconol base in streptomysin molecule
Afterwards, just become dihydrostreptomycin, antibacterial efficacy is roughly the same with streptomysin.But the antibiotic has certain poison secondary people
Effect, if remaining excessive streptomysin in food, can cause to seriously endanger to human body, such as damage vestibular nerve and cochlea mind
Led to dysacousis, streptomysin also has potential teratogenesis, mutagenesis and carcinogenesis.
Currently, both at home and abroad for the existing a large amount of research of streptomysin and the remaining detection technique of dihydrostreptomycin, commonly
Detection method has enzyme-linked immunization, spectrophotometry, liquid chromatography and liquid chromatography tandem mass spectrometry etc..Enzyme-linked immunization is easy
False positive or false negative result are generated, Screening analysis is usually used in；Liquid chromatography cannot efficiently separate streptomysin and double hydrogen strepto-s
Element, and sensitivity is lower, measurement and confirmatory analysis while can not achieve streptomysin and dihydrostreptomycin, thus be difficult to solve it
The problem of mixing residual；Liquid Chromatography-Tandem Mass Spectrometry is current mainstream detection method, but before correlation technique reported in the literature
It handles more complicated, often needs the purification by solid phase extraction column twice, reagent consumption is big, and needs using ion pair
Reagent, mass detector easy to pollute make mass spectrographic reproducibility and deterioration of sensitivity.When purification condition is bad, the peak type of determinand
Very poor and sensitivity is very low, and purified treatment is the difficult point of streptomysin measurement.
Summary of the invention
The purpose of the present invention is to provide a kind of high sensitivities, accurate, economic, durable streptomysin detection method, with solution
Certainly the problems mentioned above in the background art.
To achieve the above object, the invention provides the following technical scheme:
A kind of streptomysin detection method, comprising the following steps:
1) sample to be tested is carried out being acidified to pH1.5-4.5, flocculant is added and is stirred, then carries out ceramic membrane mistake
Filter；
2) filtrate for obtaining step 1) is adjusted pH to 6.0-8.0 with sodium hydroxide, is moved with macroporous absorbent resin
State is adsorbed to get saturated resin；
3) saturated resin in step 2 is desorbed with the aqueous sulfuric acid of 3-15%；Obtained stripping liquid ion
The mixed bed desalination of exchanger resin composition neutralizes to obtain refined liquid；
4) refined liquid is filtered with the nanofiltration membrane of 200-500Da, is dried with nitrogen, counted and contained by volume with 0.5ml
The acetonitrile of 0.2% formic acid and the solution dissolved residue of water, wherein count by volume, acetonitrile: water=70:30 is surveyed for LC-MS/MS
It is fixed.
As a further solution of the present invention: in step 1), flocculant is poly-aluminium and polyacrylamide, and composition ratio is
1:0.5-5, additive amount are the 0.05-1% of sample to be tested quality.
As a further solution of the present invention: in step 2, the flow velocity of Dynamic Adsorption is 0.3-3BV/h, macroporous absorption tree
The adsorbance of rouge is 150-350g/L.
As a further solution of the present invention: any in macroporous absorbent resin D303, D308, D310 in step 2
It is a kind of.
As a further solution of the present invention: in step 3), desorption flow velocity is 0.05-3BV/h.
As a further solution of the present invention: in step 4), in LC-MS/MS,
Chromatographic column: ACQUITY UPLC BEH HILIC column be 100mm × 2.1mm, 1.7 μm；Column temperature: 40 DEG C；Sample introduction body
Product: 10uL；Mobile phase: A is 5mM formic acid aqueous ammonium, and formic acid aqueous ammonium contains 0.2% formic acid, and B is acetonitrile solution containing 0.2%
Formic acid；Gradient elution: 0min, A 25%；0-0.5min, A 25%-60%；0.5-5.5min, A 60%；5.5-6min, A
For 60%-25%；6-9min, A 25%；Flow velocity: 0.32mL/min；
Ion source: electric spray ion source；Scanning mode: cation scanning；Detection mode: multiple-reaction monitoring；Electron spray electricity
Pressure: 5000V；Ion source temperature: 550 DEG C；Focus voltage: 150V；Remove cluster voltage: 80V；Import voltage: 7V.
Compared with prior art, the beneficial effects of the present invention are:
After the present invention carries out treatment and purification to sample to be tested, numerous interference impurity are eliminated, reagent dosage is few, without using
Ion-pairing agent.To establish the quick of Determination of Streptomycin Residues amount detection, high sensitivity, accurate, economic, durable confirmation and fixed
Amount method.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of streptomysin detection method, comprising the following steps:
1) sample to be tested is carried out being acidified to pH1.5, flocculant is added and is stirred, then carries out ceramic membrane filter.Wadding
Solidifying agent is poly-aluminium and polyacrylamide, and composition ratio 1:0.5, additive amount is the 0.05% of sample to be tested quality；
2) filtrate for obtaining step 1) is adjusted pH to 6.0 with sodium hydroxide, carries out dynamic suction with macroporous absorbent resin
It is attached to get saturated resin.The flow velocity of Dynamic Adsorption is 0.3BV/h, and the adsorbance of macroporous absorbent resin is 150g/L.Macroporous absorption
Resin is D303；
3) saturated resin in step 2 is desorbed with 3% aqueous sulfuric acid；Desorption flow velocity is 0.05BV/h.?
To stripping liquid spent ion exchange resin composition mixed bed desalination neutralize to obtain refined liquid；
4) refined liquid is filtered with the nanofiltration membrane of 200Da, is dried with nitrogen, counted by volume with 0.5ml containing 0.2% first
The acetonitrile of acid and the solution dissolved residue of water, wherein count by volume, acetonitrile: water=70:30 is measured for LC-MS/MS.Chromatography
Column: ACQUITY UPLC BEH HILIC column be 100mm × 2.1mm, 1.7 μm；Column temperature: 40 DEG C；Sampling volume: 10uL；Flowing
Phase: A is 5mM formic acid aqueous ammonium, and formic acid aqueous ammonium contains 0.2% formic acid, and B is the formic acid that acetonitrile solution contains 0.2%；Gradient
Elution: 0min, A 25%；0min, A 25%；0.5min, A 60%；5.5min, A 60%；6min, A 25%；Flow velocity:
0.32mL/min.Ion source: electric spray ion source；Scanning mode: cation scanning；Detection mode: multiple-reaction monitoring；Electron spray
Voltage: 5000V；Ion source temperature: 550 DEG C；Focus voltage: 150V；Remove cluster voltage: 80V；Import voltage: 7V.
Embodiment 2
In the embodiment of the present invention, a kind of streptomysin detection method, comprising the following steps:
1) sample to be tested is carried out being acidified to pH4.5, flocculant is added and is stirred, then carries out ceramic membrane filter.Wadding
Solidifying agent is poly-aluminium and polyacrylamide, and composition ratio 1:5, additive amount is the 1% of sample to be tested quality；
2) filtrate for obtaining step 1) is adjusted pH to 8.0 with sodium hydroxide, carries out dynamic suction with macroporous absorbent resin
It is attached to get saturated resin.The flow velocity of Dynamic Adsorption is 3BV/h, and the adsorbance of macroporous absorbent resin is 350g/L.Macroporous absorption tree
Rouge is D308；
3) saturated resin in step 2 is desorbed with 15% aqueous sulfuric acid；Desorption flow velocity is 3BV/h.It obtains
Stripping liquid spent ion exchange resin composition mixed bed desalination neutralize to obtain refined liquid；
4) refined liquid is filtered with the nanofiltration membrane of 500Da, is dried with nitrogen, counted by volume with 0.5ml containing 0.2% first
The acetonitrile of acid and the solution dissolved residue of water, wherein count by volume, acetonitrile: water=70:30 is measured for LC-MS/MS.Chromatography
Column: ACQUITY UPLC BEH HILIC column be 100mm × 2.1mm, 1.7 μm；Column temperature: 40 DEG C；Sampling volume: 10uL；Flowing
Phase: A is 5mM formic acid aqueous ammonium, and formic acid aqueous ammonium contains 0.2% formic acid, and B is the formic acid that acetonitrile solution contains 0.2%；Gradient
Elution: 0min, A 25%；0.5min, A 60%；5.5min, A 60%；6min, A 25%；9min, A 25%；Flow velocity:
0.32mL/min.Ion source: electric spray ion source；Scanning mode: cation scanning；Detection mode: multiple-reaction monitoring；Electron spray
Voltage: 5000V；Ion source temperature: 550 DEG C；Focus voltage: 150V；Remove cluster voltage: 80V；Import voltage: 7V.
Embodiment 3
In the embodiment of the present invention, a kind of streptomysin detection method, comprising the following steps:
1) sample to be tested is carried out being acidified to pH2, flocculant is added and is stirred, then carries out ceramic membrane filter.Flocculation
Agent is poly-aluminium and polyacrylamide, and composition ratio 1:2, additive amount is the 0.5% of sample to be tested quality；
2) filtrate for obtaining step 1) is adjusted pH to 7.0 with sodium hydroxide, carries out dynamic suction with macroporous absorbent resin
It is attached to get saturated resin.The flow velocity of Dynamic Adsorption is 1BV/h, and the adsorbance of macroporous absorbent resin is 250g/L.Macroporous absorption tree
Rouge is D310；
3) saturated resin in step 2 is desorbed with 5% aqueous sulfuric acid；Desorption flow velocity is 13BV/h.It obtains
Stripping liquid spent ion exchange resin composition mixed bed desalination neutralize to obtain refined liquid；
4) refined liquid is filtered with the nanofiltration membrane of 300Da, is dried with nitrogen, counted by volume with 0.5ml containing 0.2% first
The acetonitrile of acid and the solution dissolved residue of water, wherein count by volume, acetonitrile: water=70:30 is measured for LC-MS/MS.Chromatography
Column: ACQUITY UPLC BEH HILIC column be 100mm × 2.1mm, 1.7 μm；Column temperature: 40 DEG C；Sampling volume: 10uL；Flowing
Phase: A is 5mM formic acid aqueous ammonium, and formic acid aqueous ammonium contains 0.2% formic acid, and B is the formic acid that acetonitrile solution contains 0.2%；Gradient
Elution: 0min, A 25%；0.3min, A 40%；3min, A 60%；5.5min, A 40%；7min, A 25%；Flow velocity:
0.32mL/min.Ion source: electric spray ion source；Scanning mode: cation scanning；Detection mode: multiple-reaction monitoring；Electron spray
Voltage: 5000V；Ion source temperature: 550 DEG C；Focus voltage: 150V；Remove cluster voltage: 80V；Import voltage: 7V.
Embodiment 4
Using negative catsup sample as bare substrate, prepare the extraction standard solution of various concentration, streptomysin it is dense
Degree is respectively 0.01,0.02,0.05,0.1 and 0.2 mg/L.The equation of linear regression of streptomysin is Y=1.8 × 106X+
5.5 × 103, related coefficient 0.9993, the range of linearity is 0.01~0.2 mg/L.
The concentration of method detection catsup streptomycin through above-described embodiment 3 is 0.15mg/L.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (1)
1. a kind of streptomysin detection method, which comprises the following steps: 1) carry out sample to be tested being acidified to pH1.5-
4.5, flocculant is added and is stirred, then carries out ceramic membrane filter；2) filtrate for obtaining step 1) is with sodium hydroxide by pH
6.0-8.0 is adjusted, carries out Dynamic Adsorption with macroporous absorbent resin to get saturated resin；3) aqueous sulfuric acid pair of 3-15% is used
Saturated resin in step 2 is desorbed；The mixed bed desalination of obtained stripping liquid spent ion exchange resin composition neutralizes to obtain
Refined liquid；4) refined liquid is filtered with the nanofiltration membrane of 200-500Da, is dried with nitrogen, counted by volume with 0.5ml containing 0.2%
The acetonitrile of formic acid and the solution dissolved residue of water, wherein count by volume, acetonitrile: water=70:30 is measured for LC-MS/MS；Step
It is rapid 1) in, flocculant is poly-aluminium and polyacrylamide, and composition ratio 1:0.5-5, additive amount is sample to be tested quality
0.05-1%；
In step 2, the flow velocity of Dynamic Adsorption is 0.3-3BV/h, and the adsorbance of macroporous absorbent resin is 150-350g/L；Step
2) any one in, in macroporous absorbent resin D303, D308, D310；In step 3), desorption flow velocity is 0.05-3BV/h；
In step 4), in LC-MS/MS, chromatographic column: ACQUITYUPLCBEHHILIC column be 100mm × 2.1mm, 1.7 μm；Column temperature: 40
℃；Sampling volume: 10uL；Mobile phase: A is 5mM formic acid aqueous ammonium, and formic acid aqueous ammonium contains 0.2% formic acid, and B is acetonitrile
Solution contains 0.2% formic acid；Gradient elution: 0min, A 25%；0-0.5min, A 25%-60%；0.5-5.5min, A 60%；
5.5-6min, A 60%-25%；6-9min, A 25%；Flow velocity: 0.32mL/min；Ion source: electric spray ion source；Scanning side
Formula: cation scanning；Detection mode: multiple-reaction monitoring；Electron spray voltage: 5000V；Ion source temperature: 550 DEG C；Focus voltage:
150V；Remove cluster voltage: 80V；Import voltage: 7V.
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| CN107884498A (en) * | 2017-11-23 | 2018-04-06 | 重庆方通动物药业有限公司 | Liquid phase chromatography analytical method that is a kind of while determining procaine, penicillin and content of streptomycin |
| CN112763592A (en) * | 2020-12-15 | 2021-05-07 | 上海明捷医药科技有限公司 | Method for measuring content of streptomycin in protein solution |
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| CN1292250C (en) * | 2005-01-06 | 2006-12-27 | 湖南纽尔科技有限公司 | Molecular engram integrally separating column for gentamycin, streptomycin and neomycin and preparing process thereof |
| CN102393425B (en) * | 2011-10-20 | 2014-04-09 | 新疆出入境检验检疫局检验检疫技术中心 | Method for detecting residual amount of streptomycin and dihydrostreptomycin in tomato sauce |
| CN103109928A (en) * | 2011-11-16 | 2013-05-22 | 李洪梅 | Research on integrated control technology of antibiotic residues in milk |
| CN104568925A (en) * | 2014-12-30 | 2015-04-29 | 中国农业科学院农产品加工研究所 | Streptomycin sulfate detection method |
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