CN108205031A - The detection method of tylosin residual potency in a kind of bacteria residue - Google Patents
The detection method of tylosin residual potency in a kind of bacteria residue Download PDFInfo
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- CN108205031A CN108205031A CN201711479821.8A CN201711479821A CN108205031A CN 108205031 A CN108205031 A CN 108205031A CN 201711479821 A CN201711479821 A CN 201711479821A CN 108205031 A CN108205031 A CN 108205031A
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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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- 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
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Abstract
The detection method of tylosin residual potency in a kind of bacteria residue, it is related to a kind of detection method of tylosin residual potency in bacteria residue, this method includes the preparation of reference substance solution, the preparation of test solution, high performance liquid chromatography Mass Spectrometry is used, by above-mentioned standard product solution and test solution injection liquid chromatography mass combined instrument, tylosin potency to be calculated according to external standard method in a manner of gradient elution later.The present invention establishes the high performance liquid chromatography mass spectrometry determination method of tylosin in bacteria residue by the experiment of preferred and test sample, standard items preparation method to chromatographic condition；By the optimization to chromatography and mass spectrometer system, the detection method of tylosin in bacteria residue is established, is shown by methodology validation experimental study：The method accuracy established is high, specificity is strong, reproducibility is good, can effectively detect tylosin potency in bacteria residue.
Description
Technical field
The present invention relates to the detection inspection technology fields of bacteria residue, and potency is remained more particularly to tylosin in a kind of bacteria residue
Detection method.
Background technology
Biofermentation is one of main production process of antibiotics, and fermented or pharmaceutical intermediate is engineered again
Side chain can synthesize antibiotics.After extracted in zymotic fluid, purification object, a large amount of bacteria residues are generated, China is anti-
Sheng Su big producers, these antibiotic generate a large amount of antibiotic bacterium dregs per annual meeting, and 40 tons of wet bacteria slags are generated according to 1 ton of antibiotic
(moisture content is 70% or so) calculates, and the wet bacteria slag generated is just up to 6,000,000 tons or so within 1 year 2009.Since the bacteria residue is mainly
The flocculant added in when inactivation mycelium, residual media, microbial metabolic products, part residual potency and extraction, drainage
Agent etc., if do not dealt carefully with, the antibiotic residue in bacteria residue can generate potential environmental risk by causing microorganism drug resistance
And Human Health Risk.In China, antibiotic bacterium dregs have been decided to be danger wastes, due to lacking the harmless treatment to bacteria residue
Technique and safety carry out going deep into system research, and antibiotic bacterium dregs are not only prohibited to be used as feed or feed addictive at present,
Do not allow to be used as agricultural fertilizer, burning disposal can only be carried out, but burning disposal cost is very high.
Tylosin has been widely used in livestock culture industry, be current veterinary antibiotic widely used in the world it
One, it is the most widely used pig feed additive.It is counted according to Minister Agriculture of China, international market is to tylosin year demand at present
For amount at 1500 tons or so, domestic annual requirement is 200 tons.According to estimates the U.S. have 590 tons of tylosin for ox every year and
The feed addictive of pig.Japan has 50% pannage to be added to tylosin.China's tylosin has used for many years, and a large amount of
Outlet is gone abroad, the super kiloton of annual export volume of tylosin.In the antibiotic raw material of outlet in 2007, veterinary antibiotic
Export value accounts for the 28% of whole antibiotic export amount of moneys, and quantity size accounts for 55%.Most of veterinary antibiotic 2007
Export volume increased than same period last year, and the year-on-year growth rate of sulfate colistin, tylosin and kitasamycin is more
Significantly.
The innocuity treatment problem of bacteria residue has seriously affected the sound development of current China's antibiotic pharmaceutical industry, as early as possible
Antibiotic bacterium dregs innocent processing and recycling production technology are researched and developed, it is very urgent.
There is no the completely detection sides about tylosin residual quantity in tylosin dreg in existing method standard
Method.
Invention content
The purpose of the invention is to provide a kind of detection method of tylosin residual potency in bacteria residue, to fill up existing
There is no the completely blank about the detection method of tylosin residual quantity in tylosin dreg in method standard.And it solves not having
There is a kind of effective influence for solving matrix effect, and the prior art quick/accurate can not detect tylosin residual quantity in bacteria residue
The problem of,
The detection method of tylosin residual potency in a kind of bacteria residue of the present invention, it is followed the steps below：
First, test sample sample preparation：
1) it weighs containing tylosin dreg, adds in extractant, after 1~5min of vortex oscillation, the assisted extraction in ultrasound
20~30min centrifuges 5~10min with 3000~4000rpm, takes supernatant, and it is spare to collect precipitation；
2) precipitation for collecting upper step adds in extractant, after vortex oscillation 1min, the assisted extraction 30min in ultrasound, with
3000~4000rpm centrifuges 10min, takes supernatant；
3) merge the supernatant of step 1) and step 2), by supernatant in 40 DEG C of water-bath rotary evaporations to the 1/4 of original volume
~1/5, inorganic phase is obtained, the acetate buffer of methanol solution and pH for 5.5, a concentration of 0.15mol/L is added in into inorganic phase
Solution；Fully shaking is vortexed, and after residue all dissolving, is transferred in centrifuge tube, obtains sample to be clean；
4) methanol and ultra-pure water are pipetted respectively to activate HLB solid-phase extraction columns, add in be clean obtained by above-mentioned processing
Sample crosses column with 1mL/min speed；After sample liquid all outflow, eluted successively with water and leacheate, drain all liquid
Body, analyte methanol are eluted and are collected, shakes up, and after crossing 0.45 μm of filter membrane, obtain test sample sample, are examined for liquid chromatography-mass spectrography
It surveys；
2nd, Specification Curve of Increasing：
It is 1.00mg/L, 5.00mg/L, 10.00mg/ that tylosin Standard Reserving Solution is diluted to mass concentration with methanol
L, the standard working solution of 20.00mg/L, 50.00mg/L, 100.00mg/L, 200.00mg/L and 500.00mg/L, using height
Effect liquid phase chromatogram-Mass Spectrometry is detected above-mentioned standard working solution, to measure peak area as ordinate, with standard
Solution concentration draws standard curve, and ask regression equation and related coefficient as abscissa；
3rd, it measures：It draws in above-mentioned standard working solution and test sample sample injection liquid chromatograph-mass spectrometer, adopts
It is examined with on-line solid phase extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph to remaining tylosin in soil
It surveys, and tylosin residual quantity is calculated according to external standard method；Complete the detection of tylosin residual potency in the bacteria residue；
Chromatographic condition：Chromatographic column：Agilent TC-C18 columns, chromatography column internal diameter be 4.6mm, column length 250mm, filler particles
A diameter of 5 μm, mobile phase：- 0.1% formic acid solution system gradient elution of methanol, Detection wavelength：290nm, sample size：20 μ L, stream
Speed：1mL/min, column temperature：30℃；
Mass Spectrometry Conditions：
ESI electric spray ion sources；
Cation scan pattern；
Acquisition mode：MRM；
Daughter ion mass-to-charge ratio：173.84
Ion source inner capillary tube voltage：3.5kV；
Orifice potential：50V；
Remove solvent temperature degree：350℃；
Ion source temperature：150℃；
Desolventizing gas flow：900L/Hr；
Taper hole throughput：60L/hr.
The present invention includes following advantageous effect：
The present invention arranges the Solid Phase Extraction-efficient liquid phase improved out about tylosin dreg residual quantity through experimental study
Chromatographic detection method, this method have preferable repeatability and practicability by verification, are the section of China's microbiological pharmacy bacteria residue
Effectively management, safety and utmostly recycling, realization low-carbon economy and cycle economic development provide foundation.Due in bacteria residue
Containing interfering substances such as a large amount of mycelium residuals, protein, amino acid and heavy metals, make its detection process mesostroma effect tight
Weight, therefore the present invention passes through the exploration and optimization that are extracted to tylosin dreg, concentrate, the processes such as purify so that bacteria residue
Middle impurity substances content is greatly reduced, and matrix interference weakens, this research being capable of high efficiency extraction Thailand by the modes such as extracting, purifying
Happy rhzomorph simultaneously removes impurity, reduces matrix interference, increases accuracy in detection, not only can protect detecting instrument simultaneously can be accurate
The content of tylosin in bacteria residue is detected.
The present invention carries out the residual quantity of bacteria residue tylosin for 500~900 μ g/g, and average recovery rate is：80.2%~
95.3%.The RSD of precision test is 4.1%~6.0%.
Tylosin can effectively be extracted from bacteria residue by the method for the present invention, as much as possible reduce its in bacteria residue
His influence of the impurity to subsequent detection, so as to improve the accuracy of detection.
Description of the drawings
Fig. 1 is the canonical plotting in the range of Examples 1 to 5 00mg/L, and the regression equation of standard curve is y=
14115.03x-25366.01；R2=0.998；
Fig. 2 is the standard items measurement chart of embodiment 100mg/L；
Fig. 3 is the standard items collection of illustrative plates of embodiment 100mg/L；
Fig. 4 is the sample collection of illustrative plates of embodiment 1g sample detections；
Fig. 5 is embodiment not into the baseline collection of illustrative plates of sample.
Specific embodiment
Specific embodiment one：The detection method of tylosin residual potency in a kind of bacteria residue of present embodiment, it is
It follows the steps below：
First, test sample sample preparation：
1) it weighs containing tylosin dreg, adds in extractant, after 1~5min of vortex oscillation, the assisted extraction in ultrasound
20~30min centrifuges 5~10min with 3000~4000rpm, takes supernatant, and it is spare to collect precipitation；
2) precipitation for collecting upper step adds in extractant, after vortex oscillation 1min, the assisted extraction 30min in ultrasound, with
3000~4000rpm centrifuges 10min, takes supernatant；
3) merge the supernatant of step 1) and step 2), by supernatant in 40 DEG C of water-bath rotary evaporations to the 1/4 of original volume
~1/5, inorganic phase is obtained, the acetate buffer of methanol solution and pH for 5.5, a concentration of 0.15mol/L is added in into inorganic phase
Solution；Fully shaking is vortexed, and after residue all dissolving, is transferred in centrifuge tube, obtains sample to be clean；
4) methanol and ultra-pure water are pipetted respectively to activate HLB solid-phase extraction columns, add in be clean obtained by above-mentioned processing
Sample crosses column with 1mL/min speed；After sample liquid all outflow, eluted successively with water and leacheate, drain all liquid
Body, analyte methanol are eluted and are collected, shakes up, and after crossing 0.45 μm of filter membrane, obtain test sample sample, are examined for liquid chromatography-mass spectrography
It surveys；
2nd, Specification Curve of Increasing：
It is 1.00mg/L, 5.00mg/L, 10.00mg/ that tylosin Standard Reserving Solution is diluted to mass concentration with methanol
L, the standard working solution of 20.00mg/L, 50.00mg/L, 100.00mg/L, 200.00mg/L and 500.00mg/L, using height
Effect liquid phase chromatogram-Mass Spectrometry is detected above-mentioned standard working solution, to measure peak area as ordinate, with standard
Solution concentration draws standard curve, and ask regression equation and related coefficient as abscissa；
3rd, it measures：It draws in above-mentioned standard working solution and test sample sample injection liquid chromatograph-mass spectrometer, adopts
It is examined with on-line solid phase extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph to remaining tylosin in soil
It surveys, and tylosin residual quantity is calculated according to external standard method；Complete the detection of tylosin residual potency in the bacteria residue；
Chromatographic condition：Chromatographic column：Agilent TC-C18 columns, chromatography column internal diameter be 4.6mm, column length 250mm, filler particles
A diameter of 5 μm, mobile phase：- 0.1% formic acid solution system gradient elution of methanol, Detection wavelength：290nm, sample size：20 μ L, stream
Speed：1mL/min, column temperature：30℃；
Mass Spectrometry Conditions：
ESI electric spray ion sources；
Cation scan pattern；
Acquisition mode：MRM；
Daughter ion mass-to-charge ratio：173.84
Ion source inner capillary tube voltage：3.5kV；
Orifice potential：50V；
Remove solvent temperature degree：350℃；
Ion source temperature：150℃；
Desolventizing gas flow：900L/Hr；
Taper hole throughput：60L/hr.
Specific embodiment two：The present embodiment is different from the first embodiment in that：Leacheate is containing volume basis
The aqueous solution that content is 2% ammonium hydroxide and volumn concentration is 5% methanol.It is other same as the specific embodiment one.
Specific embodiment three：The present embodiment is different from the first embodiment in that：On-line solid phase extraction-super-pressure
The Mass Spectrometry Conditions of liquid chromatogram-triple quadrupole bar liquid phase mass spectrograph are using reaction of high order monitoring pattern, using positive ion mode
The parent ion and fragment of tylosin are detected.It is other same as the specific embodiment one.
Specific embodiment four：The present embodiment is different from the first embodiment in that：Step 1) and 2) extractant are equal
It is 90% acetonitrile solution for mass percentage, pH=4.It is other same as the specific embodiment one.
Specific embodiment five：The present embodiment is different from the first embodiment in that：Tylosin dreg and extraction
The mass volume ratio of agent is 1g：10mL.It is other same as the specific embodiment one.
Specific embodiment six：The present embodiment is different from the first embodiment in that：Precipitation and the quality of extractant
Volume ratio is 1g：10mL.It is other same as the specific embodiment one.
Specific embodiment seven：The present embodiment is different from the first embodiment in that：Methanol solution delays with sodium acetate
The volume ratio for rushing solution is 1:9.It is other same as the specific embodiment one.
Specific embodiment eight：The present embodiment is different from the first embodiment in that：In step 4) methanol with it is ultrapure
The volume ratio 1 of water:1.It is other same as the specific embodiment one.
Specific embodiment nine：The present embodiment is different from the first embodiment in that：Water and leacheate in step 4)
Volume ratio 1:1.It is other same as the specific embodiment one.
Specific embodiment ten：The present embodiment is different from the first embodiment in that：Tylosin is residual in bacteria residue
Allowance is 500~900 μ g/g.It is other same as the specific embodiment one.
Specific embodiment 11：The present embodiment is different from the first embodiment in that：Tylosin in bacteria residue
Residual quantity is 500~900 μ g/g.It is other same as the specific embodiment one.
The content of present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
Contract sample can also realize the purpose of invention.
Beneficial effects of the present invention are verified by following embodiment：
Embodiment 1
The detection method of tylosin residual potency, includes the following steps in a kind of bacteria residue of the present embodiment：
Instrument：On-line solid phase extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph (U.S. Waters, Open
Architecture UPLC,Xevo TQ MS)。
Chromatographic condition：Chromatographic column is Agilent TC-C18 columns (4.6mm × 250mm, 5 μm), mobile phase：Methanol -0.1%
Formic acid solution system gradient elution, gradient elution program (A) methanol；(B) 0.1% formic acid：Time 0min, 45%A, 55%B；
3min, 45%A, 55%B；6min, 90%A, 10%B；8min, 45%A, 55%B.Detection wavelength：290nm, sample size：20μ
L, flow velocity：1mL/min, column temperature：30℃.
Mass Spectrometry Conditions：
ESI electric spray ion sources；
Cation scan pattern；
Acquisition mode：MRM；
Daughter ion mass-to-charge ratio：173.84
Ion source inner capillary tube voltage：3.5kV；
Orifice potential：50V；
Remove solvent temperature degree：350℃；
Ion source temperature：150℃；
Desolventizing gas flow：900L/Hr；
Taper hole throughput：60L/hr.
Mass Spectrometry Conditions use reaction of high order monitoring pattern (MRM), use positive ion mode to the parent ion of tylosin with
And fragment is detected.
1) test sample sample preparation：Sample 1g (being accurate to 0.0001g) is weighed, in 50mL polypropylene centrifuge tubes, is added in
10mL extractants (pH=4,90% acetonitrile solution), vortex oscillation 1min, the assisted extraction 30min in ultrasound, with 3000~
4000rpm centrifuges 10min, takes supernatant.Repetitive operation is primary (adding in the operation that extractant obtains supernatant), merges supernatant
Liquid.Extracting solution is transferred to 50mL polypropylene centrifuge tubes, adds 0.5mL in 40 DEG C of water-bath rotary evaporations to surplus 2mL inorganic phases
Methanol solution and pH be the sodium acetate buffer solution of 5.5 0.15mol/L in 10mL centrifuge tubes, fully shaking is vortexed
1min after residue all dissolving, is transferred to 10mL polypropylene centrifuge tubes.5mL methanol is pipetted respectively, 5mL ultra-pure waters consolidate HLB
Phase extraction column is activated, and adds in above-mentioned processing gained sample, column is crossed with 1mL/min speed, after sample liquid all outflow, use
5mL water, 5mL leacheate (aqueous solution of 5% methanol Han 2% ammonium hydroxide) eluted, drain all liquid, analyte is used
5mL methanol is eluted and is collected.It shakes up, after crossing 0.45 μm of filter membrane, test sample is obtained, for liquid chromatographic detection.
2) standard items Drawing of Curve：With methanol by Standard Reserving Solution be diluted to mass concentration be 1.00mg/L, 5.00mg/L,
10.00mg/L, 20.00mg/L, 50.00mg/L, 100.00mg/L, 200.00mg/L and 500.00mg/L standard solution use
Tablets by HPLC-MS is detected above-mentioned standard working solution, to measure peak area as ordinate, with right
The concentration of standard solution answered draws standard curve, asks regression equation and related coefficient as abscissa；
3) it measures：It draws in above-mentioned standard product solution and each 10 μ L injections liquid chromatograph-mass spectrometer of test solution,
Tylosin residual potency is calculated according to external standard method.
The detection method of tylosin residual potency, experiment process are found in the bacteria residue of the present embodiment：In bacteria residue impurity compared with
More, existing method cannot effectively detect tylosin, and detected level is small, and sensitivity is low.It to quantify and control, it is difficult；Together
When sample handling processes it is complicated, cumbersome so that part tylosin loses.By many experiments, according to the property of each impurity, carry
It takes, purification of samples, while using gradient elution mode, micro tylosin is effectively detected, and obtain and efficiently separate, effectively examined
Survey the tylosin potency in bacteria residue.
Verification result shows：The average recovery rate of accuracy test is：80.2%~95.3%.The RSD of precision test
Respectively 4.1%~6.0%；Linear relationship is good, correlation coefficient r2=0.998；The negative interference of specificity experiment investigation.Examination
It verifies that bright established method accuracy is high, specificity is strong, reproducibility is good, can effectively detect tylosin residual effect in bacteria residue
Valency.
Embodiment 2
Tylosin remains analyte detection in bacteria residue, it is followed the steps below：
Instrument：On-line solid phase extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph (U.S. Waters, Open
Architecture UPLC,Xevo TQ MS)。
Chromatographic condition：Chromatographic column：Agilent TC-C18 columns (4.6mm × 250mm, 5 μm), mobile phase take methanol-
0.1% formic acid solution system gradient elution, gradient elution program (A) methanol；(B) 0.1% formic acid：Time 0min, 45%A,
55%B；3min, 45%A, 55%B；6min, 90%A, 10%B；8min, 45%A, 55%B.Detection wavelength：290nm, sample introduction
Amount：20 μ L, flow velocity：1mL/min, column temperature：30℃.
Mass Spectrometry Conditions：
ESI electric spray ion sources；
Cation scan pattern；
Acquisition mode：MRM；
Daughter ion mass-to-charge ratio：173.84
Ion source inner capillary tube voltage：3.5kV；
Orifice potential：50V；
Remove solvent temperature degree：350℃；
Ion source temperature：150℃；
Desolventizing gas flow：900L/Hr；
Taper hole throughput：60L/hr.
1) test sample sample preparation：Sample 1g (being accurate to 0.0001g) is weighed, in 50mL polypropylene centrifuge tubes, is added in
10mL extractants (pH=4,90% acetonitrile solution), vortex oscillation 1min, the assisted extraction 30min in ultrasound, with 3000~
4000rpm centrifuges 10min, takes supernatant.Repetitive operation is primary (adding in the operation that extractant obtains supernatant), merges supernatant
Liquid.Extracting solution is transferred to 50mL polypropylene centrifuge tubes, adds 0.5mL in 40 DEG C of water-bath rotary evaporations to surplus 2mL inorganic phases
Methanol solution and pH be the sodium acetate buffer solution of 5.5 0.15mol/L in 10mL centrifuge tubes, fully shaking is vortexed
1min after residue all dissolving, is transferred to 10mL polypropylene centrifuge tubes.5mL methanol is pipetted respectively, 5mL ultra-pure waters consolidate HLB
Phase extraction column is activated, and adds in above-mentioned processing gained sample, column is crossed with 1mL/min speed, after sample liquid all outflow, use
5mL water, 5mL leacheate (aqueous solution of 5% methanol Han 2% ammonium hydroxide) eluted, drain all liquid, analyte is used
5mL methanol is eluted and is collected.It shakes up, liquid chromatographic detection is supplied after crossing 0.45 μm of filter membrane.
2) standard items Drawing of Curve：With methanol by Standard Reserving Solution be diluted to mass concentration be 1.00mg/L, 5.00mg/L,
10.00mg/L, 20.00mg/L, 50.00mg/L, 100.00mg/L, 200.00mg/L and 500.00mg/L standard solution use
Tablets by HPLC-MS is detected above-mentioned standard working solution, to measure peak area as ordinate, with right
The concentration of standard solution answered draws standard curve, asks regression equation and related coefficient as abscissa；
3) it measures：It draws in above-mentioned standard product solution and each 10 μ L injections liquid chromatograph-mass spectrometer of test solution,
Tylosin residual potency is calculated according to external standard method.
Method validation：
By certain processing, in reduced levels, the mark-on in blank sample is dense for tylosin residual quantity in bacteria residue
Degree respectively 10,50,200mg/kg.Analysis detection is carried out using this experimental method, laboratory repeats 3 times, and that acquires returns
Yield and its standard deviation, the content of tylosin is calculated by formula (1) in sample.
In formula：The content of tylosin in X-sample, unit are every gram of microgram (μ g/g)；
The concentration of tylosin in C-sample solution, unit are milligrams per liter (μ g/mL)；
M-sample quality, unit are gram (g)；
V-sample solution volume, unit are milliliter (mL)
When mark-on level is in 10-200mg Kg-1When, the tylosin average recovery rate ranging from 79.9%- of bacteria residue
93.2%, Standard deviation-Range is 4.1%~6.0% (n=3), and it is 0.061 μ g/ that LOD, which is calculated, as 0.048 μ g/mg, LOQ
mg.And the absolute difference of measurement result independent twice obtained under the conditions of repeatability must not exceed the 10% of arithmetic mean of instantaneous value.
Claims (10)
1. the detection method of tylosin residual potency in a kind of bacteria residue, it is characterised in that it is followed the steps below：
First, test sample sample preparation：
1) weigh containing tylosin dreg, add in extractant, after 1~5min of vortex oscillation, in ultrasound assisted extraction 20~
30min centrifuges 5~10min with 3000~4000rpm, takes supernatant, and it is spare to collect precipitation；
2) precipitation for collecting upper step adds in extractant, after vortex oscillation 1min, the assisted extraction 30min in ultrasound, with 3000
~4000rpm centrifuges 10min, takes supernatant；
3) merge the supernatant of step 1) and step 2), by supernatant in 40 DEG C of water-bath rotary evaporations to the 1/4~1/ of original volume
5, inorganic phase is obtained, the sodium acetate buffer solution of methanol solution and pH for 5.5, a concentration of 0.15mol/L is added in into inorganic phase；
Fully shaking is vortexed, and after residue all dissolving, is transferred in centrifuge tube, obtains sample to be clean；
4) methanol and ultra-pure water are pipetted respectively to activate HLB solid-phase extraction columns, add in above-mentioned processing gained sample to be clean,
Column is crossed with 1mL/min speed；After sample liquid all outflow, eluted successively with water and leacheate, drain all liquid, point
Analysis object methanol is eluted and is collected, and is shaken up, and after crossing 0.45 μm of filter membrane, obtains test sample sample, is detected for liquid chromatography-mass spectrography；
2nd, Specification Curve of Increasing：
With methanol by tylosin Standard Reserving Solution be diluted to mass concentration be 1.00mg/L, 5.00mg/L, 10.00mg/L,
The standard working solution of 20.00mg/L, 50.00mg/L, 100.00mg/L, 200.00mg/L and 500.00mg/L, using efficient
Liquid Chromatography-Mass Spectrometry is detected above-mentioned standard working solution, molten with standard to measure peak area as ordinate
Liquid concentration draws standard curve, and ask regression equation and related coefficient as abscissa；
3rd, it measures：Draw above-mentioned standard working solution and test sample sample injection liquid chromatograph-mass spectrometer in, using
Line Solid Phase Extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph is detected to remaining tylosin in soil,
And tylosin residual quantity is calculated according to external standard method；Complete the detection of tylosin residual potency in the bacteria residue；
Chromatographic condition：Chromatographic column：Agilent TC-C18 columns, chromatography column internal diameter be 4.6mm, column length 250mm, filler particles diameter
It is 5 μm, mobile phase：- 0.1% formic acid solution system gradient elution of methanol, Detection wavelength：290nm, sample size：20 μ L, flow velocity：
1mL/min, column temperature：30℃；
Mass Spectrometry Conditions：
ESI electric spray ion sources；
Cation scan pattern；
Acquisition mode：MRM；
Daughter ion mass-to-charge ratio：173.84
Ion source inner capillary tube voltage：3.5kV；
Orifice potential：50V；
Remove solvent temperature degree：350℃；
Ion source temperature：150℃；
Desolventizing gas flow：900L/Hr；
Taper hole throughput：60L/hr.
2. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that elution
The aqueous solution that it is 2% ammonium hydroxide containing volumn concentration that liquid, which is, and volumn concentration is 5% methanol.
3. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that online
The Mass Spectrometry Conditions of Solid Phase Extraction-ultrahigh pressure liquid phase chromatography-triple quadrupole bar liquid phase mass spectrograph are to use reaction of high order monitoring pattern,
The parent ion and fragment of tylosin are detected using positive ion mode.
4. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that step
And 2) 1) extractant is mass percentage for 90% acetonitrile solution, pH=4.
5. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that Thailand is happy
The mass volume ratio of rhzomorph bacteria residue and extractant is 1g：10mL.
6. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that precipitation
Mass volume ratio with extractant is 1g：10mL.
7. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that methanol
The volume ratio of solution and sodium acetate buffer solution is 1:9.
8. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that step
4) volume ratio 1 of methanol and ultra-pure water in:1.
9. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that step
4) volume ratio 1 of water and leacheate in:1.
10. the detection method of tylosin residual potency in a kind of bacteria residue according to claim 1, it is characterised in that bacteria residue
The residual quantity of middle tylosin is 500~900 μ g/g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711479821.8A CN108205031A (en) | 2017-12-29 | 2017-12-29 | The detection method of tylosin residual potency in a kind of bacteria residue |
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