CN106153741A - The detection method of tetracycline antibiotics in a kind of Lac Bovis seu Bubali - Google Patents
The detection method of tetracycline antibiotics in a kind of Lac Bovis seu Bubali Download PDFInfo
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Abstract
The invention provides the detection method of tetracycline antibiotics in a kind of Lac Bovis seu Bubali, including sample extraction, sample purification and Instrumental Analysis step, sample extraction step is: add Na in milk sample to be measured2EDTA-acetic acid aqueous solution, centrifuging and taking supernatant after mixing, it is sample extracting solution.The detection method of tetracycline antibiotics in the Lac Bovis seu Bubali that the present invention provides, result is accurate, simple and efficient to handle.
Description
Technical field
The present invention relates to a kind of food test method, particularly to the method for inspection of a kind of Lac Bovis seu Bubali veterinary drug residue.
Background technology
Tetracycline antibiotics includes tetracycline, oxytetracycline, chlortetracycline and doxycycline, due to its broad-spectrum antimicrobial effect, during being commonly used in milk cattle cultivating, in order to prevent and treat milch cow digestive system infection and to promote milch cow growth.But in order to prevent medicine from entering human body with food chain, consumer health is produced threat, European Union and the Ministry of Agriculture of China tetracycline antibiotics residue limits in Lac Bovis seu Bubali is all made strict rules.
The method being presently used for detecting tetracycline antibiotics is a lot, substantially can be divided into microbial method, euzymelinked immunosorbent assay (ELISA), high performance capillary electrophoresis, electrochemical methods, high performance liquid chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS).In HPLC method and LC-MS method, the pre-treating method that sample-pretreating method generally provides according to " Tetracyclines residue of veterinary drug quantity measuring method-liquid chromatography-mass spectrography/mass spectrography and high performance liquid chromatography in GB/T21317-2007 animal derived food ", uses Na2EDTA-Mellvaine buffer solution carries out sample extraction, and this extracting solution preparation steps is loaded down with trivial details, causes taking a lot of time in sample pre-treatments step, is unfavorable for the daily quick detection of enterprise.
Summary of the invention
It is an object of the invention to provide the detection method of tetracycline antibiotics in a kind of Lac Bovis seu Bubali, result is accurate, simple and efficient to handle.
The detection method of tetracycline antibiotics in Lac Bovis seu Bubali disclosed by the invention, including sample extraction, sample purification and Instrumental Analysis step, sample extraction step is: adds Na2EDTA-acetic acid aqueous solution, centrifuging and taking supernatant after mixing in milk sample to be measured, is sample extracting solution.
In Lac Bovis seu Bubali in a kind of exemplary embodiment of the detection method of tetracycline antibiotics, sample extraction step is: weigh milk sample 5g to be measured, adds 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, centrifugal after mixing, take supernatant, be sample extracting solution.
In Lac Bovis seu Bubali in a kind of exemplary embodiment of the detection method of tetracycline antibiotics, sample extraction step is: weigh milk sample 5g to be measured in 50mL centrifuge tube, add 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, eddy mixer mixes 30s, then it was centrifuged 8 minutes under 10000r/ minute, take 5mL supernatant, be sample extracting solution.
In Lac Bovis seu Bubali in a kind of exemplary embodiment of the detection method of tetracycline antibiotics, sample purification step is: take HLB solid-phase extraction column, activates respectively with 3 to 5mL methanol and 3 to 5mL ultra-pure water and balances；Again sample extracting solution is less than 2mL/ minute through HLB solid-phase extraction column, the flow velocity of solid-phase extraction device；After sample extracting solution is all by HLB solid-phase extraction column, washs HLB solid-phase extraction column with 3 to 5mL5% methanol aqueous solution, discard whole effluent；Again with 3 to 5mL methanol-eluted fractions HLB solid-phase extraction column, and collect eluent in centrifuge tube；Then the nitrogen under 40 DEG C of water-baths of the eluent in centrifuge tube is dried up, add 1.0mL constant volume liquid vortex and dissolve, finally with 0.22 μm organic system membrane filtration, be i.e. purified rear sample；The wherein compound method of constant volume liquid: take 10mL acetonitrile, be settled to 100mL with water.
In Lac Bovis seu Bubali in a kind of exemplary embodiment of the detection method of tetracycline antibiotics, Instrumental Analysis step uses tablets by HPLC-MS, and chromatographic condition is: chromatographic column is ACQUITY UPLC BEH C18 post, 2.1 × 100mm, 1.7 μm；Column temperature is 40 DEG C；Sampling volume is 10 μ L；Mobile phase A is acetonitrile, and Mobile phase B is 0.1% formic acid-water；Gradient elution: 0～1.0 minute, 15%A；1.0～2.0 minutes, 15%A～25%A；2.0～2.5 minutes, 25%A～30%A；2.5～3.0 minutes, 30%A；3.0～3.5 minutes, 30%A～40%A；3.5～4.5 minutes, 40%A～80%A；4.5～5.0 minutes, 80%A～15%A, flow velocity was 0.4mL/ minute；Mass Spectrometry Conditions is: ionization mode is electron spray positive ion mode, and scanning of the mass spectrum mode is multiple-reaction monitoring, and ionization voltage is 3.00kV, ion source temperature is 120 DEG C, and going solvent gas temperature is 400 DEG C, and removing solvent gas is N2, flow is 500L/h, and collision gas is Ar, and flow is 50L/h.
The detection method of tetracycline antibiotics in the Lac Bovis seu Bubali that the present invention provides, result is accurate, simple and efficient to handle；Reagent can also be saved, thus reduce analysis cost, be more beneficial for environmental conservation.The method of the present invention is applicable not only to the detection of fresh cow milk, is applied equally to the detection of reconstituted milk or milk powder.
Detailed description of the invention
In order to technical characteristic, purpose and the effect of invention are more clearly understood from, the detailed description of the invention of the present invention is described in conjunction with following example.
Embodiment
1
。
1, experimental apparatus
Ultra Performance Liquid Chromatography-QQ-TOF mass spectrometry instrument: be furnished with electric spray ion source (ESI) (TQD U.S. Waters)；Solid-phase extraction device (U.S. Supelco).
2, experiment reagent and material
Tetracycline, oxytetracycline, chlortetracycline, doxycycline standard substance (Germany Dr. Ehrenstorfer
GmbH company)；
Methanol, acetonitrile are chromatographically pure；Glacial acetic acid (top grade is pure)；Use for laboratory water is deionized water；HLB solid-phase extraction column (60mg/3mL, waters company of the U.S.).
3, the preparation of solution
3.1, standard solution
Accurately weigh appropriate tetracycline, oxytetracycline, chlortetracycline, doxycycline standard substance, be configured to the standard reserving solution of 100mg/L ,-18 DEG C of refrigerator storage with methanol.During use, it is diluted to the standard working solution of debita spissitudo as required with 10% acetonitrile solution；
3.2, sample extracting solution
0.1mol/L Na2EDTA-acetic acid aqueous solution: add 2ml glacial acetic acid in 1000ml volumetric flask, be settled to scale with ultra-pure water, add 37.23g disodiumedetate, dissolve and shake up.Preparation steps about needs 5 minutes；
0.2% acetic acid aqueous solution: take 2mL glacial acetic acid, be settled to 1L with water；
Leacheate: take 5mL methanol, be settled to 100mL with water；
Constant volume liquid: take 10mL acetonitrile, be settled to 100mL with water.
4, chromatographic condition
Chromatographic column: ACQUITY UPLC BEH C18 post (2.1 × 100mm, 1.7 μm)；Column temperature: 40 DEG C；Sampling volume: 10 μ L；Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid-water；Gradient elution: 0～1.0 minute, 15%A；1.0～2.0 minutes, 15%A～25%A；2.0～2.5 minutes, 25%A～30%A；2.5～3.0 minutes, 30%A；3.0～3.5 minutes, 30%A～40%A；3.5～4.5 minutes, 40%A～80%A；4.5～5.0 minutes, 80%A～15%A；Flow velocity: 0.4mL/ minute.
5, Mass Spectrometry Conditions
Ionization mode: electron spray positive ion mode (ES+)；
Scanning of the mass spectrum mode: multiple-reaction monitoring (MRM)；
Ionization voltage (kv): 3.00；
Ion source temperature (DEG C): 120；
Remove solvent gas temperature (DEG C): 400；
Remove solvent gas (N2) flow (L/h): 500；
Collision gas (high-purity Ar) flow (L/h): 50；
The mass spectrometry parameters such as taper hole voltage, collision energy and qualitative and quota ion are as shown in table 1:
The mass spectrometry parameters of table 1 Instrumental Analysis sets
| Compound | Parent ion (m/z) | Daughter ion 1(m/z) | Collision energy 1(eV) | Daughter ion 2(m/z) | Collision energy 2(eV) | Residence time (s) | Taper hole voltage (V) |
| Oxytetracycline | 461.2 | 426.1* | 22 | 443.2 | 13 | 0.1 | 22 |
| Tetracycline | 445 | 409.7* | 22 | 426.8 | 17 | 0.1 | 22 |
| Chlortetracycline | 478.8 | 443.6* | 27 | 461.6 | 23 | 0.1 | 22 |
| Doxycycline | 445 | 427.7* | 23 | 153.6 | 38 | 0.1 | 22 |
Note: * is quota ion.
6, sample pre-treatments
6.1, the extraction of sample
Weigh milk sample 5g(and be accurate to 0.0001g) in 50mL centrifuge tube, add 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, mixes 30s on eddy mixer, then under 10000r/ minute centrifugal 8 minutes, takes 5mL supernatant in another centrifuge tube, to be clean；
6.2, the purification of sample
Take HLB solid-phase extraction column (50mg/3mL), respectively by 3mL methanol and the activation of 3mL water and balance, by above-mentioned supernatant to be clean through solid-phase extraction column, on solid-phase extraction device, coutroi velocity is within 2mL/ minute, after sample liquid is all by solid-phase extraction column, washs HLB solid-phase extraction column with 3mL 5% methanol aqueous solution leacheate, discard whole effluent, finally with 3mL methanol-eluted fractions solid-phase extraction column, and collect eluent in centrifuge tube.Then being dried up by the nitrogen under 40 DEG C of water-baths of the eluent in centrifuge tube, add 1.0mL constant volume liquid vortex and dissolve, cross 0.22 μm organic system filter membrane, LC-MS/MS analyzes.Purifying step needs 30 minutes.
Preparation steps and purifying step need 35 minutes altogether.
Embodiment
2
。
Sample is milk powder.The present embodiment is only otherwise varied in " extractions of 6.1 samples ", " 6.2, the purification of sample " step of embodiment 1, and remaining step is all identical with embodiment 1.
6.1, the extraction of sample
Weigh powdered milk sample 1g(and be accurate to 0.0001g) in 50mL centrifuge tube, add 5mL water dissolution, add 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, mixes 30s on eddy mixer, then under 10000r/ minute centrifugal 8 minutes, takes 5mL supernatant in another centrifuge tube, to be clean.
6.2, the purification of sample
Take HLB solid-phase extraction column (50mg/3mL), respectively by 4mL methanol and the activation of 4mL water and balance, by above-mentioned supernatant to be clean through solid-phase extraction column, on solid-phase extraction device, coutroi velocity is within 2mL/ minute, after sample liquid is all by solid-phase extraction column, washs HLB solid-phase extraction column with 4mL 5% methanol aqueous solution leacheate, discard whole effluent, finally with 4mL methanol-eluted fractions solid-phase extraction column, and collect eluent in centrifuge tube.Then being dried up by the nitrogen under 40 DEG C of water-baths of the eluent in centrifuge tube, add 1.0mL constant volume liquid vortex and dissolve, cross 0.22 μm organic system filter membrane, LC-MS/MS analyzes.Purifying step needs 40 minutes.
Preparation steps and purifying step need 45 minutes altogether.
Embodiment
3
。
Sample is milk powder.The present embodiment is only otherwise varied in " extractions of 6.1 samples ", " 6.2, the purification of sample " step of embodiment 1, and remaining step is all identical with embodiment 1.
6.1, the extraction of sample
Weigh powdered milk sample 1g(and be accurate to 0.0001g) in 50mL centrifuge tube, add 5mL water dissolution, add 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, mixes 30s on eddy mixer, then under 10000r/ minute centrifugal 8 minutes, takes 5mL supernatant in another centrifuge tube, to be clean.
6.2, the purification of sample
Take HLB solid-phase extraction column (50mg/3mL), respectively by 5mL methanol and the activation of 5mL water and balance, by above-mentioned supernatant to be clean through solid-phase extraction column, on solid-phase extraction device, coutroi velocity is within 2mL/ minute, after sample liquid is all by solid-phase extraction column, washs HLB solid-phase extraction column with 5mL 5% methanol aqueous solution leacheate, discard whole effluent, finally with 5mL methanol-eluted fractions solid-phase extraction column, and collect eluent in centrifuge tube.Then being dried up by the nitrogen under 40 DEG C of water-baths of the eluent in centrifuge tube, add 1.0mL constant volume liquid vortex and dissolve, cross 0.22 μm organic system filter membrane, LC-MS/MS analyzes.Purifying step needs 50 minutes.
Preparation steps and purifying step need 55 minutes altogether.
Embodiment
4
(comparative example).
The present embodiment is only otherwise varied in 6.1 and 6.2 steps of embodiment 1, and remaining step is all identical with embodiment 1.
6.1, the extraction of sample
Weigh milk sample 5g(and be accurate to 0.0001g) in 50mL centrifuge tube, add Na2EDTA-Mellvaine buffer solution 20mL, mixes 30s on eddy mixer, then under 10000r/ minute centrifugal 8 minutes, takes 5mL supernatant in another centrifuge tube, to be clean.
Wherein Na2The compound method of EDTA-Mcllvaine buffer solution is: weigh 21.01g citric acid, dissolves with ultra-pure water, is settled to 1000ml, is configured to the citric acid solution of 0.1mol/L；Weigh 28.41g disodium hydrogen phosphate, with water dissolution and be settled to 1000ml, be configured to the disodium phosphate soln of 0.2mol/L；The citric acid solution of 1000ml0.1mol/L is mixed with the disodium phosphate soln of 625ml0.2mol/L, and with sodium hydroxide or salt acid for adjusting pH value to 4.0, is configured to Mcllvaine solution；Weigh 60.5g disodiumedetate to put in 1625ml Mcllvaine solution so that it is dissolve and mix.30 minutes preparation time.
6.2, the purification of sample
Take HLB solid-phase extraction column (50mg/3mL), respectively by 5mL methanol and the activation of 5mL water and balance, take 10ml extracting solution and cross HLB solid-phase extraction column with the speed of 1/s, after sample liquid flows completely out, successively with 5ml water and 5ml5% methanol-water solution drip washing, decompressing and extracting 5 minutes under 2.0kPa, finally with 10ml methanol-ethyl acetate eluting.Being dried up by nitrogen under 40 DEG C of water-baths of eluent, add 1.0mL and determine the dissolving of solution vortex, cross 0.22 μm organic system filter membrane, LC-MS/MS analyzes.Purifying step needs 50 minutes.
Preparation steps and purifying step need 80 minutes altogether.
Embodiment
5
(compliance test result).
Standard curve linear: according to the method compound concentration in embodiment 1 be 0.005 μ g/mL, the standard working solution of six Concentraton gradient of 0.01 μ g/mL, 0.02 μ g/mL, 0.05 μ g/mL, 0.10 μ g/mL, 0.20 μ g/mL, detect according to the instrument condition in embodiment 1, with the peak area Y of determinand quota ion to its mass concentration X(μ g/L) obtain the standard curve of tetracycline, oxytetracycline, chlortetracycline and doxycycline, its correlation coefficient (R2) closer to 1, then explanation linear relationship is the best.As shown in table 2 below:
Table 2 standard curve correlation coefficient
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | |
| Oxytetracycline | 0.996381 | 0.992833 | 0.993207 | 0.996216 |
| Tetracycline | 0.993264 | 0.999071 | 0.996238 | 0.99712 |
| Chlortetracycline | 0.992142 | 0.998653 | 0.99447 | 0.994524 |
| Doxycycline | 0.990533 | 0.992145 | 0.99574 | 0.996588 |
By upper table it can be seen that compared with Example 4, oxytetracycline, tetracycline, chlortetracycline and doxycycline standard curve and embodiment 4(GB in embodiment 1-3) effect is suitable, all has good linear relationship.
Embodiment
6
(compliance test result).
Method recovery test: not detect the Lac Bovis seu Bubali/milk powder of tetracycline antibiotics as blank sample, is response rate experiment, spiked levels 10 μ g/L, 20 μ g/L, 50 μ g/L and 100 μ g/L respectively to blank sample mark-on.The ult rec recording tetracycline antibiotics is as shown in table 3, and the method response rate can meet the needs of detection.
The table 3 method response rate (%)
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | |
| Oxytetracycline 10 μ g/L | 95.4~105.7 | 92.3~96.8 | 93.4~102.1 | 88.6~110.4 |
| Oxytetracycline 20 μ g/L | 93.2~99.1 | 93.3~104.5 | 92.8~98.6 | 94.0~98.7 |
| Oxytetracycline 50 μ g/L | 92.4~110.6 | 86.1~95.2 | 90.7~97.2 | 91.5~99.8 |
| Oxytetracycline 100 μ g/L | 89.7~107.9 | 94.5~99.8 | 94.3~105.6 | 87.9~96.8 |
| Tetracycline 10 μ g/L | 84.3~96.5 | 79.5~94.1 | 87.2~95.6 | 82.1~97.0 |
| Tetracycline 20 μ g/L | 90.4~97.2 | 82.7~94.8 | 84.6~97.2 | 92.6~99.4 |
| Tetracycline 50 μ g/L | 84.1~98.0 | 85.9~97.4 | 80.7~96.8 | 90.8~104.2 |
| Tetracycline 100 μ g/L | 92.3~99.8 | 90.4~102.5 | 88.3~98.8 | 88.1~97.7 |
| Chlortetracycline 10 μ g/L | 75.2~90.5 | 74.2~89.5 | 77.0~91.4 | 86.3~91.1 |
| Chlortetracycline 20 μ g/L | 78.4~93.2 | 83.6~93.6 | 86.1~95.3 | 72.4~87.5 |
| Chlortetracycline 50 μ g/L | 82.9~94.7 | 80.5~93.7 | 82.5~92.8 | 86.5~92.8 |
| Chlortetracycline 100 μ g/L | 80.6~97.4 | 76.0~88.4 | 79.7~90.4 | 71.0~87.9 |
| Doxycycline 10 μ g/L | 90.4~98.0 | 92.7~104.6 | 88.3~98.5 | 86.8~94.3 |
| Doxycycline 20 μ g/L | 88.6~96.7 | 88.5~97.2 | 90.6~100.7 | 91.5~112.4 |
| Doxycycline 50 μ g/L | 90.9~109.8 | 90.0~98.4 | 88.6~97.3 | 92.4~98.6 |
| Doxycycline 100 μ g/L | 86.9~99.2 | 89.4~99.7 | 91.4~98.5 | 90.1~97.5 |
By upper table it can be seen that embodiments of the invention 1-3 is compared with existing method (embodiment 4), the response rate of oxytetracycline, tetracycline, chlortetracycline and doxycycline is the highest, has all reached the effect suitable with existing method.
Embodiment
7
(compliance test result).
Method precision is tested: the mark-on sample duplicate detection 6 times to spiked levels each in embodiment 8 (10 μ g/L, 20 μ g/L, 50 μ g/L and 100 μ g/L) respectively, calculates the relative standard deviation (RSD%) of testing result, the results are shown in Table shown in 4.
Table 4 method precision (RSD%)
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | |
| Oxytetracycline 10 μ g/L | 5.22 | 2.11 | 5.42 | 6.22 |
| Oxytetracycline 20 μ g/L | 3.59 | 5.36 | 5.83 | 4.36 |
| Oxytetracycline 50 μ g/L | 6.46 | 4.87 | 4.96 | 5.43 |
| Oxytetracycline 100 μ g/L | 7.90 | 3.05 | 5.2 | 4.46 |
| Tetracycline 10 μ g/L | 6.33 | 8.69 | 4.67 | 7.88 |
| Tetracycline 20 μ g/L | 5.42 | 6.20 | 4.63 | 4.84 |
| Tetracycline 50 μ g/L | 7.58 | 5.49 | 6.29 | 7.31 |
| Tetracycline 100 μ g/L | 4.55 | 5.19 | 5.89 | 6.55 |
| Chlortetracycline 10 μ g/L | 9.60 | 4.91 | 5.17 | 5.97 |
| Chlortetracycline 20 μ g/L | 8.55 | 5.74 | 7.24 | 8.25 |
| Chlortetracycline 50 μ g/L | 5.29 | 6.39 | 5.8 | 6.59 |
| Chlortetracycline 100 μ g/L | 8.64 | 7.21 | 7.22 | 6.54 |
| Doxycycline 10 μ g/L | 4.58 | 6.59 | 3.58 | 7.03 |
| Doxycycline 20 μ g/L | 3.26 | 4.90 | 4.37 | 5.69 |
| Doxycycline 50 μ g/L | 6.58 | 3.34 | 7.34 | 6.42 |
| Doxycycline 100 μ g/L | 7.20 | 5.13 | 6.19 | 7.32 |
By upper table it can be seen that prior embodiment 4 method precision can meet general analysis requirements of one's work, embodiments of the invention 1-3 precision on the whole is suitable with embodiment 4.
Embodiments of the invention 1-3 compares with prior embodiment 4, and preparation steps and purifying step can save the operating time of 56%, 43% and 31% respectively, substantially increase work efficiency, reduce the labor intensity of staff.
In this article, " schematically " expression " serves as example, example or explanation ", and any embodiment being described herein as " schematically " should not be construed to a kind of preferred or more advantage technical scheme.
In this article, the restriction on the mathematics of the also non-critical such as " equal ", " identical " and/or geometry meaning, also comprise it will be appreciated by those skilled in the art that and the error of the permission such as production or use.Except as otherwise noted, numerical range herein not only includes the gamut in two end points, also includes being contained in some subranges therein.
It is to be understood that, although this specification describes according to each embodiment, but the most each embodiment only comprises an independent technical scheme, this narrating mode of description is only for clarity sake, those skilled in the art should be using description as an entirety, technical scheme in each embodiment can also form, through appropriately combined, other embodiments that it will be appreciated by those skilled in the art that.
The a series of detailed description of those listed above is only for illustrating of the possible embodiments of the present invention; they also are not used to limit the scope of the invention; all equivalent embodiments made without departing from skill of the present invention spirit or change; as feature combination, split or repeat, should be included within the scope of the present invention.
Claims (5)
1. a detection method for tetracycline antibiotics in Lac Bovis seu Bubali, including sample extraction, sample purification and Instrumental Analysis step, it is characterised in that described sample extraction step is: add Na in milk sample to be measured2EDTA-acetic acid aqueous solution, centrifuging and taking supernatant after mixing, it is sample extracting solution.
The detection method of tetracycline antibiotics in Lac Bovis seu Bubali the most according to claim 1, wherein said sample extraction step is: weigh milk sample 5g to be measured, adds 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, centrifugal after mixing, take supernatant, be sample extracting solution.
The detection method of tetracycline antibiotics in Lac Bovis seu Bubali the most according to claim 1, wherein said sample extraction step is: weigh milk sample 5g to be measured in 50mL centrifuge tube, adds 0.1mol/L Na2EDTA-acetic acid aqueous solution 20mL, mixes 30s on eddy mixer, was then centrifuged 8 minutes under 10000r/ minute, takes 5mL supernatant, be sample extracting solution.
The detection method of tetracycline antibiotics in Lac Bovis seu Bubali the most as claimed in any of claims 1 to 3, wherein said sample purification step is: take HLB solid-phase extraction column, activates respectively with 3 to 5mL methanol and 3 to 5mL ultra-pure water and balances；Again described sample extracting solution is less than 2mL/ minute through HLB solid-phase extraction column, the flow velocity of solid-phase extraction device；After described sample extracting solution is all by HLB solid-phase extraction column, washs HLB solid-phase extraction column with 3 to 5mL5% methanol aqueous solution, discard whole effluent；Again with 3 to 5mL methanol-eluted fractions HLB solid-phase extraction column, and collect eluent in centrifuge tube；Then the nitrogen under 40 DEG C of water-baths of the described eluent in centrifuge tube is dried up, add 1.0mL constant volume liquid vortex and dissolve, finally with 0.22 μm organic system membrane filtration, be i.e. purified rear sample；The compound method of wherein said constant volume liquid: take 10mL acetonitrile, be settled to 100mL with water.
The detection method of tetracycline antibiotics in Lac Bovis seu Bubali the most as claimed in any of claims 1 to 3, wherein said Instrumental Analysis step uses tablets by HPLC-MS, chromatographic condition is: chromatographic column is ACQUITY UPLC BEH C18 post, 2.1 × 100mm, 1.7 μm；Column temperature is 40 DEG C；Sampling volume is 10 μ L；Mobile phase A is acetonitrile, and Mobile phase B is 0.1% formic acid-water；Gradient elution: 0～1.0 minute, 15%A；1.0～2.0 minutes, 15%A～25%A；2.0～2.5 minutes, 25%A～30%A；2.5～3.0 minutes, 30%A；3.0～3.5 minutes, 30%A～40%A；3.5～4.5 minutes, 40%A～80%A；4.5～5.0 minutes, 80%A～15%A；Flow velocity is 0.4mL/ minute；Mass Spectrometry Conditions is: ionization mode is electron spray positive ion mode, and scanning of the mass spectrum mode is multiple-reaction monitoring, and ionization voltage is 3.00kV, and ion source temperature is 120 DEG C, and going solvent gas temperature is 400 DEG C, and removing solvent gas is N2, flow is 500L/h, and collision gas is Ar, and flow is 50L/h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109752466A (en) * | 2017-11-02 | 2019-05-14 | 上海安谱实验科技股份有限公司 | A method of it is detected for a variety of matrix tetracycline compounds |
| CN112526054A (en) * | 2020-12-31 | 2021-03-19 | 镇江华大检测有限公司 | Method for measuring residual quantity of tetracycline veterinary drugs in animal-derived food |
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