CN101290306B - Milk and milk product tetracycline antibiotic residual quantity checking method - Google Patents
Milk and milk product tetracycline antibiotic residual quantity checking method Download PDFInfo
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Abstract
The invention relates to a method for detecting the residue amount of terracycline antibiotics in milk and dairy products. The method utilizes an ultra performance liquid chromatography - electrospray tandem triple quadrupole mass spectrometer to determine the residue amount of the terracycline antibiotics. The method is as follows: a sample is extracted from Na2EDTA-McIlvaine buffer solution (pH4.0); proteins are removed by trichloroacetic acids; a columella is extracted through an HLB solid phase and then purified and enriched; the sample is separated by a chromatographic column, with the column temperature of 30 DEG C; gradient elution is performed by utilization of water solution (v/v) which contains 0.1 percent of methanoic acids and acetonitrile as a moving phase; and quantitative detection is performed by adoption of the multi-reaction monitoring means. The detection limit of instruments is between 1.0 and 2.0 mu g/kg; a related coefficient r reaches over 0.999 within the linear range of between 1 and 100 mu g/kg; and the recovery rate is between 81.7 and 100.7 percent (the addition levels are 10 mu g/kg, 50 mu g/kg and 100 mu g/kg). The method has the advantages of quickness, accuracy, high sensitivity and wide application scope.
Description
Technical field
The present invention relates to a kind of method that detects antibiotic content in the milk and milk products, the detection method of teracycline antibiotic residues amount belongs to analysis technical field in particularly a kind of milk and milk products.
Background technology
TCs is all very sensitive to most of gram-positive bacterias and negative bacterium, be mainly used in the control of brucellosis, anthrax, Escherichia coli, ARI, mammitis etc., because of it has the antibacterial effect of wide spectrum and cheap price, become antibiotic principal item for animals commonly used in recent years.But this medicine can be residual in the livestock and poultry body, residual Tetracyclines is to people's lung, stomach and intestine, kidney, skin and bone in the cow's milk, especially maximum to the infringement of children's tooth, can influence fetal bone, dental growth, cause illnesss such as imperfect dentinogenesis, bone tissue growth be slow.Be the protection human health; control the residual of TCs (comprising tetracycline, aureomycin, terramycin etc.); U.S. FDA regulation tetracycline, the residual quantity of terramycin in milk are respectively less than 80 μ g/L and 30 μ g/L, and tetracycline, the residual quantity of terramycin in milk of China's regulation should be less than 100 μ g/L.Therefore, need seek easy, quick, accurately detect the detection method of teracycline antibiotic residues amount in the milk and milk products, could satisfy the strict day by day residual requirement of limiting the quantity of, thereby guarantee the health and the safety of people's milk drink.
The method that detects tetracycline has a lot, and for example microbial method, high performance liquid chromatography also have thin-layered chromatography (TLC method), ultraviolet spectrophotometry, fluorophotometric method, vapor-phase chromatography, efficient capillary zone electrophoresis method etc. in addition.At present, the detection method that TCs is commonly used in the milk and milk products mainly is a high performance liquid chromatography, utilize high performance liquid chromatograph (UV-detector) to measure the residual quantity of TCs, but adopt this method, detection time is longer, a general sample detection finishes and needs about 30min, and high performance liquid chromatography detects the detection limit higher (generally being higher than 10 μ g/kg) of TCs.
Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer can be according to the feature fragment of characteristic ion and the characteristic ion fracture generation thus fingerprint as a certain target compound in quantitative test, it is chosen from the blend sample of complex matrices carry out quantitatively, therefore highly sensitive strong with selectivity, be the goldstandard of target compound quantitative test.It is a food security, the reference instrument of various international regulations defineds such as pharmacokinetics.Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer can carry out quantitatively at parent ion and the daughter ion that target compound ionization produces, compare with traditional high performance liquid chromatography, selectivity and sensitivity have been improved greatly to target compound, and shortened analysis time greatly, detecting a sample only needs about 4min.Ultra Performance Liquid Chromatography-mass spectrometric application prospect of electron spray series connection triple quadrupole bar is very extensive.
Summary of the invention
The detection method that the purpose of this invention is to provide teracycline antibiotic residues amount in a kind of milk and milk products.
The detection method of teracycline antibiotic residues amount of the present invention comprises the steps:
1) the TCs solution of a plurality of variable concentrations of preparation, and it is detected respectively, according to measured data, draw the content of TCs and the canonical plotting between the testing result;
2) sample is used with the same method of step 1) detected, obtain testing result;
3) with step 2) in testing result and described canonical plotting comparison, obtain TCs content in the sample;
4) checkout equipment is Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer.
Above-mentioned TCs is: terramycin, tetracycline and aureomycin.
Above-mentioned preparation process 1) solvent of solution is preferably in: methyl alcohol.
The TCs solution content of above-mentioned a plurality of variable concentrations is preferably: 100ng/mL, 50ng/mL, 20ng/mL, 1ng/mL, 0.1ng/mL, the standard solution of 0ng/mL.
Preferably, the disposal route of sample is as follows above-mentioned described step 2):
A) the Na2EDTA-McIlvaine buffer solution of sample and pH4.0 adds according to the ratio of quality and volume ratio 1: 4-10, shake well 1-5min at ambient temperature, leave standstill 10-20min, fully extract, after adding the trichloroacetic acid mixing of 1-5mL50%, removing albumen, is 6000rpm/min at rotating speed, temperature is centrifugal 5-15min under 4 ℃ the condition, after the filtration filtrate;
B) use 5.0mL methyl alcohol to pass through the solid phase extractions pillar with the flow velocity of 1.0～2.0mL/min, cross post with the 10.0mL ultrapure water with the flow velocity of 1.0～2.0mL/min again, be under the condition of 1.0～2.0mL/min through little column purification of solid phase extractions and enrichment with above-mentioned gained filtrate at flow velocity, cross this post with 5% the methanol aqueous solution of 3.0mL with the flow velocity of 1.0～2.0mL/min then, the solid phase extractions pillar is drained with vacuum pump;
C) add the 6.0mL methanol-eluted fractions in the solid phase extractions pillar of enrichment target substance, elution flow rate 1.0～2.0mL/min collects eluent, dries up under 40 ℃ of water-baths with nitrogen, and with 50% dissolve with methanol of 1.0mL, vortex is through 0.22 μ m filtering with microporous membrane.
Described solid phase is separated pillar and is preferably HLB (hydrophilic-lipophilic balance) solid phase extractions pillar.
Preferably, above-mentioned Ultra Performance Liquid Chromatography-mass spectrometric chromatography separating method of electron spray series connection triple quadrupole bar is as follows:
Chromatographic column: ACQUITY UPLC BEH C18 chromatographic column (2.1mm * 50mm, I.D., the 1.7 μ m) post of this (Waters) company of water, column temperature: 30 ℃, flow velocity: 0.3mL/min; Sampling volume: 5 μ L; Eluent gradient is as follows:
B(％)
A (%) graded
Time, (min) contained 0.1% formic acid
The acetonitrile curve
Aqueous solution
0.00 10 90
2.50 30 70 6
3.50 50 50 6
4.50 10 90 6
5.00 10 90 1
Wherein, 1 is instant the variation, and 6 is linear change.
Preferably, above-mentioned Ultra Performance Liquid Chromatography-mass spectrometric mass spectrum condition of electron spray series connection triple quadrupole bar is as follows:
Ionization mode: ES (+), ionization voltage: 3.50KV, taper hole voltage: 22V, source temperature: 120 ℃, the temperature degree desolvates: 350 ℃, the airshed of desolvating: 500L/hr, acquisition mode: multiple-reaction monitoring (MRM):
The quasi-molecular ion taper hole voltage collision energy residence time
Compound title feature fragmention
[M+H]+ (V) (eV) (S)
426.2
* 20 0.05
Terramycin 461.4 24
443.2 12 0.05
410.1
* 20 0.05
445.3 25
West ring plain 427.2 13 0.05
Aureomycin 444.0
*22 0.05
461.4 30
462.0 15 0.05
Wherein,
*Be expressed as and be quota ion.
TCs detection method of the present invention has following advantage:
1) detection time is short.A sample only needs about 4min.
2) highly sensitive.Detection limit only is 1.0～2.0 μ g/kg, can satisfy international and domestic to the particularly detection requirement of trace amount tetracycline antibiotics of TCs content.
3) selectivity to target compound is strong, accurate quantification.Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer carries out quantitative test according to the parent ion of target compound and the feature daughter ion that produces through collision, have only the target compound ability that satisfies these two characteristic mass selections simultaneously detected, and selected feature sliver ion must be to produce from selected parent ion, thereby got rid of the interference phenomenon of other molecule that may exist in the sample.Method of the present invention adopts the ESI+ ion gun of triple quadrupole bar, parent ion and two daughter ions of terramycin, tetracycline and aureomycin have been determined, greatly reduce and utilize the high effective liquid chromatography for measuring TCs probability of false sun (the moon) property testing result may occur, thereby reach purpose the target compound accurate quantification.
4) under institute's chromatogram of the present invention and mass spectrum condition, measure, in the experimental concentration scope, correlation coefficient r can reach more than 0.999, and the recovery is between 81.7～100.7%, and in a few days the relative standard deviation (RSD) with the day to day precision experiment is respectively 2.48% and 3.96%.This method can be got rid of interfering component and extract target substance from complex matrices, still is that the sample of complex matrices is all suitable equally for having simple matrix, has advantage applied widely.
Description of drawings
Fig. 1 is the total ion current spectrogram of terramycin, tetracycline and the aureomycin of one embodiment of the present of invention, its horizontal ordinate express time (min), ordinate is represented the size of ion flow response signal intensity, and the ion flow intensity of expression terramycin, tetracycline, three kinds of different target compounds of aureomycin accounts for the number percent of total ion current.
Fig. 2 is the terramycin parent ion 461.4 of one embodiment of the present of invention and the chromatogram of quantitative daughter ion 426.2.Its horizontal ordinate express time (min), ordinate is represented the size of ion flow response signal intensity, the ion flow intensity of expression terramycin accounts for the number percent of total ion current.
Fig. 3 is the tetracycline parent ion 445.3 of one embodiment of the present of invention and the chromatogram of quantitative daughter ion 410.1.Its horizontal ordinate express time (min), ordinate is represented the size of ion flow response signal intensity, the ion flow intensity of expression tetracycline accounts for the number percent of total ion current.
Fig. 4 is the aureomycin parent ion 479.4 of one embodiment of the present of invention and the chromatogram of quantitative daughter ion 444.1.Its horizontal ordinate express time (min), ordinate is represented the size of ion flow response signal intensity, the ion flow intensity of expression aureomycin accounts for the number percent of total ion current.
Fig. 5 is the terramycin canonical plotting of one embodiment of the present of invention.Wherein, horizontal ordinate is represented the concentration (ng/mL) of terramycin, and ordinate is represented ion response signal intensity (mv).
Fig. 6 is the tetracycline canonical plotting of one embodiment of the present of invention.Wherein, horizontal ordinate is represented the concentration (ng/mL) of tetracycline, and ordinate is represented ion response signal intensity (mv).
Fig. 7 is the aureomycin canonical plotting of one embodiment of the present of invention.Wherein, horizontal ordinate is represented the concentration (ng/mL) of aureomycin, and ordinate is represented ion response signal intensity (mv).
Embodiment
Embodiment 1 utilizes Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure the content of terramycin, tetracycline and aureomycin in the cow's milk.
1) preparation of standard solution:
Precision takes by weighing terramycin, tetracycline and each 10mg of aureomycin standard items, with methanol constant volume in the brown volumetric flask of 10mL, mixing, obtain the standard inventory solution that terramycin, tetracycline and chlortetracycline concentration are 1.00mg/mL, be positioned over-18 ℃ of refrigerators and keep in Dark Place, can preserve for 4 week at least.During use, be diluted to 100ng/mL, 50ng/mL, 20ng/mL, 1ng/mL, the standard solution of 0.1ng/mL.
2) preparation of reagent:
(a) preparation of extract:
0.2mol/L disodium phosphate soln: accurately take by weighing the 28.41g sodium hydrogen phosphate, the water dissolving is settled to 1000mL.
0.1mol/L citric acid solution: accurately take by weighing the 21.01g citric acid, the water dissolving is settled to 1000mL.
McIlvaine buffer solution: the 1000mL0.1mol/L citric acid solution is mixed with 0.2mol/L disodium phosphate soln 625mL, regulate pH=4.0 with NaOH.
0.1mol/LNa2EDTA-McIlvaine buffer solution: take by weighing the 60.5g disodium ethylene diamine tetraacetate and put into the 1625mLMcIlvaine damping fluid, make its dissolving, shake up.
(b) preparation of moving phase:
Mobile phase A: acetonitrile
Mobile phase B: 0.1% aqueous formic acid
3) preparation of sample:
Take by weighing the 5.00g milk sample, place the 50mL polypropylene centrifuge tube, add 25mLNa2EDTA-McIlvaine buffer solution and extract, at ambient temperature shake well 2min, leave standstill 15min, fully extract, remove deproteinized, homogeneous after adding 2mL50% trichloroacetic acid mixing again, the 6000rpmin temperature is centrifugal 10min under 4 ℃ the condition, filter with quantitative filter paper, extract once with 20mLNa2EDTA-McIlvaine buffer solution again, filter the merging supernatant.
With 5.0mL methyl alcohol with the flow velocity of 1.0～2.0mL/min by this (Waters) Oasis of company of water
HLB (hydrophilic-lipophilic balance) solid phase extractions pillar (packing quality: 60mg; The column jecket volume: 3ml), cross post with the 10.0mL ultrapure water with the flow velocity of 1.0～2.0mL/min again, purpose is an activation solid phase extractions pillar.With above-mentioned gained filtrate be at flow velocity under the condition of 1.0～2.0mL/min through little column purification of solid phase extractions and enrichment, cross this post removal of impurities with 5% methanol aqueous solution with the flow velocity of 1.0～2.0mL/min when waiting fast doing with 3.0mL.With vacuum pump the solid phase extractions pillar is drained.Add the 6mL methanol-eluted fractions, dry up for 40 ℃ with nitrogen then, 50% dissolve with methanol with 1mL, vortex is used for Ultra Performance Liquid Chromatography (the ACQUITYTM UPLC of Waters)-electron spray series connection triple quadrupole bar mass spectrometer (Quattro Premier XE of U.S. Waters behind 0.22 μ m filtering with microporous membrane
MicroMass) analyze.
4) detecting instrument: Ultra Performance Liquid Chromatography (the ACQUITYTM UPLC of Waters)-electron spray series connection triple quadrupole bar mass spectrometer (the Quattro Premier XE of U.S. Waters
MicroMass) ".
5) chromatographic condition:
Chromatographic column: the ACQUITY UPLC BEH C18 post (50mm * 2.1mm I.D., 1.7 μ m) of this (Waters) company of water; Column temperature: 30 ℃, flow velocity: 0.3mL/min; Sampling volume: 5 μ L; Eluent gradient sees Table 1.
Table 1 chromatographic resolution gradient
Annotate: 1 is instant the variation, and 6 is linear change.
6) mass spectrum condition:
Ionization mode: ES (+), ionization voltage: 3.50KV, source temperature: 120 ℃, the temperature degree desolvates: 350 ℃, the airshed of desolvating: 500L/hr, acquisition mode: multiple-reaction monitoring (MRM) specifically sees Table 2:
The mass spectrum condition of table 2 Tetracyclines
Annotate:
*Be expressed as and be quota ion.
7) detect step:
A) drafting of typical curve: utilize Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure terramycin, tetracycline and the aureomycin standard solution of variable concentrations.Extract total ion current as shown in Figure 1.The retention time of terramycin is 1.55min as seen from Figure 1, and the retention time of tetracycline is 1.76min, and the retention time of aureomycin is 2.49min.
From the total ion current figure of Fig. 1, extract the parent ion of terramycin, tetracycline and aureomycin and each ion pair chromatogram of daughter ion self-quantitatively respectively, the results are shown in Figure 2～Fig. 4.Wherein, Fig. 2 s is the chromatogram of terramycin parent ion 461.4 and quantitative daughter ion 426.2, and Fig. 3 is the chromatogram of tetracycline parent ion 445.3 and quantitative daughter ion 410.1, the chromatogram of Fig. 4 aureomycin parent ion 479.4 and quantitative daughter ion 444.1.
Utilize Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure terramycin, tetracycline and the aureomycin standard solution (100ng/mL of variable concentrations, 50ng/mL, 20ng/mL, 1ng/mL, 0.1ng/mL) detect, according to the gained data, the drawing standard curve map the results are shown in Figure 5～Fig. 7.Wherein, Fig. 5 is the terramycin canonical plotting, and Fig. 6 is the tetracycline canonical plotting, and Fig. 7 is the aureomycin canonical plotting.
According to the canonical plotting of terramycin, tetracycline and aureomycin, determine linear equation and linearly dependent coefficient, the concentration that equals 3 o'clock correspondences with signal to noise ratio (S/N ratio) is detection limit, concrete outcome sees Table 3.
The linear equation of table 3 terramycin, tetracycline and aureomycin and detection limit
Adopt multiple-reaction monitoring (MRM) acquisition mode, in the experimental concentration scope, correlation coefficient r=0.9999, the recovery is between 81.7～100.7%, and in a few days the relative standard deviation (RSD) with the day to day precision experiment is respectively 2.48% and 3.96%.
B) sample determination:
Utilize the testing sample that Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar Mass Spectrometer Method prepares, obtain the collection of illustrative plates of terramycin, tetracycline and aureomycin, respectively the result of the corresponding signal value of terramycin, tetracycline and aureomycin is inserted in the typical curve equation, can obtains the concentration (ng/mL) of terramycin in the sample, tetracycline and aureomycin respectively.Behind the working sample, the response Y that obtains terramycin in the sample is 983, the typical curve equation of substitution terramycin: Y=44.0573x-86.1172, and then the concentration of terramycin is 24.3 nanograms/milliliter (ng/mL) in this sample; The response Y that records tetracycline is 1021, the typical curve equation of substitution tetracycline: Y=54.685x-33.7211, and then the concentration of tetracycline is 19.3 nanograms/milliliter (ng/mL) in this sample; The response Y that records aureomycin is 784, the typical curve equation of substitution aureomycin: Y=25.9306x-37.6191, then the concentration of aureomycin is 31.7 nanograms/milliliter (ng/mL) in this sample, then in this sample three kinds of antibiotic total concentrations for being 75.3 nanograms/milliliter (ng/mL).
Embodiment 2 utilizes Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure the content of terramycin, tetracycline and aureomycin in the sour milk.
1) preparation of standard solution:
Precision takes by weighing terramycin, tetracycline and each 10mg of aureomycin standard items, with methanol constant volume in the brown volumetric flask of 10mL, mixing, obtain the standard inventory solution that terramycin, tetracycline and chlortetracycline concentration are 1.00mg/mL, be positioned over-18 ℃ of refrigerators and keep in Dark Place, can preserve for 4 week at least.During use, be diluted to 50ng/mL, 20ng/mL, 10ng/mL, 1ng/mL, the standard solution of 0.1ng/mL.
2) preparation of reagent:
(a) preparation of extract:
0.2mol/L disodium phosphate soln: accurately take by weighing the 28.41g sodium hydrogen phosphate, the water dissolving is settled to 1000mL.
0.1mol/L citric acid solution: accurately take by weighing the 21.01g citric acid, the water dissolving is settled to 1000mL.
McIlvaine buffer solution: the 1000mL0.1mol/L citric acid solution is mixed with 0.2mol/L disodium phosphate soln 625mL, regulate pH=4.0 with NaOH.
0.1mol/LNa2EDTA-McIlvaine buffer solution: take by weighing the 60.5g disodium ethylene diamine tetraacetate and put into the 1625mLMcIlvaine damping fluid, make its dissolving, shake up.
(b) preparation of moving phase:
Mobile phase A: acetonitrile
Mobile phase B: 0.1% aqueous formic acid
3) preparation of sample:
Take by weighing 3.00g sour milk sample, place the 50mL polypropylene centrifuge tube, add 25mLNa2EDTA-McIlvaine buffer solution and extract, at ambient temperature shake well 4min, leave standstill 20min, fully extract, remove deproteinized, homogeneous after adding 4mL50% trichloroacetic acid mixing again, the 6000rpmin temperature is centrifugal 15min under 4 ℃ the condition, filter with quantitative filter paper, extract once with 20mLNa2EDTA-McIlvaine buffer solution again, filter the merging supernatant.
With 5.0mL methyl alcohol with the flow velocity of 1.0～2.0mL/min by solid phase extractions pillar (the BONDELUT PLEXA solid phase extraction column of the U.S., packing quality: 60mg; The column jecket volume: 3ml), cross post with the 10.0mL ultrapure water with the flow velocity of 1.0～2.0mL/min again, purpose is the activating solid pillar.With above-mentioned gained filtrate be at flow velocity under the condition of 1.0～2.0mL/min through little column purification of solid phase extractions and enrichment, cross this post removal of impurities with 5% methanol aqueous solution with the flow velocity of 1.0～2.0mL/min when waiting fast doing with 3.0mL.With vacuum pump the solid phase extractions pillar is drained.Add the 6mL methanol-eluted fractions, dry up for 40 ℃ with nitrogen then, 50% dissolve with methanol with 1mL, vortex is used for Ultra Performance Liquid Chromatography (the ACQUITYTM UPLC of Waters)-electron spray series connection triple quadrupole bar mass spectrometer (Quattro Premier XE of U.S. Waters behind 0.22 μ m filtering with microporous membrane
MicroMass) analyze.
4) detecting instrument: Ultra Performance Liquid Chromatography (the ACQUITYTM UPLC of Waters)-electron spray series connection triple quadrupole bar mass spectrometer (the Quattro Premier XE of U.S. Waters
MicroMass).
5) chromatographic condition:
Chromatographic column: the ACQUITY UPLC BEH C18 post (50mm * 2.1mm I.D., 1.7 μ m) of this (Waters) company of water; Column temperature: 30 ℃, flow velocity: 0.3mL/min; Sampling volume: 5 μ L; Eluent gradient sees Table 4.
Table 4 chromatographic resolution gradient
Annotate: 1 is instant the variation, and 6 is linear change.
6) mass spectrum condition:
Ionization mode: ES (+), ionization voltage: 3.50KV, source temperature: 120 ℃, the temperature degree desolvates: 350 ℃, the airshed of desolvating: 500L/hr, acquisition mode: multiple-reaction monitoring (MRM) specifically sees Table 5:
The mass spectrum condition of table 5 Tetracyclines
Annotate:
*Be expressed as and be quota ion.
7) detect step:
A) drafting of typical curve: utilize Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure terramycin, tetracycline and the aureomycin standard solution of variable concentrations.Extract total ion current figure.
From total ion current figure, extract the parent ion of terramycin, tetracycline and aureomycin and each ion pair chromatogram of daughter ion self-quantitatively respectively.
Utilize Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer to measure terramycin, tetracycline and the aureomycin standard solution (50ng/mL of variable concentrations, 20ng/mL, 1ng/mL 0.1ng/mL) detects, according to the gained data, the drawing standard curve map.
According to the canonical plotting of terramycin, tetracycline and aureomycin, determine linear equation and linearly dependent coefficient, the concentration that equals 3 o'clock correspondences with signal to noise ratio (S/N ratio) is detection limit, concrete outcome sees Table 6.
The linear equation of table 6 terramycin, tetracycline and aureomycin and detection limit
Adopt multiple-reaction monitoring (MRM) acquisition mode, in the experimental concentration scope, correlation coefficient r reaches 0.999, and the recovery is between 81.7%～100.7%, and in a few days the relative standard deviation (RSD) with the day to day precision experiment is respectively 3.45% and 5.98%.
B) sample determination: utilize the testing sample that Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar Mass Spectrometer Method prepares, obtain the collection of illustrative plates of terramycin, tetracycline and aureomycin, respectively the result of the corresponding signal value of terramycin, tetracycline and aureomycin is inserted in the typical curve equation, can obtains the concentration (ng/mL) of terramycin in the sample, tetracycline and aureomycin respectively.Behind the working sample, the response Y that obtains terramycin in the sample is 537, the typical curve equation of substitution terramycin: Y=44.1625x-88.1150, and then the concentration of terramycin is 14.1ng/mL in this sample; The response Y that records tetracycline is 1363, the typical curve equation of substitution tetracycline: Y=54.586x-34.1128, and then the concentration of tetracycline is 25.6ng/mL in this sample; The response Y that records aureomycin is 985, the typical curve equation of substitution aureomycin: Y=26.0506x-38.7823, and then the concentration of aureomycin is 39.3ng/mL in this sample, then three kinds of antibiotic total concentrations are 79.0ng/mL in this sample.
Claims (3)
1. the detection method of a teracycline antibiotic residues amount is characterized in that, comprises the steps:
1) use methyl alcohol as solvent, the standard solution for preparing terramycin, tetracycline and the aureomycin of a plurality of variable concentrations, and it is detected respectively, according to measured data, draw the content of terramycin, tetracycline and aureomycin and the canonical plotting between the testing result;
Ultra Performance Liquid Chromatography-electron spray series connection triple quadrupole bar mass spectrometer is used in described detection;
Described mass spectrometric chromatography separating method is as follows:
Chromatographic column: the ACQUITY UPLC BEH C18 chromatographic column of Waters, specification is 2.1mm * 50mm, I.D, 1.7 μ m, 30 ℃ of column temperatures, flow velocity 0.3mL/min; Sampling volume 5 μ L; Eluent gradient is as follows:
Described mass spectrometric mass spectrum condition is as follows:
Ionization mode ES, ionization voltage 3.50KV, 120 ℃ of source temperatures, 350 ℃ of the temperature degree of desolvating, the airshed of desolvating 500L/hr, multiple-reaction monitoring mass spectrum condition is as follows:
Wherein, * is expressed as quota ion;
2) sample is detected with the same method of step 1), obtain testing result;
3) with step 2) in testing result and described canonical plotting comparison, obtain TCs content in the sample.
2. the disposal route of sample is as follows detection method as claimed in claim 1, wherein said step 2):
A) Na of sample and pH4.0
2EDTA-McIlvaine buffer solution adds according to quality and 1: 4～1: 10 ratio of volume ratio, shake well 1～5min at ambient temperature, leave standstill 10～20min, fully extract, after adding 50% the trichloroacetic acid mixing of 1～5mL, removing albumen, is 6000rpm/min at rotating speed, temperature is centrifugal 5～15min under 4 ℃ the condition, after the filtration filtrate;
B) use 5.0mL methyl alcohol to pass through the solid phase extractions pillar with the flow velocity of 1.0～2.0mL/min, cross post with the 10.0mL ultrapure water with the flow velocity of 1.0～2.0mL/min again, be under the condition of 1.0～2.0mL/min through little column purification of solid phase extractions and enrichment with above-mentioned gained filtrate at flow velocity, cross this post with 5% the methanol aqueous solution of 3.0mL with the flow velocity of 1.0～2.0mL/min then, the solid phase extractions pillar is drained with vacuum pump;
C) add the 6.0mL methanol-eluted fractions in the solid phase extractions pillar of enrichment target substance, elution flow rate 1.0～2.0mL/min collects eluent, dries up under 40 ℃ of water-baths with nitrogen, and with 50% dissolve with methanol of 1.0mL, vortex is through 0.22 μ m filtering with microporous membrane.
3. detection method as claimed in claim 2, wherein said solid phase extractions pillar is preferably hydrophilic-lipophilic balance solid phase extractions pillar.
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| CN101419203B (en) * | 2008-10-24 | 2011-06-01 | 江苏大学 | Method for separating trace amount tetracycline antibiotics in enrichment environment |
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