CN106872588A - The detection method of TCs in a kind of water sample - Google Patents
The detection method of TCs in a kind of water sample Download PDFInfo
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- CN106872588A CN106872588A CN201510920092.XA CN201510920092A CN106872588A CN 106872588 A CN106872588 A CN 106872588A CN 201510920092 A CN201510920092 A CN 201510920092A CN 106872588 A CN106872588 A CN 106872588A
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- G—PHYSICS
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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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a kind of detection method of TCs in water sample, water sample is pre-processed using magnetic material first, high performance liquid chromatography and tandem mass spectrum are then passed through again to determine the content of the TCs in filtrate.The method that the present invention is provided can detect the TCs of trace or ultra trace in water body, and easy to operate, quick, environmental protection, reliable results, detection limit are low, favorable reproducibility.
Description
Technical field
The present invention relates to a kind of assay method of antibiotic, the inspection of TCs in more particularly to a kind of water sample
Survey method.
Background technology
TCs (Tetracycline antibiotics, TCs) is the class wide spectrum produced by actinomyces
Antibiotic, including tetracycline, terramycin, aureomycin etc., is widely used in treatment and the livestock and poultry breeding industry of human diseases.By
A large amount of in TCs use, and its incomplete metabolism in biological living and be drained into water environment, therefore
The amount of TCs is progressively accumulated in water environment, may be excited the selective hereditary variation of microorganism and be caused appearance anti-
Raw element resistance pathogen, causes harm to environmental microorganism population, and and then produces irreversible destruction to water body system.
In the prior art, there is XRF, capillary electrophoresis, immune for detecting the method for TCs
Analytic approach, thin-layered chromatography, liquid chromatography and tablets by HPLC-MS etc..Such as High Performance Liquid Chromatography/Mass Spectrometry
Combination method has separation and detection feature, is more and more used for the detection of teracycline antibiotic residues.It is existing disclosed
Detection method, seems helpless for the detection of trace in water body or the TCs of ultra trace.
The existing research on Tetracyclines residual, biases toward analysis detection.Due to the characteristics of, foundation fast and effectively divide
Particularly significant is monitored to antibiotic residual contamination from beneficiation technologies.
Therefore, it is hopeful to develop a kind of method of the TCs that can detect trace or ultra trace in water body.
The content of the invention
It is an object of the invention to provide a kind of TCs that can detect trace or ultra trace in water body
Method.
The present invention provides following technical scheme：
A kind of detection method of TCs in water sample, methods described comprises the following steps：
(1) pretreatment of sample：
Magnetic material is added toward water sample, magnetic solid phase extraction is carried out；Extraction is filtered water sample and obtains magnetic material after terminating；It is past
The eluant, eluent of pH=9~11 is added in magnetic material carries out ultrasonic wash-out, forms eluent；Eluent is carried out into membrane filtration,
Obtain filtrate；
(2) detection method：
Filtrate is set to pass through high performance liquid chromatography and tandem mass spectrum to determine the tetracycline antibiotics in filtrate；
The condition of the liquid chromatogram is：
Chromatographic column：ZORBAX Eclipse Plus C18 [(3.0~5.0mm) × (100~250mm), 1.8 μm], flowing
Phase：The acetic acid aqueous solution of acetonitrile and 1% volumetric concentration, column temperature：25~35 DEG C, flow velocity：0.3~0.5mL/min, sample size：5~
10 μ L, the Mass Spectrometry Conditions are：
Dry temperature degree：280~320 DEG C, dry gas stream speed：1~5l/min, atomization gas pressure：40~45psi, capillary
Tube voltage：3000~4000V, spray nozzle voltage：100~2000V；
The TCs includes selected from epitetracyclin (ETC), terramycin (OTC), tetracycline (TC), goes first
One kind, more than two or three of combination in aureomycin (DMC), aureomycin (CTC) and dehydration tetracycline (ATC).
The detection method of TCs, needs first to enter sample before sample detection in the water sample that the present invention is provided
Row pretreatment.Preprocess method includes：Magnetic material is added toward water sample, magnetic solid phase extraction is carried out；Extraction terminates rear filter water
Sample obtains magnetic material；The eluant, eluent of pH=9~11 is added toward magnetic material carries out ultrasonic wash-out, forms eluent；To wash
De- liquid carries out membrane filtration, obtains filtrate.Preferably, the consumption of magnetic material is 1~25mg magnetic materials/100mL water samples,
Eluant, eluent be selected from pH=9~11 ammonia spirit, extraction temperature be 10~60 DEG C, extraction time be 1~60min, filter membrane for≤
0.45 μm of filter membrane.
The detection method of TCs in the water sample that the present invention is provided, after sample pretreatment is good, by efficient
Liquid chromatogram and tandem mass spectrum are determining the content of the tetracycline antibiotics in filtrate.
The test condition of the liquid chromatogram is：
Chromatographic column：ZORBAX Eclipse Plus C18 [(3.0~5.0mm) × (100~250mm), 1.8 μm], flowing
Phase：The acetic acid aqueous solution of acetonitrile and 1% volumetric concentration, column temperature：25~35 DEG C, flow velocity：0.3~0.5mL/min, sample size：5~
10 μ L, gradient elution.
Preferably, chromatographic column：ZORBAX Eclipse Plus C18(3.0mm × 100mm, 1.8 μm), mobile phase：Second
Nitrile and 1% (v/v) acetic acid aqueous solution, column temperature：35 DEG C, flow velocity：0.3mL/min, sampling volume：10μL.
Above-mentioned gradient elution, it is preferred that carried out according to table 1 below：
Table 1
The mass spectrographic test condition is：
Dry temperature degree：280~320 DEG C, dry gas stream speed：1~5l/min, atomization gas pressure：40~45psi, capillary
Tube voltage：3000~4000V, spray nozzle voltage：100~2000V.
Preferably, temperature degree is dried：300 DEG C, dry gas stream speed：5l/min, atomization gas pressure：45psi, capillary electricity
Pressure：3500V, spray nozzle voltage：1000V.
Preferably, in the mass spectrographic test condition, gas, atomization gas and sheath gas are dried and is high pure nitrogen, data are adopted
Integrated mode：Many reaction detections (MRM).
The detection method of TCs in the water sample that the present invention is provided, the content of the TCs is pressed
Calculated according to external standard method method.
Brief description of the drawings
Fig. 1 is the MRM spectrograms of TCs in sample 1, wherein：1 is epitetracyclin (ETC), and 2 is terramycin
(OTC)。
Fig. 2 is the MRM spectrograms of TCs in sample 2, wherein：1 is epitetracyclin (ETC), and 2 is terramycin
(OTC), 3 is tetracycline (TC), and 4 is dehydration tetracycline (ATC).
Fig. 3 is the MRM spectrograms of TCs in sample 3, wherein：1 is terramycin (OTC), and 2 is dehydration tetracycline
(ATC)。
Fig. 4 is the MRM spectrograms of TCs in sample 4, wherein：1 is epitetracyclin (ETC), and 2 is tetracycline
(TC)。
Fig. 5 is the MRM spectrograms of TCs in sample 5, wherein：1 is terramycin (OTC), and 2 is demethylchlortetra cylinum
(DMC), 3 is aureomycin (CTC).
Specific embodiment
The present invention is further described with reference to specific embodiment, but does not limit the invention to these tools
Body implementation method.One skilled in the art would recognize that present invention encompasses being potentially included in Claims scope
All alternatives, improvement project and equivalents.
The preparation of embodiment 1, standard liquid
Six kinds of 1000mgL-1The Standard Stock solutions of TCs：Epitetracyclin (ETC) is weighed respectively, soil
Mycin (OTC), tetracycline (TC), demethylchlortetra cylinum (DMC), aureomycin (CTC), dehydration tetracycline (ATC) each 10mg (is accurate to
0.0001g) in 10mL brown volumetric flasks, scale is dissolved to methyl alcohol, shaken up.Each Standard Stock solutions lucifuge, -4 DEG C
Refrigerator in preserve, it is standby.
1000μg·L-1Six kinds of TCs hybrid standard stock solutions：By 1000mgL-1Difference to Fourth Ring
Plain (ETC), terramycin (OTC), tetracycline (TC), demethylchlortetra cylinum (DMC), aureomycin (CTC), dehydration tetracycline (ATC) mark
Quasi- stock solution is respectively with methyl alcohol stepwise dilution to 10mgL-1, obtain epitetracyclin (ETC), terramycin (OTC), tetracycline
(TC), demethylchlortetra cylinum (DMC), aureomycin (CTC) and dehydration tetracycline (ATC) Standard Stock solutions.1mL is respectively taken in 10mL palm fibres
In color tolerance measuring bottle, scale is dissolved to methyl alcohol, shaken up, preserved in lucifuge, -4 DEG C of refrigerator, it is standby.
Embodiment 2, sample pretreatment
100mL water samples remove suspension through 0.45 μm of filter membrane, and add about 1.5mg magnetic materials, in 40 DEG C of waters bath with thermostatic control
Middle heating stirring 15min carries out magnetic solid phase extraction, and the magnetic material after extraction completely is collected using magnet, discards the water after extraction
Sample.Then eluted with 10mL ammoniacal liquor (pH=11) ultrasound 10min, and wash-out solution is collected under magnet effect, through 0.45 μ
After m membrane filtrations, filtrate is obtained stand-by.
The linear dependence of embodiment 3, method and sensitivity
According to the method described in embodiment 1, appropriate six kinds of tetracycline mixed standard solutions (1000 μ gL are accurately measured-1), with methanol dilution, obtain mass concentration and be respectively 1,5,10,20,50 and 100 μ gL-1Series standard solution, test table
Bright, six kinds of tetracyclines are in 1~100 μ gL-1Mass concentration in the range of quota ion peak area (y) and mass concentration (x)
Linear relationship is good, correlation coefficient r2>=0.995, detection limit (LOD, S/N=3) and quantitative limit (LOQ, S/N=10) scope are divided
It is not 2.44~25.21ngL-1With 8.14~84.03ngL-1.(being shown in Table 2)
Table 2 six kinds of equations of linear regression of tetracycline, coefficient correlation, detection limit and quantitative limits
| TCs | Equation of linear regression | Detection limit/(ng/L) | Quantitative limit/(ng/L) | |
| Epitetracyclin | Y=26781.9x+25238.9 | 0.9975 | 9.20 | 30.67 |
| Terramycin | Y=26322.5x+53807.5 | 0.9984 | 3.49 | 11.64 |
| Tetracycline | Y=31525.8x+12857.2 | 0.9993 | 4.78 | 15.92 |
| Demethylchlortetra cylinum | Y=11614.6x-372.3 | 0.9991 | 25.21 | 84.03 |
| Aureomycin | Y=8760.0x+2799.7 | 0.9993 | 20.98 | 69.93 |
| Dehydration tetracycline | Y=25278.8x+30483.6 | 0.9967 | 2.44 | 8.14 |
The rate of recovery and precision of embodiment 4, method
It is 10 μ gL in concentration known-1Water sample in add the method for standard liquid and carry out the measure of recovery of standard addition.Plus
Enter mixed mark (the 100 μ gL of six kinds of tetracyclines of different volumes-1), it is configured to 30,50 and 70 μ gL-1Water sample carry out 3 levels
Concentration actual sample mark-on is tested, and each sample carries out 6 parallel laboratory tests.HPLC- is carried out after being processed by sample pretreating method
MS/MS is determined, and calculates the rate of recovery.Respectively in sample treatment same day different time points and different disposal number of days sample introduction analysis (n=
6), calculate in a few days and day to day precision.The recovery of standard addition of six kinds of tetracyclines is all higher than 80%, withinday precision and in the daytime precision
Degree is respectively smaller than 2.5% and 7.1%.The rate of recovery and precision of method the results are shown in Table 3.
Knowable to rate of recovery data, the sample pretreating method that the application is provided has the rate of recovery higher, disclosure satisfy that
Test request.
The rate of recovery and precision of 3 six kinds of tetracyclines of table
Embodiment 5, high performance liquid chromatography-tandem mass (HPLC-MS/MS) testing conditions
(1) test condition of liquid chromatogram is：
Chromatographic column：ZORBAX Eclipse Plus C18(3.0mm × 100mm, 1.8 μm)；
Mobile phase：A is acetonitrile, and B is 1% (v/v) acetic acid aqueous solution, gradient elution；
Column temperature：35℃；
Flow velocity：0.3mL/min；
Sampling volume：10μL；
Gradient elution is carried out according to the following table：
Table 4
(2) mass spectrographic test condition is：
Dry temperature degree：300℃；
Dry gas stream speed：5l/min；
Atomization gas pressure：45psi；
Sheath temperature degree：250℃；
Sheath gas：10l/min；
Capillary voltage：3500V；
Spray nozzle voltage：1000V；
Dry gas, atomization gas and sheath gas and be high pure nitrogen；
Data acquisition scheme：Many reaction detections (MRM).
The measure of embodiment 6, actual sample
According to high performance liquid chromatography-tandem mass (HPLC-MS/MS) testing conditions described in embodiment 5, to Hangzhou river
Obtain carrying out test analysis in sample 1~5 in section, it is determined that the wherein residual concentration of tetracycline antibiotic.Experimental data is shown in Table 5.
Test spectrogram is shown in accompanying drawing 1~5.
6 kinds of measure of tetracycline (n=3) in the wastewater sample of table 5
Note：ND is not detect.
From table 5 and accompanying drawing 1-5, the method that the present invention is provided can accurately measure TCs in water sample
Content, easy to operate, quick, environmental protection, reliable results, detection limit are low, favorable reproducibility.
Claims (7)
1. in a kind of water sample TCs detection method, it is characterised in that methods described comprises the following steps：
(1) pretreatment of sample：
Magnetic material is added toward water sample, magnetic solid phase extraction is carried out；Extraction is filtered water sample and obtains magnetic material after terminating；Toward magnetic
The eluant, eluent of pH=9~11 is added in material carries out ultrasonic wash-out, forms eluent；Eluent is carried out into membrane filtration, is obtained
Filtrate；
(2) detection method：
Filtrate is set to pass through high performance liquid chromatography and tandem mass spectrum to determine the tetracycline antibiotics in filtrate；
The test condition of the liquid chromatogram is：
Chromatographic column：ZORBAX Eclipse Plus C18 [(3.0~5.0mm) × (100~250mm), 1.8 μm], mobile phase：
The acetic acid aqueous solution of acetonitrile and 1% volumetric concentration, column temperature：25~35 DEG C, flow velocity：0.3~0.5mL/min, sample size：5~10 μ
L, gradient elution；
The mass spectrographic test condition is：
Dry temperature degree：280~320 DEG C, dry gas stream speed：1~5l/min, atomization gas pressure：40~45psi, capillary electricity
Pressure：3000~4000V, spray nozzle voltage：100~2000V；
The TCs includes selected from epitetracyclin, terramycin, tetracycline, demethylchlortetra cylinum, aureomycin and takes off
One kind in water tetracycline, more than two or three of combination.
2. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the step (1)
In the pretreatment of sample, the consumption of magnetic material is 1~25mg magnetic materials/100mL water samples, and eluant, eluent is selected from pH=9~11
Ammonia spirit, extraction temperature is 10~60 DEG C, and extraction time is 1~60min, and filter membrane is≤0.45 μm of filter membrane.
3. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the step (2)
The test condition of liquid chromatogram is：
Chromatographic column：ZORBAX Eclipse Plus C18(3.0mm × 100mm, 1.8 μm), mobile phase：Acetonitrile and 1% (v/v) second
Aqueous acid, column temperature：35 DEG C, flow velocity：0.3mL/min, sampling volume：10μL.
4. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the step (2)
The test condition of liquid chromatogram is：Gradient elution is carried out according to the following table：
5. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the step (2)
Mass spectrographic test condition is：Dry gas, atomization gas and sheath gas and be high pure nitrogen, data acquisition scheme：Many reaction detections
(MRM)。
6. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the step (2)
Mass spectrographic test condition is：Dry temperature degree：300 DEG C, dry gas stream speed：5l/min, atomization gas pressure：45psi, capillary electricity
Pressure：3500V, spray nozzle voltage：1000V.
7. according to the detection method of TCs in the water sample described in claim 1, it is characterised in that the tetracycline
The content of class antibiotic is calculated according to external standard method method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507313A (en) * | 2018-10-26 | 2019-03-22 | 桂林理工大学 | A method of tetracycline antibiotics in separation analysis large volume environmental water sample |
| CN110470780A (en) * | 2019-09-12 | 2019-11-19 | 中国农业科学院农业质量标准与检测技术研究所 | The discrimination method of protein feed raw material admixture Terramycin-type waste water |
| CN112067729A (en) * | 2020-08-19 | 2020-12-11 | 南京白云环境科技集团股份有限公司 | Method for analyzing three tetracycline antibiotics in water environment through HLB (hydrophile-lipophile balance) solid-phase extraction-high performance liquid chromatography |
| CN113945658A (en) * | 2021-10-13 | 2022-01-18 | 深圳技师学院（深圳高级技工学校） | Method for simultaneously determining content of 7 antibiotics in water sample |
-
2015
- 2015-12-11 CN CN201510920092.XA patent/CN106872588A/en active Pending
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| GÜLBAHAR AKKAYA SAY˘GILI 等: "Development and physicochemical characterization of a new magnetic nanocompositeas an economic antibiotic remover", 《PROCESS SAFETY AND ENVIRONMENTAL PROTECTION》 * |
| 赵晓亚 等: "高效液相色谱-串联质谱法对化妆品中9种四环素类药物的同时测定", 《分析测试学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507313A (en) * | 2018-10-26 | 2019-03-22 | 桂林理工大学 | A method of tetracycline antibiotics in separation analysis large volume environmental water sample |
| CN110470780A (en) * | 2019-09-12 | 2019-11-19 | 中国农业科学院农业质量标准与检测技术研究所 | The discrimination method of protein feed raw material admixture Terramycin-type waste water |
| CN112067729A (en) * | 2020-08-19 | 2020-12-11 | 南京白云环境科技集团股份有限公司 | Method for analyzing three tetracycline antibiotics in water environment through HLB (hydrophile-lipophile balance) solid-phase extraction-high performance liquid chromatography |
| CN113945658A (en) * | 2021-10-13 | 2022-01-18 | 深圳技师学院（深圳高级技工学校） | Method for simultaneously determining content of 7 antibiotics in water sample |
| CN113945658B (en) * | 2021-10-13 | 2023-10-31 | 深圳技师学院（深圳高级技工学校） | Method for simultaneously measuring contents of 7 antibiotics in water sample |
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Application publication date: 20170620 |