CN109856268B - Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography - Google Patents
Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography Download PDFInfo
- Publication number
- CN109856268B CN109856268B CN201910080673.5A CN201910080673A CN109856268B CN 109856268 B CN109856268 B CN 109856268B CN 201910080673 A CN201910080673 A CN 201910080673A CN 109856268 B CN109856268 B CN 109856268B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- antibiotics
- high performance
- performance liquid
- liquid chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a method for rapidly detecting multiple antibiotics by utilizing high performance liquid chromatography, which greatly shortens the detection time and can synchronously search out ten antibiotics, wherein the antibiotics comprise sulfonamide antibiotics, beta-lactam antibiotics and quinolone antibiotics. The detection process is simplified, the detection cost is reduced, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of antibiotic detection, in particular to a method for rapidly detecting multiple antibiotics by utilizing high performance liquid chromatography.
Background
The antibiotic not only can kill bacteria, but also has good inhibiting and killing effects on other pathogenic microorganisms such as mould, mycoplasma, chlamydia, spirochete, rickettsia and the like, can be a substance generated in the growth and reproduction processes of certain microorganisms, and is used for treating diseases except for direct extraction; and also completely or partially synthesized. However, with the progress of science and technology, the production process of antibiotics is mature day by day, a large amount of antibiotics are put into the lives of people and are widely used, according to incomplete statistics, about 75% of antibiotics cannot be utilized by people or animals, but are discharged into the environment in the form of raw medicines or metabolites through urine, excrement and the like, so that the drug resistance level in the environment is improved, the antibiotics become important novel environmental pollutants, and the ecological environment safety and the human health are seriously harmed.
The Handan City Fuyan river and the Qin river have complex components, and the trace amount of antibiotics exists, so that the problem of local area is how to accurately detect various antibiotics in the water samples of the Fuyan river and the Qin river so as to carry out environmental early warning and effective radical treatment on the antibiotics.
In patent CN107561187A, a method for synchronously detecting multiple antibiotics in polluted water is disclosed, the method adopts high performance liquid chromatography tandem mass spectrometry to detect the content of antibiotics, and can synchronously detect 20 kinds of antibiotics, the adopted instrument is high in cost, and the detection time is long, so that for the continuously changing water to be treated, the detection efficiency needs to be improved, the detection time is reduced, and the detection accuracy is improved.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting multiple antibiotics by using high performance liquid chromatography. By adopting the technical scheme of the invention, a high performance liquid chromatograph is utilized to accurately analyze a plurality of antibiotics within 14min, wherein the plurality of antibiotics comprise sulfonamide antibiotics, beta-lactam antibiotics and quinolone antibiotics.
The invention relates to a method for rapidly detecting multiple antibiotics by high performance liquid chromatography, wherein the antibiotics comprise sulfonamide antibiotics, beta-lactam antibiotics and quinolone antibiotics, and the method comprises the following steps:
(1) firstly, selecting a water sample to be detected containing multiple antibiotics;
(2) filtering the water sample to be detected, and then adding Na2-EDTA and adjusting the pH to 3;
(3) introducing the water sample to be detected treated in the step (2) into the activated HLB column, and enriching the water sample to be detected;
(4) eluting the enriched HLB small column by using an eluant to obtain an eluent, and concentrating the eluent to obtain a concentrated solution;
(5) the concentrated solution is subjected to constant volume to 1mL by using methanol, a diluted water sample to be detected is transferred, and the antibiotics are detected by using a high performance liquid chromatography, wherein the high performance liquid chromatography adopts a Shim-pack GIST C18 chromatographic column, the column temperature is 40 ℃, the sample injection volume is 30 mu L, a mobile phase adopted by the high performance liquid chromatography adopts a formic acid aqueous solution with the concentration of 0.1% as a mobile phase A, the methanol as a mobile phase B and the acetonitrile as a mobile phase C, and a gradient elution mode is adopted, and the flow rate of the mobile phase is 0.8 mL/min;
(6) calculating the peak area corresponding to the antibiotics in the water sample, and quantitatively calculating according to a standard curve to obtain the content of various antibiotics in the water sample, wherein the standard curve is prepared by precisely weighing trimethoprim, sulfadiazine, sulfamethoxazole, cefaclor, cefixime, cefazolin, fleroxacin, norfloxacin, ciprofloxacin and lomefloxacin, 5mg of each standard substance is respectively fixed to the volume of 10mL by methanol, and the standard substance is prepared into a single-standard stock solution with the volume of 0.5 mg/mL. Taking 1mL of single-standard stock solution, using methanol to fix the volume to 25mL, preparing 20mg/L mixed standard stock solution for later use, storing at 4 ℃, and using methanol to dilute the mixed standard stock solution into a standard series with the following gradient concentration: 1. mu.g/mL, 0.6. mu.g/mL, 0.5. mu.g/mL, 0.4. mu.g/mL, 0.3. mu.g/mL, 0.2. mu.g/mL, 0.1. mu.g/mL, followed by detection according to the chromatographic procedure of step (5).
Preferably, the step (2) is to filter the water sample by using a filter membrane with the pore diameter of 0.45 μm, and Na2The amount of EDTA added was 0.5 g/L.
Preferably, 4mol/L hydrochloric acid is used for adjusting the pH of the water sample in the step (2).
Preferably, the activation method of the HLB column in step (3) is to sequentially pass 5mL of methanol and 5mL of 0.1% formic acid aqueous solution through the HLB column to perform activation treatment.
Preferably, the speed of the water sample to be detected in the step (3) passing through the HLB column is 5 mL/min.
Preferably, the step (4) of eluting the enriched HLB microcolumn specifically comprises: and (3) eluting the enriched HLB small column by using 6mL of methanol aqueous solution with the mass concentration of 5%, and eluting by using 6mL of methanol after vacuum drying for 20 min.
Preferably, the eluent in the step (5) is concentrated by natural blowing to near dryness in a fume hood.
Preferably, the sulfa antibiotic is trimethoprim, sulfadiazine, sulfamethoxazole, the beta-lactam antibiotic is cefaclor, cefixime, cefazolin, quinolone antibiotic is fleroxacin, norfloxacin, ciprofloxacin, lomefloxacin.
Preferably, the gradient elution mode of the mobile phase in the step (6) is as follows: 0-6min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 8:1: 1; 6-8min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 13:5: 2; 8-9min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 6:3: 1; 9-13min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 5:4: 1; 13-14min, wherein the volume ratio of the mobile phase A to the mobile phases B and C is 8:1: 1.
Preferably, the detection wavelength of the high performance liquid chromatograph is 270 nm.
Compared with the prior art, the invention has the following beneficial effects: the method for detecting multiple antibiotics has the advantages that the detection time is greatly shortened, and ten antibiotics can be synchronously searched out, wherein the ten antibiotics comprise sulfonamide antibiotics, beta-lactam antibiotics and quinolone antibiotics. The detection process is simplified, the detection cost is reduced, and the detection efficiency is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
Figure 1 is a chromatographic separation chart for detecting a water sample of the present invention for the treatment of the disease of the human body, namely, the human body, of the present invention;
FIG. 2 is a standard graph of cefaclor;
FIG. 3 is a standard graph of sulfadiazine;
FIG. 4 is a standard graph of fleroxacin;
FIG. 5 is a standard curve of trimethoprim;
FIG. 6 is a standard graph of norfloxacin;
FIG. 7 is a standard graph of ciprofloxacin;
FIG. 8 is a standard curve of cefixime;
FIG. 9 is a standard graph of lomefloxacin;
FIG. 10 is a standard curve of cefazolin;
FIG. 11 is a standard graph of sulfamethoxazole.
Detailed Description
Examples
A method for rapidly detecting multiple antibiotics by high performance liquid chromatography comprises the following steps: an LC-2030 Shimadzu autoinjection high performance liquid chromatography system is adopted, wherein a chromatographic column of Shim-pack GIST C18 is adopted, the column temperature is 40 ℃, the sample injection volume is 30 mu L, a mobile phase adopted by the high performance liquid chromatography is a formic acid aqueous solution with the concentration of 0.1 percent as a mobile phase A, methanol as a mobile phase B, acetonitrile as a mobile phase C, a gradient elution mode is adopted, the flow rate of the mobile phase is 0.8mL/min, and the gradient elution sequence is as follows: 0-6min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 8:1: 1; 6-8min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 13:5: 2; 8-9min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 6:3: 1; 9-13min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 5:4: 1; 13-14min, the volume ratio of the mobile phase A to the mobile phase B and the mobile phase C is 8:1:1, and the detection wavelength is 270 nm.
The method for respectively detecting the Handan city Fuyang river water sample and the Qin river water sample comprises the following steps:
(1) firstly, selecting a water sample to be detected of Handan City Fuyang river or Qin river water sample;
(2) filtering the water sample to be detected, and then adding Na2EDTA and adjusting the pH value of the water sample to 3 by using 4mol/L hydrochloric acid, wherein the water sample is filtered by using a filter membrane with the pore diameter of 0.45 mu m, and Na2-the amount of EDTA added is 0.5 g/L;
(3) introducing the water sample to be detected treated in the step (2) into the activated HLB column, and enriching the water sample to be detected, wherein the speed of the water sample to be detected passing through the HLB column is 5mL/min, and the activation method of the HLB column is to sequentially use 5mL of methanol and 5mL of formic acid aqueous solution with the mass concentration of 0.1% to perform activation treatment on the water sample to be detected passing through the HLB column;
(4) eluting the enriched HLB small column to obtain an eluent, which specifically comprises the following steps: eluting the enriched HLB (hydrophile-lipophile balance) small column by using 6mL of methanol aqueous solution with the mass concentration of 5%, eluting by using 6mL of methanol after vacuum drying for 20min to obtain eluent, and concentrating the eluent to obtain concentrated solution;
(5) the concentrated solution is subjected to constant volume to 1mL by using methanol, a diluted water sample to be detected is transferred, and the antibiotics in the water sample are detected by using high performance liquid chromatography according to the program, wherein figure 1 shows the chromatographic separation of the water sample of the Fuyanghe;
(6) determination of the standard curve: precisely weighing trimethoprim, sulfadiazine, sulfamethoxazole, cefaclor, cefixime, cefazolin, fleroxacin, norfloxacin, ciprofloxacin and lomefloxacin, wherein 5mg of each standard substance is respectively prepared into a single standard stock solution with the standard substance of 0.5mg/mL by diluting the volume to 10mL with methanol. Taking 1mL of single-standard stock solution, using methanol to fix the volume to 25mL, preparing 20mg/L mixed standard stock solution for later use, storing at 4 ℃, and using methanol to dilute the mixed standard stock solution into a standard series with the following gradient concentration: 1. mu.g/mL, 0.6. mu.g/mL, 0.5. mu.g/mL, 0.4. mu.g/mL, 0.3. mu.g/mL, 0.2. mu.g/mL, 0.1. mu.g/mL. Performing HPLC determination according to the chromatographic program, and simultaneously drawing a standard curve by taking the peak area as the ordinate and the concentration as the abscissa to obtain a linear regression equation thereof, as shown in FIGS. 2 to 11.
The standard curve established by the method shows that the linear relation between the mass concentration and the peak area of 10 antibiotics in the range of 0.1-1 mu g/mL is good, and R is2>0.99, can accurately detect the content of 10 antibiotics in the water sample and can effectively separate. The antibiotics in the water samples of Handan City, Fu Yan river and Qin river were quantitatively analyzed by combining with the standard curve, and the results are shown in Table 1.
TABLE 1
Meanwhile, the method has higher precision and high accuracy, the invention transfers the mixed standard working solution with the concentration of 1 mug/mL, samples 30 mug L each time, samples 5 times continuously, measures according to the high phase chromatographic procedure, records the retention time of each target substance, and calculates the precision. The relative standard deviation values calculated for the 10 antibiotics based on retention time are shown in table 2.
TABLE 2
The principles and embodiments of the present invention have been described herein using specific examples, which are provided only to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (8)
1. A method for rapidly detecting multiple antibiotics by utilizing high performance liquid chromatography is characterized in that the antibiotics comprise sulfonamide antibiotics, beta-lactam antibiotics and quinolone antibiotics, and the method comprises the following steps:
(1) firstly, selecting a water sample to be detected;
(2) filtering the water sample to be detected, and then adding Na2-EDTA and adjusting the pH to 3;
(3) introducing the water sample to be detected treated in the step (2) into the activated HLB column, and enriching the water sample to be detected;
(4) eluting the enriched HLB small column by using an eluant to obtain an eluent, and concentrating the eluent to obtain a concentrated solution;
(5) the concentrated solution is subjected to constant volume to 1mL by using methanol, a diluted water sample to be detected is transferred, and the antibiotics are detected by using a high performance liquid chromatography, wherein the high performance liquid chromatography adopts a Shim-pack GIST C18 chromatographic column, the column temperature is 40 ℃, the sample injection volume is 30 mu L, a mobile phase adopted by the high performance liquid chromatography adopts a formic acid aqueous solution with the concentration of 0.1% as a mobile phase A, the methanol as a mobile phase B and the acetonitrile as a mobile phase C, and a gradient elution mode is adopted, and the flow rate of the mobile phase is 0.8 mL/min;
(6) calculating the peak area corresponding to the antibiotics in the water sample, and quantitatively calculating according to a standard curve to obtain the content of the multiple antibiotics in the water sample;
the sulfonamide antibiotics are trimethoprim, sulfadiazine, sulfamethoxazole, the beta-lactam antibiotics are cefaclor, cefixime, cefazolin, and quinolone antibiotics are fleroxacin, norfloxacin, ciprofloxacin, and lomefloxacin;
the gradient elution mode of the mobile phase in the step (5) is as follows: 0-6min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 8:1: 1; 6-8min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 13:5: 2; 8-9min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 6:3: 1; 9-13min, wherein the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 5:4: 1; 13-14min, wherein the volume ratio of the mobile phase A to the mobile phases B and C is 8:1: 1.
2. The method for rapid detection of multiple antibiotics by high performance liquid chromatography according to claim 1, wherein the water sample is filtered by a filter membrane with a pore size of 0.45 μm in step (2), and Na is added2The amount of EDTA added was 0.5 g/L.
3. The method for rapid detection of multiple antibiotics by using high performance liquid chromatography according to claim 1, wherein the pH of the water sample is adjusted by using 4mol/L hydrochloric acid in the step (2).
4. The method for rapid detection of multiple antibiotics by high performance liquid chromatography according to claim 1, wherein the activation method of the HLB column in step (3) is to sequentially perform activation treatment on 5mL of methanol and 5mL of 0.1% formic acid aqueous solution by mass through the HLB column.
5. The method for rapid detection of multiple antibiotics by using high performance liquid chromatography according to claim 1, wherein the speed of the water sample to be tested in step (3) passing through the HLB column is 5 mL/min.
6. The method for rapidly detecting multiple antibiotics by using high performance liquid chromatography according to claim 1, wherein the step (4) of eluting the enriched HLB columella specifically comprises: and (3) eluting the enriched HLB small column by using 6mL of methanol aqueous solution with the mass concentration of 5%, and eluting by using 6mL of methanol after vacuum drying for 20 min.
7. The method for rapid detection of multiple antibiotics by high performance liquid chromatography according to claim 1, wherein the concentration method of the eluate in the step (4) is natural air blowing to near dryness.
8. The method for rapid detection of multiple antibiotics by high performance liquid chromatography according to claim 1, wherein the detection wavelength of the high performance liquid chromatograph is 270 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080673.5A CN109856268B (en) | 2019-01-28 | 2019-01-28 | Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080673.5A CN109856268B (en) | 2019-01-28 | 2019-01-28 | Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109856268A CN109856268A (en) | 2019-06-07 |
| CN109856268B true CN109856268B (en) | 2021-07-09 |
Family
ID=66896425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910080673.5A Active CN109856268B (en) | 2019-01-28 | 2019-01-28 | Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109856268B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816568B (en) * | 2020-11-02 | 2023-02-03 | 中国科学院沈阳应用生态研究所 | Method for simultaneously detecting multiple antibiotics |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940925B (en) * | 2014-04-16 | 2015-08-12 | 环境保护部华南环境科学研究所 | High performance liquid chromatography detects method and the application of sulfa antibiotics fast |
| CN105241990A (en) * | 2015-09-25 | 2016-01-13 | 沈阳药科大学 | Method for determining 10 kinds of antibiotics in water environment through combination of sample pre-treatment technology and HPLC-MS |
| CN105424825B (en) * | 2015-11-03 | 2017-08-22 | 沈阳药科大学 | A kind of method that separation and concentration determines 12 kinds of left drugs in water environment |
| CN107561187A (en) * | 2017-09-08 | 2018-01-09 | 中国科学院生态环境研究中心 | A kind of method of Multiple Classes of Antibiotics in synchronous detection polluted-water |
-
2019
- 2019-01-28 CN CN201910080673.5A patent/CN109856268B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109856268A (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | A review of pretreatment and analytical methods of biogenic amines in food and biological samples since 2010 | |
| McCalley | Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques | |
| Ziarrusta et al. | Determination of fluoroquinolones in fish tissues, biological fluids, and environmental waters by liquid chromatography tandem mass spectrometry | |
| Chen et al. | Liquid chromatography of active principles in Sophora flavescens root | |
| CN107543877A (en) | A kind of method that SPE liquid chromatography tandem mass spectrometry determines six 24 kinds of antibiotic of class in water body simultaneously | |
| CN105651894B (en) | Method for determining antibiotics in environmental soil | |
| CN107490649A (en) | The screening method of 62 kinds of antibacterials in a kind of farm animal excrement | |
| CN109342632A (en) | The method that microwave abstracting-Solid Phase Extraction pre-treatment combination LC-MS technology detects 15 kinds of antibiotic in aquaculture bed mud simultaneously | |
| CN105651922B (en) | Method for determining PPCPs in environmental water sample | |
| CN107543876A (en) | A kind of method that SPE liquid chromatography tandem mass spectrometry detects 9 kinds of estrogenic chemicalses in water body simultaneously | |
| CN105784858B (en) | Method for measuring PPCPs in environmental soil | |
| Zhang et al. | Separation and purification of flavonoid from ginkgo extract by polyamide resin | |
| CN109856268B (en) | Method for rapidly detecting multiple antibiotics by using high performance liquid chromatography | |
| Chen et al. | Trace analysis of 28 antibiotics in plant tissues (root, stem, leaf and seed) by optimized QuEChERS pretreatment with UHPLC-MS/MS detection | |
| CN109142597A (en) | The pre-treating method of Tilmicosin in a kind of detection Swine plasma | |
| Guedes‐Alonso et al. | Molecularly imprinted solid‐phase extraction coupled with ultra high performance liquid chromatography and fluorescence detection for the determination of estrogens and their metabolites in wastewater | |
| CN113466381A (en) | Method for measuring antibiotics in human urine by using solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry | |
| CN101561421A (en) | Method for detecting oxytetracycline in domestic sewage by solid phase extracted-high performance liquid chromatography method | |
| CN104280473A (en) | Analysis method of 5-hydroxymethylfurfural in linezolid injection | |
| CN102033111A (en) | Antiphase high efficiency liquid chromatography method for segregating polymyxin E methyl sodium sulfnate and analog thereof | |
| Qian et al. | A high‐throughput screening method for determination of multi‐antibiotics in animal feed | |
| CN101639467A (en) | Method for determining amount of residual florfenicol in aquatic product by using high-efficiency liquid chromatogram fluorescence method | |
| CN108732272B (en) | HPLC detection method for benzyl chloride residue in enoxaparin sodium | |
| CN108802243B (en) | Method for simultaneously detecting bentazone, 2,4-D, 2, 4-dichlorophen, 2,4, 6-trichlorophenol and pentachlorophenol in water | |
| CN107884502B (en) | Method for detecting residual quantity of abamectin in soil |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |