Method for rapidly detecting various antibiotics in environmental water body by combining online solid-phase extraction-liquid chromatography-triple quadrupole mass spectrometry

Abstract

The invention discloses a method for rapidly detecting various antibiotics in an environmental water body by online solid-phase extraction-liquid chromatography-triple quadrupole mass spectrometry, which comprises the following steps: pre-treating a sample, enriching through an online solid-phase extraction column, eluting and separating through liquid chromatography after the solid-phase extraction and enrichment are finished, and detecting the antibiotics in the environmental water body. And detecting the various antibiotics by combining triple quadrupole mass spectrometry, and obtaining the concentration of the antibiotics in the sample according to the standard curve. According to the method, on-line solid-phase extraction and ultra-high performance liquid chromatography-triple quadrupole mass spectrometry are combined, all analysis steps are automatically completed by an instrument after a sample is simply pretreated, full-automatic operation of enrichment, purification, separation and determination of the environmental water body sample is realized, 31 antibiotics in the environmental water body can be rapidly detected and analyzed, and the method is suitable for industrial production. The whole determination time is only 35 min, the detection limit reaches the ng / L level, due to automatic operation, the reproducibility precision is better than that of a traditional manual pretreatment method, and errors caused by manual operation are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry. Background Art

[0002] Antibiotics are secondary metabolites or synthetic analogs produced by bacteria, molds or other microorganisms. They are widely used in the treatment of infectious diseases in humans and animals, and are also widely used in aquaculture and livestock and poultry breeding. With the widespread use of antibiotics, a large number of antibiotics can enter water bodies through sewage treatment plants, surface runoff, seepage, etc. The antibiotics with high usage at present are mainly sulfonamides, tetracyclines, quinolones, macrolides, and β-lactams. Antibiotics remaining in water bodies can accumulate for a long time, causing damage to microbial communities in water and affecting aquatic organisms. At the same time, they can destroy the ecosystem and enter the human body through the food chain, posing a high risk to both ecology and human health. In addition, the long-term residue of antibiotics in the environment can increase the drug resistance of pathogens. Therefore, the development of new technologies for detecting the content of antibiotics in water environments is of great practical significance for correctly assessing the degree of antibiotic pollution in water bodies and providing technical support and data support for environmental management.

[0003] Since there are many types of antibiotics entering the water environment and their content is low, and there are large differences in polarity, solubility and acidity coefficient of different antibiotics, there are many difficulties in the simultaneous enrichment and efficient detection of multiple antibiotics. Efficient and high-precision detection of antibiotics in water is the key to conducting in-depth research, so it is necessary to establish a method for simultaneous detection of trace antibiotics in water. At present, the detection methods of antibiotics include microbial detection, enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among them, microbial detection, capillary electrophoresis and CC-MS are less used in antibiotic testing. LC is suitable for the detection of high-concentration antibiotics, but is not sensitive enough for the determination of low-concentration antibiotics. In addition, solid phase extraction, solid phase microextraction and liquid-liquid extraction are usually used for the pretreatment of antibiotic samples in water environments to purify and concentrate the analytes, thereby obtaining sensitive and more reproducible results. However, the above pretreatment methods all require manual extraction experiments, which are labor-intensive and take a long time for pretreatment and analysis.

[0004] Therefore, it is very necessary to develop an online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry method that can solve the above problems and quickly detect multiple antibiotics in environmental water. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry, so as to conveniently, efficiently and quickly determine multiple antibiotics in water.

[0006] The objective of the present invention is achieved by comprising the following steps: pre-treating the sample, enriching it through an online solid phase extraction column, eluting and separating it through liquid chromatography after the solid phase extraction enrichment is completed, detecting multiple antibiotics by triple quadrupole mass spectrometry, and obtaining the concentration of the antibiotics in the sample according to a standard curve.

[0007] Preferably, the pretreatment is to remove suspended particles from the water sample through a 0.22 μm glass fiber filter membrane, add Na2EDTA, adjust the pH of the water sample to 2-3, take a water sample (5 mL) and add an internal standard to indicate the recovery rate; wherein the internal standard is tetracycline-d6, doxycycline-d3, sulfadiazine-d4, sulfamethoxazole-d4, norfloxacin-d5, ciprofloxacin hydrochloride-d8, roxithromycin-d7, and azithromycin-d3.

[0008] There are 31 kinds of antibiotics described in the present invention, specifically: (1) Tetracyclines: tetracycline, oxytetracycline, chlortetracycline; (2) Quinolones: cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, lomefloxacin, norfloxacin, ofloxacin, sarafloxacin, flumequine, nalidixic acid; (3) Sulfonamides: sulfachloropyridazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfapyridine, sulfathiazole; (4) Macrocyclic esters: clarithromycin, azithromycin, erythromycin A, lincomycin, roxithromycin, clindamycin; (5) Chloramphenicol: florfenicol, chloramphenicol.

[0009] Preferably, the online solid phase extraction column is an HLB column (polystyrene-divinylbenzene, 2.1×12.5 mmi.d., 15 μm), and the sample loading volume of the online solid phase extraction column during solid phase extraction enrichment is 0.5 mL to 1.5 mL.

[0010] Preferably, the mobile phase conditions for solid phase extraction enrichment in the online solid phase extraction column are: 0min to 4min, the mobile phase A accounts for 100%; 4min to 10min, the mobile phase B accounts for 50%, and the mobile phase C accounts for 50%; 10min to 15min, the mobile phase A accounts for 100%; wherein, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B contains acetonitrile, and the mobile phase C is methanol; The mobile phase flow rate was set as follows: 0.4 mL / min for 0 min to 0.25 min, 1 mL / min for 0.25 min to 5 min, and 1.2 mL / min for 5 min to 15 min.

[0011] Preferably, the mobile phase conditions for elution separation by liquid chromatography are as follows: 0min~4min initial mobile phase A1 accounts for 98%, B1 accounts for 2%; 4min~5min mobile phase A1 accounts for 85%, B1 accounts for 15%; 5min~12min mobile phase A1 accounts for 80%, B1 accounts for 20%; 12min~16min mobile phase A1 accounts for 60%, B1 accounts for 40%; 16min~18min mobile phase A1 accounts for 40%, B1 accounts for 60%; 18min~20min mobile phase A1 accounts for 2%, B1 accounts for 98%; Among them, the mobile phase A1 contains 0.1% formic acid + 4.5mmol / L ammonium formate aqueous solution + 0.5mmol / L ammonium fluoride aqueous solution, and the mobile phase B1 contains methanol + acetonitrile, and the volume ratio of methanol to acetonitrile is 1:2; The mobile phase flow rate for elution separation in liquid chromatography was set at 0.45 mL / min.

[0012] Preferably, the standard curve establishment process is: measure the response values ​​of a series of mass concentrations of antibiotic standard solutions and the response values ​​of the internal standard, and then establish a calibration curve with the mass concentration of the target as the horizontal axis and the ratio of its corresponding response value to the response value of the internal standard and the product of the concentration of the internal standard as the vertical axis.

[0013] Preferably, the chromatographic column for elution separation is 18 C liquid chromatography column, specification: 150mm×3.0mm, 2.7μm; column temperature of the chromatography column is 40℃.

[0014] Preferably, the gradient elution program for online solid phase extraction is shown in Table 1; the liquid chromatography gradient elution program is shown in Table 2; Table 1: Online solid phase extraction gradient elution program Table 2: Liquid chromatography gradient elution program .

[0015] Preferably, the conditions for triple quadrupole mass spectrometry detection are: Ionization mode: Electrospray positive ion source and electrospray negative ion source scan simultaneously; Capillary voltage: 3500V, nozzle voltage: 500V; Atomizer pressure 40psi; Drying gas temperature: 300°C, sheath gas temperature: 350°C; Drying gas flow rate: 10 L / min, sheath gas flow rate: 11 L / min; Monitoring mode: Multiple reaction monitoring (MRM) mode.

[0016] Compared with the prior art, the present invention has the following technical effects: 1. The method of the present invention combines online solid phase extraction with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The sample only needs simple pretreatment and the instrument automatically completes all analysis steps, realizing the fully automated operation of environmental water sample enrichment, purification, separation and determination. It can quickly detect and analyze 31 kinds of antibiotics in environmental water. The entire determination time only takes 35 minutes, and the detection limit reaches ng / L level. Due to the automated operation, the reproducibility and precision are better than the traditional manual pretreatment method, reducing the error caused by human operation; 2. The present invention has the advantages of small demand for environmental water samples, simple pretreatment process, high detection sensitivity, short determination time, high recovery rate, etc., and can be applied to the detection of ultra-trace antibiotics in environmental water; 3. The present invention realizes the rapid enrichment and elution of antibiotics in water through an online solid phase extraction column. The whole process takes only 15 minutes, thereby improving the detection sensitivity. 4. The present invention separates different antibiotics by optimizing liquid chromatography conditions to meet the requirements of analytical monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 chromatogram of 31 antibiotics in Example 1; Figure 2 is the chromatogram of tetracycline antibiotics in Example 1; Figure 3 is the chromatogram of sulfonamide antibiotics in Example 1; Figure 4 is the chromatogram of quinolone antibiotics in Example 1; Figure 5 The chromatogram of the macrocyclic ester antibiotics in Example 1 is shown in FIG. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0019] Example 1 The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry of this embodiment is applied to a water sample to detect a water sample, comprising the following steps: S1. Pre-treat the sample, specifically, remove suspended particles from the water sample through a 0.22 μm glass fiber filter, add Na2EDTA, add hydrochloric acid to adjust the pH of the water sample to 2-3, take a water sample (5 mL) and add internal standards to indicate the recovery rate. The internal standards are tetracycline-d6, doxycycline-d3, sulfadiazine-d4, sulfamethoxazole-d4, norfloxacin-d5, ciprofloxacin hydrochloride-d8, roxithromycin-d7, and azithromycin-d3; S2, enrichment was performed by an online solid phase extraction column, the online solid phase extraction column was an HLB column (polystyrene-divinylbenzene, 2.1×12.5 mm id, 15μm), mobile phase A, mobile phase B and mobile phase C are mixed in proportion, and the sample volume when the online solid phase extraction column is used for solid phase extraction enrichment is 0.5mL~1.5mL; the mobile phase conditions when the online solid phase extraction column is used for solid phase extraction enrichment are: 0min~4min mobile phase A accounts for 100%; 4min~10min mobile phase B accounts for 50%, and mobile phase C accounts for 50%; 10min~15min mobile phase A accounts for 100%; wherein mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is acetonitrile, and mobile phase C is methanol; the mobile phase flow rate is set to: 0min~0.25min mobile phase flow rate is 0.4mL / min, 0.25min~5min mobile phase flow rate is 1mL / min, and 5min~15min mobile phase flow rate is 1.2mL / min; the gradient elution program during online solid phase extraction is shown in Table 1; Table 1: Online solid phase extraction gradient elution program S3. After solid phase extraction and enrichment are completed, elution and separation are performed by liquid chromatography; the chromatographic column for elution and separation is 18C liquid chromatography column, specification 150mm×3.0mm, 2.7μm; column temperature of the chromatographic column is 40℃, mobile phase A1 and mobile phase B1 are mixed in proportion; mobile phase conditions for elution separation of liquid chromatography are: 0min~4min initial mobile phase A1 accounts for 98%, B1 accounts for 2%; 4min~5min mobile phase A1 accounts for 85%, B1 accounts for 15%; 5min~12min mobile phase A1 accounts for 80%, B1 accounts for 20%; 12min~16min mobile phase A1 accounts for 60% , B1 accounts for 40%; 16min~18min mobile phase A1 accounts for 40%, B1 accounts for 60%; 18min~20min mobile phase A1 accounts for 2%, B1 accounts for 98%; wherein, mobile phase A1 contains 0.1% formic acid + 4.5mmol / L ammonium formate aqueous solution + 0.5mmol / L ammonium fluoride aqueous solution, mobile phase B1 contains methanol + acetonitrile, and the volume ratio of methanol to acetonitrile is 1:2; the mobile phase flow rate during elution separation by liquid chromatography is set to 0.45mL / min; the liquid chromatography gradient elution program is shown in Table 2; Table 2: Liquid chromatography gradient elution program S4. Multiple antibiotics were detected by triple quadrupole mass spectrometry, and the concentration of antibiotics in the sample was obtained according to the standard curve. The conditions for triple quadrupole mass spectrometry detection were as follows: ionization mode: simultaneous scanning of electrospray positive ion source and electrospray negative ion source; capillary voltage: 3500V, nozzle voltage: 500V; nebulizer pressure 40psi; drying gas temperature: 300℃, sheath gas temperature: 350℃; drying gas flow rate: 10L / min, sheath gas flow rate: 11L / min; monitoring mode: multiple reaction monitoring (MRM) mode; the ion source conditions of 31 antibiotics were optimized. The specific mass spectrometry parameters are shown in Table 3, and the chromatograms of 31 antibiotics are shown in Figure 1 ; Among them, the chromatogram of tetracycline antibiotics is detailed in Figure 2 , the chromatogram of sulfonamide antibiotics is shown in Figure 3 , the chromatogram of quinolone antibiotics is shown in Figure 4 , see the chromatogram of macrocyclic ester antibiotics for details. Figure 5 The process of establishing the standard curve is as follows: the response values ​​of a series of mass concentrations of the antibiotic standard solution and the response values ​​of the internal standard are measured, and then the mass concentration of the target is used as the abscissa, and the ratio of the corresponding response value to the response value of the internal standard and the product of the concentration of the internal standard are used as the ordinate to establish a calibration curve; Table 3: Mass spectrometry parameters of 31 antibiotics in MRM mode Test results: Use ultrapure water to prepare 1 ng / ~100 ng / L antibiotic standard solution, test under the above instrument conditions, record the peak area, and draw the peak area-concentration standard curve; the results are shown in Table 4; Table 4: Peak area-concentration standard curves of 31 antibiotics Under this condition, the actual samples were extracted in this embodiment to conduct precision and accuracy experiments, and the results are shown in Table 5; Table 5: Experimental results

Claims

1. A method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry, characterized in that The following steps are involved: After the sample is pretreated, it is enriched by an online solid phase extraction column. After the solid phase extraction enrichment is completed, it is eluted and separated by liquid chromatography. A triple quadrupole mass spectrometer is used to detect multiple antibiotics, and the concentration of antibiotics in the sample is obtained according to the standard curve.

2. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The pretreatment is to remove suspended particles from the water sample through a 0.22 μm glass fiber filter membrane, adjust the pH of the water sample to 2-3 after adding Na2EDTA, and add an internal standard to the water sample to indicate the recovery rate.

3. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that There are 31 kinds of antibiotics, specifically: (1) Tetracyclines: tetracycline, oxytetracycline, chlortetracycline; (2) Quinolones: cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, lomefloxacin, norfloxacin, ofloxacin, sarafloxacin, flumequine, nalidixic acid; (3) Sulfonamides: sulfachloropyridazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfapyridine, sulfathiazole; (4) Macrocyclic esters: clarithromycin, azithromycin, erythromycin A, lincomycin, roxithromycin, clindamycin; (5) Chloramphenicol: florfenicol, chloramphenicol.

4. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The online solid phase extraction column is an HLB column, and the sample loading volume of the online solid phase extraction column during solid phase extraction enrichment is 0.5 mL to 1.5 mL.

5. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The mobile phase conditions for solid phase extraction enrichment in the online solid phase extraction column are as follows: 0min to 4min, the mobile phase A accounts for 100%; 4min to 10min, the mobile phase B accounts for 50%, and the mobile phase C accounts for 50%; 10min to 15min, the mobile phase A accounts for 100%; wherein, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B contains acetonitrile, and the mobile phase C is methanol; The mobile phase flow rate was set as follows: 0.4 mL / min for 0 min to 0.25 min, 1 mL / min for 0.25 min to 5 min, and 1.2 mL / min for 5 min to 15 min.

6. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The mobile phase conditions for elution separation by liquid chromatography are as follows: 0min~4min, the initial mobile phase A1 accounts for 98%, and B1 accounts for 2%; 4min~5min, the mobile phase A1 accounts for 85%, and B1 accounts for 15%; 5min~12min, the mobile phase A1 accounts for 80%, and B1 accounts for 20%; 12min~16min, the mobile phase A1 accounts for 60%, and B1 accounts for 40%; 16min~18min, the mobile phase A1 accounts for 40%, and B1 accounts for 60%; 18min~20min, the mobile phase A1 accounts for 2%, and B1 accounts for 98%; Among them, the mobile phase A1 contains 0.1% formic acid + 4.5mmol / L ammonium formate aqueous solution + 0.5mmol / L ammonium fluoride aqueous solution, and the mobile phase B1 contains methanol + acetonitrile, and the volume ratio of methanol to acetonitrile is 1:2; The mobile phase flow rate for elution separation in liquid chromatography was set at 0.45 mL / min.

7. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The process of establishing the standard curve is as follows: the response values ​​of a series of mass concentrations of antibiotic standard solutions and the response values ​​of the internal standard are measured, and then a calibration curve is established with the mass concentration of the target as the horizontal coordinate and the ratio of the corresponding response value to the response value of the internal standard and the product of the concentration of the internal standard as the vertical coordinate.

8. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The chromatographic column used for elution separation is 18 C liquid chromatography column, specification: 150mm×3.0mm, 2.7μm; column temperature of the chromatography column is 40℃.

9. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The gradient elution program for online solid phase extraction is shown in Table 1; the gradient elution program for liquid chromatography is shown in Table 2; Table 1: Online solid phase extraction gradient elution program Table 2: Liquid chromatography gradient elution program 。 10. The method for rapid detection of multiple antibiotics in environmental water by online solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry according to claim 1, characterized in that The conditions for triple quadrupole mass spectrometry detection were: Ionization mode: Electrospray positive ion source and electrospray negative ion source scan simultaneously; Capillary voltage: 3500V, nozzle voltage: 500V; Atomizer pressure 40psi; Drying gas temperature: 300°C, sheath gas temperature: 350°C; Drying gas flow rate: 10 L / min, sheath gas flow rate: 11 L / min; Monitoring mode: Multiple reaction monitoring mode.

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