Method for detecting macrolide antibiotics in water body

Abstract

The invention discloses a method for detecting macrolide antibiotics in a water body, which adopts a dispersive solid-phase extraction method and comprises the following steps: S1, synthesizing a material to obtain a metal organic framework material Ce-MOF of cerium; s2, sample collection: collecting river and lake water samples and transporting the river and lake water samples to a laboratory for analysis; s3, sample pretreatment; s4, analyzing the sample, detecting the macrolide antibiotics through a liquid chromatography tandem mass spectrometer, and adopting an ion electrospray positive ion mode ESI < + > and a multi-reaction monitoring MRM mode. Compared with traditional solid-phase extraction, the method has the advantages that the operation process is simplified, the complexity and time cost of sample treatment are reduced, the accuracy and repeatability are improved, the method is more environment-friendly, and the experimental detection cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of water quality detection, and in particular to a method for detecting macrolide antibiotics in water. Background Art

[0002] Macrolide antibiotics (MA) are a class of antimicrobial drugs with a 12-16 carbon lactone ring in their molecular structure. Macrolide antibiotics include roxithromycin, clarithromycin, josamycin, tylosin, etc. They are widely used in the treatment of human and animal diseases and are often detected in water bodies. However, the presence of these antibiotics may not only cause health problems for aquatic organisms and humans, but also promote the spread of antibiotic resistance genes. Therefore, the development of an accurate, rapid and efficient detection method is a demand for current technological development.

[0003] At present, the commonly used instrumental methods for antibiotic detection include: liquid chromatography-tandem mass spectrometry, high performance liquid chromatography, capillary electrophoresis, etc. Among them, liquid chromatography-tandem mass spectrometry has strong specificity and high sensitivity, and is more suitable for trace analysis of antibiotics in the environment. The pretreatment methods of antibiotics in water bodies include online solid phase extraction, solid phase extraction, dispersed liquid-liquid microextraction, and ionic liquid membrane microextraction. Among them, solid phase extraction is the most commonly used method for the determination of antibiotics in water. Although the solid phase extraction method can achieve the purpose of enriching and concentrating the target, there are the following problems: 1. Limited selection of adsorption materials: Adsorption materials commonly used in traditional solid phase extraction, such as C18, polymers or silica gel, have limited selectivity and adsorption capacity for certain macrolide antibiotics, resulting in low extraction efficiency.

[0004] 2. Complicated operation steps: Traditional solid phase extraction usually requires multiple operation steps, such as equilibration, loading, elution, etc. These steps are time-consuming and prone to introduce operational errors, affecting the reproducibility of the results.

[0005] 3. Large sample processing volume: In order to improve the detection sensitivity, it is often necessary to process a larger volume of water samples, which not only increases the extraction time, but also for some equipment, processing large volume samples may cause an increased burden on the equipment.

[0006] 4. High cost: The solid phase extraction process often requires expensive solid phase extraction columns and a large amount of organic solvents for elution, which not only increases costs but also poses a potential threat to the environment.

[0007] 5. Insufficient sensitivity and selectivity: Due to the limitations of traditional adsorption materials, it may be difficult to effectively distinguish certain macrolide antibiotics with similar structures, which affects the selectivity and sensitivity of the method. Summary of the invention

[0008] The purpose of the present invention is to design a method for detecting macrolide antibiotics in water in view of the deficiencies of the prior art, simplify the operation steps by using dispersed solid phase extraction technology, and use Ce-MOF to reduce cost consumption, improve adsorption efficiency, enhance selectivity, reduce sample processing volume, and improve sensitivity. The following technical solutions are adopted: A method for detecting macrolide antibiotics in water, using dispersed solid phase extraction (DSPE), comprises the following steps: S1: Material synthesis: Accurately weigh 0.217g-4.34g of cerium nitrate and dissolve it in 1-20mL of aqueous solution to prepare a 0.5mol / L cerium nitrate solution, and adjust the pH value of the solution to 3.5 with dilute nitric acid. Accurately weigh 0.105g-1.31g of 1,3,5-trimethylbenzene carboxylic acid and add it to a mixed solution of 40-500mL of water and ethanol, with a mass ratio of water to ethanol of 3:1. Slowly add the prepared 1-5mL 0.5 mol / L cerium nitrate solution to 40-200mL 0.5 mmol / L In a mixed solution of 1,3,5-trimethylbenzene trimesic acid in water and ethanol, the volume ratio of cerium nitrate solution to 1,3,5-trimethylbenzene trimesic acid solution is 1:40, and the mixture is stirred vigorously for 10-20 minutes at room temperature, and a large amount of white precipitate is produced. After filtering, washing with anhydrous ethanol and water for several times, and drying at 40-60°C, a cerium metal organic framework material, namely Ce-MOF, is obtained; S2: Sample collection: 12 samples of river and lake water were collected in Yangzhou area. Specifically, 0.25-1L water sample was collected at a depth of 0-50 cm below the water surface using a vertical sampler, 1-5mL methanol and 5-25mg ascorbic acid were added, and filled into a 0.25-1L clean brown glass bottle, which was then stored at 0-4°C and transported to the laboratory for analysis within 24h; S3: Sample pretreatment: Place 25-50 mg of Ce-MOF in a 50-100 mL centrifuge tube, add 50-100 mL of water sample containing the target compound, then add 4-8 g of sodium chloride, and use hydrochloric acid to adjust the pH of the water sample to 6.5-7.5. After being evenly dispersed, place it in an oscillator for 20-30 minutes, then centrifuge it at 4000 rpm for 10-15 minutes, discard the supernatant, and then add 2-4 mL of methanol solution containing 0.2% ammonia water to elute the macrolide antibiotics adsorbed on the adsorbent Ce-MOF, vortex and elute for 5-10 minutes, collect the eluate and filter it through a 0.22 μm filter membrane, blow the eluate to near dryness with nitrogen blower at 35-40°C, then dilute it to 0.1-1 mL with methanol-water solution, the volume ratio of methanol-water solution is 1:1, add internal standard solution roxithromycin-D7, transfer it to a chromatographic bottle, and then analyze it with ultra-high performance liquid chromatography-tandem mass spectrometry; S4: Sample analysis: Four macrolide antibiotics were detected by liquid chromatography-tandem mass spectrometry using electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) mode.

[0009] Preferably, the four macrolide antibiotics in step 4 are roxithromycin, clarithromycin, josamycin and tylosin.

[0010] Preferably, the specific mass spectrometry parameters of the liquid chromatography tandem mass spectrometer in step 4 are as follows: capillary voltage 3.0 kV, ion source temperature 150°C, desolvation temperature 500°C, desolvation and cone gases are both nitrogen, and flow rates are 1000 L / h and 50 L / h, respectively; the chromatographic column model used is 1.7 μm, 2.1 mm×50 mm ACQUITY UPLC BEH C18, the column temperature is 35°C, the sample injection volume is 5 μL, and the flow rate is 0.4 mL / min.

[0011] Preferably, the four macrolide antibiotics are subjected to liquid phase gradient elution in ESI+ mode, and the mobile phases for elution are phase A: an aqueous solution of 0.1% formic acid and 5 mmol / L ammonium formate solution, and phase B: pure acetonitrile.

[0012] Compared with the closest prior art, the technical solution provided by the present invention has the following beneficial effects: 1. Simplified operation process: Compared with traditional solid phase extraction, the steps are simplified, reducing the complexity and time cost of sample processing; 2. Improve accuracy and repeatability: According to the experimental data, the recovery rate and precision of the method are within the acceptable range, which proves the accuracy and reliability of the method; 3. Environmentally friendly: The amount of Ce-MOF materials and solvents used is relatively small, which is more environmentally friendly than traditional organic solvent extraction; 4. Reduce experimental detection costs: Since the synthesis cost of Ce-MOF is relatively low and it can be mass-produced through chemical synthesis methods, its use in solid phase extraction helps to reduce material costs compared to commercially available solid phase extraction columns; at the same time, dispersed solid phase extraction technology reduces the use of organic solvents, further reducing solvent costs in experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a comparison chart of the recovery rates of the dispersed solid phase extraction method DSPE of the present invention and the traditional solid phase extraction method SPE. DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] See also Figure 1 , the present invention provides the following technical solutions: Embodiment 1: A method for detecting macrolide antibiotics in water, using dispersed solid phase extraction (DSPE), comprises the following steps: S1: Material synthesis: Accurately weigh 0.217g-4.34g of cerium nitrate and dissolve it in 1-20mL of aqueous solution to prepare a 0.5mol / L cerium nitrate solution, and adjust the pH value of the solution to 3.5 with dilute nitric acid. Accurately weigh 0.105g-1.31g of 1,3,5-trimethylbenzene carboxylic acid and add it to a mixed solution of 40-500mL of water and ethanol, with a mass ratio of water to ethanol of 3:1. Slowly add the prepared 1-5mL 0.5 mol / L cerium nitrate solution to 40-200mL 0.5 mmol / L In a mixed solution of 1,3,5-trimethylbenzene trimesic acid in water and ethanol, the volume ratio of cerium nitrate solution to 1,3,5-trimethylbenzene trimesic acid solution is 1:40, and the mixture is stirred vigorously for 10-20 minutes at room temperature, and a large amount of white precipitate is produced. After filtering, washing with anhydrous ethanol and water for several times, and drying at 40-60°C, a cerium metal organic framework material, namely Ce-MOF, is obtained; S2: Sample collection: 12 samples of river and lake water were collected in Yangzhou area. Specifically, 0.25-1L water sample was collected at a depth of 0-50 cm below the water surface using a vertical sampler, 1-5mL methanol and 5-25mg ascorbic acid were added, and filled into a 0.25-1L clean brown glass bottle, which was then stored at 0-4°C and transported to the laboratory for analysis within 24h; S3: Sample pretreatment: Place 25-50 mg of Ce-MOF in a 50-100 mL centrifuge tube, add 50-100 mL of water sample containing the target compound, then add 4-8 g of sodium chloride, and use hydrochloric acid to adjust the pH of the water sample to 6.5-7.5. After being evenly dispersed, place it in an oscillator for 20-30 minutes, then centrifuge it at 4000 rpm for 10-15 minutes, discard the supernatant, and then add 2-4 mL of methanol solution containing 0.2% ammonia water to elute the macrolide antibiotics adsorbed on the adsorbent Ce-MOF, vortex and elute for 5-10 minutes, collect the eluate and filter it through a 0.22 μm filter membrane, blow the eluate to near dryness with nitrogen blower at 35-40°C, then dilute it to 0.1-1 mL with methanol-water solution, the volume ratio of methanol-water solution is 1:1, add internal standard solution roxithromycin-D7, transfer it to a chromatographic bottle, and then analyze it with ultra-high performance liquid chromatography-tandem mass spectrometry; S4: Sample analysis: Four macrolide antibiotics were detected by liquid chromatography-tandem mass spectrometry using electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) mode.

[0016] Preferably, the four macrolide antibiotics in step 4 are roxithromycin, clarithromycin, josamycin and tylosin.

[0017] Preferably, the specific mass spectrometry parameters of the liquid chromatography tandem mass spectrometer in step 4 are as follows: capillary voltage 3.0 kV, ion source temperature 150°C, desolvation temperature 500°C, desolvation and cone gases are both nitrogen, and flow rates are 1000 L / h and 50 L / h, respectively; the chromatographic column model used is 1.7 μm 2.1 mm×50 mm ACQUITY UPLC BEH C18, the column temperature is 35°C, the sample injection volume is 5 μL, and the flow rate is 0.4 mL / min.

[0018] Preferably, the four macrolide antibiotics are subjected to liquid phase gradient elution in ESI+ mode, and the mobile phases for elution are phase A: an aqueous solution of 0.1% formic acid and 5 mmol / L ammonium formate solution, and phase B: pure acetonitrile.

[0019] Example 2 A method for detecting macrolide antibiotics in water, using dispersed solid phase extraction (DSPE), comprises the following steps: S1: Material synthesis: Accurately weigh 0.217 g of cerium nitrate and dissolve it in 1 mL of aqueous solution to prepare a 0.5 mol / L cerium nitrate solution, and adjust the pH value of the solution to 3.5 with dilute nitric acid. Accurately weigh 0.105 g of 1,3,5-trimethylbenzene trimesic acid and add it to 40 mL of a mixed solution of water and ethanol (V / V = 3:1). Slowly add 1 mL of the prepared 0.5 mol / L cerium nitrate solution to a mixed solution of water and ethanol containing 0.5 mmol / L 1,3,5-trimethylbenzene trimesic acid, and vigorously stir for 10 minutes at room temperature. A large amount of white precipitate is produced. After filtering, washing with anhydrous ethanol and water several times, and drying at 50°C, a cerium metal organic framework material (Ce-MOF) is obtained.

[0020] S2: Sample collection: In order to study the accuracy and reliability of this method, river and lake water samples were collected in Yangzhou area. A vertical sampler was used to collect 1L of water sample at a depth of 50 cm below the water surface. 5mL of methanol and 500 mg of ascorbic acid were added and filled into a 1L clean brown glass bottle. The bottle was then stored at 4°C and transported to the laboratory for analysis within 24h.

[0021] S3: Sample pretreatment: 25 mg of Ce-MOF was placed in a 50 mL centrifuge tube, 50 mL of water sample containing the target compound was added, and then 4.0 g of sodium chloride was added, and the pH of the water sample was adjusted to 7.0 with hydrochloric acid. After being evenly dispersed, it was placed in an oscillator for 20 min, and then centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and then 2 mL of methanol (volume ratio) solution containing 0.2% ammonia water was added to elute the macrolide antibiotics adsorbed on the adsorbent Ce-MOF, and vortexed for 5 min. The eluate was collected and filtered through a 0.22 μm filter membrane, and the eluate was nitrogen blown to near dryness at 40°C with a nitrogen blower, and then the volume was made up to 0.1 mL with methanol-water solution (volume ratio of 1:1), and the internal standard solution roxithromycin-D7 was added, and the mixture was transferred to a chromatographic bottle, and then analyzed by ultra-performance liquid chromatography-tandem mass spectrometry.

[0022] S4: Sample analysis: Four macrolide antibiotics (roxithromycin, clarithromycin, josamycin, and tylosin) were detected in electrospray positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode. The specific mass spectrometry parameters are as follows: capillary voltage 3.0 kV; ion source temperature 150°C, desolvation temperature 500°C; desolvation and cone gas are both nitrogen, with flow rates of 1000L / h and 50 L / h, respectively. The chromatographic column model used is ACQUITY UPLC BEHC18 (1.7μm, 2.1mm×50mm), the column temperature is 35°C, the sample injection volume is 5 μL, and the flow rate is 0.4 mL / min. The mobile phases are 0.1% formic acid and 5mmol / L ammonium formate in aqueous solution in phase A and pure acetonitrile in phase B. The liquid phase gradient elution conditions are shown in Table 1.

[0023] Table 1 Liquid phase gradient elution conditions of four macrolide antibiotics in ESI+ mode Comparative Example 1 A method for detecting macrolide antibiotics in water, using a conventional solid phase extraction (SPE) method, comprises the following steps: S1: No material synthesis steps S2: Sample collection: 12 samples of river and lake water were collected in Yangzhou. Specifically, 1-4L of water samples were collected at a depth of 0-50 cm below the water surface using a vertical sampler, filled into 1-4L clean brown glass bottles, and then stored at 0-4°C, transported to the laboratory, filtered through a 0.45μm glass fiber filter, and analyzed within 24h; S3: Sample pretreatment: Accurately transfer 500 mL of the sample, adjust the pH to 6.0 (±0.2) with formic acid, add 0.1 g of disodium tetraacetate, mix well, and wait for extraction and purification. Use HLB solid phase extraction column for extraction and purification, and activate the HLB solid phase extraction column with 5 mL of methanol, 5 mL of water, and 5 mL of water (pH=6) in sequence. Take the water sample through the column, and control the flow rate not to exceed 5 mL / min. After the water sample has passed through the small column, rinse with 10 mL of water, dry under reduced pressure, and elute with 8 mL of methanol. Collect the eluate and blow it to near dryness with nitrogen at 45 °C. Add 1 mL of methanol-water solution (volume ratio of 1:1) to re-dissolve, then add the internal standard roxithromycin-D7, pass through a 0.22 μm regenerated cellulose filter, and provide liquid chromatography-tandem mass spectrometry for determination.

[0024] S4: Sample analysis: Four macrolide antibiotics (roxithromycin, clarithromycin, josamycin, and tylosin) were detected in electrospray positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode. The specific mass spectrometry parameters are as follows: capillary voltage 3.0 kV; ion source temperature 150°C, desolvation temperature 500°C; desolvation and cone gas are both nitrogen, with flow rates of 1000L / h and 50 L / h, respectively. The chromatographic column model used is ACQUITY UPLC BEH C18 (1.7μm, 2.1mm×50 mm), the column temperature is 35°C, the sample injection volume is 5 μL, and the flow rate is 0.4 mL / min. The mobile phases are 0.1% formic acid and 5 mmol / L ammonium formate in aqueous solution in phase A and pure acetonitrile in phase B. The liquid phase gradient elution conditions are shown in Table 1.

[0025] Comparison of recovery rate between Example 2 and Comparative Example 1 like Figure 1 As shown in the figure, the recovery rate experiment of the dispersed solid phase extraction method (DSPE) of this method is compared with the traditional solid phase extraction method (SPE). The closer the recovery rate experimental value is to 100%, the higher the accuracy of the method is, and the smaller the error fluctuation is, the better the reproducibility of the method is.

[0026] The recoveries of four macrolide antibiotics, roxithromycin, clarithromycin, josamycin and tylosin, were compared between dispersed solid phase extraction (DSPE) and conventional solid phase extraction (SPE).

[0027] As can be seen from the figure, the recovery rate obtained by using Ce-MOF material for dispersed solid phase extraction is closer to 100% and the error fluctuation is smaller, indicating that this method has good accuracy and reproducibility.

[0028] In summary, the present invention uses dispersed solid phase extraction (DSPE) to detect macrolide antibiotics in water, which has the following advantages: Simplified operation steps: Dispersed solid phase extraction technology avoids the multi-step operation in traditional solid phase extraction by directly adding dispersed Ce-MOF powder into water samples, simplifies the experimental process, and improves the convenience and reproducibility of the operation.

[0029] Reduce cost consumption: The method of synthesizing Ce-MOF is simple, and the raw materials are cheap. Compared with the consumables required for solid phase extraction, the cost is lower. The dispersed solid phase extraction technology used can use less organic solvent to complete the extraction process. Compared with traditional solid phase extraction technology, it greatly reduces the use of organic solvents, is environmentally friendly and reduces costs.

[0030] Improve detection reproducibility: Since the operation of dispersed solid phase extraction is simple and direct, it reduces human operation errors and improves the reproducibility and accuracy of the experiment.

[0031] Improve adsorption efficiency: Ce-MOF has a high specific surface area and porous structure, which can provide more active adsorption sites, thereby significantly improving the adsorption efficiency of four macrolide antibiotics: roxithromycin, clarithromycin, josamycin, and tylosin.

[0032] Reduce sample processing volume: Due to the efficient adsorption capacity of Ce-MOF, efficient extraction can be achieved in a smaller volume of water sample, which not only reduces the processing time but also reduces the burden on the equipment.

[0033] Improved sensitivity: The high adsorption efficiency and excellent selectivity of Ce-MOF significantly improve the detection sensitivity, and even at low concentration levels, it can achieve effective detection of macrolide antibiotics. The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field can still modify or replace the specific implementation of the present invention. Any modification or equivalent replacement that does not deviate from the spirit and scope of the present invention is within the scope of protection of the claims of the present invention to be approved.

Claims

1. A method for detecting macrolide antibiotics in water, using a dispersed solid phase extraction method, characterized in that: The following steps are involved: S1: Material synthesis: Accurately weigh 0.217g-4.34g of cerium nitrate and dissolve it in 1-20mL of aqueous solution to prepare a 0.5 mol / L cerium nitrate solution, and adjust the pH value of the solution to 3.5 with dilute nitric acid. Accurately weigh 0.105g-1.31g of 1,3,5-trimethylbenzene carboxylic acid and add it to a mixed solution of 40-500mL of water and ethanol, with a mass ratio of water to ethanol of 3:

1. Slowly add the prepared 1-5mL 0.5 mol / L cerium nitrate solution to 40-200mL 0.5 mmol / L In a mixed solution of 1,3,5-trimethylbenzene trimesic acid in water and ethanol, the volume ratio of cerium nitrate solution to 1,3,5-trimethylbenzene trimesic acid solution is 1:40, and the mixture is stirred vigorously for 10-20 minutes at room temperature, and a large amount of white precipitate is produced. After filtering, washing with anhydrous ethanol and water for several times, and drying at 40-60°C, a cerium metal organic framework material, namely Ce-MOF, is obtained; S2: Sample collection: 12 samples of river and lake water were collected in Yangzhou area. Specifically, 0.25-1L water sample was collected at a depth of 0-50 cm below the water surface using a vertical sampler, 1-5mL methanol and 5-25mg ascorbic acid were added, and filled into a 0.25-1L clean brown glass bottle, which was then stored at 0-4°C and transported to the laboratory for analysis within 24h; S3: Sample pretreatment: Place 25-50 mg of Ce-MOF in a 50-100 mL centrifuge tube, add 50-100 mL of water sample containing the target compound, then add 4-8 g of sodium chloride, and use hydrochloric acid to adjust the pH of the water sample to 6.5-7.

5. After being evenly dispersed, place it in an oscillator for 20-30 minutes, then centrifuge it at 4000 rpm for 10-15 minutes, discard the supernatant, and then add 2-4 mL of methanol solution containing 0.2% ammonia water to elute the macrolide antibiotics adsorbed on the adsorbent Ce-MOF, vortex and elute for 5-10 minutes, collect the eluate and filter it through a 0.22 μm filter membrane, blow the eluate to near dryness with nitrogen blower at 35-40°C, then dilute it to 0.1-1 mL with methanol-water solution, the volume ratio of methanol-water solution is 1:1, add internal standard solution roxithromycin-D7, transfer it to a chromatographic bottle, and then analyze it with ultra-high performance liquid chromatography-tandem mass spectrometry; S4: Sample analysis: Four macrolide antibiotics were detected by liquid chromatography-tandem mass spectrometry using electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) mode.

2. A method for detecting macrolide antibiotics in water according to claim 1, characterized in that: The four macrolide antibiotics in step 4 are roxithromycin, clarithromycin, josamycin and tylosin.

3. A method for detecting macrolide antibiotics in water according to claim 1, characterized in that: The specific mass spectrometry parameters of the liquid chromatography tandem mass spectrometer in step 4 are as follows: capillary voltage 3.0 kV, ion source temperature 150°C, desolvation temperature 500°C, desolvation and cone gases are both nitrogen, and the flow rates are 1000 L / h and 50 L / h, respectively; the chromatographic column model used is 1.7 μm, 2.1 mm×50 mm ACQUITY UPLC BEH C18, the column temperature is 35°C, the sample injection volume is 5 μL, and the flow rate is 0.4 mL / min.

4. A method for detecting macrolide antibiotics in water according to claim 3, characterized in that: The four macrolide antibiotics were subjected to liquid phase gradient elution in ESI+ mode, and the mobile phases for elution were phase A: an aqueous solution of 0.1% formic acid and 5 mmol / L ammonium formate solution, and phase B: pure acetonitrile.

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