Rapid determination method of solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry of antibiotics in aquaculture fresh water

Abstract

The invention relates to a solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry rapid determination method for antibiotics in aquaculture fresh water, which comprises the following steps: (1) adding an internal standard solution into aquaculture fresh water to be determined, uniformly mixing, filtering the obtained water sample, and adjusting the pH value; (2) activating a small solid-phase extraction column, enriching the water sample obtained in the step (1) through the small solid-phase extraction column, then leaching and eluting, collecting an eluent, blowing the eluent until the eluent is nearly dry, and fixing the volume by using methanol and water; and (3) feeding the solution obtained after volume metering in the step (2) into ultra-high performance liquid chromatography-tandem mass spectrometry for detection. The invention establishes a method for simultaneously detecting 37 antibiotic residues in aquaculture fresh water by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, the method comprises six common antibiotics such as tetracyclines, macrolides, fluoroquinolones, phenols, sulfonamides and cephalospores, the detection is convenient and fast, and the accuracy is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of water body antibiotic detection, and relates to a rapid determination method for antibiotics in aquaculture fresh water by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry. Background Technique

[0002] Pond aquaculture is the most common artificial aquaculture system. The high-density and intensive aquaculture mode makes related diseases of aquatic organisms occur frequently, leading to the widespread use of antibiotics in disease prevention and treatment, resulting in high-concentration residues of antibiotics in aquaculture ponds. In many countries, the total amount of antibiotics used in aquaculture far exceeds that used in human medicine. It has even been found in research that there is a situation of illegal and large-scale use of human antibiotics in the freshwater aquaculture industry. However, only 20%-30% of the antibiotics used in large quantities are absorbed by aquatic organisms, and most of the drugs are discharged into the environment in the form of the original or metabolites. The remaining antibiotics will cause environmental pollution and bacterial drug resistance, and pose a threat to human health. However, at present, there is no unified national standard for the detection methods and substances to be detected of antibiotics in aquaculture fresh water in China, and the related methods established have certain limitations. In terms of the selection of substances to be detected, the types of target antibiotics are few, and most of them are veterinary antibiotics, ignoring the use of human antibiotics and the residues of antibiotic-related metabolites. In terms of the detection steps, the volume of the sample required by the research method is generally high, so the detection cycle is long and the cost is high. Therefore, establishing a method that can effectively separate, enrich and detect various trace antibiotics in the aquaculture environment of freshwater products, and studying the residual characteristics of antibiotics in the aquaculture area, is of great significance for food safety supervision, environmental risk assessment and bacterial drug resistance detection.

[0003] A study published by Li Zhenjin et al. in the journal "Asian Journal of Ecotoxicology" in 2020 (Research on the Optimization of Antibiotic Detection Methods and Residual Characteristics in Aquaculture Water and Sediments) mentioned the analysis of 15 antibiotics by the UPLC-MS / MS method. This method ignores some antibiotics prohibited from being added to edible aquatic products stipulated by the state at present, and also ignores the detection of some human antibiotics, etc.

[0004] A study published by Lin Qi et al. in the journal "Modern Preventive Medicine" in 2018 (Determination of 16 Quinolone Antibiotics in Water by Automatic Solid Phase Extraction-High Performance Liquid Chromatography-Tandem Mass Spectrometry) used the UPLC-MS / MS method for quantitative analysis of antibiotics. However, this method only detects and studies quinolone antibiotics, ignoring the situation of the residue of various types of antibiotics in current aquaculture fresh water. In addition, the detection limit and quantification limit of quinolone antibiotics in this method are higher than those of this experiment, so it may cause a decrease in the positive detection rate of samples.

[0005] Chinese Patent CN202210167998.9 discloses a rapid determination method of antibiotics in sewage by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, which detects 27 kinds of antibiotics (fluoroquinolones, phenols, tetracyclines, macrolides and sulfonamides), but this method cannot cover the detection of cephalosporin antibiotics at the same time. Summary of the Invention

[0006] The purpose of the present invention is to provide a rapid determination method of antibiotics in aquaculture fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, so as to realize the rapid detection of 37 kinds of antibiotic residues including six major categories of commonly used antibiotics such as fluoroquinolones, phenols, tetracyclines, macrolides, sulfonamides and cephalosporins in aquaculture fresh water, etc.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] A rapid determination method of antibiotics in aquaculture fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0009] (1) Add an internal standard solution to the aquaculture fresh water to be tested, mix evenly, then filter the obtained water sample, and adjust the pH value;

[0010] (2) Activate the solid-phase extraction cartridge, then enrich the water sample obtained in step (1) through the solid-phase extraction cartridge, and then perform rinsing and elution, collect the eluate and blow it to near dryness, and then make a constant volume with methanol and water;

[0011] (3) Send the solution obtained after constant volume in step (2) into ultra-high performance liquid chromatography-tandem mass spectrometry for detection, and that's it.

[0012] Furthermore, the internal standard substances contained in the internal standard solution include D3-azithromycin, D5-chloramphenicol, D7-roxithromycin, D4-sulfamethoxazole, D6-tetracycline, D8-ciprofloxacin, D4-cephalapirin and D3-cefotaxime.

[0013] Furthermore, the concentration of the internal standard solution is 100 μg / L, and the volume ratio of the internal standard solution to the aquaculture fresh water to be tested is 1:4000.

[0014] Furthermore, the antibiotics contained in the aquaculture fresh water to be tested include at least one of fluoroquinolones, phenols, tetracyclines, macrolides, sulfonamides and cephalosporins.

[0015] Even further, the fluoroquinolones include ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin;

[0016] The phenols include florfenicol, chloramphenicol, and thiamphenicol;

[0017] The tetracyclines include doxycycline, tetracycline, chlortetracycline, and oxytetracycline;

[0018] The macrolides include azithromycin, roxithromycin, clarithromycin, erythromycin monohydrate, tilmicosin, and spiramycin;

[0019] The sulfonamides include trimethoprim, sulfadiazine, sulfamethoxazole, sulfadimidine, acetylsulfamethazine, and acetylsulfamethoxazole;

[0020] The cephalosporins include cephalexin, cefazolin, cephapirin, cefapirin benzoate, cefprozil, cefotaxime, cefoperazone, ceftiofur, cefetamet pivoxil, and cefotaxime.

[0021] Furthermore, a 0.7 μm glass fiber filter membrane is used during filtration;

[0022] After filtration, the pH value is adjusted to 7.0 - 8.5.

[0023] Furthermore, during enrichment, the flow rate of the water sample is 4 - 6 mL / min.

[0024] Furthermore, ultrapure water is used for rinsing; methanol is used for elution; and the eluate is blown with nitrogen for 30 minutes.

[0025] Furthermore, the chromatographic conditions during detection are as follows: ACQUITY UPLC T3, 100 mm × 2.1 mm, 1.8 μm; column temperature 35°C; flow rate 0.35 mL / min; injection volume: 10 μL; mobile phase A: 40% methanol - acetonitrile, mobile phase B: 0.2% formic acid - water.

[0026] Furthermore, the mass spectrometry conditions during detection are as follows: ionization mode, electrospray positive ion source ESI + and electrospray negative ion source ESI - simultaneously scanned; ion source voltage, 4500 V; temperature, 400°C; curtain gas, 30 psi; spray gas, 55 psi; auxiliary heating gas, 55 psi; monitoring mode, multiple reaction monitoring MRM mode;

[0027] The mass spectrometry parameters are as follows:

[0028]

[0029]

[0030]

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) It can detect 37 kinds of antibiotics at one time, and the target antibiotics include six major categories of commonly used antibiotics, namely fluoroquinolones, phenols, tetracyclines, macrolides, sulfonamides and cephalosporins, covering human-use antibiotics, veterinary antibiotics and human-veterinary shared antibiotics. In addition to the parent compounds, this method also includes the detection of antibiotic metabolites, greatly expanding the substances to be detected in the existing technology and improving the detection efficiency.

[0033] (2) It greatly reduces the volume of the samples required in the pretreatment steps. By repeatedly optimizing the experimental pretreatment steps and processes, while ensuring a high recovery rate, the sample loading volume required for pretreatment is reduced to 200 mL, which is 1 / 5 to 1 / 10 of the sample loading volume required in the existing technology. It greatly reduces the time required for the experiment and improves the work efficiency, providing convenience for detection experiments and scientific research with a large number of samples.

[0034] (3) It adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and continuously optimizes the mass spectrometry conditions. While ensuring the stability of the spike recovery rate, all 37 antibiotics in six categories can be detected within one method, and the time of the entire detection method is compressed as much as possible. Finally, all 37 antibiotic substances can peak within 11 minutes, and the quality of the peak shape diagram is relatively high.

[0035] (4) The isotope internal standard method has high accuracy. After the method is established, experimental verification is carried out, and the 37 antibiotics have a good linear relationship within the measurement range. The test results are consistent with the local drug types and in line with the actual situation; the detected concentrations of parallel samples are stable, with small errors, ensuring the accuracy of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the total ion current chromatogram in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will be described in detail below with reference to the drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0038] In the following embodiments, unless otherwise specified for raw materials or processing techniques, it means that they are all conventional commercially available raw materials or conventional processing techniques in the art.

[0039] Example 1:

[0040] (1) Materials and Methods

[0041] 1.1 Instruments and Reagents

[0042] Instruments: Ultra-high performance liquid chromatograph (Waters, UK), equipped with a mass spectrometer (ABSCIEX QTRAP 6500 + , USA); Solid-phase extraction instrument (Anpel, China); Nitrogen evaporator (LabTech, China); Ultra-pure water instrument (Milli-Q, USA); Oasis HLB solid-phase extraction column 3cc, 60mg (Waters, USA); 0.7μm glass fiber filter membrane (Whatman, USA); Chromatographic column: ACQUITY UPLC T3 (100mm x 2.1mm, 1.8μm).

[0043] Standards: (1) Fluoroquinolones: Ciprofloxacin, Ofloxacin, Norfloxacin, Pefloxacin, Enrofloxacin, Danofloxacin, Difloxacin, Lomefloxacin; (2) Phenols: Florfenicol, Chloramphenicol, Thiamphenicol; (3) Tetracyclines: Doxycycline, Tetracycline, Chlortetracycline, Oxytetracycline; (4) Macrolides: Azithromycin, Roxithromycin, Clarithromycin, Erythromycin-hydrate, Tilmicosin, Spiramycin; (5) Sulfonamides: Trimethoprim, Sulfadiazine, Sulfamethoxazole, Sulfamethazine, Acetylsulfamethazine, Acetylsulfamethoxazole; (6) Cephalosporins: Cefalexin, Cefazolin, Cefapirin, Cefaclor, Cefprozil, Cefotaxime, Cefoperazone, Cefotaxime, Cefetamet pivoxil, Cefazoxime; (7) Internal standards: D3-Azithromycin, D5-Chloramphenicol, D7-Roxithromycin, D4-Sulfamethoxazole, D6-Tetracycline, D8-Ciprofloxacin, D4-Cefapirin, D3-Cefotaxime (Dr.Ehrenstorfer, Germany).

[0044] Reagents: Methanol (chromatographic grade, Supelco, USA), Acetonitrile (chromatographic grade, Supelco, USA), Formic acid (chromatographic grade, Aladdin, USA).

[0045] 1.2 Methods

[0046] 1.2.1 Preparation of solutions

[0047] Accurately weigh 10mg of each standard, dissolve it with methanol and make up the volume to 10mL in a brown volumetric flask. The standard stock solution is stored in a -20°C refrigerator. Appropriately draw an amount of the standard stock solutions of 37 antibiotics, dilute them with a methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1mg / L; Appropriately draw an amount of the standard stock solutions (1000ppm) of 8 antibiotics (i.e., internal standards), dilute them with an acetonitrile solution to prepare a mixed internal standard working solution with a total concentration of 1mg / L, store it in a 4°C refrigerator for later use, and dilute it to the required concentration when in use.

[0048] 1.2.2 Pretreatment method

[0049] Accurately measure 200 mL of aquaculture fresh water sample, add 50 μL of internal standard working solution with a concentration of 100 μg / L respectively, mix well, filter twice through a 0.7 μm glass fiber filter membrane, and adjust the pH to 7. Use an automatic solid-phase extraction instrument for sample enrichment and purification. Activate the solid-phase extraction cartridge with 2 mL of methanol and 2 mL of ultrapure water in sequence, enrich the water sample at a flow rate of 4 - 6 mL / min. After the sample loading is completed, wash the cartridge with 3 mL of ultrapure water, and then elute with 4 mL of methanol. Blow the eluate with nitrogen for 30 minutes, and make the volume constant to 0.5 mL with methanol-water (V:V = 1:1). After vortex oscillation, transfer it to an injection vial for determination.

[0050] 1.2.3 Analytical method

[0051] (1) Chromatographic conditions: ACQUITY UPLC T3 (100 mm x 2.1 mm, 1.8 μm); column temperature 35 °C; flow rate 0.35 mL / min; injection volume: 10 μL. Mobile phase A: 40% methanol - acetonitrile, mobile phase B: 0.2% formic acid - water. The gradient elution conditions are shown in Table 1.

[0052] Table 1 Gradient elution conditions

[0053] Time (min) Mobile Phase A (%) Mobile Phase B (%) 1.0 10 90 7.0 98 2 9.5 98 2 9.8 10 90 11.0 10 90

[0054] (2) Mass spectrometry conditions: Ionization mode: electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) are scanned simultaneously; ion source voltage: 4500 V; temperature: 400 °C; curtain gas: 30 psi; spray gas: 55 psi; auxiliary heating gas: 55 psi; monitoring mode: multiple reaction monitoring (MRM) mode. The mass spectrometry parameters are shown in Table 2.

[0055] Table 2 Mass spectrometry parameters of 37 antibiotic drugs and 6 internal standard substances

[0056]

[0057]

[0058]

[0059] (2) Method validation

[0060] 2.1 Standard curve and method detection limit

[0061] Using the peak areas of the antibiotics and their internal standards as the ordinate and the concentrations of the antibiotics as the abscissa for linear regression, within the linear range of 1 - 100 μg / L, the linear relationships of 37 antibiotics are good. The method detection limit (MDL) is 0.001 ng / L - 0.271 ng / L, and the method quantification limit (MQL) is 0.002 ng / L - 0.903 ng / L. The linear equations, correlation coefficients, method detection limits, and method quantification limits of each analyte are shown in Table 3.

[0062] Table 3 Linear parameters of 37 antibiotic drugs, as well as method detection limits and method quantification limits

[0063]

[0064]

[0065]

[0066] In addition, the peak shapes of all target antibiotics and their internal standard substances are as shown in Table 4 below.

[0067] Table 4 Quantitative and qualitative peak shapes of all antibiotics

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] 2.2 Accuracy and precision of the method

[0081] Antibiotic standard solutions were added to the samples to prepare spiked samples with concentrations of 20 and 200 ng / L. The spiked recovery experiments were carried out according to the above pretreatment method. Six parallel samples were set for each concentration gradient. The results are shown in Table 5. The results indicate that the spiked recovery rates of the method are in the range of 62.17%-116.49%, and the relative standard deviations are all lower than 15%.

[0082] Table 5 Spiked recovery rates at low and high levels of the detection method (n = 6)

[0083]

[0084]

[0085] 2.3 Detection of actual samples

[0086] In the summer of 2020, water samples were collected from aquaculture ponds in a village in Zhejiang. A total of 3 aquaculture waters were sampled, including fresh water samples from shrimp ponds, fish ponds, and bullfrog ponds. Sampling activities were carried out once in the morning and once in the afternoon at each sampling point. After mixing, they were waiting to be tested. Three parallel samples were set for each sample to be tested, and blank samples were set throughout the experiment for quality control of the experiment.

[0087] Detection was carried out according to the above experimental method. The results are shown in Table 6. The results indicate that a total of 20 antibiotics were detected, including 6 fluoroquinolones (ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin, difloxacin, and lomefloxacin), 6 macrolides (azithromycin, roxithromycin, clarithromycin, anhydroerythromycin, tilmicosin, and spiramycin), 4 sulfonamides (trimethoprim, sulfadiazine, sulfamethoxazole, and sulfamerazine), 2 phenols (thiamphenicol and florfenicol), and 2 cephalosporins (cefprozil and cefotaxime), indicating that a large amount of antibiotics are used in aquaculture.

[0088] A relatively large number of antibiotic species were detected in the fresh water of shrimp ponds, a total of 17 species. 15 antibiotics were detected in the fresh water of fish ponds and bullfrog ponds respectively, showing a relatively high distribution level of quinolone antibiotics and a relatively low usage rate of tetracyclines. This is consistent with the results of an aquaculture area in Shanghai (Research on the optimization of antibiotic detection methods and residue characteristics in aquaculture water and sediments). Among them, the antibiotics azithromycin, clarithromycin, and roxithromycin, which are only for human use, were detected in different aquaculture water bodies, and the detection rates were relatively high, indicating that rural aquaculture farmers have irregular antibiotic use behaviors.

[0089] Table 6 Antibiotic concentrations (ng / L) in fresh water samples from aquaculture ponds in a village in Zhejiang Province

[0090]

[0091]

[0092] In addition, it should be noted that when various cephalosporin antibiotics were added to sewage samples based on the method of CN202210167998.9, it was found that the spike recovery rate was poor and it could not be incorporated into the final method. The specific spike recovery rates of cephalosporin antibiotics in sewage are as follows:

[0093] Antibiotic Name Low Concentration Spike High Concentration Spike Average Recovery Rate % Cefalexin 55.24 32.54 43.89 Cefazolin 17.56 23.58 20.57 Cephapirin 11.25 21.35 16.30 Cefaclor 147.58 260.23 203.91 Cefprozil 111.78 270.36 191.07 Cefotaxime 43.68 52.37 48.03 Cefoperazone 14.25 60.54 37.40 Ceftiofur 47.89 57.12 52.51 Cefetamet Pivoxil 5.41 10.77 8.09 Ceftizoxime 14.34 20.34 17.34

[0094] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A rapid determination method for antibiotics in cultured fresh water by solid phase extraction - ultra high performance liquid chromatography - tandem mass spectrometry, characterized in that, It includes the following steps: (1) Add an internal standard solution to the fresh aquaculture water to be tested. After mixing evenly, filter the obtained water sample and adjust the pH value. (2) Activate the solid-phase extraction cartridge, then enrich the water sample obtained in step (1) through the solid-phase extraction cartridge. Then, perform rinsing and elution, collect the eluate and blow it to near dryness, and then make a constant volume with methanol and water. (3) Send the solution obtained after constant volume in step (2) into an ultra-high performance liquid chromatography-tandem mass spectrometry for detection, and that's it.

2. The rapid determination method of antibiotics in aquaculture freshwater by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The internal standard substances contained in the internal standard solution include D3-azithromycin, D5-chloramphenicol, D7-roxithromycin, D4-sulfamethoxazole, D6-tetracycline, D8-ciprofloxacin, D4-cefapirin, and D3-cefotaxime.

3. The rapid determination method of antibiotics in cultured fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein, The concentration of the internal standard solution is 100 μg / L, and the volume ratio of the internal standard solution to the fresh aquaculture water to be tested is 1:4000.

4. A rapid determination method of antibiotics in cultured fresh water by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The antibiotics contained in the fresh aquaculture water to be tested include at least one of fluoroquinolones, phenols, tetracyclines, macrolides, sulfonamides, and cephalosporins.

5. The rapid determination method of antibiotics in cultured fresh water by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The fluoroquinolones include ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, and lomefloxacin. The phenols include florfenicol, chloramphenicol, and thiamphenicol. The tetracyclines include doxycycline, tetracycline, chlortetracycline, and oxytetracycline. The macrolides include azithromycin, roxithromycin, clarithromycin, erythromycin monohydrate, tilmicosin, and spiramycin. The sulfonamides include trimethoprim, sulfadiazine, sulfamethoxazole, sulfamethazine, acetylsulfamethazine, and acetylsulfamethoxazole. The cephalosporins include cephalexin, cefazolin, cefapirin, cefaloram, cefprozil, cefotaxime, cefoperazone, ceftiofur, cefetamet pivoxil, and cefazoxime.

6. The rapid determination method of antibiotics in cultured fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein, Use a 0.7-μm glass fiber filter membrane during filtration. Adjust the pH value to 7.0 - 8.5 after filtration.

7. The rapid determination method of antibiotics in cultured fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein During enrichment, the flow rate of the water sample is 4 - 6 mL / min.

8. The rapid determination method of antibiotics in cultured fresh water by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, Use ultrapure water for rinsing; use methanol for elution; blow the eluate with nitrogen for 30 minutes.

9. The rapid determination method of antibiotics in freshwater aquaculture by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The chromatographic conditions during detection are: ACQUITY UPLC T3, 100 mm × 2.1 mm, 1.8 μm; column temperature 35°C; flow rate 0.35 mL / min; injection volume: 10 μL; mobile phase A: 40% methanol-acetonitrile, mobile phase B: 0.2% formic acid-water.

10. A rapid determination method of antibiotics in cultured fresh water by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions during the detection process are as follows: ionization mode, electrospray positive ion source ESI + and electrospray negative ion source ESI - simultaneous scanning; ion source voltage, 4500 V; temperature, 400 °C; curtain gas, 30 psi; nebulizing gas, 55 psi; auxiliary heating gas, 55 psi; monitoring mode, multiple reaction monitoring MRM mode; The mass spectrometry parameters are:

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