Synchronous detection method for 42 antibiotics in estuary multi-environment medium

Abstract

The invention belongs to the technical field of antibiotic detection, and particularly discloses a synchronous detection method for 42 antibiotics in estuary multi-environment media. The method comprises the following steps: mixing an estuary sample to be detected, a phosphate buffer solution and a methanol-acetonitrile solution of formic acid, carrying out multiple times of ultrasonic extraction, combining extract liquids obtained by the multiple times of ultrasonic extraction, sequentially carrying out pH value regulation and concentration, and then adding water for dilution, so as to obtain an extraction diluent; then carrying out purification treatment on the extraction diluent by adopting a solid-phase extraction method to obtain a to-be-detected solution; adding five antibiotic internal standards into the to-be-detected solution, fixing the volume, detecting the to-be-detected solution by adopting an ultra-high performance liquid chromatography tandem mass spectrometer, and performing data processing on a detection result to obtain the concentration of 42 antibiotics in the to-be-detected estuary sample. The test method disclosed by the invention can be used for trace detection of antibiotics in a complex environmental medium in an estuary region, has the characteristics of low detection limit, good reproducibility and relatively high recovery rate, and can meet analysis requirements.

Description

Technical Field

[0001] The invention relates to the technical field of antibiotic detection, and in particular to a method for synchronously detecting 42 kinds of antibiotics in multiple environmental media of an estuary. Background Art

[0002] Antibiotics are a class of secondary metabolites produced by certain microorganisms (including bacteria, fungi, and actinomycetes) or higher animals and plants during their life activities. They have anti-pathogen or other activities and are chemical substances that can interfere with the developmental functions of other cells. Antibiotics are widely used. In addition to being used to treat bacterial infections, they can also be used for anti-fungal, anti-tumor, and immunosuppression. Since their advent, they have played an important role in human health and animal husbandry. However, due to the irrational use of antibiotics by humans, antibiotic pollution problems have emerged in an endless stream. Studies have shown that 40% to 90% of antibiotics are discharged directly or indirectly into the environment through original drugs or active ingredients. At present, antibiotics have been widely detected in various environmental media such as the global atmosphere, water bodies, sediments, and organisms. These antibiotics entering the environment may pose a threat to the ecosystem, leading to changes in the structure and diversity of biological communities, and even the production of drug-resistant bacteria, which has attracted widespread attention from the international community.

[0003] With the progress of pollution control actions, higher requirements have been put forward for the rapid, accurate, sensitive and reliable monitoring and analysis of antibiotic content in various sources and multiple environmental matrices. Only by accurately monitoring the content of antibiotics can we accurately assess their potential ecological and health risks, trace the source of pollution, and explore the migration and transformation mechanism. Some antibiotics have been included in the priority control list in many countries and regions due to their high persistence and use history, high frequency of detection and potential ecological and health risks. For example, the top five priority controlled antibiotics in global rivers include amoxicillin, sulfamethoxazole, erythromycin dehydrate, trimethoprim and sulfapyridine. However, at present, my country has only established standard methods for the detection of a certain type of antibiotics for cosmetics, food and water bodies. Existing literature and patents have increased the types of antibiotics detected in environmental media such as soil and sludge, but there are many types of antibiotics and their physical and chemical properties vary greatly. Existing methods are difficult to achieve simultaneous and efficient detection of different antibiotics, and there are limitations in practical applications. Compared with water bodies and the atmosphere, solid matrices such as soil, sediment (including sludge) and dust are relatively complex and more likely to accumulate antibiotics, especially in the complex ecosystem of estuaries, which are severely affected by the interaction between salt and fresh water and human activities. How to separate and detect trace amounts of antibiotics from the complex environmental media in the estuary area and ensure the precision and accuracy of the measurement is one of the problems that urgently need to be solved.

[0004] At present, highly sensitive liquid chromatography triple quadrupole tandem mass spectrometry is widely used to detect antibiotics, but it is necessary to enrich and concentrate the antibiotics in environmental samples in advance to reduce the interference of impurities. However, the pretreatment conditions for the extraction and purification of antibiotics in complex environmental media vary greatly, and most studies use solid phase extraction pretreatment that requires a large amount of solvent and nitrogen blowing, which is time-consuming. It is urgent to establish an efficient, rapid, highly sensitive, and low-detection limit antibiotic simultaneous extraction and analysis method for complex environmental matrices, which is of great significance for the comprehensive evaluation of the pollution status of antibiotics in estuarine environmental media. Summary of the invention

[0005] In view of this, the present invention provides a method for the simultaneous detection of 42 antibiotics in multiple environmental media in estuaries, so as to solve the problem that existing detection methods are difficult to achieve simultaneous and efficient detection of many different antibiotics, especially for trace detection in complex environmental media in estuary areas; and the problem that the pretreatment conditions for the extraction and purification of antibiotics in existing complex environmental media vary greatly, are difficult to unify, and the pretreatment process is complicated.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary comprises the following steps:

[0008] 1) Mixing the estuary sample to be tested, phosphate buffer and methanol-acetonitrile solution of formic acid and performing ultrasonic extraction multiple times, combining the extracts obtained by the multiple ultrasonic extractions, adjusting the pH value and concentrating them in sequence, and then diluting them with water to obtain an extraction dilution;

[0009] 2) using solid phase extraction to purify the extracted dilution solution to obtain a test solution;

[0010] 3) After adding 5 kinds of antibiotic internal standards to the test solution, the volume was fixed, and the fixed volume of the test solution was tested by ultra-high performance liquid chromatography tandem mass spectrometry. The test results were processed to obtain the concentrations of 42 kinds of antibiotics in the estuary test sample;

[0011] The 42 antibiotics are tylosin, spiramycin, roxithromycin, josamycin, azithromycin, clarithromycin, oleandomycin, tiamulin, chlortetracycline, oxytetracycline, tetracycline, doxycycline, methoxythiocarbazine, lincomycin, moxifloxacin, difloxacin, sparfloxacin, sarafloxacin, fleroxacin, marbofloxacin, enrofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, norfloxacin, pazufloxacin, sulfamethoxazole, sulfaquinoxaline, trimethoprim, sulfachloropyridazine, sulfamethoxazole, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfathiazole, sulfadiazine, sulfapyridine, sulfacetamide, chloramphenicol, thiamphenicol, and florfenicol.

[0012] The five antibiotic internal standards were sulfamethoxazole-d4, tetracycline-d6, roxithromycin-d7, fleroxacin-d3, and chloramphenicol-d5.

[0013] Preferably, the estuarine sample to be tested is estuarine sediment and / or estuarine tidal flat sediment;

[0014] The particle size of the estuary sample to be tested is ≤100 mesh.

[0015] Preferably, the mass volume ratio of the estuary sample to be tested, the phosphate buffer and the methanol-acetonitrile solution of formic acid is 2 g: 5 mL: 5 mL;

[0016] The pH value of the phosphate buffer is 3;

[0017] The volume concentration of formic acid in the methanol-acetonitrile solution of formic acid is 5%, and the volume ratio of methanol to acetonitrile is 1:1.

[0018] Preferably, the ultrasonic extraction in step 1) is performed 2 to 3 times;

[0019] The time of each ultrasonic extraction is independently 15 minutes, and the power of ultrasonic extraction is independently 35-45 kHz.

[0020] Preferably, the pH value of the extracts in step 1) is adjusted to 5 after being combined;

[0021] The concentration temperature in step 1) is 37-40° C., and the concentration is carried out by nitrogen blowing, and the nitrogen blowing pressure is 3-4 psi.

[0022] Preferably, the volume content of the organic solvent in the extraction dilution liquid obtained after the dilution is ≤5%.

[0023] Preferably, the purification process in step 2) is as follows:

[0024] The HLB solid phase extraction column is activated and balanced, and then the extraction diluent is passed through the HLB solid phase extraction column at a flow rate of 0.8 to 1.2 drops / s, and then the first elution and the second elution are performed in sequence to complete the purification process; wherein, the eluent obtained from the second elution is the test solution.

[0025] Preferably, the activation balance is to add methanol solution and water in sequence; the reagent for the first elution is ultrapure water; the reagent for the second elution is formic acid / methanol solution and ammonia / methanol solution in sequence;

[0026] The volume concentration of formic acid in the formic acid / methanol solution is 1%; the volume concentration of ammonia water in the ammonia water / methanol solution is 5%.

[0027] Preferably, the liquid chromatography conditions of the ultra-high performance liquid chromatography tandem mass spectrometer in step 3) are as follows: the chromatographic column is an ACQUITY UPLC BEH C18 column: 2.1 mm×50 mm×1.7 μm, the column flow rate is 0.4 mL / min, and the injection volume is 2 μL;

[0028] The mobile phase A in the positive ion mode was 0.1% formic acid water, and the mobile phase B was methanol / acetonitrile: v / v, 3 / 7; the mobile phase A in the negative ion mode was ultrapure water, and the mobile phase B was acetonitrile; the mobile phase gradient elution conditions in the positive ion mode were: 0-0.5 min, 10% B phase, 0.5-3 min, B phase increased to 40%, 3-6 min, B phase increased to 70%, 6-8 min, B phase increased to 95%, 8-10 min, B phase decreased to 10%; the mobile phase gradient elution conditions in the negative ion mode were: 0-0.2 min, 5% B phase, 0.2-4 min, B phase increased to 90%, 4-5 min, B phase maintained at 70%, 5-6.5 min, B phase decreased to 5%;

[0029] The mass spectrometry conditions were as follows: electrospray ion source, ionization mode was ESI negative ion mode, nebulizer gas was nitrogen, carrier gas was high-purity argon, mass spectrometry scanning mode was multiple reaction ion monitoring, ion source temperature was 150°C, desolvation temperature was 500°C, and flow rate was 800L / h.

[0030] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0031] The detection limit of the solid sample detection method of the present invention is 0.002-0.192 ng / g, the matrix spike recovery rate is 53.6-109.6%, and the relative standard deviation of the parallel samples is 0.05-5.89%. In general, the detection method of the present invention has a low detection limit, good reproducibility, and a high recovery rate, which can meet the analysis requirements. The present invention discloses a method for detecting antibiotics in a complex environmental matrix with high efficiency, rapidity, high sensitivity, and high recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The recovery rates of antibiotics by different extractants in Example 1 and Comparative Example 1;

[0034] Figure 2The recovery rates of antibiotics at different ultrasonic times in Example 1, Example 2 and Comparative Example 2;

[0035] Figure 3 The recovery rates of antibiotics at different ultrasonic times in Example 1 and Comparative Example 3;

[0036] Figure 4 The recovery rates of antibiotics with different elution solvent compositions in Example 1 and Comparative Example 4;

[0037] Figure 5 The recovery rate of antibiotics with different elution solvent dosages in Example 1 and Comparative Example 5. DETAILED DESCRIPTION

[0038] The present invention provides a method for synchronously detecting 42 antibiotics in multiple environmental media of an estuary, comprising the following steps:

[0039] 1) Mixing the estuary sample to be tested, phosphate buffer and methanol-acetonitrile solution of formic acid and performing ultrasonic extraction multiple times, combining the extracts obtained by the multiple ultrasonic extractions, adjusting the pH value and concentrating them in sequence, and then diluting them with water to obtain an extraction dilution;

[0040] 2) using solid phase extraction to purify the extracted dilution solution to obtain a test solution;

[0041] 3) After adding 5 kinds of antibiotic internal standards to the test solution, the volume was fixed, and the fixed volume of the test solution was detected by ultra-high performance liquid chromatography tandem mass spectrometry. The test results were processed to obtain the concentrations of 42 antibiotics in the estuary test samples.

[0042] In the present invention, the 42 antibiotics are tylosin, spiramycin, roxithromycin, josamycin, azithromycin, clarithromycin, oleandomycin, tiamulin, chlortetracycline, oxytetracycline, tetracycline, doxycycline, methoxythiocarbazine, lincomycin, moxifloxacin, difloxacin, sparfloxacin, sarafloxacin, fleroxacin, marbofloxacin, enrofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, norfloxacin, pazufloxacin, sulfamethoxazole, sulfaquinoxaline, trimethoprim, sulfachloropyridazine, sulfamethoxazole, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfathiazole, sulfadiazine, sulfapyridine, sulfacetamide, chloramphenicol, thiamphenicol, and florfenicol;

[0043] The five antibiotic internal standards included sulfamethoxazole-d4, tetracycline-d6, roxithromycin-d7, fleroxacin-d3, and chloramphenicol-d5.

[0044] In the present invention, the estuary sample to be tested is estuary sediment and / or estuary tidal flat sediment; the estuary is the mouth of a river entering the sea.

[0045] In the present invention, the particle size of the estuary sample to be tested is ≤100 mesh, specifically, it can be 100 mesh, 120 mesh, 150 mesh, or 200 mesh.

[0046] In the present invention, the mass volume ratio of the estuary sample to be tested, the phosphate buffer and the methanol-acetonitrile solution of formic acid is 2g:5mL:5mL.

[0047] In the present invention, the pH value of the phosphate buffer is 3.

[0048] In the present invention, the volume concentration of formic acid in the methanol-acetonitrile solution of formic acid is 5%, and the volume ratio of methanol to acetonitrile is 1:1.

[0049] In the present invention, the ultrasonic extraction in step 1) is performed 2 to 3 times.

[0050] In the present invention, the time of each ultrasonic extraction is independently 15 minutes, and the power of ultrasonic extraction is independently 35-45 kHz, specifically 36 kHz, 38 kHz, 40 kHz, 42 kHz, and 44 kHz.

[0051] In the present invention, the pH value of the extracts in step 1) is adjusted to 5 after being combined; preferably, ammonia water is added to adjust the pH value.

[0052] In the present invention, the concentration temperature in step 1) is 37-40°C, specifically 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C; the concentration is carried out by nitrogen blowing, and the nitrogen blowing pressure is 3-4psi, specifically 3.2psi, 3.4psi, 3.5psi, 3.6psi, 3.8psi.

[0053] In the present invention, the volume content of the organic solvent in the extracted dilution liquid obtained after dilution is ≤5%, and can be specifically 1%, 2%, 3%, or 4%.

[0054] In the present invention, the purification process in step 2) is as follows:

[0055] The HLB solid phase extraction column is activated and balanced, and then the extraction diluent is passed through the HLB solid phase extraction column at a flow rate of 0.8 to 1.2 drops / s, and then the first elution and the second elution are performed in sequence to complete the purification treatment; wherein, the eluent obtained by the second elution is the test liquid; the specific flow rate can be 0.9 drops / s, 1 drop / s, and 1.1 drops / s.

[0056] In the present invention, the activation equilibrium is to add methanol solution and water in sequence; the reagent for the first elution is ultrapure water; the reagent for the second elution is formic acid / methanol solution and ammonia water / methanol solution in sequence.

[0057] In the present invention, the volume concentration of formic acid in the formic acid / methanol solution is 1%; the volume concentration of ammonia water in the ammonia water / methanol solution is 5%.

[0058] In the present invention, in the second elution, the usage ratio of formic acid / methanol solution, ammonia water / methanol solution and the estuary sample to be tested in step 1) is 2 mL: 2 mL: 2 g.

[0059] In the present invention, the liquid chromatography conditions of the ultra-high performance liquid chromatography tandem mass spectrometer in step 3) are as follows: the chromatographic column is an ACQUITYUPLC BEH C18 column: 2.1 mm×50 mm×1.7 μm, the column flow rate is 0.4 mL / min, and the injection volume is 2 μL;

[0060] The mobile phase A in the positive ion mode was 0.1% formic acid water, and the mobile phase B was methanol / acetonitrile: v / v, 3 / 7; the mobile phase A in the negative ion mode was ultrapure water, and the mobile phase B was acetonitrile; the mobile phase gradient elution conditions in the positive ion mode were: 0-0.5 min, 10% B phase, 0.5-3 min, B phase increased to 40%, 3-6 min, B phase increased to 70%, 6-8 min, B phase increased to 95%, 8-10 min, B phase decreased to 10%; the mobile phase gradient elution conditions in the negative ion mode were: 0-0.2 min, 5% B phase, 0.2-4 min, B phase increased to 90%, 4-5 min, B phase maintained at 70%, 5-6.5 min, B phase decreased to 5%;

[0061] The mass spectrometry conditions were as follows: electrospray ion source, ionization mode was ESI negative ion mode, nebulizer gas was nitrogen, carrier gas was high-purity argon, mass spectrometry scanning mode was multiple reaction ion monitoring, ion source temperature was 150°C, desolvation temperature was 500°C, and flow rate was 800L / h.

[0062] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0063] Example 1

[0064] (1) Sample pretreatment: The sediments from the Yangtze River Estuary were freeze-dried, ground, and passed through a 100-mesh sieve. The sieved samples were then stored at -20°C.

[0065] (2) Extraction of organic matter: 2 g of the pretreated sediment sample was placed in a 50-mL polypropylene centrifuge tube, and 5 mL of phosphate buffer (containing phosphoric acid and sodium dihydrogen phosphate, pH 3) and 5 mL of a methanol-acetonitrile mixture with a volume concentration of 5% formic acid (1:1, v:v) were added. The tube was vortexed and mixed at 1000 rpm for 1 min. Ultrasonic extraction was performed at a power of 40 kHz for 15 min in a water bath at room temperature, and then centrifuged at 5000 rpm for 6 min. The ultrasonic extraction and centrifugation were repeated three times, and the supernatants were combined.

[0066] (3) Concentration of organic matter: The combined supernatant was aspirated with a glass needle filter and filtered through a 0.45 μm organic phase nylon filter to remove particulate matter. The filtrate was poured into a parallel quantitative concentrator and ammonia water was added to adjust the pH to 5. The filtrate was concentrated by nitrogen blow-through in a 40°C water bath (2 h) to obtain 1 mL of concentrated solution at a nitrogen blow-through pressure of 4 psi. The concentrated solution was diluted with 20 mL of ultrapure water, and the volume content of the organic solvent in the diluted solution was 4.8%.

[0067] (3) Organic separation and purification: Activate the balanced HLB column (500 mg, 6 mL) with 6 mL of methanol and 6 mL of ultrapure water in turn. Keep the column head moist during the addition of solvent for activation to remove the solvent and impurities remaining on the column. Use a glass pipette to repeatedly absorb the above dilution to rinse the inner wall of the concentration cup (to reduce the error), and then use a pipette to transfer the dilution to the HLB column. Let the dilution flow through the HLB column under negative pressure at a rate of 1 drop / s. Elute the solid phase extraction column with 10 mL of ultrapure water. At this time, all the target objects are fixed on the HLB column, and the previous samples and eluents are discarded. Continue to vacuum for 30 minutes to drain the HLB column to allow the impurities and eluent to flow out completely. The HLB column was eluted with 2 mL of 1% formic acid / methanol and 2 mL of 5% ammonia / methanol solution in turn, and the eluate was collected in a 15 mL glass centrifuge tube. The collected eluate was concentrated to 0.5 mL by nitrogen blowing on a parallel quantitative concentrator. The concentrate was aspirated with a glass syringe and passed through a 0.22 μm organic phase filter membrane, then transferred to a 2 mL brown glass injection vial, 20 ng of a mixture of 5 antibiotic internal standards (sulfamethoxazole-d4, tetracycline-d6, roxithromycin-d7, fleroxacin-d3, chloramphenicol-d5) was added, and then the volume was made up to 1 mL with 10% methanol aqueous solution and stored at 4°C for testing.

[0068] (4) Determination of antibiotics: Ultra-high performance liquid chromatography tandem mass spectrometry was used to detect the test solution. The liquid chromatography conditions were as follows: the chromatographic column was an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm × 1.7 μm), the column flow rate was 0.4 mL / min, and the injection volume was 2 μL. The mobile phase A in the positive ion mode was 0.1% formic acid water, the mobile phase B was methanol / acetonitrile (v / v, 3 / 7), and the mobile phase gradient elution conditions were as follows: 0-0.5 min, 10% B phase, 0.5-3 min, B phase increased to 40%, 3-6 min, B phase increased to 70%, 6-8 min, B phase increased to 95%, 8-10 min, B phase decreased to 10%; the mobile phase A in the negative ion mode was ultrapure water, and the mobile phase B was acetonitrile. The mobile phase gradient elution conditions were: 0-0.2 min, 5% phase B, 0.2-4 min, phase B increased to 90%, 4-5 min, phase B maintained at 70%, 5-6.5 min, phase B decreased to 5%. The mass spectrometry conditions were: electrospray ion source, ESI negative ion mode, multiple reaction ion monitoring (MRM), desolvation gas flow rate of 800 L / h, desolvation gas temperature of 500°C, cone gas flow rate of 150 L / h, nebulizer gas of nitrogen, and carrier gas of high-purity argon.

[0069] Drawing of standard curve: 42 kinds of antibiotics were mixed with standard solutions of 1μg / L, 2μg / L, 5μg / L, 10μg / L, 20μg / L, 50μg / L and 100μg / L respectively using methanol-water solution (the above concentrations represent the concentration of each antibiotic). The same liquid chromatography conditions and mass spectrometry conditions were used for ultra-high performance liquid chromatography-tandem mass spectrometry detection, and the standard curve of the target antibiotic concentration and peak area was drawn according to the internal standard method. The correlation coefficient R of the obtained 42 kinds of antibiotics standard curves was 2 All of them are greater than 0.99, which can be used for actual sample testing and accurate quantitative analysis.

[0070] The mass spectrometry parameters and chromatographic peak time of the antibiotics in Example 1 are shown in Table 1.

[0071] Table 1 Antibiotic mass spectrometry parameters and chromatographic peak time

[0072]

[0073]

[0074] The method of steps (1) to (4) was used for the qualitative and quantitative analysis of antibiotics in the sediments of the Yangtze River Estuary. Data quality control was performed using parallel sample detection, matrix spike and other methods, that is, at least one blank sample, one matrix spike and one parallel sample were made for every 10 samples. No sample was added to the blank test, and the other conditions were the same as the detection conditions of the sample to be tested. The blank test was repeated 7 times, and the standard deviation of the target content values ​​of 7 parallel determinations was calculated, and multiplied by the t value (6 degrees of freedom) when the confidence level was 99% to obtain the method detection limit (MDL). The actual sample was randomly selected, 40 ng of 42 kinds of target mixed standard solutions (20 μL, mixed standard concentration was 2000 μg / L) were added, and the matrix spike test was performed. The method for the sample to be tested was repeated 3 times. The actual sample was randomly selected, 2 parallel samples were weighed, and the determination was repeated 3 times according to the above method, and the relative standard deviation of the parallel samples was calculated. The experimental results of each type of sample were analyzed and processed, and the results are shown in Table 2.

[0075] Table 2 Test results of MDL, matrix spike recovery and relative standard deviation (RSD) of antibiotics in Example 1

[0076]

[0077]

[0078] As shown in Table 2, the detection limits of 42 antibiotics in the sediment sample detection method provided by the present invention are 0.002-0.192 ng / g, the matrix spike recovery is 53.6-109.6%, and the relative standard deviation (RSD) of the parallel samples is 0.05-5.89%, which can meet the analytical requirements for the detection of antibiotics in sediments.

[0079] Comparative Example 1

[0080] The extraction solution of Example 1 was replaced by methanol, 5% formic acid in methanol / acetonitrile solvent in equal volumes, 5% formic acid in methanol, acetonitrile, 5% formic acid in acetonitrile, and methanol / acetonitrile (v:v) (the above concentrations and ratios are all volume concentrations and volume ratios), and other conditions were the same as in Example 1. The recovery rates of antibiotics by different extraction solutions are shown in Figure 1. Figure 1 shown.

[0081] Comparative Example 2

[0082] The number of ultrasonic times in the extraction in Example 1 was replaced from 3 times to 1 time, and the other conditions were the same as in Example 1.

[0083] Example 2

[0084] The number of ultrasonic times in the extraction of Example 1 was replaced by 2 times instead of 3 times, and the other conditions were the same as those of Example 1. The recovery rate of antibiotics with different ultrasonic times is shown in Figure 2. Figure 2 shown.

[0085] Comparative Example 3

[0086] The ultrasonic time in the extraction of Example 1 was replaced from 15 min to 10 min and 20 min, and the other conditions were the same as those in Example 1. The recovery rate of antibiotics at different ultrasonic times was as follows: Figure 3 shown.

[0087] Comparative Example 4

[0088] The elution solvent (reagent for the second elution) of the organic solid phase extraction purification in Example 1 was replaced by 1% formic acid / methanol + 5% ammonia / methanol in equal volumes with 1% formic acid / methanol, 5% ammonia methanol, methanol + 5% ammonia methanol, and methanol / acetonitrile (1:1) (the above concentrations and ratios are all volume concentrations and volume ratios), and other conditions were the same as in Example 1. The recovery rate of antibiotics by different elution solvents is shown in Figure 1. Figure 4 shown.

[0089] Comparative Example 5

[0090] The amount of elution solvent (reagent for the second elution) in Example 1 was replaced by 4 mL to 6, 8, and 10 mL, and the other conditions were the same as in Example 1. The recovery rate of antibiotics with different elution solvent amounts is shown in Figure 2. Figure 5 shown.

[0091] Depend on Figures 1 to 3 It can be seen that the ultrasonic extraction method used in the pretreatment method provided by the present invention is low in price and easy to promote and apply, and the optimized ultrasonic time only takes 15 minutes, and a good recovery rate can be achieved after 3 ultrasonic times. Figure 4 It can be seen that the addition of 1% formic acid methanol in the elution solvent used in the present invention can improve the recovery rate of tetracycline antibiotics. Figure 5 It can be seen that the solid phase extraction method of the present invention greatly reduces the amount of organic solvent used and further shortens the nitrogen blowing concentration time.

[0092] The method of the present invention can also be applied to estuarine water bodies. Specifically, the Yangtze River Estuary sediments in Example 1 are replaced with estuarine water bodies, and "the above-mentioned solid sample concentrate is diluted with 5 mL of ultrapure water, and the above-mentioned dilution is repeatedly aspirated with a glass pipette to rinse the inner wall of the concentration cup, and then the dilution is transferred to the HLB column with a pipette, and the dilution is passed through the HLB column (500 mg, 6 mL) at a rate of 1 drop / s under negative pressure" is replaced with "the environmental water body after passing through the 0.45 μm aqueous polyethersulfone filter membrane is directly loaded with formic acid after adjusting the pH to 5, and the liquid is passed through the HLB column (500 mg, 6 mL) at a rate of 1 drop / s under negative pressure", and the other conditions are the same as in Example 1.

[0093] Table 3 Test results of MDL, matrix spike recovery and relative standard deviation (RSD) of antibiotics in estuarine water

[0094]

[0095]

[0096] As can be seen from Table 3, the method detection limit of 42 antibiotics in the water sample detection method provided by the present invention is 0.01-1.82 ng / L, the matrix spike recovery rate is 50.8-167.6%, and the relative standard deviation (RSD) of parallel samples is 0.12-18.9%, which can meet the analytical requirements for water antibiotic detection.

[0097] The detection method of the present invention takes a short time, has good separation and purification effects on antibiotics in a variety of complex environmental matrix samples, has little interference from impurities, has a high recovery rate, and has good repeatability, and has good application value, especially for the analysis and detection of large quantities of environmental samples.

[0098] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0099] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneous detection of 42 antibiotics in multiple environmental media in an estuary, characterized in that: The steps include: 1) Mixing the estuary sample to be tested, phosphate buffer and methanol-acetonitrile solution of formic acid and performing ultrasonic extraction multiple times, combining the extracts obtained by the multiple ultrasonic extractions, adjusting the pH value and concentrating them in sequence, and then diluting them with water to obtain an extraction dilution; 2) using solid phase extraction to purify the extracted dilution solution to obtain a test solution; 3) After adding 5 kinds of antibiotic internal standards to the test solution, the volume was fixed, and the fixed volume of the test solution was tested by ultra-high performance liquid chromatography tandem mass spectrometry. The test results were processed to obtain the concentrations of 42 kinds of antibiotics in the estuary test sample; The 42 antibiotics are tylosin, spiramycin, roxithromycin, josamycin, azithromycin, clarithromycin, oleandomycin, tiamulin, chlortetracycline, oxytetracycline, tetracycline, doxycycline, methoxythiocarbazine, lincomycin, moxifloxacin, difloxacin, sparfloxacin, sarafloxacin, fleroxacin, marbofloxacin, enrofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, norfloxacin, pazufloxacin, sulfamethoxazole, sulfaquinoxaline, trimethoprim, sulfachloropyridazine, sulfamethoxazole, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfathiazole, sulfadiazine, sulfapyridine, sulfacetamide, chloramphenicol, thiamphenicol, and florfenicol. The five antibiotic internal standards were sulfamethoxazole-d4, tetracycline-d6, roxithromycin-d7, fleroxacin-d3, and chloramphenicol-d5.

2. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 1, characterized in that: The estuary sample to be tested is estuary sediment and / or estuary tidal flat sediment; The particle size of the estuary sample to be tested is ≤100 mesh.

3. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 2, characterized in that: The mass volume ratio of the estuary sample to be tested, phosphate buffer and methanol-acetonitrile solution of formic acid is 2 g: 5 mL: 5 mL; The pH value of the phosphate buffer is 3; The volume concentration of formic acid in the methanol-acetonitrile solution of formic acid is 5%, and the volume ratio of methanol to acetonitrile is 1:

1.

4. A method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to any one of claims 1 to 3, characterized in that: The number of ultrasonic extractions in step 1) is 2 to 3 times; The time of each ultrasonic extraction is independently 15 minutes, and the power of ultrasonic extraction is independently 35-45 kHz.

5. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 4, characterized in that: The pH value of the combined extracts in step 1) is adjusted to 5; The concentration temperature in step 1) is 37-40° C., and the concentration is carried out by nitrogen blowing, and the nitrogen blowing pressure is 3-4 psi.

6. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 5, characterized in that: The volume content of the organic solvent in the extracted dilution liquid obtained after the dilution is ≤5%.

7. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 4 or 5, characterized in that: The operation of the purification process described in step 2) is as follows: The HLB solid phase extraction column is activated and balanced, and then the extraction diluent is passed through the HLB solid phase extraction column at a flow rate of 0.8 to 1.2 drops / s, and then the first elution and the second elution are performed in sequence to complete the purification process; wherein, the eluent obtained from the second elution is the test solution.

8. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 7, characterized in that: The activation balance is to add methanol solution and water in sequence; the reagent for the first elution is ultrapure water; the reagent for the second elution is formic acid / methanol solution and ammonia / methanol solution in sequence; The volume concentration of formic acid in the formic acid / methanol solution is 1%; the volume concentration of ammonia water in the ammonia water / methanol solution is 5%.

9. The method for simultaneous detection of 42 antibiotics in multiple environmental media of an estuary according to claim 8, characterized in that: The liquid chromatography conditions of the ultra-high performance liquid chromatography tandem mass spectrometer in step 3) are as follows: the chromatographic column is an ACQUITY UPLC BEH C18 column: 2.1 mm×50 mm×1.7 μm, the column flow rate is 0.4 mL / min, and the injection volume is 2 μL; The mobile phase A in the positive ion mode was 0.1% formic acid water, and the mobile phase B was methanol / acetonitrile: v / v, 3 / 7; the mobile phase A in the negative ion mode was ultrapure water, and the mobile phase B was acetonitrile; the mobile phase gradient elution conditions in the positive ion mode were: 0-0.5 min, 10% B phase, 0.5-3 min, B phase increased to 40%, 3-6 min, B phase increased to 70%, 6-8 min, B phase increased to 95%, 8-10 min, B phase decreased to 10%; the mobile phase gradient elution conditions in the negative ion mode were: 0-0.2 min, 5% B phase, 0.2-4 min, B phase increased to 90%, 4-5 min, B phase maintained at 70%, 5-6.5 min, B phase decreased to 5%; The mass spectrometry conditions were as follows: electrospray ion source, ionization mode was ESI negative ion mode, nebulizer gas was nitrogen, carrier gas was high-purity argon, mass spectrometry scanning mode was multiple reaction ion monitoring, ion source temperature was 150°C, desolvation temperature was 500°C, and flow rate was 800L / h.

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