Quantitative determination of multiple antibiotics in groundwater by high performance liquid chromatography coupled with high resolution mass spectrometry

Abstract

This invention relates to the field of antibiotic detection technology, specifically to a high-performance liquid chromatography-electrostatic orbital trap (HPLC-EORT) method for quantitative detection of multiple antibiotics in groundwater. The method includes the following steps: solid-phase extraction of the groundwater sample to obtain a test solution; adding an antibiotic internal standard to the test solution and making up to a fixed volume; and then using HPLC-EORT high-resolution mass spectrometry to detect the concentration of multiple antibiotics in the groundwater. This invention establishes a standardized pretreatment procedure to ensure effective extraction of various compounds and minimize losses. Simultaneously, it optimizes the gradient elution procedure of liquid chromatography and specific settings of mass spectrometry parameters to achieve optimal separation and detection of 83 antibiotics in groundwater. This invention can reduce the detection limit of the testing method to the ppt level; the atomic weight of the quantified ions is accurate to four decimal places, improving the resolution and accuracy of screening multiple antibiotics in groundwater.

Description

Technical Field

[0001] This invention relates to the field of antibiotic detection technology, specifically to a high-resolution mass spectrometry method for quantitative detection of multiple antibiotics in groundwater using high-performance liquid chromatography-electrostatic orbital trap. Background Technology

[0002] Currently, most countries face water quality problems. Naturally safe drinking water is becoming increasingly scarce, and even sources considered safe for drinking water are not immune to pollution. Exogenous chemicals entering the aquatic environment include not only persistent organic pollutants (POPs) that have already received significant attention, but also emerging organic pollutants that are already widely present in various environmental media. Antibiotics, as a major component of pharmaceuticals and personal care products among emerging organic pollutants, have been reported in soil, sediments, sludge, groundwater, wastewater, tap water, surface water (lakes, streams, rivers, oceans), plants, and aquatic animals.

[0003] Antibiotics are frequently detected in various aquatic environments at ng / L levels due to their widespread use and high-frequency discharge. Compared to other pollutants in the aquatic environment, antibiotics have more complex structures due to the presence of multiple functional groups. Currently, there is no completely unified standard method for the detection of antibiotics internationally. However, Europe and the United States widely adopt a series of analytical guidelines and recommended procedures based on technologies such as GC-MS and LC-MS / MS, and sample pretreatment is mostly based on relevant US EPA and ISO specifications. my country's standard system in this field is still under development. Existing methods mostly rely on the detection regulations for specific categories of drugs and pollutants in the Ministry of Ecology and Environment (HJ) and national standards (GB), such as high-performance liquid chromatography and gas chromatography-mass spectrometry. The systematic nature and coverage still need further improvement and expansion.

[0004] While liquid chromatography-mass spectrometry (LC-MS / MS) is a powerful tool for detecting trace antibiotics in groundwater, it does have some significant drawbacks in practical applications, mainly in terms of technical complexity, cost, and the professional requirements for operators. (1) Technical limitations: The matrix effect has a significant impact. Complex coexisting substances in groundwater (such as humic acid and inorganic salts) can inhibit or enhance the ionization efficiency of the target analytes, leading to inaccurate quantitative results; it is highly dependent on the pretreatment process. Water samples usually require enrichment and purification (such as solid phase extraction), which is a complicated process, and any improper operation may introduce errors or cause loss of the target analytes.

[0005] (2) Method development and optimization: The process of establishing methods is complex and time-consuming. It is necessary to optimize chromatographic separation conditions and mass spectrometry parameters (such as collision energy) for different types of antibiotics (such as sulfonamides and quinolones), which is a huge workload.

[0006] (3) Instrument and operation requirements: The purchase and maintenance costs of instruments are high: High-precision liquid chromatographs and mass spectrometers are expensive, and the subsequent maintenance, consumables (such as chromatographic columns and high-purity solvents) and calibration costs are considerable.

[0007] (4) High requirements for the professional competence of operators: From sample pretreatment and instrument operation to data analysis and interpretation, professional knowledge and experience are required; otherwise, it is difficult to guarantee the reliability of the results.

[0008] (5) Quality control challenges: Strict quality control is required to offset matrix effects and losses during sample processing. Correction must be performed using isotope internal standards, which increases the complexity and cost of the method.

[0009] (6) The detection limit and accuracy cannot fully meet current needs. The detection limit of the current triple quadrupole liquid chromatography-mass spectrometry is generally between 0.012 and 0.024 μg / L, which is difficult to fully meet the standards (such as the minimum standard for perfluorinated compounds in drinking water in the United States is 0.004 μg / L); the atomic weight of the quantitative ions is accurate to two decimal places, which can easily lead to errors in compound identification.

[0010] Chinese patent application CN119915935A discloses a method for the simultaneous detection of 42 antibiotics in multiple environmental media in estuaries. This patent employs high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) to simultaneously detect 42 antibiotics in complex and heterogeneous estuarine environments. However, this method focuses more on antibiotics commonly found in aquatic environments (especially estuaries) and easily adsorbed by particulate matter, and is not suitable for relatively simple and clean groundwater environments containing more polar antibiotics, metabolites, or novel antibiotics. Summary of the Invention

[0011] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a high-performance liquid chromatography-electrostatic orbital trap high-resolution mass spectrometry method for the quantitative detection of multiple antibiotics in groundwater. This method solves the problems of insufficient sensitivity, throughput, and accuracy faced by traditional methods when simultaneously detecting multiple antibiotics.

[0012] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A high-performance liquid chromatography-electrostatic orbital trap high-resolution mass spectrometry method is provided for the quantitative detection of multiple antibiotics in groundwater, which includes the following steps: After adjusting the pH of the groundwater sample to be tested, solid-phase extraction was performed to obtain the test solution. After adding an antibiotic internal standard to the test solution and making up the volume, the test solution was analyzed by high-performance liquid chromatography-electrostatic orbital trap high-resolution mass spectrometry to obtain the concentration of multiple antibiotics in the groundwater. The antibiotics include: SGD, SCT, SMZ, SSM, SDZ, STZ, SPD, SMR, TMP, SMO, SMP, SMTZ, SFM, SCP, SMX, SMM, SDX, SSZ, SBZ, SPZ, SDM, and SQX. , SNT, AETM, AZM, CTM, CDM, ETM, JSM, LM, LCM, OLM, RTM, SPM, TIL, TYL, CIP, DAN, DIF, ENO, ENR, FLE, FLU, LOM, NDA, NOR, OFL, ORB, OLA, PEF, SAR, SPA, EACT C, EATC, ECTC, EOTC, ETC, ACTC, ATC, CTC, DCTC, DC, ICTC, OTC, TC, AMX, AMP, CEF, CED, CFL, CEP, CEZ, CPV, CFT, CEL, CER, CLX, DSX, DLX, NAF, OXA, PCG, PCV, SMX-d4, CIP-d8, TC-d6, RTM-d7, AMX-d4 and CEL-d5.

[0013] Furthermore, concentrated H2SO4 was added to adjust the pH value of the groundwater sample to 3.0.

[0014] Furthermore, the specific method for the solid-phase extraction treatment is as follows: Glass fiber membrane filtration was used. 50 μL of internal standard and 500 mg of Na2EDTA were added to every 2 L of filtrate. Solid phase extraction was performed using an Oasis HLB solid phase extraction column. The extraction column was then activated with methanol and ultrapure water, followed by rinsing with ultrapure water, drying, and elution with methanol. The eluent was stored in a clean glass tube, dried with high-purity nitrogen, and then diluted to volume with methanol solution. After filtration through a nylon membrane, the solution was transferred to a glass sample vial.

[0015] Furthermore, the liquid chromatography reference conditions are as follows: Mobile phase A: 1 / 1000 formic acid solution; Mobile phase B: methanol; Column temperature: 35 ℃; Injection volume: 5.0 μL.

[0016] Furthermore, the liquid chromatography mobile phase gradient elution program is as follows: .

[0017] Furthermore, the mass spectrometry reference conditions are as follows: Ion source: Electrospray ionization source, positive ion mode; Monitoring method: Parallel reaction detection; Other instrument conditions are as follows: a) Drying gas temperature: 350 ℃; b) Drying gas flow rate: 5 L / min; c) Sheath gas temperature: 380 ℃; d) Sheath gas flow rate: 10 L / min; e) Capillary voltage: 3.5 kV; f) Nozzle voltage: 500 V; g) Nebulizer pressure: 2.4 × 10⁻⁶ 5 Pa; The ion transmission tube and atomization temperature are both 40℃, and the spray voltage is 3.5KV-4.0KV.

[0018] Furthermore, the parallel reaction detection conditions are as follows:

[0019]

[0020] .

[0021] The beneficial effects of this invention are as follows: This invention establishes a standardized pretreatment procedure applicable to antibiotics with different properties, including sulfonamides, quinolones, macrolides, tetracyclines, and β-lactams. This includes optimization of sample pH adjustment, extraction, and elution steps to ensure effective extraction and minimize loss of various compounds. Simultaneously, it optimizes specific combinations of liquid chromatography gradient elution programs and mass spectrometry parameters (such as resolution and collision energy) to achieve optimal separation and detection of 83 antibiotics in groundwater. This invention lowers the detection limit of the testing method to the ppt (ng / L) level; the atomic weight of quantitative ions is accurate to four decimal places, improving the resolution and accuracy of screening multiple antibiotics in groundwater. Attached Figure Description

[0022] Figure 1 This is a chromatogram of the total ion current of sulfonamide antibiotics in the examples; Figure 2 This is an example diagram of ion channels after extraction of the parent and daughter ions of sulfonamide antibiotics in the embodiments; Figure 3 The recovery rates of 83 antibiotics and 6 internal standard compounds in the examples are shown. Figure 4 The results of antibiotic testing in groundwater in the Beijing-Tianjin-Hebei region are shown in the examples. Detailed Implementation

[0023] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0024] Example (a) Sample collection, preservation and pretreatment methods A 5L groundwater sample was collected from each sampling point and stored in a brown glass bottle. Three drops of concentrated H₂SO₄ were added to the sample bottle beforehand to adjust the pH to 3.0. All samples were immediately sent to the laboratory and stored in a refrigerator at 0–4°C.

[0025] For solid-phase extraction (SPE), a glass fiber membrane was used for filtration. 50 μL of internal standard and 500 mg of Na₂EDTA were added to every 2 L of filtrate. SPE was performed using an Oasis HLB SPE column, followed by activation of the column with 10 mL of methanol and 10 mL of ultrapure water. The sample loading rate was 15 mL / min. -1 Then, the extraction column was rinsed with 10 mL of ultrapure water, dried for 30 min, and then eluted with 10 mL of methanol. The eluent was stored in a clean glass tube, dried with high-purity nitrogen, and then diluted to volume with 1 mL of methanol solution. After filtration through a 0.22 μm nylon filter membrane, the eluent was transferred to a 2 mL glass vial.

[0026] Table 1. Chinese names of antibiotics in groundwater in China

[0027] (II) Instrument Reference Method (1) Reference conditions for liquid chromatography Mobile phase A: 1 / 1000 formic acid solution; Mobile phase B: methanol; Gradient elution program and flow rate are shown in Table 2. Column temperature: 35 ℃; injection volume: 5.0 μL.

[0028] Table 2 Gradient elution program for liquid chromatography mobile phase

[0029] (2) Mass spectrometry reference conditions Ion source: Electrospray ionization (ESI), positive ion mode; Monitoring method: Parallel reaction detection (PRM).

[0030] Other instrument conditions are as follows: a) Drying gas temperature: 350 ℃; b) Drying gas flow rate: 5 L / min; c) Sheath gas temperature: 380 ℃; d) Sheath gas flow rate: 10 L / min; e) Capillary voltage: 3.5 kV; f) Nozzle voltage: 500 V; g) Nebulizer pressure: 2.4 × 10⁻⁶ 5 The multiple reaction monitoring (MRM) conditions for the target compound (Pa (35 psi); h) are shown in Table 4. The ion transfer tube and atomization temperature were both 40 °C, and the spray voltage was 3.5 kV-4.0 kV.

[0031] Table 3. Abbreviations and Chemical Formulas of Target Antibiotics

[0032] Table 4 Target Antibiotic PRM Mode Parameters

[0033] (III) Calibration Method (1) Establishment of the standard curve Solid-phase extraction: Take a certain amount of the target compound standard working solution and prepare a standard series with at least 5 concentration points using methanol. The mass concentrations of the target compound are 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 50.0 μg / L, and so on. For the 100 μg / L and 200 μg / L (reference concentrations) standards, take 1.0 mL of the prepared standard series, add 50.0 μL of the internal standard working solution, mix well and test.

[0034] According to the instrument reference conditions, standard series solutions were measured sequentially from low concentration to high concentration. A standard curve was established with the mass concentration of the target compound as the abscissa and the product of the ratio of its corresponding response value to the internal standard response value and the internal standard concentration as the ordinate.

[0035] (2) Standard reference spectrum Under the reference conditions of this instrument, the total ion chromatograms of some antibiotics are shown below. Figure 1-2 .

[0036] (3) Sample determination The sample was measured under the same instrument reference conditions as those used for the standard curve determination.

[0037] (4) Blank test The blank sample was measured using the same instrument reference strip as the sample sample.

[0038] (iv) Actual sample test results The methods in steps (1) to (4) were used for the qualitative and quantitative analysis of 83 antibiotics in 50 groundwater samples from the Beijing-Tianjin-Hebei region. Parallel sample testing and matrix spiking were used for data quality control, meaning that for every 20 samples, at least one blank sample, one matrix-spiked sample, and one parallel sample were prepared. No test sample was added to the blank test, and all other conditions were the same as those for the test sample. The limit of detection and limit of quantitation were calculated and determined under the conditions of an instrument signal-to-noise ratio (S / N) of 3 and 10, respectively. Actual samples were randomly selected, and 100 μg / L of a mixed standard solution of 83 target substances was added. A matrix spiking test was performed, and the method was repeated three times according to the test sample method. Two parallel samples were randomly selected and weighed, and the method was repeated three times. The relative standard deviation (RSD) of the parallel samples was calculated. The experimental results for each type of sample were analyzed and processed, and the results are shown in Table 5.

[0039] Table 5. Limit of Detection, Recovery Rate and Relative Standard Deviation of Target Antibiotics

[0040] As shown in the table above, this method has a low detection limit, which meets the requirements for the detection of trace antibiotics in groundwater; the recovery rate is at the same level as other related research methods; after parallel testing, it was found that, except for a few types of antibiotics, the test method meets the quality control requirements.

[0041] In summary, this invention establishes a standardized pretreatment procedure applicable to antibiotics with different properties, including sulfonamides, quinolones, macrolides, tetracyclines, and β-lactams. This includes optimization of sample pH adjustment, extraction, and elution steps to ensure effective extraction and minimize loss of various compounds. Simultaneously, it optimizes specific combinations of liquid chromatography gradient elution programs and mass spectrometry parameters (such as resolution and collision energy) to achieve optimal separation and detection of 83 antibiotics in groundwater. This invention lowers the detection limit of the testing method to the ppt (ng / L) level; the atomic weight of quantitative ions is accurate to four decimal places, improving the resolution and accuracy of screening multiple antibiotics in groundwater.

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0043] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A high-resolution mass spectrometry method for quantitative detection of multiple antibiotics in groundwater using high-performance liquid chromatography-electrostatic orbital trap, characterized in that, Includes the following steps: The groundwater sample to be tested was subjected to solid-phase extraction to obtain the test solution; After adding an antibiotic internal standard to the test solution and making up the volume, the test solution was analyzed by high-performance liquid chromatography-electrostatic orbital trap high-resolution mass spectrometry to obtain the concentration of multiple antibiotics in the groundwater. The antibiotics include: SGD, SCT, SMZ, SSM, SDZ, STZ, SPD, SMR, TMP, SMO, SMP, SMTZ, SFM, SCP, SMX, SMM, SDX, SSZ, SBZ, SPZ, SDM, and SQX. , SNT, AETM, AZM, CTM, CDM, ETM, JSM, LM, LCM, OLM, RTM, SPM, TIL, TYL, CIP, DAN, DIF, ENO, ENR, FLE, FLU, LOM, NDA, NOR, OFL, ORB, OLA, PEF, SAR, SPA, EACT C, EATC, ECTC, EOTC, ETC, ACTC, ATC, CTC, DCTC, DC, ICTC, OTC, TC, AMX, AMP, CEF, CED, CFL, CEP, CEZ, CPV, CFT, CEL, CER, CLX, DSX, DLX, NAF, OXA, PCG, PCV, SMX-d4, CIP-d8, TC-d6, RTM-d7, AMX-d4 and CEL-d5.

2. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 1, characterized in that, Concentrated H2SO4 was added to adjust the pH of the groundwater sample to 3.

0.

3. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 1, characterized in that, The specific method for the solid-phase extraction treatment is as follows: Glass fiber membrane filtration was used. 50 μL of internal standard and 500 mg of Na2EDTA were added to every 2 L of filtrate. Solid phase extraction was performed using an Oasis HLB solid phase extraction column. The extraction column was then activated with methanol and ultrapure water, followed by rinsing with ultrapure water, drying, and elution with methanol. The eluent was stored in a clean glass tube, dried with high-purity nitrogen, and then diluted to volume with methanol solution. After filtration through a nylon membrane, the solution was transferred to a glass sample vial.

4. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 1, characterized in that, The liquid chromatography reference conditions are as follows: Mobile phase A: 1 / 1000 formic acid solution; Mobile phase B: methanol; Column temperature: 35 ℃; injection volume: 5.0 μL.

5. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 4, characterized in that, The liquid chromatography mobile phase gradient elution procedure: 。 6. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 1, characterized in that, The mass spectrometry reference conditions are as follows: Ion source: Electrospray ionization source, positive ion mode; Monitoring method: Parallel reaction detection; Other instrument conditions are as follows: a) Drying gas temperature: 350 ℃; b) Drying gas flow rate: 5 L / min; c) Sheath gas temperature: 380 ℃; d) Sheath gas flow rate: 10 L / min; e) Capillary voltage: 3.5 kV; f) Nozzle voltage: 500 V; g) Nebulizer pressure: 2.4 × 10⁻⁶ 5 Pa; The ion transmission tube and atomization temperature are both 40℃, and the spray voltage is 3.5KV-4.0KV.

7. The method for quantitative detection of multiple antibiotics in groundwater by high-performance liquid chromatography-electrostatic orbital trap mass spectrometry according to claim 6, characterized in that, The parallel reaction detection conditions are as follows: ； ； 。

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