Liquid chromatography-mass spectrometry detection method and system for trace target object in seawater

Abstract

The invention belongs to the field of liquid chromatography-mass spectrometry detection, and discloses a liquid chromatography-mass spectrometry detection method for trace target objects in seawater, which comprises the following steps: firstly, configuring and precisely calibrating parameters such as flow velocity, column temperature, ion source voltage and the like for a liquid chromatography system, a chromatographic column, an automatic sampler and a tandem mass spectrometer; then, a methanol-water (containing 0.1% formic acid) system is adopted to implement linear gradient elution, polar difference component separation is considered, and analysis duration is compressed; a sample is subjected to solid-phase extraction, purification and concentration, then is automatically injected, is subjected to chromatographic separation and then enters an electrospray ion source; a mass spectrum end selects a dynamic multi-reaction monitoring (dMRM) mode, characteristic ion pairs are collected in a preset time window, and selective scanning and baseline noise suppression are achieved; and finally, utilizing retention time matching and internal standard correction to obtain a pg.L <-1 > magnitude quantitative result.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of liquid chromatography-mass spectrometry detection technology, and in particular relates to a liquid chromatography-mass spectrometry detection method and system for trace target substances in seawater. Background Art

[0002] Liquid chromatography-mass spectrometry (LC-MS) is a detection technology that separates compounds through liquid chromatography and combines it with qualitative analysis by mass spectrometry. It is suitable for the analysis of highly polar, non-volatile and thermally unstable compounds.

[0003] Traditional methods have insufficient coverage, low recovery rates for trace target compounds (detection limits must reach ng / L) in complex matrices (such as seawater), and insufficient sensitivity.

[0004] The closest existing technology is a solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS / MS) method for the detection of new-generation neonicotinoid insecticides in surface water. After enriching one liter of freshwater sample using a C18 solid-phase extraction column, the researchers separated the target compounds on a reversed-phase UHPLC system at a flow rate of 0.30 mL / min and used a triple quadrupole mass spectrometer for quantification under a fixed MRM channel. The detection limit can reach 0.1 ng / L. This method has been verified in quantitative linearity, recovery rate and precision in samples from multiple rivers and is considered to be an optimized solution for the detection of trace neonicotinoids in freshwater.

[0005] However, this technology has not been evaluated for ion suppression in high-salinity seawater matrices. Literature indicates that high concentrations of inorganic salts in seawater can significantly reduce ESI-MS response, triggering severe matrix effects and leading to quantitative bias. Furthermore, it uses static MRM channels with limited scan time allocation, making it difficult to maintain high sensitivity in multi-residue scenarios. dMRM, on the other hand, can significantly improve detection efficiency through time window scheduling. Furthermore, this method operates only in positive ion mode and lacks rapid positive-negative polarity switching, making it incapable of synchronously capturing some highly polar or easily deprotonated metabolites. Therefore, existing solutions still have significant technical gaps in high-salt matrix adaptability, dynamic monitoring channel management, and polarity compatibility. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a liquid chromatography-mass spectrometry method for detecting trace targets in seawater.

[0007] The present invention is achieved by providing a liquid chromatography-mass spectrometry method for detecting trace amounts of target substances in seawater, characterized in that the liquid chromatography-mass spectrometry method for detecting trace amounts of target substances in seawater specifically comprises:

[0008] S1: Configure instrument parameters such as liquid chromatography, chromatographic column, mass spectrometer, and autosampler;

[0009] S2: gradient elution;

[0010] S3: Mass spectrometry detection was performed in dynamic multiple reaction monitoring (dMRM) scanning mode.

[0011] Furthermore, the core parameters of the S1 instrument configuration are as follows:

[0012] (1) Liquid chromatography: binary pump (GT120A), flow rate: 0.40 mL / min; column temperature: 40°C;

[0013] (2) Chromatographic column: ZORBAX SB-Phenyl (2.1×100 mm, 1.8 μm) high salt resistant matrix;

[0014] (3) Mass spectrometer: triple quadrupole (Agilent 6470), ion source: AJS ESI (positive / negative ion mode switch);

[0015] (4) Automatic sampler: G7187B Multisampler, injection volume: 2.00 μL; needle wash: standard flush (3 s).

[0016] Furthermore, the S2 gradient elution procedure is as follows: the initial methanol ratio is 30%, increased to 60% at 3 minutes, increased to 80% at 5 minutes, increased to 90% at 10 minutes, increased to 100% methanol at 11 minutes (maintained for 5 minutes), and then returned to the initial conditions.

[0017] Furthermore, the S3 ion source parameters are as follows:

[0018] Drying gas temperature: 250°C, atomizing gas pressure: 30 psi, capillary voltage: +3000 V (positive ion) / -2500 V (negative ion).

[0019] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0020] By adopting a phenyl salt-tolerant stationary phase column, optimizing the gradient elution curve, and using a dynamic multiple reaction monitoring scanning mode, this method achieves a sub-nanogram detection limit and a wide linear range in a high-salt seawater matrix, significantly improving the sensitivity and quantitative accuracy of trace targets. It also solves the problem of high detection limits in traditional methods due to salt suppression effects and co-mobile phase interference, ensuring reliable detection under complex matrix conditions.

[0021] The sample pretreatment phase incorporates a segmented solid-phase extraction (SPE) and deuterated internal standard dual calibration strategy, balancing the recovery of both weakly and strongly polar compounds and correcting for matrix effects. This minimizes both intra- and extra-laboratory variability and improves method precision. Compared to single-solvent extraction or offline concentration methods, this technology can enrich multiple contaminants within the same sample preparation process, reducing sample loss and increasing experimental throughput.

[0022] In terms of instrument configuration, precise matching of flow rate, column temperature, ion source temperature, and voltage ensures chromatographic peak separation while minimizing ion suppression. Rapid switching between positive and negative ions and multi-channel dMRM monitoring enable highly selective identification of structurally similar compounds, such as isomers and metabolites. This coupled strategy significantly shortens single analysis time and improves the efficiency of both on-site emergency monitoring and high-frequency routine monitoring.

[0023] This method can be seamlessly integrated into marine environmental monitoring and risk assessment systems: a closed loop from on-site portable preconcentration to high-precision laboratory analysis to cloud-based data decision-making, providing technical support for marine ecological health assessment, pollution source tracing, and regulatory compliance. Compared with existing detection solutions, this method offers advantages such as higher sensitivity, greater matrix adaptability, faster analysis speed, and a wider range of applicable targets, possessing significant application and promotion value and social and environmental benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a flow chart of a liquid chromatography-mass spectrometry method for detecting trace targets in seawater provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Example 1

[0027] One liter of coastal surface seawater was collected and filtered through a 0.7 μm / 0.45 μm cascade filter to remove particulate matter. The sample was then enriched using a C18 solid-phase extraction column. The eluent was concentrated to 200 μL of methanol:water (1:1) using nitrogen purging. The liquid chromatography system was configured with a 2.1 mm × 100 mm C18 column (1.7 μm particle size); a flow rate of 0.30 mL min-1, a column temperature of 35°C, an automatic injection volume of 5 μL, and ESI. + The source voltage was 3.5 kV and the atomizing gas pressure was 40 psi.

[0028] Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B consisted of methanol with 0.1% formic acid: 10% B from 0–1 min, ramped linearly to 95% B from 1–8 min, held for 8–10 min, and returned to 10% B at 10.1 min for a 3-min equilibration. Mass spectrometry employed dMRM, with retention time windows (±0.5 min) set for 12 PAHs, including naphthalene, phenanthrene, and pyrene. Precursor / product ion transitions, such as 128→102 and 202→126, were collected at collision energies of 15–35 eV. The method detection limit was 5–20 pg·L⁻¹, with a relative standard deviation of ≤6%.

[0029] Example 2

[0030] 500 mL of nearshore high-salinity seawater was collected and concentrated using an Oasis WAX solid-phase extraction column (eluent: methanol / 0.1% NH4OH). The volume was then nitrogen-purged to 100 μL of 100% methanol. The liquid phase system was connected to a PFAS-specific C18 column (50 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.25 mL / min. -1 , column temperature 40℃; injection volume 10μL; ESI-source voltage 2.8kV, desolvation 450℃, 900L·h-1.

[0031] Mobile phase A consisted of 5 mM NH₄OAc in water, and mobile phase B consisted of methanol: 40% B from 0–0.5 min, linearly increasing to 95% B from 0.5–5 min and maintaining this concentration for 5–7 min; then decreasing to 40% B from 7.1 min and equilibrating for 4 min. dMRM monitoring was performed for 10 PFAS, including PFOA and PFOS, with a retention window of ±0.3 min, ion transitions such as 499→80 and m / z 413→369, and a collision energy of 20–40 eV. The method had a detection limit of 2–8 pg·L⁻¹, and matrix effects were observed. 13 <10% after correction with C8-PFOA internal standard.

[0032] like Figure 1 As shown, an embodiment of the present invention provides a liquid chromatography-mass spectrometry method for detecting trace targets in seawater, the method specifically comprising:

[0033] S1: Configure instrument parameters such as liquid chromatography, chromatographic column, mass spectrometer, and autosampler;

[0034] S2: gradient elution;

[0035] S3: Mass spectrometry detection was performed in dynamic multiple reaction monitoring (dMRM) scanning mode.

[0036] The core parameters of the S1 instrument configuration are as follows:

[0037] (1) Liquid chromatography: binary pump (GT120A), flow rate: 0.40 mL / min; column temperature: 40°C;

[0038] (2) Chromatographic column: ZORBAX SB-Phenyl (2.1×100 mm, 1.8 μm) high salt resistant matrix to improve separation;

[0039] (3) Mass spectrometer: triple quadrupole (Agilent 6470), ion source: AJS ESI (positive / negative ion mode switch);

[0040] (4) Automatic sampler: G7187B Multisampler, injection volume: 2.00 μL; needle wash: standard flush (3 s).

[0041] The S2 gradient elution procedure is as follows:

[0042] Time (min) Mobile phase A (%) (water) Mobile phase B (%) (methanol) Function 0.00 95.0 5.0 Initial balance 1.00 90.0 10.0 Weakly polar substances eluted 5.00 70.0 30.0 Separation of medium polar substances 10.00 9.0 100.0 Elution of highly polar substances and metabolites 12.00 9.0 100.0 Ensures elution of highly retained compounds 12.10 95.0 5.0 System balance

[0043] The initial methanol ratio was 30%, which was increased to 60% at 3 minutes, 80% at 5 minutes, 90% at 10 minutes, and 100% methanol at 11 minutes (maintained for 5 minutes), and then returned to the initial conditions.

[0044] The S3 ion source parameters are as follows:

[0045] Drying gas temperature: 250°C, atomizing gas pressure: 30 psi, capillary voltage: +3000 V (positive ion) / -2500 V (negative ion).

[0046] After the monitoring vessel docked, seawater samples collected daily were immediately transported to the local environmental monitoring station for filtration (using pre-ignited GF / F glass fiber filters of known weight, Whatman, Maidstone, UK) and OUVF extraction. One liter of filtered seawater was spiked with 1 ng of deuterated internal standards (Sulfoxaflor-d3, Imidaclothiz-d4, E-Nitenpyram-d3, and Clothianidin-d3). Solid-phase extraction (SPE) was performed using an Oasis HLB solid-phase extraction cartridge (6 mL / 500 mg) at a trickle flow rate. The target compounds were eluted with 3 × 4 mL of a methanol / dichloromethane mixture (50:50, v:v). The eluate was concentrated to near dryness under a gentle stream of high-purity nitrogen, reconstituted with 200 μL of methanol, and spiked with Acetamiprid-d3 as an injection internal standard. The final extract was stored in 2 mL glass vials at −20°C and transported to our laboratory for instrumental analysis.

[0047] Extracts containing NNIs were analyzed using an Agilent 1290 Infinity II ultra-high performance liquid chromatography coupled to an Agilent 6470 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was equipped with an AJS-ESI source (Agilent Jet Stream electrospray ionization source) in positive ion mode. The injection volume was 2 μL, and chromatographic separation was performed on an Agilent ZORBAX SB-Phenyl column (2.1 × 100 mm, 1.8 μm) with a column oven temperature set at 40°C. The mobile phase consisted of milli-Q water (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.30 mL / min. A gradient elution program was used: starting with 30% methanol, increasing to 60% at 3 minutes, 80% at 5 minutes, 90% at 10 minutes, and 100% methanol at 11 minutes (held for 5 minutes), followed by a return to the starting conditions. Data were acquired in dynamic multiple reaction monitoring (dMRM) mode. An Agilent 8890 gas chromatograph system was coupled to an Agilent 7010B mass spectrometer. Mass spectrometry was performed in 70 eV electron impact (EI) mode, with separation on an Agilent HP-5MS UI capillary column (30 m × 0.25 mm × 0.25 μm). The column oven temperature was programmed as follows: 60°C for 1 minute, then increased to 120°C at 40°C / min, and finally to 300°C at 5°C / min (held for 7.5 minutes). Quantification was performed in dMRM mode.

[0048] This implementation utilizes post-collection seawater filtration and solid-phase extraction pretreatment, coupled with ultra-performance liquid chromatography-tandem mass spectrometry detection in a dynamic multiple reaction monitoring (MRM) mode for the qualitative and quantitative analysis of trace targets of varying types. By optimizing the sampling volume, preconcentration process, internal standard strategy, and gradient elution procedure, high sensitivity and matrix effect control are achieved. The following four examples each propose differentiated processing and detection procedures for specific targets or field conditions to meet diverse research or monitoring needs.

[0049] Example 1 Joint Detection of Multiple Residues of Nitrogen Heterocyclic Pesticides

[0050] One liter of pelagic seawater was collected from the ocean floor and immediately removed suspended particles on-site using a calcined filter membrane. Pretreatment with hydrophobic packing solid-phase extraction was performed using a 1:1 ratio of methanol to dichloromethane (volume ratio). The eluent was concentrated to near dryness using a 4-ml solution three times. The eluent was then reconstituted with 200 μl of methanol and a deuterated internal standard was added to complete sample preparation. Chromatographic separation was performed using a salt-tolerant phenyl stationary phase column with a flow rate of 0.4 ml / min and a column temperature of 40°C. A gradient program was used with an initial methanol concentration of 30% over 33 minutes, increasing to 65%, 80%, 91%, 100%, and then maintaining the concentration for five minutes before returning to the starting concentration. The mass spectrometer ion source was operated at a drying temperature of 250°C, a nebulizer pressure of 30 psi, and a capillary voltage of 3,000V in positive mode. Dynamic multiple reaction monitoring (DMRM) was used to configure multiple channels to cover the conversion of each pesticide precursor ion and characteristic fragments. Data processing included calibration curves and sample response comparisons to enable simultaneous quantification of multiple residues. This example is suitable for the simultaneous monitoring of trace levels of multiple nitrogen heterocyclic pesticides in seawater.

[0051] Example 2 Large volume on-site pre-concentration combined with online concentration detection

[0052] To improve the sensitivity of ultra-trace target detection, five liters of seawater were collected on-site. Continuous solid-phase extraction and an online modular concentration device were used to initially remove solid particles from the sample through a pre-burned filter membrane. The sample was then pre-concentrated using a large-capacity solid-phase extraction column at a constant drip flow rate. After multiple elutions, it was concentrated to near dryness, re-dissolved in a small volume of solvent, and the corresponding internal standard was added. The chromatographic separation conditions were consistent with those in Example 1, with the same flow rate and gradient program, but an extended retention time stage was added to improve the elution efficiency of weakly retained compounds. The mass spectrometer settings were synchronized with the dynamic multiple reaction monitoring mode, but the monitoring window was adjusted for ultra-low content targets and the acquisition time was extended to enhance the signal-to-noise ratio. This example is suitable for on-site monitoring or rapid response to environmental emergencies requiring extremely high sensitivity.

[0053] Example 3 Broad spectrum analysis and dual-mode fusion under complex matrices

[0054] To address the presence of both weakly and strongly polar trace compounds in seawater, one liter of seawater was collected and pretreated for organic interference removal, including liquid-liquid separation or recent membrane adsorption cleaning. Segmented solid-phase extraction was then performed, followed by collection of the weakly and strongly polar fractions. The fractions were then reconstituted and injected using an optimized gradient. For the weakly polar fraction, the gradient started with 40% methanol, increasing to 70% over three minutes, to 90% over seven minutes, and then recovering. For the strongly polar fraction, the gradient started with 20% methanol, increasing to 50% over four minutes, to 80% over eight minutes, to 100% over ten minutes, and then maintaining the concentration before recovering. Chromatography was optimized in the laboratory using the same salt-tolerant stationary phase column to achieve separation of both target compounds. The mass spectrometer was operated in dynamic multiple reaction monitoring mode, but separate monitoring channels were set for the two components and acquired simultaneously to achieve broad-spectrum screening and quantification. Data processing began with matrix-matched calibration and then internal standard correction. This example is suitable for the combined analysis of multiple compound types in complex marine samples.

[0055] Example 4 On-site portable small-scale combined with remote automated monitoring

[0056] A portable concentrator and a small liquid chromatography-mass spectrometry module are deployed on an offshore platform or vessel. Two liters of seawater are collected. After rough filtration on-site, the sample is pre-concentrated using a modular solid-phase extraction cartridge. The sample is concentrated to a small volume before being transferred to a portable liquid chromatography system for separation. The gradient program is optimized within the limits of the portable device: a starting methanol ratio of 30%, increasing to 80% over five minutes, then to 100% over ten minutes, then maintaining for three minutes before returning to normal. Simplified ion source parameters are set: drying temperature set to 200°C, nebulizer gas pressure set to 20 psi, capillary voltage set to 2,500V in positive mode, and 2,000V in negative mode. Dynamic multiple reaction monitoring mode is used to automatically acquire target signals, and the results are uploaded to a cloud platform in real time via a remote data transmission module. The cloud automatically processes and provides alerts. This example is suitable for rapid, real-time screening in remote waters or on mobile monitoring platforms.

[0057] Four examples propose optimization strategies for multi-residue detection of nitrogen heterocyclic pesticides, high-sensitivity preconcentration in large volumes, broad-spectrum analysis in complex matrices, and portable, remote, automated monitoring in the field. These strategies are based on a unified core process but incorporate differentiated adjustments in sample volume, pretreatment methods, gradient programs, and mass spectrometry monitoring design to meet the needs of different target types and application scenarios. During research or application, preconcentration parameters and separation conditions can be further fine-tuned based on the specific physical and chemical properties of the target and field conditions to ensure detection sensitivity and accuracy.

[0058] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for detecting trace targets in seawater, characterized in that: The following steps are involved: Step 1: Configure the liquid chromatography system. Set the flow rate to 0.4 ml / min. Set the column temperature to 40 degrees Celsius. The column stationary phase is a phenyl salt-tolerant matrix. Switch the electrospray ionization source to positive and negative modes. Automatic injection volume: 2 μl. Use a standard flush time of 3 seconds for needle cleaning. Step 2: Separate according to the gradient elution program: the initial mobile phase methanol ratio is 30% in 3 minutes, increased to 60% in 5 minutes, increased to 80% in 10 minutes, increased to 90% in 11 minutes, increased to 100% and maintained for 5 minutes, and then restored to the initial mobile phase ratio; Step 3: The ion source drying temperature was set to 250 degrees Celsius, the nebulizer gas pressure was set to 30 pounds per square inch, the capillary voltage was 3,000 volts in positive ion mode, and 2,500 volts in negative ion mode. The mass spectrometer was tested in dynamic multiple reaction monitoring mode. Step 4: Filter the collected seawater and use a pre-burned glass fiber filter to remove suspended particles. Then perform solid phase extraction and use a hydrophobic high-throughput filler to load the sample. The flow rate is a trickle flow mode. The elution solvent is methanol and dichloromethane in a volume ratio of 1:

1. The elution volume is three times, 4 ml each time. Step 5: The eluate was concentrated to near dry weight under a gentle high-purity nitrogen stream, dissolved in 200 μL of methanol, and then added with the injection internal standard and stored at -20°C. Step 6: Inject the reconstituted sample for analysis and perform peak identification, quantification and qualitative analysis, and finally output the target concentration results.

2. The method according to claim 1, characterized in that The specifications of the chromatographic column are 2.1 mm x 100 mm, and the particle size of the filling particles is 1.8 microns.

3. The method according to claim 1, characterized in that The filtration uses a glass fiber filter membrane with a known weight and a pre-burned treatment to remove suspended particles in seawater.

4. The method according to claim 1, characterized in that The solid phase extraction step uses a hydrophobic high-throughput filler to load the sample and then elutes in a trickle flow mode. The elution solvent is methanol and dichloromethane in a volume ratio of 1:1, and the elution volume is three times 4 ml each time.

5. The method according to claim 1, characterized in that: The concentration step uses a gentle high-purity nitrogen stream to concentrate to near dryness, then re-dissolve in 200 μl of methanol and add an internal standard.

6. The method according to claim 1, characterized in that Dynamic multiple reaction monitoring mode involves setting up multiple monitoring channels in the mass spectrometer for the conversion of target ions and internal standards into characteristic fragment ions to achieve highly sensitive detection.

7. A system for detecting trace targets in seawater, characterized in that: It includes a sampling device, a filtering device, a solid phase extraction device, a liquid chromatography-mass spectrometry analysis device and a data processing device. The configuration parameters of the analysis device are consistent with the configuration described in step 1 of claim 1, and is used to execute the detection method.

8. The system according to claim 7, characterized in that: The analytical device has a liquid phase pump flow rate of 0.4 ml per minute, a chromatographic column temperature of 40 degrees Celsius, an ion source drying temperature of 250 degrees Celsius, a nebulizer gas pressure of 30 pounds per square inch, a capillary voltage of 3,000 volts in positive mode and 2,500 volts in negative mode, and adopts a dynamic multiple reaction monitoring mode.

9. The system according to claim 7, characterized in that: The data processing device includes a peak identification module, a quantitative calculation module and a result output module. The quantitative calculation module calculates the concentration of the target substance based on a calibration curve.

10. The system according to claim 7, wherein: The gradient elution program starts with a mobile phase methanol ratio of 30%, increases to 60% in 3 minutes, increases to 80% in 5 minutes, increases to 90% in 10 minutes, increases to 100% in 11 minutes, maintains for 5 minutes, and then returns to the starting mobile phase ratio for separating targets of different polarities.

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