A rapid and accurate method for simultaneously determining the content of multiple new pollutants in water quality

Abstract

This invention discloses a rapid and accurate method for simultaneously determining the content of multiple new pollutants in water, belonging to the field of new pollutant detection technology. The method involves adding a compound salt and water sample to a narrow-necked, pear-shaped, flat-bottomed flask and shaking well. An internal standard solution is then added to the flask, and after shaking again, an organic extraction solvent is added. The flask is then subjected to high-frequency extraction in a vortex mixing extraction device, followed by standing. The upper organic phase in the flask is transferred, centrifuged, and the supernatant is used for ESI-HPLC-MS / MS-MRM analysis. The advantages of this invention are: by synergistically enhancing extraction through compound salting-out and mixed solvents, combined with techniques such as phenyl column chromatography and rapid gradient elution, it solves the problem of simultaneous extraction, separation, and quantification of new pollutants. Pretreatment eliminates the need for concentration, dissolution, and dehydration steps, achieving non-co-current separation of three types of substances within 10 minutes, significantly shortening the analysis time. This method is suitable for the simultaneous rapid screening and quantitative analysis of new pollutants in water bodies.

Description

Technical Field

[0001] This invention relates to the field of new pollution detection technology, and in particular to a rapid and accurate method for simultaneously determining the content of multiple new pollutants in water. Background Technology

[0002] New pollutants refer to substances that have recently emerged or become a concern, meaning substances with a relatively short history of production or use, or whose hazards were discovered relatively late. These substances pose significant risks to the ecological environment or human health, but are not yet included in management or existing management measures are insufficient to effectively control their risks. Organic UV absorbers, organophosphates, and tributyltin compounds are key indicators for screening new pollutants. Among them, organic UV absorbers, as light stabilizers, are widely used in personal care products and industrial products. They have endocrine-disrupting effects, genotoxicity, and reproductive toxicity, and have been identified as substances of very high concern by the European Chemicals Agency. They are a new type of "quasi" persistent organic pollutant, including 2-(2H)-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol (UV-320) and 2-(5-chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol (UV-327); organophosphates are mainly... Widely used as flame retardants and plasticizers in industrial additives, these compounds have potential endocrine disrupting, neurotoxic, and carcinogenic effects. They can be released into the environment through production, use, and disposal, posing a threat to ecosystems and human health. Examples include xylenol phosphate (1:3) ester (TXP) and tris(2,3-dibromopropionic acid) phosphate (TDBPP). Tributyltin compounds (TBTs) are mainly used as preservatives and heat stabilizers. They are extremely toxic to aquatic organisms and are among the most potent known endocrine disruptors, classifying them as persistent organic pollutants.

[0003] Currently, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and gas chromatography-tandem mass spectrometry (GC-MS) are commonly used methods for organic analysis. Liquid chromatography-tandem inductively coupled plasma mass spectrometry is also used for organic analysis. However, when analyzing ultraviolet absorbers, organophosphates, and tributyltin compounds, these methods are mostly only applicable to one or a single substance from these three categories. This is because these three categories of substances have a wide range of polarities: ultraviolet absorbers are weakly polar, organophosphates are moderately polar, and tributyltin is moderately polar. Existing technologies all detect single substances individually, which makes it difficult to solve problems such as polarity incompatibility, the contradiction between separation efficiency and speed, and conflicting ionization conditions. Secondly, existing quantitative methods are mostly single-mode and cannot be designed with strategies to address pretreatment losses, matrix benefits, and ionization stability for different types of substances. Therefore, existing technologies mostly employ gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS) to determine benzotriazole UV absorbers in aquatic products, dispersive solid-phase extraction-high performance liquid chromatography (HPLC) to determine four types of benzotriazole UV absorbers, and liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) to determine organotin compounds in water. Among these methods, liquid-liquid extraction uses a large amount of organic solvent, requiring subsequent concentration and conversion to methanol or acetonitrile before analysis. For wastewater samples, an additional purification step is needed (2.5 hours for extraction and dehydration of 6 samples per batch, 1.5 hours for concentration and conversion to solvent, 1 hour for purification, and another 1 hour for concentration and conversion to solvent, totaling approximately 6 hours). Solid-phase extraction also requires concentration followed by conversion to methanol or acetonitrile before analysis (4 hours for extraction of 6 samples per batch, 1 hour for concentration and conversion to solvent, totaling approximately 5 hours).

[0004] The above analysis process not only requires three different methods and three on-machine analyses, but also requires different types of pretreatment and multiple types of analytical instruments. In addition to the large investment in instruments, the process is also lengthy and time-consuming, which is inefficient and makes it difficult to achieve simultaneous detection of ultra-trace amounts and high sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid and accurate method for simultaneously determining the content of multiple new pollutants in water. This method is fast, efficient, and accurate. It utilizes a micro-volume liquid-liquid extraction system for pretreatment, combined with a chromatographic optimization strategy based on a rapid gradient program, and employs a categorized internal / external standard combination quantitative method. This method enables the detection of new pollutants such as organic ultraviolet absorbers, organophosphates, and tributyltin compounds within 10 minutes of a single injection. It is highly efficient and accurate, providing strong technical support for the rapid screening of new pollutants in the context of new pollutant treatment.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A rapid and accurate method for simultaneously determining the content of multiple new pollutants in water quality includes the following steps:

[0008] S1. Add the compound salt and water sample containing multiple new pollutants to a narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle at a ratio of 50 g / L, and shake well to form an unsaturated solution.

[0009] S2. Add UV320-D4 internal standard solution to a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask, shake well again, and then add organic extraction solvent.

[0010] S3. Place the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle in a vortex mixing extraction device for high-frequency extraction and let it stand. Add pure water dropwise so that the liquid levels of the aqueous phase and the organic phase are located at the narrow neck of the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle.

[0011] S4. Transfer the upper organic phase from the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle, centrifuge it, and then take the supernatant for ESI-HPLC-MS / MS-MRM analysis.

[0012] Furthermore, several new pollutants have emerged, including organic ultraviolet absorbers UV-320 and UV-327, organophosphate compounds TXP and TDBPP, and TBTs.

[0013] Furthermore, in the ESI-HPLC-MS / MS-MRM analysis and detection:

[0014] The high-performance liquid chromatography (HPLC) conditions were as follows: a phenyl column, a column temperature of 35℃, a mobile phase of 0.1% formic acid in 1 mmol / L ammonium formate aqueous solution / 0.1% formic acid in methanol solution, a flow rate of 0.5 mL / min, an injection volume of 10 μL, and a gradient elution program.

[0015] The mass spectrometry conditions were: spray voltage 5500V, curtain gas 30psi, ion source temperature 600℃, and nebulizing gas and nebulization auxiliary gas both 50psi.

[0016] Quantitative calculation by category: UV-320 and UV-327 were quantified using the internal standard method with UV-320-D4 as the internal standard, while TXP, TDBPP, and TBTs were quantified using the external standard method. The external standard method used a matrix-matched standard curve, and the blank matrix was prepared by removing the target analytes from the water sample after it was processed by a C18 solid phase extraction column.

[0017] Furthermore, the gradient elution program was as follows: from 0 to 0.2 min, the mobile phase of 0.1% formic acid methanol solution was linearly increased from 20% to 99%, maintained for 7 min, and then decreased to 20% and maintained for 10 min.

[0018] Furthermore, the high-performance liquid chromatography conditions also include a column temperature gradient program executed synchronously with the gradient elution program. The column temperature gradient program involves a linear increase in column temperature from 35°C to 45°C from 0 to 0.2 min and maintaining this temperature for 7 min, followed by a linear decrease to 35°C from 7 to 10 min.

[0019] Furthermore, the parameters of MRM are:

[0020] UV-320: precursor ion 324.2 Da, quantitative daughter ion 268.3 Da, qualitative daughter ion 212.3 Da, cone voltage 120 V, collision voltages 31.73 V and 38.25 V respectively;

[0021] UV-327: precursor ion 358.4 Da, quantitative daughter ion 302.0 Da, qualitative daughter ion 246.3 Da, cone voltage 124 V, collision voltages 32.11 V and 41.13 V respectively;

[0022] TXP: precursor ion 411.2 Da, quantitative daughter ion 194.0 Da, qualitative daughter ion 179.2 Da, cone voltage 240 V, collision voltages 41.0 V and 54.0 V respectively;

[0023] TDBPP: precursor ion 698.6 Da, quantitative daughter ion 299.0 Da, qualitative daughter ion 99.0 Da, cone voltage 50 V, collision voltages 23.0 V and 51.0 V respectively;

[0024] TBTs: precursor ion 291.0 Da, quantitative daughter ion 179.0 Da, qualitative daughter ion 235.2 Da, cone voltage 30 V, collision voltages 18.07 V and 12.08 V respectively;

[0025] UV-320-D4: Parent ion 328.3 Da, Quantitative daughter ion 272.2 Da, Cone voltage 160 V, Collision voltage 33.01 V.

[0026] Furthermore, in step S1, the composite salt includes sodium chloride and magnesium sulfate, with a mass ratio of sodium chloride to magnesium sulfate of 4:1. The sodium chloride and magnesium sulfate are mixed and then dried at 500°C for 2 hours, sealed and cooled before use.

[0027] Furthermore, in step S2, the volume ratio of the organic extractant to the water sample is 1:100, and the organic extractant includes n-hexane and ethyl acetate, wherein the volume ratio of n-hexane to ethyl acetate is 9:1.

[0028] Furthermore, in step S3, the vortex frequency of the vortex mixing extraction device is 1000~2000 r / min, the extraction time is 5~10 min, and the settling time is 5-15 min.

[0029] The present invention has the following advantages:

[0030] 1. By utilizing a phenyl column combined with a mobile phase and a rapid gradient program, the problem of simultaneous separation of three types of substances with large polarity ranges is solved, achieving non-co-current separation of the three types of substances within 10 minutes. Compared with existing single-substance detection methods, the detection efficiency is significantly improved. Combined with micro-volume high-magnification extraction, the detection sensitivity is high.

[0031] 2. A micro-volume liquid-liquid extraction system using a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask with vortex mixing is employed. Combined with compound salting-out and mixed solvent synergistic extraction, no concentration, solvent transfer, or dehydration steps are required. This solves the problems of large solvent consumption, cumbersome procedures, and easy sample emulsification associated with traditional separatory funnel extraction. The extraction ratio reaches 100 times, which greatly shortens the analysis time while ensuring the detection of pollutants at ng / L concentration levels in water. It can achieve simultaneous high recovery of weakly polar, moderately polar, and moderately strongly polar new pollutants, and has the advantages of efficient, rapid, and accurate detection.

[0032] 3. Based on the different pretreatment loss patterns, matrix effect degree, and ionization stability of the three types of substances, a classified internal / external standard combination quantitative method is adopted. The matrix interference and pretreatment loss are further reduced by compound salting out and mixed solvents. The internal standard method ensures the quantitative accuracy of organic ultraviolet absorbers, while the external standard method ensures the detection stability of organophosphates and tributyltin compounds. This eliminates systematic errors and significantly improves the recovery rate and quantitative accuracy under ultra-trace detection. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the process of the present invention.

[0034] Figure 2 The effect of different flow rates on the response of the target compound.

[0035] Figure 3 The effect of different chromatographic columns on the response of target compounds.

[0036] Figure 4 The effect of different curtain gas and spray temperatures on the response of target compounds.

[0037] Figure 5 The effect of different spray voltages on the response of the target compound.

[0038] Figure 6 This is the standard spectrum of the target compound.

[0039] Figure 7 The effects of different amounts of compound salts, extraction frequency, and extraction time on the recovery rate of the target compound were investigated. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0041] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0042] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0043] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0044] Example 1

[0045] refer to Figure 1 The flowchart shown illustrates a rapid and accurate method for simultaneously determining the levels of multiple emerging pollutants in water, comprising the following steps:

[0046] S1. Add 10g of compound salt and 200mL of water sample containing multiple new pollutants to a 250mL narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle, and shake well to form an unsaturated solution.

[0047] Among them, several new pollutants include organic ultraviolet absorbers such as 2-(2H)-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol (UV-320) and 2-(5-chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol (UV-327); organophosphate compounds such as xylenol phosphate (1:3) ester (TXP) and tris(2,3-dibromopropionic acid) phosphate (TDBPP); and tributyltin compounds (TBTs). The complex salt is sodium chloride and anhydrous magnesium sulfate in a specific mass ratio. The mixture is prepared in a 4:1 ratio and pre-mixed before addition. It is then dried at 500℃ for 2 hours, sealed and cooled before use. The compound ratio of sodium chloride and anhydrous magnesium sulfate is used to synergistically enhance the salting-out effect and demulsification ability. This can significantly improve the extraction efficiency of weakly polar organic UV absorbers and improve the partitioning effect of moderately polar organic phosphate compounds. At the same time, it avoids the inhibitory effect of high salt on the recovery rate of target substances. High temperature treatment can completely remove adsorbed water, residual organic impurities and potential mass spectrometry interference substances from the salt, and prevent external pollution from affecting the sensitivity of pg / L ultra-trace detection.

[0048] S2. Add 1 μL of 1.0 mg / LUV320-D4 internal standard solution to a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask, shake well again, and then add 2 mL of organic extraction solvent.

[0049] Dichloromethane and n-hexane are commonly used extraction solvents for liquid-liquid extraction in chromatographic analysis. Dichloromethane is a moderately polar solvent, while n-hexane is a non-polar solvent. Both can be used to extract five novel pollutants from three classes in this study. However, dichloromethane is denser than water and lies at the bottom of the aqueous phase during liquid-liquid extraction. Since this study uses a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask for extraction, the upper organic phase is easier to transfer precisely; therefore, n-hexane remains the primary solvent. To simultaneously extract weakly and moderately polar targets and improve the recovery rate of pollutants with a wide polarity range, the polarity of the extraction solvent was fine-tuned. A 9:1 volume ratio of n-hexane to ethyl acetate was used as the extraction phase. Without changing the layering position or affecting the transfer, the introduction of a small amount of ethyl acetate slightly increases the polarity, enhancing the extraction of polar substances in TXP and TDBPP through hydrogen bonding. Simultaneously, it works with the formic acid / ammonium formate mobile phase to improve ionization efficiency, achieving simultaneous high recovery of novel pollutants. A smaller extraction solvent volume results in a higher sample concentration factor, which is more conducive to ultra-trace detection of target substances in water. However, too little extraction solvent will lead to an excessively thin upper organic phase layer, making it difficult to accurately remove. Considering both concentration efficiency and operational feasibility, this study selected an extraction solvent to water sample volume ratio of 1:100, that is, 200 mL of water sample was supplemented with 2 mL of organic extraction solvent.

[0050] S3. Place the narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask in the vortex mixing extraction device, control the vortex frequency to 1500 r / min, extract for 10 min to allow the organic phase and aqueous phase to reach a fully stirred state, let stand for 5 min, and add pure water dropwise so that the liquid surface of the aqueous phase and organic phase is located at the narrow neck of the narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask.

[0051] In actual environmental water samples, the concentrations of the three types of new pollutants mentioned above are often low, resulting in an excessively thin organic phase that cannot be accurately transferred. Therefore, a vortex mixing extraction device is used in conjunction with a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask to avoid the need for liquid-liquid extraction using a separatory funnel. Using a traditional separatory funnel would result in a large amount of organic solvent used, multiple steps of dehydration, concentration, and solvent transfer, easy sample emulsification, and cumbersome operation. This embodiment utilizes the structural features of a narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle. The flat-bottomed structure allows the pear-shaped bottle to be stably placed on the vortex apparatus. The high-speed shear force of the vortex achieves thorough mixing of the aqueous and organic phases without the need for a stirring rotor, thus avoiding the adsorption of the target analyte and the introduction of impurities. After the organic extraction solvent separates into layers in the aqueous sample, the addition of pure water to the bottle can precisely push the separation interface to the narrow neck. The thin organic phase, which was not visible in the main body of the bottle, forms a distinct liquid column in the narrow neck, which can be accurately removed using a Pasteur dropper. Using the micro-volume organic solvent used in this application for direct extraction, at an extraction concentration factor of 100 times, the extraction time for the same amount of sample can be reduced by 7 times (the extraction from liquid to concentrate using a separatory funnel takes about 4 hours, while the device of this invention, including the liquid removal operation, takes about 30 minutes).

[0052] S4. Transfer the upper organic phase from the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle using a Pasteur pipette. Centrifuge at 8000 rpm for 10 min using a microcentrifuge. Transfer the supernatant to a sample vial for analysis. Separate the supernatant using high-performance liquid chromatography (HPLC) column analysis, employing electrospray ionization (ESI)-HPLC-tandem triple quadrupole mass spectrometry (MS / MS) in positive ion mode with multiple reaction monitoring (MRM).

[0053] Due to the significant differences in the physicochemical properties of the three types of novel pollutants mentioned above, simultaneous detection faces challenges such as polarity incompatibility, contradictions between separation efficiency and speed, and conflicts in ionization conditions. This application employs a combination of a phenyl column, a composite modified mobile phase, and an ultrafast gradient to address the large polarity range and ionization conflicts, achieving rapid separation and efficient detection within 10 minutes.

[0054] The high-performance liquid chromatography (HPLC) conditions were as follows: a phenyl column, a column temperature of 35℃, a mobile phase of 0.1% formic acid in 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid in methanol (B), a flow rate of 0.5 mL / min, an injection volume of 10 μL, and a gradient elution program. The gradient elution program was as follows: from 0 to 0.2 min, the mobile phase of 0.1% formic acid in methanol solution linearly increased from 20% to 99%, maintained for 7 min, then decreased to 20% and maintained for 10 min. The column temperature gradient program was executed synchronously with the gradient elution program. The column temperature gradient program was as follows: from 0 to 0.2 min, the column temperature linearly increased from 35℃ to 45℃ and maintained for 7 min, then linearly decreased to 35℃ from 7 to 10 min. The injector employs a multi-channel needle washing mode. Washing solutions R0 and R1 are both methanol-water (1:1 volume ratio of methanol to water), while washing solution R2 uses either methanol-water (1:1 volume ratio of methanol to water) or acetonitrile-water (1:1 volume ratio of acetonitrile to water). This simultaneous cleaning of the inside and outside of the injection needle effectively removes UV residues, eliminates peak shape interference, and results in more accurate and sensitive detection. In the experiment, using methanol or acetonitrile as washing solution R2 did not significantly affect the detection results. The experiment verified that methanol-water washing only on the outside of the needle easily produces significant injection residues, while using isopropanol-water as the washing solution affects the peak shape of the target analyte.

[0055] The mass spectrometry conditions were: spray voltage 5500V, curtain gas 30psi, ion source temperature 600℃, and nebulizing gas and nebulization auxiliary gas both 50psi.

[0056] The categorical quantitative calculation was performed using a combination of internal and external standard methods. Because UV320 and UV327 are easily adsorbed by glassware during extraction, resulting in large fluctuations in loss, subsequent ESI ionization is significantly affected by the water sample. The recovery rate and ionization efficiency of TXP and TDBPP are relatively problematic, but their matrix effect has been largely eliminated in the pretreatment. TBTs, however, exhibit good retention under the micro-volume extraction method described in this application, and their metallic properties give them strong ionization specificity and weak matrix interference. Therefore, in this embodiment, the organic ultraviolet absorbers UV-320 and UV-327 were quantified using the internal standard method with UV-320-D4 as the internal standard, while TXP, TDBPP, and TBTs were quantified using the external standard method. The external standard method used a matrix-matched standard curve, and the blank matrix was prepared by removing the target analytes from the water sample using a C18 solid-phase extraction column.

[0057] The parameters of MRM are:

[0058] UV-320: precursor ion 324.2 Da, quantitative daughter ion 268.3 Da, qualitative daughter ion 212.3 Da, cone voltage 120 V, collision voltages 31.73 V and 38.25 V respectively, and the abundance ratio of quantitative ion to qualitative ion ranges from 1.2 to 1.4:1.

[0059] UV-327: precursor ion 358.4 Da, quantitative daughter ion 302.0 Da, qualitative daughter ion 246.3 Da, cone voltage 124 V, collision voltages 32.11 V and 41.13 V respectively, and the abundance ratio of quantitative ion to qualitative ion ranges from 1.0 to 1.2:1.

[0060] TXP: precursor ion 411.2 Da, quantitative daughter ion 194.0 Da, qualitative daughter ion 179.2 Da, cone voltage 240 V, collision voltages 41.0 V and 54.0 V respectively, and the abundance ratio of quantitative ion to qualitative ion ranges from 1.1 to 1.3:1.

[0061] TDBPP: precursor ion 698.6 Da, quantitative daughter ion 299.0 Da, qualitative daughter ion 99.0 Da, cone voltage 50 V, collision voltages 23.0 V and 51.0 V respectively, and the abundance ratio of quantitative ion to qualitative ion ranges from 3.0 to 3.2:1;

[0062] TBTs: precursor ion 291.0 Da, quantitative daughter ion 179.0 Da, qualitative daughter ion 235.2 Da, cone voltage 30 V, collision voltages 18.07 V and 12.08 V respectively, and the abundance ratio of quantitative ion to qualitative ion ranged from 6.5 to 7.5:1.

[0063] UV-320-D4: Parent ion 328.3 Da, Quantitative daughter ion 272.2 Da, Cone voltage 160 V, Collision voltage 33.01 V.

[0064] The above MRM parameters were obtained by ion scanning of standard solutions: UV320, UV320-D4, UV327, TXP, TDBPP, and TBTs standard solutions with a concentration of 1 μg / mL were prepared using organic extractants (n-hexane and ethyl acetate) as solvents. Nitrogen was used as the collision gas and curtain gas, and zero-level air was used as the nebulizer gas and ion source auxiliary gas. The above standard solutions with a concentration of 1 μg / mL were injected into each solution. The precursor ion was scanned in negative ion mode and positive ion mode. No ion characteristic peaks were found in negative ion mode, while molecular ion characteristic peaks of the above compounds were obtained in positive ion mode. The obtained characteristic molecular ions were used as precursor ions for daughter ion scanning, and the cone voltage and collision voltage were optimized to obtain the above MRM parameters.

[0065] Example 2

[0066] In this embodiment, the composition of the mobile phase was adjusted to study the effect of different flow rates on the response of the target compound, in order to determine the high-performance liquid chromatography (HPLC) conditions. Specifically, a mixed standard solution of UV320, UV327, TXP, TDBPP, and TBTs with a concentration of 20 ng / mL was prepared using an organic extractant as the solvent; a phenyl column was used, with the column temperature set at 35°C and the flow rate at 0.5 mL / min. A fixed gradient elution program was used: from 0 to 0.2 min, mobile phase B linearly increased from 20% to 99%, maintained for 7 min, then decreased to 20% and maintained for 10 min. A column temperature gradient program was executed synchronously with the gradient elution program: from 0 to 0.2 min, the column temperature linearly increased from 35°C to 45°C and maintained for 7 min, then linearly decreased to 35°C from 7 to 10 min. The mobile phase used was 0.1% formic acid 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid methanol solution (B), methanol / water, 0.1% formic acid aqueous solution (A) / 0.1% formic acid methanol solution (B), 1 mmol / L ammonium formate aqueous solution (A) / 1 mmol / L ammonium formate methanol solution (B), 1 mmol / L ammonium formate 0.1% formic acid aqueous solution (A) / methanol solution (B), and 1 mmol / L ammonium formate 0.1% formic acid aqueous solution (A) / acetonitrile (B). The above 20 ng / mL mixed standard solutions were injected for analysis.

[0067] For compounds that generate molecular ions in positive ion mode, adding a certain concentration of acid or acidic salt to the mobile phase can generally increase their ionization efficiency. However, an acidic mobile phase is detrimental to the retention of these target compounds on the chromatographic column, potentially reducing their response. Therefore, this application adds 1 mmol / L ammonium formate and 0.1% formic acid to the mobile phase. This adjusts the retention time of TXP / TDBPP through ion-pair interactions and stabilizes the chromatographic behavior of TBTs through protonation, thereby improving the ESI ionization efficiency of all substances. Specific results are as follows: Figure 2As shown, with water / methanol as the mobile phase, the peak area responses of the three new pollutants were all poor. When 0.1% formic acid was added to the mobile phase, the peak area responses of TBTs, TDBBP, UV320, and UV327 increased, while the response of TXP decreased slightly. When 1 mmol / L ammonium formate was added to the mobile phase, the peak area responses of TDBBP, UV320, UV327, and TXP increased significantly, while TBTs decreased somewhat. When only 1 mmol / L ammonium formate and 0.1% formic acid were added to mobile phase A, the responses of all compounds increased, but the increases in the responses of TDBPP and TXP were not as significant as those when 1 mmol / L ammonium formate was added to the mobile phase. When 1% formic acid was added and mobile phase B was replaced with acetonitrile, the responses of TXP, UV320, TDBBP, and TBTs increased significantly, while the response of UV320 was relatively low. When 1 mmol / L ammonium formate and 0.1% formic acid were added to mobile phase A and 0.1% formic acid was added to mobile phase B, the responses of all compounds increased. TBTs, which had the lowest overall response, had the highest response when using this mobile phase, and the responses of the other compounds were also relatively high when using this mobile phase. Therefore, this embodiment comprehensively considers the influence of five different mobile phases on the peak area response of the target compounds and selects 0.1% formic acid and 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid and methanol solution (B) as the mobile phase for analyzing five new pollutants in three categories.

[0068] Example 3

[0069] This embodiment, based on the mobile phase determined in Example 2, only adjusts different chromatographic columns to study the effect of different chromatographic columns on the response of the target compound. Specifically, the mobile phase is 0.1% formic acid 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid methanol solution (B). The chromatographic columns used are a Kinetex biphenyl LC (100×3.0 mm, 2.6 μm) phenyl column, a Chemalin C18 (2.1×50 mm, 3 μm) and ACQUITYUPLC@BEH C18 (100×2.1, 1.7 μm) carbon-based column, and an Agilent Eclipse PAH (50×2.1 mm, 1.8 μm) polycyclic aromatic hydrocarbon column. The above 20 ng / mL mixed standard solution was injected for analysis.

[0070] Because the target compounds have different polarities, their sensitivity and resolution vary on chromatographic columns of different polarities. This invention does not involve isomers; the monitored precursor ion has a mass-to-charge ratio greater than unit resolution, which a triple quadrupole mass spectrometer can fully resolve. Accurate quantification can be performed without achieving baseline separation between the target compounds; therefore, peak area response is the primary factor considered. Results are as follows... Figure 3As shown, C18 columns with different polarities, polycyclic aromatic hydrocarbon (PAH)-specific columns, and biphenyl columns were investigated. The influence of different specifications of C18 columns on analytical sensitivity was also discussed. The column plots show that for organic UV absorbers (UV320, UV327), among columns with similar particle sizes—Kinetex biphenyl LC (100×3.0 mm, 2.6 μm), Chemalin C18 (2.1×50 mm, 3 μm), and Agilent Eclipse PAH (50×2.1 mm, 1.8 μm)—the PAH-specific column performed the worst, while the biphenyl column performed the best. However, compared to ultra-high performance liquid chromatography (UHPLC) columns with smaller particle sizes, the UHPLC column showed a higher response. For organophosphates (TXP, TDBPP), the biphenyl column performed the best among the four columns studied. In principle, phenyl columns possess retention properties for weakly polar substances (UV320, UV327) and moderately to strongly polar substances (TBTs) containing benzene rings / heterocyclic compounds. This reduces the excessive retention of UV320 and UV327, causing their peaks to elute earlier, while simultaneously enhancing the stable retention of TBTs. Further analysis of TBTs showed that among the three types of columns with the same particle size, the PAH-specific column performed best, but not as well as the smaller-particle-size ultra-high performance liquid chromatography (UHPLC) column; the biphenyl column performed better. However, in actual sample testing, because smaller-particle-size UHPLC columns are more prone to clogging, a Kinetex biphenyl (3.0 × 100 mm, 2.6 μm) phenyl column was used to analyze five novel pollutants from three categories.

[0071] Having solved the retention and separation issues by selecting a phenyl column as the chromatographic column, a rapid gradient elution program was then employed to quickly elute the weakly polar UV320 and UV327 within 7 minutes, while ensuring the separation of moderately polar and moderately polar substances. Simultaneously, the column temperature gradient program, in synergy with the phenyl column, accelerated the elution of the weakly polar organic UV absorber, preventing co-elution with high-concentration matrix, and stabilized the separation effect of moderately polar substances, achieving co-elution separation of the three types of substances within a 10-minute analysis cycle.

[0072] Example 4

[0073] Based on the mobile phase and chromatographic column determined in Example 3, this embodiment adjusts the curtain gas and spray temperatures respectively to study the effects of different curtain gas and spray temperatures on the response of the target compound.

[0074] If the curtain gas pressure is too low, the matrix ion removal rate may be insufficient, leading to low detection sensitivity; if the curtain gas pressure is too high, target ion loss may occur, reducing sensitivity. Spray temperature is a core parameter for target compound ionization. Its functions are twofold: first, it reduces intermolecular forces, promoting the transition from liquid to gaseous state, improving ionization efficiency, and thus increasing analytical sensitivity; second, it accelerates the evaporation of the mobile phase solvent, reducing solvent molecule encapsulation of target ions, preventing the formation of solvent cluster ions, increasing the ion throughput into the mass analyzer, and improving analytical sensitivity. Taking UV320 and UV327 as examples, specifically, with a mobile phase of: 0.1% formic acid 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid methanol solution (B), using Kinetex... A biphenyl (3.0×100mm, 2.6μm) column was used as the chromatographic column, and the mixed standard solution was reconfigured: a mixed standard solution of UV320 and UV327 with a concentration of 20 ng / mL was prepared using an organic extractant as the solvent; the above 20 ng / mL mixed standard solution of UV320 and UV327 was injected and analyzed with an ion source spray voltage of 5500V, an ion source temperature of 600℃, an nebulizer gas of 50psi, a nebulizer gas of 50psi, and curtain gas of 30psi, 35psi, 40psi, 45psi, 50psi, and 55psi respectively; the above 20 ng / mL mixed standard solution of UV320 and UV327 was then injected and analyzed with a curtain gas of 30psi, a spray voltage of 5500V, an nebulizer gas of 50psi, a nebulizer gas of 50psi, and a spray temperature of 350℃, 400℃, 450℃, 500℃, 550℃, 600℃, and 650℃ respectively. The results are as follows Figure 4 As shown, the responses of UV320 and UV327 decreased with the increase of the curtain gas. Therefore, 30 psi was selected as the curtain gas parameter in this embodiment. With the increase of the ion source temperature, the responses of UV320 and UV327 increased. Since the new pollutants have good thermal stability, the increase of spray temperature is conducive to the ionization of target compounds, allowing more target ions to enter the mass analyzer. However, excessively high temperature can also lead to the decomposition of thermally unstable compounds, thereby reducing the sensitivity of the analysis. Therefore, a spray temperature of 600°C was used as the ion source temperature in this embodiment.

[0075] Example 5

[0076] This embodiment, based on the mobile phase, column, curtain gas, and spray temperature determined in Example 4, only adjusts the spray voltage to study the effect of different spray voltages on the response of the target compound. Specifically, the mobile phase was 0.1% formic acid in 1 mmol / L ammonium formate aqueous solution (A) / 0.1% formic acid in methanol solution (B). A Kinetex biphenyl (3.0 × 100 mm, 2.6 μm) column was used as the chromatographic column. The curtain gas was 30 psi, the spray temperature was 600 °C, the nebulizer gas was 50 psi, and the nebulizer gas was 50 psi. The spray voltages were 3500 V, 4000 V, 4500 V, 5000 V, and 5500 V, respectively. A mixed standard solution of 20 ng / mL of UV320, UV327, TXP, TDBPP, and TBTs prepared in Example 2 was used for injection analysis. The results are as follows: Figure 5 As shown, with the increase of spray voltage, the peak area response of UV327, TXP, TDBPP, and TBTs increases slowly, while the response of UV320 is relatively better. Therefore, a spray voltage of 5500V was used as the spray voltage for the ionization of the target compound.

[0077] Example 6

[0078] This embodiment, based on Example 5, determines the instrumental analysis method to ensure the accuracy of qualitative and quantitative analysis. Specifically, a gradient standard solution is prepared: Based on the mixed standard solution of UV320, UV327, TXP, TDBPP, and TBTs with a concentration of 20 ng / mL prepared in Example 2, a series of mixed standard solutions with concentrations of 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, and 5.00 ng / mL are prepared by serial dilution using an organic extractant. Then, a UV320-D4 internal standard solution with a mass concentration of 1 μg / mL is added to achieve a concentration of 1 ng / mL. Each mixed standard solution in the step is measured sequentially from low to high concentration. Qualitative analysis is based on retention time, quantitative ion abundance ratio, and qualitative ion abundance ratio. The results are as follows: Figure 6 As shown in the standard spectrum (5 ng / mL), the analysis of five new pollutants in three categories can be completed within 10 minutes with a single injection, with high signal sensitivity and good peak shape.

[0079] Further, a standard curve was established with the ratio of the mass concentration of organic ultraviolet absorbers (UV320, UV327) to the concentration of internal standard (UV320-D4) as the x-axis and the ratio of the corresponding peak area response value to the internal standard response value as the y-axis. A standard curve was also established with the mass concentration of tributyltin compounds (TBTs) and two organophosphate compounds (TXP, TDBPP) as the x-axis and the peak area response as the y-axis. The retention time, linear range, regression equation and correlation coefficient r of the target compounds were then obtained. The results are shown in Table 1.

[0080] Table 1. Retention time, linear range, regression equation, and correlation coefficient r of the target compound.

[0081]

[0082] As shown in Table 1, there is a significant linear correlation between the mass concentrations of the five new pollutants in the three categories and the response values, with correlation coefficients (r) not lower than 0.999, far exceeding the technical requirements of environmental monitoring and analysis methods. Specifically, the organic ultraviolet absorbers (UV320, UV327) were quantified using the internal standard method, and the intercept of the regression equation was close to 0, indicating that the UV320-D4 internal standard effectively corrected for pretreatment losses and matrix effects. Tributyltin compounds (TBTs) and organophosphate compounds (TXP, TDBPP) were quantified using the external standard method, and the correlation coefficients remained above 0.999, indicating stable recovery rates and weak matrix interference in the micro-volume extraction system, allowing for accurate quantification without internal standard correction. The linear range covers 0.01–5.00 μg / L, simultaneously meeting the needs for ultra-trace screening and high-concentration emergency monitoring of new pollutants in surface water, groundwater, domestic sewage, and industrial wastewater, demonstrating excellent quantitative accuracy and applicability.

[0083] Example 7

[0084] The process steps were consistent with those in Example 1. Multiple 200 mL water samples were taken, each containing 2 μL of a mixed standard solution of UV320, UV327, TXP, TDBPP, and TBTs at a concentration of 20 ng / mL. In single-group experiments, the addition amounts of different composite salts (0 g, 10 g, 20 g), extraction times (5 min, 10 min), and extraction frequencies (1000 r / min, 1500 r / min, 2000 r / min) were adjusted. The addition of the composite salt (a mixture of sodium chloride and anhydrous magnesium sulfate at a mass ratio of 4:1) enhanced the salting-out effect and synergistically demulsified the extract, reducing the degree of emulsification and facilitating the smooth extraction of the organic phase for subsequent operations when a higher concentration factor was obtained. The addition of the composite salt also affected the recovery rate. The results of the effect on the recovery rate are shown below. Figure 7As shown, when different amounts of compound salt were added for wastewater sample extraction, the recoveries of the five new pollutants after adding 0, 10, and 20 g of compound salt were 18.6%–72.6%, 62.7%–102%, and 34.1%–102%, respectively. The addition of compound salt had little effect on TDBPP and TBTs, maintaining a relatively high level overall, while having a greater effect on TXP, UV320, and UV327. Without the addition of compound salt, the spiked recoveries of TXP, UV320, and UV327 were all low. When the amount was increased to 10 g, the recoveries increased to 62.7%–102%, but when the amount was increased to 20 g, the recoveries of UV320 and UV327 decreased, while TXP remained relatively unchanged. Regarding the selection of different extraction times, 5 min and 1 min... At 0 min, the recoveries of the five new pollutants were 30.6%–102% and 62.6%–102%, respectively. The impact on TDBPP, TXP, and TBTs was relatively small, with high recoveries achieved. However, the impact on UV320 and UV327 was significant. With different extraction frequencies, the spiked recoveries of the five new pollutants in wastewater at 1000 r / min, 1500 r / min, and 2000 r / min were 43.4%–103%, 62.6%–102%, and 60.1%–121%, respectively. When the extraction frequency increased from 1000 r / min to 1500 r / min, the spiked recoveries improved significantly. However, when the frequency was increased to 2000 r / min, the recovery effect did not improve significantly and even decreased.

[0085] Example 8

[0086] This embodiment studies the detection limit, precision, and accuracy of this method when analyzing five new pollutants from three categories in different water samples. The detection limit experiment was conducted using a blank spiked method: Before adding the mixed standard solution (a mixed standard solution of UV320, UV327, TXP, TDBPP, and TBTs at a concentration of 20 ng / mL) to multiple water samples (surface water, groundwater, and wastewater), 1 μL of 1 μg / mL UV320-D4 standard working solution was added to each sample. The TXP and TDBPP spiked concentrations for the blank samples were 0.6 ng / L, and the TBTs, UV320, and UV327 spiked concentrations were 0.4 ng / L. Seven parallel samples were prepared for the blank spikes, and pure water was used instead of water samples to prepare laboratory blank samples. For the precision and accuracy experiments, the TBTs spiked concentrations were 0.4, 2.0, and 25 ng / L, respectively, and the other four compounds were spiked at concentrations of 0.4, 2.0, and 20 ng / L, respectively. For low concentrations (0.4 ng / L), groundwater was used for spikes, for medium concentrations (2.0 ng / L), surface water was used for spikes, and for high concentrations (20 ng / L and 25 ng / L), wastewater was used for spikes. Six parallel samples were prepared for each spiked concentration, and two parallel samples were prepared simultaneously for the unspecified samples. Water was used instead of water samples to prepare laboratory blank samples. The process steps were the same as in Example 1 for pretreatment and instrumental analysis. The measured values ​​of blank samples, blank spiked samples, and detection limits are shown in Table 2.

[0087] Table 2. Blank spiked values ​​and limits of detection for the target compounds.

[0088]

[0089] As shown in Table 2, after synergistic optimization of microvolume extraction, chromatography, mass spectrometry, and classification quantification, the method of this application showed small fluctuations in the seven parallel blank spiked values ​​for five new pollutants in three categories, with a standard deviation of 0.025~0.045 ng / L. This indicates that the method has good precision and high repeatability in the pretreatment and instrument analysis processes. The detection limit is 100-200 pg / L lower, and the quantification limit is 400-800 pg / L, which is a significant improvement over existing technologies. It achieves ultra-trace detection at the pg / L level. This high sensitivity is due to the 100-fold one-step concentration of microvolume extraction, the efficient separation of phenyl column and rapid gradient, the maximization of ionization efficiency of the best mass spectrometry parameters, and the error correction of the classification quantification strategy. It can effectively meet the needs of early screening and risk warning of new pollutants in environmental water samples.

[0090] The measured values ​​of actual water samples, laboratory blanks, and spiked actual water samples, as well as the spiked recovery rate and relative standard deviation of actual samples, are shown in Table 3.

[0091] Table 3. Laboratory blank, actual water sample and spiked values ​​of the target compound, spiked recovery rate and relative standard deviation of the actual sample.

[0092]

[0093] Note: ND means Not Detected.

[0094] As shown in Table 3, none of the five novel pollutants in the three categories were detected in the blank sample, indicating that there was no exogenous pollution during the experiment, meeting the blank requirements for ultra-trace detection. The average recoveries of six parallel determinations of actual water samples with low (0.4 ng / L), medium (2.0 ng / L), and high (20.0 ng / L, 25 ng / L) concentrations were between 100-125%, 80.0-95.0%, and 83.5-104%, respectively, with relative standard deviations between 0-14%, 3.0-8.6%, and 1.0-16%, respectively. These values ​​are far below the standard limits, indicating that the method has good precision and stable repeatability. It can efficiently adapt to the simultaneous detection needs of multiple novel pollutants in various water bodies, including surface water, groundwater, domestic sewage, and industrial wastewater, demonstrating excellent accuracy and applicability.

[0095] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid and accurate method for simultaneously determining the content of multiple new pollutants in water, characterized in that, Includes the following steps: S1. Add the compound salt and water sample containing multiple new pollutants to a narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle at a ratio of 50 g / L. Shake well to form an unsaturated solution. The compound salt is sodium chloride and magnesium sulfate, and the mass ratio of sodium chloride to magnesium sulfate is 4:

1. The multiple new pollutants are organic ultraviolet absorbers UV-320 and UV-327, organophosphate compounds TXP and TDBPP, and tributyltin compounds TBTs. S2. Add UV320-D4 internal standard solution to a narrow-mouthed, long-necked, pear-shaped, flat-bottomed flask, shake well again, and then add an organic extractant, wherein the organic extractant is n-hexane and ethyl acetate, and the volume ratio of n-hexane to ethyl acetate is 9:

1. S3. Place the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle in a vortex mixing extraction device for high-frequency extraction and let it stand. Add pure water dropwise so that the liquid levels of the aqueous phase and the organic phase are located at the narrow neck of the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle. S4. Transfer the upper organic phase from the narrow-mouthed, long-necked, pear-shaped, flat-bottomed bottle, centrifuge, and take the supernatant for ESI-HPLC-MS / MS-MRM analysis. The high-performance liquid chromatography conditions are as follows: chromatographic column is a phenyl column, column temperature is 35℃, mobile phase is 0.1% formic acid 1mmol / L ammonium formate aqueous solution / 0.1% formic acid methanol solution, flow rate is 0.5mL / min, injection volume is 10μL, and gradient elution is performed. The gradient elution program is as follows: from 0 to 0.2 min, the mobile phase 0.1% formic acid methanol solution linearly increases from 20% to 99%, maintains it for 7 min, then decreases to 20% and maintains it for 10 min. The mass spectrometry conditions were: spray voltage 5500V, curtain gas 30psi, ion source temperature 600℃, and nebulizing gas and nebulization auxiliary gas both 50psi.

2. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 1, characterized in that, In the ESI-HPLC-MS / MS-MRM analysis and detection: Quantitative calculation by category: UV-320 and UV-327 were quantified using the internal standard method with UV-320-D4 as the internal standard, while TXP, TDBPP, and TBTs were quantified using the external standard method. The external standard method used a matrix-matched standard curve, and the blank matrix was prepared by removing the target analytes from the water sample after it was processed by a C18 solid phase extraction column.

3. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 2, characterized in that, The high performance liquid chromatography conditions also include a column temperature gradient program that is executed synchronously with the gradient elution program. The column temperature gradient program is that the column temperature is linearly increased from 35°C to 45°C from 0 to 0.2 min and maintained for 7 min, and then linearly decreased to 35°C from 7 to 10 min.

4. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 1, characterized in that, The parameters of the MRM are: UV-320: precursor ion 324.2 Da, quantitative daughter ion 268.3 Da, qualitative daughter ion 212.3 Da, cone voltage 120 V, collision voltages 31.73 V and 38.25 V respectively; UV-327: precursor ion 358.4 Da, quantitative daughter ion 302.0 Da, qualitative daughter ion 246.3 Da, cone voltage 124 V, collision voltages 32.11 V and 41.13 V respectively; TXP: precursor ion 411.2 Da, quantitative daughter ion 194.0 Da, qualitative daughter ion 179.2 Da, cone voltage 240 V, collision voltages 41.0 V and 54.0 V respectively; TDBPP: precursor ion 698.6 Da, quantitative daughter ion 299.0 Da, qualitative daughter ion 99.0 Da, cone voltage 50 V, collision voltages 23.0 V and 51.0 V respectively; TBTs: precursor ion 291.0 Da, quantitative daughter ion 179.0 Da, qualitative daughter ion 235.2 Da, cone voltage 30 V, collision voltages 18.07 V and 12.08 V respectively; UV-320-D4: Parent ion 328.3 Da, Quantitative daughter ion 272.2 Da, Cone voltage 160 V, Collision voltage 33.01 V.

5. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 1, characterized in that, In step S1, the sodium chloride and magnesium sulfate are mixed and dried at 500°C for 2 hours, then sealed and cooled for later use.

6. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 1, characterized in that, In step S2, the volume ratio of organic extractant to water sample is 1:

100.

7. The method for rapidly and accurately determining the content of multiple new pollutants in water simultaneously according to claim 1, characterized in that, In step S3, the vortex frequency of the vortex mixing extraction device is 1500 r / min, the extraction time is 10 min, and the settling time is 5 min.

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