Non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry

Abstract

The present invention relates to the field of pollutant detection, and in particular to a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry, comprising: completing the collection of raw sewage samples; completing the pretreatment of a plurality of test samples; completing the pretreatment of quality control samples; obtaining total ion chromatograms of test samples and total ion chromatograms of quality control samples; performing data processing and identifying pollutant information based on database matching and secondary mass spectrometry matching; performing information comparison according to a pollutant database to determine conventional pollutants or new pollutants; determining new pollutants based on metabolite response values ​​and peak time information, wherein new pollutants include persistent organic pollutants, antibiotics and endocrine disruptors; judging whether the new pollutant exists in a single test based on the number of test samples with the new pollutant, thereby improving the screening accuracy of new pollutants in sewage.

Description

Technical Field

[0001] The present invention relates to the field of pollutant detection, and in particular to a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry. Background Art

[0002] High-throughput screening technology is a technology that can quickly and efficiently detect and analyze a large number of samples based on biological, chemical, physical and other means. High-throughput non-targeted screening can be achieved through chromatography-mass spectrometry technology to identify the chemical structure of pollutants.

[0003] Chinese patent publication number: CN107179366B, discloses a high-throughput non-targeted screening and identification method for chlorinated organic pollutants in sediments, wherein the sediment sample is first pre-treated to obtain a target detection sample, which is a liquid; then the target detection sample is tested on a machine, and a high-performance liquid chromatography is set Time-of-flight mass spectrometry conditions, and finally, based on the primary mass spectrometry and secondary mass spectrometry information of each substance, a non-targeted screening analysis method is used to determine the type of substance. It uses high-throughput screening technology to identify and identify a single type of pollutant in a specific matrix. Limited by the type and number of pollutants in the standard sample library, the number of pollutants screened and identified is relatively small, and the sample pretreatment is relatively complex, so the accuracy is low when dealing with the screening of new pollutants in sewage. Summary of the invention

[0004] To this end, the present invention provides a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry, so as to overcome the problems in the prior art of low accuracy in screening new pollutants in sewage due to the limitations of the types and quantities of pollutants in the standard sample spectral library, the relatively small number of pollutants screened and identified, and the relatively complex sample pretreatment.

[0005] To achieve the above object, the present invention provides a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry, comprising:

[0006] Determine a number of points with the same flow rate in the preset water area and determine a number of collection points at equal intervals vertically downward from each point, and complete the collection of raw sewage samples based on each collection point;

[0007] Complete the preprocessing of several test samples;

[0008] Complete the pre-processing of quality control samples;

[0009] Chromatographic conditions and mass spectrometry conditions are set, and each test sample and the components separated and discharged by the chromatograph are respectively fed into the mass spectrometer, and the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample are obtained based on the negative ion continuous scanning of the mass spectrometer;

[0010] Data processing was performed on the total ion chromatograms of the test samples and the total ion chromatograms of the quality control samples, and the information of the pollutants was identified based on database matching and secondary mass spectrum matching;

[0011] Compare information with pollutant database to determine conventional pollutants or new pollutants;

[0012] Identify new pollutants based on metabolite response values ​​and peak time information, including persistent organic pollutants, antibiotics and endocrine disruptors;

[0013] For a single new pollutant, whether the new pollutant exists in a single test is determined based on the number of test samples containing the new pollutant.

[0014] Furthermore, the preprocessing process of the detection sample includes:

[0015] Mix the raw sewage samples collected from various collection points;

[0016] Transfer raw sewage samples into glass centrifuge tubes;

[0017] Mix 60% acetonitrile aqueous solution with the raw sewage sample;

[0018] The mixed liquid sample is subjected to low temperature ultrasonic extraction;

[0019] The filtrate is obtained by filtering the liquid sample after low-temperature ultrasonic extraction using a hydrophobic PTFE filter membrane;

[0020] The filtrate was transferred to a brown injection bottle to complete the pretreatment of the test sample.

[0021] Furthermore, the process of identifying the pollutant information includes:

[0022] The total ion chromatograms of the test samples and the total ion chromatograms of the quality control samples were subjected to baseline filtering, peak identification, integration, retention time correction and peak alignment analysis respectively;

[0023] Obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information;

[0024] Perform characteristic peak comparison and identification, match the MS mass spectrum information and MS / MS mass spectrum information with the database, wherein the MS mass error is set to less than 10ppm;

[0025] Contaminants were identified based on the MS / MS matching scores.

[0026] Furthermore, for a single new pollutant, it is determined whether the new pollutant exists in a single test based on the number of test samples containing the new pollutant, wherein:

[0027] If the number of test samples with new pollutants is zero, it is determined that there are no new pollutants in the single test, and the result of the single test is output;

[0028] If the number of test samples with new pollutants is one, it is determined that new pollutants exist in a single test, and the new pollutant test is repeated after increasing the number of test samples;

[0029] If the number of test samples containing new pollutants is greater than one, it is determined that new pollutants exist in a single test and whether the single test meets the preset standard is determined based on the pollutant test fluctuation evaluation value.

[0030] Furthermore, the process of determining whether a single test meets the preset standard according to the pollutant detection fluctuation evaluation value includes:

[0031] The fluctuation evaluation value is compared with the first preset evaluation value threshold and the second preset evaluation value threshold respectively,

[0032] If the fluctuation evaluation value is less than the first preset evaluation value threshold, it is determined that the single test meets the preset standard and the result of the single test is output;

[0033] If the fluctuation evaluation value is greater than or equal to the first preset evaluation value threshold and less than the second preset evaluation value threshold, the reason why the single detection does not meet the preset standard is determined based on the measured maximum concentration difference of the single new pollutant;

[0034] If the fluctuation evaluation value is greater than or equal to the second preset evaluation value threshold, it is determined that the single test does not meet the preset standard and the mixing ratio of 60% acetonitrile aqueous solution and raw sewage sample in the next batch of test samples is reduced according to the difference between the fluctuation evaluation value and the second preset evaluation value threshold;

[0035] The pollutant detection fluctuation evaluation value is determined based on the variance value of detecting a single new pollutant.

[0036] Furthermore, the reduction in the mixing ratio of the 60% acetonitrile aqueous solution and the raw sewage sample is positively correlated with the fluctuation evaluation difference, wherein the fluctuation evaluation difference is the difference between the fluctuation evaluation value and the second preset evaluation value threshold.

[0037] Furthermore, the reasons why a single test does not meet the preset standard are determined based on the maximum concentration difference of a single new pollutant measured, including:

[0038] If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the duration of low-temperature ultrasonic extraction does not meet the standard, and the duration of low-temperature ultrasonic extraction of the next batch of test samples is increased;

[0039] If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the mass spectrometry scanning range does not meet the standard, and the mass spectrometry scanning range of the next batch of test samples is increased.

[0040] Furthermore, the chromatographic conditions include: chromatographic column: ACQUITY UPLC BEH C18 column, 2.1 mm×100 mm, 1.7 um, Waters, Ireland; mobile phase A: acetonitrile; mobile phase B: formic acid aqueous solution; flow rate: 0.3 mL / min; injection volume: 10 uL; column temperature: 40°C.

[0041] Furthermore, the mass spectrometry conditions include: scanning range: 50m / z-1500m / z; cone voltage: 30.0V; sheath gas flow rate: 40arb; auxiliary gas flow rate: 10arb; heating temperature: 400°C; capillary temperature: 320°C; spray voltage: -2800V; S-lens voltage: 50V; collision energy: 70000; Full MS resolution: 70000; MS2 resolution: 17500.

[0042] Furthermore, the data processing of the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample is completed by MassLynx software.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention simplifies the sample pretreatment process to the greatest extent, the pretreatment method is simply optimized, the accuracy and breadth of detection are increased, and the sample is directly fixed to volume after being diluted with a certain proportion of organic solvent, and then filtered into a sampling vial using a hydrophobic PTFE filter membrane with a pore size of 0.22 um, thereby minimizing the loss of pollutants in the sample pretreatment extraction process.

[0044] Furthermore, the present invention adopts MassLynx mass spectrometry software, which can be used to collect, analyze, extract peaks, align, and identify standard mass and accurate mass numbers as well as MS and MS / MS data, and ultimately obtain a data matrix containing retention time, peak area, mass-to-charge ratio, and identification information, to clarify the type and category of pollutants, and combine with comparative analysis of quality control samples to screen out new pollutants in sewage. Sample screening covers multiple types of new pollutants such as persistent organic pollutants, endocrine disruptors, and antibiotics.

[0045] Furthermore, the present invention sets up a number of test samples and quality control samples, wherein the quality control sample is a blank control group, so as to combine the data processing of the control experimental samples to carry out bioinformatics analysis, improve the analysis accuracy, and ensure the accuracy of high-throughput screening.

[0046] Furthermore, the present invention determines whether a single pollutant is detected in a single test through the number of test samples containing new pollutants, optimizes the number of test samples, and determines the cause of poor detection when it is determined that the stability of the detection is poor, thereby dynamically optimizing the detection method accordingly and improving the identification accuracy of new pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a flow chart of a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to an embodiment of the present invention;

[0048] Figure 2 A flow chart of a pre-processing process for detecting a sample according to an embodiment of the present invention;

[0049] Figure 3 This is a flow chart of determining whether a new pollutant exists in a single test based on the number of test samples containing new pollutants according to an embodiment of the present invention;

[0050] Figure 4 This is a flow chart of an embodiment of the present invention for determining whether a single detection meets a preset standard according to a pollutant detection fluctuation evaluation value;

[0051] Figure 5 It is a total ion chromatogram of a sample detected in an embodiment of the present invention;

[0052] Figure 6 This is the total ion chromatogram of the quality control sample of the embodiment of the present invention. DETAILED DESCRIPTION

[0053] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] Those skilled in the art will appreciate that the method of the present invention for determining a single parameter mentioned above may be to select the value with the highest proportion as the preset standard parameter based on data distribution, use weighted summation to use the obtained value as the preset standard parameter, substitute each historical data into a specific formula and use the value obtained by using the formula as the preset standard parameter or other selection methods, as long as the system of the present invention can clearly define different specific situations in the single determination process through the obtained values.

[0055] It should be pointed out that the data in this embodiment are all results obtained through preliminary experimental verification by the method described in the present invention before conducting this test. The preset values ​​can be adjusted according to the specific usage, as long as the method described in the present invention can clearly define the different specific situations in the single determination process through the obtained numerical values.

[0056] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0057] See also Figure 1 , Figure 2 , Figure 3 as well as Figure 4 As shown, they are respectively a flow chart of a method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to an embodiment of the present invention; a flow chart of a pretreatment process of a detection sample according to an embodiment of the present invention; a flow chart of determining whether a new pollutant exists in a single detection based on the number of detection samples with new pollutants according to an embodiment of the present invention; a flow chart of determining whether a single detection meets a preset standard according to a pollutant detection fluctuation evaluation value according to an embodiment of the present invention; Figure 5 It is a total ion chromatogram of a sample detected in an embodiment of the present invention; Figure 6 This is the total ion chromatogram of the quality control sample of the embodiment of the present invention.

[0058] The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry in an embodiment of the present invention comprises:

[0059] Step S1, determining a number of points with the same flow rate in a preset water area and determining a number of collection points at equal intervals vertically downward from each point, and completing the collection of raw sewage samples based on each collection point;

[0060] Step S2, completing preprocessing of a number of test samples;

[0061] Step S3, completing the preprocessing of the quality control sample;

[0062] Step S4, setting chromatographic conditions and mass spectrometry conditions, respectively feeding each test sample and the components separated and discharged by the chromatograph into the mass spectrometer, and obtaining a total ion chromatogram of the test sample and a total ion chromatogram of the quality control sample based on the negative ion continuous scanning of the mass spectrometer, wherein the mass spectrometer is continuously scanned for data acquisition, and a mass spectrum is obtained for each scan, and all ion intensities in each mass spectrum are added to obtain a total ion current intensity, and the total ion chromatogram is drawn with time as the horizontal axis and the total ion intensity as the vertical axis;

[0063] Step S5, respectively processing the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample and identifying the information of the pollutants based on database matching and secondary mass spectrum matching;

[0064] Step S6, determining conventional pollutants or new pollutants by comparing information with a pollutant database, wherein the pollutant database contains information on conventional pollutants;

[0065] Step S7, combined with the data processing of quality control samples, bioinformatics analysis is carried out to analyze and mine the changes and differential calibration of metabolites in crops under BPs exposure, and new pollutants are determined through metabolite response values ​​and peak time information, where new pollutants include persistent organic pollutants, antibiotics and endocrine disruptors;

[0066] Step S8, for a single new pollutant, determining whether the new pollutant exists in a single test based on the number of test samples containing the new pollutant.

[0067] Specifically, the preprocessing process of the test sample includes:

[0068] Step S201, mixing the raw sewage samples collected at each collection point;

[0069] Step S202, transferring the raw sewage sample into a glass centrifuge tube;

[0070] Step S203, mixing 60% acetonitrile aqueous solution with the original sewage sample at a mixing mass ratio of 3:2;

[0071] Step S204, performing low-temperature ultrasonic extraction on the mixed liquid sample, the extraction time is 15 minutes;

[0072] Step S205, using a hydrophobic PTFE filter membrane to filter the liquid sample after low-temperature ultrasonic extraction to obtain a filtrate, wherein the filtration pore size of the PTFE filter membrane is 0.22 um;

[0073] Step S206, transferring the filtrate to a brown sample injection bottle to complete the pretreatment of the test sample.

[0074] Specifically, the pretreatment process of quality control samples includes:

[0075] Step S301, transferring a pure water sample into a glass centrifuge tube;

[0076] Step S302, mixing a 60% acetonitrile aqueous solution with a pure water sample at a mixing mass ratio of 3:2;

[0077] Step S303, performing low-temperature ultrasonic extraction on the mixed liquid sample, and the extraction time is 15 minutes;

[0078] Step S304, using a hydrophobic PTFE filter membrane to filter the liquid sample after low-temperature ultrasonic extraction to obtain a filtrate;

[0079] Step S305, transferring the filtrate to a brown injection bottle to complete the pretreatment of the quality control sample.

[0080] Specifically, step S5 includes:

[0081] S501, performing baseline filtering, peak identification, integration, retention time correction and peak alignment analysis on the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample respectively;

[0082] S502, obtaining a data matrix containing information such as retention time, mass-to-charge ratio and peak intensity;

[0083] S503, performing characteristic peak comparison and identification, matching the MS mass spectrum information and the MS / MS mass spectrum information with the database, wherein the MS mass error is set to be less than 10 ppm;

[0084] S504, scoring the contaminants according to the secondary mass spectrum matching.

[0085] Specifically, for a single new pollutant, whether a new pollutant exists in a single test is determined based on the number of test samples containing the new pollutant, wherein:

[0086] If the number of test samples with new pollutants is zero, it is determined that there are no new pollutants in the single test, and the result of the single test is output;

[0087] If the number of test samples with new pollutants is one, it is determined that new pollutants exist in a single test, and the new pollutant test is repeated after increasing the number of test samples;

[0088] If the number of test samples containing new pollutants is greater than one, it is determined that new pollutants exist in a single test and whether the single test meets the preset standard is determined based on the pollutant test fluctuation evaluation value.

[0089] Specifically, the pollutant detection fluctuation evaluation value is determined based on the variance value of detecting a single new pollutant.

[0090] Specifically, the process of determining whether a single test meets the preset standard based on the pollutant detection fluctuation evaluation value includes:

[0091] The fluctuation evaluation value is compared with the first preset evaluation value threshold and the second preset evaluation value threshold respectively,

[0092] If the fluctuation evaluation value is less than the first preset evaluation value threshold, it is determined that the single test meets the preset standard and the result of the single test is output;

[0093] If the fluctuation evaluation value is greater than or equal to the first preset evaluation value threshold and less than the second preset evaluation value threshold, the reason why the single detection does not meet the preset standard is determined based on the measured maximum concentration difference of the single new pollutant;

[0094] If the fluctuation evaluation value is greater than or equal to the second preset evaluation value threshold, it is determined that the single test does not meet the preset standard and the mixing ratio of 60% acetonitrile aqueous solution and raw sewage sample in the next batch of test samples is reduced according to the difference between the fluctuation evaluation value and the second preset evaluation value threshold;

[0095] Among them, the first preset evaluation value threshold is set to 0.27, and the second preset evaluation value threshold is set to 0.64.

[0096] Specifically, the reduction in the mixing ratio of the 60% acetonitrile aqueous solution and the original sewage sample is positively correlated with the fluctuation evaluation difference, wherein the fluctuation evaluation difference is the difference between the fluctuation evaluation value and the second preset evaluation value threshold, wherein the positive correlation is, for example, a linear positive correlation, and the slope of the linear correlation is not specifically limited, as long as the greater the fluctuation evaluation difference, the greater the reduction in the mixing ratio.

[0097] Specifically, the reasons why a single test does not meet the preset standard based on the maximum concentration difference of a single new pollutant measured include:

[0098] If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the duration of low-temperature ultrasonic extraction does not meet the standard, and the duration of low-temperature ultrasonic extraction of the next batch of test samples is increased;

[0099] If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the mass spectrometry scanning range does not meet the standard, and the mass spectrometry scanning range of the next batch of test samples is increased;

[0100] The increase in the duration of low-temperature ultrasonic extraction of the next batch of test samples is positively correlated with the duration difference, wherein the duration difference is the difference between the preset concentration difference threshold and the maximum concentration difference;

[0101] Among them, the increase in the mass spectrometry scanning range of the next batch of test samples is positively correlated with the scanning difference, wherein the scanning difference is the difference between the maximum concentration difference and the preset concentration difference threshold. It should be emphasized that the mass spectrometry scanning range of the increased test samples does not exceed 1500m / z;

[0102] Among them, the maximum concentration difference is the difference between the highest concentration and the lowest concentration of a single new pollutant in different test samples.

[0103] Among them, the preset concentration difference threshold is set to 2.24 mg / L.

[0104] Specifically, the chromatographic conditions include: chromatographic column: ACQUITY UPLC BEH C18 column, 2.1 mm×100 mm, 1.7 um, Waters, Ireland; mobile phase A: acetonitrile; mobile phase B: formic acid aqueous solution; flow rate: 0.3 mL / min; injection volume: 10 uL; column temperature: 40°C.

[0105] Specifically, the mass spectrometry conditions include: scanning range: 50m / z-1500m / z; cone voltage: 30.0V; sheath gas flow rate: 40arb; auxiliary gas flow rate: 10arb; heating temperature: 400°C; capillary temperature: 320°C; spray voltage: -2800V; S-lens voltage: 50V; collision energy: 70000; Full MS resolution: 70000; MS2 resolution: 17500.

[0106] Specifically, the data processing of the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample is completed by MassLynx software.

[0107] Based on the method of non-targeted screening of new pollutants in rural sewage by high-resolution mass spectrometry, a total of 86 perfluorinated compounds, 91 nafils, 20 lipid hormones, 23 sex hormones, 138 antibiotics, 42 cephalosporins, 192 pesticides and 41 glucocorticoids were screened in rural sewage, as shown in Table 1 for details.

[0108] Table 1 Screening results of some pollutants

[0109] Serial number Element Mass-to-charge ratio m / z 1 * Erythromycin 734.462 2 * Oxymetholone 333.2381 3 *cadaverine (1,5-diaminopentane) 103.1289 4 Methyltestosterone 303.2371 5 *2-(Perfluorohexyl)ethyl methacrylate 433.0431 6 *Avermectin 873.5054 7 *Acephate 184.013 8 *Acetamiprid 223.0665 9 *Alclometasone dipropionate 521.239 10 * Ancinonide 503.2493 11 Arachidonic acid 305.2443 12 *Atrazine 216.1086 13 *Triazolidine 438.1862 14 Bentazone 241.0664 15 *Sildenafil benzyl 551.2485 16 * Betamethasone 393.215 17 *Bolatestosterone 317.2525 18 * Boldenone 287.1971 19 *Carbofuran 222.1209 20 Cefpirome 515.1191 21 *Cefuroxime 384.0431 22 * Chlorimuron-methyl 415.0481 23 *Guidenafil 555.312 24 *Ciprofloxacin 332.1314 25 * Clothianidin 250.0187 26 Daminozide 161.0966 27 *Demethylpiperazinesildenafil sulfonic acid 393.113 28 *Desethylcarbadenafil 425.2367 29 Fluorometholone acetate 243.0988 30 * Dimethyl erythridine 467.276 31 *Dinotefuran 204.1169 32 * Epitestosterone 289.2123 33 Estriol 289.1763 34 * Ethinyl estradiol 297.1812 35 * Fenbuterol 208.1294 36 * Cypermethrin 350.1823 37 * Fleroxacin 370.1423 38 *Fluocin 249.057 39 Fluorochloridone 312.0137 40 Fluoxymesterone 337.2115 41 *Gentamicin 478.3265 42 * Glufosinate 199.0823 43 *Perfluorodecane sulfonic acid 600.9477 44 Hydrocortisone 363.21 45 * Hydroxychlordenafil 391.1553 46 * Icaridin 230.1698 47 Imidacloprid 257.0758 48 *Kanamycin 240.1383 49 *Latamoxef 521.1108 50 *Azone 282.2793 51 *Lecithin 758.562 52 *Linoleic acid 281.246 53 *pyraclostrobin 373.0763 54 * Cyfluthrin 415.0482 55 *Methandrostenol 301.2142 56 *Methoprene 311.2529 57 Methyl linoleate 295.2601 58 *Methocarb 166.0831 59 *Mometasone furoate 521.1407 60 * Nitrosdenafil 630.2264 61 *DEET 192.1444 62 Nandrolone Phenylpropionate 407.2549 63 * Nandrolone 275.2051 64 *N-Ethyl perfluorooctanesulfonamideethanol 572.0203 65 Nitrofurantoin 239.0479 66 * Capsaicin 294.2018 67 * Ofloxacin 362.1473 68 *Omethoate 214.0385 69 * Hydroxymethyltestosterone 319.2246 70 *Pefloxacin 334.1473 71 * Pendimethalin 282.1396 72 *Phoxim 299.0582 73 *Piconazole 401.2507 74 *Praziquantel 313.1979 75 Prednicarbate 489.2575 76 Prednisone 359.1808 77 * Progesterone 315.2281 78 *Promethazine 242.1472 79 *Propamocarb 189.16 80 Carbosulfan 210.1065 81 * Propylthiophene hydroxybutyric acid sildenafil 535.2192 82 * Benthiocarb 252.1354 83 *Roxithromycin 837.5258 84 *Allethrin 303.1895 85 Sildenafil N-oxide 491.2135 86 *Silver Perfluorooctanoate 520.873 87 Methylprednisolone acetate 417.2318 88 * Spermine 203.2173 89 Spirotetramat 374.1942 90 Sulfadiazine 315.091 91 *Sulfadimethoxine (sulfadimethoxine) 279.0902 92 Sulfluramid 527.9932 93 *Tetrabutylphosphonium2H,2H-Perfluorodecanoate 737.2314 94 * Tobramycin 468.269 95 *Triamcinolone acetonide acetate 477.2294 96 * Trimethoprim 291.1526 97 *Tryptamine 161.1053 98 *Acetylvardenafil 467.2743 99 * Vardenafil 489.2199 100 * Estradiol 273.1839

[0110] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry. It is characterized in that include: Determine a number of points with the same flow rate in the preset water area and determine a number of collection points at equal intervals vertically downward from each point, and complete the collection of raw sewage samples based on each collection point; Complete pre-processing of several test samples; Complete the pre-processing of quality control samples; Chromatographic conditions and mass spectrometry conditions are set, and each test sample and the components separated and discharged by the chromatograph are respectively fed into the mass spectrometer, and the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample are obtained based on the negative ion continuous scanning of the mass spectrometer; Data processing was performed on the total ion chromatograms of the test samples and the total ion chromatograms of the quality control samples, and the information of the pollutants was identified based on database matching and secondary mass spectrum matching; Compare information with pollutant database to determine conventional pollutants or new pollutants; Identify new pollutants based on metabolite response values ​​and peak time information, including persistent organic pollutants, antibiotics and endocrine disruptors; For a single new pollutant, determine whether the new pollutant exists in a single test based on the number of test samples containing the new pollutant; The pretreatment process of the test sample includes: mixing the raw sewage samples collected at each collection point; transferring the raw sewage samples into a glass centrifuge tube; mixing a 60% acetonitrile aqueous solution with the raw sewage sample; performing low-temperature ultrasonic extraction on the mixed liquid sample; filtering the liquid sample after low-temperature ultrasonic extraction using a hydrophobic PTFE filter membrane to obtain a filtrate; transferring the filtrate to a brown injection bottle to complete the pretreatment of the test sample; For a single new pollutant, determine whether there is a new pollutant in a single test based on the number of test samples with the new pollutant, wherein if the number of test samples with the new pollutant is zero, determine that there is no new pollutant in the single test, and output the result of the single test; if the number of test samples with the new pollutant is one, determine that there is a new pollutant in the single test, and increase the number of test samples before retesting the new pollutant; if the number of test samples with the new pollutant is greater than one, determine that there is a new pollutant in the single test, and determine whether the single test meets the preset standard based on the pollutant test fluctuation evaluation value; The pollutant detection fluctuation evaluation value is determined based on the variance value of a single new pollutant detected; The process of determining whether a single detection meets the preset standard based on the pollutant detection fluctuation evaluation value includes: comparing the fluctuation evaluation value with the first preset evaluation value threshold and the second preset evaluation value threshold respectively; if the fluctuation evaluation value is less than the first preset evaluation value threshold, then determining that the single detection meets the preset standard and outputting the result of the single detection; if the fluctuation evaluation value is greater than or equal to the first preset evaluation value threshold and less than the second preset evaluation value threshold, determining the reason why the single detection does not meet the preset standard based on the measured maximum concentration difference of the single new pollutant; if the fluctuation evaluation value is greater than or equal to the second preset evaluation value threshold, then determining that the single detection does not meet the preset standard and reducing the mixing ratio of 60% acetonitrile aqueous solution and original sewage sample in the next batch of detection samples according to the difference between the fluctuation evaluation value and the second preset evaluation value threshold.

2. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 1, It is characterized in that The process of conducting the pollutant information identification includes: The total ion chromatograms of the test samples and the total ion chromatograms of the quality control samples were subjected to baseline filtering, peak identification, integration, retention time correction and peak alignment analysis respectively; Obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity information; Perform characteristic peak comparison and identification, match the MS mass spectrum information and MS / MS mass spectrum information with the database, wherein the MS mass error is set to less than 10ppm; Contaminants were identified based on the MS / MS matching scores.

3. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 1, It is characterized in that The reduction in the mixing ratio of the 60% acetonitrile aqueous solution and the raw sewage sample is positively correlated with the fluctuation evaluation difference, wherein the fluctuation evaluation difference is the difference between the fluctuation evaluation value and the second preset evaluation value threshold.

4. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 3, It is characterized in that The reasons why a single test does not meet the preset standard based on the maximum concentration difference of a single new pollutant measured include: If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the duration of low-temperature ultrasonic extraction does not meet the standard, and the duration of low-temperature ultrasonic extraction of the next batch of test samples is increased; If the maximum concentration difference is less than the preset concentration difference threshold, it is determined that the reason for not meeting the preset standard is that the mass spectrometry scanning range does not meet the standard, and the mass spectrometry scanning range of the next batch of test samples is increased.

5. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 1, It is characterized in that The chromatographic conditions include: chromatographic column: ACQUITY UPLC BEH C18 column, 2.1 mm×100 mm, 1.7 um, Waters, Ireland; mobile phase A: acetonitrile; mobile phase B: formic acid aqueous solution; flow rate: 0.3 mL / min; injection volume: 10 uL; column temperature: 40°C.

6. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 1, It is characterized in that The mass spectrometry conditions include: scanning range: 50m / z-1500m / z; cone voltage: 30.0V; sheath gas flow rate: 40arb; auxiliary gas flow rate: 10arb; heating temperature: 400°C; capillary temperature: 320°C; spray voltage: -2800V; S-lens voltage: 50V; collision energy: 70000; Full MS resolution: 70000; MS2 resolution: 17500.

7. The method for non-targeted screening of new pollutants in rural sewage based on high-resolution mass spectrometry according to claim 1, It is characterized in that The data processing of the total ion chromatogram of the test sample and the total ion chromatogram of the quality control sample is completed by MassLynx software.

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