A method for non-targeted screening of perfluoro- and polyfluoroether carboxylic acids in soil and sediments

Abstract

The application discloses a method for non-target screening of perfluoro and polyfluoro ether carboxylic acids in soil and sediments, which comprises the following steps: pretreatment of soil samples by using a modified solid-liquid extraction method; detection of the samples by using an ultra-high performance liquid chromatography tandem high-resolution mass spectrometry system; conversion of the original data format; extraction of the sample primary chromatographic peak information and secondary mass spectrometry fragment information by using R language; sample peak source intraparticle cracking labeling and sample peak isotopic pattern recognition labeling by using a Matlab algorithm; and spectrum analysis and structure inference according to the secondary mass spectrometry fragment information of the compounds, and reference to relevant literatures and databases. Compared with a traditional perfluorinated compound non-target identification method, the method has higher recovery rate and sensitivity for perfluoro and polyfluoro ether carboxylic acids, is compatible with traditional perfluorinated compounds, can identify more types of new fluorine-containing substitutes, and has wide practicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, and particularly relates to a method for non-targeted screening of perfluoro and polyfluoro ether carboxylic acids in soil and sediment. BACKGROUND

[0002] Perfluoro and polyfluoro alkyl compounds (PFAS) are a class of synthetic organic compounds in which all or part of the hydrogen atoms on the carbon skeleton are replaced by fluorine atoms. They are widely used in many aspects of human life, such as textiles, outdoor clothing, non-stick pots, food packaging, furniture coatings, aviation hydraulic oil, fuel cells, etc. However, these compounds are difficult to hydrolyze, photolyze and biodegrade, which leads to their widespread detection in global environmental media, organisms and human bodies. In addition, animal model studies have found that PFAS has hepatotoxicity, neurotoxicity, reproductive and developmental toxicity, immunotoxicity, and endocrine disrupting effects, etc. Given the environmental persistence, bioaccumulation and multiple toxicity effects of PFAS, it poses potential harm to the global ecological environment and human health. In the past decade, the international community has launched corresponding management actions against PFAS. However, as the industrial application of perfluorinated compounds becomes more widespread, more and more perfluorinated compound substitutes have entered industrial production.

[0003] Currently, the research on PFAS mainly focuses on traditional compounds such as PFOS and PFOA. The research on new PFAS substitutes is still insufficient, especially after the ban of PFOA, the hexafluoropropylene oxide dimer and trimer have become important substitutes in the industry. The similar compounds are numerous and complex in structure (different chain lengths, uncertain ether bond positions, containing branched chains, halogen heteroatoms, etc.), and it is urgent to develop non-targeted identification methods for such unknown compounds.

[0004] For complex matrices such as soil and sediment, due to their large fluctuations in acidity and alkalinity, complex composition and difficulty in pollutant enrichment, the target substances need to be extracted and purified before they can be detected. During sample pretreatment, the ether oxygen bond in perfluoro and polyfluoro ether carboxylic acids will have an electron-withdrawing effect on the carboxyl group in its structure, increasing its polarity, making it difficult to obtain satisfactory recovery rate using traditional liquid-liquid extraction methods (such as using methyl tert-butyl ether). Even under normal temperature conditions, common aprotic solvents (acetonitrile, acetone and dimethyl sulfoxide) will degrade ether-containing PFAS within a certain time, resulting in a decrease in recovery rate.

[0005] In addition, due to the thermal instability of the C-O ether bond, perfluoro and polyfluoro ether carboxylic acids are prone to C-O ether bond rupture, i.e. "in-source fragmentation", during mass spectrometry ionization at high temperature. In a high-resolution mass spectrometry system, the in-source fragmentation phenomenon weakens the signal of the original substance, resulting in low signal response in the mass spectrum. Traditional instrument analysis methods for perfluorinated compounds often fail to effectively identify such substances due to the neglect of the parent ion signal. In addition, the in-source fragmentation process produces a variety of in-source fragmentation product chromatographic peaks, i.e. interference chromatographic peaks, making it more difficult to effectively identify perfluorinated compound chromatographic peaks.

[0006] The structural complexity of perfluoro and polyfluoro ether carboxylic acids also lies in the presence of halogen heteroatom substitution. Due to the inability to a priori obtain the position and number of substituted atoms, it is difficult to identify them specifically. In addition, substances containing halogen elements will form corresponding isotope peaks, which will cause noise interference and increase the difficulty of identification and spectrum analysis if not labeled and identified. SUMMARY

[0007] In view of the above deficiencies in the prior art, the purpose of the present application is to provide a method for non-target screening of perfluoro and polyfluoro ether carboxylic acids in soil and sediments. This method innovates in sample pretreatment, instrument analysis, data processing, etc., and can quickly and efficiently identify perfluoro and polyfluoro ether carboxylic acids in soil and sediments.

[0008] To achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is:

[0009] A method for non-target screening of perfluoro and polyfluoro ether carboxylic acids in soil and sediments is provided, which comprises the following steps:

[0010] Step 1, using an improved solid-liquid extraction method to pretreat the soil or sediment sample;

[0011] Step 2, detecting the sample by ultra-high performance liquid chromatography tandem high-resolution mass spectrometry, collecting the FullScan information and ddMS2 secondary fragment information of the sample;

[0012] Step 3, using ProteoWizard software to convert the data source file into mzML format and mgf format;

[0013] Step 4, using R language to extract the primary chromatographic peak information and secondary mass spectrometry fragment information of the sample;

[0014] Step 5, using Matlab algorithm for sample peak in-source fragmentation labeling and sample peak isotope pattern identification labeling;

[0015] Step 6, according to the source cracking peak labeling information, isotopic pattern peak labeling information, compound secondary mass spectrum fragment information, deconvolution and structure inference are carried out to obtain the structure of perfluorinated and polyfluorinated ether carboxylic acid.

[0016] Further, the sample pretreatment method of step 1 is specifically as follows: taking a soil or sediment sample, adding potassium hydroxide methanol solution, vortexing, ice bath ultrasonic, oscillation, centrifugation, taking the supernatant, repeating extraction twice, combining the supernatants of the two times of extraction, nitrogen blowing to dryness, adding water to constant volume, and adjusting the pH to 7 with acetic acid solution; sequentially activating the solid phase extraction column with ammonia water methanol solution, methanol and water, and uniformly feeding the extract; eluting with acetic acid / ammonium acetate buffer, sequentially eluting with methanol and ammonia water methanol solution, nitrogen blowing the second eluent to dryness, constant volume with methanol aqueous solution, and detecting after centrifugation.

[0017] Further, in step 2, the secondary fragment information includes the retention time, accurate molecular weight and relative intensity value of the fragment.

[0018] Further, in step 2, the determination is carried out in negative ion mode using an ESI ion source.

[0019] Further, in step 2, 5mM ammonium acetate methanol solution and 5mM ammonium acetate aqueous solution are used as the mobile phase, and gradient elution is used for sampling.

[0020] Further, in step 4, the "patRoon" package of R language is used to extract the chromatographic peaks in the sample, specifically, the findFeaturesOpenMS function in the patRoon package is used to extract the peaks from the mzML data file, and the write.csv function is used to export the peak list data after recognition.

[0021] Further, in step 4, first save the mgf format file as a txt file, then use R language to filter out fragment peaks with a relative intensity of less than 10%, and retain at most 20 peak fragments with the largest intensity, and export the filtered information using the write.csv function.

[0022] Further, in step 5, according to the regularity of the m / z corresponding to a certain chromatographic peak appearing in the primary spectrum / secondary spectrum, it is judged that the chromatographic peak belongs to the original m / z or the source cracking product, and the core discrimination standard of the source cracking peak is whether it appears as a fragment in the secondary fragment spectrum of another larger m / z at the same retention time.

[0023] Further, the source cracking recognition algorithm specifically includes the following sub-steps:

[0024] Step 5-1, import the Raw raw data file, and use R language to extract the primary chromatographic peak data mz i, rt i and MS2 secondary spectrum data mz j , rt j , f j，k ; mz i represents the mass-to-charge ratio of the ion corresponding to the i th chromatographic peak, rt i represents the retention time of the i th chromatographic peak; mz j represents the mass-to-charge ratio of the j th parent ion, rt j represents the fragmentation time of the j th parent ion, f j，k represents the mass-to-charge ratio of the k th fragment after the secondary fragments generated by the j th parent ion are sorted in descending order of relative abundance;

[0025] Step 5-2, using matlab algorithm to traverse each primary chromatographic peak, find chromatographic peaks that meet the following conditions within a certain error range:

[0026] ΔRT = abs (rt i -rt j ) < 0.2 min

[0027] Δmz = abs (f j，k -mz i ) < 5 mDa

[0028] Δmz' = mz j -mz i > 1 Da

[0029] At the same retention time, the secondary fragment with the same mass-to-charge ratio as the chromatographic peak can be generated by the parent ion with a larger mass-to-charge ratio; in the code, ΔRT calculates the difference between the retention time of the primary chromatographic peak and the parent ion, and the error is required to be less than 0.2 min; Δmz calculates the difference between the mass-to-charge ratio of the primary chromatographic peak and the fragment of the parent ion, and the error is required to be less than 5 mDa; Δmz' calculates the difference between the mass-to-charge ratio of the parent ion and the mass-to-charge ratio of the corresponding parent ion of the primary chromatographic peak, and the difference between the mass-to-charge ratio of the parent ion and the mass-to-charge ratio of the primary chromatographic peak is required to be not less than 1 Da; if the primary chromatographic peak and the parent ion meet the above conditions, it is determined that the primary chromatographic peak is an in-source fragmentation fragment peak generated by the parent ion.

[0030] Further, in step 5, whether a certain chromatographic peak may contain halogen heteroatoms is judged according to the peak area ratio of the chromatographic peak to its corresponding suspected isotope chromatographic peak; taking chlorine element as an example, if the peak area of a certain chromatographic peak satisfies [M]-:[M+2]- = 3:1, the substance corresponding to the chromatographic peak may contain one chlorine atom.

[0031] The beneficial effects of the present application are:

[0032] (1) The soil sample pretreatment method developed by the present application adopts potassium hydroxide methanol solution as an extractant and a low-temperature ice bath extraction method, has good extraction effect, high recovery rate, reduces the degradation of ether PFAS in the solvent, the SPE solid-phase extraction process can remove the complex matrix in the soil and sediment as much as possible while enriching the target, reduces the matrix effect, and is suitable for new fluorine-containing compounds such as perfluoro and polyfluoro ether carboxylic acids in complex environmental media. The quantification limit of the method developed by the present application can reach 0.02-0.1 ng / g, has high sensitivity, the method recovery rate (n=5) is between 78.8% and 125.3%, and the relative standard deviation of the inter-day precision (n=5x5) is within 10%.

[0033] (2) The ultra-high performance liquid chromatography tandem high-resolution mass spectrometer instrument analysis method developed by the present application optimizes the ion source temperature, reduces the ion source temperature used for detecting traditional perfluorinated compounds from 400 DEG C to 150 DEG C, compared with the traditional method, the low-temperature condition reduces the cracking of ether PFAS in the ion source, is conducive to finding more perfluoro and polyfluoro ether carboxylic acids, and has a wide detection range.

[0034] (3) The source cracking identification method for perfluorinated compounds developed by the present application can efficiently mark the source cracking product peaks existing in the chromatographic peaks of the sample based on the cracking phenomenon in the mass spectrum, and find the corresponding parent ions, thereby significantly reducing the noise interference and structure inference difficulty.

[0035] (4) The isotope pattern marking algorithm developed by the present application can flexibly and efficiently screen the isotope chromatographic peaks in the sample and mark them, and improves the screening efficiency of perfluoro and polyfluoro ether carboxylic acids containing halogen heteroatoms. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a data format conversion flowchart in the present application;

[0037] Figure 2 It is a principle diagram of the source cracking identification algorithm in the present application;

[0038] Figure 3 It is a case diagram of the source cracking substances found in the present application. DETAILED DESCRIPTION

[0039] The specific embodiments of the present application are described below, so that those skilled in the art can understand the present application, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all inventions utilizing the concept of the present application are within the scope of protection.

[0040] Examples

[0041] A 1 g soil sample from an industrial contaminated area was placed in a 15 mL polypropylene centrifuge tube (n = 5), 10 mL of 10 mM potassium hydroxide methanol solution was added to the tube, vortexed and mixed, then ultrasonic extraction for 1 h in an ice bath, shaken for 12 h, centrifuged at 4400 rpm for 15 min to take the supernatant, repeated extraction twice, combined the supernatant of the two extractions, dried under nitrogen at 40 °C, added 10 mL of water to dissolve, and adjusted the pH to 7 with 0.2% (v / v) acetic acid solution. The Oasis-WAX solid phase extraction column (6 cc / 150 mg) (Waters, MA, USA) was activated with 8 mL of 0.5% (v / v) ammonia water methanol solution, 4 mL of methanol and 4 mL of water in turn, and the extract was uniformly loaded. Eluted with 4 mL of 5 mM acetic acid / ammonium acetate buffer (pH = 4.0), 4 mL of methanol, 4 mL of 0.5% (v / v) ammonia water methanol solution in turn, the second eluent was dried under nitrogen at 40 °C, and 200 μL of 1:1 (v / v) methanol water solution was added to constant volume, centrifuged and detected on the instrument.

[0042] Detection was performed using Vanquish Flex Ultra-High Performance Liquid Chromatograph ultra-high performance liquid chromatography tandem Orbitrap Exploris 120 (Thermo Scientific, USA) high resolution mass spectrometer, and secondary mass spectrum information was collected in data-dependent acquisition mode. The specific system condition parameters were as follows:

[0043] Chromatographic column: Poroshell 120EC-C18 (150 mm x 3 mm, 2.7 μm, Agilent, CA, USA)

[0044] Mobile phase: A: 5 mM ammonium acetate aqueous solution, B: 5 mM ammonium acetate methanol solution

[0045] Negative ion voltage: -3000 V

[0046] Sheath gas flow: 50 Arb

[0047] Auxiliary gas flow: 12.5 Arb

[0048] Sweep gas flow: 0 Arb

[0049] Ion transfer tube temperature: 150 °C

[0050] Atomizer temperature: 200 °C

[0051] The raw file data source file is converted into mzML format and mgf format using ProteoWizard software. The findFeaturesOpenMS function in the patRoon package of R language version 4.2.0 is used for peak extraction of the mzML data file, and the write.csv function is used to export the peak list data after identification. The mgf format file is saved as a txt file, and then the R language is used to filter out the fragment peaks with a relative intensity of less than 10%, and at most 20 peak fragments with the largest intensity are retained. The filtered information is exported using the write.csv function.

[0052] The in-source fragmentation peaks in the sample peak list are identified and labeled using Matlab code, and the isotopic pattern peaks are identified and labeled.

[0053] According to the in-source fragmentation peak labeling information, the isotopic pattern peak labeling information, and the suspected perfluorinated compound fragment information, the spectrum is solved and the structure is inferred to obtain the suspected structure of the perfluoro and polyfluoro ether carboxylic acid.

[0054] For example Figure 3 C8HClF 14 O4(m / z = 460.9262) substance, which produces C6ClF 12 O2 fragments and forms a 366.9395 chromatographic peak at the same retention time. During the in-source fragmentation labeling process, each chromatographic peak is traversed, and for the chromatographic peak with m / z1 = 366.9395, the retention time rt1 = 11.85 min, all secondary spectra in the range of 11.85 ± 0.2 min are searched, and it is found that the f 2,1 = 366.9403 fragment is contained in the secondary spectrum with m / z2 = 460.9262, rt2 = 11.86 min, and the two chromatographic peaks satisfy the conditions:

[0055] ΔRT = abs(rt1-rt2) = 0.01 min < 0.2 min

[0056] Δmz = abs(f 2，1 -mz1) = 0.8 mDa < 5 mDa

[0057] Δmz' = mz2-mz1 = 93.9859 Da > 1 Da

[0058] Therefore, the m / z1 chromatographic peak is identified as an in-source fragmentation peak by the above algorithm, and its parent ion mass is determined to be 460.9262.

[0059] In addition, after isotope identification and labeling, there is a chromatographic peak of m / z3=462.9241 at a retention time of 11.85, and the ratio of the peak area s of the chromatographic peak m / z2=460.9262 to the peak area s' of the chromatographic peak m / z3=462.9241 is:

[0060]

[0061] Therefore, it is speculated that the substance may contain 1 chlorine atom.

[0062] According to other MS2 mass spectrum fragment information and literature, database reference, the structure of the substance is determined, the interference of the impurity peak is reduced, and the identification efficiency is improved.

[0063] Based on the above, by applying the method of the present application, the rapid screening of perfluoro and polyfluoro ether carboxylic acids in soil and sediment samples can be realized without complex pretreatment methods, and the response signal of the compound in the mass spectrum is significantly improved by adjusting the mass spectrum parameters according to the properties of the compound. The in-source fragmentation identification algorithm proposed in the present application can quickly find new perfluoro and polyfluoro ether carboxylic acids existing in massive data. More importantly, this algorithm is not only effective for perfluorinated compounds, but also can be used for screening other substances that are prone to in-source fragmentation in mass spectrometry. Based on the analysis method process and parameter setting of the present application, the analysis method process and parameter setting of the present application can be flexibly applied and integrated into other existing analysis methods, which promotes the comprehensive understanding of PFAS substances and deepens the understanding of the mass spectrometry behavior of new pollutants. It has important significance for the subsequent pollution control and regional ecological protection.

[0064] Compared with the traditional non-target identification method of perfluorinated compounds, the present method has higher recovery rate and sensitivity for perfluoro and polyfluoro ether carboxylic acids, and can identify more types of new fluorine-containing substitutes while being compatible with traditional perfluorinated compounds, and has wide practicability.

[0065] It is apparent to those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, but can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, and the scope of the present application is defined by the appended claims rather than the foregoing description, and all changes falling within the meaning and range of equivalents of the essential elements of the claims are intended to be embraced therein.

[0066] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes every feature described. The specification can include implicit combinations of explicitly mentioned features and / or explicit combinations of implicitely mentioned features. Each embodiment depends on the explicit combinations of features and / or the implicit combinations of features made specifically within that embodiment, and each such embodiment can be combined with every other such embodiment to create further embodiments.

Claims

1. A method of non-targeted screening of soils and sediments for perfluoro- and polyfluoroether carboxylic acids, characterized in that, Comprising the following steps: Step 1, pretreatment of soil or sediment samples by improved solid-liquid extraction method; Step 2, detection of the sample by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry, collecting FullScan information and ddMS2 secondary fragment information of the sample; Step 3, converting the data source file into mzML format and mgf format using ProteoWizard software; Step 4, extracting the primary chromatographic peak information and secondary mass spectrometry fragment information of the sample using R language; Step 5, using Matlab algorithm for sample peak in-source fragmentation labeling and sample peak isotope pattern recognition labeling; Step 6, according to the in-source fragmentation peak labeling information, isotope pattern peak labeling information and compound secondary mass spectrometry fragment information, deconvolution and structure inference are carried out to obtain the structure of perfluorinated and polyfluorinated ether carboxylic acid; In step 5, according to the regularity of the m / z corresponding to a chromatographic peak appearing in the primary spectrum / secondary spectrum, it is judged whether the chromatographic peak belongs to the original m / z or the in-source fragmentation product, and the core discrimination standard of in-source fragmentation peak is whether it appears as a fragment in the secondary fragment spectrum of another larger m / z at the same retention time; The in-source fragmentation labeling algorithm specifically includes the following sub-steps: Step 5-1, import Raw raw data file, use R language to extract the first level chromatographic peak data , , , ; represents the mass-to-charge ratio of the ion corresponding to the i-th chromatographic peak, represents the retention time of the i-th chromatographic peak; represents the mass-to-charge ratio of the j-th parent ion, represents the fragmentation time of the j-th parent ion, represents the mass-to-charge ratio of the k-th fragment after the secondary fragments generated by the j-th parent ion are sorted in descending order of relative abundance; Step 5-2, using matlab algorithm to traverse each primary chromatographic peak, and finding chromatographic peaks within a certain error range that meet the following conditions: ； ； ； At the same retention time, the secondary fragment with the same mass-to-charge ratio as the chromatographic peak can be generated by the parent ion with a larger mass-to-charge ratio; in the code, The difference between the retention time of the primary chromatographic peak and the parent ion is calculated, and the error is required to be less than 0.2 min; The difference between the mass-to-charge ratio of the primary chromatographic peak corresponding to the mass-to-charge ratio and the fragment of the parent ion is calculated, and the error is required to be less than 5 mDa; The difference between the mass-to-charge ratio of the parent ion and the mass-to-charge ratio of the primary chromatographic peak corresponding to the parent ion is calculated, and the difference between the mass-to-charge ratio of the parent ion and the mass-to-charge ratio of the primary chromatographic peak is required to be not less than 1 ; if the primary chromatographic peak and the parent ion meet the above conditions, it is determined that the primary chromatographic peak is an in-source fragmentation fragment peak generated by the parent ion; The sample pretreatment method of step 1 is as follows: take soil or sediment samples, add potassium hydroxide methanol solution, vortex uniformly, ice bath ultrasonic, oscillation, centrifugal take supernatant, repeat extraction twice, combine the supernatants of two times, nitrogen blow to dry, add water to constant volume, and adjust the pH to 7 with acetic acid solution; activate the solid phase extraction column with ammonia water methanol solution, methanol and water in turn, and sample the extract liquid uniformly; elute with acetic acid / ammonium acetate buffer, elute with methanol and ammonia water methanol solution in turn, and then elute with methanol and water; centrifuge and detect.

2. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In step 2, the secondary fragment information includes the retention time, accurate molecular weight and relative intensity value of the fragment.

3. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In step 2, the determination is carried out in negative ion mode using ESI ion source.

4. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In step 2, 5 mM ammonium acetate methanol solution and 5 mM ammonium acetate aqueous solution are used as mobile phase, and gradient elution is used for sampling.

5. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In step 4, the "patRoon" package of R language is used to extract chromatographic peaks in the sample, specifically the findFeaturesOpenMS function in the patRoon package is used to extract peaks from the mzML data file, and the write.csv function is used to export the peak list data after recognition.

6. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In step 4, first save the mgf format file as a txt file, then use R language to filter out fragment peaks with a relative intensity of less than 10%, and retain at most 20 peak fragments with the largest intensity, and export the filtered information using the write.csv function.

7. The method of non-targeted screening of soil and sediments for perfluoro- and polyfluoroether carboxylic acids according to claim 1, characterized in that, In Step 5, whether a certain chromatographic peak possibly contains a halogen atom is determined according to the peak area ratio of the chromatographic peak to the corresponding suspected isotope chromatographic peak; taking chlorine as an example, if the peak area of a certain chromatographic peak satisfies [M] - :[M+2] - = 3:1, then the substance corresponding to the chromatographic peak possibly contains one chlorine atom.

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