Mass spectrometric method for the detection of perfluoroalkyl or polyfluoroalkyl substances (PFAS)

Abstract

A method for determining the presence of a perfluoro- or polyfluorochemical (PFC), such as a PFAS compound, in a sample is described. The method involves generating sample ions from the sample using electron impact ionization (El), direct laser ionization, or field desorption ionization, and mass-analyzing the sample ions using a mass analyzer to generate mass spectral data. The mass analyzer has a mass accuracy of approximately ≤ 100 ppm and a resolution of approximately ≥ 10,000. The method further includes determining whether an ion peak is present in the mass spectral data exhibiting an accurate mass-to-charge ratio within ± 100 ppm of a first accurate mass-to-charge ratio (m / z). The first accurate mass-to-charge ratio (m / z) is the exact mass-to-charge ratio of an ion that indicates the presence of a PFC in the sample.The procedure further includes determining whether a PFC is present in the sample if it is determined that an ion peak is present which has an accurate mass-to-charge ratio within ± 100 ppm of the first accurate mass-to-charge ratio m / z.

Description

Field of invention

[0001] The present invention relates to the field of mass spectrometry and in particular to mass spectrometry methods for the detection of per- and polyfluoroalkyl substances (PFAS), especially, but not exclusively, per- and polyfluoroalkyl substances (PFAS). State of the art

[0002] Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals used in many products, such as fire-fighting foam, chrome plating, waterproof textiles, and others. Due to their unique properties, including heat resistance, water repellency, and high durability, they are widely used in industry and manufacturing. However, these chemicals are currently attracting particular attention because of their persistence in the environment and the risk of adverse health effects.

[0003] Over 4,700 PFAS substances have been identified. PFAS consist of a carbon chain, particularly an alkyl chain, in which hydrogen atoms are wholly or partially replaced by fluorine atoms. PFAS therefore have multiple carbon-fluorine bonds. A detailed list of highly fluorinated compounds can be found in the Swedish Chemicals Agency's 7 / 15 report, "Occurrence and Use of Highly Fluorinated Substances and Alternatives," which includes 2,060 identified substances with a CAS number and an estimate of substances without a CAS number.

[0004] There is a great need for the identification and accessible screening of PFAS and PFCs. In 2021, the U.S. Environmental Protection Agency (EPA) published a strategic roadmap regarding PFAS as an interagency approach to addressing the environmental impacts of PFAS.

[0005] Currently, non-mass spectrometry-based techniques are most commonly used for the detection of PFAS and PFCs. For example, CN113791057 describes a high-throughput screening method for perfluorinated compounds using a fluorescence sensor array, WO2021 / 142455 describes the screening / analysis of fluorocarbons using X-ray photoelectron spectroscopy, and US11,002,691 describes a method for detecting fluorinated chemicals in liquids. These non-mass spectrometry-based methods carry the risk of either not being sensitive enough or producing false-positive results due to the low specificity of the technique.

[0006] Other known methods use a targeted mass spectrometry approach. For example, CN105784881 describes a method for determining the presence of isomers of perfluorinated compounds in soils and / or plants, and CN105467026 describes a method for detecting perfluorinated compounds in soils and sediments. However, these methods do not provide a general approach for PFAS screening.

[0007] WO202183894 describes a method for the parallel analysis of an evaporated sample under two different ionization conditions, which enables the simultaneous characterization of a chromatographically separated compound under two different ionization conditions, such as hard ionization by electron impact (El) or soft ionization by chemical ionization (CI).

[0008] It is assumed that there is still room for improvement in facilities and procedures for mass analysis. Brief description

[0009] A first aspect concerns a method for determining the presence of a perfluoro- or polyfluoro-compound (PFC) in a sample, wherein the method comprises: Generating sample ions from a sample, either: (i) using electron impact ionization (El), direct laser ionization or field desorption ionization; or (ii) using electrospray ionization (ESI) or chemical ionization (CI) and electron-induced dissociation (EID); Mass analysis of the sample ions using a mass analyzer to generate mass spectral data, wherein the mass analyzer has a mass accuracy of approximately ≤ 100 ppm and a resolution of approximately ≥ 10,000; Determine whether an ion peak is present in the mass spectral data whose exact mass-to-charge ratio lies within ± 100 ppm of a first exact mass-to-charge ratio m / z; where the first exact mass-to-charge ratio m / z is the exact mass-to-charge ratio of an ion indicating the presence of a PFC in the sample; and If it is determined that an ion peak is present whose exact mass-to-charge ratio lies within ± 100 ppm of the first exact mass-to-charge ratio m / z: Determine whether a PFC is present in the sample.

[0010] The PFC can typically be a PFAS compound. Existing mass spectrometric methods for identifying PFAS compounds use library queries, but these methods suffer from library incompleteness, as there are many thousands of different PFAS compounds. Furthermore, existing methods generally use soft ionization techniques such as electrospray ionization (ESI), which do not produce ions that are generally indicative of the presence of a PFAS, such as the CF3 cation.

[0011] According to the embodiments, electron impact ionization (El) or other similar "hard" ionization techniques are specifically used to generate ions that generally indicate the presence of a PFAS compound, such as the CF3 cation. Alternatively, a soft ionization technique such as electrospray ionization (ESI) or chemical ionization (CI) together with electron-induced dissociation (EID) is used to generate ions that generally indicate the presence of a PFAS compound, such as the CF3 cation. As described in more detail below, these ions have a slightly lower exact mass than their nominal mass, making them distinguishable from other ions with the same nominal mass when a mass spectrometer with sufficiently high mass accuracy and high resolution (such as an electrostatic orbitrap ion trap mass spectrometer) is used.

[0012] Sample ions can be generated from the sample by ionizing it via electron impact ionization (El), direct laser ionization, or field desorption ionization. In particular, sample ions can be generated from the sample using electron impact ionization (EI).

[0013] Alternatively, the sample ions can be generated from the sample by ionizing the sample via electrospray ionization (ESI) or chemical ionization (CI) to generate precursor ions, and then fragmenting the precursor ions via electron-induced dissociation (EID) to generate the sample ions. In these embodiments, the electrospray ionization (ESI) or chemical ionization (CI) can be operated in a negative ionization mode.

[0014] The mass analyzer can be an electrostatic orbitrap ion trap mass analyzer, a time-of-flight (ToF) mass analyzer, a multi-reflection time-of-flight (mrToF) mass analyzer, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer.

[0015] The mass analyzer can be an electrostatic orbitrap ion trap mass analyzer.

[0016] If the PFC is a PFAS, then the ion that indicates the presence of a PFC is an ion that indicates the presence of a PFAS in the sample.

[0017] The ion that indicates the presence of a PFAS may be a CF3 cation, where the first accurate mass-to-charge ratio is m / z= 68.99466.

[0018] The ion that indicates the presence of a PFAS may be a CF3CF2 cation, where the first accurate mass-to-charge ratio is m / z= 118.99147.

[0019] The ion that indicates the presence of a PFAS may be a CF3CF cation, where the first accurate mass-to-charge ratio is m / z= 130.99147.

[0020] Prior to the mass analysis step of the sample ions, the procedure may further include: isolating sample ions with mass-to-charge ratios within a mass-to-charge ratio range that is aligned with the nominal mass-to-charge ratio of the ion, indicating the presence of a PFC or PFAS.

[0021] The step of isolating sample ions can include isolating sample ions using a mass filter, for example a quadrupole mass filter.

[0022] The mass-to-load ratio range can be ≤ 10 m / z or ≤ 5 m / z.

[0023] The ion that indicates the presence of a PFAS may be a CF3 cation, with the first accurate nominal mass-to-charge ratio being 69.

[0024] The ion indicating the presence of a PFAS can be a CF3CF2 cation with a nominal mass-to-charge ratio of 119.

[0025] The ion indicating the presence of a PFAS can be a CF3CF cation with a nominal mass-to-charge ratio of 131.

[0026] The step of determining whether an ion peak is present may include determining whether an ion peak exists whose intensity exceeds a signal-to-noise ratio threshold. The signal-to-noise ratio threshold is approximately 50.

[0027] The procedure can also include the chromatographic separation of the sample prior to the step of generating sample ions from the sample.

[0028] The step of chromatographic separation of the sample can include separating the sample using liquid chromatography (LC) or gas chromatography (GC).

[0029] The procedure may also include: Dividing the chromatographically separated sample into a first part and a second part, wherein the sample ions are first sample ions generated by ionization of the first part of the sample using a first ion source, wherein the mass spectral data are first mass spectral data, and wherein the method further comprises: Generating a second sample ions from the second part of the sample using a second, different ion source; and Mass analysis of the ions of the second sample to generate second mass spectral data.

[0030] The splitting of the chromatographically separated sample into the first part and the second part can involve a continuous splitting of the chromatographically separated sample into the first part and the second part. In these embodiments, the first sample ions and the second sample ions can be generated simultaneously.

[0031] Alternatively, the division of the chromatographically separated sample into the first part and the second part may comprise: alternating the following steps (i) and (ii): (i) passing the chromatographically separated sample to the first ion source; and then (ii) passing the chromatographically separated sample to the second ion source. In these embodiments, the generation of the first sample ions and the second sample ions may be staggered in time (i.e., the first sample ions and the second sample ions are not generated simultaneously).

[0032] The second ion source can be an electrospray ion source (ESI) or a chemical ionization source (CI).

[0033] The first and second sample ions can be mass-analyzed using the same mass analyzer. In these embodiments, the mass analysis of the first and second sample ions can be staggered in time.

[0034] The first sample ions can be mass-analyzed using a first mass analyzer, and the second sample ions can be mass-analyzed using a second, different mass analyzer. In these embodiments, the mass analysis of the first and second sample ions can be performed either simultaneously or sequentially.

[0035] The procedure may also include: If it is determined that an ion peak is present whose exact mass-to-charge ratio lies within ± 100 ppm of the first exact mass-to-charge ratio m / z: Determine a chromatographic retention time related to the ion peak; and Characterize or identify the PFCs or PFAS present in the sample using the second mass spectral data at or approximately at (e.g., closest in time) the determined chromatographic retention time.

[0036] The method may further include querying a library of spectral data of PFC or PFAS compounds to characterize or identify the PFC or PFAS present in the sample. The method may further include comparing the second mass spectral data with spectral data in the library to find matching data in the library that characterize or identify the PFC or PFAS.

[0037] The sample can be an environmental sample, for example a water sample or a soil sample, possibly dissolved in a solvent.

[0038] The procedure may further include: if it is determined that an ion peak whose exact mass-to-charge ratio lies within ± 100 ppm of the first exact mass-to-charge ratio m / z is not present in the mass spectral data: determining that a PFC or PFAS is not present in the sample. It may be determined that an ion peak is not present if an ion peak is not present or if an ion peak is present whose intensity is below the threshold of the signal-to-noise ratio.

[0039] The method can further include adding a known concentration of a labeled sample to the unlabeled sample, such that the sample ions include both sample ions from the unlabeled sample and sample ions from the labeled sample. The labeled sample can, for example, include a labeled sample enriched with C13. The step of generating sample ions (when using a "hard" ionization source) can result in sample fragment ions (i.e., fragment ions originating from the unlabeled sample) and labeled sample fragment ions (i.e., fragment ions originating from the labeled sample). If a "soft" ionization source is used (as described above), the method can also include fragmenting the ions generated by the second ion source to produce sample fragment ions (i.e., fragment ions originating from the unlabeled sample) and labeled sample fragment ions (i.e., fragment ions originating from the labeled sample).Fragment ions originating from the labeled sample) can be generated. In both cases, the fragment ions can be mass-analyzed to generate mass spectral data of the sample fragment ions and the labeled sample fragment ions. In the mass spectral data, one or more pairs of corresponding fragment ions can be identified (each pair comprising an unlabeled fragment and its corresponding C13-enriched labeled fragment), and the relative intensity of each pair of corresponding fragment ions can be determined. The relative intensity(s) can be used to quantitatively determine the PFC concentration present in the sample.

[0040] Another aspect is a non-volatile, computer-readable storage medium that stores computer software code which, when executed on a processor, performs the procedure described above.

[0041] Another aspect concerns a control system for an analytical instrument, such as a mass spectrometer, wherein the control system is configured to cause the analytical instrument to perform the procedure described above.

[0042] Another aspect concerns an analytical instrument, such as a mass spectrometer, which includes the control system described above.

[0043] The analyzer may include a first ion source, which may be an electron impact ionization (El) source, a direct laser ionization (DLE) source, or a field desorption ionization (DDE) source. The analyzer may also include a second, different ion source, which may be an electrospray ionization (ESI) source or a chemical ionization (CI) source.

[0044] The analyzer may include a mass analyzer located downstream of the ion source(s). The mass analyzer has a mass accuracy of approximately ≤ 100 ppm and a resolution of approximately ≥ 10,000. The mass analyzer may be an electrostatic orbitrap ion trap mass analyzer, a time-of-flight (ToF) mass analyzer, a multi-reflection time-of-flight (mrToF) mass analyzer, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer. The analyzer may include a second, different mass analyzer, which can be any mass analyzer, such as an electrostatic orbitrap ion trap mass analyzer, a time-of-flight (ToF) mass analyzer, a multi-reflection time-of-flight (mrToF) mass analyzer, a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, an ion trap mass analyzer, or a quadrupole mass analyzer.

[0045] The analyzer may include a mass filter located downstream of the ion source and upstream of the mass analyzer. The mass filter may be a quadrupole mass filter.

[0046] The analytical instrument may include a chromatographic separation device coupled to the ion source. The chromatographic separation device may be a liquid chromatography (LC) or a gas chromatography (GC) device. The sample may be supplied to the ion source(s) by the chromatographic separation device.

[0047] A second aspect concerns a method for determining the presence of a compound in a sample, wherein the method comprises: Generating sample ions from a sample by: ionizing the sample by electrospray ionization (ESI) or chemical ionization (CI) to generate negatively charged precursor ions, wherein the electrospray ionization (ESI) or chemical ionization (CI) is performed in negative ionization mode, and fragmenting the precursor ions by electron-induced dissociation (EID) to generate the sample ions, wherein the sample ions contain at least one desired positively charged fragment ion indicating the presence of the compound in the sample; Mass analysis of the sample ions using a mass analyzer to generate mass spectral data; Determine whether an ion peak corresponding to the desired positively charged fragment ion is present in the mass spectral data; and If it is determined that an ion peak is present that corresponds to the desired positively charged fragment ion: Determine whether the compound is present in the sample.

[0048] This aspect can, and in embodiments does, include any one or more of the optional features described herein. For example, the mass analyzer can be configured as described above and elsewhere herein.

[0049] In this aspect, the compound can be a nonpolar compound, such as a lipid, a hydrocarbon, or a perfluoro- or polyfluoroalkyl substance (PFAS), such as a perfluoroalkyl substance (PFAS). The removal of an electron with excess energy from the negatively charged precursor ion during electron-induced dissociation (EID) leads to a negatively charged fragment ion as well as the characteristic positively charged fragment ion. Description of the drawings

[0050] Various embodiments are now described in more detail with reference to the attached illustrations, showing the following: Fig. shows a mass spectrum of a polyfluorinated alcohol recorded with an Orbitrap™ instrument coupled to a gas chromatography (GC) interface; Fig. shows the detection of the CF3 cation from a polyfluorinated alcohol separated by GC using an Orbitrap™ device; Fig. schematically shows an analyzer configured according to embodiments; Fig. schematically shows an analyzer configured according to embodiments; Fig. schematically shows an analyzer configured according to embodiments; Fig. schematically shows an analyzer configured according to embodiments; Fig. schematically shows an analyzer configured according to embodiments; Fig. schematically shows a method according to embodiments; and Fig. schematically shows the soft ionization and electron impact dissociation (EID) of a PFAS compound, perfluorononanoic acid (PFNA). Detailed description

[0051] The embodiments relate to a screening method for per- and polyfluoroalkyl substances (PFAS), such as per- and polyfluoroalkyl substances (PFAS). This general screening tool can detect low concentrations of PFAS or PFCs in the environment, for example in groundwater or soil.

[0052] Until now, there has been no general, mass spectrometry-based screening method for PFCs. Existing mass spectrometric methods typically use a targeted identification approach based on a list of known PFAS or PFC compounds. Such targeted approaches struggle to identify unknown PFCs or new types of synthetic substances, such as perfluorinated polyethers. Previously unknown PFCs are highly likely to go undetected using such targeted identification approaches.

[0053] PFAS and PFCs exhibit high hydrophobicity and are only minimally likely to be efficiently ionized using common soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Consequently, such soft ionization techniques offer relatively low sensitivity for PFAS and PFC compounds. A general ionization process that favors the ionization of the hydrophobic residue and the high electron density of the perfluoroalkyl chain offers higher sensitivity compared to standard "soft" ESI.

[0054] The list of per- or polyfluorinated substances (PFAS) is expected to be further expanded in the future based on current and future knowledge about the environmental risks and toxicity of certain PFAS and PFC compounds. These new classes of substances include, for example, perfluorinated ethers. Although PFCs cannot be detected by targeted searches for perfluorinated alkyl substances, it can be assumed that these new classes uniformly possess a hydrophobic molecular residue, represented by the following two formulas: CF₂CF₃ (fluorinated ethyl) or CF₃ (fluorinated methyl).

[0055] Electron impact ionization (El) is an ionization technique in which high-energy electrons interact with atoms in the solid or gaseous phase, producing electron-deficient ions. This ionization technique is considered a hard or aggressive ionization method because its characteristic energy of 70 eV exceeds the energy required for ionization-induced fragmentation. El ionization is frequently used for various classes of organic molecules. Unlike softer ionization techniques, El ionization typically results in extensive fragmentation, which is known to contribute to compound-specific libraries for El ionization mass spectrometry.

[0056] To date, the presence of PFAS- or PFC-specific electron impact ionization fragments has not been used to screen for the presence of poly- or perfluorinated compounds within a mass spectrometric approach.

[0057] Embodiments relate to a method for the accurate screening of polyfluorinated or perfluorinated compounds (PFCs), wherein a sample is ionized by means of “hard” electron impact ionization (El) and is examined for the presence of ions at m / z = 68.99466 with a mass accuracy of at least 100 ppm.

[0058] The CF3 fragment consists of three fluorine atoms and one carbon atom and is positively charged due to the electron deficiency of the cation. Compared to the relatively high molecular weight of perfluorinated or polyfluorinated compounds, the CF3 cation itself is a fragment ion with a relatively low mass and a nominal mass-to-charge ratio of 69. Although such a fragment ion with a mass-to-charge ratio of 69 is known to form under harsh ionization conditions, the exact mass identity of the CF3 cation has not yet been used to confirm the presence of poly- or perfluorinated compounds when a sample is ionized under harsh electron impact (El) conditions.

[0059] The reason for using highly specific mass detection lies partly in the unique property of the fluorine atom, whose exact mass is less than its nominal mass of m / z = 19. A further advantage of selecting the CF3 fragment ion, or more generally a fragment ion containing one or more fluorine atoms, is the relatively high intensity of the fluorine isotope. 19 F, since fluorine (along with 20 other elements) is known to be a mononuclidean element. Such a single nuclide has a characteristic single atomic mass.

[0060] Therefore, in embodiments, the exact mass of 68.99466, which exclusively represents the presence of a CF3 cation, is used to exclude all false-positive ions with the same nominal mass of m / z 69. Such false-positive ions could be present during the CF3 cation screening. A comparison of the exact masses of CF3 and various false-positive ions at m / z 69 is shown in Table 1. Monoisotope Masse Delta-m / z * 1.000 ppm CF3 68,9946612 0 0 PF2 68,9700195 -24,6417 -357,2 C3H4N2H 69,00472 10,0588 145,8 C3H3NO 69,02092 26,2588 380,6

[0061] Table 1: Exact monoisotopic positive mass-to-charge ratios of four different chemical compounds that have the same nominal mass-to-charge ratio of 69

[0062] As can be seen from Table 1, the difference between the exact monoisotopic mass of the CF3 cation and that of the three potential false-positive positively charged fragment ions is more than 100 ppm. Using a high-precision, high-resolution mass analyzer, the resulting mass spectrum can therefore be used to distinguish the presence of the CF3 cation from other ions with the same nominal mass.

[0063] Electron impact ionization (El) of PFAS offers the advantage of higher specificity in the ionization of the alkyl chain, since the interaction of the electron with the higher electron density of the perfluorinated residue leads to a higher probability of ionization of the hydrophobic chain. In contrast, "softer" ionization techniques (such as ESI or MALDI) favor the ionization of more hydrophilic parts of the PFAS. Therefore, if no hydrophilic functional groups are present in the analyzed PFAS compound, it is unlikely that such substances can be detected using the aforementioned softer ionization techniques.

[0064] Accordingly, it is understood that the embodiments relate to a method for screening for PFAS and PFCs by a mass spectrometric, data-independent approach using electron impact ionization (El). Electron impact ionization (El) in the positive ion polarity mode leads to electron-deficient radical cations that are typically rapidly cleaved. Accordingly, El mass spectra of PFAS and PFC compounds show a high intensity of the CF3 cation at a nominal mass of m / z 69. Due to the mass-deficient nature of the monoisotopic mass of the fluorine atom, the exact mass of the CF3 cation is just below the nominal mass of 69, namely at m / z = 68.99466.

[0065] In reference electrochemical mass spectra of perfluorooctanoic acid (PFOA, CAS registration number 335-67-1), the intact mass of m / z 414, representing the formula C8HF15O2, is not observable. The highest MS signal at m / z 395 indicates the loss of one fluorine atom from the intact mass. The signal at m / z 69 has a relative intensity of approximately 55%. This signal is among the three highest-intensity signals in the reference spectra of PFOA.

[0066] Fig. This shows an El mass spectrum of a randomly selected polyfluorinated alcohol, acquired using an Orbitrap™ Exploris™ 240 instrument coupled to a gas chromatography (GC) interface. The diamond indicates the signal corresponding to the CF3 cation with the calculated exact mass of m / z 68.99466.

[0067] The detection of the exact mass of the CF3 cation is in Fig. To better illustrate the confirmed presence of CF3, an enlarged mass range of m / z = 68.98400–69.00500 is shown. Fig. The dotted box covers the mass range from 68.988 to 69.001, which corresponds to an exact mass range of the expected theoretical mass-to-charge ratio of the CF3 cation plus / minus the exact detection range of 100 ppm for non-targeted screening for the presence of the CF3 cation.

[0068] The in Fig. The CF3 cation shown was acquired with a resolution of 60,000 at m / z 200, corresponding to a resolving power of 102,000 for the CF3 cation. The CF3 cation recorded in the mass spectrum shown agrees with the exact mass of the calculated monoisotopic mass of 68.99466. The mass spectrum of the CF3 cation shown in the Fig. The polyfluorinated alcohol shown was scanned in a single scan with a measured signal-to-noise ratio of S / N= 2.58e3.

[0069] There are various practical applications for the high-precision screening method for PFAS or PFC compounds.

[0070] Fig. Figure 1 shows a first embodiment in which a direct screening of a sample 100 is carried out using hard El ionization in an ionization device 101, followed by high-precision and high-resolution mass detection in an MS detector 102 of at least one compound-specific fragment mass-to-charge ratio to confirm the presence of a per- or polyfluorinated compound.

[0071] Fig. A second embodiment is shown in which a direct screening of a sample 100 is carried out using hard El ionization in an ionization device 101, followed by the isolation of a compound-specific fragment ion in an isolation device 111, for example a quadrupole mass filter, and subsequently a high-precision and high-resolution mass detection in an MS detector 102 of at least one compound-specific fragment mass-to-charge ratio to confirm the presence of a per- or polyfluorinated compound.

[0072] Fig. represents a further embodiment comprising chromatographic or other applicable separation or purification of a sample 100 by a separation or purification device 201, followed by hard El ionization in an ionization device 202 and high-precision and high-resolution mass detection in an MS detector 203 for at least one compound-specific fragment mass-to-charge ratio to confirm the presence of a per- or multiply fluorinated compound.

[0073] Fig. Figure 3 shows a further embodiment comprising chromatographic or other suitable separation or purification of a sample 100 by a separation or purification device 301, followed by splitting the sample for simultaneous analysis under hard and soft ionization conditions by at least two different ionization devices 302 and 312 connected to two different mass analyzers 303 and 313. The soft ionization of the sample in the soft ionization source 312 preferably ionizes an intact polyfluorinated or perfluorinated molecule. The retention time of the confirmed presence of a per- or polyfluorinated compound in the mass detector 303 is used to selectively identify the polyfluorinated or perfluorinated compound.In these embodiments, the two types of ionization can be carried out simultaneously during the same chromatographic run, either by continuously splitting the chromatographic eluent between the two sources or by rapidly switching the chromatographic eluent between the two ion sources.

[0074] Fig. Figure 401 shows a further embodiment comprising chromatographic or other applicable separation or purification of a sample 100 by a separation or purification device 401, followed by splitting the sample for simultaneous analysis under hard and soft conditions by at least two different ionization devices 402 and 412. Both ionization devices 402 and 412 are connected to a single mass analyzer 403. Again, the two types of ionization can be performed simultaneously during the same chromatographic run, either by continuously splitting the chromatographic eluent between the two sources or by rapidly switching the chromatographic eluent between the two ion sources. Alternatively, the hard and soft ionization and detection can be performed in two separate (e.g., consecutive) chromatographic runs.In these embodiments, the detection of ions from both ion sources can be achieved by continuously and rapidly switching between the two ion sources.

[0075] This indicates that the procedure for non-targeted screening for poly- or perfluorinated compounds with a perfluorinated CF3 residue may include the following steps: 1. Hard electron impact ionization (El) of a sample under investigation that may contain a poly- or perfluorinated compound; and 2. Unambiguous detection of a fragment with hard ionization, such as in particular electron impact ionization, leading to at least one known perfluorinated fragment ion; wherein a. Unique identification includes the signal intensity of at least one CF3-specific poly- and / or perfluorinated fragment such as CF3 (m / z 68.99466), CF3CF2 (m / z 118.99147) or CF3CF=CF (130.99147) above a significant S / N threshold, e.g. higher than 50; b. Unique identification is achieved through accurate mass detection within a predetermined precise mass range of the expected precise mass plus / minus a mass accuracy of less than approximately 100 ppm; and c. The resolving power of the mass detection device is high enough to ensure the unambiguous identification of the presence of the fragmentation.

[0076] Fig. Figure 5 shows a screening procedure according to one embodiment, wherein a screening range for selected ion monitoring (SIM) is used at m / z 69 ± 5 when a sample is mass spectrometrically analyzed using a high-resolution, mass-accurate (HRAM) instrument (step 501). The resulting mass spectrum is analyzed to determine whether an ion peak at m / z 68.99466 ± 100 ppm has been detected (step 502). If this is not the case, no PFAS / PFC compound has been detected in the sample. However, if an ion peak is present, the presence of a PFAS / PFC compound in the sample is confirmed (step 503).

[0077] As also described above, in addition to the unambiguous screening for the presence of known El-induced fragment ions, chromatographic separation of the sample can be performed to further improve the compound-specific identification of the per- or polyfluorinated compound. The chromatographically separated sample is then independently ionized by a soft ionization technique, such as electrospray ionization (ESI) or chemical ionization (CI). Such separation can be carried out in parallel with or after the electron impact ionization (EI) of the separated sample, provided the eluent retention time is reproducible.

[0078] Therefore, in some embodiments, the screening method is combined with a chromatographic separation such as gas chromatography (GC) or liquid chromatography (LC) or another fluid-based separation device, and the repeatable retention time feature is used to further confirm the identity of the substance.

[0079] Embodiments offer a non-targeted screening method for poly- and / or perfluorinated compounds, e.g., from environmental samples. The screening method, which includes El ionization, is highly sensitive and highly specific for PFCs and / or PFAS. The selection of compound-specific fragment ions can be adapted for screening subclasses of PFAS and PFCs, provided that further class-specific El fragment ions are selected.

[0080] In some embodiments, a known concentration of a C13-enriched, isotopically labeled sample is added to the analyzed sample, and the corresponding relative intensities between fragment ions of the sample and the isotopically enriched sample are used to internally determine the concentration of the PFC or PFAS.

[0081] The high-precision, mass-resolved screening method for compound-specific fragment ions, such as m / z 68.99466, which specifically characterizes the CF3 cation, is applicable to high-precision mass spectrometric instruments such as orthogonal time-of-flight (ToF) instruments, Orbitrap™ instruments, FT-ICR instruments, and multireflection time-of-flight (mrToF) instruments. With less accurate mass analyzers or instruments with lower resolution (typically offering nominal mass detection), such as quadrupoles or ion traps, the identification of false-positive ions cannot be ruled out. Therefore, embodiments can utilize any type of high-precision mass analyzer, such as an Orbitrap™ mass analyzer, ToF analyzer, mrToF analyzer, or FT-ICR analyzer, that provides at least the specified resolution to unambiguously confirm the presence of the compound-specific fragment ion.

[0082] In particular, the resolving power at a mass-to-charge ratio of 69 should be higher than approximately 50% of the ±100 ppm range to enable positive identification in screening for the CF3 cation at m / z 68.99466. The corresponding FWHM is (68.988 - 69.001) / 2 = 0.0069. This minimum FWHM value corresponds to a resolving power of 10,000. The presence of ions with a nominal mass of m / z 69 itself indicates ions other than the compound-specific CF3 cation. Therefore, the detection of ions with a nominal mass of 69 alone is not specific enough for screening poly- or perfluorinated compounds.

[0083] Although the CF3 cation is particularly useful in the procedures described here, other poly- or perfluorinated specific fragment ions with a higher mass-to-charge ratio than m / z 68.99466 can also be included, e.g., in a list of specific screening candidates.

[0084] Although electron impact ionization is particularly suitable for the methods described here, other hard ionization techniques such as direct laser ionization and field desorption ionization can produce a similar ionization of nonpolar compounds and activation of the compounds, leading to fragmentation.

[0085] Alternatively, a soft ionization technique such as electrospray ionization (ESI) or chemical ionization (CI) together with electron-induced dissociation (EID) can be used to generate ions that uniquely indicate the presence of a PFAS compound, e.g., the CF3 cation. In certain embodiments, an electrospray ionization (ESI) source is configured to ionize the eluent from a liquid chromatography (LC) separation device, and the ions thus generated are subjected to EID.

[0086] To ionize PFAS compounds with ESI, the more sensitive electrospray polarity is the negative ionic polarity (based on the fact that the functional chemical group is a carboxylic acid, sulfonamic acid, or sulfonic acid). The perfluoroalkyl residue is not directly involved in the ESI ionization of PFAS. Ionization of PFAS in positive ESI is possible, but less sensitive.

[0087] Thus, embodiments use a soft ionization of the compounds in negative polarity and subsequently an electron impact of the PFAS residue to form CF3 (or analogous) longer, positively charged C m F m -to create residues, i.e. a "charge reversal".

[0088] The following formula is a more general formula for PFAS with a carbocyclic acid representing the head of an amphiphilic compound: CF3C m F m nCO2H, where m and n are integer values. Soft ionization (e.g., ESI) in negative polarity yields the PFAS anion: CF3C m F m nCO2 - => CF3C m F m nCOO - , and the electron impact dissociation (EID) of the PFAS anion yields: CF3C m F m nCOO - CF3 + .

[0089] Fig. The figure shows the soft ionization of perfluorononanoic acid, which generates a negatively charged ion at m / z 463. Electron impact ionization of the amphiphilic compound leads to the generation of (i) a negatively charged hydrophilic fragment and (ii) the positively charged perfluorinated cation CF3, which in turn indicates the presence of PFAS.

[0090] In cases where the CF3 cation is not detectable (e.g., if the CF3 cation is not captured), the loss of the exact mass of the CF3 cation can be used to confirm the presence of the perfluorinated residue.

[0091] This "charge reversal" process is considered novel and inherently advantageous, and applicable to compounds other than PFAS. Advantageously, this "charge reversal process" enables the generation of characteristic positively charged fragment ions from certain compounds, even when electrospray ionization (ESI) is operated in its more sensitive negative ionization mode. Thus, a second aspect is provided, as described above.

[0092] In general, the compound can be a nonpolar compound, such as a lipid, a hydrocarbon, or a perfluoro- or polyfluoroalkyl substance (PFAS), such as a perfluoroalkyl substance (PFAS). The removal of an electron from the negatively charged precursor ion during electron-induced dissociation (EID) leads to a negatively charged fragment ion as well as the characteristic positively charged fragment ion.

[0093] The negatively charged precursor ion can be captured simultaneously with the positively charged fragment ion of interest in a fragmentation device, e.g. by a quadrupole capture potential using radio frequency.

[0094] Although the method can be used to analyze PFAS compounds, this “charge reversal” can also be applied to other amphiphilic ionic species, as long as the negatively charged head of the molecule is not involved in the electron collision and thus remains unaffected, and only the more hydrophobic residue interacts with the electron hitting the molecule.

[0095] Although the present invention has been described with reference to various embodiments, it is understood that various modifications can be made without deviating from the scope of protection of the invention as set out in the attached claims. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] CN 113791057

[0005] WO 2021 / 142455

[0005] US 11,002,691

[0005] CN 105784881

[0006] CN 105467026

[0006] Cited non-patent literature

[0000] PFAS and PFCs. In 2021

[0004]

Claims

[1] Method for detecting the presence of a perfluoroalkyl or polyfluoroalkyl substance (PFAS) in a sample, the method comprising: Generating sample ions from a sample, either: (i) using electron impact ionization (El), direct laser ionization or field desorption ionization; or (ii) using electrospray ionization (ESI) or chemical ionization (CI) and electron-induced dissociation (EID); Mass analysis of the sample ions using a mass analyzer to generate mass spectral data, wherein the mass analyzer has a mass accuracy of approximately ≤ 100 ppm and a resolution of approximately ≥ 10,000; Determine whether an ion peak is present in the mass spectral data whose exact mass-to-charge ratio lies within ± 100 ppm of a first exact mass-to-charge ratio m / z; where the first exact mass-to-charge ratio m / z is the exact mass-to-charge ratio of an ion indicating the presence of a PFAS in the sample; and If it is determined that an ion peak is present whose exact mass-to-charge ratio is within ± 100 ppm of the first exact mass-to-charge ratio m / z: Determine that a PFAS is present in the sample; where: The ion indicating the presence of a PFAS is a CF3 cation with a nominal mass-to-charge ratio of 69 and the first exact mass-to-charge ratio m / z = 68.99466; and / or The ion indicating the presence of a PFAS is a CF3CF2 cation with a nominal mass-to-charge ratio of 119 and the first exact mass-to-charge ratio m / z = 118.99147; and / or The ion indicating the presence of a PFAS is a CF3CF cation with a nominal mass-to-charge ratio of 131 and the first exact mass-to-charge ratio m / z = 130.99147. [2] Method according to claim 1, wherein the sample ions are generated from the sample by means of electron impact ionization (El). [3] Method according to claim 1 or 2, wherein the mass analyzer may be an electrostatic orbitrap ion trap mass analyzer, a time-of-flight (ToF) mass analyzer, a multi-reflection time-of-flight (mrToF) mass analyzer or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer. [4] Method according to claim 3, wherein the mass analyzer is an electrostatic orbitrap ion trap mass analyzer. [5] Method according to any of the preceding claims, further comprising, prior to the step of mass analysis of the sample ions: isolating sample ions with mass-to-charge ratios within a mass-to-charge ratio range, the center of which is the nominal mass-to-charge ratio of the ion that indicates the presence of a PFAS. [6] Method according to claim 5, wherein the step of isolating sample ions comprises isolating sample ions using a mass filter, for example a quadrupole mass filter. [7] Method according to claim 5 or 6, wherein the mass-to-load ratio range has a width ≤ 10 m / z or ≤ 5 m / z. [8] Method according to any one of claims 5 to 6, wherein: The ion indicating the presence of a PFAS is a CF3 cation, and the mass-to-charge ratio range is centered on a mass-to-charge ratio of 69; and / or The ion indicating the presence of a PFAS is a CF3CF2 cation, and the mass-to-charge ratio range is centered on a mass-to-charge ratio of 119; and / or The ion indicating the presence of a PFAS is a CF3CF cation, and the mass-to-charge ratio range is centered on a mass-to-charge ratio of 13. [9] Method according to one of the preceding claims, wherein the step of determining whether an ion peak is present comprises determining whether an ion peak is present whose intensity is above a threshold of the signal-to-noise ratio. [10] Method according to any of the preceding claims, further comprising the chromatographic separation of the sample prior to the step of generating sample ions from the sample. [11] Method according to claim 10, wherein the step of chromatographic separation of the sample comprises separating the sample by liquid chromatography (LC) or gas chromatography (GC). [12] Method according to claim 10 or 11, further comprising: Dividing the chromatographically separated sample into a first part and a second part, wherein the sample ions are first sample ions generated from the first part of the sample using a first ion source, wherein the mass spectral data are first mass spectral data, and wherein the method further comprises: Generating a second sample ions from the second part of the sample using a second, different ion source; and Mass analysis of the ions of the second sample to generate second mass spectral data. [13] Method according to claim 12, wherein the second ion source is an electrospray ionization source (ESI) or a chemical ionization source (CI). [14] Method according to claim 12 or 13, wherein: the first sample ions and the second sample ions are mass-analyzed using the same mass analyzer; or The first sample ions are mass-analyzed with a first mass analyzer, and the second sample ions are mass-analyzed with a second, different mass analyzer. [15] Method according to one of claims 12, 13 or 14, further comprising: If it is determined that an ion peak is present whose exact mass-to-charge ratio lies within ± 100 ppm of the first exact mass-to-charge ratio m / z: Determine a chromatographic retention time related to the ion peak; and Characterize and / or identify the PFAS present in the sample using the second mass spectral data related to the determined chromatographic retention time. [16] Method according to any of the preceding claims, further comprising: when it is determined that an ion peak whose exact mass-to-charge ratio is within ± 100 ppm of the first exact mass-to-charge ratio m / z is not present in the mass spectral data: Determine that a PFAS is not present in the sample. [17] Method according to any of the preceding claims, wherein the sample comprises a known concentration of labeled sample, wherein the sample ions comprise sample fragment ions and labeled sample fragment ions, and wherein the method further comprises: Comparing the intensities of the sample fragments with the intensities of the labeled sample fragments; and Determining the PFAS concentration present in the sample based on the reference(s). [18] Method according to claim 17, wherein the labeled sample is a labeled sample enriched with C13. [19] Non-transitory computer-readable storage medium that stores computer program code which, when executed on a processor, performs the method according to any of the preceding claims. [20] Control system for an analyzer, wherein the control system is configured to cause the analyzer to perform the method according to any one of claims 1 to 18. [21] Analytical instrument, such as a mass spectrometer, comprising the control system according to claim 20.

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