Method of mass spectrometry for the detection of perfluoroalkyl or polyfluoroalkyl substances (PFAS)

Abstract

A method of determining the presence of a perfluoroalkyl or polyfluoroalkyl substance (PFAS) or a compound comprising a perfluoro-alkylated substructure in a sample using a charge inversion is provided. The method comprises ionising a sample under negative soft ionisation conditions at atmospheric pressure to produce negatively charged precursor ions, fragmenting precursor ions by interaction with electrons and / or photons to produce positively charged fragments ions, and mass analysing the positively charged fragments ions using a mass analyser so as to produce positive fragment mass spectral data. The method comprises determining whether, in the positive fragment mass spectral data, an ion peak is present that correspond to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample. When it is determined that an ion peak is present that corresponds to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample: the method comprises determining that a PFAS or a compound comprising a perfluoro-alkylated substructure is present in the sample.

Description

[0001] METHOD OF MASS SPECTROMETRY FOR THE DETECTION OF PERFLUOROALKYL OR POLYFLUOROALKYL SUBSTANCES (PFAS)

[0002] Field of the invention

[0003] The present invention relates to the field of mass spectrometry, and in particular to mass spectrometric methods of detecting compounds of interest.

[0004] Background

[0005] Per- and poly-fluoroalkyl substances (PFAS) are a group of synthetic chemicals that have been used in many products such as firefighting foam, chrome-plating, waterproof textiles, and other products. They have been widely used in industry and manufacturing due to their unique properties, including being heat-resistant, able to repel water, and highly robust. However, these chemicals are currently attracting particular attention due to their persistence in the environment and risk of adverse health impacts.

[0006] More than 4700 identified PFAS substances are known. PFAS comprise a carbon chain, in particular an alkyl chain, in which hydrogen atoms are entirely or partly replaced by fluorine atoms. Thus, PFAS have multiple carbon-fluorine bonds. A detailed list of highly fluorinated compounds is given in the KEMI 7 / 15 report, Occurrence and use of highly fluorinated substances and alternatives, covering 2060 identified substances with a CAS number and an estimation of substances without a CAS number.

[0007] There is a great need for the identification and accessible screening of PFAS and PFC. In 2021, the EPA (US) environmental protection agency released a PFAS strategic roadmap as a whole-of-agency approach addressing the impact of PFAS on the environment.

[0008] Currently, non-mass spectrometry-based techniques are most commonly utilised for screening of PFAS and PFC. For example, in CN113791057 describes a perfluorinated compound high-throughput screening method using a fluorescence sensor array, WO2021 / 142455 describes screening / analysis of fluorocarbons using x-ray photoelectron spectroscopy, and US11,002,691 describes a method for detecting fluorinated chemicals in liquid. These non-MS techniques have the risk of either not being sensitive enough or providing false positive results because of the low specificity of the technique. Other known methods use a targeted mass spectrometric approach. For example, CN 105784881 describes a method of determining the presence of perfluorinated compound isomers in soil and / or plants, CN 105467026 describes a method for detecting perfluorinated compounds in soil and sediments, and CN117871729 describes a method for non-targeted screening of PFAS in electroplating sludge. However, these methods do not provide a general PFAS screening approach.

[0009] It is believed that there remains scope for improvements to apparatus and methods for mass analysis.

[0010] Summary

[0011] A first aspect provides a method of determining the presence of a perfluoroalkyl or polyfluoroalkyl substance (PFAS) or a compound comprising a perfluoro-alkylated substructure in a sample, the method comprising: ionising a sample under negative soft ionisation conditions at atmospheric pressure to produce negatively charged precursor ions; fragmenting precursor ions by interaction with electrons and / or photons to produce positively charged fragments ions; mass analysing the positively charged fragments ions using a mass analyser so as to produce positive fragment mass spectral data; determining whether, in the positive fragment mass spectral data, an ion peak is present that correspond to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample; and when it is determined that an ion peak is present that corresponds to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample: determining that a PFAS or a compound comprising a perfluoro-alkylated substructure is present in the sample.

[0012] Thus, embodiments provide a non-targeted screening for PFAS and related compounds which relies on a charge inversion to produce ions characteristic of these compounds. Existing mass spectrometric methods of identifying PFAS compounds use library searches, but these methods suffer from the incompleteness of libraries as there are many thousands of different PFAS compounds. The method may comprise, before the step of fragmenting precursor ions (and after the step of ionising): isolating negatively charged precursor ions having mass to charge ratios within a mass to charge ratio window centred at a mass to charge ratio of at least one negatively charged precursor ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure.

[0013] The step of isolating negatively charged precursor ions may comprise isolating negatively charged precursor ions using a mass filter such as a quadrupole mass filter. The mass filter may use a mass to charge ratio window having a width <5 m / z or <2 m / z.

[0014] The step of ionising may be done by an ion source operating in negative ionisation mode, and the method may comprise transmitting the negatively charged precursor ions from the ion source to the mass filter.

[0015] The step of fragmenting may be done within a fragmentation device. The method may comprise transmitting the isolated negatively charged precursor ions of interest from the mass filter to the fragmentation device. The method may comprise trapping the negatively charged precursor ions within the fragmentation device, e.g. by RF pseudopotential barrier(s).

[0016] The step of fragmenting precursor ions may comprise fragmenting negatively charged precursor ions by interaction with electrons and / or photons while negatively charged precursor ions remain trapped within the fragmentation device. The method may comprise trapping the positively charged fragment ions within the fragmentation device, e.g. by the RF pseudopotential barrier(s). Thus, in embodiments, negatively charged precursor ions are trapped simultaneously with positively charged fragment ions in the fragmentation device, e.g. by RF pseudopotential barrier(s).

[0017] The method may further comprise, before the step of isolating negatively charged precursor ions: mass analysing the negatively charged precursor ions using a mass analyser so as to produce negative precursor ion (MS1) mass spectral data; and identifying an ion peak in the negative precursor ion mass spectral data that corresponds to the at least one negatively charged precursor ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample. In other words, once this ion peak has been identified in a negative MS1 mass spectrum, it may be targeted in a positive MS2 scan by isolating the negatively charged precursor ion and fragmenting it by interaction with electrons and / or photons to produce positively charged fragments ions.

[0018] The method may further comprise collisionally activating negatively charged precursor ions to produce at least one negatively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample. This step can be performed before or after the step of isolating negatively charged precursor ions.

[0019] The step of mass analysing negatively charged precursor ions may be performed using a mass analyser operating in a negative polarity mode. The method may comprise transmitting negatively charged precursor ions from the ion source (optionally via the mass filter and / or via the fragmentation device) to the mass analyser for mass analysis.

[0020] The step of mass analysing positively charged fragments ions may be performed using the same mass analyser operating in a positive polarity mode. The method may comprise transmitting positively charged fragment ions from the fragmentation device to the mass analyser for mass analysis.

[0021] In some embodiments, a mass (or m / z) difference between adjacent fragment ions is consistent with an accurate mass difference of 49.997 (indicating the presence of CF2). Thus, the at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample may comprise two or more positively charged fragment ions having a mass (or m / z) difference consistent with an accurate mass difference of 49.997.

[0022] Corresponding, the method may comprise: determining whether, in the positive fragment mass spectral data, two or more ion peaks are present that correspond to positively charged fragment ions having a mass (or m / z) difference consistent with an accurate mass difference of 49.997; and when it is determined that two or more ion peaks are present that correspond to positively charged fragment ions having a mass (or m / z) difference consistent with an accurate mass difference of 49.997 present: determining that a PFAS or a compound comprising a perfluoro-alkylated substructure is present in the sample.

[0023] The mass analyser may have a mass accuracy < about 100 ppm and a resolving power > about 10,000. The mass analyser is an electrostatic orbital ion trap mass analyser, a Time-of-Flight (ToF) mass analyser, a multi-reflection Time-of-Flight (mrTOF) mass analyser, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyser.

[0024] The step of ionising may comprise ionising the sample using electrospray ionisation (ESI) or chemical ionisation (Cl).

[0025] The step of fragmenting precursor ions by interaction with electrons may comprise fragmenting the precursor ions using electron induced dissociation (EID). The electrons may have an energy exceeding the binding energy of the C-F bond and / or of the C-C bond. This provides suitable conditions for the charge inversion.

[0026] The step of fragmenting precursor ions by interaction with photons may comprise fragmenting the precursor ions using multiphoton laser excitation.

[0027] Fragmentation operational parameters may be set to be more selective for PFAS compounds and / or compounds comprising a perfluoro-alkylated substructure relative to other compounds. For example, operational parameters of the EID ion source (i.e. electron energy, electron emission current and / or exposure time of the precursor ions to the electrons) may be set to be more selective for PFAS compounds and / or compounds comprising a perfluoro-alkylated substructure relative to other compounds.

[0028] The sample may comprise a known concentration of a reference standard, and the method may further comprise: comparing an intensity of the at least one positively charged fragment ion to an intensity of at least one reference standard ion; and determining a concentration of PFAS or the compound comprising a perfluoro-alkylated substructure present in the sample based on the comparison. In other words, the intensity of the measured fragment signal can be utilised to determine the quantity of PFAS present in the sample by applying a reference standard with known concentration.

[0029] The step of determining whether an ion peak is present may comprise determining whether an ion peak is present that has an intensity above a signal to noise ratio threshold. The signal to noise ratio threshold may be about 50.

[0030] The method may comprise determining whether, in the mass spectral data, an ion peak is present that has an accurate mass to charge ratio within ± 100 ppm of a first accurate mass to charge ratio m / z, wherein the first accurate mass to charge ratio m / z is the accurate mass to charge ratio of an ion indicative of the presence of a perfluoroalkyl or polyfluoroalkyl substance (PFAS) or a compound comprising a perfluoro-alkylated substructure in the sample; and when it is determined that an ion peak is present that has an accurate mass to charge ratio within ± 100 ppm of the first accurate mass to charge ratio m / z: determining that a perfluoroalkyl or polyfluoroalkyl substance (PFAS) or a compound comprising a perfluoro- alkylated substructure is present in the sample.

[0031] The method may further comprise: when it is determined that an ion peak that has an accurate mass to charge ratio within ± 100 ppm of the first accurate mass to charge ratio m / z is not present in the mass spectral data: determining that a PFAS or a compound comprising a perfluoro-alkylated substructure is not present in the sample. It may be determined an ion peak is not present when an ion peak is not present, or when an ion peak is present that has an intensity below the signal to noise ratio threshold.

[0032] The sample may be an environmental sample, such as a water sample or a soil sample, optionally dissolved in a solvent.

[0033] A second aspect provides a method of determining the presence of a compound of interest in a sample, the method comprising: ionising a sample under negative soft ionisation conditions at atmospheric pressure to produce negatively charged precursor ions; fragmenting precursor ions by interaction with electrons and / or photons to produce positively charged fragments ions; mass analysing the positively charged fragments ions using a mass analyser so as to produce positive fragment mass spectral data; determining whether, in the positive fragment mass spectral data, an ion peak is present that correspond to at least one positively charged fragment ion indicative of the presence of the compound of interest in the sample; and when it is determined that an ion peak is present that corresponds to at least one positively charged fragment ion indicative of the presence of the compound of interest in the sample: determining that the compound of interest is present in the sample.

[0034] This aspect can, and in embodiments does, include any one or more or each of the optional features described herein. The method and the instrument may be configured as described above and elsewhere herein. Thus, for example, the method may comprise simultaneously trapping negatively charged precursor ions with positively charged fragment ions in a fragmentation device, e.g. as described above and elsewhere herein. The method may comprise, before the step of fragmenting precursor ions: isolating negatively charged precursor ions having mass to charge ratios within a mass to charge ratio window centred at a mass to charge ratio of at least one negatively charged precursor ion indicative of the presence of the compound of interest, e.g. as described above and elsewhere herein. The method may further comprise, before the step of isolating negatively charged precursor ions: mass analysing the negatively charged precursor ions using a mass analyser so as to produce negative precursor ion mass spectral data; and identifying an ion peak in the negative precursor ion mass spectral data that corresponds to the at least one negatively charged precursor ion indicative of the presence of the compound of interest, e.g. as described above and elsewhere herein. A further aspect provides a non-transitory computer readable storage medium storing computer software code which when executed on a processor performs the method described above.

[0035] A further aspect provides a control system for an analytical instrument, the control system configured to cause the analytical instrument to perform the method described above.

[0036] A further aspect provides an analytical instrument, such as a mass spectrometer, comprising the control system described above.

[0037] The analytical instrument may comprise an ion source, which may comprise an electrospray (ESI) ion source or a chemical ionisation (Cl) ion source. The analytical instrument may comprise a fragmentation device, such as an electron induced dissociation (EID) fragmentation device or a laser, arranged downstream of the ion source. The analytical instrument may comprise a mass analyser arranged downstream of the fragmentation device. The mass analyser may have a mass accuracy < about 100 ppm and a resolving power > about 10,000. The mass analyser may be an electrostatic orbital ion trap mass analyser, a Time-of-Flight (ToF) mass analyser, a multi-reflection Time-of-Flight (mrTOF) mass analyser, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyser. The analytical instrument may comprise a mass filter such as a quadrupole mass filter arranged downstream of the ion source and upstream of the fragmentation device.

[0038] Description of the drawings

[0039] Various embodiments will now be described in more detail with reference to the accompanying Figures, in which:

[0040] Figure 1 shows schematically an analytical instrument in accordance with embodiments;

[0041] Figure 2 shows schematically detail of an analytical instrument in accordance with embodiments;

[0042] Figure 3 illustrates a workflow of soft PFAS ionisation in negative polarity, isolation, charge inversion followed by fragmentation of the PFAS;

[0043] Figure 4 shows an MS1 mass spectrum of Pierce™ FlexMix™ acquired in negative polarity using the instrument depicted in Figure 2; Figure 5 shows an MS2 mass spectrum of the isolated 318.9 m / z negative precursor ion after electron irradiation acquired in positive polarity using the instrument depicted in Figure 2;

[0044] Figure 6 shows an MS1 mass spectrum of perfluoro octanoic acid (PFOA) acquired in negative polarity using the instrument depicted in Figure 2;

[0045] Figure 7A shows an MS2 mass spectrum of the isolated 369 m / z negative precursor ion after electron irradiation acquired in positive polarity using the instrument depicted in Figure 2, and Figure 7B shows an MS2 mass spectrum of the isolated 369 m / z negative precursor ion after electron irradiation acquired in positive polarity using the instrument depicted in Figure 2;

[0046] Figure 8 shows an MS2 mass spectrum of the isolated 413 m / z negative precursor ion after electron irradiation acquired in positive polarity using the instrument depicted in Figure 2; and

[0047] Figure 9 shows MS2 mass spectra of the isolated 369 m / z negative precursor ion after electron irradiation acquired in positive polarity using the instrument depicted in Figure 2.

[0048] Detailed description

[0049] Figure 1 illustrates schematically an analytical instrument, such as a mass spectrometer, that may be used in conjunction with the methods described herein. As shown in Figure 1, the instrument includes an ion source 10, a mass filter 20, a fragmentation device 30, and a mass analyser 40.

[0050] The ion source 10 is configured to generate ions from a sample. The ion source 10 can be any suitable ion source, such as an electrospray ionisation (ESI) ion source, a chemical ionisation (Cl) ion source, or similar.

[0051] The mass filter 20 is arranged downstream of the ion source 10 and is configured to receive ions from the ion source 10. The mass filter 20 is configured to filter the received ions according to their mass to charge ratio (m / z). The mass filter 20 may be configured such that received ions having m / z within an m / z transmission window (or “isolation window”) of the mass filter are onwardly transmitted by the mass filter, while received ions having m / z outside the m / z transmission window are attenuated by the mass filter, i.e. are not onwardly transmitted by the mass filter. The width and / or the centre m / z of the transmission window may be controllable (variable), e.g. by suitable control of RF and / or DC voltage(s) applied to electrodes of the mass filter 20. Thus, for example, the mass filter 20 may be operable in a transmission mode of operation, whereby most or all ions within a relatively wide m / z window are onwardly transmitted by the mass filter 20, and a filtering mode of operation, whereby only ions within a relatively narrow m / z window (centred at a desired m / z) are onwardly transmitted by the mass filter 20. The mass filter 20 can be any suitable type of mass filter, such as a quadrupole mass filter.

[0052] The fragmentation device 30 is arranged downstream of the mass filter 20 and is configured to receive most or all ions transmitted by the mass filter 20. The fragmentation device 30 may be configured to selectively fragment some or all of the received ions, i.e. so as to produce fragment ions. The fragmentation device 30 may be operable in a fragmentation mode of operation, whereby most or all received ions are fragmented so as to produce fragment ions (which may then be onwardly transmitted from the fragmentation device 30), and a non-fragmentation mode of operation, whereby most or all received ions are onwardly transmitted without being (deliberately) fragmented. It would also be possible for a non-fragmentation mode of operation to be implemented by causing ions to bypass the fragmentation device 30. The fragmentation device 30 may also be operable in one or more intermediate modes of operation, e.g. whereby the degree of fragmentation is controllable (variable). The fragmentation device 30 can be any suitable type of fragmentation device, such as an electron induced dissociation (EID) fragmentation device, a laser, or similar.

[0053] The mass analyser 40 is arranged downstream of the fragmentation device 30 and is configured to receive ions from the fragmentation device 30. Thus, the mass analyser 40 may receive unfragmented precursor ions and / or fragment ions, depending on the mode of operation of the fragmentation device 30. The mass analyser 40 is configured to analyse the received ions so as to determine their mass to charge ratio (m / z) and / or mass, i.e. to produce a mass spectrum of the ions. The mass analyser 40 can be any suitable type of mass analyser, such as an ion trap mass analyser, an electrostatic orbital trap mass analyser (such as an Orbitrap™ FT mass analyser as made by Thermo Fisher Scientific), a time-of-flight (ToF) mass analyser such as a multi-reflecting time-of-flight (MR-ToF) mass analyser, or a quadrupole mass analyser. Numerous other types of mass analyser are possible.

[0054] In embodiments, the mass analyser is a High Resolution and Accurate Mass (“HRAM”) mass analyser. Thus, the mass analyser may have a mass accuracy < about 100 ppm, < about 50 ppm, < about 20 ppm, or < about 10 ppm, and a resolving power (at 200 m / z) > about 5,000, > about 10,000, > about 20,000, > about 50,000, or > about 100,000. It should be noted that Figure 1 is merely schematic, and that the instrument can, and in embodiments does, include any number of one or more additional components such as ion optical devices. For example, the instrument may include one or more ion transfer stage(s) arranged between any of the illustrated components, e.g. including an atmospheric pressure interface and / or one or more ion guides, lenses and / or other ion optical devices configured such that some or all of the ions can be transmitted appropriately through the instrument. The ion transfer stage(s) may include any suitable number and configuration of ion optical devices, for example optionally including one or more ion guides, lenses and / or other ion optical devices.

[0055] As also shown in Figure 1 , the instrument is under the control of a control unit 50, such as an appropriately programmed computer, which controls the operation of various components of the instrument and, for example, sets the voltages to be applied to the various components of the instrument. The control unit 50 may also receive and process data from various components including the analyser(s).

[0056] The instrument may be operable in various mode of operation. In particular, the instrument may be a tandem mass spectrometer operable in an MS1 mode of operation and an MS2 mode of operation.

[0057] In the MS1 (or “full mass scan”) mode of operation, the mass filter 20 is operated in its transmission mode of operation and the fragmentation device 30 is operated in its nonfragmentation mode of operation, e.g. so that a wide m / z range (e.g. full mass range) of unfragmented (“precursor” or “parent”) ions are analysed by the analyser 40 to produce an MS1 spectrum.

[0058] In the MS2 mode of operation, the mass filter 20 is operated in its filtering mode of operation and the fragmentation device 30 is operated in its fragmentation mode of operation, e.g. so that a selected narrow m / z range of precursor ions are fragmented and the resulting fragment (“product” or “daughter”) ions are analysed by the analyser 40 to produce an MS2 spectrum.

[0059] Figure 2 shows in more detail an example instrument that may be operated in accordance with embodiments. It will be understood that the instrument shown in Figure 2 is a non-limiting example, and that numerous variations are possible.

[0060] As shown in Figure 2, the instrument’s ion source 10 is an electrospray ionisation (ESI) ion source. The instrument includes a vacuum interface, which includes a transfer tube 21, an ion funnel 22, a quadrupole pre-filter ion guide 23, and a “bent flatapole” ion guide 24. The bent flatapole ion guide 24 may be of the design described in US patent No. 9,536,722.

[0061] The instrument also includes a mass filter in the form of a quadrupole mass filter 26, an ion trap 30a in the form of a curved linear ion trap (“C-Trap”), and a fragmentation device 30b in the form of an ion routing multipole (“I RM”). Ions from the ion source 10 can be accumulated in the C-Trap 30a and / or fragmentation device 30b by opening and closing a gating electrode located in a charge detector assembly 27, which is arranged between the C-Trap 30a and the mass filter 26.

[0062] The instrument also includes a mass analyser 40 in the form of an orbital ion trap mass analyser. As shown in Figure 2, the orbital trap 40 comprises an inner electrode 41 elongated along the orbital trap axis and a split pair of outer electrodes 42, 43 which surround the inner electrode 41 and define therebetween a trapping volume. The pair of outer electrodes 42, 43 also function as detection electrodes to detect an image current induced by the oscillation of the ions in the trapping volume and thereby provide a detected signal. The detected signal can be processed using Fourier transformation to obtain a mass spectrum of ions within the trap.

[0063] Once accumulated in the ion trap 30a and / or fragmentation device 30b, ions can be ejected into the mass analyser 40 for analysis. Ions collected in the ion trap 30a can either be ejected orthogonally to the mass analyser 40 without entering the fragmentation device 30b, or the ions can be transmitted axially to the fragmentation device 30b for processing before the processed ions are returned to the ion trap 30a for subsequent orthogonal ejection to the mass analyser 40. The processing may comprise, for example, fragmenting the ions in the fragmentation device 30b by interaction with electrons and / or photons, and / or collisions with a collision gas in the fragmentation device 30b (e.g. in the MS2 mode of operation), or further cooling the ions by collisions with a gas at lower energies that do cause the ions to fragment (e.g. in the MS1 mode of operation).

[0064] In Figure 2, the fragmentation device 30b is a higher collision energy dissociation (HCD) fragmentation device. The instrument has been modified for electron induced dissociation (EID) by the addition of an RF ion trap 31 having an electron gun adjacent to the HCD fragmentation cell 30b.

[0065] Embodiments provide a screening method for specific compound detection and quantitation by applying a selective and specific charge inversion. Particular embodiments provide a PFAS screening method. Per- and polyfluoroalkyl substances (PFAS) and compounds comprising a perfluoroalkylated substructure have been referred to be the “forever chemicals”, and are a group of nearly 15,000 synthetic organofluorine compounds. PFAS may be defined as a chemical substance that contains at least one of the following three substructures:

[0066] • R-(CF2)-CF(R')R", where both the CF2 and CF moieties are saturated carbons.

[0067] • R-CF2OCF2-R', where R and R' can either be F, O, or saturated carbons.

[0068] • CF3C(CF3)R'R", where R' and R" can either be F or saturated carbons.

[0069] Embodiments provide a non-targeted screening for PFAS compounds. The method uses a mass spectrometer that is configured to determine the mass to charge ratio (m / z) of ions in both (positive and negative) polarities. Embodiments of the method involve:

[0070] • Soft ionisation at atmospheric conditions of at least one PFAS compound having at least one perfluorated alkyl chain.

[0071] • Detection of the negatively charged precursor ion m / z and the fragment ions thereof produced from the at least one PFAS compound.

[0072] • Harsh interaction of the ionised PFAS compound with an energised electron beam, causing stripping of electrons from the ionised PFAS precursor ion, leading to a charge inversion and fragmentation of the PFAS ions releasing perfluoro-carbon daughter ions.

[0073] • Detection the of the positively charged daughter ions with a highly mass accurate and highly resolved (HRAM) mass spectrometer to enhance specificity by determining the accurate mass of the perfluorated compound.

[0074] Figure 3 shows schematically a workflow according to embodiments, comprising soft ionisation of a PFAS compound in negative polarity, isolation of a PFAS precursor ion, and charge inversion of the PFAS precursor ion, resulting in positively charged PFAS- characteristic fragments. The PFAS fragment ions are detected in positive ion mode. The interaction of the highly energetic electron(s) with a perfluorated alkyl backbone removes a binding electron and weakens specifically Carbon-Carbon bonds resulting in a positively charged perfluorated cation. The electron interaction is very fast and produces positively charged perfluorated alkyl chain fragments.

[0075] This charge inversion is highly selective for the PFAS compound class. All member of the PFAS class share the property of having a high electron density at the fluorated tail, and independent of the length or the size of the perfluorated alkyl chain, can be used confirm the presence of PFAS without the need for a PFAS-specific database. When the generated daughter ions are detected under highly accurate and highly resolved mass spectrometric conditions, the presence of PFAS can be even more reliably confirmed. The detected accurate masses are highly specific for PFAS compounds because of the combination of the number of pure isotopes of n-times Fluorine atoms (n-times 18.999 amu) and the Carbon atoms having atomic mass of m-times 12.000. The isotopic pattern of the PFAS-fragments is unique independent of the detected molecular mass, because the element Fluorine is isotopically pure and therefore the isotopic pattern of PFAS only includes the isotope of the number of Carbon atoms.

[0076] Experiments to investigate this charge inversion concept were performed using an Orbitrap™ Exploris™ 480 instrument equipped with an additional RF ion trap 31 with an electron gun, placed behind the fragmentation cell 30b. This is illustrated by Figure 2.

[0077] The instrument control software was modified to run a method comprising ionisation, charge inversion and detection of product ions with inversed charge. The target compound was ionised using electrospray ionisation (ESI) in negative polarity. The voltages of the ion optics were set for transmitting and isolating negatively charged ion towards the collision cell 30b.

[0078] Irradiation of the PFAS compound ions with electrons released from an electron gun was achieved in a modified collision cell. Product ions were subsequentially transferred to the Orbitrap™ mass analyser 40 for detection of the fragment ions in positive polarity.

[0079] Figure 4 shows a precursor ion mass spectrum of Pierce™ FlexMix™ ions acquired in negative polarity while the ion optics were tuned for the lower mass range with the modified Orbitrap™ Exploris™ instrument.

[0080] The spectrum provides a base peak at m / z 318.9 representing the perfluorated charged alkyl tail anion with formula C6F13 which is spontaneously built while being transferred throughout the ion optics from the ESI source 10 towards the C-Trap 30a and ejected to the Orbitrap™ mass analyser 40. The negatively charged precursor ion of such an organic acid is stabilised by the presence of several Fluorine atoms attached to the alkyl chain and favours a carbon dioxide loss from its negatively charged precursor ion at m / z

[0081] 362.9 (C7F13O2). The signal at m / z 362.9 itself can be assigned to the deprotonated perfluoro heptanoic acid which is a known compound from Pierce™ FlexMix™.

[0082] Figure 5 shows a mass spectrum acquired in positive polarity using the modified Orbitrap™ Exploris™ instrument of the isolated negatively charged precursor ions at m / z

[0083] 318.9 after electron irradiation. The C-Trap 30a and Orbitrap™ mass analyser 40 voltage settings in this experiment for the detection of the product ion spectrum were set for the exclusive transmission of positively charged ions. In the resulting product ion spectrum shown in Figure 5, several cations can be assigned as fragment ions of the precursor ion indicating the successful fragmentation of the precursor ion. The design of the experiment did not allow transmission of negatively charged ions.

[0084] Thus, in embodiments negatively charged precursor ions are generated by the ion source 10 and transmitted to the mass filter 26. The mass filter 26 is configured to isolate negatively charged precursor ions of interest, and the isolated negatively charged precursor ions of interest are transmitted to a fragmentation device 30b (via the C-trap 30a). The negatively charged precursor ions are trapped within the fragmentation device by RF pseudopotential barrier(s), and are fragmented by interaction with electrons while negatively charged precursor ions remain trapped within the fragmentation device. The resulting positively charged fragments ions are also trapped within the fragmentation device by the RF pseudopotential barrier(s). Thus, in embodiments, negatively charged precursor ions are trapped simultaneously with positively charged fragment ions in the same trapping volume (i.e. in the fragmentation device), e.g. by RF pseudopotential barrier(s). Next, the positively charged fragment ions are removed from the fragmentation device and are transmitted to the mass analyser 40 (via the C-trap 30a) for mass analysis.

[0085] Another PFAS sample, particularly perfluoro octanoic acid (PFOA), was measured and the charge inversion was repeated.

[0086] Figure 6 shows an MS1 mass spectrum obtained in negative polarity of perfluoro octanoic acid (PFOA, formula: C7F17COOH). The peak marked with a star is m / z 413 (PFOA), and the peak marked with a diamond is m / z 369 (PFOA minus CO2).

[0087] Figure 7A shows an MS / MS spectrum of m / z 369 in positive polarity after charge inversion and EID fragmentation. Figure 7B is identical to the Figure 7A but shows m / z labels with four digits. Figure 8 shows an MS / MS spectrum of m / z 413 in positive polarity after charge inversion and EID fragmentation.

[0088] Figure 9 shows a zoom into the MS / MS data showing the fragment m / z 281 , which is in fact m / z 280.9819. By applying high resolution mass analysis, this fragment can be differentiated from the siloxane fragment at m / z 281.0507. In addition, the presence of the13C isotope at m / z 281.9847 including the isotopic ratios of in the MS / MS data further confirms the presences of a perfluorated alkyl compound.

[0089] For all three MS / MS spectra, the obtained fragments follow a similar pattern: all analysed sample provide after charge inversion the fragments C4F7, C5F9, and C6F11. For the longer PFAS sample, the C7F13 is observed as well. The accurate mass difference between adjacent fragment ions is 49.997 amu reflecting the loss of CF2.

[0090] It will thus be appreciated that embodiments provide a method involving a charge inversion of PFAS compounds being ionised under soft ESI condition in negative polarity, and a compound-specific mass spectrometry detection of positively charged fragment ions originating from a perfluorinated alkyl chain via charge inversion. Embodiments provide selective detection, because the method not only ionises the compound of perfluorated alkyl but also converts the negatively charged precursor ions into positively charged fragment ions. Embodiments enable specific detection of the PFAS fragment ions in the presence of other ionised organic compounds on the assumption that only the perfluorated tail of PFAS result in the formation of positively charged fragment ions.

[0091] Embodiments provide a non-targeted screening for the identification of PFAS compounds or metabolites thereof. The accurate mass determination of (i) the negatively charged precursor ion and (ii) the positively charged fragment ion(s) enables the selective determination of the presence of PFAS in an analysed sample. The method allows for fast screening without the need for a baseline separated chromatographic separation of the PFAS.

[0092] The method can provide an approximate quantity of the PFAS compounds present in an analysed sample. While the method does not yield exact measurements, it offers results with a known threshold of certainty, thereby allowing for a reliable estimation within predefined confidence limits. If a known standard is analysed, the method offers the possibility to quantify the presence of the total amount of all PFAS without the need of detailed knowledge of the presence of the individual PFAS compounds.

[0093] The charge inversion and fragmentation use a well-focused beam of electrons with energies exceeding the binding anergy of the C-F or C-C bond. The ion-electron beam interaction is applied under vacuum in a discontinuous mode of operation. Charge inversion of the positively charged PFAS fragment ions can be executed under atmospheric conditions or in any other compartment throughout the ion transfer of the PFAS analyte in the mass spectrometer. Because the electron-ion interaction resulting in charge inversion of the precursor ion is fast and needs only a few microseconds, it can be applied in a run-through experiment.

[0094] The method can be particularly beneficial where PFAS precursor ions or anionic fragment ions are obscured by other anions, as positive PFAS fragment ions may still be clearly observed. In some embodiments, the EID operational parameters (i.e. electron energy, electron emission current and / or exposure time of the precursor ions to the electrons) are set to be more selective for PFAS compounds relative to other species.

[0095] Although embodiments have been described above with respect to EID fragmentation, it is believed that multiphoton laser excitation can also lead to positive fragment ions via the charge inversion process. In these embodiments, sufficient energy is given to the anion to remove an electron (i.e. an energy greater than or equal to the electron affinity) in order to ionise and fragment the resulting neutral molecule (i.e. ionisation energy plus fragmentation energy). This requires, in general, more than 10 eV. Considering a UV, visible or IR laser, each photon has less than 10 eV energy. Therefore multiphoton absorption may be used to generate positive fragments. In these embodiments, a laser may be used to provide a sufficiently high photon density. Multiphoton activation has the advantage that it can avoid potential neutralisation of cation product ions (i.e. where too many electrons overlap with a cation and neutralise it so that it can no longer be trapped), which can otherwise result in fewer product ions.

[0096] An alternative approach while using an electron gun is to apply a DC gradient along the RF ion guide so that the cation product ions are moved away from the electron beam.

[0097] The “charge inversion” process described herein may be applicable to compounds other than PFAS compounds. Advantageously, the “charge inversion” process allows for the production of characteristic positively charged fragment ions from certain compounds even while using electrospray ionisation (ESI) operating in its more sensitive negative ionisation mode. Thus, a second aspect is provided, as described above. The negatively charged precursor ions can be trapped simultaneously with the positively charged fragment ions of interest in the same trapping volume (i.e. in the fragmentation device), e.g. by an RF quasipotential (as described above).

[0098] Although the present invention has been described with reference to various embodiments, it will be understood that various changes may be made without departing from the scope of the invention as set out in the accompanying claims.

Claims

CLAIMS1. A method of determining the presence of a perfluoroalkyl or polyfluoroalkyl substance (PFAS) or a compound comprising a perfluoro-alkylated substructure in a sample, the method comprising: ionising a sample under negative soft ionisation conditions at atmospheric pressure to produce negatively charged precursor ions; fragmenting precursor ions by interaction with electrons and / or photons to produce positively charged fragments ions; mass analysing the positively charged fragments ions using a mass analyser so as to produce positive fragment mass spectral data; determining whether, in the positive fragment mass spectral data, an ion peak is present that correspond to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample; and when it is determined that an ion peak is present that corresponds to at least one positively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample: determining that a PFAS or a compound comprising a perfluoro-alkylated substructure is present in the sample.

2. The method of claim 1, wherein the method comprises simultaneously trapping negatively charged precursor ions with positively charged fragment ions in a fragmentation device.

3. The method of claim 1 or 2, wherein the method further comprises, before the step of fragmenting precursor ions: isolating negatively charged precursor ions having mass to charge ratios within a mass to charge ratio window centred at a mass to charge ratio of at least one negatively charged precursor ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure.

4. The method of claim 3, wherein the step of isolating negatively charged precursor ions comprises isolating negatively charged precursor ions using a mass filter such as a quadrupole mass filter.

5. The method of claim 3 or 4, wherein the method further comprises, before the step of isolating negatively charged precursor ions: mass analysing the negatively charged precursor ions using a mass analyser so as to produce negative precursor ion mass spectral data; and identifying an ion peak in the negative precursor ion mass spectral data that corresponds to the at least one negatively charged precursor ion indicative of the presence of a PFAS or a compound comprising a perfluoro-alkylated substructure in the sample.

6. The method of claim 5, wherein: the step of mass analysing negatively charged precursor ions is performed using a mass analyser operating in a negative polarity mode; and the step of mass analysing positively charged fragments ions is performed using the same mass analyser operating in a positive polarity mode.

7. The method of any one of the preceding claims, wherein a mass difference between fragment ions is consistent with an accurate mass of 49.997 indicating the presence of CF2.

8. The method of any one of the preceding claims, further comprising collisionally activating negatively charged precursor ions to produce at least one negatively charged fragment ion indicative of the presence of a PFAS or a compound comprising a perfluoro- alkylated substructure in the sample.

9. The method of any one of the preceding claims, wherein the mass analyser has a mass accuracy < about 100 ppm and a resolving power > about 10,000.

10. The method of any one of the preceding claims, wherein the mass analyser is an electrostatic orbital ion trap mass analyser, a Time-of-Flight (ToF) mass analyser, a multireflection Time-of-Flight (mrTOF) mass analyser, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyser.

11. The method of claim 10, wherein the mass analyser is an electrostatic orbital ion trap mass analyser.

12. The method of any one of the preceding claims, wherein the step of ionising comprises ionising the sample using electrospray ionisation (ESI) or chemical ionisation (Cl).

13. The method of any one of the preceding claims, wherein the step of fragmenting precursor ions by interaction with electrons comprises fragmenting the precursor ions using electron induced dissociation (EID).

14. The method of any one of the preceding claims, wherein the electrons have an energy exceeding the binding energy of the C-F bond and / or of the C-C bond.

15. The method of any one of the preceding claims, wherein the step of fragmenting precursor ions by interaction with photons comprises fragmenting the precursor ions using multiphoton laser excitation.

16. The method of any one of the preceding claims, wherein fragmentation operational parameters are set to be more selective for PFAS compounds and / or compounds comprising a perfluoro-alkylated substructure relative to other compounds.

17. The method of any one of the preceding claims, wherein the sample comprises a known concentration of a reference standard, and wherein the method further comprises: comparing an intensity of the at least one positively charged fragment ion to an intensity of at least one reference standard ion; anddetermining a concentration of PFAS or the compound comprising a perfluoroalkylated substructure present in the sample based on the comparison.

18. A method of determining the presence of a compound of interest in a sample, the method comprising: ionising a sample under negative soft ionisation conditions at atmospheric pressure to produce negatively charged precursor ions; fragmenting precursor ions by interaction with electrons and / or photons to produce positively charged fragments ions; mass analysing the positively charged fragments ions using a mass analyser so as to produce positive fragment mass spectral data; determining whether, in the positive fragment mass spectral data, an ion peak is present that correspond to at least one positively charged fragment ion indicative of the presence of the compound of interest in the sample; and when it is determined that an ion peak is present that corresponds to at least one positively charged fragment ion indicative of the presence of the compound of interest in the sample: determining that the compound of interest is present in the sample.

19. The method of claim 18, wherein the method comprises simultaneously trapping negatively charged precursor ions with positively charged fragment ions in a fragmentation device.

20. A non-transitory computer readable storage medium storing computer software code which when executed on a processor performs the method of any one of the preceding claims.

21. A control system for an analytical instrument, the control system configured to cause the analytical instrument to perform the method of any one of claims 1 to 19.

22. An analytical instrument, such as a mass spectrometer, comprising the control system of claim 21.

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