Detection of PFAS compounds in animal tissues

Abstract

A method of detecting one or more PFAS, e.g. perfluorobutanoic acid (PFBA), in a sample of animal tissue, comprises spraying electrically charged solvent droplets onto the tissue sample to generate an

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from and the benefit of United Kingdom patent application No. 2316090.6 filed on 20 October 2023. The entire contents of this application are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to the use of mass spectrometers for detecting Per-and polyfluoroalkyl substances. BACKGROUND Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic organofluorine chemical compounds that have been widely used since the mid-20th century. These substances have been found to take a long time to break down, and to have built up in the environment to the extent that they have been found to be present in animals and food products. This has led to health concerns for humans and other wildlife. SUMMARY From a first aspect the present invention provides a method of detecting one or more PFAS in a sample of animal tissue, comprising: spraying electrically charged solvent droplets onto the sample of animal tissue so as to generate analyte ions from analyte in the animal tissue; passing the analyte ions into a mass spectrometer; and mass analysing the analyte ions so as to determine if the one or more PFAS is in the animal tissue. Although it is known to detect PFASs using mass spectrometry, such techniques have been used to detect these substances when they are present at high concentrations. However, it is not expected that PFASs would have built up to such high concentrations during the lifetime of an animal. It has been found that the ionisation technique of the present invention is relatively efficient at generating ions of PFASs that are present in animal tissue, whilst providing relatively little background noise to the ion signal. This enables the PFASs to be detected in animal tissue even when they are present in the low concentrations that are expected to occur during the lifetime of the animal. The term PFAS, as used herein, is defined as a fluorinated substance that contains at least one fully fluorinated methyl or methylene carbon atom (without any H / CI / Br / l atom attached to it). For example, the PFASs detected according to the present invention may be chemicals having at least a perfluorinated methyl group (-CF3) or a perfluorinated methylene group (-CF2-), or may be chemicals having at least two adjacent carbon atoms, where one carbon is fully fluorinated and the other is at least partially fluorinated. For the avoidance of doubt, the term PFAS used herein refers to a per- or polyfluoroalkyl substance, whereas the term PFASs refers to multiple different per- or polyfluoroalkyl substances. The method may comprise using a Desorption Electrospray Ionisation (DESI) source to spray the electrically charged solvent droplets onto the sample of animal tissue. The sample of animal tissue may be a section of tissue (i.e. a layer sliced off the animal tissue to be analysed). The animal tissue may be a food product. The animal tissue may be pork, beef, chicken or fish. Said mass analysing may comprise mass analysing the analyte ions in a tandem quadrupole mass spectrometer that is set to mass analyse one or more MRM transition that would be expected to be observed if the one or more PFAS is present in the sample of animal tissue. Accordingly, the mass spectrometer may comprise: a first mass filter that is set to selectively transmit a precursor ions species; a fragmentation device that fragments the transmitted precursor ion species so as to form fragment ion species; a second mass filter that is set to selectively transmits one of the fragment ion species; wherein the precursor ion species is an ion species expected to be generated by said spraying step if said one or more PFAS is present in the sample, and the fragment ions species is an ion species expected to be generated in said fragmentation device by fragmenting the PFAS precursor ion species. The method may comprise detecting if one or more of multiple different PFASs are present in the sample of animal tissue, wherein the mass spectrometer mass analyses different MRM transitions that would be expected to be observed if different respective PFASs were present in the sample of animal tissue. The mass spectrometer may mass analyse a plurality of different MRM transitions that would be expected to be observed for each PFAS of said one or more PFAS, if said one or more PFAS were present in the sample of animal tissue. The method may comprise determining that the PFAS is present in the sample if ions are detected for at least one of the MRM transitions expected to be observed for that PFAS. The method may only determine that the PFAS is present in the sample if ions are detected for multiple MRM transitions expected for that PFAS. The one or more PFAS may only identified as being present in the sample if ions of that PFAS, or ions derived therefrom, are detected by the mass spectrometer with an intensity that is above a pre-selected threshold value; and / or the one or more PFAS may only be identified as being present in the sample if ions of that PFAS, or ions derived therefrom, are detected by the mass spectrometer with a signal to noise ratio that meets a threshold criterion. For example, the one or more PFAS may only be identified as being present in the sample if fragment ions for the MRM transition are detected by the mass spectrometer with an intensity that is above a pre-selected threshold value; and / or the one or more PFAS may only be identified as being present in the sample if fragment ions for the MRM transition are detected by the mass spectrometer with a signal to noise ratio that meets a threshold criterion. For instance, the PFAS may only be identified as being present in the sample if fragment ions for the MRM transition are detected by the mass spectrometer with a signal to noise ratio that is 1:3 or better. As described above, the present invention enables PFASs to be detected in animal tissue even when a PFAS is present at a relatively low concentration. Accordingly, the method may comprise detecting and identifying that the PFAS is present in the sample when it is present in the sample at a concentration that is at or below the concentration that would be present if the PFAS has been deposited onto the sample at a concentration of 200 pg / pL and the sample was otherwise free of the PFAS. Alternatively, the method may comprise detecting and identifying that the PFAS is present in the sample when it is present in the sample at a concentration that is at or below the concentration that would be present if the PFAS has been deposited onto the sample at a concentration of 150 pg / pL, 100 pg / pL, 80 pg / pL, 60 pg / pL, 40 pg / pL, 30 pg / pL, 10 pg / pL, 5 pg / pL, or 1 pg / pL. The method may comprise moving the spray of electrically charged solvent droplets relative to the sample of animal tissue, and generating an ion image of the sample that shows the locations in the sample at which the PFAS has been detected. The ion image may be generated by correlating the ion signal detected by the mass spectrometer for the PFAS ions (or ions derived therefrom, such as fragment ions) with the location on the sample at which the solvent droplets are being directed whilst the ion signal is detected. The ion image may also indicate the location of the animal tissue. For example, the step of generating the ion image may comprise correlating the ion signal detected by the mass spectrometer for a lipid or fatty acid (or ions derived therefrom, such as fragment ions) with the location on the sample at which the solvent droplets are being directed whilst the ion signal is detected. For example, the mass analysing step may comprise mass analysing the analyte ions in a tandem quadrupole mass spectrometer that is set to mass analyse one or more MRM transition that would be expected to be observed if the lipid or fatty acid is present in the sample of animal tissue. If the one or more PFAS is detected as being present in the sample, the method may display an indication on an electronic display that the one or more PFAS has been detected. The one or more PFAS may be any one, or any combination, of the substances in the group of: (i) Perfluorobutanoic Acid (PFBA); (ii) the ammonium salt of hexafluoropropylene oxide dimer acid (e.g. GenX); (iii) Perfluorobutane Sulfonic Acid (PFBS); (iv) Perfluorohexane Sulfonic Acid (PFHxS); (v) Perfluorooctanoic Acid (PFOA); (vi) Perfluorooctane Sulfonic Acid (PFOS); (vii) Perfluorotridecanoic acid (PFTriDA); (viii) Perfluoropentanoic Acid (PFPeA); (ix) Perfluorobutane sulfonamide (FBSA); (x) Perfluorohexanoic Acid (PFHxA); (xi) 4:2 Fluorotelomer Sulfonic Acid (4:2 FTS); (xii) Perfluoropentane Sulfonic Acid (PFPeS); (xiii) Perfluoroheptanoic Acid (PFHpA); (xiv) Perfluorohexane sulfonamide (FHxSA); (xv) 6:2 Fluorotelomer Sulfonic Acid (6:2 FTS); (xvi) Perfluorooctane sulfonaminde (FOSA); (xvii) Perfluoroheptane Sulfonic Acid (PFHpS); (xviii) Perfluorononanoic Acid (PFNA); (xix) Perfluorodecanoic Acid (PFDA); (xx) 8:2 Fluorotelomer Sulfonic Acid (8:2 FTS); (xxi) Perfluorononane Sulfonic Acid (PFNS); (xxii) N-Methylperfluorooctanesulfonamidoacetic acid (N-MeFOSAA); (xxiii) N-ethyl perfluorooctane sulfonamido acetic acid (N-EtFOSAA); (xxiv) Perfluorodecane Sulfonic Acid (PFDS); (xxv) perfluorododecanoic acid (PFDoDA). The method may comprise determining if the sample comprises any one, any subset, or all of the PFASs listed above. The method may comprise determining if the sample comprises the above listed PFASs by monitoring one or more of the MRM transitions shown in the drawings for that PFAS. For example, the method may comprise determining if PFBA is present in the sample by monitoring the MRM transition corresponding to precursor ions of PFBA having m / z=212.9 being fragmented to form fragment ions having m / z=169. Alternatively or additionally, the method may comprise determining if PFBA is present in the sample by monitoring the MRM transition corresponding to precursor ions of PFBA having m / z=212.9 being fragmented to form fragment ions having m / z=19. Although specific mass to charge ratios are given for the precursor and product ions in each of the MRM transitions listed herein, it will be appreciated that the precursor ion mass to charge ratio and / or the fragment ion mass to charge ratio that is monitored in each MRM transition may have a mass to charge ratio that is up to 1 Dalton (e.g. up to 0.5 Dalton) above or below the value listed. From a second aspect the present invention provides a method of screening a plurality of products containing animal tissue for the presence of one or more PFAS, the method comprising performing the method described hereinabove on each of the products. The method may be a high throughput screening method. The products may be food products; and / or the animal tissue may be pork, beef, chicken or fish. If said one or more PFAS is determined to be present in the product, the method may further comprise determining the concentration or total amount of each of the one or more PFAS in the product. If said one or more PFAS is not determined to be present in the product, the method may not subject the product to the step of determining the concentration or total amount of each of the one or more PFAS in the product. In other words, the method may only select products for the step of determining the concentration or total amount of each of the one or more PFAS in the product if said one or more PFAS is determined to be present in the product. The method may comprise using a mass spectrometer to detect ions of the one or more PFAS, or ions derived therefrom, and to quantify the number of ions detected; and determining the concentration or total amount of each of the one or more PFAS in the product based on the number of ions detected. The mass spectrometer may be the tandem mass spectrometer described herein. It has been recognised that mass spectrometry ionisation techniques other than DESI ionisation may be preferred for determining the concentration or total amount of each of the one or more PFAS in the product. Accordingly, for this step the method may comprise subjecting a sample of the product to an ionisation technique other than DESI, and mass analysing the resulting ions in the mass spectrometer so as to quantify the number of ions detected. The sample may be subjected to various preparation steps prior to being subjected to the ionisation technique. For example, the sample may be placed in a solvent and / or subjected to liquid chromatography prior to ionisation. However, it is contemplated that a DESI ionisation source may be used when quantifying the number of ions and determining the concentration or total amount of each of the one or more PFAS in the product based on the number of ions detected. Although embodiments have been described in which the sample is, or contains, animal tissue, it is also contemplated that the techniques described herein may be used to detect PFASs in other types of samples. Accordingly, from a third aspect the present invention provides a method of detecting one or more PFAS in a sample, comprising: spraying electrically charged solvent droplets onto the sample so as to generate analyte ions from analyte in the sample; passing the analyte ions into a mass spectrometer; and mass analysing the analyte ions so as to determine if the one or more PFAS is in the sample. The method may have any of the features described herein in relation to the first aspect of the present invention, except that the sample may be a sample other than animal tissue (i.e. it may or may not be or contain animal tissue). The sample may be a solid sample, e.g. a non-animal tissue such as from a plant or vegetable, or other foodstuff. Alternatively, the sample may be a liquid sample, e.g. a beverage such as water. A fourth aspect of the present invention provides a method of screening a plurality of samples for the presence of one or more PFAS, the method comprising performing the method described in relation to the third aspect of the invention on each of the samples. The method may have any of the features described herein in relation to the second aspect of the present invention, except wherein the plurality of products described in relation to the second aspect are, more generally, a plurality of samples (i.e. which may or may not contain animal tissue). For example, the samples may be liquid samples. The liquid samples may be spotted onto a slide, such as one or more glass slide, and subjected to the ionisation thereon. BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which: Figs. 1A and 1B illustrate how a DESI ion source ionises analyte in a sample; Fig. 2 shows a sample of animal tissue that has been spotted with different concentrations of PFASs; Figs. 3-5 show the results of experiments in which the sample shown in Fig. 2 was analysed by DESI imaging and tandem mass spectrometry; Fig. 6 shows a table summarising the results of the experiments shown in Figs. 3-5; and Fig. 7 shows the intensity at which the fragment ions where detected when analysing different concentrations of PFOS. DETAILED DESCRIPTION Embodiments of the present invention use desorption electrospray ionisation (“DESI”) mass spectrometry in order to mass analyse samples. Such techniques may be employed for direct analysis of a sample surface, and may be performed at ambient pressure, i.e. not under vacuum. This technique enables a sample to be analysed in its native state, with minimal or no prior sample preparation. As such, DESI techniques allow direct and fast analysis of samples. Figs. 1A and 1B illustrate how a DESI ion source ionises analyte in a sample. Fig. 1A shows the sprayer 1 of a DESI ion source arranged to ionise a sample 2 that is arranged on a sample slide 3. The sprayer directs a spray of electrically charged droplets 4 onto a surface of the sample. This is performed by supplying the sprayer with a solvent 5, and optionally also a nebulising gas 6 such as nitrogen, and a voltage from a voltage source 7. The solvent may be supplied to a central capillary of the sprayer, and the nebulising gas may be supplied to a surrounding capillary. The voltage supply is connected to the sprayer so as to electrify the solvent such that the ejected solvent droplets are electrically charged. As shown in Fig. 1B, the charged droplets impact 4 on the sample surface and cause molecules 8 in the sample to be desorbed and ionised so as to form desorbed analyte ions 9. Referring back to Fig. 1A, these ions travel into an atmospheric pressure interface 10 of a mass spectrometer, e.g. via a transfer capillary 11, which may be heated. According to embodiments of the present invention, the analyte ions are mass analysed by tandem mass spectrometry, also known as MS / MS. In such techniques the mass spectrometer may be operated so as to monitor for one or more Multiple Reaction Monitoring (MRM) transitions. When monitoring for an MRM transition a first mass filter is set to isolate a particular mass to charge ratio that is intended to correspond to a particular precursor ion species, and to transmit it to a fragmentation device that fragments these precursor ions to form fragment ion species. A second mass filter is set to isolate a particular mass to charge ratio that is intended to correspond to a particular fragment ion species, and to transmit those ions to an ion detector. If ions are detected then it can be determined with relatively high confidence what the precursor ions species is and therefore what is in the sample. In embodiments of the present invention, a DESI ion source is used to ionise analyte present in a section of porcine liver tissue, and a tandem mass spectrometer is used to determine if PFASs are present in the tissue. In order to evaluate the limit of detection of several PFASs, a generic multi-PFAS standard was spotted onto a porcine liver tissue section, which was then analysed in an ion imaging mode using a DESI XS ion source coupled with a Xevo (TM) TQ Absolute mass spectrometer, both from Waters Corporation. The PFASs standard contained the PFASs that are regulated, wherein the different PFASs were present in the standard at the same concentration. The standard was then diluted using PFAS-free solvent, so as to prepare five samples having different concentrations of each PFAS, i.e. 100 pg / pL, 50 pg / pL, 10 pg / pL, 5 pg / pL, and 1 pg / pL. The samples having different concentrations where then spotted onto different locations on the tissue section, as shown in Fig. 2. Prior to analysis of the tissue section, PFAS-free methanol was used to clean the surfaces upstream of the mass spectrometer inlet orifice that come into contact with the sample. For example, the glass side on which the tissue section is placed and the movable DESI stage were cleaned with PFAS-free methanol. The cryostat used to prepare the tissue section was also cleaned with PFAS-free methanol prior to use. The glass slide with the tissue section mounted thereon was then loaded onto the movable DESI stage and analysed in a negative ion mode. The DESI source was operated by spraying a solvent solution of 95:5 MeOH:H2O at a flow rate of 2 pL / min. PFAS-free methanol was used as the solvent for the DESI source. The DESI stage was moved beneath the DESI sprayer such that the DESI sprayer was scanned across the tissue section so as to desorb analyte ions from the tissue section. The analyte then passed into a capillary transfer capillary that was heated to 100 degrees centigrade, which conveyed the analyte ions to the inlet orifice of the mass spectrometer. The mass spectrometer operated in a tandem quadrupole (MSMS) mode so as to scan the quadrupole mass filters such that 20 MRM transitions were monitored in each scan. Five such scans were performed per second, although it will be appreciated that a different number of MRM transitions and / or different scan durations could be used. The intensities of the fragment ions that were transmitted by the second quadrupole were measured and these values were assigned to the locations on the tissue section that their precursor ions were generated, i.e. based on their time of detection and the position of the sample stage at that time, so as to generate an ion image. The spectrometer was operated such that the pixel size in each image was 50 pm. Fig. 3 shows the results from a first experiment in which the section of tissue shown in Fig. 2 was analysed by DESI imaging and tandem mass spectrometry (i.e. MS / MS). Fig. 3 shows 18 different ion images of the same section of tissue, where the different ion images represent the intensities detected for different, respective MRM transitions. More specifically the different ion images illustrate the positions on the tissue section at which different, respective fragment ions were detected as having originated from. The MRM transition illustrated for any given one of the ion images is listed above that image, and the greater that the brightness in each image, the higher the intensity with which ions were detected. For example, the image in the top left corner of Fig. 3 illustrates the intensity of ions detected across the tissue section for the MRM transition in which ions having a mass to charge ratio of 212.9 (i.e. PFBA ions) were fragmented to form ions having a mass to charge ratio of 169. The level of brightness in this image indicates the intensity with which the m / z=169 fragment ions were detected. Similarly, the second image down in the leftmost column of images represents the intensity of ions detected across the tissue section for the MRM transition in which ions having a mass to charge ratio of 212.9 (i.e. PFBA ions) were fragmented to form ions having a mass to charge ratio of 19. The third image down in the left-most column of images represents the intensity of ions detected across the tissue section for the MRM transition in which ions having a mass to charge ratio of 285 (i.e. GenX ions) were fragmented to form ions having a mass to charge ratio of 169. The fourth image down in the left-most column of images represents the intensity of ions detected across the tissue section for the MRM transition in which ions having a mass to charge ratio of 285 (i.e. GenX ions) were fragmented to form ions having a mass to charge ratio of 119. The bottom ion image in the right-most column illustrates the detection of m / z= 303.25 fragment ions that were generated by fragmenting m / z=885.6 precursor ions. This transition was selected as these fragment ions are ions that would be derived from a lipid that is expected to be in the tissue section, and which therefore helps spatially map the tissue section. Similarly, the MRM transition illustrated in the penultimate ion image in the right-most column was selected to help spatially map the presence of a fatty acid that is expected to be present in the tissue section. This image illustrates the detection of m / z=283.4 fragment ions that were generated by fragmenting m / z=327.2 precursor ions. Fig. 4 shows the results from a second experiment that was the same as that described in relation to Fig. 3, except that different MRM transitions for the PFASs were analysed. Fig. 4 shows the ion images for 16 different PFAS MRM transitions, and also shows ion images for the lipid and fatty acid MRM transitions. Fig. 5 shows the results from a third experiment that was also the same as that described in relation to Figs. 3 and 4, except that different MRM transitions for the PFASs were analysed. Fig. 5 shows the ion images for 18 different PFAS MRM transitions. Ion images for the lipid and fatty acid MRM transitions are not shown in Fig. 5. Fig. 6 shows a table summarising the results of the experiments shown in Figs. 3-5. In particular the first column indicates the PFAS analysed by each MRM transition. The second column indicates the mass to charge ratio of the precursor ion species for each MRM transition, and the third column indicates the mass to charge ratio of the fragment ion species for each MRM transition. The fourth column indicates the limit of detection of the DESI technique for each PFAS. The limit of detection listed for each PFAS is the lowest of the sample concentrations that were spotted onto the tissue section, as shown in Fig. 1, but for which an ion signal is detected. The fifth column in the table indicates the signal to noise ratio for the concentration that was identified as being the limit of detection. The sixth column indicates the next highest concentration, of those concentrations shown in Fig. 1, that is above the concentration identified as being the limit of detection. The final column in the table indicates the signal to noise ratio for the concentration in the sixth column. This data may be useful for determining the sample concentrations that meets the limit of quantification, which may be determined to be sample concentrations that provide a signal to noise that meets a certain criterion, such as being 1:10 or better. The table shows that PFTrDS was not detected in any of the MRM transitions monitored at any of the concentrations spotted onto the tissue section, although other MRM transitions and / or concentrations would be expected to detect PFTrDS. The table also shows that FHxSA was not detected at concentrations below 50 pg / pL that were spotted on tissue. PFBA, PFPeA, FBSA and FOSA were detected in the 10 pg / pL and / or 50 pg / pL spots. The remaining PFSA compounds were detected in the 1 pg / pL and / or 5 pg / pL spots. Some PFAS results showed almost no background signal. Fig. 7 shows the intensity at which the fragment ions where detected when analysing different concentrations of PFOS, each at the MRM transition corresponding to m / z= 498.9 precursor ions being fragmented to form m / z=99 fragment ions. Three sets of peaks are shown corresponding to three scans of the DESI ion source over the sample. As reported in Fig. 6, PFOS was detected at a concentration of only 5 pg / pL and at a signal to noise ratio of 1:8. This shows that the DESI technique is able to meet the limit of detection at a low concentration of PFOS. Similarly, the table shows that PFOS was detected at a concentration of only 10 pg / pL with a signal to noise ratio of 1:20, which shows that the DESI technique is able to meet the limit of detection at a relatively low concentration of PFOS. This is a significant improvement over known mass spectrometry techniques for identifying PFAS compounds. For example, when using MALDI mass spectrometry the limit of detection has been shown to be 100 pg / pL and the limit of quantification has been shown to be 1000 pg / pL, e.g. see Yang, Chunxue, et al. "In situ detection and imaging of PFOS in mouse kidney by matrix-assisted laser desorption / ionization imaging mass spectrometry", Analytical chemistry 91.14 (2019): 8783-8788. For this reason, mass spectrometry technique have been used to examine PFAS compounds at high concentrations, such as 1500 to 150000 pg / pL, e.g. see Bian, Yu, et al. "Tissue distribution study of perfluorooctanoic acid in exposed zebrafish using MALDI mass spectrometry imaging", Environmental Pollution 293 (2022): 118505. The PFASs have been described herein using their commonly known abbreviations. However, for the avoidance of doubt their full chemical names and formulae are provided below: PFBA: Perfluorobutanoic acid PFPeA: Perfluoro-pentanoic acid FBSA: Perfluorobutane sulfonamide PFBS: Perfluorobutanesulfonic acid PFHxA: Perfluorohexanoic acid 4:2 FTS: 4:2 Fluorotelomer sulfonic acid 6:2 FTS: 6:2 fluorotelomer sulfonic acid R 8:2 FTS: 8:2 Fluorotelomer sulfonic acid F PFPeS: Perfluoropentane sulfonic acid PFHpA: perfluoroheptanoic acid: FHxSA: Perfluorohexanesulfonamide 10 PFOA: Perfluorooctanoic acid F Ff I PFHpS: Perfluoroheptanesulfonic acid 15 F; F I XY-’ / \ PFNA: Perfluorononanoic acid 20 FOSA: Perfluorooctanesulfonamide F F F F ,F 0 v* ?*■ M J* F F E p F F F F V V V V P F 7<;Sx p p p p p F 0 w F F F F F F F F F F F F p F F F F F F X X XXX Y Y' Y X” 25 X v >• X \s / SS X >' OH PFOS: Perfluorooctanesulfonic acid P P P P P E P PO PFDA: Perfluorodecanoic acid F F F I F' PFNS: Perfluorononanesulfonic acid s p' p p sr 0 'X X X A X X X X C’H X F F / >fX ■\FEFEFEFEF V V V V V P pp pp pp pp pp / / 7\ 7\ A 7k / X. z\ / \ / \ / \ / / qh FFFFFFFFO N-MeFOSAA: N-Methylperfluorooctanesuifonamidoacetic acid N~EtFOSAA: N-ethyl perfluorooctane sulfonamide acetic acid R F R. F F. F E F F F F F F F F F O I i| CHS O f. F R F E F Fx F k X.XL X.xX X XXX X / / S-N^( k 0 'CH;} DPnQ1 Dzarfh dfrtns / ' JH E F E F E F E F F. F xxxXXx / X A A X A a Xh MM. M. KM pM. MMM MK MM. MM M- MMK > o / mH H r- H r* F r- H H H f } F f \ / A’l PFDoDA: perfluorododecanoic acid F F F F F F F F F F V V X X I XXX X X "0H F F F F F F F F F PFTriDa: Perfluorotridecanoic acid F F F F F F F FF FF F PFTrDS: PerfiuoroWdecane sulfonic add ssssssssssss ......... Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims. For example, although the PFASs have been described as being detected by monitoring MRM transitions in a tandem quadrupole mass spectrometer, it is contemplated that the PFAS ions may be detected via other forms of mass spectrometry. For example, a Time-of-Flight mass analyser may be used to detect the PFAS precursor and / or fragment ions. Although the use of DESI mass spectrometry to detect PFASs in animal tissues has been described, it is contemplated that such techniques may alternatively be used to detect PFASs in other types of samples, such as samples of other biological material such as plants. Additionally, or alternatively, the sample may be a solid or liquid sample.

Claims

1. A method of detecting one or more PFAS in a sample of animal tissue, comprising: spraying electrically charged solvent droplets onto the sample of animal tissue so as to generate analyte ions from analyte in the animal tissue;passing the analyte ions into a mass spectrometer; andmass analysing the analyte ions so as to determine if the one or more PFAS is in the animal tissue.

2. The method of claim 1, comprising using a Desorption Electrospray Ionisation (DESI) source to spray the electrically charged solvent droplets onto the sample of animal tissue.

3. The method of claim 1 or 2, wherein the animal tissue is a food product.

4. The method of claim 1, 2 or 3, wherein the animal tissue is pork, beef, chicken orfish.

5. The method of any preceding claim, wherein said mass analysing comprises mass analysing the analyte ions in a tandem quadrupole mass spectrometer that is set to mass analyse one or more MRM transition that would be expected to be observed if the one or more PFAS is present in the sample of animal tissue.

6. The method of any preceding claim, comprising detecting if one or more of multiple different PFASs are present in the sample of animal tissue, wherein the mass spectrometer mass analyses different MRM transitions that would be expected to be observed if different respective PFASs were present in the sample of animal tissue.

7. The method of any preceding claim, wherein the mass spectrometer mass analyses a plurality of different MRM transitions that would be expected to be observed for each PFAS of said one or more PFAS, if said one or more PFAS were present in the sample of animal tissue.

8. The method of claim 5, 6 or 7, comprising determining that the PFAS is present in the sample if ions are detected for at least one of the MRM transitions expected to be observed for that PFAS.

9. The method of any preceding claim, wherein the one or more PFAS is only identified as being present in the sample if ions of that PFAS, or ions derived therefrom,are detected by the mass spectrometer with an intensity that is above a pre-selected threshold value; and / orwherein the one or more PFAS is only identified as being present in the sample if ions of that PFAS, or ions derived therefrom, are detected by the mass spectrometer with a signal to noise ratio that meets a threshold criterion.

10. The method of any preceding claim, comprising detecting and identifying that the PFAS is present in the sample when it is present in the sample at a concentration that is at or below the concentration that would be present if the PFAS has been deposited onto the sample at a concentration of 200 pg / pL and the sample was otherwise free of the PFAS.

11. The method of any preceding claim, comprising moving the spray of electrically charged solvent droplets relative to the sample of animal tissue, and generating an ion image of the sample that shows the locations in the sample at which the PFAS has been detected.

12. The method of any preceding claim, wherein if the one or more PFAS is detected as being present in the sample, the method displays an indication on an electronic display that the one or more PFAS has been detected.

13. The method of any preceding claim, wherein the one or more PFAS is any one, or any combination, of the substances in the group of: (i) Perfluorobutanoic Acid (PFBA); (ii) the ammonium salt of hexafluoropropylene oxide dimer acid (e.g. GenX); (iii) Perfluorobutane Sulfonic Acid (PFBS); (iv) Perfluorohexane Sulfonic Acid (PFHxS); (v) Perfluorooctanoic Acid (PFOA); (vi) Perfluorooctane Sulfonic Acid (PFOS); (vii) Perfluorotridecanoic acid (PFTriDA); (viii) Perfluoropentanoic Acid (PFPeA); (ix) Perfluorobutane sulfonamide (FBSA); (x) Perfluorohexanoic Acid (PFHxA); (xi) 4:2 Fluorotelomer Sulfonic Acid (4:2 FTS); (xii) Perfluoropentane Sulfonic Acid (PFPeS); (xiii) Perfluoroheptanoic Acid (PFHpA); (xiv) Perfluorohexane sulfonamide (FHxSA); (xv) 6:2 Fluorotelomer Sulfonic Acid (6:2 FTS); (xvi) Perfluorooctane sulfonaminde (FOSA); (xvii) Perfluoroheptane Sulfonic Acid (PFHpS); (xviii) Perfluorononanoic Acid (PFNA); (xix) Perfluorodecanoic Acid (PFDA); (xx) 8:2 Fluorotelomer Sulfonic Acid (8:2 FTS); (xxi) Perfluorononane Sulfonic Acid (PFNS); (xxii) N-Methylperfluorooctanesulfonamidoacetic acid (N-MeFOSAA); (xxiii) N-ethyl perfluorooctane sulfonamido acetic acid (N-EtFOSAA); (xxiv) Perfluorodecane Sulfonic Acid (PFDS); (xxv) perfluorododecanoic acid (PFDoDA).

14. A method of screening a plurality of products containing animal tissue for the presence of one or more PFAS, the method comprising performing the method of any preceding claim on each of the products.

15. The method of claim 14, wherein the products are food products; and / or wherein the animal tissue is pork, beef, chicken or fish.

16. The method of claim 14 or 15, wherein if said one or more PFAS is determined to be present in the product, the method further comprises determining the concentration or total amount of each of the one or more PFAS in the product.

17. The method of claim 16, comprising using a mass spectrometer to detect ions of the one or more PFAS, or ions derived therefrom, and to quantify the number of ions detected; and determining the concentration or total amount of each of the one or more PFAS in the product based on the number of ions detected.

18. The method of claim 17, comprising subjecting a sample of the product to an ionisation technique other than DESI, and mass analysing the resulting ions in the mass spectrometer so as to quantify the number of ions detected.

19. A method of detecting one or more PFAS in a sample, comprising:spraying electrically charged solvent droplets onto the sample so as to generate analyte ions from analyte in the sample;passing the analyte ions into a mass spectrometer; andmass analysing the analyte ions so as to determine if the one or more PFAS is in the sample.

20. A method of screening a plurality of samples for the presence of one or more PFAS, the method comprising performing the method of claim 19 on each of the samples.Application No: GB2415480.9Claims searched: 1 to 20Examiner:Geoff HolmesDate of search: 14 March 2025Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X 1,2, 9, 11-14, 16, 17, 19 & 20 at least Environmental Science &Technology Vol. 54, No. 10, 23 April 2020, TT Wang et al., "Uptake and Translocation of Perfluorooctanoic Acid (PFOA) and Perfluorooctanesulfonic Acid (PFOS) by Wetland Plants: Tissue- and Cell-Level Distribution Visualization with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) and Transmission Electron Microscopy Equipped with Energy Dispersive Spectroscopy (TEM-EDS)", pages 6009-6020; see column 1 on page 6011 in particular v A 1,2, 9, 11-14, 16, 17, 19 & 20 at least Environment International Vol.

156. May 2021, W Mei et al., "Per- and polyfluoroalkyl substances (PFASs) in the soil-plant system: Sorption, root uptake, and translocation", pages 106642 1-9; see section 3.1.2 on page 5 X 1,2, 9, 11-14, 16, 17, 19 & 20 at least CN 107121487 A [CHINESE ACADEMY OF INSPECTION &QUARANTINE] see Figure 2 ¶graphs 16 &17 in particularCategories:X Document indicating lack of novelty or inventive step. A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family. E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:Search of GB, EP, WO &US patent documents classified in the following areas of the UKCX:Worldwide search of patent documents classified in the following areas of the IPC:____________ H01JThe following online and other databases have been used in the preparation of this search report: SEARCH-PATENT, SEARCH-NPLInternational Classification:Subclass Subgroup Valid From HOU 0049 / 00 01 / 01 / 2006 HOU 0049 / 16 01 / 01 / 2006

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