Ion mobility resolved isotope pattern analysis

Abstract

A method of combined ion mobility and mass spectrometry analysis comprises the steps of generating analyte ions, introducing the analyte ions into an ion mobility separator and into a mass analyzer according to their ion mobility and carrying out at least one mass spectrometry measurement on said analyte ions, whereby a mass spectrum comprising m / z-values and associated intensities of the analyte ions is obtained, obtaining a mobility-integrated mass spectrum, convoluting the mobility-integrated mass spectrum with at least one reference isotope pattern being indicative of isotopes of at least one compound of interest and / or at least one compound class of interest, whereby a quantitative measure for the presence of said reference isotope pattern in the mobility-integrated mass spectrum is obtained, and wherein the presence of the reference isotope pattern in the mobility-integrated mass spectrum is indicative of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample.

Description

[0001] TITLE

[0002] ION MOBILITY RESOLVED ISOTOPE PATTERN ANALYSIS

[0003] TECHNICAL FIELD

[0004] The present invention relates to a method of combined ion mobility and mass spectrometry analysis according to claim 1 , to the use of such a method for identifying a target compound in a sample and / or for the analysis of a complex sample according to claim 17, and to an apparatus for carrying out said method according to claim 18.

[0005] PRIOR ART

[0006] US 7,838,826 B1 (M. A. Park, 2008) and the corresponding patent family members presents a small ion mobility analyzer / spectrometer which has become known under the acronym “TIMS” analyzer / spectrometer (TIMS = trapped ion mobility spectrometry). The terms ion mobility analyzer and ion mobility spectrometer are used interchangeably here. A TIMS analyzer comprises a gas flow that drives ions against a counter-acting electric field barrier such that the ions are at first trapped along the axis of the TIMS analyzer. The ions are confined in the radial direction by an electric RF field. After transferring ions from an ion source to the electric field barrier, the height of the electric field barrier or the gas velocity is adjusted such that ion species are released from the electric field barrier in the sequence of their mobility.

[0007] Commonly, the length of the ion mobility separation unit of a TIMS analyzer amounts to about five centimeters only. In a small tube with an inner diameter of about eight millimeters, a radial RF quadrupole field is generated to hold ions near to the axis. A gas flow inside a tube drives ions entrained in the gas flow against a ramped counter-acting electric DC field barrier where the ions are trapped and separated according to their mobilities at locations on the field ramp at which the friction force of the moving gas equals the counter-acting force of the electric DC field on the ramp. After loading the TIMS with ions, the height of the electric DC field barrier is decreased; this scan releases the ion species in the sequence of their mobility. Unlike many other trials to build small ion mobility spectrometers, the small device by M. A. Park has already achieved, with reduced scan speeds, ion mobility resolutions up to RmOb = 400, which is extraordinarily high.

[0008] Regarding the theoretical basis of TIMS, see the research article “Fundamentals of Trapped Ion Mobility Spectrometry”, K. Michelmann, J. A. Silveira, M. E. Ridgeway and M. A. Park, J. Am. Soc. Mass Spectrom., (2015) 26: 14-24.

[0009] US 8,766,176 B2 proposes improvements of the scan modes for TIMS analyzers by application of non-linear scans to achieve a linear mobility scale, a constant resolution along the mobility scale, or a temporal zoom. Furthermore, US 9,984,864 B2 describes a spatial zoom.

[0010] A TIMS analyzer with parallel ion accumulation is described in US 9,683,964; it improves the utilization of the ions from the ion source to nearly 100%. Importantly, many ions sources, such as electrospray ion sources produce ions continuously. TIMS with parallel accumulation can also be operated to collect and analyze ions continuously - that is, TIMS can operate at 100% duty cycle. Barring pseudopotential or space charge effects, substantially all ions of the ion source are collected and analyzed without loss. TIMS with parallel ion accumulation further provides the possibility to prolong the ion accumulation and scan duration, thereby increasing the ion mobility resolution so as to separate and detect more ion species.

[0011] The ions are collected in an accumulator unit, preferably almost identical to the scanning unit, at a ramp of an electric DC field barrier such that they get spatially separated by their ion mobility along the ramp. Therefore, the accumulated ions are less influenced by space charge than in other types of accumulator units. Of greatest importance, however, is the unique feature of a TIMS analyzer that a longer accumulation period permits to increase the mobility resolution by choosing correspondingly longer mobility scan durations, e.g. 100 milliseconds scan duration with an ion mobility resolution of RmOb = 75 instead of 20 milliseconds scan duration with RmOb = 30. As a consequence of the higher number of ions collected and the better ion mobility resolution, more ion species can be detected and measured. Once an ion mobility scan is completed (optionally after twenty to some hundred milliseconds), the accumulated ions are transferred (in about a millisecond) from the accumulation unit to the scanning unit, and the next ion mobility scan can be started. In total, a skilled practitioner will appreciate that it will be possible to achieve a measurement rate of 300 to 450 ion species per second. If TIMS with parallel ion accumulation is installed in tandem mass spectrometer (MS / MS instrument) an MS-MS instrument, 300 to 450 characteristic fragment ion spectra per second may be measured quantitatively.

[0012] Some improvements for higher amounts of stored ions in selected regions of ion mobility, particularly for ions of low ion mobility, are given in US 9,304,106 B1. The higher loading capacity is based on non-linear electric DC field ramps, with flatter field ramps for ion species of interest, in order to diminish the effect of space charge for these ion species. But for precise ion mobility analyses of low abundant ion species in complex mixtures the influence of the space charge is still high.

[0013] TIMS extends conventional liquid chromatography-mass spectrometry (LC-MS) and bioanalytical workflows, such as proteomic, lipidomic, metabolomic, drug metabolism, structure elucidation or other methods, with an additional ion mobility dimension. In addition to the general benefits of the additional signal separation, the implementation of TIMS in the Bruker timsTOF family of instruments has demonstrated that collisional cross section (CCS) values of analytes, i.e. peptides, lipids, or metabolites, are highly reproducible. The additional separation dimension already allows in data dependent acquisition (DDA) methods to identify precursors of isomeric compounds to be scheduled for fragmentation individually. As omics workflows aim at covering as much of the chemical complexity of a sample with tandem-MS spectra by DDA, TIMS can already greatly improve the analytical coverage of complex samples.

[0014] WO-A-2019096852 relates to use of an isobaric label in mass spectrometry (MS) analysis using data-independent acquisition (DIA), wherein said isobaric label comprises or consists of a group which fragments in the mass spectrometer (i) at an energy below the energy required for fragmenting analyte-derived precursor ions and / or a higher conversion rate than said precursor ions; and (ii) at said energy according to (i) and when coupled to a precursor ion, at a single site within said group, to yield a first moiety and a second moiety, said second moiety being coupled to said precursor ion. It proposes the use of a trapped ion mobility spectrometry- time of flight (timsTOF) instrument, equipped with parallel / serial fragmentation (PASEF); see, e.g. Meier et al. 2015, doi: 10.1021 / acs.jproteome.5b00932.

[0015] US-A-2019371585 relates to selection of precursors from a measured mobility-mass map for tandem mass spectrometry and is based on processing a peak list from measured signals and clustering these peaks in the mobility-mass space.

[0016] US-A-2022034840 discloses an apparatus and a method of data independent combined ion mobility and mass spectroscopy analysis which includes introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectroscopy measurement on said fragment ions. The IMS and mass filter are controlled in a synchronized manner to carry out a plurality of IM scans, wherein adjacent mass windows in said IM scan that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane.

[0017] US-A-2022034840 discloses an apparatus and a method of data independent combined ion mobility and mass spectroscopy analysis which includes introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectroscopy measurement on said fragment ions. The IMS and mass filter are controlled in a synchronized manner to carry out a plurality of IM scans, wherein adjacent mass windows in said IM scan that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane.

[0018] For metabolism or biomarker studies, it is important to be able to trace the path of biochemical reactions in complex biological samples. This is especially relevant in the context of drug molecule development. For approval by authorities, all metabolites of a drug need to be identified, even if only very low abundant. Therefore, sensitivity and specificity of analytical methods become important.

[0019] SUMMARY OF THE INVENTION

[0020] It is an object of the present invention to provide an analysis method that allows to analyse an analyte with enhanced sensitivity and specificity.

[0021] This object is achieved with a method according to claim 1. That is, a method of combined ion mobility and mass spectrometry analysis is provided, wherein said method comprises the steps of:

[0022] (i) Generating analyte ions from one or more analytes in a sample with an ion source;

[0023] (ii) Introducing the analyte ions into an ion mobility separator and separating the analyte ions according to their ion mobility, whereby ion-mobility-signals of the analyte ions are obtained;

[0024] (iii) Introducing the analyte ions into a mass analyzer according to their ion mobility, and carrying out at least one mass spectrometry measurement on said analyte ions, whereby a mass spectrum comprising m / z-values and associated intensities of the analyte ions is obtained;

[0025] (iv) summing the intensities of the mass spectrum of the analyte ions for each m / z-value across all ion mobility signals, whereby a mobility-integrated mass spectrum comprising accumulated intensities of the analyte ions for each m / z-value is obtained, and

[0026] (v) convoluting the mobility-integrated mass spectrum with at least one reference isotope pattern being indicative of isotopes of at least one compound of interest and / or at least one compound class of interest, whereby a quantitative measure for the presence of said reference isotope pattern in the mobility-integrated mass spectrum is obtained, and wherein the presence of the reference isotope pattern in the mobility-integrated mass spectrum is indicative of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample. That is, the quantitative measure is indicative of the presence of at least one compound of interest and / or at least one compound class of interest in the sample.

[0027] That is, a comparison between an isotope pattern being indicative of isotopes of at least one compound of interest and / or of at least one compound class of interest with data obtained from a combination of ion-mobility separation and mass spectrometry allows the analysis, in particular an identification and / or a screening of said at least one compound of interest and / or said at least one compound class of interest and / or their metabolites, fragments, etc. (see further below).

[0028] In particular, in the present invention the mass spectral information obtained from the mass spectrometry measurement on the analyte ions is analyzed by summing the intensities across all ion mobility signals resulting in a mass spectrum with accumulated intensities across the ion mobility dimension for each m / z signal, i.e. the mobility-integrated mass spectrum is obtained. Said resulting mobility-integrated mass spectrum is then convoluted with a reference isotope being indicative of isotopes of at least one compound of interest and / or at least one compound class of interest. Thereby, a quantitative measure for the presence of the reference isotope pattern in said mobility-integrated mass spectrum is generated.

[0029] Said quantitative measure is indicative of the presence of compounds of interest and / or of compound classes of interest matching the isotope reference pattern in the sample while reducing false positive findings from overlapping isotopic patterns of isobaric compounds with different ion mobilities. The quantitative measure is preferably based on a comparison between the accumulated intensities of the mobility-integrated mass spectrum and intensities at specified mass differences of the isotope pattern.

[0030] In particular, the quantitative measure is preferably determined by summing intensities from the mobility-integrated mass spectrum only for m / z-values where a normalized intensity difference Idbetween the measured intensities of the analyte ions and intensities at specified mass differences of the isotope pattern is less than a tolerance threshold It.

[0031] The accumulated intensities of the mobility-integrated mass spectrum and the intensities of the isotope pattern are preferably normalized to a common scale prior to calculating the intensity difference Id.

[0032] The quantitative measure is particularly preferably determined as:

[0033] M = i Im(J + dt) (Ja = \Im(j + di) - Ir(di) \ Id< It), where:

[0034] / iterates over all mass values of the mobility-integrated mass spectrum and i iterates over all mass differences c / ;of the reference isotope pattern.

[0035] Imis the normalized intensity of the mobility-integrated mass spectrum at mass j + di.

[0036] Iris the normalized intensity of the reference isotope pattern at a mass difference dt.

[0037] Idis the absolute difference between Imand Ir.

[0038] Itis a tolerance threshold for the absolute intensity difference Id.

[0039] Id< Itis the condition that filters the intensity differences Idbased on the tolerance threshold It.

[0040] If the absolute difference Idis smaller than the tolerance threshold or tolerance value It, it is considered a match, i.e. the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample is confirmed. If the absolute difference Idexceeds It, it is considered a mismatch, i.e. the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample is denied.

[0041] This tolerance threshold preferably is a value, in which case the tolerance threshold is a tolerance value. Statements regarding the tolerance threshold preferably likewise apply to the tolerance value and vice versa. Additionally or alternatively, the tolerance threshold is preferably chosen by an operator and / or is determined or set based on an instrument performance and / or data quality and / or is preferably in the percent range. Additionally or alternatively, the tolerance threshold or tolerance value, respectively, can be a fraction and / or a percentage value.

[0042] The quantitative measure M preferably is a value.

[0043] Hence, the quantitative measure ean be seen as the summed intensity of all measured signals of the mobility-integrated mass spectrum, where each normalized intensity difference Idbetween the normalized intensity of the mobility-integrated mass spectrum and the normalized intensity of the reference isotope pattern at a mass difference is below the tolerance threshold or tolerance value Itin the event of a matching association, i.e. the compounds of interest and / or of compound classes of interest matching the isotope reference pattern in the sample, which is indicative of the presence of compounds of interest and / or of compound classes of interest matching the isotope reference pattern in the sample. On the other hand, if the normalized intensity difference Idbetween the normalized intensity of the mobility-integrated mass spectrum and the normalized intensity of the reference isotope pattern at a mass difference is at or above the tolerance threshold or tolerance value It, there is a mismatching association, i.e. the compounds of interest and / or of compound classes of interest are mismatching the isotope reference pattern in the sample, which is indicative of the absence of compounds of interest and / or of compound classes of interest matching the isotope reference pattern in the sample.

[0044] At this point it is noted that any statements made with respect to at least one compound of interest preferably likewise apply to one compound of interest, two or more compounds of interest, at least one compound class of interest, one compound class of interest, or two or more compound classes of interest and vice versa.

[0045] Various compounds of interest are conceivable, for instance pesticides or herbicides.

[0046] A significant improvement in sensitivity and specificity can further be achieved by taking into account the ion mobility, which is required to be identical for all isotopes of the compound of interest and / or the compound class of interest matching the reference isotope pattern.

[0047] To this end it is preferred that the method further comprises the step of:

[0048] - comparing the ion mobility signals associated with the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern with one another, and wherein matching ion mobilities signals are further indicative of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample.

[0049] Said further method step is preferably performed in the event of a matching association and / or the intensity difference Idbetween the normalized intensity of the mobility-integrated mass spectrum and the normalized intensity of the reference isotope pattern at a mass difference being below the tolerance threshold or tolerance value It.

[0050] Hence, additional analysis steps can be conducted, wherein it is ensured that signals of the mobility-integrated spectrum or parts thereof matching the reference isotope pattern originate from a single compound of interest and / or single compound class of interest. In doing so, the ion mobilities of said signals can be compared to one another. In the event that the ion mobilities fall within the full-width-half-maximum of peaks given by the mobility resolution of the apparatus, it is confirmed that said signals originate from a single compound of interest and / or single compound class of interest, i.e. a matching of ion mobilities is determined. In other words, the criterion of matching ion mobility of said signals is applied on the mobility resolved measured data as a filter. Said additional analysis steps are preferably performed in the event of a matching association and / or in the event of the intensity difference Idbetween the normalized intensity of the mobility-integrated mass spectrum and the normalized intensity of the reference isotope pattern at a mass difference being below the tolerance threshold or tolerance value It.

[0051] The reference isotope pattern can be defined by a list of m / z-differences between m / z- values of the isotopes, also called mass offsets, and respective experimentally determined intensity ratios.

[0052] That is, the reference isotope pattern preferably comprises m / z-differences between m / z- values of the isotopes of the compounds of interest and / or the compound classes of interest. When comparing the mobility-integrated mass spectrum of the analyte ions associated with matching ion-mobilities, m / z-differences between m / z-values of the mobility-integrated mass spectrum of the analyte ions are preferably compared with the m / z-differences of the reference isotope pattern.

[0053] The reference isotope pattern preferably comprises intensity ratios associated with the m / z- differences that are determined from intensities associated with the m / z-values of the isotopes of the compounds of interest and / or the compound classes of interest. When comparing the mobility-integrated mass spectrum of the analyte ions associated with matching ion mobilities, intensity ratios associated with the m / z-differences of the mobility- integrated mass spectrum of the analyte ions are preferably compared with the intensity ratios associated with the m / z-differences of the reference isotope pattern.

[0054] Hence, the method can be seen as comparing, for instance screening, the mobility- integrated mass spectrum or the data obtained from the combination of ion-mobility separation and mass spectrometry of the analyte ions or its fragments (see further below) with an isotope pattern comprising m / z-differences and associated intensity ratios and possibly also further parameters such as tolerances of compounds of interest and / or compound classes of interest.

[0055] The reference isotope pattern that is indicative of isotopes of the compounds of interest and / or the compound classes of interest can be determined experimentally by carrying out a mass spectrometry measurement. This can also be an isotope-labelled compound of interest and / or isotope-labelled compound class of interest, or even a mixture of unlabelled and / or labelled compounds and / or compound classes of interest. Alternatively, the reference isotope pattern indicative of isotopes of the compounds and / or compound classes of interest can likewise be theoretically determined, preferably based on the structural or molecular formula of the compounds and / or compound classes of interest.

[0056] The compounds and / or compound classes of interest can be isotope-labelled by e.g.13C or14C and / or can comprise atoms introducing a characteristic shift in the reference isotope pattern, for instance heteroatoms such as Br or Cl.

[0057] At least part of the mobility-integrated mass spectrum of the analyte ions is determined to match the reference isotope pattern when said part of the mobility-integrated mass spectrum exhibits the reference isotope pattern.

[0058] In particular, at least part of the mobility-integrated mass spectrum of the analyte ions is determined to match the reference isotope pattern when said part of the mobility-integrated mass spectrum comprises m / z-differences and optionally further associated intensity ratios that correspond to the m / z-differences and optionally further associated intensity ratios of the reference isotope pattern.

[0059] It is noted that various types of measurements that utilize both ion mobility and mass spectrometry are conceivable such as IM-MS (Ion Mobility-Mass Spectrometry) or MS / MS (Tandem Mass Spectrometry) with Ion Mobility.

[0060] In any case, the analyte, before being introduced into the ion source, is preferably separated from the sample comprising the analyte in a liquid chromatography separation unit.

[0061] Hence, the method according to the invention preferably relates to a method of combined liquid chromatography (LC), ion mobility and mass spectrometry analysis. As such, it is preferred that the analytes resulting from an LC separation are introduced into the ion source, whereby the analyte ions are generated, and thereafter introduced into the ion mobility separator that separates the analyte ions according to the ion mobility, and thereafter introduced into the mass analyzer based on their ion mobility.

[0062] It is preferred to acquire a series of mass spectra of the analyte ions by the mass analyzer while the ion mobility separator scans through a full ion mobility range suitable for the sample.

[0063] That is, a series of mass spectra are preferably acquired by the mass analyzer while the ion mobility separator scans through a defined ion-mobility range, i.e. an ion-mobility range of ion-mobility data of ions of the compounds of interest and / or of the compound class of interest such, that each acquired mass spectrum contains signals of analyte ions of a certain ion mobility only. In other words, a series of mass spectra are acquired while ramping the ion mobility separator.

[0064] A recording time during which the mass spectra are acquired is preferably recorded, and wherein a Base Peak Chromatogram (BPC) is derived from the most intense m / z-signal of each mass spectrum as a function of the recording time. This is provided as reference chromatogram to allow the user to compare to the computed isotope pattern chromatogram detailed above.

[0065] An isotope pattern chromatogram is derived from signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern as a function of the recording time and ion mobility.

[0066] That is, an isotope pattern chromatogram is determined from the series of mass spectra of the analyte ions associated with ion-mobility data falling within the full-width-half-maximum of peaks given by the mobility resolution of the instrument and exhibiting the "reference isotope pattern". To this end the mass-to-charge (m / z) signals of each mass spectrum of the mobility-integrated mass spectrum is compared with the given reference isotope pattern in relative m / z-differences and relative signal intensity ratios. If a match is found the intensity of all matching measured m / z signals is added, whereby the reference isotope pattern chromatogram is derived. The reference isotope pattern chromatogram preferably comprises the ion intensities of the most intense analyte ions as a function of the recording time.

[0067] A mobilogram can be derived for signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern and furthermore having matching ion mobilities as a function of the inverse, reduced ion-mobility 1 / o of the analyte ions. That is, a relevant criterion is that the signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern also exhibit identical ion mobilities.

[0068] That is, and as it is well-known in the field of the invention, the ion mobility K, a physical quantity, of the analyte ions can be obtained from the ion mobility separations of the analyte ions. Said ion mobility K preferably provides the ion-mobility-data of the analyte ions. After making adjustments to account, for instance, for the standard gas density, the ion mobilities can be expressed as reduced ion-mobilities Ko.

[0069] Collision-cross-sections (CCS) can be derived from signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern and furthermore having matching collision-cross-sections as a function of the inverse, reduced ion-mobility of the analyte ions. That is, a relevant criterion is that the signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern also exhibit identical collision-cross-sections. In particular, the CCS can be derived by applying the Mason-Schamp equation from the measured ionmobility or from the inverse, reduced mobility 11 Ko, as it is well-known in the art.

[0070] At least part of the analyte ions, after having been separated in the ion mobility separator according to their ion mobility, can be fragmented into fragment analyte ions. At least one mass spectrometry measurement on said fragment analyte ions is carried out, whereby a fragment mass spectrum comprising m / z-values and associated intensities of the fragment analyte ions is obtained. In the event that the fragment analyte ions originate from analyte ions being associated with the mobility-integrated mass spectrum or part thereof that matches the reference isotope pattern and additionally also in the event that the ion mobilities of said fragment analyte ions match the ion mobilities of said analyte ions, the fragment mass spectrum of said fragment analyte ions are preferably compared with the reference isotope pattern. A match between the fragment mass spectrum of the fragment analyte ions and the reference isotope pattern is indicative of the presence of fragments of the compound of interest and / or of fragments of the compound class of interest in the sample. That is, the fragment mass spectrum of the fragment analyte ions originating from a precursor matching the reference isotope pattern can be also compared with the reference isotope pattern.. A match between the fragment mass spectrum of the fragment analyte ions and the reference isotope pattern is indicative of the presence of fragments of the compounds and / or the compound classes of interest.

[0071] That is, the analyte ions can be subjected to fragmentation. A mass spectrum of the generated fragment analyte ions is preferably acquired and compared with the reference isotope pattern. That is, the reference isotope pattern can be used to analyse, for instance screen, the combined ion-mobility (IM) and mass-spectrometry (MS) data or the LC-IM-MS data not only of the "unfragmented" analyte ions, also called precursor analyte ions in the following, but also of its fragments, wherein a match between the fragment analyte ions and the reference isotope pattern is indicative of a presence of fragments of the compounds or compound classes of interest. To this end the mass spectrum of the "unfragmented" analyte ions or the precursor analyte ions, also called precursor spectrum, and the mass spectrum of the fragment analyte ions are preferably compared with the same reference isotope pattern and for the same ion mobilities.

[0072] To this end various fragmentations are conceivable. For instance, only certain mass ranges could be fragmented, in the order of the extent of the reference isotope pattern and / or in the order of a few m / z. However, it is likewise conceivable that the complete and / or a wide mass range is fragmented.

[0073] The fragment analyte ions can be associated with the precursor analyte ions through the information stored by the mass analyzer in the raw data. Both precursor and fragment ions exhibit the same ion mobility.

[0074] The method can be configured for using a data-dependent acquisition (DDA). Thereby, one or more of the precursor analyte ions can be selectively fragmented into the fragment analyte ions based on one or more precursor ion profiles such as their intensity and / or m / z values falling within a mass window in the precursor spectrum.

[0075] That is, one or more of the precursor analyte ions can be selectively fragmented into the fragment analyte ions. Said selection can be based on a precursor ion profile such as intense m / z-intensity signals of the precursor analyte ions in the precursor spectrum. For instance, the precursor analyte ions can be selected sequentially for fragmentation in order of decreasing intensity. Additionally or alternatively, one or more of the precursor analyte ions can be selected based on their m / z values falling within a m / z window in the precursor spectrum.

[0076] To this end it is particularly preferred to select one or more of the precursor analyte ions based on their intensity in the precursor spectrum and to filter them, preferably sequentially based on their intensity such as a decreasing intensity, by selectively releasing the precursor analyte ions into a mass filter which selectively transmits precursor analyte ions having m / z values falling within a controllable mass window. Thereafter, the precursor analyte ions transmitted through said mass filter can be fragmented to generate the fragment analyte ions, and a mass spectrometry measurement can then be carried out on each of said fragment analyte ions, wherein each fragment analyte ion is associated with the mass window and the ion-mobility data of the precursor analyte ions. This approach allows to only fragment precursor analyte ions that meet signal intensity, ion mobility and m / z criteria.

[0077] Alternatively, it is likewise conceivable that the method is configured to use data- independent acquisition (DIA).

[0078] That is, and in contrast to DDA, in DIA the precursor spectrum is preferably not analyzed with regard to a precursor ion profile such as intense m / z signals. Instead, it is preferred to filter and fragment m / z ranges of the precursor spectrum that falls within predefined m / z and mobility intervals. That is, the analyte ions are preferably recorded in a precursor spectrum, and wherein the analyte ions are fragmented into the fragment analyte ions based on their m / z values in the precursor spectrum falling within one or more predefined m / z windows.

[0079] For illustrative purposes, concrete examples for using DDA and DIA are given in the following. Example 1 : DDA

[0080] (1) A precursor spectrum of the analyte ions is acquired;

[0081] (2) Said precursor spectrum is analysed with regard to intense m / z-intensity signals;

[0082] (3) One or more of these intense m / z-intensity signals are selected;

[0083] (4) A first signal of these selected intense m / z-intensity signals is filtered in a suitable, small mass range and fragmented, and a fragment mass spectrum is acquired;

[0084] (5) Step (4) is repeated for the further selected intense m / z-intensity signals.

[0085] Example 2: DIA:

[0086] (1) A precursor spectrum of the analyte ions is acquired;

[0087] (2) The precursor spectrum is filtered and m / z ranges of predefined values are fragmented, and a fragment spectrum is acquired.

[0088] That is, in the DIA Example the precursor spectrum is not analyzed with regard to intense m / z-intensity signals but filtered and fragmented for m / z ranges of predefined values. To this end the mass range can extend to only part of the full mass range.

[0089] In another aspect, the method as described above is used to identify at least one compound of interest and / or at least one compound class of interest in a sample and / or for the analysis of a complex sample, including samples in proteomics and metabolomics.

[0090] In yet another aspect, an apparatus for carrying out the method as described above is provided. The apparatus comprises an ion source, an ion mobility separator, and a mass analyzer. The apparatus can comprise further components, for instance a liquid chromatography separation unit or a mass filter.

[0091] Any statements made with respect to the method of combined ion mobility and mass spectrometry analysis preferably likewise apply to the apparatus for carrying out said method and vice versa.

[0092] Various ion sources, ion mobility separators, a mass analyzers and liquid chromatography separation units are conceivable and are well-known in the art. For instance, the ion source can be an electro-spray being configured for electro-spray ionization. The ion mobility separator can be a trapped ion mobility spectrometer (TIMS), a drift tube ion mobility spectrometer, or a travelling-wave ion mobility spectrometer. The mass analyzers could be a TOF mass analyzer. The mass filter could be an RF quadrupole mass filter, etc.

[0093] BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0095] Fig. 1 shows a general overview of an apparatus comprising a liquid chromatography unit, an electro spray, an ion mobility spectrometer and a mass spectrometer for performing a combined liquid chromatography, ion mobility and mass spectrometry analysis (LC-IM-MS) according to the invention;

[0096] Fig. 2 shows an overview of conceivable method steps of the method according to the invention;

[0097] Fig. 3 shows the structural formula of the exemplary compound of interest Arylex;

[0098] Fig. 4 shows a mass spectrum of the exemplary compound of interest Arylex;

[0099] Fig. 5 shows chromatograms that were derived in the method according to the invention;

[0100] Fig. 6 shows mass spectra that were acquired in the method according to the invention;

[0101] Fig. 7 shows a mobilogram that was derived from the chromatogram depicted in figure 5c;

[0102] Fig. 8 shows a mass spectrum that was acquired outside the ion-mobility range that was applied in figures 5c and 6;

[0103] Fig. 9 shows a mobilogram of figure 7 after applying the Mason-Schamp equation to calculate the collision-cross-section (CCS).

[0104] DESCRIPTION OF PREFERRED EMBODIMENTS

[0105] Aspects of the invention are now illustrated with reference to the figures.

[0106] In particular, figure 1 depicts a general overview of an apparatus configured to perform the method of combined ion mobility and mass spectrometry analysis according to the invention. Here, said apparatus comprises a liquid chromatography (LC) unit that separates an analyte from a sample, an ion source in the form of an electro spray that is configured to generate analyte ions from the analyte by electro-spray ionization, an ion mobility separator or an ion mobility spectrometer, respectively, that is configured to separate the analyte ions according to their ion mobility, and a mass analyzer or mass spectrometer, respectively, that is configured to acquire mass spectra of the analyte ions. After their separation of the analyte ions by the ion mobility separator, a fragmentation into fragment analyte ions can occur but not need to, which is indicated by “fragmentation yes / no”.

[0107] Figure 2 lists method steps of an example of a combined LC-IM-MS analysis according to the invention. That is, in step S1 , an isotope labelling such as14C or characteristic atoms such as Br or Cl are introduced in a labelling experiment into a compound of interest and / or of the compound class of interest and its fragments or metabolites. In step S2, a reference isotope pattern of the compound and / or compound class of interest is defined from the labelling experiment. Here, said reference isotope pattern is based on a table of m / z offset or difference values and associated intensities as well as tolerance thresholds Itas will be explained in greater detail below. In step S3, an ion mobility range of ion-mobility data of the compound and / or compound classes of interest and its fragments or metabolites is defined. Collision-cross-section (CCS) predictions or reference measurements can be performed to obtain the inverse, reduced mobility 1 / K0interval of the compound of interest and / or the compound class of interest. In step S3, combined LC-IM-MS or LC-IM-MS / MS measurements can be performed in order to aquire LC-IM-MS or LC-IM-MS / MS datasets from a complex sample exhibiting the compound of interest and / or the compound class of interest and their fragments or metabolits. Any complex matrix can be resolved by LC and ion mobility. In step S4, a data processing of individual spectra is performed and the convolution with the reference isotope pattern calculates an intensity value as quantitative measure that highlights the presence of the compounds and / or compound classes of interest, i.e. a data processing of individual spectra is performed, wherein the spectra are compared with the reference isotope pattern, and wherein a match is indicative of the presence of a target compound, i.e. a compound of interest and / or a compound class of interest, or its fragments or metabolits, which is highlighted. In particular, in step S4, the intensities of the mass spectrum of the analyte ions for each m / z-value across all ion mobility signals of the LC-IM-MS or LC-IM-MS / MS datasets are summed up, whereby a mobility- integrated mass spectrum comprising accumulated intensities of the analyte ions for each m / z-value is obtained. Then, the mobility-integrated mass spectrum is convoluted with the reference isotope pattern being indicative of isotopes of at least one compound of interest and / or at least one compound class of interest, whereby a quantitative measure for the presence of said reference isotope pattern in the mobility-integrated mass spectrum is obtained. The ion mobility signals associated with the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern are compared with one another in step S5. Matching ion mobilities are a further indication of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample. To this end, a matching ion mobility is confirmed if the compared ion mobilities equal one another within the full-width-half-maximum of the measurement peaks that is given by the mobility resolution of the apparatus. In the event that also fragments shall be analyzed it is conceivable to use tandem-MS-spectra to search for the reference isotope pattern, i.e. to search for the labeling pattern found in fragments.

[0108] An exemplary compound of interest is Arylex, an agrochemical containing a Cl heteroatom leading to a characteristic reference isotope pattern. The structural formula of which is shown in figure 3.

[0109] In this case, the reference isotope pattern is based on the following m / z-differences and associated relative intensities, table 1 :

[0110] These values are calculated from a mixture of the compound of interest shown in figure 4 and its heavy carbon labeled variant where 612C atoms were replaced by13C atoms.

[0111] Moreover, in the depicted examples an intensity tolerance of 30% and a m / z tolerance of 2 mDa have been applied.

[0112] However, it is likewise conceivable that the reference isotope pattern is derived experimentally, in particular from a mass spectrum of the compound of interest such as the mass spectrum of Arylex shown in figure 4.

[0113] Figure 5 depicts in panel (a) a base peak chromatogram of an LC-IM-MS run performed with the compound of interest Arylex. The base peak chromatogram is acquired by first separating the sample by liquid chromatography (LC) into individual analytes. Next the analytes are introduced into an ion-mobility spectrometer followed by the mass spectrometer acquiring a series of mass spectra. During this process the ion-mobility spectrometer is scanning through a defined mobility range thus that each recorded mass spectrum contains only signals of analytes of a certain mobility. Also, the time the spectra were measured is recorded. Taking the most intense signal (base peak) of each mass spectrum and depicting it against the time gives this base peak chromatogram (BPC).

[0114] The ion-mobility information is not yet used for this chromatogram. Instead, it comprises signals from all mobilities.

[0115] The result of comparing or convoluting, respectively, the mobility-integrated mass spectra with the reference isotope pattern defined in table 1 , i.e. in a sense the result of applying a reference isotope pattern filter algorithm, on the chromatogram extraction is shown in panel (b) of figure 5. This chromatogram is calculated from the mass spectra acquired and shown in panel (a) of figure 5. It is calculated by searching each mass spectrum for mass-to-charge (m / z) signals matching the given reference isotope pattern in relative m / z-differences and relative signal intensity ratios. If a match is found the intensity of all matching measured m / z signals is added making up this chromatogram. A match corresponds here to a sequence of m / z signals with the same m / z differences and the same intensity ratios.

[0116] Figure 6a shows a measured mass spectrum, flagged with matching m / z-intensity signals. That is, figure 6 shows the mass spectrum (a) of the peak of the Reference Isotope Pattern Filter chromatogram shown in fig 5c at 10 min. This spectrum was extracted from m / z signals only, matching the given pattern in relative m / z-differences and relative signal intensity ratios and within a mobility range of 0.80 - 0.85 V s / cm2. Additionally, the matching m / z signals are flagged with a symbol (o). A matching pattern may occur multiply in a mass spectrum, however, all of the m / z-differences and intensity ratios of the given pattern must have a match.

[0117] These flagged m / z signals are also used in the fragmentation step between the ion mobility spectrometer and the mass spectrometer. In this fragmentation step all other ions except those with the flagged m / z ratio are first discarded and the remaining ions (those in (a)) then fragmented. This produces then a fragment mass spectrum (b). Here again the given reference isotope pattern of relative m / z-differences and relative signal intensity ratios is applied and used for just flagging matching m / z signals with a symbol (o).

[0118] This proves that the specific pattern of relative m / z-differences and relative signal intensity ratios of the analyte can also be observed after fragmentation.

[0119] Applying the ion-mobility information by only using mass spectra of a mobility range of 0.80 - 0.85 V s / cm2is shown in panel (c) of figure 5. This chromatogram is calculated from the same acquired mass spectra, however now only spectra of this defined mobility range are used for the chromatogram calculation.

[0120] Comparing the Reference Isotope Pattern Filter chromatogram of figure 5 with (c) and without (b) mobility filtering shows that applying the mobility filter successfully filters out unwanted signals, chromatographic peaks between 4 and 7 min.

[0121] Figure 7 shows the mobilogram taken from spectra of the peak at 10 min in fig. 5c. The mobilogram is calculated from selected m / z-intensity signals of acquired mass spectra in dependency of the mobility. To do so the ion mobility spectrometer is filtering out ions of a specific, small mobility window only, while scanning this mobility window over a certain range.

[0122] A typical scan of such a mobility window takes less time than a chromatogram peak as in figure 5. Typically, each mobility scan then comprises several hundred mass spectra, each of a unique mobility. The mobility is measured as inverse, reduced mobility 1 / K0.

[0123] This particular mobilogram is taken from m / z-intensity signals only, which match the reference isotope pattern filter and are also used in figure 5c and figure 6a.

[0124] The mobilogram shows a dominant peak in the mobility range 0.80 - 0.85 V s / cm2- the same mobility range used for the Reference isotope pattern Filter chromatogram in figure 5c.

[0125] It also shows an additional, smaller peak at 0.9 V s / cm2. This peak results from the same m / z-intensity signals as the dominant peak, just from another analyte of different mobility. This m / z-intensity signals may or may not satisfy the reference isotope pattern filter criteria used for the dominant peak. In any case they are not taken into account for the mobility filtered chromatogram and compound mass spectra, yet producing results not being overlayed with signals of same m / z values yet different mobility.

[0126] Figure 8 shows the mass spectrum taken at this additional peak at 0.9 V s / cm2. These m / z- intensity signals are being successfully filtered out by the mobility range which was applied in figure 5c and 6.

[0127] Figure 9 shows the mobilogram of figure 7 after applying the Mason-Schamp equation to calculate the collision cross section from the inverse, reduced mobility 1 / o. For this formula an average mass of m / z 349.0 and a charge state of 1+ was applied. The average mass was taken from the flagged signals in figure 6a.

[0128] Hence, in summary it is noted that the present invention improves the confident detection and identification of analyte ions and their fragments such as metabolites with effective background removal by using a combination of both ion mobility separation and high- resolution accurate mass spectrometry (HRAM). We exploit the presence of unique isotope patterns detectable in the raw data of the HRAM analysis.

[0129] The defined reference isotope pattern is used to screen LC-IM-MS data and calculate a derived extracted isotope trace representing a quantitative signal of the target compounds and its metabolites which exhibit the specified reference isotope pattern. Thereby matrix background can be removed and the compounds containing the isotope pattern at the defined ion mobility are highlighted.

[0130] Moreover, CCS-enabled measurements are enabled that allow to screen other complex samples such as pesticides or herbicides and their metabolites in complex matrices like soil or plant material.

[0131] The novel ion mobility capabilities allow them to significantly improve signal-to-noise ratio of the analysis and therefore find low abundant signals even in strong backgrounds.

Claims

1. CLAIMS1. A method of combined ion mobility and mass spectrometry analysis, comprising the steps of:- Generating analyte ions from one or more analytes in a sample with an ion source;- Introducing the analyte ions into an ion mobility separator and separating the analyte ions according to their ion mobility, whereby ion mobility-signals of the analyte ions are obtained;- Introducing the analyte ions into a mass analyzer according to their ion mobility, and carrying out at least one mass spectrometry measurement on said analyte ions, whereby a mass spectrum comprising m / z-values and associated intensities of the analyte ions is obtained;- summing the intensities of the mass spectrum of the analyte ions for each m / z-value across all ion mobility signals, whereby a mobility-integrated mass spectrum comprising accumulated intensities of the analyte ions for each m / z-value is obtained, and- convoluting the mobility-integrated mass spectrum with at least one reference isotope pattern being indicative of isotopes of at least one compound of interest and / or at least one compound class of interest, whereby a quantitative measure for the presence of said reference isotope pattern in the mobility-integrated mass spectrum is obtained, and wherein the presence of the reference isotope pattern in the mobility-integrated mass spectrum is indicative of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample.

2. The method according to claim 1 , wherein the quantitative measure is based on a comparison between the accumulated intensities of the mobility-integrated mass spectrum and intensities at specified mass differences of the isotope pattern.

3. The method according to any one of the preceding claims, wherein the quantitative measure is determined by summing intensities from the mobility-integrated mass spectrum only for m / z-values where a normalized intensity difference Idbetween the measured intensities of the analyte ions and intensities at specified mass differences of the isotope pattern is less than a tolerance threshold It.

4. The method according to any one of the preceding claims, further comprising the step of:- comparing the ion mobility signals associated with the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern with one another, and wherein matching ion mobilities are further indicative of the presence of the at least one compound of interest and / or the at least one compound class of interest in the sample.

5. The method according to any one of the preceding claims, wherein the reference isotope pattern comprises m / z-differences between m / z-values of isotopes of the compound of interest and / or of the compound class of interest, and wherein m / z-differences between m / z-values of the mobility-integrated mass spectrum of the analyte ions are compared with the m / z-differences of the reference isotope pattern.

6. The method according to claim 5, wherein the reference isotope pattern further comprises intensity ratios associated with the m / z-differences that are determined from intensities associated with the m / z-values of the isotopes of the compound of interest and / or the compound class of interest, and wherein intensity ratios associated with the m / z-differences of the mobility-integrated mass spectrum of the analyte ions are compared with the intensity ratios associated with the m / z-differences of the reference isotope pattern.

7. The method according to any one of the preceding claims, wherein the reference isotope pattern being indicative of isotopes of the compound of interest and / or the compound class of interest is determined experimentally by carrying out a mass spectrometry measurement on an isotope-labelled compound of interest and / or an isotopelabelled compound class of interest, or wherein the reference isotope pattern is theoretically determined preferably based on the structural or molecular formula of the compound of interest and / or the compound class of interest.

8. The method according to any one of the preceding claims, wherein the mobility- integrated mass spectrum of the analyte ions or part thereof matches the reference isotope pattern when said mobility-integrated mass spectrum or said part thereof exhibits thereference isotope pattern, and in particular, when said mobility-integrated mass spectrum or part thereof comprises m / z-differences and optionally further associated intensity ratios that correspond to the m / z- differences and optionally further associated intensity ratios of the reference isotope pattern.

9. The method according to any one of the preceding claims, wherein the analyte, before being introduced into the ion source, is separated from the sample in a liquid chromatography separation unit.

10. The method according to any one of the preceding claims, wherein a series of mass spectra of the analyte ions are acquired by the mass analyzer while the ion mobility separator scans through the full ion mobility range suitable for the sample.11 . The method according to claim 10, wherein a recording time during which the mass spectra are acquired is recorded, and wherein an isotope pattern chromatogram is derived from signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern as a function of the recording time and the ion mobility.

12. The method according to claim 10, wherein a mobilogram is derived for signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern and furthermore having matching ion mobilities as a function of the inverse, reduced ion-mobility of the analyte ions.

13. The method according to any one of claims 10 to 12, wherein collision-cross- sections are derived from signals of each mass spectrum of the mobility-integrated mass spectrum or part thereof matching the reference isotope pattern and furthermore having matching collision-cross-sections as a function of the inverse, reduced ion-mobility of the analyte ions.

14. The method according to any one of the preceding claims, wherein at least part of the analyte ions, after having been separated in the ion mobility separator according to their ion mobility, are fragmented into fragment analyte ions, wherein at least one mass spectrometry measurement on said fragment analyte ions is carried out, whereby a fragment mass spectrum comprising m / z-values and associated intensities of the fragment analyte ions is obtained,wherein, in the event that the fragment analyte ions originate from analyte ions being associated with the mobility-integrated mass spectrum or part thereof that matches the reference isotope pattern and furthermore in the event that the ion mobilities of said fragment analyte ions match the ion mobilities of said analyte ions, the fragment mass spectrum of said fragment analyte ions are compared with the reference isotope pattern, and wherein a match between the fragment mass spectrum of the fragment analyte ions and the reference isotope pattern is indicative of the presence of fragments of the compound of interest and / or of fragments of the compound class of interest in the sample.

15. The method according to claim 14, using data-dependent acquisition (DDA), and / or wherein the analyte ions are precursor analyte ions, and the mass spectrum of the analyte ions is a precursor spectrum, and wherein one or more of the precursor analyte ions are selectively fragmented into the fragment analyte ions based on one or more precursor ion profiles such as their intensity and / or m / z values falling within a mass window in the precursor spectrum.

16. The method according to claim 14, using data-independent acquisition (DIA), and / or wherein the analyte ions are recorded in a precursor spectrum, and wherein the analyte ions are fragmented into the fragment analyte ions based on their m / z values in the precursor spectrum falling within one or more predefined m / z windows.

17. Use of the method of any one of the preceding claims for identifying at least one compound of interest and / or at least one compound class of interest in a sample and / or for the analysis of a complex sample, including samples in proteomics and metabolomics.

18. An apparatus for carrying out the method according to any of the preceding claims, said apparatus comprising:- an ion source,- an ion mobility separator, and- a mass analyzer, and- optionally a liquid chromatography separation unit.

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