CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from and the benefit of United Kingdom patent application No. 1613235.9 filed on 1 August 2016. The entire content of this application is incorporated herein by reference. FIELD OF THE INVENTION

The present invention relates generally to mass and/or ion mobility spectrometers and in particular to mass and/or ion mobility spectrometers in which ions are reacted or fragmented as they pass through a drift cell such as an ion mobility separator drift tube.

BACKGROUND

Ion-molecule reactions can be used to investigate the structure and chemical properties of analytes.

A population of ions may be pulsed into an atmospheric pressure drift tube and driven through a reactive buffer gas or a buffer gas comprising a reactive additive. As ions transit through the drift tube, ion-molecule reactions can occur due to the interaction of ions with reactive molecules in the buffer gas. Ions that exit the drift tube may then be mass analysed.

The reactive gas or dopant can be chosen to react only with specific analyte ions of interest, e.g. having specific chemical properties. For example, unsaturated bonds in lipids may be located using ozonolysis. Ions which do not have these specific characteristics will not react with the buffer gas and will pass through the drift tube intact and un-attenuated.

These un-attenuated ions may be of little interest, and may give rise to significant interferences in the acquired data. For example, in the analysis of biological samples, in addition to the lipid compounds of interest, many endogenous, non-lipid compounds can be present, often in excess, which can give rise to significant interferences. To obtain a usable spectrum of the reaction products for a particular precursor ion (e.g. to obtain a usable spectrum of the ozonolysis product ions for a particular precursor lipid ion), tandem mass spectrometry ("MS-MS") is typically employed.

However, it may not always be desirable to use tandem mass spectrometry, since it involves mass selecting a particular precursor ion, and can therefore have a low duty cycle and can be time consuming. In addition, in order to select a particular precursor ion, its mass to charge ratio ("m/z") must firstly be known.

It can also be the case that interferences are present in acquired data where tandem mass spectrometry techniques are used.

Reference is made to US 2015/0034813, the article Ozonolysis of

Phospholipid Double Bonds during Electrospray Ionization: A New Tool for

Structure Determination", J. Am. Chem. Soc, 2006, 128, 58-59, and to the article "The Kinetic Ion Mobility Mass Spectrometer: Measurements of Ion-Molecule Reaction Rate Constants at Atmospheric Pressure", J. Phys. Chem., 1992, 96, 6680-6687.

It is desired to provide an improved method of mass and/or ion mobility spectrometry. SUMMARY

According to an aspect, there is provided a method comprising:

passing precursor ions into a drift region or cell such that at least some ions are separated according to their ion mobility;

causing at least some of the precursor ions within the drift region or cell to dissociate, fragment or react so as to produce reaction product ions;

mass analysing ions that emerge from the drift region or cell and/or ions derived from ions that emerge from the drift region or cell so as to produce one or more data sets; and

identifying one or more reaction product ions in the one or more data sets based on an ion mobility peak width, profile or shape; and/or

identifying one or more reaction product ions in the one or more data sets and/or identifying one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions based on a change in ion intensity between two or more data sets. Various embodiments described herein are directed to methods of mass and/or ion mobility spectrometry, in which precursor ions are passed into a drift region or cell such that at least some ions separate according to their ion mobility. At least some of the precursor ions within the drift region or cell are caused to dissociate, fragment or react to produce reaction product ions. Ions that emerge from the drift region or cell and/or ions derived from ions that emerge from the drift region or cell are then mass analysed so as to produce one or more data sets.

According to various embodiments, one or more reaction product ions are identified in the one or more data sets based on an ion mobility drift time peak width, profile or shape. According to various embodiments, one or more reaction product ions are identified in the one or more data sets and/or one or more precursor ions that are associated with one or more reaction product ions are identified in the one or more data sets based on a change in ion intensity between two or more data sets.

Thus, as will be described in more detail below, reaction product ions and/or precursor ions that are associated with one or more reaction product ions (i.e.

reactive precursor ions) can be identified in the one or more data sets. This then allows the reaction product ions and/or the reactive precursor ions to be

distinguished and isolated from non-reactive precursor ions and/or other

background ions, which may otherwise interfere with the reaction product ions and/or reactive precursor ions of interest.

Accordingly, a simplified data set may be produced, e.g. comprising only the identified reaction product ions and/or reactive precursor ions.

As such, a usable reaction product spectrum can be produced, e.g. without requiring mass selection, and in particular without using tandem mass spectrometry ("MS-MS"). In addition, the techniques according to various embodiments can allow an entire population of precursor ions generated from a sample (e.g. eluting from a chromatography system) to be analysed, thereby increasing the coverage of ions analysed and reducing analysis time.

A simplified data set may also be produced from a data set acquired using tandem mass spectrometry or other techniques.

Various embodiments of the techniques described herein are particularly useful in the analysis of complex mixtures, e.g. where precursor ions of interest may be relatively less intense compared to relatively intense background or other ions, and where the background or other ions have a dissimilar chemical nature to the precursor ions of interest, and are not therefore susceptible to dissociation, fragmentation or reaction in the drift region or cell.

For example, according to various embodiments, reaction products and associated precursor ions of unsaturated lipids may be recognised, and unwanted co-eluting background ions discarded, thereby producing simplified ozonolysis spectra, e.g. without requiring mass selection. This allows the entire population of lipids eluting from a chromatograph to be captured without bias, thereby increasing the coverage of lipids interrogated and saving analysis time.

It will accordingly be appreciated that various embodiments provide an improved method of mass and/or ion mobility spectrometry.

Causing at least some of the precursor ions within the drift region or cell to dissociate, fragment or react may comprise supplying a reactive buffer gas or a reactive buffer gas additive to the drift region or cell.

Causing at least some of the precursor ions within the drift region or cell to dissociate, fragment or react may comprise causing at least some of the precursor ions within the drift region or cell to photo-fragment.

Identifying one or more reaction product ions in the one or more data sets may comprise comparing an ion mobility peak width, profile or shape in the one or more data sets to an expected or threshold ion mobility drift time peak width, profile or shape.

The method may comprise varying the reaction time and/or rate for causing precursor ions within the drift region or cell to dissociate, fragment or react between two or more different reaction times and/or rates.

At least one of the two or more data sets may comprise a data set acquired using one of the two or more different reaction times and/or rates.

At least one of the two or more data sets may comprise a data set acquired using another of the two or more different reaction times and/or rates.

Identifying one or more reaction product ions in the one or more data sets and/or identifying one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions may comprise identifying one or more reaction product ions in the one or more data sets and identifying one or more precursor ions in the one or more data sets that are associated with the one or more identified reaction product ions.

The method may comprise identifying one or more analytes using the one or more data sets. The method may comprise generating one or more simplified data sets on the basis of the identification or identifications.

The method may comprise passing unreactive calibrant ions into the drift region or cell.

According to an aspect, there is provided a method of identifying ions in one or more data sets acquired by passing precursor ions into a drift region or cell such that at least some ions are separated according to their ion mobility, causing at least some of the precursor ions within the drift region or cell to dissociate, fragment or react so as to produce reaction product ions, and mass analysing ions that emerge from the drift region or cell and/or ions derived from ions that emerge from the drift region or cell so as to produce the one or more data sets, the method comprising:

identifying one or more reaction product ions in the one or more data sets based on an ion mobility peak width, profile or shape; and/or

identifying one or more reaction product ions in the one or more data sets and/or identifying one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions based on a change in ion intensity between two or more data sets.

According to an aspect there is provided a spectrometer comprising:

a drift region or cell configured to cause ions to separate according to their ion mobility;

a mass analyser configured to mass analyse ions that emerge from the drift region or cell and/or ions derived from ions that emerge from the drift region or cell so as to produce one or more data sets; and

a processing system;

wherein the spectrometer is configured such that precursor ions within the drift region or cell are caused to dissociate, fragment or react so as to produce reaction product ions; and

wherein the processing system is configured:

to identify one or more reaction product ions in the one or more data sets based on an ion mobility peak width, profile or shape; and/or

to identify one or more reaction product ions in the one or more data sets and/or to identify one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions based on a change in ion intensity between two or more data sets. The spectrometer may comprise a gas supply configured to supply a reactive buffer gas or a reactive buffer gas additive to the drift region or cell.

The spectrometer may comprise a photo-fragmentation device configured to cause at least some ions within the drift region or cell to photo-fragment.

The processing system may be configured to identify one or more reaction product ions in the one or more data sets by comparing an ion mobility peak width, profile or shape in the one or more data sets to an expected or threshold ion mobility drift time peak width, profile or shape.

The spectrometer may be configured to vary the reaction time and/or rate for causing precursor ions within the drift region or cell to dissociate, fragment or react between two or more different reaction times and/or rates.

At least one of the two or more data sets may comprise a data set acquired using one of the two or more different reaction times and/or rates.

At least one of the two or more data sets may comprise a data set acquired using another of the two or more different reaction times and/or rates.

The processing system may be configured to identify one or more reaction product ions in the one or more data sets and to identify one or more precursor ions in the one or more data sets that are associated with the one or more identified reaction product ions.

The processing system may be configured to identify one or more analytes using the one or more data sets.

The processing system may be configured to generate one or more simplified data sets on the basis of the identification or identifications.

The spectrometer may comprise a device configured to pass unreactive calibrant ions into the drift region or cell.

According to an aspect, there is provided a computer program comprising computer software code for performing the method as described above when the program is run on data processing means.

According to an aspect, there is provided a method of identifying mass spectral peaks arising from reaction product ions and/or corresponding precursor ions emerging or eluting from an ion-molecule or other reaction drift tube coupled with a down-stream mass spectrometer in the presence of non-reactive background ions, the method comprising:

identifying reaction product ions based on their characteristic drift (elution) time profile or shape and/or identifying reaction product ions and associated precursor ions by comparing the signal intensity between two or more data sets acquired using different (ion-molecule or other) reaction times and/or rates.

The drift tube or reaction cell may be a linear DC field or a travelling DC wave ion mobility device. Ions may be confined within the drift tube or reaction cell using RF confinement or non-RF confinement.

The method may comprise repetitively switching between two or more reaction times and/or rates, e.g. by altering the driving force, residence time and/or reagent gas concentration. 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:

Fig. 1 shows a mass spectrometer according to various embodiments; Fig. 2A shows schematically a reconstructed mass mobility spectrum where ions do not react with a reactant gas; Fig. 2B shows schematically a reconstructed mass mobility spectrum where ions react with a reactant gas for a relatively long reaction time; and Fig. 2C shows schematically a reconstructed mass mobility spectrum where ions react with a reactant gas for a relatively short reaction time; and

Fig. 3 shows schematically a mass to charge ratio versus drift time heat map for the data of Fig. 2B.

DETAILED DESCRIPTION

Various embodiments are directed to a method in which precursor ions are passed into (and/or through) a drift cell or drift region such that at least some ions, e.g. at least some of the precursor ions, separate according to their ion mobility. At least some of the precursor ions within the drift cell or region are caused to dissociate, fragment or react to produce reaction product ions within the drift cell or region. Ions that emerge from the drift cell or region and/or ions derived from ions that emerge from the drift cell or region are then analysed, e.g. mass analysed, so as to produce one or more data sets. The precursor ions may comprise any suitable ions, and may be generated in any suitable manner. The precursor ions may be generated by (and the spectrometer may comprise) an ion source, e.g. upstream of the drift cell or region.

The ion source may comprise any suitable ion source. The ion source may comprise a continuous or a pulsed ion source. The ion source may be selected from the group consisting of: (i) an Electrospray ionisation ("ESI") ion source; (ii) an Atmospheric Pressure Photo lonisation ("APPI") ion source; (iii) an Atmospheric Pressure Chemical lonisation ("APCI") ion source; (iv) a Matrix Assisted Laser Desorption lonisation ("MALDI") ion source; (v) a Laser Desorption lonisation ("LDI") ion source; (vi) an Atmospheric Pressure lonisation ("API") ion source; (vii) a Desorption lonisation on Silicon ("DIOS") ion source; (viii) an Electron Impact ("El") ion source; (ix) a Chemical lonisation ("CI") ion source; (x) a Field lonisation ("Fl") ion source; (xi) a Field Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma ("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") ion source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xv) a Desorption Electrospray lonisation ("DESI") ion source; (xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; (xviii) a Thermospray ion source; (xix) an Atmospheric Sampling Glow Discharge lonisation ("ASGDI") ion source; (xx) a Glow Discharge ("GD") ion source; (xxi) an Impactor ion source; (xxii) a Direct Analysis in Real Time ("DART") ion source; (xxiii) a Laserspray lonisation ("LSI") ion source; (xxiv) a Sonicspray lonisation ("SSI") ion source; (xxv) a Matrix Assisted Inlet lonisation ("MAN") ion source; (xxvi) a Solvent Assisted Inlet lonisation ("SAN") ion source; (xxvii) a Desorption Electrospray lonisation ("DESI") ion source; (xxviii) a Laser Ablation Electrospray lonisation ("LAESI") ion source; and (xxix) Surface Assisted Laser Desorption lonisation ("SALDI").

The spectrometer may comprise a chromatography or other separation device, e.g. upstream of and coupled to the ion source. The chromatography separation device may comprise a liquid chromatography or a gas chromatography device. Alternatively, the separation device may comprise: (i) a Capillary

Electrophoresis ("CE") separation device; (ii) a Capillary Electrochromatography ("CEC") separation device; (iii) a substantially rigid ceramic-based multilayer microfluidic substrate ("ceramic tile") separation device; or (iv) a supercritical fluid chromatography separation device. The precursor ions that are passed into the drift cell or region may comprise most or all of the ions generated by the ion source. Alternatively, the ions generated by the ion source may be filtered and/or dissociated, fragmented or reacted so as to generate precursor ions which may then be passed into the drift cell or region.

The ions may be filtered by (and the spectrometer may comprise) any suitable ion filtering device, such as a mass filter, e.g. which is configured to filter ions according to their mass to charge ratio. The mass filter may be selected from the group consisting of: (i) a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter; and (viii) a Wien filter. The filtering device may be arranged upstream of the drift cell or region and downstream of the ion source.

The ions may be dissociated, fragmented or reacted by (and the

spectrometer may comprise) any suitable collision, fragmentation or reaction device, which may be arranged upstream of the drift cell or region and downstream of the ion source.

The collision, fragmentation or reaction device may be selected from the group consisting of: (i) a Collisional Induced Dissociation ("CID") fragmentation device; (ii) a Surface Induced Dissociation ("SID") fragmentation device; (iii) an Electron Transfer Dissociation ("ETD") fragmentation device; (iv) an Electron Capture Dissociation ("ECD") fragmentation device; (v) an Electron Collision or Impact Dissociation fragmentation device; (vi) a Photo Induced Dissociation ("PID") fragmentation device; (vii) a Laser Induced Dissociation fragmentation device; (viii) an infrared radiation induced dissociation device; (ix) an ultraviolet radiation induced dissociation device; (x) a nozzle-skimmer interface fragmentation device; (xi) an in-source fragmentation device; (xii) an in-source Collision Induced

Dissociation fragmentation device; (xiii) a thermal or temperature source fragmentation device; (xiv) an electric field induced fragmentation device; (xv) a magnetic field induced fragmentation device; (xvi) an enzyme digestion or enzyme degradation fragmentation device; (xvii) an ion-ion reaction fragmentation device; (xviii) an ion-molecule reaction fragmentation device; (xix) an ion-atom reaction fragmentation device; (xx) an ion-metastable ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation device; (xxii) an ion- metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion- metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an Electron lonisation Dissociation ("EID") fragmentation device.

Ions may be trapped, i.e. accumulated, before they are passed into the drift cell or region. This can facilitate increased duty cycle operation, e.g. where the drift tube is configured to separate each group of ions of plural consecutive groups of ions in turn. The spectrometer may comprise an ion trap or trapping region for this purpose, which may be arranged upstream of the drift cell or region and

downstream of the ion source. Additionally or alternatively, the collision, fragmentation or reaction device may be used to trap ions.

The precursor ions that are passed into the drift cell or region may comprise precursor ions of interest, together with other precursor ions, such as background ions and/or other undesired ions, e.g. precursor ions of little or no interest. For example, the precursor ions that are passed into the drift cell or region may comprise targeted precursor ions, e.g. precursor ions that have some specific chemical property or properties (and that may therefore be susceptible to dissociation, fragmentation or reaction in the drift cell), together with non-targeted precursor ions, e.g. precursor ions that do not have the specific chemical property or properties (and that may therefore be unsusceptible to dissociation,

fragmentation or reaction in the drift cell).

Various embodiments of the techniques described herein are particularly useful in the analysis of complex mixtures, e.g. where precursor ions of interest may be relatively less intense compared to relatively intense background or other ions, and where the background or other ions have a dissimilar chemical nature to the precursor ions of interest, and are not therefore susceptible to dissociation, fragmentation or reaction in the drift cell.

The drift cell or drift region according to various embodiments may comprise any suitable device that is configured to cause ions to separate according to their ion mobility. The drift cell or region may comprise a drift tube, and may be configured such that ions can be passed through the drift tube such that they separate according to their ion mobility as they pass through the drift tube, e.g. along its (axial) length.

A buffer gas may be provided to the drift cell or region, e.g. from a gas source. Ions within the drift cell or region may be caused to separate according to their ion mobility by being urged through the buffer gas. The buffer gas may be substantially static, or may be caused to flow in a direction substantially opposite to the direction in which the ions are urged through the drift cell or region, e.g. along the axial direction.

Ions may be radially confined within the drift cell or region, i.e. confined in the direction(s) orthogonal to the direction in which the ions are passed (urged) through the drift cell or region, i.e. orthogonal to the axial direction.

Ions may be radially confined within the drift cell or region by a pseudo- potential well or barrier. In this case, the drift cell or region may comprise a plurality of electrodes or electrode groups which may each have an aperture or opening through which ions may be transmitted in use. One or more RF voltages may be applied to the electrodes so as to confine ions radially within the ion guide.

Opposite phases of the RF voltage may be applied to axially adjacent electrodes or electrode groups. Other arrangements would be possible.

Additionally or alternatively, ions may be radially confined within the drift cell or region using non-RF confinement, e.g. using a radial DC electric field or otherwise.

An axial electric field may be provided in the drift cell or region, e.g. which may be configured to urge ions through the drift cell or region (and through or against the buffer gas). The axial electric field may comprise a static DC electric field, e.g. which may vary linearly along the axial length of the drift cell or region. Additionally or alternatively, the axial electric field may comprise a travelling DC wave. In this case, a DC voltage may be successively applied to each electrode or electrode group of the drift tube in order, e.g. starting from one end of the drift tube and progressing to the other end of the drift tube, i.e. such that ions are urged along the drift cell or region in the axial direction.

At least some of the precursor ions within the drift cell or region are caused to dissociate, fragment or react to produce reaction product ions, i.e. as they are passed into and/or as they pass through the drift cell or region.

Precursor ions may be caused to dissociate, fragment or react within the drift cell or region in any suitable manner. For example, the buffer gas that is provided to the drift cell or region may comprise a reactive buffer gas. Alternatively, a reactive additive or dopant may be added to the (otherwise unreactive) drift cell buffer gas.

The reactive buffer gas or reactive buffer gas additive may be selected so as to cause at least some targeted precursor ions, e.g. precursor ions that have some specific chemical property or properties, to dissociate, fragment or react (and such that substantially fewer non-targeted precursor ions, e.g. precursor ions that do not have the specific chemical property or properties, dissociate, fragment or react, or such that non-targeted precursor ions do not dissociate, fragment or react). That is, the reactive buffer gas or reactive buffer gas additive may comprise a chemically selectively reactive reagent gas or gas additive.

Suitable reactive gases include, for example, ozone, which may be used for gas phase ozonolysis, e.g. for the location of unsaturated lipid double bonds, and deuterated gases or vapours, which may be used for gas phase hydrogen- deuterium exchange ("HDX"). A number of other reactive gases could be used. Suitable unreactive buffer gasses include, for example, nitrogen and (clean) air.

Additionally or alternatively, photo-fragmentation may be used to cause the ions to fragment within the drift cell or region. For example, a laser or other light source, e.g. that is configured to emit light having a selected wavelength or wavelength range, may be used to (selectively) photo-fragment (targeted) ions as they pass through the drift cell or region.

Other selective dissociation or fragmentation processes can be utilised, e.g. as the ions travel into and/or through the drift cell or region.

According to various embodiments, the reaction product ions are created within the drift cell or region. At least some reaction product ions may be created from precursor ions as the precursor ions are passed into the drift cell or region, i.e. immediately upon the precursor ions entering the drift cell or region. At least some reaction product ions may be created as the precursor ions are passed through, e.g. as the precursor ions are urged through, the drift cell or region.

As such, at least some or all of the reaction product ions may be separated according to their ion mobility, e.g. as they pass through (i.e. are urged through) the (remainder of the) drift tube.

The reaction product ions may comprise any suitable ions that result from the precursor ions being dissociated, fragmented or reacted in the drift cell or region. The reaction product ions may comprise first generation reaction products ions, second generation reaction product ions (e.g. that result from multiple-step reaction processes), and/or third or higher generation reaction product ions.

Ions that emerge from the drift cell or region and/or ions derived from ions that emerge from the drift cell or region are analysed, e.g. mass analysed. That is, un-reacted (un-fragmented) precursor ions (e.g. including non-reactive precursor ions and/or any remaining reactive precursor ions from the initial population of precursor ions passed into the drift cell or region that have not reacted), together with reaction product ions, and optionally together with other background ions, and/or ions derived from these ions, are analysed.

Accordingly, the spectrometer may comprise a mass analyser, e.g.

downstream of the drift cell or region. The mass analyser may comprise any suitable mass analyser, such as for example, a time of flight ("ToF") mass analyser, e.g. an orthogonal time of flight ("ToF") mass analyser.

The mass analyser may be selected from the group consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance ("ICR") mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance ("FTICR") mass analyser; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadro-logarithmic potential distribution; (x) a Fourier

Transform electrostatic mass analyser; (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; and (xiv) a linear acceleration Time of Flight mass analyser.

The ions that are analysed may comprise ions that emerge from the drift cell or region and/or ions derived from ions that emerge from the drift cell or region.

The ions that emerge from the drift cell or region may be filtered and/or dissociated, fragmented or reacted, e.g. by a second ion filtering device and/or a second collision, fragmentation or reaction device, which may be arranged downstream of the drift cell or region and upstream of the analyser, i.e. before the ions are analysed.

It should be noted that post-reaction, second generation product ions of the reaction product ions themselves will exhibit the same (e.g. relatively broad) drift time width, profile or shape as the associated reaction product ion profile at the same drift time, and so can be associated with specific reaction product ions. Thus, according to various embodiments, ions that emerge from the drift cell or region are dissociated, fragmented or reacted to produce second generation product ions and the second generation product ions are analysed (together with other ions) so as to produce the one or more data sets.

In these embodiments, the one or more reaction product ions that are identified in the one or more data sets based on an ion mobility peak width, profile or shape (as described further below) may comprise one or more second generation product ions (i.e. that have been produced from reaction product ions).

Additionally or alternatively, in these embodiments, the method may further comprise identifying one or more second generation product ions (i.e. that have been produced from reaction product ions) in the one or more data sets that are associated with (i.e. derived from) one or more reaction product ions, e.g. based on the ion mobility peak width, profile or shape and/or on the ion mobility drift time of the ions.

Ions may be trapped, i.e. accumulated, before they are analysed. This can facilitate increased duty cycle operation, e.g. where the analyser is configured to analyse each group of ions of plural consecutive groups of ions in turn. The spectrometer may comprise a second ion trap or trapping region for this purpose, which may be arranged downstream of the drift cell or region and upstream of the analyser. Additionally or alternatively, the second collision, fragmentation or reaction device may be used to trap ions before they are analysed.

The spectrometer may be operated in various modes of operation including, for example, a mass spectrometry ("MS") mode of operation; a tandem mass spectrometry ("MS/MS") mode of operation; a multiple fragmentation or reaction stage ("MS ^N ") mode of operation; a Multiple Reaction Monitoring ("MRM") mode of operation; a Data Dependent Analysis ("DDA") mode of operation; a Data

Independent Analysis ("DIA") mode of operation; or a Quantification mode of operation.

The one or more data sets may comprise one or more two dimensional drift time ("DT") versus mass to charge ratio ("m/z") data sets. The one or more data sets may comprise plural two dimensional drift time ("DT") versus mass to charge ratio ("m/z") data sets, where each data set corresponds to a different

chromatographic retention time ("RT") or retention time range. According to various embodiments, one or more reaction product ions are identified in the one or more data sets based on an ion mobility drift time peak width, profile or shape.

According to various embodiments, the dissociation, fragmentation or reaction process occurs for precursor ions as they are passed into and/or as they pass through (i.e. are urged through) the drift tube. The dissociation, fragmentation or reaction process may occur for the ions of the initial population of precursor ions that is passed into the drift cell or region at different points (axial positions) along the drift cell or region (as the ions are passed (urged) through the drift cell or region), i.e. at different transit times of the ions through the drift cell or region, and may occur over a significant proportion of the length of the drift cell or region, i.e. during a significant proportion of the transit time of the ions through the drift cell or region.

As such, reaction product ions will be caused to transit along different lengths of the drift cell or region, i.e. will experience different transit times through the drift cell or region, e.g. depending on where in the drift cell or region the particular reaction product ion is created.

Therefore, the resulting ion mobility drift time peak width of product ions will typically be relatively broad, e.g. when compared with the ion mobility drift time peak width of precursor ions or conventional ion mobility peak widths.

In addition, the dissociation, fragmentation or reaction process may have some reaction probability. Relatively more precursor ions may be dissociated, fragmented or reacted per unit time when there are relatively more precursor ions present in the drift cell or region, e.g. since there is a relatively high chance of ions interacting with the reactant molecules or otherwise being dissociated, fragmented or reacted. Relatively fewer precursor ions may be dissociated, fragmented or reacted per unit time when there are relatively fewer precursor ions present in the drift cell or region, i.e. as the precursor ion population decreases.

Therefore, the ion mobility drift time peak shape of product ions may be relatively unsymmetrical or skewed, e.g. when compared with the ion mobility drift time peak of precursor ions or conventional ion mobility peaks.

The width and shape of the ion mobility drift time peak will depend on the rate at which the reaction proceeds, as well as on the ion mobility of the associated product ion(s). According to various embodiments, the ion mobility drift time peak width, profile or shape of ions present in the one or more data sets is compared with an expected (e.g. theoretically calculated or previously measured) or other threshold ion mobility drift time peak width, profile or shape. Reaction product ions may be identified on the basis of the comparison. For example, where an ion mobility drift time peak width, profile or shape is larger, wider or less symmetrical than an expected or other threshold ion mobility drift time peak width, profile or shape, then it may be determined that the peak corresponds to reaction product ion(s).

According to various embodiments, one or more reaction product ions are identified in the one or more data sets and/or one or more precursor ions that are associated with one or more reaction product ions are identified in the one or more data sets based on a change in ion intensity between two or more data sets.

According to various embodiments, the reaction time and/or rate, and hence the conversion efficiency of precursor ions into reaction product ions is controlled, e.g. altered or varied. This may be done in any suitable manner.

According to various embodiments, the transit time of ions through the drift cell or region is controlled, e.g. altered or varied. This may be done, for example, by controlling (altering or varying) the urging force experienced by ions through the drift cell or region. For example, the magnitude of the static DC field within the drift cell or region, and/or the velocity, the amplitude, the amplitude ramp, and/or the velocity ramp of a travelling DC wave within the drift cell or region may be controlled, e.g. altered or varied. Additionally or alternative, the gas flow, and/or a pseudo- potential driving force, or combinations of the above may be controlled, e.g. altered or varied.

Additionally or alternatively, the reaction rate may be controlled (altered or varied), e.g. by controlling the concentration of reactant gas, and/or the total pressure of the reactant and buffer gas. Other arrangements would be possible.

According to various embodiments, the reaction time and/or rate is repeatedly switched between two or more different values, e.g. such that

consecutive data sets are acquired using different drift cell reaction times and/or rates.

Sets of data acquired using the different reaction times and/or rates, e.g. consecutive sets of data having the same or similar chromatographic retention times, may then be compared. Thus, according to various embodiments, identifying one or more reaction product ions in the one or more data sets and/or identifying one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions (i.e. one or more reacted precursor ions) comprises comparing an ion intensity between two or more data sets.

According to various embodiments, the two or more data sets that are used in the identification may comprise two or more data sets that are acquired using different reaction times and/or rates.

The ion peak intensity of non-reactive precursor ions, or other non-reactive background ions, will be substantially unchanged between respective data sets.

In contrast, reactive precursor ion peaks will be substantially more intense in data set(s) produced using relatively shorter reaction times or slower reaction rates. Similarly, reaction product ion peaks will be substantially less intense in data set(s) produced using relatively shorter reaction times or slower reaction rates.

Accordingly, by comparing data sets acquired using different reaction times and/or rates, reactive and non-reactive ions can be differentiated.

In addition reaction product ions can be associated with their corresponding precursor ion(s). That is, two or more data sets may be compared to determine the precursor ion(s) associated with identified reaction product ions, i.e. to determine the precursor ions(s) from which identified reaction product ions are derived.

Thus, according to various embodiments, identifying one or more reaction product ions in the one or more data sets and/or identifying one or more precursor ions in the one or more data sets that are associated with one or more reaction product ions comprises identifying one or more reaction product ions in the one or more data sets and identifying one or more precursor ions in the one or more data sets that are associated with the one or more identified reaction product ions.

Where reaction product ions are formed by multiple-step reaction processes, and where the final reaction product ions and/or intermediate ions are present in the data set(s), for relatively longer reaction times or faster reaction rates, some of the reaction product ion peaks may decrease in intensity and others may increase. Therefore, according to various embodiments, reaction product ions may be identified (recognised) by a decrease or increase in ion intensity, e.g. compared to non-reactive ions which will not substantially change in intensity or will change significantly less in intensity with respect to different reaction times and/or rates. Thus, according to various embodiments, where an ion intensity of a particular ion peak changes or changes by more than a threshold amount between the two or more data sets, then it may be determined that the ion peak corresponds to a reaction product ion and/or a reactive precursor ion. Where an ion intensity of a particular ion peak does not change, or changes less than a threshold amount between the two or more data sets, then it may be determined that the ion peak corresponds to other than a reaction product ion and/or a reactive precursor ion, e.g. corresponds to a non-reactive ion.

In these embodiments, where the reaction time and/or rate is switched between two or more different values, the two or more different values may both be non-zero, or alternatively, one of the values could effectively be zero, i.e. the reaction time and/or rate could be such that there is no dissociation, fragmentation or reaction of ions (of interest).

According to various embodiments, one or more reaction product ions and/or one or more precursor ions that are associated with one or more reaction product ions may be identified in the one or more data sets based on a mass to charge ratio and/or ion mobility, drift time or collision cross section (CCS) difference, e.g. corresponding to a known or predicted neutral loss or gain.

In this regard, chemically selective reaction or dissociation can lead to reaction product ions which have a characteristic constant neutral loss or gain in mass to charge ratio (m/z) in comparison to the associated precursor ion.

Interrogation of the data to look for these characteristic losses and/or gains can be used to locate or confirm the presence of reactive species. These methods may be used in combination with the methods described above to improve confidence in identification of product ions and/or associated precursor ions.

In addition these constant losses and gains in mass to charge ratio (m/z) cause broadly deterministic shifts in drift time for product ions compared to precursor ions due to the strong correlation between mass to charge ratio (m/z) and ion mobility. These drift time differences or collision cross section (CCS)

differences may also be used to improve confidence in identification of precursor, product ion pairs.

Thus, according to various embodiments, where a particular (neutral loss or gain) mass to charge ratio and/or ion mobility, drift time or collision cross section (CCS) difference between two or more ion peaks is present in one or more of the data sets, then it may be determined that the ion peaks corresponds to a reaction product ion and/or a reactive precursor ion.

According to various embodiments, one or more precursor ions that are associated with (i.e. that give rise to) one or more reaction product ions may be identified (and associated with its reaction product ions and/or ions derived from the reaction product ions) based on the chromatographic retention time ("RT") of the ions. For example, precursor ions and reaction product ions that have the same or similar chromatographic retention time ("RT") may be determined to be associated with one another.

According to various embodiments, the identification(s) may comprise processing the one or more data sets, e.g. using a software algorithm.

According to various embodiments, ion peaks which are determined to arise from reaction product ions and reacted precursor ions may be selected, and other ion peaks, e.g. which do not arise from reaction products, may be rejected, e.g. so as to produce one or more simplified data sets.

Additionally or alternatively, the analyte (e.g. one or more specific compounds) may be identified on the basis of the ion identification or identifications.

In this regard, the mass to charge ratio ("m/z") and ion mobility drift time, peak width, profile and/or shape of reaction product ions and/or associated precursor ions will be characteristic of the analyte. As such, according to various embodiments, the one or more (two dimensional mass to charge ratio versus drift time ("m/z, DT")) data sets may be used (optionally together with the reaction time, the reactant gas choice and concentration) as a fingerprint to locate and/or confirm the presence of a target analyte in a sample.

In embodiments where precursor ions that are associated with one or more reaction product ions (i.e. reactive precursor ions) are identified, the identification may be used as desired. For example, the identification of reactive precursor ions may be used to trigger, inform or control a subsequent analysis, e.g. a Data

Dependent Acquisition ("DDA"). In one such embodiment, identified reactive precursor ions may be selected (e.g. filtered) (i.e. passed through a (upstream) mass filter with a relatively narrow mass to charge ratio window centred on the mass to charge ratio of the identified reactive precursor ions) and then subjected to further analysis such as tandem mass spectrometric or other analysis. Selecting identified reactive precursor ions, e.g. using a mass filer, in this manner will reduce interferences and simplify the obtained data sets. Additionally or alternatively, identification of reaction product ions may be used to trigger, inform or control a subsequent analysis, e.g. a Data Dependent Acquisition ("DDA"). For example, identified reaction product ions may be selected (e.g. filtered) (i.e. passed through a (downstream) mass filter with a relatively narrow mass to charge ratio window centred on the mass to charge ratio of the identified reaction product ions) and then subjected to further analysis such as tandem mass spectrometric or other analysis.

It will be appreciated that various embodiments are directed to the use of an atmospheric pressure drift tube to investigate reaction kinetics. A population of ions may be pulsed into the drift tube and driven through a reactive buffer gas, or buffer gas additive, e.g. using an electrostatic force or electro dynamic force. During the transit time, selective ion molecule reactions may occur due to the interaction of ions with reactive neutrals in the buffer gas.

The reactant may be chosen to react only with specific analyte ions having specific chemical properties. Ions which do not have these specific characteristics will not react with the buffer gas and may pass through the drift tube intact and un- attenuated.

Reaction product ions have a different mass to charge ratio ("m/z") value and therefore a different ion mobility compared to the associated precursor ion, and so elute at different ion mobility drift times ("DT") compared to the precursor ion.

Since, according to various embodiments, fragmentation or reaction of the precursor ion population occurs over a significant proportion of the transit time through the drift cell, the eluting peak profile of the product ions is broad compared to the profile of the remaining precursor ion population. The width and shape of the profile reflects the rate at which the reaction proceeds. Ions from other species which do not react with the buffer gas will have peak shapes which are more typical of conventional ion mobility peak profiles.

The ion mobility drift tube may be combined with a down-stream mass spectrometer, such as an orthogonal time-of-f light mass spectrometer, e.g. to produce two dimensional drift time versus mass to charge ratio ("DT-m/z") data, e.g. at rates capable of profiling chromatographic separations.

Reconstructed mobility profiles of specific mass to charge ratio ("m/z") ranges may be generated, e.g. to allow drift time profiles to be examined.

By comparing the measured mobility peak profile to an expected (e.g.

theoretically calculated or previously measured) profile for species eluting at different drift times, reaction product ions, e.g. which may arise from ion-molecule reactions of precursor ions during transit through the drift cell, may be distinguished from ions which have not reacted or remaining un-reacted precursor ions.

According to various embodiments, the reaction time and hence the conversion efficiency of precursor ions into reaction product ions can be controlled, e.g. by controlling the transit time of ions through the drift cell (i.e. reaction cell).

Data may be acquired repetitively using two or more different reaction times, e.g. under two or more different settings of the driving force that urges ions through the drift cell (reaction cell). The two sets of data may be compared, e.g. at each retention time, to determine the precursor ion associated with the identified reaction product ions, i.e. to determine the precursor ion from which the identified reaction product ions are derived.

According to various embodiments, in a travelling wave ion mobility device, the speed, the amplitude, the amplitude ramp, and/or the velocity ramp may be altered between two different values, e.g. to produce consecutive drift time ("DT") versus mass to charge ratio ("m/z") data sets (i.e. spectra), i.e. where the ions analysed in consecutive data sets have experienced different transit times through the drift cell.

Un-reactive background species, which may be identified or confirmed by their characteristic ion mobility separation ("IMS") peak shape, will be substantially unchanged in intensity, whereas reactive precursor ions will be substantially more intense in the data set(s) produced using the shorter reaction time (shorter drift time).

Reaction product ions, which again may be identified or confirmed by their characteristic ion mobility separation ("IMS") peak shape, will be less intense in the data set(s) produced using the at the shorter reaction time.

These effects can be used both to differentiate between reactive and non- reactive species, and also to associate reaction product ions with their precursor ions.

According to various embodiments, the data set(s) may be interrogated in the drift time ("DT") domain, or the chromatographic retention time ("RT") domain, or in both dimensions simultaneously. The RT profile of the product ions and precursor ions may be used to associate precursor ions with product ions. The combination of these approaches allows precursor ions and associated reaction product ions to be identified and extracted at each chromatographic retention time. Some reaction product ions may be formed by multiple-step reaction processes, and both the final reaction product ions and intermediate ions may be present in the data set(s) (i.e. in the mass spectra and ion mobility spectra). In this case, for relatively longer reaction times, some of the reaction product ions will decrease in intensity and others will increase. Therefore, reaction product ions may be recognised by a decrease or increase in ion intensity, e.g. compared to un- reactive species which will not substantially change in intensity or will change significantly less in intensity with respect to reaction time.

The specific ion-molecule reaction or reactions that are used according to various embodiments, may comprises any suitable ion-molecule reaction(s).

For example, gas phase ozonolysis may be used, e.g. for the location of unsaturated lipid double bonds. In this reaction, the olefinic bonds are converted to ozonides which subsequently dissociate to produce product ions characteristic of the double bond position. Ozonolysis may be used in reactions with other types of ions. For example, oxidation of Tyrosine and Histidine amino acid residues in peptides and proteins may be performed using Ozone as a reactant gas.

Characterisation of protein structure by this method may be performed in a similar way to known hydrogen deuterium exchange ("HDX") techniques.

Other reactions that may be used in accordance with various embodiments include hydrogen deuterium exchange ("HDX"), e.g. for characterisation of protein structure and/or folding or small molecule characterisation, and proton transfer, e.g. for charge state manipulation.

Many other specific selective gas phase reactions can be used in

accordance with various embodiments.

Fig. 1 shows a block diagram of a spectrometer according to various embodiments.

The spectrometer may comprise an ion source 1 , an optional mass filter 2 downstream of the ion source 1 , an optional ion trap and/or gas cell 3 downstream of the ion source 1 and the mass filter 2, a drift or reaction cell 4 downstream of the ion source 1 and the gas cell 3, an optional ion trap and/or gas cell 5 downstream of the drift or reaction cell 4, and a mass analyser 6 downstream of the reaction cell 4 and the gas cell 5.

As shown in Fig. 1 , the spectrometer may also comprise a control system and/or a processing system. The control system and the processing system may be at least partially provided as a single (the same) (integrated) system, or as distinct (separate) systems. The control system may be configured to control the operation of the spectrometer, e.g. in the manner of the various embodiments described herein. The control system may comprise suitable control circuitry that is configured to cause the spectrometer to operate in the manner of the various embodiments described herein. The processing system may comprise suitable processing circuitry configured to perform any one or more or all of the necessary processing and/or post-processing operations in respect of the various

embodiments described herein.

Ions may be produced in the ion source 1 and may optionally be mass selected by the mass filter 2. The mass filter may comprise, for example, a quadrupole mass filter.

The gas cell 3 may act as both a collision cell, e.g. for pre-reaction fragmentation and/or as an ion trap, e.g. for trapping ions prior to releasing them into the reaction cell 4, e.g. in order to facilitate high duty cycle operation.

Buffer gas containing a reactive component may be introduced into the reaction drift cell 4. The pressure in this region may be similar to or equal to a pressure typically used for ion mobility separations. For example, the gas pressure may be between 0.1 and 10 mbar. This means that ions transiting the drift cell 4 will separate according to their ion mobility.

Ions accumulated in the ion trap/gas cell 3 may be released into the reaction cell 4 at an initial time (i.e. 7=0). Precursor ions and product ions formed during transit through the reaction cell 4 elute from the reaction cell 4 at times T governed by their ion mobilities in the buffer gas.

Ions eluting from the reaction cell 4 can optionally be subjected to post reaction dissociation, for example collisionally induced dissociation ("CID"), in gas cell 5, before being directed to the mass analyser 6 for mass analysis. The mass analyser 6 may comprises, for example, an orthogonal time of flight ("ToF") mass analyser.

The reaction time experienced by ions may be varied by changing the driving force through reaction cell 4. For example, where a DC field is used to urge ions through the reaction cell 4, the magnitude of the DC field may be changed. Where a travelling DC wave is used to urge ions through the reaction cell 4, the amplitude and/or velocity of the travelling wave may be changed. Other driving forces such as the gas flow, a pseudo-potential driving force, or combinations of the above may be changed to vary the reaction time

experienced by ions.

The reaction rate may also be varied, e.g. by controlling the concentration of reactant gas, and/or the total pressure of the reactant plus buffer gas, e.g. while using the same driving force, or changing the driving force.

Where the reaction time and/or rate is switched between two or more different values, the two or more different values may both be non-zero, or alternatively, one of the values could effectively be zero, i.e. the reaction time and/or rate could be such that there is no dissociation, fragmentation or reaction of ions (of interest) (e.g. one of the different reaction rates and/or dissociation energies could be effectively zero reaction rate or a dissociation energy at which there is no dissociation of the analyte of interest).

Figs. 2A, 2B and 2C show schematically reconstructed mass mobility spectra, and illustrate the characteristics of reactant product ions, non-reactive product ions from post reaction collisionally induced dissociation ("CID"), and non- reactive background ions.

Fig. 2A shows schematically a mobility spectrum acquired without a reactant present in the drift cell 4, i.e. with an ion mobility separation ("IMS") buffer gas present in the drift cell 4, under "normal" conditions.

The largest peak 7 in Fig. 2A corresponds to the reconstructed mass mobility peak for a precursor ion.

Another peak 8 is present at the same drift time ("DT"), which corresponds to a post-reaction (e.g. CID) product ion having a different mass to charge ratio ("m/z") to the precursor ion, but substantially the same ion mobility drift time.

The other two peaks 9 in Fig. 2A corresponds to two species which could correspond to pre-ion mobility separation ("pre-IMS") CID reaction product ions of the precursor ion 7, and/or background ions that are unrelated to the precursor ion 7.

These peaks 9 may be due, for example, to different precursor ion types

(e.g. different lipid classes) giving rise to product ions (e.g. CID product ions), which are not reactive to the buffer gas (e.g. do not contain double bonds and are not therefore reactive to Ozone). These fragment ions may be produced by performing pre-reaction collision, fragmentation or reaction (e.g. CID), e.g. in gas cell 3. These fragment ions may pass through the reaction cell 4 un-attenuated and without elution profile distortion.

In various embodiments, such fragment ions may be characteristic fragment ions (e.g. characteristic CID fragment ions), and may be used to locate or confirm specific species classes (e.g. specific lipid classes).

Fig. 2B shows schematically a mobility spectrum for the same mixture of ions as shown in Fig. 2A, but where a reactant gas is present in the drift cell 4. In Fig. 2B, the absolute drift times of the ions are illustrated as being unchanged for simplicity. However, in reality, the addition of a reactant gas, e.g. at the same pressure as the buffer gas used in Fig. 2A, will change the absolute drift times.

As shown in Fig. 2B, the precursor ion peak 7 has reduced in intensity due to the precursor ions reacting during their transit through the drift cell 4. Similarly, the intensity of the post-reaction product ion peak 8 (which may be a post reaction CID product of remaining un-reacted precursor ions), has reduced in proportion to the reduction of the precursor ion peak 7.

The other two peaks 9 are unchanged, suggesting that the ions do not react as they pass through the drift cell 4.

The additional peaks 10 present in Fig. 2B correspond to two reaction product ions which have a lower mass to charge ratio ("m/z") and hence a higher ion mobility than the precursor ion 7. These may be produced throughout the transit time of the precursor ions through the drift cell 4, and so appear broader and less symmetrical compared to the un-reacted product ion peaks 9.

The additional peak 11 present in Fig. 2B corresponds to a reaction product ion which has a lower ion mobility than the precursor ion 7, and so appears at a higher drift time ("DT"). Such ions may arise, e.g., from the formation of a precursor-reactant adduct during the transit of ions through the drift cell 4. Again, the reaction product ion is readily distinguishable due to its relatively broad, and asymmetrical peak shape.

It should be noted that post-reaction, second generation (e.g. CID) product ions of the reaction product ions themselves will exhibit the same broad drift time profile as the associated reaction product ion profile at the same drift time, and so can be associated with specific reaction product ions.

Thus, product ions formed in the drift cell may be dissociated in a post drift cell dissociation device and the precursor ions (i.e. the reaction product ions) may be correlated with the associated (second generation) product ions based on the characteristic peak shape and drift time ("DT") of the ions. This is analogous to MS- MS-MS, where the first generation product ions are formed in the drift cell and identified by their IMS peak shape and drift time ("DT"). First generation product ions may then be associated with the corresponding second generation product ions based on their characteristic peak shape and drift time ("DT").

It is also possible to dissociate ions before the drift cell and to then identify reaction product ions of the first generation product ions and to optionally dissociate these ions post reaction (this is analogous to MS-MS-MS-MS).

In addition, chromatographic retention time ("RT") and/or chromatographic peak shape may also be used to correlate first, second and/or third generation product ions with their corresponding precursor ion(s).

In various embodiments, data is acquired under different conditions of dissociation and/or reaction, e.g. within chromatographic timescales. This generates multiple data sets which can be compared to extract the drift time ("DT") and retention time ("RT") profiles required to correlate precursor ions and product ions.

Fig. 2C shows schematically a mobility spectrum for the same mixture of ions as shown in Figs. 2A and 2B, but under conditions whereby the precursor ion has taken less time to traverse the drift cell 4, and so has reacted for less time relative to Fig. 2B. As shown in Fig. 2C, this results in shorter drift times for all the species in the data.

As also shown in Fig. 2C, the remaining un-reacted precursor ion peak 7 and associated post reaction fragment ion peak 8 both increase in intensity, since less precursor ions are converted into reaction product ions.

Un-reactive ion peaks 9 do not substantially change in intensity, although their drift times do change.

Reaction product ion peaks 10 and 11 reduce in intensity. Based on the intensity change alone, it is possible to distinguish the precursor ion and all product ions related to the precursor ion from un-reactive background ions.

According to various embodiments, in addition to correlating reaction product ions and reactive precursor ions based on their intensity and elution profiles (drift time profiles), in individual two-dimensional drift time versus mass to charge ratio ("DT, m/z") spectra, ion peaks arising from the same component can be correlated, e.g. based on their chromatographic elution profile or elution time. According to various embodiments, any calibration standard, e.g. internal or external calibration standard, which is used to calibrate the spectrometer, may be selected be substantially non-reactive with respect to the reaction cell 4. A calibration standard may be introduced continuously or periodically, e.g. during an experiment and may pass through the reaction device 4. Selecting an unreactive calibration standard beneficially ensures that the calibration standard can be relatively straightforwardly distinguished from the reaction product ions, thereby minimising interference and ensuring that the calibration standard remains substantially unchanged in intensity regardless of the reaction time chosen.

As described above, the reaction time may be switched repetitively and continuously between two different values.

According to various embodiments, the reaction time may be adjusted for particular target species, e.g. with known chromatographic retention (elution) time ranges. In this way, the reaction times, and hence total reaction efficiency, may be tailored for specific analytes, e.g. based on known or previously determined rates of reaction. A combination of these approaches may be used.

Additionally or alternatively, the reaction time may be adjusted dynamically, e.g. based on the acquired data, to produce a desired reaction efficiency. The reaction time may be controlled as part of a feedback process, e.g. to ensure that the desired conversion of precursor ions to product ions is maintained.

According to various embodiments, real time interrogation of the drift time ("DT") versus mass to charge ratio ("m/z") spectra for the different reaction times may be used to locate likely reactive precursor ions. Reactive precursor ions (precursor ions which react in the cell) may be identified by the methods described above. Reactive precursor ions may be of interest. For example, in the case of ozonolysis and lipids it is likely that these reactive ions will be unsaturated lipid species.

Based on identification of these candidate precursor ions, the mass filter 2 may be switched to transmit a narrow mass to charge ratio ("m/z") range, e.g.

including this candidate mass to charge ratio ("m/z") value. These ions may then be analysed as desired, e.g. in a tandem mass spectrometry (MS-MS) mode of operation or otherwise. Accordingly, various embodiments may be used to drive data dependant mass selection, thereby reducing interferences and simplifying the reaction product ion spectra. It will be appreciated that the mass to charge ratio ("m/z") and elution profiles of reaction product ions and associated precursor ions are characteristic of the analyte. Therefore, according to various embodiments, the reaction time, the reactant gas choice and concentration, and the two dimensional mass to charge ratio versus drift time ("m/z, DT") data, may be used as a fingerprint to locate and/or confirm the presence of a target analyte in a sample.

According to various embodiments, a library of two dimensional data may be created, e.g. using standards.

Additionally or alternatively (e.g. if the chemistry governing the reaction product ions and reaction rates are known), a theoretical library of expected mass to charge ratios ("m/z") and elution profiles may be created, e.g. in silico, and used to locate and/or confirm targeted species. As described above, the ion mobility drift time peak width of product ions will be relatively broad due to reactions occurring throughout the drift region. As such, the calculated ion mobility elution profile (ion mobility drift time peak width) for the library may depend on the actual ion collision cross section (CCS) and its reaction rate.

Fig. 3 shows schematically the two dimensional mass to charge ratio ("m/z") versus drift time ("DT") 'heat map' plot for the data of Fig. 2B.

According to various embodiments, identification of reaction product ions and/or precursor ions, and/or the association of reaction product ions and their precursor ions at different retention times may be achieved in a "continuous beam flow through" manner, i.e. without generating drift time profiles, by repetitively changing the reaction time, e.g. as described above, and by monitoring the intensity of the acquired mass spectral peaks at each retention time.

Ions with the same retention time profile may be associated together as relating to a single component eluting from the chromatogram, and un-reactive background ions may be distinguished from reactant ions and precursor ions, e.g. based on their intensity difference between the data acquired using the two different reaction times.

In this way, simplified spectra of reaction product ions and associated precursor ions can be generated without interferences from non-reactive species, thereby facilitating efficient additional post-processing, such as analyte identification, etc. Non-reactive ions from unwanted background components may be recognised and removed from the data due to the invariant relative intensity of these species with respect to reaction time. Although above embodiments have been described primarily in terms of reacting the precursor ions using a reactive buffer gas or a reactive buffer gas additive, it would additionally or alternatively be possible to fragment or react the ions as they transit the drift cell 4 using one or more other techniques.

For example, selective photo-fragmentation processes can be (and in various embodiments is) performed during the transit of ions through the drift cell 4. Other selective fragmentation processes can be utilised as the ions travel through the drift cell 4.

Chemically selective reaction or dissociation can lead to product ions which have a characteristic constant neutral loss or gain in mass to charge ratio (m/z) in comparison to the associated precursor ion. Interrogation of the data to look for these characteristic losses and gains can be used to locate or confirm the presence of reactive species. These methods may be used in combination with the methods described above to improve confidence in identification of product ions and associated precursor ions.

In addition these constant losses and gains in mass to charge ratio (m/z) cause broadly deterministic shifts in drift time for product ions compared to precursor ions due to the strong correlation between mass to charge ratio (m/z) and ion mobility. These drift time differences or collision cross sections (CCS) may also be used to improve confidence in identification of precursor, product ion pairs.

It will be appreciated that various embodiments are directed to the de- convolution of ion-molecule reaction products or other reaction product ions.

Various embodiments are directed to a method of identifying mass spectral peaks arising from reaction product ions and/or corresponding precursor ions emerging or eluting from an ion-molecule reaction or other reaction drift tube coupled with a down-stream mass spectrometer in the presence of non-reactive background ions. The method may comprise identifying reaction product ions by their characteristic drift time profile or shape and/or identifying reaction product ions and their associated precursor ions by comparing the signal intensity between two or more data sets acquired using different reaction times and/or rates.

The reaction cell may comprise a linear DC field or a travelling DC wave ion mobility device, and ions may be confined within the reaction cell by RF

confinement or non-RF confinement. The method may comprise repetitively switching between two or more reaction times, e.g. by altering the driving force, residence time or reagent gas concentration.

According to various embodiments, ion mobility separation is performed in the presence of a chemically selectively reactive reagent gas or gas additive, or at the same time as some other selective fragmentation process.

Acquired two dimensional drift time ("DT") versus mass to charge ratio ("m/z") data may be processed using a software algorithm, e.g. to identify and extract mass to charge ratio ("m/z") peaks which arise from reactive precursor ions and reaction product ions and to reject peaks which do not arise from reactive precursor ions and reaction product ions, such as, for example, endogenous background species, and/or post- or pre-ion mobility separator ("IMS") separation fragmentation or dissociation products.

The methods according to various embodiments enable a simplified mass spectrum of reaction product ions, e.g. at a particular chromatographic retention time, to be produced.

The identification of reaction product ions according to various embodiments can be particularly useful in the analysis of complex mixtures, e.g. where analytes of interest may be relatively minor components in the presence of intense background ions which have a dissimilar chemical nature to the analyte ions of interest, and hence do not substantially react with the buffer gas or other reaction process.

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.