WO2025045340A1

WO2025045340A1 - Improvements in and relating to ion spectrometry

Info

Publication number: WO2025045340A1
Application number: PCT/EP2023/073436
Authority: WO
Inventors: Andrew Entwistle; Patrick Knight; Stephane Moreau; Ann-Christin NIEHOFF; Pauline GRIFFEUILLE; Franck SAINT-MARCOUX; Souleiman EL BALKHI
Original assignee: Shimadzu Research Laboratory (Europe) Ltd
Priority date: 2023-08-25
Filing date: 2023-08-25
Publication date: 2025-03-06

Abstract

An ion spectrometry apparatus for analysing ions from a sample comprising a pre-set target compound. An ambient pressure ionization (API) source ionizes the sample to generate ions. The API source generates the ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby. An ion mobility analysis unit receives ions periodically from the API ion source as generated during successive ion generation time intervals. It transmits ions from amongst the received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied to its electrodes during an ion transmission time interval. Transmission of ions received from the sample is permitted according to ion mobility and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage is selected to permit transmission of ions from the pre-set target compound and to prevent transmission of other ions amongst said ions from the sample.

Description

IMPROVEMENTS IN AND RELATING TO ION SPECTROMETRY

Field of the Invention

The present invention relates to ion spectrometry, such as mass spectrometry or ion mobility spectrometry, and particularly, although not exclusively, to ion spectrometry involving ambient ionisation methods.

Background

Ambient ionisation methods allow for rapid analysis of solid or liquid samples, generally with little or no sample preparation. Some methods can also be used for imaging or localised sampling (e.g., for clinical settings). However, the samples are often complex and produce many ions which generate complex and overlapping spectra. This makes data interpretation difficult and can also lead to interferences from isomeric ions etc. In liquid chromatography mass spectrometry (LC-MS) methods the spectral complexity is overcome by adding an additional separation stage before the mass spectrometry (MS) stage. This cannot be done in ambient ionisation as the sample is introduced directly to the mass spectrometer.

The present invention has been devised in light of the above considerations.

Summary of the Invention

At its most general the invention proposes performing spectrometry of ions generated by an ambient pressure ionization (API) source that are pre-filtered by an ion mobility analyser/spectrometer unit before undergoing detection, or downstream mass spectrometry. The API source may be a periodic or pulsed ion source. It may be configured to generate ions periodically during a plurality of successive ion generation time intervals (e.g., ion pulses) separated by intervening time intervals in which no ions are generated.

The inventors have found that with pre-filtering by ion mobility it is possible to efficiently identify suitable operating parameters for an ion mobility analyser/spectrometer which very effectively remove (filter-out) ion interferences/species amongst ions from a sample under study that are not those from a target compound of interest for which an ion spectrum is desired (e.g., a chromatogram, a mass spectrum, an ion mobility spectrum, etc.). Removal of interferences, results in removal of unwanted spectral peaks which may otherwise swamp or overwhelm a ion spectrum containing much fewer and/or smaller spectral peals for the target compound, rendering it difficult or impossible to interpret correctly.

Ambient pressure ionization (API) sources, also known as atmospheric pressure ionization (API) sources, may be used to ionize the sample at ambient (e.g., atmospheric) pressure and then transfer the ions into the ion spectrometer (e.g., mass spectrometer, ion mobility spectrometer). API refers to ionisation at atmospheric pressure, whereas ambient ionisation methods (Al) in generally are methods in which ions are formed in an ion source outside an ion spectrometer (e.g., mass spectrometer), often without sample preparation or separation. The combination of an API source with a downstream mass spectrometer may provide an ambient ionization mass spectrometry (AIMS) device. AIMS devices and methods have grown into a group of emerging analytical systems that allow rapid, real-time, high-throughput, in vivo, and in situ analysis in areas such as biomedicine, pharmaceuticals, and forensic sciences to name but a few. Many AIMS techniques have been devised in the past two decades, as discussed in review by reference [1] cited below.

Electro Spray Ionization (ESI) is a common API technique. Here, an electrospray is created by applying a suitably large electric potential between a metal inlet needle and a skimmer in an API source. Theory suggests that a mechanism for the ionization process is as follows. As the liquid leaves the nozzle, the electric field generated by the gradient of the electric potential between the metal inlet needle and the skimmer induces a net charge on small droplets of a solvent containing the sample under study. As the solvent evaporates, the charge density at the surface of the droplet increases because the droplets shrink, finally reaches a size at which Coulomb forces of repulsion between charged particles in the droplet exceed the surface tension maintaining the droplet. This causes the droplet to explode. Multiply charged analyte ions are thereby produced.

Probe electrospray ionization (PESI) is technique that can be used in ambient ionization mass spectrometry (AIMS). This technique, the metal inlet needle is moved back and forth along an axis (usually down along a vertical axis) such that the tip of the needle periodically touches to the sample. After extracting sample material at the needle tip, the needle is then moved away from the sample and a high voltage is applied to the needle, typically when at its greatest separation from the sample, so as to generate electrospray ions. Due to the discontinuous sampling and generation of electrospray, a sequence of time-separated electrospray pulses are created rendering the PESI, in effect, an example of a pulsed ion source.

A pulsed or periodic ionisation source may be considered to include a reference to an ion source configured to generate said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby. A review of ion injection methods and pulsed ion sources is provided in reference [4] identified below.

Other examples of pulsed or periodic ion sources exist. For example, the measurement of the mobility for an ion begins when ions from an ion source are introduced into the drift region of an “ion mobility spectrometry” (IMS) system. The ions may be introduced continuously or periodically. Periodic or pulsed ion sources are a traditional configuration of many IMS systems, which may employ ion shutters. The performance of an ion shutter is controlled by the drift velocities of ions within the drift region of an IMS system. Periodic or pulsed ion sources are also an inherent feature of configurations using travellingwave methods for the transport of ions as discrete ion clusters within travelling potential wells.

The term “ion mobility spectrometry” (IMS) refers to the methods and apparata used to characterise ions from sample substances according to the mobility with which those ions drift through a buffer gas. The ions may be entrained in a buffer gas flow and/or may be driven by an external electric field. Ion drift velocity (v) may thereby depend upon ion mobility through the buffer gas, and in some applications such as FAIMS and DMS discussed below, different electric fields may be applied to exploit this property of the velocity. Ion mobility measurements involve injecting an ion ensemble into a “drift region”, in some but not all cases using an ion shutter at the entrance to control the timing of injection process. An ion detector is provided downstream of the output end of the drift region, immediately or following further analytical steps such as mass spectrometry. Within the drift region of Field Asymmetric waveform IMS (FAIMS) cells, an “analytical gap” is formed transversely between two parallel electrodes, or parallel sets of segmented electrodes. Ions are moved longitudinally along the drift region, within the analytical gap between the electrodes, towards an output port, whilst typically entrained in a flow of neutral buffer or “carrier” gas (commonly molecular nitrogen). Simultaneously, the ions are pulled perpendicularly to the electrodes by electric field of some intensity (E) generated by an appropriate voltage waveform applied across the analytical gap. The buffer gas is typically refreshed continuously to maintain a constant pure environment.

All IMS measurements pertain to the vectorial mean rather than (commonly much larger) instant scalar ion velocities. E.g., the median absolute velocity of N2 ions at the 25° C temperature is ~450 m/s while the vectorial mean may be only v = 4 m/s. The vectorial mean velocity values are commonly normalised by dividing them by the value of the perpendicular electric field E, to produce the ion mobility K. This quantity characterizes ions in a gas of defined composition and depends on the gas temperature, T, and pressure, P. For near-thermal ions in the “low-field limit”, the K value is inversely proportional to the orientationally- averaged ion-molecule collision cross-section (integral).

Unlike Linear IMS based on the absolute K values typically captured in the low-field limit, FAIMS relies on the evolution of K as a function of E at constant gas particle number density, N (the number of particles per unit volume). This can normally be represented as the dependence of K on the E/N ratio (known as the “reduced field intensity”) expressed in units of Townsend (1 Td = 10 ¹⁷ V x cm²).

K(E/N) = K₀(l + a(E/N)) where the “alpha-function” a can be expanded in an infinite series over even powers of E/N with the coefficients a_2n (n = 1, 2, ...) characteristic of a given ion-gas molecule pair and gas temperature: a(E/N) = <z₂ x (E/N)² + a₄ x (E/N)⁴ + ••• + a_2n x (E/N)²ⁿ

The electrodes and thus the analytical gap can be planar or curved. An asymmetric oscillatory electric field is established in the analytical gap using a voltage applied across the electrode pair. The voltage is controlled to vary in time according to a predefined waveform. The waveform must have zero 1^st moment (i.e., equal areas in the positive and negative polarities), hence the high-field segment is shorter duration than the low-field segment. The maximum separation field (ED) is determined by the waveform amplitude (“dispersion voltage”, I/D) and analytical gap width, g, between opposing electrodes. For example, with g = 2 mm a rectangular waveform with 2:1 aspect ratio and I/D = 4 kV produces fields of 20 and 10 kV/cm in the two segments. Had the K value been independent of E (meaning a = 0), all ions would have oscillated with no net drift across the analytical gap and could emerge from the drift region to be detected. As K actually depends on E, in each waveform cycle some ions experience a net drift towards one of the electrodes and would be neutralized on contact with the electrode. To prevent the neutralisation of ions of selected ion mobility, a fixed compensation voltage (Vc) is provided across the analytical gap, g, producing compensation field (Ec) which is superposed on the waveform to offset the net displacement that would otherwise be experienced by the selected ions as they progress along the drift region. This equilibrates the path of the selected ions in the drift region and thus permits them to pass along a longitudinal pathway fully through the drift region of the ion mobility device and onwards to a downstream detector or further analytical device (e.g., mass spectrometer). That outcome is possible for a given Vc only for an ion species with the appropriate displacement in response to the applied dispersion voltage, I/D, depending on the difference between ion mobility, K, values in high-field and low-field segments of the waveform profile. This method provides ion mobility filtering, and ion separations are based on differences in ion mobility, leading to the name “Differential Mobility Spectrometry” (DMS), also known as FAIMS.

By varying/scanning the value of the compensation voltage, Vc, one may reveal the distribution/intensity of ions entering, and subsequently exiting, the drift region in the form of a spectrum as a function of the varying compensation voltage, Vc. By varying/scanning the value of both the compensation voltage, Vc, and the dispersion voltage, I/D, one may produce a VC(I/D) curve for each ion species, which describes the distribution/intensity of ions entering, and subsequently exiting, the analytical gap as a 2-dimensional function of both c and I/D as independent variables spanning two respective coordinate dimensions, (e.g., in an x-y plane of a graph) and the distribution/intensity of ions described as a third coordinate dimension (e.g., in a z-dimension of the graph, or as a heat map spanning the x-y plane of a graph). This data reveals ion separations based on differences in ion mobility.

In a first aspect, the invention may provide a mass spectrometer apparatus for analysing ions from a sample comprising a target compound, comprising: an ambient pressure ionization (API) source arranged to ionize the sample at an ambient atmospheric pressure to generate ions for analysis by the mass spectrometer, wherein the ambient pressure ionization source is configured to generate said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; an ion mobility analysis unit configured to receive ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; a mass analysis unit comprising a mass analyser arranged to receive ions from the ion mobility analysis unit, and an ion detector arranged to detect ions from the mass analyser. For example, the mass analysis unit may be arranged to in communication with the ion mobility analysis unit either to receive ions directly from the latter or indirectly to receive ions therefrom via intervening ion optics etc., before the mass analyser. These ion optics may be part of the mass spectrometer but not part of the mass analysis unit (e.g., other than, in addition to, ion optics of a mass analysis unit, such as a quadrupole or ToF optics).

The ion mobility analysis unit is configured to: vary the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitor the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, select from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof which permits transmission through the ion mobility analysis unit of ions from the target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

In a second aspect, the invention may provide an ion spectrometry apparatus for analysing ions from a sample comprising a target compound, comprising: an ambient pressure ionization (API) source arranged to ionize the sample at an ambient atmospheric pressure to generate ions for analysis, wherein the ambient pressure ionization source is configured to generate said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; an ion mobility analysis unit configured to receive ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; an ion detector arranged to detect ions from the ion mobility analysis unit; wherein the ion mobility analysis unit is configured to: vary the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to said electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitor the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, select from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof which permits transmission through the ion mobility analysis unit of ions from the target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample. In each of these aspects, an efficient process is provided for searching for suitable combinations of a dispersion voltage and a concurrent compensation voltage to be applied thereby to the electrodes of the ion mobility analysis unit, during an ion transmission time interval, for use in filtering out ion interferences.

The following preferable, desirable and/or optional features of the invention are applicable to the invention in its first aspect and in its second aspect described above.

The target compound may be a pre-set target compound, e.g., of known identity or properties.

Alternatively, the user may obtain the identity of the target compound after an initial experiment indicating the presence of the compound (e.g., via data independent acquisition) or implementation of the invention may reveal/indicate the presence of an unknown ion/compound that the user wants to investigate further. By setting/selecting the values of the dispersion voltage and compensation voltage the user sets the ion(s) that will be transmitted, but the target compound may or may not have been pre-set/known beforehand.

The ambient pressure ionization (API) source may comprise an ambient ionisation source.

The ion mobility analysis unit may be configured to vary the values of both the dispersion voltage and the compensation voltage such that none of the values of the dispersion voltage are repeated as between two successive ion transmission time intervals.

The ion mobility analysis unit may be configured to vary the values of both the dispersion voltage and the compensation voltage such that all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

The ion mobility analysis unit may be configured to vary the values of both the dispersion voltage and the compensation voltage such that some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

The ion mobility analysis unit may be configured to vary the values of both the dispersion voltage and the compensation voltage such that none of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

Desirably, each ion transmission time interval may be synchronised to coincide with a respective ion generation time interval.

Successive ion transmission time intervals may be separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit.

Successive ion transmission time intervals may be separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit. The intervening time intervals that separate successive ion generation time intervals may be synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

The target compound may be a pre-set target compound comprising Cortisol.

The ambient pressure ionization (API) source may comprise a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process.

The ion mobility analysis unit may be configured to receive ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and to transmit ions from amongst the received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval. The transmission by the ion mobility analysis unit of ions received from the sample may be permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage may be selected to permit transmission of ions from the target compound and to prevent transmission of other ions amongst said ions from the sample. The pre-set combination of a dispersion voltage and a concurrent compensation voltage may comprise voltages that are be applied throughout a finite interval of time and which remain unchanged during that finite interval of time. The finite interval of time may be substantially equal in duration to an ion generation time interval.

The ion mobility analysis unit may be configured to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage, wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval. The pre-set combinations of a dispersion voltage and a concurrent compensation voltage may each comprise voltages that are be applied throughout a respective one of a plurality of separate finite interval of times such that each distinct combination remains unchanged during that respective finite interval of time. The sum of the durations of the plurality of finite intervals of time may be substantially equal in duration to an ion generation time interval. The values of the dispersion voltage and the compensation voltage corresponding to each pre-set combination may be constant throughout the duration of a respective time sub-interval within an ion transmission time interval. The sum of the respective time subintervals of the plurality of distinct pre-set combinations may collectively define (correspond to) the ion transmission time interval of which each sub-interval forms a part.

In a third aspect, the invention may provide a method for mass spectrometry for analysing ions from a sample comprising a target compound, the method comprising: providing an ambient pressure ionization (API) source and therewith ionising the sample at an ambient atmospheric pressure to generate ions for analysis by mass spectrometry, and by the ambient pressure ionization source generating said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; providing an ion mobility analysis unit and therewith receiving ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; providing a mass analysis unit comprising a mass analyser and an ion detector and, by the mass analyser, receiving ions from the ion mobility analysis unit and, by the ion detector, detecting ions from the mass analyser; wherein the method further comprises: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof which permits transmission through the ion mobility analysis unit of ions from the target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

In a fourth aspect, the invention may provide a method for ion spectrometry for analysing ions from a sample comprising a target compound, the method comprising: providing an ambient pressure ionization (API) source and therewith ionising the sample at an ambient atmospheric pressure to generate ions for analysis, and by the ambient pressure ionization source generating said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; providing an ion mobility analysis unit and therewith receiving ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; providing an ion detector and, by the ion detector, detecting ions from the ion mobility analysis unit; wherein the method further comprises: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof which permits transmission through the ion mobility analysis unit of ions from the target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

The following preferable, desirable and/or optional features of the invention are applicable to the invention in its third aspect and in its fourth aspect described above.

The ambient pressure ionization (API) source provided according to the method may comprise an ambient ionisation source.

The method may comprise varying the values of both the dispersion voltage and the compensation voltage such that none of the values of the dispersion voltage are repeated as between two successive ion transmission time intervals.

The method may comprise varying the values of both the dispersion voltage and the compensation voltage such that all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

The method may comprise varying the values of the compensation voltage such that some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

The method may comprise varying the values of the compensation voltage such that none of the values of the compensation voltages are repeated as between two successive ion transmission time intervals.

In the method, preferably or optionally, each ion transmission time interval may be synchronised to coincide with a respective ion generation time interval.

In the method, preferably or optionally, successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit. Desirably, successive ion transmission time intervals are separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit.

Preferably or optionally, the intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

Desirably, the target compound may be a pre-set target compound comprising Cortisol.

Preferably, the ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process.

The method may comprise the following steps: by the ion mobility analysis unit, receiving ions periodically from the ion source as generated during successive ion generation time intervals, and transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval. Transmission by the ion mobility analysis unit of ions received from the sample may be permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage may be selected to permit transmission of ions from the target compound and to prevent transmission of other ions amongst said ions from the sample. The pre-set combination of a dispersion voltage and a concurrent compensation voltage may comprise voltages that are be applied throughout a finite interval of time and which remain unchanged during that finite interval of time. The finite interval of time may be substantially equal in duration to an ion generation time interval.

The ion mobility analysis unit may be configured to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage, wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval. The pre-set combinations of a dispersion voltage and a concurrent compensation voltage may each comprise voltages that are be applied throughout a respective one of a plurality of separate finite interval of times such that each distinct combination remains unchanged during that respective finite interval of time. The sum of the durations of the plurality of finite intervals of time may be substantially equal in duration to an ion generation time interval. The values of the dispersion voltage and the compensation voltage corresponding to each pre-set combination may be constant throughout the duration of a respective time sub-interval within an ion transmission time interval. The sum of the respective time subintervals of the plurality of distinct pre-set combinations may collectively define (correspond to) the ion transmission time interval of which each sub-interval forms a part. In a fifth aspect, the invention may provide a mass spectrometer apparatus for analysing ions from a sample comprising a pre-set target compound, comprising: an ambient pressure ionization (API) source arranged to ionize the sample at an ambient atmospheric pressure to generate ions for analysis by mass spectrometry, wherein the ambient pressure ionization source is configured to generate said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby. For example, the mass analysis unit may be arranged to in communication with the ion mobility analysis unit either to receive ions directly from the latter or indirectly to receive ions therefrom via intervening ion optics etc., before the mass analyser. These ion optics may be part of the mass spectrometer but not part of the mass analysis unit (e.g.. other than, in addition to, ion optics of a mass analysis unit, such as a quadrupole or ToF optics). The mass spectrometer apparatus comprises an ion mobility analysis unit is configured to receive ions periodically from the ion source as generated during successive ion generation time intervals, and to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; a mass analysis unit comprising a mass analyser arranged to receive ions from the ion mobility analysis unit, and an ion detector arranged to detect ions from the mass analyser; wherein transmission by the ion mobility analysis unit of ions received from the sample is permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage is selected to permit transmission of ions from the pre-set target compound and to prevent transmission of other ions amongst said ions from the sample.

In this aspect, an efficient process is provided for filtering out ion interferences associated with the sample and/or pre-set target compound by applying a pre-set combination (e.g., pre-set for the sample and/or pre-set target compound) of a dispersion voltage and a concurrent compensation voltage to the electrodes of the ion mobility analysis unit, during an ion transmission time interval.

The ion mobility analysis unit may be configured to transmit ions from amongst the received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage, wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval. The values of the dispersion voltage and the compensation voltage corresponding to each pre-set combination may be constant throughout the duration of a respective time sub-interval within an ion transmission time interval. The sum of the respective time sub-intervals of the plurality of distinct pre-set combinations may collectively define (correspond to) the ion transmission time interval of which each subinterval forms a part. The pre-set combination of a dispersion voltage and a concurrent compensation voltage may be provided by the user. The ion mobility analysis unit may be configured to determine said pre-set combination of a dispersion voltage and a concurrent compensation voltage by a process comprising: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a said pre-set combination thereof which permits transmission through the ion mobility analysis unit of ions from the pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

The ion mobility analysis unit may be configured to vary the values of both the dispersion voltage and the compensation voltage such that none of the values of the compensation voltages are repeated as between two successive ion transmission time intervals.

Desirably, each ion transmission time interval is synchronised to coincide with a respective ion generation time interval.

Preferably, successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit.

Successive ion transmission time intervals may be separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit. Desirably, the intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

The pre-set target compound may be provided by the user. The pre-set target compound preferably comprises Cortisol.

Preferably, ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process.

In a sixth aspect, the invention may provide a method for mass spectrometry for analysing ions from a sample comprising a pre-set target compound, the method comprising: providing an ambient pressure ionization (API) source and therewith ionising the sample at an ambient atmospheric pressure to generate ions for analysis by mass spectrometry, and by the ambient pressure ionization source generating said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; providing an ion mobility analysis unit configured and therewith receiving ions periodically from the ion source as generated during successive ion generation time intervals, and transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; providing a mass analysis unit comprising a mass analyser and an ion detector and, by the mass analyser, receiving ions from the ion mobility analysis unit and, by the ion detector, detecting ions from the mass analyser; wherein transmission by the ion mobility analysis unit of ions received from the sample is permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage is selected to permit transmission of ions from the pre-set target compound and to prevent transmission of other ions amongst said ions from the sample.

For example, the method may comprise providing the mass analysis unit arranged to in communication with the ion mobility analysis unit either to receive ions directly from the latter or indirectly to receive ions therefrom via intervening ion optics etc., before the mass analyser. These ion optics may be part of the mass spectrometer but not part of the mass analysis unit (e.g., other than, in addition to, ion optics of a mass analysis unit, such as a quadrupole or ToF optics).

The ambient pressure ionization (API) source provided according to the method may comprise an ambient ionisation source. The method may comprise, by ion mobility analysis unit, transmitting ions from amongst the received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval. The values of the dispersion voltage and the compensation voltage corresponding to each pre-set combination may be constant throughout the duration of a respective time sub-interval within an ion transmission time interval. The sum of the respective time sub-intervals of the plurality of distinct pre-set combinations may collectively define (correspond to) the ion transmission time interval of which each sub-interval forms a part.

The pre-set combination of a dispersion voltage and a concurrent compensation voltage may be provided by the user. The method may comprise determining said pre-set combination of a dispersion voltage and a concurrent compensation voltage by a process comprising: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a said pre-set combination thereof which permits transmission through the ion mobility analysis unit of ions from the pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

The method may comprise varying the values of both the dispersion voltage and the compensation voltage such that some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

The method may comprise varying the values of both the dispersion voltage and the compensation voltage such that none of the values of the compensation voltages are repeated as between two successive ion transmission time intervals.

Desirably, each ion transmission time interval is synchronised to coincide with a respective ion generation time interval. Desirably, successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit.

Preferably, or optionally, successive ion transmission time intervals are separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit.

Desirably, the intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

The pre-set target compound may be provided by the user. Preferably, the pre-set target compound comprises Cortisol.

Preferably, the ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1A schematically shows a mass spectrometry apparatus comprising an ambient pressure ionization unit, an ion mobility analysis unit and a mass spectrometer unit.

Figure 1 B schematically shows an ion spectrometry apparatus comprising an ambient pressure ionization unit, an ion mobility analysis unit and an ion detector unit.

Figure 2 schematically shows trajectories of ions within the drift region of an ion mobility analysis unit and associated dispersion and compensation voltage waveforms.

Figures 3A and 3B show schematic representations (two different perspectives) of a combination of a PESI ion source with a vDMS ion mobility analysis unit and a mass spectrometer (MS). Figure 4 shows an example of a signal/time plot of an ion output signal from a PESI-MS apparatus operating in single-ion monitoring mode.

Figure 5 shows an arrangement of concurrent ion beam intensity output from a PESI ion source, a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit downstream of the PESI ion source, and varying/scanned values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 6 shows an arrangement of concurrent ion beam intensity output from a PESI ion source, a variety of dispersion voltages (DV) applied to electrodes of a vDMS ion mobility analysis unit downstream of the PESI ion source, and varying/scanned values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 7 shows an arrangement of concurrent ion beam intensity output from a PESI ion source, a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit downstream of the PESI ion source, and varying values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 8 shows an arrangement of concurrent ion beam intensity output from a PESI ion source, a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit downstream of the PESI ion source, and varying/scanned values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 9 shows ion signal-vs-CV plots for Cortisol at various Ed/N values.

Figure 10 shows schematically a method to combine PESI-vDMS data describing compensation voltage (CV) variation/scan data and dispersion voltage (DV) (or Ed/N values) variation data into CVDV data sets/plots.

Figure 11 shows data describing compensation voltage (CV) variation/scan data and dispersion voltage (DV) (or Ed/N values) variation data of Figure 10 after combining into a CVDV data sets/plots (Ed/N values). Figure 12 shows an arrangement of concurrent ion beam intensity signals from a PESI ion source, a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit, and a value of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 13 shows an arrangement of concurrent ion beam intensity signals from a PESI ion source, varying values of a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit, and varying values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit.

Figure 14 shows an arrangement of concurrent ion beam intensity signals from a PESI ion source, varying values of a dispersion voltage (DV) applied to electrodes of a vDMS ion mobility analysis unit, and varying values of a compensation voltage (VC) applied to electrodes of the vDMS ion mobility analysis unit..

Figure 15A shows steps in a method for selecting a combination of a compensation voltage (CV) and a dispersion voltage (DV).

Figure 15B shows steps in a method for generating a mass spectrum for ions of a target compound according to the selected combination of a compensation voltage (CV) and a dispersion voltage (DV).

Figure 15C shows steps in an experimental method.

Figure 16 shows data describing an example chromatogram for PESI-SIM and PESI-vDMS-SIM of Cortisol in blank artificial saliva.

Figures 17 and 18 show data describing full scan MS spectra of artificial saliva by a PESI-MS apparatus (Fig. 17) and by a PESI-vDMS-MS apparatus (Fig. 18) optimised for transmission of Cortisol ions.

Figures 19 and 20 show data describing full scan MS spectra of artificial saliva spiked with Cortisol by a PESI-MS apparatus (Fig. 19) and by a PESI-vDMS-MS apparatus (Fig. 20) optimised for transmission of Cortisol ions. Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Ion mobility spectrometry (IMS), such as differential mobility spectrometry (DMS) [ref. 2] or field asymmetric waveform ion mobility spectrometry (FAIMS) [ref. 3] are established methods to separate different types of ions according to differences of their mobility through a gas in response to the application of a force to the ions via an electric field intensity. These mobility differences depend on the physical and chemical properties of ions and gas particles (e.g., neutral particles such as atoms and/or molecules), but are only weakly correlated with the ion mass. The resulting strong orthogonality of this method relative to mass spectrometry (MS) makes FAIMS/MS a powerful analytical approach.

Referring to Figure 1 A, a mass spectrometer apparatus 1 is shown. The mass spectrometer apparatus comprises an ion mobility analysis unit 3 configured to implement field asymmetric ion mobility spectrometry (FAIMS), also known as a differential field mobility spectrometry, DMS. The mass spectrometer also apparatus comprises an atmospheric pressure ion source (API) 10 comprising a probe electrospray ionization (PESI) source configured to provide ions from a sample, and a first vacuum region 12 containing a differential ion mobility assembly comprising a drift region defined between electrodes 14 separated by an analytical gap (g). Ions 25 are moved longitudinally along the drift region, within the analytical gap between the electrodes, on a pathway towards an output port 16, whilst entrained in a flow of neutral buffer or “carrier” gas (commonly molecular nitrogen). A first vacuum pump 26 is configured in communication with the first vacuum region and is arranged to maintain a gas pressure P1 therein.

The drift region extends along an ion optical axis towards a second vacuum region 15 containing amass analysis unit (analyzer/spectrometer). The first vacuum region is in ion-flow communication with the second vacuum region via a skimmer 16 defining an ion outlet port of the first vacuum region and simultaneously an ion inlet of the second vacuum region. Accordingly, ions 28 transmitted by the FAIMS device enter the second vacuum region and the mass analysis unit within it. The mass analysis unit comprises a mass analyser 29 for receiving ions 28 transmitted by the ion mobility analysis unit and an ion detector 31 for detecting ions output by the mass analyser and for producing an ion intensity output signal 35 accordingly.

A power supply unit 20 of the ion mobility analysis unit provides a voltage source and is configured to apply a dispersion voltage, V_D, and a DC compensation voltage, V_c, to one or more of the electrodes 14 to generate between them a dispersion electric field, E_D, and a compensation field, E_c, across the analytical gap (g). The power supply unit 20 comprises switches (not shown) configured to switch to provide the AC dispersion voltage waveform, V_D, such as described below with reference to Figure 2, that alternates between adjustable voltage values, and the DC compensation voltage, V_c.

A control unit 33 of the ion mobility analysis unit is configured to control the power supply unit 20 to apply a dispersion voltage, V_D, and compensation voltage, V_c, to the electrodes 14 of the ion mobility analysis unit. In particular, the control unit is configured to control the power supply unit to supply the electrodes 14 of the drift region with a selected asymmetric AC waveform 34 of the dispersion voltage, V_D, and a selected DC compensation voltage, V_c, 36 described below with reference to Figure 2. The selected combination of dispersion voltage and compensation voltage is non-changing or is chang ing/varying according to the control of the control unit 33. The selected combination is determined by the control unit 33 according to the ion intensity output signal 35 from the ion detector of the mass analysis unit, as will be described in more detail below.

The dispersion voltage, V_D, consists of the high field (HF) segment of the waveform 34. This dispersion voltage, V_D, creates a dispersion electric field:

E_D = -(dVo(x)/dx) = -V_D/g which spans the analytical gap, g, all along the drift region and extends in a direction perpendicular to the longitudinal axis of the drift region and alternating in polarity. The DC compensation voltage, V_c, also generates a compensation electric field:

E_c = — (dV_c(x)/dx) = -V_c/g which spans the analytical gap, g, all along the drift region and extends in a direction perpendicular to the longitudinal axis of the drift region.

A second vacuum pump 27 is configured in communication with the second vacuum region and is arranged to maintain a gas pressure P2 therein, which is lower than the first gas pressure (i.e., P2 < P1) so as to create a pressure differential which induces a flow of buffer gas along the drift region. The first vacuum region is located before the second vacuum region on the ion optical axis such that in use ions generated from the sample undergo differential ion mobility analysis by the differential ion mobility assembly before undergoing mass spectral analysis by the mass spectrometer.

A gas flow former 18 comprises a capillary providing gas flow communication from the atmospheric pressure ion source (API) 10 to the first vacuum region. The capillary is configured to establish in the first vacuum region a flow of buffer gas (e.g., N2 molecules) entrained with ions generated from the sample, the flow of buffer gas being directed along the drift region (between electrodes 14).

Referring to Figure 2, there is schematically illustrates the basic principles and the mechanism for IMS separation based on the non-linear ion mobility dependence on electric field and pressure. Ions are entrained in a stream 30 of buffer gas directed along the axis of a drift region defined between two (or more) opposing electrodes 32. A high frequency asymmetric AC waveform 34 is either applied to one of the two opposing electrodes, or alternatively half of the amplitude of the waveform may be applied simultaneously to each electrode in opposite respective polarities, the net effect is the same. This is known as the dispersion voltage, V_D. It is responsible for causing spatial separation of ions according to differences in ion mobility through the buffer gas within which the ions are entrained. The dispersion electric field, E_D = ~(dV_D(x) / dx) , generated by the dispersion voltage induces a motion in the ions in a direction of the dispersion electric field extending from one of the two electrodes to the other electrode. Combined with the concurrent drift motion of the ions in the direction of buffer gas flow, the resulting path of ions follows a zig-zag shape as the polarity of the dispersion voltage, V_D, alternates between positive and negative values such that the dispersion electric field, E_D, alternates between opposite directions across the gas drift direction.

Superimposed to the waveform is a slow compensation DC voltage waveform 36 comprising a succession of "sawtooth" DC ramps. This is known as the compensation voltage, V_c. The frequency of the asymmetric waveform 34 of the dispersion voltage, V_D, usually spans between a few hundreds of KHz to ~1 MHz, while that of the "sawtooth" DC ramp 36 typically repeats at a rate that is much slower to repeat than is the RF waveform. The amplitude of the asymmetric waveform of the dispersion electric field when the IMS is operated at ambient pressure (or at below ambient pressure in vDMS), is limited by the breakdown limit of the gas flowing within a given electrode geometry and for a parallel plate IMS system the electric field does not generally exceed 3 kV mm'¹.

Still referring to Figure 2, separation of ions is possible using waveforms substantially different to the pure rectangular waveform. A family of waveforms based on quasi-sinusoidal variations of the voltage as a function of time are widely used; these are the bi-sinusoidal, the clipped sinusoidal or other substantially rectangular waveforms. Asymmetric waveforms are designed so that the area of the positive pulse portion of one waveform cycle matches that of the negative pulse portion, i.e., Ai = A2. For this particular arrangement of time-dependent electric fields, an ion with no mobility dependence on variations in electric field will therefore be transmitted at zero compensation voltage. The waveform is characterized by its duty cycle 38, usually defined as the width of the short positive pulse part, TH, of one wave cycle, divided by the full duration of one waveform cycle which defines the waveform period T (i.e., defined as: d = TH/T). The sum of the width of the short positive pulse part, TH, and the width of the long negative pulse part, TL, of the wave cycle is equal to the full duration, T, of one waveform cycle.

There exist optimum duty cycles for separating certain types of ions. For example, the type A and C ions are best separated in the IMS spectrum when the duty cycle is d ~ 0.33. B-type ions exhibit a more complex behaviour and having the ability to vary the duty cycle during the course of an experiment is essential for enhancing instrument performance.

Also shown in Figure 2 are a stable ion trajectory 40 transmitted successfully through the drift region and a second ion trajectory hitting the top DMS electrode 42. Successful transportation of the lost ion 42 would require the appropriate compensation voltage, V_c, 36 to be applied to the IMS electrode to compensate for the small average displacement, Ax, 46 introduced per waveform cycle. By varying/scanning the compensation voltage, V_c, ions with different non-linear mobility dependencies on electric field and pressure are successively transported through the drift region gap and can be detected/monitored e.g., by an ion detector 31 of the mass spectrometer of Figure 1 A.

Referring to Figure 1A, the ambient pressure ionization (API) source 10 may comprise any pulsed or periodic ion source, as discussed above. However, for illustration, the ambient pressure ionization (API) source 10 in the following examples may be a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process at an ambient atmospheric pressure to generate ions comprising ions from a pre-set target compound for transfer into the mass analyser 29 via the ion mobility analysis unit 3. The ambient pressure ionization source is configured to generate these ions periodically (e.g., as a pulsed ion source) during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby.

Figures 3A and 3B show schematic representations (two different perspectives) of a combination of a PESI ion source with a vDMS ion mobility analysis unit and a mass spectrometer (MS). The apparatus comprises a PESI source 201 comprising a PESI needle and needle holder. The needle holder is configured to periodically insert and retract the PESI needle from a sample (not shown) arranged upon a sample plate (not shown) held underneath the needle holder upon a sample plate holder 203. The needle holder, needle and sample plate holder collectively define an example of an API source such as item 10 of Figure 1A. A desolvation line 204 is arranged adjacent to the sample holder in order to receive ions from the sample as generated by the PESI needle 202 in use (such as item 18 of Figure 1 A). The desolvation line is arranged in communication with the drift region of a vDMS ion mobility analysis unit (such as item 3 of Figure 1 A) as defined by an analytical gap formed between two parallel electrode plates 205 of the ion mobility analysis unit (such as item 14 of Figure 1 A). A mass spectrometer 206 including ion optics (ion guides, mass analysers/filters such as item 29 of Figure 1A, collision cell) and detector (such as item 31 of Figure 1A) is arranged in communication with the ion mobility analysis unit 205 so as to receive ions that have been transmitted through the drift region defined between the electrodes 205 of the ion mobility analysis unit.

Figure 4 shows an example of a signal/time plot of an ion intensity output signal (such as item 35 of Figure 1 A) from the ion detector of the PESI- vDMS-MS when the mass spectrometer apparatus 206 is operating in single-ion monitoring mode. The ratio of on-phase to off-phase of the PESI ion source (201 , 202) is ~1 .4 in this case. Here ‘on-phase’ refers to the time intervals in which ions are generated by the PESI ion source such that successive ion intensity pulses (120, 121) are detected by the ion detector. Conversely, ‘off-phase’ refers to intervening time intervals in which no ions are generated by the PESI ion source, such as the intervening time interval between the two ion pulses (120, 121) of Figure 4 in which no ion detection signal is produced.

As described above, the short duration of each one of the individual ‘on-phase’ time intervals places constraints upon the number of different ion species that can be analysed by the mass analyser of the mass spectrometer within that relatively short time interval as a result of the time required for ions to first pass through the drift region defined between the electrode plates (205, 14) of the ion mobility analysis unit. In a typical PESI experiment the needle cycle time, as between ‘on-phase’ and ‘off-phase’, is in the order of hundreds of milliseconds, producing an ion intensity detection signal peak (120, 121) with a width of ~100ms. However, in method optimisation experiments, one may typically wish to scan the value of the compensation voltage V_c applied by the ion mobility analysis unit for a given value of the applied dispersion voltage V_D and record the ion intensity detection signal. Each separate combination of the compensation voltage and dispersion voltage corresponds to one ‘setting’ of the ion mobility device for which it is typically desired to obtain data relating to a target ion of interest which is transmitted through the ion mobility analysis unit according to that respective ‘setting’. However, sufficient ion intensity detection signal data must be obtained at each setting to achieve a reliable signal-to-noise ratio and thereby permit the data to be used reliably for analysis.

In order to scan the compensation voltage through all desired ‘settings’ would require an initial knowledge of the desired ‘settings’ and a rapid continuous scanning of the compensation voltage through a range of values encompassing those desired ‘settings’. An example of this is shown schematically in Figure 5, Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) comprises ‘on-phase’ time intervals 101a in which ions are output by the PESI source, separated in time by intervening ‘off- phase’ time intervals 101 b in which no ions are output. The ion beam intensity of the PESI source has a periodicity is related to the PESI needle cycle time as discussed above. A dispersion voltage 102 is set to a constant value for all needle cycles and the value of the compensation voltage 103 concurrently undergoes a rapid continuous scanning 103a through a range of values encompassing the desired ‘settings’ during the ‘on-phase’ time intervals 101 a of the PESI ion source in synchrony with it. In the ‘off- phase’ the compensation voltage 103b is reset to the starting value or to some non-transmitting value. However, the time duration/width of each ion intensity output/signal peak present during the ‘on-phase’ has been found by the inventors to be too short to allow sufficient ion intensity detection signal data to be obtained at each ‘setting’ through which the compensation voltage must be rapidly scanned.

To address this problem, the invention in one aspect provides a means to initially identify an appropriate combination (‘settings’) of dispersion voltage and compensation voltage values for analysis of ions from a target compound within a sample which enhance the signal-to-noise ratio of the ion intensity detection signals by reducing the noise arising from non-target ions (interferences). The invention in another aspect provides a means to apply one or more of the resulting identified combination (‘settings’) of dispersion voltage and compensation voltage values within the time duration/width of each ion intensity signal peak present during the ‘on-phase’. More than one such identified combination may be efficiently applied as a sequence of settings either all within the time duration/width of the same ion intensity signal peak present during the ‘on-phase’, or separately within the time duration/width of the successive separate respective ion intensity signal peaks present during separate ‘on-phases’.

Settings Search Stage

An example of this is shown schematically in Figure 6. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. A dispersion voltage 104 is varied to be one of a plurality of different values (104a, 104b, 104c, 104d) that each remain constant during one respective needle cycle time 101a during the ‘on-phase’ time intervals 101 a of the PESI ion source in synchrony with it. The value of the compensation voltage 105 concurrently undergoes a continuous scanning 105a through a range of values during the ‘on-phase’ time intervals 101a of the PESI ion source in synchrony with it. In the ‘off-phase’ the compensation voltage 105b is reset to the starting value or to some non-transmitting value, whereas the dispersion voltage is set to a new value different from a preceding value it during the immediately preceding ‘on-phase’ time interval 101a. In this way, the value of the dispersion voltage is varied in a step-wise manner through a plurality of different values, each of which remains constant while a continuous scan of the value of a concurrent compensation voltage is performed - each compensation scan passing through the same range of compensation voltage values in each such successive scan. As a result, prospective ‘settings’ for the ion mobility analysis unit are searched for during successive ‘on-phases’ of the PESI ion source.

Another example of this is shown schematically in Figure 7. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. A compensation voltage 107 is varied to be one of a plurality of different values (107a, 107b, 107c, 107d) that each remain constant during one respective needle cycle time 101a during the ‘on-phase’ time intervals 101a of the PESI ion source in synchrony with it. The value of the dispersion voltage 106 concurrently remains constant during all of the ‘on-phase’ time intervals 101a and the intervening ‘off- phase’ intervals 101 b of the PESI ion source, remaining unchanging throughout. In the ‘off-phase’ the compensation voltage 107 is reset to the starting value 107e or to some non-transmitting value, whereas the dispersion voltage remains unchanged. In this way, the value of the compensation voltage is varied in a stepwise manner through a plurality of different values, each of which remains constant while the value of a concurrent compensation voltage is held constant throughout. As a result, prospective ‘settings’ for the ion mobility analysis unit are searched for during successive ‘on-phases’ of the PESI ion source. The process may then be repeated with the dispersion voltage set to a new value.

Yet another example of this is shown schematically in Figure 8. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. The value of the dispersion voltage 108 remains constant during all of the ‘on-phase’ time intervals 101 a and the intervening ‘off-phase’ intervals 101 b of the PESI ion source, remaining unchanging throughout. The value of the compensation voltage 109 concurrently undergoes a continuous scanning 109a through a range of values during each ‘on-phase’ time interval 101a of the PESI ion source in synchrony with it. In the ‘off-phase’ the compensation voltage 109e is reset to the starting value or to some non-transmitting value, whereas the dispersion voltage remains unchanged. The compensation voltage 109 is varied/scanned to pass through one of a plurality of different continuous ranges of values (109a, 109b, 109c, 109d) that each include a repeated respective sub-range of values (110a, 110b, 110c) that overlaps with an end part of the range of values spanned by a preceding range of values of compensation voltage as scanned during a preceding ‘on-phase’ time interval 101a of the PESI ion source. However, each continuous range of values of the compensation voltage also spans a unique respective sub-range of values, which spans values not included in the preceding range of values, within one respective needle cycle time 101a during the ‘on-phase’ time intervals 101a of the PESI ion source in synchrony with it. The partial overlap assists in re-sampling data for settings previously scanned in a preceding ‘on-phase’ time interval 101 a of the PESI ion source, so as to increase the signal-to-noise ratio obtainable using the obtained data. The process may then be repeated with the dispersion voltage set to a new value.

A common feature of these three examples is that at least some of the values of the concurrent dispersion and compensation voltages (‘settings’) are not repeated as between two successive ion transmission time intervals of the ion mobility analysis unit, which are synchronised with the ‘on-phase’ time intervals 101a of the PESI ion source. This means that a range of ‘settings’ can be scanned efficiently over successive ‘on-phase’ time intervals 101 a of the PESI ion source in a manner that permits an increase the signal-to-noise ratio obtainable using the obtained data.

Referring to Figure 1A, the control unit 33 of the ion mobility analysis unit 3 is configured to control the power source unit 20 in this way, according to any of these three examples (or another appropriate way), to vary the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes 14 during a given ion transmission time interval are not also applied during a preceding ion transmission time interval. This means that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals.

The control unit monitors 33 the intensity of ions detected by the ion detector 31 , via the ion detection signal 35 input to it from the ion detector, and thereby records the received detection signals in conjunction with the applied values of the dispersion voltage and compensation voltage. According to the monitored intensity of detected ions, the control unit selects from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof (a ‘setting’) which permits transmission through the ion mobility analysis unit of ions from a pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst the ions from the sample.

The control unit 33 operates in either one of two states as follows:

(i) In a first state when scanning the ‘settings’ during the ‘settings search stage’ described above, to identify a suitable dispersion voltage and compensation voltage combination for pre-set use later;

(ii) In a second state when applying pre-set suitable dispersion voltage and compensation voltage combinations that has been identified during the ‘settings search stage’.

When the control unit is in either one of two states, the mass analyser may be operated in any known mode suitable forthat kind of mass analyser for either/both the settings search stage and the later stage, to produce the ion detection signal 35 (e.g., for input to the control unit in the first state etc.)

Figure 9 shows an example of recorded ion detection signal data each of which corresponds to a respective one combination of a given dispersion voltage and a scanned range of different concurrent compensation voltages collectively corresponding to a scan of ‘settings’. Seven different dispersion voltage values are applied and the same scanned range of different concurrent compensation voltages is applied in each separate case so as to vary/scan the dispersion voltage is a stepwise manner. These settings were scanned during a ‘settings search stage’ described above. These data were obtained with the control unit 33 in the first state (i) described above and with the mass analyser operating in SIM mode (continuously transmitting a single mass in this case). The ion detection signal data according to the first five different ‘settings’ (401 , 402, 403, 404, 405) show only a single ion intensity peak. However, ion detection signal data (406, 407) according to each one of two different ‘settings’ shows two well- separated ion intensity peaks (408a, 409) in which a new relatively small peak (409) has separated from the larger single peak 408 present alone in the data (401 , 402, 403, 404, 405) obtained for the other ‘settings’. The larger single peak 408 obtained for the other ‘settings corresponds to detection of ions of a target compound concurrently with other ions (interferences) from the sample. The larger peak 408a obtained for the final two ‘settings corresponds to detection of ions of a target compound alone, and the remaining larger peak 409 of the two peaks corresponds to other ions (interferences) from the sample. This indicates that the settings associated with the last two signal data (406, 407) is appropriate for filtering out interference ions from amongst the ions delivered from the API unit 10 (PESI unit) and for transmitting to the mass analyser 29 those ions from the target compound for which mass analysis is wanted, with the control unit 33 in the second state (ii) described above.

In this way, concurrently with varying/scanning the value of both the compensation voltage, l/c, and also the value of the dispersion voltage, I/D, the control unit 33 records the intensity of ions detected by the ion detector 31 according to corresponding/consequential variations in the ion detection signal 35 it receives from the ion detector. As is shown schematically in Figure 10, the control unit combines or merges the recorded intensity data (Figure 9) of detected ions arising from the various different values of the dispersion voltage I/D covering all of the ‘settings’ 124 applied during the ‘settings search stage’. This data merging 126 results in a data set 128 that describes a distribution/intensity of ions exiting the drift region in the form of a 2-dimensional (2D) mobility spectrum that maps the varying compensation voltage, Vc, as a function, VC(I/D), of the value of the dispersion voltage. This 2D spectrum describes the distribution/intensity of ions entering, and subsequently exiting, the analytical gap of the ion mobility analysis unit as a 2-dimensional function with both Vc and I/D as independent variables spanning two respective coordinate dimensions, (e.g., in an x-y plane of a graph) and the distribution/intensity of ions described as a third coordinate dimension (e.g., in a z-dimension of the graph, or as a heat map spanning the x-y plane of a graph). This data reveals those combinations of Vc and I/D for which ions from the target compound which enter the ion mobility analysis unit in an ion cloud collectively and concurrently with other ions from the sample, are separated from those other ions based on differences in ion mobility, such that the ions from the target compound exit the ion mobility analysis unit whereas the other ions do not.

As noted above, the control unit is arranged to select a given combination of values of the dispersion voltage and compensation voltage from amongst the plurality of such values describing the 2-dimensional function, VC(I/D). This selection may be implemented in any suitable way as would be readily apparent to the skilled person. One example is to train a machine learning algorithm to identify a separation of ion intensity peaks into a peak containing ions from a target compound, and a peak containing other ions from a sample. Such a separation is revealed by the heat-map of the 2-dimensional function, VC(I/D), shown in Figure 11 . The trained algorithm may then be applied to select appropriate combinations of values of the dispersion voltage and compensation voltage. Another example is to apply a thresholding algorithm to the data describing the 2-dimensional function, I/C(I/D), whereby for a given value of the dispersion voltage (or reduced field intensity, Ed/N), and a concurrent range of corresponding compensation voltage values spanning those applied by the ion mobility analysis unit in conjunction with the given value of the dispersion voltage, the thresholding algorithm determines if a pre-set threshold value of the 2-dimensional function, VC(I/D), is exceeded. Exceeding such a threshold (i.e., I/C(I/D) > Threshold) when the threshold is set to a suitable threshold value, indicates the presence of an ion intensity peak at that combination of compensation and dispersion voltage setting. Exceeding the threshold more than once at disparate compensation voltage values that are separated by intervening compensation voltage values for which the threshold is not exceeded, indicates the existence of two separate such ion intensity peaks. The thresholding algorithm may then be applied to select appropriate combinations of values of the dispersion voltage and compensation voltage associated with the existence of one of the two separate such ion intensity peaks. Other appropriate ion intensity peak detection algorithms and methods such as would be readily available to the skilled person, may be used by the control unit to select a given combination of values of the dispersion voltage and compensation voltage. By selecting those settings associated with that one peak, ions associated with the peak may be transmitted by the ion mobility analysis unit whereas ions associated with the other peak, which occurs for different settings values of Vc and Vd, are not transmitted. The user may confirm that the transmitted ions of the selected peak (selected settings) contain the target ions of interest. Of course, the user may change the setting to correspond to those associated with the other one of the two ion intensity peaks should that contain the target ions of interest.

These selection methods enable selection of an appropriate combination of values of the dispersion voltage and compensation voltage that permits transmission through the ion mobility analysis unit of ions from a pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst the ions from the sample.

For example, as shown in Figure 11 , settings associated with the ion detection signal data 401 according to the first five different ‘settings’ (401) described above with reference to Figure 9, reveals a combination of Vc and I/D for which ions from the target compound will be transmitted by the ion mobility analysis unit along with other ions from the sample within the same ion responsible for the ion intensity peak 408 due to having substantially the same ion mobility for the ‘settings’ in question. Here, the settings are such that both Vc and I/D are relatively small values. By contrast, the ion detection signal data 407 according to the sixth or seventh ‘settings’ (406, 407) described above with reference to Figure 9, reveals a combination of Vc and I/D for which ions from the target compound will be transmitted by the ion mobility analysis unit alone, and other ions from the sample within the same ion cluster responsible for the separate ion intensity peak 408a would not be transmitted due to significantly different ion mobility for the ‘settings’ in question. These settings are appropriate for use with the mass analyser in the second state (ii) described above.

Applying Pre-Set Settings

Figure 12 shows an example of the application of one pre-set combination/setting of Vc and I/D, the preset settings having been determined according to the ‘settings search stage’ described above. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. A constant dispersion voltage 1 11 and a constant compensation voltage 112 are applied to the electrodes of the ion mobility analysis unit according to a pre-set ‘setting’ as determined during a previous ‘settings search stage’ described above. Each of the dispersion and compensation voltages remain constant during all needle cycle times 101 a during the ‘on-phase’ time intervals 101 a of the PESI ion source and during all intervening ‘off-phase’ time intervals 101 b of the PESI ion source.

Figure 13 shows an example of the application of four different pre-set combination/setting of Vc and I/D, the pre-set settings each having been determined according to the ‘settings search stage’ described above. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. All four pre-set ‘settings’ (Vc and 1/D value combinations) are sampled in each PESI cycle. In preferred examples, at least 12 pre-set ‘settings’ (Vc and I/D value combinations) are sampled in each PESI cycle which may also optionally allow a user to repeat the same one pre-set Vc, VD ‘settings’ pair within the same one PESI cycle (101) many times to permit the user to obtain good definition of the peak shape for PESI ion intensity output 101 which makes calculating the peak area for quantitation purposes easier. The values of the dispersion and compensation voltages is controlled to switch to a non-transmitting values (113e, 114e) in the ‘off-phase’ 101 b of the PESI ion source. In this way, the values of the dispersion voltage and the compensation voltage are synchronised so as to mutually comply with a respective one of four different pre-set ‘settings’ values ( c and VD value combinations) throughout the duration of four respective time sub-intervals that collectively make up the overall ion transmission time interval of the ion mobility analysis unit. Each preset ‘setting’ corresponds to the transmission by the ion mobility analysis unit of a respective one of four different target compounds within the sample, and the concurrent non-transmission of other ions (interferences) from the sample. The result is that the ion mobility analysis unit transmits ions throughout its ion transmission time interval and does so by separately transmitting in sequence the ions from four different target compounds. For example, a first target compound is transmitted in response to the first Vc and I/D value combination (113a, 114a), whereas ions from other target compounds and from interferences are not transmitted. A second target compound is transmitted in response to the subsequent second Vc and VD value combination (113b, 114b), whereas ions from other target compounds and from interferences are not transmitted. A third target compound is transmitted in response to the subsequent second Vc and I/D value combination (1 13c, 114c), whereas ions from other target compounds and from interferences are not transmitted. A fourth target compound is transmitted in response to the subsequent second Vc and I/D value combination (113d, 114d), whereas ions from other target compounds and from interferences are not transmitted. The values of the dispersion and compensation voltages are subsequently switch to a non-transmitting value (113e, 114e) in the ‘off- phase’ 101 b of the PESI ion source. The cycle repeats when the subsequent ‘on-phase’ 101 a of the PESI ion source begins.

Figure 14 shows another example of the application of four different pre-set combination/setting of Vc and I/D, the pre-set settings each having been determined according to the ‘settings search stage’ described above. Here, the ion intensity 101 of the ion beam produced by the PESI source (120, 121) is shown. This is described above with reference to Figure 5. A respective one of each of the four pre-set ‘settings’ (Vc and VD value combinations) is sampled in a respective one of each of four successive PESI cycles. The values of the dispersion and compensation voltages is controlled to switch to a non-transmitting values (115b, 116b) in the ‘off-phase’ 101 b of the PESI ion source.

In this example, the values of the dispersion voltage and the compensation voltage are synchronised so as to mutually comply with a single pre-set ‘setting’ values (Vc and I/D value combination) throughout the duration of a respective one of a plurality of successive ion transmission time intervals of the ion mobility analysis unit. Each pre-set ‘setting’ corresponds to the transmission by the ion mobility analysis unit of a respective one of a plurality of different target compounds within the sample, and the concurrent nontransmission of other ions (interferences) from the sample. The result is that the ion mobility analysis unit transmits ions throughout each ion transmission time interval and does so by separately transmitting in sequence the ions from a respective one of a plurality of different target compounds. For example, a first target compound is transmitted in response to the first Vc and VD value combination (115a, 116a), whereas ions from other target compounds and from interferences are not transmitted. A second target compound is transmitted in response to the subsequent second Vc and D value combination (115c, 116c), whereas ions from other target compounds and from interferences are not transmitted. A third target compound is transmitted in response to the subsequent second c and VD value combination (115d, 116d), whereas ions from other target compounds and from interferences are not transmitted. A fourth target compound is transmitted in response to the subsequent second Vc and I/D value combination (115e, 116e), whereas ions from other target compounds and from interferences are not transmitted. The values of the dispersion and compensation voltages are subsequently switch to a nontransmitting value (115b, 116b) in the ‘off-phase’ 101 b of the PESI ion source. The cycle repeats when the subsequent ‘on-phase’ 101a of the PESI ion source begins.

Figure 15A shows the steps in a method for performing the ‘Settings Search Stage’ described above, as follows:

Step 301 : Periodically generate ions using the API source (e.g., PESI source).

Step 302: Receive ions at the ion mobility analysis unit periodically from API source.

Step 303: Vary the values of Vc and VD possible defining ‘settings’ combinations, as between successive ion transmission time intervals of the ion mobility analysis unit in synchrony with the ion generation time intervals of the API source, including non-repeating combinations.

Step 304: Monitor the detected ion intensity of transmitted ions from the ion mobility analysis unit. Step 305: Select a combination of Vc and I/D which permits transmission of ions from target compound and prevents transmission of other ions from the sample

Figure 15B shows the steps in a method for performing the stage of ‘Applying Pre-Set Settings’ described above, as follows:

Step 310: Periodically generate ions from sample including target compound (e.g., Cortisol) using

API source (e.g., PESI source).

Step 311 : Receive ions at the ion mobility analysis unit periodically from API source.

Step 312: Transmit ions from the target compound through ion mobility analysis unit according to a pre-set combination of Vc and D applied to the ion mobility analysis unit which permits transmission of ions from target compound and prevents transmission of other ions from the sample.

Step 313: Receive, at a mass spectrometer unit, transmitted ions from the ion mobility analysis unit.

Step 314: Generate a mass spectrum of ions from target compound, for analysis.

Examples

EXAMPLE 1

The methods and apparata described herein have been found to be particularly effective in the detection and/or analysis of Cortisol as a target compound within a sample, such as (but not limited to) a sample of saliva, blood or urine for example.

Direct analysis offers rapid (timely) analysis of samples, such as samples comprising Cortisol. Ion mobility-mass spectrometry reduces the interference from matrix compounds (chemical noise) and allows the separation of isomeric/isobaric compounds of interest. Together these technologies provide a rapid analysis method for Cortisol detection and/or analysis with minimal sample preparation and high data quality without interferences.

Cortisol levels may be analysed in a clinical context, specifically for the diagnosis and monitoring of Cushing syndrome, Addison disease, congenital adrenal hyperplasia, and other diseases of the adrenal and pituitary system. This includes adrenal crisis, a potentially fatal condition which requires rapid diagnosis and treatment. There is also an interest in monitoring Cortisol levels as a marker of (chronic) stress. As such, a need has been identified for rapid, accurate and robust monitoring of Cortisol in saliva, blood and urine.

Typical methods in clinical laboratories include biochemical assays (BCA), immunological assays (IMA) and liquid chromatography (LC)-mass spectrometry (LCMS). BCA and IMA suffer from issues with interferences and cross reactivity. LCMS may resolve some of these interferences, but doing so takes a long time (e.g. 5 mins per sample). Sample preparation for LCMS assays can also be complex and time consuming. The present invention, in any of its aspects, has been found to be quick, reliable and efficient in the detection and/or analysis of a target compound within a sample, and particularly so for Cortisol, which may be especially needed in any of the following circumstances:

• Time critical analysis (e.g., during suspected adrenal crisis)

• High throughput analysis (e.g., clinical core labs)

• Point of care use

The prior methods suffer from long analysis time and complicated sample preparation, making them are not suitable.

PESI methods for ion generation in mass spectrometry are fast and simple (little/no sample preparation, no LC optimisation). However, PESI (and other ion source methods) suffers from interferences as there is no chromatographic separation. These can lead to inaccurate results. The invention aims to address the problem interferences found in PESI. The inventors have found that this can be addressed effectively by incorporating an ion mobility dimension. For example, vDMS is a fast-filtering method of IMS and has been found to be well suited to targeted MS analysis.

As discussed above, PESI is a type of ambient ionisation/direct analysis ionisation source for mass spectrometry. By directly probing the sample, there is no delay from chromatography etc. However, in PESI there is no separation/fractionation of the chemical components of the sample. This can lead to interference from isomeric/isobaric compounds in the matrix. As also discussed above, vDMS is an ion mobility method which separates ions in the gas phase based on their (differential mobility) in electric fields. It has been found that vDMS can efficiently remove (e.g., isomeric/isobaric) such chemical interferences by filtering for a specific ion. In combination of PESI, ion mobility filtering and subsequent mass analysis is able to provide the following benefits:

• Rapid analysis of Cortisol in saliva/blood etc (PESI allows rapid sampling, vDMS means only very simple sample preparation is needed).

• Interferences from matrix chemicals are removed by vDMS filtering.

• Isomeric/isobaric species of interest can be separated in the vDMS dimension and analysed separately, without interference.

The methods of the invention may be applied to the detection of Cortisol within a sample as illustrated in the method steps shown in Figure 15C.

Step 320: Prepare samples (e.g., by diluting in methanol or other organic solvent mixture). Ideally a high concentration of analyte of interest in matrix matched blank.

Step 321 : Optimise MS settings for ion of interest. Set the MS settings (e.g., source voltage, target m/z, MRM transition(s), CID gas pressure and CID voltages, etc.) to achieve desired sensitivity and selectivity. Step 322: Optimise vDMS conditions, including compensation voltage and dispersion voltage

‘settings’, as discussed above, to remove interfering signals (ideally with standards spiked into real matrix). The vDMS conditions are optimised to achieve required separations and improvements in the S/N ratio. Other parameters that may also be optimised, optionally, include: buffer gas pressure, speed, temperature, gas makeup; dispersion voltage waveform aspect ratio, frequency.

Step 323: Optionally, having established the optimum vDMS conditions in the previous step, perform a compensation voltage scan to detect and correct for any drift in the necessary value thereof. This may be performed intermittently by the user. This corrects for any variation in the optimum settings between the default conditions determined in the previous step and those required on the end user system (caused by e.g., variation in ambient pressure, solvent quality or mechanical variation).

Step 324: Generate PESI-vDMS-MS method files. Generally the method may include MS settings

(e.g., target m/z, MRM transition(s), various MS instrument parameters etc.), PESI settings (e.g., cycle time, voltages etc.) and vDMS conditions including compensation and dispersion voltage settings and other optional settings (e.g., buffer gas pressure, temperature, etc) and record them in a format that allows collective synchronisation of the PESI, vDMS and MS stages according to their settings.

Step 325: Optionally, acquire QC/control samples to confirm performance. Known negative matrix matched blanks and various spiked matrix matched blanks/real samples may thereby be tested to confirm the level of interference and signal response is acceptable.

Step 326: Begin quantitation, when ready. Quantitation experiment methods (e.g., external calibrators or isotope dilution) are described in prior art literature and are well known to the person skilled in the art. They are not repeated here.

In PESI-MS quantitation can be performed via external calibration or using isotope dilution. Due to substantial sample to sample variation in signal for PESI-MS the use of isotope dilution calibration may be preferred, although external calibration with internal standard can also be used successfully.

Preparation of the samples is preferably done with a large concentration of analyte of interest in matrix matched blank.

The setting of MS parameters, in Step 321 , may be as appropriate to monitor the analyte of interest (e.g., Cortisol). Selection of conditions for which the analyte of interest is separated from any interferences may comprise a process of using two samples: a blank and positive. A comparison of ion detection signals (Fig .1 , item 35) associated with each may thereby confirm which signal is the analytes. This may be further confirmed by MRM steps etc.). The benefit of such a comparison is to allow one to check if there is any underlying interference at the same compensation voltage and dispersion voltage ‘settings’ in the blank sample. In cases where multiple conditions of compensation voltage and dispersion voltage ‘settings’ achieve the same separation, it is optimal to pick the one that gives the highest signal. The waveform aspect ratio, frequency and the gas speed can also be optimised for the chosen setting to either improve resolution of overlapping peaks or to find the point at which optimal signal can be found while resolution is maintained.

Sample types:

Saliva is a suitable sample type for detection of Cortisol. Blood (whole blood, serum, plasma), urine and hair are each a suitable sample type for detection of Cortisol.

Sample volumes (prior to any preparation) e.g. 50 pL of sample (e.g., blood, saliva, urine etc.) are preferred.

Figure 16 shows and example chromatogram for PESI-MS and PESI-vDMS-MS of Cortisol in blank artificial saliva. The data sub-group 501 corresponds to the output obtained when the vDMS unit of the ion mobility analysis unit 3 was switched ‘off’. An interfering signal is observed. The data sub-group 502 corresponds to the output when the vDMS ion mobility analysis unit 3 (see Fig. 1 A) was switched ‘on’ and pre-set compensation voltage and dispersion voltage ‘settings’ were applied as optimised for transmission of Cortisol ions. Interfering signals are reduced very significantly.

Figures 17 and 18 show a full scan mass spectrum of saliva by PESI-MS (Fig. 17) and PESI-vDMS-MS (Fig. 18) of Cortisol. The spectrum 503 of Figure 17 corresponds to the output obtained when the vDMS unit of the ion mobility analysis unit 3 was switched ‘off’. An interfering signal is observed. The spectrum 504 of Figure 18 corresponds to the output when the vDMS ion mobility analysis unit 3 (see Fig. 1A) was switched ‘on’ and pre-set compensation voltage and dispersion voltage ‘settings’ were applied as optimised for transmission of Cortisol ions. Interfering signals are reduced very significantly.

Figures 19 and 20 show a full scan mass spectrum of artificial saliva spiked with Cortisol by PESI-MS (Fig. 19) and PESI-vDMS-MS optimised for transmission of Cortisol ions (Fig. 20). The spectral peak 507 of a Cortisol ion is present in the signal. All other ions are considered to be non-target compounds. Note that some of the signal loss for the Cortisol peak in 502 is presumably a result of interfering ions being filtered out (Fig. 11) by the vDMS assembly of the ion mobility analysis unit 3 (see Fig. 1 A).

EXAMPLE 2

Figure 1 B shows an ion spectrometry apparatus for analysing ions from a sample comprising a target compound. The ion spectrometry apparatus comprising an ambient pressure ionization unit, an ion mobility analysis unit and an ion detector unit. The ion spectrometry apparatus has all of the features illustrated in the mass spectrometry apparatus 1 of Figure 1 A, with the exception that the mass analyser 15, second vacuum pump 27, second vacuum chamber 15 and port 16, are omitted. Accordingly, the ion spectrometry apparatus is an ion mobility spectrometer apparatus and provides no mass spectrometry function. In the present example, the ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process. The ion spectrometry apparatus 1 B may comprise the combination of a PESI ion source (201 , 202, 203, 204) with a vDMS ion mobility analysis unit 205 such as shown in Figure 3A and Figure 3B, with the mass spectrometer 206 removed/omitted, and replaced with the ion detector for receiving the output of the ion mobility analysis unit 205. The operation of the PESI source and ion mobility analysis unit is described above with reference to Figure 3A and Figure 3B.

The ion spectrometry apparatus 1 B comprises an ion mobility analysis unit 3B configured to implement field asymmetric ion mobility spectrometry (FAIMS), also known as a differential field mobility spectrometry, DMS. The mass spectrometer also apparatus comprises an atmospheric pressure ion source (API) 10B comprising a probe electrospray ionization (PESI) source configured to provide ions from a sample, and a vacuum region 12B containing a differential ion mobility assembly comprising a drift region defined between electrodes 14B separated by an analytical gap (g). Ions 25B are moved longitudinally along the drift region, within the analytical gap between the electrodes, on a pathway towards an ion detector 31 B, whilst entrained in a flow of neutral buffer or “carrier” gas (commonly molecular nitrogen). A vacuum pump 26B is configured in communication with the vacuum region and is arranged to maintain a gas pressure P therein.

The drift region extends along an ion optical axis towards the ion detector 31 B. Accordingly, ions 28B transmitted by the FAIMS device reach the ion detector 31 B, for producing an ion intensity output signal 35B accordingly.

A power supply unit 20B of the ion mobility analysis unit provides a voltage source and is configured to apply a dispersion voltage, V_D, and a DC compensation voltage, V_c, to one or more of the electrodes 14B to generate between them a dispersion electric field, E_D, and a compensation field, E_c, across the analytical gap (g). The power supply unit 20B comprises switches (not shown) configured to switch to provide the AC dispersion voltage waveform, V_D, such as described above with reference to Figure 2, that alternates between adjustable voltage values, and the DC compensation voltage, V_c.

A control unit 33B of the ion mobility analysis unit is configured to control the power supply unit 20B to apply a dispersion voltage, V_D, and compensation voltage, V_c, to the electrodes 14B of the ion mobility analysis unit. In particular, the control unit is configured to control the power supply unit to supply the electrodes 14B of the drift region with a selected asymmetric AC waveform 34 of the dispersion voltage, V_D, and a selected DC compensation voltage, V_c, 36 described above with reference to Figure 2. The selected combination of dispersion voltage and compensation voltage is non-changing or is changing/varying according to the control of the control unit 33B. The selected combination is determined by the control unit 33B according to the ion intensity output signal 35B from the ion detector of the mass analysis unit, as has been described above.

E_c = — (dV_c(x)/dx) = -V_c/g which spans the analytical gap, g, all along the drift region and extends in a direction perpendicular to the longitudinal axis of the drift region. A flow of buffer gas along the drift region is provided such that in use ions generated from the sample undergo differential ion mobility analysis by the differential ion mobility assembly. A gas flow former 18B comprises a capillary providing gas flow communication from the atmospheric pressure ion source (API) 10 to the vacuum region. The capillary is configured to establish in the first vacuum region a flow of buffer gas (e.g., N2 molecules) entrained with ions generated from the sample, the flow of buffer gas being directed along the drift region (between electrodes 14B). The basic principles and the mechanism for IMS separation based on the non-linear ion mobility dependence on electric field and pressure, is described above with referring to Figure 2.

The ambient pressure ionization (API) source 10B ionizes the sample at an ambient atmospheric pressure to generate ions for analysis by the ion mobility analysis unit 3B, which is configured to generate these ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby. Examples are as described above with reference to figures 6 to 8.

The ion mobility analysis unit receives ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals. It transmits ions from amongst those received ions through a drift region between its electrodes. Transmission is conditional upon the combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval. The ion detector detects ions from the ion mobility analysis unit that have been transmitted by it.

In particular, in a ‘settings search stage’ as described above, the ion mobility analysis unit is configured to vary the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to its electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals.

The control unit 33B of the ion mobility analysis unit monitors the intensity of ions detected by the ion detector 31 B according to the varied values of the dispersion voltage and compensation voltage and, based on the monitored intensity of detected ions, it selects from amongst the varied values of the dispersion voltage and compensation voltage a combination thereof which permits transmission through the ion mobility analysis unit of ions from the target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample. The selection process is the same as that described above with reference to Figure 9, Figure 10 and Figure 11 .

This process is the same as that described above with reference to the operation of the apparatus illustrated in Figure 1A. The explanation of examples of the functions of the ion mobility analysis unit 3, the ionisation source 10, the first vacuum chamber 12, the electrodes 14, the gas flow former 18, the first vacuum pump 26, the ion detector 31 , the power supply unit 20 and the control unit 33 of the apparatus of Figure 1A are applicable to the example described above with reference to Figure 1 B in relation to the functions of the ion mobility analysis unit 3B, the ionisation source 10B, the first vacuum chamber 12B, the electrodes 14B, the gas flow former 18B, the first vacuum pump 26B, the ion detector 31 B, the power supply unit 20B and the control unit 33B.

The control unit 33B may vary the values of both the dispersion voltage and the compensation voltage, in a ‘settings search stage’ as described above, such that:

(i) none of the values of the dispersion voltage are repeated as between two successive ion transmission time intervals; and/or,

(ii) all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals; and/or,

(iii) some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals; and/or,

(iv) none of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

Examples are as described above with reference to figures 6 to 8.

Each ion transmission time interval is synchronised to coincide with a respective ion generation time interval. Successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit. Successive ion transmission time intervals may be separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit. The intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

In a process of applying pre-set settings, such as setting that have been selected during a previous ‘settings search stage’, the ion mobility analysis unit 1 B is configured to receive ions periodically from the ambient pressure ionization source as generated during successive ion generation time intervals, and under the control of the control unit 33B, to transmit ions from amongst the received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval. The transmission by the ion mobility analysis unit of ions received from the sample is permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage may be selected to permit transmission of ions from the target compound and to prevent transmission of other ions amongst said ions from the sample. Each pre-set combination of a dispersion voltage and a concurrent compensation voltage comprises voltages that are be applied throughout a finite interval of time and which remain unchanged during that finite interval of time, such as is described above with reference to figures 12 to 14. The finite interval of time may be substantially equal in duration to an ion generation time interval.

Under the control of the control unit 33B, the ion mobility analysis unit transmit may transmit ions from amongst received ions through its drift region according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage. Respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval. The pre-set combinations of a dispersion voltage and a concurrent compensation voltage may each comprise voltages that are be applied throughout a respective one of a plurality of separate finite interval of times (see Fig. 13, Fig. 14) such that each distinct combination remains unchanged during that respective finite interval of time. The sum of the durations of the plurality of finite intervals of time are substantially equal in duration to an ion generation time interval. The values of the dispersion voltage and the compensation voltage corresponding to each pre-set combination are constant throughout the duration of a respective time sub-interval within an ion transmission time interval. The sum of the respective time sub-intervals of the plurality of distinct pre-set combinations collectively define (correspond to) the ion transmission time interval of which each sub-interval forms a part.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[1] Ting-Hao Kuo, Ewelina P. Dutkiewicz, Jiying Pei, and Cheng-Chih Hsu: “Ambient Ionization Mass Spectrometry Today and Tomorrow: Embracing Challenges and Opportunities" . Anal. Chem. 2020, 92, 3, 2353-2363. Publication Date: 11^th December 2019

[2] I. A. Buryakov, et al., Int. J. Mass Spectrom. Ion Processes 1993, 128, 143

[3] R. W. Purves, et al., Rev. Sci. Instrum. 1998, 69, 4094

[4] G.A. Eiceman et al.: “Ion Mobility Spectrometry". CRC Press, 2016. ISBN 13:978-1-138-19948-4. See Chapter 5: “Ion Injection and Pulsed Sources"

Claims

Claims:

1 . A mass spectrometer apparatus for analysing ions from a sample comprising a pre-set target compound, comprising: an ambient pressure ionization (API) source arranged to ionize the sample at an ambient atmospheric pressure to generate ions for analysis by the mass spectrometer, wherein the ambient pressure ionization source is configured to generate said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; an ion mobility analysis unit configured to receive ions periodically from the ion source as generated during successive ion generation time intervals, and to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; a mass analysis unit comprising a mass analyser arranged to receive ions from the ion mobility analysis unit, and an ion detector arranged to detect ions from the mass analyser; wherein transmission by the ion mobility analysis unit of ions received from the sample is permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage is selected to permit transmission of ions from the pre-set target compound and to prevent transmission of other ions amongst said ions from the sample.

2. A mass spectrometer apparatus according to any preceding claim wherein the ion mobility analysis unit is configured to transmit ions from amongst said received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage, wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval.

3. A mass spectrometer apparatus according to any preceding claim wherein the ion mobility analysis unit is configured to determine said pre-set combination of a dispersion voltage and a concurrent compensation voltage by a process comprising: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector thereby according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a said pre-set combination thereof which permits transmission through the ion mobility analysis unit of ions from the pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

4. A mass spectrometer apparatus according to claim 3 wherein the ion mobility analysis unit is configured to vary the values of both the dispersion voltage and the compensation voltage such that none of the values of the dispersion voltage are repeated as between two successive ion transmission time intervals.

5. A mass spectrometer apparatus according to any of claims 3 and 4 wherein the ion mobility analysis unit is configured to vary the values of both the dispersion voltage and the compensation voltage such that all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

6. A mass spectrometer apparatus according to any of claims 3 and 4 wherein the ion mobility analysis unit is configured to vary the values of both the dispersion voltage and the compensation voltage such that some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

7. A mass spectrometer apparatus according to any of claims 3 and 4 wherein the ion mobility analysis unit is configured to vary the values of both the dispersion voltage and the compensation voltage such that none of the values of the compensation voltages are repeated as between two successive ion transmission time intervals.

8. A mass spectrometer apparatus according to any of claims 3 to 7 wherein each ion transmission time interval is synchronised to coincide with a respective ion generation time interval.

9. A mass spectrometer apparatus according to any of claims 3 to 8 wherein successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit.

10. A mass spectrometer apparatus according to claim 9 wherein successive ion transmission time intervals are separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit.

11 . A mass spectrometer apparatus according to any of claims 3 to 10 wherein the intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

12. A mass spectrometer apparatus according to any preceding claim wherein the pre-set target compound comprises Cortisol.

13. A mass spectrometer apparatus according to any preceding claim wherein the ambient pressure ionization (API) source comprises an ambient ionisation source.

14. A mass spectrometer apparatus according to any preceding claim wherein the ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process.

15. A method for mass spectrometry for analysing ions from a sample comprising a pre-set target compound, the method comprising: providing an ambient pressure ionization (API) source and therewith ionising the sample at an ambient atmospheric pressure to generate ions for analysis by mass spectrometry, and by the ambient pressure ionization source generating said ions periodically during a plurality of successive ion generation time intervals separated by intervening time intervals in which no ions are generated thereby; providing an ion mobility analysis unit configured and therewith receiving ions periodically from the ion source as generated during successive ion generation time intervals, and transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a pre-set combination of a dispersion voltage and a concurrent compensation voltage applied thereby to the electrodes during an ion transmission time interval; providing a mass analysis unit comprising a mass analyser and an ion detector and, by the mass analyser, receiving ions from the ion mobility analysis unit and, by the ion detector, detecting ions from the mass analyser; wherein transmission by the ion mobility analysis unit of ions received from the sample is permitted according to a mobility of respective ions within the drift region, and the pre-set combination of concurrent values of the dispersion voltage and the compensation voltage is selected to permit transmission of ions from the pre-set target compound and to prevent transmission of other ions amongst said ions from the sample.

16. A method according to claim 14 comprising, by ion mobility analysis unit, transmitting ions from amongst said received ions through a drift region between electrodes thereof according to a plurality of distinct pre-set combinations of a dispersion voltage and a concurrent compensation voltage wherein respective pre-set combinations are applied separately and sequentially thereby to the electrodes during an ion transmission time interval.

17. A method according to claim 15 or 16 comprising determining said pre-set combination of a dispersion voltage and a concurrent compensation voltage by a process comprising: varying the values of the dispersion voltage and/or of the compensation voltage such that concurrent values of the dispersion voltage and the compensation voltage that are applied to the electrodes during a given ion transmission time interval are not also applied during a preceding ion transmission time interval, such that at least some of the values of the concurrent dispersion and compensation voltages are not repeated as between two successive ion transmission time intervals; monitoring the intensity of ions detected by the ion detector according to said varied values of the dispersion voltage and compensation voltage; and, according to the monitored intensity of detected ions, selecting from amongst the varied values of the dispersion voltage and compensation voltage a said pre-set combination thereof which permits transmission through the ion mobility analysis unit of ions from the pre-set target compound and which prevents transmission through the ion mobility analysis unit of other ions amongst said ions from the sample.

18. A method according to any of claims 15 to 17 comprising varying the values of both the dispersion voltage and the compensation voltage such that none of the values of the dispersion voltage are repeated as between two successive ion transmission time intervals.

19. A method according to any of claims 15 to 18 comprising varying the values of both the dispersion voltage and the compensation voltage such that all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

20. A method according to any of claims 15 to 19 comprising varying the values of both the dispersion voltage and the compensation voltage such that some but not all of the values of the compensation voltage are repeated as between two successive ion transmission time intervals.

21 . A method according to any of claims 15 to 20 comprising varying the values of both the dispersion voltage and the compensation voltage such that none of the values of the compensation voltages are repeated as between two successive ion transmission time intervals.

22. A method according to any of claims 15 to 21 wherein each ion transmission time interval is synchronised to coincide with a respective ion generation time interval.

23. A method according to any of claims 15 to 22 wherein successive ion transmission time intervals are separated by intervening time intervals in which no ions are transmission by the ion mobility analysis unit.

24. A method according to claim 23 wherein successive ion transmission time intervals are separated by said intervening time intervals in which no compensation voltage is applied to the electrodes of the ion mobility analysis unit.

25. A method according to any of claims 15 to 24 wherein the intervening time intervals that separate successive ion generation time intervals are synchronised to coincide with the intervening time intervals that separate successive ion transmission time intervals.

26. A method according to any of claims 15 to 25 wherein the pre-set target compound comprises Cortisol.

27. A method according to any of claims 15 to 26 wherein the ambient pressure ionization (API) source comprises an ambient ionisation source.

28. A method according to any of claims 15 to 27 wherein the ambient pressure ionization (API) source comprises a probe electrospray ionisation (PESI) unit configured to ionize the sample according to a probe electrospray ionisation process.