GB2539871A - A method of mass spectrometry - Google Patents

Abstract

A method of quantitation of compounds of interest within a sample comprises performing a liquid chromatography separation of the sample, passing the liquid chromatography eluant into a mass spectrometer and performing ion mobility analysis and mass spectrometry analysis on the sample. Compounds of interest suspected to be present in the sample which coelute in the liquid chromatography separation and have substantially the same mass are identified. Data corresponding to each of the compounds of interest are identified by ion mobility separation of the compounds, and quantitation is performed on the compounds of interest from the data. The compounds of interest may be isomers, protomers, epimers or conformers. If the compounds of interest are only separated partially by ion mobility, the method may further comprise obtaining fragment mass spectrometry data by repeatedly switching modes of operation and using the intensities of fragment ions relating to the compounds of interest to provide quantitation information.

Description

A METHOD OF MASS SPECTROMETRY

The present invention relates to the field of mass spectrometry. More specifically a method of quantitation by mass spectrometry

BACKGROUND ART

In many industries, the screening of samples for compounds of interest and to determine the quantity (or concentration) of these compounds of interest within the sample can provide vital information. Liquid Chromatography and mass spectrometry are often used to enable the user to perform the analysis of this type.

In some instances, there may be different isomers, or other coeluting species which may also have the same mass to charge ratio that a user may want to differentiate, identify and quantify. Determining the presence of these species can cause a major problem for a user - it is entirely possible the user may not even realise that there are two different coeluting species. Determining the amount of these different species present is even more problematic.

It is commonly accepted that it is not possible to accurately quantify species which coelute in the given conditions, which also have the same m/z. Conventionally, a user would try to change the conditions that were used for the chromatography, in order to separate these species. This can be tricky, complicated and time consuming, and in some instances may be impossible to do.

There is therefore desired to be a method of quantifying co-eluting species in a quicker, easier and reliable method.

According to a first embodiment of the invention, there is provided a method of quantitation of compounds of interest within a sample comprising the steps of Performing a Liquid chromatography separation of the sample to produce a liquid chromatography eluant. Passing the liquid chromatography eluant into a mass spectrometer and performing Ion mobility analysis and Mass spectrometry analysis on the sample to produce LC, Ion mobility and Mass Spectrometry data. Identifying two or more compounds of interest suspected to be present in the sample which coelute in the liquid chromatography separation and have substantially the same mass. Identifying the LC, Ion Mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest by the at least partial separation of the compounds of interest by ion mobility and performing quantitation of the two or more compounds of interest from the LC, Ion mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest.

Separation of the coeluting compounds of interest within the sample by Ion mobility is particularly advantageous as this would remove the problems of changing Chromatography conditions, to try to separate the coeluting compounds of interest.

In some embodiments the method may use a ion mobility triple quadrupole mass spectrometer. The method may comprise a multiple reaction monitoring experiment.

In some embodiments the method comprises an ion mobility apparatus, a quadrupole and a Time of Flight mass spectrometer.

The quadrupole may be arranged to perform mass selection. In other embodiments, the quadrupole may be arranged to perform mass filtering

In some embodiments the ion mobility measurement may be performed before the sample passes the quadrupole. In other embodiments the ion mobility measurement may be performed after the sample passes the quadrupole.

In some embodiments the separation of the compounds of interest by ion mobility may be only partial.

In some embodiments the method may further comprise switching from a first mode of operation where the mass spectrometry data represents substantially parent ion data and a second mode of operation where the mass spectrometry data represents substantially fragment ion data.

The step of Identifying the LC, Ion Mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest may further comprise identifying fragment mass spectrometry data that relate to a first compound of interest and identifying fragment mass spectrometry data from a fragment not produced by the first compound of interest data that relates to a second ion of interest.

The method may further comprise using the intensities of the fragment mass spectrometry data that relate to a first compound of interest and identifying fragment mass spectrometry data from a fragment not produced by the first compound of interest data that relates to a second ion of interest to provide quantitation information on the two or more compounds of interest.

The mass spectrometer may comprise: (a) an ion source selected from the group consisting of: (i) an Electrospray ionisation ("ESI") ion source; (ii) an Atmospheric Pressure Photo Ionisation ("APPI") ion source; (iii) an Atmospheric Pressure Chemical Ionisation ("APCI") ion source; (iv) a Matrix Assisted Laser Desorption Ionisation ("MALDl") ion source; (v) a Laser Desorption Ionisation ("LDI") ion source; (vi) an Atmospheric Pressure Ionisation ("API") ion source; (vii) a Desorption Ionisation on Silicon ("DIOS") ion source; (viii) an Electron Impact ("El") ion source; (ix) a Chemical Ionisation ("Cl") ion source; (x) a Field Ionisation ("FI") ion source; (xi) a Field Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma ("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") ion source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xv) a Desorption Electrospray Ionisation ("DESI") ion source; (xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption Ionisation ion source; (xviii) a Thermospray ion source; (xix) an Atmospheric Sampling Glow Discharge Ionisation (“ASGDI”) ion source; (xx) a Glow Discharge (“GD”) ion source; (xxi) an Impactor ion source; (xxii) a Direct Analysis in Real Time ("DART") ion source; (xxiii) a Laserspray Ionisation ("LSI") ion source; (xxiv) a Sonicspray Ionisation ("SSI") ion source; (xxv) a Matrix Assisted Inlet Ionisation ("MAM") ion source; (xxvi) a Solvent Assisted Inlet Ionisation ("SAM") ion source; (xxvii) a Desorption Electrospray Ionisation (“DESI”) ion source; and (xxviii) a Laser Ablation Electrospray Ionisation (“LAESI”) ion source; and/or (b) one or more continuous or pulsed ion sources; and/or (c) one or more ion guides; and/or (d) one or more ion mobility separation devices and/or one or more Field Asymmetric Ion Mobility Spectrometer devices; and/or (e) one or more ion traps or one or more ion trapping regions; and/or (f) one or more collision, fragmentation or reaction cells selected from the group consisting of: (i) a Collisional Induced Dissociation ("ClD") fragmentation device; (ii) a Surface Induced Dissociation ("SID") fragmentation device; (iii) an Electron Transfer Dissociation ("ETD") fragmentation device; (iv) an Electron Capture Dissociation ("ECD") fragmentation device; (v) an Electron Collision or Impact Dissociation fragmentation device; (vi) a Photo Induced Dissociation ("PID") fragmentation device; (vii) a Laser

Induced Dissociation fragmentation device; (viii) an infrared radiation induced dissociation device; (ix) an ultraviolet radiation induced dissociation device; (x) a nozzle-skimmer interface fragmentation device; (xi) an in-source fragmentation device; (xii) an in-source Collision Induced Dissociation fragmentation device; (xiii) a thermal or temperature source fragmentation device; (xiv) an electric field induced fragmentation device; (xv) a magnetic field induced fragmentation device; (xvi) an enzyme digestion or enzyme degradation fragmentation device; (xvii) an ion-ion reaction fragmentation device; (xviii) an ion-molecule reaction fragmentation device; (xix) an ion-atom reaction fragmentation device; (xx) an ion-metastable^ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an Electron Ionisation Dissociation (“EID”) fragmentation device; and/or (g) a mass analyser selected from the group consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance ("ICR") mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance ("FTICR") mass analyser; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadro-logarithmic potential distribution; (x) a Fourier Transform electrostatic mass analyser; (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; and (xiv) a linear acceleration Time of Flight mass analyser; and/or (h) one or more energy analysers or electrostatic energy analysers; and/or (i) one or more ion detectors; and/or 0) one or more mass filters selected from the group consisting of: (i) a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter; and (viii) a Wien filter; and/or (k) a device or ion gate for pulsing ions; and/or (l) a device for converting a substantially continuous ion beam into a pulsed ion beam.

The mass spectrometer may comprise either: (i) a C-trap and a mass anaiyser comprising an outer barrei-iike eiectrode and a coaxiai inner spindie-iike electrode that form an electrostatic field with a quadro-logarithmic potential distribution, wherein in a first mode of operation ions are transmitted to the C-trap and are then injected into the mass analyser and wherein in a second mode of operation ions are transmitted to the C-trap and then to a collision cell or Electron Transfer Dissociation device wherein at least some ions are fragmented into fragment ions, and wherein the fragment ions are then transmitted to the C-trap before being injected into the mass analyser; and/or (ii) a stacked ring ion guide comprising a plurality of electrodes each having an aperture through which ions are transmitted in use and wherein the spacing of the electrodes increases along the length of the ion path, and wherein the apertures in the electrodes in an upstream section of the ion guide have a first diameter and wherein the apertures in the electrodes in a downstream section of the ion guide have a second diameter which is smaller than the first diameter, and wherein opposite phases of an AC or RF voltage are applied, in use, to successive electrodes.

The mass spectrometer may further comprise a device arranged and adapted to supply an AC or RF voltage to the electrodes. The AC or RF voltage optionally has an amplitude selected from the group consisting of: (i) about < 50 V peak to peak; (ii) about 50-100 V peak to peak; (iii) about 100-150 V peak to peak; (iv) about 150-200 V peak to peak; (v) about 200-250 V peak to peak; (vi) about 250-300 V peak to peak; (vii) about 300-350 V peak to peak; (viii) about 350-400 V peak to peak; (ix) about 400-450 V peak to peak; (x) about 450-500 V peak to peak; and (xi) > about 500 V peak to peak.

The AC or RF voltage may have a frequency selected from the group consisting of: (i) < about 100 kHz; (ii) about 100-200 kHz; (iii) about 200-300 kHz; (iv) about 300-400 kHz; (v) about 400-500 kHz; (vi) about 0.5-1.0 MHz; (vii) about 1.0-1.5 MHz; (viii) about 1.5-2.0 MHz; (ix) about 2.0-2.5 MHz; (x) about 2.5-3.0 MHz; (xi) about 3.0-3.5 MHz; (xii) about 3.5-4.0 MHz; (xiii) about 4.0-4.5 MHz; (xiv) about 4.5-5.0 MHz; (xv) about 5.0-5.5 MHz; (xvi) about 5.5-6.0 MHz; (xvii) about 6.0-6.5 MHz; (xviii) about 6.5-7.0 MHz; (xix) about 7.0-7.5 MHz; (xx) about 7.5-8.0 MHz; (xxi) about 8.0-8.5 MHz; (xxii) about 8.5-9.0 MHz; (xxiii) about 9.0-9.5 MHz; (xxiv) about 9.5-10.0 MHz; and (xxv) > about 10.0 MHz.

The mass spectrometer may comprise a chromatography or other separation device upstream of an ion source. The chromatography separation device may comprise a liquid chromatography or gas chromatography device. The separation device may comprise: (i) a Capillary Electrophoresis (“CE”) separation device; (ii) a Capillary Electrochromatography (“CEC”) separation device; (iii) a substantially rigid ceramic-based multilayer microfluidic substrate (“ceramic tile”) separation device; or (iv) a supercritical fluid chromatography separation device.

The ion guide may be maintained at a pressure selected from the group consisting of: (i) < about 0.0001 mbar; (ii) about 0.0001-0.001 mbar; (iii) about 0.001-0.01 mbar; (iv) about 0.01-0.1 mbar; (v) about 0.1-1 mbar; (vi) about 1-10 mbar; (vii) about 10-100 mbar; (viii) about 100-1000 mbar; and (ix) > about 1000 mbar.

The analyte ions may be subjected to Electron Transfer Dissociation (“ETD”) fragmentation in an Electron Transfer Dissociation fragmentation device. Analyte ions may be caused to interact with ETD reagent ions within an ion guide or fragmentation device.

In order to effect Electron Transfer Dissociation optionally either: (a) analyte ions are fragmented or are induced to dissociate and form product or fragment ions upon interacting with reagent ions; and/or (b) electrons are transferred from one or more reagent anions or negatively charged ions to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (c) analyte ions are fragmented or are induced to dissociate and form product or fragment ions upon interacting with neutral reagent gas molecules or atoms or a non-ionic reagent gas; and/or (d> electrons are transferred from one or more neutral, non-ionic or uncharged basic gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (e) electrons are transferred from one or more neutral, non-ionic or uncharged superbase reagent gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charge analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (f) eiectrons are transferred from one or more neutral, non-ionic or uncharged alkali metal gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (g) electrons are transferred from one or more neutral, non-ionic or uncharged gases, vapours or atoms to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions, wherein the one or more neutral, non-ionic or uncharged gases, vapours or atoms are selected from the group consisting of: (i) sodium vapour or atoms; (ii) lithium vapour or atoms; (iii) potassium vapour or atoms; (iv) rubidium vapour or atoms; (v) caesium vapour or atoms; (vi) francium vapour or atoms; (vii) Ceo vapour or atoms; and (viii) magnesium vapour or atoms.

The multiply charged analyte cations or positively charged ions may comprise peptides, polypeptides, proteins or biomolecules.

In order to effect Electron Transfer Dissociation, optionally: (a) the reagent anions or negatively charged ions are derived from a polyaromatic hydrocarbon or a substituted polyaromatic hydrocarbon; and/or (b) the reagent anions or negatively charged ions are derived from the group consist ing of. (i) anthracene; (ii) 9,10 diphenyl-anthracene; (iii) naphthalene; (iv) fluorine; (v) phenanthrene; (vi) pyrene; (vii) fluoranthene; (viii) chrysene; (ix) triphenylene; (x) perylene; (xi) acridine; (xii) 2,2' dipyridyl; (xiii) 2,2' biquinoline; (xiv) 9-anthracenecarbonitrile; (xv) dibenzothiophene; (xvi) 1,10'-phenanthroline; (xvii) 9' anthracenecarbonitrile; and (xviii) anthraquinone; and/or (c) the reagent ions or negatively charged ions comprise azobenzene anions or azobenzene radical anions.

The process of Electron Transfer Dissociation fragmentation may comprise interacting analyte ions with reagent ions, wherein the reagent ions comprise dicyanobenzene, 4-nitrotoluene or azulene.

Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:

Figure 1 is an illustration of a mass spectrometer suitable for performing the present invention; and

Figure 2 is a computer screen print showing results of a method according to the invention.

Figure 1 illustrates a mass spectrometer 10 suitable for use with the invention. A sample is injected into a liquid Chromatography system, where the sample is subjected to Liquid Chromatography separation. Once this has happened, in the instance of this instrument, the separated sample of interest is passed into the instrument at an injection inlet 12. The sample is sprayed from a needle into the ionisation chamber 14. Ionisation of the sample may occur, to form sample ions. The ionised sample will pass out of the ionisation chamber, and the ions will flow towards a first vacuum region 16. The will transfer through the first vacuum region, into the stepwave ion guide 18. The stepwave ion guide will then guide the ions along the ion guide initially in a large cross section area 20 and then, focus the ions into a smaller cross section in the off axis part 22 of the guide. The ions will then be transferred into a further ion guide 24, where the ions are transmitted through to a quadrupole mass filter 26.

The quadrupole mass filter can be used in a transmission mode, so that all the ions entering, pass through the filter, and passes into the Triwave chamber 28. Once the ions are passed into the Triwave chamber 28 they are collected in bunches within the trap cell 30 within the Triwave chamber 28. A bunch of ions in the trap cell, will then be released through the helium cell 32, into the ion mobility separator 34. The ions will then temporally separate according to their ion mobility within the mobility separator, and as ions exit the separator, they are passed into a transfer cell 36, where ions of small ranges of ion mobility are collected in groups, and passed through the transfer cell, several lenses 38 and into a Tof pusher region 40. Each group of ions of small mobility ranges can then be pulsed out of the ToF pusher region into a flight tube 42, into a reflectron 44, in where they are reflected back to a detection system 46, where the flight times of the ions are recorded, together with the small range of mobility of the ions. A second, consecutive, analysis may then be performed along a similar basis, except, after the ions have been separated into the groups of small ranges of ion mobility in the separator 34, energy is provided to the ions within the transfer cell 36, to induce fragmentation of the ions in each group, to provide fragment ions. These fragment ions are kept in the small groups according to the mobility of the parent ions, and are passed into the Tof pusher region 40. Similarly, each group of fragment ions from the parent ions of small mobility ranges can then be pulsed out of the ToF pusher region into a flight tube 42, into a reflectron 44, in where they are reflected back to a detection system 46, where the flight times of these fragment ions are recorded, together with the small range of mobility of the parent ions that produced the fragment ions.

The information produced from each small mobility range in the first and the second analysis may be combined, to provide parent and fragment ion information for all the ions where the small range of mobility in the first and the second analysis matches.

In some embodiments, the quadrupole 26 may be used in a transmission mode, so that all or most of the ions are passed through the quadrupole.

In other embodiments, the quadrupole 26 may be used in a mass section mode, so that only the ions of a single mass, or small mass range are passed through the quadrupole.

The instrument described above performs ion mobility separation after the ions have been passed into the quadrupole. This allows the user to select ions of a particular mass to charge ratio prior to the ion mobility separation. In some modes of operation, this can be very useful where there may be ions of similar mobility but different mass in the sample.

In other embodiments, the ion mobility separation may be performed before the quadrupole. This allows the user to separate ions of different mobilities prior to mass selection. This can be useful where ions of a particular mobility and mass are of interest, but need to be separated from other ions of a different mobility, but similar mass.

The present invention uses ion mobility separation to separate co-eluting species of the same m/z.

In some embodiments the coeluting species may be isomer, protomers, epimers or conformers.

In other embodiments the coeluting species may be of different chemical make up.

In many applications isomers, or even protomers can occur (where a ionization takes place on more than one site of a molecule, giving rise to two mobility separated species), epimers, conformers are also isomeric. They have the same elemental composition and hence the same exact mass.

In applications generated the inventors have separated isomeric species using ion mobility, even though they coelute chromatographically. Even using accurate mass measurement, it would not be possible to distinguish coeluting isomeric species. If possible, a chromatographic separation could be developed to separate the positional isomers shown. However in the case of protomers, where they are formed during the process of ionization, there is no means of pwroducing chromatographic separation. In addition, where using different chromatographic techniques, such as micro fluidic chromatography, the peak capacity of the column may not be sufficient to enable separation of isomers to be developed. Use of ion mobility and a characteristic drift time, enables calibration curves for individual isomers to be generated and then within a sample, it will be possible to calculate the individual concentrations of coeluting isomeric species. A first example of the invention, is shown in Figure 2 where under the chromatographic conditions used, vitexin and isovitexin coelute. In negative ion mode the 6C glycoside isomers (isoorientin/orientin) and 8C glycoskJe isomers (isovitexin/vitexin), can be separated using ion mobility. They can also be identified from their collision cross section values obtained using nitrogen based travelling ion wave mobility (™CCSn2 )-The software produced component summary for PassiHora incarnata below shows coeluting vitexin and isovitexin (retention time 8.15/8.14 minutes), with mass measurement error <2ppm and collision cross section errors<0.5% are presented. The ion mobility trace and ion mobility data viewer 3D plot of drift time/vs retention time are presented, illustrating the ion mobility separation of these two isomeric species.

The component summary, also shows the calculated concentration (pg/pL) generated for the isomers vitexin and isovitexin, Passiflora incarnata extract. Using accurate mass only it would not be possible to distinguish the isomers. The calibration curves are formed on the basis of precursor ion intensity.

This process would be applicable for all IMS/MS instruments where the resolution of the IMS is sufficiently good to produce at least partial separation of the components of that co-elute and have the same M/Z values. Quantitation may be performed using precursor ion intensity, or from fragment ion intensities.

The methods according to the invention may be combined with ion mobility and any number of other mass spectrometry techniques.

In some embodiments the invention may be used in a Multiple Reaction monitoring mode where a first quadrupole is set to a parent ion mass, then fragmentation occurs in a reaction or collision cell, and a second quadrupole is set to look for a specific fragment ion indicative of the ion of interest.

In some embodiments, where the species of interest are of the same M/Z and may to have the same fragmentation pattern, the instrument may simply be held in a single MRM mode. In this instance the ion current could simply be measured, and provided, at least partial separation of the species is achieved by IMS, quantitation may be performed.

In other embodiments where the fragmentation patterns for the two species of interest are different, then the instrument may need to be set with two MRMs. Both these MRMs need to be performed, alternating between them. In this instance the ion current for the relevant MRM may give an indication of the quantity of the relevant species.

In some embodiments, where the identity of any species of interest within the sample needs confirming, as well as the quantity of the species, once they have been identified, the MSe functionality, may be used. MSe functionality may comprise consecutively switching between a first mode where the parent ions are analysed, then the fragment ions are analysed in a second mode. In these embodiments all the ions that are in the sample are detected with their ion mobility, and their LC retention time in the first mode. In the second mode the fragment ions of all these parent ions are detected by the instrument with the retention time, and ion mobility of the parent ions.

From this information, even with only partial separation of the two it should be possible to deduce which of the parent ions and fragment ions correspond with each other. From this information, the identity of the ions may be possible to be deduced. The quantity of each of the parent ions present in the sample can be calculated from the spectra provided that there is at least partial separation of the parent Ions by Ion mobility.

Given that the two parent ions overlap, the quantitation of the substances can not immediately be done from only the parent ion data. However, if the fragment ion information is used, it can be identified where the overlap begins, and ends, and by checking the proportion of fragment and parent ions, and working out the contribution to the overall peak from each of the overlapping parent ions quantitation data can be calculated in an accurate manner.

Once data has been acquired, and the identification of the peaks has been performed, quantitation of the coeluting species (analytes of interest) is performed. Typically, quantitation of the species is performed from the parent ion spectra, though, this can also be done using the fragment ion spectra.

Peaks are detected by finding local maxima in the m/z vs LC data vs Ion mobility data.

The properties of these peaks, including their m/z (mass spectrometry data) and range in retention time (LC data) and drift time (ion mobility data) are measured. A flag is also set if they are being interfered with by another peak, or if they contain data where the detector is saturated.

Given a charge state the peak's ion mobility can be converted to a CCS. So a target component plus a charged adduct can be identified by screening these peak's mass spectrometry data, LC data and CCS (or ion mobility data) values against the target's expected values.

The response of the peaks can then be calculated using several methods - two examples of possible methods are indicated below:-

In a first exemplary method, the response of a peak may be calculated by examining each scan in the region defined by the range in LC data and ion mobility data. Spectral peaks are detected in each scan, and the area of each peak at the appropriate mass spectrometry data is summed to give the response. If there are any interfering peaks with slightly different m/z values in each retention time and drift time scan, if they are not identifiable from any fragment data, will be excluded from the response estimate.

In a second exemplary method, the response of a peak can be calculated by creating a drift specific extracted ion chromatogram (DS XIC) over the Drift time range and at the appropriate m/z. After integrating this chromatogram, the area or height of the chromatographic peak within the retention time range can be used as the response. If there are interfering peaks the integration can be manually adjusted to improve the response estimate.

In some embodiments, if there are several standard samples which contain a known amount of a target component, then a calibration curve can be constructed. This calibration curve can be used to convert a response of the target in an analyte sample to an amount.

It will be appreciated by those skilled in the art that any number of combinations of the aforementioned features and/or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein.

Claims (12)

1. A method of quantitation of compounds of interest within a sampie comprising;-Performing a Liquid chromatography separation of the sampie to produce a liquid chromatography eluant; Passing the iiquid chromatography eluant into a mass spectrometer and performing ion mobiiity analysis and Mass spectrometry analysis on the sampie to produce LC, Ion mobility and Mass Spectrometry data; Identifying two or more compounds of interest suspected to be present in the sample which coelute in the liquid chromatography separation and have substantially the same mass; Identifying the LC, Ion Mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest by the at least partial separation of the compounds of interest by ion mobiiity; Performing quantitation of the two or more compounds of interest from the LC, Ion mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest.

2. The method of claim 1 where the method uses a ion mobility triple quadrupole mass spectrometer.

3. The method of claim 1 wherein the method comprises a multiple reaction monitoring experiment.

4. The method of claim 1 where the method comprises an ion mobility apparatus, a quadrupole and a Time of Flight mass spectrometer.

5. The method of claim 4 where the quadrupole is arranged to work in a mass selection mode.

6. The method of claim 4 where the quadrupole is arranged to work in a transmission mode.

7. The method of claim 4 where the ion mobility measurement is performed before the sample passes the quadrupole.

8. The method of claim 4 where the ion mobility measurement is performed after the sample passes the quadrupole.

9. The method of claim 1 wherein the separation of the compounds of interest by ion mobility is only partial.

10. The method of claim 9 further comprises switching from a first mode of operation where the mass spectrometry data represents substantially parent ion data and a second mode of operation where the mass spectrometry data represents substantially fragment ion data.

11. The method of claim 10 wherein the step of Identifying the LC, Ion Mobility and Mass Spectrometry data corresponding to each of the two or more compounds of interest further comprises identifying fragment mass spectrometry data that relate to a first compound of interest and identifying fragment mass spectrometry data from a fragment not produced by the first compound of interest data that relates to a second ion of interest.

12. The method of claim 12 further comprising using the intensities of the fragment mass spectrometry data that relate to a first compound of interest and identifying fragment mass spectrometry data from a fragment not produced by the first compound of interest data that relates to a second ion of interest to provide quantitation information on the two or more compounds of interest.