Method of screening a sample for the presence of one or more known compounds of interest and a mass spectrometer performing this method

Abstract

A method of screening a sample for the presence of one or more known compounds of interest is disclosed. A fragmentation device is repeatedly switched between a fragmentation mode of operation and a non-fragmentation mode of operation. A determination is made whether a candidate parent ion of interest is present in a non-fragmentation data set and whether one or more corresponding fragment ions of interest are present in a fragmentation data set. A further determination is made to check if the candidate parent ion of interest and the one or more corresponding fragment ions of interest have substantially similar elution or retention times and/or ion mobility drift times.

Claims

1. A method of screening a sample for a presence of one or more known compounds of interest with a mass spectrometer comprising a fragmentation, collision or reaction device and a mass analyser, said method comprising: repeatedly switching said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; selecting a known compound of interest, and identifying a candidate parent ion of interest associated with said known compound of interest; determining if said candidate parent ion of interest is present in a first data set obtained when said fragmentation, collision or reaction device was operated in said first mode of operation; and determining if one or more fragment ions of interest corresponding to said candidate parent ion of interest are present in a second data set obtained when said fragmentation, collision or reaction device was operated in said second mode of operation; wherein if said candidate parent ion of interest is determined to be present in said first data set and one or more corresponding fragment ions of interest are also determined to be present in said second data set then said method further comprises determining whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that said known compound of interest is present in said sample.

2. A method as claimed in claim 1, wherein said mass analyser comprises a Time of Flight mass analyser, a Fourier Transform Ion Cyclotron Resonance mass analyser or an electrostatic orbital mass analyser.

3. A method as claimed in claim 1, wherein said elution or retention time comprises an elution or retention time from a chromatographic column.

4. A method as claimed in claim 1, wherein said ion mobility drift times correspond with ion mobility drift times of ions through an ion mobility spectrometer or separator arranged upstream of said fragmentation, collision or reaction device.

5. A method as claimed in claim 1, wherein said fragmentation, collision or reaction device comprises a Collision Induced Dissociation fragmentation device, an Electron Transfer Dissociation fragmentation device, an Electron Capture Dissociation fragmentation device or a Surface Induced Dissociation fragmentation device.

6. A method as claimed in claim 1, wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially different elution or retention times or ion mobility drift times then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

7. A method as claimed in claim 1, wherein if said candidate parent ion of interest is determined to have a lower charge state that one or more of said corresponding fragment ions of interest then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

8. A method as claimed in claim 1, wherein if said candidate parent ion of interest is determined to have an unexpected isotopic distribution then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

9. A method as claimed in claim 1, wherein if fragment ions of interest corresponding with said candidate parent ion of interest are determined to have an unexpected isotopic distribution then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

10. A method as claimed in claim 1, wherein if said candidate parent ion of interest or corresponding fragment ions of interest are determined to have an elution or retention time falling outside of an expected time window then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

11. A method as claimed in claim 1, wherein if said candidate parent ion of interest or corresponding fragment ions of interest are determined to have an ion mobility drift time falling outside of an expected time window then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

12. A method as claimed in claim 1, wherein if a ratio of an intensity of said candidate parent ion of interest to one or more of said fragment ions of interest falls outside an expected range then said candidate parent ion of interest is rejected, downgraded in status or reduced in significance as a candidate parent ion of interest.

13. A method as claimed in claim 1, further comprising determining an intensity of or quantifying said known compound of interest.

14. A method of screening a sample for a presence of one or more known compounds of interest with a mass spectrometer comprising a fragmentation, collision or reaction device and a mass analyser, said method comprising: repeatedly switching said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; selecting a known compound of interest, and identifying one or more fragment ions of interest associated with said known compound of interest; determining if said one or more fragment ions of interest are present in a second data set obtained when second fragmentation, collision or reaction device was operated in said second mode of operation; determining if a candidate parent ion of interest corresponding to said one or more fragment ions of interest is present in a first data set obtained when said fragmentation, collision or reaction device was operated in said first mode of operation; wherein if said one or more fragment ions of interest are determined to be present in said second data set and said corresponding candidate parent ion of interest is also determined to be present in said first data set then said method further comprises determining whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that said known compound of interest is present in said sample.

15. A method of screening a sample for a presence of one or more known compounds of interest with a mass spectrometer comprising a fragmentation, collision or reaction device and a mass analyser, said method comprising: repeatedly switching said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; determining if a candidate parent ion of interest is present in a first data set obtained when said fragmentation, collision or reaction device was operated in said first mode of operation; and determining if one or more corresponding fragment ions of interest are present in a second data set obtained when said fragmentation, collision or reaction device was operated in said second mode of operation; wherein if said candidate parent ion of interest is determined to be present in said first data set and one or more corresponding fragment ions of interest are also determined to be present in said second data set then said method further comprises determining whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if a ratio of an intensity of said candidate parent ion of interest to one or more of said fragment ions of interest falls within an expected range then a determination is made that a known compound of interest is present in said sample.

16. A method of screening a sample for a presence of one or more known compounds of interest with a mass spectrometer comprising a fragmentation, collision or reaction device and a mass analyser, said method comprising: repeatedly switching said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; determining if a candidate parent ion of interest is present in a first data set obtained when said fragmentation, collision or reaction device was operated in said first mode of operation; and determining if one or more corresponding fragment ions of interest are present in a second data set obtained when said fragmentation, collision or reaction device was operated in said second mode of operation; wherein if said candidate parent ion of interest is determined to be present in said first data set and one or more corresponding fragment ions of interest are also determined to be present in said second data set then said method further comprises determining whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that a known compound of interest is present in said sample; determining the intensity of or quantifying said known compound of interest either: (i) by summing an intensity of an isotopic distribution of said parent ion of interest; or (ii) by summing an intensity of all fragment ions which are determined as corresponding with said parent ion of interest.

17. A mass spectrometer for screening a sample for a presence of one or more known compounds of interest, said mass spectrometer comprising: a fragmentation, collision or reaction device; a mass analyser; and a control system arranged and adapted: (i) to switch repeatedly said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; (ii) to select a known compound of interest, and identify a candidate parent ion of interest associated with said known compound of interest; (iii) to determine if said candidate parent ion of interest is present in a first data set obtained when said fragmentation, collision or reaction device was operated in said first mode of operation; and (iv) to determine if one or more fragment ions of interest corresponding to said candidate parent ion of interest are present in a second data set obtained when said fragmentation, collision or reaction device was operated in said second mode of operation; wherein if said candidate parent ion of interest is determined to be present in said first data set and one or more corresponding fragment ions of interest are also determined to be present in said second data set then said control system further determines whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that said known compound of interest is present in said sample.

18. A mass spectrometer for screening a sample for a presence of one or more known compounds of interest, said mass spectrometer comprising: a fragmentation, collision or reaction device; a mass analyser; and a control system arranged and adapted: (i) to switch repeatedly said fragmentation, collision or reaction device between a first mode of operation and a second mode of operation, wherein in said first mode of operation parent ions are not substantially fragmented within said fragmentation, collision or reaction device or are arranged to bypass said fragmentation, collision or reaction device and are then subsequently mass analysed by said mass analyser and wherein in said second mode of operation parent ions are substantially fragmented within said fragmentation, collision or reaction device to form a plurality of fragment ions which are then subsequently mass analysed by said mass analyser; (ii) to select a known compound of interest and identify one or more fragment ions of interest associated with said known compound of interest; (iii) to determine if said one or more fragment ions of interest are present in a second data set obtained when second fragmentation, collision or reaction device was operated in said second mode of operation; and (iv) to determine if a candidate parent ion of interest corresponding to said one or more fragment ions of interest is present in a first data set obtained when said fragmentation device, collision or reaction was operated in said first mode of operation; wherein if said one or more fragment ions of interest are determined to be present in said second data set and said corresponding candidate parent ion of interest is also determined to be present in said first data set then said control system further determines whether or not said candidate parent ion of interest and said one or more fragment ions of interest have substantially similar elution or retention times or ion mobility drift times; wherein if said candidate parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that said known compound of interest is present in said sample.

19. A method of mass spectrometry comprising: mass analysing a sample and acquiring a parent ion data set and a fragment ion data set; identifying a compound of interest and a parent ion of interest associated with said compound of interest; and determining if said parent ion of interest is present in said parent ion data set and a fragment ion corresponding to said parent ion of interest is present in said fragment ion data set; wherein if said parent ion of interest and a corresponding fragment ion are present in said data sets then said method further comprises confirming whether or not said parent ion of interest and said corresponding fragment ion have: (i) substantially a same elution or retention time; (ii) substantially a same ion mobility drift time; or (iii) substantially a same elution or retention time and substantially a same ion mobility drift time; wherein if said parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times, then a determination is made that said compound of interest is present in said sample.

20. A mass spectrometer comprising: a fragmentation, collision or reaction device; a mass analyser; and a control system arranged and adapted: (i) to mass analyse a sample and acquire a parent ion data set and a fragment ion data set; (ii) to identify a compound of interest and a parent ion of interest associated with said compound of interest; and (iii) to determine if said parent ion of interest is present in said parent ion data set and a fragment ion corresponding to said parent ion of interest is present in said fragment ion data set; wherein if said control system determines that said parent ion of interest and a corresponding fragment ion are present in said data sets then said control system further confirms whether or not said parent ion of interest and said corresponding fragment ion have: (i) substantially a same elution or retention time; (ii) substantially a same ion mobility drift time; or (iii) substantially a same elution or retention time and substantially a same ion mobility drift time; wherein if said parent ion of interest and said one or more fragment ions of interest are determined to have substantially similar elution or retention times or ion mobility drift times then a determination is made that said compound of interest is present in said sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments of the present invention will now be described, by way of example only, together with other arrangements given for illustrative purposes only and with reference to the accompanying drawings in which:

(2) FIG. 1 is a schematic drawing of a mass spectrometer according to an embodiment of the present invention;

(3) FIG. 2 shows a schematic of a valve switching arrangement during sample loading and desalting and the inset shows desorption of a sample from an analytical column;

(4) FIG. 3A shows a fragment or daughter ion mass spectrum and FIG. 3B shows a corresponding parent or precursor ion mass spectrum when a mass filter allowed parent or precursor ions having a mass to charge ratio greater than 350 to be transmitted;

(5) FIG. 4A shows a mass chromatogram showing the time profile of various mass ranges, FIG. 4B shows a mass chromatogram showing the time profile of various mass ranges, FIG. 4C shows a mass chromatogram showing the time profile of various mass ranges, FIG. 4D shows a mass chromatogram showing the time profile of various mass ranges, and FIG. 4E shows a mass chromatogram showing the time profile of various mass ranges;

(6) FIG. 5 shows the mass chromatograms of FIGS. 4A-4E superimposed upon one another;

(7) FIG. 6 shows a mass chromatogram of 87.04 (Asparagine immonium ion);

(8) FIG. 7 shows a fragment T5 from ADH sequence ANELLINVK MW 1012.59;

(9) FIG. 8 shows a mass spectrum for a low energy spectra of a tryptic digest of β-Caesin;

(10) FIG. 9 shows a mass spectrum for a high energy spectra of a tryptic digest of β-Caesin;

(11) FIG. 10 shows a processed and expanded view of the same spectrum as in FIG. 9;

(12) FIG. 11A shows a full ion chromatogram of a sample, FIG. 11B shows a list of compounds of potential interest which may be screened for, and FIG. 11C shows an extracted parent ion chromatogram (LHS), a parent ion mass spectrum (upper RHS) and a corresponding fragment ion mass spectrum (lower RHS);

(13) FIG. 12A shows a full ion chromatogram of a sample, FIG. 12B shows a list of compounds of potential interest which may be screened for, and FIG. 12C shows an extracted parent ion chromatogram (LHS), a parent ion mass spectrum (upper RHS) and a corresponding fragment ion mass spectrum (lower RHS); and

(14) FIG. 13A shows a full ion chromatogram of a sample, FIG. 13B shows a list of compounds of potential interest which may be screened for, and FIG. 13C shows an extracted parent ion chromatogram (LHS), a parent ion mass spectrum (upper RHS) and a corresponding fragment ion mass spectrum (lower RHS).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

(15) A preferred embodiment will now be described with reference to FIG. 1. A mass spectrometer 6 is provided which preferably comprises an ion source 1 preferably an Electrospray ionization source. An ion guide 2 is preferably provided downstream of the ion source 1. A quadrupole rod set mass filter 3 is preferably provided downstream of the ion guide 2 and upstream of a collision, fragmentation or reaction device 4. According to an embodiment an orthogonal acceleration Time of Flight mass analyser 5 preferably incorporating a reflectron is preferably provided downstream of the collision, fragmentation or reaction device 4. The ion guide 2 and the mass filter 3 may be omitted if necessary. The mass spectrometer 6 is preferably interfaced with a chromatograph, such as a liquid chromatograph (not shown) so that the sample entering the ion source 1 may be taken from the eluent of the liquid chromatograph.

(16) The quadrupole rod set mass filter 3 is preferably disposed in an evacuated chamber which is preferably maintained at a relatively low pressure e.g. less than 10.sup.−5 mbar. The rod electrodes comprising the mass filter 3 are connected to a power supply which generates both RF and DC potentials which determine the range of mass to charge values that are transmitted by the mass filter 3.

(17) The collision, fragmentation or reaction device 4 preferably comprises either a Collision Induced Dissociation (“CID”) fragmentation device, a Surface Induced Dissociation (“SID”) fragmentation device, an Electron Transfer Dissociation fragmentation device or an Electron Capture Dissociation fragmentation device.

(18) According to an embodiment the collision, fragmentation or reaction device 4 may comprise an Electron Capture Dissociation fragmentation device. According to this embodiment multiply charged analyte ions are preferably caused to interact with relatively low energy electrons. The electrons preferably have energies of <1 eV or 1-2 eV. The electrons are preferably confined by a relatively strong magnetic field and are directed so that the electrons collide with the analyte ions which are preferably confined within an RF ion guide which is preferably arranged within the collision, fragmentation or reaction device 4. An AC or RF voltage is preferably applied to the electrodes of the RF ion guide so that a radial pseudo-potential well is preferably created which preferably acts to confine ions radially within the ion guide so that the ions can interact with the low energy electrons.

(19) According to another embodiment the collision, fragmentation or reaction device 4 may comprise an Electron Transfer Dissociation fragmentation device. According to this embodiment positively charged analyte ions are preferably caused to interact with negatively charged reagent ions. The negatively charged reagent ions are preferably injected into an RF ion guide or ion trap located within the fragmentation device 4. An AC or RF voltage is preferably applied to the electrodes of the RF ion guide so that a radial pseudo-potential well is preferably created which preferably acts to confine ions radially within the ion guide so that the ions can interact with the negatively charged reagent ions. According to a less preferred embodiment negatively charged analyte ions may alternatively be arranged to interact with positively charged reagent ions.

(20) According to another embodiment the collision, fragmentation or reaction device 4 may comprise a Surface Induced Dissociation fragmentation device. According to this embodiment ions are preferably directed towards a surface or target plate with a relatively low energy. The ions may, for example, be arranged to have an energy of 1-10 eV. The surface or target plate may comprise stainless steel or more preferably the surface or target plate may comprise a metallic plate coated with a monolayer of fluorocarbon or hydrocarbon. The monolayer preferably comprises a self-assembled monolayer. The surface or target plate may be arranged in a plane which is substantially parallel with the direction of travel of ions through the Surface Induced Dissociation fragmentation device in a mode of operation wherein ions are not fragmented. In a mode of operation wherein it is desired to fragment ions, the ions may be deflected onto or towards the surface or target plate so that the ions impinge the surface or target plate at a relatively shallow angle with respect to the surface of target plate. Fragment ions are preferably produced as a result of the analyte ions colliding with the surface or target plate. The fragment ions are preferably directed off or away from the surface or target plate at a relatively shallow angle with respect to the surface or target plate. The fragment ions are then preferably arranged to assume a trajectory which preferably corresponds with the trajectory of ions which are transmitted through or past the Surface Induced Dissociation fragmentation device in a mode of operation wherein ions are not substantially fragmented.

(21) The collision, fragmentation or reaction device 4 may comprise an Electron Collision or Impact Dissociation fragmentation device wherein ions are fragmented upon collisions with relatively energetic electrons e.g. wherein the electrons have >5 eV.

(22) According to other embodiments the collision, fragmentation or reaction device 4 may comprise a Photo Induced Dissociation (“PID”) fragmentation device, a Laser Induced Dissociation fragmentation device, an infrared radiation induced dissociation device, an ultraviolet radiation induced dissociation device, a thermal or temperature source fragmentation device, an electric field induced fragmentation device, a magnetic field induced fragmentation device, an enzyme digestion or enzyme degradation fragmentation device, an ion-ion reaction fragmentation device, an ion-molecule reaction fragmentation device, an ion-atom reaction fragmentation device, an ion-metastable ion reaction fragmentation device, an ion-metastable molecule reaction fragmentation device, an ion-metastable atom reaction fragmentation device, an ion-ion reaction device for reacting ions to form adduct or product ions, an ion-molecule reaction device for reacting ions to form adduct or product ions, an ion-atom reaction device for reacting ions to form adduct or product ions, an ion-metastable ion reaction device for reacting ions to form adduct or product ions, an ion-metastable molecule reaction device for reacting ions to form adduct or product ions or an ion-metastable atom reaction device for reacting ions to form adduct or product ions.

(23) According to another embodiment the collision, fragmentation or reaction device may form part of the ion source 1. For example, the collision, fragmentation or reaction device may comprise a nozzle-skimmer interface fragmentation device, an in-source fragmentation device or an ion-source Collision Induced Dissociation fragmentation device.

(24) The collision, fragmentation or reaction device 4 may comprise a quadrupole or hexapole rod set ion guide in order to confine ions. The ion guide may be enclosed in a substantially gas-tight casing (other than a small ion entrance and exit orifice) into which a gas such as helium, argon, nitrogen, air or methane may be introduced at a pressure of between 10.sup.−4 and 10.sup.−1 mbar, preferably 10.sup.−3 mbar to 10.sup.−2 mbar. Suitable RF potentials for the electrodes comprising the collision, fragmentation or reaction device 4 may be provided by a power supply (not shown).

(25) Ions generated by the ion source 1 are preferably transmitted by the ion guide 2 and pass via an interchamber orifice 7 into a vacuum chamber 8 housing the mass filter 3 and the collision, fragmentation or reaction device 4. The ion guide 2 is preferably maintained at a pressure intermediate to that of the ion source 1 and the vacuum chamber 8. In the embodiment shown, ions may be mass filtered by the mass filter 3 before entering the collision, fragmentation or reaction device 4. However, mass filtering is not essential to the present invention. In a mode of operation ions are preferably fragmented or reacted within the collision, fragmentation or reaction device 4 so that a plurality of fragment, product, daughter or adduct ions are preferably produced. Fragment, product, daughter or adduct ions exiting the collision, fragmentation or reaction device 4 preferably pass into the Time of Flight mass analyser 5 arranged downstream of the collision, fragmentation or reaction device 4. Other ion optical components, such as further ion guides and/or electrostatic lenses, may be present (which are not shown in the figures or described herein) in order to maximise ion transmission between various parts or stages of the mass spectrometer. Various vacuum pumps (not shown) may be provided for maintaining optimal vacuum conditions in the mass spectrometer. The Time of Flight mass analyser 5 incorporating a reflectron preferably operates in a known way by measuring the transit time or time of flight of ions. Ions are preferably injected as a packet of ions into the drift or time of flight region of the mass analyzer 5. The ions become temporally separated and their mass to charge ratios can be determined by measuring the transit time or time of flight of ions through the drift or time of flight region.

(26) A control system (not shown) preferably provides control signals for the various power supplies (not shown) which respectively provide the necessary operating potentials for the ion source 1, ion guide 2, quadrupole mass filter 3, collision, fragmentation or reaction device 4 and the Time of Flight mass analyser 5. These control signals preferably determine the operating parameters of the instrument, for example the mass to charge ratios transmitted through the mass filter 3 and the operation of the mass analyser 5. The control system is preferably controlled by signals from a computer (not shown) which may also be used to process the mass spectral data acquired. The computer may also display and store mass spectra produced from the analyser 5 and receive and process commands from an operator. The control system may be set to perform various methods automatically and make various determinations without operator intervention, or may optionally require operator input at various stages.

(27) The control system is preferably arranged to switch, vary or alter the collision, fragmentation or reaction device 4 back and forth between at least two different modes. If the collision, fragmentation or reaction device 4 comprises an Electron Capture Dissociation fragmentation device then the electron source or beam may be switched ON in a fragmentation mode of operation and may be switched OFF in a non-fragmentation mode of operation (or may be left ON or switched OFF in a non-fragmentation mode in which ions are directed to bypass the device, so the ions are not fragmented.) If the collision, fragmentation or reaction device 4 comprises an Electron Transfer Dissociation fragmentation device 4 then reagent ions may be injected into an ion guide or ion trap comprising analyte ions in a fragmentation mode of operation and substantially no reagent ions may be injected into the ion guide or ion trap in a non-fragmentation mode of operation. If the collision, fragmentation or reaction device 4 comprises a Surface Induced Dissociation fragmentation device then the analyte ions may be directed so that they collide or impinge upon the surface or target plate in a fragmentation mode of operation and the analyte ions may be directed straight past the surface or target plate in a non-fragmentation mode of operation so that the analyte ions do not collide or impinge upon the surface of target plate.

(28) The control system preferably switches the collision, fragmentation or reaction device 4 between modes according to an embodiment approximately once every second. When the mass spectrometer is used in conjunction with an ion source being provided with an eluent separated from a mixture by means of liquid or gas chromatography, the mass spectrometer 6 may be run for several tens of minutes over which period of time several hundred high fragmentation or reaction mass spectra and several hundred low fragmentation or reaction mass spectra may be obtained.

(29) At the end of the experimental run the data which has been obtained is preferably analysed. According to an arrangement parent or precursor ions and fragment, product, daughter or adduct ions may be recognised on the basis of the relative intensity of a peak in a mass spectrum obtained when the collision, fragmentation or reaction device 4 was in one mode compared with the intensity of the same peak in a mass spectrum obtained approximately a second later in time when the collision, fragmentation or reaction device 4 was in another mode.

(30) Mass chromatograms for each parent and fragment, product, daughter or adduct ion may be generated and fragment, product, daughter or adduct ions may be assigned to parent or precursor ions on the basis of their relative elution times.

(31) Since all the data is acquired and subsequently processed then all fragment, product, daughter or adduct ions may be associated with a parent or precursor ion by closeness of fit of their respective elution times. This allows all the parent or precursor ions to be identified from their fragment, product, daughter or adduct ions irrespective of whether or not they have been discovered by the presence of a characteristic fragment, product, daughter or adduct ion or characteristic “neutral loss”.

(32) An attempt may be made to reduce the number of parent or precursor ions of interest. A list of possible (i.e. not yet finalised) candidate parent or precursor ions may be formed by looking for parent or precursor ions which may have given rise to a predetermined fragment, product, daughter or adduct ion of interest e.g. an immonium ion from a peptide. Alternatively, a search may be made for parent and fragment, product, daughter or adduct ions wherein the parent or precursor ion could have fragmented into a first component comprising a predetermined ion or neutral particle and a second component comprising a fragment, product, daughter or adduct ion. Various steps may then be taken to further reduce/refine the list of possible candidate parent or precursor ions to leave a number of final candidate parent or precursor ions which are then subsequently identified by comparing elution times of the parent and fragment, product, daughter or adduct ions. As will be appreciated, two ions could have similar mass to charge ratios but different chemical structures and hence would most likely fragment differently enabling a parent or precursor ion to be identified on the basis of a fragment, product, daughter or adduct ion.

(33) As noted above, some embodiments provide substantial non-fragmentation during alternate, interleaved, periods by switching the operation of a fragmentation device and/or by bypassing a fragmentation device. Alternating fragmentation or reaction of a sample is accomplished in any suitable manner. For example, sample molecules and/or ions may optionally be admitted to a device within which a sample stream is alternately fragmented and not fragmented. Alternatively, a sample stream may be admitted to and bypass a device, to again provide fragmented or reacted material from the device and not fragmented material that bypasses the device. Thus, “pseudo-chromatograms” are obtainable for product ions and such chromatograms will be well, if not perfectly, aligned in time with the chromatograms for precursor ions, since the mass spectra for precursor and product ions are collected essentially simultaneously due to the interleaved periods of fragmentation and non-fragmentation of the sample stream.

(34) As described next, some embodiments entail bypassing of a fragmentation device. Thus, for example, a sample having a temporal variation may optionally be analysed by alternately passing sample material to a fragmentation device and bypassing the device to alternately fragment and not fragment.

(35) In the following, for convenience, a “fragmentation device” refers generally to any suitable fragmentation device, reaction device, collision device, or a device suitable for converting precursor ions into product ions or some other form for a desired analysis.

(36) A method for analyzing a sample comprising a mixture of components is disclosed comprising: forming precursor ions from the components of a sample; alternately causing precursor ions to pass to and to bypass a fragmentation device, to form product ions from at least some of the precursor ions that pass to the device, and to form substantially fewer product ions from precursor ions that bypass the device; and alternately obtaining mass spectra from product ions received from the fragmentation device, and from precursor ions that bypassed the fragmentation device.

(37) Causing precursor ions to pass to and to bypass the fragmentation device optionally comprises alternating for a few minutes to several tens of minutes to an hour or more. The collection of data, in this manner, optionally is determined by the amount of time required to deliver one sample to a mass spectrometer, for example, from a chromatographic module.

(38) During the analysis of one sample, any where from tens to hundreds to thousands of mass spectra may be collected. The duration, or period, of each alternation between fragmentation and non-fragmentation is optionally a fraction of a second to about one second to about several seconds. One duration preferably includes at least one mass spectrum, preferably more, such as about 10 spectra. Preferably, the alternation cycle is sufficiently fine to resolve temporal changes of interest in the sample. For example, where the sample is delivered from a chromatograph, alternating in a time span approximately equals one tenth of a chromatographic peak width.

(39) Particularly preferred embodiments of the present invention will now be described with reference to FIGS. 11-13. These embodiments preferably relate to MRM-type analyses performed using a Time of Flight mass analyser.

(40) According to the preferred embodiment the above described apparatus and methods can be extended to support Single Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) type analyses on a Time of Flight mass analyser rather than as conventionally performed on a triple-quadrupole instrument.

(41) As is well known in the art of mass spectrometry, SRM and MRM studies are commonly used in mass spectrometric quantitation and/or monitoring of preselected or known compounds through use of apparatus that includes at least two quadrupoles operated as mass filters/analysers. The first quadrupole is used to select precursor or parent ions having a mass to charge ratio associated with the compound of interest. These precursor or parent ions are then fragmented, and the second quadruple is used to select product or fragment ions having a particular mass to charge which have been fragmented from the filtered precursor or parent ions. The dual selection/filtering process provides confident observation of the compound of interest.

(42) In a typical SRM analysis, as performed on a tandem quadrupole instrument such as a triple quadrupole instrument, the presence of a specific compound of interest is monitored over time. SRM plots of relative intensity versus time are usually relatively simple and usually contain only a single peak. This characteristic makes a typical SRM plot useful for sensitive and specific quantitation.

(43) A SRM experiment may be accomplished by specifying the precursor or parent mass or mass to charge ratio of the compound for MS/MS fragmentation and then monitoring for a specific single fragment ion. The specific experiment is typically known as a “transition” and may be represented in the form parent mass.fwdarw.fragment mass (e.g. 534.fwdarw.375).

(44) It is therefore possible to quantitatively observe the presence of a specific known component of interest in a sample stream. This conventional approach is limited, however, by the requirement to select a compound of interest prior to an analysis and then collect data only for that compound. Accordingly, potentially useful data may be wasted.

(45) According to embodiments of the present invention the limitations of conventional SRM/MRM and similar analyses may be mitigated by time alignment and full data sets as described above.

(46) Embodiments of the present invention preferably provide various advantages in comparison to prior tandem quadrupole based approaches. In particular, according to the preferred embodiment several compounds of interest can be monitored simultaneously, since no selection of a single precursor ion from a sample stream is required during analysis. Furthermore, more than one product or fragment ion can be monitored in association with a precursor or parent ion of interest because no selection of a single product or fragment ion is required during analysis (i.e. data is obtained substantially for all precursor or parent ions and for all product or fragment ions.)

(47) FIGS. 11-13 are screenshots of displays of example analyses of two compounds of interest present in a single sample where all data was collected during the analysis of one temporally (chromatographically) varying sample. An Electrospray ion source was used for precursor ion production and the data was collected for a sample run that lasted for approximately 13 minutes, as illustrated by the full chromatograms shown in FIGS. 11A, 12A and 13A.

(48) FIGS. 11B, 12B and 13B list all compounds of potential interest which may be screened for. The left hand figure of FIGS. 11C, 12C and 13C is an extracted parent ion chromatogram for a precursor or parent ion associated with a selected compound of interest. The two right-hand figures of FIGS. 11C, 12C and 13C are two mass spectra. The upper mass spectrum corresponds with a precursor ion associated with a particular compound and the lower mass spectrum corresponds with a product ion associated with the precursor ion. Accordingly, the upper mass spectrum comprises a “low-fragmentation” mass spectrum and the lower mass spectrum comprises a “high-fragmentation” mass spectrum collected at a time very close to the time when the upper mass spectrum was obtained.

(49) The analysis system preferably includes a database of precursor or parent ions and their expected mass to charge ratio values and one or more (preferably four) product or fragment ions preferably associated with each precursor ion and the expected mass to charge ratio values of the product or fragment ions.

(50) Extracted parent ion chromatograms are preferably produced by extracting intensity data for a particular selected precursor or parent ion whose association with a compound of interest has been confirmed through time alignment confirmation with one or more product ions associated with the selected precursor. The intensity as a function of time of the confirmed precursor or parent ion is obtained from the series of mass spectra that were collected as the sample was analysed over time. The extracted data may then be depicted as a graph of intensity or relative intensity versus time as shown in the extracted parent ion chromatograms shown in FIGS. 11C, 12C and 13C.

(51) Extracted ion chromatograms for precursor ions and/or product ions may be produced. Also, a summed chromatogram may be produced if more than one precursor-type is available and utilized, or if multiple isotopes are obtained for a particular precursor.

(52) As illustrated in FIG. 11B, after data collection from a sample a determination was made to monitor and quantify the presence of Imidacloprid® in the sample stream. Imidaclopriad® is a moderately toxic insecticide manufactured by Bayer Cropscience (Bayer AG)®.

(53) Parent or precursor ions having an expected mass to charge ratio of 256.0601 and product or fragment ions having an expected transition mass to charge ratio of 209.0594 were selected for display. In this example, based on empirical data, the known compound was expected to have a chromatographic elution time (i.e. retention time) of 2.72 minutes. Correct association between the precursor or parent ions and observed product or fragment ion(s) was confirmed by time-aligned association. That is, both precursor or parent and product or fragment ions were confirmed as having expected mass to charge ratio values within an error tolerance and also having substantially the same retention time (within an error tolerance.)

(54) Availability of an expected retention time is not essential but advantageously permits windowing or filtering of the data. Optionally, without any knowledge of expected retention time(s), all data may be surveyed for precursor or parent ions having an expected mass to charge ratio for a selected compound of interest. The association of potential precursor or parent ions with the compound may then be confirmed by confirming that one or more product or fragment ions are also present in association with the same retention time.

(55) FIGS. 12A-C and 13A-C illustrate an analysis wherein Flufenacet® which is an herbicide available from Bayer Corporation® was selected after data collection for monitoring in the sample data. Precursor or parent ions having an expected mass to charge ratio of 364.0743 and product or fragment ions having an expected transition mass to charge ratio of 194.0981 were expected in association with Flufenacet®. As indicated in the list, based on empirical data, the compound was expected to have a chromatographic elution time (i.e. retention time) of 7.02 minutes.

(56) Upon examining the data for candidate precursor or parent ions, two candidate precursor or parent ions having potentially correct mass to charge ratio values appeared. These two candidate precursor or parent ions appear in the list labeled as scan 961 and scan 982. The two candidate precursor or parent ions had intensity maxima associated with different scan times, each scan time corresponding to a particular retention time. Confirmation of the correct candidate precursor or parent ions as being the correct precursor or parent of Flufenacet® was then obtained by confirming which of the candidate parent ions was time-aligned i.e. shared a retention time association with one or more of the expected product ions.

(57) As illustrated in FIGS. 12B and 12C, the 961 scan was confirmed to contain the correct precursor or parent ion for Flufenacet® because, as depicted in the upper parent ion mass spectrum and the lower fragment ion mass spectrum of FIG. 12C, the parent ion scans at and near 961 contained the expected precursor or parent ion and the fragment ion scan at 960 contained the expected product ions. The extracted parent ion chromatogram was generated by seeking and confirming precursor or parent ions at all times to then graph the intensity of the precursor ion as a function of chromatographic elution time.

(58) FIGS. 13A-13C depicts precursor or parent ion and product or fragment ion mass spectra for the second candidate parent ion. As can be seen from the lower product or fragment ion mass spectrum (corresponding to or near to scan 982) as shown in FIG. 13C, the expected product or fragment ion having a mass to charge ratio of 194.0981 is absent. Therefore, the second candidate precursor or parent ion is deemed not to be associated with the compound of interest i.e. Flufenacet®.

(59) The correct association between an observed candidate precursor or parent ion and a compound of interest is then confirmed by confirmation of time-alignment between the candidate precursor or parent ion and observed product or fragment ion(s) having expected mass to charge ratio values. With regard to the two candidate parent ions being detected and indicated in the list, the association of one with Flufenacet® was confirmed as indicated in the list with a superscript “e” in FIG. 13B.

(60) Referring to a precursor or parent ion as mass M1 which is fragmented to produce a product or fragment ion mass M2, embodiments of the invention preferably provide an intensity versus time plot of ion M2 (or M1 or both M1 and M2) for quantitative experiments.

(61) For example, the acquired data (or peak list) may be processed in a chronological manner and then the intensity of M2 may be recorded in a new function only if certain conditions are met. The conditions can, for example, indicate that both mass M2 and mass M1 must be present in the high and low energy acquisitions respectively, and furthermore that their retention times must also agree to within normal or user defined limits. As a further optional condition, their masses or mass to charge ratios may be required to be within a specific tolerance and optionally their isotope clusters, ion mobility and chromatographic peak shapes may also be required to meet user defined criteria. Furthermore, the ratio of the intensity of the parent ion to the intensity of our or more corresponding fragment ions may be required to fall within a predetermined range in order for a positive confirmation to be made that a known compound is present in the sample. Optionally, thresholds for M1 and M2 may also be used to avoid selection of noise.

(62) If M2 and M1 meet these conditions then the resultant output is the intensity of M2. However, if M1 and M2 do not met the conditions then the resultant output is preferably zero.

(63) The preferred embodiment may, therefore, be considered as involving the conditional logical operation AND of high-fragmentation and low-fragmentation data. The output of the process may be considered as being the product of this logical AND operation and the intensity of M2.

(64) Further embodiments are contemplated wherein, for example, an entire M2 (fragment ion) isotopic cluster may be utilized. For example, multiple isotopes or other related fragment ions (M2, M3, M4 . . . ) in the high energy function may be summed and may be used in place of the intensity of M2.

(65) The output data according to preferred embodiment preferably emulates MRM but differs from conventional tandem quadrupole MRM in that no parent ion isolation is preferably either performed or required. In order to improve specificity/selectivity, exact mass and/or conditional isotope ratios may be used.

(66) The output data may be used for quantification and/or for exact mass screening. According to an embodiment one exact mass (e.g. M1) at high resolution and a related mass (M2) may be used to improve the accuracy in comparison to prior approaches.

(67) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.