(19) TZZ Z__T

(11) EP 2 667 401 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 27.11.2013 Bulletin 2013/48 H01J 49/00 (2006.01) H01J 49/16 (2006.01) H01J 49/10 (2006.01) H01J 49/12 (2006.01) (21) Application number: 13181095.4

(22) Date of filing: 27.07.2009

(84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • Brown, Jeffery Mark HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Hyde, Cheshire SK14 6LT (GB) PT RO SE SI SK SM TR • Green, Martin Raymond Designated Extension States: Bowdon, Cheshire WA14 3EE (GB) AL BA RS • Pringle, Steven Derek Darwen, BB3 3PS (GB) (30) Priority: 28.07.2008 GB 0813777 05.08.2008 US 86213 P (74) Representative: Jeffrey, Philip Michael Dehns (62) Document number(s) of the earlier application(s) in St Bride’s House accordance with Art. 76 EPC: 10 Salisbury Square 09784792.5 / 2 308 076 London EC4Y 8JD (GB) (71) Applicant: Micromass UK Limited Wythenshawe Remarks: Manchester M23 9LZ (GB) This application was filed on 20-08-2013 as a divisional application to the application mentioned under INID code 62.

(54) Glow discharge ion source

(57) A mass spectrometer is disclosed comprising a glow discharge. The resulting reagent ions may be used glow discharge device within the initial vacuum chamber for Electron Transfer Dissociation of analyte ions gener- of the mass spectrometer. The glow discharge device ated by an atmospheric pressure ion source. Other em- may comprise a tubular electrode 14 located within an bodiments are contemplated wherein the ions generated isolation valve 15 which is provided in the vacuum cham- by the glow discharge device may be used to reduce the ber.Reagent vapour may beprovided throughthe tubular charge state of analyte ions by Proton Transfer Reaction electrode 14 which is then subsequently ionised by the or may act as lock mass or reference ions. EP 2 667 401 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 667 401 A1 2

Description is necessary simultaneously to confine both positive ions and electrons at near thermal kinetic energies. Electron [0001] The present invention relates to an ion source, Capture Dissociation has been demonstrated using Fou- a mass spectrometer, a method of generating ions and rier Transform Ion Cyclotron Resonance ("FT-ICR") a method of mass spectrometry. 5 mass analysers which use a superconducting magnet [0002] Electrospray ionisation ion sources are well to generate large magnetic fields. However, such mass known and may be used to convert neutral peptides elut- spectrometers are very large and are prohibitively ex- ing from an HPLC column into gas-phase analyte ions. pensive for the majority of mass spectrometry users. In an aqueous acidic solution, tryptic peptides will be ion- [0007] As an alternative to Electron Capture Dissoci- ised on both the amino terminus and the side chain of 10 ation it has been demonstrated that it is possible to frag- the C-terminal amino acid. As the peptide ions proceed mentpeptide ions by reacting negatively charged reagent to enter a mass spectrometer the positively charged ami- ions with multiply charged analyte cations in a linear ion no groups hydrogen bond and transfer protons to the trap. The process of reacting positively charged analyte amide groups along the backbone of the peptide. ions with negatively charged reagent ions has been re- [0003] It is known to fragment peptide ions by increas- 15 ferred to as Electron Transfer Dissociation ("ETD"). Elec- ing the internal energy of the peptide ions through colli- tron Transfer Dissociation is a mechanism wherein elec- sions with a collision gas. The internal energy of the pep- trons are transferred from negatively charged reagent tide ions is increased until the internal energy exceeds ions to positively charged analyte ions. After electron the activation energy necessary to cleave the amide link- transfer, the charge-reduced peptide or analyte ion dis- ages along the backbone of the molecule. This process 20 sociates through the same mechanisms which are be- of fragmenting ions by collisions with a neutral collision lieved to be responsible for fragmentation by Electron gas is commonly referred to as Collision Induced Disso- Capture Dissociation i.e. it is believed that Electron ciation ("CID"). The fragment ions which result from Col- Transfer Dissociation cleaves the amine bond in a similar lision Induced Dissociation are commonly referred to as manner to Electron Capture Dissociation. As a result, the b-type and y-type fragment or product ions, wherein b- 25 product or fragment ions which are produced by Electron type fragment ions contain the amino terminus plus one Transfer Dissociation of peptide analyte ions comprise or more amino acid residues and y-type fragment ions mostly c-type and z-type fragment or product ions. contain the carboxyl terminus plus one or more amino [0008] One particular advantage of Electron Transfer acid residues. Dissociation is that such a process is particularly suited [0004] Other methods of fragmenting peptides are30 for the identification of post-translational modifications known. An alternative method of fragmenting peptide ("PTMs") since weakly bonded PTMs like phosphoryla- ions is to interact the peptide ions with thermal electrons tionor glycosylation willsurvive the electroninduced frag- by a process known as Electron Capture Dissociation mentation of the backbone of the amino acid chain. ("ECD"). Electron Capture Dissociation cleaves the pep- [0009] It is known to perform Electron Transfer Disso- tide in a substantially different manner to the fragmenta- 35 ciation by mutually confining cations and anions in a 2D tion process which is observed with Collision Induced linear ion trap which is arranged to promote ion-ion re- Dissociation. In particular, Electron Capture Dissociation actions between reagent anions and analyte cations. The cleaves the backbone N- Cα bond or the amine bond and cations and anions are simultaneously trapped within the the resulting fragment ions which are produced are com- 2D linear ion trap by applying an auxiliary axially confining monly referred to as c- type and z-type fragment or prod- 40 RF pseudo-potential barrier at both ends of the 2D linear uct ions. Electron Capture Dissociation is believed to be quadrupole ion trap. non-ergodic i.e. cleavage occurs before the transferred [0010] Another method of performing Electron Trans- energy is distributed over the entire molecule. Electron fer Dissociation is known wherein a fixed DC axial po- Capture Dissociation also occurs with a lesser depend- tential is applied at both ends of a 2D linear quadrupole ence on the nature of the neighbouring amino acid and 45 ion trap in order to confine ions having a certain polarity only the N-side of proline is 100% resistive to Electron (e.g. reagent anions) within the ion trap. Ions having an Capture Dissociation cleavage. opposite polarity (e.g. analyte cations) to those confined [0005] One advantage of fragmenting peptide ions by within the ion trap are then directed into the ion trap. The Electron Capture Dissociation rather than by Collision analyte cations will react with the reagent anions already Induced Dissociation is that Collision Induced Dissocia- 50 confined within the ion trap. tion suffers from a propensity to cleave Post Translational [0011] It is known that when multiply charged (analyte) Modifications ("PTMs") making it difficult to identify the cations are mixed with (reagent) anions then loosely site of modification. By contrast, fragmenting peptide ions bound electrons may be transferred from the (reagent) by Electron Capture Dissociation tends to preserve Post anions to the multiply charged (analyte) cations. Energy Translational Modifications arising from, for example, 55 is released into the multiply charged cations and the mul- phosphorylation and glycosylation. tiply charged cations may be caused to dissociate. How- [0006] However, the technique of Electron Capture ever, some of the (analyte) cations may not dissociate Dissociation suffers from the significant problem that it but may instead be reduced in charge state. The cations

2 3 EP 2 667 401 A1 4 may be reduced in charge by one of two processes. First- wherein the atmospheric pressure ion source may be ly, the cations may be reduced in charge by Electron substituted with a sub-atmospheric pressure ion source Transfer ("ET") of electrons from the anions to the cati- such as a sub-atmospheric pressure Electrospray ioni- ons. Secondly, the cations may be reduced in charge by sation ion source. Proton Transfer ("PT") of protons from the cations to the 5 [0017] It will be understood by those skilled in the art anions. Irrespective of the process, an abundance of that corona discharge devices and arc or spark discharge charged reduced product ions are observed within mass devices should not be construed as comprising "a glow spectra and give an indication of the degree of ion-ion discharge device" within the meaning of the present ap- reactions (either ET or PT) that are occurring. plication. Typical operating parameters for glow dis- [0012] In bottom-up or top-down proteomics Electron 10 charge operation are that the glow discharge device is Transfer Dissociation experiments may be performed in operated at sub-atmospheric pressure preferably in the order to maximize the information available by maximiz- range 0.01-15 mbar. The voltage applied to the pin or ing the abundance of dissociated product ions within electrode of the glow discharge device once a glow dis- mass spectra. The degree of Electron Transfer Dissoci- charge has been initiated is preferably in the range ation fragmentation depends upon the conformation of 15 100-2000 V and the applied current is preferably in the the cations (and anions) together with many other instru- range 0.1 mA - 100 mA. By contrast, corona discharge mental factors. It can be difficult to know a priori the op- devices are typically operated at atmospheric (or near timal parameters for every anion-cation combination atmospheric) pressure i.e. at pressures > 900 mbar. For from an LC run. a corona discharge device the applied current is typically [0013] A problem with known mass spectrometers20 in the range 5-15 mA and the applied voltage once a which incorporate an Electron Transfer Dissociation frag- corona discharge has been established is typically in the mentation cell is that the reagent anions which are used range 3000-7000 V. to cause analyte cations to fragment are generated by [0018] A corona discharge may be considered to com- an additional ion source (e.g. an Atmospheric Pressure prise a gas discharge where the geometry confines the Chemical lonisation ion source) which is arranged along- 25 gas ionising processes to high-field ionisation region(s) sidean ion source (e.g. an Electrospray ion source) which around the active electrode(s). As all discharge forms is used to ionise the sample of interest. Locating two ion have ionisation regions, the distinguishing feature of a sources in close proximity to the inlet to the mass spec- corona discharge may be considered to be the existence trometer is problematic. Furthermore, the known ar- of a low field drift region connecting the ionisation region rangement can suffer from the problem of cross-talk. A 30 (s) with the eventual low field passive electrodes. In this yet further problem with the known arrangement is that driftregion ions andelectrons drift and react with neutrals, the reagents which are produced from the Atmospheric but with too low energy to ionise and too low density to Pressure Chemical lonisation ion source may be carci- react with other ionised particles. In unipolar conduction nogenic. Another problem with the known arrangement coronas the drifting ions/electrons will be of the corona is that the sensitivity of the Atmospheric Pressure Chem- 35 polarity (i.e. no plasma) and their space charge field will ical lonisation ion source for generating the reagent ions be the dominating factor in determining both the corona is relatively low. current/voltage characteristic and the current density dis- [0014] It is therefore desired to provide an improved tribution in the discharge gap. mass spectrometer and an improved method of gener- [0019] In a low- density, low-temperature glow (or plas- ating reagent ions for use in Electron Transfer Dissocia- 40 ma) discharge (such as that used according to the pre- tion. ferred embodiment) the voltage applied to the plasma is [0015] According to an aspect of the present invention greater than the ionisation potential of the gas used. Most there is provided a mass spectrometer comprising: of the plasma voltage drop is near the cathode where the majority of ionisation occurs. The discharge is sustained a vacuum chamber; 45 by secondary electrons emitted when ions or recombi- an atmospheric pressure ion source for generating nation radiation impact on the cathode. Electrons are ac- first ions, wherein first ions generated by the atmos- celerated away from the cathode and ionise neutral gas pheric pressure ion source are transmitted, in use, in the discharge. into the vacuum chamber via a sampling cone or first [0020] The glow discharge device preferably compris- aperture; and 50 es an electrode or pin and the mass spectrometer pref- a glow discharge device for generating second ions, erably further comprises a voltage device for supplying wherein second ions generated by the glow dis- or applying a DC and/or RF voltage to the electrode or charge device are either generated within the vacu- pin in order to cause or generate a glow discharge. um chamber or are transmitted into the vacuum [0021] The mass spectrometer preferably further com- chamber without being transmitted through the sam- 55 prises one or more dispensing devices for dispensing pling cone or first aperture. one or more reagents in proximity to the glow discharge device so that the one or more reagents are ionised, in [0016] Less preferred embodiments are contemplated use, by a glow discharge caused or generated by the

3 5 EP 2 667 401 A1 6 glow discharge device. agents and/or one or more lock mass reagents through [0022] The one or more reagents preferably comprise the tube. one or more Electron Transfer Dissociation reagents [0031] The glow discharge device is preferably oper- and/or one or more Proton Transfer Reaction reagents ated in a continuous or pulsed manner. According to a and/or one or more lock mass or calibration reagents. 5 particularly preferred embodiment the glow discharge [0023] The glow discharge device may according to a device is operated in a pulsed manner alternately with less preferred embodiment be housed in a housing ad- the analyte ion source (which preferably comprises either jacent the vacuum chamber and second ions generated an atmospheric pressure ion source such as an Electro- by the glow discharge device may pass from the housing spray ion source or a sub-atmospheric pressure ion through a second aperture into the vacuum chamber. 10 sourcesuch as a sub- atmospheric pressure Electrospray [0024] The mass spectrometer may comprise either a ion source). solid, powdered, partially solid or gel substance, a volatile [0032] According to an embodiment the glow dis- liquid or a gas which is arranged or supplied in proximity charge device is maintained or operated in use in a mode to the glow discharge device so that ions are sputtered, of operation at a potential selected from the group con- extracted or otherwise released from the substance, liq- 15 sisting of: (i) < - 1 kV; (ii) -900 to -800 V; (iii) -800 to -700 uid or gas. According to an embodiment the substance V; (iv) -700 to -600 V; (v) -600 to -500 V; (vi) -500 to -400 may comprise caesium iodide which when subjected to V; (vii) -400 to -300 V; (viii) -300 to -200 V; (ix) -200 to a glow discharge results in the release of caesium lock -100 V; (x) -100 to 0 V; (xi) 0 to 100 V; (xii) 100 to 200 V; mass ions. Fluoranthene reagent vapour may be ionised (xiii) 200 to 300 V; (xiv) 300 to 400 V; (xv) 400 to 500 V; by the preferred glow discharge device to provide refer- 20 (xvi) 500 to 600 V; (xvii) 600 to 700 V; (xviii) 700 to 800 ence ions according to an embodiment of the present V; (xix) 800 to 900 V; (xx) 900 to 1000 V; and (xxi) > 1 kV. invention. [0033] According to an embodiment the glow dis- [0025] The mass spectrometer preferably further com- charge device is operated, in use, at a pressure selected prisesa supplydevice for supplying one or more reagents from the group consisting of (i) > 0.001 mbar; (ii) > 0.01 and/or one or more Proton Transfer Reaction reagents 25 mbar; (iii) > 0.1 mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > and/or one or more Electron Transfer Dissociation rea- 100 mbar; (vii) < 0.001 mbar; (viii) < 0.01 mbar; (ix) < 0.1 gents and/or one or more lock mass reagents in proximity mbar; (x) < 1 mbar; (xi) < 10 mbar; (xii) < 100 mbar; (xiii) to the glow discharge device. 0.001-0.01 mbar; (xiv) 0.01-0.1 mbar; (xiv) 0.1-1 mbar; [0026] The mass spectrometer preferably further com- (xv) 1-10 mbar; and (xvi) 10-100 mbar. According to the prises an isolation valve arranged in the vacuum cham- 30 preferred embodimentthe glow dischargedevice is main- ber. The isolation valve is preferably arranged down- tained at a pressure in the range 0.01-20 mbar. stream of the sampling cone or first aperture. [0034] The mass spectrometer preferably further com- [0027] According to a less preferred embodiment the prises an Electron Transfer Dissociation fragmentation glow discharge device may be located upstream of the cell arranged in a second vacuum chamber, wherein the isolation valve. According to a particularly preferred em- 35 second vacuum chamber is located downstream of the bodiment the glow discharge device may be located with- vacuumchamber and wherein,in use, at least some Elec- in the isolation valve or downstream of the isolation valve. tron Transfer Dissociation reagent ions generated by the Other embodiments are contemplated wherein the glow glow discharge device are caused to interact with at least discharge device may be located in a sub-atmospheric some analyte ions within the Electron Transfer Dissoci- pressure region within a nozzle- skimmer interface of the 40 ation fragmentation cell so as to cause at least some of mass spectrometer. the analyte ions to fragment by Electron Transfer Disso- [0028] The isolation valve preferably comprises a first ciation. rotatable port wherein when the isolation valve is rotated [0035] According to another aspect of the present in- to a first position then the vacuum chamber downstream vention there is provided a method of mass spectrometry of the isolation valve is in fluid communication with the 45 comprising: sampling cone or first aperture and when the isolation valve is rotated to a second position then the vacuum providing a vacuum chamber; chamber downstream of the isolation valve is no longer providing an atmospheric pressure ion source for in fluid communication with the sampling cone or first generating first ions; aperture. The isolation valve preferably enables a vacu- 50 generating first ions by the atmospheric pressure ion um to be maintained within the mass spectrometer when source and transmitting the first ions into the vacuum not in use. chamber via a sampling cone or first aperture; [0029] The glow discharge device preferably compris- providing a glow discharge device for generating es a tube having a sharpened or pointed end. second ions; and [0030] The mass spectrometer preferably further com- 55 generating second ions by the glow discharge de- prisesa supplydevice for supplying one or more reagents vice, wherein the second ions are generated either and/or one or more Electron Transfer Dissociation rea- within the vacuum chamber or are transmitted into gents and/or one or more Proton Transfer Reaction re- the vacuum chamber without being transmitted

4 7 EP 2 667 401 A1 8

through the sampling cone or first aperture. an atmospheric pressure ion source for generating analyte ions; [0036] According to another aspect of the present in- a vacuum chamber; vention there is provided a mass spectrometer compris- a nozzle-skimmer interface separating the vacuum ing: 5 chamber from the atmospheric pressure ion source, wherein at least some analyte ions generated by the an atmospheric pressure ion source for generating atmospheric pressure ion source are transmitted, in analyte ions; and use, through the nozzle-skimmer interface into the a glow discharge device for generating reagent ions vacuum chamber; and/or reference ions, wherein the glow discharge 10 an isolation valve located in the vacuum chamber; device is located within a vacuum chamber of the a glow discharge device for generating reagent ions mass spectrometer. and/or reference ions, wherein the glow discharge device is located within or downstream of the isola- [0037] The glow discharge device is preferably main- tion valve; and tained at sub- atmospheric pressure. According to an em- 15 a device for supplying reagent to the glow discharge bodiment of the present invention the glow discharge de- device. vice is maintained at a pressure < 200 mbar, further pref- erably < 100 mbar. [0042] The device preferably comprises a device for [0038] The mass spectrometer preferably further com- supplying reagent vapour to the glow discharge device. prises: 20 According to a particularly preferred embodiment the re- agent vapour may be supplied through a hollow tube (i) an Electron Transfer Dissociation reaction cell which forms the glow discharge device. wherein reagent ions are caused to interact with an- [0043] According to another aspect of the present in- alyte ions within the reaction cell in order to cause vention there is provided a method of mass spectrometry at least some of the analyte ions to fragment by Elec- 25 comprising: tron Transfer Dissociation; and/or (ii) a Proton Transfer Reaction reaction cell wherein providing an atmospheric pressure ion source for reagent ions are caused to interact with analyte ions generating analyte ions; within the reaction cell in order to reduce the charge providing a vacuum chamber; state of at least some of the analyte ions by Proton 30 providing a nozzle-skimmer interface separating the Transfer Reaction. vacuum chamber from the atmospheric pressure ion source wherein at least some analyte ions generated [0039] According to another aspect of the present in- by the atmospheric pressure ion source are trans- vention there is provided a method of mass spectrometry mitted through the nozzle- skimmer interface into the comprising: 35 vacuum chamber; providing an isolation valve located in the vacuum providing an atmospheric pressure ion source for chamber; generating analyte ions; and generating reagent ions and/or reference ions using using a glow discharge device for generating reagent a glow discharge device, wherein the glow discharge ions and/or reference ions, wherein the glow dis- 40 device is located within or downstream of the isola- charge device is located within a vacuum chamber tion valve; and of the mass spectrometer. supplying reagent to the glow discharge device.

[0040] The method preferably further comprises: [0044] According to another aspect of the present in- 45 vention there is provided a mass spectrometer compris- (i) causing reagent ions to interact with analyte ions ing: within an Electron Transfer Dissociation reaction cell in order to cause at least some of the analyte ions a vacuum chamber; to fragment by Electron Transfer Dissociation; a sub-atmospheric pressure ion source for generat- and/or 50 ing analyte ions; and (ii) causing the reagent ions to interact with analyte a glow discharge device for generating reagent or ions within a Proton Transfer Reaction reaction cell referenceions, wherein thereagent or reference ions in order to reduce the charge state of at least some generated by the glow discharge device are gener- of the analyte ions by Proton Transfer Reaction. ated within the vacuum chamber. 55 [0041] According to another aspect of the present in- [0045] According to another aspect of the present in- vention there is provided a mass spectrometer compris- vention there is provided a method of mass spectrometry ing: comprising:

5 9 EP 2 667 401 A1 10

providing a vacuum chamber; transmitted, in use, through the nozzle-skimmer in- generating analyte ions using a sub-atmospheric terface into the first vacuum chamber; pressure ion source; and a glow discharge device for generating Electron using a glow discharge device to generate reagent Transfer Dissociation reagent ions, wherein the glow or reference ions, wherein the reagent or reference 5 discharge device is located within or downstream of ions generated by the glow discharge device are the nozzle-skimmer interface and within the first vac- generated within the vacuum chamber. uum chamber wherein the glow discharge device is maintained in use at a pressure in the range 0.01-100 [0046] According to another aspect of the present in- mbar; vention there is provided a mass spectrometer compris- 10 a device for supplying reagent vapour to the glow ing: discharge device so that Electron Transfer Dissoci- ation reagent ions are formed in use by the glow an atmospheric or sub-atmospheric pressure ion discharge device; and source for generating analyte ions; an Electron Transfer Dissociation fragmentation cell a vacuum chamber; 15 arranged in a second vacuum chamber, wherein the a nozzle-skimmer interface separating the vacuum second vacuum chamber is located downstream of chamber from the ion source wherein at least some the first vacuum chamber and wherein, in use, at analyte ions generated by the ion source are trans- least some of the Electron Transfer Dissociation re- mitted, in use, through the nozzle- skimmer interface agent ions are caused to interact with at least some into the vacuum chamber; 20 analyte ions within the Electron Transfer Dissocia- a glow discharge device for generating reagent ions tion fragmentation cell so as to cause at least some and/or reference ions, wherein the glow discharge of the analyte ions to fragment by Electron Transfer device is located within or downstream of the nozzle- Dissociation. skimmer interface and is maintained in use at a sub- atmospheric pressure; and 25 [0049] According to another aspect of the present in- a device for supplying reagent vapour to the glow vention there is provided a method of mass spectrometry discharge device. comprising:

[0047] According to another aspect of the present in- providing an atmospheric pressure ion source for vention there is provided a method of mass spectrometry 30 generating analyte ions; comprising: providing a first vacuum chamber; providing a nozzle-skimmer interface separating the providing an atmospheric or sub-atmospheric pres- first vacuum chamber from the ion source; sure ion source for generating analyte ions; transmitting at least some analyte ions generated by providing a vacuum chamber; 35 the ion source through the nozzle- skimmer interface providing a nozzle-skimmer interface separating the into the first vacuum chamber; vacuum chamber from the ion source wherein at using a glow discharge device to generate Electron least some analyte ions generated by the ion source Transfer Dissociation reagent ions, wherein the glow are transmitted through the nozzle-skimmer inter- discharge device is located within or downstream of face into the vacuum chamber; 40 the nozzle-skimmer interface and within the first vac- generating reagent ions and/or reference ions using uum chamber; a glow discharge device, wherein the glow discharge maintaining the glow discharge device at a pressure device is located within or downstream of the nozzle- of 0.01-100 mbar; skimmer interface and is maintained at a sub- atmos- supplying reagent vapour to the glow discharge de- pheric pressure; and 45 vice so that Electron Transfer Dissociation reagent supplying reagent vapour to the glow discharge de- ions are formed by the glow discharge device; vice. providing an Electron Transfer Dissociation frag- mentation cell arranged in a second vacuum cham- [0048] According to another aspect of the present in- ber, wherein the second vacuum chamber is located vention there is provided a mass spectrometer compris- 50 downstream of the first vacuum chamber; and ing: causing at least some of the Electron Transfer Dis- sociation reagent ions to interact with at least some an atmospheric pressure ion source for generating analyte ions within the Electron Transfer Dissocia- analyte ions; tion fragmentation cell so that at least some of the a first vacuum chamber; 55 analyte ions are caused to fragment by Electron a nozzle-skimmer interface separating the first vac- Transfer Dissociation. uum chamber from the ion source wherein at least some analyte ions generated by the ion source are [0050] According to another aspect of the present in-

6 11 EP 2 667 401 A1 12 vention there is provided a computer program executable (i) to cause the glow discharge device to generate by the control system of a mass spectrometer comprising reagent or reference ions within the vacuum cham- a vacuum chamber, an atmospheric pressure ion source ber. for generating first ions and a glow discharge device for generating second ions, the computer program being ar- 5 [0054] According to another aspect of the present in- ranged to cause the control system: vention there is provided a computer program executable by the control system of a mass spectrometer comprising (i) to cause first ions generated by the atmospheric an atmospheric or sub- atmospheric pressure ion source pressure ion source to be transmitted into the vacu- for generating analyte ions, a vacuum chamber, a nozzle- um chamber via a sampling cone or first aperture; 10 skimmer interface separating the vacuum chamber from and the ion source, a glow discharge device for generating (ii) to cause second ions to be generated by the glow reagent ions and/or reference ions, wherein the glow dis- discharge device wherein the second ions are either charge device is located within or downstream of the noz- generated within the vacuum chamber or are trans- zle-skimmer interface and a device for supplying reagent mitted into the vacuum chamber without being trans- 15 vapour to the glow discharge device, the computer pro- mitted through the sampling cone or first aperture. gram being arranged to cause the control system:

[0051] According to another aspect of the present in- (i) to cause at least some analyte ions generated by vention there is provided a computer program executable the ion source to be transmitted through the nozzle- by the control system of a mass spectrometer comprising 20 skimmer interface into the vacuum chamber; and an atmospheric pressure ion source and a glow dis- (ii) to maintain the glow discharge device at a sub- charge device located within a vacuum chamber of the atmospheric pressure. mass spectrometer, the computer program being ar- ranged to cause the control system: [0055] According to another aspect of the present in- 25 vention there is provided a computer program executable (i) to cause the atmospheric pressure ion source to by the control system of a mass spectrometer comprising generate analyte ions; and an atmospheric pressure ion source for generating ana- (ii) to cause the glow discharge device to generate lyte ions, a first vacuum chamber, a nozzle-skimmer in- reagent ions and/or reference ions. terface separating the first vacuum chamber from the ion 30 source, a glow discharge device for generating Electron [0052] According to another aspect of the present in- Transfer Dissociation reagent ions, wherein the glow dis- vention there is provided a computer program executable charge device is located within or downstream of the noz- by the control system of a mass spectrometer comprising zle-skimmer interface and within the first vacuum cham- an atmospheric pressure ion source for generating ana- ber, a device for supplying reagent vapour to the glow lyte ions, a vacuum chamber, a nozzle- skimmer interface 35 discharge device so that Electron Transfer Dissociation separating the vacuum chamber from the atmospheric reagent ions are formed by the glow discharge device pressure ion source, an isolation valve located in the vac- and an Electron Transfer Dissociation fragmentation cell uum chamber, a glow discharge device, wherein the glow arranged in a second vacuum chamber, wherein the sec- discharge device is located within or downstream of the ond vacuum chamber is located downstream of the first isolation valve and a device for supplying reagent to the 40 vacuum chamber, the computer program being arranged glow discharge device, the computer program being ar- to cause the control system: ranged to cause the control system: (i) to cause at least some analyte ions generated by (i) to cause at least some analyte ions generated by the ion source to be transmitted through the nozzle- the atmospheric pressure ion source to be transmit- 45 skimmer interface into the first vacuum chamber; ted through the nozzle-skimmer interface into the (ii) to maintain the glow discharge device at a pres- vacuum chamber; and sure in the range of 0.01-100 mbar; and (ii) to cause the glow discharge device to generate (iii) to cause at least some of the Electron Transfer reagent ions and/or reference ions. Dissociation reagent ions to interact with at least 50 some analyte ions within the Electron Transfer Dis- [0053] According to another aspect of the present in- sociation fragmentation cell so as to cause at least vention there is provided a computer program executable some of the analyte ions to fragment by Electron by the control system of a mass spectrometer comprising Transfer Dissociation. a vacuum chamber, a sub-atmospheric pressure ion source for generating analyte ions and a glow discharge 55 [0056] According to another aspect of the present in- device, the computer program being arranged to cause vention there is provided a computer readable medium the control system: comprising computer executable instructions stored on the computer readable medium, the instructions being

7 13 EP 2 667 401 A1 14 arranged to be executable by a control system of a mass arranged to be executable by a control system of a mass spectrometer a vacuum chamber, an atmospheric pres- spectrometer comprising a vacuum chamber, a sub-at- sure ion source for generating first ions and a glow dis- mospheric pressure ion source for generating analyte charge device for generating second ions, the computer ions and a glow discharge device, the computer program program being arranged to cause the control system: 5 being arranged to cause the control system:

(i) to cause first ions generated by the atmospheric (i) to cause the glow discharge device to generate pressure ion source to be transmitted into the vacu- reagent or reference ions within the vacuum cham- um chamber via a sampling cone or first aperture; ber. and 10 (ii) to cause second ions to be generated by the glow [0060] According to another aspect of the present in- discharge device wherein the second ions are either vention there is provided a computer readable medium generated within the vacuum chamber or are trans- comprising computer executable instructions stored on mitted into the vacuum chamber without being trans- the computer readable medium, the instructions being mitted through the sampling cone or first aperture. 15 arranged to be executable by a control system of a mass spectrometer comprising an atmospheric or sub-atmos- [0057] According to another aspect of the present in- pheric pressure ion source for generating analyte ions, vention there is provided a computer readable medium a vacuum chamber, a nozzle- skimmer interface separat- comprising computer executable instructions stored on ing the vacuum chamber from the ion source, a glow the computer readable medium, the instructions being 20 discharge device for generating reagent ions and/or ref- arranged to be executable by a control system of a mass erenceions, wherein the glow discharge device islocated spectrometer comprising an atmospheric pressure ion within or downstream of the nozzle-skimmer interface source and a glow discharge device located within a vac- and a device for supplying reagent vapour to the glow uum chamber of the mass spectrometer, the computer discharge device, the computer program being arranged program being arranged to cause the control system: 25 to cause the control system:

(i) to cause the atmospheric pressure ion source to (i) to cause at least some analyte ions generated by generate analyte ions; and the ion source to be transmitted through the nozzle- (ii) to cause the glow discharge device to generate skimmer interface into the vacuum chamber; and reagent ions and/or reference ions. 30 (ii) to maintain the glow discharge device at a sub- atmospheric pressure. [0058] According to another aspect of the present in- vention there is provided a computer readable medium [0061] According to another aspect of the present in- comprising computer executable instructions stored on vention there is provided a computer readable medium the computer readable medium, the instructions being 35 comprising computer executable instructions stored on arranged to be executable by a control system of a mass the computer readable medium, the instructions being spectrometer comprising an atmospheric pressure ion arranged to be executable by a control system of a mass source for generating analyte ions, a vacuum chamber, spectrometer comprising an atmospheric pressure ion a nozzle-skimmer interface separating the vacuum source for generating analyte ions, a first vacuum cham- chamber from the atmospheric pressure ion source, an 40 ber, a nozzle-skimmer interface separating the first vac- isolation valve located in the vacuum chamber, a glow uum chamber from the ion source, a glow discharge de- discharge device, wherein the glow discharge device is vice for generating Electron Transfer Dissociation rea- located within or downstream of the isolation valve and gent ions, wherein the glow discharge device is located a device for supplying reagent to the glow discharge de- within or downstream of the nozzle-skimmer interface vice, the computer program being arranged to cause the 45 and within the first vacuum chamber, a device for sup- control system: plying reagent vapour to the glow discharge device so that Electron Transfer Dissociation reagent ions are (i) to cause at least some analyte ions generated by formed by the glow discharge device and an Electron the atmospheric pressure ion source to be transmit- Transfer Dissociation fragmentation cell arranged in a ted through the nozzle-skimmer interface into the 50 second vacuum chamber, wherein the second vacuum vacuum chamber; and chamber is located downstream of the first vacuum (ii) to cause the glow discharge device to generate chamber, the computer program being arranged to cause reagent ions and/or reference ions. the control system:

[0059] According to another aspect of the present in- 55 (i) to cause at least some analyte ions generated by vention there is provided a computer readable medium the ion source to be transmitted through the nozzle- comprising computer executable instructions stored on skimmer interface into the first vacuum chamber; the computer readable medium, the instructions being (ii) to maintain the glow discharge device at a pres-

8 15 EP 2 667 401 A1 16

sure in the range of 0.01-100 mbar; and mbar; or (x) < 0.1 mbar. (iii) to cause at least some of the Electron Transfer [0069] The glow discharge source may be arranged to Dissociation reagent ions to interact with at least operate at a pressure: (i) 1-100 mbar; (ii) 0.5-50 mbar; some analyte ions within the Electron Transfer Dis- (iii) 0.2-20 mbar; (iv) 0.1-10 mbar; (v) 0.1-100 mbar; (vi) sociation fragmentation cell so as to cause at least 5 0.2-50 mbar; (vii) 0.5-20 mbar; (viii) 1-10 mbar; or (ix) 2-5 some of the analyte ions to fragment by Electron mbar. Transfer Dissociation. [0070] The glow discharge source preferably compris- es an electrode which is preferably held at a potential [0062] The computer readable medium is preferably (preferably with respect to its surrounding chamber) of: selected from the group consisting of: (i) a ROM; (ii) an 10 (i) > 50 V; (ii) > 100 V; (iii) > 200 V; (iv) > 500 V; (v) > EAROM; (iii) an EPROM; (iv) an EEPROM; (v) a flash 1000 V; (vi) < 1000 V; (vii) < 500 V; (viii) < 200 V; (ix) < memory; (vi) an optical disk; (vii) a RAM; and (viii) a hard 100 V; (x) 50-1000 V; and (xi) 100-500 V. disk drive. [0071] The glow discharge ion source may be operated [0063] According to a preferred embodiment of the substantially simultaneously or alternately with an At- present invention a multi- purpose glow discharge device 15 mospheric Pressure lonisation ion source. The Atmos- or ion source is provided. According to an embodiment pheric Pressure lonisation ion source is preferably oper- reagent ions are generated by the glow discharge ion ated at substantially atmospheric pressure. source within a vacuum chamber of the mass spectrom- [0072] Further embodiments are contemplated where- eter. The reagent ions are preferably generated within in the ion source which is used to generate analyte ions the main body of the mass spectrometer and advanta- 20 may comprise a sub-atmospheric pressure ion source geously the glow discharge source preferably does not such as a sub-atmospheric pressure Electrospray ion interfere with the positioning or operation of an atmos- source. The sub-atmospheric pressure ion source used pheric pressure ion source which is preferably used to to generate analyte ions may be operated at a pressure: generate analyte ions. (i) < 100 mbar; (ii) < 50 mbar; (iii) < 20 mbar; (iv) < 10 [0064] The reagent ions which are generated by the 25 mbar; (v) < 5 mbar; (vi) < 2 mbar; (vii) < 1 mbar; (viii) < glow discharge ion source are preferably transmitted 0.5 mbar; (ix) < 0.2 mbar; (x) < 0.1 mbar; or (xi) < 800 from the vacuum chamber or housing in which they are mbar. The sub-atmospheric pressure ion source may be generated to a collision cell which is preferably arranged arranged to operate at a pressure: (i) 1-100 mbar; (ii) in a downstream vacuum chamber of the mass spec- 0.5-50 mbar; (iii) 0.2-20 mbar; (iv) 0.1-10 mbar; (v) trometer. The reagent ions are preferably interacted with 30 0.1-100 mbar; (vi) 0.2-50 mbar; (vii) 0.5-20 mbar; (viii) analyte ions within the collision cell. The analyte ions are 1-10 mbar; (ix) 2-5 mbar; or (x) 100-800 mbar. According preferably caused to fragment by ion-ion reaction with to an embodiment the sub-atmospheric pressure ion the reagent ions by a process of Electron Transfer Dis- source may be operated at a higher (or lower) pressure sociation (ETD). The analyte ions may also be reduced than that of the glow discharge ion source. in charge state by Proton Transfer Reactions (PTR) ei- 35 [0073] The Atmospheric Pressure lonisation ion ther within the collision cell or within a further collision cell. source may, for example, comprise an Electrospray ion [0065] The glow discharge ion source may additionally source, an APCI ion source, an APPI ion source, a DESI or alternatively be used to provide ions for calibration or ion source, a DART ion source or an Atmospheric Pres- lock mass correction. sure MALDI ion source. [0066] According to an aspect of the present invention 40 [0074] The mass spectrometer or mass analyser may there is provided a mass spectrometer comprising an comprise either a quadrupole mass analyser, 3D or Paul atmospheric pressure ion source and a glow discharge ion trap mass analyser, 2D or linear ion trap mass ana- source which is preferably provided or maintained at an lyser, a Time of Flight mass analyser, an orthogonal ac- intermediate or sub- atmospheric pressure. The glow dis- celeration Time of Flight mass analyser, a magnetic sec- charge source is preferably used to generate reagent 45 tor mass analyser, a Fourier Transform mass spectrom- ions. The reagent ions may be used within a reaction cell eter ("FT-MS"), a Fourier Transform Ion Cyclotron Res- of a mass spectrometer to induce dissociation or frag- onance ("FT-ICR") mass analyser, a Fourier Transform mentation of organic analyte ions by Electron Transfer electrostatic ion trap, or an orbitrap (RTM) mass analys- Dissociation. er. [0067] The glow discharge source may also be used 50 [0075] The operating pressure of the mass spectrom- to generate reference ions. The reference ions may be eter is preferably arranged to be: (i) < 10-3 mbar; (ii) < used,for example, to calibrate the mass scale of themass 10-4 mbar; (iii) < 10-5 mbar; (iv) < 10-6 mbar; (v) < 10-7 spectrometer or may be used to correct the calibration mbar; (vi) < 10-8 mbar; (vii) < 10-9 mbar; or (viii) < 10-10 of the mass scale of the mass spectrometer. mbar. [0068] The glow discharge source is preferably ar-55 [0076] The mass spectrometer preferably includes a ranged to be operated at a pressure: (i) < 100 mbar; (ii) gas collision cell. The gas collision cell may be used to < 50 mbar; (iii) < 20 mbar; (iv) < 10 mbar; (v) < 5 mbar; induce decomposition or fragmentation of organic ana- (vi) < 2 mbar; (vii) < 1 mbar; (viii) < 0.5 mbar; (ix) < 0.2 lyte ions by collision with gas molecules, interaction with

9 17 EP 2 667 401 A1 18 electrons, interaction with reagent ions, interaction with tation device; (xv) a magnetic field induced fragmentation negatively charged reagent ions, interaction with metast- device; (xvi) an enzyme digestion or enzyme degradation able atoms, interaction with metastable molecules, or in- fragmentation device; (xvii) an ion-ion reaction fragmen- teraction with metastable ions. tation device; (xviii) an ion-molecule reaction fragmenta- [0077] According to an embodiment the mass spec- 5 tion device; (xix) an ion- atom reaction fragmentation de- trometer may comprise one or more ion sources selected vice; (xx) an ion-metastable ion reaction fragmentation fromthe groupconsisting of: (i) an Electrosprayionisation device; (xxi) an ion-metastable molecule reaction frag- ("ESI") ion source; (ii) an Atmospheric Pressure Photo mentation device; (xxii) an ion-metastable atom reaction lonisation ("APPI") ion source; (iii) an Atmospheric Pres- fragmentation device; (xxiii) an ion-ion reaction device sureChemical lonisation ("APCI") ion source; (iv) a Matrix 10 for reacting ions to form adduct or product ions; (xxiv) an Assisted Laser Desorption lonisation ("MALDI") ion ion-molecule reaction device for reacting ions to form ad- source; (v) a Laser Desorption lonisation ("LDI") ion duct or product ions; (xxv) an ion-atom reaction device source; (vi) an Atmospheric Pressure lonisation ("API") for reacting ions to form adduct or product ions; (xxvi) an ion source; (vii) a Desorption lonisation on Silicon ("DI- ion-metastable ion reaction device for reacting ions to OS") ion source; (viii) an Electron Impact ("EI") ion15 form adduct or product ions; (xxvii) an ion-metastable source; (ix) a Chemical lonisation ("CI") ion source; (x) a molecule reaction device for reacting ions to form adduct Field lonisation ("FI") ion source; (xi) a Field Desorption or product ions; (xxviii) an ion-metastable atom reaction ("FD") ion source; (xii) an Inductively Coupled Plasma device for reacting ions to form adduct or product ions; ("ICP") ion source; (xiii) a Fast Atom Bombardment and (xxix) an Electron lonisation Dissociation ("EID") ("FAB") ion source; (xiv) a Liquid Secondary Ion Mass 20 fragmentation device. Spectrometry ("LSIMS") ion source; (xv) a Desorption [0083] According to an embodiment the mass spec- Electrospray lonisation ("DESI") ion source; (xvi) a Nick- trometer may comprise a mass analyser selected from el-63 radioactive ion source; (xvii) an Atmospheric Pres- the group consisting of: (i) a quadrupole mass analyser; sure Matrix Assisted Laser Desorption lonisation ion (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul source; (xviii) a Thermospray ion source; (xix) an Atmos- 25 or 3D quadrupole mass analyser; (iv) a Penning trap pheric Sampling Glow Discharge lonisation ("ASGDI") mass analyser; (v) an ion trap mass analyser; (vi) a mag- ion source; (xx) a Glow Discharge ("GD") ion source; (xxi) netic sector mass analyser; (vii) Ion Cyclotron Reso- a sub-atmospheric pressure Electrospray ionisation ion nance ("ICR") mass analyser; (viii) a Fourier Transform source; and (xxii) a Direct Analysis in Real Time ("DART") Ion Cyclotron Resonance ("FTICR") mass analyser; (ix) ion source. 30 an electrostatic or orbitrap mass analyser; (x) a Fourier [0078] The mass spectrometer may comprise one or Transform electrostatic or orbitrap mass analyser; (xi) a more continuous or pulsed ion sources. Fourier Transform mass analyser; (xii) a Time of Flight [0079] The mass spectrometer may comprise one or mass analyser; (xiii) an orthogonal acceleration Time of more ion guides. Flight mass analyser; and (xiv) a linear acceleration Time [0080] According to an embodiment the mass spec- 35 of Flight mass analyser. trometer may further comprise one or more ion mobility [0084] According to an embodiment the mass spec- separation devices and/or one or more Field Asymmetric trometer may further comprise one or more energy ana- Ion Mobility Spectrometer devices. lysers or electrostatic energy analysers. [0081] The mass spectrometer may comprise one or [0085] According to an embodiment the mass spec- more ion traps or one or more ion trapping regions. 40 trometer may further comprise one or more ion detectors. [0082] According to an embodiment the mass spec- [0086] According to an embodiment the mass spec- trometer mayfurther compriseone or morecollision, frag- trometer may further comprise one or more mass filters mentation or reaction cells selected from the group con- selected from the group consisting of: (i) a quadrupole sisting of: (i) a Collisional Induced Dissociation ("CID") mass filter; (ii) a 2D or linear quadrupole ion trap; (iii) a fragmentation device; (ii) a Surface Induced Dissociation 45 Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; ("SID") fragmentation device; (iii) an Electron Transfer (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Dissociation ("ETD") fragmentation device; (iv) an Elec- Time of Flight mass filter; and (viii) a Wein filter. tron Capture Dissociation ("ECD") fragmentation device; [0087] According to an embodiment the mass spec- (v) an Electron Collision or Impact Dissociation fragmen- trometer may further comprise a device or ion gate for tation device; (vi) a Photo Induced Dissociation ("PID") 50 pulsing ions. fragmentation device; (vii) a Laser Induced Dissociation [0088] According to an embodiment the mass spec- fragmentation device; (viii) an infrared radiation induced trometer may further comprise a device for converting a dissociation device; (ix) an ultraviolet radiation induced substantially continuous ion beam into a pulsed ion dissociation device; (x) a nozzle- skimmer interface frag- beam. mentation device; (xi) an in- source fragmentation device; 55 [0089] According to an embodiment the mass spec- (xii) an in- sourceCollision Induced Dissociation fragmen- trometer may comprise a C-trap and an orbitrap mass tation device; (xiii) a thermal or temperature source frag- analyser comprising an outer barrel- like electrode and a mentation device; (xiv) an electric field induced fragmen- coaxial inner spindle-like electrode, wherein in a first

10 19 EP 2 667 401 A1 20 mode of operation ions are transmitted to the C- trap and the body of an isolation valve which is located in the are then injected into the orbitrap mass analyser and first vacuum chamber of a mass spectrometer; wherein in a second mode of operation ions are trans- Fig. 8 shows an Electron Transfer Dissociation frag- mitted to the C- trap and then to a collision cell or Electron mentation spectrum of triply charged substance-P Transfer Dissociation device wherein at least some ions 5 which has been subjected to fragmentation by are fragmented into fragment ions, and wherein the frag- azobenzene reagent ions generated by a glow dis- ment ions are then transmitted to the C- trap before being charge source according to an embodiment of the injected into the orbitrap mass analyser. present invention; [0090] According to an embodiment the mass spec- Fig. 9A shows a calibration mass spectrum per- trometer may comprise a stacked ring ion guide compris- 10 formed using perfluorotripentylamine (FC70) and ing a plurality of electrodes each having an aperture caesium ions which were generated by a glow dis- through which ions are transmitted in use and wherein charge source according to an embodiment of the the spacing of the electrodes increases along the length present invention and Fig. 9B shows an experimental of the ion path, and wherein the apertures in the elec- mass spectrum, a reference mass spectrum and a trodes in an upstream section of the ion guide have a 15 determination of the residual mass errors after cali- first diameter and wherein the apertures in the electrodes bration. in a downstream section of the ion guide have a second diameter which is smaller than the first diameter, and [0092] An embodiment of the present invention will wherein opposite phases of an AC or RF voltage are now be described with reference to Fig. 1 which shows applied, in use, to successive electrodes. 20 an atmospheric pressure Electrospray ionisation ion [0091] Various embodiments of the present invention source 7 arranged adjacent to the inlet and sample cone will now be described, by way of example only, and with 8 of a mass spectrometer. A glow discharge source com- reference to the accompanying drawings in which: prising an electrode or pin 4 is preferably provided in a housing adjacent to a first vacuum chamber 3 of the mass Fig. 1 shows an embodiment of the present invention 25 spectrometer. The electrode or pin 4 is preferably con- wherein a glow discharge ion source for generating nected to an external high voltage supply. The housing reagent ions is provided in a housing adjacent to the is preferably maintained at a relatively high or interme- first vacuum chamber of a mass spectrometer; diate pressure and the application of a relatively high Fig. 2 shows an embodiment of the present invention voltage to the electrode or pin 4 preferably causes a glow wherein two reagents may be supplied to the glow 30 discharge 1 to occur within the housing. discharge ion source which is provided in a housing [0093] A volatile reagent 6 is preferably fed into the adjacent to the first vacuum chamber of a mass spec- housing and is preferably injected into the glow discharge trometer; volume 1 which is preferably formed within the housing. Fig. 3 shows an embodiment of the present invention The flow of reagent 6 is preferably controlled by a valve wherein a solid reagent is provided within a housing 35 or micro-dosing device 5. When a high voltage is applied adjacent to the first vacuum chamber of the mass to the discharge electrode or pin 4 a glow discharge is spectrometer and wherein part of the solid reagent preferably initiated which preferably ionises the reagent is ionised by a glow discharge to provide lock mass which is fed into the housing so that a plurality of reagent ions for calibrating the mass spectrometer; ions are formed within the housing. According to an em- Fig. 4 shows an embodiment of the present invention 40 bodiment a means of enhancing the extraction of reagent wherein a glow discharge is initiated directly in the ions from the glow discharge volume 1 or otherwise from first vacuum chamber of a mass spectrometer and the housing into the first vacuum chamber 3 via an ap- wherein a volatile reagent may be fed into the first erture 2 is preferably provided. For example, an electric vacuum chamber downstream of the electrode field may be maintained between the housing and the which is used to generate the glow discharge; 45 first vacuum chamber 1 in order to urge reagent ions from Fig. 5 shows a tune page from a mass spectrometer the housing into the first vacuum chamber 1. Additional and shows mass spectra showing caesium lock pumping (not shown) may be provided to the housing in mass ions generated by a preferred glow discharge which the glow discharge volume 1 is generated in order ion source and analyte ions of Leucine Enkephalin to minimise the flow of neutral reagent molecules from generated by an Electrospray ion source; 50 the housing via the aperture 2 into the first vacuum cham- Fig. 6 shows a tune page from a mass spectrometer ber 3 and the other ion optic sections of the mass spec- and shows mass spectra showing caesium and dii- trometer. Reagent ions which are generated within the odomethane lock mass ions generated by a pre- housing and which pass via the aperture 2 into the first ferred glow discharge ion source and analyte ions vacuum chamber 3 are then preferably onwardly trans- of Leucine Enkephalin generated by an Electrospray 55 mitted through an extraction cone 10 into a second vac- ion source; uum chamber. The second vacuum chamber preferably Fig. 7 shows an embodiment wherein a discharge comprises an ion guide 11 which is preferably arranged pin for generating a glow discharge is provided within to transmit ions through the second vacuum chamber.

11 21 EP 2 667 401 A1 22

The reagent ions (preferably reagent anions) are then ionised to produce reagent ions for use in Electron Trans- preferably onwardly transmitted to a collision cell (not fer Dissociation such as azobenzene are carcinogenic. shown) located in a further vacuum chamber down- An important advantage therefore of the preferred em- stream of the second vacuum chamber. bodiment is that reagents which are used to generate [0094] According to a preferred embodiment of the 5 reagent ions are not sprayed or otherwise emitted exter- present invention the reagent ions which are created nal to the inlet aperture of the mass spectrometer (and within the housing and which are preferably onwardly hence in the vicinity of a user) but instead are sprayed transmitted to the collision cell are preferably caused to or otherwise injected internally within a vacuum chamber interact with analyte ions (preferably analyte cations). of the mass spectrometer. As a result, the exposure of a The reagent ions are preferably caused to interact with 10 user to potentially carcinogenic reagents is significantly analyte ions so as to cause the analyte ions to fragment reduced. by a process of Electron Transfer Dissociation ("ETD") [0100] A yet further advantageous feature of the pre- so that a plurality of fragment, daughter or product ions ferred embodiment is that the preferred glow discharge are formed as a result of the Electron Transfer Dissoci- device significantly improves the sensitivity and intensity ation process. According to another less preferred em- 15 of generated reagent ions compared with a conventional bodiment the reagent ions which are generated may be mass spectrometer. reacted with analyte ions in order to reduce the charge [0101] It is also contemplated that at least some rea- state of the analyte ions by Proton Transfer Reaction gents may be used both as a source of reagent ions for without substantially fragmenting the analyte ions. Electron Transfer Dissociation (and/or Proton Transfer [0095] According to an alternative or additional embod- 20 Reaction) and also as a source of lock mass or reference iment, the glow discharge ion source 1 may act as a ions for calibrating the mass spectrometer. Analyte ions source of ions which are preferably used for mass cali- generated by the ion source 7 are preferably drawn bration of the mass spectrometer. A particularly preferred through the sample cone 8 of the mass spectrometer into aspect of the present invention is that a glow discharge the first vacuum chamber 3 of the mass spectrometer ion source located within the body of the mass spectrom- 25 which is preferably pumped by a vacuum pump 9. The eter may be used to generate both reagent ions for use first vacuum chamber 3 and the inlet into the mass spec- in an Electron Transfer Dissociation and/or Proton Trans- trometer are preferably heated. The analyte ion source fer Reaction cell and also calibration or lock mass ions andinitial stages of themass spectrometer maycomprise for calibrating the mass spectrometer. a z-spray (RTM) ion source. [0096] The glow discharge source is preferably oper- 30 [0102] Fig. 2 shows another embodiment of the ated in a pulsed manner wherein a high voltage pulse is present invention wherein two different reagents 6a,6b preferably only applied to the electrode or discharge pin may be introduced into the housing in which the glow 4 during a lock mass cycle or during a reagent introduc- discharge volume 1 is generated. According to an em- tion cycle. A lock mass compound is preferably only ad- bodiment one of the reagents 6a may comprise a reagent mitted into the housing during a lock mass cycle. Like- 35 which is used to generate reagent ions which are used wise, a reagent is preferably only admitted into the hous- for ion-ion reactions such as Electron Transfer Dissoci- ing during a reagent introduction cycle. ation. The other reagent 6b may be used to generate [0097] Analyte ions are preferably generated by a sep- calibrant or lock mass ions. The selection of one species arate atmospheric pressure ion source such as an Elec- of reagent ions is preferably performed or controlled by trospray lonisation ion source 7 as shown in Fig. 1. The 40 use of one or more valves or micro-dosing devices 5a, ion source for generating analyte ions is preferably ex- 5b. Alternatively, a mass selective device such as a re- ternal to the main body of the mass spectrometer. solving quadrupole rod set mass filter may be arranged [0098] A known conventional mass spectrometer com- or otherwise provided downstream of the glow discharge prises two ion sources. The first ion source comprises source 1 in order to filter out any undesired ions and/or an Electrospray ion source for generating analyte ions 45 to transmit onwardly only desired ions. and the second ion source comprises an Atmospheric [0103] Further embodiments are contemplated where- Pressure Chemical lonisation ion source for generating in three, four, five, six or more than six different reagents reagent ions or calibration ions. The necessity to use two may be selectively introduced into the housing in which atmospheric pressure ion sources is problematic and the glow discharge 1 is created. A flow of gas may be makes the ion source geometry relatively complex. The 50 admitted towards the discharge volume or otherwise two ion sources can also interfere with each other and more generally into the housing in which the glow dis- can cause problems due to cross- talk. The preferred em- charge 1 is created. The gas may comprise an inert make bodiment is therefore particularly advantageous in that up gas which is preferably introduced in order to increase only a single Electrospray ion source needs to be pro- the pressure within the discharge chamber or housing vided. This simplifies the ion source geometry and re- 55 relative to the first vacuum stage or chamber 3. Alterna- moves any problem of cross-talk between ion sources. tively, the gas may comprise a chemical ionisation (CI) [0099] A particularly serious problem with known mass gas which is preferably provided in order to enhance the spectrometers is that many of the reagents which are ionisation of the reagent in the discharge chamber or

12 23 EP 2 667 401 A1 24 housing. The gas may flow past or through the reagent is preferably applied to the electrode or pin 4 via the elec- as a means of controlling the flow of reagent neutrals into trical connection 13 and this preferably causes a glow the glow discharge. The addition of gas into the housing discharge to be created within the first vacuum chamber in which the glow discharge 1 is generated in combination 3. with suitable or appropriate differential pumping prefer- 5 [0108] Other embodiments are contemplated wherein ably enables the discharge chamber or housing in which the volatile reagent 6 may be replaced with a solid such the glow discharge 1 is generated to be interfaced to as caesium iodide (Csl) which is preferably located within other vacuum regions of the mass spectrometer. the first vacuum chamber 3. The block of caesium iodide [0104] The first vacuum chamber 3 and the inlet into is preferably provided within the first vacuum chamber 3 the mass spectrometer as shown in Fig. 2 may be heated. 10 adjacent the region wherein a glow discharge 1 is formed The analyte ion source and initial stages of the mass within the first vacuum chamber 3 by the application of spectrometer may comprise a z- spray (RTM) ion source. a high voltage to the electrode or pin 4. [0105] An alternative embodiment for producing lock [0109] The first vacuum chamber 3 and the inlet into mass ions is shown in Fig. 3. According to this embodi- the mass spectrometer shown in Fig. 4 may be heated. ment a solid highly ionic material 12 such as caesium 15 The analyte ion source and initial stages of the mass iodide (Csl) may be placed or located within the housing spectrometer may comprise a z- spray (RTM) ion source. which is located adjacent to the first vacuum chamber 3 [0110] Fig. 5 shows a tune page from a modified Wa- of the mass spectrometer. When a glow discharge 1 is ters Q-Tof premier (RTM) mass spectrometer and cor- produced within the housing, the glow discharge 1 pref- responding mass spectra. The modified mass spectrom- erably causes caesium ions to be released from the sur- 20 eter was operated in positive ion mode and was arranged face of the caesium iodide block. The caesium ions are substantially as shown in Fig. 4 except that caesium lock preferably sputtered and are preferably extracted from mass ions were generated from a solid block of caesium the discharge volume 1 for use as a means of lock mass iodide which was provided within the first vacuum cham- correction. A reagent introduction system comprising re- ber 3 adjacent electrode or pin 4. The glow discharge agent 6, a fluid flow path and a valve 5 may also be pro- 25 source 1 was generated using an ESCi (RTM) power vided in order to introduce reagent into the housing. Re- supply operating in constant current mode with approx- agent ions are preferably created by the glow discharge imately 5 mA of discharge current at a voltage of approx- 1 within the housing and may be onwardly transmitted imately +700V. Leucine Enkephalin was used as a test for use as Electron Transfer Dissociation and/or Proton analyte and was ionised by a conventional Electrospray Transfer Reaction reagent ions. Therefore, according to 30 ion source. The corresponding mass spectrum for the this embodiment both calibrant or lock mass ions and test analyte having a mass to charge ratio of 556 is shown also reagent ions may be generated within the housing in Fig. 5. Caesium lock mass calibration ions having a and may be onwardly transmitted into the first vacuum mass to charge ratio of 133 which were generated by the stage 3 and subsequent vacuum stages of the mass glow discharge ionising the caesium iodide block were spectrometer. According to an embodiment a resolving 35 used to calibrate the mass spectrometer. A correspond- quadrupole rod set mass filter may be provided upstream ing mass spectrum showing the caesium lock mass ions of an ion-ion reaction cell and downstream of the glow is also shown in Fig. 5. Alternating acquisitions of 1 s discharge source 1 to ensure that only desired reagent duration were acquired with an inter-spectrum delay of ions (preferably reagent anions) are introduced into the 0.1 sec. reaction cell. The reagent ions preferably interact with 40 [0111] Fig. 6 shows a further tune page and corre- analyte ions (preferably analyte cations) and preferably sponding mass spectra which were obtained when Diio- cause the analyte ions to fragment by means of Electron domethane vapour was additionally introduced into the Transfer Dissociation. mass spectrometer in a manner substantially as shown [0106] The first vacuum chamber 3 and the inlet into in Fig. 4 as an additional calibration compound. A block the mass spectrometer shown in Fig. 3 may be heated. 45 of caesium iodide was also provided within the first vac- The analyte ion source and initial stages of the mass uum chamber 3 adjacent the glow discharge 1 so that spectrometer may comprise a z- spray (RTM) ion source. caesium ions were also released as a source of reference [0107] Fig. 4 shows a further embodiment wherein a ions. Mass spectra for the analyte ions as generated by glow discharge is initiated directly within the first vacuum an Electrospray ion source and the two lock mass ions region 3 of the mass spectrometer rather than in a hous- 50 as generated by a glow discharge device according to ing adjacent to the first vacuum chamber 3. According to an embodiment of the present invention are shown in the embodiment shown in Fig. 4 a volatile reagent 6 is Fig. 6. preferably fed via a valve 5 directly into the first vacuum [0112] Fig. 7 shows a further embodiment of the chamber 3 of the mass spectrometer. The reagent is pref- present invention wherein a discharge pin 14 is incorpo- erably introduced into the first vacuum chamber 3 at a 55 rated into an isolation valve 15 located within the first location downstream of an electrode or pin 4. The elec- vacuum chamber of the mass spectrometer. The isola- trode or pin 4 is preferably connected via an electrical tion valve 15 is located between a skimmer cone 17 and connection 13 to a high voltage source. A high voltage an extraction cone 18 of the mass spectrometer. The

13 25 EP 2 667 401 A1 26 discharge pin 14 preferably comprises a stainless steel and formed the glow discharge device. A high voltage capillary tube (0.0625" O.D. x 0.5 mm I.D.) which is pref- was applied to the electrode or pin 14 in order to induce erably arranged to have a sharp or pointed end. Accord- a glow discharge which resulted in the ionisation of the ing to the embodiment shown in Fig. 7 vapour from a azobenzene vapour to form azobenzene reagent anions. reagent cell 16 is preferably emitted via a reagent vapour 5 The azobenzene reagent ions when interacted with triply outlet tube 19 in close proximity to the sharp or pointed charged substance-P ions in an Electron Transfer Dis- end of the discharge pin 14. The reagent cell may com- sociation cell located in a downstream vacuum chamber prise a vial of reagent crystals e.g. azobenzene or caused the triply charged substance-P ions to fragment fluoranthene. It will be understood that the isolation valve by Electron Transfer Dissociation. 15 has a cylindrical bore or port which when aligned with 10 [0117] Fig. 9A shows a mass spectrum obtained ac- the central cylindrical bore of the first vacuum chamber cording to an embodiment of the present invention 3 allows ions to pass from the analyte sampling cone 17 wherein calibration was performed using perflourotrip- towards the extraction cone 18 which leads into the sec- entylamine("FC70") ions andcaesium ions. The perfluor- ond vacuum chamber. However, when the mass spec- otripentylamine ("FC70") ions and caesium ions were trometer is not operational the isolation valve may be 15 generated by introducing perfluorotripentylamine vapour rotated by 90° so that the cylindrical bore or port of the through a tubular pin 14 located within the isolation valve isolation valve 15 is no longer in alignment with the cy- of a mass spectrometer in a manner substantially as lindrical bore of the first vacuum chamber 3. As a result, shown in Fig. 7 and as described above. The end of the the isolation valve 15 acts to seal the vacuum chambers tubular pin 14 was pointed and formed the glow discharge of the mass spectrometer from the atmosphere and20 device. A high voltage was applied to the electrode or therefore assists in maintaining a low pressure within the pin 14 in order to induce a glow discharge which resulted mass spectrometer when the mass spectrometer is not in the ionisation of the perfluorotripentylamine vapour to operational. It will be appreciated that maintaining a low form perfluorotripentylamine reference ions. The caesi- pressure within the mass spectrometer significantly re- um reference ions were generated by coating the region duces the start- up time when operation of the mass spec- 25 around the end of the tubular pin 14 with caesium iodide. trometer is desired to be resumed. [0118] Other embodiments are contemplated wherein [0113] According to a preferred embodiment of the other reagents for calibration/lock mass may be intro- present invention the reagent cell may 16 be positioned duced into the glow discharge device including perfluor- such that reagent vapour flows down and through the okerosine ("PFK"), perfluorotrihexylamine, perfluorotrib- tube which preferably forms the discharge pin 14 and 30 utylamine ("FC43"), diiodomethane and iodotetrafluoro- emerges from the tube at the sharp or pointed end of the propane. discharge pin 14. According to an embodiment the rea- [0119] The upper mass spectrum in Fig. 9B shows an gent cell 16 may comprise a vial of reagent crystals e.g. nominal mass spectrum obtained from experimental data azobenzene or flouranthene may be provided in solid prior to calibration of the mass spectrometer. The mass form within the reagent cell 16 which is preferably con- 35 or mass to charge ratio values were calculated using an nected to the tube. A make-up gas (which is preferably estimated calibration method wherein the mass is pro- inert) such as nitrogen may also be used. The make-up portional to the time of flight squared. Perfluorotrip- gas may be arranged to flow past the crystals which may entylamine ("FC70") ions were generated by an Electro- be held at room temperature ( ∼ 20°C). The make- up gas spray ionisation ion source and were admitted into the may be supplied at a flow rate of around 20 ml/min. 40 mass spectrometer and were subsequently mass ana- [0114] According to the preferred embodiment oxygen lysed. is preferably substantially prevented from flowing [0120] The middle mass spectrum shows a reference through the discharge region as this can cause a loss of or theoretical mass spectrum for perfluorotripentylamine reagent signal. According to the preferred embodiment and is based upon reference data. the source volume may be purged with nitrogen. 45 [0121] The mass spectrometer was then calibrated [0115] According to an embodiment a voltage of -500 more accurately by applying a higher order (fourth order) V may be applied to an electrode 20 which is in electrical time of flight polynomial curve to the experimental data. contact with the discharge pin 14 so that the discharge The RMS of the residual errors of the least squares fitting pin is preferably maintained at a voltage of -500 V in order of the fourth order polynomial curve against the experi- to generate a glow discharge. 50 mental data are shown in the lower figure and was de- [0116] Fig. 8 shows an ETD fragmentation spectrum termined to be 0.6 ppm. which was obtained by interacting triply charged sub- [0122] Although according to the preferred embodi- stance-P ions with azobenzene reagent anions. The ment reagent ions are preferably generated either in a azobenzene reagent anions were formed by introducing housing adjacent to the first vacuum chamber 3 or alter- azobenzene reagent vapour through a tubular pin 14 lo- 55 natively directly in the first vacuum chamber 3, according cated within the isolation valve of a mass spectrometer to other less preferred embodiments reagent ions may in a manner substantially as shown in Fig. 7 and as de- be generated in a housing adjacent to a second or sub- scribed above. The end of the tubular pin 14 was pointed sequent vacuum chamber or alternatively may be gen-

14 27 EP 2 667 401 A1 28 erated in a second or subsequent vacuum chamber 4. A mass spectrometer as claimed in claim 1, 2 or 3, which is preferably arranged downstream of the first vac- wherein said glow discharge device is housed in a uum chamber 3. For example, it is contemplated that a housingadjacent said vacuum chamber and wherein glow discharge ion source may be provided in the same second ions generated by said glow discharge de- vacuum chamber as an Electron Transfer Dissociation 5 vice pass from said housing through a second aper- reaction cell and/or the same vacuum chamber as a Pro- ture into said vacuum chamber. ton Transfer Reaction reaction cell. [0123] According to the preferred embodiment of the 5. A mass spectrometer as claimed in any preceding present invention the glow discharge device comprises claim, further comprising either a solid, powdered, a pin or electrode 4,14 to which a DC and/or RF voltage 10 partially solid or gel substance, a volatile liquid or a is applied in order to generate a glow discharge 1. gas which is arranged or supplied in proximity to said [0124] Although the present invention has been de- glow discharge device so that ions are sputtered, scribed with reference to the preferred embodiments, it extracted or released from said substance, liquid or will be understood by those skilled in the art that various gas. changes in form and detail may be made to the particular 15 embodiments discussed above without departing from 6. A mass spectrometer as claimed in claim 4, wherein the scope of the invention as set forth in the accompa- said substance comprises caesium iodide. nying claims. 7. A mass spectrometer as claimed in any preceding 20 claim, wherein said lock mass or calibration reagent Claims comprises perfluorotripentylamine ("FC70"), per- fluorokerosine ("PFK"), perfluorotrihexylamine, per- 1. A mass spectrometer comprising: fluorotributylamine ("FC43"), diiodomethane, io- dotetrafluoropropane or fluoranthene. a vacuum chamber; 25 an atmospheric pressure ion source for gener- 8. A mass spectrometer as claimed in any preceding ating first ions, wherein first ions generated by claim, further comprising a supply device for supply- said atmospheric pressure ion source are trans- ing one or more lock mass reagents in proximity to mitted, in use, into said vacuum chamber via a said glow discharge device. sampling cone or first aperture; 30 a glow discharge device for generating second 9. A mass spectrometer as claimed in any preceding ions, wherein second ions generated by said claim, wherein said glow discharge device compris- glow discharge device are either generated es a tube having a sharpened or pointed end. within said vacuum chamber or are transmitted into said vacuum chamber without being trans- 35 10. A mass spectrometer as claimed in claim 9, further mitted through said sampling cone or first aper- comprising asupply devicefor supplying one or more ture; lock mass reagents through said tube. one or more dispensing devices for dispensing one or more reagents in proximity to said glow 11. A mass spectrometer as claimed in any preceding discharge device so that said one or more rea- 40 claim, wherein said glow discharge device is oper- gents are ionised, in use, by a glow discharge ated in a continuous or pulsed manner. caused or generated by said glow discharge de- vice; 12. A mass spectrometer as claimed in any preceding wherein said one or more reagents comprise claim, wherein said glow discharge device is main- one or more lock mass or calibration reagents 45 tained or operated in a mode of operation at a po- for mass calibrating the mass spectrometer. tential selected from the group consisting of: (i) < - 1 kV; (ii) -900 to -800 V; (iii) -800 to -700 V; (iv) -700 2. A mass spectrometer as claimed in claim 1, wherein to - 600 V; (v) -600 to -500 V; (vi) -500 to -400 V; (vii) saidsecond ions are used to calibrate themass scale -400 to -300 V; (viii) -300 to -200 V; (ix) - 200 to -100 of the mass spectrometer or to correct the calibration 50 V; (x) -100 to 0 V; (xi) 0 to 100 V; (xii) 100 to 200 V; of the mass scale of the mass spectrometer. (xiii) 200 to 300 V; (xiv) 300 to 400 V; (xv) 400 to 500 V; (xvi) 500 to 600 V; (xvii) 600 to 700 V; (xviii) 700 3. A mass spectrometer as claimed in claim 1 or 2, to 800 V; (xix) 800 to 900 V; (xx) 900 to 1000 V; and wherein said glow discharge device comprises an (xxi) > 1 kV. electrodeor pin and wherein said mass spectrometer 55 further comprises a voltage device for supplying or 13. A mass spectrometer as claimed in any preceding applying a DC and/or RF voltage to said electrode claim, wherein said glow discharge device is oper- or pin in order to cause or generate a glow discharge. ated, in use, at a pressure selected from the group

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consisting of (i) > 0.001 mbar; (ii) > 0.01 mbar; (iii) > 0.1 mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > 100 mbar; (vii) < 0.001 mbar; (viii) < 0.01 mbar; (ix) < 0.1 mbar; (x) < 1 mbar; (xi) < 10 mbar; (xii) < 100 mbar; (xiii) 0.001-0.01 mbar; (xiv) 0.01-0.1 mbar; (xiv) 0.1-1 5 mbar; (xv) 1-10 mbar; (xvi) 10-100 mbar; and (xvii) 0.01-20 mbar.

14. A method of mass spectrometry comprising: 10 providing a vacuum chamber; providing an atmospheric pressure ion source for generating first ions; generating first ions by said atmospheric pres- sure ion source and transmitting said first ions 15 into said vacuum chamber via a sampling cone or first aperture; providinga glow discharge devicefor generating second ions; generating second ions by said glow discharge 20 device, wherein said second ions are generated either within said vacuum chamber or are trans- mitted into said vacuum chamber without being transmitted through said sampling cone or first aperture; 25 dispensing one or more reagents in proximity to said glow discharge device so that said one or more reagents are ionised by a glow discharge caused or generated by said glow discharge de- vice; 30 wherein said one or more reagents comprise one or more lock mass or calibration reagents for mass calibrating the mass spectrometer.

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