Differentielle Ionen-Mobilität als Vorfilter für LC-MS/MS

Transcription

1 Differentielle Ionen-Mobilität als Vorfilter für LC-MS/MS Nicole Kohl 1, Dr. Stefanie Kreppenhöfer 2, Dr. Karl Schmeer 1 1 Grünenthal GmbH, Aachen, 2 ABSciex, Darmstadt 1

2 Ion Mobility MS Definition and Differences Advantages: Separation of isomers, isobars, and conformers Reduction of chemical noise Measurement of ion sizes structurally similar ions and ions of the same charge state can be separated into families of ions which appear along a unique mass-mobility correlation line. Types of IMS (1) drift-time ion mobility spectrometry (DTIMS), DTIMS provides the highest IMS resolving power and is the only IMS method which can directly measure collision crosssections (2) aspiration ion mobility spectrometry (AIMS), AIMS is a low resolution mobility separation method but can monitor ions in a continuous manner (3) differential-mobility spectrometry (DMS) or field-asymmetric waveform ion mobility spectrometry (FAIMS) offer continuous-ion monitoring capability as well as orthogonal ion mobility separation in which high-separation selectivity can be achieved (4) traveling-wave ion mobility spectrometry (TWIMS) a low resolving power but has good sensitivity and is well integrated into a commercial mass spectrometer. 2

3 BB Schneider, TR Covey, SL Coy, EV Krylov, Erkinjon G Nazarov Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry, International Journal of Mass Spectrometry 298 (2010)

4 SelexION TM Orifice Plate Removable Interface Heater DMS Cell (polished surface on cell and orifice to provide a gas tight seal) Analytical gab: (space between electrodes) 30mm x 10mm x 1mm With a drag of 2.8 l/min, the residence time of a particle in the gab is 6.5 ms (to be added on pause time; standard setting: 20 ms) Jet-Stream Area 2 3 Torr Throttle Gas constant vacuum drag 2.8 l/ min 760 Torr Curtain plate with DMS- Heater and Sensor (Temperature adjustable) to establish a constant gas temperature. Heat exchanger Throttle gas (Bleed gas) used to throttle the flow through the gab i.e. increasing the residence time and improving resolution. Curtain Gas 4

5 Field Asymmetric Waveform COV (mostly -20 to 30 V, possible range -100 to 100 V) E [V/cm] t H E(H) SV (<4kV) -0.5 t [s] E(L) -1.0 t L 5

6 Differential Mobility E [V/cm] t [s]

7 Compensation Voltage (CV) MHz, F = 0 Separation Voltage (SV) 6 MHz, F = 90 BB Schneider, TR Covey, SL Coy, EV Krylov, Erkinjon G Nazarov Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry, International Journal of Mass Spectrometry 298 (2010)

8 5200 SV: 3500 V parent 400 SV: 3000 V but with modifier parent parent + O parent + O 2800 parent -methyl parent -methyl parent methyl +O 400 parent methyl +O COV, Volts COV, Volts 8

9 Comparison (using a validated method) 1.8e e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 ohne Selexion e4 7.8e e4 6.0e4 5.0e4 4.0e4 3.0e4 mit Selexion e Time, min 9

10 Comparison (using a validated method) 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 S/N = 7.9 Peak Int.(Subt.)=2.8e+3 Ymax=1.3e+3 cps Ymin=9.8e+2 cps - Noise -! 3.93! ohne Selexion 4.04!- Noise! - S/N = 58.8 Peak Int.(Subt.)=2.6e+4 Ymax=1.1e+3 cps Ymin=6.1e+2 cps e4 1.4e4 1.2e4 1.0e Ymax=9.0e cps Ymin=2.0e+1 cps!- Noise! - mit Selexion S/N = 51.4 Peak Int.(Subt.)=3.6e+3!- Noise! - S/N = Time, min 4.35 Peak Int.(Subt.)=1.5e+4 Ymax=1.4e+2 cps Ymin=5.0e+1 cps 10

11 Parent with and without Selexion e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 1.8e4 1.6e4 1.4e4 Instrumentation Agilent 1200 API 5500, ESI+ eluent A: eluent B: column: trivial pursuit, with guard col. gradient: t [min] % B inj: vol.: 10 µl flow: 700 µl/min 4.80 parent in plasma without Selexion with Selexion 1.2e4 1.0e4 800 valve switch Time, min 11

12 Parent with and without Selexion S/N without Selexion: 67 with Selexion: 134 parent in plasma valve switch without Selexion with Selexion Time, min 12

13 Parent with and without Selexion Parent compound ("1 / x" weighting): without Selexion: y = x r = with Selexion: y = x r = Analyte Area / IS Area Analyte Conc. / IS Conc. 13

14 Metabolite with and without Selexion 3.1e4 3.0e e4 2.6e4 2.4e4 2.2e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 800 metabolite in plasma (not optimized) without Selexion with Selexion Time, min 14

15 Metabolite with and without Selexion Metabolite ("1 / x" weighting): without Selexion: y = x r = with Selexion: y = x r = Analyte Area / IS Area Analyte Conc. / IS Conc. 15

16 Modifier M M M M Analyte M M M M M M M M Analyte M M M M 9. LC/MS-Diskussionstreffen, Wuppertal 16

17 2.2e5 2.1e5 2.0e5 separation voltage (SV): 2000 V 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 L-Adrenalin, Epinephrine 183,20 Th 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 3,4-Dihydroxyphenethylamin, Dopamine 153,18 Th 7.0e4 6.0e4 5.0e4 4.0e4 L-( )-Noradrenalin, (R)-( )-Norepinephrine 169,18 Th 3.0e4 1.0e4 Serotonin, 5-Hydroxytryptamin 176,22 Th COV, Volts 9. LC/MS-Diskussionstreffen, Wuppertal Cortisol, 11β-Hydroxycortison 362,47 Th 17

18 9.0e5 Modifier 8.0e5 7.0e5 L-Adrenalin, Epinephrine 183,20 Th 6.0e5 5.0e5 4.0e5 3,4-Dihydroxyphenethylamin, Dopamine 153,18 Th 3.0e5 L-( )-Noradrenalin, (R)-( )-Norepinephrine 169,18 Th 2.0e5 1.0e5 Serotonin, 5-Hydroxytryptamin 176,22 Th COV, Volts Cortisol, 11β-Hydroxycortison 362,47 Th 18

19 2.0e5 9.0e5 8.0e5 2.0e5 3,4-Dihydroxyphenethylamin, Dopamine 153,18 Th 1.8e5 1.6e5 1.4e5 7.0e5 1.2e5 L-Adrenalin, Epinephrine 183,20 Th 6.0e5 1.0e5 5.0e5 4.0e5 1.0e5 8.0e4 6.0e4 4.0e4 3.0e5 2.0e5 L-( )-Noradrenalin 169,18 Th COV, Volts 1.0e COV, Volts COV, Volts 19

20 2 mm column 1.00e6 5.00e5 XIC of +MRM (6 pairs): Neurotransmitter without Selexion, without modifier 0 8.0e4 6.0e4 4.0e4 XIC of +MRM (6 pairs): Neurotransmitter with Selexion, without modifier XIC of +MRM (6 pairs): Neurotransmitter with Selexion, with modifier (iso-prop) 1.5e4 1.0e4 500?? Time, min 20

21 2 mm vs. 1 mm column 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 1.0e4 XIC of +MRM (6 pairs): Neurotransmitter with Selexion 2 mm column 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 1.0e4 XIC of +MRM (6 pairs): Neurotransmitter with Selexion 1 mm column Time, min 21

22 1 mm column 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 1.0e4 with selexion 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 with selexion and modifier (MeOH) 4.0e4 3.0e4 1.0e Time, min 22

23 1 mm column, modifier 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 1.0e4 3.0e4 MeOH 2.5e4 1.5e4 1.0e4 500 Iso-prop Time, min 23

24 Area, counts 6.7e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 1.5e4 1.0e e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 Calibrations (without ISTD) Corticosteron: ("1 / x" weighting): y = 314 x (r = ) Concentration, ng/ml Cortisol: ("1 / x" weighting): y = 351 x (r = ) Area, counts 1.03e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e e4 5.0e4 4.5e4 4.0e4 Noradrenalin: ("1 / x" weighting): y = 523 x (r = ) Concentration, ng/ml Serotonin: ("1 / x" weighting): y = 2.64e+003 x (r = ) 5.0e4 3.5e4 Area, counts 4.5e4 4.0e4 3.5e4 Area, counts 3.0e4 2.5e4 3.0e4 2.5e4 1.5e4 1.5e4 1.0e4 1.0e Concentration, ng/ml Concentration, ng/ml Area, counts 2.2e5 2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 1.0e4 Dopamin: ("1 / x" weighting): y = 1.04e+003 x e-005 (r = ) Concentration, ng/ml 5.0e5 4.8e5 4.6e5 4.4e5 4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 9. LC/MS-Diskussionstreffen, 4.0e4 Area, counts Wuppertal Adrenalin: ("1 / x" weighting): y = 2.45e+003 x (r = ) Concentration, ng/ml 24

25 Modfier and Signal +MRM / Da mit Selexion 1.24e5 1.20e5 1.15e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Serotonin, 5-Hydroxytryptamin 176,22 Th N 2 methanol acetonitrile iso-propanol Time, min 25

26 Relation of compensation voltage (CV) to a) Proton affinity, (PA), in kcal*mol b) mass (m) of the employed modifier. c) A linear combination of both parameters yields a linear relationship of [log(m) + log(pa-100) with compensation voltage. V Blagojevic, A Chramow, BB Schneider, TR Covey, DK Bohme Differential Mobility Spectrometry of Isomeric Dipeptides: Modifier and Field Effects on Ion Mobility and stability Analytical Chemistry 2011, 83,

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28 Thus.. separation depends on Nebulizer gas flow Curtain gas flow Temperature Throttle gas flow Flow rate the Modifier Polartiy Electron density Size 9. LC/MS-Diskussionstreffen, Wuppertal 28

29 to buy, or not to buy 29

30 Pro A very powerful tool, that may a) add an other dimension to the analysis, or b) remove distortions c) separate even of very similar compounds Quick and easy to mount and to remove Good implementation into analyst (1.5) 30

31 Still many things to learn. a) especially on modifiers (modifier mixtures?) b) theory so far not entirely understood. Optimization for each analyte required. Thus, method optimization may take some time. Electrodes need to be cleaned, intervals depend on the matrix Contra 31

32 Acknowledgements Holm Sommer, Sciex Engelbert Wagner, Sciex Andreas Müller, AµMass Rolf Terlinden, Grünenthal Klaus Pusecker, Grünenthal 9. LC/MS-Diskussionstreffen, Wuppertal 32

33 Bibliography General IMS 1. AB Kanu, P Dwivedi, M Tam, L Matz HH Hill: Ion mobility mass spectrometry J. Mass Spectrom. 2008; 43: 1 22 DMS 1. EV Krylov, EG Nazarov: Electric field dependence of the ion mobility International Journal of Mass Spectrometry 285 (2009) BB Schneider, TR Covey, SL Coy, EV Krylov, EG Nazarov: Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry International Journal of Mass Spectrometry 298 (2010) Evgeny V. Krylov, Stephen L. Coy, John Vandermey, Bradley B. Schneider, Thomas R. Covey et al.: Selection and generation of waveforms for differential mobility spectrometry REVIEW OF SCIENTIFIC INSTRUMENTS 81, BB Schneider, TR Covey, SL Coy, EV Krylov, EG Nazarov Control of chemical effects in the separation process in a differential mobility mass spectrometer system European Journal of Mass Spectrometry 16, (2010) SL Coy, EV Krylov, BB Schneider, TR Covey, DJ Brenner, et al. Detection of radiation-exposure biomarkers by differential mobility prefiltered mass spectrometry (DMS-MS) International Journal of Mass Spectrometry 291 (2010) LC/MS-Diskussionstreffen, Wuppertal 33

34 thank you grazie danke shukran eucaristw gracias 謝 謝 gracas merci dank je спасибо 9. LC/MS-Diskussionstreffen, Wuppertal 34