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Introduction
Identification and Quantification of In Vitro and In Vivo metabolites are a routine procedure for the Pharma industry.
Knowledge of low level, potentially toxic metabolites is required earlier and earlier in the drug discovery progress to avoid costly development of ill-fated drug candidates.
The chemical noise of metabolism samples results in the need for potentially time consuming sample clean-up reducing through-put and increasing cost.
Instrumentation and conditions
- Accela uHPLC
- 200uL/min
- Hypersil Gold 50mm x 1.9um column
- FAIMS
- 50:50 He:N2 carrier gas
- Compensation Voltages -13.0V and -15.5V
- Inner Electrode 80°C
- Outer Electrode 100°C
- LXQ Linear Ion Trap Mass Spectrometer
- 2 CVs with Data Dependant most intense ion
- Dynamic Exclusion on
| Time |
Water + 0.1% Formic Acid |
ACN + 0.1%
Formic Acid |
| 0 |
95% |
5% |
| 1 |
95% |
5% |
| 1.5 |
75% |
25% |
| 7.5 |
15% |
85% |
| 8 |
15% |
85% |
Samples
Samples were T=0 and T= 15mins of 5µm Loperamide in rat liver microsomes.
Once the samples were taken the reaction was quenched with an equal volume of ACN.
FAIMS on Ion Traps: A New Separation Scheme
FAIMS on LXQ
Diagram of High Field Asymmetric Waveform Ion Mobility Spectrometry
Experimental Method for Track I (Ion Max) and Track II (FAIMS) Mass Spectral Approaches.
Experimental Method for Track I (Ion Max) and Track II (FAIMS) Mass Spectral Approaches.
Track I: Ion MAX
Source and chromatographic conditions were adjusted to give optimal S/N for the majority of ion of interest. Full scan MS and data dependent MS/MS spectra were collected (Track I).
Track II: FAIMS
The experimental sequence described above for Track I: Ion Max was replicated in the Track II sequence except that an atmospheric pressure FAIMS device was inserted between the ionization source and the mass spectrometer.
CV Scan between -40V and +10V for Direct Infusion of a 5 µM Incubation of Loperamide and its Metabolites
Chemical noise Reduction
Compilation of Analytical Results for Ion Max and FAIMS for a 5 µM Microsome Incubation of Loperamide at t= 15min
| Compound |
Ion Max S/N |
FAIMS S/N |
Ratio |
| Parent: 477.2 |
769 |
2460 |
3.2 |
| M1: 479.2 –CH3,+OH |
40,91 |
82,153 |
2.1,1.7 |
| M2: 493.2 +OH |
--,112 |
18, 201 |
∞, 1.8 |
| M3: 463.1 -CH3 |
1069 |
4735 |
4.4 |
| M4: 455.1 -OH,-6H |
24 |
86 |
3.6 |
| M5: 441.2 -OH,-6H,-CH3 |
52 |
107 |
2.1 |
T= 15 mins sample - NoFAIMS
T= 15 mins sample -FAIMS
Conclusions
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High-Field Asymmetric-waveform Ion Mobility Spectrometry (FAIMS) has been implemented in the LC-data dependent full scan MS/MS analysis
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FAIMS provides an orthogonal separation based on shape, size, charge and can be used to dramatically reduce chemical noise due, for example, to solvent clusters, dosing vehicles, or endogenousmatrix
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In these studies we observe significant reduction in Full Scan MS chemical noise at mass-to-charge ratios below 300
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In addition, implementation of FAIMS in this analysis resulted in an improvement of between 2X and 4X vs standard Ion Max for the XIC peak area S/N ratios
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Finally, but most notably, FAIMS reveals a second hydroxylated metabolite, M2*, which was undetected in the Track I (standard Ion Max) experiments. Without FAIMS, full scan chemical noise at the mass-to-charge of interest makes peak assignment impossible for this low level metabolite.
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