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Introduction to Owlstone ultraFAIMS

Technology Overview and Applications

Danielle Toutoungi

on 27 January 2017

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Transcript of Introduction to Owlstone ultraFAIMS

Compensation field
may be distinguished at higher E
100% DF
Compensation field
So species “co-eluting” at lower E
Owlstone ultraFAIMS

What is FAIMS?
FAIMS is a technique derived from ion mobility spectrometry that separates ions based on changes in size & shape
Applying an electric field to an ion in a buffer gas causes it to drift in the direction of the field
Ion velocity
Electric field strength
v = K E
At low fields, the drift velocity is proportional to the electric field strength and the constant of proportionality is known as the mobility (K)
[Purves R W, Guevremont R, Anal. Chem. 1999, 71, 2346-2357]
At higher electric fields, the ion mobility is not constant but varies with electric field

Separation speed is determined by the ion residence time

Typically sized FAIMS devices have residence time of ~10ms to ~100ms - hardly compatible with UHPLC and data-dependent FAIMS filtering
Ultra-fast FAIMS fundamentals
What is different about ultraFAIMS?
The Owlstone ultraFAIMS device
Each device consists of a set of parallel gaps in a metal substrate that forms the electrodes
Higher fields ensure good resolution despite the big reduction in ion residence time
In a FAIMS device, ions pass between electrodes, across which a transverse alternating electric field (the dispersion field, DF) is applied
A DC field (compensation field, CF) can be superimposed onto the DF waveform to oppose a specific drift velocity, allowing selected ions to pass through the filter electrodes
Principles of FAIMS separation
FAIMS separation depends on all terms
Higher fields also increase separation flexibility
Higher terms are more important at greater E, and ions with close α1 often have dissimilar α2 (J. Phys. Chem. A 110, 2663, 2006)
Assuming the MS can acquire multiple samples during the CF sweep, the FAIMS device provides
of the components in the sample
In a FAIMS-MS hybrid system, ions emerging from the FAIMS device pass into the inlet of the Mass Spectrometer for analysis
compensation field
FAIMS-MS Analysis
UltraFAIMS is designed to provide
How do we achieve residence time

1 ms
and still achieve good separation and sensitivity?
The solution is to reduce drift length and hence residence time...
but increase dispersion field to compensate
The field is increased by going to narrower channels
(the physics of electrical breakdown in vapour is such that narrower channels can sustain higher electric fields)
The Owlstone ultraFAIMS devices are produced using micromanufacturing techniques to produce gaps of
100 microns and below

This allows maximum electric fields of
more than double
those in millimetre-scale devices
at constant t
at constant R
75% DF
ultraFAIMS-MS Interface Schematics
The device has been interfaced with an Agilent Jetstream ionisation source
Transmission and Resolution
Overall peak capacity (without modifiers) now up to 15-20
Variable chip sizes allows easy performance adjustment
Compensation Field
Open area 5mm
Open area 7.5mm
Open area 9.5mm
Residence time: 120 microseconds*
Higher sensitivity
Residence time: 190 microseconds*
Best overall performance
Residence time: 235 microseconds*
Highest peak capacity
CF stability & sensitivity to source/sample
CF drift due to solvent/matrix composition is known to be a problem for many FAIMS designs, particularly for higher flow applications
Compensation field
Infusion expt - low flow, high DF
Morphine in ACN: water
Flow rate = 0.05ml/min
10-90% ACN:water
DF = 270Td
Peak position stable with solvent composition
Preliminary experiments with drug spiked into human plasma shows similar behaviour at similar flow rates.
LC injection expt - high flow, lower DF
Flow rate = 0.4ml/min
DF = 220Td
0.25CV scans/sec
No appreciable sensitivity to solvent/matrix composition with ultraFAIMS & Agilent Jet Stream
What can we do with high speed?
Fast stepping of CF & DF
(2ms & 10ms)
allows use with ultra-high speed chromatography
Within the LC peak duration, several FAIMS scans can be performed
During each FAIMS scan, many MS spectra can be acquired
1-5s per peak
Up to 10 FAIMS scans/sec
50-100 steps/scan (2-100ms each)
Single MRM data point or TOF spectrum per step
Real-time CF/DF scanning & hopping
Two approaches for real time data acquisition and method optimization:
Hopping between multiple MRM transitions during QQQ MS acquisition
Real time optimization of CF & DF during injection
15 complete CF scans during 2s FWHM of an LC peak
What can you do with ultraFAIMS?
Separation of pharmaceutical excipients
Protonated 2-hydroxy-4-octyloxybenzophenone (HOBP, m/z 327.1955) and the n=7 oligomer of PEG 400 (m/z 327.2013) are sufficiently close in mass (17.7ppm mass difference) that they can not be resolved by the TOF mass analyzer (required resolution ~ 130K)
An ultraFAIMS sweep at 48kV/cm produces good separation – allowing more accurate mass measurement of each ion and a clean product ion spectrum
Separation of peptide charge states
Different charge states tend to separate well in ultraFAIMS - the high DF exposes the differences in high-field mobility of the different charge states
The behaviour is consist across different peptides (both standards and tryptic digests)
Singly charged ions can be selected as a group
Ion trap mass spectrum of bradykinin
without FAIMS separation
Selective enhancement of the [M+H]+ (m/z 1060) ion at a
CV of +1.4V , DF 60kV/cm
Selective enhancement of the [M+2H]2+ (m/z 531) ion at a
CV of +3.5V, DF 60kV/cm
Ion trap mass spectra alpha-1-acid glycoprotein (AAG) tryptic digest (73pmol µl-1) –
without FAIMS both 1+ and 2+ charge states are present
Using appropriate FAIMS CV, the doubly charged ions can be filtered out
, leaving just a spectrum of singly charged ions
Assisting protein identification
The top 20 ions in the spectra list generated from the FAIMS-filtered spectra was searched against the SwissProt protein PMF database using the MASCOT31 search engine
Similar approach to that used in MALDI-TOFMS
Alternatively, multiply charged peptide ions can be individually selected and fragmented and the resulting spectrum used to identify the peptide
Optimum CV for transmission of doubly charged m/z 877 ion applied (+3V), excludes singly charged species and other doubly charged peptide ions
Selection of parent ions prior to in-source CID
The ultraFAIMS device can be used to select a subset of ions which can then be fragmented using in-source CID - the pre-selection produces simplified fragment ion spectra that can be used for identification or quantification without the need for tandem MS
FISCID analysis of tryptic peptides in plasma
TIC of tryptic peptides from LC analysis of human plasma
The complex nature of the sample results in co-elution of peptides e.g. here around RT=3.5mins, 3 peptides co-elute
SICs of 3 of the tryptic peptide ions
LC-MS spectrum at 3.5mins
without FAIMS separation
LC-MS spectrum at 3.5mins
with FAIMS at DV=47kV/cm and CV=2.5-2.6V
- showing preferential transmission of the m/z 480 peptide
LC-in source CID-MS spectrum at 3.5mins
without FAIMS separation
UltraFAIMS allows pre-selection of peptides prior to fragmentation...
LC-in source CID-MS spectrum at 3.5mins
with FAIMS at DV=47kV/cm and CV=2.5-2.6V
- showing a far simpler fragment pattern
This results in a simpler fragment spectrum, which increases the probability of identification
TOF fragmentor voltage at 75V
TOF fragmentor voltage at 340V
Peptide identification was carried out via the MASCOT search engine, searched against the SwissProt protein database. All ions with greater than 10% of the base peak intensity were included in the search peak list.
Peptide quantification by LC-FISCID-MS
Gramcidin S (m/z 571) spiked into human plasma sample at different concentrations
FAIMS used to select the Gramcidin S ion for transmission or fragmentation (fragments at m/z 311,424, 685, 798)
The FISCID technique can also be used for quantitative analysis
Improved metabolite quantification
Direct infusion of an IAG standard was used to optimise FAIMS transmission of IAG
The quantitative performance of the prototype LC-FAIMS-MS system was evaluated by the analysis of (R/S) Ibuprofen 1-β-O acylglucuronide (IAG) metabolite spiked into urine
Higher LDR observed with FAIMS separation, by over 3 orders of magnitude
Improved intra-day reproducibility was observed with FAIMS pre-selection of the [IAG-H]- ion
The absolute intensity of the [IAG-H]- peak is reduced ~50% because of lower FAIMS transmission, but is compensated by an improvement in signal to noise ratio, which lowers the LOQ from 0.018 to 0.010 µg/ml
The incorporation of a FAIMS separation in the LC-MS analysis significantly reduced chemical interference from urine
Urine blank
IAG spiked urine sample
Metabolite identification
The ability to select the metabolite based on differential mobility results in the enhancement of IAG derived fragment ion peaks in mass spectrum (bottom) compared with LC-CID-MS without FAIMS (top)
LC-FISCID-MS analysis of IAG in urine, monitoring the m/z 205 fragment of the FAIMS-selected [IAG-H]- ion (bottom), shows enhanced selectivity compared to FAIMS off LC-CID-MS (top)
(V) aglycone m/z 205
Four fragments are produced from [IAG – H]- by CID
(II) decarboxylated di-dehydrated glucuronate m/z 113
(III) dehydrated glucuronate m/z 175
(IV) glucuronate m/z 193
- IAG fragments are difficult to locate due to the complexity of the mass spectrum and poor signal to noise as a result of other ions in the matrix
- signal-to-noise ratios for the fragment ions increased by approximately 2-fold, enhancing the response of the IAG fragments relative to other interfering peaks to aid identification
Selection of large proteins from a complex matrix
Large (>30kDa) proteins such as BSA tend to behave as type A ions in the extreme ultraFAIMS dispersion fields, whereas smaller proteins and peptides behave as type C ions - this means they can be readily separated by selection of appropriate CF

With FAIMS selection at CF = 0
- the non-BSA ions over the whole mass range are suppressed by 10000 times, allowing detection of nearly the full charge state distribution of BSA ions
Mass spectra for a solution of ubiquitin and trace of BSA in a complex matrix
with no FAIMS
Compensation field /Td
Improved accurate mass measurement
We selected an analyte (2-hydroxy-4-(octyloxy)benzophenone with the same nominal mass – 327 – as one of the PEG400 ions - and applied a FAIMS DF of 48kV/cm
Extracted chromatogram for 326.3-330.3
Sample: 2-hydroxy-4-(octyloxy)benzophenone (0.01mg/ml) + PEG400 (0.1mg/ml) in 50:50 ACN:water + >0.1% formic acid
2-hydroxy-4-(octyloxy)benzophenone has m/z 327.1960
The two ions were well resolved in the FAIMS spectrum, allowing each to be individually selected for accurate mass measurement - reducing the mass error in each case
Separation & quantification of isobaric PGIs
Use of modifiers to enhance selectivity
ESI-FAIMS-MS spectrum of a mixture of the three PGI’s overlaid with individual components
UltraFAIMS can be used to enable rapid and direct analysis of PGIs using thermal desorption in conjunction FAIMS-MS or FISCID-MS.
The 3 PGIs were partially resolved at a DF of 228Td
The chip was set to constantly transmit at a compensation field of 1.3 Td to preselect NNDMT. A thermal desorption profile for the molecular ion of NNDMT (m/z 136.1121) was obtained
50 ng of NNDMT was spiked into 50 mg of starch to simulate a PGI concentration of 1 ppm. This was placed into a TD tube and thermally desorbed.
This profile shows a peak-to-peak signal to noise ratio of 11.8:1 indicating that this response is in the quantifiable range.
The calculated limit of detection for NNDMT is
250 ppb
. Since the threshold for toxicological concern equates to a control limit of ~1.5ppm, this LOD is sufficient to make TD-FAIMS-MS suitable for fast toxicological profiling of APIs.
J.Reynolds et al, ISIMS poster presentation 2012
The addition of solvent vapours and modifier gases to the carrier gas can enhance peak separation in ultraFAIMS, as well as in macro-scale FAIMS devices - this is likely to open up a wide range of further applications
Effect of adding increasing concentration of carbon dioxide (top row) and methanol (bottom row) on separation of the [M-H]- ions of three positional isomers of phthalic acid. The figures on the right illustrate the best separation achieved.
C. Beekman et al, IMSC 2012 poster presentation
By ramping the CF over a range of values, all the ions in the sample can be transmitted through the filter in sequence, producing a CF spectrum
MS inlet
The use of multiple channels ensures total ion capacity is not reduced
Batch micro-fabrication techniques allow extremely precise control over ion channel dimensions on a micron scale.
The device is small, stable and mechanically robust
Transmission as shown in table (blue fields indicate typical operating condition)
*for 1.7L/min MS inlet flow
Ugarov et al., ASMS presentation, 2012
Ugarov et al., ASMS presentation, 2012
L.J. Brown et al, Anal. Chem., 2012, 84 (9), pp 4095–4103
L.J. Brown et al, Anal. Chem., 2012, 84 (9), pp 4095–4103
Ugarov et al, ASMS presentation, 2012.
L.J. Brown et al., Anal. Chem. 2010, 82, 9827–9834
L.J. Brown et al., Anal. Chem. 2010, 82, 9827–9834
S/N ratio 100:1 (m/z1060); 1000:1 (m/z 531))
S/N ratio 1000:1
S/N ratio 10000:1
Multiply charged ions can be selected as a group or individually
L.J. Brown et al., Anal. Chem. 2010, 82, 9827–9834
AAG was identified as the top hit with a significant confidence score of 61 (where 56 or above is deemed statistically significant at a 95% confidence interval)
Ion trap MS/MS spectrum of m/z 877 ion
L.J. Brown et al., Anal. Chem. 2010, 82, 9827–9834
L.J. Brown et al., Anal. Chem. 2010, 82, 9827–9834
With the CV of 2.5-2.6 V applied, human serum albumin (HSA) was identified as the top hit, the only significant match, with a confidence score of 34 (where 27 or above was deemed statistically significant at a 95% confidence interval).
With no FAIMS separation, LC-in source CID-MS yielded no significant hits on the database.
L.J. Brown et al., Anal. Chem., 2012, 84 (9), pp 4095–4103
L.J. Brown et al., Anal. Chem., 2012, 84 (9), pp 4095–4103
L.J. Brown et al., Anal. Chem., 2012, 84 (9), pp 4095–4103
RSDs (n=6, at 0.45ug/ml) were 5.1% for precursor, and <15% for fragments
Peak areas for all ions produce a linear response (R2>0.99) between 0.45-9.0ug/ml
R. Smith et al, J. Chromatogr. A, 2012 (under review)
R. Smith et al, J. Chromatogr. A, 2012 (under review)
R. Smith et al, J. Chromatogr. A, 2012 (under review)
A. Shvartsburg et al., Anal Chem. 2012 Sep 4;84(17):7297-300
J.Reynolds et al, ISIMS poster presentation 2012
J.Reynolds et al, ISIMS poster presentation 2012
For more information - or to discuss potential applications testing, please contact us at
The mobility is related to the size and shape of the ion, and different ions drift at different speeds for a given field
The alternating asymmetric high/low electric field also creates a net drift, but now the drift velocity depends on the difference between the high- and low-field mobility of the ion
Separation speed is inversely proportional to residence time, so a doubling of enables
16 to 64
times faster separation without loss of resolution
L.J. Brown et al., Anal. Chem., 2012, 84 (9), pp 4095–4103
500um gap - max field ~5V/um

100um gap - max field ~10V/um
Large protein
Small protein
Full transcript