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COMPARISON OF CCS(N2) MEASUREMENTS OBTAINED FROM TWO DIFFERENT T-WAVE ION MOBILITY SYSTEMS WITH DIRECT MEASUREMENTS USING A DRIFT TUBE ION MOBILITY SYSTEM Kevin Giles, Martin Palmer, Keith Richardson, Nick Tomczyk
Waters MS Technologies Centre, Wilmslow, UK
OVERVIEW
Favourable comparison of two alternative
geometry T-wave ion mobility systems (IMS QTof
and Q IMS Tof) with a drift tube ion mobility system and literature values
Best correlation between measured CCS(N2)
values from drift tube and calibrated T-wave IM devices achieved when CCS calibrant and analyte
charge state are matched
Improved reproducibility of measured CCS(N2)
values with novel pressure feedback control
Good correlation of measured and theoretically
derived CCS(N2) values
INTRODUCTION
With recent developments in ion mobility (IM) separation instrumentation, interest has increased in the determination of
collisional cross-sections (CCS) of various classes of
compounds. These CCS measurements can be used to augment screening of complex samples both by reducing
interfering effects of matrix ions and as an additional identification criterion1. Additionally they assist in structural
confirmation of isoforms of protein complexes2 and structural isomers of small molecules. The widely used T-Wave IM
system relies on calibration to provide CCS values as there is no direct analytical solution for the complex motion of ions
through the device. The efficacy of T-wave CCS calibration is investigated by comparison of CCS values obtained from a
linear-field drift tube IM system, theoretical calculations and available literature values.
METHODS
Instrumentation
All CCS data were measured in nitrogen drift gas (CCS(N2)).
The instruments used for this study were a SYNAPT G2-Si, a modified SYNAPT G2-Si where the T-wave IM cell was replaced
with a linear field drift tube and a novel geometry T-wave IM instrument, Vion IMS QTof, a schematic of which is shown in
Figure 1.
SYNAPT G2-Si and Vion IMS QTof
All data were acquired chromatographically with an AQUITY I-Class and electrospray ionisation. A simple linear gradient of
acidified water and acetonitrile was used to separate analytes
of interest. The T-wave IM systems were calibrated using a mixture of
acetaminophen and poly-D,L-Alanine (Sigma-Aldrich) using the automated calibration routine in the acquisition control
software. The Vion IMS QTof calibration also corrects for mass dependant transmission time post IM separation.
Furthermore when data from the two T-wave IM systems and the DT SYNAPT G2-Si are compared to the literature values
good correlation is observed, see Figure 4. This further demonstrates that the CCS(N2) values determined
by a calibrated T-wave system (of either geometry) are
equivalent to those obtained by a linear field drift tube IM system.
Figure 8 shows the % deviation from the mean for each
analyte.
Comparison of Measured and Theoretically Derived CCS(N2) Values
Excellent correlation has been observed for experimentally measured and theoretically derived CCS(N2) values3, see
Table 4 for acetaminophen.
CONCLUSION
Excellent correlation has been observed between in
house and literature drift tube CCS(N2) values.
It has been demonstrated that the CCS(N2) values
determined by calibrated T-wave systems (of either geometry) are equivalent to those obtained by a linear
field drift tube IM systems.
The term ‘deviation’ should be used to describe
observed variation between experimentally measured CCS(N2) values not ‘error’ or ‘accuracy’ as these terms
imply that a given experimentally observed value is correct and free from measurement error with respect
to another experimentally observed value.
The charge state of a calibration should ideally be
matched to intended analytes, however for targeted screening purposes, the calibration method just needs
to be consistent between method development and analysis.
A novel dynamic IM pressure adjustment results in
improved robustness of observed CCS(N2) values.
Good correlation observed between theoretically
derived and experimentally determined CCS(N2) values.
References
1. Goscinny S. et al, 61st ASMS Conference, Minneapolis, MN, 9th-13th June 2013.
2. Robinson C et al , Annu. Rev. Phys. Chem. 2015, 66, 453-474
3. Paizs B, Bangor University, personal communication
4. Campuzano I et al , Anal. Chem. 2012, 84, 1026-1033
5. McLean J et al, Anal. Chem. 2014, 86, 2107-2116
6. Bush M, http://depts.washington.edu/bushlab/ccsdatabase/
7. Dodds, E. et al Anal. Chem. 2014, 86, 11396-11402
In order to prevent this pressure effect occurring in Vion IMS
QTof a real time feedback loop has been implemented where the trap and IM pressure are continuously measured and any
changes in the pressure are compensated for by adjusting the nitrogen flow rate delivered by the trap and IM mass flow
controllers. Once active the real time pressure monitor corrects for
changes in source pressure conditions every 100ms. This phenomenon can be observed in Figure 6, where measured
drift time values of a small molecule mix are compared. The sample mix was infused into a t-piece along with either 250µL/
min 8/2 acetonitrile/water or 800µL/min 1/9 acetonitrile/water delivered by an LC system to mimic a significant change in LC
gradient and hence source pressure conditions. A shift in the measured drift time is observed when the post solvent
composition is changed and the dynamic pressure control
(DPC) is disabled (blue in Figure 6) whereas no shift is observed when the DPC is enabled (pink in Figure 6).
A further benefit of dynamic pressure control is illustrated in Figures 7 and 8, which demonstrate long term stability of
measured CCS(N2) values of 400 replicate injections of a drug-like small molecule mix. A high degree of precision is obtained
over the 33 hours taken to acquire these data, which would not be achieved without the dynamic pressure control
preventing pressure drift. The measured CCS(N2) %RSD are
summarised in Table 3.
IM T-wave parameters:
SYNAPT G2-Si velocity ramp 1000 to 350ms-1 at 40V Vion IMS QTof velocity ramp 850 to 350ms-1 at 60V
Linear field drift tube (DT) SYNAPT G2-Si
All CCS data were acquired by direct infusion at 3µL/min into
the electrospray source. Each sample was analysed at three different N2 IM gas pressures (circa 1.8, 2.0 and 2.4 mBar). At
each gas pressure the drift time was measured at eight individual linear fields to remove the constant offset from the
drift time and the CCS was calculated using the Mason-Schamp
equation. The CCS value is reported as the mean obtained from the three gas pressures.
Typical linear fields were between 2 and 12 Vcm-1 with a drift tube length of 25.5cm.
Theoretical Calculation of CCS(N2) Values
In order to obtain energy minimised structures, an initial
structural and protomer search was carried out. This was followed by a full density functional theory structural
optimisation and charge distribution calculation3. CCS(N2)
values were calculated using a nitrogen optimised version of MOBCAL4.
Sample Preparation
Eight classes of compounds were analysed across all three
platforms, the classes and number of components are summarised in Table 1.
All samples for chromatographic analysis were prepared in mobile phase A (0.1% aqueous formic acid), samples for direct
infusion were prepared in 1:1 water:acetonitrile + 0.1% formic
acid.
RESULTS
Comparison of T-wave systems and Linear Field Drift Tube
The data obtained from the DT Synapt G2-Si compares
favourably with those values available in the literature5,6, see Figure 2. Representative CCS(N2) values for a selection of
drug-like small molecules are shown in Table 2. Good correlation can be observed in Table 2 between the two
calibrated T-wave systems and the linear field drift tube. This can also be observed in Figure 3 for all compounds.
As demonstrated above, good CCS(N2) reproducibility can be
achieved across multiple IM MS system platforms, however, it should be noted that within all of these values there will be a
small degree of experimental variance for all techniques and as such no one value is necessarily ‘correct’ and as such, terms
like ‘error’ and ‘accuracy’ should be used with caution when comparing experimentally derived CCS values.
Effect of Charge State on Calibrated CCS(N2)
Measurements
It has been demonstrated by Dodds et al7 that on a calibrated
T-wave IM system to achieve the most comparable CCS(N2) values with those obtained with a linear field drift tube system
the charge state of the calibrant and analyte should ideally be matched.
This effect can be observed in Figure 5. This shows the measured CCS(N2) values (from Vion IMS QTof) for a series of
a singly and doubly charged ions when a calibration created from a singly charged series (blue trace) or doubly charged
series (red trace) is applied to the acquired data. The T-wave IM CCS(N2) values for those components whose
charge state matches the charge state of the calibrant exhibit the smallest deviation with respect to the linear field drift tube
measured CCS(N2) values. Matching charge is advised to obtain the best absolute CCS(N2)
values, however for targeted screening purposes, the
calibration method just needs to be consistent between method development and analysis.
Measured CCS(N2) Reproducibility of Vion IMS QTof system
In order to maintain drift time (and hence CCS) reproducibility it is important to have constant IM cell parameters, such as
fields, pressures etc.. Any external factors affecting these conditions may have a detrimental effect on experimental
precision. An aspect of the geometry of an IM QTof is that any changes
in pressure in the source region of the instrument (e.g. due to different LC flow rates or composition) will cause a
corresponding change in the trap and IM pressure. This change in pressure will manifest itself as a change in overall drift time
and hence measured CCS(N2). This can also result in different measured CCS(N2) values if data are compared between an
infusion and chromatographic acquisition or using when
different LC conditions to those used during calibration or generation of library CCS(N2) values.
These effects are not observed on the Synapt platform (geometry Q IM Tof) owing to the high pressure ion mobility
cell is isolated from the source by a high vacuum quadrupole region.
Figure 1. Schematic of Vion IMS QTof.
Table 1. List of compound classes analysed.
Compound Class Number of Analytes
Drug-like small molecule 37
Pesticide 13
Saccharide 2
Phosphazine 9
Peptide (z=1 to 5) 84
Synthetic peptide 12
Tetra-alkyl ammonium salts 10
Synthetic polymer 10
Table 2. CCS(N2) values for selected drug-like small molecules.
Name m/z
CCS(N2) / Å2
Vion IMS QTof
SYNAPT G2-Si
DT SYNAPT G2-Si
Sulfadimethoxine 311.08 168.8 168.5 167.9
Terfenadine 472.32 229.8 230.4 227.7
Leucine
Enkephalin 556.28 229.9 230.8 229.3
Reserpine 609.28 251.0 252.5 253.2
Erythromycin 734.47 265.8 264.1 265.1
Figure 5. Effect of calibration charge state on a mix of singly
and doubly charged ions
Table 3. CCS(N2) reproducibility on Vion IMS QTof for 400
replicate injections.
Mean CCS(N2) / Å2 %RSD
Caffeine 135.4 0.2%
Sulfadimethoxine 168.3 0.2%
Verapamil 212.0 0.2%
Terfenadine 230.3 0.1%
Leucine Enkephalin 230.8 0.2%
Reserpine 251.0 0.1%
Figure 2. Differences between DT SYNAPT G2-Si measured
CCS(N2) values and those obtained by Bush and McLean.
Bush drift tubeMcLean drift tube
Figure 3. Differences between DT SYNAPT G2-Si measured
CCS(N2) values and those obtained on Vion IMS QTof and a standard SYNAPT G2-Si.
Synapt G2-SiVion IMS QTof
Figure 4. Absolute (upper trace) and difference (lower trace) of measured CCS(N2) values from DT SYNAPT G2-Si, standard SYNAPT
G2-Si, Vion IMS QTof and those obtained by Bush and McLean.
Bush drift tubeSynapt drift tubeMcLean drift tubeSynapt G2-SiVion IMS QTof
Figure 8. Measured CCS(N2) reproducibility on Vion IMS QTof of
various drug-like small molecules.
CaffeineLeu-enkephalinReserpineSulfadimethoxineTerfenadineVerapamil
Figure 7. Measured CCS(N2) reproducibility on Vion IMS QTof of
various drug-like small molecules.
Reserpine - 609.28 m/z : 252 Å2 - 0.10 % RSD
Leu-enkephalin – 556.27 m/z : 230 Å2 - 0.15 % RSD
Terfenadine – 472.32 m/z : 230 Å2 - 0.12 % RSD
Verapamil – 455.29 m/z : 211 Å2 - 0.17 % RSD
Sulphadimethoxine – 311.08 m/z : 168 Å2 - 0.15 % RSD
Caffine – 195.08 m/z : 136 Å2 - 0.22 % RSD
Figure 6. Dynamic pressure control minimising drift time shift
for changes in chromatographic conditions.
Dynamic pressure controlDisabledEnabled
Table 4. Experimental and theoretical CCS(N2) values for
acetaminophen.
Vion IMS QTof
SYNAPT G2-Si
DT SYNAPT G2-Si
Bush DT
Experimental
CCS(N2) / Å2
130.8 128.9 130.2 130.4
Theoretical
CCS(N2) / Å2
130.3 130.3 130.3 130.3
% deviation 0.38% -1.07% -0.08% 0.08%