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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%