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1 COMPARISON OF CCS(N 2 ) MEASUREMENTS OBTAINED FROM TWO DIFFERENT T-WAVE ION S WITH DIRECT MEASUREMENTS USING A DRIFT TUBE ION 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(N 2 ) values from drift tube and calibrated T-Wave IM devices achieved when CCS calibrant and analyte charge state are matched INTRODUCTION With recent developments in ion mobility (IM) separation instrumentation, interest has increased in the determination of collision 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 criterion 1. Additionally they assist in structural confirmation of isoforms of protein complexes 2 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. TO DOWNLOAD A COPY OF THIS POSTER NOTE, VISIT 1

2 METHODS Instrumentation All CCS data were measured in nitrogen drift gas (CCS(N 2 )). 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. Figure 1. Schematic of Vion IMS QTof. SYNAPT G2-Si and Vion IMS QTof All data were acquired chromatographically with an ACQUITY 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. IM T-wave parameters: SYNAPT G2-Si Vion IMS QTof velocity ramp 1000 to 350ms -1 at 40V 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 N 2 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(N 2 ) 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 calculation 3. CCS(N 2 ) values were calculated using a nitrogen optimised version of MOBCAL 4. ION S WITH DIRECT MEASUREMENTS USING A DRIFT TUBE ION 2

3 Table 1. List of compound classes analysed. 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 literature 5,6, see Figure 2. Representative CCS(N 2 ) 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. Figure 3. Differences between DT SYNAPT G2- Si measured CCS(N 2 ) values and those obtained on Vion IMS QTof and a standard SYNAPT G2-Si. 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(N 2 ) values determined by a calibrated T-Wave system (of either geometry) are equivalent to those obtained by a linear field drift tube IM system. Table 2. CCS(N 2 ) values for selected drug-like small molecules. Figure 4. Absolute (upper trace) and difference (lower trace) of measured CCS(N 2 ) values from DT SYNAPT G2-Si, standard SYNAPT G2-Si, Vion IMS QTof and those obtained by Bush and McLean. Figure 2. Differences between DT SYNAPT G2-Si measured CCS(N 2 ) values and those obtained by Bush and McLean. As demonstrated above, good CCS(N 2 ) 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. ION S WITH DIRECT MEASUREMENTS USING A DRIFT TUBE ION 3

4 Effect of Charge State on Calibrated CCS(N 2 ) Measurements It has been demonstrated by Dodds et al 7 that on a calibrated T-Wave IM system to achieve the most comparable CCS(N 2 ) 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(N 2 ) 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(N 2 ) 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(N 2 ) values. Matching charge is advised to obtain the best absolute CCS (N 2 ) values, however for targeted screening purposes, the calibration method just needs to be consistent between method development and analysis. 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). Figure 5. Effect of calibration charge state on a mix of singly and doubly charged ions Measured CCS(N 2 ) 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(N 2 ). This can also result in different measured CCS(N 2 ) 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(N 2 ) 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 6. Dynamic pressure control minimising drift time shift for changes in chromatographic conditions. 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(N 2 ) %RSD are summarised in Table 3. ION S WITH DIRECT MEASUREMENTS USING A DRIFT TUBE ION 4

5 CONCLUSION Figure 7. Measured CCS(N 2 ) reproducibility on Vion IMS QTof of various drug-like small molecules. Table 3. CCS(N 2 ) reproducibility on Vion IMS QTof for 400 replicate injections. Figure 8 shows the % deviation from the mean for each analyte. Excellent correlation has been observed between in house and literature drift tube CCS(N 2 ) values. It has been demonstrated that the CCS (N 2 ) 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(N 2 ) 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(N 2 ) values. Good correlation observed between theoretically derived and experimentally determined CCS(N 2 ) values. Comparison of Measured and Theoretically Derived CCS(N 2 ) Values Excellent correlation has been observed for experimentally measured and theoretically derived CCS (N 2 ) values 3, see Table 4 for acetaminophen. References 1. Goscinny S. et al, 61st ASMS Conference, Minneapolis, MN, 9th-13th June Robinson C et al, Annu. Rev. Phys. Chem. 2015, 66, Paizs B, Bangor University, personal communication 4. Campuzano I et al, Anal. Chem. 2012, 84, McLean J et al, Anal. Chem. 2014, 86, Bush M, ccsdatabase/ 7. Dodds, E. et al Anal. Chem. 2014, 86, Table 4. Experimental and theoretical CCS(N 2 ) values for acetaminophen Waters Corporation 5