Comparability of Steroid Collision Cross Sections Using Three Different IM-HRMS Technologies: An Interplatform Study

Abstract

Steroids play key roles in various biological processes and are characterized by many isomeric variants which makes their unambiguous identification challenging. Ion mobility-mass spectrometry (IM-MS) has been proposed as a suitable platform for this application, particularly using collision cross section (CCS) databases obtained from differ-ent commercial IM-MS instruments. CCS is foreseen as an ideal additional identification parameter for steroids as long-term repeatability and interlaboratory reproducibility of this measurand are excellent and matrix effects are negligible. While excellent results were demonstrated for individual IM-MS technologies, a systematic comparison of CCS derived from all major commercial IM-MS technologies has not been performed. To address this gap, a comprehensive interlabor-atory comparison of 142 CCS values derived from drift tube (DTIM-MS), traveling wave (TWIM-MS) and trapped ion mo-bility (TIM-MS) platforms using a set of 87 steroids was undertaken. Besides delivering three instrument-specific CCS databases, systematic comparisons revealed excellent interlaboratory performance for 95% of the ions with CCS biases within ±1% for TIM-MS and within ±2% for TWIM-MS with respect to DTIM-MS values. However, a small fraction of ions (<1.5 %) showed larger biases of up to 7% indicating that differences in the ion conformation sampled on different in-strument types need to be further investigated. Systematic differences between CCS derived from different IM-MS analyz-ers and implications on the applicability for non-targeted analysis are critically discussed. To the best of our knowledge this is the most comprehensive interlaboratory study comparing CCS from three different IM-MS technologies for analysis of steroids and small molecules in general.

Critical evaluation of the role of external calibration strategies for IM-MS

“…In context of small molecules, steroid analysis is of special interest due to the large number of possible isomers, and benefits of IM-MS for steroid analysis were demonstrated previously [7,13,15,23,24]. From the analytical applications perspective, the comparability of CCS N2 for steroid analysis using three different IM-MS technologies was recently investigated, and interlaboratory bias of < 2% for the majority of the investigated ions was demonstrated [14]. Nevertheless, large deviations (up to 7%) of CCS values derived from TWIM-MS and TIM-MS to DT CCS N2 values have also been reported [14,25].…”

“…In IM-MS, analyte ions are separated based on opposing forces of an applied electric field and collisions with a neutral buffer gas (typically nitrogen) before entering the MS analyser [8,11]. As a derived property, the collision cross section (CCS) of an ion can be calculated with excellent interlaboratory precisions of typically in the range of 1-2% reported in several studies [12][13][14]. Moreover, the Published in the topical collection Making Waves in Analytical Chemistry with guest editors Johanna Irrgeher, Evelyn Rampler, and Teresa Steininger-Mairinger.…”

“…From the analytical applications perspective, the comparability of CCS N2 for steroid analysis using three different IM-MS technologies was recently investigated, and interlaboratory bias of < 2% for the majority of the investigated ions was demonstrated [14]. Nevertheless, large deviations (up to 7%) of CCS values derived from TWIM-MS and TIM-MS to DT CCS N2 values have also been reported [14,25]. In addition to the possibility of fundamental differences in ion conformations generated and sampled by the different IM-MS instruments, systematic bias of TW CCS N2 values compared to other IM-MS instruments is evident and may have its origin in the applied external calibration [14,25].…”

“…Nevertheless, large deviations (up to 7%) of CCS values derived from TWIM-MS and TIM-MS to DT CCS N2 values have also been reported [14,25]. In addition to the possibility of fundamental differences in ion conformations generated and sampled by the different IM-MS instruments, systematic bias of TW CCS N2 values compared to other IM-MS instruments is evident and may have its origin in the applied external calibration [14,25]. Alongside analytically challenging examples such as ions with complex arrival time distributions and the high level of effort required for computational prediction of CCS values using density functional theory (DFT) for large datasets [26], this issue leaves the use of CCS N2 values as an IM-MS technology-independent identification parameter in a currently unsatisfactory position [14].…”

“…In addition to the possibility of fundamental differences in ion conformations generated and sampled by the different IM-MS instruments, systematic bias of TW CCS N2 values compared to other IM-MS instruments is evident and may have its origin in the applied external calibration [14,25]. Alongside analytically challenging examples such as ions with complex arrival time distributions and the high level of effort required for computational prediction of CCS values using density functional theory (DFT) for large datasets [26], this issue leaves the use of CCS N2 values as an IM-MS technology-independent identification parameter in a currently unsatisfactory position [14]. Therefore, to further investigate the effect of the applied calibration approach and especially the role of reference values used for TW CCS N2 calibration, alternative external calibration and internal correction approaches are explored in the present work.…”