MP135 Evaluation of a Cyclic Mobility-Enabled Research Platform with Multiple-Pass Ion Geometry for Metabolite Characterisation Studies
Richard Clayton, Catherine Holdsworth, Nick Tomczyk, Kevin Giles, Jakub Ujma, Daniel Weston
Covance, Harrogate, North Yorkshire, United Kingdom
Waters Corporation, Wilmslow, Cheshire, United Kingdom
Introduction
The performance of routine ion-mobility in a production mass spectrometer has shown great utility in mapping the chemical space of the metabolite characterisation experiment, capable of discriminating between isomeric metabolites in complex samples. In this proof-of-concept work, the use of a cyclic mobility-enabled research platform is discussed with the aim of providing greatly increased ion-mobility resolution and analytical flexibility, whilst assessing the added value and impact for metabolite characterisation in support of a biotransformation-led endpoint. Criteria for assessment included the more confident assignment of metabolite structure, particularly for previously unobserved metabolites, the ability to discriminate between isomeric (often co-eluting) metabolites, and the reduction of false negatives at trace levels.
Methods
Initial proof-of-concept experiments were carried out using nefazodone incubated with cryopreserved hepatocytes and relevant cofactors (37 °C, from 0 to 240 min.) prior to termination with acetonitrile, centrifugation and supernatant dilution. Samples were introduced using direct injection via a reversed-phase UPLC gradient (Waters Acquity FTN and BSM, flow rate of 400 µL/min, Waters HSS-T3 column (2.1 x 50 mm (1.7 µm) held at 40 °C) into a modified SYNAPT HDMS system fitted with a prototype cyclic ion-mobility device, operated in positive-ion electrospray (ESI) mode. Multiple-pass ion geometry was used to investigate the benefit of increased mobility resolution, the subsequent improvement in data quality and the added value for metabolite characterization experiments, all within a duty-cycle compatible with ultra-fast UPLC separation.
Preliminary Data or Plenary Speakers Abstract
Co-eluting isomeric metabolites were detected and characterised previously on a Waters Vion IMS Q-Tof ion-mobility mass spectrometer using routine HDMSE with precise CCS measurement, where fit-for-purpose ion mobility resolution was shown to be adequate in partially resolving two distinct metabolites. When using the cyclic mobility-enabled platform, with the same chromatographic conditions, preliminary data illustrates how the increased resolution of the cyclic data allows mobility separation of multiple drug metabolite isomers, even for co-eluting metabolites. This brings an expanded chemical space in which the assessment of chromatographic peak purity for complex samples can be made more robustly, particularly at trace levels, where potential false negatives can occur. By increasing the number of passes taken by the ion population around the cyclic device, the mobility resolution increased until baseline separation of multiple isomers was achieved, at biologically-relevant concentrations with little-to-no loss in ion transmission compared with a single pass. Being able to detect and distinguish co-eluting isomeric metabolites using high resolution ion mobility allows for comprehensive assessment of the overall metabolite profile, largely independent of chromatographic performance and species type. These data could increase confidence in the likelihood that a newly-detected (partially-characterized) isomeric metabolite was genuine and distinguishable from other isomers, as it would occupy a point in an expanded chemical space, allowing more confident biotransformation support.
Novel Aspect
Use of a novel cyclic mobility-enabled research platform to provide increased mobility resolution and added value within metabolite characterisation