Measuring the Integrity of Gas-Phase Conformers of Sodiated 25-Hydroxyvitamin D3 by Drift Tube, Traveling Wave, Trapped, and High-Field Asymmetric Ion Mobility.

Quantitation of the serum concentration of 25-hydroxyvitamin D is a high-demand assay that suffers from long chromatography time to separate 25-hydroxyvitamin D from its inactive epimer; however, ion mobility spectrometry can distinguish the epimer pair in under 30 ms due to the presence of a unique extended or "open" gas-phase sodiated conformer, not shared with the epimer, reducing the need for chromatographic separation. Five ion mobility mass spectrometers utilizing commercially available IMS technologies, including drift tube, traveling wave, trapped, and high-field asymmetric ion mobility spectrometry, are evaluated for their ability to resolve the unique open conformer. Additionally, settings for each instrument are evaluated to understand their influence on ion heating, which can drive the open conformer into a compact or "closed" conformer shared with the epimer. The four low-field instruments successfully resolved the open conformer from the closed conformer at baseline or near-baseline resolution at typical operating parameters. High-field asymmetric ion mobility was unable to resolve a unique peak but detected two peaks for the epimer, in contrast to the low-field methods that detected one conformer. This study seeks to expand the instrument space by highlighting the potential of each platform for the separation of 25-hydroxyvitamin D epimers.

Separation of biologically relevant isomers on an Orbitrap mass spectrometer using high-resolution drift tube ion mobility and varied drift gas mixtures.

RATIONALE: Atmospheric pressure drift tube ion mobility is a powerful addition to the Orbitrap mass spectrometer enabling direct separation of isomers. Apart from offering high resolving power in a compact design, it also facilitates optimization of the separation gas, as shown here for a series of biologically relevant isomer pairs. METHODS: An Excellims MA3100 High-Resolution Atmospheric Pressure Ion Mobility Spectrometer (HR-IMS) was coupled to a Thermo Scientific™ Q Exactive™ Focus hybrid quadrupole-Orbitrap™ mass spectrometer, using an Excellims Directspray™ Electrospray Ionization source and a gas mixture setup to provide various drift gases (air, CO2 and mixtures). This instrument combination was used to separate isomers of eight pairs of metabolites and gangliosides, optimizing drift gas conditions for best separation of each set. RESULTS: All but one of the isomers pairs provided could be partially or fully separated by the HR-IMS-MS combination using ion mobility drift times. About half of the separated compounds showed significantly better analytical separation when analyzed in a mixture of CO2 and air rather than air or CO2 alone. Resolving power of up to 102 was achieved using the 10 cm atmospheric drift tube ion mobility add-on for the Orbitrap mass spectrometer. CONCLUSIONS: The present analysis demonstrates the usefulness of using atmospheric drift tube IMS on an Orbitrap mass spectrometer to characterize the isomeric composition of samples. It also highlights the potential benefits of being able to quickly optimize the drift gas composition to selectively maximize the mobility difference for isomer separation.