Resolving isobaric interferences in direct infusion tandem mass spectrometry

Rationale: The co-fragmentation of precursors in direct infusion (DI) tandem high-resolution mass spectrometry (HRMS) can complicate the fragment spectra and consequently lead to false hits during compound identification.

Methods: The method herein described, termed IQAROS (incremental quadrupole acquisition to resolve overlapping spectra), modulates the intensities of precursors and fragments by stepwise movement of the quadrupole isolation window over the mass-to-charge (m/z) range of the precursors. The modulated signals are then deconvoluted by a linear regression model to reconstruct the fragment spectra with less interference. The hardware to demonstrate the use of IQAROS was an orbitrap with electrospray ionization (ESI) or secondary electrospray ionization (SESI), although the method can also be applied to other ionization techniques or mass analyzers.

Results: Assessing the performance of IQAROS with isobaric standards revealed that the reconstructed fragment spectra match with spectra acquired from the pure standards and that more compounds were correctly identified compared with the classical approach with the quadrupole centered at the m/z value of the precursor of interest. Moreover, the strength of IQAROS is exemplified by the identification of two isobaric biomarkers directly from a breath sample with SESI-HRMS.

Conclusions: With IQAROS, cleaner fragment spectra of co-fragmenting isobars during DI-HRMS analysis can be obtained. IQAROS can easily be set up by the standard graphical user interface of the instrument. Therefore, it facilitates the characterization of features of interest in samples analyzed by DI-HRMS, for example, in high-throughput or real-time metabolomics.