Evaluation of ion mobility, uni- and multidimensional liquid chromatography for non-target screening of phenolic compounds in wheat flag leaves

Non-target screening (NTS) of plant secondary metabolites is analytically challenging due to the complexity of mixtures with structurally similar compounds and isomers. This study evaluates the added value of ion mobility spectrometry (IMS) and comprehensive two-dimensional liquid chromatography (LC × LC) for enhancing separation, mass spectral quality, chromatographic structure and from access to collisional cross section (CCS) values in NTS workflows. Four LCHRMS configurations were compared: RPLCHRMS, HILICHRMS, RPLC × HILICHRMS, and HILIC × RPLCHRMS, each with and without travelling wave (TW)IMS. Data were acquired in data-independent acquisition (DIA) mode. Phenolic compounds in wheat flag leaf extracts were analyzed as a representative case. The results show that mass spectral purity improved up to 74 times when hyphenating RPLC with IMS for peaks with intensities <1.5 × 10⁴. The compound groups: flavones, flavan-3-ols, benzoic acid derivatives and phenolic acids were separated into distinct retention time regions in RPLC × HILIC, while co-eluting in RPLC and HILIC. Only two of ten isomeric pairs were partially resolved by IMS, with resolutions of 0.50 and 0.63, whereas RPLC achieved resolution values of 29 and 13 for the same pairs. Notably, the only isomers with IMS resolution ≥ 0.5 had m/z > 447. We showed that large [Formula: see text] deviations (-9.47 %) were observed when several deprotomers of a molecule were created and separated in IMS. Deprotomers are not accommodated for in the machine learning prediction models, which limits their use for such molecules. The complementary strengths of IMS and LC × LC should be exploited strategically to address specific analytical goals in plant metabolite screening.