Broad‐spectrum separations in metabolomics using enhanced polar LC stationary phases: A dedicated evaluation using plant metabolites

Abstract

Original PaperBroad-spectrum separations in metabolomics using enhanced polar LC stationary phases: A dedicated evaluation using plant metabolitesIn metabolomics, major efforts are invested in the development of suitable analytical approaches. A tendency towards the use of LC-MS is nowadays very obvious. A great majority of the metabolites of interest are polar to highly polar in nature. We focus on the so-called , extended polarity' reversed LC phases, developed specifically to allow better retention characteristics for polar compounds. Several of these phases (Atlantis dC18, Inertsil ODS-3, Zorbax XDB, Alltima HP C18) are tested for different column dimension variations (0.5, 1.0, 2.1 mm id) in a specific LC-MS metabolomics setting. Important chromatographic and mass spectrometric quality parameters such as capacity factor, separation efficiency, peak symmetry, sensitivity, and mass accuracy are taken into account. All phases show adequate retention of polar compounds and also perform well with highly aqueous mobile phase compositions. On comparing 1.0 and 2.1 mm id columns, it is clear that the potential gain in sensitivity is not achieved. Using a Lockspraym device, accurate mass measurement with a Q-TOF micro is feasible within a mass range of 12 ppm if signal intensities of compound and lockmass are equated. Finally, the extended polarity RP approach in metabolomics experiments is endorsed using real plant extracts.

Evaluation of hydrophilic interaction chromatography versus reversed‐phase chromatography in a plant metabolomics perspective

“…In previous results [11], matrix suppression could be demonstrated in the polar bulk of the chromatogram when analysing complex A. thaliana extracts on RP stationary phases. ME is defined as the alteration of ionisation efficiency by the presence of coeluting substances.…”

“…It consists of spherical silica particles that are covalently bonded with carbamoyl groups. This column is now matched to the Atlantis dC18 column, designated as the best choice for our RP LC-MS metabolomics set-up [11]. This specialty RP phase enhances retentive performance in 100% aqueous conditions through prevention of phase pore dewetting, causing better retention of polar analytes.…”

“…The introduction of extended polarity RP material partially solved this problem. However, in these approaches, matrix suppression in the polar front of the RP analysis appears even more as a point of concern [11]. The very high aqueous mobile phase indeed causes a low ionisation efficiency in ESI MS of the most polar metabolites which, consequently, is also prone to a substantial variability [12].…”

Joint GC–MS and LC–MS platforms for comprehensive plant metabolomics: Repeatability and sample pre-treatment

“…This composition was then maintained for 5 final minutes after which the whole system was allowed to re-equilibrate at initial conditions. This generic gradient had separately been optimized [17].…”

LC–MS based global metabolite profiling: the necessity of high data quality

“…These metabolites experience increased ion suppression/enhancement effects due to overlapping molecule signals and extremely poor selectivity (Kloos et al 2013), a problem which can be solved through polarity-based fractionation strategies during sample preparation (Whiley et al 2012). It should however be noted that additional RP column materials are available, which to some extent can retain polar to semi-polar metabolites (Bruce et al 2008;T'Kindt et al 2007). Moreover, researchers have also used the addition of cationic ion-pairing agents such as tributylamine or hexylamine to the mobile phase in RP-LC-MS to afford retention of anionic polar metabolites (Coulier et al 2006;Lu et al 2011) as well as alterative pentafluorophenylpropyl-based columns displaying both RP and normal-phase retention characteristics (Bell et al 2005;Yang et al 2013).…”