Abstract
Hydrophilic Interaction Liquid Chromatography (HILIC) chromatography is widely applied in metabolomics as a complementary strategy to reverse phase chromatography. Nevertheless, it still faces several issues in terms of peak shape and compounds ionization, limiting the automatic de-convolution and data semi-quantification performed through dedicated software. A way to improve the chromatographic and ionization performance of a HILIC method is to modify the electrostatic interactions of the analytes with both mobile and stationary phases. In this study, using a ZIC-HILIC chromatographic phase, we evaluated the performance of ammonium fluoride (AF) as additive salt, comparing its performance to ammonium acetate (AA). Three comparative criteria were selected: (1) identification and peak quality of 34 standards following a metabolomics-specific evaluation approach, (2) an intraday repeatability test with real samples and (3) performing two real metabolomics fingerprints with the AF method to evaluate its inter-day repeatability. The AF method showed not only higher ionization efficiency and signal-to-noise ratio but also better repeatability and robustness than the AA approach. A tips and tricks section is then added, aiming at improving method replicability for further users. In conclusion, ammonium fluoride as additive salt presents several advantages and might be considered as a step forward in the application of robust HILIC methods in metabolomics.
Highlights
Hydrophilic Interaction Liquid Chromatography (HILIC [1]) has gained popularity in LC-HRMS metabolomics due to its complementarity with Reverse Phase (RP) approaches [2]
HILIC chromatographic separation is commonly achieved through an organic to aqueous gradient, consisting of acetonitrile (ACN) and ultra-pure water (H2 O), assisted with small volatiles salts as additives, like ammonium acetate or ammonium formate [4], to improve chromatographic peak shape
As electrostatic interaction is one of the leading process governing HILIC retention [4,26], the change of the additive salt might result in very different apparent retention factor of the stationary phase
Summary
Hydrophilic Interaction Liquid Chromatography (HILIC [1]) has gained popularity in LC-HRMS metabolomics due to its complementarity with Reverse Phase (RP) approaches [2]. Thanks to the water-mediated separation of the analytes, the HILIC technology allows to extend the separative abilities of liquid chromatography to very polar metabolites, increasing the metabolome coverage, while maintaining the robustness and the repeatability (critical parameters in metabolomics) at similar level than the RP chromatography [3]. HILIC chromatographic separation is commonly achieved through an organic to aqueous gradient, consisting of acetonitrile (ACN) and ultra-pure water (H2 O), assisted with small volatiles salts as additives, like ammonium acetate or ammonium formate [4], to improve chromatographic peak shape. Metabolites 2019, 9, 292; doi:10.3390/metabo9120292 www.mdpi.com/journal/metabolites pKa (pH 4.7 for ammonium acetate and pH 3.2 for ammonium formate, [5]). These methodological adjustments allow to achieve efficient chromatographic separation for the majority of the eluting peaks. Ammonium formate and ammonium acetate have limited solubility in organic solvents [6,7]
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