Improved Sensitivity in Hydrophilic Interaction Liquid Chromatography-Electrospray-Mass Spectrometry after Removal of Sodium and Potassium Ions from Biological Samples

Abstract

Inorganic ions, such as sodium and potassium, are present in all biological matrices and are sometimes also added during sample preparation. However, these inorganic ions are known to hamper electrospray ionization -mass spectrometry (ESI-MS) applications, especially in hydrophilic interaction liquid chromatography (HILIC) where they are retained and can be detected as adducts and clusters with mobile phase components or analytes. The retention of inorganic ions leads to co-elution with analytes and as a result ion-suppression, extensive adduct formation and problems with reproducibility. In the presented work, a sample preparation method using cation exchange solid phase extraction (SPE) was developed to trap Na+ and K+ ions from human blood plasma and head and neck cancer cells for the analysis of small cationic, anionic as well as neutral organic analytes. The investigated analytes were small, hydrophilic compounds typically in focus in metabolomics studies. The samples were analyzed using full-scan HILIC-ESI-quadrupole time of flight (QTOF)-MS with an untargeted, screening approach. Method performance was evaluated using multivariate data analysis as well as relative quantifications, spiking of standards to evaluate linearity of response and post-column infusion to study ion-suppression. In blood plasma, the reduction of sodium and potassium ion concentration resulted in improved sensitivity increased signal intensity for 19 out of 28 investigated analytes, improved linearity of response, reduced ion-suppression and reduced cluster formation as well as adduct formation. Thus, the presented method has significant potential to improve data quality in metabolomics studies.

HILIC-MS for Untargeted Profiling of the Free Glycation Product Diversity

“…Compared with model systems, biological samples contain complex endogenous metabolites, large amounts of salts (urine), high diversity of lipids (plasma), and gut microbiota metabolites (feces). These metabolites and salts can affect both chromatographic separation and electrospray ionization [39,40]. Therefore, we further evaluated the performance of the optimized method on analyzing glycation products with complex biological extracts.…”

Predicting Preferences for Adduct Formation in Electrospray Ionization: The Case Study of Succinic Acid

“…In addition to the array of experimental setups and analyte classes, this also has a downside�the obtained spectra are often complicated due to ionization by adduct formation with metal or small organic ions. 1,2 The appearance of multiple adducts makes sample identification more problematic, in particular considering that they do not fragment well in collision-induced dissociation. 3−5 This is why, in metabolomics, the adducts are typically regarded as unwanted contaminants.…”

“…Although the most common way for ion formation in ESI is protonation in positive mode and deprotonation in negative mode, multiple other ionization processes are known, such as adducts or homo- and heteromultimer formation. In addition to the array of experimental setups and analyte classes, this also has a downsidethe obtained spectra are often complicated due to ionization by adduct formation with metal or small organic ions. , The appearance of multiple adducts makes sample identification more problematic, in particular considering that they do not fragment well in collision-induced dissociation. − This is why, in metabolomics, the adducts are typically regarded as unwanted contaminants . Fortunately, since many features are mutually related, statistically grouping correlated features reduces the total number of feature groups .…”

All Ion Fragmentation Analysis Enhances the Untargeted Profiling of Glucosinolates in Brassica Microgreens by Liquid Chromatography and High-Resolution Mass Spectrometry

“…18 In this context, reversedphase liquid chromatography (RPLC) has been widely employed for the separation of GSL. 14 Although proposed as a possible alternative, 19 hydrophilic interaction liquid chromatography (HILIC) may suffer from chromatographic interferences introduced by ion clusters 20 arising from salts occurring in the plant extracts. Looking at the MS detection, the possible presence of several coextracted phytochemicals makes highresolution mass spectrometry (HRMS) a very interesting tool for the untargeted profiling of GSL.…”