Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts

Elmsjö, Albert; Haglöf, Jakob; Engskog, Mikael K.R.; Erngren, Ida; Nestor, Marika; Arvidsson, Torbjörn; Pettersson, Curt

doi:10.1016/j.chroma.2018.05.007

Cited by 17 publications

(11 citation statements)

References 46 publications

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“…Inorganic alkali metal ions, e.g., sodium and potassium ions, are present in all biological matrices and are sometimes also added during sample processing and preparation. These ions cause a number of issues in electrospray ionization-mass spectrometry (ESI-MS) and can severely hamper the analysis [1][2][3][4][5][6]. The use of reversed phase (RP) liquid chromatography (LC) prior to MS detection often mitigates some of the problems caused by non-volatile inorganic ions, as they generally elute with the void volume.…”

Section: Introductionmentioning

confidence: 99%

“…The use of reversed phase (RP) liquid chromatography (LC) prior to MS detection often mitigates some of the problems caused by non-volatile inorganic ions, as they generally elute with the void volume. However, when using hydrophilic interaction liquid chromatography (HILIC) the inorganic ions are retained in the chromatographic system and can be detected as cluster ions with mobile phase additives, other inorganic ions, endogenous components of the sample including analytes [2,5,7]. The retention of inorganic ions in HILIC analyses have been studied and utilized for the analysis of counter ions in pharmaceutical formulations, particularly using zwitterionic stationary phases where buffer concentration as well as the organic content of the mobile phase have been concluded to the most important factors to adjust the retention and selectivity of ionic species [8,9].…”

Section: Introductionmentioning

confidence: 99%

“…The retention of inorganic ions in HILIC analyses have been studied and utilized for the analysis of counter ions in pharmaceutical formulations, particularly using zwitterionic stationary phases where buffer concentration as well as the organic content of the mobile phase have been concluded to the most important factors to adjust the retention and selectivity of ionic species [8,9]. Different HILIC stationary phases have different selectivities towards inorganic ions which was investigated by Elmsjö et al [5]; they found that the amide and sulfobetaine stationary phases were preferred for untargeted metabolomics analyses as the retention time range of the inorganic ions were narrower as compared to the bare silica phase meaning that their impact on co-eluting metabolites could be minimized. The co-elution with analytes affects adduct formation, ion-suppression, and thereby sensitivity and reproducibility.…”

Section: Introductionmentioning

confidence: 99%

“…McMillan et al [10] reported that as many as 28% of the features (m/z-retention time pairs) in a metabolomics data set were in fact salt clusters and that the proportion of those in the datasets were generally higher using HILIC than RP. The issues caused by inorganic ions have been investigated in targeted quantitative LC-MS/MS applications as well as in untargeted/screening applications e.g., metabolomics [2,5,7]. However, solutions to these problems have not yet been introduced or investigated.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2021

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“…This technique benefits from a variety of columns with different stationary phases, which allows separating a large number of distinct polar compounds. Frequently used columns are reversed phased (RP) C18 or C8 [ 17 , 18 ] and hydrophilic interaction chromatography (HILIC) [ 19 ]. The variety of these columns offers additional information depending on the composition of the stationary phase and the selected pH range [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

A Metabolomics Workflow for Analyzing Complex Biological Samples Using a Combined Method of Untargeted and Target-List Based Approaches

et al. 2020

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In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.

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Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

Hemmer,

Manier,

Wagmann

et al. 2024

Metabolomics

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Introduction Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose. Objectives The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices. Methods Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others. Results All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column. Conclusion The results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix. Graphical abstract

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Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts

Cited by 17 publications

References 46 publications

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

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

A Metabolomics Workflow for Analyzing Complex Biological Samples Using a Combined Method of Untargeted and Target-List Based Approaches

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

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