Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.

Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies.

Cuykx M ，Negreira N ，Beirnaert C ，Van den Eede N ，Rodrigues R ，Vanhaecke T ，Laukens K ，Covaci A ... -  《-》

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices.

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features (< 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes.

Iturrospe E ，Da Silva KM ，Talavera Andújar B ，Cuykx M ，Boeckmans J ，Vanhaecke T ，Covaci A ，van Nuijs ALN ... -  《-》

Cross platform solutions to improve the zebrafish polar metabolome coverage using LC-QTOF MS: Optimization of separation mechanisms, solvent additives, and resuspension solvents.

Untargeted metabolomics has been widely used for studies with zebrafish embryos. Until now, the number of analytical approaches to determine metabolites in zebrafish is limited, and there is a lack of consensus on the best platforms for comprehensive metabolomics analysis of zebrafish embryos. In addition, the capacity of these methods to detect metabolites is unsatisfactory and the confidence level for identifying compounds is relatively low. To improve the metabolome coverage, we mainly focused on the optimization of separation mechanisms, mobile phase additives, and resuspension solvents based on liquid chromatography (LC) coupling to high-resolution mass spectrometry (HRMS) techniques. Moreover, the procedures for optimizing methods were assessed when taking metabolite profiles in both positive and negative ionization modes into account. Four LC columns were studied: C18, T3, PFP, and HILIC. In positive ionization mode, it was strongly recommended to employ the HILIC approach operated at the neutral condition, which led to the presence of more than 4700 features and the annotation of 151 metabolites, mainly zwitterionic and basic compounds, in comparison to reverse phase (RP)-based methods with less than 1000 features. In negative ionization mode, the PFP column operated at 0.02% acetic acid showed the best performance in terms of metabolite coverage: 3100 metabolic features were detected and 218 metabolites were annotated in zebrafish embryos. Metabolite profiles mainly contained acidic and zwitterionic compounds. HILIC-based platforms were complementary to RP columns when analyzing highly polar metabolites. Additionally, it was preferable to reconstitute zebrafish extracts in 100% water for analysis of metabolites on RP columns, with a 20-30% increase in the number of identified metabolites compared to a 50% water in methanol solution. However, water/methanol (1:9, v/v), as resuspension solution, was advantageous over water/methanol (1:1, v/v) for HILIC analysis showing an 8-15% increase in detected metabolites. In total 336 polar metabolites were annotated by the combination of the optimized HILIC (positive) and PFP (negative) approaches. The largest metabolome coverage of polar metabolites in zebrafish embryos was obtained when three approaches were combined (negative PFP and HILIC, and HILIC positive) resulting in more than 420 annotated compounds.

Xu M ，Legradi J ，Leonards P 《-》

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma.

To increase metabolome coverage in global LC-MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59-776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

Sonnenberg RA ，Naz S ，Cougnaud L ，Vuckovic D ... -  《-》

Evaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics.

Wernisch S ，Pennathur S 《-》