Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics

Abstract

In order to increase metabolite coverage in LC–MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC–MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds.

Ultra‐high performance liquid chromatography‐quadrupole/time‐of‐flight mass spectrometry‐based lipidomics reveals key lipid molecules as potential therapeutic targets of Polygonum cuspidatum against hyperlipidemia in a hamster model

“…To access the data stability and reproducibility, PCA on all samples was performed. As shown in Figure 2C,D, quality control samples clustered tightly, indicating the whole analytical process was stable and repeatable [21].…”

Miniaturized Two-Dimensional Heart Cutting for LC–MS-Based Metabolomics

“…After a classical sample injection and elution in the first dimension, the second step was the trapping of the non-retained analytes on a trapping column (Figure 1). Using this trapping step from the front solvent elution, 28 lipophilic metabolites were eluted through the trapping column using a six-port valve. The onset and completion of the trapping step were optimized on the mixture of standards when bile acid isomers and fatty acid standards were eluted in the first HILIC dimension.…”

“…Obviously, heart-cutting is becoming of interest for nontargeted metabolomics analysis. Recently, Pirttilä validated a single-injection HILIC-RP method by applying the heart-cutting principle to plasma and then performing nontargeted metabolomics analyses of guinea pig perilymph samples. The advantages of a 2D heart-cutting method over a conventional LC–MS method in terms of metabolic coverage and polarity variety were demonstrated by Feng through a simultaneous analysis of the metabolome and lipidome in rat plasma.…”