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

Kristian Pirttilä,Mikael Hedeland,Göran Laurell,Curt Pettersson

doi:10.3390/metabo11050295

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.

Highlights

One of the main issues that still plague untargeted metabolomics is the immense chemical diversity as well as the wide concentration range of the metabolites that make up biological samples [1,2]
One of the most powerful of such techniques used today is liquid chromatography hyphenated with high-resolution mass spectrometry (LC–HRMS) [6,7]
We have shown that the method is fit for purpose for application in untargeted metabolomics analysis of biological samples

Summary

Introduction

One of the main issues that still plague untargeted metabolomics is the immense chemical diversity as well as the wide concentration range of the metabolites that make up biological samples [1,2]. Achieving complete chemical coverage with a single analytical technique is a practical impossibility as each technique comes with its own inherent limitations. With this in mind, the aim in developing an analytical method for untargeted metabolomics instead becomes that of maximizing the chemical coverage obtained within the bounds of the techniques employed. One of the most powerful of such techniques used today is liquid chromatography hyphenated with high-resolution mass spectrometry (LC–HRMS) [6,7] This is due to the large variation of possible combinations of columns and mobile phases, giving the separation technique the potential to cover a very wide range of chemical properties. The combination of different techniques increases the total number of analyses performed per sample, leading to a higher demand on instrument time, thereby increasing costs

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Conclusion