A combined flow injection/reversed-phase chromatography-high-resolution mass spectrometry workflow for accurate absolute lipid quantification with 13C internal standards

Harald Schoeny; Evelyn Rampler; Yasin El Abiead; Felina Hildebrand; Olivia Zach; Gerrit Hermann; Gunda Koellensperger

doi:10.1039/d0an02443k

A combined flow injection/reversed-phase chromatography-high-resolution mass spectrometry workflow for accurate absolute lipid quantification with ¹³C internal standards

Analyst. 2021 Apr 26;146(8):2591-2599. doi: 10.1039/d0an02443k.

Authors

Harald Schoeny¹, Evelyn Rampler², Yasin El Abiead¹, Felina Hildebrand¹, Olivia Zach¹, Gerrit Hermann³, Gunda Koellensperger²

Affiliations

¹ Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria. gunda.koellensperger@univie.ac.at.
² Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria. gunda.koellensperger@univie.ac.at and Vienna Metabolomics Center (VIME), University of Vienna, Althanstrasse 14, 1090 Vienna, Austria and Chemistry Meets Microbiology, Althanstrasse 14, 1090 Vienna, Austria.
³ Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria. gunda.koellensperger@univie.ac.at and ISOtopic solutions, Waehringer Strasse 38, 1090 Vienna, Austria.

PMID: 33734229
DOI: 10.1039/d0an02443k

Abstract

We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.