Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry

Abstract

Rationale: The present work shows comprehensive chromatographic methods and MS conditions that have been developed based on the chemical properties of each lipid subclass to detect low-abundance molecular species. This study shows that the developed methods can detect low- and/or very-low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipid (GP) method; dihydroceramide (dhCer) and dihydrosphingosine/sphinganine (dhSPB) in the sphingolipid (SP) method; and lysophosphatidic acid (LPA), LPI, LPG and sphingosine-1-phosphate (SPBP) in the lysolipid method. Methods: An optimised method for the extraction of lysolipids in plasma is used in addition to Folch extraction. Then, four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches are described here. Three of the methods use a tertiary pumping system to enable the inclusion of a gradient for analyte separation (pumps A and B) and an isocratic wash (pump C). This wash solution elutes interfering compounds that could cause background signal in the subsequent injections, reducing column lifetime. Results: Semi-quantitative values for 37 lipid subclasses are reported for a plasma sample (NIST SRM 1950). Furthermore, the methods presented here enabled the identification of 338 different lipid molecular species for GPs (mono- and diacyl-phospholipds), SPs, sterols and glycerolipids. The methods have been validated, and the reproducibility is presented here. Conclusions: The comprehensive analysis of the lipidome addressed here of glycerolipids, GPs, sterols and SPs is in good agreement with previously reported results, in the NIST SRM 1950 sample, by other laboratories. Ten lipid subclasses LPS, LPI, alkyl-lysophosphatidic acid/alkenyl-lysophosphatidic acid, alkyl-lysophosphatidylethanolamine/alkenyl-lysophosphatidylethanolamine, dhCer (d18:0), SPB (d18:1), dhSPB (d18:0) and SPBP (d18:2) have been detected using this comprehensive method and are uniquely reported here.