Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry

Abstract

Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has been a subject of dramatic developments over the past decade. This review focuses on state of the art in LC-MS-based lipidomics, covering all the steps of global lipidomic profiling. On the basis of review of 185 original papers and application notes, we can conclude that typical LC-MS-based lipidomics methods involve: (1) extraction using chloroform/MeOH or MTBE/MeOH protocols, both with addition of internal standards covering each lipid class; (2) separation of lipids using short microbore columns with sub-2-μm or 2.6-2.8-μm (fused-core) particle size with C18 or C8 sorbent with analysis time <30 min; (3) electrospray ionization in positive- and negative-ion modes with full spectra acquisition using high-resolution MS with capability to MS/MS. Phospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidylglycerols) followed by sphingomyelins, di- and tri-acylglycerols, and ceramides were the most frequently targeted lipid species.

Keywords: Acylglycerol; Biological system; Comprehensive analysis; Extraction method; Global lipidomic profiling; LC-MS; Lipidomics; Liquid chromatography-mass spectrometry; Metabolomics; Phospholipid.

Fig. 1

Number of original papers published over 11 years dedicated to lipidomics and different instrumental platforms. Scopus (www.scopus.com) and Web of Knowledge (www.webofknowledge.com) databases used for citation analysis.

Fig. 2

LC-MS-based lipidomic analysis.

Fig. 3

Focus of LC-MS-based lipidomics studies: (A) analyzed matrices; and, (B) extraction protocols.

Fig. 4

Use of (A) chromatographic systems, (B) ionization modes, and (C) mass analyzers in LC-MS-based lipidomics papers.

Fig. 5

RP-UHPLC-ESI(+/–)-QTOFMS chromatograms of human plasma extract separated on an Acquity UPLC HSS T3 (100 × 2.1 mm I.D.; 1.8 μm) column with a gradient elution using mobile phase ACN/H₂O (40:60), 10 mM ammonium acetate and IPA/ACN (90:10), 10 mM ammonium acetate at a flow-rate of 0.4 mL/min at 55°C. {Adapted and reproduced with permission from [26]}.

Fig. 6

RP-HPLC-ESI(–)-orbital ion trap MS extracted ion chromatograms of the standard PA(34:1), m/z 673.5 (A + C) and PS(36:1), m/z 788.5 (B + D) separated on a Waters X-Bridge C18 (150 × 1.0 mm I.D.; 3.5 μm) column. (A + B) Mobile phase A: ACN/MeOH/H₂O (19:19:2) with 0.2% formic acid and 0.028% ammonia; mobile phase B: IPA with 0.2% formic acid and 0.028% ammonia at a flow-rate of 20 μL/min and temperature 50°C. (C) Phosphoric acid added into the mobile phase A and the injector-rinsing solvent. (D) Mobile phase A: IPA/MeOH/H₂O (5:1:4) with 0.2% formic acid, 0.028% ammonia, and 5 μM phosphoric acid; mobile phase B: IPA with 0.2% formic acid and 0.028% ammonia at a flow-rate of 20 μL/min and temperature 25°C. {Reproduced with permission from [27]}.

Fig. 7

NP-HPLC-ESI(–)-MS/MS (QqQ) chromatograms of rat-brain extract separated on a Phenomenex Luna 3 μm Silica (150 × 2 mm I.D.; 3 μm) column with a gradient elution using mobile phase chloroform/MeOH/triethylamine/acetic acid (80:19:0.5:0.5) and chloroform/MeOH/H₂O/triethylamine/acetic acid (60:33.5:5.5:1:0.065) at a flow-rate of 0.2 mL/min at room temperature. {Reproduced with permission from [28]}.

Fig. 8

RP-UHPLC-ESI(+)-IM-QTOFMS analysis of a human plasma lipid extract separated on an Acquity UPLC HSS T3 (100 × 2.1 mm I.D.; 1.8 μm) column with a gradient elution using mobile phase ACN/H₂O (60:40), 10 mM ammonium formate and IPA/ACN (90:10), 10 mM ammonium formate. (a) A chromatogram with ion mobility separation turned on. (b) Corresponding driftogram with orthogonal ion mobility separation. {Reproduced with permission from [46]}.

Fig. 9

Creation, validation, and application of in-silico generated MS/MS spectra in LipidBlast. (a) New lipid compound structures were generated using in-silico methods. Lipid core structure scaffolds were connected via a linker to fatty acyls with different chain lengths and different degrees of unsaturation. Asterisks denote connection points. (b) Reference tandem spectra (top) were used to simulate mass spectral fragmentations and ion abundances of the in-silico spectra (bottom). The compound shown is PC(16:0/ 16:1) at precursor m/z 732.55 [M + H]⁺. (c) For lipid identification, MS/MS spectra obtained from LC-MS/MS or direct-infusion experiments were submitted to LipidBlast. An m/z precursor-ion filter first filtered the data, and a subsequent product-ion match generated a library hit score that reflects the level of confidence for compound annotation. {Reproduced with permission from [56]}.

Fig. 10

Focus on particular lipid classes in LC-MS-based lipidomics.