An improved SPE method for fractionation and identification of phospholipids (original) (raw)
Related papers
Electrophoresis, 2018
This article is protected by copyright. All rights reserved. 2 (CM), , endoplasmic reticulum (ER), ethanol (EtOH), ether-insoluble PLS (EIP), free fatty acids (FFA), hexane/isopropanol (HIP), high-abundance proteins (HAPs), limit of detection (LOD), low-abundance proteins (LAPs), lyso-phosphatidylethanolamine (lyPE), lysophospholipids (LyPLS), methanol (MetOH), methyl-tert-buthyl ether (MTBE), origin of replication (oriC), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phospholipids (PLS), pressurized liquid extraction (PLE), solid phase extraction (SPE), sphingomyelin (SM), supercritical fluids (SCF), thin-layer chromatography (TLC), triacylglicerol (TG), very-low density lipoproteins (VLDL).
Rapid Communications in Mass Spectrometry, 2008
One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a novel HPLC-MS/MS method that separates molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid fingerprinting using full scan mass spectrometry, as well as lipid class specific scanning. Excellent separation of isobaricand even isomeric species is achieved, and original levels of lysolipids can be determined without interference from lysolipids formed from diacyl species by source fragmentation. Using this method, more than 400 phospholipid species were identified and quantified in crude lipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.
Journal of Chromatography A, 2012
A hydrophilic interaction liquid chromatography (HILIC) -ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60 mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain wellshaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R 2 , 0.990-0.999), retention time stability (RSD < 1%), within day repeatability (RSD, 5-13%), between day variation (RSD, 7-14%) and recoveries (99.6-115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.
Liquid Chromatography/Mass Spectrometry of Phospholipids using Electrospray Ionization
Analytical Chemistry, 1994
An improved technique for phospholipid molecular species analysis was developed using high-performance liquid chromatography/mass spectrometry with the electrospray interface. Using the 0.5% ammonium hydroxide in a water-methanolhexane mixture and a C-18 column, complex mixtures of phospholipid molecular species were separated and detected mainly as protonated or natriated molecular species. The response was linear over 2 orders of magnitude, allowing quantification of each molecular species. In comparison to the existing LC/MS techniques, marked improvement in sensitivity was observed. The present quantification limit is approximately 0.5 pmol before split (5 fmol after 1/100 split). The relative responses were more dependent on the head group identity rather than fatty acyl composition within a phospholipid class. In general, phosphatidylcholine (PC) species are most sensitively detected followed by phosphatidylethanolamine (PE) species. The sensitivity of phosphatidylserine (PS) in the positive ion mode is approximately 20 times less in comparison to PC under our condition. Phospholipids are one of the major constituents in the cell membrane bilayer. Membrane phospholipids are a complex mixture of molecular species containing a variety of fatty acyl and head groupcompositions (Scheme 1). It is widely accepted that chemical and physical properties of cell membranes are largely dependent on the phospholipid composition.1 Maintenance of tightly regulated membrane environment appears to be important for many membrane functions including transport and endocytosis2J as well as activity of membrane bound enzyme^.^ In addition, specific pools of phospholipids serve as reservoirs for polyunsaturated fatty acids that can be metabolized to various biologically important mediatoms Therefore, altering the phospholipid profile may bring about significant biological consequences. Analysis of phospholipid molecular species often requires laborious procedures including separation by column,6 argentation thin-layer (TLC)7 or high-performance liquid chromatography (HPLC).8 Identification of separated comt Section of Mass Spectrometry.
Journal of chromatography. B, Biomedical applications, 1994
An improved high-performance liquid chromatographic method for the separation and determination of radioactively labelled cellular phospholipids is described. The method is based on separation of phospholipids on a 250 x 4 mm I.D. LiChrospher DIOL 100 (5 microns) column, fitted with a 50 x 4 mm I.D. LiChrospher Si 60 (5 microns) precolumn and a gradient of 5% H3PO4 and acetonitrile. It allows the determination of small amounts of labelled phosphatidylcholine and sphingomyelin due to the sharp elution profile in spite of long retention times.
Hydrophilic interaction liquid chromatography (HILIC), coupled to tandem mass spectrometry, can be used to separate and determine various polar lipid classes. The development of an HILIC chromatographic separation of several molecular species among five phospholipid classes (PC, PE, PG, PI and PS) is reported here. In this method, a gradient with acetonitrile and 40 mM ammonium acetate buffer was employed. The initial composition was 95 % of acetonitrile, then this proportion was decreased to 70 % in order to elute all the compounds of interest for a total running time of 11 mins. Furthermore, mobile phase pH can affect the ionizable character of the compounds, according to their pKa values, and also the stationary phase charge state. The influence of such a parameter on both retention times and resolution was evaluated. Besides, the response of different kinds of internal standards (post-extraction standard addition) was evaluated in four different biological matrices, two microalg...
Analysis of phospholipids using electrospray ionisation tandem mass spectrometry
Lipid Technology, 2007
Phospholipids have widespread use as surfactants and emulsifying agents, and as ingredients with nutritional benefits. We describe an approach for their analysis, based on separation by reversed-phase HPLC and identification and quantification by electrospray ionization MS and MS/MS. It enables the identification of phospholipids by assessing their polar head and fatty acid moieties, as well as their quantitative analysis.