Light-scattering detection of phospholipids resolved by HPLC

Reviews in Analytical Chemistry, 2012

Phospholipids (PLs) are a class of compounds essential for living organisms, including human beings. This paper presents a brief review concerning the extraction as well as thin-layer chromatographic separation and determination of phospholipids. After a short introduction regarding phospholipids and their specifi c characteristics, the extraction techniques will be discussed. Then, phospholipid separation and system detection for thin-layer chromatography (TLC) will be presented. The focus will be on phospholipids that are most abundant in biological systems; that is why chosen examples of PLs determination in biological samples will be revealed as well as general PL determination schemes. In the Summary section, there will also be a short discussion about advantages and disadvantages of PL determination with the use of TLC.

Journal of Chromatography B: Biomedical Sciences and Applications, 1986

The seven major phospholipid classes of pulmonary surfactant were cleanly separated within 45 min by high-performance liquid chromatography (HPLC) on microparticulate silica (5 pm). Phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine (PC) were separated with an isocratic mobile phase of acetonitrile-8 5% phosphoric acid (99: 1). Lysophosphatidylcholine and sphingomyelin were subsequently eluted by changing the composition of the mobile phase to acetonitrilemethanol-85% phosphoric acid (97:2:1). Phospholipids were detected by ultraviolet absorbance at 203 nm and were quantitated by integratioGf peaks. When integrator counts were corrected by using calibration curves obtained from &romatograms of commercial phospholipids and surfactant-derived PC, the HPLC analysis-gave results comparable to those obtained by thin-layer chromatography.

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).

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 Separation Science, 2013

This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel-aminopropyl-silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high-resolution LC-MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP-HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP-HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC-MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified.

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.

Food Reviews International, 2012

PLs include milk and dairy products, meat, fish, eggs, cereals, and oils. Both the technological aspects and analytical parameters affecting the concentration and quantitative determination of PLs in food matrices are evaluated. In particular, different extraction, purification, and chromatographic conditions were extensively investigated; moreover, wherever possible, the results obtained are reported and compared with other detection methods.

Biomedical Chromatography, 2013

A new phospholipid-specific spray reagent is described. A new phospholipid-specific spray reagent, which is a modification of the Dittmer-Lester reagent, is described in authors' studies. The difference between these two reagents is in the addition of tin (II) chloride to the proposed spray reagent. The use of the described spray reagent together with an image analysis technique allows not only for qualitative, but also for quantitative, determination of major phospholipid classes. Separation of phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was conducted on an HPTLC plate with a mixture of chloroform, methanol and 25% ammonia solution in a volume ratio of 65:25:4 as mobile phase. The calibration curves were linear in the ranges of 5.0-25.0, 1.5-15.0 and 1.0-20.0 mg/spot for PC, PS and PE, respectively. The use of the new spray reagent resulted also in lower limits of detection than the standard molybdenum method for the investigated phospholipids. The proposed method was used to determine the amount of PS in the dietary supplement 'Session', and of PS, PE and PC in biological samples, with good results.

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...