The impact of phospholipids and phospholipid removal on bioanalytical method performance

Abstract

Phospholipids (PLs) are a component of cellmembranes, biological fluids and tissues. These compounds are problematic for the bioanalytical chemist, especially when PLs are not the analytes of interest. PL interference with bioanalysis highly impacts reverse-phase chromatographic methods coupled with mass spectrometric detection. Phospholipids are strongly retained on hydrophobic columns, and can cause significant ionization suppression in the mass spectrometer, as they outcompete analyte molecules for ionization. Strategies for improving analyte detection in the presence of PLs are reviewed, including in-analysis modifications and sample preparation strategies. Removal of interfering PLs prior to analysis seems to be most effective atmoderating thematrix effects fromthese endogenous cellular components, and has the potential to simplify chromatography and improve column lifetime. Products targeted at PL removal for sample pre-treatment, as well as products that combine multiplemodes of sample preparation (i.e. Hybrid SPE), show significant promise inmediating the effect on PL interference in bioanalysis.

Untargeted Metabolomics-Based Screening Method for Inborn Errors of Metabolism using Semi-Automatic Sample Preparation with an UHPLC- Orbitrap-MS Platform

“…It must be noted that we did not investigate performance of semi-automated sample preparation in comparison to manual sample processing, but, from a practical point of view, automation did facilitate integration of sampling handling into the lab information system. An additional advantage of the 96-well Phree filter plates that we employed, is selective binding of phospholipids, which, if not removed, may cause ion suppression [24].…”

Sample preparation for large‐scale bioanalytical studies based on liquid chromatographic techniques

“…The ME phenomenon was originally observed in case of electrospray ionization, when MS responses of organic bases decreased with an increase in concentrations of other organic bases (Kebarle & Tang, ). The most abundant phospholipids, namely glycerophosphocholines and lysophosphatidylcholines, are supposed to induce serious ion suppression effects in LC‐MS analysis, caused by the competition for space on the surface of droplets formed during the ESI process (Bylda et al, ; Carmical & Brown, ; Little, Wempe, & Buchanan, ). ME can be defined as the percentage by which the response of an analyte in the injected sample solvent is altered by the matrix.…”

Sample Preparation Strategies for the Effective Quantitation of Hydrophilic Metabolites in Serum by Multi-Targeted HILIC-MS/MS

“…Endogenous phospholipids contained in serum (with approximately 17,000 different types of lipids and fatty acids being present ) in significant levels are considered to be a real problem in HILIC (Hydrophilic Interaction Liquid Chromatography) systems. In HILIC mode, these species (Glycerophosphocholines (PC), sphingomyelins (SM), lysophosphatidylcholines (LPC), phosphatidylglycerols (PG), phosphatidylinositols (PI), and phosphatidylethanolamines (PE)) are eluted based on their ionizable moieties and appear in a wide range of retention times efficiency [18,19]. Even though phospholipids may not co-elute with the analytes, they can accumulate on the column and may elute in an unpredictable manner in subsequent analyses coverage [20].…”