Interest of Fluorescence Derivatization and Fluorescence Probe Assisted Post-column Detection of Phospholipids: A Short Review

Abstract

Phospholipids are essential constituents of all living cell membranes. There are many analytical methods available for the quantitative and qualitative determination of phospholipids, but since these molecules lack chromophores, common absorbance based methods are of limited use. Beside mass spectrometry, some less specific approaches that are routinely used are evaporative light scattering detection or fluorescence, which exhibit sufficient sensitivity. Here, we focus on fluorescence, which remains an interesting way to quantify phospholipids. Two ways of detecting phospholipids by fluorescence are possible coupled with separation techniques such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE): firstly, pre-column derivatization procedures and secondly, probe assisted post-column detection with suitable fluorescence reagents. In both cases, the common purpose is to increase the detection sensitivity. It is shown that, whereas pre-column derivatization is characterized by selectivity due to the chemical functionality of the analyte involved in the derivatization process, in supramolecular post-column derivatization, the selectivity only proceeds from the capacity of the lipid to involve supramolecular assemblies with a fluorescence probe. The aim of this review is to summarize available experiments concerning fluorescence detection of phospholipids. The interest and limitation of such detection approaches are discussed.

Investigating the possible use of a tetra (hydroxyphenyl) porphyrin as a fluorescence probe for the supramolecular detection of phospholipids

“…Lipids can be detected after HPLC separation by means of a dye introduced via a post-column device [4]. In practice, an aqueous post-column reagent containing the dye (and a surfactant in order to assure dye solubility) is mixed with the chromatographic effluent before the detector.…”

“…Phospholipids are molecules that lack chromophores making a sensitive UV detection impossible. Usual detection techniques after chromatographic separation are mass spectrometry, evaporating light scattering detector (ELSD) and fluorescence detection by means of a covalent or a non-covalent association with a fluorescent reagent [4]. In this study, we are interested in the use of polarity probes in order to indirectly detect lipids.…”

Development of an HPLC post-column antioxidant assay forSolidago canadensisradical scavengers

“…Online post-column methods are employed in the rapid identification of antioxidants and determination of their activity (Kosar et al 2004). One highly effective technique developed and modified by different researchers are methods based on a model oxidation system that combines HPLC separation methods with the online post-column detection of radical-scavenging compounds (Ibrahim et al 2010;Raudonis et al 2012;Camenzuli et al 2013). Model oxidation systems of ABTS and DPPH radicals have been extensively used for screening antioxidants from plant extracts, and they possess important advantages when compared with colorimetric methods (Pukalskas et al 2002;Kosar et al 2004;Ali et al 2014).…”

Lipidomic mass spectrometry and its application in neuroscience

“…Another approach is specific chemical derivatization that selectively reacts to specific or class specific lipids eliminating them from the total spectra upon chemical derivatization. Thus, mass spectra with and without chemical derivatization enables distinguishing specific lipids from scans performed in time domain resolving instruments [30][31][32] . The time domain instruments produce a comprehensive dataset of MS precursor ions and the MS/MS spectra comprising all fragment ions derived from all lipid precursor ions [33] .…”

“…Derivatization enhances detection of specific lipids or members of lipid classes on time domain resolved instruments. Derivatization also often assists localization of lipids on cell and organelle membranes or even identification of lipid bound proteins in the membranes [31,32,39] .…”