| The objective of this research was to develop and optimize chromatographic separations for three groups of lipid classes: neutral lipids and free fatty acids; ceramides and sphingoid bases; and anionic phospholipids. Phospholipids are essential components of cell membranes and sphingolipids are important due to their role as intracellular second messengers. Both ceramides and sphingoid bases have been implicated as important second messengers in cell transduction pathways regulating cell proliferation, differentiation, secretion and apotosis. The study of these lipid intermediates has been of high interest in recent years in determining mechanisms of intracellular signal transduction pathways and how these pathways are regulated. Many sphingolipid and phospholipid processes have still not been determined and the development of analytical methodologies for lipids is a key feature of these studies.; High performance liquid chromatography (HPLC) using silica columns is now the primary method for analyzing lipids. The analysis of charged compounds by HPLC has been difficult due to the interactions of charged groups with silanols of the silica gel stationary phases. Therefore rapid, direct, and quantitative techniques are needed to separate lipid classes. Evaporative light scattering detection (ELSD) is a semi-universal mass detector which has great potential for the analysis of lipids. Lipids can be analyzed which do not have a UV absorbing chromophore and gradients can be used. Analytes are quantified with the use of external standards. An other detector, Fourier transform infrared spectrophometry (FTIR), was recently interfaced with HPLC and has shown great promise for the analysis of lipids.; In the present work, HPLC/ELSD and HPLC/FTIR methods were developed for the analysis of a variety of lipid classes. Method development activities were based upon the transfer of thin layer chromatography solvent systems with silica gel plates to HPLC methods. Fatty acids were separated from triglycerides, diglycerides, and monoglycerides with a polyvinylalcohol-silica (PVA-Si) column. Two different types of stationary phases were investigated to separate the sphingoid bases and ceramides with mobile phase modifiers of formic acid and triethylamine. The phospholipids were resolved as sharp peaks over the complete linear range on a PVA-Si column with a binary mobile phase of chloroform, methanol, water, and ammonium hydroxide. All lipids were resolved as lipid classes by normal phase-HPLC with rapid assays of 20 minutes or less. |
