Quantification of phospholipids and sphingomyelin by HPLC-ELSD

Abstract: Phospholipids are amphiphilic molecules that are the main component of cellular lipid bilayer membranes that are crucial to the function of all eukaryotic cells. Phospholipids can also act as signaling molecules and can be broken down for energy and their functional head groups can impact metabolism. Phospholipids play an important role in the human diet and are found in high concentrations in some animal and plant sources. The main extraction methods and analytical determination methods are Folch extraction method and HPLC-ELSD. Here we use a modified Folch extraction for various matrices including human plasma as well as dairy products followed by filtration and injection onto an HPLC. HPLC conditions utilize a silica column and a chloroform/methanol solvent system to separate four major phospholipids; phophatidylethanolamine (PE), phosphatidylinositol (PI), phophatidylserine (PS), and phophatidylcholine (PC) as well as Sphingomyelin (SM) and Dioleoylphosphatidylglycerol (DOPG) as an internal standard. HPLC is coupled with evaporative light scattering detector (ELSD) optimized for quantification of phospholipids.