HPLC Analytical Methods for the Separation of Molecular Species of Fatty Acids in Diacylglycerol and Cellular Phospholipids

Separation of PLs or the lipid mediators DAG and PA into individual molecular species is a complex multistep procedure. For example, first, phospholipid classes must be quantitatively isolated in amounts large enough for further analysis. In Chapter 22 , we described the multiple steps and analytical methods necessary to achieve this fractionation with good resolution and recoveries. Once pure phospholipid fractions (PC, PE, PI, and CL) or pure DAG or PA are obtained, analyses of their molecular species have customarily been performed in a series of steps that include partial hydrolysis, derivative formation, and/or a combination of several different types of chromatographic procedures (
1
,
2
). These methods would ideally be simple enough to be performed routinely, but at the same time, the methods have to be rigorously controlled qualitatively and quantitatively to show that the fractions of fatty acids collected are not changed by the method used. It is also important to differentiate changes in molecular species of fatty acids because of dietary manipulations. These are not easy tasks. Length and unsaturation of fatty acids, the double-bond position in the chain or in the PL molecule, and the proportions and the topographically heterogenous distribution of lipid constituents inside the cell membrane, are all factors responsible for the structural and dynamic properties of the membrane edifice.