Light‐scattering detection of phospholipids resolved by HPLC

An improved method for the analysis of phospholipids by normal-phase HPLC is described. Addition of methanol and acetonitrile to a gradient based on 2-propanol/hexane/water promoted a rapid separation of major classes of bovine surfactant phospholipids (PL) by using a conventional silica column. The use of an ELSD permitted an accurate analysis of a mixture of PL. Calibration curves were linear within the range of 5-40 microg with detection limits below 1 microg for PE and PC, and CV ranged from 0.6 to 9.6%. PL present in surfactant homogenates were separated by a solid-phase extraction (SPE) procedure before HPLC analysis. This methodology gave a recovery of 95% and combined SPE-HPLC and quantification of biological PL within a 30-min run. The use of ELSD detection of the eluted compounds was precise, linear, and sensitive.