Separation and quantitation of peroxidized phospholipids using high-performance thin-layer chromatography with tetramethyl-p-phenylenediamine detection.

A simple method for the selective determination of phospholipid hydroperoxide (PLOOH) families in complex lipid populations has been developed. Referred to as HPTLC-TPD, the method is based on PLOOH separation by normal-phase high-performance thin-layer chromatography, followed by spray detection with N,N,N',N'-tetramethyl-p-phenylenediamine and densitometric scanning of the purple bands. Parental phospholipids and alcohol analogues are unreactive. Calibration curves, dynamic ranges, and detection limits were established for hydroperoxide standards prepared from phospatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. For all PLOOH classes, responsiveness was linear out to at least 10 nmol of sample load, the detection limit being 0.1-0.2 nmol. HPTLC-TPD data were validated by subjecting duplicate samples to more complex column chromatography with reductive-mode electrochemical detection. General applicability of the new technique was demonstrated using lipid extracts from two test systems: (i) photoperoxidized liposomal membranes and (ii) tumor cells that had been oxidatively stressed with the respiratory inhibitor antimycin A. HPTLC-TPD provides a convenient, specific, and highly sensitive means for quantifying individual PLOOH families in complex natural mixtures.