Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids.
We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3-diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBD-aminocaproyl (C6-NBD) or NBD-aminododecanoyl (C12-NBD) acyl chains were synthesized and analyzed by the following HPLC methods. An isocratic normal-phase method permitted resolution of a mixture of the 1,2-(palmitoyl, C6-NBD)-analogs of triacylglycerol, diacylglycerol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in less than 10 min, while a mixture of the (C6-NBD)-labeled analogs of ceramide, glucocerebroside, and sphingomyelin was separated in approximately 15 min. This method also detected various (C6-NBD)-phosphatidylcholine and -phosphatidylethanolamine molecules which differed only in their nonfluorescent acyl (oleoyl or palmitoyl) chains, and readily separated nonfluorescent dipalmitoylphosphatidylcholine from both (C6-NBD)- and (C12-NBD)-phosphatidylcholine derivatives. An isocratic reverse-phase system permitted separation of isomers of fluorescent phosphatidylcholine, -ethanolamine, -glycerol, -inositol, -serine, and phosphatidic acid in which the NBD-fatty acid was present in either the sn-1 or sn-2 position of the glycerol backbone.
D009327:4-Chloro-7-nitrobenzofurazan; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008055:Lipids / Q000302:isolation & purification*; D010069:Oxadiazoles; D013050:Spectrometry, Fluorescence; D013107:Sphingolipids / Q000032:analysis
D008055:Lipids; D010069:Oxadiazoles; D013107:Sphingolipids; D009327:4-Chloro-7-nitrobenzofurazan