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High-Performance Liquid Chromatographic Analysis of Phospholipids from Different Sources with Combined Fluorescence and Ultraviolet Detection

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
220
Issue
1
Identifiers
DOI: 10.1006/abio.1994.1315
Disciplines
  • Physics

Abstract

Abstract An isocratic high-performance liquid chromatographic (HPLC) system was developed for the separation of major phospholipid classes, i.e., phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. Phospholipids were detected with ultraviolet absorption at 205 nm and subsequent fluorescence detection. Fluorescence of the phospholipids (excitation, 340 nm; emission, 460 nm) was achieved by postcolumn formation of mixed micelles with 1,6-diphenyl-1,3,5-hexatriene. For ultraviolet absorption there were great differences depending on the saturation of phospholipid fatty acids but for fluorescence the sensitivity was almost identical for all phospholipids except phosphatidylinositol and lysophosphatidylcholine. Dipalmitoylphosphatidylcholine showed nearly no ultraviolet but good fluorescence response. Ultraviolet to fluorescence ratio was characteristic for different phospholipids and for identical phospholipids from different sources. Quantification of phosphatidylcholine and phosphatidylethanolamine with HPLC using N-monomethylphosphatidylethanolamine (dioleoyl) as an internal standard gave the same results as phospholipid phosphorus quantification after thin-layer chromatography.

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