High-performance liquid chromatography of phospholipids using deuterated solvents for infrared detection

Infrared detection of chromatographic effluents offers the advantage of direct on-line quantitation of lipid fractions. However, infrared detection imposes limitations on the solvent systems that can be used for chromatography. Methanol and water, which are essential ingredients in the mobile phase for the successful chromatography of phospholipids, do not have spectral transmittance windows in the infrared region. Substituting deuterated methanol and deuterium oxide for methanol and water allowed infrared detection because they had lower infrared absorbance than their hydrogenated counterparts. We report a method that is suitable for the quantitative analysis of phosphatidylethanolamine, phosphatidylcholine and sphingomyelin in the tissue extracts. The lipid separation was accomplished on a micropraticulate silica gel column. Phosphatidylethanolamine and phosphatidylcholine were eluted isocratically with chloroform-acetonitrile-methanol-deuterium oxide (136:25:34:5.9) and detected at a wavelength of 5.75 microns. For the analysis of sphingomyelin, chloroform-acetonitrile-deuterated methanol-deuterium oxide (130:24:37.6:7.0) was used as the mobile phase, and the detection was at a wavelength of 6.15 microns.