Quantitative determination of lecithin and sphingomyelin in amniotic fluid

A quantitative method for lecithin and sphingomyelin is presented. In this method, which may be calibrated with a primary standard phosphate salt or pure phospholipids, 2 mL of amniotic fluid is extracted, and the solvent passed through a column of hydroxyapatite to remove all phospholipids except lecithin and sphingomyelin. After evaporation, the residue is treated with methanolic sodium hydroxide, which hydrolyzes lecithin to inorganic phosphate and a methyl ester. The unreacted sphingomyelin is removed by extraction, and the solvent evaporated. Both fractions are digested and assayed for phosphate by reduced phosphomolybdate. The total time required is 2 h. Analytical recoveries were 91-97%, and excellent agreement was found for controls. Amniotic fluid specimens were assayed to illustrate application of the proposed method. If this method is used for obtaining a lecithin/sphingomyelin ratio, a new ranges must be established, as this method is not equivalent to thin-layer chromatographic ones.