We describe a method for analyzing the choline-containing phospholipids, namely phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin that is highly sensitive, easy to perform, and very reliable. The method employs enzymic hydrolysis of choline from these phospholipids by phospholipase D with subsequent oxidation of choline by choline oxidase and generation of hydrogen peroxide. Hydrogen peroxide is then reduced by peroxidase with p-hydroxy-phenylacetic acid acting as an oxygen acceptor to form a fluorescent product which is stable for at least 24 h without loss of linearity. The analysis requires a 5 microL sample volume and demonstrates linearity over a wide range (133 mumol/L-6.65 mmol/L). The CVs are less than 3%.
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