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Journal of Lipid Research, Vol 31, 157-159, Copyright © 1990 by Lipid Research, Inc.

ARTICLES

An HPLC procedure for separating polyphosphoinositides on hydroxylapatite

LE Boyle, LA Sklar and AE Traynor-Kaplan
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.

We describe a method for separation of several phosphoinositides by high performance liquid chromatography (HPLC) for the purposes of identification, analysis, and possible purification of cell phospholipid extracts. The phosphoinositides were separated on an hydroxylapatite column using a solvent system consisting of tetrahydrofuran-ethanol-water with a gradient of triethylamine phosphate ranging from 1 to 100 mM. Increasing triethylamine phosphate concentrations over a series of isocratic steps resulted in the resolution of [3H]phosphoinositide standards (phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5- bisphosphate) and their lyso-derivatives. The eluted peaks were collected and quantitated using scintillation counting, and the samples of the eluates were subjected to further analysis by thin-layer chromatography to verify their integrity and identity.
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