Abstract
A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.
Highlights
high-performance liquid chromatography (HPLC) grade glass-distilled acetonitrile, chloroform, and methanol were obtained from MCB Manufacturing Chemists Inc. (Cincinnati, OH)
Biochemicals Inc. (Milwaukee, WI), while PI, PS, and sphingomyelin (SPH) were purchased from Avanti Biochemicals Inc. (Birmingham, AL). [ l-'4C]Oleic acid and [3H]arachidonic acid were obtained from New England Nuclear Inc. (Boston, MA) and [ 1-'4C]dipalmitoyl phosphatidylcholine was purchased from Applied Science Inc. (State College, PA)
Preliminary studies demonstrated that the main phospholipid classes can be separated on an Ultrasphere-Si column with a mobile phase containing acetonitrile, methanol, and sulfuric acid
Summary
MATERIALSHPLC grade glass-distilled acetonitrile, chloroform, and methanol were obtained from MCB Manufacturing Chemists Inc. (Cincinnati, OH). HPLC grade glass-distilled acetonitrile, chloroform, and methanol were obtained from MCB Manufacturing Chemists Inc. Analytical ACS grade sulfuric acid was purchased from Fisher Scientific Co. (Fair Lawn, NJ), and 12% BF3 in methanol (w/v) was obtained from Supelco, Inc. Phosphatidylcholine (PC), PE, and l-oleoyl-L-a-lysolecithi(nLPC) were obtained from P. (Milwaukee, WI), while PI, PS, and sphingomyelin (SPH) were purchased from Avanti Biochemicals Inc. [ l-'4C]Oleic acid and [3H]arachidonic acid were obtained from New England Nuclear Inc. (Boston, MA) and [ 1-'4C]dipalmitoyl phosphatidylcholine was purchased from Applied Science Inc.
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