Separation of neutral lipid,free fatty acid and phospholipid classes by normal phase HPLC |
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Authors: | James G Hamilton Karen Comai |
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Affiliation: | (1) Present address: Department of Pharmacology, Hoffmann-La Roche Inc., 151 Rutgers Place, 07110 Nutley, NJ;(2) Present address: 2976 Heather Bow, 34235 Sarasota, FL |
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Abstract: | Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid
and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10
min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100∶5∶0.02) proved effective
in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled
triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic
acid (70∶30∶0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride.
A mobile phase consisting of hexane/isopropanol/acetic acid (100∶2∶0.02) effectively separated free fatty acid, cholesterol,
1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid
classes were separated using methylteriarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5∶8∶2) as the mobile phase.
The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine
were each greater than 96%. |
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