Separation and determination of phospholipids in plasma employing thin-layer chromatographic plate with concentration zone or solid phase extraction.

ABSTRACT

Two efficient extraction methods for phospholipids have been established employing a TLC plate with a concentration zone or a solid-phase extraction cartridge. First, small amounts of five authentic phospholipids, 10 to 100 micrograms each, were separated on a TLC plate with a concentration zone. These phospholipids were not adsorbed to the silica gel of this TLC plate and the substrates were recovered in good yields. Second, a solid-phase extraction cartridge containing NH2-modified silica gel was able to retain all of the authentic phospholipids among C18-, diol-, and CN-modified silica gel and silica gel beds. Elution of neutral and acidic phospholipids from the NH2-cartridge was carried out with a chloroform-methanol mixture and a chloroform-methanol-28% ammonium mixture containing 0.05 M ammonium acetate, respectively. Recovery tests on the above two methods were carried out by conventional capillary gas chromatography. Thus, not less than 95% of each phospholipid was recovered from the TLC plate or NH2-cartridge in a 10 to 100 micrograms sample range. Also, the standard deviation of repeatability of recovery for phosphatidylcholine by solid phase extraction at a 10 micrograms sample range was favorable at sigma = 2.8. This method of separation was applied to determination of the fatty acid compositions of phospholipids in small amounts of plasma.