Separation of lipid classes by solid phase extraction free fatty acid, and phospholipid fractions.

Hee-Yong Kim' and Norman Salem, Jr.

Fatty acid analysis Standard bovine brain PS obtained from Avanti Polar Lipid was transmethylated
before and after solid phase extraction using BF3-methanol (14% w/v) at 100°C for 20 min according to
Morrison and Smith (5). After extraction three times with hexane, the extracts were dried under
nitrogen and redissoved in hexane. Fatty acid methyl esters were injected into an HP5880A gas
chromatograph equipped with a flame ionization detector.

PRODUCTION OF TRIACYLGLYCERIDES, FATTY

ACIDS, AND THEIR DERIVATIVES
For the analysis of total lipids, fatty acids were converted to methyl esters by methanolysis followed by gas
chromatography (GC) (Brandl et al. 1988; Waltermann et al.

2000). 10 mg of lyophilized cells was resuspended in 1 ml of methanol containing 15% (v/v)

H 2 SO 4 and 1 ml of chloroform. Methanolysis was carried out in a 100 0 C heating block for

2.5 h. After cooling to room temperature, 0.5 ml of deionized water was added to the solution and then vortexed for 1
min. The organic phase containing fatty acid methyl esters

(FAMEs) was analyzed by using an Agilent 6850 series II GC system equipped with an

Agilent DB-Wax column (30 m x 0.32 mm, 0.5 μm thick film) with hydrogen as carrier gas.

A 2 μl portion of the organic phase was injected with a 30:1 split ratio using the autosampler.