TABLE 1.

Optimized conditions for preparation of FAMEs from individual lipid classes with conc. HCl/methanol/toluene

Lipid Class	HCl Conc.	Time	Yield of FAMEa
Mild methanolysis/methylation at 45°C
	%		%
SE	1.2	16 h	98.2 ± 0.7
TG	1.2	8 h	98.8 ± 0.7
	0.6	14 h	97.8 ± 0.2
PhL	1.2	8 h	96.7 ± 0.2b
FFA	0.5	20 min	98.4 ± 0.1c
	0.2	60 min	99.1 ± 0.2
Rapid methanolysis/methylation at 100°C
	%		%
SE	1.2	90 min	96.8 ± 0.3d,e
TG	1.2	30 min	97.3 ± 0.3e
PhL	1.2	30 min	96.5 ± 0.5
FFA	0.2	5 min	98.4 ± 0.3

Molecular species of lipid classes analyzed were cholesteryl oleate as SE, glyceryl trioleate as TG, dioleoyl PC as PhL, and oleic acid as FFA.

^aAfter reaction of each lipid class, a hexane solution of methyl heptadecanoate as an internal standard and water were added to the reaction mixture. The yield of FAME in the hexane layer was determined by GC. Each value of yield is the average of three determinations.

^bMethanolysis of PhL with 0.6% HCl for 14 h at 45°C also gave a similar yield.

^cA similar yield, 97.9%, was obtained with 1.2% HCl.

^dArtifacts were produced from cholesterol. A shorter reaction time, 60 min, gave a yield of 96.5%.

^eFor comparison, anhydrous methanolic 5% HCl was prepared prior to use by mixing 2 ml of acetyl chloride and 20 ml of chilled methanol. SE and TG were each dissolved in toluene and heated at 100°C for 90 and 60 min, respectively, with the anhydrous reagent. The HCl concentration of the reaction mixture was 4.5%. The yields of FAME were 99.0 ± 0.8% for SE and 99.6 ± 0.1% for TG.