commercial reagents contained 1–4% water. Anhydrous metha-
Here, we propose two procedures for derivatization of
fatty acyl residues with the reagent: one is for mild reac-
tion, and the other is for rapid reaction. All types of fatty
acids with O-ester linkages and FFAs were converted al-
most quantitatively into the corresponding methyl esters
in one-step reactions.
nolic 5% (w/v) HCl was also prepared by mixing acetyl chloride
with methanol (3, 9). To a lipid sample in a screw-capped glass
test tube was added 2.0 ml of anhydrous methanolic HCl, and the
mixture was heated at 100°C for 1 h in a boiling water bath. After
cooling, 1 ml of water was added, and then FAMEs were extracted
with 1 ml of hexane.
Mild methanolysis/methylation and rapid methanolysis/methylation
using conc. HCl. Commercial conc. HCl (35%, w/w; 9.7 ml) was
diluted with 41.5 ml of methanol to make 50 ml of 8.0% (w/v)
HCl. This HCl reagent contained 85% (v/v) methanol and 15%
(v/v) water that was derived from conc. HCl and was stored in a
refrigerator.
MATERIALS AND METHODS
Reagents
Fatty acids, TG, and cholesterol esters were purchased from
Avanti Polar Lipids (Alabaster, AL), Matreya (Pleasant Gap, PA),
Nu-Chek-Prep (Elysian, MN), and Sigma-Aldrich (St. Louis, MO).
Phosphatidylcholine (PC; dioleoyl) was synthesized according to
Ref. 13. Glass-distilled solvents were purchased from Sigma-
Aldrich and Wako Pure Chemical Industries (Osaka, Japan). An-
hydrous methanolic HCl reagents, HCl (35%, w/w), acetyl
chloride, methyl acetate, and 50% BF3 in methanol were of re-
agent grade.
A lipid sample was placed in a screw-capped glass test tube
(16.5 × 105 mm) and dissolved in 0.20 ml of toluene. To the lipid
solution, 1.50 ml of methanol and 0.30 ml of the 8.0% HCl solu-
tion were added in this order. The final HCl concentration was
1.2% (w/v) or 0.39 M, which corresponded to 0.06 ml of conc.
HCl in a total volume of 2 ml. Addition of mixed 1.2% HCl/
methanol/toluene solution to SE or TG samples should be
avoided because of the low solubilities of these lipids in water-
containing methanol. The tube was vortexed and then incubated
at 45°C overnight (14 h or longer) for mild methanolysis/methy-
lation or heated at 100°C for 1 h for rapid reaction. The reaction
time at 100°C was extended up to 1.5 h for samples containing
SE. After cooling to room temperature, 1 ml of hexane and 1 ml
of water were added for extraction of FAMEs. The tube was vor-
texed, and then the hexane layer was analyzed by GC directly or
after purification through a silica gel column.
TLC and GC
Reaction products of methanolysis/methylation were analyzed
by TLC on silica gel. Lipids separated were visualized by spraying
50% (w/w) sulfuric acid and then heating at 135°C. FAMEs were
analyzed with a Shimadzu 2014 gas chromatograph equipped
with a column of SUPELCOWAX 10 (0.53 mm × 30 m) at a col-
umn temperature of 215°C or 225°C.
Preparation of FAMEs from blood on a micro scale. FAMEs were also
prepared from one drop of whole blood spotted on filter paper
with 1.2% HCl/methanol/toluene under the mild conditions.
The procedure was a modification of a KOH/methanol method
(6), which had been developed for fatty acid analysis of blood
glycerolipids. Blood (0.025 ml) was spotted onto a small piece of
filter paper (1.5 × 1.5 cm; Whatman 3MM) that had been washed
with acetone containing 0.05% 2,6-di-tert-butyl-p-cresol. Each
piece, once it had dried, was put in a screw-capped test tube, to
which 0.2 ml of toluene, 1.5 ml of methanol, and 0.3 ml of the
8% HCl reagent were added sequentially and then incubated at
45°C overnight. To the reaction mixture were added 1 ml of
hexane and 0.2 ml of water, and the tube was vortexed. FAMEs in
the hexane layer were purified through a silica gel cartridge
column.
Preparation of FAMEs
Lipid substrates and FAME products. FAMEs were prepared from
1 mg of cholesteryl oleate, 1 mg of glyceryl trioleate, 1 mg of di-
oleoyl PC, 1 mg of oleic acid, 0.05 mg of cis-9,10-methyleneocata-
decanoic acid (a cyclopropane fatty acid), 0.05 mg of conjugated
linoleic acids that were mainly composed of cis-9,trans-11 and
trans-10,cis-12 isomers, 1 mg of olive oil, 1 mg of soybean oil, 1 mg
of linseed oil, 0.5 mg of fish oil (Pacific saury), 0.3 mg of blood
lipids that had been extracted by the method of Bligh and Dyer
(14) in the presence of 0.05% (w/v) 2,6-di-tert-butyl-p-cresol as an
antioxidant, and 0.025 ml of whole blood. The formation of
FAMEs was mainly investigated with cholesteryl oleate because
SEs are the most resistant to transesterification of lipid classes
having ester linkages (7). The internal standard was methyl hep-
tadecanoate or methyl tricosanoate. FAMEs formed from biologi-
cal materials were purified on cartridge columns packed with 200
mg of silica gel (No.102021; Merck, Darmstadt, Germany) prior
to GC. The silica gel cartridge was conditioned with 3 ml of hex-
ane, charged with FAMEs dissolved in 1 ml of hexane, and washed
with 3 ml of hexane. FAMEs were eluted with 3 ml of 1.5% (v/v)
methyl acetate in hexane.
Conventional method 1: saponification followed by BF3-catalyzed
methylation (Sap/BF3). A lipid sample in a screw-capped glass tube
(16.5 × 105 mm) was hydrolyzed with 1 ml of 1 M KOH in 70%
ethanol at 90°C for 1 h. The reaction mixture was acidified with
0.2 ml of 6 M HCl, and then 1 ml of water was added. FFAs re-
leased were extracted with 1 ml of hexane. After evaporation of
the hexane in vacuo, the FFAs were methylated with 1 ml of 10%
BF3 in methanol at 37°C for 20 min. Water was added to the solu-
tion, and then FAMEs were extracted with 1 ml of hexane.
Conventional method 2: anhydrous methanolic HCl method. HCl
concentrations of commercial anhydrous solutions of HCl/
methanol were 5% (w/v) according to the manufacturers’ speci-
fications, but the precise concentrations determined by titration
were 2.3–3.2%. Karl-Fischer titration indicated that some of these
RESULTS AND DISCUSSION
Mild methanolysis/methylation at 45°C
Reaction conditions were investigated for mild meth-
anolysis and methylation. Figure 1A shows that the
methanolysis of cholesteryl oleate at 45°C for 16 h pro-
ceeded almost quantitatively in the presence of 1.2–1.6%
HCl, which corresponded to 0.06–0.08 ml of conc. HCl
in a total volume of 2 ml. The HCl concentration of
2.0% increased the amount of cholesteryl oleate that
did not undergo methanolysis, presumably by lowering
the solubility of the substrate. The formation of FFA was
stimulated as the concentration of HCl increased. This
would be due to an increase in water content. In the
presence of 1.2% HCl, cholesteryl oleate required reac-
tion times longer than 14 h for completion of metha-
nolysis at 45°C (Fig. 1B).
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Journal of Lipid Research Volume 51, 2010