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J Am Oil Chem Soc (2011) 88:1275–1283
advantages of minimal sample preparation and high sen-
sitivity (method LOD = 5 ng/g). However, time-of-flight
mass spectrometers are not common instruments in most
laboratories, and the sodium content of the mobile phase
requires cleaning of the instrument on a daily basis and
more extensive cleaning on a weekly basis. Masukawa
et al. [6] developed and later refined [9] a method for
quantifying glycidyl esters in edible oils using a single
quadrupole mass spectrometer, an instrument that is more
affordable and user-friendly, but sample preparation
requires two solid-phase extraction and solvent evaporation
steps which add significant costs for time and materials on
a per sample basis.
temperature over a 24 h period. The progress of the reac-
1
tion was monitored by TLC and H NMR. After the
reaction was complete, the reaction mixture was diluted
with 20 mL hexane and washed with aqueous sodium
bisulfite (2% w/w), aqueous sodium bicarbonate (10%
w/w), water and aqueous saturated sodium chloride. The
organic layer was dried over anhydrous Na SO and con-
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centrated in vacuo to yield a colorless oil. The oil was
purified on a silica gel column (60–200 mesh, 100 mL bed
volume) using gradient elution of ethyl acetate (0–20%
v/v) in hexane. The fractions containing glycidyl esters
were concentrated, and each product yielded a colorless oil
that solidified on cooling. Final products were character-
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Therefore, the objective of this research was to develop
a fast, accurate and rugged screening method for glycidyl
esters in edible oils using minimal sample preparation and
a single quadrupole mass spectrometer.
ized using H NMR and C NMR, and purity was deter-
mined using GCMS.
Internal Standard Solution Preparation
Deuterated glycidyl palmitate was diluted with acetone to a
target concentration of 200 ng/mL. This internal standard
solution was used in the direct LC–MS method for sample
and calibration standard preparation.
Experimental Procedures
Reagents
HPLC grade methanol and acetone were purchased from
Fisher Scientific (Pittsburgh, PA, USA). HPLC grade
acetonitrile was from EMD Chemicals, Inc. (Gibbstown,
NJ, USA).
Calibration Standard Solution Preparation
Mixed standards containing each of the seven glycidyl
esters were prepared by dilution with d31-glycidyl palmi-
tate internal standard solution in acetone. Individual glyc-
idyl ester concentrations ranged from 10 to 400 ng/mL and
were corrected for purity.
Standards
Glycidyl stearate was purchased from TCI America
(
Portland, OR, USA). Glycidyl linolenate, glycidyl linole-
Samples
ate, glycidyl oleate and glycidyl palmitate were purchased
from Wako Pure Chemical Industries (Tokyo, Japan).
Glycidyl esters of lauric acid, myristic acid and fully-
deuterated (d31) palmitic acid were synthesized individu-
ally using a two-step chemical procedure in which the allyl
ester was the initial product followed by conversion to the
glycidyl ester. Deuterated palmitic acid was included in
these experiments as an internal standard. A detailed
description of the synthesis is provided elsewhere [8].
Briefly, allyl alcohol, toluene and Amberlyst 15 were
refluxed with the respective fatty acid in an oil bath for
Samples included refined, bleached, de-waxed, deodorized
corn oil (RBWD Corn); refined, bleached, de-waxed,
deodorized canola oil (RBWD Canola); refined, bleached,
deodorized mid-oleic sunflower oil (RBD sunflower);
refined, bleached soy oil (RB Soy); refined, bleached,
deodorized palm oil (RBD Palm); and palm kernel oil.
Sample Preparation for Direct LC–MS Method
An aliquot of oil (*0.25 g) was accurately weighed into a
glass centrifuge tube, and 5 mL of the internal standard
solution in acetone were added. If necessary, samples were
placed in a heating block set at 65 °C after addition of the
internal standard solution to melt the oil and ensure sample
homogeneity. Sample amounts were increased for repro-
ducibility assays, but the sample:solvent ratio was main-
tained at 1:20 (1.25 g diluted with 25 mL internal standard
solution). Sample extracts were assayed directly using LC–
MS without further clean-up. Sample fortification, when
performed, utilized the same final volume (5 mL), internal
2
4 h. The reaction mixture was diluted with 20 mL hexane
and filtered to remove Amberlyst 15. The reaction mixture
was washed with water and saturated sodium chloride, and
the organic layer was dried over anhydrous Na SO and
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concentrated in vacuo to yield a colorless oil that solidified
on cooling (allyl ester). This material was mixed with
dichloromethane and cooled in an ice bath for 5–10 min
before meta-chloroperbenzoic acid was added in small
amounts. After the addition was complete, the reaction
mixture was stirred and allowed to slowly warm to room
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