6
102 J. Agric. Food Chem., Vol. 54, No. 16, 2006
Zamora et al.
Table 1. Retention Indices and Mass Spectra of Compounds
tafluorobenzyl)hydroxylamine hydrochloride, and N,O-bis(trimethylsilyl)-
trifluoroacetamide were purchased from Aldrich (Milwaukee, WI). All
other chemicals were analytical grade and purchased from reliable
commercial sources.
a
Determined in This Study
compound
retention index
mass spectrum
4
,5-Epoxy-2-decenal was prepared from 2,4-decadienal as described
benzaldehyde
phenylacetaldehyde
phenylpyruvic acid
960
1049
2015
106 (100), 105 (98), 77 (89), 51 (37)
120 (23), 92 (22), 91 (100), 65 (16)
431 (3), 416 (5), 189 (14), 181 (46),
previously (15). Briefly, 3-chloroperoxybenzoic acid (25 mmol) was
dissolved in chloroform (175 mL), washed with three 100 mL portions
of buffer (0.2 M Na
2
4
HPO ‚12H
2
O adjusted to pH 7.5 with 0.1 M citric
(derivatized)
116 (34), 91 (23), 73 (100)
acid monohydrate) followed by three 100 mL portions of water, and
dried with anhydrous sodium sulfate. This solution was added slowly
a
Structures for these compounds are given in Scheme 1.
(25 mL every 10 min) to a solution of 2,4-decadienal (3.0 g, 19.7 mmol)
in chloroform (29 mL), which was stirred at room temperature. The
reaction mixture was then stirred overnight and, finally, washed with
three 100 mL portions of buffer (0.2 M Na
2
HPO
4
2
‚12H O adjusted to
pH 7.5 with 0.1 M citric acid monohydrate) followed by three 100 mL
portions of water to remove 3-chlorobenzoic acid. The organic solution
was dried with anhydrous sodium sulfate and concentrated under
vacuum. The residue was fractionated by column chromatography using
hexane/acetone (95:5) as eluent. 4,5-Epoxy-2-decenal was obtained
chromatographically pure. Additional confirmations of identity and
1
13
purity were obtained by H and C NMR and GC-MS.
,5-Epoxy-2-decenal/Phenylalanine Reaction Mixtures. A solution
4
of 0-31 µmol of 4,5-epoxy-2-decenal and 25 µmol of phenylalanine
in 500 µL of acetonitrile-sodium citrate or sodium phosphate buffer
(
2:1) was heated at 37 or 60 °C. Incubated mixtures were analyzed for
phenylacetaldehyde and phenylpyruvic contents. For phenylacetalde-
hyde analysis, incubated samples (75 µL) were diluted with 125 µL of
acetonitrile-water (2:1) and 25 µL of internal standard solution [337
µg of 3-(Z)-nonenol in 1 mL of methanol] and analyzed by GC-MS.
Phenylpyruvic acid was determined according to Lee et al. (16) using
a slightly modified procedure. Briefly, incubated samples (100 µL) were
diluted with 200 µL of acetonitrile-water (2:1) and 50 µL of internal
standard solution [386 µg of stearic acid in 1 mL of methanol] and
treated with 4.6 mg of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine
hydrochloride. This reaction mixture was maintained for 2 h at 37 °C
and then taken to dryness. The obtained residue was derivatized with
Figure 1. Effect of pH on phenylacetaldehyde (
nylpyruvic acid ( and ) formation in the reaction of 4,5-epoxy-2-decenal
with phenylalanine (Phe) in acetonitrile/buffer (2:1) after 21 h at 37 C.
The employed buffers were 50 mM sodium citrate buffer for pH 2.15
and ) and 50 mM sodium phosphate buffer for pH 6 8 ( and
0 and O) and phe-
4
3
°
−6
(
0
4
−
O
3
).
RESULTS
Effect of pH in the Formation of Both Phenylacetaldehyde
and Phenylpyruvic Acid in 4,5-Epoxy-2-decenal/Phenylala-
nine Reaction Mixtures. As described previously (1), direct
injection of epoxydecenal/phenylalanine reaction mixtures al-
lowed detection of phenylacetaldehyde by GC-MS. However,
when the incubated reaction mixtures were derivatized, the
presence of phenylpyruvic acid was also observed. Both
compounds were identified unambiguously by comparison with
the retention indices and mass spectra of authentic compounds.
Retention indices and mass spectra of compounds determined
in this study are collected in Table 1.
1
°
50 µL of N,O-bis(trimethylsilyl)trifluoroacetamide for 30 min at 60
C and analyzed by GC-MS.
Phenylpyruvic Acid/Phenylalanine Reaction Mixtures. A solution
of 0.5 µmol of phenylpyruvic acid and 25 µmol of phenylalanine in
00 µL of acetonitrile-sodium citrate buffer (2:1), pH 3.0, was heated
5
at 60 °C. Incubated mixtures were analyzed for phenylacetaldehyde
and benzaldehyde contents. Incubated samples (75 µL) were diluted
with 125 µL of acetonitrile-water (2:1) and 25 µL of internal standard
solution [337 µg of 3-(Z)-nonenol in 1 mL of methanol] and analyzed
by GC-MS.
Both carbonyl derivatives of the amino acid were produced
simultaneously in the reaction mixtures to an extent that
depended on the pH. Figure 1 shows the effect of pH in the
production of both compounds after 21 h at 37 °C. Both
phenylpyruvic acid and phenylacetaldehyde were produced to
a higher extent at pH 2-4, and the amount of phenylpyruvic
acid was ∼2.5 times the amount of phenylacetaldehyde. This
preference for the formation of phenylpyruvic acid was reduced
at a higher pH. The rest of this study was carried out at pH 3
because the highest amounts of both compounds were produced
at this pH.
GC-MS Analyses. GC-MS analyses were conducted with a
Hewlett-Packard 6890 GC Plus coupled with an Agilent 5973 MSD
(Mass Selective Detector-Quadrupole type). A fused silica HP5-MS
capillary column (30 × 0.25 mm i.d.; coating thickness 0.25 µm) was
used. Working conditions were as follows: carrier gas, helium (1 mL/
min at constant flow); injector temperature, 250 °C; oven temperature,
from 70 (1 min) to 240 °C at 5 °C/min and then to 325 °C at 10 °C/
min; transfer line to MSD, 280 °C; ionization EI, 70 eV.
Determination of Phenylacetaldehyde, Phenylpyruvic acid, and
Benzaldehyde Contents. Quantification of phenylacetaldehyde and
benzaldehyde was carried out by preparing standard curves over a
concentration range of 2-60 nmol of phenylacetaldehyde or 5-60 nmol
of benzaldehyde in the 225 µL of solution prepared for GC-MS injection
Effect of 4,5-Epoxy-2-decenal Concentration in the For-
mation of Both Phenylacetaldehyde and Phenylpyruvic Acid.
The amount of phenylacetaldehyde and phenylpyruvic acid
produced depended on the concentration of the epoxyalkenal
added, therefore confirming the role of this lipid oxidation
product in the degradation of the amino acid. Figure 2 shows
the formation of both phenylacetaldehyde and phenylpyruvic
acid after 21 h at pH 3 and 37 °C as a function of epoxyalkenal
concentration. The concentration of both carbonyl derivatives
increased linearly (r > 0.995, p < 0.0001) with the concentration
of epoxyalkenal, but both lines were not parallel, and a higher
amount of epoxyalkenal favored the production of phenylpyruvic
(see above). For each curve five different concentration levels of the
aldehyde were used. Phenylacetaldehyde and benzaldehyde contents
were directly proportional to the aldehyde/internal standard area ratio
(
r > 0.99, p < 0.0001). The coefficients of variation within this range
were lower than 5%.
Quantification of phenylpyruvic acid was carried by preparing
standard curves over a concentration of 15-400 nmol of phenylpyruvic
acid in the 100 µL of sample used in the derivatization reaction (see
above). For each curve five different concentration levels of the
aldehyde were used. Phenylacetaldehyde content was directly propor-
tional to the aldehyde/internal standard area ratio (r > 0.99, p < 0.0001).
The coefficients of variation within this range were lower than 5%.