600
A. SATAKE et al.
Table 1. Components of the 13C-Labeled Ester Mixture
Yuzu fruits were collected from 10 local wholesale
markets from northern to southern Japan in Novem-
ber 1999, and their cold-pressed oils (CPO) were pre-
pared as described in a previous paper.10) Lemon
CPO was prepared from commercially sold fruits by
the same method. Authentic chemicals for mass spec-
trometry were obtained from the commercial sources
mentioned previously.11)
Conc.
Molecular
weight
Compound
z
(w w)
W
Pentyl acetate
Hexyl acetate
Heptyl acetate
Octyl acetate
Nonyl acetate
Decyl acetate
2.36
2.64
2.96
3.23
3.54
3.80
131.2
145.0
159.2
173.3
187.3
201.3
Gas chromatography-mass spectrometry (GC-
MS). Gas chromatography combined with mass spec-
trometry was used for identifying the volatile compo-
nents. The analysis was carried out with a Shimadzu
GC-17A linked with a Shimadzu QP-5050 at an MS
ionization voltage of 70 eV, accelerating voltage of
octanol, nonanol or decanol in benzene was mixed
with 82 mmol 1-13C-acetic acid. The reaction mixture
was re‰uxed for 1.5 hr with a small amount of p-tol-
uene sulfonic acid. The reaction proceeded almost
perfectly, and mixtures containing the esters of the
9
1500 V, and ion source temperature of 250 C. The
GC column was DB-Wax fused-silica capillary type
5–10 carbon number at 2.6–3.8z (w w) were ob-
W
(60 m 0.25 mm i.d., 0.25
m
m ˆlm thickness; J & W
tained (Table 1). The synthesized 13C-labeled esters
were determined by GC and GC-MS. Non-labeled es-
ters were synthesized as well.
×
Scientiˆc, Folsom, CA, U.S.A). The column temper-
ature was programmed from 70 C (2 min hold) to
100 C at a rate of 2 C min. The column was cleaned
by heating to 230
temperature was 250
carrier gas at a ‰ow rate of 0.8 ml min. An oil sam-
9
9
9
W
9C before each run. The injector
Synthesis of limonene. 4-Acetyl-1-methylcyclo-
9
C, and helium was used as the
hexene was synthesized by the method described by
13)
14)
Lutz et al
.
and Fray et al
.
Ninety mmol of SnCl4
W
ple of 0.2
m
l was injected at a split ratio of 1:50.
was added to 120 ml of benzene while stirring at 3 C,
9
and then 550 mmol of isoprene was added. The reac-
tion mixture was added to 500 mmol of methyl vinyl
ketone over a 15-min period, and then stirred con-
Identiˆcation of the components. Each component
was initially identiˆed by the GC retention index and
the NIST library connected to the QP-5050 mass
tinuously for 2 hr at 5–109C. The mixture was succes-
spectrometer, as described in previous papers.10,11)
A
sively washed with an NaCl aq. solution and KOH
aq. solution, and dried with sodium sulfate. After
drying, the mixture was evaporated to 31.4 g (a yield
JNM-LA400 spectrometer (Jeol, Tokyo) was em-
ployed for recording the 13C-NMR spectra, CDCl3
being used as a solvent.
of 45.4
GC-MS. The purity of 4-acetyl-1-methylcyclohexene
was 96 by GC, containing 4 of 3-acetyl-1-
methylcyclohexene.
z
). Identiˆcation was carried out by GC and
Determination of the isotope ratio. The following
10 monoterpene compounds were examined in the
z
z
determination of isotope ratios:
sabinene, myrcene,
limonene, -terpinene,
a
-pinene,
-phellandrene, -terpinene,
b-phellandrene and terpino-
b
-pinene,
13C-Labeled limonene, 1-methyl-4-(1-methyl-(2-
13C)-ethenyl)-cyclohexene, was synthesized by means
of the Witting-reaction.15) The labeling reagent,
a
a
g
lene. Selected ion monitoring (SIM) of GC-MS was
performed in order to estimate the intensities of the
4.9 mmol 13CH3P(C6H5)3I, was added at 20
9
C over
5 min to a solution of 4.9 mmol NaH in 5 ml of
DMSO, which had been prepared while stirring at
ion peaks of each molecule (m z 136) and of its iso-
W
tope (m z 137). The total intensity of each compound
below 70
After adding 5.0 mmol of acetyl-1-methylcyclo-
hexene at 20 C over 5 min, the reaction mixture was
9
C, and the mixture stirred for 20 min.
W
7
×
was regulated to achieve about 8.0 10 in MS, the
mass spectrometry scanning interval being 0.1 sec.
The isotope ratio (Ir) of each peak was calculated by
the following equation:
9
stirred for 2.5 hr at room temperature. Extraction
was performed with pentane and, after drying with
sodium sulfate, the solvent was removed to obtain
＝
Ir (Intensity of an isotope peak of m z 137)
W
the ˆnal product of 0.14 g (a yield of 20.9
z).
(Intensity of a molecular peak of m z 136)
W
W
Results and Discussion
×
100
where the intensity is the mean value from quintupli-
cate measurements.
Accuracy of the isotope ratio by ordinary GC-MS
In principle, it is possible to obtain the isotope
ratio from MS data. The authors have previously
shown, in fact, a practical use for the isotope ratio
from mass spectrometry.10) However, more detailed
analytical conditions need to be found to achieve
Synthesis of esters. The 13C-labeled esters were
synthesized by a conventional method.12) A solution
of 9 mmol of butanol, pentanol, hexanol, heptanol,
Satake, Atsushi
