Instrumental analysis of terminal-conjugated dienes for reexamination of the sex pheromone secreted by a nettle moth, parasa lepida lepida

Article abstract of DOI:10.1271/bbb.90047

Conjugated dienyl compounds make one of the main groups of lepidopteran sex pheromones, and GC has been frequently used to determine the configurations of the double bonds. However, the separation of two geometric isomers of a terminal-conjugated diene, such as 7,9-decadien-1-ol secreted by a nettle moth Parasa lepida lepida (Limacodidae), is assumed to be difficult. In order to clarify the chromatographic separation of the terminal dienes, 7,9-decadienyl and 9,11-dodecadienyl compounds (alcohols, acetates, and aldehydes) were analyzed by GC and HPLC. On a capillary GC column, the (E)-isomers flowed out slightly faster than the corresponding (Z)-isomers, but their peaks almost overlapped. On the other hand, HPLC equipped with an ODS column completely separated the two geometric isomers examined and the (Z)-isomers eluted from the column faster than the (E)-isomers without dependence on a functional group. In addition to undergoing direct HPLC analysis without derivatization, the dienyl alcohols were converted into 3,5-dinitrobenzoates and analyzed by LC-ESI-MS operated under the same reversed-phase condition. The two separated geometric isomers were sensitively monitored by negative ions at m=z 211, M, M+1, M+17, and M+31, which were characteristically derived from the benzoates. Based on these results, a pheromone extract of P. l. lepida was examined, and it was confirmed that the female moths exclusively produced the (Z)-isomer of the 7,9-diene. Furthermore, a GC-EAD analysis and a field evaluation with both geometrical isomers indicated that the mating communication of P. l. lepida is predominantly mediated with the (Z)-isomer.

Full text of DOI:10.1271/bbb.90047

Biosci. Biotechnol. Biochem., 73 (5), 1156–1162, 2009
Instrumental Analysis of Terminal-Conjugated Dienes for Reexamination
of the Sex Pheromone Secreted by a Nettle Moth, Parasa lepida lepida
MD. Azharul ISLAM,1 Rei YAMAKAWA,1 Nguyen Duc DO,1 Naoko NUMAKURA,1
y
1;
Toshiro SUZUKI,2 and Tetsu ANDO
1Graduate School of Bio-Applications and Systems Engineering (BASE),
Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
2Agricultural Technology Institute of Gigu Prefecture, Gifu, Gifu 501-1152, Japan
Received January 19, 2009; Accepted February 12, 2009; Online Publication, May 7, 2009
[doi:10.1271/bbb.90047]
Conjugated dienyl compounds make one of the main
groups of lepidopteran sex pheromones, and GC has
been frequently used to determine the configurations of
the double bonds. However, the separation of two
geometric isomers of a terminal-conjugated diene, such
as 7,9-decadien-1-ol secreted by a nettle moth Parasa
lepida lepida (Limacodidae), is assumed to be difficult.
In order to clarify the chromatographic separation of
the terminal dienes, 7,9-decadienyl and 9,11-dodeca-
dienyl compounds (alcohols, acetates, and aldehydes)
were analyzed by GC and HPLC. On a capillary GC
column, the (E)-isomers flowed out slightly faster than
the corresponding (Z)-isomers, but their peaks almost
overlapped. On the other hand, HPLC equipped with an
ODS column completely separated the two geometric
isomers examined and the (Z)-isomers eluted from the
column faster than the (E)-isomers without dependence
on a functional group. In addition to undergoing direct
HPLC analysis without derivatization, the dienyl alco-
hols were converted into 3,5-dinitrobenzoates and
analyzed by LC-ESI-MS operated under the same
reversed-phase condition. The two separated geometric
isomers were sensitively monitored by negative ions
at m=z 211, M, M+1, M+17, and M+31, which were
characteristically derived from the benzoates. Based on
these results, a pheromone extract of P. l. lepida was
examined, and it was confirmed that the female moths
exclusively produced the (Z)-isomer of the 7,9-diene.
Furthermore, a GC-EAD analysis and a field evalua-
tion with both geometrical isomers indicated that the
mating communication of P. l. lepida is predominantly
mediated with the (Z)-isomer.
have been identified from many species. In the case of
terminal-conjugated dienes, 7,9-decadienyl, 9,11-dodec-
adienyl, and 11,13-tetradecadienyl compounds have
been found in 12 species in the families of Tortricidae
(six species), Limacodidae (four species), Gelechiidae
(one species), and Noctuidae (one species). Furthermore,
male attraction of about 20 other species has been
discovered by field screening tests with synthetic
dienes.2,3) In addition to these Type I pheromones,
structure-related but unique terminal-conjugated dienyl
compounds, the esters of (E)-7,9-decadienoic acid, have
been identified in three species of Limacodidae.4,5) The
larvae of the Limacodidae species known as nettle
caterpillars have spines that contain a poison that causes
serious irritation and inflammation in humans. Their
pheromones can be useful to monitor male moths in the
field. In Japan, more than 35 species are found, but their
sex pheromones have not been studied, except for
Parasa lepida lepida Cramer. This species is a defoliator
of trees, such as cherry and persimmon, in fruit orchards
and parks. (Z)-7,9-Decadien-1-ol (Z7,9-10:OH) was
identified from the pheromone gland extract.6) Although
experiments with insects in this family are still limited in
number, previous identifications suggest that the termi-
nal diene structure is characteristic of the pheromones of
Limacodidae females, including Japanese species.
In pheromone analyses, GC-MS has frequently been
used to determine chemical structures, particularly the
positions and configurations of unsaturated bonds.
However, separation of two geometric isomers of a
terminal-conjugated diene on a GC column has been
assumed to be difficult since the first identification of
lepidopteran sex pheromone components with a terminal-
conjugated dienyl structure, (Z)-9,11-dodecadienyl ace-
tate (Z9,11-12:OAc) and its (E)-isomer in Diparopsis
castanea (Noctuidae). These two geometric isomers
could not be separated even with a capillary GC column,
and their mixing ratio was thus surmised on the basis of
a reaction between the natural pheromone extract and
tetracyanoethylene and finally by a field evaluation of
two synthetic isomers.7,8) Since then, many new capil-
lary columns with high resolution and theoretical plates
have been developed, but the separation of two geo-
metric isomers of terminal-conjugated dienes has not
been examined in depth to the best of our knowledge.
Key words: sex pheromone of Lepidoptera; terminal-
conjugated diene; geometric isomer separa-
tion; liquid chromatography; electrospray
ionization
Lepidopteran sex pheromones are mainly composed
of C10–C18 fatty alcohols and their derivatives, acetates
and aldehydes, including one, two, or three carbon-
carbon double bonds, which are classified as Type I
pheromones.1) Among them, conjugated dienyl com-
pounds are one of the important chemical groups, in that
pheromone components unsaturated at various positions
y
To whom correspondence should be addressed. Tel/Fax: +81-42-388-7278; E-mail: antetsu@cc.tuat.ac.jp

Terminal-Conjugated Dienyl Pheromones
1157
terminal abdominal segments of 2- to 3-d-old virgin females were cut
off during the scotophase and soaked in hexane for 30 min. This extract
was subjected to instrumental analysis without purification.
In the case of a recent study with a nettle moth,
P. l. lepida, direct comparison between the tRs of a
natural pheromone and only one synthetic standard with
a Z configuration (Z7,9-10:OH) was done. The config-
uration of the pheromone component was estimated
from the structure of a monoenyl derivative produced by
its partial reduction.6) Mass-rearing methods for every
Limacodidae species are not established in Japan, and
the population densities of these species are not very
high. At the start of studies on Limacodidae phero-
mones, we recognized that it was necessary to establish
a convenient technique to determine the configuration,
because the available amounts of the natural phero-
mones might be very limited. Hence, we first analyzed
synthetic 7,9-decadienyl and 9,11-dodecadienyl com-
pounds by GC and HPLC in order to clarify the
chromatographic separation of the pheromone candi-
dates, and we examined their LC-MS analysis using
electrospray ionization (ESI). These results were sub-
sequently applied to inspection of the (E)-isomer of
Z7,9-10:OH (E7,9-10:OH) in the pheromone extract of
P. l. lepida as a first step. Furthermore, we evaluated the
effect of E7,9-10:OH on male attraction by Z7,9-10:OH
in the field. The results are presented in this report.
Chemicals. (Z)-9,11-Dodecadien-1-ol (Z9,11-12:OH), its (E)-
isomer (E9,11-12:OH), and their functional derivatives had been
synthesized in our previous studies.11) With reference to their syn-
thesis, Z7,9-10:OH and E7,9-10:OH were synthesized as shown in
Scheme 1A and B, respectively. One hydroxyl group of 1,7-heptane-
diol (1) was protected as a tetrahydropyranyl (THP) ether, and another
hydroxyl group was reacted on Swern oxidation to produce aldehyde
(2), which was coupled with a ylide derived from allyltriphenylphos-
phonium bromide using butyllithium as base to yield a Z and E mixture
of THP ether of 7,9-decadien-1-ol (3). After removal of the (E)-isomer
by treatment with tetracyanoethylene, the unreacted (Z)-isomer was
deported to obtain the objective Z7,9-10:OH. On the other hand, C7
alkanal (2) was converted into C9 (E)-2-alkenal (4) in three steps: a
coupling reaction with (carbomethoxymethylene)triphenylphosphor-
ane, reduction with LiAl(OEt)2H2, and Swern oxidation. The aldehyde
(4) was coupled with an ylide derived from methyltriphenylphospho-
nium iodide, and E7,9-10:OH was obtained after deprotection. 1H-
NMR (ꢀ, ppm): Z7,9-10:OH, ꢃ1.35 (6H, m), 1.56 (2H, tt, J ¼ 7, 7 Hz),
2.19 (2H, dt, J ¼ 7, 7 Hz), 3.62 (2H, t, J ¼ 7 Hz), 5.08 (1H, d, J ¼
10 Hz), 5.18 (1H, d, J ¼ 17 Hz), 5.45 (1H, dt, J ¼ 11, 8 Hz), 6.00 (1H,
dd, J ¼ 11, 11 Hz), 6.63 (1H, ddd, J ¼ 17, 11, 10 Hz). E7,9-10:OH,
ꢃ1. 35 (6H, m), 1.56 (2H, tt, J ¼ 7, 7 Hz), 2.08 (2H, dt, J ¼ 7, 7 Hz),
3.63 (2H, t, J ¼ 7 Hz), 4.95 (1H, d, J ¼ 10 Hz), 5.08 (1H, d, J ¼
17 Hz), 5.70 (1H, dt, J ¼ 15, 7 Hz), 6.04 (1H, dd, J ¼ 15, 10 Hz), 6.31
(1H, ddd, J ¼ 17, 10, 10 Hz). 13C-NMR (ꢀ, ppm): Z7,9-10:OH, 25.6
(C-3), 27.6 (C-6), 29.0 and 29.5 (C-4 and C-5), 32.7 (C-2), 62.9 (C-1),
116.8 (C-10), 129.2 (C-8), 132.3 (C-9), 132.8 (C-7). E7,9-10:OH, 25.6
(C-3), 29.0 and 29.1 (C-4 and C-5), 32.5 (C-6), 32.7 (C-2), 63.0 (C-1),
114.7 (C-10), 131.0 (C-8), 135.4 (C-7), 137.3 (C-9). The 13C-signal
assignments were achieved with reference to the published data for
Z9,11-12:OH and E9,11-12:OH.11) An aliquot of each isomer was
separately acetylated with acetic anhydride in pyridine to yield acetyl
derivatives (Z7,9-10:OAc and E7,9-10:OAc), and another aliquot was
oxidized with pyridinium chlorochromate to yield aldehyde derivatives
(Z7,9-10:Ald and E7,9-10:Ald). (E,E)-8,10-Dodecadien-1-ol (E8,E10-
12:OH), (Z)-7-dodecen-1-ol (Z7-12:OH), and (E)-11-tetradecen-1-ol
(E11-14:OH), used for spectral comparison in LC-MS analysis, were
supplied by Shin-Etsu Chemical (Tokyo).
Materials and Methods
Instuments. 1H and 13C NMR spectra were recorded with a Jeol AL
300 FT spectrometer at 300.4 and 75.45 MHz, respectively, with
CDCl3 solutions containing TMS as internal standard. GC analysis was
carried out on an HP-5890 Series II gas chromatograph (Hewlett-
Packard, Wilmington, DE, USA) equipped with a flame ionization
detector (FID), a split/splitless injector, and a DB-23 column (0.25 mm
ID ꢀ 30 m, 0.25 mm film, J & W Scientific, Folsom, CA, USA). The
column temperature program was 50 ꢁC for 2 min, 10 ꢁC/min to
160 ꢁC, and 4 ꢁC/min to 220 ꢁC. The carrier gas was He. GC-MS was
conducted in EI mode (70 eV) with an HP5973 mass spectrometer
system (Hewlett-Packard) equipped with the same GC system and
column. Measurements were carried out under the same conditions as
those in the GC analysis. HPLC was conducted with a Jasco PU-980
liquid chromatograph equipped with an integrator (System Instrument
Chromatocorder 21J), a UV detector (Jasco UV-970, 240 nm), and a
reversed phase column (Senshu Pak PEGASIL ODS-5, 2.0 mm ꢀ
25 cm, Senshu-Kagaku, Tokyo). Water (30–38%) in MeOH was used
as the eluent at a flow rate of 0.20 ml/min. LC-MS analysis was carried
out with a Shimadzu LCMS-2010 equipped with an ESI interface and
the same ODS column as the above HPLC analysis under the following
conditions: eluent, water including 0.1% formic acid (20–25%) in
MeOH; flow rate, 0.20 ml/min. The mass spectra were recorded in
negative ion mode, and the following parameters were used: prove
potential, ꢂ3:50 kV; CDL temperature, 200 ꢁC. The electroantenno-
graphic (EAG) activities of synthetic Z7,9-10:OH and E7,9-10:OH
were measured using GC, HP-5890 Series II, equipped with an EAG
detector (GC-EAD).9) The effluent from the column was split into two
lines, which were led to FID and EAD at a ratio of 1:1.10) While
samples were injected in splitless mode, the other GC conditions were
the same as those in the analysis by FID. An antenna was excised at the
base from a P. l. lepida male, and a few distal segments were cut off.
Each end of the antenna was attached to a droplet of saline solution on
an electrode of the EAD device. EAG responses at 0.1, 1, and 10 ng
injections were measured for each compound with at least five different
antennae.
Esterification of alcohols with benzoic acid. Z7,9-10:OH (10 mg,
65 mmol) dissolved in CCl4 (0.5 ml) was stirred and mixed with
pyridine (0.1 ml) and 3,5-dinitrobenzoyl chloride (30 mg, 130 mmol) at
room temperature. After stirring for 30 min, the reaction mixture was
poured into water (10 ml), extracted with hexane, and washed with 1 N
HCl and a saturated aqueous solution of NaHCO3. The hexane extract
was fractionated using a TLC plate, and 3,5-dinitrobenzoate of Z7,9-
10:OH (Z7,9-10:ODNB), which was used as an authentic sample of
LC-MS analysis, was obtained in 83% yield. 1H-NMR (ꢀ, ppm): ꢃ1.35
(6H, m), 1.83 (2H, tt, J ¼ 7, 7 Hz), 2.18 (2H, dt, J ¼ 7, 7 Hz), 4.45
(2H, t, J ¼ 7 Hz), 5.08 (1H, d, J ¼ 10 Hz), 5.18 (1H, d, J ¼ 17 Hz),
5.45 (1H, J ¼ 11, 8 Hz), 6.00 (1H, dd, J ¼ 11, 11 Hz), 6.63 (1H, ddd,
J ¼ 17, 11, 10 Hz), 9.16 (2H, d, J ¼ 2 Hz), 9.23 (1H, t, J ¼ 2 Hz).
13C-NMR (ꢀ, ppm): 25.9, 27.7, 28.9, 29.0, 29.5, 67.2, 116.8, 122.3,
129.2, 129.4, 132.3, 132.9, 134.2, 148.7, 162.6. In the same manner,
3,5-dinitrobenzoates of E7,9-10:OH, Z9,11-12:OH, E8,E10-12:OH,
Z7-12:OH, E11-14:OH, and cetyl alcohol (16:OH) were prepared for
spectral analysis of the LC-MS measurements. For small-scale
derivatization, Z7,9-10:OH (10 mg) dissolved in CCl4 (100 ml) was
treated with pyridine (50 ml) and 3,5-dinitrobenzoyl chloride (1 drop,
about 5 mg) for 30 min at room temperature. After the usual workup,
Z7,9-10:ODNB was analyzed by LC-MS. After removal of the solvent,
the pheromone extract of P. l. lepida (2 female equivalent, FE) was
also treated with 3,5-dinitrobenzoyl chloride in the same manner and
analyzed.
Insects and pheromone extraction. Cocoons of the blue-striped nettle
grub moth P. l. lepida were collected in a persimmon orchard in Gifu
Prefecture, Japan, from February to March, and individually placed in
plastic caps, which were set outdoors in the shade. In early April, the
caps were moved to an incubator kept at 25 ꢁC with a 16L-8D
photocycle. The moths, which appeared in May, were sexed, and the
Field test. The synthetic lures were evaluated in persimmon
orchards in Gifu Prefecture from 2006 to 2008. Rubber septa (white
rubber, 8 mm OD, Sigma-Aldrich, St. Louis, MO, USA) were used as
dispensers, and synthetic compounds (>97% purity, without contam-

1158
M. A. ISLAM et al.
A
B
i, ii
iii
OHC
OTHP
HO
OH
74%
61%
2
1
iv, v
45%
OTHP
OH
OH
3
Z7, 9-10:OH
vi, vii, ii
43%
viii, iv
78%
OHC
2
OTHP
E7, 9-10:OH
4
Scheme 1. Synthetic Routes to (Z)-7,9-Decadien-1-ol (Z7,9-10:OH, A) and (E)-7,9-Decadien-1-ol (E7,9-10:OH, B).
i, 2,3-dihydropyran/p-TsOH; ii, (COCl)2, DMSO, Et3N/CH2Cl2; iii, CH2 = CHCH = PPh3/THF; iv, p-TsOH/EtOH; v, (CN)2C =
C(CN)2/benzene; vi, CH3OCOCH = PPh3/benzene; vii, LiAlH2(OEt)2/ether; viii, CH2 = PPh3/THF.
Table 1. Separation of 7,9-Decadienyl and 9,11-Dodecadienyl Compounds by GC and HPLC
(A) GC analysisa
tR (min)
(B) HPLC analysisb
tR (min)
Compound
C10 7,9-diene
C12 9,11-diene
Resolution
Resolution
(E)-isomer
(Z)-isomer
(E)-isomer
(Z)-isomer
alcohol
acetate
(7,9-10:OH)
(7,9-10:OAc)
(7,9-10:Ald)
(9,11-12:OH)
(9,11-12:OAc)
(9,11-12:Ald)
13.96
13.82
12.59
16.30
16.04
14.78
14.00
13.86
12.62
16.35
16.12
14.82
0.64
0.61
0.54
0.69
0.71
0.70
21.37
36.80
36.30
26.94
65.00
55.45
19.59
33.75
33.25
24.61
58.30
48.80
1.24
1.89
1.45
1.55
1.77
1.61
aldehyde
alcohol
acetate
aldehyde
aSeparation on a DB-23column (0.25 mm ꢀ 30 m) detected by FID. Column temperature program: 50 ꢁC for 2 min, 10 ꢁC/min to 160 ꢁC, and 4 ꢁC/min to 220 ꢁC.
bSeparation on an ODS-5 column (2.0 mm ID ꢀ 25 cm) detected by UV (240 nm). Flow rate, 0.20 ml/min. Eluent, 38% water in MeOH for 7,9-decadien-1-ol, 36%
water in MeOH for 7,9-decadienyl acetate and 7,9-decadienal, and 30% water in MeOH for all 9,11-dodecadienyl compounds.
ination of the geometric isomer) dissolved in hexane (100 ml) were
applied to them. Each lure was placed at the center of a sticky board
trap (30 ꢀ 27 cm bottom plate with a roof, Takeda Chemical, Tokyo).
Plural traps were used for each lure, and other traps including the septa
treated with only hexane (100 ml) were tested as control. The traps were
placed at least 7 m apart at about 1.5 m above the ground.
column (Fig. 1B). Although the geometric separation
of the alcohol (Z7,9-10:OH and E7,9-10:OH) in this
figure is not sufficient due to the use of an inappropriate
eluent (36% water), their complete separation was
accomplished with a more polar solvent (38% water,
Table 1B).
Results
LC-ESI-MS analysis of benzoates derived from pher-
omonal alcohols
GC and HPLC separation of terminal-conjugated
dienes
Since the geometrical isomers were separable on
reversed-phase HPLC, LC-MS was applied to the
analysis. Although dienyl alcohols were not detected
by ESI-MS, ionization was successfully achieved by
derivatization into 3,5-dinitrobenzoates. The benzoates
showed Mꢂ, some negative molecular-related ions at
m=z M+1, M+17, and M+31, and a characteristic
fragment ion at m=z 211 (C7H3N2O6, acyl moiety of
3,5-dinitrobenzoates). In Fig. 2A and B show the mass
spectra of Z7,9-10:ODNB and Z9,11-12:ODNB (3,5-
dinitrobenzoates of Z7,9-10:OH and Z9,11-12:OH),
respectively. Mass chromatograms of a 1:1 mixture of
the former benzoate and its (E)-isomer (Fig. 2C) con-
firmed that these ions were made by ESI of the injected
compounds. The formation of ions at M+17 and M+31
cannot be simply explained by addition of the solvent,
water and methanol, respectively. In order to examine
the universal production of the molecular-related ions,
3,5-dinitrobenzoates of other dienyl, monoenyl, and
saturated alcohols (E8,E10-10:OH, Z7-12:OH, E11-
14:OH, and 16:OH) were also analyzed under the same
conditions. In the ESI-MS analysis, these benzoates
showed spectral patterns similar to those of the terminal-
conjugated dienes (Table 2), indicating that pheromonal
With regard to the geometric isomers of 7,9-deca-
dienyl and 9,11-dodecadienyl compounds (alcohols,
acetates, and aldehydes), their separation by GC and
HPLC was examined. Table 1A shows the tRs of the GC
analysis with a DB-23 capillary column and resolution
calculated from the shapes of the peaks. The (E)-isomer
of the conjugated dienes always flowed from the polar
column faster than the corresponding (Z)-isomers with-
out dependence on the functional group at the opposite
terminal position. While two peak tops were always
detected for an approximately 1:1 mixture of two
geometric isomers, as shown in the chromatogram of
7,9-decadienyl compounds (Fig. 1A), the values of
resolution were less than 0.8 in every dienyl compound
analyzed. The peaks of the two isomers almost over-
lapped. In the case of HPLC, the (E)-isomers always
eluted later than the corresponding (Z)-isomers from an
ODS column, and the values of resolution were larger
than 1.2, as shown in Table 1B. The conjugated dienes
were sensitively detected with a UV detector, and
chromatograms with two isolated peaks of geometric
isomers were recorded on the basis of the analysis of
each 7,9-decadienyl compound by the reversed-phase

Terminal-Conjugated Dienyl Pheromones
1159
A
B
E7,9-10:OH
E7,9-10:OH
Z7,9-10:OH
Z7,9-10:OH
E7,9-10:OAc
Z7,9-10:OAc
Z7,9-10:OAc
E7,9-10:OAc
E7,9-10:Ald
Z7,9-10:Ald
Z7,9-10:Ald
E7,9-10:Ald
12.5
13.5
13.0
14.0
t
R (min)
20.0
30.0
40.0
tR (min)
10.0
Fig. 1. Chromatographic Separation of Geometric Isomers of 7,9-Decadienyl Compounds (About 50 ng Each): Alcohol (Z7,9-10:OH and E7,9-
10:OH), Acetate (Z7,9-10:OAc and E7,9-10:OAc), and Aldehyde (Z7,9-10:Ald and E7,9-10:Ald).
A, GC with a DB-23 column (0.25 mm ID ꢀ 30 m) detected by FID. Column temperature program: 50 ꢁC for 2 min, 10 ꢁC/min to 160 ꢁC, and
4 ꢁC/min to 220 ꢁC. B, HPLC with an ODS-5 column (2.0 mm ID ꢀ 25 cm) detected by UV (240 nm). Eluent, 36% water in MeOH; flow rate,
0.20 ml/min.
A
C
379
O
Z7,9-10:ODNB
E7,9-10:ODNB
NO2
O
349
NO2
211
365
379
365
200
250
300
350
400
m/z
407
B
349
O
NO2
O
348
211
377
NO2
393
211
30.0
40.0
R (min)
t
200
250
300
350
400
m/z
Fig. 2. Analysis of 3,5-Dinitrobenzoates of Dienyl Alcohols by LC-ESI-MS with an ODS-5 Column (2.0 mm ID ꢀ 25 cm).
A, Mass spectrum of the ester of Z7,9-10:OH (Z7,9-10:ODNB). B, Mass spectrum of the ester of Z9,11-12:OH (Z9,11-12:ODNB). C, Selected
ion monitoring (SIM) of Z7,9-10:ODNB and E7,9-10:ODNB (1:1 mixture) recorded at m=z 379 (M+31), 365 (M+17), 349 (M+1), 348 (M), and
211. Eluent, mixture of water with 0.1% formic acid (25%) and MeOH (75%); flow rate, 0.20 ml/min.
Table 2. LC-ESI-MS Analysis of 3,5-Dinitrobenzoates of Pheromonal Fatty Alcoholsa
m=z of ion (relative intensity)c
tR
Compoundb
Mꢂ
[M+1]ꢂ
[M+17]ꢂ
[M+31]ꢂ
ꢂ
(min)
C7H3N2O6
Z7,9-10:ODNB
E7,9-10:ODNB
Z9,11-12:ODNB
E8,E10-12:ODNB
Z7-12:ODNB
E11-14:ODNB
16:ODNB
32.53
34.60
36.70
41.19
43.65
49.62
58.65
211 (27)
211 (16)
211 (22)
211 (19)
211 (25)
211 (16)
211 (14)
348 (17)
348 (24)
376 (41)
376 (26)
378 (35)
406 (23)
436 (38)
349 (55)
349 (33)
377 (45)
377 (61)
379 (48)
407 (51)
437 (63)
365 (36)
365 (14)
393 (26)
393 (14)
395 (15)
423 (24)
453 (12)
379 (100)
379 (100)
407 (100)
407 (100)
409 (100)
437 (100)
467 (100)
aMeasured on an ODS-5 column (2.0 mm ID ꢀ 25 cm). Eluent, 25% water (including 0.1% formic acid) in MeOH for the esters of C10 alcohols and 20% water (0.1%
formic acid) in MeOH for the esters of C12 alcohols. Flow rate, 0.20 ml/min. Prove Potential, ꢂ3:50 kV. CDL temperature, 200 ꢁC.
b3,5-Dinitrobenzoates of (Z)-7,9-decadien-1-ol (Z7,9-10:OH), (E)-7,9-decadien-1-ol (E7,9-10:OH), (Z)-9,11-dodecadien-1-ol (Z9,11-12:OH), (E,E)-8,10-dodecadien-
1-ol (E8,E10-10:OH), (Z)-7-dodecen-1-ol (Z7-12:OH), (E)-11-tetradecen-1-ol (E11-14:OH), and hexadecan-1-ol (16:OH).
cCalculated with abundance of the ion at m=z M+31 as base peak (100%).

1160
M. A. ISLAM et al.
A
B
C
Z7,9-10:OH
E7,9-10:OH
Z7,9-10:OH
E7,9-10:OH
Z7,9-10:ODNB
E7,9-10:ODNB
i
i
i
ii
ii
ii
iii
iii
iii
iv
iv
iv
40.0
tR (min)
25.0
30.0
35.0
10.0
15.0
20.0
tR (min)
tR (min)
Fig. 3. Instrumental Analysis of a Pheromone Extract of P. l. lepida Females and the Synthetic Standards.
A, GC analysis with a DB-23 column (0.25 mm ID ꢀ 30 m) detected by FID. Column temperature program: 50 ꢁC for 2 min, 10 ꢁC/min to
160 ꢁC, and 4 ꢁC/min to 220 ꢁC. B, HPLC analysis with an ODS-5 column (2.0 mm ID ꢀ 25 cm) detected by UV (240 nm). Eluent, 36% water in
MeOH; flow rate, 0.20 ml/min. C, LC-ESI-MS analysis with an ODS-5 column (2.0 mm ID ꢀ 25 cm) conducted in SIM mode at m=z 379
(M+31). Eluent, water with 0.1% formic acid (25%) and MeOH (75%); flow rate, 0.20 ml/min. In the analyses of A and B, mixtures of synthetic
Z7,9-10:OH and E7,9-10:OH (50 ng + 20 ng [i], 50 ng + 5 ng [ii], and 50 ng + 0 ng [iii]) and a pheromone gland extract (0.5 FE [iv]) were
injected. In the analysis of C, mixtures of synthetic Z7,9-10:ODNB and E7,9-10:ODNB (50 ng + 20 ng [i], 50 ng + 5 ng [ii], and 50 ng + 0 ng
[iii]) and a pheromone gland extract treated with 3,5-dinitrobenzoyl chloride (0.5 FE [iv]) were injected.
alcohol can be analyzed sensitively by focusing on the
diagnostic ions for LC-ESI-MS measurement in selected
ion monitoring (SIM) mode after derivatization to
benzoates.
250
a
200
ab
ab
150
Analysis of pheromone extracts of P. l. lepida females
Figure 3 shows chromatograms of a mixture of
b
b
100
50
0
synthetic Z7,9-10:OH and E7,9-10:OH and the natural
pheromone of P. l. lepida females, which were analyzed
by GC, HPLC, and LC-MS. On the GC analysis of
the 5:2 mixture, the occurrence of the (E)-isomer
was recorded only as a shoulder of the peak of the
(Z)-isomer, and its exact composition could not be
estimated. On the other hand, separation on the
reversed-phase HPLC column was sufficient to detect
a minute amount of the (E)-isomer on a quantitative
basis. In addition to the HPLC analysis of the alcohol at
UV 240 nm without derivatization, LC-ESI-MS analysis
of Z7,9-10:ODNB monitored at the ion at m=z 379
(M+31) revealed that the pheromone gland extract
included Z7,9-10:OH exclusively.
b
0.1
1
10
Injection (µg)
Fig. 4. EAG Responses (Mean mV ꢄ SE, n = 6) of P. l. lepida
Males against Synthetic Z7,9-10:OH (Solid Bars) and E7,9-10:OH
(Open Bars).
The responses of each compound were measured by individual
injections (0.1, 1, and 10 ng) into GC-EAD, which delivered about
half of the injected compound to the male antenna. Bars super-
scripted by a different letter indicate significant difference at
p < 0:05 by the Tukey-Kramer test.
EAG activity of synthetic 7,9-decadien-1-ol
Field tests with 7,9-decadienyl compounds
The EAG activities of synthetic Z7,9-10:OH and
E7,9-10:OH were measured by GC-EAD attached with a
male antenna of P. l. lepida. The antenna responded not
only to Z7,9-10:OH, a naturally occurring isomer, but
also to E7,9-10:OH, and its responses increased dose-
dependently, as shown in Fig. 4. While the activities of
the two isomers were not statistically significantly
different at the same dose, the average activity values
of the (Z)-isomer were always larger than those of the
(E)-isomer, indicating that the former natural phero-
mone component stimulated the antenna about 10 times
more strongly than the latter.
Since GC-MS and GC-EAD analyses of a gland
extract of P. l. lepida females indicated no other
pheromone components (data not shown), the effects
of some structure-related compounds on male attraction
by Z7,9-10:OH were examined in order to find a
compound acting as a minor component. The tested
compounds, however, showed no synergist effect in the
field. In the case of the geometric isomer, the activity of
Z7,9-10:OH was reduced by mixing. The number of
males captured by traps baited with a 9:1 mixture of
Z7,9-10:OH and E7,9-10:OH was smaller than that by
traps baited with only the natural pheromone compo-

Terminal-Conjugated Dienyl Pheromones
1161
Table 3. Attraction of Parasa lepida lepida Males by Lures Baited
with Synthetic Z7,9-10:OH and E7,9-10:OH in a Persimmon Orcharda
by LC-ESI-MS equipped with the same ODS column.
The two separated geometric isomers could be sensi-
tively monitored by characteristic negative ions at m=z
211, M, M+1, M+17, and M+31, which were derived
from the benzoates (Fig. 2 and Table 2), indicating that
LC-MS is one of the most reliable analytical methods for
terminal-conjugated dienyl pheromones. On the basis of
these results, a pheromone extract of P. l. lepida was
examined, and it was confirmed that the female moths
exclusively produced the (Z)-isomer of the 7,9-diene
(Fig. 3).
Lure components (mg/rubber septum)
Captured males
Z7,9-10:OH
E7,9-10:OH
/trap/nightb
Total
0.50
0.45
0.25
0.00
0.00
0.00
0.05
0.25
0.50
0.00
0:73 ꢄ 0:04a
0:35 ꢄ 0:13b
0:08 ꢄ 0:08c
0:08 ꢄ 0:08c
0:00 ꢄ 0:00
38
18
4
4
0
aTested from August 21 to September 3 in 2007 using four traps for each
lure.
Although lepidopteran sex pheromones are volatile
and LC-MS has not been frequently applied in their
identification, we analyzed a chiral epoxy component
extracted from a geometrid species using LC-ESI-MS.
The LC was equipped with a chiral column, which
showed better resolution of the enantiomers than a chiral
GC column, and the stereochemistry of an epoxy
pheromone was successfully revealed.12) In addition,
LC-MS demonstrated its advantage for the analysis of
heat-unstable 3,13- and 2,13-dienals, pheromone candi-
dates of clearwing moths. We treated the aldehydes with
2,4-dinitrophenylhydrazine, and the hydrazones pro-
duced were analyzed by LC-MS equipped with atmo-
spheric pressure chemical ionization.13) This study with
terminal-conjugated dienes revealed the value of LC-MS
analysis. For pheromone research dealing with rather
small and less-polar molecules, LC-MS is a useful
instrument, compensating for the drawbacks of GC-MS
analysis.
P. l. lepida, recorded in Southeast Asia, is regarded as
exotic in Japan. Lately, its habitat has started to spread
from south to north in Honshu, the main island of Japan,
and the larvae have been found more frequently in an
orchard in Gifu Prefecture than other nettle moths
peculiar to Japan, such as the Oriental nettle moth
Monema flavescens Walker. In our preliminary monitor-
ing from April to October in 2007 and 2008, adults were
caught mainly in June and August, indicating bivoltin-
ism (data not shown). The lure baited with 0.5 mg of
Z7,9-10:OH was replaced with a fresh one every month,
and the number of captured males clearly increased
just after the replacement. The pheromone component
is more volatile than other long-chain pheromones
and might be easily released. In order to establish a
reliable monitoring tool, further field examination is
necessary.
bMean ꢄ SE. Values within each column followed by a different letter are
significantly different at p < 0:05 by the Tukey-Kramer test.
Table 4. Attraction of Parasa lepida lepida Males by Lures Baited
with Synthetic Z7,9-10:OH and the Functional Derivatives in a
Persimmon Orcharda
Lure components (mg/rubber septum)
Z7,9-10:OH Z7,9-10:OAc Z7,9-10:Ald /trap/nightb Total
(A) July 5-14, 2006
1.00
Captured males
0.00
1.00
0.00
0.00
0.00
0.00
1.00
0.00
1:89 ꢄ 0:78
0:00 ꢄ 0:00
0:00 ꢄ 0:00
0:00 ꢄ 0:00
34
0
0.00
0.00
0.00
0
0
(B) Aug. 29-Sept. 4, 2008
0.50
0.50
0.50
0.50
0.50
0.00
0.00
0.10
0.50
0.00
0.00
0.00
0.00
0.00
0.00
0.10
0.50
0.00
1:25 ꢄ 0:42a 30
0:00 ꢄ 0:00
0:00 ꢄ 0:00
0
0
0:50 ꢄ 0:17b 12
0:67 ꢄ 0:33ab 16
0:00 ꢄ 0:00
0
aTested using two traps in 2006 and four traps in 2008 for each lure.
bMean ꢄ SE. Values within each column followed by a different letter are
significantly different at p < 0:05 by the Tukey-Kramer test.
nent. The 1:1 mixture scarcely attracted males as
the single component lure of E7,9-10:OH (Table 3).
The functional derivatives of the alcohol pheromone,
Z7,9-10:OAc and Z7,9-10:Ald, did not attract any males
either. The acetate strongly inhibited the activity of
Z7,9-10:OH, and no males were captured by traps baited
with a 5:1 mixture of Z7,9-10:OH and Z7,9-10:OAc.
The inhibitory activity of the aldehyde was indicated,
but some males were attracted even by the 1:1 mixture
of Z7,9-10:OH and Z7,9-10:Ald (Table 4).
Discussion
On the other hand, field evaluations of structure-
related compounds showed that mixing of E7,9-10:OH
or Z7,9-10:OAc strongly inhibited male attraction by the
synthetic pheromone (Tables 3 and 4). The male moths
might detect these compounds as offensive orders,
possibly pheromone components of other species. This
result suggests the use of pure Z7,9-10:OH as the lure
for a monitoring trap. In the sample of Z7,9-10:OH,
contamination of the (E)-isomer might happen easily
because of the chemical instability of the Z double bond.
Selective synthesis of Z7,9-10:OH, which excludes the
formation of the geometric isomer, is difficult. We
utilized the synthetic route, as shown in Scheme 1,
where the (E)-isomer was completely removed from a
mixture of two geometric isomers by treatment with
tetracyanoethylene. It is noteworthy that, in a small-
scale experiment, desirable separation of them is
On GC analysis equipped with a polar capillary
column (DB-23), the (E)-isomers of 7,9-decadienyl and
9,11-dodecadienyl compounds (alcohols, acetates, and
aldehydes) flowed out slightly faster than the corre-
sponding (Z)-isomers, but their peaks almost overlapped.
On the other hand, a reversed-phase HPLC column
(ODS) completely separated the two geometric isomers,
and the (Z)-isomers always eluted from the column faster
than the (E)-isomers (Fig. 1 and Table 1). The conju-
gated dienyl compounds were sensitively tracked with a
UV detector. However, if an unknown material with
similar UV absorption occurred in a pheromone extract,
their differentiation could not be accomplished by the
usual HPLC system alone. Therefore, the dienyl alcohols
were converted into 3,5-dinitrobenzoates and analyzed

1162
M. A. ISLAM et al.
6) Wakamura S, Tanaka H, Masumoto Y, Sawada H, and
Toyohara N, Appl. Entomol. Zool., 42, 347–352 (2007).
7) Nesbitt BF, Beevor PS, Cole RA, Lester R, and Poppi RG,
Nature, 244, 208–209 (1973).
straightforwardly achieved by preparative HPLC with an
ODS column (Fig. 1), as established in this study. These
procedures should be of use in preparing pure isomers of
terminal-conjugated dienyl compounds.
8) Nesbitt BF, Beevor PS, Cole RA, Lester R, and Poppi RG,
J. Insect Physiol., 21, 1091–1096 (1975).
9) Struble DL and Arn H, ‘‘Techniques in Pheromone Research,’’
eds. Hummel HE and Miller TA, Springer-Verlag, New York,
pp. 161–178 (1984).
References
1) Ando T, Inomata S, and Yamamoto M, Top. Curr. Chem., 239,
51–96 (2004).
10) Inomata S, Watanabe A, Nomura M, and Ando T, J. Chem.
Ecol., 31, 1429–1442 (2005).
2) Ando T, Internet database (2009), http://www.tuat.ac.jp/
~antetsu/LepiPheroList.htm.
11) Ando T, Kurotsu Y, Kaiya M, and Uchiyama M, Agric. Biol.
Chem., 49, 141–148 (1985).
3) El-Sayed AM, Internet database (2008), http://www.pheroba-
se.com/
12) Yamazawa H, Yamamoto M, Karasawa KI, Pu G-Q, and Ando
T, J. Mass Spectrom., 38, 328–332 (2003).
4) Sasaerila Y, Gries G, Gries R, and Boo TC, Chemoecology, 10,
193–199 (2000).
13) Islam MDA, Yamamoto M, Sugie M, Naka H, Tabata J, Arita
Y, and Ando T, J. Chem. Ecol., 33, 1763–1773 (2007).
5) Siderhurst MS, Jang EB, Hara AH, and Conant P, Entomol. Exp.
Appl., 125, 63–69 (2007).