GC-MS and LC-MS analyses for unraveling the diversity of lepidopteran communication systems

23rd ISCE Annual MeetingSymposium on Insect Semiochemicals I

Analysis, Structures, Synthesis, ……

Graduate School of BASE,Tokyo University of

Agriculture and Technology, Tokyo 184-8588, JapanE-mail: antetsu@cc.tuat.ac.jp

Tetsu ANDO

Jena, Germany (July 23, 2007)

Lepidoptera: ca. 150,000 speciesVariety of chemical structures Blending of multiple components

Small insects Low contentIdentification by GC-EAD

GC-MS

Produced by ♀ to attract ♂

Main factor for reproductive isolationSpecies specific

Pheromones of 580 species have been reported.

HPLC and LC-MS ?

Lepidopteran sex pheromones

i) Monoenyl compounds

Diamondback moth

Type I sex pheromones Pest insects in Japan

Pine caterpillar

Cherry treeborer

Mulberry pyralid

OAc

OH

H

O

OH

OAc

OAc

OAc

Z11-16:OAcZ11-16:AldZ11-16:OH

Z5,E7-12:OH

Z3,Z13-18:OAc

E3,Z13-18:OAc

E10,E12,Z14-16:OAc

iii) Trienyl compounds

ii) Dienyl compounds

Double bond position, counting from the functional group

C12

C14

C16

C18

2 3 4 5 6 8 15141312111097C10

Chain length 16 17

(C13 and C15 chain compounds are not included.)

Double-bond positions of dienes and trienes

(A) MTAD (4-methyl-1,2,4-triazoline-3,5-dione) derivative

Determination of double-bond positions

N NN OOCH3

N NN OOCH3

R2 R1R1R2

GC-MS

Derivatization is difficult for natural pheromones in a trace amount.

If conjugated dienes specifically show characteristic fragment ionson the direct GC-MS analysis, derivatization is not necessary.

R1R2 R1

R2

R1R2

(B) Diimide reduction DMDS derivative

NH2NH2

H2O2

DMDSR1

R2SCH3

SCH3

R1R2

SCH3

SCH3

GC-MS

Mass spectra of conjugated dienes (C12 alcohols)

5,7-diene

4,6-diene

3,5-diene

2,4-diene

1,3-diene

5,7-diene

6,8-diene

7,9-diene

8,10-diene

9,11-diene

123110

10996

9582

8168

6754

In the case of the a terminal conjugated diene, identification of a natural pheromone can be accomplished without derivatization.

Diagnostic ions of conjugated dienes

Fragment ion B

A+ (m/z) 123 109 95 81 67

5,7- 4,2- 3,5- 2,4- 1,3-diene diene diene diene diene

B+ (m/z) 110 96 82 68 54

Fragment ion A

In the case of the a terminal conjugated diene, identification of a natural pheromone can be accomplished without derivatization.

Identification of dienyl pheromones

5,7-diene

4,6-diene

3,5-diene

2,4-diene

1,3-diene

123110

10996

9582

8168

6754

OAcOH

Pine caterpillar [Lasiocampidae] Chrysanthemum goldenplusia [Noctuidae: Plusiinae]

Z5,E7-12:OH E5,Z7-12:OAc

E8,E10-12:OAcOAc

[Tortricidae: Olethreutinae]

Soybean pod borer Centroxena sp.

Identification of dienyl pheromones

5,7-diene

4,6-diene

3,5-diene

2,4-diene

1,3-diene

123110

10996

9582

8168

6754

E8,E10-12:OAcOAc

[Tortricidae: Olethreutinae]

Soybean pod borer Centroxena sp.

OAcOH

Pine caterpillar [Lasiocampidae] Chrysanthemum goldenplusia [Noctuidae: Plusiinae]

Z5,E7-12:OH E5,Z7-12:OAcOAcOH

Pine caterpillar [Lasiocampidae] Chrysanthemum golden plusia [Noctuidae]

Z5,E7-12:OH E5,Z7-12:OAc

E8,E10-12:OAcOAc

[Tortricidae: Olethreutinae]

Soybean pod borer Centroxena sp.

Identification of 1,3-dienes

Darna trima (Sasaerila et al., 2000)Malaysia

Setothosea asigna (Sasaerila et al., 1997)Indonesia

E9,11-12:AldH

O

O

O

O

O E7,9-10:Acyl derivatives

Oil palm defoliatorsParasa lepida

OHZ7,9-10:OH

[Limacodidae] Nettle moths

Identification of 1,3-dienes

Parasa lepida

[Limacodidae]

OHZ7,9-10:OH

30 40 50 Rt (min)

(A) GC-MS analysis (TIC)

Synthetic mixture

Z-isomerE-isomer

DB-23 column (0.25 mm X 30 m)

(B) HPLC analysis (UV 240 nm)Column: ODS 2.0 mm X 25 cm Eluent: 45 % H2O

in MeOH (0.2 ml/min)

Synthetic mixture

Extract (0.2 FE)

E-isomer

Z-isomer

Pheromones of Sesiidae species

3,13-Diene

OH OAc Ald

2,13-Diene

4 3 03 3 00 1 ? 00 0 0

0 0 00 5 3

Number of species

Z3,Z13E3,Z13Z3,E13E3,E13

Z2,Z13E2,Z13

Z2 compounds ?

E13 compounds ?

Z3 or E3 aldehydes ?

Do any females produce

M. japona

Macroscelesia spp.

M. longipes

H

O

E2,Z13-18:Ald

Double bondsPosition Configuration

Cool on-column injection, DB-23 column (0.25 mm X 30 m)

GC analysis of 3,13- and 2,13-dienals

20 22Rt (min)

21 23

★

★

★

★

(A) 3,13-18:Ald

Z3,Z13

E3,Z13

Z3,E13

E3,E13

E2,Z13 E2,E13

Peaks without any isomerization ★ Isomerized to E2,Z13-18:Ald ★ Isomerized to E2,E13-18:Ald

OAc

OH

(B) 2,13-18:Ald

20 22Rt (min)

21 23

★

★

Z2,Z13

E2,Z13

Z2,E13

E2,E13

E2,Z13 E2,E13

OAc OH

GC-MS is not suitable forthe analysis of the dienals.

LC and LC-MS analyses of the dienals

20

ZZ EZ ZEEE

ZZ EZ ZEEE3,13-18:Ald

2,13-18:Ald235 nm

215 nm

Column: ODS 2.0 mm X 25 cmEluent: 16 % H2O in CH3CN (0.2 ml/min)

Pheromone extractof M. japona 5 FE

50 ng

250 ng

(A) LC analysis

Rt (min)30 403525 Rt (min)15 2520 30

ZZ

ZE EE

EZ

ZZZE

EZ EE

50 ng

50 ng

2.5 FE

dienals

-

(B) LC-MS (APCI) analysisof DNP derivatives

([M-1] , m/z 443)

NHNH2

NO2O2N

Hill et al., (1981) J. Chem. Ecol., 7: 655saltmarsh caterpillar moth (Estigmene acrea: Arctiidae)

Z3,Z6,epo9-21:H+ Z9,Z12,Z15-18:Ald+ Z9,Z12-18:Ald (27:6:1)

(A) First Identification

O

O

OO

OO

Biston robustum (Geometridae)

O

epo6,Z9-19:H

Z3,epo6,Z9-19:H

Milionia basalis(Geometridae)

epo3,Z6,Z9-19:H

Penina nuda(Lymantriidae)

Z3,epo6,Z9-21:H

epo3,epo6,Z9-21:H

Type II sex pheromones

(B) Identification in Japan

Diagnostic ions of GC-MS analysis (EI)

RO

RO

RO

M-29

M-58

M-72-H

79-H

11197-H

M-83M-69M-29

-H

M-69M-29M-109

10879

122

-H-H

M-123-H

i) Monoepoxydienes

RO

RO

M-100

99-H

M-114-H

124

M-125-H

M-111

ii) Monoepoxymonoenes

RO O

RO O

RO O

M-85M-87 (-2H)

128 167

M-69

111 M-125 M-125

M-139-H

ii) Diepoxymonoenes

Analysis by LC-TOF MS (ESI)

RO

RO

RO

M-57M-71

123

M-123

+ H

- H

+ H

Column: ODS (2.1 mm X 15 cm)Eluent: 20-5% H2O in MeOHSpray tip potential: +3,400 VNozzle potential: +120 V

[M+NH4]+ 296[M+H]+ 279[M-OH]+ 261

C19 compounds(R=C4H9) m/z150 250 300

0

50

100

200

221

155123

207

0

50

100

150 250 300200m/z

150 200 250 3000

50

100

m/z

279261

296

279261

296

279261

296

X 5

X 5

X 5

3,4-epoxide (C19)

6,7-epoxide (C19)

9,10-epoxide (C19)

221207

155

LC-TOF MS (ESI) analysis of pheromones ①

RO

RO

RO

M-57M-71

123

M-123

+ H

- H

+ H

Column: ODS (2.1 mm X 15 cm)Eluent: 20-5% H2O in MeOHSpray tip potential: +3,400 VNozzle potential: +120 V

[M+NH4]+ 282[M+H]+ 265[M-OH]+ 247

C18 compounds(R=C3H7) m/z150 250 300

0

50

100

200

207

141123

193

0

50

100

150 250 300200m/z

150 200 250 3000

50

100

m/z

265

247

282

265247

282

265247

282

X 5

X 5

X 5

3,4-epoxide (C18)

6,7-epoxide (C18)

9,10-epoxide (C18)

207193

141

6,7-epoxide3,4-epoxide

9,10-epoxide

16 17 18 19 20 Rt (min)

(A) Synthetic mixture

247265282

207

141123

193

m/z

247265282

207

141123

193

m/z(B) Extract of the mulberry looper

LC-TOF MS (ESI)

RO

RO

RO

M-57M-71

123

M-123

+ H

- H

+ H

Column: Chiralcel OJ-REluent: 10% H2O in MeOHSpray tip potential: +3,400 VNozzle potential: +120 V

[M+NH4]+ 296[M+H]+ 279[M-OH]+ 261

C1９ compounds(R=C4H9)

221207

155

analysis of pheromones ②

Rt (min)

296

221261

279TICUV

207

123155

3R,4S3S,4R

30 35 40

m/z

296

221261

279TICUV

207

123155

m/z

(A) Racemic mixture of 3,4-epoxide

(B) Extract of the giant looper

ConclusionEI-Mass spectra of Type I pheromones (conjugated

dienes) and Type II pheromones (polyunsaturatedhydrocarbons and their epoxy derivatives) showeddiagnostic fragment ions for the structure determination.

The diversity of lepidopteran communication systems willbe understood in detail using these elegant(?) and smart(?)techniques.

LC and LC-MS are useful tools for unstableand inapplicable components on GC analysis.

Stereochemistry of epoxy pheromones can bedetermined by LC-MS with a chiral column.

AcknowledgmentsCo-workersDrs. H. Naka, Y. Arita and K. Tsuchida

Identification of the pheromones from Sesiidae speciesDrs. F. Komai, M. Kinjo and H. Ishitani

identification of the pheromones from Olethreutinae speciesDrs. K. Ohtani, G.-Q. Pu and K. I. Karasawa

Identification of the pheromone from Geometridae species

Chemical Ecology Laboratory of TUATDr. X.-R. Qin Dr. Witjaksono Dr. S. Inomata Dr. M. YamamotoDr. A. OnoDr. H. YamazawaDr. L. V. Vang

T. NishidaT. NakazawaK. MatsuokaM. D. A. IslamN. NumakuraM. Sugie