From wasps to ion chemistryThe outer surface of the insect cuticle (epicuticle) is covered by a layer of low volatility hydrocarbons. Thislayer is impermeable and thus limits water loss from the insect body. Furthermore, according to severalauthors, it is also a barrier to pathogens. In some species the mixture of hydrocarbons is used in intraspecific recognition, for example, recognition of sexual partners. In social insects it has beendemonstrated that the hydrocarbon mixture is used to recognize individuals belonging to the same colony. In a society of wasps of the genus Polistes, the workers and foundresses are therefore capable of discriminating among individuals with differing hydrocarbon signatures, and act aggressively in responseto these individuals to prevent exploitation of their food reserves. To identify saturated and unsaturated hydrocarbons present on the cuticle of these insects, we usedpositive chemical ionization (PICI) with acetonitrile as the reagent gas in a GC/MS system. This gas, in the presence of hydrocarbons, favours the formaton of the cation (M + C2H2N)+. The abundance of this ion(M + 40)+ increases in proportion to the chain length of the hydrocarbon (in contrast, in EI, the oppositeoccurs). In the presence of unsaturated hydrocarbons, in addition to the ion (M + 40)+, the ion (M + 54)+ isalso apparent. Furthermore, PICI spectra with acetonotrile of these hydrocarbons allow for the determination of the position of the double bond: a pair of ions appear with distinct intensities and withmasses corresponding to the resultant fragments after cleavage of the chain in the position of the doublebond.The basic mechanism of the formation of these products and diagnostic ions is still unclear, but we havedemonstrated that PICI with acetonotrile allows for the identification both of the molecular weight of hydrocarbons and the position of the double bond in mono-unsaturated alkenes.

Bibl.: - RCM, 11, 857-862 (1997)- J. Mass Spectrom. 32 (1997)

FROM WASPSTO ION CHEMISTRY

G. Moneti, G. Pieraccini, F. Dani, S. Turillazzi, D. Favretto, P. Traldi

•The insect cuticle is covered by a layerof lipids. This layer protect the insectfrom infection and to reduce water loss. •In social insects (wasps, bees, ants, and termites) cuticular compounds also allowindividuals to recognize each other. Thus, individuals are able to discriminate colony members on the basis of the cuticular signature. •In social insects the major compoundsfound on the cuticle are hydrocarbons. These are usually long-chained (C20 toC37) and may be saturated or unsaturated. •In social wasps (Polistes sp.) cuticularhydrocarbons have been found to differbetween colonies within species and allow colony members to recognizenestmates. Furthermore, the nest hasbeen shown to be an important source of these hydrocarbons as well as glandspresent in the wasps.

COMPARISON OF EXTRACTION BY HEXANE AND SPME FROM LEGS OF Vespa crabro

3 legs3 legs

hexane

EXANE

•We compared the classical solventextraction method (with hexane) forhydrocarbons with solid phase micro-extraction (SPME) in the headspace. •Results from SPME analyses compare well with those of the classical solventmethod•SPME was a simpler and more efficient method for analysing cuticularhydrocarbons. •An added advantage of the SPME fibre is that extractions can be madedirectly off live wasps allowing for the analysis of changes in individuals over time.

CUTICULAR HYDROCARBON PROFILE OF A WASP SOCIAL PARASITEBEFORE AND AFTER USURPATION OF A NEST OF Polistes dominulus

42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00

5000000

1e+07

1.5e+07

2e+07

Time-->

Abundance

42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00

5000000

1e+07

1.5e+07

2e+07

2.5e+07

Time-->

42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00

5000000

1e+07

1.5e+07

2e+07

2.5e+07

3e+07

3.5e+07

Time-->

Abundance

PARASITE BEFORE USURPATION

PARASITE AFTER 3h ON NEST

HOSTPolistes dominulus

C

B

A•Polistes sulcifer is a species of wasp that has lost the ability toestablish nests and workers asother social wasps do. Instead thisspecies is a social parasite. Ittakes over the nests of otherPolistes species and forces the host workers to raise its eggs. Using SPME on live individuals, wehave shown that this species isable to integrate itself into nests of the host by sequentially changingthe cuticular hydrocarbon profile tomatch that of the host.•A - The hydrocarbon profile of the parasite before usurpation of a nest.•B - The hydrocarbon profile of the host species, Polistes dominulus•C - The cuticular signature of the parasite 40 days after it has takenover the nest of the host

Abundance

Polistes dominulus: cuticular hydrocarbons

1

18-23

13

11

8

7

6

432

17

2415

16

29

26

28

33 34

35-38

59 12

14

2527 30

32

31

10

}

}

1. C252. 13-/ 11-/ 9-, meC253. C264. 2-meC265. C27:16. C277. 13-/ 11- meC278. 7-meC279. 5-meC2710. 9, y dimetil-C2711. 3-meC2712. 5, 15 dimetil-C2713. C2814. 14-/ 13-/ 12- meC2815. 2-meC2816. C2917. 15-/,13- meC2918. 7-meC2919. 5-meC2920. 11, y dimetil-C2921. 7, y dimetil-C2922. 3-meC2923. 5, y dimetil-C2924. C3025. x, y dimetil-C2926. Unknown27. C3128. 15-/, 13-, meC3129. 7-meC3130. 13, 17 dimetil-C3131. 7, 15 dimetil-C3132. 5, 15 dimetil-C3133. x, y dimetil-C3334. Unknown35. 17-/, 15-/, 13-, meC3336. 13, y dimetil-C3337. 7, y dimetil-C3338. 5, y dimetil-C33

Abundance

0

40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00

•Chromatogram after SMPE of a live individual of Polistes dominulus•The cuticular signature consists mainly of saturated hydrocarbons ranging from chain length C25 to C33. •Many of these are mono- or di-methylated, unsaturated hydrocarbons are present in very small quantities

Wing of Polistes dominulus

•A - In the EI NIST library the molecular ions of hydrocarbonsdecrease in proportion with the length of the carbon chain. Thismeans that identification of hydrocarbons with more than 28 carbon atoms is very difficult byMS.•B - Instead, in CI with CH3CN the intensity of (M+40) ion increaseswith an increase in chain length•C-Therefore,long-chainedhydrocarbons can have (M+40) ionsmore intense than any other ions in the spectrum•D - This means that even if thesecompounds are present in smallquantities, they are identifiablebecause the (M+40) ions are verybig.

CHEMICAL IONIZATION WITH ACETONITRILE

42

C2H4N

C2H2N

40

C3H4N54

100%

25 30 35 40 45 50 55 60 65 m/z

H2C C N NCH2C HC C NH C C NH2

a b c d

C NH2

C C

NH2C

H HC

NH2C

H

C

H

NHC

e f g h

•In CI, acetonitrile is introduced into the ion trap as the reagent gas. Together with the molecularion of acetonitrile (m/z 42), the ions m/z 40 and m/z 54 are also present.• The ion m/z 40 is the ion that forms an attachment with the hydrocarbons•This ion can be written in eight structutally different configurations (a-h), but is probably present in the forms a or c.

Saturated hydrocarbon in CI with CH3CN,13CH3CN, C2H3CN and CH3CH2CN

0%

25%

50%

75%

100% 71

140 182 210 238 280

364C23 with CH3CN (M+40)

0%

25%

50%

75%

100% 85

211 267 295

365C23 with 13CH3CN (M+41)

100 150 200 250 300 3500%

25%

50%

75%

100% 85

171 213 240 312

366C23 with C2H3CN(M+42)

100 150 200 250 300 350Mass0%

25%

50%

75%

100% 71

223 311 366

C23 with CH3CH2CN

(M+42)?

A

• We demonstrated that it wasthe acetenotrile reagent gas that formed the attachmentswith the hydrocarbons byusing stable isotopes of acetenotrile like 13C and 2H asreagent gases.• In the figures A-C the sameC23 hydrocarbon formsattachments with the differentreagent gases•In D it can be seen thatproprionitrile does not reactwith the hydrocarbon

B

C

D

SATURATED HYDROCARBONs IN CI WITH CH3CN: THE ABUNDANCE OF (M+40)+ INCREASES AS CHAIN LENGTH INCREASES

•Chemical ionization of saturated hydrocarbonswith acetonitrile revealsthat the ion (M+40)+

increases in intensity asthe chain length of the hydrocarbon increases.

•Thus long chainedhydrocarbons such as C31 have very visible (M+40)+

ions and thus makes themvery easy to identify.

SATURATED HYDROCARBON IN CI WITH CH3CN: INCREASE IN PRESSURE OF CH3CN INCREASES INTENSITY OF (M+40)

1750%

25%

50%

75%

100% 71

85

97 112 126 140 154 168 196 224 238(M+40)

C14 150° 30 x 10-6

750%

25%

50%

75%

100% 71

85

98 112 126 140 154 168 182 196 210

238(M+40)

C14 150° 60 x 10-6

Mass0%

25%

50%

75%

100% 85

182 224 294

378(M+40)

C24 150° 30 x 10-6

0%

25%

50%

75%

100% 71

140 196 280

378(M+40)C24 150° 60 x 10-6

• We have also demonstratedthat an increase in the pressure of the acetonitrilereagent gas increases the intensity of the (M+40)+ ion at constant temperatures.

•Doubling the pressure of the gas doubles the intensity of the (M+40)+

100 150

200

250

300

350

0%

25%

50%

75%

100%

7185

99

140 168 182 196 210 224 238 252 266 280 294 308 322

378

0%

25%

50%

75%

100% 71

140 168 196 280

378C24 a 150°C

C24 a 130°C

SATURATED HYDROCARBON IN CI WHIT CH3CN: AS INCREASE IN THE TEMPERATURE OF THE TRAP DECREASES (M+40)

(M+40)

(M+40)•In contrast, when the temperature of the trap isincreased the intensity of the (M+40)+ ion decreases.

•Thus, the most suitablemethod for acquiring intense (M+40)+ ions is at lowertemperatures.

0%

25%

50%

75%

100% 71

140 168 210 252 280 322

378C24 a 170°C(M+40)

0%

25%

50%

75%

100% 71

168 224 252 280 336

378

C24 a 190°C

(M+40)

•Chemical ionization withacetonitrile also allows for the identification of mono-unsaturatedhydrocarbons and the position of the double bond in thesecompounds.•In CI of these hydrocarbons a new reaction product is detectedat m/z 356 corresponding to(M+54)+. Thus mono-unsaturatedhydrocarbons have both (M+40)+

and (M+54)+ ions while saturatedones have only (M+40)+

•Furthermore, the presence of pairs of peaks of high abundance(e.g. m/z 180 and 250 in figure) displays the location of the doublebond.•These peaks can be explainedby the scheme above. The reactive species at m/z 54 (k) can add to both CH groups of the double bond: by further cleavageof the addition products j and l are formed.

H3C (CH2 )m

)CH2(n

CH3

CH3n(CH2 )

m)CH2(H3C k+

+k

H3C (CH2 )m

)CH2(n

CH3

k CH (CH2) CH3n+

H3C (CH2) CH km+

+ k ( k = 54 )

j l

100 150 200 250 300 350 m/z0%

25%

50%

75%

100% 85

180 250

362

71

97

111124 376

j l

[ M+

40 ]+

[ M+

54 ]+

(9) C23H46

[ C9H

18 +

54

]

[ C14

H28

+ 5

4 ]

m/z 54

m/z 180

100 150 200 250 300 350m/z

0%

25%

50%

75%

100% 85

180 250

362

71

97

111

124 376

j l

[ M+

40 ]+

[ M+

54 ]+

(9) C23H46

[ C9H

18 +

54

]

[ C14

H28

+ 5

4 ]

CI of ALKANES, ALKENES and ALKADIENES whitACETONITRILE

0%

25%

50%

75%

100% 71

182 224 266

36485

0%

25%

50%

75%

100% 85

180 250

362

71

376

100 150 200 250 300 3500%

25%

50%

75%

100% 85

152 278

362

71

376

100 150 200 250 300 350 Mass

0%

25%

50%

75%

100% 97

178 250 290

360

85

71374

138

[ C6H

12 +

54

]

[ C9H

16 +

54

]

[ C14

H28

+ 5

4 ]

[ C17

H32

+ 5

4 ]

( 6,9 ) C23H44

[ M +

40

]+

[ M +

54

]+

[ C7H

14 +

54

]

[ C16

H32

+ 5

4 ]

( 7 ) C23H46

[ M +

40

]+

[ M +

54

]+

( 9 ) C23H46

[ C9H

18 +

54

]

[ C14

H28

+ 5

4 ]

[ M +

40

]+

[ M +

54

]+

C23H48 [ M + 40 ]+•Chemical ionization withacetonitrile as the reagent gas thus allows for the identificationof both saturated and unsaturated hydrocarbons of any chain length. For example:•A - Alkanes only display (M+40)+

•B and C - Alkenes possess both(M+40)+ and (M+54)+ ions. Peaks of high abundancedisplay the position of the doublebond: m/z 180 and 250 for(9)C23:1 (B) and m/z 152 and 278 for (7)C23:1 (C).•D - Alkadienes possess alsoboth (M+40)+ and (M+54)+ ions. Peaks of high abundance alsoallow for the determination of the position of the double bonds.

IDENTIFICATION OF ALKENESpaolo\paolo08.ms

0%

10%

20%

30%

40%

50%

111 180228

264305 335

376

5 10 15 20 25 30 35 minutes0

100

200

300

400

500600kInt

19.7 19.8 19.9 20.0 20.1 20.2 20.3 20.4 minutes5.0

7.5

10.0

12.5

15.0

17.5

kInt paolo08.ms RIC

0%

10%

20%

30%

40%

50%

60%

111 166243

278315 336

376

150 200 250 300 350 Mass0%

10%

20%

30%

40%

50%

124152

175 239292

325

376

390

390

390

(9)C24:1 (8)C24:1(7)C24:1

[ C9H

18 +

54

]

[ C15

H30

+ 5

4 ]

[ C8H

16 +

54

]

[ C16

H32

+ 5

4 ]

[ C7H

14 +

54

]

[ C17

H34

+ 5

4 ]

(7)C24:1

(8)C24:1

(9)C24:1

TICGC/MS in CI con CH3CN

•Another major advantage of chemical ionization withacetonitrile as the reagentgas is the fact that alkenescan be identified when theyare present in very smallamounts. This can be seenin the top figure.•CI allows for the detection and identification of the alkenes (9)C24:1, (8)C24:1, and (7)C24:1, which are present in very low amounts.•The added advantage is that derivatization does not have to be performed. Derivatization has been found to change the retention time of these compounds and thus the low abundances of the compounds makes themimpossible to find.