A METHOD FOR ANALYSING SMALL-SIZE SPECIMENS IN

GC-MS

S. TURILLAZZI
1
, M. F. SLEDGE
1
, S. CREMER
2
, J. HEINZE
2


1
Dipartimento di Biologia Animale e Genetica, Università di Firenze, Via Romana
17, Firenze, I-50125, Italy
2
Lehrstuhl für Biologie 1, Universitaet Regensburg, Universitaetsstrasse 31, 93040
Regensburg, Germany


ABSTRACT

A particular Solid Injector needle, suitable for GC-MS analyses of small specimens,
is described together with its application in a study on ants.


KEY WORDS: GC-MS, solid injector, ants

INTRODUCTION

Cuticular lipids have various functions in insects. Their principal
function is to contribute to the reduction of water loss, while they may also
prevent a barrier to pathogens entry into the body. It is now known that they
also play important communicative roles in both solitary and social insects.
These roles include recognition of sexes, and in the social insects nest-mate
recognition.
Cuticular lipids are relatively straightforward to analyse chemically,
and methods for their extraction have especially proven to be simple.
Classically, sampling has relied mainly on extraction by means of apolar
solvents of the entire insect body. Recently, several alternative methods have
been employed to overcome some of the shortcomings of solvent extraction.
These include the use of solid phase microextraction fibres (SPME) to
extract hydrocarbons from the headspace of heated samples (MONETI et al.
1997), SPME to sample live individuals (MONNIN et al. 1998) and a less
expensive method (utilized for social wasps, TURILLAZZI et al. 1999)
which consists in the collection of cuticular lipids by means of small pieces
of cotton rubbed on the body of the insect. The substances are then extracted
in a solvent and injected into the GC-MS. Despite these advances, several
problems still exist especially as the abovementioned methods are suitable
for moderate to large sized insects for suitable amounts of lipids to be
extracted. Individual information on smaller insects has proven to be more
difficult, however. A method for analysing samples of reduced size (such as
pieces of cuticle or single exocrine glands) was first developed by
BAGNÈRES and MORGAN (1990) (the solid injector) and consisted of a
inserting sealed glass capillaries (containing the sample) into the (modified)
GC injector. The foremost problem of this method is the need to modify the
injector port of the GC. It is also a relatively time consuming process as the
port has to be opened frequently to be cleaned. In order to overcome these
complications we modified the general idea of solid injection and created a
special injection needle, which does not require any modification of the GC
and which is easily cleaned in hexane, which can be used to analyse small






















Figure 1. The solid injection needle and details of sample loading and injection into
the GC-MS. Samples are inserted into the groove, which is then moved to align with
two holes that allow passage of the carrier gas over the sample in the GC once
injected.

Piston
Needle
Position 1
Position 2
Position 4
Position 3
Sample
solid sample including body parts, glands or even small size insects. We
used the ant Cardiocondyla wroughtonii as our analytical specimen.
Individuals of this species measure between 1 and 2 mm in length and were
therefore suitable for assessment of our analytical tool.

METHODS

The solid injector
The design of the needle was based on the SPME fibre, an already
existing analytical tool that can be injected into GC injector ports. A hole
(approx. 3 mm x 1 mm) was made midway up the outer covering and a
groove corresponding in size also made in the internal steel piston (Fig. 1).
Samples are positioned in the groove when it is placed in line with the
opening on the external shaft (position 1). The piston is turned to close the
opening in the exterior shaft (position 2). Two further holes (approx. 0.2 mm
x 0.2 mm) were made towards near the exit point of the outer shaft. The
holes allow for the GC carrier gas to pass over the sample. Before injection
into the GC, the internal piston is pushed down in such a way that the groove
holding the sample is aligned with these two holes (but not exposing the
sample) (position 3). On insertion into the GC injector port, the internal
piston is turned to expose the sample to the two holes (position 4). We
called the solid injector SDIP (SoliD Injection Probe).

Analytical methods
Chemical analyses were made of five colony member types of C.
wroughtonii: workers, ergatoid and winged males, winged (virgin) queens
and de-alate reproductive queens. Samples of these individuals (except
reproductive queens) were taken at 1 day and 10 days after emergence.
For solid injections, we first removed the heads of individuals to avoid
contamination from glands such as the Postpharyngeal gland. These were
then placed in the solid injection needle and injected into the GC-MS as
described above. To ensure that no memory effects occurred in our method,
we cleaned the needle by immersing it after each run into hexane in an
ultrasonic bath for 10 minutes.
We compared our method to two currently accepted methods of
analytical extraction. Solvent extraction was performed by immersing
specimens individually into 10 µl of heptane for 2 minutes. Of this 2 µl were
injected into the
































Figure 2. Total ion chromatograms of three extraction methods for cuticular
hydrocarbons in the ant Cardiocondyla wroughtonii. Only solid injection (A)
produced well defined and usable results (as seen in the abundances of the
various peaks) when compared to extraction by SPME (B) and solvent (C).
In many cases, no peaks were produced in the latter two methods. Each peak
represents one or more hydrocarbons.


30.00 35.00 40.00 45.00 50.00 55.00
1x10
6

B
C
30.00 35.00 40.00 45.0
0
50.00 55.00
8.5x10
6

A
32.00 36.00 40.00

44.00 48.00 52.00
6x10
6

A
b
u
n
d
a
n
c
e

A
b
u
n
d
a
n
c
e
A
b
u
n
d
a
n
c
e
Time
Time
Time
GC-MS. Secondly, we performed SPME headspace extraction of heated
samples as described by MONETI et al. (1997). Samples were heated at
170°C for 20 min, during which time extraction was performed with a 7 µm
polydimethylsiloxane (PDMS) fibre. Identification of cuticular compounds
was performed on the basis of their mass spectra produced by electron
ionisation (EI) mass spectrometry using a Hewlett Packard (Palo Alto, CA,
USA) 5890A gas chromatograph coupled to an HP 5971A mass selective
detector (using 70 eV electron impact ionisation). A fused silica capillary
column coated with 5% diphenyl–95% dimethyl polysiloxane (Restek,
Bellefonte, PA, USA; Rtx-5MS, 30m x 0.25mm x 0.5 µm) was used. The
injector port and transfer line were set at 280ºC and the carrier gas was
helium (at 12 psi). The temperature protocol for solvent injections was as
follows: 70–150ºC at a rate of 30 ºC / min (held for 5 min), and 150–320 ºC
at 5 ºC / min (held for 13 min). For SPME and SDIP injections the
temperature protocol was: from 150 °C - 200 °C at a rate of 5 °C / min.,
from 200 °C to 260 at 2 °C / min, and from 260 °C to 310 °C at 10 °C / min.
Injections were performed in splitless mode.

RESULTS

Solid injection proved to be a superior method of extracting
hydrocarbons (Fig. 2A) when compared to SPME headspace (Fig. 2B) and
solvent extractions (Fig. 3B). Total ion chromatograms of solid injection
displayed well defined peaks and a total of 44 hydrocarbons were identified
positively by their mass spectra. On the other hand, extraction by solvent and
SPME proved to be unsatisfactory, with insufficient hydrocarbons being
extracted in many cases (Fig. 2A and B). Only when individuals were pooled
together, for example, were acceptable results produced.

DISCUSSION

Our method demonstrates that reliable chemical information of
cuticular hydrocarbons can be collected from individuals of specimens of
very reduced size. Our simple method ensures that samples do not have to be
pooled to recover sufficient information, as had to be performed with SPME
headspace and solvent extraction (and at the same time losing valuable
information on individuals). The simplicity of our method further guarantees
that the injector inlet of the GC does not have to be modified (see
BAGNÈRES and MORGAN 1990). The needle is injected as for any other
type of syringe. The inlet does also not have to be cleaned after each
injection, while we observed no memory effects in our method,
demonstrating that the needle itself was sufficiently cleaned after each run.
One of the drawbacks of our method is the fact that hydrocarbons may have
been extracted from body parts other than the cuticle. This, though, is also a
negative aspect of the other two methods where it is also possible for
contamination to originate from within the insect body. Only SPME of live
individuals and use of the method by TURILLAZZI et al. (1998) can
guarantee that hydrocarbons are extracted from the cuticle only.
Our method will prove to be extremely useful for providing individual
chemical information for social insects of reduced size. This will be very
important for understanding communication processes such as inter- and
intracolony recognition of individuals.


ACKNOWLEDGEMENTS

We thank Giancarlo Bucelli and Gloriano Moneti (Centro Interdipartimentale
di Spettrometria di Massa, Università di Firenze) for their help. Funding was
provided by the MURST “60%” and Italian CNR “40%” funds, as well as through
the research network “Social evolution” of the Universities of Aarhus, Firenze,
Keele, Sheffield, Uppsala, Würzburg and the ETH of Zürich financed by the
European commission via the Training and Mobility of Researchers (TMR)
programme.


REFERENCES

BAGNÈRES, A. G. & MORGAN, E. D. 1990. A simple method for analysis of
insect cuticular hydrocarbons. J. Chem. Ecol. 16, 3263-3276.
MONETI, G., DANI, F. R., PIERACCINI, G. & TURILLAZZI, S. 1997. Solid-
phase microextraction of insect epicuticular hydrocarbons for gas
chromatographic/mass spectrometric analysis. Rapid Commun. Mass Spectrom. 11,
857–862.
MONNIN, T., MALOSSE, C. & PEETERS, C. 1998 Solid-phase microextraction
and cuticular hydrocarbon differences related to reproductive activity in queenless
ant Dinoponera quadriceps. J. Chem. Ecol. 24, 473-490.
TURILLAZZI, S., SLEDGE, M. F. & MONETI, G. 1998 Use of a simple method
for sampling cuticular hydrocarbons from live social wasps. Ethol. Ecol. Evol. 10,
293-297.








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