Journal of Chromatography B, 937 (2013) 67–78

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Review

The use of chromatographic techniques for the separation and the

identification of insect lipids
∗
˛
Magdalena Cerkowniak, Alan Puckowski, Piotr Stepnowski, Marek Gołebiowski
University of Gdańsk, Faculty of Chemistry, Institute for Environmental and Human Health Protection, Department of Environmental Analysis, Sobieskiego
18/19, 80-952 Gdańsk, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The paper presents the state of knowledge on the chromatographic techniques used in the analysis of
Received 19 February 2013 insect lipids. Lipids are one of the forms of protection against entomopathogenic fungi. Their composi-
Accepted 16 August 2013 tion may include a number of different compounds, such as long chain hydrocarbons, waxes, alcohols,
Available online 26 August 2013
aldehydes and free fatty acids. These compounds may be presented in different amounts depending on
the species of insects, living environment, lifestyle, seasons, etc. The most commonly used techniques
Keywords:
in the analysis of lipids of insects are: gas chromatography, high performance liquid chromatogra-
Lipids
phy, and combined techniques such as gas chromatography–mass spectrometry (GC–MS) and liquid
Entomopathogenic fungi
Insects
chromatography–mass spectrometry (LC–MS). Compounds present in the cuticular lipids of insects may
Combined analytical techniques serve diverse functions such as minimizing transpiration. It was also found that extracts of insects possess
Chromatography antifungal effects. Determination of lipid profiles and biological activity of the identified compounds can
Extraction effectively contribute to the knowledge of the defense mechanisms of insects, and thus, impact the
development of new methods of combating harmful insects.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
2. Biological methods for controlling harmful insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3. The composition of the cuticular lipids of insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4. Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5. Liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
6. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
7. Combined techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

1. Introduction interest in these animals was already developing in the XLVIIth cen-
tury BC, when breeding of the silkworm was launched in China [1].
Insects (Insecta) represent the most numerous group of animals, Currently reducing the number of harmful insects is a major
which is estimated by some to consist of about 1 million species problem, especially in agriculture and forestry [2]. This problem
(several thousand more identified each year). From the very begin- appeared with changes in crops technology. Intensive protection
ning humans had been using insects as food. Then they had made against weeds and diseases, and heavy fertilization promote the
use of them in the production of drugs [1]. Human contact with development of different species of insects, including pests of
insects seems to have utilitarian ground. It is not surprising that economically important grain. Therefore it is necessary to search
for effective methods of controlling insects which present a real
threat. Regrettably, the pejorative impact of chemical pesticides
was revealed not only on the natural environment, but also on
∗ Corresponding author. Tel.: +48 585235398. human beings [3,4]. It is therefore important to search for numerous
˛
E-mail address: goleb@chem.univ.gda.pl (M. Gołebiowski). environmentally friendly methods of controlling harmful insects.

1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2013.08.023
68 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78
Therefore, it is important to know all the mechanisms of defense
of insects, including lipids. Lipids are secreted by the exocrine
system to the surface of insect cuticle and may have biological activ-
ity. Chromatographic methods such as liquid chromatography (e.g.
LLSD detector) or gas chromatography (e.g. with MS detector) are
generally used to identify lipids.
2. Biological methods for controlling harmful insects
Lately biological methods, such as the use of bioinsecticides,
are becoming more and more popular in the fight against pests
[5]. Entomopathogenic fungi are parasitic organisms which tar-
get insects, causing lethal infections. These life forms are used
to regulate the population of harmful insects. So far there are
more than 400 known species of such nature, and 1800 defined
relationships between fungi and certain insect species [6]. Ento-
mopathogenic fungi are natural pathogens of insects and they can
cause disease of many species, therefore they have great insec-
ticidal potential. Fungi belonging to the Entomophthorales order
have high species specificity – some species are infected by the
fungi, while other are resistant to infection. Due to this trait these
organisms are considered a promising source of new generation
bioinsecticides. Fungi imperfecti (Deuteromycetes) are most com-
monly used as insecticides. These are filamentous fungi, which
include about 2000 taxa. Some of them are dangerous to humans
because of the mycotoxins, which in plants and animals can cause
mycosis [7]. Attempts to use these fungi have already been taken in
the mid-nineteenth century [5]. In literature you can now find many
examples of successful insect population reduction achieved by the
use of appropriate entomopathogenic fungi. For example, lethal
effects of Microsporidium Octosporea muscaedomesticae infection
were found among adult specimens of the Lucilia cuprina fly [8,9].
The most frequently used entomopathogenic fungi are: Metarhiz-
ium anisopliae, Beauveria bassiana, and Nomurea rileyi [10]. Not only
fungi but also bacteria can act as parasites of insects. For example,
Serratia sp. has a lethal effect on the population of Lucilia sericata
flies [11]. However there are obstructions preventing the infection
of insects by entomopathogenic fungi, which are related to both:
the morphological barrier – cuticle, and the bacterial flora existing
on it. The cuticle is an exoskeleton covering the bodies of insects,
and it is constructed mainly of chitin fibers embedded in a matrix of
proteins. Its structure is often additionally impregnated with phe-
nols, lipids, calcium carbonate and metal ions such as Zn, Mn, and
Fe [12]. The cuticle is formed by several layers and each layer has
different chemical structure and properties [13]. In order to use
entomopathogenic fungi as insecticides, one must first consider the
mechanism of infection. It is important to penetrate both: mechan-
ical barriers – such as the cuticle, and various kinds of biologically
active compounds. Chemicals, such as hydrocarbons [14], waxes
[15], alcohols [16], and free fatty acids [17], are present in the outer
and inner layers of the cuticle, what is more some of them, such as
free fatty acids play very important role in the defense of insects.
These compounds may be present in different amounts depend-
ing on the species of insects, living environment, the availability
of food, seasons, etc. Their main function is to minimize transpi-
ration, though some of them also perform as pheromones. It was
also found that the lipids of insects possess antifungal properties
[17–20]. For example, free fatty acids present in insect lipids are
biologically active compounds. Certain short-chain fatty acids can
block the absorption of phosphorus and thiamine by fungi, thereby
preventing the development of mold spores – for example caprylic
acid is such an inhibitor. It has been found that cis-unsaturated
fatty acids possess strong inhibitory properties [21,22]. Saturated
(SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty
acids, together with glycerol all contribute to the structure of cell
membranes. They are components of membrane phospholipids and
precursors of eicosanoids (paracrine hormones: prostaglandins).
The biologically active compounds found on insect bodies can
affect entomopathogenic fungi [23,24]. For example, tests were
conducted on Conidiobolus coronatus fungi by applying increasing
doses of fatty acids (from C5:0 to C20:1 ). At all applied concentra-
tions (0.1–0.0001%, w/v) growth was inhibited by C16:0 , C16:1 , C18:0 ,
C18:1 , C18:2 , C18:3 , C20:0 and C20:1 [24]. Also the mixtures of fatty
acids were tested against bacterial (Bacillus cereus, Bacillus subtilis,
Enterococcus faecalis, Citrobacter freundii, Pseudomonas aeruginosa,
Pseudomonas fluorescens) and fungal strains (Paecilomyces lilacinus,
Paecilomyces fumosoroseus, Lecanicillium lecanii, M. anisopliae, B.
bassiana (Tve-N39), B. bassiana (Dv-1/07)). One of the four analyzed
mixtures of Forcipomyia nigra was significantly effective, especially
toward B. cereus and E. faecalis. This mixture contained C14:0 , C16:1 ,
C16:0 , C18:2 , C18:1 , and C18:0 fatty acids. However, The most effective
acids against bacterial growth were C9:0 , C10:0 and C16:1 , whereas
C14:0 , C16:0 , C18:0 and C18:1 demonstrated rather poor antifungal
activity and did not inhibit the growth of bacteria [19]. Cuticular
and internal lipids obtained from Calliphora vomitoria were tested
for their potential antimicrobial activity. The minimal inhibitory
concentrations of extracts against reference strains of bacteria
(B. subtilis, Rhodococcus equi, Staphylococcus aureus, Escherichia
coli, Klebsiella pneumoniae, Proteus vulgaris) and fungi (Aspergillus
niger, Candida albicans, Candida lipolytica, Candida tropicalis) were
determined. Antimicrobial activity was the strongest against Gram-
positive bacteria. All the lipid extracts obtained from larvae, pupae,
adult males and females were equally effective against all the fungal
strains examined [25].
3. The composition of the cuticular lipids of insects
Lipids (gr. Lipos – fat) are components of plant and animal orga-
nisms and are characterized by good solubility in organic solvents,
such as ether. They are hardly soluble in water. This is what dis-
tinguishes the lipid solubility of the remaining three groups of
biological substances: carbohydrates, proteins or nucleic acids. In
the last 50 years have seen a rapid growth of research on lipids. The
reason for this development was to realize the important role they
play in many aspects of chemistry. The modernization of analytical
techniques has also become a stimulus for further research into the
biological and technological functions of lipid [26,27]. The surfaces
of all higher plants and animals are covered with a layer of cuticular
lipids, which usually consist of recurring long-chain aliphatic com-
pounds. Their role is primarily to protect against excessive loss of
body water and deter predators. Surface waxes of plants are used
to produce essential oils, perfumes, and medicines, if they have the
appropriate properties [28]. In the case of insect lipids they form
not only the surface protective layer, but also are responsible for
the interaction between species. Individuals of different sexes on
the basis of the composition of surface lipids evaluate each other
as potential partners for breeding. In addition, surface waxes pro-
tects against various insect pathogens, such as fungi [17,28]. The
compounds on the cuticles are mainly fatty acids, hydrocarbons,
aldehydes, alcohols, and waxes (Table 1). The general formulas of
lipids found in insects are presented in Table 2.
The fatty acids were identified in the cuticular lipids of Apis mel-
lifera. They contained from 16 to 36 carbon atoms in the alkyl chain
and included saturated entities only [36]. The major fatty acids in
the cuticular lipids were tetracosanoic (29%), hexacosanoic (12%)
and octacosanois (11%) acids. Smaller amounts (<10%) of the fatty
acids in the range C30:0 to C36:0 and C16:0 and C18:0 were present.
Hydrocarbons are present on the surface of insects very
often [37–39]. Based on the analysis of lipids of several fly
species: Aldrichina grahami, Chrysomya megacephala, L. sericata,
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78 69

Table 1
The number of carbon atoms in the compounds, which were identified in insects.

Species of insect Fatty acids Aldehydes Alcohols Alkanes Waxes Ref.

Aleurothrixus floccosus C10 –C28 C30 –C34 C12 –C38 C27 –C33 C28 –C54 [29]
Aleyrodes singularis C10 –C26 C25 –C34 C18 –C34 – C26 –C32 [30]
Bemisia argentifolii C14 –C28 C28 –C34 C29 –C34 – C40 –C64 [31]
Heliothis virescens C16 –C22 C24 –C30 C26 –C32 – C32 –C52 [32]
Helicoverpa zea C5 –C15 C26 –C32 C26 –C30 – C28 –C42 [32]
Liposcelis bostrychophila C16 –C20 C15 –C17 – C21 –C34 – [33]
Semidalis flinti C14 –C28 – C16 –C34 – C34 –C44 [34]

Achoetandrus rufifacies, Boettcherisca peregrina, and Parasarcophaga
crassipalpis, it was concluded that hydrocarbon profiles are char-
acteristic for each species [40]. The percentage concentrations
of a few groups of chemical compounds in insect cuticle are:
16.85–52.31% for monoethyl-alkanes, 1.13–4.78% for diethyl-
alkanes, and 2.07–3.76% for n-alkanes. Some compounds are absent
only in specific species, for example pentacosane is not found in A.
grahami lipids – while it was present in all other species of flies
listed above.
Waxes are lipid components of many species of insects. For
instance a 52-carbon wax constitutes 82% of Drosicha corpulenta
cuticle lipids [41]. A high wax content was also found in Helicov-
erpa zea (38% of lipids) – the composition of lipids of this insect are
as follows: hydrocarbons (19%), alcohols (17%), diols (16%), alde-
hydes (2–5%), and esters of alcohols/oxo alcohols [32]. Analysis of
the lipid composition of Aleurodicus dugesii showed that they con-
sist of a mixture of waxes containing from 30 to 60 carbon atoms,
wherein the most abundant wax (64%) was a 44-carbon compound
[15]. In the case of A. dugesii waxes dominated the exuviae extracts,
but the nymph lipid extracts contained primarily aldehydes.
Alcohols represent another group of biologically active com-
pounds noticeable on the surface of the cuticle. The properties
of these compounds originate from the presence of the hydroxyl
group and the alkyl chain length. The solubility of alcohol in water
decreases with the elongation of the alkyl chain, therefore alco-
hols also co-create the protective barrier which is the cuticle. Free
alcohols found in insect lipids are usually saturated, primary, with
an even number of carbon atoms. For example in the lipids of Dia-
pheromera femorata the most abundant are primary alcohols – C26:0
and C28:0 . C23:0 , and C24:0 alcohols were also found, but in lesser
quantities [42]. In addition there are some interesting observations
concerning the alcohol content in lipids of Aleyrodes singularis adult
insects and their exuviae. The adult specimens predominant lipid
alcohol is C32:0 , whereas the exuviae specimens main alcohol is
C26:0 [30].
Sterols and their esters are present in insect lipids in small
quantities. These substances are synthesized by insects from
phytosterols (i.a. ␤-sitosterol, stigmasterol, campesterol, and bras-
sicasterol). Sterols constitute a large share of hormones, which
regulate body functions. The most common insect sterol lipid is
Table 2
The most common components of insects lipids [32,35].
Component Formula
n-Alkanes H3 C[CH2 ]nCH3
Ketones R1 COR2
II-row alkohols R1 CH(OH)R2
␤-Diketones R1 COCH2 COR2
Esters R1 COOR2
I-row alkohols RCH2 OH
Aldehydes RCHO
Carboxylic acids RCOOH
Dicarboxylic acids HOOC[CH2 ]nCOOH
␻-Hydroxylic acids HOCH2 [CH2 ]nCOOH
R1 and R2 – chains containing from 10 to 20 carbon atoms (or longer).
cholesterol. Cholesterol is a precursor of insect hormones. This
fact is confirmed by studies which focus on the effects of sterols
in the development of insects. Experiments were conducted on
Coleomegilla maculata to verify whether and to what extent, given
food (corn pollen containing different amounts of sterols: ␤-
sitosterol, cholesterol, ergosterol) will have an impact on the
condition of the insects [43]. The results showed a strong corre-
lation between the amount of administered sterols and an increase
in insect population. The correlation also occurred between, pro-
portionally decreasing the content of sterols in the insect diet over
time, and the growth of C. maculata exuviae [43]. In addition to
cholesterol in insect lipids there are also other sterols (stigmasterol
and sitosterol). Stigmasterol was found in the lipids of Melanoplus
species, whereas both mentioned sterols were found in the lipids of
Solenopsis invicta and Solenopsis richterii [44]. The number of sterol
esters in the lipids of insects is usually about half of the number of
sterols. Approximately 3% of the sterol esters were identified in the
lipids of Ceutorhynhus assimilis [45].
The presence of methyl and ethyl esters of carboxylic acids
was found in the lipids of Schistocera gregaria locusts. A
chloroform extract contained 9,12-methyloctadecadienoate, and
methyloctadecanoate methyl esters. Five esters were identified
in a methanolic extract: ethyldecanoate, methylpentadecanoate,
9-methylheptadecanoate, ethyl-2-hydroxy-9-octadecenoate, and
3,7-dimethyl-2,6-octadecadienoate [46].
Long-chain aldehydes are rarely components of the insect
cuticle. They are identified as constituents of mixtures of free
long-chain alcohols (22–34 carbon atoms). Aldehydes such as tri-
conatal (48.8%) and octacosanal were identified in the lipids of
Acyrthosiphon pisum [47]. The most abundant aldehyde found in
the lipids of A. singularis (whitefly) adult insects was composed of
32 carbon atoms [30]. However, aldehydes found in the exuviae of
those insects are: C26 , C28 , C30 and C32 .
Depending on the stage of development of the insect, the lipid
composition is slightly different and it serves different purposes.
Larvae and pupae cuticle functions only as a temporary cover of
the body. During the shedding up to 90% of the cuticle is degraded
by enzymes. Larvae lead a very active lifestyle, which involves
mainly eating and rapid weight gain. Their body cover is not fully
developed and rigid as it is in the case of adult insects. However it
can extend only to a certain degree, which necessitates shedding.
Sometimes the environment may induce some modifications, such
as the need to change the cuticle to enable penetration of more dry
habitats, in cases when the environmental resources are too poor.
The pupae are on the other hand very idle – the insects do not feed
and have a rather limited ability to move. In this stage of the insect
life a transformation takes place involving the reconstruction of
the body into an adult insect body (imago). Adult insects are fully
developed forms, where the age, sex and state of growth have great
impact on the lipid profile of an insect–which in turn defines their
resistance to infections of entomopathogenic fungi.
The first step in the analysis of insect lipids is extraction. After-
wards a group analysis is performed in order to separate the
mixture into groups of compounds, which in the final step are sub-
jected to qualitative and quantitative analysis. Table 3 contains data
70 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78

Table 3
Examples of the use of chromatographic techniques in the analysis of insects lipids composition.

Species of insect/analytes Extraction method Analytical techniques Ref.

Analytes: standard – HPLC [48]

solutions of cholesterol Detector: UV-DAD and LLSD
and its metabolites Phase: hexane (A) and 2-propanol (B)
Gradient: from 99.6% to 79.3% (A)
Flow rate: 1 ml/min for 56 min
Column: ␮-Porasil (300 mm × 3.9 mm, 10 ␮m)

Helicoverpa virescens, H. zea Chloroform (30 s) HPTLC [32]

Analytes: alcohols, Phase: hexane/diethyl ether/formic acid (80:20:1, v/v/v)
aldehydes, waxes, Plate: silica gel G (250 ␮m in thickness) (20 cm × 20 cm)
hydrocarbons Recover: chloroform, 5% methanol in chloroform (alcohols, oxoalcohols, diols)

GC-FID and GC–MS

Column: HP Ultra-1 (0.2 mm × 12 m, 1 ␮m)
Conditions: column temp. 150–320 ◦ C
Ramp rate: 3 or 4 ◦ C/min
Final temperature hold: 20–40 min

Aleyrodes singularis Hexane (1.5 min), HPTLC [30]

Analytes: waxes, chloroform (30 s) Phase: hexane/diethyl ether/formic acid (80:20:1, v/v/v)
alcohols, aldehydes, Visualized: 5% concentrated sulfuric acid in 95% ethanol
hydrocarbons

GC–MS
Column: HP Ultra-1 (0.2 mm × 12 m, 1 ␮m)
Conditions: column temp. 150–320 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: up to 200 min

Dinoponera quadriceps Pentane (2 min) SPME-GC [49]

Analytes: hydrocarbons Fiber: 7-␮m polydimethylosiloxane
Extraction: 2 min
Desorption: 5 min directly to GC
Column: HP-5 (30 m × 0.32 mm, 0.25 ␮m)
Conditions: column temp. isotherm 260 ◦ C for 15 min, and ramp to 300 ◦ C
Ramp rate: 5 ◦ C/min
Final temperature hold: 22 min

GC–MS
Column: DB5-MS (25 m × 0.32 mm, 0.4 ␮m)
Conditions: column temp. 200–310 ◦ C
Ramp rate: 5 ◦ C/min

Bemisia argentifolii Hexane (1 min), HPTLC [31]

Analytes: waxes, chloroform (1 min) Phase: hexane/diethyl ether/formic acid (80:20:1, v/v/v)
hydrocarbons, Plate: silica gel HPTLC (150 ␮m in thickness) (10 cm × 10 cm)
aldehydes, alcohols Recover: solution of 5% conc. H2 SO4 in 95% ethanol followed by heating to
180–200 ◦ C

GC-FID and GC–MS

Column: HP Ultra-1 (0.2 mm × 12.5 m)
Conditions: column temp. 150–320 ◦ C
Ramp rate: 3 or 4 ◦ C/min
Final temperature hold: 20–40 min

Aleurodicus dugesii Hexane (1.5 min), HPTLC [15]

Analytes: waxes, chloroform (1 min) Phase: hexane/diethyl ether/formic acid (80:20:1, v/v/v)
hydrocarbons, Visualized: solution of 5% concentrated sulfuric acid in 95% ethanol, allowing
aldehydes, alcohols the ethanol to evaporate, heating at 150 ◦ C for 10 min, and then at 250 ◦ C for
10–20 min

GC–MS
Column: HP Ultra (0.2 mm × 12 m, 1 ␮m)
Conditions: column temp. 150–320 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: up to 200 min

Semidalis flinti Hexane (1.5 min), HPTLC [34]

Analytes: hydrocarbons, chloroform (1 min) Phase: hexane/diethyl ether/formic acid (80:20:1, v/v/v)
free fatty acids, alcohols Visualized: solution of 5% sulfuric acid in 95% ethanol, allowing the ethanol to
evaporate, heating at 150 ◦ C for 10 min, and then at 250 ◦ C for 10–20 min

GC–MS
Column: HP Ultra-1 (0.2 mm × 12 m, 1 ␮m)
Conditions: column temp. 150–320 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: up to 200 min

Table 3 (Continued)
Species of insect/analytes
Cockroaches: Periplaneta
brunnea, P. fuliginosa, P.
australiasiae, P.
americana
Analytes: hydrocarbons
Calliphoridae
Analytes: hydrocarbons
Flies: Aldrichina grahami,
Chrysomya megacephala,
Lucilia sericata,
Achoetandrus rufifacies,
Boettcherisca peregrina,
Parasarcophaga
crassipalpis
Analytes: hydrocarbons
(n-alkanes, mono-methyl
alkanes, di-methyl
alkanes, n-alkenes)
Bagrada hilaris
Analytes: hydrocarbons
Anopheles gambiae,
Anopheles arabiensis
Analytes: hydrocarbons
Acanthoscelides obtecus
(Say)
Analytes: hydrocarbons,
free fatty acids, methyl
and ethyl esters of
carboxylic acids,
aldehydes,
triacylglycerols, alcohols,
sterols
Neobellieria bullata,
Drosophila melanogaster,
Arabidophis thaliana
Analytes: waxes,
hydrocarbons
Bumble-bees of the
Bombus genus
Analytes:
triacylglycerols
M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78 71
Extraction method Analytical techniques Ref.
Dichloromethane GC-FID and GC–MS [50]
(2 min) Column: CP Sil5-CB (0.25 mm × 25 m, 0.25 ␮m) and CP Sil8-CBMS
(0.25 mm × 25 m, 0.25 ␮m)
Conditions: column temp. 100–320 ◦ C
Ramp rate: 20 ◦ C/min to 210 ◦ C, 4 ◦ C/min to 320 ◦ C
Hexane GC-FID [51]
Column: HT5 (0.22 mm × 25 m, 0.1 ␮m)
Conditions: column temp. 60–320 ◦ C
Ramp rate: 10 ◦ C/min to 200 ◦ C, 3 ◦ C/min to 320 ◦ C
Final temperature hold: 6 min
Hexane (15 min) GC-FID and GC–MS [40]
Column: 30-m fused film silica capillary column (0.25 mm × 30 m, 0.25 ␮m)
Conditions: column temp. 50–270 ◦ C
Ramp rate: 25 ◦ C/min to 200 ◦ C, 3 ◦ C/min to 270 ◦ C
Final temperature hold: 20 min
– SPME-GC–MS [52]
Fiber: PDMS, 100 ␮m, PA, 80 ␮m, CW-DVB, 65 ␮m
Extraction: 2 min
Desorption: 2 min
Column: HP5-MS 0.2 mm × 30 m, 0.25 ␮m
Conditions: column temp. isotherm 150 ◦ C for 2 min, and ramp to 280 ◦ C
Ramp rate: 5 ◦ C/min
Final temperature hold: 10 min
Hexane (5 min) GC-FID and GC–MS [53]
Column: DB-1 0.32 mm × 15 m, 0.1 ␮m
Conditions: column temp. 75–320 ◦ C
Ramp rate: 25 ◦ C/min to 150 ◦ C, 8 ◦ C/min to 320 ◦ C
Final temperature hold: 15 min
Petroleum ether HPLC-LLSD [54]
(10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
dichloromethane
(5 min)
GC-FID and GC–MS
Column: DB-1 (30 m × 0.25 mm, 0.5 ␮m)
Conditions: column temp. 30–320 ◦ C
Ramp rate: 4 ◦ C/min
Dichloromethane MALDI-TOF [55]
(1 min) The acceleration voltage was 20 kV and ion extraction pulse was 200 ns.
Desorption and ionization were achieved using a nitrogen UV laser (337.1 nm,
4 ns pulse of 300 J, maximum frequency 20 Hz). Matrix ions were suppressed
below m/z 300. Spectra were averaged from 800 (WEs) or 1200 (HCs) laser
shots
Chloroform/methanol HPLC/APCI-MS [56]
(1:1, v/v), TLC Detector: APCI-MS
(hexane/diethyl Phase: acetonitrile/2-propanol
ether/formic acid Gradient: 0 min: 100% of A, flow rate 1 ml/min; 108 min: 30% of A, 70% of B,
(80:20:1, v/v/v)) 1 ml/min; 150 min: 5% of A, 95% of B, 0.5 ml/min; 165 min: 5% of A, 95% of B,
0.5 ml/min, 177–100% of A, 0.5 ml/min; 180 min: 100% of A, 1 ml/min
Column: Nova-Pak C18 (300 mm × 3.9 mm, 150 mm × 3.9 mm, a particle size
of 4 ␮m)
MALDI-MS
Desorption and ionization were achieved using a nitrogen UV laser (337.1 nm,
with a 4 ns pulse of 300 J, and the maximum frequency of 20 Hz) with the laser
power adjusted to 50–60%. The matrix ions were suppressed below m/z 200.
The data were collected from a m/z of 500–1300
72 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78

Table 3 (Continued)

Species of insect/analytes Extraction method Analytical techniques Ref.

Osmia lignaria, Megachile Hexane (60 s), GC-FID [57]

rotundata chloroform (30 s) Column: ATTM -1HT (0.25 mm × 15 m, 0.1 ␮m) and DB-1MS (0.2 mm × 12.5 m,
Analytes: hydrocarbons 0.33 ␮m)
and waxes Conditions: column temp. 75 ◦ C isotherm for 0.5 min, and ramp to 320 ◦ C
Ramp rate: 25 ◦ C/min to 225 ◦ C, 10 ◦ C/min to 300 ◦ C, 25 ◦ C/min to 320 ◦ C
Final temperature hold: 45 min

Dendrolimus pini moults Petroleum ether HPLC-LLSD [58]

Analytes: carboxylic (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
acids (methyl esters), 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
dehydroabietic acid dichloromethane
(5 min)

GC-FID and GC–MS

Column: DB-1 (30 m × 0.25 mm, 0.15 ␮m)
Conditions: column temp. 50 ◦ C isotherm for 10 min, and ramp to 310 ◦ C
Ramp rate: 3 ◦ C/min
Final temperature hold: 20 min

Calliphora vicina (larvae Petroleum ether HPLC-LLSD [59]

and pupae) (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
Analytes: free fatty acids 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
dichloromethane
(5 min)

GC-FID and GC–MS

Column: DB-1 (30 m × 0.25 mm, 0.15 ␮m)
Conditions: column temp. 60 ◦ C isotherm for 10 min, and ramp to 320 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: 20 min

Calliphora vicina, Petroleum ether HPLC-LLSD [17]

Dendrolimus pini, Galleria (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
mellonella 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
Analytes: free fatty acids dichloromethane
(5 min)

GC-FID and GC–MS

Column: DB-1 (30 m × 0.25 mm, 0.15 ␮m)
Conditions: column temp. 30 ◦ C ramp to 320 ◦ C
Ramp rate: 4 ◦ C/min

Lucilla sericata (larvae, Petroleum ether HPLC-LLSD [14]

pupae, male and female) (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
Analytes: n-alkane 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
dichloromethane
(5 min)

GC-FID and GC–MS

Column: DB-1 (30 m × 0.25 mm, 0.15 ␮m)
Conditions: column temp. 80 ◦ C isotherm for 8 min, and ramp to 320 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: 10 min

Calliphora vomitoria Petroleum ether GC–MS [25]

(larvae, pupae, male and (10 s) (bp Column: Rtx-5 (30 m × 0.25 mm, 0.15 ␮m)
female) 38–58 ◦ C), Conditions: column temp. 80 ◦ C isotherm for 8 min, and ramp to 310 ◦ C
Analytes: fatty acids dichloromethane Ramp rate: 4 ◦ C/min
(5 min) Final temperature hold: 10 min

Calliphora vomitoria Petroleum ether HPLC-LLSD [60]

(larvae, pupae, male and (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
female) 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
Analytes: fatty acid dichloromethane
methyl esters and (5 min)
alcohols

GC–MS
Column: Rtx-5 (30 m × 0.25 mm, 0.15 ␮m)
Conditions: column temp 80 ◦ C isotherm for 8 min, and ramp to 310 ◦ C
Ramp rate: 4 ◦ C/min
Final temperature hold: 20 min
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78 73

on the most frequently applied extraction methods and analytical thin-layer chromatography (TLC) [65,66], and high performance
techniques in the analysis of insect lipids, with particular reference thin layer chromatography (HPTLC) are commonly used [67]. The
to combined gas chromatography–mass spectrometry (GC–MS). latter was exploited in order to identify Semidalis flinti cuticle waxes
[34], along with the determination of cuticle lipid composition of
Bemisia argentifolii nymphs and adult insects [31]. For these pur-
4. Extraction
poses silica gel HPTLC plates of the following measurements were
used: 10 cm × 10 cm and 150 ␮m thickness. The mobile phase was
The basic method of insect lipid extraction involves the use of
a mixture of hexane/diethyl ether/formic acid (80:20:1; v/v/v). The
organic solvents. The principle of the method is transferring the
obtained groups of chemical compounds were then analyzed by GC
analyte to a liquid phase (the organic solvent) which is immiscible
and GC–MS.
with the matrix. This method requires significant amounts of a
A commonly used technique for insect lipid analysis is column
solvent, which must be subsequently evaporated in order to con-
chromatography [51,68–70]. After extraction the analytes are sep-
centrate the sample. In addition, the extracted volatile compounds
arated on a column (e.g. 6 cm × 0.5 cm), and eluted with a nonpolar
may be evaporated together with the solvent, which affects the
solvent (e.g. hexane).
analysis result. For extraction of insect lipids numerous methods
Another important analytical technique is high performance liq-
are available. For extraction of insect lipids numerous methods are
uid chromatography (HPLC). It is particularly useful in the case
available. Using a combination of solvents, and long enough extrac-
of less volatile compounds. A number of different detectors are
tion time could lead to the isolation of internal lipids, whereas a
used in HPLC: spectrophotometer (UV–vis), diode array detector
too short extraction time of surface lipids would not be sufficient.
spectrophotometer (UV–vis DAD), fluorescence, refractive index,
Therefore, two-step extraction using solvents of different polarity
conductivity, and laser light-scattering detector (LLSD).
is commonly applied to isolate the surface lipid without internal
HPLC with UV–vis and LLSD detection is a preliminary analysis
lipids extraction. Despite the use of different methods of extrac-
technique, aimed primarily at the separation of the tested mixture
tion and analysis, the results obtained are comparable due to the
for single compounds or groups of compounds (HPLC-LLSD). Thanks
use of internal standard method. Table 3 presents examples of the
to the use of HPLC it is possible to collect fractions of groups of
use of various solvents in the extraction of insect lipids. A com-
compounds, which are then passed on to further analysis. HPLC
monly used solvent is hexane, which was used for the extraction of
serves a preliminary identification of compounds, mainly based
cuticular lipids found in A. dugesii [61]. Hydrocarbons and waxes
on the comparison of retention times of the tested analytes and
were successfully identified in the lipids of this insect. Chloro-
solutions of well known standards. It is necessary to use other com-
form was also used in the extraction, resulting in a higher yield
plementary techniques, because the identification using only liquid
of surface lipids. Examples confirming the wide range of applica-
chromatography is not reliable enough and honest (even if the
tions of hexane are contained in the work of James S. Buckner.
retention times of analyte and standard are the same). Mass spec-
The author performed extractions of lipids of Osmia lignaria and
trometry is generally used for this purpose, because it completes
Megachile rotundata. The extraction was performed by immersion
chromatographic techniques and gives the answer to the analytes
in hexane for 60 s, followed by an extraction in chloroform also for
structure.
60 s [57]. Results showed that almost 64% of the cuticle lipids of O.
The separation and analysis of the insect lipids are performed
lignaria are C25 –C31 alkenes. In the case of M. rotundata, 48% of the
in normal- and reverse-phase chromatography (NP and RP). Nor-
cuticle lipids were C23 –C33 alkenes, and 45% were alkanes of the
mal phase is such a system in which the stationary phase is
same chain length. For the extraction of lipids also other solvents
the more polar than mobile phase and the reverse phase sys-
such as petroleum ether (bp 38–58 ◦ C) and dichloromethane are
tem in which the stationary phase occurs is less polar than the
used. These two solvents were used for the extraction of lipids from
mobile phase. Solvents used in normal phase are: hexane, isooc-
Calliphora vicina, Dendrolimus pini, and Galleria mellonella, wherein
tane, chloroform, dichloromethane and ethyl acetate. Similarly,
the first extraction was performed in 10 s and the second one in
in the reverse phase solvents used are: methanol, acetonitrile,
5 min [17]. The use of two solvents with different polarity ensures
isopropanol, tetrahydrofuran and water (water buffers). Apply-
achieving better extraction efficiency.
ing normal phase chromatography one can successfully perform
Another popular technique for lipid extraction is solid phase
the separation of analytes starting from the least polar hydrocar-
microextraction (SPME) [62]. The advantages of this technique are
bons, waxes, sterol esters, triacylglycerols, free fatty acids, alcohols,
primarily limiting the amount of necessary analytical operations,
sterols and polar phospholipids.
elimination of toxic solvents and also reduction of the time of anal-
For example normal phase high performance liquid chro-
ysis. The SPME method, however, requires paying special attention
matography combined with a laser light-scattering detector
to the purity of the absorbent fiber. In order to obtain reproducible
(HPLC-LLSD) was used for the group analysis of Calliphora viv-
results using SPME it is essential to control the parameters of sorp-
ina, D. pini, G. mellonella and Acanthoscelides obtecus lipids
tion and desorption of analytes on the fiber. This method is often
[17,54,58,59]. The lipids were separated on an analytical column
used in the analysis of insect cuticle hydrocarbons. It was applied
– Econosil Silica (250 mm × 4.6 mm) with hexane (phase A), and
for several species, including: Dinoptera quadriceps, Bagrada hilaris,
dichloromethane/acetone (phase B) as the mobile phase. The fol-
and Coptotermes formosanus [49,52,63,64]. The procedure is not
lowing gradient was used in HPLC: from 100% phase A, to 100%
destructive, which means that it enables repeated analysis of the
phase B in 30 min. Carbon dioxide was used as the nebulization gas
same samples.
in the detector.
Fig. 1 presents an exemplary chromatogram obtained by HPLC-
5. Liquid chromatography LLSD lipid analysis of A. obtecus.
As many as 7 groups of compounds were obtained from the
Group analysis is a very important step in the whole analytical extracts of A. obtecus lipids. The most abundant groups were
process. Usually before qualitative and quantitative determination triacylglycerols, saturated and unsaturated carboxylic acids, and
is possible, the analytes need to be separated from the sample. a significant amount of hydrocarbons. Aldehydes, esters of fatty
This task is difficult because most samples have a highly devel- acids, alcohols and sterols were found in smaller quantities.
oped matrix. Therefore if one does not use solvent extraction, Various groups of compounds were collected and analyzed by
other separation techniques need to be applied. In group analysis GC–MS, which confirmed the initial identification on the basis
74 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78
Fig. 1. HPLC-LLSD chromatogram: 1: hydrocarbons; 2: aldehydes; 3: methyl and
ethyl esters of fatty acids; 4: triacylglycerols; 5: free fatty acids; 6: alcohols; 7:
sterols.
Reprinted from [54] with the permission of Elsevier B.V. All rights reserved.
of characteristic mass spectra and by comparing the obtained
retention times with the retention times of standards. Reversed
phase (RP) HPLC is also applicable to the identification of lipids
– it is effective in the analysis of saturated and unsaturated fatty
acids and their methyl esters [71]. HPLC with UV-DAD and LLSD
detectors was used for the analysis of cholesterol and its metabo-
lites [48]. For these procedures hexane and 2-propanol were used
as the mobile phase (from 99.6% to 79.3% of hexane, flow rate
1 ml/min for 56 min). The elution conditions were identical for
both detectors, nitrogen was used as the nebulization gas (LLSD).
More analytes were identified using the LLSD detector in com-
parison with the UV-DAD. Much more stable and better baseline
was achieved with LLSD detector than using UV detector (Fig. 2).
As a result of HPLC analysis using both detectors the following
compounds have been identified: cholesterol, cholestan, 7-
ketocholesterol, 7␣-hydroxycholesterol, 7␤-hydroxycholesterol,
20-hydroxycholesterol and 25-hydroxycholesterol [48] (Table 3).
6. Gas chromatography
Chromatographic methods, such as most of the instrumental
techniques are the techniques of the comparative solutions requir-
ing calibration patterns. Analysis is done by comparison of the
area or height of the analyte signal with the area or the height of
the signal of standard. The quantitative analysis is based on the
proportion of the signal field and/or the height of the analyte signal
to the amount (mass or concentration) in the sample. Measured
parameter: the area or height of the signal is a function of the
analyte in the mixture. The most common method of qualitative
analysis is a method of the internal standard. It involves the addi-
tion of an appropriate amount of standard, which is well separated
from all the constituents of the sample in current conditions
of analysis; however, physico-chemical properties do not differ
from the analyte. Standard cannot be previously present in the
Fig. 2. HPLC of cholesterol and its oxidization products on a (␮-Porasil (10-
␮m) column (300 mm × 3.9 mm I.D.)) with (A and B) diode-array UV detection
at (A) 206 nm and (B) 233 nm and (C) laser light-scattering detection. Peaks: 1:
cholestane; 2: cholesterol; 3: 20-hydroxycholesterol; 4: 25-hydroxycholesterol; 5:
␣-epoxide; 6: ␤-epoxide; 7: 7-ketocholesterol; 8: 7␤-hydroxycholesterol; 9: 7␣-
hydroxycholesterol; 10: cholestanetriol.
Reprinted from [48] with the permission of Elsevier B.V. All rights reserved.
sample. Analysis of the obtained chromatogram consists typically
in determine the retention time of the individual compounds and
establishing area of signals. With such a universal method for the
quantitative analysis which is method of the internal standard, it
is possible to compare qualitative analytical results obtained from
two extraction methods from the same individuals. The results are
expressed as mean ± standard deviation of three analyses.
The use of gas chromatography techniques often requires trans-
forming analytes into derivatives. The most popular reagents used
for this purpose are: N,O-bis(trimethylosilyl)trifluoroacetamide
(BSTFA), and trimethylchlorosilane (TMCS). Silylation is mainly
used in the case of: alcohols, alkaloids, amines, carboxylic acids,
phenols and steroids, though there are other compounds suscep-
tible to this process [72]. The reaction mechanism is based on a
nucleophilic attack (SN2) resulting in the substitution of a hydro-
gen atom in certain groups: OH, SH, NH, COOH, NOH, SOH,
POH, CONH2 and NH2 . For example, the trimethylsilyl (TMSi)
ethers of alcohols or fatty acids are obtained by the addition of
100 ␮l of a mixture of 85% bis(trimethylsilyl)acetamide and 15%
chlorotrimethylsilane (TMCS) (TMCS is used as catalyst) to 1 mg
of lipid extract. Obtained mixture is kept at 100 ◦ C for 1 h [73].
The fatty acids can be also analyzed as fatty acid methyl esters.
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78
For example, the fatty acids can be methylated by the addition
of diazomethane. The N-methyl-N-nitroso-p-toluenesulfonamide
is diluted in diethyl ether and then KOH, which is dissolved in 10 ml
ethanol, is added. After 5 min, the ether solution of diazomethane
is evaporated over a water bath. Then fatty acids are dissolved in
a small amount of anhydrous diethyl ether to which small por-
tions of the diazomethane solution are subsequently added [73].
The free fatty acids can be also methylated by the addition of 14%
BF3 in methanol for 1 h at 80 ◦ C in sealed ampoules [73]. The result-
ing methyl esters are ideal derivatives for the characterization of
carboxylic acids. They are easily described by GC and readily iden-
tified by interpretation of mass spectra, but the methyl esters can
readily arise as artifacts during the extraction of lipids [74]. The
use of methanol as an extraction solvent for lipids may cause arti-
ficial methyl esters of fatty acids in certain lipids in the presence of
some organic or inorganic materials [75]. Wax esters and triacyl-
glycerols are often hydrolyzed and then the hydrolysis products are
silylized. Compounds are hydrolyzed by use of a solution of KOH in
99.8% ethanol (0.5 mol dm−3 , 3 h at 70 ◦ C) [76]. After evaporation of
the ethanol under a stream of nitrogen the hydrolysis products are
silylated as described above.
The presence of numerous isomers (positional and geometric)
in insect lipids makes qualitative analysis difficult. The reten-
tion times and the mass spectra of these compounds are usually
very similar. In this case, equivalent chain lengths (ECL) for
identification of fatty acid derivatives are applied. The ECL val-
ues increase with increasing distance of the double bond from
the carboxyl group. Mass spectra both of positional isomers
are identical, so identification by GC/MS these isomers requires
transforming analytes into derivatives. Additionally, compounds
with increasing alkyl-chain branching elutes before a linear sat-
urated compounds and retention time of the E-isomer is shorter
than of the Z-isomer on a polar column. For the GC–MS loca-
tion of double bonds, the methyl esters or alkenes are reacted
with dimethyl disulphide (DMDS) [77]. A small sample is dis-
solved in 50 ␮l dimethyl disulphide, and 50 ␮l carbon disulphide
is added with 300 ␮g iodine, the mixture is kept at 60 ◦ C for 40 h
in a sealed vial. Next, the aqueous Na2 S2 O3 is added and the
organic phase is extracted and evaporated in a stream of nitrogen
[78].
Insect lipids are examined primarily by gas chromatogra-
phy, in particular GC-FID since the flame ionization detector
is used for the analysis of most organic compounds. The uni-
versality of this technique is confirmed by numerous examples
mentioned in Table 3. For the analysis of cuticle lipids of
O. lignaria and M. rotundata, GC-FID was used with capil-
lary columns: ATTM-1HT (0.25 mm × 15 m × 0.1 ␮m) and DB-1MS
(0.2 mm × 12.5 m × 0.33 ␮m). As a result of these examinations
the percent composition of lipids (containing mainly long-chain
alkanes, alkenes, and waxes) was determined as follows: 95.0% of
lipids were hydrocarbons and 4.6% were esters [57]. The analysis
of hydrocarbons of Anopheles gambiae and Anopheles arabien-
sis (the most common malaria-carrying mosquito species) was
also performed applying GC-FID with a capillary column DB-1MS
(0.32 mm × 15 m × 0.1 ␮m) [53]. This technique was useful in iden-
tifying the differences in hydrocarbon profiles of different species
of mosquitoes (M- and S-forms), caused by allopatric speciation.
Hydrocarbon profiles of mosquitoes from three different loca-
tions in Burkina Faso (villages Gountry and Baloungen – separated
from each other by about 80 km, located near the capital city of
Ougadougou; a small sample was collected at Bama-VK7, located
at the periphery of the rice cultivation area of Vallee du Kou,
about 400 km SW from the former villages). Intra-taxonomic differ-
ences, in the concentrations of the same hydrocarbons, were found
not only between the allopatric taxa but also in both sympatric
ones.
75
7. Combined techniques
Gas chromatography does not always allow an unambiguous
identification of analytes, making it necessary to use coupled tech-
niques. Also mass spectrometry often gives no definite answer to
the structure of the tested compounds and, therefore, is usually
used in combination with other techniques such as chromatogra-
phy. Gas chromatography–mass spectrometry (GC–MS) and liquid
chromatography–mass spectrometry (LC–MS) represent coupled
techniques, which are a definite help in qualitative and quantitative
analysis.
Lipids analysis often poses major problems due to their very
large composition. Using the retention time as an independent vari-
able is not practical because of the minor shifts and partial peak
coelution. Such a mixture contains a number of analytes of different
groups of compounds with different physicochemical properties.
Combined techniques are great practical application in order to
identify lipids qualitatively and quantitatively. Detector MS/MS
may find use to the analysis of especially strongly complex matri-
ces.
Mass spectrometry is a physical technique, which is based
on measuring the mass to charge ratios of charged molecules or
molecule fragments, created by ionizing chemical compounds [79].
Currently, this technique is often used in chemistry, biochemistry,
medicine and biology, especially for the identification of chemi-
cal compounds and their mixtures, for determining the structure
of chemical compounds and determining their elemental compo-
sition [80]. The basis of the detection by mass spectrometry in the
formation of ions from neutral compounds and then testing the
compounds produced after degradation. The result of this type of
detection is a mass spectrum, which provides information about the
resulting fragment ions, isotopic ions and, where appropriate, the
molecular ion. In GC/MS two potential methods exist for ionization.
The choice of ionization method depends on the nature of the sam-
ple and the type of information required from the analysis. Electron
ionization (EI) is widely used in mass spectrometry for analysis of
relatively volatile compounds. EI is known as a “hard” ionization
method, so it is very good for producing fragment ions which are
useful for identification of compounds, but the molecular ion does
not appear or is a smaller peak in the spectrum. For example, the
characteristics fragment ions of methyl esters are at m/z 74 and
87 and characteristics ions of fatty acids are at m/z 60 and 73, and
molecular ions in these mass spectra have small intensity. Chem-
ical ionization (CI) is applied to similar samples, but this method
of ionization is used to enhance the abundance of the molecular
ion, which represented the molecular weight of the compound. For
both ionization modes, the molecular weight range is 40–800 Da.
The use of GC/MS often requires transforming analytes into
derivatives. In case of the analysis of fatty acids, which have low
volatility, it is necessary to transform them into methyl derivatives
first [81]. For example long chain fatty acids derived from insect
exocrine glands were subjected to GC–MS analysis [82]. In order to
optimize the method some aspects of the process were revised –
such as injector temperature, different ways of dosing the sample,
and the effect of using solid phase microextraction (SPME) on the
obtained quantitative results. An inappropriate selection of param-
eters resulted in low detection of fatty acids. Another example of the
use of GC–MS is the analysis of locust cuticle lipids. The compounds
identified were: hydrocarbons, long chain fatty acids, methyl and
ethyl esters. Prior to GC–MS analysis the methanolic extract was
subjected to derivatization (to transform the fatty acids into methyl
derivatives) [46]. Currently GC–MS is the most common separation
technique for the analysis of insect lipids [14,40,53,57,83]. Exam-
ples of the application of GC–MS for the analysis of insect lipids
are included in Table 3. Exemplary total ion current (TIC) of lipids
extracted from A. singularis is pictured in Fig. 3.
76 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78

Fig. 3. CGC–MS total ion current trace of the lipid extract from exuviae of A. singularis. Ald, aldehyde; Alc, alcohol; AcE, acetate ester. The number indicates the number of
carbon atoms in the backbone of the molecule or of the alcohol moiety of the acetate ester. For the wax esters the number indicates the total number of carbon atoms in
the ester. The hydrocarbons are designated with a number or a number and uppercase letter. The letters A and B indicate one and two methyl branches, respectively, on the
backbone. The letter, A%, indicates one methyl branch near the end of the molecule. If the A% component eluted before a B component, it is a 2- or 4-methylalkane; if it eluted
after a B component, it is a 3-methylalkane. The presence of a double bond is indicated by the carbon number followed by ‘:1’.
Reprinted from [30] with the permission of Elsevier B.V. All rights reserved.

Another example of the use of coupled techniques for

the analysis of insect lipids is high performance liquid
chromatography–mass spectrometry. This technique found
its use for the analysis of triacylglycerols. HPLC with atmospheric-
pressure chemical ionization (HPLC/APCI-MS) was used for the
analysis of triacylglycerols in the lipids of bumble bees (Bombus
terrestris, B. lucorum, B. lapidarius, B. pratorum, B. sylvarum, B.
ruderatus, B. pomorum, B. subterraneus, B. campestris, B. bohemicus,
and B. rupestris) [56] (Fig. 4). For this purpose the following mobile
phase was used: (A/B) acetonitrile/2-propanol in a gradient from
100% phase A to 5% phase A, and then return to 100% phase A. As
a result of these analyzes, it was found that among the triacylgly-
cerols one-unsaturated fatty acids with 18–16 carbon atoms were
the most abundant. The same characteristic triacylglycerols were
found in the lipids of 11 species of the Bombus family. A MALDI-MS
analysis was also performed in the mass range of 600–950 Da. The
HPLC/APCI-MS results provided information on chemotaxonomy,
phylogenetic levels, and the relationships between lipids and
their secondary metabolites. HPLC/ACPI-S results have provided
a significant amount of information because of high separation
power of chromatography and mass spectrometry. This allows us
to obtain detailed information about almost all the components
of the triacylglycerols mixtures. The advantage of MALDI-MS is its
rapid analysis and data evaluation. However this technique pro-
vides information only about the molecular weights of the mixture
components, not about the precursor ion for fragmentation. The
MALDI-MS is suitable for cases when only a pattern of the TG
composition is important in large sets of samples, without the
need for detailed information on the TGs’ structures. A principal
component analysis (PCA) and the identification of compounds
specific for certain species were also possible. MS/MS HPLC also
can be used in the analysis of lipid [84]. The use of tandem mass
spectrometry improves the resolution, improves the specificity
Fig. 4. The HPLC/APCI-MS chromatogram of the triacylglycerols isolated from the
and sensitivity in identifying the components of the mixture. By fat bodies of the Psithyrus bohemicus (a) and P. campestris (b) males. The numbers
choosing one of the molecular ions as the primary ion the spectrum above represent the ECN values.
of product ions are easily obtained. Changes in the end groups can Reprinted from [56] with the permission of Elsevier B.V. All rights reserved.
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78
cause significant changes in the spectra of product ions, allowing
the identification of the end groups [80].
8. Conclusions
This paper describes the function and lipid composition of
insects and also a review of recently used methods of analysis of
insect cuticle lipids. The lipids of insects contain many compounds,
which belong to different groups. The most common are: hydro-
carbons, carboxyl acids, ketones, alcohols and aldehydes. These
compounds are inhibitors of fungal growth, although sometimes
they may be nutrients for entomopathogenic fungi. Cuticle lipids
of insects also reduce transpiration, and some of them can act as
pheromones. The most commonly used techniques in the analysis
of insect lipids are: gas chromatography, liquid chromatography,
and combined techniques such as gas chromatography–mass spec-
trometry (GC–MS) and liquid chromatography–mass spectrometry
(LC–MS). Qualitative and quantitative analysis of insect lipids
consisted of four steps: (1) extraction of analytes into an organic
solvents, (2) separation by TLC, column chromatography and HPLC,
(3) derivatization, and (4) determination by GC, HPLC and combined
techniques.
Acknowledgments
Financial support was provided by the Polish Ministry of Science
and Higher Education for 2010–2013 grant: N N303 504238, DS
530-8110-D195-12 and UMO-2012/05/N/NZ4/02210.
References
[1] J. Boczek, Insects and People, PWN, Warsaw, 1990, 9, 23, 24 pp.
[2] H. Malinowski, Prog. Plant Prot. 49 (2009).
[3] Centers for Disease Control and Prevention (CDC), Morb. Mortal. Wkly. Rep. 60
(2011) 1269.
[4] Y. Katsuda, Top. Curr. Chem. 314 (2012) 1.
[5] J.J. Lipa, Outline of Insect Pathology, PWRiL, Warsaw, 1967, 342 pp.
[6] L. Jankevica, Latvijas Entomologs 41 (2004) 60.
[7] A. Borowska, Deuteromycetes, Hyphomycetes, Dematiaceae phialoconidiae,
Mycota, vol. XVI, PWN, Warszawa – Kraków, 1986 (in polish).
[8] D.J. Cooper, D.E. Pinnock, S.M. Bateman, J. Aust. Enromol. Soc. 22 (1983) 292.
[9] C.J. Smallbridge, D.J. Cooper, D.E. Pinnock, J. Invertebr. Pathol. 66 (1995) 196.
[10] N. Pedrini, R. Crespo, M.P. Juárez, Comp. Biochem. Physiol. 146 (2007) 124.
[11] M. O’Callaghan, M.L. Garnham, T.L. Nelson, D. Baird, T.A. Jackson, J. Invertebr.
Pathol. 68 (1996) 22.
[12] B. Chen, X. Peng, W. Wang, J. Zhang, R. Zhang, Micron 33 (2002) 571.
[13] M.P. Juárez, G.M. Calderón Fernandez, Comp. Biochem. Physiol. 147 (2007)
711.
˛
[14] M. Gołebiowski, ˛
M. Paszkiewicz, A. Grubba, D. Gasiewska, M.I. Boguś, E. Włóka,
W. Wieloch, P. Stepnowski, Bull. Entomol. Res. 102 (2012) 453.
[15] D.R. Nelson, T.P. Freeman, J.S. Buckner, Comp. Biochem. Physiol. 125 (2000)
265.
˛
[16] M. Gołebiowski, M.I. Boguś, M. Paszkiewicz, W. Wieloch, E. Włóka, P. Step-
nowski, Lipids 47 (2012) 613.
˛
[17] M. Gołebiowski, E. Maliński, M.I. Boguś, J. Kumirska, P. Stepnowski, Insect
Biochem. Mol. Biol. 38 (2008) 619.
[18] R.E. Leucona, J.M. Clement, G. Riba, C. Joulie, P. Juarez, J. Econ. Entomol. 90
(1997) 119.
[19] A. Urbanek, R. Szadziewski, P. Stepnowski, J. Boros-Majewska, I. Gabriel, M.
˛
Dawgul, W. Kamysz, D. Sosnowska, M. Gołebiowski, J. Insect Physiol. 58 (2012)
1265.
˛
[20] M. Gołebiowski, M. Dawgul, W. Kamysz, M.I. Boguś, W. Wieloch, E. Włóka, M.
Paszkiewicz, E. Przybysz, P. Stepnowski, J. Exp. Biol. 215 (2012) 3419.
[21] Ch. Wang, J. Xing, Ch. Chin, J.S. Peters, Physiol. Mol. Plant Pathol. 61 (2002) 151.
[22] S.E. Barnes, D. Moore, Mycol. Resp. 101 (1997) 662.
˛
[23] M.I. Boguś, E. Kedra, J. Bania, M. Szczepanik, M. Czygier, P. Jabłoński, A. Pasz-
taleniec, J. Samborski, J. Mazgajska, A. Polanowski, J. Insect Physiol. 53 (2007)
909.
˛
[24] M.I. Boguś, M. Czygier, M. Gołebiowski, ˛
E. Kedra, J. Kucińska, J. Mazgajska, J.
Samborski, W. Wieloch, E. Włóka, Exp. Parasitol. 125 (2010) 400.
˛
[25] M. Gołebiowski, M. Cerkowniak, M.I. Boguś, E. Włóka, M. Dawgul, W. Kamysz,
P. Stepnowski, J. Insect Physiol. 59 (2013) 416.
[26] I. Żak, Lipidy i pochodne, Chemia medyczna, Wydawnictwo ŚAM, Katowice,
2001.
[27] J. Gmiński, D. Polańska, K. Pucicka-Hoffmann, Lipidy, Biochemia: Skrypt dla
studentów Wydziału Lekarskiego, Wydawnictwo ŚAM, Katowice, 1999.
77
˛
[28] B. Szafranek, M. Paszkiewicz, M. Gołebiowski, P. Stepnowski, Gas chromato-
graphic analysis of plant and insect surface compounds: cuticular waxes and
terpenoids, in: B. Salih, Ö. Çelikbiçak (Eds.), Gas Chromatography in Plant Sci-
ence, Wine Technology, Toxicology and Some Specific Applications, InTech,
Rijeka, Croatia, 2012, ISBN 978-953-51-0127-7, pp. 39–72.
[29] D.R. Nelson, G. Walker, J. Buckner, C. Fatland, Comp. Biochem. Physiol. B 117
(1997) 241.
[30] D.R. Nelson, M. Guershon, D. Gerling, Comp. Biochem. Physiol. B 119 (1988)
655.
[31] J.S. Buckner, M.M. Hagen, D.R. Nelson, Comp. Biochem. Physiol. B 124 (1999)
201.
[32] J.S. Buckner, M.C. Mardaus, D.R. Nelson, Comp. Biochem. Physiol. 114 (1996)
207.
[33] R. Howard, J. Lord, J. Chem. Ecol. 29 (2003) 615.
[34] D.R. Nelson, T.P. Freeman, J.S. Buckner, K.A. Hoelmer, Ch.G. Jackson, J.R. Hagler,
Comp. Biochem. Physiol. 136 (2003) 343.
[35] H. Hart, L.E. Craine, D.J. Hart, Chemia organiczna, Krótki kurs, Wydawnictwo
lekarskie PZWL, Warszawa, 2006.
[36] G.J. Blomquist, A.J. Chu, S. Remaley, Insect Biochem. 10 (1980) 313.
[37] E. Dubis, E. Maliński, E. Hebanowska, E. Synak, K. Pihlaja, J. Nawrot, J. Szafranek,
Comp. Biochem. Physiol. B 88 (1987) 681.
[38] E. Hebanowska, E. Maliński, E. Dubis, P. Oksman, K. Pihlaja, J. Nawrot, J.
Szafranek, Comp. Biochem. Physiol. B 95 (1990) 699.
˛
[39] M. Gołebiowski, E. Maliński, J. Nawrot, J. Szafranek, P. Stepnowski, Comp.
Biochem. Physiol. B 147 (2007) 288.
[40] G. Ye, K. Li, J. Zhu, G. Zhu, C. Hu, J. Med. Entomol. 44 (2007) 450.
[41] A. Hashimoto, S. Kitaoka, Appl. Entomol. Zool. 17 (1982) 453.
[42] J.D. Warthen, E.C. Uebel, W.R. Lusby, V.E. Adler, Insect Biochem. 11 (1981)
467.
[43] L. Pilorget, J. Buckner, J.G. Lundgren, J. Insect Physiol. 56 (2010) 81.
[44] J.B. Lok, E.W. Cupp, G.J. Blomquist, Insect Biochem. 5 (1975) 821.
[45] I. Richter, H. Krain, Lipids 15 (1980) 580.
[46] S. Jarrold, D. Moore, U. Potter, A.K. Charnley, Mycol. Res. 111 (2007) 240.
[47] J.S. Buckner, Cuticular polar lipids of insects, in: D.W. Stanley-Sameulson, D.R.
Nelson (Eds.), Insect Lipids: Chemistry, Biochemistry and Biology, University
of Nebraska Press, Lincoln, 1993, pp. 227–270.
[48] S. Kermasha, S. Kubow, M. Goetghebeur, J. Chromatogr. A 685 (1994) 229.
[49] T. Monnin, Ch. Malosse, Ch. Peeters, J. Chem. Ecol. 24 (3) (1998) 473.
[50] I. Said, G. Costagliola, I. Leoncini, C. Rivault, J. Insect Physiol. 51 (2005)
995.
[51] O. Roux, Ch. Gers, L. Legal, Biochem. Syst. Ecol. 34 (2006) 406.
[52] De Pasquale, S. Guarino, E. Peri, G. Alonzo, S. Colazza, Anal. Bioanal. Chem. 389
(2007) 1259.
[53] B. Caputo, F.R. Dani, G.L. Horne, S.N. Fale, A. Diabate, S. Turillazzi, M. Coluzzi, C.
Costantini, A.A. Priestman, V. Petrarca, A. della Torre, Insect Biochem. Mol. Biol.
37 (2007) 389.
˛
[54] M. Gołebiowski, E. Maliński, J. Nawrot, P. Stepnowski, J. Stored Prod. Res. 44
(2008) 386.
[55] V. Vrkoslav, A. Muck, J. Cvačka, A. Svatoš, J. Am. Soc. Mass Spectrom. 21 (2009)
220.
[56] E. Kofronova, J. Cvačka, V. Vrkoslav, R. Hanus, P. Jiroš, J. Kindl, O. Hovorka, I.
Valterová, J. Chromatogr. B 877 (2009) 3878.
[57] J.S. Buckner, T.L. Pitts-Singer, Ch. Guédot, M.M. Hagen, Ch.L. Fatland, W.P. Kemp,
Comp. Biochem. Physiol. 153 (2009) 200.
˛
[58] M. Gołebiowski, M.I. Boguś, M. Paszkiewicz, P. Stepnowski, J. Insect Physiol. 56
(2010) 391.
˛
[59] M. Gołebiowski, Lipids 47 (2012) 1001.
˛
[60] M. Gołebiowski, M. Cerkowniak, M. Dawgul, W. Kamysz, M.I. Boguś, P. Step-
nowski, Parasitology 140 (2013) 972.
[61] D.R. Nelson, Ch.L. Fatland, J.S. Buckner, T.P. Freeman, Comp. Biochem. Physiol.
123 (1999) 137.
[62] J. Zhou, Y. Qi, Y. Hou, J. Zhao, Y. Li, X. Xue, L. Wu, J. Zhang, F. Chen, J. Chromatogr.
B 879 (2011) 2533.
[63] J.M. Bland, W.L.A. Osbrink, M.L. Cornelius, A.R. Lax, C.B. Vigo, J. Chromatogr. A
932 (2001) 119.
[64] M.D. Ginzel, J.A. Moreira, A.M. Ray, J.G. Millar, L.M. Hanks, J. Chem. Ecol. 32
(2006) 435.
[65] M.C. Mardaus, J.S. Buckner, Insect Biochem. Mol. Biol. 27 (1997) 551.
[66] C.C. Gomes, J.R. Trigo, Á.E. Eiras, J. Chem. Ecol. 34 (2008) 636.
[67] M. Miyazaki, L. Maoc, G. Hendersonc, R.A. Laine, J. Chromatogr. B 877 (2009)
3175.
[68] T.T. Bailock, G.J. Blomquist, Biochem. Biophys. Res. Commun. 68 (1976)
841.
[69] E. Dubis, E. Maliński, E. Hebanowska, M. Świecka, ˛ J. Nawrot, K. Pihlaja, J.
Szafranek, Z. Warnke, Comp. Biochem. Physiol. B 88 (1987) 911.
[70] J.G. Stoffolano, E. Schauber, C.M. Yin, J.A. Tillman, G.J. Blomquist, J. Insect Physiol.
43 (1997) 1065.
[71] J.T. Lin, T.A. McKeon, A.E. Stafford, J. Chromatogr. A 699 (1995) 85.
[72] C. Schummer, O. Delhomme, B.M.R. Appenzeller, R. Wennig, M. Millet, Talanta
77 (2009) 1473.
[73] S. Hamilton, R.J. Hamilton, P.A. Sewell, in: R.J. Hamilton, S. Hamilton (Eds.),
Lipid Analysis. A Practical Approach, Oxford University Press, Oxford, 1992, pp.
13–64.
[74] A.K. Lough, L. Felikski, G.A. Garton, J. Lipid Res. (1962) 478.
[75] I. Brondz, D. Ekeberg, K. Høiland, D.S. Bell, A.R. Annino, J. Chromatogr. A 1148
(2007) 1.
78 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78

[76] H. Brüschweiler, A. Hautfenne, Pure Appl. Chem. 62 (1990) 781. [81] L.S. Basconcillo, B.E. McCarry, J. Chromatogr. B 871 (2008) 22.
[77] R.P. Evershed, in: R.J. Hamilton, S. Hamilton (Eds.), Lipid Analysis. A Prac- [82] R. Maile, F.R. Dani, G.R. Jones, E.D. Morgan, D. Ortius, J. Chromatogr. A 816 (1998)
tical Approach, Oxford University Press, Oxford, 1992, pp. 263–308, ISBN 169.
0199630984. ˛
[83] M. Gołebiowski, M.I. Boguś, M. Paszkiewicz, P. Stepnowski, Anal. Bioanal. Chem.
[78] M. Vincenti, G. Guglielmetti, G. Cassani, C. Tonini, Anal. Chem. 59 (1987) 1520. 39 (2011) 3177.
[79] O.D. Sparkman, Mass Spectrometry Desk Reference, Global View Publishing, [84] C. Blais, T. Blascob, A. Mariaa, C. Dauphin-Villemanta, R. Lafont, J. Chromatogr.
Pittsburgh, PA, 2000, 106 pp., ISBN 0-9660813-2-3. B 878 (2010) 925.
[80] R.A.W. Johnstone, M. Rose, Mass Spectrometry. Handbook for Chemists and
Biochemists, PWN, Warsaw, 2001.