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: [email protected] (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 membranes. They are components of membrane phospholipids and
of insects, including lipids. Lipids are secreted by the exocrine precursors of eicosanoids (paracrine hormones: prostaglandins).
system to the surface of insect cuticle and may have biological activ- The biologically active compounds found on insect bodies can
ity. Chromatographic methods such as liquid chromatography (e.g. affect entomopathogenic fungi [23,24]. For example, tests were
LLSD detector) or gas chromatography (e.g. with MS detector) are conducted on Conidiobolus coronatus fungi by applying increasing
generally used to identify lipids. 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
2. Biological methods for controlling harmful insects acids were tested against bacterial (Bacillus cereus, Bacillus subtilis,
Enterococcus faecalis, Citrobacter freundii, Pseudomonas aeruginosa,
Lately biological methods, such as the use of bioinsecticides, Pseudomonas fluorescens) and fungal strains (Paecilomyces lilacinus,
are becoming more and more popular in the fight against pests Paecilomyces fumosoroseus, Lecanicillium lecanii, M. anisopliae, B.
[5]. Entomopathogenic fungi are parasitic organisms which tar- bassiana (Tve-N39), B. bassiana (Dv-1/07)). One of the four analyzed
get insects, causing lethal infections. These life forms are used mixtures of Forcipomyia nigra was significantly effective, especially
to regulate the population of harmful insects. So far there are toward B. cereus and E. faecalis. This mixture contained C14:0 , C16:1 ,
more than 400 known species of such nature, and 1800 defined C16:0 , C18:2 , C18:1 , and C18:0 fatty acids. However, The most effective
relationships between fungi and certain insect species [6]. Ento- acids against bacterial growth were C9:0 , C10:0 and C16:1 , whereas
mopathogenic fungi are natural pathogens of insects and they can C14:0 , C16:0 , C18:0 and C18:1 demonstrated rather poor antifungal
cause disease of many species, therefore they have great insec- activity and did not inhibit the growth of bacteria [19]. Cuticular
ticidal potential. Fungi belonging to the Entomophthorales order and internal lipids obtained from Calliphora vomitoria were tested
have high species specificity – some species are infected by the for their potential antimicrobial activity. The minimal inhibitory
fungi, while other are resistant to infection. Due to this trait these concentrations of extracts against reference strains of bacteria
organisms are considered a promising source of new generation (B. subtilis, Rhodococcus equi, Staphylococcus aureus, Escherichia
bioinsecticides. Fungi imperfecti (Deuteromycetes) are most com- coli, Klebsiella pneumoniae, Proteus vulgaris) and fungi (Aspergillus
monly used as insecticides. These are filamentous fungi, which niger, Candida albicans, Candida lipolytica, Candida tropicalis) were
include about 2000 taxa. Some of them are dangerous to humans determined. Antimicrobial activity was the strongest against Gram-
because of the mycotoxins, which in plants and animals can cause positive bacteria. All the lipid extracts obtained from larvae, pupae,
mycosis [7]. Attempts to use these fungi have already been taken in adult males and females were equally effective against all the fungal
the mid-nineteenth century [5]. In literature you can now find many strains examined [25].
examples of successful insect population reduction achieved by the
use of appropriate entomopathogenic fungi. For example, lethal
effects of Microsporidium Octosporea muscaedomesticae infection 3. The composition of the cuticular lipids of insects
were found among adult specimens of the Lucilia cuprina fly [8,9].
The most frequently used entomopathogenic fungi are: Metarhiz- Lipids (gr. Lipos – fat) are components of plant and animal orga-
ium anisopliae, Beauveria bassiana, and Nomurea rileyi [10]. Not only nisms and are characterized by good solubility in organic solvents,
fungi but also bacteria can act as parasites of insects. For example, such as ether. They are hardly soluble in water. This is what dis-
Serratia sp. has a lethal effect on the population of Lucilia sericata tinguishes the lipid solubility of the remaining three groups of
flies [11]. However there are obstructions preventing the infection biological substances: carbohydrates, proteins or nucleic acids. In
of insects by entomopathogenic fungi, which are related to both: the last 50 years have seen a rapid growth of research on lipids. The
the morphological barrier – cuticle, and the bacterial flora existing reason for this development was to realize the important role they
on it. The cuticle is an exoskeleton covering the bodies of insects, play in many aspects of chemistry. The modernization of analytical
and it is constructed mainly of chitin fibers embedded in a matrix of techniques has also become a stimulus for further research into the
proteins. Its structure is often additionally impregnated with phe- biological and technological functions of lipid [26,27]. The surfaces
nols, lipids, calcium carbonate and metal ions such as Zn, Mn, and of all higher plants and animals are covered with a layer of cuticular
Fe [12]. The cuticle is formed by several layers and each layer has lipids, which usually consist of recurring long-chain aliphatic com-
different chemical structure and properties [13]. In order to use pounds. Their role is primarily to protect against excessive loss of
entomopathogenic fungi as insecticides, one must first consider the body water and deter predators. Surface waxes of plants are used
mechanism of infection. It is important to penetrate both: mechan- to produce essential oils, perfumes, and medicines, if they have the
ical barriers – such as the cuticle, and various kinds of biologically appropriate properties [28]. In the case of insect lipids they form
active compounds. Chemicals, such as hydrocarbons [14], waxes not only the surface protective layer, but also are responsible for
[15], alcohols [16], and free fatty acids [17], are present in the outer the interaction between species. Individuals of different sexes on
and inner layers of the cuticle, what is more some of them, such as the basis of the composition of surface lipids evaluate each other
free fatty acids play very important role in the defense of insects. as potential partners for breeding. In addition, surface waxes pro-
These compounds may be present in different amounts depend- tects against various insect pathogens, such as fungi [17,28]. The
ing on the species of insects, living environment, the availability compounds on the cuticles are mainly fatty acids, hydrocarbons,
of food, seasons, etc. Their main function is to minimize transpi- aldehydes, alcohols, and waxes (Table 1). The general formulas of
ration, though some of them also perform as pheromones. It was lipids found in insects are presented in Table 2.
also found that the lipids of insects possess antifungal properties The fatty acids were identified in the cuticular lipids of Apis mel-
[17–20]. For example, free fatty acids present in insect lipids are lifera. They contained from 16 to 36 carbon atoms in the alkyl chain
biologically active compounds. Certain short-chain fatty acids can and included saturated entities only [36]. The major fatty acids in
block the absorption of phosphorus and thiamine by fungi, thereby the cuticular lipids were tetracosanoic (29%), hexacosanoic (12%)
preventing the development of mold spores – for example caprylic and octacosanois (11%) acids. Smaller amounts (<10%) of the fatty
acid is such an inhibitor. It has been found that cis-unsaturated acids in the range C30:0 to C36:0 and C16:0 and C18:0 were present.
fatty acids possess strong inhibitory properties [21,22]. Saturated Hydrocarbons are present on the surface of insects very
(SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty often [37–39]. Based on the analysis of lipids of several fly
acids, together with glycerol all contribute to the structure of cell 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 cholesterol. Cholesterol is a precursor of insect hormones. This
crassipalpis, it was concluded that hydrocarbon profiles are char- fact is confirmed by studies which focus on the effects of sterols
acteristic for each species [40]. The percentage concentrations in the development of insects. Experiments were conducted on
of a few groups of chemical compounds in insect cuticle are: Coleomegilla maculata to verify whether and to what extent, given
16.85–52.31% for monoethyl-alkanes, 1.13–4.78% for diethyl- food (corn pollen containing different amounts of sterols: ␤-
alkanes, and 2.07–3.76% for n-alkanes. Some compounds are absent sitosterol, cholesterol, ergosterol) will have an impact on the
only in specific species, for example pentacosane is not found in A. condition of the insects [43]. The results showed a strong corre-
grahami lipids – while it was present in all other species of flies lation between the amount of administered sterols and an increase
listed above. in insect population. The correlation also occurred between, pro-
Waxes are lipid components of many species of insects. For portionally decreasing the content of sterols in the insect diet over
instance a 52-carbon wax constitutes 82% of Drosicha corpulenta time, and the growth of C. maculata exuviae [43]. In addition to
cuticle lipids [41]. A high wax content was also found in Helicov- cholesterol in insect lipids there are also other sterols (stigmasterol
erpa zea (38% of lipids) – the composition of lipids of this insect are and sitosterol). Stigmasterol was found in the lipids of Melanoplus
as follows: hydrocarbons (19%), alcohols (17%), diols (16%), alde- species, whereas both mentioned sterols were found in the lipids of
hydes (2–5%), and esters of alcohols/oxo alcohols [32]. Analysis of Solenopsis invicta and Solenopsis richterii [44]. The number of sterol
the lipid composition of Aleurodicus dugesii showed that they con- esters in the lipids of insects is usually about half of the number of
sist of a mixture of waxes containing from 30 to 60 carbon atoms, sterols. Approximately 3% of the sterol esters were identified in the
wherein the most abundant wax (64%) was a 44-carbon compound lipids of Ceutorhynhus assimilis [45].
[15]. In the case of A. dugesii waxes dominated the exuviae extracts, The presence of methyl and ethyl esters of carboxylic acids
but the nymph lipid extracts contained primarily aldehydes. was found in the lipids of Schistocera gregaria locusts. A
Alcohols represent another group of biologically active com- chloroform extract contained 9,12-methyloctadecadienoate, and
pounds noticeable on the surface of the cuticle. The properties methyloctadecanoate methyl esters. Five esters were identified
of these compounds originate from the presence of the hydroxyl in a methanolic extract: ethyldecanoate, methylpentadecanoate,
group and the alkyl chain length. The solubility of alcohol in water 9-methylheptadecanoate, ethyl-2-hydroxy-9-octadecenoate, and
decreases with the elongation of the alkyl chain, therefore alco- 3,7-dimethyl-2,6-octadecadienoate [46].
hols also co-create the protective barrier which is the cuticle. Free Long-chain aldehydes are rarely components of the insect
alcohols found in insect lipids are usually saturated, primary, with cuticle. They are identified as constituents of mixtures of free
an even number of carbon atoms. For example in the lipids of Dia- long-chain alcohols (22–34 carbon atoms). Aldehydes such as tri-
pheromera femorata the most abundant are primary alcohols – C26:0 conatal (48.8%) and octacosanal were identified in the lipids of
and C28:0 . C23:0 , and C24:0 alcohols were also found, but in lesser Acyrthosiphon pisum [47]. The most abundant aldehyde found in
quantities [42]. In addition there are some interesting observations the lipids of A. singularis (whitefly) adult insects was composed of
concerning the alcohol content in lipids of Aleyrodes singularis adult 32 carbon atoms [30]. However, aldehydes found in the exuviae of
insects and their exuviae. The adult specimens predominant lipid those insects are: C26 , C28 , C30 and C32 .
alcohol is C32:0 , whereas the exuviae specimens main alcohol is Depending on the stage of development of the insect, the lipid
C26:0 [30]. composition is slightly different and it serves different purposes.
Sterols and their esters are present in insect lipids in small Larvae and pupae cuticle functions only as a temporary cover of
quantities. These substances are synthesized by insects from the body. During the shedding up to 90% of the cuticle is degraded
phytosterols (i.a. ␤-sitosterol, stigmasterol, campesterol, and bras- by enzymes. Larvae lead a very active lifestyle, which involves
sicasterol). Sterols constitute a large share of hormones, which mainly eating and rapid weight gain. Their body cover is not fully
regulate body functions. The most common insect sterol lipid is 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
Table 2 as the need to change the cuticle to enable penetration of more dry
The most common components of insects lipids [32,35].
habitats, in cases when the environmental resources are too poor.
Component Formula The pupae are on the other hand very idle – the insects do not feed
n-Alkanes H3 C[CH2 ]nCH3 and have a rather limited ability to move. In this stage of the insect
Ketones R1 COR2 life a transformation takes place involving the reconstruction of
II-row alkohols R1 CH(OH)R2 the body into an adult insect body (imago). Adult insects are fully
␤-Diketones R1 COCH2 COR2 developed forms, where the age, sex and state of growth have great
Esters R1 COOR2
impact on the lipid profile of an insect–which in turn defines their
I-row alkohols RCH2 OH
Aldehydes RCHO resistance to infections of entomopathogenic fungi.
Carboxylic acids RCOOH The first step in the analysis of insect lipids is extraction. After-
Dicarboxylic acids HOOC[CH2 ]nCOOH wards a group analysis is performed in order to separate the
␻-Hydroxylic acids HOCH2 [CH2 ]nCOOH
mixture into groups of compounds, which in the final step are sub-
R1 and R2 – chains containing from 10 to 20 carbon atoms (or longer). 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
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78 71

Table 3 (Continued)

Species of insect/analytes Extraction method Analytical techniques Ref.

Cockroaches: Periplaneta Dichloromethane GC-FID and GC–MS [50]

brunnea, P. fuliginosa, P. (2 min) Column: CP Sil5-CB (0.25 mm × 25 m, 0.25 ␮m) and CP Sil8-CBMS
australiasiae, P. (0.25 mm × 25 m, 0.25 ␮m)
americana Conditions: column temp. 100–320 ◦ C
Analytes: hydrocarbons Ramp rate: 20 ◦ C/min to 210 ◦ C, 4 ◦ C/min to 320 ◦ C

Calliphoridae Hexane GC-FID [51]

Analytes: hydrocarbons 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

Flies: Aldrichina grahami, Hexane (15 min) GC-FID and GC–MS [40]
Chrysomya megacephala, Column: 30-m fused film silica capillary column (0.25 mm × 30 m, 0.25 ␮m)
Lucilia sericata, Conditions: column temp. 50–270 ◦ C
Achoetandrus rufifacies, Ramp rate: 25 ◦ C/min to 200 ◦ C, 3 ◦ C/min to 270 ◦ C
Boettcherisca peregrina, Final temperature hold: 20 min
Parasarcophaga
crassipalpis
Analytes: hydrocarbons
(n-alkanes, mono-methyl
alkanes, di-methyl
alkanes, n-alkenes)

Bagrada hilaris – SPME-GC–MS [52]

Analytes: hydrocarbons 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

Anopheles gambiae, Hexane (5 min) GC-FID and GC–MS [53]

Anopheles arabiensis Column: DB-1 0.32 mm × 15 m, 0.1 ␮m
Analytes: hydrocarbons 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

Acanthoscelides obtecus Petroleum ether HPLC-LLSD [54]

(Say) (10 s) (bp Column: Econosil silica (250 mm × 4.6 mm, 5 ␮m)
Analytes: hydrocarbons, 38–58 ◦ C), Phase: n-hexane and dichloromethane with the addition of acetone (15%)
free fatty acids, methyl dichloromethane
and ethyl esters of (5 min)
carboxylic acids,
aldehydes,
triacylglycerols, alcohols,
sterols

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

Neobellieria bullata, Dichloromethane MALDI-TOF [55]

Drosophila melanogaster, (1 min) The acceleration voltage was 20 kV and ion extraction pulse was 200 ns.
Arabidophis thaliana Desorption and ionization were achieved using a nitrogen UV laser (337.1 nm,
Analytes: waxes, 4 ns pulse of 300 J, maximum frequency 20 Hz). Matrix ions were suppressed
hydrocarbons below m/z 300. Spectra were averaged from 800 (WEs) or 1200 (HCs) laser
shots

Bumble-bees of the Chloroform/methanol HPLC/APCI-MS [56]

Bombus genus (1:1, v/v), TLC Detector: APCI-MS
Analytes: (hexane/diethyl Phase: acetonitrile/2-propanol
triacylglycerols 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- 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
lites [48]. For these procedures hexane and 2-propanol were used
at (A) 206 nm and (B) 233 nm and (C) laser light-scattering detection. Peaks: 1:
as the mobile phase (from 99.6% to 79.3% of hexane, flow rate cholestane; 2: cholesterol; 3: 20-hydroxycholesterol; 4: 25-hydroxycholesterol; 5:
1 ml/min for 56 min). The elution conditions were identical for ␣-epoxide; 6: ␤-epoxide; 7: 7-ketocholesterol; 8: 7␤-hydroxycholesterol; 9: 7␣-
both detectors, nitrogen was used as the nebulization gas (LLSD). hydroxycholesterol; 10: cholestanetriol.
More analytes were identified using the LLSD detector in com- Reprinted from [48] with the permission of Elsevier B.V. All rights reserved.
parison with the UV-DAD. Much more stable and better baseline
was achieved with LLSD detector than using UV detector (Fig. 2).
sample. Analysis of the obtained chromatogram consists typically
As a result of HPLC analysis using both detectors the following
in determine the retention time of the individual compounds and
compounds have been identified: cholesterol, cholestan, 7-
establishing area of signals. With such a universal method for the
ketocholesterol, 7␣-hydroxycholesterol, 7␤-hydroxycholesterol,
quantitative analysis which is method of the internal standard, it
20-hydroxycholesterol and 25-hydroxycholesterol [48] (Table 3).
is possible to compare qualitative analytical results obtained from
two extraction methods from the same individuals. The results are
6. Gas chromatography expressed as mean ± standard deviation of three analyses.
The use of gas chromatography techniques often requires trans-
Chromatographic methods, such as most of the instrumental forming analytes into derivatives. The most popular reagents used
techniques are the techniques of the comparative solutions requir- for this purpose are: N,O-bis(trimethylosilyl)trifluoroacetamide
ing calibration patterns. Analysis is done by comparison of the (BSTFA), and trimethylchlorosilane (TMCS). Silylation is mainly
area or height of the analyte signal with the area or the height of used in the case of: alcohols, alkaloids, amines, carboxylic acids,
the signal of standard. The quantitative analysis is based on the phenols and steroids, though there are other compounds suscep-
proportion of the signal field and/or the height of the analyte signal tible to this process [72]. The reaction mechanism is based on a
to the amount (mass or concentration) in the sample. Measured nucleophilic attack (SN2) resulting in the substitution of a hydro-
parameter: the area or height of the signal is a function of the gen atom in certain groups: OH, SH, NH, COOH, NOH, SOH,
analyte in the mixture. The most common method of qualitative POH, CONH2 and NH2 . For example, the trimethylsilyl (TMSi)
analysis is a method of the internal standard. It involves the addi- ethers of alcohols or fatty acids are obtained by the addition of
tion of an appropriate amount of standard, which is well separated 100 ␮l of a mixture of 85% bis(trimethylsilyl)acetamide and 15%
from all the constituents of the sample in current conditions chlorotrimethylsilane (TMCS) (TMCS is used as catalyst) to 1 mg
of analysis; however, physico-chemical properties do not differ of lipid extract. Obtained mixture is kept at 100 ◦ C for 1 h [73].
from the analyte. Standard cannot be previously present in the The fatty acids can be also analyzed as fatty acid methyl esters.
 M. Cerkowniak et al. / J. Chromatogr. B 937 (2013) 67–78 75

For example, the fatty acids can be methylated by the addition 7. Combined techniques
of diazomethane. The N-methyl-N-nitroso-p-toluenesulfonamide
is diluted in diethyl ether and then KOH, which is dissolved in 10 ml Gas chromatography does not always allow an unambiguous
ethanol, is added. After 5 min, the ether solution of diazomethane identification of analytes, making it necessary to use coupled tech-
is evaporated over a water bath. Then fatty acids are dissolved in niques. Also mass spectrometry often gives no definite answer to
a small amount of anhydrous diethyl ether to which small por- the structure of the tested compounds and, therefore, is usually
tions of the diazomethane solution are subsequently added [73]. used in combination with other techniques such as chromatogra-
The free fatty acids can be also methylated by the addition of 14% phy. Gas chromatography–mass spectrometry (GC–MS) and liquid
BF3 in methanol for 1 h at 80 ◦ C in sealed ampoules [73]. The result- chromatography–mass spectrometry (LC–MS) represent coupled
ing methyl esters are ideal derivatives for the characterization of techniques, which are a definite help in qualitative and quantitative
carboxylic acids. They are easily described by GC and readily iden- analysis.
tified by interpretation of mass spectra, but the methyl esters can Lipids analysis often poses major problems due to their very
readily arise as artifacts during the extraction of lipids [74]. The large composition. Using the retention time as an independent vari-
use of methanol as an extraction solvent for lipids may cause arti- able is not practical because of the minor shifts and partial peak
ficial methyl esters of fatty acids in certain lipids in the presence of coelution. Such a mixture contains a number of analytes of different
some organic or inorganic materials [75]. Wax esters and triacyl- groups of compounds with different physicochemical properties.
glycerols are often hydrolyzed and then the hydrolysis products are Combined techniques are great practical application in order to
silylized. Compounds are hydrolyzed by use of a solution of KOH in identify lipids qualitatively and quantitatively. Detector MS/MS
99.8% ethanol (0.5 mol dm−3 , 3 h at 70 ◦ C) [76]. After evaporation of may find use to the analysis of especially strongly complex matri-
the ethanol under a stream of nitrogen the hydrolysis products are ces.
silylated as described above. Mass spectrometry is a physical technique, which is based
The presence of numerous isomers (positional and geometric) on measuring the mass to charge ratios of charged molecules or
in insect lipids makes qualitative analysis difficult. The reten- molecule fragments, created by ionizing chemical compounds [79].
tion times and the mass spectra of these compounds are usually Currently, this technique is often used in chemistry, biochemistry,
very similar. In this case, equivalent chain lengths (ECL) for medicine and biology, especially for the identification of chemi-
identification of fatty acid derivatives are applied. The ECL val- cal compounds and their mixtures, for determining the structure
ues increase with increasing distance of the double bond from of chemical compounds and determining their elemental compo-
the carboxyl group. Mass spectra both of positional isomers sition [80]. The basis of the detection by mass spectrometry in the
are identical, so identification by GC/MS these isomers requires formation of ions from neutral compounds and then testing the
transforming analytes into derivatives. Additionally, compounds compounds produced after degradation. The result of this type of
with increasing alkyl-chain branching elutes before a linear sat- detection is a mass spectrum, which provides information about the
urated compounds and retention time of the E-isomer is shorter resulting fragment ions, isotopic ions and, where appropriate, the
than of the Z-isomer on a polar column. For the GC–MS loca- molecular ion. In GC/MS two potential methods exist for ionization.
tion of double bonds, the methyl esters or alkenes are reacted The choice of ionization method depends on the nature of the sam-
with dimethyl disulphide (DMDS) [77]. A small sample is dis- ple and the type of information required from the analysis. Electron
solved in 50 ␮l dimethyl disulphide, and 50 ␮l carbon disulphide ionization (EI) is widely used in mass spectrometry for analysis of
is added with 300 ␮g iodine, the mixture is kept at 60 ◦ C for 40 h relatively volatile compounds. EI is known as a “hard” ionization
in a sealed vial. Next, the aqueous Na2 S2 O3 is added and the method, so it is very good for producing fragment ions which are
organic phase is extracted and evaporated in a stream of nitrogen useful for identification of compounds, but the molecular ion does
[78]. not appear or is a smaller peak in the spectrum. For example, the
Insect lipids are examined primarily by gas chromatogra- characteristics fragment ions of methyl esters are at m/z 74 and
phy, in particular GC-FID since the flame ionization detector 87 and characteristics ions of fatty acids are at m/z 60 and 73, and
is used for the analysis of most organic compounds. The uni- molecular ions in these mass spectra have small intensity. Chem-
versality of this technique is confirmed by numerous examples ical ionization (CI) is applied to similar samples, but this method
mentioned in Table 3. For the analysis of cuticle lipids of of ionization is used to enhance the abundance of the molecular
O. lignaria and M. rotundata, GC-FID was used with capil- ion, which represented the molecular weight of the compound. For
lary columns: ATTM-1HT (0.25 mm × 15 m × 0.1 ␮m) and DB-1MS both ionization modes, the molecular weight range is 40–800 Da.
(0.2 mm × 12.5 m × 0.33 ␮m). As a result of these examinations The use of GC/MS often requires transforming analytes into
the percent composition of lipids (containing mainly long-chain derivatives. In case of the analysis of fatty acids, which have low
alkanes, alkenes, and waxes) was determined as follows: 95.0% of volatility, it is necessary to transform them into methyl derivatives
lipids were hydrocarbons and 4.6% were esters [57]. The analysis first [81]. For example long chain fatty acids derived from insect
of hydrocarbons of Anopheles gambiae and Anopheles arabien- exocrine glands were subjected to GC–MS analysis [82]. In order to
sis (the most common malaria-carrying mosquito species) was optimize the method some aspects of the process were revised –
also performed applying GC-FID with a capillary column DB-1MS such as injector temperature, different ways of dosing the sample,
(0.32 mm × 15 m × 0.1 ␮m) [53]. This technique was useful in iden- and the effect of using solid phase microextraction (SPME) on the
tifying the differences in hydrocarbon profiles of different species obtained quantitative results. An inappropriate selection of param-
of mosquitoes (M- and S-forms), caused by allopatric speciation. eters resulted in low detection of fatty acids. Another example of the
Hydrocarbon profiles of mosquitoes from three different loca- use of GC–MS is the analysis of locust cuticle lipids. The compounds
tions in Burkina Faso (villages Gountry and Baloungen – separated identified were: hydrocarbons, long chain fatty acids, methyl and
from each other by about 80 km, located near the capital city of ethyl esters. Prior to GC–MS analysis the methanolic extract was
Ougadougou; a small sample was collected at Bama-VK7, located subjected to derivatization (to transform the fatty acids into methyl
at the periphery of the rice cultivation area of Vallee du Kou, derivatives) [46]. Currently GC–MS is the most common separation
about 400 km SW from the former villages). Intra-taxonomic differ- technique for the analysis of insect lipids [14,40,53,57,83]. Exam-
ences, in the concentrations of the same hydrocarbons, were found ples of the application of GC–MS for the analysis of insect lipids
not only between the allopatric taxa but also in both sympatric are included in Table 3. Exemplary total ion current (TIC) of lipids
ones. 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 77

cause significant changes in the spectra of product ions, allowing ˛

[28] B. Szafranek, M. Paszkiewicz, M. Gołebiowski, P. Stepnowski, Gas chromato-
the identification of the end groups [80]. graphic analysis of plant and insect surface compounds: cuticular waxes and
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