Journal of Chromatography B, 881–882 (2012) 69–75 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb Profiling and characterization of volatile secretions from the European stink bug Graphosoma lineatum (Heteroptera: Pentatomidae) by two-dimensional gas chromatography/time-of-flight mass spectrometry Miloslav ˇ Sanda 1 , Petr ˇ Zᡠcek 1 , Ludvík Streinz, Martin Draˇ cínsk ´ y, Bohumír Koutek ∗ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic a r t i c l e i n f o Article history: Received 15 August 2011 Accepted 27 November 2011 Available online 6 December 2011 Keywords: Graphosoma lineatum Pentatomidae Volatile secretion GC × GC/TOF-MS Profiling (E)-4-oxohex-2-enal a b s t r a c t An efficient method combining the headspace solid-phase microextraction (HS-SPME) sampling pro- cedure and comprehensive two-dimensional gas-chromatography/time-of-flight mass spectrometry (GC × GC/TOF-MS) was established to study the volatile secretion components of stink bugs (Heteroptera: Pentatomidae). The combined power of this approach is illustrated by the identification of fifty-seven compounds in the secretion of a European stink-bug representative, Graphosoma lineatum. (E)-4-oxohex- 2-enal and (E)-dec-2-enal were found to be the major components in the adult bug secretions followed by lower amounts of n-alkenal (C 5 –C 12 ), n-alkenyl acetate (C 5 –C 11 ), n-alkane (C 11 –C 17 ) homologs, dien- als and other compounds. More than thirty known compounds have been identified that had not been described before in G. lineatum adults. Of these compounds, (E)-4-oxohex-2-enal is of particular interest, since its isolation and identification, while calling some previous reports into question, clearly demon- strates a potential ability of our approach to yield artifact-free secretion profiles. © 2011 Elsevier B.V. All rights reserved. 1. Introduction A wide variety of insect species have developed chemi- cal defense mechanisms that significantly contribute to their widespread success in ecosystems [1,2]. Typical representatives of such species are the pentatomid bugs (Heteroptera: Pentatomidae), commonly known as “stink bugs”, because in response to distur- bance or aggression they produce large quantities of foul-smelling odorous volatiles. The pentatomids are one of the four largest families of Heteroptera comprising approximately 4500 species worldwide [3]. Their volatile secretions released from exocrine glands, such as the metathoracic glands (MTG) in adults or dor- sal abdominal glands in nymphs [4,5], act mainly as defensive means against predators/parasitoids and/or as aggregation/alarm pheromones [1,6]. Since chemical defense mechanisms have a profound impact on the entire biology of an insect species (e.g. adaptations in morphology, physiology, niche use, behavior, etc.) [1], considerable research effort has been directed at isolating and characterizing the volatile secretion components in a variety of stink bugs [7–10]. However, the data are difficult to compare, mainly owing to the use of diverse experimental designs and ∗ Corresponding author. Tel.: +420 220183201; fax: +420 220183582. E-mail addresses: sanda@uochb.cas.cz (M. ˇ Sanda), zacek@uochb.cas.cz (P. ˇ Zᡠcek), streinz@uochb.cas.cz (L. Streinz), dracinsky@uochb.cas.cz (M. Draˇ cínsk ´ y), koutek@uochb.cas.cz (B. Koutek). 1 These authors contributed equally to this work. analytical methods, so that numerous questions concerning the role that most of the components may play in chemical com- munication systems within the Pentatomidae family still remain to be answered. It is often unclear which chemicals are defen- sive against predators, which elicit a dispersal behavior among conspecifics and whether those chemical identities change as the insects pass through different life stages. Apart from this, minor constituents have yet to receive the same level of attention as the major ones to provide more detailed chemical information about the systems. One of the stink bugs widely distributed in Europe is the stri- ated shield bug Graphosoma lineatum (Linneaus), a 1–1.2 cm long bug occurring on umbelliferous plants. The adults of G. lineatum are mostly recognized by their conspicuously red (epidermis) and black (melanized cuticle) striated coloration. Previous attempts to identify the compounds secreted by Graphosoma have involved a variety of analytical approaches including a solvent extraction of either whole insects or MTGs [7,8], trapping the volatile compounds in MeOH or using a solid-phase microextraction (SPME) followed by conventional (one-dimensional) gas chromatographic (GC) sep- aration combined with mass spectrometry (MS) [9,10]. While most of these studies [7,8,10] indicate that, besides hydrocarbons, the composition of G. lineatum secretions primarily includes saturated aldehydes and (E)-alk-2-enals with either (E)-dec-2-enal [7,8] or (E)-hex-2-enal [10] being the most abundant components, some investigators have found a predominance of (Z)-alkenals over the corresponding (E)-isomers and a relatively large amount of fura- nones [9,10]. 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.11.043