## 3 Volatile Pheromones
### Analytical Approaches to Unravel Pheromone Composition
#### 3.4.1 Conventional Chemical Ecology Approaches
Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOFMS) and desorption electrospray ionization (DESI) mass spectrometry are soft ionization techniques developed for the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules). However, they have been applied very sporadically for studies of the fruit fly pheromone components [276]. High performance liquid chromatography (HPLC) coupled to a MS detector is at present the favoured technique for the study of polar non-volatile tephritid semiochemicals. Other types of detectors that are commonly used with HPLC separation are UV and ELSD (evaporative light scattering) detectors [277; 278; 279; 280]. Normal phase and reversed phase HPLC separation are still popular in lipid analysis. The mobile phases usually consist of methanol, propan-2-ol, or _n_-hexane. Chromatographic columns with C18-phases still prevail. A few separations were reported on C8-columns. In a recent metabolomic study of _B. dorsalis_ larvae, liquid chromatography-mass spectrometry (LC-MS) and GC-MS were applied for the characterisation of endogenous metabolite changes and biochemical effects of azadirachtin [281]. NMR spectroscopy is one of the most informative, but least sensitive, modern spectrometric methods. Fourier transform \({}^{1}\)H and \({}^{13}\)C NMR spectroscopy require approximately 100 \(\upmu\)g and 10 mg pure material, respectively (50 \(\mathrm{ng}-1\)\(\upmu\)g with very expensive NMR-nanoprobe). Since many insect pheromones are only present in nanogram amounts, a very high number of individuals are required. NMR is widely used to determine the absolute configuration of optically active compounds (semiochemicals), either using chiral shift reagents, or by converting enantiomers to diastereoisomers prior to spectroscopic study, or using chiral solvent. The use of NMR for absolute identification of semiochemicals has two main disadvantages: isolation of the pure substance is required, and an enantioenriched or a homochiral synthetic standard is needed. In modern semiochemical research, NMR spectroscopy is normally used only when insufficient structural information is provided by more sensitive methods [282]. Baker and Heath [283] applied NMR spectroscopy for the identification of lactone pheromone components emitted by _A. suspensa_ and _A. ludens_. The chemical synthesis of tephritid pheromones is a key step towards producing pheromone compounds for use in bioassays (usually very low amounts), and for insect pest monitoring and management purposes. Moreover, their absolute configuration is an important determinant of their biological activities [284]. In 1973, Jacobson and colleagues isolated and synthetized the first medfly pheromone components, i.e., methyl (_E_)-non-6-enoate and (_E_)-non-6-en-1-ol that were attractive eliciting also sexually excitatory response to females in laboratory trials [215]. Three additional major components of the medfly pheromone, namely ethyl (_E_)-oct-3-enoate, geranyl acetate, and (_E_,_E_)-\(\alpha\)-farnesene were isolated and synthetized later [225]. In 2010, Olszewski and Grison [285] reported a novel and versatile synthetic approach to the preparation of (_E_)-non-6-en-1-ol. Since the first successful studies on the isolation and synthesis of the medfly pheromone components, several efforts have been made to characterise the volatiles emitted by _Bactrocera_ and _Rlagoletis_ species, mainly using the enantioselective approach. Moreover, in the case of chiral pheromones, which occur in tephritids, the synthesis and following bioassays using stereoisomers are essential for recognition of the stereochemistry-bioactivity relationships. This knowledge also has practical implications for pest control as in most chiral pheromones only one enantiomer is biologically active [284]. In the case of _B.