## 5 Cuticular Hydrocarbons
### Analytical Approaches to Trace CH Profiles  
Preparation of samples for gas chromatographic analysis of tephritid CHs is usually made by solvent extraction of whole insect bodies with solvents such as pentane, hexane, dichloromethane, and chloroform [90; 427]. The most widely used method for recovering tephritid CHs is hexane washing [274; 421; 428]. This process may contaminate the cuticular sample with other materials, such as those from the exocrine glands. To collect only the CH fraction of the extract, the hexane solution is placed on a short chromatographic column and the CHs eluted with a small volume of hexane. For pre-cleaning of the CHs, thin layer chromatography (TLC) has proven useful in identifying novel lipid pheromones. TLC plates consist of glass or aluminium coated with an adsorbent layer of silica gel. Using TLC, components of the chemical extract can be separated into discrete fractions according to hydrophobicity [429; 430]. After the hydrocarbon fraction has been collected, the solution must be concentrated to a suitable volume before the analysis. For the determination of the final concentration of the sample, the number of insects extracted must also be known. Another reported method involves continuous extraction of the insect in a refluxing solvent [431]. This procedure also requires column chromatography and re-concentration of solutes. Solvent extraction procedure involves several steps, in which more volatile compounds may be lost during the process, and moreover the use of high-purity solvents is required. Besides, the extraction and sample preparation account for most of the analysis time [428]. The solvent-free SPME technique has been used for the analysis of cuticular components of _Trupane vicina_ (Wulp), an Asteraceae-feeding tephritid [432]. SPME polydimethylsiloxane coated fibre (PDMS) was used to wipe samples from various body parts of male _T. vicina_ and the subsequent GC-MS analyses showed that 1-nonanol, the male-specific compound, was concentrated on the abdomens of males exhibiting pleural distension [432]. In _A. ludens_, a PDMS fibre was used to rub down the male wing CHs, which were subsequently analysed by GC-MS [415]. Recently, the direct immersion-SPME (DI-SPME) coupled with GC-MS analyses was employed for characterisation of _C. capitata_ semiochemicals in three different mating stages. This study demonstrated that medfly compound compositions were not significantly different before and during mating. However, new chemical compounds were generated after mating, such as (_Z_)-tricos-9-ene and hentriacontane among ten other components [260]. Female medflies seem to discriminate mated from virgin conspecifics and express higher rates of aggressive behaviour against the later [433]. Considering the recent findings, it is plausible to argue that changes in CHs of mated females drive the differential aggressive interactions between virgin and mated female medflies. The method of choice for the analysis of the CH profile in tephritids has been GC-MS or GC\(\times\)GC-MS using two different ionization techniques: electron (EI) and chemical ionization (CI) [93; 289; 427; 434; 435]. EI-MS is the primary tool for assessing the location of methyl branch points in long-chain alkanes, but it is often difficult to identify the molecular ion. The EI mass spectra interpretation allows complete identification of a compound, but often microscale reactions or derivativzations are necessary to provide additional structural information [436]. A mass spectrum also provides a 'finger print' of a compound, which can be compared with the libraries and mass spectra registries such as the NIST library, the Wiley/NBS registry of mass spectral data, and the published retention indices [437; 438]. Mass spectra of an CI-MS yields the (M-1)\({}^{+}\) ion as an intense peak (sometimes as the only peak), but when the peak consists of mixture of isomeric methyl or dimethyl alkanes, placement of methyl group position becomes more difficult [435].