4.2.1. Extraction

Modern analytical equipment easily allows for the analysis of CHC from single specimens.

The most used method for extracting CHC is, by far, solvent extraction. This can be done using the following protocol.

  1. Transfer a single bee that has been anesthetised by chilling (see the section on Standard methods for immobilising, terminating, and storing adult Apis mellifera in the BEEBOOK miscellaneous paper (Human et al., 2013), into a small glass container with a convenient amount of an apolar solvent (e.g. 1 ml of hexane HPLC grade).
    Use clean forceps to avoid any possible contamination. Glass containers that are used need to be carefully washed, rinsed with hexane and kept overnight at high temperature before use; other materials should not be used to avoid contamination.
  2. Leave the specimens for extraction at room temperature.
    The duration of the extraction can vary and preliminary tests are advisable to determine the best duration according to the amount of compounds obtained. In general, 2-10 min can be regarded as a good compromise between extraction efficiency and the need to keep the working time reasonably short.
  3. Transfer the extract into a vial and store at -20 °C until use.
  4. In case an absolute quantification of CHC has to be carried out, add a convenient internal standard to the sample before extraction (see section above for more details on this subject).

Such extracts are normally suitable for analysis without further processing apart from solvent evaporation under a stream of nitrogen to concentrate the sample to 1 insect equivalent in 1 µl of hexane).

Other extraction methods can be used to sample CHC; in particular Nazzi et al. (2002 and 2004) used solid phase microextraction (SPME) to study short chain hydrocarbons (C8 – C10) released by honey bee pupae infested by the parasitic mite Varroa destructor; Nazzi et al. (2002) rinsed the gelatin capsules used for rearing the bees from larval stage to emergence, to sample the hydrocarbons released during pupation. The use of SPME for extracting CHC from living specimens is increasingly being used in other insects and details of this method are available (Bland et al., 2001). For more details on other extraction methods see section 2.2.2 above.