Solid phase microextraction was developed by Arthur and Pawliszyn in 1989 to analyse rapid sample preparation, for laboratory conditions and on-site arrangements, and to provide an efficient method towards integration of sample preparation with detection systems and separation (Arthur and Pawliszyn, 1990). In accordance with the principle of SPME, the technique employs a small volume of polymeric extracting phase coated on the outside of a metal alloy solid support or fused silica (Pawliszyn, 1997). The most important part of SPME is a solid fibre support coated with a thin layer of a polymeric stationary phase that is used to extract the analyses by concentrating them from the sample. The fibre is located inside the needle, which serves to protect the fibre coating from damage by injecting it into vial/injector septa penetrations (Pawliszyn, 1997). In general, SPME has been used routinely in combination with GC-FID and GC-MS. However, to analyse non-volatile chemical and thermally labile compounds not amenable to GC-MS, significant improvements were made in direct coupling of SPME with HPLC and LC-MS. Recently, SPME is widely applicable in both targeted and non-targeted quantitative and qualitative analyses of organic compounds from various gaseous, liquid, solid environmental, food and biological matrices.
The SPME principle relies on placing a thin polymeric coating coated on the outside of a fused- silica fibre directly towards the sample matrix. As soon as the coated fibre was placed in contact with the sample matrix, analyses partition by absorption from the sample matrix to the extracting phase (Pawliszyn, 1997). The amount of analysis extracted by the SPME coating does further increase with extraction time within the limits of error. This means sampling under equilibrium conditions provides the maximum sensitivity achievable with the SPME (Ouyang et al., 2011). However, SPME extraction can be interrupted if sufficient sensitivity is achieved for a particular application of interest. Once the extraction process is completed, concentrated
22
extracts are injected into the separation system either through the GC injection port, or by solvent desorption (Nerín et al., 2009).
The preferred extraction mode of SPME implementation in complex sample analysis is headspace solid phase microextraction (HS-SPME), where fibre coating is exposed to the headspace above the simple, complex aqueous sample or solid sample (Pawliszyn, 1997). The sensitivity limitation of HS-SPME when highly polar compounds and high molecular weight are concerned may be overcome by increasing the extraction temperature of samples (Lord and Pawliszyn, 2000). During direct immersion (DI-SPME), the fibre coating is exposed and completely immersed inside the sample matrix. Thus, this extraction mode is favourable for analyses having low-to-medium volatility and high-to-medium polarity (Pawliszyn, 1997). Given this, SPME technique features small sample amount requirements, automation capability and quantitative analytical results for gaseous, as well as the ability to produce high quality qualitative, aqueous and solid real-life samples that are highly complex. Therefore, this technique has been capable of overcoming several drawbacks encountered during the implementation of traditional sample preparation methods. The metabolomics studies conducted on the hyphenated SPME-GC-FID-GC-MS system will also be introduced to highlight the advantages of this multidimensional analytical approach in the analysis of volatile and non-volatiles complex sample characteriszation.
1.4.2 Application of HS-SPME
Although insect produced volatiles have been isolated by extraction of insect glands or from whole insects (Drijfhout et al., 2000), some volatiles are synthesiszed and released immediately from the insects rather than being stored in the body. The glands contain inhibitory substances that may also complicate purification and the isolation of the active chemicals by the instruments (Griepink et al., 2000). In contrast to this, volatile chemicals using the headspace technique from the air surrounding the insect can be collected (Figure 1.3).
This allows for gains to be made not only on the qualitative structure but also in the relative concentration of the compounds. The values of multicomponent volatiles are usually decipherable for biological activity. The headspaces analysis has been used widely in the collection of volatiles and pheromones from organisms and substrates (Blight, 1990). Two
23
approaches have been used in the collection of the volatiles. The first approach is a trap and purge approach, where the chemical fraction accumulates from the gaseous flow stream through the matrix onto a suitable trapping medium, and the second approach is taking the samples from the gaseous flow stream and passing them the matrix (Bicchi et al., 2008). The volatiles samples are recovered either by thermal desorption or by solvent elution to the GC-FID or GC-MS systems. The volatile collecting from insects should be conducted over a period of several days, depending on the numbers of samples and in order to obtain enough material for the volatiles analysis (Drijfhout et al., 2000). In addition to the abovementioned collection methods, SPME technique has been used successfully to eliminate solvents and to create a short extraction time (Lord and Pawliszyn, 2000; Kataoka et al., 2000).
Volatile organic compounds can be profiled and quantified using many different extraction methods, including microwave-assisted hydro distillation, solvents, and HS-SPME (Reddy and Guerrero, 2004). The latter, is considered the best technique for extracting volatiles from food products, plants, fruits, and insects (González‐Mas et al., 2009). Flamini et al. (2003) used solid- phase micro-extraction as a sampling technique in the determination of volatiles emitted by flowers, isolated flower parts, and pollen. Djozan et al. (2005) investigated different SPME fibres efficiently using pencil lead fibre from insect scent glands.
Cai et al. (2007) used SPME to determine the characteristic odorants from Harmonia axyridis beetles using in vivo solid-phase microextraction and multidimensional gas chromatography- mass spectrometry–olfactometry. Rochat et al. (2000) have used solid-phase microextraction in the identification of the highly volatile pheromones of two Rhinoceros beetles, Scapanes australis and Strategus aloeus (Coleoptera, Scarabaeidae, Dynastinae). Villaverde et al. (2007) used SPME to detect the volatile defensive capillary secretions of Tribolium castaneum (Herbst) i.e., gas chromatography (SPME-CGC). Stoppacher et al. (2010) identified and profiled the volatile metabolites of the biocontrol fungus Trichoderma atroviride using the HS- SPME-GC-MS technique. Furthermore, Alfaro et al. (2010) studied the effect of the sex, age and mating status of medfly C. capitata using SPME fibre. The SPME fibres consist of a fibre coated with an adsorbent that extracts volatile organic compounds from the gas-surrounding solid or liquid samples (Jones and Oldham, 1999).
24
Figure 1. 3. SPME device: (a) SPME fiber holder, (b) section of SPME holder and fiber assembly (de Koning et al., 2009)
1.5 Direct immersion Solid Phase Microextraction (DI-SPME)