## 2 SPME Techniques
### In-tube Solid-Phase Microextraction  
The more recent in-tube (IT) SPME was developed by [PERSON] and [PERSON] [45] for application with HPLC or liquid chromatography (LC)-MS because fiber SPME could barely withstand aggressive HPLC solvent conditions [6,45]. In-tube SPME overcomes fiber-related drawbacks such as fragility, low sorption capacity and bleeding from thick-film coatings. It uses an open tubular fused-silica capillary in which the extraction phase is either an inner surface coating or a sorbent bed. Consequently, in-tube SPME devices exhibit considerably higher mechanical stability than fiber SPME devices. Another reason for the development of in-tube SPME was the lack of automation in the use of SPME coupled to HPLC [2]. By means of automation it is possible to perform extraction, desorption and injection simultaneously [6,12]. The advantages of automated systems compared to manual techniques are shorter total analysis times as well as higher accuracy and precision [2]. A drawback of in-tube SPME is the tendency of the capillary to clog up. This can be avoided by working with samples without interfering phases like particles or macromolecules [2]. Moreover, the enrichment factor is reduced compared to fiber SPME [2]. [PERSON] [30] and [PERSON] [46] provided an overview of automated sample preparation using in-tube SPME and applications of this technique in environmental, clinical and food analysis, mainly for the determination of polar and thermolabile compounds. In-tube SPME can be categorized into methods using _extraction coatings_ where the coating is used as an internal extraction phase immobilized in the capillary wall and methods using _extraction fillings_ where the extraction phase is a sorbent packing, as demonstrated in Figure 1[47]. Figure 1: “Classical” solid-phase microextraction (SPME) fiber compared with in-tube fibers, adapted from [PERSON] _et al._ (2009) [2], reprinted with permission of Springer Verlag. The latter techniques are particularly similar to the previously developed solid-phase extraction (SPE) and use different sorbent types. Both in-tube SPME techniques can be easily applied with existing autosampler systems. Several in-tube devices with extraction fillings have been developed, firstly in 2004 by [PERSON] _et al._[48], and are now commercially available, for example as in-tube extraction (ITEX) [2]. There are two modes of extraction in in-tube SPME. In the static mode, the analytes are transferred to the stationary phase by mere diffusion, while in the dynamic mode, the sample is actively drawn through the tube by repeated draw/eject cycles [49]. When the equilibrium is virtually reached, the extracted analytes are desorbed from the capillary either by direct transfer to the liquid chromatographic column or by a stream of the mobile phase. In case of a stronger attachment of the analytes to the capillary, desorption is achieved by a static desorption solvent which is subsequently injected into the chromatographic system [50]. The desorbed analytes are carried through the column and separated and detected by a mass selective detector [9]. Even though in-tube SPME was originally developed for HPLC applications, it can also be used with other instrumental equipment, for example capillary electrophoresis (CE) or GC [30]. On-line coupling to GC can be achieved by open-tubular trapping (OTT), which is mainly applied to HS samples [30]. In OTT, desorption of the analytes is carried out with a small amount of solvent or by thermal desorption. However, OTT is characterized by a complex instrumental setup and unfavorable sampling conditions, such as high pressure drop from long traps and limited flow rates [2]. In-tube SPME techniques have been developed further and modifications like _wire-in-tube and fiber-in-tube techniques_ have emerged [51]. The wire-in-tube technique contains an additional stainless steel wire inserted into the extraction capillary.