## 2 Exhaled Breath (EB) Analysis
### EB Analysis Experimental Layout
#### 2.1.2 Pre-Concentration  
As already mentioned, the volatile composition of interest in EB represents less than 1% of its composition and can range from umol L\({}^{-1}\) to fmol L\({}^{-1}\)[25, 35]. Therefore, small interfering compounds could affect the analytical results. To minimize this effect, an intermediate step between sampling and analysis is sometimes necessary and advantageous to increase the concentration level of the target analytes over the possible interfering compounds. There are several enrichment techniques available, such as cryogenic trapping, usually used with the canister breath sampling, and adsorption in different thermal desorption tubes (TD-tubes), sorbent traps and coated fibres. The adsorption option requires a thermodesorption step, being usually followed by gas chromatography combined with mass spectrometry (GC/MS). A wide range of TD-tubes, also known as sorbent tubes, are available with different _strength_ abilities to retain VOCs, working temperatures and hydrophobicity (reviewed in [52, 53]). Therefore, applications of TD-tubes are also very broad and include, among others, environmental control [54], natural products characterisation [55], and breath research for disease diagnosis (as reported in [43, 56, 57, 58]) (Table 1). Among the TD sorbents, Tenax tubes are quite popular for breath analysis as they allow a good performance in terms of pre-concentration and transport of breath samples before processing [43, 56, 57, 58, 59]). The multibed versions of TD-tubes, in which different sorbents with increasing strength are packed sequentially, are particularly suitable for breath analysis given the broad range in volatility of the VOCs present in EB samples [52]. Among the EB sample concentration methods, Solid-Phase Microextraction (SPME) is the most popular adsorption methodology [32, 35, 45, 60, 61]. This technique, particularly its headspace variant (HS-SPME), in which the extraction of the analytes belonging to solid or liquid sample is taken from the headspace, has gained tremendous importance with regard to the EB sampling. Based on the equilibrium partitioning of the analyte in the sample matrix and stationary phase, the HS-SPME extraction/concentration is more effective from the moment the analyte reaches the equilibrium concentration in the fibre, and its concentration in the extraction fibre remains constant [62; 63; 64]. The HS-SPME device is composed of a coated, silica fibre, with a thin layer (5-100 nm) of a suitable polymeric adsorbent. Many types of adsorbent are available in the market, for example, PDMS, CAR, DVB, combinations of these, and others. The selection of the fibre type and its thickness is usually done according to the polarity and molecular weight of the VOCs. In EB analysis, the CAR/PDMS stationary phases have shown great performance, however, the search for improved materials is still ongoing [62; 63; 64; 65]. Additional factors affecting SPME performance, such as temperature and time of extraction, sample pH and ionic strength, are very important and must be optimized to obtain the best results. A more detailed overview about this experimental optimization can be found elsewhere (reviewed in [4; 66; 67; 68]). An emerging and promising alternative that combines the EB sampling and pre-concentration steps in a single device is the Needle Trap Device (NTD) [69; 70; 71; 72]. The NTD is a trap device composed of a needle containing a sorbent material packed inside. The sorbent constitution is variable and includes Carboxen (CAR), Divinylbenzene (DVB), Polydimethylsiloxane (PDMS), mixtures of these, and other sorbents (similar to the composition of SPME and TD-tubes). In the NTD, the sample can be actively drawn in and out by diffusion, gas-tight syringe or automated devices, such as vacuum pumps. The sensitivity that can be achieved using NTDs is comparable to SPME. Unlike SPME, NTD is an exhaustive methodology, allowing an increase in the concentration of several compounds by using more sample volume.