## 2 Exhaled Breath (EB) Analysis
### EB Analysis Experimental Layout
#### 2.1.1 EB Sampling  
EB sampling is one of the most important steps in breath analysis and there are a number of parameters that researchers should pay attention, in order to avoid wrongful assumptions about the origin of the compounds identified. These parameters include the type and the number of breath collections, the portion of breath used, the EB storage and the interference of environmental VOCs from the collection room (reviewed in [24,32,33]). Breath collection can be achieved through a single breath or  
Figure 1: Generic layout for exhaled breath (EB) analysis. Abbreviations used: GC—gas chromatography, MEPS—microextraction by packed sorbent, POC—point of care, PTR-MS—proton transfer reaction with mass spectrometry, PTR-TOF-MS—proton transfer reaction with time-of-flight mass spectrometry, SIFT-MS—Selected ion flow tube mass spectrometry, NTDs—Needle Trap Devices, MVA—multivariated analysis, PCA—Principal Component Analysis, PLS—Partial least-square, VOCs—volatile organic compounds  
multiple breaths; both show associated advantages, which should be taken into account before choosing one of the two. For screening of potential compounds associated with a given disease and determination of a specific set of biomarkers, multiple breath analysis is required in order to achieve the best results, since it is more reproducible in terms of composition of the sample (reviewed in [24]). However, a single breath tends to be less time consuming and thus more acceptable for patients. EB can also be sampled in its full composition (total breath sample or mixed expiratory air) or, alternatively, only the alveolar air may be sampled. The first choice is more prone to contaminations, since the sampling control is very deficient. The patient just breathes for the sample collection device and therefore the risk of contamination with exogenous compounds from the oral cavity and dilutions in dead space are higher and may compromise the analysis (reviewed in [24]). These problems are reflected in the variation of the number of compounds and their concentration, jeopardizing the analytical reproducibility. In contrast, the alveolar air is richer in volatile blood-borne compounds [34]. Therefore the use of an alveolar air sampling approach, which can easily be achieved using a simple Capnograph to monitor the expiratory CO\({}_{2}\) (Figure 2), is more accurate, assuring a more reliable sampling and consistent sample quality for within- and between-subject comparisons [34, 35]. It would, therefore, be highly desirable that standardized procedures could be adopted for generic EB analysis as it was done for measurement of exhaled lower respiratory nitric oxide (NO) and nasal NO. EB sampling can be performed directly or indirectly according to the type of analysis to be performed; we will see in more detail in the next section. In the off-line approach, EB has to be stored and concentrated before it is analysed. This procedure allows enrichment of the sample in the target analytes before its analysis [32]. EB storage can be made in the following ways [24, 36]. The first approaches used Tedlar(r) bags and canisters. The sampling bags are made from chemically inert materials as polyvinyl fluoride (PVF), perfluoroalkoxy polymer (PFA), polytetrafluoroethylene (PTFE) or  
Figure 2: Exhaled CO2 monitoring by normal Capnograph: (**A**)—initial of alveolar sampling; (**B**)—final of alveolar sampling; (**C**)—final of exhalation. Differentiation phases of breathing: (**I**)-inspiratory phase; (**II**)-mixing phase; (**III**)-alveolar phase (adapted from [34, 35]). polyvinylidene chloride (PVDC) [37]. These bags are impermeable to gas diffusion if covered with an aluminium foil [38] and their utilization is easy as they just need to be connected to a sample collection tube for EB collection. Tedlar(r) bags are reusable provided they are extensively purged with nitrogen flows to avoid problems of contamination and background VOCs [27, 32, 36, 37, 39], such as some phenols and \(N\), \(N\)-dimethylacetamide.