## 2 Results and Discussion
### Possible Cause and Origin of Detected Volatiles
Tetradecanal levels are elevated in exhaled breath from patients with ventilator-associated pneumonia (VAP) in comparison to healthy individuals [36]. Benzaldehyde is a compound found in saliva, hair, and fingernails samples [37]; however, its origin can be exogenous, including gasoline vehicle emissions [38]. (E)-2-ocrenal and (E)-2-nonenal are expected to be biomarkers of inflammation and/or other carcinogenesis associated processes since they are identified in the headspace of human epithelial cervical carcinoma cells [39]. (E)-2-nonenal is also found as the cucumber odor in the cucumber/farinaceous subgroup of mushrooms with farinaceous odors [40]. In the case of \"halitosis\" (HA) group, the profiles were characterized by an increased number of volatile sulfur compounds (6 VOCs) and esters (11 VOCs). Predominantly, esters are fatty acid derivatives (released from bacteria), exhibiting a distinctly increased volatility compared to their parent fatty acids [26]. Among them, the most notable were methyl dodecanoate, isopropyl tetradecanoate, 2-ethylhexyl decanoate, and methyl octadecanoate. Volatile sulfur compounds are the most important biomarkers of halitosis detected in breath and saliva [1; 41; 42]. They are produced by bacteria located in the mouth, such as _P. gingivalis_, _Solobacterium moorei_, _P. intermedia_, _F. nucleatum_, and _T. danticola_[1; 7; 43]. Sulfur-containing amino-acids, such as cysteine, cysteine, and methionine, are the main substrates yielding VSCs found in saliva or gingival fluid. Three VSCs (hydrogen sulfide, methyl mercaptan, and dimethyl sulfide) are the most referred biomarkers of bad breath in literature [1; 3; 4]. In our work, we found 10 VSCs occurred in the samples of patients with halitosis. However, the distribution of VSCs among the three investigated groups is presented in Table 1.  
Three volatiles, dimethyl disulfide, dimethyl trisulfide, and dimethyl sulfone, were present in control (HE) and diseased groups (AB and HA). Exclusive sulfur-containing compounds for halitosis group were methyl thiolacetate, dimethyl pentasulfide, allyl thiocyanate, allyl isothiyanate, S-methyl pentanetthioate, and thiolan-2-one. These compounds could be potential disease biomarkers and were generated by bacteria incubated from saliva in \"limited\" (by closed seals of headspace vials) aerobic conditions that allow the growth of predominantly aerobic bacteria or even anaerobic species. Our work modified and improved the salivary incubation test with the incorporation of HS-SPME-GC/MS, instead of organoleptic examination and usage of portable sulfide monitor. The main advantages of the proposed method were high sensitivity and that it could detect a wider spectrum of VOCs at low concentration compared to organoleptic measurement and usage of chemical sensors and portable sulfide monitors. This technique was also highly objective, reproducible, and reliable. Moreover, incubation of saliva gives samples less influenced by food, smoking, and scented cosmetics compared to the gas chromatography technique used alone for the analysis of exhaled \"fresh\" breath. The disadvantages of the method were time-consuming analyses and the requirement of being carried out by a skilled operator. The costs of GC/MS apparatus could not also be neglected. On the other hand, these drawbacks could be diminished by the high sensitivity of measurements and potential capability to investigate and incorporate new indicators of other dysfunctions and diseases as targets. This methodology also allowed to assess several biomarkers in a single chromatographic run, enabling personalized diagnosis in a sole assay. Saliva collection protocols may also alter the chemical composition of saliva; hence, the important issue is to maintain high standards of sampling. The procedures of the collection of oral fluid involve non-stimulated (draining, spitting, suction, and adsorption into swab) and stimulated (with chemical or masticatory stimulus) techniques.