## 1 Introduction  
Colorectal cancer (CRC) is a leading cause of cancer-related death [1; 2; 3; 4] and the third most prevalent malignancy worldwide [5]. Only an early diagnosis can increase the 5-year survival rate up to 90% [4].  
The volatilome [6] is represented by the volatile fraction of metabolome generated within the human organism and reflects the metabolic processes in the body, which may change in presence of cancer [7; 8; 9]. Wherever cancer is in the body, the volatile organic compounds (VOCs) released by the tumor tissue into the bloodstream are subsequently eliminated through the lung alveoli as components of exhaled air [10; 11], making breath analysis an attractive, promising and non-invasive means for the diagnosis and follow-up of the disease [12].  
Several investigations have confirmed that different patterns of VOCs are exhaled by patients affected by different tumors [13; 14; 15; 16] and our previous studies pointed out that selected VOCs could discriminate CRC patients from normal individuals [16; 17]. Specifically, three VOCs (benzaldehyde, ethylbenzene and tetradecane) were found to be useful to detect differences among patterns of substances secreted by cancerogenic tissues compared to healthy mucosa, in the same individual. If reflected in the pattern of exhaled VOCs of the patients, those differences could lead to a simple, non-invasive, unexpensive method for early diagnosis of the disease based on the direct breath analysis [16].  
Solid phase microextraction (SPME) is a simple, sensitive and cost-effective [18; 19; 20; 21; 22; 23; 24; 25] technique for the extraction of volatile and semi-volatile compounds by means of a fiber coated with a proper polymeric phase. The extraction can be performed in the headspace (HS-SPME) of solid/liquid matrixes, exposing to the gas phase the polymeric film that adsorbs the volatiles, or by direct immersion (DI-SPME) of the fiber in a small volume of aqueous samples [20; 21]. As the SPME mechanism is based on the equilibrium of analytes among different phases, the extraction of the compounds is greatly influenced by the vapor pressure in the vial, which depends on extraction temperature, equilibrium and extraction time [22]. Therefore, these parameters must be carefully optimized [22; 23; 24; 25], together with other important variables, including ionic strength, pH, sample volume, uniformity and stirring speed [22; 23; 24; 25]. Recently, our research group successfully applied HS-SPME to find correlation among the VOCs released by the surgical resected tissue of patients affected by CRC and the VOCs contained in their exhaled breath, collected in sorbent tubes and desorbed with a thermal desorber [17]. This study was then aimed to optimize and compare two different extraction protocols, based on HS- and DI-SPME, respectively, for the extraction of seven target VOCs secreted by surgical resected tissues of the colonic mucosa of colorectal carcinoma patients, whose concentration likely depends on the presence of the disease. All the relevant parameters were carefully optimized and the methods were validated using the ideal conditions. The extracted compounds were then subsequently analyzed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. Both optimized protocols were then employed for the analysis of colon tissues (healthy tissue and tumor) from CRC patients, allowing to extract and identify 31 secreted VOCs. Besides, the DI-SPME approach permitted to perform kinetic studies, being independent from the metabolic evolution of the ex vivo systems, monitoring over time the emission of the seven selected VOCs. Therefore, both systems described could be useful to understand which metabolites released come directly from the healthy or tumor tissue of the same patient, but the DI-SPME system, described here for the first time, could provide more important and useful knowledge for understanding CRC metabolism.