# New Challenges in (Bio)Analytical Sample Treatment Procedures for Clinical Applications
## 3 Sample Preparation Techniques for Diagnostic Purposes
### Solidphase Microextraction (SPME)
Thanks to the rapidity and simplicity of this SPME method, with the advantages of minimal sample handling and high sensitivity, this procedure might be considered a valuable tool for the diagnosis of cancerous and neurological diseases correlated with the urinary levels of analyzed amines [40]. Among the markers proposed for the diagnosis of oncological diseases, the scientific literature proposes the analysis of volatile organic compounds (VOCs) as a valid alternative due to their importance in the identification and differentiation of cancer samples. In particular, the determination of urinary VOCs has been proposed as alternative cancer biomarkers for lung, bladder, and breast cancer. [PERSON] et al. validated a new HS-SPME method to obtain a urine volatile metabolomics profile useful for renal cell carcinoma (RCC). These compounds generally require a high efficiency of extraction that depends on the sample preparation method selected. In this study, the authors validated an HS-SPME method after the optimization of different factors that may influence the results, such as the pH of the urine sample, fiber coating, time of incubation, and time and temperature of extraction. They tested different conditions (three pH levels, time, and temperatures of extraction, temperature of incubation) and fibers (five types of SPME fibers); amongst these, the best results were obtained with the exposition of 2 mL of a urine sample with optimal pH 2.0. The use of an acidic pH value confirmed a better HS-SPME extraction in terms of the total of compounds and total chromatographic peak area, but strong acidic conditions may determine some errors due to the chemical degradation of some pH-sensitive compounds. Moreover, the fiber with the highest and better performance for urinary volatile metabolites was divinylbenzene/polyddimethylsiloxane (DVB/PDMS). The optimal analytical performance was also obtained with the addition of 0.59 g of NaCl, 9 min of incubation, and extraction at 68 \({}^{\circ}\)C in 24 min. This method was simple, solvent-free, inexpensive, and fast, with a combination of extraction and preconcentration of a sample with minimal pre-treatment [41]. [PERSON] et al. validated an SPME method to obtain preconcentration of VOCs in the urine sample of patients with prostate cancer already confirmed by prostatic puncture biopsy for subsequent non-target GC-MS analysis. In this procedure, 3 mL of urine with 0.3 g of sodium chloride was placed inside the headspace vial. The extraction equilibrium was obtained with stirring (250 rpm) on the hotplate. The 100-\(\upmu\)m-thick polydimethylsiloxane fibers were used for SPME. These fibers were treated at 250 \({}^{\circ}\)C for 15 min to remove impurities before sample analysis. The fibers were placed in the headspace above the gas of the urine samples for 20 min at 50 \({}^{\circ}\)C and then were inserted into the hot GC injector with the adsorbed analytes, which were desorbed for 4 min in splitless modes. After this procedure, the samples were analyzed with GC-MS. This method showed a very high sensitivity, specificity, and accuracy, and might be a good approach in non-invasive prostate cancer screening [42]. Blood steroid levels are often required for the diagnosis of various endocrinological diseases involving the pituitary-adrenal axis, such as hypercortisolism and Cushing's syndrome. In addition to blood, saliva also represents a biological sample of considerable interest for diagnostic purposes, due to the presence of these hormones in free form with concentration correlated to that present in the blood. Often the saliva concentration of endogenous steroids is very low, and it is necessary to employ a highly sensitive analytical technique. [PERSON] et al. validated a method for the quantification of endogenous steroids (cortisol, testosterone, and progesterone, E1, E2, and E3) in saliva with in vivo SPME.